WO2000053755A2 - 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
WO2000053755A2
WO2000053755A2 PCT/US2000/000376 US0000376W WO0053755A2 WO 2000053755 A2 WO2000053755 A2 WO 2000053755A2 US 0000376 W US0000376 W US 0000376W WO 0053755 A2 WO0053755 A2 WO 0053755A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
prol
pro
antι
polypeptide
Prior art date
Application number
PCT/US2000/000376
Other languages
French (fr)
Other versions
WO2000053755A3 (en
Inventor
Avi J. Ashkenazi
Kevin P. Baker
Audrey Goddard
Austin L. Gurney
Kenneth J. Hillan
Margaret Ann Roy
Colin K. Watanabe
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/US1999/028313 external-priority patent/WO2000032221A2/en
Priority claimed from PCT/US1999/030911 external-priority patent/WO2000075316A1/en
Priority claimed from PCT/US2000/000219 external-priority patent/WO2000053753A2/en
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to AU24952/00A priority Critical patent/AU2495200A/en
Priority to KR1020017011399A priority patent/KR20030002292A/en
Priority to JP2000603377A priority patent/JP2004513602A/en
Priority to PCT/US2000/004342 priority patent/WO2000078961A1/en
Priority to EP00907314A priority patent/EP1263948A2/en
Priority to CA002361840A priority patent/CA2361840A1/en
Priority to AU28837/00A priority patent/AU2883700A/en
Priority to PCT/US2000/004341 priority patent/WO2000053756A2/en
Priority to EP00913764A priority patent/EP1220905A2/en
Priority to CA002362427A priority patent/CA2362427A1/en
Priority to JP2000603379A priority patent/JP2004516227A/en
Priority to KR1020017011406A priority patent/KR20010103046A/en
Priority to PCT/US2000/005841 priority patent/WO2000053758A2/en
Priority to AU35144/00A priority patent/AU3514400A/en
Priority to CA002383254A priority patent/CA2383254A1/en
Priority to AT00916675T priority patent/ATE478145T1/en
Priority to AU37743/00A priority patent/AU3774300A/en
Priority to JP2001500766A priority patent/JP2004522402A/en
Priority to EP00916675A priority patent/EP1210418B1/en
Priority to PCT/US2000/008439 priority patent/WO2000073454A1/en
Priority to DK00939307T priority patent/DK1212417T3/en
Priority to EP00939307A priority patent/EP1212417B1/en
Priority to CA002376116A priority patent/CA2376116A1/en
Priority to PCT/US2000/013705 priority patent/WO2000073445A2/en
Priority to JP2001500757A priority patent/JP4297317B2/en
Priority to AT00939307T priority patent/ATE393825T1/en
Priority to PT00939307T priority patent/PT1212417E/en
Priority to ES00939307T priority patent/ES2307515T3/en
Priority to EP07005022A priority patent/EP1870464A3/en
Priority to PCT/US2000/014941 priority patent/WO2000073348A2/en
Priority to JP2001500672A priority patent/JP2003524406A/en
Priority to EP00941164A priority patent/EP1185648B1/en
Priority to EP07005021A priority patent/EP1873244A3/en
Priority to CA002373915A priority patent/CA2373915A1/en
Priority to AT00941164T priority patent/ATE357518T1/en
Priority to ES00941164T priority patent/ES2287020T3/en
Priority to EP07004855A priority patent/EP1820860A3/en
Priority to EP07005023A priority patent/EP1867719A3/en
Priority to DK00941164T priority patent/DK1185648T3/en
Priority to PCT/US2000/015264 priority patent/WO2000073452A2/en
Priority to PCT/US2000/020710 priority patent/WO2001009327A2/en
Priority to AU63910/00A priority patent/AU6391000A/en
Publication of WO2000053755A2 publication Critical patent/WO2000053755A2/en
Priority to JP2001550255A priority patent/JP4280444B2/en
Priority to EP00982096A priority patent/EP1244784A2/en
Priority to PCT/US2000/030952 priority patent/WO2001049715A2/en
Priority to CA002390685A priority patent/CA2390685C/en
Priority to AU19167/01A priority patent/AU1916701A/en
Priority to CA002491610A priority patent/CA2491610A1/en
Priority to CA2709291A priority patent/CA2709291A1/en
Priority to EP06000589A priority patent/EP1661997A1/en
Priority to CA002496312A priority patent/CA2496312A1/en
Priority to EP06000588A priority patent/EP1690873A3/en
Priority to CA002491258A priority patent/CA2491258A1/en
Priority to EP05025102A priority patent/EP1672070A3/en
Priority to CA002494705A priority patent/CA2494705A1/en
Priority to EP06000583A priority patent/EP1686134A3/en
Priority to PCT/US2000/032678 priority patent/WO2001040466A2/en
Priority to EP06000587A priority patent/EP1690872A3/en
Priority to CA002491433A priority patent/CA2491433A1/en
Priority to EP00983846A priority patent/EP1250426A2/en
Priority to EP06000585A priority patent/EP1661996A1/en
Priority to EP06000582A priority patent/EP1666495A1/en
Priority to EP10005292A priority patent/EP2228446A1/en
Priority to CA002391455A priority patent/CA2391455A1/en
Priority to AU20554/01A priority patent/AU2055401A/en
Priority to CA002492070A priority patent/CA2492070A1/en
Priority to EP06000581A priority patent/EP1666494A1/en
Priority to EP06000584A priority patent/EP1669371A3/en
Priority to CA002490909A priority patent/CA2490909A1/en
Priority to EP06000586A priority patent/EP1688497A1/en
Priority to CA002490853A priority patent/CA2490853A1/en
Priority to JP2001542531A priority patent/JP2004522404A/en
Priority to CA002492049A priority patent/CA2492049A1/en
Publication of WO2000053755A3 publication Critical patent/WO2000053755A3/en
Priority to US09/918,585 priority patent/US20030060406A1/en
Priority to US09/929,769 priority patent/US6914130B2/en
Priority to US09/941,992 priority patent/US20030082546A1/en
Priority to US09/946,374 priority patent/US20030073129A1/en
Priority to US09/978,193 priority patent/US20030073624A1/en
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Priority to US09/978,298 priority patent/US20030134785A1/en
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Priority to US10/124,824 priority patent/US20030077659A1/en
Priority to US10/125,922 priority patent/US7309762B2/en
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Priority to US10/125,924 priority patent/US7342097B2/en
Priority to US10/125,931 priority patent/US20030199063A1/en
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Priority to US10/137,867 priority patent/US20030207349A1/en
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Priority to US10/139,980 priority patent/US7247710B2/en
Priority to US10/140,020 priority patent/US20030207415A1/en
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Priority to US10/140,023 priority patent/US20030207416A1/en
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Priority to US10/140,024 priority patent/US20040058424A1/en
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Priority to US10/140,864 priority patent/US20030207419A1/en
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Priority to US10/140,925 priority patent/US20030073215A1/en
Priority to US10/140,928 priority patent/US20030068798A1/en
Priority to US10/140,809 priority patent/US20030207418A1/en
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Priority to US10/141,756 priority patent/US7488586B2/en
Priority to US10/141,755 priority patent/US7297764B2/en
Priority to US10/141,760 priority patent/US7342104B2/en
Priority to US10/141,701 priority patent/US20030207421A1/en
Priority to US10/142,425 priority patent/US20030207424A1/en
Priority to US10/143,114 priority patent/US20030036180A1/en
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Priority to US10/146,792 priority patent/US20030207428A1/en
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Priority to JP2002341509A priority patent/JP4017507B2/en
Priority to US11/021,329 priority patent/US20050159591A1/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

  • cancers 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)
  • a cell proliferates under conditions in which normal cells would not grow Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness Alteration of gene expression is intimately related to the uncontrolled cell growth and de-differen nation 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 tumoi 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
  • tumoi suppression genes which would normally function to prevent malignant cell growth, and/
  • 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 particulai 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
  • erbB2 also known as he ⁇ 2, oi c-erbB-2
  • p 185 HER2 HER2
  • HER2 transmembi ane glycoprotein receptor
  • EGFR epidermal giowth factor receptor
  • 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 tumo ⁇ genesis 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 PR0212, PRO290. PR0341 , PR0535, PR0619, PR0717, PRO809 PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030 PRO 1097, PROl 107, PRO l 1 1 1 , PRO l 153, PROl 182 PROl 184, PROl 187, PRO 1281 , PR023 PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide
  • the isolated antibody specifically binds to a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809.
  • the antibody induces the death of a cell which expresses a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717 PRO809, PRO830 PR0848, PR0943.
  • PR039 PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide Often, the cell that expresses the PR0212, PRO290, PR0341 PR0535, PR0619, PR0717 PRO809, PRO830, PRO848, PRO943, PRO 1005, PROl 009 PRO 1025 PRO I 030 PRO 1097, PRO l 107, PROl 11 1, PROl 153, PROl 182, PRO l 184, PROl 187, PR01281 , PR023, PR039 PRO834, PRO1317, PRO1710 PRO2094, PR02145 or PR02198 polypeptide is a tumor cell that overexpresses the polypeptide as compared to a normal cell of the same tissue type
  • the antibody is a
  • the invention concerns isolated nucleic acid molecules which encode anti- PR0212, ant ⁇ -PRO290, ant ⁇ -PR0341 , ant ⁇ -PR0535, ant ⁇ -PR0619, ant ⁇ -PR0717, anti PRO809, ant ⁇ -PRO830, anti PR0848, anti PR0943, ant ⁇ -PRO1005, ant ⁇ -PRO1009, anti PRO 1025, ant ⁇ -PRO1030, ant ⁇ -PRO1097, anti- PRO1 107, anti-PROl l l l , anti PR01 153, ant ⁇ -PROH82, anti-PROl 184, anti-PROl 187, ant ⁇ -PRO!
  • the invention concerns a method for producing an ant ⁇ -PR0212, anti PRO290, ant ⁇ -PRO341 , ant ⁇ -PRO535, ant ⁇ PRO619, ant ⁇ -PRO717, ant ⁇ -PRO809, ant ⁇ PRO830, ant ⁇ PR0848, ant ⁇ PR0943, ant ⁇ -PRO1005, ant ⁇ -PRO1009, ant ⁇ -PRO1025, anti PRO 1030, anti PRO 1097, anti-PROl 107, anti PROl 1 11 , anti-PROl 153, anti-PRO 1 182, anti PROl 184, anti-PROl 187, anti PR01281 , ant ⁇ -PR023, ant ⁇ -PR039 ant ⁇ -PR0834, anti-PRO l 317, anti-PROl 710, ant ⁇ -PRO2094, anti PR02145 or ant ⁇ -PR02! 98 antibody, wherein the method comprises culturing a host cell transformed with a nucleic acid molecule which encodes
  • the invention fui ther concerns antagonists of a PR0212, PRO290, PR0341 , PR0535 PR0619, PR0717,
  • PRO809, PRO830, PR0848, PR0943 PRO1005, PRO1009, PRO1025, PRO 1030, PRO1097, PROl 107,
  • PROl 1 11 PROl 153 PROl 182, PROl 184, PROl 187, PRO 1281 PR023 PR039 PR0834 PRO1317,PRO1710
  • PRO2094, PR02145 or PR02198 polypeptide that inhibit one or more of the biological and/or lmmunoiogical functions or activities of a PR0212 PRO290 PR0341 , PR0535 PR06I 9 PR0717 PRO809, PRO830, PR0848 PR0943, PRO1005, PRO1009, PRO 1025, PRO 1030, PRO I 097, PRO l 107 PRO l 1 1 1 , PROl 153, PROl 182 PR01 184 PROl 187, PR01281 PR023 PR039, PR0834, PR01317, PRO1710 PRO2094, PR02145 or PR02198 polypeptide
  • the invention concerns an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule encoding a PR0212.
  • the isolated 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 amp
  • the invention provides a method for determining the presence of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide in a sample suspected of containing a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO l 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR
  • the method comprises exposing the cell to an ant ⁇ -PR0212, ant ⁇ -PRO290, anti- PR0341 , ant ⁇ -PR0535, ant ⁇ -PR0619, ant ⁇ -PR0717, ant ⁇ -PRO809, ant ⁇ -PRO830, ant ⁇ -PR0848, ant ⁇ -PR0943, anti- PRO1005, ant ⁇ -PRO1009, ant ⁇ -PRO1025.
  • ant ⁇ -PRO1030 ant ⁇ -PRO 1097, anti-PRO l 107, anti-PROl 1 1 1 , anti- PRO 1 153 anti-PRO 1 182, anti-PRO 1 184, anti-PRO 1 187, anti-PRO 12 1 ant ⁇ -PR023, ant ⁇ -PR039, ant ⁇ -PR0834 anti-PRO 1 17, anti-PRO 1710, ant ⁇ -PRO2094, ant ⁇ -PR02145 or ant ⁇ -PR02198 antibody and determining binding of the antibody to the cell
  • the present invention concems a method ot diagnosing tumoi in a mammal, comprising detecting the level of expression of a gene encoding a PR0212, PRO290, PR0341 , PR0535, PRO ⁇ 19, PRO717,PRO809,PRO830 PRO848, PRO943, PRO I 005, PRO1009 PRO 1025 PRO 1030, PRO 1097, PRO l 107, PROl l l , PRO1 153, PRO 1 182, PRO 1 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710 PRO2094, PR02145 or PR02198 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 ant ⁇ -PR0212, ant ⁇ -PRO290, ant ⁇ -PR0341 , ant ⁇ -PR0535, an H-PR0619, ant ⁇ -PR0717, ant ⁇ -PRO809, ant ⁇ -PRO830, ant ⁇ -PR0848, ant ⁇ -PR0943, anti-PROl 005, anti-PRO 1009, anti-PRO 1025, anti PRO1030, ant ⁇ -PRO1097, anti-PROl 107, anti-PROl 1 1 1 , anti-PROl 153, ant ⁇ -PROH 82, anti-PROl 184, anti- PROl 187, anti-PRO 1281 , ant ⁇ -PR023, ant ⁇ -PR039, ant ⁇ -PR0834, anti-PRO 1317, anti-PRO 1710, ant ⁇ -PRO2094, ant ⁇ -PR02145 or ant ⁇ -PR02198 antibody with
  • test sample is usually obtained from an individual suspected to have neoplastic cell giovv th or proliferation (e g cancerous cells)
  • the present invention concerns a cancer diagnostic kit comprising an ant ⁇ -PR0212, ant ⁇ -PRO290, ant ⁇ -PR0341 , ant ⁇ -PR0535, anti-PRO ⁇ 19, ant ⁇ -PR0717, ant ⁇ -PRO809, ant ⁇ -PRO830, ant ⁇ -PR0848, ant ⁇ -PRO943, ant ⁇ -PRO1005, ant ⁇ -PRO1009, ant ⁇ -PRO1025, ant ⁇ -PRO1030 anti-PRO 1097, anti-PRO l 107 anti- PROl 1 1 1 , anti-PROl 153, anti-PROl 182, anti-PRO 1 184, anti-PRO 1 187, anti-PRO 1281 , ant ⁇ -PR023, ant ⁇ -PR039, ant ⁇ -PR0834, anti-PRO 1317, anti-PRO 1710, ant ⁇ -PRO2094, ant ⁇ -PR02145 oi ant ⁇ -PR02198 antibody and a carriei (e g , a buffer)
  • the invention concerns a method for inhibiting the growth of tumoi cells comprising exposing tumor cells which express a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830 PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide to an effective amount of an agent which inhibits a biological and/or immunological activity and/or the expression of a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848.
  • the agent preferably is an ant ⁇ -PR0212, ant ⁇ -PRO290, ant ⁇ -PR0341 , ant ⁇ -PR0535, ant ⁇ -PR0619, ant ⁇ -PR0717, anti- PRO809, ant ⁇ -PRO830, ant ⁇ -PR0848, ant ⁇ -PR0943, ant ⁇ -PRO1005, ant ⁇ -PRO1009, ant ⁇ -PRO1025,ant ⁇ -PRO1030, anti-PROl 097, anti-PRO 1 107, anti-PROl 1 1 1 , anti-PRO 1 153, anti-PROl 182, anti
  • 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 tumoi cells and the label on the container indicates that the composition can be used for treating conditions characterized by overexpression ot a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717. PRO809, PRO830. PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PROl 107,PROl 1 1 1 , PROl 153, PRO l 182, PROl 184, PRO! 187, PRO l 281 , PR023, PR039.
  • the active agent in the composition is an agent which inhibits an activity and/or the expression of a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO1009, PRO1025, PRO1030, PRO 1097, PROl 107 PROl 1 1 1 PROl 153, PROl 182, PROl 184, PROl 187 PROl 281 PR023, PR039, PR0834, PR01317.
  • the active agent is an ant ⁇ -PR0212, ant ⁇ -PRO290, ant ⁇ -PR0341 , ant ⁇ -PR0535. ant ⁇ -PR0619, anti- PR0717 ant ⁇ -PRO809 ant ⁇ -PRO830, ant ⁇ -PR0848, ant ⁇ -PR0943, anti-PROl 005. anti-PRO 1009, anti-PRO 1025, anti-PRO 1030, anti-PRO 1097, anti-PRO 1 107, anti-PRO 1 1 1 1 , anti-PRO 1 153, anti-PROl 182.
  • the invention also provides a method for identifying a compound that inhibits an activity of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PROl 025, PRO 1030, PRO 1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281.
  • PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide comprising contacting a candidate compound with a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809.
  • the invention provides a method for identifying a compound that inhibits the expression of a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830. PR0848, PR0943.
  • this method comprises the steps of (a) contacting cells and a candidate compound to be screened under conditions suitable tor allowing expression ofthePRO212.PRO290,PRO341,PRO535,PRO619,PRO717,PRO809,PRO830.PRO848,PRO943,PRO1005, PROI009, PRO1025, PRO1030. PRO1097, PROl 107, PROl 111 PROl 153, PROl 182, PRO! 184, PROl 187, PRO 1281 , PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182.
  • 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 PR0212, PRO290, 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 y et more preferably at least about 89% sequence identity, yet moi e 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, ⁇ et more preferably at least about 93% sequence identity, yet moie prefei ably 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
  • 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 compnsing a nucleotide sequence encoding a PR0212, PRO290, PR0341 , PR0535, PR06 I 9, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097.
  • Another embodiment is directed to fragments of a PR0212, PRO290, PR0341 , PR0535, PR06 I 9 PR0717 PRO809, PRO830 PR0848, PR0943, PRO 1005 PRO 1009, PRO 1025.
  • PRO 1030 PRO 1097, PRO l 107, PROl 1 1 1.
  • PROl 710, PRO2094, PR02145 or PR02198 polypeptide-encoding nucleotide sequence may be detei mined in a loutine manner by aligning the PR0212, PRO290, PR0341 , PR0535, PR0619. PR0717.
  • PRO l 1 1 1.
  • the invention provides isolated PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PROl 097, PRO 1 107, PROl 1 1 1 , PROl 153, PROH 82, PR01 184, PR01 187, PR01281 , PR023, PR039 PRO834, PRO1317, PRO1710 PRO2094, PR02145 or PR02198 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified
  • the invention concerns an isolated PR0212, PRO290, PR0341 , PR0535, PR0619 PR0717, PRO809, PRO830, PR0848 PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PROl 097, PRO 1 107 PROl 1 1 1 , PROl 153 PROl 182, PROl 184, PROl 187, PRO 1281 PR023 PR039, PR0834 PRO1317, PRO17 I0, PRO2094, PR02145 or PR02198 polypeptide comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, moie 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 identit , yet more preferably at least about 89%
  • the invention concerns an isolated PR0212, PRO290, PR0341 , PR0535, PR0619, 5 PR0717, PRO809, PRO830, PR0848, PR0943, PROl 005, PRO1009, PROl 025, PRO1030, PROl 097, PROl 107, PROl 1 1 1 , PROl 153, PRO 1 182, PROl 184, PROl 187 PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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
  • 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
  • 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 PR0212, PRO290, PR0341 , PR0535, PR0619,
  • PRO809, PRO830, PR0848, PR0943 PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PRO 1 107 PROl 11 1 , PROl 153, PROl 182, PROl 184,PROH 87, PRO1281 , PRO23, PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 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
  • PR0212 PRO290 PR0341 PR0535 PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO1025 PROl 030, PROl 097, PRO 1 107, PROl 1 1 1 , PRO 1 153 PR01 182 PRO l 184, PRO l 187 PR01281 , PR023 PR039, PR0834 PR01317 PRO 1710, PRO2094 PR02145 oi PR02198 polypeptide having a full-length amino acids, yet more prefei ably at least about 98% positives and yet more preferably at least about 99% positives when compared with the amino acid sequence ot a PR0212 PRO290 PR0341 , PR0535 PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO1025 PROl 030, PROl 097, PRO 1 107, PROl 1 1 1 , PRO 1 153 PR01 182 PRO
  • the invention provides an isolated PR02 I 2 PRO290, PR0341 , PR0535, PR0619 PR0717 PRO809 PRO830 PR0848 PR0943 PRO 1005 PRO1009 PRO I 025 PRO I 030 PRO 1097, PRO l 107 PROl 111, PROl 153, PRO 1182, PROl 184.PR01187,PRO1281,PRO23.PRO39,PRO834,PRO1317,PRO17i0, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PRO 1182, PRO 1184, PRO 1187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 orPR02198 polypeptide and recovering the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROH53,PR01182, PROl
  • Another aspect of the invention provides an isolated PR0212, PRO290, PR0341, PR0535, PR0619.
  • 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097.
  • the invention concerns antagonists of a native PR0212. PRO290, PR0341. PR0535. PR0619, PR0717, PRO809, PRO830, PR0848. PR0943, PROl 005.
  • PRO 1030. PRO1097,PRO1107,PROlllI,PRO1153,PRO1182,PROI184,PRO1187.PRO1281,PRO23,PRO39,PRO834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide as defined herein. In a particular embodiment.
  • the antagonist is an anti-PR0212, anti-PRO290, anti-PR0341, anti-PR0535, anti-PR0619, anti-PR0717, anti- PRO809, anti-PRO830. anti-PR0848. anti-PR0943, anti-PRO 1005, anti-PRO 1009, anti-PRO 1025, anti-PRO 1030. anti-PRO 1097, anti-PRO 1107, anti-PRO 1111, anti-PRO 1153, anti-PRO 1182, anti-PRO 1184, anti-PRO 1187, anti- PRO 1281. anti-PR023. anti-PR039. anti-PR0834. anti-PRO 1317. anti-PRO 1710, anti-PRO2094. anti-PR02145 or anti-PR02198 antibody or a small molecule.
  • the invention concerns a method of identifying antagonists to a PR0212,
  • PROl 710, PRO2094, PR02145 or PR02198 polypeptide Preferably, the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030.
  • PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PR01184, PR01187, PR01281, PR023, PR039, PR0834, PROL317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide is a native PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281, PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide
  • the invention concerns a composition of matter comprising a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PROl 710, PRO2094, PR02145 orPR02198 polypeptide, or an antagonist of aPR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PR0
  • the carrier is a pharmaceutically acceptable carrier
  • Another embodiment of the present invention is directed to the use of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717.
  • PRO 1030, PRO 1097, PROl 107, PROllll PROl 153, PROl 182,PR01184,PR01187,PR01281.PR023.PR039.PR0834 PROl 317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide, or an antagonist thereof as hereinbefore described, or an ant ⁇ -PR0212, ant ⁇ -PRO290 ant ⁇ -PR0341, ant ⁇ -PR0535, ant ⁇ -PR06I9.
  • ant ⁇ -PRO2094, ant ⁇ -PR02145 or ant ⁇ -PR02198 antibody for the preparation of a medicament useful in the tieatment of a condition which is responsive to the PR0212, PRO290,PRO341 PR0535.PR0619,PR0717 PRO809 PRO830,PRO848.PRO943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184 PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PROI710, PRO2094.
  • PR02145 oi PR02198 polypeptide an antagonist thereof or an ant ⁇ -PR0212, ant ⁇ -PRO290, ant ⁇ -PR034I.
  • 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 coh, yeast, or Baculovirus-infected insect cells
  • a process for producing any of the herein described polypeptides is further provided and comprises culturmg host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture
  • the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence
  • Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulm
  • 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 ohgonucleotide 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 PR0212, wherein the nucleotide sequence (SEQ ID NO 1 ) is a clone designated herein as DNA30942-1 134 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 PR0212 polypeptide as derived from the coding sequence of SEQ ID NO 1
  • Figures 3A through 3B show the nucleotide sequence (SEQ ID NO 6) ot a cDNA containing a nucleotide sequence encoding native sequence PRO290, wherein the nucleotide sequence (SEQ ID NO 6) is a clone designated herein as DNA35680-1212 Also presented in bold font and underlined aie the positions of the respective start and stop codons
  • Figure 4 shows the amino acid sequence (SEQ ID NO 7) of a native sequence PRO290 polypeptide as derived from the coding sequence ot SEQ ID NO 6
  • Figure 5 shows the nucleotide sequence (SEQ ID NO 9) of a cDNA containing a nucleotide sequence encoding native sequence PR0341 wherein the nucleotide sequence (SEQ ID NO 9) is a clone designated herein as DNA26288- 1239 Also presented in bold font and underlined are the positions ot the lespective start and stop codons
  • Figure 6 shows the amino acid sequence (SEQ ID NO 10) ot a native sequence PR0341 polypeptide as derived from the coding sequence of SEQ ID NO 9
  • Figure 7 shows the nucleotide sequence (SEQ ID NO 1 ) of a cDNA containing a nucleotide sequence encoding native sequence PR0535, wherein the nucleotide sequence (SEQ ID NO ! 3) is a clone designated herein as DNA49143-1429 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 14) of a native sequence PR0535 polypeptide as derived from the coding sequence of SEQ ID NO 13
  • Figure 9 shows the nucleotide sequence (SEQ ID NO 15) ot a cDNA containing a nucleotide sequence encoding native sequence PR0619, wherein the nucleotide sequence (SEQ ID NO 15) is a clone designated herein as DNA49821 - 1562 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 16) ot a native sequence PR0619 polypeptide as derived from the coding sequence of SEQ ID NO 15
  • Figure 1 1 shows the nucleotide sequence (SEQ ID NO 17) of a cDNA containing a nucleotide sequence encoding native sequence PR0717, wherein the nucleotide sequence (SEQ ID NO 17) is a clone designated herein as DNA50988-1 26 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 12 shows the ammo acid sequence (SEQ ID NO 18) of a native sequence PR0717 polypeptide as derived from the coding sequence of SEQ ID NO 17
  • Figure 13 shows the nucleotide sequence (SEQ ID NO 22) of a cDNA containing a nucleotide sequence encoding native sequence PRO809, wherein the nucleotide sequence (SEQ ID NO 22) is a clone designated herein as DNA57836-1338 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 23) of a native sequence PRO809 polypeptide as derived from the coding sequence of SEQ ID NO 22
  • Figure 15 shows the nucleotide sequence (SEQ ID NO 24) ot a cDNA containing a nucleotide sequence encoding native sequence PRO830, wherein the nucleotide sequence (SEQ ID NO 24) is a clone designated herein as DNA56866-1342 Also presented in bold font and undeihned aie the positions of the respective start and stop codons
  • Figuie 16 shows the amino acid sequence (SEQ ID NO 25) of a native sequence PRO830 polypeptide as derived from the coding sequence of SEQ ID NO 24
  • Figuie 17 shows the nucleotide sequence (SEQ ID NO 26) ot a cDNA containing a nucleotide sequence encoding native sequence PR0848, wherein the nucleotide sequence (SEQ ID NO 26) is a clone designated herein as DNA59839- 1461 Also presented in bold font and undei lined ai e the positions of the respective start and stop codons Figuie 18 shows the amino acid sequence (SEQ ID NO 27) of a native sequence PR0848 polypeptide as derived from the coding sequence of SEQ ID NO 26
  • Figures 19A through 19B show the nucleotide sequence (SEQ ID NO 28) ot a cDNA containing a nucleotide sequence encoding native sequence PR0943 w herein the nucleotide sequence (SEQ ID NO 28) is a clone designated heiein as DNA52192- 1369 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 29) of a native sequence PR0943 polypeptide as derived from the coding sequence of SEQ ID NO 28
  • Figure 21 shows the nucleotide sequence (SEQ ID NO 33) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1005 wherein the nucleotide sequence (SEQ ID NO 33) is a clone designated herein as DNA57708 141 1 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 34) of a native sequence PRO 1005 polypeptide as derived from the coding sequence of SEQ ID NO 33
  • Figure 23 show the nucleotide sequence (SEQ ID NO 35) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1009, wherein the nucleotide sequence (SEQ ID NO 35) is a clone designated herein as DNA57129-1413 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 36) of a native sequence PRO 1009 polypeptide as derived from the coding sequence of SEQ ID NO 35
  • Figure 25 shows the nucleotide sequence (SEQ ID NO 37) ot a cDNA containing a nucleotide sequence encoding native sequence PRO 1025, wherein the nucleotide sequence (SEQ ID NO 37) is a clone designated herein as DNA59622 1334 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 38) of a native sequence PRO 1025 polypeptide as derived from the coding sequence of SEQ ID NO 37
  • Figure 27 shows the nucleotide sequence (SEQ ID NO 39) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1030, wherein the nucleotide sequence (SEQ ID NO 39) is a clone designated herein as DNA59485 1336 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 40) of a native sequence PRO 1030 polypeptide as denved from the coding sequence of SEQ ID NO 39
  • Figure 29 shows the nucleotide sequence (SEQ ID NO 41 ) of a cDNA containing a nucleotide sequence encoding native sequence PROl 097, wherein the nucleotide sequence (SEQ ID NO 41 ) is a clone designated herein as DNA59841 1460 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 30 shows the amino acid sequence (SEQ ID NO 42) of a native sequence PRO 1097 polypeptide as derived from the coding sequence of SEQ ID NO 41
  • Figure 31 shows the nucleotide sequence (SEQ ID NO 43) ot a cDNA containing a nucleotide sequence encoding nat ⁇ e sequence PRO 1 107 whei ein the nucleotide sequence (SEQ ID NO 43) is a clone designated herein as DNA59606- 1471 Also presented in bold font and underlined are the positions ot the respecm e stai t and stop codons
  • Figure 32 shows the amino acid sequence (SEQ ID NO 44) oi a native sequence PRO l 107 polypeptide as denved fiom the coding sequence of SEQ ID NO 4
  • Figure 33 shows the nucleotide sequence (SEQ ID NO:45) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1 1 1 1 , wherein the nucleotide sequence (SEQ ID NO:45) is a clone designated herein as DNA58721 -1475. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 34 shows the amino acid sequence (SEQ ID NO:46) of a native sequence PROl 1 1 1 polypeptide as derived from the coding sequence of SEQ ID NO:45.
  • Figure 35 shows the nucleotide sequence (SEQ ID NO:48) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1 153, wherein the nucleotide sequence (SEQ ID NO:48) is a clone designated herein as DNA59842-1502. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 36 shows the amino acid sequence (SEQ ID NO:49) of a native sequence PROl 153 polypeptide as derived from the coding sequence of SEQ ID NO:48.
  • Figure 37 shows the nucleotide sequence (SEQ ID NO:50) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1 182, wherein the nucleotide sequence (SEQ ID NO:50) is a clone designated herein as DNA59848-1512. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 38 shows the amino acid sequence (SEQ ID NO:51 ) of a native sequence PROl 182 polypeptide as derived from the coding sequence of SEQ ID NO:50.
  • Figure 39 shows the nucleotide sequence (SEQ ID NO:52) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1 184, wherein the nucleotide sequence (SEQ ID NO:52) is a clone designated herein as DNA59220- 1514. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 40 shows the amino acid sequence (SEQ ID NO:53) of a native sequence PROl 184 polypeptide as derived from the coding sequence of SEQ ID NO:52.
  • Figure 41 shows the nucleotide sequence (SEQ ID NO:54) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1 187, wherein the nucleotide sequence (SEQ ID NO:54) is a clone designated herein as DNA62876-1517. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 42 shows the amino acid sequence (SEQ ID NO:55) of a native sequence PRO l 187 polypeptide as derived from the coding sequence of SEQ ID NO:54.
  • Figures 43A through 43B show the nucleotide sequence (SEQ ID NO:56) of a cDNA containing a nucleotide sequence encoding native sequence PROl 281 , wherein the nucleotide sequence (SEQ ID NO:56) is a clone designated herein as DNA59820-1549. Also presented in bold font and underlined are the positions of the respective start and stop codons.
  • Figure 44 shows the amino acid sequence (SEQ ID NO:57) of a native sequence PRO 1281 polypeptide as derived from the coding sequence of SEQ ID NO:56.
  • Figure 45 shows the nucleotide sequence (SEQ ID NO:64) of a cDNA containing the nucleotide sequence encoding native sequence PR023, wherein the nucleotide sequence (SEQ ID NO:64) is a clone designated herein as DNA36640.
  • Figure 46 shows the amino acid sequence (SEQ ID NO 65) of a native sequence PR023 polypeptide as derived from the coding sequence of SEQ ID NO 64 Also presented is a further truncated form of a native sequence PR023 polypeptide
  • Figure 47 shows the nucleotide sequence (SEQ ID NO 66) of a cDNA containing the nucleotide sequence encoding native sequence PR039, wherein the nucleotide sequence (SEQ ID NO 66) is a clone designated as DNA36651
  • Figure 48 shows the amino acid sequence (SEQ ID NO 67) of a native sequence PR039 polypeptide as derived from the coding sequence of SEQ ID NO 66
  • Figure 49 shows the nucleotide sequence (SEQ ID NO 68) of a cDNA containing the nucleotide sequence encoding native sequence PR0834, wherein the nucleotide sequence (SEQ ID NO 68) is a clone designated herein as DNA56538
  • Figure 50 shows the amino acid sequence (SEQ ID NO 69) of a native sequence PR0834 polypeptide as derived from the coding sequence of SEQ ID NO 68 Also presented is a pro-polypeptide of the PR0834 polypeptide
  • Figures 51 A through 51B show the nucleotide sequence (SEQ ID NO 70) of a cDNA containing the nucleotide sequence encoding native sequence PROl 317, wherein the nucleotide sequence (SEQ ID NO 70) is a clone designated herein as DNA71 166 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 52 shows the amino acid sequence (SEQ ID NO 71 ) of a native sequence PR01317 polypeptide as derived from the coding sequence of SEQ ID NO 70
  • Figure 53 shows the nucleotide sequence (SEQ ID NO 72) of a cDNA containing the nucleotide sequence encoding native sequence PRO 1710 wherein the nucleotide sequence (SEQ ID NO 72) is a clone designated herein as DNA82331 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 54 shows the amino acid sequence (SEQ ID NO 73) of a native sequence PROl 710 polypeptide as derived from the coding sequence of SEQ ID NO 72 Also presented is a pro polypeptide of the PRO l 710 polypeptide
  • Figures 55A through 55B show the nucleotide sequence (SEQ ID NO 74) of a cDNA containing the nucleotide sequence encoding native sequence PRO2094, wherein the nucleotide sequence (SEQ ID NO 74) is a clone designated herein as DNA83123 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 56 shows the amino acid sequence (SEQ ID NO 75) ot a native sequence PRO2094 polypeptide as derived from the coding sequence of SEQ ID NO 74
  • Figure 57 shows the nucleotide sequence (SEQ ID NO 76) of a cDNA containing the nucleotide sequence encoding native sequence PR02145 whei ein the nucleotide sequence (SEQ ID NO 76) is a clone designated hei ein as DNA88050 Also presented in bold font and underlined are the positions of the iespective stait and stop codons
  • Figure 58 shows the amino acid sequence (SEQ ID NO 77) of a native sequence PR02145 polypeptide derived from the coding sequence of SEQ ID NO 76
  • Figures 59A through 59B show the nucleotide sequence (SEQ ID NO 78) ot a cDNA containing the nucleotide sequence encoding nativ e sequence PR02198 wherein the nucleotide sequence (SEQ ID NO 78) is a clone designated herein as DNA88153 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 60 shows the amino acid sequence (SEQ ID NO 79) of a native sequence PR02198 polypeptide derived from the coding sequence of SEQ ID NO 78
  • Figure 61 is a map of Chromosome 4 showing the mapping region of DNA50988-1326
  • Figure 62 is a map of Chromosome 2 showing the mapping region of DNA57708-141 1
  • Figure 63 is a map of Chromosome 6 showing the mapping region of DNA83123
  • Figure 64 is a map of Chromosome 16 showing the mapping region of DNA88153
  • 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 "amphcon "
  • mRNA messenger RNA
  • i e the level of gene expression
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues
  • cancer and “cancerous” refer 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, ghoblastoma, 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
  • treatment' refers to both therapeutic treatment and prophylactic or preventative measuies
  • 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" ot cancer includes all phenomena that compromise the well-being ot the patient This includes, without limitation, abnormal or uncontrollable cell giowth metastasis, interference with the normal functioning of neighboring cells, release ot cytokines or other secretory pioducts at abnormal levels, suppression oi aggravation ot 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 Pi eferably, the mammal is human
  • Car ⁇ ei s as used herein include pharmaceutically acceptable cai ⁇ ei s 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 oi immunoglobuhns, hydrophilic polymers such as polyvinylpyrrohdone, amino acids such as glycine, glutamine, asparagine, arginme or lysine monosacchandes, disacchandes, and other carbohydrates including glucose mannose, or dextnns, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counte ⁇ ons such as sodium, and
  • 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 , I P5 , Y 9 " 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 ad ⁇ amycin, doxorubicin, epirubicin, 5-fluorourac ⁇ l, cytosine arabinoside (' Ara C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e g , pachtaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rh ⁇ ne-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastme, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincnstine, vinorelbme, carboplatin, temposide, daunomycin, carminomycin
  • the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressmg such genes in S phase
  • growth inhibitory agents include agents that block cell cycle progression (at a place othei than S phase), such as agents that induce Gl arrest and M phase arrest
  • Classical M phase blockers include the vincas (vincnstine and vinblastme), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin etoposide and bleomycin
  • agents that arrest Gl also spill over into S-phase arrest for example DNA alkylatmg agents such as tamoxifen, prednisone, dacarbazine mechlorethamine, cisplatin, methotrexate 5 fluorouracil, and aia-C
  • DNA alkylatmg agents such as tamoxifen, prednisone, dacarbazine mechlorethamine, cisplatin, methotrexate 5
  • Doxorubicin' is an anthracychne antibiotic
  • the full chemical name ot doxorubicin is (8S-c ⁇ s) 10 [(3 amino 2,3,6-t ⁇ deoxy ⁇ -L-lyxo hexapyranosyl)oxy]-7 8 9 10 tetiahydro 6,8, 1 1 -t ⁇ hydroxy 8-(hyd ⁇ o ⁇ yacet ⁇ l) 1 methoxy 5 12-naphthacened ⁇ one
  • cytokine is a generic term tor pioteins leleased by one cell population which act on another cell as intercellular mediatoi s
  • lymphokines include 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, proinsuhn, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH).
  • FSH follicle stimulating hormone
  • TSH thyroid stimulating hormone
  • LH luteinizing hormone
  • hepatic growth factor hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor- ⁇ and - ⁇ , mulle ⁇ an-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothehal growth factor, integrin, thrombopoietin (TPO), nerve growth factors such as NGF- ⁇ , platelet- growth factor, transforming growth factors (TGFs) such as TGF- ⁇ and TGF- ⁇ , insulin-like growth factor-I and -II, erythropoietin (EPO), osteoinductive factors, interferons such as interferon - ⁇ , - ⁇ , and - ⁇ , colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocy te-CSF (G- CSF), interleukins (IL
  • the prodrugs of this invention include, but are not limited to, phosphate- containing prodrugs, thiophosphate-containmg prodrugs, sulfate-containing prodrugs, peptide-containin prodrugs, D-amino acid-modified prodrugs, glysocylated prodrugs, R-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-contaimng prodrugs, 5- fluorocytosine and other 5-fluorou ⁇ d ⁇ ne prodrugs which can be converted into the more active cytotoxic free drug
  • cytotoxic drugs that can be denvatized 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 growth, 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 mhibitoiy, cytostatic and/or cytotoxic effect and/or apoptosis of the target cells
  • An “effective amount " of a PRO 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 "theiapeuticalK effective amount of a PRO polypeptide antagonist for purposes of treatment of tumor may be determined empirically and in a routine manner
  • a “growth inhibitory amount” of a PRO antagonist is an amount capable of inhibiting the growth of a cell, especially tumor, e g , cancer cell, either in vitio or in vivo
  • a “growth inhibitory amount" of a PRO antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
  • a "cytotoxic amount” of a PRO antagonist is an amount capable of causing the destruction of a cell, especially tumor, e g , cancer cell, either in vitio ox in vivo
  • a "cytotoxic amount" of a PRO antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
  • PRO polypeptide and "PRO” as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (t e , PRO/number) refers to specific polypeptide sequences as described herein
  • PRO/number polypeptide and “PRO/number” wherein the term “number” is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein)
  • the PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods
  • a “native sequence PRO polypeptide” comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means The term "native sequence PRO polypeptide” specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e g , an extracellular domain sequence), naturally-occurring variant forms (e g , alternatively spliced forms) and naturally-occurring allehc variants of the polypeptide.
  • the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures Start and stop codons are shown in bold font and underlined in the figures However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionme residues designated herein as am o acid position 1 in the figures, it is conceivable
  • the PRO polypeptide "extracellular domain' or "ECD” refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains
  • a PRO polypeptide ECD will have less than 1 % of such transmembrane and/oi cytoplasmic domains and preferably will have less than 0 5% of such domains
  • any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain
  • the exact boundaries of a transmembrane domain may vary but most hkeh by no more than about 5 amino acids at either end of the domain as initially identified herein
  • an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on eithei side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide.
  • Table 1 provides the complete souice code for the ALIGN-2 sequence comparison computei program This source code may be uiutinely compiled toi use on a UNIX operating system to piovide the ALIGN-2 sequence comparison computer program
  • 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 PRO 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 PRO-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
  • Page 1 of day. h -. include ⁇ std ⁇ o.h >
  • 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 >J[2], /* jmp index for a path */ static nc[2], /* number at start of current line */ static m[2], /* current elem number — for gapping */ static s ⁇ z[2], static char *ps[21 , /* ptr to current element */ static char *po[2], /* ptr to next output char slot */ static char out[2][P LINE] /* output line */ static char star[P LINE], /* set by stars() *//
  • *ps[ ⁇ ] toupper(*ps[ ⁇ ]), po[ ⁇ ] + + , ps[ ⁇ ] + + ,
  • *py++ toupper(*px); if ( ⁇ ndex("ATGCU",*(py-l))) natgc + + ;
  • Page2ofnwsubr.c ...readjmps if ⁇ 0 && dx[dmax] offset && fj) ⁇
  • Percent (%) amino acid sequence identity' with lespect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO 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 ammo acid sequence identity can be achieved in various ways that are within the skill in the art, tor instance, using publicly available computei software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % amino acid sequence identity values are 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
  • % 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
  • NCBI-BLAST2 Altschul et l Nucleic Acids Res , 25 3389-3402 ( 1997)
  • % amino acid sequence identity of a given amino acid sequence A to, with, oi against a given amino acid sequence B is calculated as follows
  • % amino acid sequence identity may also be determined 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.
  • a % amino acid sequence identity value is determined by dividing (a) the number of matching identical ammo 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 (; 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
  • the amino acid sequence A is the comparison amino acid sequence of interest and the ammo acid sequence B is the amino acid sequence of the PRO polypeptide of interest
  • a PRO variant polynucleotide w ill have at least about 80% nucleic acid sequence identity, moie pieterably at least about 81 % nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, moie preferably at least about 83%- nucleic acid sequence identity, more pi eferably at least about 84% nucleic acid sequence identity more preferably at least about 85% nucleic acid sequence identity, moie preferably at least about 86% nucleic acid sequence identity .
  • nucleic acid sequence identity with the nucleic acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an
  • PRO variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in length, more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more
  • Percent (%) nucleic acid sequence identity with respect to the PRO 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 PRO 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 oi Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % 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
  • the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows
  • the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follow s
  • a % nucleic acid sequence identity value is detei mined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence ot 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 ot interest (/ e , the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant
  • PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7), Figure 6 (SEQ ID NO 10), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 16), Figure 12 (SEQ ID NO 18), Figure 14 (SEQ ID NO 23), Figure 16 (SEQ ID NO 25), Figure 18 (SEQ ID NO 27), Figure 20 (SEQ ID NO 29), Figure 22 (SEQ ID NO 34), Figure 24 (SEQ ID NO 36), Figure 26 (SEQ ID NO 38), or Figure 28 (SEQ ID NO 40), Figure 30 (SEQ ID NO 42), Figure 32 (SEQ ID NO 44), Figure 34 (SEQ ID NO 46), Figure 36 (SEQ ID NO 49), Figure 38 (SEQ ID NO 51 ), Figure 40 (SEQ ID NO 53), Figure 42 (SEQ ID NO 2 (
  • 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 ammo acid residue of interest or are a preferred substitution (as defined in
  • 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 heiem means polypeptide that has been identified and separated and/or lecovered from a component ot its natural environment
  • the isolated polypeptide is free ot association with all components with which it is natuially associated Contaminant components of its natural environment are matenals that would typically mterteie ith diagnostic or therapeutic uses foi 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 ammo acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO natural environment will not be present Ordinari
  • an "isolated" nucleic acid molecule encoding a PRO polypeptide or an “isolated " nucleic acid encoding an anti-PRO 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 PRO-encoding nucleic acid or the anti-PRO-encoding nucleic acid Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated
  • An isolated PRO-encoding nucleic acid molecule or an anti- PRO-encodmg 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 PRO-encoding nucleic acid molecule or the anti-PRO- encoding nucleic acid molecule as it exists in natural cells
  • an isolated nucleic acid molecule encoding a PRO polypeptide or an anti-PRO antibody includes PRO-nucleic acid molecules and anti-PRO-n
  • 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 nbosome 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 ot the polypeptide
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transciiption ot the sequence
  • a nbosome 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 secretoiy leader, contiguous and in reading phase
  • enhancers do not have to be contiguous Linking is accomplished by hgation at convenient restriction sites If such sites do not exist, the synthetic ohgonucleotide adaptors or linkers are used in accordance with conventional practice
  • antibody is used in the broadest sense and specifically covei s, for example, single anti- PR0212, ant ⁇ -PRO290. ant ⁇ -PR0341 , ant ⁇ -PR0535. ant ⁇ -PR0619 ant ⁇ -PR0717, ant ⁇ -PRO809, ant ⁇ -PR083 ⁇ . anti- PR0848, ant ⁇ -PR0943. anti-PRO 1005, anti-PROl 009 anti-PRO 1025, anti-PRO 1030, anti-PRO 1097, anti- PROl 107, anti-PRO l 1 1 1 , anti-PROl 153, anti-PROl 182. anti-PRO l 184, anti-PRO l 187, anti-PRO 1281 , anti- PR023.
  • “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 01 M sodium chlo ⁇ de/0 0015 M sodium c ⁇ trate/0 1 % sodium dodecyl sulfate at 50°C, (2) employ during hybridization a denaturing agent, such as formamide, foi example, 50% (v/v) formamide with 0 1 % bovine serum album ⁇ n/0 1 % F ⁇ coll/0 1 % polyvinylpyrrohdone/ 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 citiate) 50 mM sodium phosphate (pH 6 8), 0 1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon speim
  • Modely stringent conditions may be identified as described by Sambrook etal Molecular Cloning A Laboratory Manual, New York Cold Spring Harbor Press 1989, and include the use ot w ashing solution and hybridization conditions (e g , temperature, ionic strength and % SDS) less stringent than those described above
  • An example ot moderately stringent conditions is overnight incubation at 37"C in a solution comprising 20% formamide. 5 x SSC ( 1 0 mM NaCl 1 mM t ⁇ sodium citrate) 50 M 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 filtei s 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
  • epitopope tagged when used heiein refers to a chimeric polypeptide comprising a PR0212 PRO290, PR0341 , PR0535. PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025. PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PRO l 187, PR01281 PR023, PR039, PR0834, PROl 317.
  • 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 PR0212.
  • PR0535 PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl H I , PROl 153, PROl 182, PROl 184, PR01 187, PRO1281, PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide, wherein "biological" activity refers to a function (either inhibitory or stimulatory) caused by a native or natui ally-occurring PR0212, PRO290, PR0341. PR0535.
  • PRO 12943 PRO 1005, PRO 1009, PRO1025, PRO1030, PRO1097, PRO l 107, PRO l 1 1 1 , PROl 153, PRO l 182, PRO l 184.
  • Bioactivity' in the context ot an antibody or another antagonist molecule that can be identified by the screening assays disclosed herein e g . an oiga c or inorganic small molecule peptide. etc
  • Bioactivity' in the context ot an antibody or another antagonist molecule that can be identified by the screening assays disclosed herein is used to refer to the ability of such molecules to bind or complex with the polypeptides encoded b ⁇ the amplified genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides w ith other cellular proteins oi otherwise interfere with the transcription or translation of a PR0212, PRO290. PR034 I , PR0535, PR0619.
  • PR0717, PRO809, PRO830, PR0848 PR0943 PRO 1005, PRO 1 09, PRO 1025 PRO 1030, PRO 1097, PROl 107 PROl 111, PROl 153, PROl 182, PROl 184,PRO1187,PRO1281.PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 polypeptide
  • a preferred biological activity is growth inhibition of a target tumor cell
  • Anothei preferred biological activity is cytotoxic activity resulting in the death of the target tumor cell
  • biological activity in the context of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PROl 005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PRO 1107, PROl 111, PROl 153, PRO 1182, PROl 184, PROl 187, PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO17
  • the phrase "lmmunological activity" means lmmunological cross-reactivity with at least one epitope of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 orPR02198 polypeptide
  • “lmmunological cross-reactivity” as used herein means that the candidate polypeptide is capable ot competitively inhibiting the qualitative biological activity of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184,PR01187, PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 polypeptide having this activity with polyclonal antisera raised against the known active PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PROl 005.
  • 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 lmmunological cross-reactivity preferably is "specific .
  • antagonist is used in the broadest sense, and includes any molecule that partially or fully blocks inhibits, or neutralizes a biological activity ot a native PR0212, PRO290, PR0341. PR0535 PR0619 PR0717 PRO809, PRO830. PR0848, PR0943, PRO1005, PRO 1009. PRO1025.
  • 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 PR0212, PRO290, PR0341 , PR0535. PR0619, PR0717, PRO809, PRO830. PR0848, PR0943, PRO1005, PRO1009 PRO 1025, PRO 1030, PRO1097.
  • PROl 107 PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094.
  • PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide A "small molecule" is defined herein to have a molecular weight below about 500 Daltons.
  • Antibodies are glycoproteins having the same structural characteristics While antibodies exhibit binding specificity to a specific antigen, immunoglobuhns 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 immunoglobuhns” are usually heterotetrame ⁇ c 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 immunoglobulm isotypes Each heavy and light chain also has regularly spaced intrachain disulfide bridges Each heavy chain has at one end a variable domain (V instruct) followed by a number of constant domains 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 refeis to the fact that certain poi tions of the variable domains differ extensivelv in sequence among antibodies and are used in the binding and specificity ot each particulai antibody for its particulai antigen
  • CDRs complementarity-determining regions
  • hypervanable regions both in the light-chain and the heavy-chain variable domains
  • the moie highly conserved portions of variable domains are called the amewoi k (FR) regions
  • the variable domains of native heavy and light chains each comprise tout FR regions largely adopting a ⁇ -sheet configuration, connected b ⁇ thiee CDRs which form loops connecting and in some cases forming part of, the ⁇ -sheet structure
  • the CDRs in each chain are held togethei in close pioximitv by the FR regions and, with the CDRs from the other chain contribute to the formation of the antigen-bindmg site of antibodies ( .
  • hypervanable region refers to the amino acid residues of an antibody which are responsible for antigen-binding
  • the hypervanable region comprises amino acid residues from a "complementarity determining region" or "CDR" (. e , residues 24-34 (Ll ).
  • 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') 2 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 contiguiation that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer Collectively, the six CDRs confer antigen-binding specificity to the antibody However, even a single variable domain (or half of an Fv compi ising 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 (CH 1 ) of the heavy chain Fab fiagments differ from Fab' fragments by the addition ot a tew residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine res ⁇ due(s) of the constant domains bear a free thiol group F(ab') .
  • antibody fragments originally were produced as pan s of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known
  • the "light chains" of antibodies (immunoglobuhns) 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 then constant domains
  • immunoglobuhns can be assigned to different classes There are five major classes of immunoglobuhns IgA, IgD, IgE, IgG and IgM and several of these may be further divided into subclasses ( ⁇ sotypes), e ⁇ > , IgG l , IgG2, IgG3 IgG4 IgA and IgA2
  • ⁇ sotypes e ⁇ >
  • IgG l IgG2
  • IgG3 IgG4 IgA IgA2
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ; 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 antigemc 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 immunoglobuhns The modifiei "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 pai
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobuhns) 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 cha ⁇ n(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 etal , Proc Natl Acad Sci USA, 81 6851-6855 [1984])
  • Humanized forms of non-human (e g , munne) antibodies are chimeric immunoglobuhns, immunoglobulm chains or fragments thereof (such as Fv, Fab, Fab' F(ab')-, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulm
  • humanized antibodies are human immunoglobuhns (recipient antibody) in which residues from a CDR ot 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 immunoglobulm are replaced by corresponding non-human residues
  • Furthermoie humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences
  • the humanized antibody includes a PRIMATIZED ' M antibody wherein the antigen-binding region ot the antibody is dei IV ed from an antibody pi oduced by immunizing macaque monkeys w ith the antigen of mteiest "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, domains which enables the sFv to form the desired structure for antigen binding Foi a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol 1 13, Rosenburg and Moore eds , Springer- Verlag
  • 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 )
  • V H heavy-chain variable domain
  • V L light chain variable domain
  • 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 Holhnger 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 withm 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 , radioiso tope 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
  • Radionuchdes that can serve as detectable labels include, for example, 1-131 I 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 adheie
  • solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass) polysaccharides (e g , agarose), polyacrylamides, polystyrene polyv inyl alcohol and silicones
  • the solid phase can compnse the well ot an assay plate, in othei s 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 ot lipids phosphohpids and/oi surfactant which is useful for delivery of a drug (such as a PR0212, PRO290 PR0341 PR0535 PR06 I 9 PR0717, PRO809 PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025 PRO 1030 PRO 1097, PRO l 107, PRO l l l l PRO 1 153, PRO 1 182, PRO 1 184 PRO 1 187, PRO 1281 , PR023, PR039, PR0834 PRO 1317, PRO 1710, PRO2094 PR02I 45 or PR02198 polypeptide oi antibody thereto and, optionally a chemotherapeutic agent) to a mammal
  • the components of the liposome are commonly arranged in a bilayer foimation, 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 immunoglobulm constant domains Structurally, the immunoadhesins comprise a fusion of an ammo acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i e , is "heterologous"), and an immunoglobulm 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 immunoglobulm constant domain sequence in the immunoadhesin may be obtained from any immunoglobulm, 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PRO1 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 and PR02198
  • cDNA encoding PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717 are examples of polypeptides.
  • PR01 182, PROl 184, PROl 187, PRO l 281 , PR023, PR039, PR0834, PROl 317, PRO1710, PRO2094, PR02145 and PR02198 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 vanants included in the foregoing definition of PR0212.
  • PR023, PR039, PR0834, PRO 1317, PRO 1710 PRO2094, PR02145 and PR02198 will be referred to as "PR0212, PRO290, PR0341. PR0535. PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005 PRO1009, PRO1025. PRO1030.
  • PRO23, PRO39, PRO834, PROI 317, PRO1710 PRO2094, PRO2145 orPRO2l 98" ⁇ egardless ot the ⁇ r origin or mode ot preparation
  • cDNA clones hav e been deposited with the ATCC The actual nucleotide sequence of the clones can readily be determined by the skilled artisan by sequencing ot the deposited clone using loutine methods in the art The predicted amino acid sequences can be determined fiom the nucleotide sequences using routine skill.
  • PRO 1281, PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 orPR02198 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.
  • PR023, PR039, PR0834, PROl 317, PRO1710, PRO2094, PR02145 andPR02198 variants can be prepared by introducing appropriate nucleotide changes into the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PROI030, PRO1097, PROl 107.
  • PRO1710, PRO2094, PR02145 or PR02198 polypeptide may alter post-translational processes of the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848.
  • PRO1045 PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll,PRO1153,PRO1182,PRO1184,PRO1187,PRO1281,PRO23.PRO39,PRO834.PRO1317,PRO1710.
  • PRO2094, PR02145 or PR02198 such as changing the number or position of giycosylation sites or altering the membrane anchoring characteristics.
  • PRO2094, PR02145 or PR02198 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 PR0212, PRO290 PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PRO l 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PROl 317,
  • 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO 1030,
  • Amino acid substitutions can be the result of replacing one ammo acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, J e , conservative ammo 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 oi mature native sequence
  • PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PRO l 153, PROl 182, PROl 184, PROl 187, PRO 1281 , PR023, PR039, PR0834, PROl 317, PRO l 710, PRO2094 PR02145 and PR02198 polypeptide fragments are piovided 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 nativ e piotein Certain fragments lack amino acid residues that are not essential for a desired biological activity of the PR0212, PRO290, PR0341 PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005.
  • An alternative approach involves generating PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830.
  • Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR.
  • PCR polymerase chain reaction
  • PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide fragments share at least one biological and/or immunological activity with the native PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005.
  • 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.
  • Val (V) lie, 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 oi 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
  • Non-conservative substitutions will entail exchanging a member of one of these classes for anothei class
  • Such substituted residues also may be introduced into the conseiv atn e substitution sites or moie preferably into the remaining (non-conserved) sites
  • v ariations can be made using methods known in the art such as ohgonucleotide mediated (site directed) mutagenesis, alanine scanning, and PCR mutagenesis Site dnected mutagenesis [Cartel et al Nucl Acids Res 13 4331 ( 1986), Zollei et al , Nucl Acids Res ]0 6487 ( 1987)], cassette mutagenesis [Wells et al Gene 34 315 ( 1985)] restriction selection mutagenesis [Wells tal Philos Trans R Soc London Sei A 317 415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PR0212, PRO290, PR0341, PR0535, PR0619.
  • PRQ212 PRO290. PRQ341. PRQ535. PRQ619. PRQ717. PRO809. PRO830. PRQ848, PRQ943, PRO1005 PRO1009, PRO1025, PRO1030, PRO1097. PROl 107, PROllll. PROl 153 PR01182.PROI184.PR01187.PR01281,PR023,PR039.PR0834,PR01317.PR01710,PR02094,PR02145 andPRQ2198
  • Covalent modifications of PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll. PROl 153, PRO1182,PRO1184.PRO1187,PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710,PRO2094,PRO2145 and PR02198 are included within the scope of this invention
  • One type of covalent modification includes reacting targeted amino acid residues of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005.
  • PR02145 or PR02198 Denvatization with bifunctional agents is useful, for instance, tor crosshnking PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848 PR0943, PRO1005, PRO1009 PRO1025, PRO 1030, PROl 097, PROl 107, PROllll, PROl 153, PROl 182, PRO 1184, PROl 187, PRO 1281 PR023 PR039.
  • Commonly used crosshnking agents include, e g , 1 , 1 -b ⁇ s(d ⁇ azoace ty! )-2-ph enylethane, glutai aldehyde.
  • Another type of covalent modification of the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 polypeptide included within the scope of this invention comprises altering the native giycosylation pattern of the polypeptide "Altering the native giycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097,PROl
  • the phrase includes qualitative changes in the giycosylation of the native proteins, involving a change in the natuie and proportions of the various carbohydrate moieties present
  • PRO1317,PRO1710,PRO2094 PR02145 or PR02198 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 oi thieonme residues to the native sequence PR0212, PRO290, PR0341, PR0535, PR0619, PR0717 PRO809 PRO830, PR0848.
  • PR0943 PRO1005, PRO1009, PROI025.
  • PROl 184 PRO 1187, PROl 281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094 PR02145 or PR02198 (foi O- hnked giycosylation sites)
  • PROl 107 PROllll PROl 153 PRO 1182, PRO 1184, PRO 1187. PRO 1281, PR023, PR039, PR0834, PRO 1317. PRO 1710, PRO2094.
  • PR02145 or PR02198 amino acid sequence may optionally be altered thiough changes at the DNA level, paiticularly by mutating the DNA encoding the PR0212, PRO290. PR0341, PR0535. PR0619. PR0717, PRO809, PRO830, PR0848, PR0943. PRO1005. PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll. PROl 153 PROl 182, PROl 184,PRO1187,PRO1281.PRO23.PRO39,PRO834,PRO1317.PRO1710,PRO2094.PRO2145 or PR02198 polypeptide at preselected bases such that codons are generated that will translate into the desned amino acids
  • PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153,PRO1182,PRO1184,PRO1187,PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 comprises linking the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717. PRO809, PRO830. PR0848, PR0943, PRO1005, PRO1009, PRO1025.
  • PRO1030 PRO1097, PROl 107, PROl 111, PROl 153.
  • PRO1025 The PR0212, PRO290, PR0341, PR0535, PR0619, PR0717. PRO809, PRO830. PR0848. PR0943. PRO1005, PRO1009. PRO1025. PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PRO 1187, PRO 1281, PR023, PR039, PR0834, PRO 1317, PRO 1710. PRO2094.
  • PR02145 or PR02198 of the present invention may also be modified in a way to form a chimeric molecule comprising PR0212, PRO290 PR0341, PR0535 PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PROI025 PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023 PR039, PR0834 PROl 317, PROl 710, PRO2094, PR02145 oi PR02198 fused to another, heterologous polypeptide or amino acid sequence
  • such a chime ⁇ c molecule comprises a fusion of the PR0212, PRO290, PR0341
  • the epitope tag is generally placed at the ammo- or carboxyl terminus of the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PROl 005, PRO1009, PRO1025, PRO1030 PRO1097 PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281, PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198
  • the chimeric molecule may comprise a fusion of the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830 PR0848, PR0943, PRO1005, PRO1009 PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PRO!
  • the Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PR0212, PRO290 PR0341 PR0535 PRO619,PRO717,PRO809,PRO830,PRO848 PR0943, PRO 1005, PRO 1009, PRO1025 PRO1030, PRO1097,PRO1107,PROllll,PRO1153,PRO1182 PR01184 PROl I87,PR01281,PR023 PR039 PR0834, PRO 1317, PRO 1710, PRO2094, PR02145
  • PRQ212 Preparation of PRQ212.
  • PR2094. PRQ2145 and PRQ2198 Polypeptides The description below relates primarily to production of PR0212, PRO290, PR0341 , PR0535, PR0619,
  • PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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.
  • PRO 1030, PRO 1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PROL317, PRO1710, PRO2094, PR02145 or PR02198 may be obtained from a cDNA library prepared from tissue believed to possess the PR0212, PRO290 PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PROl 281 , PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 mRNA and to express it at a detectable level Accordingly, human-
  • Probes such as antibodies to the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PR01 184, PR01 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide, or oligonucleotides 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 et al , Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1989) An alternative means to isolate the gene encoding PR0212, PRO290, PR0341 , PR0535, PR
  • the ohgonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized
  • the ohgonucleotide 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 p P-labeled ATP biotinylation or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supi a
  • Sequences identified in such library screening methods can be compai ed 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 ammo acid or nucleotide level
  • Sequence identity within defined regions ot the molecule or across the full-length sequence can be determined using methods known in the art and as descnbed 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 proceduies as described in Sambrook et al supia, to detect precursoi s 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848.
  • 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,
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells
  • Suitable prokaryotes include but are not limited to eubacte ⁇ a, such as Gram-negative oi Gram-positive organisms, for example, Enterobacte ⁇ aceae such as E coli
  • E co strains are publicly available, such as E co Kl 2 strain MM294 (ATCC 31 ,446), E coh X 1776 (ATCC 31 ,537), E coh strain W31 10 (ATCC 27,325) and E co strain K5 772 (ATCC 53,635)
  • Othei suitable prokaryotic host cells include Enterobactenaceae such as Eschei i chia, e g , E co , Enterobactei , E ⁇ ⁇ ua, Klebs ⁇ ella, P ⁇ oteus, Salmonella, e e , Salmonella t phi
  • 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 pti ⁇ p oA El 5 (argF-lac)169 degP ompTkan'
  • E. coli W31 10 strain 37D6 which has the complete genotype tonA ptr3 phoA El 5 (argF-lac)169 degP ompT rbs7 ilvG kari ;
  • coli W3110 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 PR0212-, PRO290-, PR0341 -, PR0535-, PR0619-, PR0717-, PRO809-, PRO830-, PR0848-, PR0943-, PRO1005-, PRO1009-, PRO1025-, PRO1030-, PRO1097-, PROl 107-, PROl l l l-, PR01153-, PROl 182-, PROl 184-, PROl 187-, PROl 281 -, PR023-, PR039-, PR0834-, PROl 317-, PROl 710-, PRO2094-, PR02145- or PR02198-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 et al, Bio/Technology, 9: 968-975 (1991 )) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al, J. Bacteriol, 737 [1983]), A", fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
  • 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 e
  • 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 K. marxianus
  • yarrowia EP 402,226
  • Pichia pastor is (EP 183,070; Sreekrishna etal, J. Basic Microbiol, 28:265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case era/., Proc. Natl. Acad. Sci.
  • 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., JJ2:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton et al, Proc. Natl. Acad. Sci.
  • 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).
  • PRO2094, PR02145 or PR02198 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 CV l 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.
  • the nucleic acid (e g , cDNA or genomic DNA) encoding PR0212, PRO290, PR0341 , PR0535, PR0619, PRO717,PRO809, PRO830, PRO848,PRO943, PRO1005,PRO1009, PRO1025, PRO1030,PRO1097,PRO1 107, PROl 111 , PROl 153, PRO1 182, PRO1 184, PRO1187, PRO1281 , PRO23, PRO39,PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 s ⁇ te(s)
  • PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide
  • the signal sequence may be a component of the vector, or it may be a part of the PR0212-, PRO290-, PR0341 -, PR0535-, PR0619-, PR0717-, PRO809-, PRO830-, PR0848-, PR0943-, PRO1005-, PRO1009-, PRO1025-, PRO1030-, PRO1097-,
  • 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 ⁇ BR322 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 , ampicilhn, neomycin, methotrexate, or tetracychne, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e g , the gene encoding D-alanine racemase for Bacilli
  • suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PR0212-, PRO290-, PR0341 -, PR0535-, PR0619-.
  • 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
  • Promoters recognized by a variety of potential host cells include the ⁇ -lactamase and lactose promoter systems [Chang etal , Nature, 275 615 (1978), Goeddel etal , Nature, 281 544 (1979)], alkaline phosphatas
  • Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S D ) sequence operably linked to the DNA encoding PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717. PRO809, PRO830. PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PRO l 184 PROl 187, PR01281 , PR023. PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198
  • suitable promoting sequences for use with yeast hosts include the promoters foi 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, L7 4900 (1978)], such as enolase, glyceraldehyde- 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase. pyruvate kinase, t ⁇ osephosphate 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, metallothionem, glyceraldehyde-3- phosphate dehydrogenase, and enzymes lesponsible for maltose and galactose utilization Suitable vectors and promoters for use in yeast expression are further described in EP 73,657 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l .
  • PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegaiovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e g , the actin promoter or an immunoglobulm promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems
  • viruses such as polyoma virus, fowlpox virus (UK 2,211
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription
  • 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
  • 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 PR0212.
  • PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PR0212, PRO290,
  • PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005. PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023 PR039, PR0834, PROl 317, PRO 1710, PRO2094, PR02145 or PR02198 in recombinant vertebrate cell culture are described in Gething et al , 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 ot 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-protem 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 lmmunohistochemical 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 lmmunohistochemical 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PR01182, PROl 184, PROl 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide or against a synthetic peptide based on the DNA sequences provided here
  • PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PROl 281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 may be recovered from culture medium or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (e g , T ⁇ ton-X 100) or by enzymatic cleavage Cells employed in expression of PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848 PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 ,
  • the following piocedures are exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation, revei se phase HPLC chromatography on silica or on a cation-exchange resin such as DEAE, chromatotocusing, 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 bind epitope-tagged forms ot the PR0212 PRO290, PR034I ,
  • PR0943 PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PROl 281 , PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198
  • the purification step(s) selected will depend, for example, on the nature of the production process used and the particular PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1 107, PROl l l l,
  • PRQ212 E Amplification of Genes Encoding the PRQ212.
  • PRQ290 PRQ341.
  • PRQ535 PRQ619, PRQ717,
  • 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
  • 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, . 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
  • 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
  • the bcl-2 protein was found to be amplified in certain types of non Hodgkm' 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
  • CGH comparative genomic hybridization
  • PCR polymerase chain i eaction
  • such genes have been identified by quantitative PCR (S Gelmim 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 TaqManTM 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), HI 57 (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 (adenocarcmoma, abbreviated as "AdenoCa")(LTl), SRCC725 (squamous cell carcinoma, abbreviated as "SqCCa)(LTl a), SRCC726 (adenocarc ⁇ noma)(LT2), SRCC727 (adenocarc ⁇
  • Colon cancer cell lines include, for example, ATCC cell lines SW480 (adenocarcmoma, SRCC776) SW620 (lymph node metastasis of colon adenocarcmoma, SRCC777) Colo320 (carcinoma, SRCC778), HT29 (adenocarcmoma, SRCC779), HM7 (a high mucm producing variant of ATCC colon adenocarcmoma cell line SRCC780, obtained from Dr Robert Warren, UCSF),CaW ⁇ Dr (adenocarcmoma SRCC78I ), HCT1 16(carunoma SRCC782), SKCOl (adenocarcmoma, SRCC783), SW403 (adenocarcmoma, SRCC784), LS I 74T (carcinoma SRCC785), Colo205 (carcinoma, SRCC828), HCT15 (carcinoma, SRCC
  • CT5 (SRCC753), CT6 (SRCC754), CT7 (SRCC755), CT9 (SRCC756), CT1 1 (SRCC757), CT18 (SRCC758), CT19 (adenocarcmoma, SRCC906), CT20 (adenocarcmoma, SRCC907), CT21 (adenocarcmoma, SRCC908), CT22 (adenocarcmoma, SRCC909), CT23 (adenocarcmoma, SRCC910), CT24 (adenocarcmoma, SRCC91 1 ), CT25 (adenocarcmoma, SRCC912), CT26 (adenocarcmoma, SRCC913), CT27 (adenocarcmoma, SRCC914),CT28 (adenocarcmoma, SRCC915), CT29 (adenocarcmoma, SRCC916), CT30 (adenoc
  • Human breast carcinoma cell lines include, for example, HBL 100 (SRCC759), MB435s (SRCC760), T47D
  • SRCC761 SRCC761
  • MB468(SRCC762) MB 175
  • SRCC763 MB361
  • BT20 SRCC765
  • MCF7 SRCC766
  • SKBR3 SRCC767
  • human breast tumor center designated SRCC 1057 [HF-000545]
  • human breast tumors designated SRCC1094, SRCC1095, SRCC1096, SRCC1097, SRCC1098, SRCC1099, SRCC 1 100.
  • SRCC 1 101 and human breast-met-lung-NS tumor designated SRCC893 [LT 32]
  • Human kidney tumor centers include SRCC989 [HF-00061 1 ] and SRCC1014 [HF-000613]
  • Human testis tumor center includes SRCC1001 [HF-000733] and testis tumor margin SRCC999 [HF- 000716]
  • Human parathyroid tumor includes SRCC 1002 [HF-000831] and SRCC 1003 [HF-000832]
  • 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 may be measured by immunological methods, such as lmmunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product
  • Antibodies useful for lmmunohistochemical 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830 PR0848. PR0943 PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PRO l 107, PRO !
  • PR0834 PRO 1317, PR01710, PRO2094, PR02145 or PR02198 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to sequence PR0212, PRO290.
  • 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 ant ⁇ -PR0212, ant ⁇ -PRO290, ant ⁇ -PR0341 , ant ⁇ -PR0535, anti-PRO ⁇ l 9, ant ⁇ -PR0717, ant ⁇ -PRO809, ant ⁇ -PRO830, ant ⁇ -PR0848, ant ⁇ -PR0943, ant ⁇ -PRO1005, ant ⁇ -PRO1009, ant ⁇ -PRO1025, anti-PRO 1030, anti- PRO1097, anti-PROl 107, anti-PROl 1 1 1 , anti-PROl 153, anti-PROl 182, anti-PROl 184, anti-PROl 187, anti- PRO 1281 , ant ⁇ -PR023, ant ⁇ -PR039, ant ⁇ -PR0834, ant ⁇ -PR01317, ant ⁇ -PRO1710, ant ⁇ -PRO2094, ant ⁇ -PR02145 or ant ⁇ -PR02198 antibodies to inhibit the expression
  • 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-immunoglobuhn 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
  • 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 ra -transfected NIH-3T3 cells, which can be transfected with the desired gene, and monitored for tumorogenic growth
  • stable tumor cells lines such as, the B 104-1 -1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and ra -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)
  • ADCC antibody-dependent cellular cytotoxicity
  • Animal models of tumors and cancers include both non recombinant and recombinant (transgemc) animals
  • Non-recombinant animal models include, for example, rodent, e g , munne models
  • Such models can be generated by intioducing tumor cells into svngeneic mice using standard techniques, e g , subcutaneous injection, tail vein injection, spleen implantation intraperitoneal implantation, implantation under the renal capsule, or orthopm implantation, e g , colon cancer cells implanted in colo c tissue (See, e g ,
  • 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, B IO.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); ras-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.
  • 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 new-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).
  • 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
  • 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.
  • the cell lines Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about lOxl O 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 mvestigational 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, 41 suppl 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, Zachar
  • Recombinant (transge c) 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 transgemc animals
  • Animals that can serve as a target for transgemc 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 et al , Cdl, 56 313-321 [1989]), electroporation of embryos (Lo, Mol
  • transgemc 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 ot Lasko et al , Proc Natl Acad Sci USA 89 6232- 636 (1992)
  • transgene expression in transgemc 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1 107, PROl l l l , PROH53, PR01 182, PR01184, PR01187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PROl 153, PROl 182, PROl 184, PRO1187, PRO1281, PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PRO2145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107,PROl 111 , PROl 153,PROH82, PR01184, PROl 187, PR01281 , PR023, PR039, PR0834, PRO 1317, PROl 710, PRO2094, PR02145 or PR02198 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. 51:503 (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 etal, 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. 113-152].
  • 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PRO l 184, PR01187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094. PR02145 or PR02198 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.
  • each cat undergoes complete clinical examination, biopsy, and is scanned by computed tomography (CT) Cats diagnosed with subhngual 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
  • 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
  • other spontaneous animal tumors such as fibrosarcoma, adenocarcmo
  • 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, (poly)pept ⁇ de- ⁇ mmunoglobuhn fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti ldiotypic 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
  • the candidate compound interacts with but does not bind to a particular PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PROl 317, PRO 1710, PRO2094, PR02145 or PR02198 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
  • protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and
  • PR023-, PR039-, PR0834-, PR01317-, PROl 710-, PRO2094-, PR02145- or PR02198-encodmg 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 tor 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 iormation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove The formation of a complex in the control react ⁇ on(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner To assay for antagonists, the PR0212, PRO290, PR0341
  • PRO830, PR0848. PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO 1 107, PRO l l l , PROl 153, PROl 182, PROl 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PROl 281, PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145
  • antagonists may be detected by combining the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097, PRO 1107, PROl 111, PROl 153,PR01182,PR01184,PR01187,PR01281,PR023,PR039,PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide and a potential antagonist with membrane- bound PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281, PR023, PR039, PR0834, PROl 317, PROl 710,
  • RNA is prepared from a cell responsive to the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PRO 1153, PRO 1182, PROl 184, PRO 1187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR
  • PRO0212 Transfected cells that are grown on glass slides are exposed to labeled PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097.
  • the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROH 84, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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.
  • PR0212, PRO290, PR0341 , PR0535, PRO619, PRO717, PRO809, PRO830, PRO848, PRO943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107,PROl 1 1 1, PROl 153, PR01 182, PROl 184, PR01 187, PR01281, PR023, PR039, PR0834, PROL317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide can bephotoaffinity-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 ohgonucleotide 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l , PROl 153, PRO 1182, PRO 1184, PRO 1187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 ohgonucleotide that binds to the fusions of immunoglobulin with the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROH84, PROH 87, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097, PRO 1 107, PROl 1 1 1 , PROl 153, PROH82, PR01 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097.
  • PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO l 107, PROl l l l , PROl 153, PRO l 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PROl 153, PROl 182, PROl 184, PROH87, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide herein, is used to design an antisense RNA ohgonucleotide of from about 10 to 40 base pairs in length.
  • a DNA ohgonucleotide 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1, PROl 153.
  • the antisense RNA ohgonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097,PRO1107, PROl l l l,PRO1153, PRO1182, PRO1184, PRO1187, PRO1281, PRO23,PRO39, PRO834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide (antisense - Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (
  • oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PRO1153, PRO1 182, PRO1 184, PRO1 187, PRO1281, PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341.
  • PRO834,PRO1317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide thereby blocking the normal biological activity of the PR0212, PRO290, PR0341 , PR0535 PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030 PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 oi PR02198 polypeptide
  • small molecules include, but are not limited to, small peptides or peptide-hke molecules, preferably soluble peptides, and synthetic non peptidyl organic or inorganic compounds
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA Ribozymes act by sequence specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g , Rossi, Current Biology, 4 469-471 (1994), and PCT publication No WO 97/33551 (published September 18, 1997)
  • Nucleic acid molecules in tnple-hehx formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides
  • the base composition of these oligonucleotides is designed such that it promotes tnple-hehx formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of punnes or py ⁇ midines on one strand of a duplex
  • base-pairing rules which generally require sizeable stretches of punnes or py ⁇ midines on one strand of a duplex
  • 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
  • antisense RNA and RNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation
  • 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
  • RNA target 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 oligonucleotides is designed such that it promotes triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches ot punnes oi py ⁇ midines on one strand of a duplex
  • Hoogsteen base pairing rules which generally require sizeable stretches ot punnes oi py ⁇ midines on one strand of a duplex
  • 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 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 PR0212, PRO290, PR0341, PR0535, PR0619, PRO717,PRO809, PRO830, PRO848, PRO943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1107 PROl 1 1 1.
  • 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 adj u vant (monophosphory 1 Lipid A, synthetic trehalose dicorynomycolate)
  • the immunization protocol may be selected by one skilled in the art without undue experimentation
  • ant ⁇ -PRO2094, ant ⁇ -PR02145 or ant ⁇ -PR02198 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 appiopnate 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 PR0212, PRO290 PR0341. PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO 1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PRO 1184, PRO 1 187, PR01281 , PR023.
  • 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 Piactice.
  • 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 01 survival of the unfused, immortalized cells
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth 01 survival of the unfused, immortalized cells
  • the parental cells lack the enzyme hypoxanthine guanine phospho ⁇ bosyl transferase (HGPRT or HPRT)
  • HGPRT or HPRT hypoxanthine guanine phospho ⁇ bosyl transferase
  • the culture medium for the hyb ⁇ domas typically will include hypoxanthine, aminopte ⁇ n, 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 munne 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 foi the production of human monoclonal antibodies [Kozbor. J Immunol . 133 3001 (1984), Brodeur etal , 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PRO1281, PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198
  • 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 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 immunoglobulm purification piocedures such as. for example, protein A-Sephaiose, 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 ohgonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains ot munne 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, oi myeloma cells that do not otherwise produce immunoglobulm 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 munne sequences [U S Patent No 4,816,567, Morrison et al , supia] or by covalently joining to the immunoglobul
  • antibodies may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , munne) antibodies
  • Humanized antibodies include human immunoglobuhns (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 such as mouse, rat or rabbit
  • Fv framework residues of the human immunoglobulm 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 immunoglobulm and all or substantially all of the FR regions are those of a human immunoglobulm consensus sequence
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulm constant region (Fc), typically that o
  • a humanized antibody has one or more ammo 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 Humamzation can be essentially performed following the method of Winter and co-workers [Jones et al , Nature, 321 522-525 ( 1986), Riechmann et al , Natuie, 332 323-327 (1988), Verhoeyen et al , Science, 239 1534- 1536 ( 1988)] , by substituting rodent CDRs or CDR sequences tor 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 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 )1 The techniques of Cole et al , and Boerner et al , are also available tor the preparation of human monoclonal antibodies (Cole etal , 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 immunoglobulm loci into transge c animals, e g , mice in which the endogenous immunoglobulm 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
  • 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, tor example, WO 88/07378 and U S Patent No 4,975,278
  • a prodrug e g , a peptidyl chemotherapeutic agent, see WO 81/01 1405
  • 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 glucuro dase, alkaline phosphatase useful for converting phosphate containing prodrugs into free drugs, arylsulfatase useful for converting sulfate containing prodiugs into free drugs, cytosine deaminase useful for con verting non-toxic 5-fluorocytos ⁇ ne into the anti -cancer drug 5-fluorourac ⁇ l, proteases, such as serratia protease, thermolysin, subtihsin, carboxypeptidases (e g carboxypeptidase G2 and carboxypeptidase A) and cathepsins (such
  • the enzymes of this invention can be covalently bound to the ant ⁇ -PR0212, ant ⁇ -PRO290, ant ⁇ -PR0341 , ant ⁇ -PR0535. anti-PRO ⁇ 19, ant ⁇ -PR0717, ant ⁇ -PRO809, ant ⁇ -PRO830, ant ⁇ -PR0848, ant ⁇ -PR0943, anti-PRO 1005, anti-PRO 1009, anti-PRO 1025, anti-PRO 1030, anti-PRO 1097, anti-PROl 107, ant ⁇ -PROl 1 1 1 , anti-PROl 153, anti PROl 182, ant ⁇ -PR01 184, ant ⁇ -PR01 187, ant ⁇ -PR01281 , ant ⁇ -PR023, ant ⁇ -PR039, ant ⁇ -PR0834, ant ⁇ -PR01317, anti-PRO 1710, ant ⁇ -PRO2094, ant ⁇ -PR02145 or ant ⁇ -PR02198 antibodies by techniques well known in the art such as the use of the hetero
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens
  • one of the binding specificities is for the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281 , PR023 , PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 immunoglobulm 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 immunoglobulm heavy and light chains, these hyb ⁇ domas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the coriect 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 et al , EMBO J , H) 3655-3659 ( 1991 ) Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulm constant domain sequences The fusion preferably is with an immunoglobulm heavy - chain constant domain, comprising at
  • the inteiface between a pair ot antibodv molecules can be engineered to maximize the percentage ot heterodimers which are lecovered from recombinant cell culture
  • the preferred interface comprises at least a part of the CH3 region ot 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 oi tryptophan)
  • Compensatory "cavities " of identical or similar size to the large side cha ⁇ n(s) are created on the interface of the second antibody molecule by replacing laige amino acid side chains with smaller ones (e g , alanine or threomne)
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e g , F(ab )-, 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').
  • Fab' 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 thiomtrobenzoate (TNB) derivatives
  • TAB thiomtrobenzoate
  • 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 coh 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 ). molecule Each Fab' fragment was separately secreted from E coh and subjected to directed chemical coupling in vitro to form the bispecific antibody The bispecific antibody thus formed was able to bind to cells overexpressmg 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 Kostelny etal , 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 Holhnger 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 (V tannin) 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
  • tnspecific 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 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 foi IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64) Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD 16) so as to focus cellular defense mechanisms to the cell expiessmg the particular polypeptide Bispecific antibodies ma ⁇ 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 radionuchde chelator, such as EOTUBE, DPTA, DOTA, or TETA Another bispecific antibody of interest binds the polypeptide and further binds tissue factor (TF)
  • TF tissue factor
  • 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 crosshnking 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 lminothiolate and methyl-4- mercaptobutynmidate and those disclosed, for example, in U S Patent No 4,676,980
  • cysteine res ⁇ due(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 1191-1 195 ( 1992) and Shopes, J Immunol , 148 2918-2922 ( 1992)
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunc tional cross-linkers as described in Wolff et al , 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-
  • 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)
  • toxin e g , an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin
  • radioactive isotope i e , a radioconjugate
  • Enzymatically active protein toxins and fragments thereof which can be used include diphtheria A chain nonbmding active fragments of diphtheria toxin, choleia toxin, botuhnus toxin, exotoxin A chain (from Pseudomonas aeiugmosa), ⁇ cin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuiites foi du proteins, dianthin proteins, Plntolaca amencana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, cuicin, crotin sapaona ⁇ a officinahs inhibitoi, gelo n, sapo ⁇ n.
  • Small molecule toxins include, for example, cahcheamicins, maytansinoids, palytoxm and CC 1065
  • a variety of radionuchdes are available for the production ot radioconjugated antibodies Examples include 21 B ⁇ ⁇ ⁇ I, "'In, 9 "Y and 1S6 Re Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl 3-(2-pyndyld ⁇ th ⁇ ol) propionate (SPDP), lminothiolane (IT), bifunctional derivatives of mnidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenz
  • 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 phosphatidylchohne, cholesterol and PEG-denvatized 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 et al 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 e? al , J National Cancer Inst . 81 ( 19) 1484 (1989)
  • chemotherapeutic agent such as Doxorubicin
  • 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 ot pharmaceutical compositions If the protein encoded by the amplified gene is intracellular and whole antibodies are used as inhibitois internalizing antibodies are preferred However, hpofections oi liposomes can also be used to deliver the antibody or an antibody fragment, into cells Where antibody fragments are used, the smallest inhibitory tiagment 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 lecombinant DNA technology (see, e g , Marasco et al , Proc Natl Acad Sci USA.
  • 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 lyophihzed formulations or aqueous solutions
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionme, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkomum chloride, benzethomum chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol,
  • 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, hydroxymethylcelluloseorgelatin-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
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyI-methacrylate), or poly(v ⁇ nylalcohol)) polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and ethyl-L glutamate, non-degiadable ethylene-vinyl acetate, degradable lactic acid glycohc acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate), and poly-D-( )-3-hydroxybutync acid While polymers such as ethylene-vinyl acetate
  • 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, bladdei, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas, sarcomas, ghoblastomas, and various head and neck tumors), leukemias and lymphoid malignancies, other disorders such as neuronal, glial, astrocytal, hypothalarmc and other glandular, macrophagal, epithelial, stromal and blastocoehc disorders, and inflammatory, angiogenic and immunologic disorders
  • the anti-tumor agents include benign or malignant tumors (e g , renal, liver, kidney, bladdei, breast, gastric,
  • chemotherapeutic agents 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 &W ⁇ lk ⁇ ns, 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 onap ⁇ stone (see, EP 616812) in dosages known for such molecules
  • the antibodies herein are co-admimstered with a growth inhibitory agent
  • the growth inhibitory agent may be administered first, followed by an antibody ot the present invention Howevei , simultaneous admimstiation or administration of the antibody ot the present invention first is also contemplated
  • suitable dosages for the growth inhibitory agent are those presently used and may be loweied 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
  • ⁇ 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 moi e, 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 It may further include other
  • tumor diagnostics or prognostics 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 quantitativ ely 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 factoi Such binding assays are performed essentially as described in section 5 above
  • 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 ot 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, Inms et al , PCR Piotocols 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, Ohgonucleotide Synthesis, IRL Press Oxford, 1984, R I Freshney Animal Cell Culture. 1987, Cohgan et al Current Protocols in Immunology 1991
  • EXAMPLE 1 Isolation of cDNA clones Encoding PRQ2 I 2
  • the extracellular domain (ECD) sequences (including the secretion signal sequence if any) from about
  • EST databases included public EST databases (e g , GenBank), a proprietary EST database (LIFESEQ ® , Incyte Phamaceuticals, Palo Alto, CA), and a proprietary EST database from Genentech
  • the search was performed using the computer program BLAST or BLAST2 [Altshul et al , Methods in Enzymology, 266 460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences 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 consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington)
  • a consensus DNA sequence was assembled relative to other EST sequences using phrap as described above
  • the ESTs included an EST proprietary to Genentech
  • the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above
  • 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 PR0212
  • 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, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe ohgonucleotide and one of the primer pairs
  • PCR primers (forward and reverse) were synthesized forward PCR primer 5'-CACGCTGGTTTCTGCTTGGAG-3' (SEQ ID NO 3) reverse PCR primer 5'-AGCTGGTGCACAGGGTGTCATG-3' (SEQ ID NO 4)
  • RNA for construction of the cDNA libraries was isolated from human fetal lung tissue
  • 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 ohgo 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 etal .
  • a suitable cloning vector such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes etal .
  • the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucelotide positions 101-103 and a stop signal at nucleotide positions 1001 -1003 ( Figure 1 , SEQ ID NO 1 )
  • the predicted polypeptide precursor is 300 amino acids long
  • SEQ ID NO 2 shows 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
  • the N-terminus ot the PR0212 sequence contains a typical secretion signal (amino acids 1 -23 of Figure 2, SEQ ID NO 2) It is believed that amino acids 1 -215 of PR0212 (Figure 2, SEQ ID NO 2) represents an extracellular domain PR0212 has one potential N-hnked giycosylation site at amino acid residue 173 ( Figure 2 , SEQ ID NO 2)
  • Clone DNA30942- 1 134 has been deposited with ATCC on September 16, 1997 and is assigned ATCC deposit
  • EXAMPLE 2 Isolation of cDNA Clones Encoding PRO290 An expressed sequence tag (EST) DNA database (LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA) was searched and an EST was identified which showed homology to NTII-l (a nerve protein which facilitates regeneration), FAN and beige
  • RNA for construction of cDNA libraries was then isolated from human fetal kidney tissue
  • the cDNA libraries used to isolate the cDNA clones encoding human PRO290 were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with ohgodT 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 pRK5D, 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
  • the human fetal kidney cDNA libraries (prepared as described above), were screened by hyb ⁇ dization with a synthetic ohgonucleotide probe 5'-TGACTGCACTACCCCGTGGCAAGCTGTTGAGCCAGCTCAGCTG-3' (SEQ ID NO 8)
  • a cDNA clone was sequenced in entirety and is herein designated as DNA35680-1212
  • the entire nucleotide sequence of clone DNA35680- 1212 is shown in Figures 3 A-3B (SEQ ID NO 6)
  • Clone DNA 35680- 1212 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 293- 295 and a stop signal at nucleotide positions 3002-3004 ( Figures 3A-3B.
  • a secondary cD ⁇ A library was generated in order to preferentially represent the 5 ends of the primary cD ⁇ A clones Sp6 R ⁇ A was generated from the primary library (described above), and this R ⁇ A was used to generate a random primed cD ⁇ A library in the vector pSST-AMY 0 using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above)
  • the double stranded cD ⁇ A was sized to 500-1000 bp, hnkered with blunt to ⁇ otI adaptors, cleaved with Sfil, and cloned into Sfil/ ⁇ otl cleaved vector pSST-AMY 0 is a cloning vector that has a yeast alcohol dehyrogenase promoter preceeding the cD ⁇ A cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehyrogenase terminator,
  • D ⁇ A from the library described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Lite Technologies, 20 ml) The bacteria and vector mixture w as then electroporated as recommended by the manufacturer Subsequently, SOC media (Life Technologies 1 ml) was added and the mixture was incubated at 37 °C for 30 minutes The transformants were then plated onto 20 standard 150 mm LB plates containing ampicilhn and incubated for 16 hours (37 °C) Positive colonies were sci aped off the plates and the D ⁇ A was isolated from the bacterial pellet using standard protocols, e g CsCl-gradient The purified D ⁇ A was then carried on to the yeast protocols below
  • the yeast methods were divided into three categories ( 1 ) Transformation of yeast with the plasmid/ cD ⁇ A combined vector, (2) Detection and isolation ot yeast colonies secreting amylase, and (3) PCR amplification ot the insert directly from the yeast colony and purification of the D ⁇ A for sequencing and further analysis
  • yeast strain used was HD56-5A (ATCC-90785) This strain has the following genotype M T alpha ura3 52 leu2 3 leu2-l 12, h ⁇ s3-1 1 , h ⁇ s3 15, MAL + , SUC + GAL +
  • yeast mutants can be employed that have deficient post-translational pathways Such mutants may have translocation deficient alleles in sec 71 sec 72 sec62, with truncated seel] being the most preferred
  • antagonists including antisense nucleotides and/or ligands which interfere with the normal operation of these genes, other proteins implicated in this post translational pathway (e g , SEC ⁇ lp, SEC72p, SEC62p, SEC63p, TDJ l p or SSAlp-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast
  • the cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tns-HCl, 1 mM EDTA pH 7 5,
  • Transformation took place by mixing the prepared cells (lOO ⁇ l) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1 ⁇ g, vol ⁇ 10 ⁇ l) in microfuge tubes The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 ⁇ l, 40% polyethylene glycol-4000, 10 mM Tns-HCl, 1 mM EDTA, 100 mM L ⁇ ,OOCCH 3 , pH 7 5) was added This mixture was gently mixed and incubated at 30°C while agitating for 30 minutes The cells were then heat shocked at 42 C C for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted and resuspended into TE (500 ⁇ l, 10 mM Tns-HCl, 1 mM EDTA pH 7 5) followed by recentrifugation The cells were then diluted into TE
  • the transformation was performed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly
  • the selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser et al , Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p 208-
  • the detection of colonies secreting amylase was perfoi med by including red starch in the selective growth media
  • Starch was coupled to the red dye (Reactive Red- 120, Sigma) as per the procedure described by Biely et al , Anal Biochem , 172 176-179 (1988)
  • the coupled starch was incorporated into the SCD-Ura agar plates at a final concentration of 0 15% (w/v), and was buffered with potassium phosphate to a pH of 7 0 (50-100 mM final concentration)
  • the underlined regions ot the oligonucleotides disclosed above annealed to the ADH promoter region and the amylase region, respectively, and amplified a 307 bp region from vector pSST-AMY 0 when no insert was present Typically, the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers Thus, the total product of the PCR reaction from an empty vector was 343 bp Howevei , signal sequence-fused cDNA resulted in considerably longer nucleotide sequences
  • a cDNA sequence isolated in the above screen is herein designated as DNA25314 Probes were then generated from the sequence of the DNA25314 molecule and used to screen a human placenta cDNA library prepared as described in paragraph 1 above
  • the cloning vector was pRK5B (pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278- 1280 ( 1991 )), and the cDNA size cut was less than 2800 bp
  • the ohgonucleotide probes 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-lenth coding region of PR0341
  • Forward and reverse PCR primers generally range from 20-30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length
  • the probe sequences are typically 40-55
  • Clone DNA49143- 1429 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 78-80 and ending at the stop codon at nucleotide positions 681-683 ( Figure 7)
  • the predicted polypeptide precursor is 201 ammo acids long ( Figure 8)
  • the full-length PR0535 protein shown in Figure 8 has an estimated molecular weight of about 22,180 daltons and a pi of about 9 68
  • Analysis of the full- length PR0535 sequence shown in Figure 8 evidences the presence of a variety of important polypeptide domains as shown in Figure 8, wherein the locations given for those important polypeptide domains are approximate as described above
  • Analysis of the full-length PR0535 sequence evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 25, a transmembrane domain from about amino acid 155 to about ammo acid 174, a potential N-glycosylation site from about amino acid 196 to about amino acid 199, and FKBP-type
  • Clone DNA49821-1562 shown in Figure 9 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 81 -83 and ending at the stop codon at nucleotide positions 450 452 ( Figure 9, SEQ ID NO 15)
  • the predicted polypeptide precursor is 123 ammo acids long including a predicted signal peptide at about ammo acids 1-20 ( Figure 10)
  • the full-length PR0619 protein shown in Figure 10 (SEQ ID NO 16) has an estimated molecular weight of about 13,710 daltons and an estimated pi of about 5 19 Clone DNA49821-1562 has been deposited with ATCC on June 16, 1998 and is assigned ATCC deposit no 209981
  • ECD extracellular domain 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 EST databases (e g , GenBank) and a proprietary EST database (LIFESEQ 0 , Incyte
  • 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 PR0717
  • 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, supt a, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe ohgonucleotide and one of the primer pairs
  • PCR primers (forward and reverse) were synthesized forward PCR primer 5'-AGCTTCTCAGCCCTCCTGGAGCAG-3' (SEQ ID NO 19) reverse PCR primer 5'-CGGGTCAATAAACCTGGACGCTTGG-3' (SEQ ID NO 20)
  • RNA for construction of the cDNA libraries was isolated from human fetal liver tissue (LIB229)
  • the cDNA libraries used to isolate the cDNA clones were constiucted by standaid methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with ohgo 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
  • DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full length PR0717 polypeptide (designated herein as DNA50988-1326 [ Figure 1 1 , SEQ ID NO 17]) and the derived protein sequence for that PR0717 polypeptide
  • the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucelotide positions 17- 19 and a stop signal at nucleotide positions 1697- 1699 (Figuie 1 1 , SEQ ID NO 17)
  • the predicted polypeptide precursor is 560 amino acids long, has a calculated molecular weight of approximately 58,427 daltons and an estimated pi of approximately 6 86
  • Analysis of the full length PR0717 sequence shown in Figure 12 suggests that PR0717 may be a novel 12 transmembrane receptor
  • the reverse complement strand of DNA50988 has a stretch that matches identically with human regulatory myosin light strand Further analysis of the full-length PR0717 sequence shown in Figure 12 (
  • EXAMPLE 7 Isolation of cDNA Clones Encoding PRO809 DNA57836-1338 was 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 methionme 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 ammo 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
  • Incyte database This EST sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e e . , GenBank) and a proprietary EST DNA database (LIFESEQ 1 Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homologies
  • the homology search was performed using the computer program BLAST or BLAST 2 (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 pi ogram phrap ' (Phil Green Umveisity of Washington, Seattle, Washington)
  • the consensus sequence obtained is herein designated DNA56418
  • Merck clone 230132 including EST H74302 from hbrai y 305 a human fetal liver spleen library
  • EXAMPLE 8 Isolation of cDNA Clones Encoding PRO830 DNA56866-1342 was identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc , (South San Francisco, CA) upon ESTs as well as clusteied 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 methionme 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 ammo acids without any stop codons If the first ATG has the required ammo acids, the second is not examined If neither meets the requirement, the candidate sequence is not scored In oi der to determine whether the EST sequence contains an authentic signal sequence the DNA
  • Candidate sequences also called initial consensus sequences, with a sufficient score were further examined with the computer program BLAST or BLAST-2 (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 progiam "phrap” (Phil Green, University of Washington, Seattle, Washington)
  • an initial consensus sequence formed from an Incyte database of sequences identified from the signal algorithm is designated herein as DNA55999 lmt
  • This sequence was extended using repeated cycles of BLAST and "phrap" to extend the sequence as far as possible using public and Incyte EST sequences to form a consensus sequence herein designated DNA55999
  • the clone including Incyte EST 2768571 was further examined DNA sequencing gave the sequence herein designated as DNA59839-1461
  • the extracellular domain (ECD) sequences (including the secietion 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 EST databases (e g , GenBank) and a proprietary EST database (LIFESEQ ® , Incyte Phamaceuticals, Palo Alto, CA)
  • the search was performed using the computer program BLAST or BLAST2 [Altshul et al , Methods in Enzymology 266 460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences 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 consensus DNA sequences with the program "phrap” (Phil Green, University of Washington, Seattle, Washington)
  • DNA36360 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above and is herein designated DNA36360 In some cases, the DNA36360 consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above
  • 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 PR0943
  • 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 etal , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe ohgonucleotide and one of the primer pairs PCR primers (forward and reverse
  • a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA36360 sequence which had the following nucleotide sequence hybridization probe
  • RNA for construction of the cDNA libraries was isolated from human fetal brain tissue
  • 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 ohgo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoiesis, and cloned in a defined orientation into a suitable cloning v ector (such as pRKB or pRKD, pRK5B is a precursoi 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
  • the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucelotide positions 150- 152 and a stop signal at nucleotide positions 1662- 1664 ( Figures 19A- 19B SEQ ID NO 28)
  • the predicted polypeptide precursor is 504 amino acids long (Figure 20), has a calculated molecular weight of approximately 54,537 daltons and an estimated pi of approximately 10 04
  • SEQ ID NO 29 evidences the presence of a variety of important polypeptide domains as shown in Figure 20, wherein the locations given for those important polypeptide domains are approximate as described above
  • Anaylsis of the full-length PR0943 sequence shown in Figure 20 evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 17, a transmembrane domain from about amino acid 376 to about amino acid 396, tyrosine kinase phosphorylation sites from about amino acid 212 to about amino acid 219 and from about
  • EXAMPLE 1 Isolation of cDNA Clones Encoding Human PRO 1005 DNA57708-141 1 was 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 methionme 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
  • Incyte cluster sequence no 49243 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
  • DNA59622-1334 was 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 methionme 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
  • the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to
  • Clone DNA59622 1334 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 67-69 and ending at the stop codon at nucleotide positions 814 816 ( Figure 25, SEQ ID NO 37)
  • the predicted polypeptide precursor is 249 amino acids long ( Figure 26)
  • the full-length PRO 1025 protein shown in Figure 26 (SEQ ID NO 38) has an estimated molecular weight of about 27,01 1 daltons and a pi of about 5 48
  • Analysis of the full-length PRO 1025 sequence shown in Figure 26 evidences the presence of important polypeptide domains as shown in Figure 26, wherein the locations given for those important polypeptide domains are approximate as described above
  • Analysis of the full-length PRO 1025 sequence shown in Figure 26 evidences the following a signal peptide from about amino acid 1 to about amino acid 14, N- glycosylation sites from about amino acid 124 to about amino acid 127 and from about amino acid 132 to about amino acid 135 , and a leucine zipper pattern from about amino
  • EXAMPLE 14 Isolation of cDNA Clones Encoding Human PROl 030 DNA59485-1336 was 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 methionme 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
  • Clone DNA59485- 1336 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 148-150 and ending at the stop codon at nucleotide positions 673-675 (Figure 27, SEQ ID NO 39)
  • the predicted polypeptide precursor is 175 amino acids long ( Figure 28)
  • the full-length PRO 1030 protein shown in Figure 28 (SEQ ID NO 40) has an estimated molecular weight of about 19,979 daltons and a pi of about 9 26
  • Analysis of the full-length PRO 1030 sequence shown in Figure 28 evidences the presence of an important polypeptide domain as shown in Figure 28, wherein the location given for that important polypeptide domain is approximate as described above
  • Analysis of the full-length PRO 1030 sequence shown in Figure 28 evidences the presence of a signal peptide from about amino acid 1 to about ammo acid 20
  • Clone DNA59485-1336 has been deposited with ATCC on June 23, 1998 and is assigned ATCC deposit no 203015
  • EXAMPLE 15 Isolation of cDNA Clones Encoding Human PRO 1097 DNA59841 -1460 was 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 charactei of the DNA nucleotides surrounding the first and optionally the second methionme 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 ammo 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
  • Clone DNA59841 - 1460 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 3 5 and ending at the stop codon at nucleotide positions 276-278 (Figure 29, SEQ ID NO 41)
  • the predicted polypeptide precursor is 91 amino acids long ( Figure 30)
  • the full-length PROl 097 protein shown in Figure 30 (SEQ ID NO 42) has an estimated molecular weight of about 10,542 daltons and a pi of about 10 04
  • Analysis of the full-length PRO 1097 sequence shown in Figure 30 evidences the presence of important polypeptide domains as shown in Figure 30, wherein the locations given for those important polypeptide domains are approximate as described above
  • Analysis of the full-length PRO 1097 sequence shown in Figure 30 evidences the following a signal peptide from about amino acid 1 to about amino acid 20, a glycoprotease family protein domain starting from about ammo acid 56, and an acyltransferase ChoActase/COT/CPT family peptide starting from
  • EST cluster sequence was then compared to 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 identity existing homologies
  • EST expressed sequence tag
  • the homology search was performed using the computer program BLAST or BLAST2 (Altshul et al Methods in Enzymolgy, 266460-480 ( 1996)) Those compai isons 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 DNA56402
  • Incyte EST no 3203694 was further examined DNA sequencing gave the full-length DNA sequence and is herein designated as DNA59606-1471 shown in Figure 31 (SEQ ID NO 43)
  • Clone DNA59606-1471 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 244-246 and ending at the stop codon at nucleotide positions 1675- 1677 ( Figure 31 , SEQ ID NO 43)
  • the predicted polypeptide precursor is 477 amino acids long ( Figure 32)
  • the full-length PROl 107 protein shown in Figure 32 (SEQ ID NO 44) has an estimated molecular weight of about 54,668 daltons and a pi of about 6 33
  • Analysis of the full-length PRO 1 107 sequence shown in Figure 32 evidences the presence of important polypeptide domains as shown in Figure 32, wherein the locations given for those important polypeptide domains are approximate as described above
  • Analysis of the full-length PROl 107 sequence shown in Figure 32 evidences the presence of the following a signal peptide from about ammo acid 1 to about amino acid 22; a transmembrane domain from about amino acid 429 to about amino acid 452, N-glycosylation sites from about amm
  • EXAMPLE 17 Isolation of cDNA Clones Encoding a Human PRO l 1 1 1 1
  • An expressed sequence tag (EST) DNA database (LIFESEQ ® . Incyte Pharmaceuticals, Palo Alto, CA) was searched and an EST was identified which showed homology to msuhn-hke giowth factor binding protein
  • RNA for construction of cDNA libraries was then isolated from human fetal brain tissue
  • the cDNA libraries used to isolate the cDNA clones encoding human PROl 1 1 1 were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with oligodT containing a Notl site, linked with blunt to Sail hemikinased adaptois, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRK5D, 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
  • the human fetal brain cDNA libraries (prepared as described above), were screened by hybridization with a synthetic ohgonu
  • DNA58721 -1475 The entire nucleotide sequence of clone DNA58721 - 1475 is shown in Figure 33 (SEQ ID NO 45)
  • Clone DNA58721 - 1475 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 57-59 and a stop signal at nucleotide positions 2016 2018 ( Figure 33, SEQ ID NO 45)
  • the predicted polypeptide precursoi is 653 ammo acids long, has a calculated molecular weight of approximately 72,717 daltons and an estimated pi of approximately 6 99
  • Analysis of the full-length PROl 111 sequence shown in Figure 34 (SEQ ID NO 46) evidences the presence of a variety of important polypeptide domains as shown in Figure 34, wherein the locations given
  • DNA59842-1502 was 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 methionme codon(s) (ATG) at the 5 -end of the sequence oi 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 fust ATG has the required ammo acids, the second is not examined If neither meets the requirement, the candidate sequence is not scoi ed In order to determine whethei the EST sequence contains an authentic signal sequence the DNA and corresponding amino acid sequences surrounding the ATG codon are
  • Clone DNA59842-1502 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 92 94 and ending at the stop codon at nucleotide positions 683-685 ( Figure 35, SEQ ID
  • the predicted polypeptide precursor is 197 ammo acids long (Figure 36)
  • the full-length PROl 153 protein shown in Figure 36 (SEQ ID NO 49) has an estimated molecular weight of about 21,540 daltons and a pi of about 8 31
  • Analysis of the full-length PRO 1 153 sequence shown in Figure 36 (SEQ ID NO 49) evidences the presence of important polypeptide domains as shown in Figure 36, wherein the locations given for those important polypeptide domains are approximate as described above
  • Analysis of the full-length PROl 153 sequence shown in Figure 36 evidences the presence of the following transmembrane domains from about amino acid 10 to about amino acid 28 and from about amino acid 85 to about amino acid 1 10, an N giycosylation site from about amino acid 38 to about amino acid 41 and N mynstoylation sites from about amino acid 5 to about amino acid 10 and from about amino acid 88 to about amino acid 93
  • Clone DNA59842 1502 has been deposited with ATCC on June 16, 1998 and is assigned ATCC deposit no 20
  • DNA59848 1512 was identified by applying a piop ⁇ etary signal sequence finding algorithm de eloped 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 ot the DNA nucleotides surrounding the first and optionally the second methionme 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
  • the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of
  • EST cluster sequence was then compared to 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 Enzymolgy.
  • Clone DNA59848- 1512 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 67-69 and ending at the stop codon at nucleotide positions 880-882 (Figure 37, SEQ ID NO 50)
  • the predicted polypeptide precursor is 271 amino acids long ( Figure 38)
  • the full-length PRO l 182 protein shown in Figure 38 (SEQ ID NO 51) has an estimated molecular weight of about 28,665 daltons and a pi of about 5 33
  • Analysis of the full-length PROl 182 sequence shown in Figure 38 evidences the presence of important polypeptide domains as shown in Figure 38, wherein the locations given for those important polypeptide domains are approximate as described above
  • Analysis of the full-length PROl 182 sequence shown in Figure 38 evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 25, an amino acid block having homology to C-type lectin domain proteins from about amino acid 247 to about amino acid 256, and an amino acid sequence block
  • EST sequence was then compared to 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 Enzvmol gy.
  • Clone DNA59220-1514 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 106-108 and ending at the stop codon at nucleotide positions 532-534 ( Figure 39, SEQ ID NO 52)
  • the predicted polypeptide precursor is 142 amino acids long ( Figure 40)
  • the full-length PROl 184 protein shown in Figure 40 (SEQ ID NO 53) has an estimated molecular weight ot about 15,690 daltons and a pi of about 9 64
  • Analysis of the full-length PROl 184 sequence shown in Figure 40 evidences the presence of an important polypeptide domain as shown in Figure 40, wherein the location given for that important polypeptide domain is approximate as described above
  • Analysis of the full-length PROl 184 sequence shown in Figure 40 evidences the presence of the following a signal peptide from about ammo acid 1 to about amino acid 38 Clone DNA59220-1514 has been deposited with ATCC on June 9, 1998 and is assigned ATCC deposit no 209962
  • EST cluster sequence was then compared to 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 Enzymolgy, 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 DNA57726
  • Clone DNA62876- 1517 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 121 -123 and ending at the stop codon at nucleotide positions 481 -483 ( Figure 41 , SEQ ID NO 54)
  • the predicted polypeptide precursor is 120 amino acids long ( Figure 42)
  • the full length PRO l 187 protein shown in Figure 42 (SEQ ID NO 55) has an estimated molecular weight of about 12,925 daltons and a pi of about 9 46
  • Analysis of the full-length PROl 187 sequence shown in Figure 42 evidences the presence of important polypeptide domains as shown in Figuie 42 wherein the locations given for those important polypeptide domains are approximate as described above
  • Analy sis of the full-length PROl 187 sequence shown in Figure 42 evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 17, and N-my ⁇ stoylation sites from about amino acid 58 to about amino acid 63, from about amino acid 63 to about
  • EXAMPLE 22 Isolation of cDNA Clones Encoding a Human PRO 1281
  • 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 EST databases (e g , GenBank) and a proprietary EST database (LIFESEQ ® , Incyte Phamaceuticals, Palo Alto, CA)
  • the search was performed using the computer program BLAST or BLAST2 [Altshul et al , Methods in Enzymology, 266 460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences 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 consensus DNA sequences with the program "phrap” (Phil Green, University of Washington, Seattle, Washington
  • DNA35720 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above and is herein designated DNA35720
  • the DNA35720 consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above
  • oligonucleotides were synthesized 1 ) to identity by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO 1281
  • 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 greatei than about 1 -1 5kbp In order to screen several libraries for a full-length clone, DNA from
  • PCR primers forward and reverse were synthesized toiward PCR primers (35720 f 1 ) 5'-TGGAAGGCTGCCGCAACGACAATC-3' (SEQ ID NO 58)
  • hybridization probe was constructed from the consensus DNA35720 sequence which had the following nucleotide sequence: hybridization probe
  • RNA for construction of the cDNA libraries was isolated from human fetal liver tissue.
  • 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
  • the predicted polypeptide precursor is 775 amino acids long (Figure 44), has a calculated molecular weight of approximately 85,481 daltons and an estimated pi of approximately 6.92.
  • Analysis of the full length PROl 281 sequence shown in Figure 44 (SEQ ID NO:57) evidences the presence of a variety of important polypeptide domains as shown in Figure 44, wherein the locations given for those important polypeptide domains are approximate as described above.
  • Anaylsis of the full-length PRO 1281 sequence shown in Figure 44 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 15; and potential N- giycosylation sites from about amino acid 138 to about amino acid 141 and from about amino acid 361 to about amino acid 364.
  • Clone DNA59820-1549 has been deposited with the ATCC on August 18, 1998 and is assigned
  • CAC2_HAECO B3A2_HUMAN, S22373, CEF38A3_2, CEC34F6_2, CEC34F6_3 and CELT22B1 1_3.
  • PRO2094-, PR02145- or PR02198-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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PROl 097, PROl 107, PROl 1 1 1 , PROl 153.PR01 182.
  • PR01184, PROl 187, PROl 281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide for example, mu ⁇ ne-human chimeric, humanized or human antibodies against a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PRO 1153, PRO 1182, PRO 1 184, PRO 1 187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide
  • the starting material for the screen was genomic DNA isolated from a variety cancers
  • the DNA is quantitated precisely, e g , fluoromet ⁇ cally
  • DNA was isolated from the cells of ten normal healthy individuals which was pooled and used as assay controls for the gene copy in healthy individuals (not shown)
  • the 5' nuclease assay for example, TaqManTM
  • real-time quantitative PCR for example, ABI P ⁇ zm 7700 Sequence Detection SystemTM (Perkin Elmer, Applied Biosystems Division, Foster City, CA)
  • the results were used to determine whether the DNA encoding PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PROl 153, PROl 182, PROl 184, PROl
  • PRO290 (DNA35680-1212): 3568Q.tm.p:
  • PR0341 (DNA26288-1239): 26288.tm.fl : 5'-CAGCTGGACTGCAGGTGCTA-3' (SEQ ID NO:98)
  • PR0619 (DNA49821 -1562): 49821.tm.f1 :
  • PRO809 (DNA57836-1338):
  • PRO830 (DNA56866-1342): 56866.tm.fl:
  • PRQ848 (DNA59839-1461): 59839.tm.fl:
  • PRQ943 (DNA52192-1369): 52192.tm.fl:
  • PRO 1005 (DNA57708-1411): 57708.tm.fl:
  • PRO1025 (DNA59622-1334): 59622.tm.fl : 5'-TGATTGGTTCCCAGTGGCTT-3' (SEQ ID NO: 128) 59622.tm.rl:
  • PRO1030 (DNA59485-1336): 59485.tm.fl :

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 degrees of invasiveness and aggressiveness Alteration of gene expression is intimately related to the uncontrolled cell growth and de-differen nation 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 tumoi 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 1 129 [1991 ] and Bishop Cell, 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 particulai 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 heι 2, oi c-erbB-2), which encodes a 185-kd transmembi ane glycoprotein receptor (p 185HER2, HER2) related to the epidermal giowth factor receptor EGFR), is overexpressed in about 25% to 30% of human bieast cancei (Slamon et al . Science, 235 177- 182 [ 1987], Slamon et al , Science, 244 707-712 f 1989])
It has been reported that gene amplification of a proto-oncogene is an event typically involved in the moie malignant forms of cancer, and could act as a predictor of clinical outcome (Schwab et al , Genes Chromosomes Cancer, 1 181 -193 [ 1990], Alitalo etal , sιφ la) 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], supi a, 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 anth racychnes (Baselga et al , Oncology, 1 1 (3 Suppl 1) 43-48 [1997]) However, despite the association of erb B2 overexpression with poor prognosis, the odds of HER2-posιtιve patients responding clinically to treatment with taxanes were greater than three times those of HER2-negatιve patients (Ibid) A recombinant humanized anti-Er bB2 (antι-HER2) monoclonal antibody (a humanized version of the munne antι-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or Herceptm™) has been clinically active in patients with ErbB2-overexpressιng 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 tumoπgenesis 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 PR0212, PRO290. PR0341 , PR0535, PR0619, PR0717, PRO809 PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030 PRO 1097, PROl 107, PRO l 1 1 1 , PRO l 153, PROl 182 PROl 184, PROl 187, PRO 1281 , PR023 PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide In one aspect, the isolated antibody specifically binds to a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809. PRO830, PR0848, PR0943, PROl 005, PRO 1009, PRO 1025, PRO 1030, PRO1097, PROl 107, PRO l 1 1 1 , PRO l 153, PROl 182, PROl 184, PROl 187, PRO 1281 , PR023 PR039, PR0834, PRO 1317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide In another aspect, the antibody induces the death of a cell which expresses a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717 PRO809, PRO830 PR0848, PR0943. PRO1005, PRO 1009 PRO1025, PRO1030 PRO1097, PRO l 107, PRO l 1 1 1 , PRO l 153 PRO 1 182, PRO 1 184, PRO 1 187, PRO 1281 , PR023. PR039 PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide Often, the cell that expresses the PR0212, PRO290, PR0341 PR0535, PR0619, PR0717 PRO809, PRO830, PRO848, PRO943, PRO 1005, PROl 009 PRO 1025 PRO I 030 PRO 1097, PRO l 107, PROl 11 1, PROl 153, PROl 182, PRO l 184, PROl 187, PR01281 , PR023, PR039 PRO834, PRO1317, PRO1710 PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide In another embodiment, the invention concerns a composition of matter which comprises an antibody which binds, preferably specifically, to a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PROl 281 , PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 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- PR0212, antι-PRO290, antι-PR0341 , antι-PR0535, antι-PR0619, antι-PR0717, anti PRO809, antι-PRO830, anti PR0848, anti PR0943, antι-PRO1005, antι-PRO1009, anti PRO 1025, antι-PRO1030, antι-PRO1097, anti- PRO1 107, anti-PROl l l l , anti PR01 153, antι-PROH82, anti-PROl 184, anti-PROl 187, antι-PRO! 281 , anti PR023, antι-PR039, anti PR0834, anti-PRO 1317, anti-PRO 1710, antι-PRO2094, antι-PR02145 or anti PR02198 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 antι-PR0212, anti PRO290, antι-PRO341 , antι-PRO535, antι PRO619, antι-PRO717, antι-PRO809, antι PRO830, antι PR0848, antι PR0943, antι-PRO1005, antι-PRO1009, antι-PRO1025, anti PRO 1030, anti PRO 1097, anti-PROl 107, anti PROl 1 11 , anti-PROl 153, anti-PRO 1 182, anti PROl 184, anti-PROl 187, anti PR01281 , antι-PR023, antι-PR039 antι-PR0834, anti-PRO l 317, anti-PROl 710, antι-PRO2094, anti PR02145 or antι-PR02! 98 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 fui ther concerns antagonists of a PR0212, PRO290, PR0341 , PR0535 PR0619, PR0717,
PRO809, PRO830, PR0848, PR0943 PRO1005, PRO1009, PRO1025, PRO 1030, PRO1097, PROl 107,
PROl 1 11 , PROl 153 PROl 182, PROl 184, PROl 187, PRO 1281 PR023 PR039 PR0834 PRO1317,PRO1710
PRO2094, PR02145 or PR02198 polypeptide that inhibit one or more of the biological and/or lmmunoiogical functions or activities of a PR0212 PRO290 PR0341 , PR0535 PR06I 9 PR0717 PRO809, PRO830, PR0848 PR0943, PRO1005, PRO1009, PRO 1025, PRO 1030, PRO I 097, PRO l 107 PRO l 1 1 1 , PROl 153, PROl 182 PR01 184 PROl 187, PR01281 PR023 PR039, PR0834, PR01317, PRO1710 PRO2094, PR02145 or PR02198 polypeptide In a further embodiment, the invention concerns an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule encoding a PR0212. PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943. PRO1005, PRO1009. PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PRO1 182, PRO 1 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PRO2145 or PR02198 polypeptide or the complement thereof The isolated 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide in a sample suspected of containing a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO l 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide, wherein the method comprises exposing the sample to an antι-PR0212, antι-PRO290, anti- PR0341 , antι-PR0535, antι-PR0619, antι-PR0717, an tι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, anti- PRO1005, antι-PRO1009, antι-PRO1025, antι-PRO1030, antι-PRO 1097, anti-PROl 107, anti-PROl 1 1 1 , anti- PROl 153, anti-PROl 182, anti-PROl 184, anti-PRO 1 187, anti-PRO 1281 , antι-PR023, antι-PR039, antι-PR0834, anti-PRO 1317, anti-PRO 1710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibody and determining binding of the antibody to a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO I 097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PR01 184, PROl 187 PR01281 , PR023, PR039, PR0834 PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide in the sample In another embodiment, the invention provides a method for determining the presence of a PR0212, PRO290, PR0341 , PR0535, PR0619, PR07 I 7, PRO809, PRO830, PR0848, PR0943 PRO 1005, PRO 1009, PRO1025, PRO 1030, PRO 1097, PROl 107, PRO! I l l , PROl 153, PROl 182, PRO l 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide in a cell wherein the method comprises exposing the cell to an antι-PR0212, antι-PRO290, anti- PR0341 , antι-PR0535, antι-PR0619, antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, anti- PRO1005, antι-PRO1009, antι-PRO1025. antι-PRO1030, antι-PRO 1097, anti-PRO l 107, anti-PROl 1 1 1 , anti- PRO 1 153 anti-PRO 1 182, anti-PRO 1 184, anti-PRO 1 187, anti-PRO 12 1 antι-PR023, antι-PR039, antι-PR0834 anti-PRO 1 17, anti-PRO 1710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibody and determining binding of the antibody to the cell
In yet another embodiment, the present invention concems a method ot diagnosing tumoi in a mammal, comprising detecting the level of expression of a gene encoding a PR0212, PRO290, PR0341 , PR0535, PROό 19, PRO717,PRO809,PRO830 PRO848, PRO943, PRO I 005, PRO1009 PRO 1025 PRO 1030, PRO 1097, PRO l 107, PROl l l l , PRO1 153, PRO 1 182, PRO 1 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710 PRO2094, PR02145 or PR02198 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 antι-PR0212, antι-PRO290, antι-PR0341 , antι-PR0535, an H-PR0619, antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, anti-PROl 005, anti-PRO 1009, anti-PRO 1025, anti PRO1030, antι-PRO1097, anti-PROl 107, anti-PROl 1 1 1 , anti-PROl 153, antι-PROH 82, anti-PROl 184, anti- PROl 187, anti-PRO 1281 , antι-PR023, antι-PR039, antι-PR0834, anti-PRO 1317, anti-PRO 1710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the antι-PR0212, antι-PRO290, antι-PR0341 , antι-PR0535, anti- PR0619, antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, antι-PRO1005, antι-PRO1009, anti-PRO 1025, anti-PRO 1030, anti-PRO 1097, anti-PRO 1 107, anti-PR01 1 1 1 , anti-PRO 1 153, anti-PRO 1 182, anti- PRO 1 184, anti-PRO 1 187, anti-PRO 1281 , antι-PR023, antι-PR039, antι-PR0834, anti-PRO 1317, anti-PRO 1710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibody and a PR0212, PRO290, PR0341 , PR0535, PR0619, PRO717, PRO809, PRO830, PRO848,PRO943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1, PROl 153.PR01182, PROl 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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, fluoπmetry, or other techniques known in the art
The test sample is usually obtained from an individual suspected to have neoplastic cell giovv th or proliferation (e g cancerous cells)
In another embodiment, the present invention concerns a cancer diagnostic kit comprising an antι-PR0212, antι-PRO290, antι-PR0341 , antι-PR0535, anti-PROό 19, antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PRO943, antι-PRO1005, antι-PRO1009, antι-PRO1025, antι-PRO1030 anti-PRO 1097, anti-PRO l 107 anti- PROl 1 1 1 , anti-PROl 153, anti-PROl 182, anti-PRO 1 184, anti-PRO 1 187, anti-PRO 1281 , antι-PR023, antι-PR039, antι-PR0834, anti-PRO 1317, anti-PRO 1710, antι-PRO2094, antι-PR02145 oi antι-PR02198 antibody and a carriei (e g , a buffer) in suitable packaging The kit preferably contains instructions for using the antibody to detect the presence of a PR0212, PRO290, PR0341 , PR0535, PR0619 PR0717. PRO809, PRO830, PR0848 PR0943 PRO1005 PRO1009. PRO 1025, PRO 1030, PRO 1097, PRO 1 107 PROl 1 1 1 , PROl 153, PRO l 182, PRO l 184 PROl 187. PROl 281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide in a sample suspected of containing the same
In yet another embodiment, the invention concerns a method for inhibiting the growth of tumoi cells comprising exposing tumor cells which express a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830 PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide to an effective amount of an agent which inhibits a biological and/or immunological activity and/or the expression of a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848. PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PRO1 182, PRO1 184,PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide, wherein growth of the tumor cells is thereby inhibited The agent preferably is an antι-PR0212, antι-PRO290, antι-PR0341 , antι-PR0535, antι-PR0619, antι-PR0717, anti- PRO809, antι-PRO830, antι-PR0848, antι-PR0943, antι-PRO1005, antι-PRO1009, antι-PRO1025,antι-PRO1030, anti-PROl 097, anti-PRO 1 107, anti-PROl 1 1 1 , anti-PRO 1 153, anti-PROl 182, anti-PROl 184, anti-PROl 187, anti- PRO 1281 , antι-PR023, antι-PR039, antι-PR0834, anti-PRO 1317, anti-PROl 710, antι-PRO2094, antι-PR02145 or antι-PR02198 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 antι-PR0212, antι-PRO290, anti- PR0341 , antι-PR0535, antι-PR0619, antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, anti- PRO 1005, anti-PROl 009, anti-PRO 1025, anti-PRO 1030, anti-PRO 1097, anti-PROl 107, anti-PROl 1 1 1 , anti- PRO 1 153, anti-PRO 1 182, anti-PRO 1 184, anti-PRO 1 187, anti-PRO 1281 , an tι-PR023, antι-PR039, antι-PR0834, antι-PR01317, antι-PRO1710, antι-PRO2094, antι-PR02145 or antι-PR02198 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 tumoi cells and the label on the container indicates that the composition can be used for treating conditions characterized by overexpression ot a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717. PRO809, PRO830. PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PROl 107,PROl 1 1 1 , PROl 153, PRO l 182, PROl 184, PRO! 187, PRO l 281 , PR023, PR039. PR0834, PROl 317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO1009, PRO1025, PRO1030, PRO 1097, PROl 107 PROl 1 1 1 PROl 153, PROl 182, PROl 184, PROl 187 PROl 281 PR023, PR039, PR0834, PR01317. PRO 1710 PRO2094, PR02145 or PR02198 polypeptide In preferred aspects, the active agent is an antι-PR0212, antι-PRO290, antι-PR0341 , antι-PR0535. antι-PR0619, anti- PR0717 antι-PRO809 antι-PRO830, antι-PR0848, antι-PR0943, anti-PROl 005. anti-PRO 1009, anti-PRO 1025, anti-PRO 1030, anti-PRO 1097, anti-PRO 1 107, anti-PRO 1 1 1 1 , anti-PRO 1 153, anti-PROl 182. anti-PRO 1 184, anti PRO 1 187, anti-PRO 1281 , an H-PR023, antι-PR039, antι-PR0834, anti-PRO 1317 anti-PRO 1710 antι-PRO2094 antι-PR02145 or antι-PR02198 antibody or an antisense ohgonucleotide The invention also provides a method for identifying a compound that inhibits an activity of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PROl 025, PRO 1030, PRO 1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281. PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide, comprising contacting a candidate compound with a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809. PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PRO 1153, PRO 1182, PRO 1184, PRO 1187, PRO 1281 , PR023 , PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097, PRO 1107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281, PR023,PR039,PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide is inhibited In a specific aspect, either the candidate compound or the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153,PR01182, PROl 184, PROl 187, PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 polypeptide under conditions suitable for the induction of a cellular response normally induced by a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717. PRO809, PRO830, PR0848, PR0943. PRO1005, PRO1009, PRO1025, PRO1030, PRO1097. PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281, PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830. PR0848, PR0943. PRO 1005, PRO 1009. PRO1025, PROI030, PRO1097, PROl 107, PROl 111. PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide in cells that expi ess the polypeptide, wherein the method comprises contacting the cells with a candidate compound and determining whether the expression of the PR0212, PRO290 PR0341, PR0535, PR0619, PRO717,PRO809,PRO830,PRO848 PR0943, PRO 1005, PRO 1009 PRO1025 PROI030 PRO 1097, PRO 1107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187. PRO 1281, PR023 PR039 PRO834,PRO1317,PRO1710, PRO2094, PR02145 orPR02198 polypeptide is inhibited In a pief erred aspect, this method comprises the steps of (a) contacting cells and a candidate compound to be screened under conditions suitable tor allowing expression ofthePRO212.PRO290,PRO341,PRO535,PRO619,PRO717,PRO809,PRO830.PRO848,PRO943,PRO1005, PROI009, PRO1025, PRO1030. PRO1097, PROl 107, PROl 111 PROl 153, PROl 182, PRO! 184, PROl 187, PRO 1281 , PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182. PR01 184, PROl 187, PR01281 , PR023, PR039, PR0834, PROl 317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO l 107, PRO l 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187 PRO 1281 , PR023 PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 ot 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 y et more preferably at least about 89% sequence identity, yet moi e 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, \et more preferably at least about 93% sequence identity, yet moie prefei ably 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 PR0212, PRO290, PR0341 , PR0535. PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PRO 1 182, PRO 1 184, PRO 1 187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide cDNA as disclosed herein, the coding sequence of a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PRO 1 187, PROl 281 , PR023, PR039, PR0834, PRO 1317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PROl 030, PRO 1097, PROl 107, PROl 1 1 1 , PROl 153, PR01182,PR01 184, PR01187, PROl 281 , PR023, PR039, PR0834, PR01317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length ammo 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 ot (a)
Another aspect of the invention provides an isolated nucleic acid molecule compnsing a nucleotide sequence encoding a PR0212, PRO290, PR0341 , PR0535, PR06 I 9, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097. PROl 107, PROl 1 1 1 , PROl 153, PRO l 182, PROl 184. PRO 1 187, PRO 1281 , PR023, PR039, PR0834, PRO l 317, PRO l 710, PRO2094 PR02I 45 or PR02198 polypeptide which is either transmembrane domain-deleted oi transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domam(s) of such polypeptide are disclosed herein Therefore soluble extracellular domains of the hei ein described PR0212 PRO290, PR0341 , PR0535. PR0619. PR0717, PRO809, PRO830, PR0848 PR0943. PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097 PRO l 107, PROl 1 1 1 , PRO l 153. PROl 182. PRO l 184. PRO l 187, PROl 281 , PR023 PR039, PR0834 PRO 1 17. PRO 1710, PRO2094, PR02145 or PR02198 polypeptides ai e contemplated
Another embodiment is directed to fragments of a PR0212, PRO290, PR0341 , PR0535, PR06 I 9 PR0717 PRO809, PRO830 PR0848, PR0943, PRO 1005 PRO 1009, PRO 1025. PRO 1030 PRO 1097, PRO l 107, PROl 1 1 1. PROl 153, PROl 182, PROl 184, PR01 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO17 l 0, PRO2094, PR02145 or PR02198 polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments ot a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009. PRO 1025, PRO 1030, PRO 1097, PRO 1 107, PROl 1 1 1 , PROl 153, PRO1 182, PRO1 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide that may optionally encode a polypeptide comprising a binding site for an antι-PR0212, antι-PRO290, antι-PR0341 , anti-PR0535, antι-PR0619, antι-PR0717, antι-PRO809, anti- PRO830, antι-PR0848, antι-PR0943, antι-PRO1005, antι-PRO1009, anti-PRO1025, antι-PRO1030, anti- PRO1097, antι-PRO1 107, anti-PROl 1 1 1 , anti-PROl 153, anti-PROl 182, anti-PROH 84, anti-PROl 187, anti- PRO 1281 , anti-PR023, antι-PR039, antι-PR0834, anti-PRO 1317, anti-PRO 1710, anti-PRO2094, antι-PR02145 or anti-PR02198 antibody or as antisense ohgonucleotide 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 pi ef erably 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 ot a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717. PRO809, PRO830, PR0848, PR0943 , PRO1005. PRO1009, PRO1025. PRO1030, PRO1097, PRO l 107, PRO l 1 1 1 , PRO l 153, PRO l 182, PROl 184, PROl 187, PRO 1281 , PR023, PR039, PR0834, PR01317. PROl 710, PRO2094, PR02145 or PR02198 polypeptide-encoding nucleotide sequence may be detei mined in a loutine manner by aligning the PR0212, PRO290, PR0341 , PR0535, PR0619. PR0717. PRO809. PRO830, PR0848, PR0943. PRO1005. PRO1009 PRO 1025, PRO 1030, PRO 1097, PROl 107, PRO l 1 1 1 , PRO l 153. PROl 182, PRO l 184, PRO l 187. PRO 1281. PR023, PR039, PR0834, PR01 317 PRO 1710, PRO2094, PR02145 or PR02198 polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a numbei of well known sequence alignment pi ograms and determining which PR0212, PRO290, PR0341 , PR0535. PR06 I 9. PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO1009. PRO1025. PRO 1030, PRO I 097, PRO 1 107. PRO l 1 1 1. PRO 1 153, PRO 1 182, PRO 1 184, PRO 1 187 PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094 PR02145 or PR02198 polypeptide encoding nucleotide sequence fragment(s) are novel All of such PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009 PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PR0128 I , PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide encoding nucleotide sequences are contemplated herein Also contemplated are the PR0212, PRO290, PR0341 , PR0535 PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097 PROl 107, PROl 1 1 1 , PROl 153, PRO 1 182, PROl 184, PROl 187, PROl 281 , PR023, PR039, PR0834, PROl 317 PRO1710, PRO2094, PR02145 or PR02198 polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROH 84, PR01 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide fragments that comprise a binding site for an antι-PR0212, antι-PRO290, anti PR0341 , anti PR0535, antι-PR0619, antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, antι-PRO1005, anti-PRO 1009, anti-PRO 1025, anti-PRO 1030, anti PRO 1097, anti-PRO 1 107, anti-PRO 1 1 1 1 , anti PRO 1 153, anti PRO 1 182, anti-PRO 1 184, anti-PRO 1 187, anti PRO 1281 , anti PR023, antι-PR039, antι-PR0834, anti-PRO 1317 antι-PRO1710, antι-PRO2094, anti PR02145 or antι-PR02198 antibody
In another embodiment, the invention provides isolated PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PROl 097, PRO 1 107, PROl 1 1 1 , PROl 153, PROH 82, PR01 184, PR01 187, PR01281 , PR023, PR039 PRO834, PRO1317, PRO1710 PRO2094, PR02145 or PR02198 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified
In a certain aspect, the invention concerns an isolated PR0212, PRO290, PR0341 , PR0535, PR0619 PR0717, PRO809, PRO830, PR0848 PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PROl 097, PRO 1 107 PROl 1 1 1 , PROl 153 PROl 182, PROl 184, PROl 187, PRO 1281 PR023 PR039, PR0834 PRO1317, PRO17 I0, PRO2094, PR02145 or PR02198 polypeptide comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, moie 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 identit , yet more preferably at least about 89% sequence identity yet more pi eferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more pi eferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity \et more pi eferably at least about 94% sequence identity, yet moie preferably at least about 95% sequence identity, yet more preterablv at least about 96% sequence identity, yet more preferably at least about 97% sequence identity yet more pieterably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to a PR0212 PRO290 PR0341 , PR0535 PR0619 PR0717 PRO809 PRO830 PR0848, PR0943, PRO 1005 PRO 1009, PRO 1 25 PRO 1030, PRO 1097 PRO1 107, PROl l l l PROl 153, PRO l 182 PRO l 184, PRO l 187 PR01281 , PR023 PR039, PR0834 PR01317 PROl 710 PRO2094 PR02145 or PR02198 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 further aspect, the invention concerns an isolated PR0212, PRO290, PR0341 , PR0535, PR0619, 5 PR0717, PRO809, PRO830, PR0848, PR0943, PROl 005, PRO1009, PROl 025, PRO1030, PROl 097, PROl 107, PROl 1 1 1 , PROl 153, PRO 1 182, PROl 184, PROl 187 PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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
10 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
15 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
In a further aspect, the invention concerns an isolated PR0212, PRO290, PR0341 , PR0535, PR0619,
20 PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PRO 1 107 PROl 11 1 , PROl 153, PROl 182, PROl 184,PROH 87, PRO1281 , PRO23, PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 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
25 85% positives, yet more preferably at least about 86% positives yet moie preferably at least about 87% positives yet more preferably at least about 88% positives yet more preferably at least about 89% positives, yet moie 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
30 more preferably at least about 97% positives, yet more prefei ably at least about 98% positives and yet more preferably at least about 99% positives when compared with the amino acid sequence ot a PR0212 PRO290 PR0341 , PR0535 PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO1025 PROl 030, PROl 097, PRO 1 107, PROl 1 1 1 , PRO 1 153 PR01 182 PRO l 184, PRO l 187 PR01281 , PR023 PR039, PR0834 PR01317 PRO 1710, PRO2094 PR02145 oi PR02198 polypeptide having a full-length amino
35 acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein an extracellular domain ot a transmembrane protein, with or without the signal peptide, as disclosed herein oi any othei specifically defined fragment of the full-length amino acid sequence as disclosed herein
In a specific aspect, the invention provides an isolated PR02 I 2 PRO290, PR0341 , PR0535, PR0619 PR0717 PRO809 PRO830 PR0848 PR0943 PRO 1005 PRO1009 PRO I 025 PRO I 030 PRO 1097, PRO l 107 PROl 111, PROl 153, PRO 1182, PROl 184.PR01187,PRO1281,PRO23.PRO39,PRO834,PRO1317,PRO17i0, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PRO 1182, PRO 1184, PRO 1187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 orPR02198 polypeptide and recovering the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROH53,PR01182, PROl 184,PRO1187,PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710,PRO2094, PR02145 or PR02198 polypeptide from the cell culture.
Another aspect of the invention provides an isolated PR0212, PRO290, PR0341, PR0535, PR0619. PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PROl 025, PRO 1030, PRO 1097, PRO 1107, PROl 111, PROl 153, PROl 182, PRO 1184, PRO 1187, PR0128I,PR023,PR039,PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097. PRO1107, PROllll, PRO1153,PRO1182,PRO1184,PRO1187,PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710,PRO2094, PR02145 or PR02198 polypeptide and recovering the PR0212, PRO290. PR0341 , PR0535, PR0619. PR0717. PRO809. PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182,PR01184,PR01187,PRO1281,PRO23,PRO39,PRO834,PRO1317.PRO!710, PRO2094. PR02145 or PR02198 polypeptide from the cell culture.
In yet another embodiment, the invention concerns antagonists of a native PR0212. PRO290, PR0341. PR0535. PR0619, PR0717, PRO809, PRO830, PR0848. PR0943, PROl 005. PRO 1009, PRO 1025. PRO 1030. PRO1097,PRO1107,PROlllI,PRO1153,PRO1182,PROI184,PRO1187.PRO1281,PRO23,PRO39,PRO834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide as defined herein. In a particular embodiment. the antagonist is an anti-PR0212, anti-PRO290, anti-PR0341, anti-PR0535, anti-PR0619, anti-PR0717, anti- PRO809, anti-PRO830. anti-PR0848. anti-PR0943, anti-PRO 1005, anti-PRO 1009, anti-PRO 1025, anti-PRO 1030. anti-PRO 1097, anti-PRO 1107, anti-PRO 1111, anti-PRO 1153, anti-PRO 1182, anti-PRO 1184, anti-PRO 1187, anti- PRO 1281. anti-PR023. anti-PR039. anti-PR0834. anti-PRO 1317. anti-PRO 1710, anti-PRO2094. anti-PR02145 or anti-PR02198 antibody or a small molecule. In a further embodiment, the invention concerns a method of identifying antagonists to a PR0212,
PRO290, PR0341. PR0535, PR0619, PR0717, PRO809. PRO830. PR0848, PR0943, PRO1005. PRO1009. PRO1025, PRO1030, PRO1097, PROl 107, PROl 111. PROl 153. PROl 182, PRO! 184, PROl 187. PR01281. PR023. PR039. PR0834, PRO 1317, PRO 1710, PRO2094. PR02145 or PR02198 polypeptide which comprise contacting the PR0212. PRO290. PR0341 , PR0535. PR0619. PR0717, PRO809. PRO830. PR0848. PR0943. PRO1005, PRO1009. PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PR01281. PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide with a candidate molecule and monitoring a biological activity mediated by said PR0212, PRO290, PR0341, PR0535, PR0619. PR0717, PRO809. PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1107, PROllll, PR01153, PR01182, PR01184, PR01187, PR01281, PR023, PR039, PR0834, PRO 1317. PROl 710, PRO2094, PR02145 or PR02198 polypeptide Preferably, the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030. PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PR01184, PR01187, PR01281, PR023, PR039, PR0834, PROL317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide is a native PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281, PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide
In a still further embodiment, the invention concerns a composition of matter comprising a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PROl 710, PRO2094, PR02145 orPR02198 polypeptide, or an antagonist of aPR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide as herein described, or an antι-PR0212, antι-PRO290, antι-PR0341 , antι-PR0535, antι-PR0619, antι-PR0717, anti- PRO809,antι-PRO830,antι-PRO848,antι-PRO943,antι-PRO1005,antι-PRO1009,antι-PRO1025,antι-PRO1030, anti-PROl 097, anti-PRO 1107, anti-PRO 1111. anti-PRO 1153, anti-PRO 1182, anti-PRO 1184, anti-PRO 1187, anti- PRO 1281, antι-PR023, antι-PR039, antι-PR0834, anti-PRO 1317, anti-PRO 1710, antι-PRO2094, antι-PR02145 or antι-PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717. PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025. PRO 1030, PRO 1097, PROl 107, PROllll PROl 153, PROl 182,PR01184,PR01187,PR01281.PR023.PR039.PR0834 PROl 317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide, or an antagonist thereof as hereinbefore described, or an antι-PR0212, antι-PRO290 antι-PR0341, antι-PR0535, antι-PR06I9. antι-PR0717, anti- PRO809, antι-PR083(), antι-PR0848, antι-PR0943, anti-PRO 1005. anti-PRO 1009 anti-PRO 1025, anti-PRO 1030, anti-PRO 1097. anti-PROl 107, anti-PROl 111. anti-PROl 153, anti-PROl 182, anti-PROl 184 anti-PRO! 187, anti- PRO 1281, antι-PR023, antι-PR039, antι-PR0834. anti-PRO 1 17, anti-PRO 171 . antι-PRO2094, antι-PR02145 or antι-PR02198 antibody, for the preparation of a medicament useful in the tieatment of a condition which is responsive to the PR0212, PRO290,PRO341 PR0535.PR0619,PR0717 PRO809 PRO830,PRO848.PRO943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184 PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PROI710, PRO2094. PR02145 oi PR02198 polypeptide, an antagonist thereof or an antι-PR0212, antι-PRO290, antι-PR034I. antι-PR0535, antι-PR0619, antι-PR0717, antι-PRO809. antι-PRO830. antι-PR0848. antι-PR0943. anti-PRO 1005 anti-PRO 1009, anti- PRO 1025, anti PRO 1030, anti-PRO 1097, anti-PROl 107, anti-PROl 1 1 1 , anti-PROl 153, anti-PRO l 182, anti PRO 1 184, anti-PRO 1 187, anti PRO 1281 , antι-PR023 antι-PR039, anti PR0834, anti-PRO 1317, anti-PRO 1710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibody
In othei 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 coh, yeast, or Baculovirus-infected insect cells A process for producing any of the herein described polypeptides is further provided and comprises culturmg host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture 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 immunoglobulm
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 ohgonucleotide 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 PR0212, wherein the nucleotide sequence (SEQ ID NO 1 ) is a clone designated herein as DNA30942-1 134 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 PR0212 polypeptide as derived from the coding sequence of SEQ ID NO 1
Figures 3A through 3B show the nucleotide sequence (SEQ ID NO 6) ot a cDNA containing a nucleotide sequence encoding native sequence PRO290, wherein the nucleotide sequence (SEQ ID NO 6) is a clone designated herein as DNA35680-1212 Also presented in bold font and underlined aie the positions of the respective start and stop codons
Figure 4 shows the amino acid sequence (SEQ ID NO 7) of a native sequence PRO290 polypeptide as derived from the coding sequence ot SEQ ID NO 6
Figure 5 shows the nucleotide sequence (SEQ ID NO 9) of a cDNA containing a nucleotide sequence encoding native sequence PR0341 wherein the nucleotide sequence (SEQ ID NO 9) is a clone designated herein as DNA26288- 1239 Also presented in bold font and underlined are the positions ot the lespective start and stop codons
Figure 6 shows the amino acid sequence (SEQ ID NO 10) ot a native sequence PR0341 polypeptide as derived from the coding sequence of SEQ ID NO 9 Figure 7 shows the nucleotide sequence (SEQ ID NO 1 ) of a cDNA containing a nucleotide sequence encoding native sequence PR0535, wherein the nucleotide sequence (SEQ ID NO ! 3) is a clone designated herein as DNA49143-1429 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 14) of a native sequence PR0535 polypeptide as derived from the coding sequence of SEQ ID NO 13
Figure 9 shows the nucleotide sequence (SEQ ID NO 15) ot a cDNA containing a nucleotide sequence encoding native sequence PR0619, wherein the nucleotide sequence (SEQ ID NO 15) is a clone designated herein as DNA49821 - 1562 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 16) ot a native sequence PR0619 polypeptide as derived from the coding sequence of SEQ ID NO 15
Figure 1 1 shows the nucleotide sequence (SEQ ID NO 17) of a cDNA containing a nucleotide sequence encoding native sequence PR0717, wherein the nucleotide sequence (SEQ ID NO 17) is a clone designated herein as DNA50988-1 26 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 12 shows the ammo acid sequence (SEQ ID NO 18) of a native sequence PR0717 polypeptide as derived from the coding sequence of SEQ ID NO 17
Figure 13 shows the nucleotide sequence (SEQ ID NO 22) of a cDNA containing a nucleotide sequence encoding native sequence PRO809, wherein the nucleotide sequence (SEQ ID NO 22) is a clone designated herein as DNA57836-1338 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 23) of a native sequence PRO809 polypeptide as derived from the coding sequence of SEQ ID NO 22 Figure 15 shows the nucleotide sequence (SEQ ID NO 24) ot a cDNA containing a nucleotide sequence encoding native sequence PRO830, wherein the nucleotide sequence (SEQ ID NO 24) is a clone designated herein as DNA56866-1342 Also presented in bold font and undeihned aie the positions of the respective start and stop codons
Figuie 16 shows the amino acid sequence (SEQ ID NO 25) of a native sequence PRO830 polypeptide as derived from the coding sequence of SEQ ID NO 24
Figuie 17 shows the nucleotide sequence (SEQ ID NO 26) ot a cDNA containing a nucleotide sequence encoding native sequence PR0848, wherein the nucleotide sequence (SEQ ID NO 26) is a clone designated herein as DNA59839- 1461 Also presented in bold font and undei lined ai e the positions of the respective start and stop codons Figuie 18 shows the amino acid sequence (SEQ ID NO 27) of a native sequence PR0848 polypeptide as derived from the coding sequence of SEQ ID NO 26
Figures 19A through 19B show the nucleotide sequence (SEQ ID NO 28) ot a cDNA containing a nucleotide sequence encoding native sequence PR0943 w herein the nucleotide sequence (SEQ ID NO 28) is a clone designated heiein as DNA52192- 1369 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 29) of a native sequence PR0943 polypeptide as derived from the coding sequence of SEQ ID NO 28
Figure 21 shows the nucleotide sequence (SEQ ID NO 33) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1005 wherein the nucleotide sequence (SEQ ID NO 33) is a clone designated herein as DNA57708 141 1 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 34) of a native sequence PRO 1005 polypeptide as derived from the coding sequence of SEQ ID NO 33 Figure 23 show the nucleotide sequence (SEQ ID NO 35) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1009, wherein the nucleotide sequence (SEQ ID NO 35) is a clone designated herein as DNA57129-1413 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 36) of a native sequence PRO 1009 polypeptide as derived from the coding sequence of SEQ ID NO 35
Figure 25 shows the nucleotide sequence (SEQ ID NO 37) ot a cDNA containing a nucleotide sequence encoding native sequence PRO 1025, wherein the nucleotide sequence (SEQ ID NO 37) is a clone designated herein as DNA59622 1334 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 38) of a native sequence PRO 1025 polypeptide as derived from the coding sequence of SEQ ID NO 37
Figure 27 shows the nucleotide sequence (SEQ ID NO 39) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1030, wherein the nucleotide sequence (SEQ ID NO 39) is a clone designated herein as DNA59485 1336 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 40) of a native sequence PRO 1030 polypeptide as denved from the coding sequence of SEQ ID NO 39
Figure 29 shows the nucleotide sequence (SEQ ID NO 41 ) of a cDNA containing a nucleotide sequence encoding native sequence PROl 097, wherein the nucleotide sequence (SEQ ID NO 41 ) is a clone designated herein as DNA59841 1460 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 30 shows the amino acid sequence (SEQ ID NO 42) of a native sequence PRO 1097 polypeptide as derived from the coding sequence of SEQ ID NO 41
Figure 31 shows the nucleotide sequence (SEQ ID NO 43) ot a cDNA containing a nucleotide sequence encoding natι\ e sequence PRO 1 107 whei ein the nucleotide sequence (SEQ ID NO 43) is a clone designated herein as DNA59606- 1471 Also presented in bold font and underlined are the positions ot the respecm e stai t and stop codons
Figure 32 shows the amino acid sequence (SEQ ID NO 44) oi a native sequence PRO l 107 polypeptide as denved fiom the coding sequence of SEQ ID NO 4 Figure 33 shows the nucleotide sequence (SEQ ID NO:45) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1 1 1 1 , wherein the nucleotide sequence (SEQ ID NO:45) is a clone designated herein as DNA58721 -1475. Also presented in bold font and underlined are the positions of the respective start and stop codons. Figure 34 shows the amino acid sequence (SEQ ID NO:46) of a native sequence PROl 1 1 1 polypeptide as derived from the coding sequence of SEQ ID NO:45.
Figure 35 shows the nucleotide sequence (SEQ ID NO:48) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1 153, wherein the nucleotide sequence (SEQ ID NO:48) is a clone designated herein as DNA59842-1502. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 36 shows the amino acid sequence (SEQ ID NO:49) of a native sequence PROl 153 polypeptide as derived from the coding sequence of SEQ ID NO:48.
Figure 37 shows the nucleotide sequence (SEQ ID NO:50) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1 182, wherein the nucleotide sequence (SEQ ID NO:50) is a clone designated herein as DNA59848-1512. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 38 shows the amino acid sequence (SEQ ID NO:51 ) of a native sequence PROl 182 polypeptide as derived from the coding sequence of SEQ ID NO:50.
Figure 39 shows the nucleotide sequence (SEQ ID NO:52) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1 184, wherein the nucleotide sequence (SEQ ID NO:52) is a clone designated herein as DNA59220- 1514. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 40 shows the amino acid sequence (SEQ ID NO:53) of a native sequence PROl 184 polypeptide as derived from the coding sequence of SEQ ID NO:52. Figure 41 shows the nucleotide sequence (SEQ ID NO:54) of a cDNA containing a nucleotide sequence encoding native sequence PRO 1 187, wherein the nucleotide sequence (SEQ ID NO:54) is a clone designated herein as DNA62876-1517. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 42 shows the amino acid sequence (SEQ ID NO:55) of a native sequence PRO l 187 polypeptide as derived from the coding sequence of SEQ ID NO:54.
Figures 43A through 43B show the nucleotide sequence (SEQ ID NO:56) of a cDNA containing a nucleotide sequence encoding native sequence PROl 281 , wherein the nucleotide sequence (SEQ ID NO:56) is a clone designated herein as DNA59820-1549. Also presented in bold font and underlined are the positions of the respective start and stop codons. Figure 44 shows the amino acid sequence (SEQ ID NO:57) of a native sequence PRO 1281 polypeptide as derived from the coding sequence of SEQ ID NO:56.
Figure 45 shows the nucleotide sequence (SEQ ID NO:64) of a cDNA containing the nucleotide sequence encoding native sequence PR023, wherein the nucleotide sequence (SEQ ID NO:64) is a clone designated herein as DNA36640. Figure 46 shows the amino acid sequence (SEQ ID NO 65) of a native sequence PR023 polypeptide as derived from the coding sequence of SEQ ID NO 64 Also presented is a further truncated form of a native sequence PR023 polypeptide
Figure 47 shows the nucleotide sequence (SEQ ID NO 66) of a cDNA containing the nucleotide sequence encoding native sequence PR039, wherein the nucleotide sequence (SEQ ID NO 66) is a clone designated as DNA36651
Figure 48 shows the amino acid sequence (SEQ ID NO 67) of a native sequence PR039 polypeptide as derived from the coding sequence of SEQ ID NO 66
Figure 49 shows the nucleotide sequence (SEQ ID NO 68) of a cDNA containing the nucleotide sequence encoding native sequence PR0834, wherein the nucleotide sequence (SEQ ID NO 68) is a clone designated herein as DNA56538
Figure 50 shows the amino acid sequence (SEQ ID NO 69) of a native sequence PR0834 polypeptide as derived from the coding sequence of SEQ ID NO 68 Also presented is a pro-polypeptide of the PR0834 polypeptide Figures 51 A through 51B show the nucleotide sequence (SEQ ID NO 70) of a cDNA containing the nucleotide sequence encoding native sequence PROl 317, wherein the nucleotide sequence (SEQ ID NO 70) is a clone designated herein as DNA71 166 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 52 shows the amino acid sequence (SEQ ID NO 71 ) of a native sequence PR01317 polypeptide as derived from the coding sequence of SEQ ID NO 70
Figure 53 shows the nucleotide sequence (SEQ ID NO 72) of a cDNA containing the nucleotide sequence encoding native sequence PRO 1710 wherein the nucleotide sequence (SEQ ID NO 72) is a clone designated herein as DNA82331 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 54 shows the amino acid sequence (SEQ ID NO 73) of a native sequence PROl 710 polypeptide as derived from the coding sequence of SEQ ID NO 72 Also presented is a pro polypeptide of the PRO l 710 polypeptide
Figures 55A through 55B show the nucleotide sequence (SEQ ID NO 74) of a cDNA containing the nucleotide sequence encoding native sequence PRO2094, wherein the nucleotide sequence (SEQ ID NO 74) is a clone designated herein as DNA83123 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 56 shows the amino acid sequence (SEQ ID NO 75) ot a native sequence PRO2094 polypeptide as derived from the coding sequence of SEQ ID NO 74
Figure 57 shows the nucleotide sequence (SEQ ID NO 76) of a cDNA containing the nucleotide sequence encoding native sequence PR02145 whei ein the nucleotide sequence (SEQ ID NO 76) is a clone designated hei ein as DNA88050 Also presented in bold font and underlined are the positions of the iespective stait and stop codons
Figure 58 shows the amino acid sequence (SEQ ID NO 77) of a native sequence PR02145 polypeptide derived from the coding sequence of SEQ ID NO 76
Figures 59A through 59B show the nucleotide sequence (SEQ ID NO 78) ot a cDNA containing the nucleotide sequence encoding nativ e sequence PR02198 wherein the nucleotide sequence (SEQ ID NO 78) is a clone designated herein as DNA88153 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 60 shows the amino acid sequence (SEQ ID NO 79) of a native sequence PR02198 polypeptide derived from the coding sequence of SEQ ID NO 78 Figure 61 is a map of Chromosome 4 showing the mapping region of DNA50988-1326
Figure 62 is a map of Chromosome 2 showing the mapping region of DNA57708-141 1 Figure 63 is a map of Chromosome 6 showing the mapping region of DNA83123 Figure 64 is a map of Chromosome 16 showing the mapping region of DNA88153
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 "amphcon " 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, ghoblastoma, 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 measuies 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" ot cancer includes all phenomena that compromise the well-being ot the patient This includes, without limitation, abnormal or uncontrollable cell giowth metastasis, interference with the normal functioning of neighboring cells, release ot cytokines or other secretory pioducts at abnormal levels, suppression oi aggravation ot 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 Pi eferably, the mammal is human
"Carπei s as used herein include pharmaceutically acceptable cai πei s 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 oi immunoglobuhns, hydrophilic polymers such as polyvinylpyrrohdone, amino acids such as glycine, glutamine, asparagine, arginme or lysine monosacchandes, disacchandes, and other carbohydrates including glucose mannose, or dextnns, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counteπons such as sodium, and/or noniomc 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, IP5, Y9" 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 adπamycin, doxorubicin, epirubicin, 5-fluorouracιl, cytosine arabinoside (' Ara C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e g , pachtaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rhόne-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastme, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincnstine, vinorelbme, carboplatin, temposide, daunomycin, carminomycin, aminopteπn, dactinomycm mitomycins, esperamicins (see U S Pat No 4,675,187), 5 FU, 6-thιoguanιne, 6-mercaptopuπne, 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 onapnstone A growth inhibitory agent" when used herein refers to a compound or composition which inhibits grow th of a cell, especially cancer cell overexpressmg any of the genes identified herein, either in vitio or in . no Thus the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressmg such genes in S phase Examples of growth inhibitory agents include agents that block cell cycle progression (at a place othei than S phase), such as agents that induce Gl arrest and M phase arrest Classical M phase blockers include the vincas (vincnstine and vinblastme), 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 alkylatmg agents such as tamoxifen, prednisone, dacarbazine mechlorethamine, cisplatin, methotrexate 5 fluorouracil, and aia-C Further information can be found in The Molecular Basis of Cancel , Mendelsohn and Israel, eds , Chapter 1 , entitled Cell cycle regulation oncogens, and antineoplastic drugs by Murakami etal (WB Saundeis Philadelphia, 1995), especially p 13
' Doxorubicin' is an anthracychne antibiotic The full chemical name ot doxorubicin is (8S-cιs) 10 [(3 amino 2,3,6-tπdeoxy α-L-lyxo hexapyranosyl)oxy]-7 8 9 10 tetiahydro 6,8, 1 1 -tπhydroxy 8-(hydιo\yacet\ l) 1 methoxy 5 12-naphthacenedιone
The term cytokine is a generic term tor pioteins leleased by one cell population which act on another cell as intercellular mediatoi s 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, proinsuhn, 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, mulleπan-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothehal 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 -α, -β, and -γ, colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocy te-CSF (G- CSF), interleukins (ILs) such as IL-1 , IL- l a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-1 1 , 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 ot 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 foim See, e g , Wilman, "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions, 14 375-382, 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-containmg prodrugs, sulfate-containing prodrugs, peptide-containin prodrugs, D-amino acid-modified prodrugs, glysocylated prodrugs, R-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-contaimng prodrugs, 5- fluorocytosine and other 5-fluorouπdιne prodrugs which can be converted into the more active cytotoxic free drug Examples of cytotoxic drugs that can be denvatized 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 growth, 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 mhibitoiy, cytostatic and/or cytotoxic effect and/or apoptosis of the target cells An "effective amount" of a PRO 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 "therapeutically effective amount' in reference to the tieatment of tumoi refei s to an amount capable of invoking one or more of the following effects ( 1 ) inhibition, to some extent, of tumoi growth including, slowing down and complete growth arrest (2) reduction in the numbei ot tumoi cells, (3) ieduction in tumor size, (4) inhibition (; e , reduction, slowing down or complete stopping) of tumor cell int il nation into peripheral organs, (5) inhibition (i e , reduction, slowing down oi complete stopping) ot metastasis, (6) enhancement ot anti-tumoi immune lesponse which may, but does not have to, lesult in the iegiession or reiection ot the tumor, and/or (7) relief to some extent of one or more symptoms associated with the disordei A "theiapeuticalK effective amount of a PRO polypeptide antagonist for purposes of treatment of tumor may be determined empirically and in a routine manner
A "growth inhibitory amount" of a PRO antagonist is an amount capable of inhibiting the growth of a cell, especially tumor, e g , cancer cell, either in vitio or in vivo A "growth inhibitory amount" of a PRO antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
A "cytotoxic amount" of a PRO antagonist is an amount capable of causing the destruction of a cell, especially tumor, e g , cancer cell, either in vitio ox in vivo A "cytotoxic amount" of a PRO antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
The terms "PRO polypeptide" and "PRO" as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (t e , PRO/number) refers to specific polypeptide sequences as described herein The terms "PRO/number polypeptide" and "PRO/number" wherein the term "number" is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein) The PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods
A "native sequence PRO polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means The term "native sequence PRO polypeptide" specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e g , an extracellular domain sequence), naturally-occurring variant forms (e g , alternatively spliced forms) and naturally-occurring allehc variants of the polypeptide In various embodiments of the invention, the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures Start and stop codons are shown in bold font and underlined in the figures However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionme residues designated herein as am o acid position 1 in the figures, it is conceivable and possible that other methionme residues located either upstream or downstream from the amino acid position 1 in the figures may be employed as the starting amino acid residue for the PRO polypeptides
The PRO polypeptide "extracellular domain' or "ECD" refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains Ordinarily, a PRO polypeptide ECD will have less than 1 % of such transmembrane and/oi cytoplasmic domains and preferably will have less than 0 5% of such domains It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain The exact boundaries of a transmembrane domain may vary but most hkeh by no more than about 5 amino acids at either end of the domain as initially identified herein Optionally, theretoie an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on eithei side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide. and nucleic acid encoding them, are comtemplated by the present invention
The approximate location ot the ' signal peptides" of the various PRO polypeptides disclosed herein are shown in the present specification and/or the accompanying figures It is noted however, that the C-terminal boundary of a signal peptide mav 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, wheiein 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 , 10 1 -6 (1997) and von Heinje et al , Nucl Acids Res , J_4 4683 4690 ( 1986)) Moreover, it is also 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 "PRO polypeptide variant" means an active PRO polypeptide as defined above or below having at least about 80% amino acid sequence identity with a full length nativ e sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added or deleted, at the N or C terminus of the full length native amino acid sequence Ordinarily, a PRO polypeptide variant will have at least about 80% amino acid sequence identity, preferably at least about 81 % ammo acid sequence identity, more preferably at least about 82% 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% ammo acid sequence identity and most preferably at least about 99% amino acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain ot a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full length PRO polypeptide sequence as disclosed herein Ordinarily, PRO 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 moie often at least about 50 amino acids in length, moie often at least about 60 amino acids in length more often at least about 70 am o acids in length moi e 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 ammo acids in length, moie often at least about 300 am o acids in length, or more
As shown below Table 1 provides the complete souice code for the ALIGN-2 sequence comparison computei program This source code may be uiutinely compiled toi use on a UNIX operating system to piovide 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 PRO 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 PRO-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 /* 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},
I*Ό*I 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}, l*G*l 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,_M, 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}, /* 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},
/*κ */ -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*/ -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* *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},
/*o*/ 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,-l,_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, 0, 0,_M, 0, 1, 6, 0,-1, 0,-2, 2, 0,-4, 0},
/*S */ 1, 0, 0, 0, 0,-3, 1,-1,-1, 0, 0,-3,-2, 1,_M, 1,-1, 0, 2, 1, 0,-1,-2, 0,-3, 0}, /* Ύ *j 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, 0,_M, 0,-1,-1, 1, 3, 0, 0,-5, 0,-3, 0},
1*15*1 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}, I* v */ 0,-2,-2,-2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2,_M,-l,-2,-2,-l, 0, 0, 4,-6, 0,-2,-2}, /* W */ -6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-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, 0, 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 < stdιo.h >
-. include < ctype.h >
/. define MAXJMP 16 /* max jumps in a diag */
/. define 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 */
-/define DINSO /* penalty for a gap */
/. define DINS1 /* penalty per base */
/. define PINSO /* penalty for a gap */ . define PINS1 /* penalty per residue */ struct mp { short nf MAXJMP]; /* size of jmp (neg for dely) */ unsigned short xfMAXJMP]; /* base no. of jmp in seq x */
}; /* limits seq to 2^16 -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 *ofile: /* 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, I* final diag */ int dna; /* set if dna: mam() */ 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: nwQ / int *xbm; /* bitmap for matching *7 long offset, /* current offset in jmp file * / struct diag *dx; /*■ holds diagonals */ struct path pp[21; /* holds path for seqs */ char *calloc(), *malloc(), *ιndex(), *strcpy(), char *getseq(), " g_calloc(),
Page 1 of nw.h /* Needleman-Wunsch alignment program *
* usage, progs filel file2
* where filel and fιle2 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'))|(1 < <('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')) | d < <('Q'-,A'))
Figure imgf000030_0001
int ac, char *av[], prog = av[0], if (ac ' = 3) { fpπntt(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"), tpπntf(stderr. Output is in the file V align out\ \n . exιt(l),
} namexfO] = av[l], namex[l] = a\[2], seqx[0] = getseq(namex[0], &len0) seqxfl] = getseq(namex[l], &lenl) xbm = (dna)7 _dbval _pbval, endgaps = 0, /* 1 to penalize endgaps =7 ofile = "ahεn out /* output file *■/ nw(), /* fill in the matrix, get the possible jmps readjmpsO Λ get the actual jmps * / pπntQ, /* print stats, alignment */ cleanup(O) unlink any tmp files */
Page 1 of nw.c /* do the alignment, return best score- maιn()
* 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 IJ , /* 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)), colO = (int *)g_calloc("to get colO" , lenl + 1 , sizeof(int)), coll = (int *)g_calloc("to get coll " , lenl + 1 , sizeof(int)), insO = (dna)'' DINSO PINSO, insl = (dna)1? DINS1 • PINS1, smax = -10000, if (endgaps) { for (col0[0] = dely[0] = -msO, yy = 1 , yy < = lenl , yy + +) { colOfyy] = delyhyy] = col0[yy-l] - insl , ndely [yy] = yy,
} col0[0] = 0, /* Waterman Bull Math Biol 84 */
} else for (yy = 1 , yy < = lenl , yy+ + ) delyhyy] = -insO,
/* fill in match matrix
*/ for (px = seqx[0]. , xx = 1 , xx < = lenO, px + + , xx+ +) {
/* initial lze first entry in col if (endgaps) { if (xx = = 1) coll fO] = delx = -(msO + insl ), else coll TO] = delx = colO[0] - insl ndelx = xx,
} else { col l fO] = 0, delx = -insO, ndelx = 0,
Pase 2 of nw.c ...nw seqx[l], yy = 1; yy < = lenl; py+ + , yy+ +) { mis = colO[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 "7 if (endgaps | [ ndely [yy] < MAXGAP) { if (colOhyy] - insO > = delyhyy]) { delyhyy] = colOhyy] - (msO + insl); ndelyhyy] = 1; } else { delyhyy] -= insl; ndely[yy] + + ;
} } else { if (colOh/y] - (msO + insl) > = delyyy]) { delyLyy] = colOh/y] - (msO + insl), ndely [yy] = 1; } else ndely[yy] + + ; }
/* update penalty for del in y seq;
* favor new del over ongong del */ if (endgaps 11 ndelx < MAXGAP) { if (coll[yy-l] - insO > = delx) { delx = coll[yy-l] - (msO + insl), ndelx = 1; } else { delx -= insl; ndelx + + ;
} } else { if tcoll[yy-l] - (msO + insl) > = delx) { delx = coll [yy-1] - (insO-. insl); ndelx = 1, } else ndelx+ + ; }
/* pick the maximum score: we're favoring
* mis over any del and delx over dely
"7
Page 3 of nw.c ...nw id = xx yy + lenl - 1, if (mis > = delx && mis > = delyh/y]) collh/y] = mis, else if (delx > = dely[yy]) { collh/y] = delx, ij = dx[ιd] ijmp, if (dx[ιd] jp n[0] && ('dna | | (ndelx > = MAXJMP && xx > dx[ιd] jp x[ιj]+MX) | | mis > dx[ιd] score+DINSO)) { dxfid] ιjmp+ + , if (++ιj >= MAXJMP) { wπtejmps(ιd), ij = dxfid] ijmp = 0, dx[ιd] offset = offset, offset += sizeof(struct jmp) + sizeof (offset), } } dx[ιd] jp n[ιj] = ndelx, dx[ιd] jp x[ιj] = xx, dx[ιd] score = delx,
} else { collfyy] = delyh/y] ij = dx[ιd] ijmp, if (dx[ιd] jp n[0] && ('dna | | (ndelyfyy] > = MAXJMP
&& xx > dx[ιd] jp x[ιj] + MX) | | mis > dx[ιd] score+DINSO)) { dx[ιd] ιjmp+ + , if (++ιj >= MAXJMP) { wnteιmps(ιd), ij = dx[ιd] ijmp = 0, dx[ιd] offset = offset, offset + = sizeoftstruct jmp) + sizeof (off set),
} } dx[ιd] jp n[ιjj = -ndely [yy], dx[ιd] jp x[ιj] = xx, dx[ιd] score = delyh/y],
} if (xx = = lenO && y> < lenl) { /* last col "7 if (endgaps) collh/y] = ιnsO + ιnsl*(lenl yy), if (colllyy] > smax) { smax = coll[yy], dmax = id } } } if (endgaps && xx < lenO) coll[yy-l] -= ιnsO + msl*(lenO xx), if (coll[y -l] > smax) { smax = coll vy 1], dmax = id
} tmp = colO, colO = coll, coll = tmp
}
(void) free((char *)ndel>), (void) free((char *)dely), (void) free((char H)col0), (void) free((char )coll),
Page 4 of nw.c /* *
* prιnt() — only routine visible outside this module
*
* static:
* getmatO — trace back best path, count matches: pπnt()
* pr_ahgn() — print alignment ot described in array p[] pπnt()
* dumpblockO ~ dump a block of lines with numbers, stars. pr_ahgn()
* nums() — put out a number line: dumpblockO
* puthneO - put out a line (name, [num], seq, [num]). dumpblockO
* stars() - -put a line of stars: dumpblockO
* stπpnameO — 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 */ pπnto print
{ int lx, ly, firstgap, lastgap, /* overlap */ if ((fx = fopen(ofile, "w")) = = 0) { fpπntf(stderr, " %s. can't write s\n", prog, ofile), cleanup(l ),
} fpπntf(fx. " < first sequence %s (length = %d)\n" , namex[0]. lenO), fpπntf(fx, ' < second sequence %s (length = %d)\n" , namex[l ], lenl), olen = 60, lx = lenO, ly = lenl , firstgap = lastgap = 0, if (dmax < lenl - 1) { /* leading gap in x "7 pplOJ .spc = firstgap = lenl - dmax - 1 ,
1\ -= pp[0] spc,
} else if (dmax > lenl - 1) { /* leading gap in y */ pp[l] .spc = firstgap = dmax - (lenl - 1),
Figure imgf000034_0001
if (dmaxO < lenO - 1) { /* trailing gap in x */ lastgap = lenO - dmaxO - 1 , lx - = lastgap,
} else if (dmaxO > lenO - 1) { /+ trailing gap in y * l lastgap = dmaxO - (lenO - 1), l\ - = lastgap,
} getmat(lx 1) , firstgap, lastgap), pr_ahgn()
Page 1 ofnwprint.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 nm, lO, il, sizO, sizl; char outx[32]; double pet; register nO, nl; register char *p0, *pl;
/* get total matches, score */
Figure imgf000035_0001
pO = seqx[0] + pp[l].sρc; pi = seqx[l] + pp[0].spc; nO = pp[l].spc + 1; nl = pp[0].spc + 1; nm = 0: while ( *p0 && *pl ) {
Figure imgf000035_0002
if (xbm[*p0-'A']&xbm[*pl-'A']) nm+ + ; if (n0++ == pp[0].x[ιO])
Figure imgf000035_0003
if(nl + + ==pp[l].x[ιl])
Figure imgf000035_0004
}
/* pet homology:
* if penalizing endgaps, base is the shorter seq ¥ else, knock off overhangs and take shorter core if (endgaps)
Ix = (lenO < lenl)? lenO . lenl; else lx = (lx < ly)? lx : ly, pet = 100.:f(double)nm/(double)lx; fpπntf(fλ, "\n"), tpπntf(tx, " < %d matches in an overlap ot 9cd: 9c .2t percent sιmιlaπty\n" nm, (nm == 1)? "es", lx, pet).
Page2ofnwprint.c fpπntf(fx, " < gaps in first sequence %d' , gapx), ...getmat if (gapx) {
(void) spπntf(outλ, " (%d %s%s)", ngapx, (dna)' "base" ' residue", (ngapx l)9 s"), fprιntf(fx, " %s", outx), fpπntf(fx, " , gaps in second sequence %d' , gapy), if (gapy) {
(void) spπntftoutx, " (%d %s%s)", ngapy, (dna)? "base" "residue", (ngapy = = l)9 "s"), fpπntf(fx, " %s", outx),
} if (dna) fpπntf(fx,
"\n < score. %d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n" smax, DMAT, DMIS, DINSO, DINS1), else fpπntf(fx,
"\n < score d (Dayhoff PAM 250 matrix, gap penalty = d + %d per resιdue)\n", smax, PINSO, PINS1), if (endgaps) fpπntf(fx,
' < endgaps penalized left endgap %d %s%s, right endgap %d %s%s\n , firstgap, (dna)9 "base' "residue", (firstgap = = l)9 " " "s" , lastgap, (dna)9 "base" "residue", (lastgap = = l)9 ' s"), else fpπntf(fx, " < endgaps not penahzed\n"),
static nm, /* matches in core — for checking */ static lmax, /* lengths of stripped file names */ static >J[2], /* jmp index for a path */ static nc[2], /* number at start of current line */ static m[2], /* current elem number — for gapping */ static sιz[2], static char *ps[21 , /* ptr to current element */ static char *po[2], /* ptr to next output char slot */ static char out[2][P LINE] /* output line */ static char star[P LINE], /* set by stars() */
/*
* print alignment ol described in struct path pp[]
*/ static pr ahgn() pr align
{ int nn, /* char count */ int more, register 1 , for (1 = 0, lmax = 0, l < 2 ι+ +) { nn = stπpname(namex[ι]), if (nn > lmax) lmax = nn nc[ι] = 1 , m[ι] = 1 , sιz[ι] = ιj[ι] = 0, ps[ι] = seqxfi], po[ι] = out[ι] ,
Page 3 of nwpπnt.c for (nn = nm = 0, moie = 1 , more, ) { .pr align for (l = more = 0, l < 2, ι + +) { /* * do we have more of this sequence7
*/ if ('*ps[ι]) continue, more + + , if (pp[ι] spc) { /* leading space */ *po[ι] + + = ' ' , pp[ι] spc -,
} else if (sιz[ι]) { /* in a gap */
*po[ι] + + = '-' , sιz[ι]-,
} else { /* we're putting a seq element
*/ *po[ι] = *ps[ι], if (ιslower(*ps[ι]))
*ps[ι] = toupper(*ps[ι]), po[ι] + + , ps[ι] + + ,
/*
* are we at next gap for this seq9
*/ if (m[ι] = = pp[ι] x[y[ι]]) { /*
* we need to merge all gaps
* at this location */ sιz[ι] = pp[ι] n[ιj[ι] + +], while (m[ι] = = pp[ι] x[ιj[ι]]) sιz[ι] + = pp[ι] n[ιj[ι] + +]
} m i + + ,
}
} if (+ +nn = = olen | | 'more && nn) { dumpblockO, for (ι = 0, l < 2, ι + +) po[ι] = out[ι], nn = 0, }
* dump a block of lines, including numbers, stars pr_ahgn() *7 static dumpblockO dumpblock
{ register l, for (ι = 0, i < 2 ι + τ ) *po[ι] = '\0
Page4 ofnwprmt.c ...dumpblock
(void) putc( \n', fx), for(ι = 0, i < 2, ι++) { if (*out[ι] && ("-ou ] '= ' || *(po[ι]) '= ' ')){ if (i ==0) nums(ι), if 0 == 0&& *out[l]) stars(), puthne(ι), if (l == 0&&*out[l]) fpπntf(fx, star), if(ι == 1) nums(ι),
}
}
/*
* put out a number line dumpblockO
*/ static nums(ιx) num. int ix, /* index in out
{ char nhne[P_LINE], register i.J. register char *pn, *px, *py, for (pn = nhne, i = 0, i < lmax + P_SPC, ι+ + , pn++)
*pn = ' , for (I = nc[ιx], py = outfix], *py, py+ + , pn+ +) { if (*py = = ' ■ 11 *py = = ) *pn = ' ', else { if(ι%10 == 0 || (l == 1 &&nc[ιx] ' = 1)) { j = (l < 0)9 -l l, for (px = pn, j, j /= 10, px-)
*px = j%10 + 0 , if (i < 0)
*px = - ,
} else
*pn ι++,
*pn = '\0 nc[ιx] = i, for (pn = nhne, *pn, pn+ +) (void) putcO-pn, fx) (voidj putc( \n', fx)
* put out a line (name [num], seq, [num]) dumpblockO
*/ static puthne(ιx) putline int ι^>
Page5ofnwpπnt.c ...putline int 1; register char *px; for (px = namexfix], i = 0; *px && *px ! = px+ + , 1 + +) (void) putc(*px , fx); for (; l < lmax + P SPC; i + +) (void) putcO ', fx);
/* these count from 1 :
* ni[] is current element (from 1)
* nc[] 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 out[0], out[l]): dumpblockO
*/ static stars() stars
{ int i; register char *p0, *pl, ex, *px; if (!*out[0] I I (*out[0] == ' '&& *(po[0]) == ' ') | | !*out[l] I I (*out[l] == ' '&& *(po[l]) == ' ')) return; px = star; for (I = lmax + P SPC; I; ι~) *px+ + = ' '; for(p0 = out[0],pl = out[l]; *p0 && *pl; p0+ +, pl + +){ if (isalpha(*p0) && isalpha(*pl)) { if (xbm[*p0-'A']&xbm[*pl-'A']) { ex = '*'; nm+ + ;
} else if (!dna &&_day[*p0-'A'][*pl-'A'] > 0) ex = '.'; else ex = ' ';
} else ex = ' '; *px+ + = ex;
}
*px+ + = '\n'; *px = '\0';
Pa eόofnwprint.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 = = '/') py = px + 1 ; if (py)
(void) strcpy(pn, py); return(strlen(pn)) ;
Page 7 ofnwprint.c /*
* cleanupO — cleanup any tmp file
* getseqO — read in seq, set dna, len, maxien
* g_ca!loc() — calloc() with error checkm
* 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 *fj, int cleanupO /* cleanup tmp file */ long lseek(),
/*
* remove any tmp file if we blow
*/ cleanup(ι) cleanup int 1,
{ if(fj)
(void) unhnk(jname), exιt(ι),
}
/*
* rea , return ptr to seq, set dna, len, maxien
* skip lines startm g with ',', '<', or ' > '
* seq in upper or 1 lower case
*/ char * getseq(file, len) getseq char *fιle, /* file name " I int *len, I* seq len */
{ char hne[1024], *pseq register char *px, *p> , int natgc, tlen,
FILE *fp, if ((fp = fopen(file,'r")) = = 0) { fpπntf(stderr,"%s can t read 9. s\n' , prog, file), exιt(l),
} tlen = natgc = 0 while (tgets(hne, 1024, fp)) { if (*hne == ',' j | *lme == '<' | | *lme == >') continue for (px = line, *px '= '\n', px++) if (ιsupper(*px) | | ιslower(*px)) tlen+ + ,
} if ((pseq = malloc((unsigned)(tlen+6))) = = 0) { fpπntf(stderr,"%s malloc() failed to get %d bvtes tor 9< s\n", prog, tlen+6, file), exιt(l).
} pseq[0] = pseq[l] = pseq [2] = pseq [3] = '\0 ,
Page 1 of nwsubr.c ..getseq py = pseq + 4;
*len = tlen; rewιnd(fp); while (fgets(hne, 1024, fp)){ if (*hne = = ' ;' | | *hne == ' < ' | | *hne == '>') continue, for (px = line ; *px '= '\n'; px+ +) { if (ιsupper(*px))
*py+ + = *pχ; else if (ιslower(*px))
*py++ = toupper(*px); if (ιndex("ATGCU",*(py-l))) natgc + + ;
}
}
*py++ = '\0';
*py = '\0';
(void) fclose(tp); dna = natgc > (tlen/3), return(pseq+4);
} char * g calloc(msg, nx, sz) g_calloc 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) { fpnntf(stderr, "%s- g_calloc() failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz); exιt(l); } } return(px).
* get final jmps from dx[] or tmp file, set pp[], reset dmax: maιn()
*/ readjmpsO readjmps { int fd = -1,
Figure imgf000042_0001
register i, j, xx; if(fj){
(void) tclose(fj), if ((fd = open name.0_RDONLY, 0)) < 0) { tpπntf(stderr, "9< s: can't open() 9fs\n", prog, ]name), cleanup(l). } } for (l = lO = il = 0, dmaxO = dmax, xx = lenO; . !-'-+){ while (1) { for (j = dx[dmax].ιjmp; j > = 0 && dx[dmax].jp.x[jj > = xx; )-)
Page2ofnwsubr.c ...readjmps if < 0 && dx[dmax] offset && fj) {
(void) lseek(fd, dx[dmax] offset, 0),
(void) read(fd, (char *)&dx[dmax] jp, sizeof (struct jmp)),
(void) read(fd, (char *)&dx[dmax] offset, sizeof(dx[dmax] offset)), dx[dmax] ijmp = MAXJMP- 1 ,
} else break,
} if (i > = = JMPS) { fpπntf(stderr, " %s too many gaps I n ahgnmenΛn prog), cleanup(l),
} if 0 > = = 0) { siz = dx[dmax] jp nfjj, xx = dxfdmax] jp x[j],
Figure imgf000043_0001
if (siz < 0) { /* gap in second seq
Figure imgf000043_0002
XX + = SIZ,
/* id = xx - yy + lenl - 1 */ pp[l] x[ιl] = xx - dmax + lenl 1 , gapy + + , ngapy = siz, /* ignore MAXGAP when doing endgaps */ siz = (-siz < MAXGAP | | endgaps)9 -siz MAXGAP ιl + + ,
} else if (siz > 0) { /* gap in first seq */
Figure imgf000043_0003
gapx + + , ngapx + = siz, /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP | | endgaps) ' siz MAXGAP, ι0 + + , }
} else break, }
/* reverse the order of jmps */ for = 0, lO- , j < lO j + + , lO- ) {
. = pp[0j n[)], pp[0] nh] = ppfO] n[ι0], pp[0] n[ι0] = i i = pplO] x[)], pp[0] xDJ = PP[0] x[ι0], pp[0] x[ι0] = .
} for = 0, ι l -, j < ιl , j + + , i l-) { i = pp[l] n|j], pp[l] n[j] = pp[lj n[ι l], pp[l] n[ι l] = i, i = PH] xDJ . PPfl] xW = PPl U x[ιl] , PPfl] xf ill = ' } if (fd > = 0)
(void) close(fd), if (fj) {
(void) unhnkOname), f] = 0, offset = 0, }
Page 3 of nwsubr.c /*
* write a filled jmp struct offset of the prev one (if any): πw() */ wπtejmps(ιx) writejmps
Figure imgf000044_0001
char *mktemp(); if(!fj){ if (mktempOname) < 0) { fpπntf(stderr, "%s: can't mktemp() %s\n", prog, jname); cleanup(l);
} if ((fj = fopentjname, "w")) == 0) { fpπntf(stderr, "%s: can't write %s\n", prog, jname); exιt(l); } }
(void) fwπte((char *)&dx[ιx].jp, sizeof(struct jmp), 1, fj); (void) fwrιte((char *)&dx[ιxj. offset, sizeof(dx[ιx]. offset), 1, fj),
Pase4ofnwsubr.c Table 2A
PRO XXXXXXXXXXXXXXX (Length = 15 ammo acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% amino acid sequence identity =
(the number of identically matching ammo 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 ammo acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 ammo acids)
% ammo acid sequence identity =
(the number of identically matching ammo acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of ammo 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%
-4v 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 lespect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO 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 ammo acid sequence identity can be achieved in various ways that are within the skill in the art, tor instance, using publicly available computei software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % amino acid sequence identity values are 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 ot A and B, and where Y is the total numbei of amino acid residues in B It will be appreciated that wheie 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 7. amino acid sequence identity ot B to A As examples of σk ammo acid sequence identity calculations Tables 2A-2B demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated ' Compai ison Protein" to the amino acid sequence designated "PRO" Unless specifically stated othei wise. all % amino acid sequence identity v alues used herein are obtained as described above using the ALIGN-2 sequence comparison computer pi ogram However, c/c amino acid sequence identity may also be determined using the sequence comparison pi ogram NCBI-BLAST2 (Altschul et l Nucleic Acids Res , 25 3389-3402 ( 1997)) The NCBI-BLAST2 sequence compai ison pi giam may be downloaded from http //www ncbi nlm nih
Figure imgf000049_0001
NCBI-BLAST2 uses several seaich paiameteis wherein all of those search parameters are set to default values including, for example, unmask = yes stιand = all. expected occurrences = 10 minimum low complexity length = 15/5 multi-pass e-value = 0 01 constant tor multi-pass = 25 dropoit tor final gapped alignment = 25 and scoring matrix = BLOSUM62
In situations where NCBI-BLAST2 is employed foi ammo acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, oi against a given amino acid sequence B (which can alternatively be phiased 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 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. ; 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 % amino acid sequence identity value is determined by dividing (a) the number of matching identical ammo 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 (; 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 ammo 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 ammo acid sequence B is the amino acid sequence of the PRO polypeptide of interest "PRO variant polypeptide" or "PRO variant nucleic acid sequence' means a nucleic acid molecule which encodes an actn e PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an exti acellular domain of a PRO polypeptide, with or without the signal peptide. as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Oidinaπly, a PRO variant polynucleotide w ill have at least about 80% nucleic acid sequence identity, moie pieterably at least about 81 % nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, moie preferably at least about 83%- nucleic acid sequence identity, more pi eferably at least about 84% nucleic acid sequence identity more preferably at least about 85% nucleic acid sequence identity, moie preferably at least about 86% nucleic acid sequence identity . more preferably at least about 87% nucleic acid sequence identity, moie preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity , moie 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 947o 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 prefei ably 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 PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Variants do not encompass the native nucleotide sequence
Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in length, more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more
"Percent (%) nucleic acid sequence identity" with respect to the PRO 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 PRO 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 oi Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % 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 authoied 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 piovided in Table 1 The ALIGN-2 pi ogram should be compiled for use on a UNIX operating system, prefeiably digital UNIX V4 0D All sequence compai ison parameters are set by the ALIGN-2 program and do not vaiy
For puiposes 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 oi comprises a certain % nucleic acid sequence identity to, with, oi 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 cei tain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follow s
100 times the fi action 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 numbei 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
Figure imgf000052_0001
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 parameteis, aie 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 detei mined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence ot 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 ot interest (/ 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, PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7), Figure 6 (SEQ ID NO 10), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 16), Figure 12 (SEQ ID NO 18), Figure 14 (SEQ ID NO 23), Figure 16 (SEQ ID NO 25), Figure 18 (SEQ ID NO 27), Figure 20 (SEQ ID NO 29), Figure 22 (SEQ ID NO 34), Figure 24 (SEQ ID NO 36), Figure 26 (SEQ ID NO 38), or Figure 28 (SEQ ID NO 40), Figure 30 (SEQ ID NO 42), Figure 32 (SEQ ID NO 44), Figure 34 (SEQ ID NO 46), Figure 36 (SEQ ID NO 49), Figure 38 (SEQ ID NO 51 ), Figure 40 (SEQ ID NO 53), Figure 42 (SEQ ID NO 55), Figure 44 (SEQ ID NO 57), Figure 46 (SEQ ID NO 65), Figure 48 (SEQ ID NO 67), Figure 50 (SEQ ID NO 69), Figure 52 (SEQ ID NO 71 ), Figure 54 (SEQ ID NO 73), Figure 56 (SEQ ID NO 75), Figure 58 (SEQ ID NO 77) or Figure 60 (SEQ ID NO 79), respectively PRO variant polypeptides may be those that are encoded by a PRO 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 ammo 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
Figure imgf000053_0001
amino acid sequence A that has or comprises a certain % positives to, with, oi against a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the numbei of amino acid residues scoring a positive value as defined above by the sequence alignment program ALIGN-2 in that pi ogram s alignment of A and B, and wheie 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 heiem means polypeptide that has been identified and separated and/or lecovered from a component ot its natural environment Preferably, the isolated polypeptide is free ot association with all components with which it is natuially associated Contaminant components of its natural environment are matenals that would typically mterteie ith diagnostic or therapeutic uses foi 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 ammo acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO 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 PRO polypeptide or an "isolated" nucleic acid encoding an anti-PRO 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 PRO-encoding nucleic acid or the anti-PRO-encoding nucleic acid Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated An isolated PRO-encoding nucleic acid molecule or an anti- PRO-encodmg 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 PRO-encoding nucleic acid molecule or the anti-PRO- encoding nucleic acid molecule as it exists in natural cells However, an isolated nucleic acid molecule encoding a PRO polypeptide or an anti-PRO antibody includes PRO-nucleic acid molecules and anti-PRO-nucleic acid molecules contained in cells that ordinarily express PRO polypeptides or express anti-PRO antibodies where, foi 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 nbosome 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 ot the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transciiption ot the sequence, or a nbosome 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 secretoiy leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by hgation at convenient restriction sites If such sites do not exist, the synthetic ohgonucleotide adaptors or linkers are used in accordance with conventional practice
The term "antibody" is used in the broadest sense and specifically covei s, for example, single anti- PR0212, antι-PRO290. antι-PR0341 , antι-PR0535. antι-PR0619 antι-PR0717, antι-PRO809, antι-PR083ϋ. anti- PR0848, antι-PR0943. anti-PRO 1005, anti-PROl 009 anti-PRO 1025, anti-PRO 1030, anti-PRO 1097, anti- PROl 107, anti-PRO l 1 1 1 , anti-PROl 153, anti-PROl 182. anti-PRO l 184, anti-PRO l 187, anti-PRO 1281 , anti- PR023. antι-PR039, antι-PR0834, anti-PRO 1317, anti-PRO 1710 antι-PRO2094, antι-PR02145 or antι-PR02198 monoclonal antibodies (including antagonist, and neutralizing antibodies), antι-PR0212, antι-PRO290, anti- PR0341 , antι-PR0535, antι-PR0619, antι-PR0717, antι-PRO809. antι-PRO830, antι-PR0848, antι-PR0943. anti- PRO 1005. anti-PRO 1009, anti-PRO 1025, anti-PRO 1030 anti-PRO 1097, anti-PROl 107, anti-PRO l 1 1 1 , anti- PROl 153, anti-PROl 182, anti-PROl 184, anti-PROl 187, anti-PROl 281 , antι-PR023, antι-PR039, antι-PR0834, anti-PROl 317, anti-PROl 710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibody compositions with polyepitopic specificity, single chain antι-PR0212, antι-PRO290, antι-PR0341 , antι-PR0535, antι-PR0619. anti- PRO? 17, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, anti-PRO 1005, anti-PRO 1009. anti-PRO 1025, anti-PRO 1030, anti-PRO 1097, anti-PROl 107, anti-PROl 1 1 1 , anti-PROl 153, anti-PROl 182, anti-PROl 184, anti- PRO 1 187, anti PRO 1281 , antι-PR023, antι-PR039, antι-PR0834, anti-PRO 1317, anti-PRO 1710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibodies, and fragments of antι-PR0212, antι-PRO290, antι-PR0341 , anti PR0535, antι-PR0619, antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, antι-PRO1005, anti-PRO 1009, anti-PRO 1025, anti-PRO 1030, anti-PRO 1097, anti-PRO 1 107, anti-PRO 1 1 1 1 , anti-PRO 1 153, anti- PRO 1 182, anti-PRO 1 184, anti-PRO 1 187, anti-PROl 281 , antι-PR023, antι-PR039, antι-PR0834, anti-PRO 1317, antι-PRO1710, antι-PRO2094, antι-PR02145 or antι-PR02198 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 geneial, 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 temperatui e The higher the degree of desired homology between the probe and hybndizable 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 lowei 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 01 M sodium chloπde/0 0015 M sodium cιtrate/0 1 % sodium dodecyl sulfate at 50°C, (2) employ during hybridization a denaturing agent, such as formamide, foi example, 50% (v/v) formamide with 0 1 % bovine serum albumιn/0 1 % Fιcoll/0 1 % polyvinylpyrrohdone/ 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 citiate) 50 mM sodium phosphate (pH 6 8), 0 1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon speim DNA (50 g/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 etal Molecular Cloning A Laboratory Manual, New York Cold Spring Harbor Press 1989, and include the use ot w ashing solution and hybridization conditions (e g , temperature, ionic strength and % SDS) less stringent than those described above An example ot moderately stringent conditions is overnight incubation at 37"C in a solution comprising 20% formamide. 5 x SSC ( 1 0 mM NaCl 1 mM tπsodium citrate) 50 M 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 filtei s 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 heiein refers to a chimeric polypeptide comprising a PR0212 PRO290, PR0341 , PR0535. PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025. PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PRO l 187, PR01281 PR023, PR039, PR0834, PROl 317. PROl 710, PRO2094, PR02145 or PR02198 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 PR0212. PRO290, PR0341 , PR0535, PRO619, PRO717, PRO809, PRO830, PRO848, PRO943, PRO1005, PRO1009, PRO 1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PROl 281 , PR023 PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptides which retain a biological and or an lmmunological activity/property of a native or naturally-occurring PR0212, PRO290, PR0341. PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl H I , PROl 153, PROl 182, PROl 184, PR01 187, PRO1281, PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide, wherein "biological" activity refers to a function (either inhibitory or stimulatory) caused by a native or natui ally-occurring PR0212, PRO290, PR0341. PR0535. PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO 1030, PRO1097, PRO l 107, PROl 1 11 , PROl 153, PROl 182, PROl 184, PROl 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710 PRO2094, PR02145 or PR02198 polypeptide other than the ability to induce the production of an antibody against an antigemc epitope possessed by a a native or naturally-occun ing PR0212, PRO290 PR0341 PR0535, PR0619 PR0717, PRO809, PRO830, PR0848. PR0943, PROl 005, PRO 1009, PRO 1025. PRO 1030, PRO 1097, PRO 1 107 PROl 1 1 1 , PROl 153, PROl 182, PR01 184, PR01 187, PRO1281 , PRO23, PRO39. PRO834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide and an "lmmunological" activity reteis to the ability to induce the production of an antibody against an antigemc epitope possessed by a native oi naturally-occurring PR0212, PRO290, PR0341 , PR0535. PR0619, PR0717, PRO809, PRO830, PR0848. PR0943, PRO 1005, PRO 1009, PRO1025, PRO1030, PRO1097, PRO l 107, PRO l 1 1 1 , PROl 153, PRO l 182, PRO l 184. PRO l 187, PR01281 PR023, PR039, PR0834, PROl 317. PROl 710, PRO2094, PR02145 or PR02198 polypeptide
"Biological activity' in the context ot an antibody or another antagonist molecule that can be identified by the screening assays disclosed herein (e g . an oiga c or inorganic small molecule peptide. etc ) is used to refer to the ability of such molecules to bind or complex with the polypeptides encoded b\ the amplified genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides w ith other cellular proteins oi otherwise interfere with the transcription or translation of a PR0212, PRO290. PR034 I , PR0535, PR0619. PR0717, PRO809, PRO830, PR0848 PR0943, PRO 1005, PRO 1 09, PRO 1025 PRO 1030, PRO 1097, PROl 107 PROl 111, PROl 153, PROl 182, PROl 184,PRO1187,PRO1281.PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 polypeptide A preferred biological activity is growth inhibition of a target tumor cell Anothei preferred biological activity is cytotoxic activity resulting in the death of the target tumor cell The term "biological activity" in the context of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PROl 005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PRO 1107, PROl 111, PROl 153, PRO 1182, PROl 184, PROl 187, PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 polypeptide means the ability of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PROl 107,PROl 111, PROl 153, PROl 182,PR01184, PROl 187,PR01281,PR023,PR039,PR0834,PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide to induce neoplastic cell growth or uncontrolled cell giowth
The phrase "lmmunological activity" means lmmunological cross-reactivity with at least one epitope of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 orPR02198 polypeptide
"lmmunological cross-reactivity" as used herein means that the candidate polypeptide is capable ot competitively inhibiting the qualitative biological activity of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184,PR01187, PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 polypeptide having this activity with polyclonal antisera raised against the known active PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PROl 005. PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PRO! 187, PRO 1281, PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 lmmunological cross-reactivity preferably is "specific . which means that the binding affinity of the immunologically cross-reactive molecule (eg , antibody) identified, to the corresponding PR0212, PRO290, PR0341. PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009 PRO1025, PRO 1030, PRO 1097, PROl 107, PROl 111, PROl 153. PROl 182, PROl 184, PROl 187, PR01281 PR023, PR039. PR0834, PROl 317. PROl 710, PRO2094, PR02145 orPR02198 polypeptide is significantly higher (preferably at least about 2-tιmes. more preferably at least about 4-tιmes, even more preferably at least about 8-tιmes, most preferably at least about 10-tιmes higher) than the binding affinity of that molecule to any olhei 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 ot a native PR0212, PRO290, PR0341. PR0535 PR0619 PR0717 PRO809, PRO830. PR0848, PR0943, PRO1005, PRO 1009. PRO1025. PRO1030, PRO1097, PROl 107 PROl 111, PROl 153, PROl 182,PROlI84,PRO1187,PROI281,PRO23.PRO39.PRO834.PRO1317,PRO1710 PRO2094, PR02145 or PR02198 polypeptide disclosed herein oi the transcription or translation thei eot 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 PR0212, PRO290, PR0341 , PR0535. PR0619, PR0717, PRO809, PRO830. PR0848, PR0943, PRO1005, PRO1009 PRO 1025, PRO 1030, PRO1097. PROl 107. PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094. PR02145 or PR02198 polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281. PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide A "small molecule" is defined herein to have a molecular weight below about 500 Daltons.
"Antibodies" (Abs) and "immunoglobuhns" (Igs) are glycoproteins having the same structural characteristics While antibodies exhibit binding specificity to a specific antigen, immunoglobuhns 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 immunoglobuhns" are usually heterotetrameπc 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 immunoglobulm isotypes Each heavy and light chain also has regularly spaced intrachain disulfide bridges Each heavy chain has at one end a variable domain (V„) 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" refeis to the fact that certain poi tions of the variable domains differ extensivelv in sequence among antibodies and are used in the binding and specificity ot each particulai antibody for its particulai antigen However, the variability is not evenly distributed throughout the vanable domains of antibodies It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervanable regions both in the light-chain and the heavy-chain variable domains The moie highly conserved portions of variable domains are called the amewoi k (FR) regions The variable domains of native heavy and light chains each comprise tout FR regions largely adopting a β-sheet configuration, connected b\ thiee CDRs which form loops connecting and in some cases forming part of, the β-sheet structure The CDRs in each chain are held togethei in close pioximitv by the FR regions and, with the CDRs from the other chain contribute to the formation of the antigen-bindmg site of antibodies ( . ee Kabat et al NIH Publ No 91 -3242 Vol I, pages 647-669 ( 1991 )) The constant domains aie not involv ed directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation ot the antibody in antibody-dependent cellular toxicitv The term "hypervai ble region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding The hypervanable region comprises amino acid residues from a "complementarity determining region" or "CDR" (. e , residues 24-34 (Ll ). 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 lmmunological Interest, 5th Ed Public Health Service, National Institute of Health, Bethesda, MD [ 1991 ] ) and/or those residues from a "hypervanable loop" (i e , residues 26-32 (Ll ), 50-52 (L2) and 91-96 (L3) m 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 hypervanable region residues as herein defined "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the mtact antibody Examples of antibody fragments include Fab, Fab', F(ab')„ and Fv fragments diabodies , linear antibodies (Zapata etal , Protein En g .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')2 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 contiguiation that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer Collectively, the six CDRs confer antigen-binding specificity to the antibody However, even a single variable domain (or half of an Fv compi ising 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 (CH 1 ) of the heavy chain Fab fiagments differ from Fab' fragments by the addition ot a tew residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine resιdue(s) of the constant domains bear a free thiol group F(ab') . antibody fragments originally were produced as pan s of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known The "light chains" of antibodies (immunoglobuhns) 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 then constant domains
Depending on the amino acid sequence ot the constant domain ot their heavy chains immunoglobuhns can be assigned to different classes There are five major classes of immunoglobuhns IgA, IgD, IgE, IgG and IgM and several of these may be further divided into subclasses (ιsotypes), e ι> , IgG l , IgG2, IgG3 IgG4 IgA and IgA2 The heavy-chain constant domains that correspond to the different classes ot immunoglobuhns aie called α, δ e, γ, and μ. respectively The subunit structures and three-dimensional configurations of ditteient classes of immunoglobuhns are well known The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ; 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 antigemc 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 immunoglobuhns The modifiei "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 pai ticular 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 (immunoglobuhns) 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 chaιn(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 etal , Proc Natl Acad Sci USA, 81 6851-6855 [1984])
"Humanized" forms of non-human (e g , munne) antibodies are chimeric immunoglobuhns, immunoglobulm chains or fragments thereof (such as Fv, Fab, Fab' F(ab')-, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulm For the most part humanized antibodies are human immunoglobuhns (recipient antibody) in which residues from a CDR ot 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 immunoglobulm are replaced by corresponding non-human residues Furthermoie 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 ot at least one and typically two, va able domains, in which all or substantially all of the CDR regions coirespond to those of a non human immunoglobulm and all or substantially all of the FR regions aie those of a human immunoglobulm sequence The humanized antibody optimally also will comprise at least a portion of an immunoglobulm constant legion (Fc), typically that ot a human immunoglobulm For further details see, Jones et al Nature. 321 522-525 ( 1986), Reichmann et al , Natuie, 332 323-329 [ 1988], and Presta Curr Op Struct Biol . 2 593-596 ( 1992) The humanized antibody includes a PRIMATIZED ' M antibody wherein the antigen-binding region ot the antibody is dei IV ed from an antibody pi oduced by immunizing macaque monkeys w ith the antigen of mteiest "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 V, domains which enables the sFv to form the desired structure for antigen binding Foi 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 Holhnger 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 withm 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 , radioiso tope 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 Radionuchdes that can serve as detectable labels include, for example, 1-131 I 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 adheie 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 polyv inyl alcohol and silicones In certain embodiments, depending on the context, the solid phase can compnse the well ot an assay plate, in othei s 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 ot lipids phosphohpids and/oi surfactant which is useful for delivery of a drug (such as a PR0212, PRO290 PR0341 PR0535 PR06 I 9 PR0717, PRO809 PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025 PRO 1030 PRO 1097, PRO l 107, PRO l l l l PRO 1 153, PRO 1 182, PRO 1 184 PRO 1 187, PRO 1281 , PR023, PR039, PR0834 PRO 1317, PRO 1710, PRO2094 PR02I 45 or PR02198 polypeptide oi antibody thereto and, optionally a chemotherapeutic agent) to a mammal The components of the liposome are commonly arranged in a bilayer foimation, 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 immunoglobulm constant domains Structurally, the immunoadhesins comprise a fusion of an ammo acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i e , is "heterologous"), and an immunoglobulm 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 immunoglobulm constant domain sequence in the immunoadhesin may be obtained from any immunoglobulm, such as IgG-1 , IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM
π_ Compositions and Methods of the Invention
A Full-length PRQ212. PRO290, PRQ341. PRQ535. PRQ619. PRQ717. PRO809. PRO830. PRQ848. PRQ943. PRO1005. PRO1009. PRO1025. PRO1030. PRO1097, PRO1 107. PROl l l l , PRQ1 153, PR01 182, PROl 184. PROl 187, PRQ1281. PRQ23. PRQ39. PRQ834. PRQ1317. PRO1710. PRO2094, PRQ2145 and PRQ2198 polypeptides
The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PRO1 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 and PR02198 In particular, cDNA encoding PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717. PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO 1025, PRO1030, PRO1097, PRO l 107, PROl 1 1 1 , PROl 153. PR01 182, PROl 184, PROl 187, PRO l 281 , PR023, PR039, PR0834, PROl 317, PRO1710, PRO2094, PR02145 and PR02198 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 vanants included in the foregoing definition of PR0212. PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO1009, PROl 025, PRO 1030, PRO 1097 PROl 107. PROl l l l , PROl 153, PROl 182, PRO l 184. PRO l 187 PRO 1281. PR023, PR039, PR0834, PRO 1317, PRO 1710 PRO2094, PR02145 and PR02198 will be referred to as "PR0212, PRO290, PR0341. PR0535. PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005 PRO1009, PRO1025. PRO1030. PRO1097, PROl 107, PRO l 1 1 1. PRO l 153, PRO l 182, PROl 184, PRO l 187 PRO1281. PRO23, PRO39, PRO834, PROI 317, PRO1710 PRO2094, PRO2145 orPRO2l 98", ιegardless ot theιr origin or mode ot preparation As disclosed in the Examples below, cDNA clones hav e been deposited with the ATCC The actual nucleotide sequence of the clones can readily be determined by the skilled artisan by sequencing ot the deposited clone using loutine methods in the art The predicted amino acid sequences can be determined fiom the nucleotide sequences using routine skill. For the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107. PROllll, PRO 1153, PRO 1182, PRO 1184, PRO 1187. PRO 1281, PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 orPR02198 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. PRQ212, PRO290. PRQ341, PRQ535. PRQ619. PRQ717. PRO809. PRO830, PRQ848. PRQ943. PROl 005, PRO 1009, PRO 1025, PRO 1030, PRO 1097. PROl 107, PROllll. PROl 153, PRO 1182, PROl 184. PROl 187. PRQ1281. PRQ23. PRQ39. PRQ834. PRQ1317. PRO1710. PRO2094. PRQ2145 and PRQ2198 Variants In addition to the full-length native sequence PR0212, PRO290, PR0341, PR0535, PR0619, PR0717,
PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PROI030, PRO1097, PROl 107, PROl 111, PROl 153,PROH82,PR01184, PROl 187,PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 and PR02198 polypeptides described herein, it is contemplated that PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1107, PROllll, PR01153, PR01182, PROH84, PROH87, PR01281, PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 and PR02198 variants can be prepared. PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281. PR023, PR039, PR0834, PROl 317, PRO1710, PRO2094, PR02145 andPR02198 variants can be prepared by introducing appropriate nucleotide changes into the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PROI030, PRO1097, PROl 107. PROl 111, PROl 153,PRO1182,PRO1184,PRO1187,PRO128I,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 DNA, and/or by synthesis of the desired PR0212, PRO290, PR0341 , PR0535. PRO619,PRO717,PRO809,PRO830,PRO848,PRO943.PRO1005,PRO1009,PRO1025,PRO1030.PRO1097. PROl 107,PROl 111, PROl 153, PROl 182,PR01184,PR01187,PR01281,PR023,PR039,PR0834.PR01317. PRO1710, PRO2094, PR02145 or PR02198 polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848. PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll,PRO1153,PRO1182,PRO1184,PRO1187,PRO1281,PRO23.PRO39,PRO834.PRO1317,PRO1710. PRO2094, PR02145 or PR02198, such as changing the number or position of giycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence PR0212. PRO290, PR0341. PR0535. PR0619. PR0717. PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097. PROl 107. PROl 111. PROl 153,PR01182, PROl 184. PROl 187, PR01281.PR023.PR039,PR0834,PR01317. PRO 1710. PRO2094, PR02145 or PR02198 or in various domains of the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848. PR0943, PRO 1005. PRO 1009, PRO 1025, PRO 1 30, PRO 1097. PRO 1107. PROl 111. PROl 153,PRO1182,PRO1184,PRO1187.PRO1281.PRO23,PRO39,PRO834,PRO1317.PRO1710. PRO2094, PR02145 or PR02198 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 PR0212, PRO290 PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PRO l 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PROl 317, PRO 1710, PRO2094, PR02145 or PR02198 that results in a change in the amino acid sequence of the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184. PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 as compared with the native sequence PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PR01 182, PROl 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO 1030, PRO1097, PROl 107, PROl 1 1 1, PROl 153, PR01 182, PROl 184, PROl 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 Guidance in determining which ami no acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005. PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PRO l 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 ammo acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, J e , conservative ammo 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 oi mature native sequence
PR0212, PRO290. PR0341 , PR0535, PR0619, PR0717, PRO809 PRO830, PR0848. PR0943. PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PRO l 153, PROl 182, PROl 184, PROl 187, PRO 1281 , PR023, PR039, PR0834, PROl 317, PRO l 710, PRO2094 PR02145 and PR02198 polypeptide fragments are piovided 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 nativ e piotein Certain fragments lack amino acid residues that are not essential for a desired biological activity of the PR0212, PRO290, PR0341 PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005. PRO1009, PRO1025. PRO1030. PRO 1097. PRO 1 107, PRO l 1 1 1 , PRO l 153. PRO 1 182 PROl 184 PRO l 187, PRO 1281. PR023. PR039 PR0834, PROl 317. PROl 710, PRO2094, PR02145 or PR02198 polypeptide
PR0212, PRO290, PR0341 , PR0535, PR0619, PR07 I 7, PRO809 PRO830, PR0848. PR0943, PRO 1005, PRO 1009. PRO1025, PRO1030. PRO1097, PROl 107, PROl 1 1 1. PRO l 153. PRO l 182. PROl 184. PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830. PR0848, PR0943, PRO 1005, PRO 1009, PROl 025, PRO 1030, PRO 1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PR01 184, PR01 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide fragments share at least one biological and/or immunological activity with the native PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005. PRO1009, PRO1025, PRO1030, PRO1097, PRO1107, PROl 1 1 1 , PROl 153, PROH82, PROH 84, PR01 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 Exemplan Preferred
Residue Substitutions Substitutions
Ala (A) val, leu, lie 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, lie, val,
Figure imgf000066_0001
Phe (F) leu, val, lie, ala, tyr leu
Pro (P) ala ala
Ser (S) thi thi
Thr (T) ser ser
Trp (W) tyr, phe tyr
Tyr (Y) trp, phe, thr, ser phe
Val (V) lie, 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 oi 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 noi leucine met, ala, val leu lie
(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 anothei class Such substituted residues also may be introduced into the conseiv atn e substitution sites or moie preferably into the remaining (non-conserved) sites
The v ariations can be made using methods known in the art such as ohgonucleotide mediated (site directed) mutagenesis, alanine scanning, and PCR mutagenesis Site dnected mutagenesis [Cartel et al Nucl Acids Res 13 4331 ( 1986), Zollei et al , Nucl Acids Res ]0 6487 ( 1987)], cassette mutagenesis [Wells et al Gene 34 315 ( 1985)] restriction selection mutagenesis [Wells tal Philos Trans R Soc London Sei A 317 415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PR0212, PRO290, PR0341, PR0535, PR0619. PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030. PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 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 ammo 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 am o 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 , 1501 (1976)] If alanine substitution does not yield adequate amounts of variant, an lsoteπc ammo acid can be used
C Modifications of PRQ212. PRO290. PRQ341. PRQ535. PRQ619. PRQ717. PRO809. PRO830. PRQ848, PRQ943, PRO1005 PRO1009, PRO1025, PRO1030, PRO1097. PROl 107, PROllll. PROl 153 PR01182.PROI184.PR01187.PR01281,PR023,PR039.PR0834,PR01317.PR01710,PR02094,PR02145 andPRQ2198
Covalent modifications of PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll. PROl 153, PRO1182,PRO1184.PRO1187,PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710,PRO2094,PRO2145 and PR02198 are included within the scope of this invention One type of covalent modification includes reacting targeted amino acid residues of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005. PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153. PROl 182, PROl 184,PRO1187,PRO1281,PRO23,PRO39,PRO834,PRO1317.PRO1710,PRO2094,PRO2145 or PR02198 polypeptide with an organic deπvatizing agent that is capable of reacting with selected side chains oi the N- or C- terminal residues of the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848. PR0943, PRO1005 PRO1009, PRO1025, PRO1030, PRO1097, PROl 107. PROllll. PROl 153, PRO 1182, PRO 1184, PRO 1187, PRO 1281 , PR023, PR039, PR0834 PRO 1317, PRO 1710, PRO2094. PR02145 or PR02198 Denvatization with bifunctional agents is useful, for instance, tor crosshnking PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848 PR0943, PRO1005, PRO1009 PRO1025, PRO 1030, PROl 097, PROl 107, PROllll, PROl 153, PROl 182, PRO 1184, PROl 187, PRO 1281 PR023 PR039. PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 oi PR02198 to a watei -insoluble support matrix oi surface tor use in the method for purifying antι-PR0212, antι-PRO290 an tι-PR0341 , antι-PR0535, antι-PR0619 antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, anti-PRO 1005, anti-PRO 1009, anti- PRO1025, anti-PRO 1030, antι-PRO1097, anti-PROl 107. anti-PROl 111, anti-PROl 153, anti-PROl 182. anti- PRO 1184, anti-PRO 1187, anti-PRO 1281, antι-PR023, antι-PR039, antι-PR0834. anti-PRO 1317, anti-PRO 1710. antι-PRO2094. antι-PR02!45 or antι-PR02198 antibodies, and vice-veisa Commonly used crosshnking agents include, e g , 1 , 1 -bιs(dιazoace ty! )-2-ph enylethane, glutai aldehyde. N-hydroxysuccinimide estei s tor example, estei s with 4-azιdosalιcyhc acid, homobitunctional lmidoesters, including disuccinimidyl esters such as 3.3'- dιthιobιs(succιnιmιdylpropιonate), bifunctional maleimides such as bιs-N-maleιmιdo-l,8-octane and agents such as methyl-3-[(p-azιdophenyl)dιthιojpropιoιmιdate
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of prohne and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the -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 amme, and amidation of any C-terminal carboxyl group
Another type of covalent modification of the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 polypeptide included within the scope of this invention comprises altering the native giycosylation pattern of the polypeptide "Altering the native giycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097,PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PR01281,PR023,PR039,PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 (either by removing the underlying giycosylation site oi by deleting the giycosylation by chemical and/or enzymatic means), and/or adding one or more giycosylation sites that are not present in the native sequence PR0212, PRO290, PR0341, PR0535. PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005. PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153,PR01182, PROl 184,PROU87,PRO1281,PRO23,PRO39.PRO834.PRO1317,PRO1710,PRO2094, PR02145 or PR02198 In addition, the phrase includes qualitative changes in the giycosylation of the native proteins, involving a change in the natuie and proportions of the various carbohydrate moieties present
Addition of giycosylation sites to the PR0212, PRO290, PR0341 , PR0535. PR0619, PR0717, PRO809. PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030. PRO1097, PROl 107. PROllll PR01153,PR01182,PR01184,PR01187,PR01281,PR023,PR039 PRO834.PRO1317,PRO1710,PRO2094 PR02145 or PR02198 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 oi thieonme residues to the native sequence PR0212, PRO290, PR0341, PR0535, PR0619, PR0717 PRO809 PRO830, PR0848. PR0943 PRO1005, PRO1009, PROI025. PRO 1030, PRO 1097, PROl 107 PROllll, PROl 153, PROl 182. PROl 184 PRO 1187, PROl 281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094 PR02145 or PR02198 (foi O- hnked giycosylation sites) The PR02I2, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830 PR0848, PR0943, PRO 1005, PRO 1009 PRO 1025 PRO 1030, PRO 1097. PROl 107 PROllll PROl 153 PRO 1182, PRO 1184, PRO 1187. PRO 1281, PR023, PR039, PR0834, PRO 1317. PRO 1710, PRO2094. PR02145 or PR02198 amino acid sequence may optionally be altered thiough changes at the DNA level, paiticularly by mutating the DNA encoding the PR0212, PRO290. PR0341, PR0535. PR0619. PR0717, PRO809, PRO830, PR0848, PR0943. PRO1005. PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll. PROl 153 PROl 182, PROl 184,PRO1187,PRO1281.PRO23.PRO39,PRO834,PRO1317.PRO1710,PRO2094.PRO2145 or PR02198 polypeptide at preselected bases such that codons are generated that will translate into the desned amino acids
Another means of increasing the number of carbohydrate moieties on the PR0212, PRO290. PR0341 , PR0535, PR0619, PR07I7, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097,PRO1107,PROl 111, PROl 153,PR01182,PR01184,PR01187,PR01281,PR023,PR039,PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 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 11 September 1987, and in Aphn and Wπston, CRC Cπt Rev Biochem , pp 259-306 (1981)
Removal of carbohydrate moieties present on the PR0212, PRO290, PR0341, PR0535, PR0619, PRO717,PRO809,PRO830,PRO848,PRO943,PRO1005,PRO1009,PRO1025,PRO1030,PRO1097, PROl 107, PROl 111, PROl 153,PR01182, PROl 184,PROH 87, PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 polypeptide may be accomplished chemically or enzymatically or by mu tational substitution of codons encoding for amino acid residues that serve as targets for giycosylation Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al , Arch Biochem Biophys .25952(1987) and by Edge et al , Anal Biochem , 118131 (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, 138350 (1987)
Another type of covalent modification of PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153,PRO1182,PRO1184,PRO1187,PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 comprises linking the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717. PRO809, PRO830. PR0848, PR0943, PRO1005, PRO1009, PRO1025. PRO1030 PRO1097, PROl 107, PROl 111, PROl 153. PROl 182, PROl 184, PROl 187,PR01281,PR023,PR039,PR0834,PR01317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide to one of a variety ot nonproteinaceous polymers, e g , polyethylene glycol (PEG), polypi opylene glycol, or polyoxyalkylenes. in the mannei set forth in U S Patent Nos 4640.835, 4,496,689, 4,301.144, 4,670.417, 4.791 , 192 or 4, 179,337
The PR0212, PRO290, PR0341, PR0535, PR0619, PR0717. PRO809, PRO830. PR0848. PR0943. PRO1005, PRO1009. PRO1025. PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PRO 1187, PRO 1281, PR023, PR039, PR0834, PRO 1317, PRO 1710. PRO2094. PR02145 or PR02198 of the present invention may also be modified in a way to form a chimeric molecule comprising PR0212, PRO290 PR0341, PR0535 PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PROI025 PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023 PR039, PR0834 PROl 317, PROl 710, PRO2094, PR02145 oi PR02198 fused to another, heterologous polypeptide or amino acid sequence In one embodiment, such a chimeπc molecule comprises a fusion of the PR0212, PRO290, PR0341
PR0535, PR0619. PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PROl 025, PRO 1030, PRO 1097, PRO 1107, PROl 111. PROl 153, PROl 182. PROl 184. PROl 187. PROl 281,PR023,PR039,PR0834. PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 ammo- or carboxyl terminus of the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PROl 005, PRO1009, PRO1025, PRO1030 PRO1097 PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281, PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 The presence of such epitope tagged forms of the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005 PRO1009, PRO1025, PRO1030 PRO1097, PROl 107, PROllll, PROl 153, PRO 1182, PRO 1184, PRO 1187 PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710,PRO2094,PRO2145 or PR02198 can be detected using an antibody against the tag polypeptide Also, provision of the epitope tag enables the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153 PROl 182, PROl 184, PROl 187, PRO 1281, PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 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 .82159-2165 (1988)], the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan etal , Molecular and Cellular Biology, 53610-3616 ( 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 et al , BioTechnology, 61204-1210 (1988)], the KT3 epitope peptide [Martin etal , Science, 255192-194 (1992)], an α-tubulm epitope peptide [Skinner etal , J Biol Chem, 26615163- 15166 ( 1991 )], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl Acad Sci USA.876393-6397 (1990)]
In an alternative embodiment, the chimeric molecule may comprise a fusion of the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830 PR0848, PR0943, PRO1005, PRO1009 PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PRO! 153 PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039 PR0834 PR01317 PROl 710, PRO2094,PRO214 oι PR02198 with an immunoglobulm or a particular region of an immunoglobulm 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 PR0212, PRO290 PR0341 PR0535 PRO619,PRO717,PRO809,PRO830,PRO848 PR0943, PRO 1005, PRO 1009, PRO1025 PRO1030, PRO1097,PRO1107,PROllll,PRO1153,PRO1182 PR01184 PROl I87,PR01281,PR023 PR039 PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide in place ot at least one variable region within an Ig molecule In a paiticularly preferred embodiment the immunoglobulm fusion includes the hinge, CH2 and CH3 or the hinge CHI, CH2 and CH3 regions of an IgGl molecule Foi the production ot immunoglobulm fusions see also US Patent No 5428130 issued June 27 199^ D. Preparation of PRQ212. PRO290. PRQ341. PRQ535. PRQ619. PRQ717. PRO809. PRO830. PRQ848. PRQ943. PRO1005. PRO1009. PRO1025. PRO1030. PRO1097. PROl 107. PROllll. PROl 153. PROl 182. PRQ1184. PROl 187. PRQ1281. PRQ23. PRQ39. PRQ834. PRQ1317, PRO1710. PRO2094. PRQ2145 and PRQ2198 Polypeptides The description below relates primarily to production of PR0212, PRO290, PR0341 , PR0535, PR0619,
PRO717,PRO809,PRO830,PRO848,PRO943,PRO1005,PRO1009,PRO1025,PRO1030,PRO1097,PRO1107, PROl 111, PROl 153, PROl 182, PROl 184,PR01187,PR01281,PR023,PR039,PR0834,PR01317,PR01710, PRO2094, PR02145 or PR02198 by culturing cells transformed or transfected with a vector containing PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PROl 281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO 1025 , PRO 1030, PRO 1097, PRO 1107, PRO 1111, PRO 1153, PRO 1182, PRO 1184, PRO 1187, PROl 281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198. For instance, the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005. PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PRO 1107, PROl 111, PROl 153,PR01182,PR01184,PR01187,PR01281,PR023,PR039.PR0834, PROl 317, PRO1710, PRO2094, PR02145 or PR02198 may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717. PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PRO1153,PRO1182,PRO1184,PRO1187,PRO128I,PRO23,PRO39,PRO834,PRO1317,PRO1710,PRO2094, PR02145orPR02198.
a. Isolation of DNA Encoding a PRQ212. PRQ290. PRQ341, PRQ535. PRQ619. PRQ717.
PRO809. PRO830. PRQ848. PRQ943. PRO1005. PRQ1009. PRO1025. PRO1030. PRO1097. PROl 107. PROllll.PRO1153.PRO1182.PRO1184.PRO1187.PRO1281.PRO23.PRO39.PRO834.PRO1317.PRO1710. PRO2094, PRQ2145 or PRQ2198 Polypeptide DNA encoding PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830. PR0848,
PR0943, PROl 005, PRO 1009, PRO 1025. PRO 1030, PRO 1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PROL317, PRO1710, PRO2094, PR02145 or PR02198 may be obtained from a cDNA library prepared from tissue believed to possess the PR0212, PRO290 PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PROl 281 , PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 mRNA and to express it at a detectable level Accordingly, human-[PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PRO1 187, PRO1281, PRO23, PRO39, PRO834,PRO1317, PRO1710, PRO2094, PRO2145 or PR02198] DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples The PR0212-, PRO290-, PR0341-, PR0535-, PR0619-, PR0717-, PRO809-, PRO830-, PR0848-, PR0943-, PRO 1005-, PRO 1009-, PRO 1025 , PRO 1030-, PRO 1097 , PRO 1 107-, PRO 1 11 1 -, PRO 1 153-, PROl 182-, PRO 1 184-, PRO 1 187-, PRO 1281 -, PR023-, PR039-, PR0834-, PRO 1317-, PRO 1710-, PRO2094 , PR02145- or PR02198-encodιng gene may also be obtained from a genomic library or by ohgonucleotide synthesis
Libraries can be screened with probes (such as antibodies to the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PR01 184, PR01 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide, or oligonucleotides 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 et al , Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1989) An alternative means to isolate the gene encoding PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PRO 1182, PRO 1 184 PRO 1 187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 is to use PCR methodology [Sambrook et al , supra, Dieffenbach et al , PCR Primer A Laboratory Manual (Cold Spring Harbor Laboratoi y Press, 1995)]
The Examples below describe techniques for screening a cDNA library The ohgonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized The ohgonucleotide 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 pP-labeled ATP biotinylation or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supi a
Sequences identified in such library screening methods can be compai ed 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 ammo acid or nucleotide level) within defined regions ot the molecule or across the full-length sequence can be determined using methods known in the art and as descnbed 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 proceduies as described in Sambrook et al supia, to detect precursoi s 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848. PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1 107, PROl l l l , PROH53, PROH82, PROH 84, PR01187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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, hposome-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 , supia, or electroporation is generally used for prokaryotes Infection with Agrobacterium tumefaciens is used tor 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 Sohngen etal , J Bact , J30 946 ( 1977) and Hsiao et al , 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, polyormthine, may also be used For various techniques for transforming mammalian cells, see, Keown et al , Methods in Enzymology 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 eubacteπa, such as Gram-negative oi Gram-positive organisms, for example, Enterobacteπaceae such as E coli Various E co strains are publicly available, such as E co Kl 2 strain MM294 (ATCC 31 ,446), E coh X 1776 (ATCC 31 ,537), E coh strain W31 10 (ATCC 27,325) and E co strain K5 772 (ATCC 53,635) Othei suitable prokaryotic host cells include Enterobactenaceae such as Eschei i chia, e g , E co , Enterobactei , Eι
Figure imgf000073_0001
ιιua, Klebsιella, Pιoteus, Salmonella, e e , Salmonella t phim i mm, Seπatia, e g , Serratia marcescans and Sluςella, as well as Bacilli such as B subtihs and B hcheniformis (e g , B hchenifoimis 41 P disclosed in DD 266,710 published 12 April 1989), Pseudomoiias such as P aeruginosa, and Sti eptotmces These examples are lllustiativ e rather than limiting Strain W31 10 is one particularly preferred host or parent host because it is a common host stiain for recombinant DNA product fermentations Preferably, the host cell secretes minimal amounts ot 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 ptiβ p oA El 5 (argF-lac)169 degP ompTkan'; E. coli W31 10 strain 37D6, which has the complete genotype tonA ptr3 phoA El 5 (argF-lac)169 degP ompT rbs7 ilvG kari ; E. coli W3110 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 PR0212-, PRO290-, PR0341 -, PR0535-, PR0619-, PR0717-, PRO809-, PRO830-, PR0848-, PR0943-, PRO1005-, PRO1009-, PRO1025-, PRO1030-, PRO1097-, PROl 107-, PROl l l l-, PR01153-, PROl 182-, PROl 184-, PROl 187-, PROl 281 -, PR023-, PR039-, PR0834-, PROl 317-, PROl 710-, PRO2094-, PR02145- or PR02198-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 et al, Bio/Technology, 9: 968-975 (1991 )) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al, J. Bacteriol, 737 [1983]), A", 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 pastor is (EP 183,070; Sreekrishna etal, J. Basic Microbiol, 28:265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case era/., 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., JJ2:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton et al, Proc. Natl. Acad. Sci. USA, 8L1470-1474 [1984]) and A. niger (Kelly and Hynes, 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PRO717, PRO809, PRO830, PRO848, PRO943, PRO1005. PRO1009, PRO1025, PRO1030, PRO1097. PROl 107. PROl 111, PROl 153, PROl 182, PRO1 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710. PRO2094, PR02145 or PR02198 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 CV l 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 (Wl 38, 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PRO717,PRO809, PRO830, PRO848,PRO943, PRO1005,PRO1009, PRO1025, PRO1030,PRO1097,PRO1 107, PROl 111 , PROl 153, PRO1 182, PRO1 184, PRO1187, PRO1281 , PRO23, PRO39,PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 sιte(s) using techniques known in the art Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence Construction of suitable vectors containing one or more of these components employs standard hgation techniques which are known to the skilled artisan The PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943,
PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212-, PRO290-, PR0341 -, PR0535-, PR0619-, PR0717-, PRO809-, PRO830-, PR0848-, PR0943-, PRO1005-, PRO1009-, PRO1025-, PRO1030-, PRO1097-, PRO1 107-, PROl l l l-, PROH53-, PROl 182- PR01 184-, PROl 187-, PR01281 -, PR023-, PR039-, PR0834-, PROl 317-, PROl 710-, PRO2094-, PR02145 or PR02198-encodιng 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, penicillmase, lpp, or heat-stable enterotoxm II leaders For yeast secretion the signal sequence may be, e g , the yeast invertase leader, alpha factor leader (including Saccharoi ces and
Figure imgf000075_0001
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 ρBR322 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 , ampicilhn, neomycin, methotrexate, or tetracychne, (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 PR0212-, PRO290-, PR0341 -, PR0535-, PR0619-. PR0717-, PRO809-, PRO830-, PR0848-, PR0943-, PRO 1005-, PRO 1009-, PRO 1025-, PRO 1030-, PRO 1097-, PRO 1107-, PRO 1 1 1 1 -, PRO 1 153-, PRO 1 182-, PRO 1 184-, PRO 1187-, PRO 1281 -, PR023-, PR039-, PR0834-, PRO 1317-, PRO 1710-, PRO2094-, PR02145- or PR02198-encodιng 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 ttp\ gene present in the yeast plasmid YRp7 [Stinchcomb et al , Nature, 282 39 (1979), Kingsman et al , Gene, 7 141 (1979), Tschemper et al , Gene, 10 157 (1980)] The 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 PR0212-, PRO290-,
PR0341 -, PR0535-, PR0619-, PR0717-, PRO809-, PRO830-, PR0848-, PR0943-, PRO1005-, PRO1009-, PRO1025-, PRO1030-, PRO1097-, PROl 107-, PROl l l l -, PROl 153-, PROl 182-, PROl 184-, PROl 187-, PR01281 -, PR023-, PR039-, PR0834-, PROl 317-, PROl 710-, PRO2094-, PR02145- or PR02198-encodιng 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 etal , Nature, 275 615 (1978), Goeddel etal , 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717. PRO809, PRO830. PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PRO l 184 PROl 187, PR01281 , PR023. PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198
Examples of suitable promoting sequences for use with yeast hosts include the promoters foi 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, L7 4900 (1978)], such as enolase, glyceraldehyde- 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase. pyruvate kinase, tπosephosphate 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, metallothionem, glyceraldehyde-3- phosphate dehydrogenase, and enzymes lesponsible for maltose and galactose utilization Suitable vectors and promoters for use in yeast expression are further described in EP 73,657 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l . PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegaiovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e g , the actin promoter or an immunoglobulm promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems
Transcription of a DNA encoding the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PR01 182, PROl 184, PR01 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 cytomegaiovirus 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 aposition 5' or 3' to the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 11 , PROl 153,PRO1182, PRO1184, PRO1 187,PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212. PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PROl 030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PROl 281. PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PR0212, PRO290,
PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005. PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023 PR039, PR0834, PROl 317, PRO 1710, PRO2094, PR02145 or PR02198 in recombinant vertebrate cell culture are described in Gething et al , 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 ot 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-protem 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 lmmunohistochemical 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 lmmunohistochemical 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PR01182, PROl 184, PROl 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against an exogenous sequence fused to PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO l 107, PROl 111, PROl 153, PROl 182,PRO1184,PRO1187,PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 DNA and encoding a specific antibody epitope
e Purification of Polypeptide Forms of PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848,
PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PROl 281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 , Tπton-X 100) or by enzymatic cleavage Cells employed in expression of PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848 PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 oi PR021 8 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830,
PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030 PRO1097, PROl 107, PROl l l l , PRO l 153 PRO1 182, PRO1 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO 1710 PRO2094, PRO2145 or PR02198 from recombinant cell proteins or polypeptides The following piocedures are exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation, revei se phase HPLC chromatography on silica or on a cation-exchange resin such as DEAE, chromatotocusing, 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 bind epitope-tagged forms ot the PR0212 PRO290, PR034I , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848. PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PROl 281 , PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1 107, PROl l l l, PR01 153, PROH 82, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 produced
E Amplification of Genes Encoding the PRQ212. PRQ290. PRQ341. PRQ535 , PRQ619, PRQ717,
PRO809. PRO830. PRQ848. PRQ943, PRO1005. PRO1009, PRO1025. PRO1030. PRO1097. PROl 107, PROl 1 1 1. PROl 153. PRQ1 182. PROl 184, PRO1 187, PRO1281 , PRO23, PRO39. PRO834. PRO1317. PRO1710, PRO2094, PRQ2145 or PRQ2198 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, . 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 fohc 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 exti achromosomal 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 tor 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 rephcative (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 Hodgkm' 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-l 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 tor DNA sequences that are present in the tumor at an increased frequency (WO 93/18,186, Gray er al . Radiation Res , 137 275 289 [1994]) As a screening method, this type of analysis has revealed a large number of recurring amphcons (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 ot coding sequences within the amphcon by standard molecular genetic techniques
The most sensitive methods to detect gene amplification are polymerase chain i eaction (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 tor 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 Gelmim 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), HI 57 (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 (adenocarcmoma, abbreviated as "AdenoCa")(LTl), SRCC725 (squamous cell carcinoma, abbreviated as "SqCCa)(LTl a), SRCC726 (adenocarcιnoma)(LT2), SRCC727 (adenocarcιnoma)(LT3), SRCC728 (adenocarcιnoma)(LT4), SRCC729 (squamous cell carcιnoma)(LT6), SRCC730 (adeno/squamous cell carcιnoma)(LT7), SRCC731 (adenocarcιnoma)(LT9), SRCC732 (squamous cell carcιnoma)(LT10), SRCC733 (squamous cell carcιnoma)(LTl l), SRCC734 (adenocarcιnoma)(LT12), SRCC735 (adeno/squamous cell carcιnoma)(LT13), SRCC736 (squamous cell carcιnoma)(LT15), SRCC737 (squamous cell carcιnoma)(LT16), SRCC738 (squamous cell carcιnoma)(LTl 7), SRCC739 (squamous cell carcιnoma)(LT18), SRCC740 (squamous cell carcιnoma)(LT19), SRCC741 (lung cell carcinoma, abbreviated as "LCCa")(LT21 ), SRCC81 1 (adenocarcιnoma)(LT22), SRCC825 (adenocarcιnoma)(LT8), SRCC886 (adenocarcιnoma)(LT25), SRCC887 (squamous cell carcinoma) (LT26), SRCC888 (adeno-BAC carcinoma) (LT27), SRCC889 (squamous cell carcinoma) (LT28), SRCC890 (squamous cell carcinoma) (LT29), SRCC891 (adenocarcmoma) (LT30), SRCC892 (squamous cell carcinoma) (LT31 ) SRCC894 (adenocarcmoma) (LT33) Also included are human lung tumors designated SRCC1 125 [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 (adenocarcmoma, SRCC776) SW620 (lymph node metastasis of colon adenocarcmoma, SRCC777) Colo320 (carcinoma, SRCC778), HT29 (adenocarcmoma, SRCC779), HM7 (a high mucm producing variant of ATCC colon adenocarcmoma cell line SRCC780, obtained from Dr Robert Warren, UCSF),CaWιDr (adenocarcmoma SRCC78I ), HCT1 16(carunoma SRCC782), SKCOl (adenocarcmoma, SRCC783), SW403 (adenocarcmoma, SRCC784), LS I 74T (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 (adenocarcmoma, SRCC906), CT20 (adenocarcmoma, SRCC907), CT21 (adenocarcmoma, SRCC908), CT22 (adenocarcmoma, SRCC909), CT23 (adenocarcmoma, SRCC910), CT24 (adenocarcmoma, SRCC91 1 ), CT25 (adenocarcmoma, SRCC912), CT26 (adenocarcmoma, SRCC913), CT27 (adenocarcmoma, SRCC914),CT28 (adenocarcmoma, SRCC915), CT29 (adenocarcmoma, SRCC916), CT30 (adenocarcmoma, SRCC917), CT31 (adenocarcmoma, SRCC918), CT32 (adenocarcmoma, SRCC919), CT33 (adenocarcmoma, SRCC920), CT35 (adenocarcmoma, SRCC921), and CT36 (adenocarcmoma, SRCC922) Also included are human colon tumor centers designated SRCC1051 [HF-000499], SRCC1052 [HF-000539], SRCC1053 [HF-000575], SRCC1054 [HF-000698], SRCC1142 [HF-000762], SRCC1 144 [HF-000789], SRCC1146 [HF-000795] and SRCC1 148[HF-00081 1 ]
Human breast carcinoma cell lines include, for example, HBL 100 (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 SRCC 1057 [HF-000545] Also included are human breast tumors designated SRCC1094, SRCC1095, SRCC1096, SRCC1097, SRCC1098, SRCC1099, SRCC 1 100. SRCC 1 101 , and human breast-met-lung-NS tumor designated SRCC893 [LT 32]
Human kidney tumor centers include SRCC989 [HF-00061 1 ] and SRCC1014 [HF-000613]
Human testis tumor center includes SRCC1001 [HF-000733] and testis tumor margin SRCC999 [HF- 000716]
Human parathyroid tumor includes SRCC 1002 [HF-000831] and SRCC 1003 [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 lmmunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful for lmmunohistochemical 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830 PR0848. PR0943 PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PRO l 107, PRO ! I l l , PRO l 153, PRO 1 182, PRO 1 184, PRO 1 187, PRO 1281 , PR023. PR039. PR0834, PRO 1317, PR01710, PRO2094, PR02145 or PR02198 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to sequence PR0212, PRO290. PR0341 , PR0535, PR0619, PR0717 PRO809 PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PR01182, PROl 184, PRO1 187, PRO1281 ,PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 antι-PR0212, antι-PRO290, antι-PR0341 , antι-PR0535, anti-PROόl 9, antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, antι-PRO1005, antι-PRO1009, antι-PRO1025, anti-PRO 1030, anti- PRO1097, anti-PROl 107, anti-PROl 1 1 1 , anti-PROl 153, anti-PROl 182, anti-PROl 184, anti-PROl 187, anti- PRO 1281 , antι-PR023, antι-PR039, antι-PR0834, antι-PR01317, antι-PRO1710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibodies to inhibit the expression of PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO 1030, PRO1097, PROl 107, PROl 1 11 , PROl 153, PR01 182, PR01184, PR01 187, PROl 281 , PR023 PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 standaid that becomes bound to the antibodies To facilitate determining the amount of standard that becomes bound, the antibodies preferably are insolubihzed 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-immunoglobuhn 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 lmmunohistochemistry, 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 ra -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 transgemc 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 transgemc 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 ot 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 cancel, colon cancer, prostate cancer, lung cancer, etc ) include both non recombinant and recombinant (transgemc) animals Non-recombinant animal models include, for example, rodent, e g , munne models Such models can be generated by intioducing tumor cells into svngeneic mice using standard techniques, e g , subcutaneous injection, tail vein injection, spleen implantation intraperitoneal implantation, implantation under the renal capsule, or orthopm implantation, e g , colon cancer cells implanted in colo c 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, B IO.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); ras-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 new-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 lOxl O6 to lOxlO7 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 In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the most thoroughly studied experimental tumors, can be used as an mvestigational 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, 41 suppl 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 cahper 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 tor 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 morphometπc method and flow cytometπc analysis
Recombinant (transge c) 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 transgemc animals Animals that can serve as a target for transgemc 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 et al , Cdl, 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, transgemc 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 ot Lasko et al , Proc Natl Acad Sci USA 89 6232- 636 (1992)
The expression of the transgene in transgemc 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1 107, PROl l l l , PROH53, PR01 182, PR01184, PR01187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PROl 153, PROl 182, PROl 184, PRO1187, PRO1281, PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PRO2145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107,PROl 111 , PROl 153,PROH82, PR01184, PROl 187, PR01281 , PR023, PR039, PR0834, PRO 1317, PROl 710, PRO2094, PR02145 or PR02198 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. 51:503 (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 etal, 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. 113-152]. 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PRO l 184, PR01187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094. PR02145 or PR02198 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 subhngual 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, adenocarcmoma, lymphoma, chrondroma, leiomyosarcoma of dogs, cats, and baboons can also be tested Of these mammary adenocarcmoma 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, (poly)peptιde-ιmmunoglobuhn fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti ldiotypic 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 tor 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-immobihzed 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PROl 317, PRO 1710, PRO2094, PR02145 or PR02198 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 transcπptional 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-actιvated promoter depends on reconstitution of GAL4 activity via protein-protein interaction Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galac tosidase 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 PR0212-, PRO290-, PR0341 -, PR0535-, PR0619-, PR0717-, PRO809-, PRO830-, PR0848-, PR0943-, PRO1005-, PRO1009-, PRO1025-, PRO1030-, PRO1097-, PRO 1 107-, PRO 1 1 1 1 -, PRO 1 153-, PRO 1 182-, PRO 1 184-, PRO 1 187-, PRO 1281 -. PR023-, PR039-, PR0834-, PR01317-, PROl 710-, PRO2094-, PR02145- or PR02198-encodmg 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 tor 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 iormation) 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 reactιon(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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809.
PRO830, PR0848. PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO 1 107, PRO l l l l , PROl 153, PROl 182, PROl 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PROl 281, PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide indicates that the compound is an antagonist to the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1107, PROllll, PROH53, PR01182, PR01184, PROH87, PR01281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide. Alternatively, antagonists may be detected by combining the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097, PRO 1107, PROl 111, PROl 153,PR01182,PR01184,PR01187,PR01281,PR023,PR039,PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide and a potential antagonist with membrane- bound PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281, PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1107, PROllll, PROH53, PROH82, PROH84, PROH87, PR01281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide can be labeled, such as by radioactivity, such that the number of PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl HI, PROl 153,PR01182, PROl 184, PROl 187,PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PRO 1153, PRO 1182, PROl 184, PRO 1187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PRO717,PRO809,PRO830,PRO848,PRO943,PRO1005,PRO1009,PRO1025,PRO1030,PRO1097,PRO1107, PROl 111, PROl 153, PROl 182, PROl 184.PR01187,PRO1281,PRO23,PRO39,PRO834,PRO1317.PRO1710, PRO2094, PR02145 or PR02198 polypeptide. Transfected cells that are grown on glass slides are exposed to labeled PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097. PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide. The PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PROl 182, PROH 84, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PRO619, PRO717, PRO809, PRO830, PRO848, PRO943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107,PROl 1 1 1, PROl 153, PR01 182, PROl 184, PR01 187, PR01281, PR023, PR039, PR0834, PROL317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide can bephotoaffinity-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 ohgonucleotide 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PRO 1182, PRO 1184, PRO 1187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 ohgonucleotide that binds to the fusions of immunoglobulin with the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROH84, PROH 87, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097, PRO 1 107, PROl 1 1 1 , PROl 153, PROH82, PR01 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097. PROl 107, PROl 111 , PROl 153, PR01 182, PROl 184, PR01 187,PR01281 , PR023, PR039, PR0834, PROL317. PROl 710, PRO2094, PR02145 or PR02198 polypeptide.
Another potential PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO l 107, PROl l l l , PROl 153, PRO l 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PROl 153, PROl 182, PROl 184, PROH87, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide herein, is used to design an antisense RNA ohgonucleotide of from about 10 to 40 base pairs in length. A DNA ohgonucleotide 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1, PROl 153. PR01182, PROl 184, PROl 187, PROl 281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide. The antisense RNA ohgonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097,PRO1107, PROl l l l,PRO1153, PRO1182, PRO1184, PRO1187, PRO1281, PRO23,PRO39, PRO834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide (antisense - Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL. 1988). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PRO1153, PRO1 182, PRO1 184, PRO1 187, PRO1281, PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341. PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO l 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184,PRO1187, PRO1281 ,PRO23, PRO39. PRO834,PRO1317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide, thereby blocking the normal biological activity of the PR0212, PRO290, PR0341 , PR0535 PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030 PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 oi PR02198 polypeptide Examples of small molecules include, but are not limited to, small peptides or peptide-hke molecules, preferably soluble peptides, and synthetic non peptidyl organic or inorganic compounds
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA Ribozymes act by sequence specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g , Rossi, Current Biology, 4 469-471 (1994), and PCT publication No WO 97/33551 (published September 18, 1997)
Nucleic acid molecules in tnple-hehx formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides The base composition of these oligonucleotides is designed such that it promotes tnple-hehx formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of punnes or pyπmidines 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 oligonucleotides is designed such that it promotes triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches ot punnes oi pyπmidines on one strand of a duplex For turthei details see, e g , PCT publication No WO 97/33551 , supia These molecules can be identified by any oi any combination of the sci eenmg 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 agentmay include the PR0212, PRO290, PR0341, PR0535, PR0619, PRO717,PRO809, PRO830, PRO848, PRO943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1107 PROl 1 1 1. PROl 153, PR01 182, PROl 184,PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 adj u vant (monophosphory 1 Lipid A, synthetic trehalose dicorynomycolate) The immunization protocol may be selected by one skilled in the art without undue experimentation
2 Monoclonal Antibodies
The antι-PR0212, antι-PRO290, antι-PR0341 , antι-PR0535, anti-PROόl 9, antι-PR0717, antι-PRO809 antι-PRO830, antι-PR0848, antι-PR0943, antι-PRO1005, antι-PRO 1009, antι-PRO1025, antι-PRO1030, anti- PROl 097, antι-PRO1 107, anti-PROl 1 1 1 , anti-PROl 153, antι-PR01 182 antι-PR01 184, anti-PROl 187, anti- PRO 1281 , antι-PR023, antι-PR039, antι-PR0834, anti-PRO 1317, anti-PRO 1710. antι-PRO2094, antι-PR02145 or antι-PR02198 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 appiopnate 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 PR0212, PRO290 PR0341. PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO 1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PRO 1184, PRO 1 187, PR01281 , PR023. PR039 PRO834, PRO1317, PRO I 710 PRO2094, PR02145 or PR02198 polypeptide, including fragments oi a fusion protein ot such piotein or a fragment thereof Generally, either peripheral blood lymphocytes ("PBLs") aie used it 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 Piactice. 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 01 survival of the unfused, immortalized cells For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoπbosyl transferase (HGPRT or HPRT), the culture medium for the hybπdomas typically will include hypoxanthine, aminopteπn, 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 munne 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 foi the production of human monoclonal antibodies [Kozbor. J Immunol . 133 3001 (1984), Brodeur etal , 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PRO1281, PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 immunoglobulm purification piocedures such as. for example, protein A-Sephaiose, 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 ohgonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains ot munne 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, oi myeloma cells that do not otherwise produce immunoglobulm 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 munne sequences [U S Patent No 4,816,567, Morrison et al , supia] or by covalently joining to the immunoglobulm 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 immunoglobulm 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 crosshnking Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosshnking 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
The antι-PR0212, antι-PRO290, antι-PR0341. antι-PR0535, anti-PROό 19, antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, antι-PRO1005, antι-PRO1009, antι-PRO1025, antι-PRO1030, anti- PRO1097, antι-PRO1107, anti-PROl 11 1 , antι-PR01 153, antι-PR01 182, anti-PROl 184, anti-PRO 1 187, anti- PROl 281 , antι-PR023, antι-PR039, antι-PR0834, anti-PRO 1317, anti-PRO 1710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibodies may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , munne) antibodies are chimeric immunoglobuhns, immunoglobulm chains or fragments thereof (such as Fv, Fab, Fab', F(ab'). or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulm Humanized antibodies include human immunoglobuhns (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 immunoglobulm 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 immunoglobulm and all or substantially all of the FR regions are those of a human immunoglobulm consensus sequence The humanized antibody optimally also will comprise at least a portion of an immunoglobulm constant region (Fc), typically that ot a human immunoglobulm [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 ai t Generally, a humanized antibody has one or more ammo 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 Humamzation can be essentially performed following the method of Winter and co-workers [Jones et al , Nature, 321 522-525 ( 1986), Riechmann et al , Natuie, 332 323-327 (1988), Verhoeyen et al , Science, 239 1534- 1536 ( 1988)] , by substituting rodent CDRs or CDR sequences tor 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 )1 The techniques of Cole et al , and Boerner et al , are also available tor the preparation of human monoclonal antibodies (Cole etal , 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 immunoglobulm loci into transge c animals, e g , mice in which the endogenous immunoglobulm 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 etal , Nature, 368 856 859 (1994), Morrison, Nature, 368 812-13 (1994), Fishwild etal , Nature Biotechnology. 14 845-51 (1996), Neuberger, Nature Biotechnology, ]4 826 (1996), Lonberg and Huszar, Intern Rev Immunol , H 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, tor 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 glucuro dase, alkaline phosphatase useful for converting phosphate containing prodrugs into free drugs, arylsulfatase useful for converting sulfate containing prodiugs into free drugs, cytosine deaminase useful for con verting non-toxic 5-fluorocytosιne into the anti -cancer drug 5-fluorouracιl, proteases, such as serratia protease, thermolysin, subtihsin, 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 neuram idase useful foi converting glycosylated prodrugs into free drugs, β-lactamase useful for converting drugs denvatized with β-lactams into free drugs, and penicillin amidases, such as penicillin Vamidase or penicillin G amidase, useful foi converting drugs denvatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups respectively, into tree di ugs 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 antι-PR0212, antι-PRO290, antι-PR0341 , antι-PR0535. anti-PROό 19, antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, anti-PRO 1005, anti-PRO 1009, anti-PRO 1025, anti-PRO 1030, anti-PRO 1097, anti-PROl 107, antι-PROl 1 1 1 , anti-PROl 153, anti PROl 182, antι-PR01 184, antι-PR01 187, antι-PR01281 , antι-PR023, antι-PR039, antι-PR0834, antι-PR01317, anti-PRO 1710, antι-PRO2094, antι-PR02145 or antι-PR02198 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, 312 604-608 ( 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281 , PR023 , PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 immunoglobulm 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 immunoglobulm heavy and light chains, these hybπdomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the coriect 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 et al , EMBO J , H) 3655-3659 ( 1991 ) Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulm constant domain sequences The fusion preferably is with an immunoglobulm heavy - chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions It is pieferred 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 immunoglobulm heavy-chain fusions and, if desired the immunoglobulm light chain, are inserted into separate expression vectors, and are co-transtected into a suitable host organism For f urthei details of generating bispecific antibodies vee, for example, Suresh e. -./ . Methods in Enzymology. 121 210 (1986)
According to another approach described in WO 96/2701 1 , the inteiface between a pair ot antibodv molecules can be engineered to maximize the percentage ot heterodimers which are lecovered from recombinant cell culture The preferred interface comprises at least a part of the CH3 region ot 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 oi tryptophan) Compensatory "cavities " of identical or similar size to the large side chaιn(s) are created on the interface of the second antibody molecule by replacing laige amino acid side chains with smaller ones (e g , alanine or threomne) 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 )-, 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'). 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 thiomtrobenzoate (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 coh 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 ). molecule Each Fab' fragment was separately secreted from E coh and subjected to directed chemical coupling in vitro to form the bispecific antibody The bispecific antibody thus formed was able to bind to cells overexpressmg 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 etal , 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 Holhnger 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 (V„) 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 V, domains of one fragment are forced to pair with the complementaiy 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, tnspecific 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 foi IgG (FcγR), such as FcγRI (CD64) FcγRII (CD32) and FcγRIII (CD 16) so as to focus cellular defense mechanisms to the cell expiessmg the particular polypeptide Bispecific antibodies ma\ 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 radionuchde chelator, such as EOTUBE, DPTA, DOTA, or TETA Another bispecific antibody of interest binds the polypeptide and further binds tissue factor (TF)
6 Heterocomugate 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 crosshnking 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 lminothiolate and methyl-4- mercaptobutynmidate 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 resιdue(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 1191-1 195 ( 1992) and Shopes, J Immunol , 148 2918-2922 ( 1992) Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunc tional cross-linkers as described in Wolff et al , 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 Immunocon l u gates
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 nonbmding active fragments of diphtheria toxin, choleia toxin, botuhnus toxin, exotoxin A chain (from Pseudomonas aeiugmosa), πcin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuiites foi du proteins, dianthin proteins, Plntolaca amencana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, cuicin, crotin sapaonaπa officinahs inhibitoi, gelo n, sapoπn. mitogellm, restπctocin, phenomycin, enomycin and the tπcothecenes Small molecule toxins include, for example, cahcheamicins, maytansinoids, palytoxm and CC 1065 A variety of radionuchdes are available for the production ot radioconjugated antibodies Examples include 21 Bι π ιI, "'In, 9"Y and 1S6Re Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl 3-(2-pyndyldιthιol) propionate (SPDP), lminothiolane (IT), bifunctional derivatives of mnidoesters (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-diazomum derivatives (such as bιs-(p dιazonιumbenzoyl)-ethylenedιamιne), diisocyanates (such as tolyene 2,6-dnsocyanate), and bis active fluorine compounds (such as 1 ,5-dιfluoro-2,4-dιnιtrobenzene) 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 tπaminepentaacetic 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 phosphatidylchohne, cholesterol and PEG-denvatized 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 et al 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 e? al , 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 ot pharmaceutical compositions If the protein encoded by the amplified gene is intracellular and whole antibodies are used as inhibitois internalizing antibodies are preferred However, hpofections oi liposomes can also be used to deliver the antibody or an antibody fragment, into cells Where antibody fragments are used, the smallest inhibitory tiagment 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 lecombinant 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 lyophihzed formulations or aqueous solutions Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionme, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkomum chloride, benzethomum 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 immunoglobuhns, hydrophilic polymers such as polyvinylpyrrohdone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosacchaπdes, disacchaπdes, and other carbohydrates including glucose, mannose, or dextnns, 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, hydroxymethylcelluloseorgelatin-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-hydroxyethyI-methacrylate), or poly(vιnylalcohol)) polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and ethyl-L glutamate, non-degiadable ethylene-vinyl acetate, degradable lactic acid glycohc acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate), and poly-D-( )-3-hydroxybutync acid While polymers such as ethylene-vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated antibodies lemain in the body for a long time, they may denature or aggregate as a result of exposuie to moistuie at 37' C, resulting in a loss of biological activity and possible changes in immunogenicity Rational stiategies 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, lyophihzing fiom 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, bladdei, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas, sarcomas, ghoblastomas, and various head and neck tumors), leukemias and lymphoid malignancies, other disorders such as neuronal, glial, astrocytal, hypothalarmc and other glandular, macrophagal, epithelial, stromal and blastocoehc 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 &Wιlkιns, 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 onapπstone (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 endothehal factoi (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-admimstered with a growth inhibitory agent For example, the growth inhibitory agent may be administered first, followed by an antibody ot the present invention Howevei , simultaneous admimstiation or administration of the antibody ot the present invention first is also contemplated Suitable dosages for the growth inhibitory agent are those presently used and may be loweied due to the combined action (synergy) of the growth inhibitory agent and the antibody herein For the prevention or ti eatment 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 moi e, 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 quantitativ ely 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 factoi Such binding assays are performed essentially as described in section 5 above
//; 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 Blvd , Manassas, VA 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 5} 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 ot 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, Inms et al , PCR Piotocols 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, Ohgonucleotide Synthesis, IRL Press Oxford, 1984, R I Freshney Animal Cell Culture. 1987, Cohgan et al Current Protocols in Immunology 1991
EXAMPLE 1 Isolation of cDNA clones Encoding PRQ2 I 2 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 EST databases (e g , GenBank), a proprietary EST database (LIFESEQ®, Incyte Phamaceuticals, Palo Alto, CA), and a proprietary EST database from Genentech The search was performed using the computer program BLAST or BLAST2 [Altshul et al , Methods in Enzymology, 266 460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences 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 consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington)
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above The ESTs included an EST proprietary to Genentech In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above
Based on the 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 PR0212 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, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe ohgonucleotide and one of the primer pairs
PCR primers (forward and reverse) were synthesized forward PCR primer 5'-CACGCTGGTTTCTGCTTGGAG-3' (SEQ ID NO 3) reverse PCR primer 5'-AGCTGGTGCACAGGGTGTCATG-3' (SEQ ID NO 4)
Additionally, a synthetic ohgonucleotide hybridization probe was constructed which had the following nucleotide sequence hybridization probe
5*-CCCAGGCACCTTCTCAGCCAGCCAGCAGCTCCAGCTCAGAGCAGTGCCAGCCC-3' (SEQ ID NO 5)
RNA for construction of the cDNA libraries was isolated from human fetal lung tissue 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 ohgo 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 etal . Science, 253 1278-1280 ( 1991 )) in the unique Xhol and Notl sites DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full length PR0212 polypeptide (designated herein as DNA30942-1 134 [Figure 1 , SEQ ID NO 1 ]) and the derived protein sequence for that PR0212 polypeptide
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucelotide positions 101-103 and a stop signal at nucleotide positions 1001 -1003 (Figure 1 , SEQ ID NO 1 ) The predicted polypeptide precursor is 300 amino acids long Analysis of the full length PR0212 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 The N-terminus ot the PR0212 sequence contains a typical secretion signal (amino acids 1 -23 of Figure 2, SEQ ID NO 2) It is believed that amino acids 1 -215 of PR0212 (Figure 2, SEQ ID NO 2) represents an extracellular domain PR0212 has one potential N-hnked giycosylation site at amino acid residue 173 (Figure 2 , SEQ ID NO 2) Clone DNA30942- 1 134 has been deposited with ATCC on September 16, 1997 and is assigned ATCC deposit no 209254 PR0212 shows some amino acid sequence identity to TNFR2 (28 7%) and to OPG (31%)
EXAMPLE 2 Isolation of cDNA Clones Encoding PRO290 An expressed sequence tag (EST) DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) was searched and an EST was identified which showed homology to NTII-l (a nerve protein which facilitates regeneration), FAN and beige
RNA for construction of cDNA libraries was then isolated from human fetal kidney tissue The cDNA libraries used to isolate the cDNA clones encoding human PRO290 were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with ohgodT 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 pRK5D, 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
The human fetal kidney cDNA libraries (prepared as described above), were screened by hybπdization with a synthetic ohgonucleotide probe 5'-TGACTGCACTACCCCGTGGCAAGCTGTTGAGCCAGCTCAGCTG-3' (SEQ ID NO 8)
A cDNA clone was sequenced in entirety and is herein designated as DNA35680-1212 The entire nucleotide sequence of clone DNA35680- 1212 is shown in Figures 3 A-3B (SEQ ID NO 6) Clone DNA 35680- 1212 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 293- 295 and a stop signal at nucleotide positions 3002-3004 (Figures 3A-3B. SEQ ID NO 6) The predicted polypeptide precursor is 1003 amino acids long, has a calculated moleculai weight ot approximately 1 12,013 daltons and an estimated pi of approximately 6 4 Analysis of the full-length PRO290 sequence shown in Figure 4 (SEQ ID NO 7) evidences sequence identity between the PRO290 amino acid sequence and FAN and beige Due to this sequence identity, it is currently believed that the PRO290 polypeptide is related to FAN and/or beige Clone DNA35680- 1212 has been deposited with ATCC on April 21 , 1998 and is assigned ATCC deposit no 209790 EXAMPLE 3 Isolation of cDNA Clones Encoding a Human PRQ341 1 Preparation of ohgo dT primed cDNA library mRNA was isolated from human placenta tissue using reagents and protocols from Invitrogen, San Diego, CA (Fast Track 2) This RNA was used to generate an ohgo dT primed cDNA library in the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script Plasmid System) In this procedure, the double stranded cDNA was sized to greater than 1000 bp and the Sall/Notl tinkered cDΝA was cloned into Xhol/Νotl cleaved vector pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an Sfil restriction enzyme site preceeding the Xhol/Notl cDΝA cloning sites
2 Preparation of random primed cDΝA library
A secondary cDΝA library was generated in order to preferentially represent the 5 ends of the primary cDΝA clones Sp6 RΝA was generated from the primary library (described above), and this RΝA was used to generate a random primed cDΝA library in the vector pSST-AMY 0 using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above) In this procedure the double stranded cDΝA was sized to 500-1000 bp, hnkered with blunt to ΝotI adaptors, cleaved with Sfil, and cloned into Sfil/Νotl cleaved vector pSST-AMY 0 is a cloning vector that has a yeast alcohol dehyrogenase promoter preceeding the cDΝA cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehyrogenase terminator, after the cloning sites Thus, cDΝAs cloned into this vector that are fused in frame with the amylase sequence will lead to the secretion of amylase from appropriately transfected yeast colonies
3 Transformation and Detection
DΝA from the library described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Lite Technologies, 20 ml) The bacteria and vector mixture w as then electroporated as recommended by the manufacturer Subsequently, SOC media (Life Technologies 1 ml) was added and the mixture was incubated at 37 °C for 30 minutes The transformants were then plated onto 20 standard 150 mm LB plates containing ampicilhn and incubated for 16 hours (37 °C) Positive colonies were sci aped off the plates and the DΝA was isolated from the bacterial pellet using standard protocols, e g CsCl-gradient The purified DΝA was then carried on to the yeast protocols below
The yeast methods were divided into three categories ( 1 ) Transformation of yeast with the plasmid/ cDΝA combined vector, (2) Detection and isolation ot yeast colonies secreting amylase, and (3) PCR amplification ot the insert directly from the yeast colony and purification of the DΝA for sequencing and further analysis
The yeast strain used was HD56-5A (ATCC-90785) This strain has the following genotype M T alpha ura3 52 leu2 3 leu2-l 12, hιs3-1 1 , hιs3 15, MAL+, SUC+ GAL+ Preferably, yeast mutants can be employed that have deficient post-translational pathways Such mutants may have translocation deficient alleles in sec 71 sec 72 sec62, with truncated seel] being the most preferred Alternatively, antagonists (including antisense nucleotides and/or ligands) which interfere with the normal operation of these genes, other proteins implicated in this post translational pathway (e g , SECόlp, SEC72p, SEC62p, SEC63p, TDJ l p or SSAlp-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast
Transformation was performed based on the protocol outlined by Gietz et al , Nucl Acid Res , 20 1425
(1992) Transformed cells were then inoculated from agar into YEPD complex media broth (100 ml) and grown overnight at 30°C The YEPD broth was prepared as described by Kaiser et al , Methods in Yeast Genetics, Cold
Spring Harbor Press, Cold Spring Harbor, NY, p 207 (1994) The overnight culture was then diluted to about 2 x 106 cells/ml (approx ODβ)0=0 1 ) into fresh YEPD broth (500 ml) and regrown to 1 x 107 cells/ml (approx
00^=04-0 5)
The cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tns-HCl, 1 mM EDTA pH 7 5,
100 mM Lι,OOCCH3), and resuspended into LiAc/TE (2 5 ml)
Transformation took place by mixing the prepared cells (lOOμl) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1 μg, vol < 10 μl) in microfuge tubes The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tns-HCl, 1 mM EDTA, 100 mM Lι,OOCCH3, pH 7 5) was added This mixture was gently mixed and incubated at 30°C while agitating for 30 minutes The cells were then heat shocked at 42CC for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted and resuspended into TE (500 μl, 10 mM Tns-HCl, 1 mM EDTA pH 7 5) followed by recentrifugation The cells were then diluted into TE
(1 ml) and ahquots (200 μl) were spread onto the selective media pieviously prepared in 150 mm growth plates
(VWR)
Alternatively, instead of multiple small reactions, the transformation was performed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly The selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser et al , Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p 208-
210 (1994) Transformants were grown at 30°C for 2-3 days
The detection of colonies secreting amylase was perfoi med by including red starch in the selective growth media Starch was coupled to the red dye (Reactive Red- 120, Sigma) as per the procedure described by Biely et al , Anal Biochem , 172 176-179 (1988) The coupled starch was incorporated into the SCD-Ura agar plates at a final concentration of 0 15% (w/v), and was buffered with potassium phosphate to a pH of 7 0 (50-100 mM final concentration)
The postive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in ordei to obtain well isolated and identifiable single colonies Well isolated single colonies positive for amylase secretion were detected by direct incorporation ot red starch into buffered SCD-Ura agar Positiv e colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly 4 Isolation of DNA by PCR Amplification
When a positive colony was isolated, a portion of it was picked by a toothpick and diluted into sterile water (30 μl) in a 96 well plate At this time, the positive colonies were either frozen and stored for subsequent analysis or immediately amplified An aliquot of cells (5 μl) was used as a template for the PCR reaction in a 25 μl volume containing 0 5 μl Klentaq (Clontech, Palo Alto, CA), 4 0 μl 10 M dNTP's (Perkin Elmer-Cetus), 2 5 μl Klentaq buffer (Clontech), 0 25 μl forward ohgo 1 , 0 25 μl reverse ohgo 2, 12 5 μl distilled water The sequence of the forward ohgonucleotide 1 was
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID NO 1 1 ) The sequence of reverse ohgonucleotide 2 was 5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID NO 12)
PCR was then performed as follows a Dentaure 92 °C, 5 minutes
b 3 cycles of Denature 92 °C, 30 seconds
Anneal 59 °C, 30 seconds Extend 72 °C, 60 seconds
3 cycles of Denature 92 °C, 30 seconds
Anneal 57 °C, 30 seconds
Extend 72 °C, 60 seconds
d 25 cycles of Denature 92 °C, 30 seconds Anneal 55 °C, 30 seconds
Extend 72 °C, 60 seconds
e Hold 4°C
The underlined regions ot the oligonucleotides disclosed above annealed to the ADH promoter region and the amylase region, respectively, and amplified a 307 bp region from vector pSST-AMY 0 when no insert was present Typically, the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers Thus, the total product of the PCR reaction from an empty vector was 343 bp Howevei , signal sequence-fused cDNA resulted in considerably longer nucleotide sequences
Following the PCR, an aliquot of the reaction (5 μl) was examined by agarose gel electrophoresis in a 1 % agarose gel using a Tπs-Borate-EDTA (TBE) buffering system as described by Sambrook et al , supi a Clones resulting in a single strong PCR product larger than 400 bp were furthei analyzed by DNA sequencing aftei purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc , Chatsworth CA)
5 Identification of Full-length Clone
A cDNA sequence isolated in the above screen is herein designated as DNA25314 Probes were then generated from the sequence of the DNA25314 molecule and used to screen a human placenta cDNA library prepared as described in paragraph 1 above The cloning vector was pRK5B (pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278- 1280 ( 1991 )), and the cDNA size cut was less than 2800 bp The ohgonucleotide probes 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-lenth coding region of PR0341 Forward and reverse PCR primers generally range from 20-30 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 order to screen several libraries for a full length clone, DNA from the libraries was screened by PCR amplification, as per Ausubele. al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using ohgonucleotide primers based upon the DNA25314 sequence
A full length clone was identified that contained a single open reading frame with an apparent translational initiation site at nucleotide positions 380-382 and a stop signal at nucleotide positions 1754-1756 (Figure 5, SEQ ID NO 9) The predicted polypeptide precursor is 458 amino acids long, has a calculated molecular weight of approximately 50,264 daltons and an estimated pi of approximately 8 17 Analysis of the full length PR0341 sequence shown in Figure 6 (SEQ ID NO 10) evidences the presence of a variety of important polypeptide domains as shown in Figure 6, wherein the locations given for those polypeptide domains are approximate as described above The full-length PR0341 sequence evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 17, transmembrane domains from about amino acid 171 to about amino acid 190, from about amino acid 220 to about amino acid 239, from about amino acid 259 to about amino acid 275, from about amino acid 286 to about amino acid 305, from about amino acid 316 to about amino acid 335, from about amino acid 353 to about amino acid 378, and from about ammo acid 396 to about amino acid 417, and potential N- glycosylation sites from about ammo acid 145 to about amino acid 147 and from about amino acid 155 to about amino acid 158 Clone DNA26288- 1239 has been deposited with ATCC on April 21 , 1998 and is assigned ATCC deposit no 209792 An analysis of the Dayhoff database (version 35 45 S issProt 35), using ALIGN-2 sequence alignment analysis of the full length sequence shown in Figure 6 (SEQ ID NO 10), evidenced sequence identity between the PR0341 amino acid sequence and the following Dayhoff sequences S75696, H69788, D69852, A69888, B64918, F64752, LPU89276 , G64962, S52977 and S44253
EXAMPLE 4
Isolation of cDNA Clones Encoding a Human PRQ535 Polypeptide DNA49143- 1429 was 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 scoie based on the character of the DNA nucleotides surrounding the first and optionally the second methionme codon(s) (ATG) at the 5 -end of the sequence or sequence fragment under consideration The nucleotides following the fust ATG must code for at least 35 unambiguous amino acids without any stop codons If the fust ATG has the requited amino acids, the second is not examined It 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 the above described signal sequence algorithm allowed identification of an EST cluster sequence from the Incyte database, designated as ss clu 12694 mit 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 is herein designated DNA48352 In light of an observed sequence homology between the DNA48352 sequence and Merck EST clone no H86994, the Merck EST clone H86994 was purchased and the cDNA insert was obtained and sequenced It was found herein that the cDNA insert encoded a full-length protein The sequence of this cDNA insert is shown in Figure 7 (SEQ ID NO 13) and is herein designated as DNA49143- 1429
Clone DNA49143- 1429 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 78-80 and ending at the stop codon at nucleotide positions 681-683 (Figure 7) The predicted polypeptide precursor is 201 ammo acids long (Figure 8) The full-length PR0535 protein shown in Figure 8 has an estimated molecular weight of about 22,180 daltons and a pi of about 9 68 Analysis of the full- length PR0535 sequence shown in Figure 8 (SEQ ID NO 14) evidences the presence of a variety of important polypeptide domains as shown in Figure 8, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PR0535 sequence evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 25, a transmembrane domain from about amino acid 155 to about ammo acid 174, a potential N-glycosylation site from about amino acid 196 to about amino acid 199, and FKBP-type peptidyl-prolyl cis-trans isomer signature sequences from about amino acid 62 to about armno acid 77, from about amino acid 87 to about ammo acid 123, and from about amino acid 128 to about amino acid 141 Clone DN A49143- 1429 has been deposited with ATCC on June 23, 1998 and is assigned ATCC deposit no 203013 An analysis of the Dayhoff database (version 35 45 S wissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 8 (SEQ ID NO 14), evidenced sequence identity between the PR0535 amino acid sequence and the following Dayhoff sequences S71237, P_R93551 , PJ128980 S71238 FKB2_HUMAN, CELC05C8J , S55383, S72485, CELC50F2_6 and S75144
EXAMPLE 5
Isolation of cDNA Clones Encoding PRQ619 DNA49821 -1562 was 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 methionme 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 ammo 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 the above described signal sequence algorithm allowed identification of an EST cluster sequence from the Incyte database, designated as 88434 (also called DNA48290) 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) In light of the information and discoveries provided herein, the clone including EST 1656694 was purchased from Incyte (from a library constructed from ureter tumor tissue, URETTUTO 1 ) and the cDNA insert was obtained and sequenced The sequence of this cDNA insert is shown in Figure 9 (SEQ ID NO 15) and is herein designated as DNA49821-1562
Clone DNA49821-1562 shown in Figure 9 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 81 -83 and ending at the stop codon at nucleotide positions 450 452 (Figure 9, SEQ ID NO 15) The predicted polypeptide precursor is 123 ammo acids long including a predicted signal peptide at about ammo acids 1-20 (Figure 10) The full-length PR0619 protein shown in Figure 10 (SEQ ID NO 16) has an estimated molecular weight of about 13,710 daltons and an estimated pi of about 5 19 Clone DNA49821-1562 has been deposited with ATCC on June 16, 1998 and is assigned ATCC deposit no 209981
An analysis of the Dayhoff database (version 35 45 S wissProt 35), using the ALIGN 2 sequence alignment analysis of the full-length sequence shown in Figure 10 (SEQ ID NO 16), evidenced sequence identity between the PR0619 ammo acid sequence and the following Dayhoff sequences S35302 D87009 , HSU93494 , HUMIGLAM5_1 D86999_2, HUMIGLYM 1 _ 1 , HUMIGLYMKE_ 1 , A29491 _ 1 , A29498_ 1 , and VPR2 JVIOUSE
EXAMPLE 6
Isolation of cDNA Clones Encoding Human PRQ717
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 EST databases (e g , GenBank) and a proprietary EST database (LIFESEQ0, Incyte
Phamaceuticals, Palo Alto, CA) The search was performed using the computer program BLAST or BLAST2 [Altshul et al , Methods in Enzymology, 266 460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences 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 consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington) A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above and is herein designated DNA42829 In some cases, the DNA42829 consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above
Based on the DNA42829 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 PR0717 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, supt a, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe ohgonucleotide and one of the primer pairs
PCR primers (forward and reverse) were synthesized forward PCR primer 5'-AGCTTCTCAGCCCTCCTGGAGCAG-3' (SEQ ID NO 19) reverse PCR primer 5'-CGGGTCAATAAACCTGGACGCTTGG-3' (SEQ ID NO 20)
Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA42829 sequence which had the following nucleotide sequence hybridization probe 5'-TATGTGGACCGGACCAAGCACTTCACTGAGGCCACCAAGATTG-3' (SEQ ID NO 21 ) RNA for construction of the cDNA libraries was isolated from human fetal liver tissue (LIB229) The cDNA libraries used to isolate the cDNA clones were constiucted by standaid methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with ohgo 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
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full length PR0717 polypeptide (designated herein as DNA50988-1326 [Figure 1 1 , SEQ ID NO 17]) and the derived protein sequence for that PR0717 polypeptide The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucelotide positions 17- 19 and a stop signal at nucleotide positions 1697- 1699 (Figuie 1 1 , SEQ ID NO 17) The predicted polypeptide precursor is 560 amino acids long, has a calculated molecular weight of approximately 58,427 daltons and an estimated pi of approximately 6 86 Analysis of the full length PR0717 sequence shown in Figure 12 (SEQ ID NO 18) suggests that PR0717 may be a novel 12 transmembrane receptor The reverse complement strand of DNA50988 has a stretch that matches identically with human regulatory myosin light strand Further analysis of the full-length PR0717 sequence shown in Figure 12 (SEQ ID NO 18) evidences the presence of a variety of important polypeptide domains as shown in Figure 12, wherein the locations given tor those important polypeptide domains are approximate as described above Transmembrane domains are from about amino acid 30 to about ammo acid 50, from about amino acid 61 to about amino acid 79, from about amino acid 98 to about amino acid 1 12, from about amino acid 126 to about amino acid 146, from about amino acid 169 to about amino acid 182, from about amino acid 201 to about amino acid 215, from about amino acid 248 to about amino acid 268, from about amino acid 280 to about amino acid 300, from about amino acid 318 to about amino acid 337, from about amino acid 341 to about amino acid 357, from about amino acid 375 to about amino acid 387, and from amino acid 420 to about amino acid 441 (Fιgurel 2, SEQ ID NO 18) N-glycosylation sites are from about amino acid 40 to about amino acid 43 and from about amino acid 43 to about amino acid 46 (Figure 12, SEQ ID NO 18) A glycosaminoglycan attachment site is from about amino acid 468 to about amino acid 471 (Figure 12, SEQ ID NO 18) Clone DNA50988 1326 has been deposited with the ATCC on April 28, 1998 and is assigned ATCC deposit no 209814
EXAMPLE 7 Isolation of cDNA Clones Encoding PRO809 DNA57836-1338 was 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 methionme 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 ammo 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 the above described signal sequence algorithm allowed identification of an EST sequence fiom the
Incyte database This EST sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e e. , GenBank) and a proprietary EST DNA database (LIFESEQ1 Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homologies The homology search was performed using the computer program BLAST or BLAST 2 (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 pi ogram phrap ' (Phil Green Umveisity of Washington, Seattle, Washington) The consensus sequence obtained is herein designated DNA56418 Based on the discoveries and information provided herein, Merck clone 230132 including EST H74302 (from hbrai y 305 a human fetal liver spleen library) was further examined DNA sequencing gave the full-length DNA sequence for PRO809 shown in Figure 13 (SEQ ID NO 22) (herein designated as DNA57836 1338) and the derived protein sequence for PRO809
Clone DNA57836- 1338 (Figure 13. SEQ ID NO 22) contains a singleopen readingframe with an apparent translational initiation site at nucleotide positions 63-65 and ending at the stop codon at nucleotide positions 858- 860 (Figure 13, SEQ ID NO 22) The predicted polypeptide precursor is 265 ammo acids long (Figure 14) The full-length PRO809 protein shown in Figure 14 (SEQ ID NO 23) has an estimated molecular weight of about 29,061 daltons and a pi of about 9 18 Analysis of the full-length PRO809 sequence shown in Figure 14 (SEQ ID NO 23) evidences the presence of important polypeptide domains as shown in Figure 14, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PRO809 sequence shown in Figure 14 evidences the following a signal peptide from about amino acid 1 to about amino acid 18, and potential N-glycosylation sites from about amino acid 86 to about amino acid 89, from about amino acid 132 to about amino acid 135, and from amino acid 181 to about amino acid 184 Clone DNA57836- 1338 has been deposited with ATCC on June 23, 1998 and is assigned ATCC deposit no 203025 Analysis of the amino acid sequence of the full length PRO809 polypeptide suggests that it possesses some sequence similarity to the hepann sulfate proteoglycan and to endothehal cell adhesion molecule- 1 More specifically, an analysis of the Dayhoff database (version 35 45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 14 (SEQ ID NO 23), evidenced sequence identity between the PRO809 amino acid sequence and the following Dayhoff sequences PGBM_MOUSE, D82082_l and PW14158
EXAMPLE 8 Isolation of cDNA Clones Encoding PRO830 DNA56866-1342 was identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc , (South San Francisco, CA) upon ESTs as well as clusteied 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 methionme 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 ammo acids without any stop codons If the first ATG has the required ammo acids, the second is not examined If neither meets the requirement, the candidate sequence is not scored In oi der 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 the above described signal sequence algonthm allowed identification ot an EST clustei sequence from the Incyte database, designated 20251 This EST cluster sequence was then compared to a variety ot expressed sequence tag (EST) databases which included public EST databases (e g , GenBank) and a propnetary
EST DNA database (LIFESEQ®, Incyte Pharmaceuticals Palo Alto, CA) to identify existing homologies The homology search was performed using the computer program BLAST or BLAST-2 (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 is herein designated DNA55733 In light of an observed sequence homology between DNA55733 sequence and Merck EST clone no H78534, from the Merck database, the Merck EST clone no H78534 was purchased and the cDNA insert was obtained and sequenced It was found herein that the cDNA insert encoded a full-length protein The sequence of this cDNA insert is shown in Fιgurel5 and is herein designated as DNA56866-1342 Clone DNA56866- 1342 (Figure 15, SEQ ID NO 24) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 154- 156 and ending at the stop codon at nucleotide positions 415- 417 (Figure 15, SEQ ID NO 24) The predicted polypeptide precursor is 87 amino acids long (Figure 16) The full- length PRO830 protein shown in Figure 16 (SEQ ID NO 25) has an estimated molecular weight of about 9,272 daltons and a pi of about 9 19 Analysis of the full-length PRO830 sequence shown in Figure 16 (SEQ ID NO 25) evidences the presence of important polypeptide domains as shown in Figure 16, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PRO830 sequence shown in Figure 16 evidences the following a signal peptide from about amino acid 1 to about amino acid 33, potential N-myπstoylation sites from about amino acid 2 to about amino acid 7 and from about amino acid 8 to about amino acid 13, and a thioredoxin family of proteins homology block from about amino acid 23 to about amino acid 39 Clone DNA56866 1342 has been deposited with ATCC on June 23, 1998 and is assigned ATCC deposit no 203023
An analysis of the Dayhoff database (version 35 45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 16 (SEQ ID NO 25), evidenced sequence identity between the PRO830 amino acid sequence and the following Dayhoff sequences HSU88154J , HSU88153J , SAPKSGENE_1 , HPU31791_5, GGCNOT2_l , CPU91421_1 , CHKESTPC09_1 PQ0769, U97553_79 and B60095
EXAMPLE 9 Isolation of cDNA Clones Encoding Human PRQ848 DNA59839-1461 was 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 fiagments 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 methionme codon(s) (ATG) at the 5 -end of the sequence oi 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
Candidate sequences, also called initial consensus sequences, with a sufficient score were further examined with the computer program BLAST or BLAST-2 (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 progiam "phrap" (Phil Green, University of Washington, Seattle, Washington) In this case, an initial consensus sequence formed from an Incyte database of sequences identified from the signal algorithm is designated herein as DNA55999 lmt This sequence was extended using repeated cycles of BLAST and "phrap" to extend the sequence as far as possible using public and Incyte EST sequences to form a consensus sequence herein designated DNA55999 Based on the discoveries and information provided herein, the clone including Incyte EST 2768571 was further examined DNA sequencing gave the sequence herein designated as DNA59839-1461
Clone DNA59839-1461 (Figure 17, SEQ ID NO 26) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 146-148 and ending at the stop codon at nucleotide positions 1946-1948 (Figure 17, SEQ ID NO 26) The predicted polypeptide precursor is 600 amino acids long (Figure 18) The full-length PR0848 protein shown in Figure 18 (SEQ ID NO 27) has an estimated molecular weight of about 68,536 daltons and a pi of about 10 20 Analysis of the full-length PR0848 sequence shown in Figure 18 (SEQ ID NO 27) evidences the presence of important polypeptide domains as shown in Figure 18, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PR0848 sequence shown in Figure 18 evidences the following a cytoplasmic domain from about amino acid 1 to about amino acid 10, a Type II transmembrane domain from about amino acid 1 1 to about amino acid 35, a lumenal catalytic domain from about amino acid 36 to about am o acid 600, a πbonucleotide reductase small subunit signature from about amino acid 481 to about amino acid 496, and N-glycosylation sites from about amino acid 300 to about amino acid 303, from about amino acid 31 1 to about amino acid 314. from about ammo acid 331 to about ammo acid 334, from about amino acid 375 to about amino acid 378. and from about amino acid 460 to about amino acid 463 (Figure 18, SEQ ID NO 27) Clone DNA59839-1461 has been deposited with ATCC on June 16, 1998 and is assigned ATCC deposit no 209988
An analysis of the amino acid sequence of the full-length PR0848 polypeptide suggests that it may be a novel sialyltransferase More specifically, an analysis of the Dayhoff database (version 35 45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 18 (SEQ ID NO 27), evidenced sequence identity between the PR0848 amino acid sequence and the following Dayhoff sequences P_R78619 (GalNAc-alpha-2, 6-sιalyltιansterase), CAAG5_CHICK (alpha-N-acetylgalactosamide alpha-2, 6- sialyltransferase), HSU 14550 , CAG6-HUMAN and P_R63217 (human alpha-2 3-sιaIyltransferase)
EXAMPLE 10
Isolation of cDNA Clones Encoding Human PRQ94 > The extracellular domain (ECD) sequences (including the secietion 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 EST databases (e g , GenBank) and a proprietary EST database (LIFESEQ®, Incyte Phamaceuticals, Palo Alto, CA) The search was performed using the computer program BLAST or BLAST2 [Altshul et al , Methods in Enzymology 266 460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences 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 consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington)
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above and is herein designated DNA36360 In some cases, the DNA36360 consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above
Based on the DNA36360 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 PR0943 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 etal , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe ohgonucleotide and one of the primer pairs PCR primers (forward and reverse) were synthesized forward PCR primer 5'-CGAGATGACGCCGAGCCCCC-3' (SEQ ID NO 30) reverse PCR primer 5'-CGGTTCGACACGCGGCAGGTG-3' (SEQ ID NO 31 )
Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA36360 sequence which had the following nucleotide sequence hybridization probe
5'-TGCTGCTCCTGCTGCCGCCGCTGCTGCTGGGGGCCTTCCCGCCGG-3' (SEQ ID NO 32)
RNA for construction of the cDNA libraries was isolated from human fetal brain tissue 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 ohgo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoiesis, and cloned in a defined orientation into a suitable cloning v ector (such as pRKB or pRKD, pRK5B is a precursoi 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
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full length PR0943 polypeptide (designated herein as DNA52192- 1369 [Figures 19 A- 19B SEQ ID NO 28J) and the derived protein sequence for that PR0943 polypeptide
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucelotide positions 150- 152 and a stop signal at nucleotide positions 1662- 1664 (Figures 19A- 19B SEQ ID NO 28) The predicted polypeptide precursor is 504 amino acids long (Figure 20), has a calculated molecular weight of approximately 54,537 daltons and an estimated pi of approximately 10 04 Analysis of the full length PR0943 sequence shown in Figure 20 (SEQ ID NO 29) evidences the presence of a variety of important polypeptide domains as shown in Figure 20, wherein the locations given for those important polypeptide domains are approximate as described above Anaylsis of the full-length PR0943 sequence shown in Figure 20 evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 17, a transmembrane domain from about amino acid 376 to about amino acid 396, tyrosine kinase phosphorylation sites from about amino acid 212 to about amino acid 219 and from about amino acid 329 to about amino acid 336, potential N-glycosylation sites from about amino acid 1 1 1 to about amino acid 114, from about ammo acid 231 to about ammo acid 234, from about amino acid 255 to about amino acid 258, and from about amino acid 293 to about amino acid 296, and an immunoglobulm and MHC protein sequence homology block from about amino acid 219 to about ammo acid 236 Clone DNA52192-1369 has been deposited with the ATCC on July 1 , 1998 and is assigned ATCC deposit no 203042
An analysis of the Dayhoff database (version 35 45 S wissProt 35), using the ALIGN-2 sequence alignment analysis of the full length sequence shown in Figure 20 (SEQ ID NO 29) evidenced sequence identity between the PR0943 amino acid sequence and the following Dayhoff sequences B49151 , A39752, FGR1_XENLA, S38579, RATHBFGFRB , TVHU2F, FGR2_MOUSE, CEK3_CHICK, P_R21080 and A27171J
EXAMPLE 1 1 Isolation of cDNA Clones Encoding Human PRO 1005 DNA57708-141 1 was 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 methionme 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 pai ameters) known to be associated with secretion signals
Use of the above described signal sequence algorithm allowed identification of an EST cluster from the Incyte database, designated herein as Incyte cluster sequence no 49243 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 oi BLAST2 (Altshul et al Methods in Enzymolgy, 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 DNA56380
In light of an observed sequence homology between the DNA56380 sequence and Merck EST no AA256657, from the Merck database, the Merck EST no AA256657 was purchased and the cDNA insert was obtained and sequenced It was found herein that the cDNA insert encoded a full-length protein The sequence of this cDNA insert is shown in Figure 21 (SEQ ID NO 33) and is herein designated as DNA57708-141 1
Clone DNA57708-141 1 (Figure 21 , SEQ ID NO 33) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 30-32 and ending at the stop codon at nucleotide positions 585 587 (Figure 21 , SEQ ID NO 33) The predicted polypeptide precursor is 185 amino acids long (Figure 22) The full-length PRO 1005 protein shown in Figure 22 (SEQ ID NO 34) has an estimated molecular weight of about 20,331 daltons and a pi of about 5 85 Analysis of the full-length PRO 1005 sequence shown in Figure 22 (SEQ ID NO 34) evidences the presence of important polypeptide domains as shown in Figure 22, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PRO 1005 sequence shown in Figure 22 evidences the following a signal peptide from about amino acid 1 to about amino acid 20, N-myristoylation sites from about amino acid 67 to about amino acid 72, from about ammo acid 1 18 to about amino acid 123, and from about amino acid 163 to about amino acid 168, and a flavodoxin protein homology from about amino acid 156 to about amino acid 174 (Figure 22, SEQ ID NO 34) Clone DNA57708 1411 has been deposited with ATCC on June 23, 1998 and is assigned ATCC deposit no 203021
An analysis of the Dayhoff database (version 35 45 SwιssProt 35), using the ALIGN-2 sequence alignment analysis of the full length sequence shown in Figure 22 (SEQ ID NO 34), evidenced sequence identity between the PRO1005 amino acid sequence and the following Dayhoff sequences DDU07187 , DDU87912_1 , CELD1007 4, A42239, DDU42597_1 , CYAG_DICDI, S50452, MRKC LEPN P R41998, and XYNA_RUMFL
EXAMPLE 12
Isolation of cDNA Clones Encoding Human PRO 1009 A cDNA clone 57129 1413 encoding a native humanPRO1009 polypeptide was identified using a yeast screen, in a human SK-Lu-1 adenocarcmoma cell line cDNA library that preferentialh represents the 5 ends of the primary cDNA clones Clone DNA57129- 1413 contains a single open reading frame with an apparent tianslational initiation site at nucleotide positions 41 -43 and ending at the stop codon at nucleotide positions 1886- 1888 (Figure 23, SEQ ID NO 35) The predicted polypeptide precursor is 615 amino acids long (Figure 24) The full length PRO 1009 protein shown in Figure 24 has an estimated molecular weight of about 68 125 daltons and a pi of about 7 82 Analysis of the full length PRO 1009 sequence shown in Figure 24 (SEQ ID NO 36) e idences the presence of a variety of important polypeptide domains as shown in Figure 24, wherein the locations given foi those important polypeptide domains are approximate as described above Analysis of the full-length PRO 1009 sequence shown in Figure 24 evidences the following a signal peptide from about amino acid 1 to about amino acid 22 transmembrane domains from about amino acid 140 to about amino acid 161 , from about amino acid 213 to about amino acid 229, and from about amino acid 312 to about amino acid 334, a putative AMP-binding domain signature from about amino acid 260 to about amino acid 271 , N myπstoylation sites from about amino acid 19 to about amino acid 24, from about amino acid 22 to about amino acid 27, from about amino acid 120 to about amino acid 125, from about amino acid 203 to about amino acid 208, from about amino acid 268 to about amino acid 273, from about ammo acid 272 to about ammo acid 277, from about amino acid 314 to about amino acid 319, from about amino acid 318 to about ammo acid 323, from about amino acid 379 to about ammo acid 384, from about amino acid 380 to about amino acid 385, and from about amino acid 409 to about amino acid 414, and an N giycosylation site from about amino acid 282 to about amino acid 285 Clone DNA57129-1413 has been deposited with ATCC on June 16, 1998 and is assigned ATCC deposit no 209977 An analysis of the Dayhoff database (version 35 45 SwissProt 35), using the ALIGN 2 sequence alignment analysis of the full length sequence shown in Figure 24 (SEQ ID NO 36), evidenced sequence identity between the PRO1009 amino acid sequence and the following Dayhoff sequences F69893, CEF28F8_2, BSY13917J7 , D69187, D69649, XCRPFB_1, E64928, YDID_ECOLI, BNACSF8 and RPU75363_2
EXAMPLE 13 Isolation of cDNA Clones Encoding Human PRO 1025
DNA59622-1334 was 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 methionme 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 the above described signal sequence algorithm allowed identification of an EST cluster sequence from the Incyte database from which a candidate sequence was formed designated herein as DNA56507 mit This EST cluster sequence was then compared to a variety of expiessed 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 m Enzvmol gy, 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 DNA56507
In light of an observed sequence homology between DNA56507 and Incvte EST 1324053 (from Incyte library LPARNOT 02 formed from cancerous human parotid gland tissue), Incv te EST 1 24053 was furthei examined DNA sequencing gave the full length DNA sequence shown in Figure 25 (SEQ ID NO 37) and is herein designated as DNA59622 1334
Clone DNA59622 1334 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 67-69 and ending at the stop codon at nucleotide positions 814 816 (Figure 25, SEQ ID NO 37) The predicted polypeptide precursor is 249 amino acids long (Figure 26) The full-length PRO 1025 protein shown in Figure 26 (SEQ ID NO 38) has an estimated molecular weight of about 27,01 1 daltons and a pi of about 5 48 Analysis of the full-length PRO 1025 sequence shown in Figure 26 (SEQ ID NO 38) evidences the presence of important polypeptide domains as shown in Figure 26, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PRO 1025 sequence shown in Figure 26 evidences the following a signal peptide from about amino acid 1 to about amino acid 14, N- glycosylation sites from about amino acid 124 to about amino acid 127 and from about amino acid 132 to about amino acid 135 , and a leucine zipper pattern from about amino acid 44 to about amino acid 65 Clone DNA59622 1334 has been deposited with ATCC on June 16, 1998 and is assigned ATCC deposit no 209984
An analysis of the Dayhoff database (version 35 45 S wissProt 35), using the ALIGN-2 sequence alignment analysis of the full length sequence shown in Figure 26 (SEQ ID NO 38), evidenced sequence identity between the PRO1025 ammo acid sequence and the following Dayhoff sequences BTU79413_1 , MMU1_1 , A42337, MMU46068_1 and S68553
EXAMPLE 14 Isolation of cDNA Clones Encoding Human PROl 030 DNA59485-1336 was 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 methionme 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 the above described signal sequence algorithm applied to Incyte fragments from the Incyte database, sequences fitting the algorithm were identified From those sequences fitting the algorithm, a sequence was derived This derived sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e g , GenBank Merck Washington University) 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 Enzvmol y . 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 DNA56450
Based upon the assembly and other discoveries and information provided herein, a clone containing Merck EST AA625485 was further examined DNA sequencing gave the full-length DNA sequence shown in Figure 27 (SEQ ID NO 39) and is herein designated as DNA59485-1336
Clone DNA59485- 1336 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 148-150 and ending at the stop codon at nucleotide positions 673-675 (Figure 27, SEQ ID NO 39) The predicted polypeptide precursor is 175 amino acids long (Figure 28) The full-length PRO 1030 protein shown in Figure 28 (SEQ ID NO 40) has an estimated molecular weight of about 19,979 daltons and a pi of about 9 26 Analysis of the full-length PRO 1030 sequence shown in Figure 28 (SEQ ID NO 40) evidences the presence of an important polypeptide domain as shown in Figure 28, wherein the location given for that important polypeptide domain is approximate as described above Analysis of the full-length PRO 1030 sequence shown in Figure 28 evidences the presence of a signal peptide from about amino acid 1 to about ammo acid 20 Clone DNA59485-1336 has been deposited with ATCC on June 23, 1998 and is assigned ATCC deposit no 203015
EXAMPLE 15 Isolation of cDNA Clones Encoding Human PRO 1097 DNA59841 -1460 was 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 charactei of the DNA nucleotides surrounding the first and optionally the second methionme 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 ammo 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 ammo acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals
Using of the above described signal sequence algorithm applied to Incyte fragments from the Incyte database, sequences fitting the algorithm were identified From those sequences fitting the algorithm, a sequence was derived This derived 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 m Enzvmol gy , 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 DNA56006 Based on the information discovered and provided herein, the Incyte clone including EST 2408105, from Incyte brain stem library 31 1 "BSTMNON02" was further examined DNA sequencing gave the full-length DNA sequence shown in Figure 29 (SEQ ID NO 41 ) and is herein designated as DNA59841 -1460
Clone DNA59841 - 1460 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 3 5 and ending at the stop codon at nucleotide positions 276-278 (Figure 29, SEQ ID NO 41) The predicted polypeptide precursor is 91 amino acids long (Figure 30) The full-length PROl 097 protein shown in Figure 30 (SEQ ID NO 42) has an estimated molecular weight of about 10,542 daltons and a pi of about 10 04 Analysis of the full-length PRO 1097 sequence shown in Figure 30 (SEQ ID NO 42) evidences the presence of important polypeptide domains as shown in Figure 30, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PRO 1097 sequence shown in Figure 30 evidences the following a signal peptide from about amino acid 1 to about amino acid 20, a glycoprotease family protein domain starting from about ammo acid 56, and an acyltransferase ChoActase/COT/CPT family peptide starting from about amino acid 49 (Figure 30, SEQ ID NO 42) Clone DNA59841-1460 has been deposited with ATCC on July 1, 1998 and is assigned ATCC deposit no 203044
EXAMPLE 16
Isolation of cDNA Clones Encoding Human PROl 107 DNA59606-1471 was 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 methionme 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 pai ameters) known to be associated with secretion signals
Use of the above described signal sequence algorithm allowed identification of an EST cluster sequence from the Incyte database, designated herein as DNA56402 inn This EST cluster sequence was then compared to 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 identity existing homologies The homology search was performed using the computer program BLAST or BLAST2 (Altshul et al Methods in Enzymolgy, 266460-480 ( 1996)) Those compai isons 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 DNA56402
In light of an observed sequence homology between the DNA56402 and an Incyte EST no 3203694, (from Incyte library PENCNOT02, 309, a caversnosum tissue library from the same donoi), Incyte EST no 3203694 was further examined DNA sequencing gave the full-length DNA sequence and is herein designated as DNA59606-1471 shown in Figure 31 (SEQ ID NO 43)
Clone DNA59606-1471 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 244-246 and ending at the stop codon at nucleotide positions 1675- 1677 (Figure 31 , SEQ ID NO 43) The predicted polypeptide precursor is 477 amino acids long (Figure 32) The full-length PROl 107 protein shown in Figure 32 (SEQ ID NO 44) has an estimated molecular weight of about 54,668 daltons and a pi of about 6 33 Analysis of the full-length PRO 1 107 sequence shown in Figure 32 (SEQ ID NO 44) evidences the presence of important polypeptide domains as shown in Figure 32, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PROl 107 sequence shown in Figure 32 evidences the presence of the following a signal peptide from about ammo acid 1 to about amino acid 22; a transmembrane domain from about amino acid 429 to about amino acid 452, N-glycosylation sites from about ammo acid 101 to about amino acid 104, from about ammo acid 158 to about amino acid 161 , from about amino acid 292 to about amino acid 295, from about amino acid 329 to about amino acid 332, from about amino acid 362 to about amino acid 365, from about amino acid 369 to about amino acid 372, from about amino acid 382 to about amino acid 385, and from about ammo acid 389 to about amino acid 392, a somatomedin B domain protein from about amino acid 69 to about amino acid 85, and a sulfatase protein region from about ammo acid 212 to about amino acid 241 Clone DNA59606-1471 has been deposited with ATCC on June 9, 1998 and is assigned ATCC deposit no 209945 Analysis of the amino acid sequence of the full-length PROl 107 polypeptide (Figure 32) suggests that it possesses significant sequence similarity to phosphodiesterase I/nucleotide pyrophosphatase, human insulin receptor tyrosine kinase inhibitor, alkaline phosphodiesterase and autotaxin, thereby indicating that PRO 1 107 may be a novel phosphodiesterase More specifically, an analysis of the Dayhoff database (version 35 45 SwissProt), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 32 (SEQ ID NO 44), evidenced sequence identity between the PROl 107 amino acid sequence and the following Dayhoff sequences AF005632_ 1. P_R79148, RNU78787 , AF060218_4, A57080 and HUMATXT
EXAMPLE 17 Isolation of cDNA Clones Encoding a Human PRO l 1 1 1 An expressed sequence tag (EST) DNA database (LIFESEQ®. Incyte Pharmaceuticals, Palo Alto, CA) was searched and an EST was identified which showed homology to msuhn-hke giowth factor binding protein
RNA for construction of cDNA libraries was then isolated from human fetal brain tissue The cDNA libraries used to isolate the cDNA clones encoding human PROl 1 1 1 were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with oligodT containing a Notl site, linked with blunt to Sail hemikinased adaptois, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRK5D, 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 The human fetal brain cDNA libraries (prepared as described above), were screened by hybridization with a synthetic ohgonucleotide probe
5'-CCACCACCTGGAGGTCCTGCAGTTGGGCAGGAACTCCATCCGGCAGATTG-3' (SEQ ID NO 47) based on the identified EST described above A cDNA clone was sequenced in entirety and is herein designated as DNA58721 -1475 The entire nucleotide sequence of clone DNA58721 - 1475 is shown in Figure 33 (SEQ ID NO 45) Clone DNA58721 - 1475 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 57-59 and a stop signal at nucleotide positions 2016 2018 (Figure 33, SEQ ID NO 45) The predicted polypeptide precursoi is 653 ammo acids long, has a calculated molecular weight of approximately 72,717 daltons and an estimated pi of approximately 6 99 Analysis of the full-length PROl 111 sequence shown in Figure 34 (SEQ ID NO 46) evidences the presence of a variety of important polypeptide domains as shown in Figure 34, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PROl 11 1 sequence evidences the presence of the following transmembrane domains from about amino acid 21 to about amino acid 40 and from about amino acid 528 to about amino acid 548, N-glycosylation sites from about amino acid 277 to about amino acid 280, from about amino acid 322 to about amino acid 325, from about amino acid 363 to about amino acid 366, from about amino acid 388 to about amino acid 391, from about amino acid 410 to about ammo acid 413, from about amino acid 434 to about amino acid 437, from about amino acid 440 to about amino acid 443, from about amino acid 447 to about amino acid 450, and from about amino acid 450 to about amino acid 453, a glycosaminoglycan attachment site from about amino acid 591 to about amino acid 594, and a leucine zipper pattern from about amino acid 271 to about am o acid 292 Clone DNA58721 -1475 has been deposited with ATCC on August 11 , 1998 and is assigned ATCC deposit no 203110
An analysis of the Dayhoff database (version 35 45 SwissProt), using the ALIGN-2 sequence alignment analysis of the full length sequence shown in Figure 34 (SEQ ID NO 46) evidenced sequence identity between the PROl l l l amino acid sequence and the following Dayhoff sequences A58532, D86983 , RNPLGPV PGS2_HUMAN, AF038127_1 , ALS_MOUSE, GPVJHUMAN, PGS2_BOVIN, ALS_PAPPA and 147020
EXAMPLE 18
Isolation of cDNA Clones Encoding a Human PRQ1 153
DNA59842-1502 was 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 methionme codon(s) (ATG) at the 5 -end of the sequence oi 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 fust ATG has the required ammo acids, the second is not examined If neither meets the requirement, the candidate sequence is not scoi ed In order to determine whethei 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 the above described signal sequence algorithm allowed identification of an EST cluster sequence from the Incyte database This EST cl uster sequence was then compared to variety of expressed sequence tag (EST) databases which included public EST databases (e g , GenBank, Merck Washington University) 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 Enzymolgy, 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 DNA56008
In light of an observed sequence homology between the DNA56008 sequence and an Incyte clone, including Incyte EST 2472409 (from library 309 (THP1NOT03) constructed from THP-1 cells derived from the blood of a monocytic leukemia patient), the Incyte clone, including Incyte EST 2472409 was purchased and the cDNA insert was obtained and sequenced It was found that the cDNA insert encoded a full-length protein The sequence of this cDNA insert is shown in Figure 35 (SEQ ID NO 48) and is herein designated as DNA59842-1502
Clone DNA59842-1502 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 92 94 and ending at the stop codon at nucleotide positions 683-685 (Figure 35, SEQ ID
NO 48) The predicted polypeptide precursor is 197 ammo acids long (Figure 36) The full-length PROl 153 protein shown in Figure 36 (SEQ ID NO 49) has an estimated molecular weight of about 21,540 daltons and a pi of about 8 31 Analysis of the full-length PRO 1 153 sequence shown in Figure 36 (SEQ ID NO 49) evidences the presence of important polypeptide domains as shown in Figure 36, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PROl 153 sequence shown in Figure 36 evidences the presence of the following transmembrane domains from about amino acid 10 to about amino acid 28 and from about amino acid 85 to about amino acid 1 10, an N giycosylation site from about amino acid 38 to about amino acid 41 and N mynstoylation sites from about amino acid 5 to about amino acid 10 and from about amino acid 88 to about amino acid 93 Clone DNA59842 1502 has been deposited with ATCC on June 16, 1998 and is assigned ATCC deposit no 209982
An analysis of the Dayhoff database (version 35 45 SwissProt), using the ALIGN 2 sequence alignment analysis of the full-length sequence shown in Figure 36 (SEQ ID NO 49), evidenced sequence identity between the PROl 153 amino acid sequence and the following Dayhoff sequences S57447, SOYHRGPC , S46965 P_P82971 , VCPHEROPH , EXTN_TOBAC MLCB2548_9, ANXAJ ABIT, JC5437 and SSGP_VOLCA
EXAMPLE 19
Isolation of cDNA Clones Encoding a Human PRO l 182
DNA59848 1512 was identified by applying a piopπetary signal sequence finding algorithm de eloped 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 ot the DNA nucleotides surrounding the first and optionally the second methionme 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 the above described signal sequence algorithm allowed identification of an EST cluster sequence from the Incyte database, designated herein as 146647 This EST cluster sequence was then compared to 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 Enzymolgy. 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 DNA56033
In light of an observed sequence homology between the DNA56033 sequence and the Incyte EST clone no 2595195, the Incyte EST clone 2595195 was purchased and the cDNA insert was obtained and sequenced It was found that the cDNA insert encoded a full-length protein The sequence of this cDN A insert is shown in Figure 37 (SEQ ID NO 50) and is herein designated as DNA59848-1512
Clone DNA59848- 1512 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 67-69 and ending at the stop codon at nucleotide positions 880-882 (Figure 37, SEQ ID NO 50) The predicted polypeptide precursor is 271 amino acids long (Figure 38) The full-length PRO l 182 protein shown in Figure 38 (SEQ ID NO 51) has an estimated molecular weight of about 28,665 daltons and a pi of about 5 33 Analysis of the full-length PROl 182 sequence shown in Figure 38 (SEQ ID NO 51 ) evidences the presence of important polypeptide domains as shown in Figure 38, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PROl 182 sequence shown in Figure 38 evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 25, an amino acid block having homology to C-type lectin domain proteins from about amino acid 247 to about amino acid 256, and an amino acid sequence block having homology to C 1 q domain proteins from about amino acid 44 to about amino acid 77 Clone DNA59848 1512 has been deposited with ATCC on August 4, 1998 and is assigned ATCC deposit no 203088
An analysis of the Dayhoff database (version 35 45 SwissProt), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figuie 38 (SEQ ID NO 51 ), evidenced sequence identity between the PROl 182 amino acid sequence and the following Dayhoff sequences PSPD_BOVIN CL43JBOVIN CONGJBOVIN, P_W 18780, P R45005, P R53257 and CELEGAP7 1 EXAMPLE 20 Isolation of cDNA Clones Encoding Human PROl 184 DNA59220-1514 was 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 methionme codon(s) (ATG) at the 5'-end of the sequence oi 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 the above described signal sequence algorithm allowed identification of an EST sequence from the Incyte database, designated herein as DNA56375 mit This EST sequence was then compared to 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 Enzvmol gy. 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 DNA56375
In light of an observed sequence homology between the DNA56375 sequence and an Incyte clone, including Incyte EST no 1428374 (from an lleum tissue library (39, SINTBSTOl )), the Incyte clone, including Incyte EST no 1428374, was further examined, and the cDNA insert was obtained and sequenced It was found herein that the cDNA insert encoded a full-length protein The sequence of this cDNA insert is shown in Figure
39 (SEQ ID NO 52) and is herein designated as DNA59220-1514
Clone DNA59220-1514 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 106-108 and ending at the stop codon at nucleotide positions 532-534 (Figure 39, SEQ ID NO 52) The predicted polypeptide precursor is 142 amino acids long (Figure 40) The full-length PROl 184 protein shown in Figure 40 (SEQ ID NO 53) has an estimated molecular weight ot about 15,690 daltons and a pi of about 9 64 Analysis of the full-length PROl 184 sequence shown in Figure 40 (SEQ ID NO 53) evidences the presence of an important polypeptide domain as shown in Figure 40, wherein the location given for that important polypeptide domain is approximate as described above Analysis of the full-length PROl 184 sequence shown in Figure 40 evidences the presence of the following a signal peptide from about ammo acid 1 to about amino acid 38 Clone DNA59220-1514 has been deposited with ATCC on June 9, 1998 and is assigned ATCC deposit no 209962
Analysis of the amino acid sequence of the full-length PRO l 184 polypeptide suggests that it possesses some sequence identity with a protein called TIM from Drosophila virihs More specifically, an analysis of the Dayhoff database (version 35 45 SwissProt), using the ALIGN 2 sequence alignment analysis of the full-length sequence shown in Figure 40 (SEQ ID NO 53), evidenced sequence identity between the PROl 184 amino acid sequence and the following Dayhoff sequences WIS l_SCHPO, F002186_1 , ATAC00239124 and MSAIPRP
EXAMPLE 21
Isolation of cDNA Clones Encoding PROl 187 DNA62876-1517 was 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 methionme 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 the above described signal sequence algorithm allowed identification of an EST cluster sequence from the Incyte database This EST cluster sequence was then compared to 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 Enzymolgy, 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 DNA57726
In light of an observed sequence homology between the DNA57726 sequence and the Incyte EST 358563 sequence, the clone (from a library constructed using RNA from synovial membrane tissue) including Incyte EST 358563 was purchased and the cDNA insert was obtained and sequenced The sequence of this cDNA insert is shown in Figure 41 (SEQ ID NO 54) and is herein designated as DNA62876 1517
Clone DNA62876- 1517 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 121 -123 and ending at the stop codon at nucleotide positions 481 -483 (Figure 41 , SEQ ID NO 54) The predicted polypeptide precursor is 120 amino acids long (Figure 42) The full length PRO l 187 protein shown in Figure 42 (SEQ ID NO 55) has an estimated molecular weight of about 12,925 daltons and a pi of about 9 46 Analysis of the full-length PROl 187 sequence shown in Figure 42 (SEQ ID NO 55) evidences the presence of important polypeptide domains as shown in Figuie 42 wherein the locations given for those important polypeptide domains are approximate as described above Analy sis of the full-length PROl 187 sequence shown in Figure 42 evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 17, and N-myπstoylation sites from about amino acid 58 to about amino acid 63, from about amino acid 63 to about amino acid 68, from about amino acid 64 to about amino acid 69, from about amino acid 83 to about amino acid 88, from about amino acid 1 1 1 to about amino acid 1 16, and from about amino acid 1 15 to about ammo acid 120 Clone DNA62876-1517 has been deposited with ATCC on August 4, 1998 and is assigned ATCC deposit no 203095
An analysis of the Dayhoff database (version 35 45 SwissProt), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 42 (SEQ ID NO 55), evidenced sequence identity between the PRO 1187 amino acid sequence and the following Dayhoff sequences MGNENDOBX_l, CELF41G3_9, AMPG_STRLI, HSBBOVHERL_2, LEEXTEN10_1 , AF029958J and P_W04957
EXAMPLE 22 Isolation of cDNA Clones Encoding a Human PRO 1281 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 EST databases (e g , GenBank) and a proprietary EST database (LIFESEQ®, Incyte Phamaceuticals, Palo Alto, CA) The search was performed using the computer program BLAST or BLAST2 [Altshul et al , Methods in Enzymology, 266 460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences 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 consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington)
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above and is herein designated DNA35720 In some cases, the DNA35720 consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above Based on the DNA35720 consensus sequence, oligonucleotides were synthesized 1 ) to identity by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO 1281 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 greatei than about 1 -1 5kbp In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel etal , Current Protocols in Molecular Biology supia, with the PCR primer pan A positive library was then used to isolate clones encoding the gene of interest using the probe ohgonucleotide and one of the primer pairs
PCR primers (forward and reverse) were synthesized toiward PCR primers (35720 f 1 ) 5'-TGGAAGGCTGCCGCAACGACAATC-3' (SEQ ID NO 58)
(35720 f2) 5'-CTGATGTGGCCGATGTTCTG-3' (SEQ ID NO 59)
(35720 f3) 5'-ATGGCTCAGTGTGCAGACAG-3' (SEQ ID NO 60) reverse PCR primers (35720.rl ) 5'-GCATGCTGCTCCGTGAAGTAGTCC-3' (SEQ ID NO:61 )
(32720.r2) 5'-ATGCATGGGAAAGAAGGCCTGCCC-3' (SEQ ID NO:62)
Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA35720 sequence which had the following nucleotide sequence: hybridization probe
5'-TGCACTGGTGACCACGAGGGGGTGCACTATAGCCATCTGGAGCTGAG-3' (SEQ ID NO:63)
RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. 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.
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full- length PRO 1281 polypeptide (designated herein as DNA59820-1549 [Figures 43A-43B, SEQ ID NO:56]) and the derived protein sequence for that PRO 1281 polypeptide.
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucelotide positions 228-230 and a stop signal at nucleotide positions 2553-2555 (Figures 43A-43B,
SEQ ID NO:56). The predicted polypeptide precursor is 775 amino acids long (Figure 44), has a calculated molecular weight of approximately 85,481 daltons and an estimated pi of approximately 6.92. Analysis of the full length PROl 281 sequence shown in Figure 44 (SEQ ID NO:57) evidences the presence of a variety of important polypeptide domains as shown in Figure 44, wherein the locations given for those important polypeptide domains are approximate as described above. Anaylsis of the full-length PRO 1281 sequence shown in Figure 44 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 15; and potential N- giycosylation sites from about amino acid 138 to about amino acid 141 and from about amino acid 361 to about amino acid 364. Clone DNA59820-1549 has been deposited with the ATCC on August 18, 1998 and is assigned
ATCC deposit no. 203129.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 44 (SEQ ID NO:57) evidenced sequence identity between the PR01281 amino acid sequence and the following Dayhoff sequences: S44860, CET24D 1J , CEC38H2_3,
CAC2_HAECO, B3A2_HUMAN, S22373, CEF38A3_2, CEC34F6_2, CEC34F6_3 and CELT22B1 1_3.
EXAMPLE 23
Gene Amplification
This example shows that the PR0212-. PRO290-, PR0341 -, PR0535-, PR0619-, PR0717-, PRO809-, PRO830-, PR0848-, PR0943-. PRO 1005-, PRO 1009-, PRO 1025-, PRO 1030-, PRO 1097-, PRO 1 107-, PRO 1 1 1 1 -,
PRO 1 153-, PRO 1 182-, PRO 1 184-. PRO 1 187-, PRO 1281 -, PR023-, PR039-, PR0834-, PRO 1317-, PRO 1710-,
PRO2094-, PR02145- or PR02198-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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PROl 097, PROl 107, PROl 1 1 1 , PROl 153.PR01 182. PR01184, PROl 187, PROl 281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide, for example, muπne-human chimeric, humanized or human antibodies against a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PRO 1153, PRO 1182, PRO 1 184, PRO 1 187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 polypeptide
The starting material for the screen was genomic DNA isolated from a variety cancers The DNA is quantitated precisely, e g , fluorometπcally 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 Pπzm 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212-, PRO290-, PR0341 -, PR0535-, PR0619-, PR0717-, PRO809-, PRO830-, PR0848-, PR0943-, PRO1005-, PRO1009-, PRO1025-, PRO1030-, PRO1097-, PROl 107-, PRO l l l l -, PR01 153-, PRO l 182-, PROl 184-, PROl 187-, PR01281-, PR023-, PR039-, PR0834-, PROl 317-, PROl 710-, PRO2094-, PR02145- or PR02198-encodιng gene Regions of PR0212, PRO290, PR0341. PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097, PROl 107, PROl l l l , PROl 153, PRO 1 182 PROH 84, PR01 187, PRO1281 , PRO23, PRO39, PRO834, PRO 1317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PROl 030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PR01 182, PROl 184, PRO 1 187, PR01281 , PR023, PR039. PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 gene amplification analysis were as follows PR0212 (DNA30942-1 134)
30942.3unt-probe:
5'-CCCTGTGCACTGATCCTGGCCC-3 ' (SEQ ID NO:80)
30942.3unt-5: 5'-AGCGGAGCGTCCGTGA-3' (SEQ ID N0:81)
30942.3unt-3:
5'-CCACTTGCACTGAAAGAGGCT-3' (SEQ ID NO:82)
30942.tm.p2:
5'-ACACGATGCGTGCTCCAAGCAGAA-3' (SEQ ID NO:83) 30942.tm.f2:
5'-CTTCTTCGCGCACGCTG-3' (SEQ ID NO:84)
30942.tm.r2:
5 -ATCACGCCGGCACCAG-3' (SEQ ID NO:85)
30942.doml .f3 5'-ACGCGGAGTGGCAGAAAC-3' (SEQ ID NO:86)
30942.doml.r3:
5'-CACTGGGCGCACACCA-3' (SEQ ID NO:87)
30942.doml.p3:
5'-TACCCCTGGCGGGACGCAGAG-3' (SEQ ID NO:88) 30942.dom4a.f4:
S'-CACCTTCTCAGCCAGCAGCT-S' (SEQ ID NO:89)
30942.dom4a.r4:
5'-GAGGAAGAGCCTGGCACATT-3' (SEQ ID NO:90)
30942.dom4a.p4: 5'-AACTGCACGGCCCTGGGCCT-3' (SEQ ID NO:91 )
30942.dom4b.f5:
5'-CCTCAATGTGCCAGGCTCTT-3' (SEQ ID NO:92)
30942.dom4b.r5:
5'-CCTGGTACCCTGGTGCTGA-3' (SEQ ID NO:93) 30942.dom4b.p5:
5'-CACCAGCTGCACTGGCTTCCCC-3' (SEQ ID NO:94)
PRO290 (DNA35680-1212): 3568Q.tm.p:
5'-CCACCAATGGCAGCCCCACCT-3' (SEQ ID NO:95) 3568Q.tm.f:
5'-GACTGCCCTCCCTGCCA-3' (SEQ ID NO:96) 35680.tm.r:
5'-CAAAAAGCCTGGAAGTCTTCAAAG-3' (SEQ ID NO:97)
PR0341 (DNA26288-1239): 26288.tm.fl : 5'-CAGCTGGACTGCAGGTGCTA-3' (SEQ ID NO:98)
26288.tm.rl :
5'-CAGTGAGCACAGCAAGTGTCCT-3' (SEQ ID NO:99)
26288.tm.pl : 5'-GGCCACCTCCTTGAGTCTTCAGTTCCCT-3' (SEQ ID NO: 100)
PR0535 (DNA49143- 1429):
49143.tm.fi :
5'-CAACTACTGGCTAAAGCTGGTGAA-3' (SEQ ID NO: 101 )
49143.tm.rl :
5'-CCTTTCTGTATAGGTGATACCCAATGA-3' (SEQ ID NO: 102) 49143.tm.pl :
5'-TGGCCATCCCTACCAGAGGCAAAA-3' (SEQ ID NO: 103)
PR0619 (DNA49821 -1562): 49821.tm.f1 :
5'-CTGAAGACGACGCGGATTACTA-3' (SEQ ID NO: 104) 49821. tm.rl :
5'-GGCAGAAATGGGAGGCAGA-3' (SEQ ID NO: 105)
49821.tm.pl :
5'-TGCTCTGTTGGCTACGGCTTTAGTCCCTAG-3' (SEQ ID NO: 106)
PR0717 (DNA50988-1326): 50988.tm.f3:
5'-CAAGCGTCCAGGTTTATTGA-3' (SEQ ID NO: 107)
50988.tm.r3:
5'-GACTACAAGGCGCTCAGCTA-3' (SEQ ID NO: 108)
50988.tm.p3: 5'-CCGGCTGGGTCTCACTCCTCC-3' (SEQ ID NO: 109)
PRO809 (DNA57836-1338):
57836.tm.fi :
5'-AGCAGCAGCCATGTAGAATGAA-3' (SEQ ID NO: 1 10) 57836.tm.r1:
5'-AATACGAACAGTGCACGCTGAT-3' (SEQIDNO:lll) 57836.tm.pl: 5'-TCCAGAGAGCCAAGCACGGCAGA-3' (SEQ ID NO: 112)
PRO830 (DNA56866-1342): 56866.tm.fl:
5'-TCTAGCCAGCTTGGCTCCAATA-3' (SEQ ID NO: 113) 56866.tm.rl:
5'-CCTGGCTCTAGCACCAACTCATA-3' (SEQ ID NO: 114) 56866.tm.pl:
5'-TCAGTGGCCCTAAGGAGATGGGCCT-3' (SEQ ID NO: 115)
PRQ848 (DNA59839-1461): 59839.tm.fl:
5'-CAGGATACAGTGGGAATCTTGAGA-3' (SEQ ID NO: 116) 59839.tm.rl:
5'-CCTGAAGGGCTTGGAGCTTAGT-3' (SEQ ID NO: 117)
59839.tm.pl:
5'-TCTTTGGCCATTTCCCATGGCTCA-3' (SEQ ID NO: 118)
PRQ943 (DNA52192-1369): 52192.tm.fl:
5'-CCCATGGCGAGGAGGAAT-3' (SEQ ID NO: 119)
52192.tm.rl:
5'-TGCGTACGTGTGCCTTCAG-3' (SEQ ID NO: 120)
52192.tm.pl: 5'-CAGCACCCCAGGCAGTCTGTGTGT-3' (SEQ ID NO: 121)
PRO 1005 (DNA57708-1411): 57708.tm.fl:
5'-AACGTGCTACACGACCAGTGTACT-3' (SEQ ID NO: 122) 57708.tm.rl: 5'-CACAGCATATTCAGATGACTAAATCCA-3' (SEQ ID NO: 123) 57708.tm.pl: 5'-TTGTTTAGTTCTCCACCGTGTCTCCACAGAA-3' (SEQ ID NO: 124) PRO1009 (DNA57129-1413):
57129.tm.fl :
5'-TGTCAGAATGCAACCTGGCTT-3' (SEQ ID NO: 125)
57129.tm.r1 :
5'-TGATGTGCCTGGCTCAGAAC-3' (SEQ ID NO: 126)
57129.tm.pl :
5'-TGCACCTAGATGTCCCCAGCACCC-3' (SEQ ID NO: 127)
PRO1025 (DNA59622-1334): 59622.tm.fl : 5'-TGATTGGTTCCCAGTGGCTT-3' (SEQ ID NO: 128) 59622.tm.rl:
5'-GTGGTGGCAGCAGCTTCCT-3' (SEQ ID NO: 129) 59622.tm.pl : 5'-CCCCACCCCCTTATAGCATCTCCCTC-3' (SEQ ID NO: 130)
PRO1030 (DNA59485-1336): 59485.tm.fl :
5'-AATCTCC AAGCCCTTCTGTCTGT-3 ' (SEQ ID NO: 131 ) 59485.tm.rl :
5'-TGCTTCCACACTAGCCAGTCTTC-3' (SEQ ID NO: 132) 59485.tm.pl :
5'-CACACTTCTTCTGGTTTCAAGTCTCAAGGCCT-3' (SEQ ID NO: 133)
PRO1097 (DNA59841 -1460): 59841. tm.fl :
5'-AAGATGCGCCAGGCTTCTTA-3' (SEQ ID NO: 134) 59841.tm.rl :
5'-CTCCTGTACGGTCTGCTCACTTAT-3' (SEQ ID NO: 135)
59841.tm.pl :
5 '-TGGCTGTC AGTCC AGTGTGC ATGG-3 ' (SEQ ID NO: 136)
PROl 107 (DNA59606-1471 ): 59606.tm.f 1 :
5'-GCATAGGGATAGATAAGATCCTGCTTTAT-3' (SEQ ID NO: 137)
59606.tm.rl :
5'-CAAATTAAAGTACCCATCAGGAGAGAA-3' (SEQ ID NO: 138) 59606.tm.pl :
5'-AAGTTGCTAAATATATACATTATCTGCGCCAAGTCCA-3' (SEQ ID NO: 139)
PROl l l l (DNA58721 -1475): 58721.tm.fl : 5'-GTGCTGCCCACAATTCATGA-3' (SEQ ID NO: 140) 58721.tm.rl:
5'-GTCCTTGGTATGGGTCTGAATTATAT-3' (SEQ ID NO:141 ) 58721.tm.pl : 5 -ACTCTCTGCACCCCACAGTCACCACTATCTC-3' (SEQ ID NO: 142)
PROl 153 (DNA59842-1502):
59842.tm.fl :
5'-CTGAGGAACCAGCCATGTCTCT-3' (SEQ ID NO: 143)
59842.tm.rl :
5'-GACCAGATGCAGGTACAGGATGA-3' (SEQ ID NO: 144) 59842.tm.pl :
5'-CTGCCCCTTCAGTGATGCCAACCTT-3' (SEQ ID NO: 145)
PROl 182 (DNA59848-1512): 59848.tm.fl :
5'-GGGTGGAGGCTCACTGAGTAGA-3' (SEQ ID NO: 146) 59848.tm.rl :
S'-CAATACAGGTAATGAAACTCTGCTTCTT-S' (SEQ ID NO: 147)
59848.tm.pl :
5'-TCCTCTTAAGCATAGGCCATTTTCTCAGTTTAGACA-3' (SEQ ID NO: 148)
PRQ1184 (DNA59220-1514): 59220.tm.f 1 :
5'-GGTGGTCTTGCTTGGTCTCAC-3' (SEQ ID NO: 149)
59220.tm.rl :
5'-CCGTCGTTCAGCAACATGAC-3' (SEQ ID NO: 150)
59220.tm.pl : 5'-ACCGCCTACCGCTGTGCCCA-3' (SEQ ID NO: 151 )
PROl 187 (DNA62876- 1517):
62876.tm.fl :
S'-CAGTAAAACCACAGGCTGGATTT-S' (SEQ ID NO: 152) 62876.tm.rl :
5'-CCTGAGAGCAAGAAGGTTGAGAAT-3' (SEQ ID NO: 153) 62876.tm.pl : 5'-TAGACAGGGACCATGGCCCGCA-3' (SEQ ID NO: 154)
PROl 281 (DNA59820-1549): 59820.tm.f 1 :
5'-TGGGCTGTAGAAGAGTTGTTG-3' (SEQ ID NO: 155) 59820.tm.rl :
5'-TCCACACTTGGCCAGTTTAT-3' (SEQ ID NO: 156) 59820.tm.pl :
5 '-CCC A ACTTCTCCCTTTTGG ACCCT-3 ' (SEQ ID NO:157)
PRQ23 (DNA36640): 36640.tm.f 1 :
5'-TTTCATGACCTCCGAGAAGA-3' (SEQ ID NO: 158) 36640.tm.rl :
5'-TTGTAGGCGTTCTTGATGATG-3' (SEQ ID NO: 159)
36640.tm.p1 :
5'-CCCCTGGTGACGCTGTTCAAAAA-3' (SEQ ID NO: 160)
PRQ39 (DNA36651 ): 36651.tm.fl :
5'-GCGCGTGGACACCAAGTC-3' (SEQ ID NO: 161 )
36651.tm.rl :
5'-CGAAGCAGCCCTTGGACA-3' (SEQ ID NO: 162)
36651.tm.pl : 5'-ACCCCAACGCGCGCAAATACAAA-3' (SEQ ID NO: 163)
PRQ834 (DNA 56538): 56538.tm.fl :
5'-GGGAGATGTCCCTGGAAGAA-3' (SEQ ID NO: 164) 56538.tm.rl : 5'-TGTCTGGCTTCACAGCACTGT-3' (SEQ ID NO: 165) 56538.tm.p l : 5'-TTTTTTTAGGTCTGCATTCACACTGGCTGT-3' (SEQ ID NO: 166) PRQ1317.DNA71166):
71166.tm.fl:
5*-CCCTAGCTGACCCCTTCA-3' (SEQ ID NO: 167)
71166.tm.rl:
5'-TCTGACAAGCAGTTTTCTGAATC-3' (SEQ ID NO: 168)
71166.tm.pl:
5'-CTCTCCCCCTCCCTTTTCCTTTGTTT-3' (SEQ ID NO: 169)
PRO1710.DNA82331): 82331.tm.fl: 5'-TGCTTCGGGCCCAATATCT-3' (SEQ ID NO: 170) 82331.tm.rl:
5'-CGACGGCAGGTAGTTCTCCT-3 ' (SEQ ID NO: 171) 82331.tm.pl: 5'-CGTGGGCACCGCCGAAGC-3' (SEQ ID NO: 172)
PRO2094 (DNA83123):
83123.tm.fl:
5'-CCCATGTTCAATGCAGTTTG-3' (SEQ ID NO: 173)
83123.tm.rl:
5'-AGTCCTTGGAGGTCCTATCCA-3' (SEQ ID NO: 174) 83123.tm.pl:
5'-AAGCAAGGCTGATAAAAAAGCTGCCC-3' (SEQ ID NO: 175)
PRO2145(DNA88050): 8805Q.tm.f 1 :
5'-GGCCTTCTCGGTCTTCAC-3' (SEQ ID NO: 176) 88050.tm.rl:
5'-AAGTTGGGAGCCGACATC-3' (SEQ ID NO: 177)
88050.tm.pl:
5'-ATCATCTGCACCATCGGCATCC-3' (SEQ ID NO: 178)
PR02198.DNA88153): 88l53.tm.f1:
5'-TGGAGGCTGCAGATAGTGA-3' (SEQ ID NO: 179)
88153.tm.rl:
5'-CGTCACCCTCGTAGTCATAGAT-3' (SEQ ID NO: 180) 88153.tm.pl : 5'-CCCAGTGTGCCGCCTTACGA-3' (SEQ ID NO: 181 )
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 ohgonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third ohgonucleotide, 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
PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830. PR0848, PR0943, PROl 005. PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PRO l 153, PROl 182, PROl 184, PROl 187,
PROl 281 , PR023, PR039, PR0834, PRO 1317, PR01710, PRO2094, PR02145 or PR02198 compounds of the invention.
Table 4 Primary Lung and Colon Tumor Profiles
Primary Tumor Stage Othei Stage Dukes Stage T Stage N Stage
Human lung tumor AdenoCa (SRCC724) [LT1 ] IIA Tl N l 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 (SRCC811 ) [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] M 1 , 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] G l A pT2 pNO
Human colon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2
Human colon AdenoCa (SRCC757) [CT1 1 ] 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 piotease and all from Qiagen. according to the manufacturer s instructions and the description below Cell ciihuie sis Cells were washed and tryps ized at a concentration of 7 5 x 10s per tip and pelleted by centi ituging at
1000 rpm tor 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 weie then suspended into 10 ml PBS Bufter CI was equilibrated at 4"C Qiagen protease #19155 was diluted into 6 25 ml cold ddH,0 to a final concentration of 20 mg/ml and equilibrated at 4"C 10 ml of G2 Buffer was prepaied by diluting Qiagen 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 centπfugmg 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.O, 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 Qiagen 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 mm , 4°C)
Solid human tumor sample preparation and hsis
Tumor samples were w eighed 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 ddH^O 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 ddH .0, followed by G2 buffer (50 ml) If tissue was still present on the generator tip, the apparatus was disassembled and cleaned Qiagen 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 mm 4°C)
Human blood pi epai atwn and hsis
Blood was drawn from healthy volunteers using standard infectious agent protocols and citrated into 10 ml samples per tip Qiagen protease was freshly prepared by dilution into 6 25 ml cold ddH-,0 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 l buffer and 30 ml ddH.O (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, 4C 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 ddH^O (4' C) Vortexing was repeated until the pellet was white The nuclei weie 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 Qiagen protease was added (200 μl) and incubated at 50 C for 60 minutes The incubation and centrifugation were repeated until the lysates were clear (c c , incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min 4' C)
Punfication of cleaied hsates
( 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 silamzed, autoclaved 30 ml Corex tubes with 15 ml QF buffer (50°C) Isopropanol ( 10 5 ml) was added to each sample, the tubes covered with parafin and mixed by repeated inversion until the DNA precipitated Samples were pelleted by centrifugation in the SS-34 rotor at 15,000 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) Quanti tation of genomic DNA and preparation for gene amplification assay The DNA levels in each tube were quantified by standard A26J A2M> spectrophotometry on a 1 20 dilution
(5 μl DNA + 95 μl ddH. O) using the 0 1 ml quartz cuvettes in the Beckman DU640 spectrophotometer A26,/A.8„ 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 Fluorometπc DNA quantitation was then performed on the diluted material (20-600 ng/ml) using the manufacturer's guidelines as modified below This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometer to warm-up for about 15 minutes The Hoechst dye working solution (#H33258, 10 μl, 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 3Zt(+) 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 fluorometπcly determined concentration was then used to dilute each sample to 10 ng/μl in ddH .O 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™ pi l ei s and probe both B-actin and GAPDH on a single plate with normal human DNA and no-template controls The diluted samples weie 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 ahquots at -80°C Ahquots which were subsequently to be used in the gene amplification assay were stored at 4°C Each 1 ml aliquot is enough tor 8 9 plates or 64 tests Gene amplification
Figure imgf000145_0001
The PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809 PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107 PRO l 1 1 1 PROl 153, PROl 182, PROl 184, PR01 187, PR01281 , PR023, PR039, PR0834, PR01317 PRO 17 I 0 PRO2094 PR02145 oi PR02198 compounds of the invention were screened in the following primary tumors and the resulting ΔCt values are reported in Tables 5A-5D.
Table 5A ΔCt values in lung and colon primary tumors and cell line models
Pnm-i \ PR0212 PRO290 P O I PROSES PR0619 PR0717 PRO809 PRO80 PR0848 luπioi
5
LT 1 — —
LT l 1 — 1 1
IT2 — —
10 LIT 14S — 104 168
L14 — —
L16 — —
15 LT7 I is — 121
1 M
-P-» <J1
1 19 - 119 I 34
Ll 1(1 1 17 — 141
20 191 202
LTI1 1 S6 I 6"* 140 169 140 I 2S 104 196 I S7
1 16 I 68
O
O
O α.
■α ω
3 C J- O c-:
C a. O
U
< c -3. o
X) o o
O c
3
Figure imgf000148_0001
Table 5A Continued ΔCt values in lung and colon primary tumors and cell line models
Pπmar> PR0212 PRO290 PR0341 PROW PR06I9 PR0717 PRO809 PRO830 PR0848 Tumoi
1-1 16 I 8S 1 12 5 48 1 12 1 6 i π
10 I 3S
1 1 17 I 16 I 22 1 33 42 1 83 1 10 1 17
3 83 1 67 1 12
15 I 06 1 38
LTI8 1 32 1 14
-f-» 20 LT 1 3 S3 2 07 2 33 I 31 ^1 3 67 1 90 2 27
2 92
3 27
25 LT21 1 I S 1 15 1 OS 1 07 1 09
CT 2 4 44 1 56 1 22
3 20
30 4 66
3 93
4 49 4 43
Figure imgf000149_0001
Figure imgf000150_0001
<n >r> o o Table 5A Continued ΔCt values in lung and colon primary tumors and cell line models
Pimiaiv PR02I2 PRO290 PR0341 PR0535 PR0619 PR0717 PRO809 PRO830 PR0848
Tumoi
CT14 168 129
127
220
252
10 238 253 245 236
CTI 1 1 110 I 00 261
15 283 241
230
4-- 108 SΩ
Cl 16 102 135 102 221
20 253 217 210
CTI7 283 126 123 331
25 297 278
Cll I 60 I 12 193 162
Figure imgf000151_0001
Table 5A Continued ΔCt values in lung and colon primary tumors and cell line models
Piiinaiy PR02I2 PRO290 PR0341 PROS35 PR06I9 PR0717 PRO809 PRO830 PR0848
Tumoi
CT4 268 103 125 219
142
211 201 222 267 240
CIS 263 I 34 187 I 10
168
211
C1 I 66 100 I 06 I 12 I 19
C17 1 38
CT9 143
121
229 146
CT11 164 16 125 180 I 13 1 14 I 13
130
203
Figure imgf000152_0001
Table 5A Continued ΔCt values in lung and colon primary tumors and cell line models
Pπni u y PR0212 PRO290 PR0341 PR0535 PR06I9 PR0717 PRO809 PRO830 PR0848 .timoi
.549 166
C ilu 1 177
HI 57
11441 257
H460 250
SKMES1 215
SW900
SW480 217
SW620 I 04 236
C olυ 12 199
273
11.29 71
HM7
WIDI 121
HCTI16 202 190
SKCOl 313
SW40 209
LSI74T 124 290
Table 5A Continued
ΔCt values in lung and colon primary tumors and cell line models
Piimary J PR02I2 PRO290 PR0341 PR0535 PR06I9 PR07I7 PRO809 PRO830 PR0848 lumor
5
LT22
LT8 I 66
Colo205 1.48
10 1 ICT 15 1.46
11CC2998 1.20
KMI2 1.67
H522 3.78
11810 184 cπ ro
Figure imgf000154_0001
Table 5B ΔCt values in lung and colon primary tumors and cell line models
Pπniai) :JR()943 PRO 1005 PRO 1009 PRO 1025 PRO 1030 PRO 1097 PROl 107 PROllll PROl 153 lUlUOI
Figure imgf000155_0001
10 LI2 123 1 13 I 1 K) I 39 LI4 179 I 49 I 01
15 16 129 I 16 LI7 1 13 I 09 158 152 20 1.19 250 205 I 07 121 144
Ul co Ll 10 107 1 15 I 05 nn 706 145
25
I 112 194 121
LΓΠ 164 230 355
I 27
30 1115 : 05 I 03 247 Ll 16 105 245
LΓΠ I 93 147
35
Ll 18 LTI9 290 40 LT21 C 2 192 200 I 03 475 I 70 111
Table 5B Continued ΔCt values in lung and colon primary tumors and cell line models
Piimaiy PR0943 PRO 1005 PROI009 PROI025 PRO1030 PROI097 PROl 107 PROllll PROl 153
Tumoi
Figure imgf000156_0001
CT8 I 37 I 29 I 12 0 CTIO 213 I 73 128 1 19 282 I 67
C I 12 I 43 192 I 28 CT 14 I 46 210 158 I 92 108 54 138 5 CTI5 202 I 46 CT16 156 157 1 II 0 CTI7 130 I 76 164 134 CT! 136 I II 157 CT4 I 06 146 I 59 5
CT5 I 88 - I 43 — I 05
2 S|
C1 141
I 75 U C 17 ... 100 — - — 116
(19 ... i n
Cl 11 280 83 — — — — 1 17 5 261 ens 130 105
Λ549
Table 5B Continued
ΔCt values in lung and colon primary tumors and cell line models
Figure imgf000157_0002
Figure imgf000157_0003
Figure imgf000157_0001
C ilu 6 11157 H44I
10 11460
SKMES1 SW900
Figure imgf000157_0004
15 Colo320 11129 I1M7 WIDI HCTI16
20 SKCOl SW403 LS 174 [ L1 2
25 LT8
Table 5B Continued ΔCt values in lung and colon primary tumors and cell line models
Piimaiy PR0941 PRO1005 PROI009 PROI025 PRO1030 PRO1097 PROl 107 PROllll PROl 153
Tumoi
5
Colo2()5
I IC C 2998 KM 12
10
α.
Table 5C ΔCt values in lung and colon primary tumors and cell line models
Figure imgf000159_0001
L T- 1 a 1 14 - ... I 33 20 149 109
10 I T2 ... ... - ... I 09 I 13
1 .4 143 137 ... ... 101
15 I 18
LI I 78 I 66 I 05
20 LI7
_. L.9 I 43 126
LΠO 168
25 i rιι
LΓI2 247 I 17 257 I 27 261 229 I 26
30 I 80
LI 13 143 222 222 226 253
L 1S 155 241 324 1 16 378 304 352
35
Ll 16 101 I 33 164 306 384
107 270 355
Table 5C Continued ΔCt values in lung and colon primary tumors and cell line models
Piimiiv PROl 182 PROl 184 PROl 187 PRO 1281 PR023 PR039 PR0834 PROl 317 PRO17I0
-i lumoi
Figure imgf000160_0001
LI 17 174
LΓIS 107 i π
1TI9 I 19
135 I (P
L121 00 20
CT2 1 15 345 266 495
C I .
crs I 12 I 13 I 34
Cl 10 215 I 89 I 94
c ri2 I 07 156
C114 258 196 I 56
CT15 I 88
CTI6 134 I 19
C 117 135
Table 5C Continued ΔCt values in lung and colon primary tumors and cell line models
Pπ aiv PROl 182 PROl 184 PROl 187 PRO 1281 PR023 PR039 PR0834 PROI3I7 PRO 1710 Tumoi
Figure imgf000161_0001
C 1 I I 06 I 53
CT4 I 22 118 I 19 228
CT5 187 270 163 267
C 16 185
C17 156 I 02 61
C19 Cl II I 40 225 272 217
CTI8
Λ549
C ilu 1 49 C ilu 6 11157 11441 I 19
H460
SKMFSI
SW900
SW480
SW620
Table 5C Continued ΔCt values in lung and colon primary tumors and cell line models
Pnm uy PROl 1 2 PROl 184 PROl 187 PRO 1281 PR023 PR039 PR0834 PROI317 PROl 710 I umor
Colo320 190 102 HT29 176 243 HM7 WiDi 1ICT1I6 SKCOl 160 128 SW4 313 129 LSI74F 243 I 05 I T22 269 I 18
Colo205 HC1I5 311 HCC 2998 144 KM 12 118 H522 IISIO
Table 5D ΔCt values in lung and colon primary tumors and cell line models
Pnmaiy PRO2094 PR02I45 PR02I98
Tumoi
LI-I I 11 LMa I II I 32 LI2 I 13 I 23 LI4
LI
LT7 210 I 28 I 1 1 13 329
L IO Ll I 1
1112 - 322
1 11 240 292
I 12
LflS I 48 I 26
LT16 — - I 69
I 117
LΓI8
LTI
LT I
CT2 - — 227
Table 5D Continued ΔCt values in lung and colon primary tumors and cell line models
Pnmaiv PRO2094 PR02I45 PR02I98
Tu oi
Figure imgf000164_0001
C 3 C 18 I 20
CHO I 26
CTl 2
10 C II4 I 3 146
CTl 5 I 16 143
CFI6 — — I 31
258
15
CTl 7 1 12 — 233
I 50 r>
CTl I 70
20 C14 I 34
CIS
C16
CI7
CI9 I 3
25 CTl I
CTl 8
Table 5D Continued ΔCt values in lung and colon primary tumors and cell line models
Pnmaiy PRO2094 PR02145 PR02198 Tumoi
AS 19 241 149 I 31 I 75
Calu 1
10 C il 6
HI57
H44I
H460
SKMES1
_-.15 SW900 σs
SW480
SW620
Colo320 210
20 HT29
HM7
WIDI
IIC1116 I 03 I 32 I 60
25 I 89
SKCOl
SW403
Table 5D Continued ΔCt values in lung and colon primary tumors and cell line models
Pnmaiy PR 2094 PR02I45 PR02I98 l moi
LS 174T LT22 I 18 I 35
CoIo2()5 HC ΓIS
HC C2998 KM12 11522 I IS 10
PR0717
PR0717 (UNQ385) (DNA50988 1326) was also reexamined along with selected tumors from the above initial screen with framework mapping Figure 61 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 along Chromosome 4, and aie used to control aneuploidy
PR0717 (UNQ385) (DNA50988 1326) was also reexamined with epicenter mapping The epicentei markers indicated in Table 7 are located in close proximity (in the genome) to DNA50988, and are used to assess the relative amplification in the immediate vicinity of Chromosome 4 wherein the molecule is located The distance between 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 WI 13023 is the marker found to be the closest to the location on Chromosome 4 where DNA50988 1326 maps DNA52192 maps to the same marker (WI 13023) at which DNA50988 maps
The ΔCt values of the described framework markers (Table 6) along with Chromosome 4 relative to PR0717 are indicated for selected tumors in Table 8 Table 9 indicates the ΔCt values for results of epicenter mapping to DNA50988, indicating the relative amplification in the region more immediate to the actual location of DNA50988 along Chromosome 4
Table 6 Framework Markers Along Chromosome 4
Figure imgf000167_0001
Table 7 Epicenter Markers Along Chromosome 4 used for DNA50988
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000169_0003
Figure imgf000169_0002
Table 8 Continued Amplification of Framework Markers Relative to DNA50988 (ΔCt)
Figure imgf000170_0001
Table 9 c-r> CO Amplification of Epicenter Markers Relative to DNA50988 (ΔCt)
10
Figure imgf000170_0002
Table 9 Continued Amplification of Epicenter Markers Relative to DNA50988 (ΔCt)
Figure imgf000171_0001
PRO 1005
PROl 005 (UNQ489) (DNA57708) was also reexamined with epicenter mapping The epicenter markers indicated in Table 10 are located in close proximity (in the genome) to DNA57708, and are used to assess the relative amplification in the immediate vicinity of Chromosome 2 wherein the 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 breakage between two markers One cR is very I oughly equivalent to 20 kilobases The marker SHGC-30955 is the marker found to be the closest to the location on Chromosome 2 where DNA57708 maps (Figure 62)
Table 1 1 indicates the ΔCt values for results of epicenter mapping relative to DNA57708, indicating the relative amplification in the region more immediate to the actual location of DNA57708 along Chromosome 2
Table 10 Epicenter Markers Along Chromosome 2 Used for DNA57708
Figure imgf000173_0001
Table 1 1 Amplification of Epicenter Markers Relative to DNA57708 (ΔCt)
Figure imgf000173_0002
PRO2094 (DNA83123) PRO2094 (DNA83123) was also reexamined along with selected tumors from the above initial screen with framework mapping Figure 63 and Table 12 indicate the chromosomal mapping ot the framework markei s that were used in the present example The framework markers are located approximateh every 20 megabases along
Chromosome 6, and are used to control aneuploidy
PRO2094 (DNA83123) was also reexamined with epicenter mapping The epicenter markers indicated in Table 13 are located in close proximity (in the genome) to DNA83123, and aie used to assess the relative amplification in the immediate vicinity of Chromosome 6 wherein the molecule is located The distance between markers is measured in centirays (cR), which is a radiation breakage unit approximately equal to a 1 % of a breakage between two markers. One cR is very roughly equivalent to 20 kilobases. The marker SHGC- 1259 is the marker found to be the closest to the location on Chromosome 6 where DNA83123 maps. DNA77568 maps to the same marker (SHGC-1259) at which DNA83123 maps.
The ΔCt values of the described framework markers (Table 12) along Chromosome 6 relative to PRO2094 is indicated for selected tumors in Table 14.
Table 15 indicates the ΔCt values for results of epicenter mapping relative to DNA83123, indicating the relative amplification in the region more immediate to the actual location of DNA83123 along Chromosome 6.
Table 12 Framework Markers Along Chromosome 6
Figure imgf000174_0001
Table 13 Epicenter Markers Along Chromosome 6 Used for DNA83123
Figure imgf000174_0002
Table 14 Amplification of Framework Maikers Relative to DNA8 I23 (Δ Ct)
Figure imgf000175_0001
Table 14 Continued Amplification of Framework Markers Relative to DNA8 123 (Δ Ct)
Figure imgf000176_0001
Table 15 Amplification of Epicenter Markers Relative to DNA83123 (Δ Ct)
—1
10
Figure imgf000177_0001
PRQ2198:
PR02198(DNA88153) was also reexamined along with selected tumors from the above initial screen with framework mapping. Figure 64 and Table 16 indicate the chromosomal mapping of the framework markers that were used in the present example. The framework markers are located approximately every 20 megabases along Chromosome 16, and are used to control aneuploidy.
PR02198 (DNA88153) was also reexamined with epicenter mapping. The epicenter markers indicated in Table 17 are located in close proximity (in the genome) to DNA88153, and are used to assess the relative amplification in the immediate vicinity of Chromosome 16 wherein the molecule is located. The distance between 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 AFM156xb8 is the marker found to be the closest to the location on Chromosome 16 where DNA88153 maps. DNA62377 maps to the same marker (AFM156xb8) at which DNA88153 maps.
The ΔCt values of the described framework markers (Table 16) along Chromosome 16 relative to PR02198 is indicated for selected tumors in Table 18.
Table 19 indicates the ΔCt values for results of epicenter mapping relative to DNA88153, indicating the relative amplification in the region more immediate to the actual location of DNA88153 along Chromosome 16.
Table 16 Framework Markers Along Chromosome 16
Figure imgf000178_0001
Table 17 Epicenter Markers Along Chromosome 16 used for DNA88153
Figure imgf000178_0002
Table 18 Amplification of Framework Markers Relative to DNA88153 (Δ Ct)
Figure imgf000179_0001
Table 18 Continued Amplification of Framework Markers Relative to DNA88153 (Δ Ct)
Figure imgf000180_0001
Table 19 Amplification of Epicenter Markers Relative to DNA88153 (Δ Ct)
Figure imgf000180_0002
Table 19 Continued Amplification of Epicenter Markers Relative to DNA88153 (Δ Ct)
Figure imgf000181_0001
DISCUSSION AND CONCLUSION
PRQ212 (DNA30942-1 134)
The ΔCt values for DNA30942-1134 in a variety of tumors are reported in Table 5 A A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5 A indicates that significant amplification of nucleic acid DNA30942-1 134 encoding PR0212 occurred (1) in primary lung tumors LTla, LT3, LT7, LT8, LT10, LT11 , LT12, LT13, LT15, LT16, LT17 and LT19, (2) in primary colon tumors CT2, CT3, CT8, CTl 0, CTl 2, CT14, CTl 5, CT) 6, CTl 7, CTl , CT4, CT5, CT6, CT7, CT9 and CTl 1 , and (3) in colon tumor cell lines SW480, SW620, WiDr, HCT1 16 and LS 174T Because amplification of DNA30942-1 134 occurs in various tumors and tumor cell lines, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PR0212 would be expected to be useful in cancer therapy
PRO290 (DNA35680-1212)
The ΔCt values for DNA35680-1212 in a variety of tumors are reported in Table 5A A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene cop) Table 5 A indicates that significant amplification of nucleic acid DNA35680-1212 encoding PRO290 occurred ( 1 ) in primary lung tumors LT1 1 , LT13, LT15, LT17, and LT19, and (2) in pπmaiy colon tumors CT2 and CTl 1 Because amplification of DNA35680- 1212 occurs in various tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e , antibodies) directed against PRO290 would be expected to be useful in cancer therapy
PRQ341 (DNA26288-1239)
The ΔCt values for DNA26288- 1239 in a variety of tumors are reported in Table 5A A ΔCt ot > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5 A indicates that significant amplification of nucleic acid DNA26288- 1239 encoding PR0341 occurred in primary lung tumors LTl 6, LTl 7, and LT21 Because amplification of DNA26288-1239 occurs in various lung tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PR0341 would be expected to be useful in cancer therapy
PRQ535 (DNA49143- 1429)
The ΔCt values for DNA49143- 1429 in a variety of tumors are reported in Table 5 A A ΔCt of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5A indicates that significant amplification of nucleic acid DNA49143- 1429 encoding PR0535 occurred (1 ) in primary lung tumors LTl 1 , LTl 3 and LTl 7, and (2) in primary colon tumors CT3, CT10, CT14, CTl 5, CTl 6, CT17, CT4, CT6 and CTl 1 Because amplification of DNA49143-1429 occurs in various tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PR0535 would be expected to be useful in cancer therapy
PRQ619 (DNA49821 -1562)
The ΔCt values for DNA49821 -1562 in a variety of tumors are reported in Table 5 A A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5A indicates that significant amplification of nucleic acid DNA49821 -1562 encoding PR0619 occurred (1 ) in primary lung tumors LT3, LT7, LT9, LTl 0, LTl 1 , LTl 2, LTl 3, LTl 5, LTl 7, LTl 8, LTl 9 and LT21 , and (2) in primary colon tumors CT2, CT3, CTl 5, CTl 6, CT 17, CTl , CT4, CT5, CT6 and CTl 1 Because amplification of DNA49821 -1562 occurs in various tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PR0619 would be expected to be useful in cancer therapy
PRQ717 (DNA50988-1326)
The ΔCt values for DNA50988-1326 in a variety of tumors are reported in Table 5 A A ΔCt of >1 was typically used as the threshold value for amplification sconng, as this represents a doubling of gene copy Table 5A indicates that significant amplification of nucleic acid DNA50988-1326 encoding PR0717 occurred (1 ) in primary lung tumors LTl 3, LTl 5 and LTl 6, (2) in primary colon tumors CT15, CTI 6 and CT17, (3) ιn lung tumor cell lines A549 Calu-1 H441 , H460 SKMES 1 , H522 and H810, and (4) in colon tumor cell lines SW620, Colo320, HT29, HCT1 16 SKCOl . SW403, LS174T Colo205, HCT15, HCC2998 and KM12
Amplification has been confirmed by framework mapping for DNA50988- 1326 ( 1 ) in primary colon tumors CT15, CTl 6 ,CT17, CTl , CT2, CT3, CT4, CT8.CT9, CTl 0, CTl 2 and CTl 4, (2) in primary lung tumors LT13, LT15 and LT16. (3) in colon tumor cell lines SW480 and HCT1 16, and (4) in lung tumor cell line A549 Epicenter mapping for DNA50988 resulted in significant amplification ( 1 ) in primary colon tumors CTl 5, CTl 6, CT17, CTl , CT2 CT3, CT4, CT8, CT9 CT10, CT12 and CT14, (2) in pπmaiy lung tumors LT13. LT15 and LTl 6, (3) in colon tumor cell lines SW480 and HCT1 16, and (4) in lung tumor cell line A549 In contrast, the amplification of the closest known framework markers (with the exception of D233 and D397) (Table 8) or epicentei markers (with the exception of D6) (Table 9) does not occui to a greatei extent than that of DNA50988 This strongly suggests that DNA50988 is the gene responsible for the amplification of the particular region on Chromosome 4 Because amplification of DNA50988 occurs in various lung and colon tumors and cell lines (especially colon), 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 DNA50988 (PR0717) would be expected to have utility in cancer therapy
PRO809 (DNA57836-1338)
The ΔCt values for DNA57836-1338 in a variety of tumors are reported in Table 5A A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5A indicates that significant amplification of nucleic acid DNA57836- 1338 encoding PRO809 occurred in primary lung tumors LTl 1 , LTl 2, LTl 3, LTl 7 and LT21 Because amplification of DNA57836 1338 occurs in various lung tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PRO809 would be expected to be useful in cancer therapy
PRO830 (DNA56866 1342)
The ΔCt values for DNA56866 1342 in a variety of tumors are reported in Table 5 A A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5 A indicates that significant amplification of nucleic acid DNA56866 1342 encoding PRO830 occurred in primary lung tumors LTl a, LTl 1 , LTl 2, LTl 7 and LTl 9 Because amplification of DNA56866 1342 occurs in various lung tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PRO830 would be expected to be useful in cancer therapy
PRQ848 (DNA59839-1461 )
The ΔCt values for DNA59839-1461 in a variety of tumors are reported in Table 5A A ΔCt of >1 was typically used as the threshold value for amplification scoring as this represents a doubling of gene cop\ Table 5A indicates that significant amplification of nucleic acid DNA59839 1461 encoding PR0848 occurred in pπman, lung tumors LTl 1 , LTl 2 and LT21 Because amplification of DNA59839- 1461 occurs in various lung tumoi s it is likely associated with tumor formation and/or growth As a result antagonists (e e antibodies) directed against PR0848 would be expected to be useful in cancer therapy
PRQ943 (DNA52192-1369)
The ΔCt values for DNA52192 1 69 in a variety of tumors are reported in Table 5B A ΔCt of >1 was typically used as the threshold value foi amplification scoring as this represents a doubling of gene
Figure imgf000183_0001
Table 5B indicates that significant amplification of nucleic acid DNA52192-1 69 encoding PR0943 occurred ( 1 ) in primary lung tumors LTl 1 , LTl 2, LTl 3 LTl 5, LTl 7 and LTl 9 and (2) in pπmaiy colon tumors CT2, CT8 CT10. CT12, CT14 CT17, CT1 CT5, CT6, CTl 1 and CT18 Because amplification of DNA52192 1369 occuι s in various tumors, it is likely associated with tumor formation and/or growth As a result antagonists (e g antibodies) directed against PR0943 would be expected to be useful in cancer therapy PRO 1005 (DNA57708- 141 1 )
The ΔCt values for DNA57708-141 1 in a variety of tumors are reported in Table 5B A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5B indicates that significant amplification of nucleic acid DNA57708- 141 1 encoding PRO 1005 occurred in primary lung tumors LTl , LTl a, LT3, LT6, LT9, LT12, LT13, LT15, and LT16
Epicenter mapping for DNA57708 resulted in confirmation of significant amplification in lung tumor cell lines HF545, HF539, HF733 and HF716 (Table 1 1 ) In contrast, the amplification of the closest known epicenter markers does not occur to a greater extent than that of DNA57708 This strongly suggests that DNA57708 is the gene responsible for the amplification of the particular region on Chromosome 2 Because amplification of DNA57708 occurs in various lung tumors and cell lines, it is highly probable to play a role in tumor formation or growth As a result, antagonists (e g , antibodies) directed against the protein encoded by DNA57708 (PROl 005) would be expected to have utility in cancer therapy
PRO1009 (DNA57129-1413)
The ΔCt values for DNA57129-1413 in a variety of tumors are reported in Table 5B A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene cop> Table 5B indicates that significant amplification of nucleic acid DN A57129- 1413 encoding PRO 1009 occurred in primary colon tumors CT2, CT3, CT8, CT10, CT12, CT14, CT15, CT16, CT17, CT4, CT5 and CT1 1 Because amplification of DNA57129-1413 occurs in various colon tumors, it is likely associated with tumor formation and or growth As a result, antagonists (e g , antibodies) directed against PRO 1009 would be expected to be useful in cancer therapy
PRO 1025 (DNA59622-1334)
The ΔCt values for DNA59622-1334 in a variety of tumors are reported in Table 5B A ΔCt ot >1 was typically used as the threshold value for amplification scoring as this represents a doubling of gene copy Table 5B indicates that significant amplification of nucleic acid DNA59622-1334 encoding PRO 1025 occurred (1 ) in primary lung tumors LT3, LT6, LT7, LT9, and LT10, and (2) in primary colon tumors CT2, CT3, CT10, CT14, CTl , CT4, CT7 and CT9 Because amplification of DNA59622- 1334 occurs in various tumoi s. it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PROl 025 would be expected to be useful in cancer therapy
PRO 1030 (DNA59485-1336) The ΔCt values for DNA59485- 1336 in a variety of tumors are reported in Table 5B A ΔCt ot > 1 was typically used as the thieshold value for amplification scoring, as this represents a doubling ot gene cop\ Table 5B indicates that significant amplification ot nucleic acid DNA59485-1336 encoding PRO 1030 occurred (1 ) in primary lung tumors LT4, LT7 LT9 and LT10, and (2) in primary colon tumors CT2, CT3 CT10. CT12, CT14, CTl 5. CTl 6, CTl 7 and CT5 Because amplification of DNA59485-1336 occurs in various tumors it is likely associated with tumor foi mation and/or growth As a result, antagonists (e g . antibodies) directed against PRO 1030 would be expected to be useful in cancer therapy
PRO 1097 (DNA59841 -1460)
The ΔCt values for DNA59841 -1460 in a variety of tumors are reported in Table 5B A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5B indicates that significant amplification of nucleic acid DNA59841 -1460 encoding PRO1097 occurred (1 ) in primary lung tumors LT2 and LT9, and (2) in primary colon tumors CT14, CT16 and CT17 Because amplification of DNA59841 -1460 occurs in various tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PRO 1097 would be expected to be useful in cancer therapy
PRO 1 107 (DNA59606- 1471 )
The ΔCt values for DNA59606-1471 in a variety of tumors are reported in Table 5B A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5B indicates that significant amplification of nucleic acid DNA59606-1471 encoding PROl 107 occurred (1 ) in primary lung tumors LT13, LT15, and LT16, and (2) m primary colon tumors CT2, CT3, CT10, CT14, CTl and CT4 Because amplification of DNA59606-1471 occurs in various tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PROl 107 would be expected to be useful in cancer therapy
PROl l l l (DNA58721 -1475)
The ΔCt values for DNA58721 -1475 in a variety of tumors are reported in Table 5B A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5B indicates that significant amplification of nucleic acid DNA58721 -1475 encoding PROl 1 1 1 occurred (1 ) in primary lung tumors LT3, LT4, LT7, LT9, LT10, LTl 1 and LTl 7, and (2) in primary colon tumors CTl 4, CT7 CTl 1 and CTl 8 Because amplification of DNA58721 -1475 occurs in various tumoi , it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PROl 1 1 1 would be expected to be useful in cancer therapy
PROl 153 (DNA59842-1502)
The ΔCt values for DNA59842-1502 in a variety of tumors are teported Table 5B A ΔCt of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling ot gene copy Table 5B indicates that significant amplification ot nucleic acid DNA59842- 1502 encoding PRO 1 153 occurred in primary lung tumors LT4 and LT7 Because amplification of DNA59842- 1502 occurs in various lung tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g . antibodies) directed against PROl 153 would be expected to be useful in cancer therapy PRO 1 182 (DNA59848- 1512)
The ΔCt values for DNA59848-1512 in a variety of tumors are reported in Table 5C A ΔCt of >1 was typically used as the threshold value tor amplification scoring, as this represents a doubling of gene copy Table 5C indicates that significant amplification of nucleic acid DNA59848- 1512 encoding PRO 1 182 occurred in primary lung tumors LTl , LT4 and LT9 Because amplification of DNA59848-1512 occurs in various lung tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PROl 182 would be expected to be useful in cancer therapy
PRO 1184 (DN A59220- 1514)
The ΔCt values for DNA59220-1514 in a variety of tumors are reported in Table 5C A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5C indicates that significant amplification of nucleic acid DNA encoding PROl 184 occurred in primary lung tumors LTla, LT4, LT6, LTl 2, LTl 6, LTl 8, LT19 and LT21 Because amplification of DNA59220-1514 occurs in various lung tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PROl 184 would be expected to be useful in cancer therapy
PRO 1187 (DNA62876- 1517)
The ΔCt values for DNA62876- 1517 in a variety of tumors are reported in Table 5C A ΔCt of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5C indicates that significant amplification of nucleic acid DNA62876- 1517 encoding PRO 1 187 occurred in primary lung tumors LT12, LT15 and LT16 Because amplification of DNA62876-1517 occurs in various lung tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PROl 187 would be expected to be useful in cancer therapy
PRQ1281 .DNA59820-1549)
The ΔCt values for DNA59820-1549 in a variety of tumoi s are reported in Table 5C A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5C indicates that significant amplification of nucleic acid DNA59820 1 49 encoding PRO 1281 occurred in primary colon tumors CT2 and CTl 2 Because amplification of DNA59820-1549 occurs in various colon tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g . antibodies) directed against PRO 1281 would be expected to be useful in cancer therapy
PRQ23 (DNA36640) The ΔCt values for DNA36640 in a variety of tumors are reported in Table 5C A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5C indicates that significant amplification of nucleic acid DNA36640 encoding PR023 occurred (1 ) in primary lung tumors LTl 3, LT15 and LTl 6, (2) in primary colon tumors CTl 7, CTl , CT4, CT5, CT7 and CTl 1 , (3) in lung tumor cell line H441 , and (4) in colon tumor cell lines Colo320, HT29, SKCO l , SW403, LS I 74T, HCT15, HCC2998 and KM 12 Because amplification of DNA36640 occurs in various tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PR023 would be expected to be useful in cancer therapy
PRQ39 (DNA36651 ) The ΔCt values for DNA36651 in a variety of tumors are reported in Table 5C A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5C indicates that significant amplification of nucleic acid DNA36651 encoding PR039 occurred (1 ) in primary lung tumors LTla, LT2, LT4, LT12, LT13, LT15 and LT16, (2) in primary colon tumors CT2, CT8, CT10, CT14, CT4, CT5, CT7 and CT11 , and (3) in colon tumor cell lines Colo320, HT29, SKCOl, SW403 and LS174T Because amplification of DNA36651 occurs in various tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PR039 would be expected to be useful in cancer therapy
PRQ834 (DNA56538)
The ΔCt values for DNA56538 in a variety of tumors are reported in Table 5C A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5C indicates that significant amplification of nucleic acid DNA56538 encoding PR0834 occurred (1) in primary lung tumor LTla, and (2) in primary colon tumors CT2, CT8, CT10, CT12, CT14, CT15, CT16, CTl , CT4, CT5, CT6 and CTl 1 Because amplification of DNA56538 occurs in various tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PR0834 would be expected to be useful in cancer therapy
PRQ1317 (DNA71 166)
The ΔCt values for DNA71 166 in a variety of tumors are reported in Table 5C A ΔCt of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5C indicates that significant amplification of nucleic acid DNA71 166 encoding PRO 1317 occurred in primary lung tumors LT 1 , LTl a, LT9, LT10 LTl 5, LTl 7 and LT22 Because amplification of DNA71 166 occurs in various lung tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PR01317 would be expected to be useful in cancer therapy
PRO1710 .DNA82331 )
The ΔCt values for DNA82331 in a variety of tumors are reported in Table 5C A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5C indicates that significant amplification ot nucleic acid DNA82331 encoding PRO 1710 occurred ( 1 ) in primary lung tumors LT12, LT13, LT15 and LT16 (2) in primary colon tumors CT2, CT8, CT10, CTI 4 CT16 CT4, CT5 CT7 and CT1 1 , (3) in lung tumor cell line Calu 1 , and (4) in colon tumoi cell line SW620 Because amplification of DNA82331 occurs in various tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g antibodies) directed against PROl 710 would be expected to be useful in cancer therapy PRO2094 . DNA83123)
The ΔCt values tor DNA83123 in a variety of tumors are reported in Table 5D A ΔCt of > 1 was typically used as the thi eshold value for amplification scoring, as this represents a doubling of gene copy Table 5D indicates that significant amplification of nucleic acid DNA83123 encoding PRO2094 occurred (1 ) in primary lung tumors LT7, LT9 and LT15. and (2) in primary colon tumors CT8, CT14, CT15, CT17, CTl , CT4 and CT9
Amplification has been confirmed by framework mapping for DNA83123 Table 14 indicates that significant amplification for DNA83123 was confirmed (1 ) in colon tumors CTl 5, CTl 6 and CTl 7, (2) in lung tumors LTl 3 and LTl 5 , (3) in colon tumor cell lines SW480, Colo320, HCT75, HCC2998, KM 12, SKCOl and SW620, and (4) in lung tumor cell lines Calu-6, HI 57 and H441 Epicenter mapping for DNA83123 (Table 15) resulted in significant amplification in kidney cell line 613, colon cell line 575 and testes cell line 733 In contrast, the amplification of the closest known framework markers (with the exception of F348) (Table 14) and epicenter markers (Table 15) does not occur to a greater extent than that of DNA83123 This strongly suggests that DNA83123 is the gene responsible for the amplification of the particular region in Chromosome 6 Because amplification of DNA83123 occurs in various tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PRO2094 would be expected to be useful in cancer therapy
PRQ2145 .DNA88050)
The ΔCt values for DNA88050 in a variety of tumors are reported in Table 5D A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5D indicates that significant amplification of nucleic acid DNA88050 encoding PR02145 occurred ( 1 ) in primary lung tumors LTl , LTl a, LT3, LT7, LT9, LTl 2, LTl 3, LTl 5 and LT8, (2) in lung tumor cell line A549, and (3) in colon tumor cell line HCT116 Because amplification of DNA88050 occurs in various tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PR02145 would be expected to be useful in cancer therapy
PRQ2198 (DNA88153)
The ΔCt values for DNA88153 in a variety of tumors are reported in Table 5D A ΔCt of > 1 was typically used as the threshold value for amplification scoring as this represents a doubling of gene copy Table 5D indicates that significant amplification of nucleic acid DNA88153 encoding PR02198 occurred ( 1 ) in primary lung tumors LTla, LT13 and LTl 6, (2) in pπmaiy colon tumors CT2, CT10, CT14, CTl 5, CT16 and CTl 7, (3) in lung tumor cell line A549, and (4) in colon tumor cell lines Colo320 and HCT1 16
Amplification has been confirmed by framework mapping tor DNA88153 (Table 18) ( 1 ) in primary colon tumor CT15, (2) in primary lung tumors LTl 3 and LTl 6, (3) in colon tumor cell lines HCC2998, KM 12, SKCO-1 , SW480 and HCT1 16, and (4) in lung tumor cell line A549 Epicenter mapping for DNA88153 (Table 19) resulted in significant amplification (1 ) in primary colon tumors CT15, CT16, CT17, CTl , CT2, CT3, CT4. CT8, CT9, CT10, CTl 2 and CTl 4, (2) in primary lung tumors LTl 3, LTl 5 and LTl 6, (3) in colon tumor cell lines SW480 and HCT1 16, and (4) in lung tumor cell line A549 In contrast the amplification ot the closest known framework markers (with the exception of P7 and PI 54) (Table 18) or epicenter markers (Table 19) does not occur to a greater extent than that of DNA This strongly suggests that DNA88153 is the gene responsible for the amplification of the particular region on Chromosome 16 Because amplification of DNA88153 occurs in various tumors, it is likely associated with tumor formation and/or growth As a result, antagonists (e g , antibodies) directed against PR02198 would be expected to be useful in cancer therapy
EXAMPLE 24 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-labeledπboprobes 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 33P-Riboprobe synthesis
6 0 μ] ( 125 mCi) of 3 P-UTP (Amersham BF 1002, S A<2000 Ci/mmol) were speed vac dried To each tube containing dried 33P-UTP, the following ingredients were added 2 0 μl 5x transcription buffer
1 O l DTT (l OO mM)
2 0 μl NTP mix (2 5 mM 10 μl, each of 10 mM GTP, CTP & ATP + 10 μl H,0) 1 0 μl UTP (50 μM) 1 0 μl Rnasin
1 0 μl DNA template (Iμg) 1 0 μl H20
1 0 μl RNA polymerase (for PCR products T3 = AS, T7 = S, usually)
The tubes were incubated at 37"C for one hour 1 0 μl RQ1 DNase were added, followed by incubation at 37"C for 15 minutes 90 μl TE (10 mM Tris pH 7 6/l mM EDTA pH 8 0) weie added, and the mixture was pipetted onto DE81 paper The remaining solution was loaded in a Mιcrocon-50 ultratiltration 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, 100 μl TE were added 1 μl of the final product v\ as pipetted on DE81 paper and counted in 6 ml of Biofluor II The probe was run on a TBE/urea gel 1 -3 μl of the probe or 5 μl of RNA Mrk III weie added to 3 μl of loading buffer After heating on a 37"C heat block for three minutes, the gel was immediately placed on ice The wells of gel were flushed, the sample loaded, and run at 180-250 volts for 45 minutes The gel was wrapped in saran wrap and exposed to XAR film with an intensifying screen in -70°C freezer one hour to overnight. 33P-Hybridization
Pretreatment of frozen sections: The slides were removed from the freezer, placed on aluminium 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%, 100% ethanol, 2 minutes each. 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 ) - human embryo, or 8 x proteinase K (100 μl in 250 ml Rnase buffer, 37°C, 30 minutes) - formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above. Prehvbridization: The slides were laid out in plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper. The tissue was covered with 50 μl of hybridization buffer (3.75g 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, the tissue was vortexed well, and incubated at 42"C for 1-4 hours. 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 were added per slide. After vortexing, 50 μl 33P mix were added to 50 μl prehybridization on slide. The slides were incubated overnight at 55°C.
Washes: Washing was done 2x 10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0.25M EDTA, Vt=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 2x10 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, Vt=4L). Results: DNA30942-1 134 (TNF-R Homolog) Expression in Human Tissues:
Expression in fetal and adult tissues was observed to give negative results. Fetal tissues examined included: placenta, umbilical cord, brain, spinal cord, eye, optic nerve, trachea, lung, heart, liver, spleen, esophagus, small intestine, pancreas, adrenals, thyroid, body wall and lower limb. Adult tissues examined included: liver. kidney, adrenals, myocardium, aorta, spleen, lymph node, pancreas, lung and skin. Expression in Lung Carcinomas, Tumor Block and Breast Carcinomas:
Expression was observed in mononuclear phagocytes in the normal chimp thymus, as well as in gastric carcinoma, colorectal cancer , breast cancer and lung cancer. Expression was observed in malignant cells in an osteosarcoma and a poorly differentiated liposarcoma. A possible signal may be found in the malignant cells of a testicular teratoma and breast cancers In one of the lung cancers, a scattered signal was seen over a high endothehal venule within pulmonary lymphoid tissue
Fetal tissues examined (El 2-E16 weeks) included placenta, umbilical cord, liver, kidney, adrenals, thyroid, lung, heart, great vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb Adult human tissues examined included liver, kidney, adrenals, myocardium, aorta, spleen, lung, skin, chondrosarcoma, eye, stomach, gastric carcinoma, colon, colonic carcinoma, renal cell carcinoma, prostate, bladder mucosa and gall bladder Acetominophen induced liver injury and hepatic cirrhosis Rhesus tissues examined included cerebral cortex (rm) and hippocampus (rm) Chimp tissues examined included thyπd, parathyroid, ovary, nerve, tongue, thymus, adrenals, gastric mucosa and salivary gland Expression in Lung Adenocarcmoma and Squamous Carcinoma
Eight adenocarcinomas and seven squamous lung carcinomas were examined Actins were strongly positive in all tumors, indicating that all are suitable for in situ hybridization analysis Expression of DNA30942 was observed in 6 of the tumors as follows
6727-95 / squamous carcinoma - strongly expressed over neoplastic epithelium 9558-95 / squamous carcinoma - expression over neoplastic epithelium
12235-95 / adenocarcmoma - expression over in situ and infiltrating tumor cells
6545-95 & 4187-96 / squamous carcinomas expression over cells in tumor stroma, no expression seen over tumor cells 12954-94 / squamous carcinoma - possible weak expression over stromal cells
C-l l lV DNA30942-pl
5'-GGATTCTAATACGACTCACTATAGGGCTCGCTGCTGTGCCTGGTGTTG-3' (SEQ ID NO 182)
C-l l lW DNA30942-p2 5'-CTATGAAATTAACCCTCACTAAAGGGACCGCTGCAGCCTCTTGATGGA-3' (SEQ ID NO 183)
DNA52192-1369 (FGF receptor-like molecule) Expression was observed in fetal skeletal muscle and long bone cartilage Elsewhere, a relatively high background signal is a problem In one fetal liver, expression appears to occur at sites of hematopoiesis The other fetal liver was negative Fetal tissues examined (El 2-E16 weeks) included placenta, umbilical cord, liver, kidney, adrenals, thyroid, lung, heart, great vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas brain, eye, spinal cord, body wall, pelvis and lower limb Adult human tissues examined included liver, kidney, adrenals, myocardium, aorta, spleen, lung, sk , chondrosarcoma, e \ e stomach, gastric carcinoma, colon, colonic carcinoma, renal cell carcinoma, prostate, bladder mucosa and gall bladdei Acetominophen induced liver injury and hepatic cirrhosis Rhesus tissues examined included cerebral cortex (rm) and hippocampus (rm) Chimp tissues examined included thyroid, parathyroid, ovary, ner\e, tongue, thymus adrenals, gastric mucosa and salivary gland D-269E FGFr p l 5'-GGATTCTAATACGACTCACTATAGGGCCGCTGACCATGTGGACCAAGG-3' (SEQ ID NO 184)
C-257I FGFr p2
5'-CTATGAAATTAACCCTCACTAAAGGGATCTGGCAGCACGGTGAGGAAG-3' (SEQ ID NO 185)
EXAMPLE 25
Use of PRQ212. PRO290. PRQ341. PRQ535. PRQ619. PRQ717. PRO809. PRO830. PRQ848. PRQ943. PRO 1005. PRO1009. PRO1025. PRO1030. PRO1097. PROl 107. PROl 1 1 1. PROl 153. PROl 182. PROl 184. PROl 187. PRO 1281 , PRQ23. PRQ39, PRQ834, PROl 317. PROl 710, PRO2094. PRQ2145 or PRQ2198 as a hybridization probe The following method describes use of a nucleotide sequence encoding a PR0212, PRO290, PR0341.
PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097, PRO 1 107, PROl 1 1 1 , PROl 153, PROl 182, PRO! 184, PROl 187, PRO 1281 , PR023, PR039, PR0834. PROl 317, PROl 710, PRO2094, PR02145 or PR02198 polypeptide as a hybridization probe
DNA comprising the coding sequence of a full-length or mature "PR0212. PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PRO 1097. PROl 107. PRO1 1 1 1 , PROl 153, PROl 182, PR01 184, PROl 187,PR01281 , PR023, PR039,PR0834, PR01317, PROl 710, PRO2094, PR02145 or PR02198" 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943. PRO1005, PRO1009, PRO 1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PRO l 153, PROl 182, PROl 184, PROl 187. PRO 1281 , PR023, PR039, PR0834. PROl 317, PRO 1710, PRO2094, PR02145 or PR02198) in human tissue cDNA libraries or human tissue genomic libraries
Hybridization and washing ot filters containing either library DNAs is performed under the following high stringency conditions Hybridization of radiolabeled PR0212-, PRO290-, PR0341 -, PR0535-, PR0619-.PR0717- PRO809-, PRO830-. PR0848-, PR0943-, PR01005-, PRO 1009-, PRO 1025-, PRO 1030-. PRO 1097-, PRO 1 107-. PRO 1 1 1 1 -, PRO 1 153-, PRO 1 182-, PRO 1 184-, PR01 187-, PRO 1281 -, PR023-, PR039-. PR0834-. PRO 1317-, PRO1710-. PRO2094-. PR02145- or PR02198-deπved probe to the filters is performed in a solution ot 50% formamide, 5x SSC, 0 1 % SDS, 0 1 % sodium pyrophosphate, 50 mM sodium phosphate, pH 6 8, 2x Denhardt solution, and 10% dextran sulfate at 42"C toi 20 hours Washing of the filteis is pei formed in an aqueous solution of O I x SSC and O l % SDS at 42°C
DNAs having a desired sequence identity with the DNA encoding full-length native sequence PR0212 PRO290. PR0341 , PR0535, PR061 . PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO1025, PRO 1030. PR01097, PR01 107. PROl l l l , PROl 153. PROl 182. PRO l 184. PRO l 187, PR01281 PR023, PR039, PR0834, PROl 317. PRO l 710. PRO2094, PR02145 or PR02198 can then be identified using standard techniques known in the art EXAMPLE 26
Expression of PRQ212, PRO290. PRQ341 , PRQ535. PRQ619. PR0717, PRO809. PRO830. PRQ848.
PRQ943, PRO1005 PRO1009. PRO1025. PRO1030. PRO1097. PROl 107. PROl 1 1 1. PROl 153. PROl 182
PROl 184 PROl 187. PRQ1281. PRQ23. PRQ39. PRQ834. PRQ1317, PRO1710. PRO2094. PRQ2145 oi PRQ2198 Polypeptides in E coh
This example illustrates preparation of an unglycosylated form of PR0212, PRO290, PR0341 , PR0535
PRO619, PRO717, PRO809, PRO830, PRO848, PRO943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097
PROl 107, PROl 1 1 1 , PROl 153. PR01 182 PROl 184. PR01 187, PR01281 , PR023, PR039, PR0834, PR01317,
PRO1710, PRO2094, PR02145 or PR02198 by recombinant expression in E co 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 ampicilhn and tetracychne 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 PR0212, PRO290, PR0341 , PR0535. PROό 19, PR0717, PRO809, PRO830
PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153,
PROl 182, PRO 1184, PRO 1 187, PR01281, PR023, PR039, PR0834, PR01317, PROl 710, PRO2094, PRO2145 or PR02198 coding region, lambda transcπptional terminator, and an argU gene
The hgation mixture is then used to transform a selected E coh strain using the methods described in Sambrook et al , supia 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 lestπction 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 PR0212 PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005 PRO 1009 PRO1025, PRO 1030, PRO 1097, PRO l 107, PROl 1 1 1 , PRO l 153, PRO l 182, PRO l 184, PROl 187, PR01281 PR023, PR039 PR0834. PRO l 317, PROl 710, PRO2094 PR02145 or PR02198 protein can then be purified using a metal chelating column under conditions that allow tight binding of the piotein
PR0619, PROl 184 and PR0834 were successfully expressed ιn £ coh in a poly-His tagged form using the following piocedure The DNA encoding PR0619 PRO l 184 and PR0834 was initially amplified using selected PCR primers The primers contained restnction enzyme sites which coπespond to the restriction enzyme sites on the selected expression vectoi . and other useful sequences providing foi 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 coh host based on sti a 52 (W31 10 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(ladq) Transformants were first grown in LB containing 50 mg/ml carbenicilhn 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 cιtrate«2H20, 1 07 g KCl, 5 36 g Difco yeast extract, 5 36g Sheffield hycase SF in 500 ml water, as well as HO mM MPOS, pH 7 3, 0 55% (w/v) glucose and 7 mM MgS04) and grown foi approximately 20-30 hours at 30°C 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 1M 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 sulfitohzation 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 4°C 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 acetomtrile was added to 2-10% final concentration The refolded protein was chromatographed on a Poros Rl/H reversed phase column using a mobile butter of 0 1 % TFA with elution with a gradient of acetomtrile from 10 to 80% Ahquots of tractions with A2M) absorbance were analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded piotein were pooled Generally, the properly refolded species of most proteins are eluted at the lowest concenti ations of acetomtrile since those species are the most compact with their hydrophobic intei iors shielded from interaction with the reversed phase resin Aggregated species are usually eluted at higher acetomtrile concentrations In addition to I esol ving mistolded forms of proteins from the desired form, the leversed phase step also removes endotoxin from the samples
Fractions containing the desired folded PR0619. PROl 184 and PR0834 piotein weie pooled and the acetomtrile removed using a gentle stream ot 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 oi by gel filtration using G25 Superfine (Pharmacia) lesins equilibrated in the formulation butter and sterile filtered
- 19: EXAMPLE 27
Expression of PR0212, PRO290. PRQ341 , PRQ535, PRQ619. PRQ717, PRO809. PRO830, PRQ848,
PRQ943. PRO 1005. PRO1009. PRO1025. PRO1030. PRO1097, PRO l 107. PROl 1 1 1. PROl 153. PROl 182.
PR01 184. PRO 1 187. PRO 1281. PRQ23. PRQ39. PRQ834. PR01317. PRO 1710. PRO2094. PRQ2145 or PRQ2198 in mammalian cells
This example illustrates preparation of a potentially glycosylated form of PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PROl 005, PRO1009, PRO1025, PRO1030, PRO 1097, PRO 1 107, PROl 1 1 1. PROl 153, PROH 82, PROl 184, PROl 187, PRO 1281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 by recombinant expression in mammalian cells The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector
Optionally, the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281 , PR023. PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943 PRO1005, PRO1009. PRO1025, PRO1030, PRO1097, PRO1 107, PRO1 1 1 1 , PROl 153, PROl 182, PR01 184, PR01 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 DNA using hgation methods such as described in Sambrook et al , supra The resulting vector is called pRK5-PR0212, pRK5-PRO290, pRK5-PR0341 , pRK5- PR0535, pRK5-PR0619, pRK5-PR0717, pRK5-PRO809, pRK5-PRO830, pRK5-PR0848, pRK5-PR0943, pRK5-PR01005, pRK5-PRO 1009, pRK5-PRO 1025, pRK5-PRO 1030, pRK5-PRO 1097, pRK5-PRO 1 107, pRK5- PRO 11 1 1, pRK5-PRO 1 153, pRK5-PRO 1 182, pRK5-PR01 184, pRK5-PRO 1 187, pRK5-PR01281 , pRK5-PR023, pRK5-PR039, pRK5-PR0834, pRK5-PR01317, pRK5-PRO1710, pRK5-PRO2094, pRK5-PR02145 or pRK5- PR02198
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-PR0212. pRK5-PRO290, pRK5-PR0341 pRK5-PR0535, pRK5-PR0619, pRK5-PR0717, pRK5-PRO809, pRK5-PRO830, pRK5-PR0848, pRK5-PR0943, pRK5-PR01005, pRK5-PR01009, pRK5-PRO 1025, pRK5-PR01030 pRK5-PRO 1097, pRK5-PRO 1 107, pRK5- PROl 1 1 1, pRK5-PR01 153, pRK5-PRO 1 182. pRK5-PRO 1 184, pRK5-PRO 1 187, pRK5-PRO 1281 , pRK5-PR023 pRK5-PR039, pRK5-PR0834, pRK5-PR01317, pRK5-PRO1710, pRK5-PRO2094, pRK5-PR02145 oi pRK5- PR02198 DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al Cell. 3J_ 543 ( 1982)] and dissolved in 500 μl of 1 mM Tns-HCl, 0 1 mM EDTA 0 227 M CaCl, To this mixtuie is added, dropwise, 500 μl of 50 mM HEPES (pH 7 35) 280 mM NaCl, 1 5 mM NaP04. and a piecipitate is allowed to form for 10 minutes at 25"C The precipitate is suspended and added to the 293 cells and allowed to settle tor about toui hours at 37"C The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added lor 30 seconds The 293 cells aie then washed with serum tree medium, fresh medium is added and the cells are incubated tor about 5 days
Approximately 24 hours after the transfections, the cultuie medium is lem ved and replaced with cultuie medium (alone) 01 cultuie medium containing 200 μCi/ml 3,S-cysteιne and 200 μCi/ml 3lS-methιonme 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281 , PR023. PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 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, PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PRO2145 or PR02198 DNA may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al , Proc Natl Acad Sci , J_2 7575 ( 1981 ) 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-PR0212, pRK5-PRO290, pRK5-PR0341 , pRK5-PR0535, pRK5-PR0619, pRK5-PR0717, pRK5-PRO809, pRK5-PRO830, pRK5-PR0848, pRK5-PR0943, pRK5-PRO1005, pRK5-PRO1009, pRK5- PRO1025, pRK5-PRO1030, pRK5-PRO1097, pRK5-PR01 107, pRK5-PR01 1 1 1 , pRK5-PR01 153, pRK5- PROl 182, pRK5-PR01 184. pRK5-PR01 187, pRK5-PR01281 , pRK5-PR023, pRK5-PR039, pRK5-PR0834, pRK5-PRO 1317, pRK5-PRO 1710, pRK5-PRO2094, pRK5-PR02145 or pRK5-PR02198 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-mtroduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0 1 μg/ml bovme transferrin After about four days, the conditioned media is centrifuged and filtered to remove cells and debris The sample containing expressed PR0212. PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PROl 005, PRO 1009, PRO 1025, PRO 1030, PRO 1097, PRO 1 107, PROl 1 1 1 , PRO l 153, PRO l 182, PR01 184, PROl 187, PR01281 , PR023. PR039. PR0834, PR01317, PROl 710, PRO2094, PR02145 or PR02198 can then be concentrated and purified by any selected method, such as dialysis and/oi column chromatography
In another embodiment PR0212, PRO290, PR0341 , PR0535 PR0619. PR0717, PRO809, PRO830 PR0848, PR0943, PRO1005, PRO 1009, PRO 1025, PRO 1030, PRO1097, PROl 107, PRO l l l l , PRO l 153, PRO1 182, PRO1 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PRO2145 or PR02198 can be expressed in CHO cells The pRK5-PR0212, pRK5-PRO290, pRK5-PR0341 , pRK5-PR0535, pRK5-PR0619. pRK5-PR0717, pRK5-PRO809, pRK5-PRO830, pRK5-PR0848. pRK5-PR0943, pRK5- PRO1005, pRK5-PRO1009, pRK5-PRO 1025. pRK5-PRO1030, pRK5-PRO 1097, pRK5-PR01 107, pRK5- PROl 1 1 l , pRK5-PR01 153. pRK5-PR01 182, pRK5-PR01 184, pRK5-PR01 187, pRK5-PR01281 , pRK5-PR023 pRK5-PR039. pRK5-PR0834, pRK5-PRO 1317, pRK5-PRO 1710, pRK5-PRO2094, pRK5-PR02145 or pRK5 PR02198 vectoi 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 leplaced with culture medium (alone) oi medium containing a radiolabel such as "S-methionine After determining the presence ot the PR0212, PRO290 PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PR01 153, PROl 182, PR01184, PROH87, PR01281 , PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02I 45 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PROl 030, PRO 1097, PROl 107, PROl 111, PROl 153, PROl 182, PR01 184, PR01187, PR01281 , PR023, PR039, PR0834, PR01317, PROl 710, PRO2094, PR02145 or PR02198 can then be concentrated and purified by any selected method
Epitope-tagged PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005. PRO1009, PRO1025, PRO1030, PRO1097. PRO1107, PROl l l l , PR01 153, PR01 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 oi PR02198 may also be expressed host CHO cells The PR0212, PRO290, PR0341 , PR0535, PR0619, PRO717, PRO809, PRO830, PRO848,PRO943, PRO1005, PRO1009, PRO1025, PRO1030,PRO1097, PRO1 107, PROl 1 11, PROl 153, PROl 182, PROl 184, PROl 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 SV40 driven vector Labeling may be performed, as described above, to verify expression The culture medium containing the expressed poly-His tagged PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943 PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187. PROl 281 , PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 orPR02198 can then be concentrated and purified by any selected method, such as by Nf +-chelate affinity chromatography Expression in CHO and/or COS cells may also be accomplished by a transient expression procedure
PR0212, PR0535, PR0943, PRO 1005, PROl 025 and PRO 1 182 were expressed in CHO cells by a stable expression procedure, whereas PR0535 and PROl 025 were also 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' ot the DNA of interest to allow the convenient shuttling of cDNA's The vector used tor 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® (Qiagen), Dosper® or Fugene® (Boehπnger Mannheim) The cells were grown as described in Lucas et al , supia Approximately 3 x 107 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 ahquoted 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 106 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 ,+ 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 1+-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/mm 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 Tπs buffer, pH 9 The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins The homogeneity w as assessed by SDS polyacrylamide gels and by N terminal am o acid sequencing by Edman degradation EXAMPLE 28
Expression of PRQ212. PRO290. PRQ341. PRQ535. PRQ619. PRQ717. PRO809. PRO830. PRQ848,
PRQ943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107. PROl 1 1 1. PROl 153. PROl 182,
PROl 184, PROl 187, PRO 1281. PRQ23. PRQ39. PRQ834, PROl 317, PROl 710. PRO2094. PRQ2145 or PRQ2198 in Yeast
The following method describes recombinant expression of PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809. PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO 1025, PRO 1030, PROl 097, PRO1107, PROl l 1 1, PROl 153, PR01 182, PROl 184, PR01 187, PR01281 , PR023, PR039, PR0834,PR01317, PROl 710, PRO2094, PR02145 or PR02198 in yeast. First, yeast expression vectors are constructed for intracellular production or secretion of PR0212,
PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PROl 281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 from the ADH2/GAPDH promoter. DNA encoding PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1, PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PROl 153, PROl 182, PROH 84, PR01187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198. For secretion, DNA encoding PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PRO1 184, PRO1 187, PRO1281 , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097, PROl 107, PRO l l l l , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO 1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO 1009, PRO1025, PRO1030, PRO 1097, PRO 1 107, PROl 1 1 1 , PROl 153, PROl 182, PR01 184, PR01 187, PR01281. PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198.
Yeast cells, such as yeast strain AB 1 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717. PRO809, PRO830, PR0848. PR0943, PRO1005, PRO1009, PRO 1025, PRO1030, PRO1097, PROl 107, PRO l l l l , PRO l 153, PRO l 182, PROl 184, PROl 187, PR01281 , PR023 PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROH 84, PR01187, PROl 281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 may further be purified using selected column chromatography resins
EXAMPLE 29 Expression of PRQ212, PRO290. PRQ341 , PRQ535, PRQ619, PRQ717. PRO809. PRO830, PRQ848, PRQ943. PRO1005, PRO1009 PRO1025. PRO1030. PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182. PROl 184, PRQ1187. PRQ1281. PRQ23, PRQ39. PRQ834, PRQ1317. PRO1710. PRO2094, PRQ2145 or
PRQ2198 in Baculovirus-infected Insect Cells The following method describes recombinant expression in Baculovirus-infected insect cells The sequence coding for PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PRO1 187, PRO1281, PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PRO2145 or PR02198 is fused upstream of an epitope tag contained within a baculovirus expression vector Such epitope tags include poly-His tags and immunoglobulm tags (like Fc regions of IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVLl 393 (Novagen) Briefly, the sequence encoding PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281 , PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 or the desired portion of the coding sequence of PR0212, PRO290, PR0341 , PR0535, PR0619, PRO717, PRO809, PRO830, PRO848, PRO943, PRO1005, PRO1009, PRO1025 PRO 1030, PRO 1097 PROl 107, PROl 1 1 1 , PROl 153, PROH 82, PR01 184 PROl 187, PR01281 , PR023, PR039, PR0834, PR01317 PRO1710, PRO2094, PR02145 or PR02198 [such as the sequence encoding the extracellular domain ot a transmembrane protein or the sequence encoding the mature protein if the protein is extracellulai ] 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 Spodoptei afrugψei da ("Sf9") cells (ATCC CRL 171 1 ) using hpofectin (commercially available from GIBCO-BRL) Aftei 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 Pi ess ( 1994) Expressed poly-His tagged PR0212, PRO290, PR0341 , PR0535 PR0619 PR0717, PRO809, PRO830
PR0848, PR0943, PRO1005, PRO 1009, PRO1025, PRO1030, PRO1097 PRO l 107, PROl l l l , PROl 153, PRO 1 182, PRO 1 184, PRO 1 187 PRO 1281 , PR023, PR039, PR0834, PRO 1 17 PRO 1710, PRO2094, PR02145 or PR02198 can then be purified, for example, by Nι2+-chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al , Nature, 362 175-179 (1993) Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes, pH 7 9, 12 5 mM MgCl . , 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 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7 8) and filtered through a 0 45 μm filter A Nr+-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 mM 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 Nι2+-NTA-conjugated to alkaline phosphatase (Qiagen) Fractions containing the eluted Hιs10-tagged PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PR01184, PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198, respectively, are pooled and dialyzed against loading buffer
Alternatively, purification of the IgG tagged (or Fc tagged) PR0212, PRO290, PR0341 , PR0535, PRO619, PRO717, PRO809, PRO830, PRO848, PRO943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107,PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PR01281, PR023, PR039, PR0834, PR01317, PROl 710, PRO2094, PRO2145 or PR02198 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 Spodopteia frugψeida ("Sf9") cells (ATCC CRL 171 1 ), using Lipofectm (Gibco BRL) pb PH IgG and pb PH His are modifications of the commercially available baculovirus expression vector ρVL1393 (Pharmingen), with modified polyhnker 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 ot infection (MOI) of 10 Cells are incubated for 3 days at 28 °C The supernatant is harvested and the expiession of the constructs in the baculovirus expression vector is detei mined by batch binding of 1 ml of supernatant to 25 ml of Ni 2+-NTA beads (QIAGEN) for histidine tagged pioteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standai d 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 +-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
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 tused upstream (5' of) of an epitope tag contained with a baculovirus expression vector Such epitope tags include poly His tags and immunoglobulm 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 pIEl 1 and pIEl 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 ot the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with pi imers complementary to the 5 and 3 regions The 5 primer may incorporate flanking (selected) iestπction enzyme sites The product is then digested with those selected restriction enzymes and subcloned into the expression vector For example, derivatives ofpIEl 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 foi confirmation
High 5 cells are grown to a confluency of 50% under the conditions of 27 °C no CO., NO pen/strep Foi each 150 mm plate, 30 μg of pIE based vector containing the sequence is mixed w ith I 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 of CellFectin (CellFECΗN (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/CellFECTTN mix is then aspirated, and the cells are washed once with Ex-Cell to remove excess CellFECΗN, 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 M 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.
PR0212. PR0535, PR0943. PRO1005, PRO1025, PROl 1 1 1 , PROl 182, PROl 187 and PR0848 were successfully expressed by the above modified baculovirus procedure incorporating high 5 cells.
EXAMPLE 30
Preparation of Antibodies that Bind PRQ212. PRO290, PRQ341 , PRQ535, PRQ619. PRQ717. PRO809.
PRO830. PRQ848. PRQ943. PRO 1005, PRO 1009. PRO1025. PRO 1030. PRO 1097. PROl 107. PROl 1 1 1 ,
PRO l 153. PROl 182. PRQ1 184. PROl 187. PRQ1281. PRQ23. PRQ39. PRQ834. PRQ1317. PRO1710. PRO2094. PRQ2145 or PRQ2198
This example illustrates preparation of monoclonal antibodies which can specifically bind PR0212, PRO290, PR0341 , PR0535, PR0619. PR0717, PRO809, PRO830, PR0848, PR0943, PRO 1005, PRO 1009, PRO1025, PRO1030, PRO1097, PROl 107, PRO! 111, PROl 153, PROl 182, PROl 184, PROl 187, PROl 281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 orPR02198
Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supia Immunogens that may be employed include purified PR0212, PRO290, PR0341, PR0535, PRO619,PRO717,PRO809,PRO830,PRO848,PRO943,PRO1005,PRO1009,PRO1025,PRO1030,PRO1097, PROl 107,PROl 111, PROl 153,PR01182, PROl 184,PR01187,PRO!281,PR023,PR039,PR0834,PR01317, PRO1710, PRO2094, PR02145 or PR02198 fusion proteins containing PR0212, PRO290, PR0341, PR0535, PRO619,PRO717,PRO809,PRO830,PRO848,PRO943,PRO1005,PRO1009,PRO1025,PRO1030,PRO1097, PROl 107,PROl 111, PROl 153,PR01182, PROl 184,PR01187,PR01281,PR023,PR039,PR0834,PR01317, PRO1710, PRO2094, PR02145 or PR02198 and cells expressing recombinant PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097, PRO 1107, PROl 111, PROl 153,PR01182, PROl 184,PR01187,PRO1281,PR023,PRO39,PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717,
PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153,PR01182,PR01184, PROl 187, PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710, PRO2094, PR02145 or PR02198 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 antι-PR0212. antι-PRO290, antι-PR0341 , antι-PR0535, antι-PR0619, antι-PR0717, anti- PRO809, antι-PRO830, antι-PR0848 antι-PR0943, anti-PROl 005, anti-PROl 009, antι-PRO1025, anti-PROl 030, anti-PR01097, anti-PROl 107, anti-PRO 1111, anti-PRO 1153, anti-PRO 1182, anti-PRO 1184, anti-PRO 1187, anti- PRO 1281, antι-PR023, antι-PR039. antι-PR0834, anti-PRO 1317, anti-PRO 1710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibodies
After a suitable antibody titer has been detected, the animals "positive" tor antibodies can be injected with a final intravenous injection of PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PRO 1182, PRO 1184, PRO 1187, PRO 1281, PR023, PR039 PR0834, PRO 1317 PRO 1710, PRO2094, PR02145 or PR02198 Three to four days later the mice are sacrificed and the spleen cells aie harvested The spleen cells are then fused (using 35% polyethylene glycol) to a selected munne myeloma cell line such as P3X63AgU 1, 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, aminopteπn, 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 tor reactivity against PR0212, PRO290, PR0341, PR0535, PR0619, PR0717. PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107. PRO1 1 1 1. PROl 153, PR01 182, PR01 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PROl 182, PROl 184, PR01 187, PRO1281 ,PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 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-PR0212, anti-PRO290, anti-PR0341 , anti-PR0535, anti-PR0619, anti-PR0717, anti- PRO809, anti-PRO830, anti-PR0848, anti-PR0943, anti-PRO1005, anti-PRO1009, anti-PRO 1025, anti-PRO 1030, anti-PRO1097, anti-PROl 107, anti-PROl 1 11 , anti-PROl 153, anti-PROl 182, anti-PROl 184, anti-PROl 187, anti- PRO 1281 , anti-PR023, anti-PR039, anti-PR0834, anti-PROl 317, anti-PROl 710, anti-PRO2094, anti-PR02145 or anti-PR02198 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 201 10-2209, USA (ATCC):
Material ATCC Deposit No.: Deposit Date
DNA30942-1 134 209254 9/16/97
DNA35680-1212 209790 4/21/98
DNA26288-1239 209792 4/21/98
DNA49143- 1429 203013 6/23/98
DNA49821-1562 209981 6/16/98
DNA50988-1326 209814 4/28/98
DNA57836-1338 203025 6/23/98
DNA56866-1342 203023 6/23/98
DNA59839-1461 209988 6/16/98
DNA52192- 1369 203042 7/1/98
DNA57708-141 1 203021 6/23/98
DNA57129-1413 209977 6/16/98
DNA59622-1334 209984 6/16/98
DNA59485-1336 203015 6/23/98
DNA59841 -1460 203044 7/1/98
DNA59606-1471 209945 6/9/98 DNA58721-1475 203110 8/1 1/98
DNA59842-1502 209982 6/16/98
DNA59848-1512 203088 8/4/98
DNA59220-1514 209962 6/9/98
DNA62876-1517 203095 8/4/98
DNA59820-1549 203129 8/18/98
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 m 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 withm 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 ithin the scope of the appended claims

Claims

WHAT IS CLAIMED IS
1 An isolated antibody that binds to a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 11 1 , PROl 153, PR01182, PROl 184, PROl 187, PROl 281 ,PR023, PR039, PR0834, PROl 317, PRO1710, PRO2094, PR02145 or PR02198 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 ot matter of Claim 9 which comprises a therapeutically effective amount of said antibody
1 1 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 PR0212. PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PRO848, PR0943, PRO1005, PRO1009 PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1, PROl 153, PROl 182, PROl 184, PR01 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO 1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PROl 153, PROl 182, PRO 1 184, PROl 187, PROl 281 , PRO23, PRO39,PRO834, PRO1317, PRO1710, PRO2094, PRO2145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l, PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PRO717, PRO809, PRO830, PRO848 PR0943, PRO 1005, PRO 1009, PRO 1025 PRO1030. PRO1097. PRO1 107, PROl 11 1 , PROl 153. PROl 182, PROl 184, PROl 187, PROl 28 l , PRO23, PRO39, PRO834, PRO1317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide in a sample suspected of containing said polypeptide, said method comprising exposing the sample to an antι-PR0212, antι-PRO290, antι-PR0341. antι-PR0535, anti-PROόl 9, anti- PR0717, antι-PRO809, antι-PRO830 antι-PR0848, antι-PR0943, anti-PRO 1005. anti-PRO 1009, anti-PRO 1025, anti-PRO 1030, anti-PROl 097, anti-PRO 1 107, anti-PRO 1 1 11 , anti-PRO 1 153, anti-PRO 1 182, anti-PRO 1 184, anti- PROl 187, anti-PROl 281 , antι-PR023. antι-PR039, antι-PR0834, anti-PRO 1317. anti-PROl 710, antι-PRO2094, antι-PR02145 or antι-PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619 PR0717, PRO809, PRO830, PR0848. PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO l 107, PROl 1 1 1 , PROl 153, PRO l 182 PRO l 184, PRO ! 187, PR01281 , PR023, PR039, PR0834, PR01317 PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PRO 1 182, PRO 1 184, PRO 1 187, PRO 1281 , PR023 , PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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-
PR0212, anti-PRO290, anti-PR0341 , anti-PR0535, anti-PROό 19, anti-PR0717, anti-PRO809, anti-PRO830, anti- PR0848, anti-PR0943, anti-PRO1005, anti-PRO1009, anti-PRO1025, anti-PRO1030, anti-PRO1097, anti- PROl 107, anti-PROl 1 1 1 , anti-PR01153, anti-PROl 182, anti-PROl 184, anti-PROl 187, anti-PR01281. anti- PR023, anti-PR039, anti-PR0834, anti-PRO 1317, anti-PRO 1710, anti-PRO2094, anti-PR02145 or anti-PR02198 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between said antibody and a PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PROl 153, PRO 1182, PRO 1 184, PRO 1 187, PRO 1281 , PR023 , PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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-PR0212, anti-PRO290, anti-PR0341 , anti-PR0535, anti- PR0619, anti-PR0717, anti-PRO809, anti-PRO830, anti-PR0848, anti-PR0943, anti-PRO1005, anti-PRO1009, anti-PRO 1025, anti-PRO 1030, anti-PROl 097, anti-PRO 1 107, anti-PRO 1 1 1 1 , anti-PRO 1 153, anti-PRO 1 182, anti- PROl 184, anti-PRO 1 187, anti-PRO 1281 , anti-PR023, anti-PR039, anti-PR0834, anti-PRO 1317, anti-PRO 1710, anti-PRO2094, anti-PR02145 or anti-PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619. PR0717, PRO809, PRO830, PR0848, PR0943.
PRO 1005, PRO1009. PRO1025, PRO1030, PRO1097, PRO l 107, PROl 1 1 1 , PROl 153, PROl 1 82. PROl 184. PROl 187, PR01281 , PR023, PR039, PR0834, PR01317. PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PROl 009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 1 1 1 , PROl 153, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 antι-PR0212, antι-PRO290, antι-PR0341 , anti-
PR0535, antι-PR0619, antι-PR0717, antι-PRO809, anti PRO830, antι-PR0848, antι-PR0943, antι-PRO1005, antι-PRO1009, antι-PRO1025, antι-PRO1030, antι-PRO1097, antι-PROl 107, antι-PROl 1 1 1, anti-PROl 153, anti- PROl 182, anti-PROl 184, anti-PROl 187, anti-PROl 281 , antι-PR023, antι-PR039, antι-PR0834, anti-PROl 317, antι-PRO1710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibody
32 The method of Claim 31 , wherein said antι-PR0212, antι-PRO290, antι-PR0341 , antι-PR0535, antι-PR0619, antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, anti-PROl 005, anti PRO1009, antι-PRO1025, antι-PRO1030, antι-PRO1097, anti-PROl 107, anti-PROl 1 1 1 , anti-PROl 153, anti- PROl 182, anti-PRO 1 184, anti PRO 1 187, anti-PRO 1281 , antι-PR023, antι-PR039, antι-PR0834, anti-PROl 317, anti-PROl 710, antι-PRO2094, antι-PR02145 or antι-PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107 PRO l 1 1 1 , PROH 53, PROl 182, PROl 184, PROl 187, PR01281 , PR023, PR039 PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 polypeptide to an effective amount ot an agent that inhibits the expression ot 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 ohgonucleotide that hybridizes to a nucleic acid which encodes the PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1107, PROllll, PROH53, PRO1182,PRO1184,PRO1187,PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710,PRO2094,PRO2145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097, PRO 1107, PROl 111, PROl 153,PR01182,PR01184,PR01187,PR01281,PR023,PR039,PR0834, PROl 317, PROl 710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROllll, PROl 153, PRO1182,PRO1184,PRO1187,PRO1281,PRO23,PRO39,PRO834,PRO1317,PRO1710,PRO2094,PRO2145 or PR02198 polypeptide
40 The article of manufacture of Claim 39, wherein said active agent is an anti PR0212, antι-PRO290, antι-PR0341 , antι-PR0535, anti-PROό 19, antι-PR0717, antι-PRO809, antι-PRO830, anti PR0848, antι-PR0943, antι-PRO1005,antι-PRO1009,antι-PRO1025,antι-PRO1030,antι-PRO1097,antι-PROl 107,antι-PROl 11 l.anti- PRO 1153, anti-PRO 1182, anti-PRO 1184, anti-PROl 187, anti-PRO 1281, antι-PR023, antι-PR039, antι-PR0834, antι-PR01317, antι-PRO1710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibody
41 The article of manufacture of Claim 39, wherein said active agent is an antisense ohgonucleotide
42 A method of identifying a compound that inhibits a biological or immunological activity of a PR0212, PRO290, PR0341, PR0535, PR0619, PR0717 PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl 111, PROl 153, PROl 182, PROl 184, PROl 187, PRO 1281, PR023, PR039, PR0834, PROl 317, PROl 710, PRO2094, PR02145 orPR02198 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 antι-PR0212, antι-PRO290, anti- PR0341 , antι-PR0535, antι-PR0619, antι-PR0717, antι-PRO809, antι-PRO830, antι-PR0848, antι-PR0943, anti- PRO1005, antι-PRO1009, antι-PRO1025, antι-PRO1030, antι-PRO1097, anti-PROl 107, anti-PROl 11 1 , anti- PRO 1 153, anti-PRO 1182, anti-PRO 1 184, anti-PRO 1 187, anti-PRO 1281 , antι-PR023 , antι-PR039, antι-PR0834, antι-PR01317, antι-PRO1710, antι-PRO2094, antι-PR02145 or antι-PR02198 antibody
44 The method of Claim 42, wherein said candidate compound or said PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PRO1 107, PROl 11 1 , PROl 153. PR01 182, PROl 184, PROl 187, PRO 1281 , PR023, PR039, PR0834, PR01317, PROl 710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341, PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097, PRO 1 107, PROl 111, PROl 153, PR01182, PR01184, PR01 187, PR01281, PR023, PR039, PR0834, PR01317, PRO1710, PRO2094, PR02145 or PR02198 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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809. PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO 1097, PRO 1 107, PROl 1 1 1 , PROl 153, PR01 182, PROl 184. PR01 187, PR01281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 ohgonucleotide
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 7), Figure 6 (SEQ ID NO 10), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 16), Figure 12 (SEQ ID NO 18), Figure 14 (SEQ ID NO 23), Figure 16 (SEQ ID NO 25), Figure 18 (SEQ ID NO:27), Figure 20 (SEQ ID NO:29), Figure 22 (SEQ ID NO:34), Figure 24 (SEQ ID NO:36), Figure 26 (SEQ ID NO:38), Figure 28 (SEQ ID NO:40), Figure 30 (SEQ ID NO:42), Figure 32 (SEQ ID NO:44), Figure 34 (SEQ ID NO:46), Figure 36 (SEQ ID NO:49), Figure 38 (SEQ ID N0:51), Figure 40 (SEQ ID NO:53), Figure 42 (SEQ ID NO:55), Figure 44 (SEQ ID NO:57), Figure 46 (SEQ ID NO:65), Figure 48 (SEQ ID NO:67), Figure 50 (SEQ ID NO:69), Figure 52 (SEQ ID NO:71 ), Figure 54 (SEQ ID NO:73), Figure 56 (SEQ ID NO:75), Figure 58 (SEQ ID NO:77) and Figure 60 (SEQ ID NO:79).
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 ), Figures 3 A-3B (SEQ ID NO:6), Figure 5 (SEQ ID NO:9), Figure 7 (SEQ ID NO: 13), Figure 9 (SEQ ID NO: 15), Figure 1 1 (SEQ ID NO: 17), Figure 13 (SEQ ID NO:22), Figure 15 (SEQ ID NO:24), Figure 17 (SEQ ID NO:26), Figures 19A- 19B (SEQ ID NO:28), Figure 21 (SEQ ID NO:33), Figure 23 (SEQ ID NO:35), Figure 25 (SEQ ID NO:37), Figure 27 (SEQ ID NO:39), Figure 29 (SEQ ID NO:41 ), Figure 31 (SEQ ID NO:43), Figure 33 (SEQ ID NO:45), Figure 35 (SEQ ID NO:48), Figure 37 (SEQ ID NO:50), Figure 39 (SEQ ID NO:52), Figure 41 (SEQ ID NO:54), Figures 43A-43B (SEQ ID NO:56), Figure 45 (SEQ ID NO:64), Figure 47 (SEQ ID NO:66), Figure 49 (SEQ ID NO:68), Figures 51A-51 B (SEQ ID NO:70), Figure 53 (SEQ ID NO:72), Figures 55A-55B (SEQ ID NO:74), Figure 57 (SEQ IDNO:76) and Figures 59A-59B (SEQ ID NO:78).
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 ), Figures 3A-3B (SEQ ID NO:6), Figure 5 (SEQ ID NO:9), Figure 7 (SEQ ID NO: 13), Figure 9 (SEQ ID NO: 15), Figure 1 1 (SEQ ID NO: 17), Figure 13 (SEQ ID NO:22), Figure 15 (SEQ ID NO:24), Figure 17 (SEQ ID NO:26), Figures 19A-19B (SEQ ID NO:28), Figure 21 (SEQ ID NO:33), Figure 23 (SEQ ID NO:35), Figure 25 (SEQ ID NO:37), Figure 27 (SEQ ID NO:39), Figure 29 (SEQ ID NO:41), Figure 31 (SEQ ID NO:43), Figure 33 (SEQ ID NO:45), Figure 35 (SEQ ID NO:48), Figure 37 (SEQ ID NO:50), Figure 39 (SEQ ID NO:52), Figure 41 (SEQ ID NO:54), Figures 43A-43B (SEQ ID NO:56), Figure 45 (SEQ ID NO:64), Figure 47 (SEQ ID NO:66), Figure 49 (SEQ ID NO:68), Figures 51 A-51B (SEQ ID NO:70), Figure 53 (SEQ ID NO:72), Figures 55A- 55B (SEQ ID NO:74), Figure 57 (SEQ IDNO:76) and Figures 59A-59B (SEQ ID NO:78).
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 209254, 209790, 209792, 203013, 209981. 209814, 203025, 203023, 209988, 203042, 203021 , 209977, 209984, 203015, 203044, 209945, 2031 10, 209982, 203088. 209962, 203095 or 203129.
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 co
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 PR0212, PRO290, PR0341 , PR0535, PR0619, PR0717, PRO809, PRO830, PR0848, PR0943, PRO1005, PRO1009, PRO1025, PRO1030, PRO1097, PROl 107, PROl l l l , PRO 1153, PRO 1182, PRO 1 184, PRO 1 187, PRO 1281 , PR023, PR039, PR0834, PRO 1317, PRO 1710, PRO2094, PR02145 or PR02198 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 7), Figure 6 (SEQ ID NO 10), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 16), Figure 12 (SEQ ID NO 18), Figure 14 (SEQ ID NO 23), Figure 16 (SEQ ID NO 25), Figure 18 (SEQ ID NO 27). Figure 20 (SEQ ID NO 29), Figure 22 (SEQ ID NO 34), Figure 24 (SEQ ID NO 36), Figure 26 (SEQ ID NO 38), Figure 28 (SEQ ID NO 40), Figure 30 (SEQ ID NO 42), Figure 32 (SEQ ID NO 44), Figure 34 (SEQ ID NO 46) Figure 36 (SEQ ID NO 49), Figure 38 (SEQ ID NO 51 ), Figure 40 (SEQ ID NO 53), Figure 42 (SEQ ID NO 55) Figure 44 (SEQ ID NO 57), Figure 46 (SEQ ID NO 65), Figure 48 (SEQ ID NO 67), Figure 50 (SEQ ID NO 69), Figure 52 (SEQ ID NO 71 ), Figure 54 (SEQ ID NO 73), Figure 56 (SEQ ID NO 75), Figure 58 (SEQ ID NO 77) and Figuie 60 (SEQ ID NO 79)
62 An isolated polypeptide scoring at least 80% positives when compared to an ammo acid sequence selected from the group consisting of the ammo acid sequence shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7), Figure 6 (SEQ ID NO 10), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 16), Figure 12 (SEQ ID NO 18), Figure 14 (SEQ ID NO 23), Figure 16 (SEQ ID NO 25), Figure 18 (SEQ ID NO 27), Figure 20 (SEQ ID NO 29), Figure 22 (SEQ ID NO 34), Figure 24 (SEQ ID NO 36), Figure 26 (SEQ ID NO 38), Figure 28 (SEQ ID NO 40) Figure 30 (SEQ ID NO 42), Figure 32 (SEQ ID NO 44), Figure 34 (SEQ ID NO 46), Figure 36 (SEQ ID NO 49), Figure 38 (SEQ ID NO 51 ), Figure 40 (SEQ ID NO 53), Figure 42 (SEQ ID NO 55), Figure 44 (SEQ ID NO 57), Figure 46 (SEQ ID NO 65), Figure 48 (SEQ ID NO 67), Figure 50 (SEQ ID NO 69), Figure 52 (SEQ ID NO 71 ), Figure 54 (SEQ ID NO 73), Figure 56 (SEQ ID NO 75), Figure 58 (SEQ ID NO 77) and Figure 60 (SEQ ID NO 79) 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 209254, 209790, 209792, 203013, 209981 , 209814, 203025, 203023, 209988, 203042, 203021 , 209977, 209984, 203015, 203044, 209945, 2031 10, 209982, 203088, 209962, 203095 or 203129
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 immunoglobulm
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 7), Figure 6 (SEQ ID NO 10), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 16), Figure 12 (SEQ ID NO 18), Figure 14 (SEQ ID NO 23), Figure 16 (SEQ ID NO 25), Figure 18 (SEQ ID NO 27), Figure 20 (SEQ ID NO 29), Figure 22 (SEQ ID NO 34), Figure 24 (SEQ ID NO 36), Figure 26 (SEQ ID NO 38), Figure 28 (SEQ ID NO 40), Figure 30 (SEQ ID NO 42), Figure 32 (SEQ ID NO 44), Figure 34 (SEQ ID NO 46), Figure 36 (SEQ ID NO 49), Figure 38 (SEQ ID NO 51 ) Figure 40 (SEQ ID NO 53), Figure 42 (SEQ ID NO 55), Figure 44 (SEQ ID NO 57), Figure 46 (SEQ ID NO 65), Figure 48 (SEQ ID NO 67), Figure 50 (SEQ ID NO 69), Figure 52 (SEQ ID NO 71 ), Figure 54 (SEQ ID NO 73), Figure 56 (SEQ ID NO 75), Figure 58 (SEQ ID NO 77) or Figure 60 (SEQ ID NO 79), 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 7), Figure 6 (SEQ ID NO 10), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 16), Figure 12 (SEQ ID NO 18), Figure 14 (SEQ ID NO 23), Figure 16 (SEQ ID NO 25), Figure 18 (SEQ ID NO 27), Figure 20 (SEQ ID NO 29) Figure 22 (SEQ ID NO 34), Figure 24 (SEQ ID NO 36), Figure 26 (SEQ ID NO 38), Figure 28 (SEQ ID NO 40), Figure 30 (SEQ ID NO 42), Figure 32 (SEQ ID NO 44), Figuie 34 (SEQ ID NO 46), Figure 36 (SEQ ID NO 49), Figure 38 (SEQ ID NO 51 ), Figure 40 (SEQ ID NO 53), Figure 42 (SEQ ID NO 55), Figure 44 (SEQ ID NO 57) Figure 46 (SEQ ID NO 65), Figure 48 (SEQ ID NO 67), Figure 50 (SEQ ID NO 69), Figure 52 (SEQ ID NO 71 ) Figure 54 (SEQ ID NO 73), Figure 56 (SEQ ID NO 75), Figure 58 (SEQ ID NO 77) or Figure 60 (SEQ ID NO 79), 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 7), Figure 6 (SEQ ID NO 10), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 16), Figure 12 (SEQ ID NO 18), Figure 14 (SEQ ID NO 23), Figure 16 (SEQ ID NO 25), Figure 18 (SEQ ID NO 27), Figure 20 (SEQ ID NO 29), Figure 22 (SEQ ID NO 34), Figure 24 (SEQ ID NO 36), Figure 26 (SEQ ID NO 38), Figure 28 (SEQ ID NO 40), Figure 30 (SEQ ID NO 42), Figure 32 (SEQ ID NO 44), Figure 34 (SEQ ID NO 46), Figure 36 (SEQ ID NO 49), Figure 38 (SEQ ID NO 51 ), Figure 40 (SEQ ID NO 53), Figure 42 (SEQ ID NO 55), Figure 44 (SEQ ID NO 57), Figure 46 (SEQ ID NO 65), Figure 48 (SEQ ID NO 67), Figure 50 (SEQ ID NO 69), Figure 52 (SEQ ID NO 71 ), Figure 54 (SEQ ID NO 73), Figure 56 (SEQ ID NO 75), Figure 58 (SEQ ID NO 77) or Figure 60 (SEQ ID NO 79), 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 7), Figure 6 (SEQ ID
NO 10), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 16), Figure 12 (SEQ ID NO 18), Figure 14 (SEQ ID NO 23), Figure 16 (SEQ ID NO 25), Figure 18 (SEQ ID NO 27), Figure 20 (SEQ ID NO 29), Figure 22 (SEQ ID NO 34), Figure 24 (SEQ ID NO 36), Figure 26 (SEQ ID NO 38), Figure 28 (SEQ ID NO 40), Figure 30 (SEQ ID NO 42), Figure 32 (SEQ ID NO 44), Figure 34 (SEQ ID NO 46), Figure 36 (SEQ ID NO 49), Figure 38 (SEQ ID NO 51), Figure 40 (SEQ ID NO 53), Figure 42 (SEQ ID NO 55), Figure 44 (SEQ ID NO 57), Figure 46 (SEQ ID NO 65), Figure 48 (SEQ ID NO 67), Figure 50 (SEQ ID NO 69), Figure 52 (SEQ ID NO 71), Figure 54 (SEQ ID NO 73), Figure 56 (SEQ ID NO 75), Figure 58 (SEQ ID NO 77) or Figure 60 (SEQ ID NO 79), 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 7), Figure 6 (SEQ ID NO 10), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 16), Figure 12 (SEQ ID NO 18), Figure 14 (SEQ ID NO 23), Figure 16 (SEQ ID NO 25), Figure 18 (SEQ ID NO 27), Figure 20 (SEQ ID NO 29), Figure 22 (SEQ ID NO 34), Figure 24 (SEQ ID NO 36), Figure 26 (SEQ ID NO 38), Figure 28 (SEQ ID NO 40), Figure 30 (SEQ ID NO 42), Figure 32 (SEQ ID NO 44), Figure 34 (SEQ ID NO 46), Figure 36 (SEQ ID NO 49), Figure 38 (SEQ ID NO 51 ), Figure 40 (SEQ ID NO 53), Figure 42 (SEQ ID NO 55), Figure 44 (SEQ ID NO 57), Figure 46 (SEQ ID NO 65), Figure 48 (SEQ ID NO 67), Figure 50 (SEQ ID NO 69), Figure 52 (SEQ ID NO 71 ), Figure 54 (SEQ ID NO 73), Figure 56 (SEQ ID NO 75), Figure 58 (SEQ ID NO 77) or Figure 60 (SEQ ID NO 79), 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 7), Figure 6 (SEQ ID NO 10), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 16), Figure 12 (SEQ ID NO 18), Figure 14 (SEQ ID NO 23), Figure 16 (SEQ ID NO 25), Figure 18 (SEQ ID NO 27), Figure 20 (SEQ ID NO 29), Figure 22 (SEQ ID NO 34), Figure 24 (SEQ ID NO 36) Figure 26 (SEQ ID NO 38), Figure 28 (SEQ ID NO 40), Figure 30 (SEQ ID NO 42), Figure 32 (SEQ ID NO 44), Figuie 34 (SEQ ID NO 46), Figure 36 (SEQ ID NO 49), Figure 38 (SEQ ID NO 51 ), Figure 40 (SEQ ID NO 53), Figuie 42 (SEQ ID NO 55), Figure 44 (SEQ ID NO 57), Figure 46 (SEQ ID NO 65), Figure 48 (SEQ ID NO 67), Figure 50 (SEQ ID NO 69), Figure 52 (SEQ ID NO 71 ), Figure 54 (SEQ ID NO 73) Figure 56 (SEQ ID NO 75), Figure 58 (SEQ ID NO 77) or Figure 60 (SEQ ID NO 79), lacking its associated signal peptide
PCT/US2000/000376 1996-11-06 2000-01-06 Compositions and methods for the treatment of tumor WO2000053755A2 (en)

Priority Applications (432)

Application Number Priority Date Filing Date Title
AU24952/00A AU2495200A (en) 1999-03-08 2000-01-06 Compositions and methods for the treatment of tumor
KR1020017011399A KR20030002292A (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/US2000/004342 WO2000078961A1 (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
CA002361840A CA2361840A1 (en) 1999-03-08 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
PCT/US2000/004341 WO2000053756A2 (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP00913764A EP1220905A2 (en) 1999-03-08 2000-03-02 Composition 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
JP2000603379A JP2004516227A (en) 1999-03-08 2000-03-02 Compositions and methods for treating immune-related diseases
KR1020017011406A KR20010103046A (en) 1999-03-08 2000-03-02 Compositions and Methods for the Treatment of Immune Related Diseases
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
CA002383254A CA2383254A1 (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
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
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
DK00939307T DK1212417T3 (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and vascularization
EP00939307A EP1212417B1 (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)
JP2001500757A JP4297317B2 (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
PT00939307T PT1212417E (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and cardiovascularization
ES00939307T ES2307515T3 (en) 1999-06-02 2000-05-17 ACTIVATION OR INHIBITION OF ANGIOGENESIS AND CARDIOVASCULARIZATION.
EP07005022A EP1870464A3 (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
JP2001500672A JP2003524406A (en) 1999-06-02 2000-05-30 Compositions and methods for inhibiting tumor cell growth
EP00941164A EP1185648B1 (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
CA002373915A CA2373915A1 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
AT00941164T ATE357518T1 (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.
EP07004855A EP1820860A3 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
EP07005023A EP1867719A3 (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
PCT/US2000/015264 WO2000073452A2 (en) 1999-06-02 2000-06-02 Compositions and methods for the treatment of immune related diseases
PCT/US2000/020710 WO2001009327A2 (en) 1999-07-28 2000-07-28 Method of preventing the injury or death of retinal cells and treating ocular diseases
AU63910/00A AU6391000A (en) 1999-07-28 2000-07-28 Method of preventing the injury or death of retinal cells and treating ocular diseases
JP2001550255A JP4280444B2 (en) 2000-01-06 2000-11-08 Compositions and methods for inhibiting neoplastic cell growth
EP00982096A EP1244784A2 (en) 2000-01-06 2000-11-08 Methods and compositions for inhibiting neoplastic cell growth
PCT/US2000/030952 WO2001049715A2 (en) 2000-01-06 2000-11-08 Methods and compositions for inhibiting neoplastic cell growth
CA002390685A CA2390685C (en) 2000-01-06 2000-11-08 Methods and compositions for inhibiting neoplastic cell growth
AU19167/01A AU1916701A (en) 2000-01-06 2000-11-08 Methods and compositions for inhibiting neoplastic cell growth
CA002491610A CA2491610A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA2709291A CA2709291A1 (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
CA002496312A CA2496312A1 (en) 1999-12-01 2000-12-01 Colon tumour marker pro4799 polypeptides and nucleic acids encoding the same
EP06000588A EP1690873A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
CA002491258A CA2491258A1 (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
CA002494705A CA2494705A1 (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
PCT/US2000/032678 WO2001040466A2 (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
CA002491433A CA2491433A1 (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
EP06000585A EP1661996A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000582A EP1666495A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP10005292A EP2228446A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptieds 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
AU20554/01A AU2055401A (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
EP06000581A EP1666494A1 (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
CA002490909A CA2490909A1 (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
CA002490853A CA2490853A1 (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
CA002492049A CA2492049A1 (en) 1999-12-01 2000-12-01 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
US09/929,769 US6914130B2 (en) 1998-06-17 2001-08-14 Compositions and methods for the diagnosis and treatment of tumor
US09/941,992 US20030082546A1 (en) 1996-11-06 2001-08-28 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
US09/978,193 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
US09/978,295 US20020156006A1 (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
US09/978,192 US20020177553A1 (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
US09/978,194 US20030195333A1 (en) 1997-10-17 2001-10-15 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
US09/978,299 US20030199435A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,564 US7195760B2 (en) 1997-10-17 2001-10-16 Anti-pro363 antibodies
US09/978,697 US20020169284A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,757 US20030083248A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,375 US7196165B2 (en) 1997-10-17 2001-10-16 PRO363 polypeptides
US09/978,544 US20030199436A1 (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
US09/978,643 US20030104998A1 (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
US09/978,681 US20030195148A1 (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
US09/978,585 US20030049633A1 (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
US09/981,915 US20030054986A1 (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
US09/978,824 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
US10/160,502 US7220835B2 (en) 1998-07-30 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
US10/165,036 US20050227342A1 (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
US10/165,247 US7112657B2 (en) 1998-10-07 2001-10-19 PRO697 polypeptides
US10/165,067 US7279553B2 (en) 1998-05-13 2001-10-19 PRO1083 polypeptides
US10/145,124 US20030190701A1 (en) 1998-04-30 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
US10/164,749 US20040029218A1 (en) 1998-10-07 2001-10-19 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
US10/164,829 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
US10/162,521 US7067628B2 (en) 1998-03-17 2001-10-19 PRO788 polypeptides
US10/166,709 US20030104536A1 (en) 1998-10-07 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
US10/017,083 US20030148376A1 (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
US10/017,085 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
US09/999,829 US20030195344A1 (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
US09/999,832 US7132283B2 (en) 1997-10-17 2001-10-24 PRO273 polypeptides
US10/017,086 US7122375B2 (en) 1997-10-17 2001-10-24 PRO274 nucleic acids
US09/999,832 US20020192706A1 (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
US10/020,445 US20030198994A1 (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
US10/013,926 US7074593B2 (en) 1998-04-01 2001-10-25 PRO 703 nucleic acids
US10/013,920 US20040006219A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,917 US7029874B2 (en) 1998-03-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,923 US7169912B2 (en) 1997-10-17 2001-10-25 PRO1017 nucleic acids
US10/016,177 US20030073131A1 (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
US10/013,921 US20030068648A1 (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
US10/013,927 US7189529B2 (en) 1997-10-17 2001-10-25 PRO792 nucleic acids
US10/013,925 US7037710B2 (en) 1997-10-17 2001-10-25 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
US09/990,456 US20020137890A1 (en) 1997-03-31 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
US09/993,687 US20020198149A1 (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
US09/993,583 US7074897B2 (en) 1997-06-16 2001-11-14 Pro943 polypeptides
US09/991,163 US20020132253A1 (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
US09/990,726 US20030054359A1 (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
US09/991,073 US20020127576A1 (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
US09/990,443 US20030054987A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,469 US20030068623A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,748 US20030069403A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,444 US6930170B2 (en) 1997-06-16 2001-11-14 PRO1184 polypeptides
US09/991,854 US20030059780A1 (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
US09/993,667 US20030022187A1 (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
US09/990,427 US20030073809A1 (en) 1997-06-16 2001-11-14 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
US09/997,857 US20030064375A1 (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
US09/997,428 US20030027162A1 (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
US09/997,573 US20030049682A1 (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
US09/997,653 US7034122B2 (en) 1997-06-16 2001-11-15 Antibodies to PRO1159 polypeptides
US09/998,156 US20030044806A1 (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
US09/997,529 US20030134284A1 (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
US09/997,641 US20030224358A1 (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
US09/997,559 US20030054403A1 (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
US09/997,601 US7189814B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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
US09/997,585 US7166282B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/998,041 US20030119001A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,349 US7034106B2 (en) 1997-06-16 2001-11-15 Pro1159 polypeptides
US09/997,542 US20030068647A1 (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
US09/997,333 US6953836B2 (en) 1997-06-16 2001-11-15 PRO844 polypeptides
US09/997,514 US7019116B2 (en) 1997-06-16 2001-11-15 PRO 1387 polypeptides
US09/997,384 US7119177B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,157 US7101687B2 (en) 1997-06-16 2001-11-16 Nucleic acids encoding PRO943
US09/990,441 US7041804B2 (en) 1997-06-16 2001-11-16 Antibodies to PRO1387 polypeptides
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
US09/991,181 US6913919B2 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,437 US20030045463A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,729 US20030059831A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,723 US20020072092A1 (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
US09/989,735 US6972185B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO844 polypeptides
US09/989,721 US20020142961A1 (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
US09/989,727 US20020072497A1 (en) 1997-06-16 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
US09/989,328 US7056736B2 (en) 1997-06-16 2001-11-19 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
US09/989,732 US7037679B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO1184 polypeptides
US09/989,726 US7018811B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO189 polypeptides
US09/989,293 US7034136B2 (en) 1997-06-16 2001-11-20 Nucleic acids encoding PRO1159 polypeptides
US09/989,731 US20020103125A1 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,724 US7060812B2 (en) 1997-06-16 2001-11-20 PRO1312 nucleic acids
US09/989,730 US7157247B2 (en) 1997-06-16 2001-11-20 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
US10/001,054 US20020192209A1 (en) 1997-09-17 2001-11-30 Methods and compositions for inhibiting neoplastic cell growth
US10/006,172 US7081514B2 (en) 1998-09-01 2001-12-06 PRO1347 polypeptides
US10/007,194 US7041805B2 (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
US10/006,818 US20030054406A1 (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
US10/006,768 US6936697B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,485 US7026448B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/007,236 US7034123B2 (en) 1998-09-01 2001-12-06 Anti-PRO1347 antibodies
US10/006,041 US6951921B2 (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
US10/006,856 US7538086B2 (en) 1998-09-01 2001-12-06 PRO1303 polypeptides
US10/006,063 US20030114652A1 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/011,833 US6951920B2 (en) 1998-09-01 2001-12-07 PRO1340 polypeptides
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
US10/011,692 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
US10/011,795 US7012131B2 (en) 1998-09-01 2001-12-07 Pro1410 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
US10/012,755 US20030096955A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,754 US7375184B2 (en) 1998-09-01 2001-12-07 PRO1382 polypeptides
US10/012,752 US7026455B2 (en) 1998-09-01 2001-12-07 Anti-pro 1343 antibodies
US10/012,101 US20030187239A1 (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
US10/012,754 US20030187191A1 (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
US10/011,671 US20030096954A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,907 US20030064925A1 (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
US10/013,906 US20030191282A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,909 US20030186318A1 (en) 1999-01-05 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
US10/013,915 US20030204053A1 (en) 1998-09-01 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
US10/013,911 US20030187193A1 (en) 1998-09-01 2001-12-10 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
US10/015,869 US7189530B2 (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
US10/015,499 US20030065142A1 (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
US10/015,653 US20030187195A1 (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
US10/015,869 US20030073130A1 (en) 1998-09-01 2001-12-11 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
US10/015,715 US7033786B2 (en) 1998-09-01 2001-12-12 Pro1340 nucleic acids
US10/015,386 US7022498B2 (en) 1998-09-01 2001-12-12 Pro 1325 nucleic acids
US10/015,391 US20030120053A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,387 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
US10/015,385 US20030195347A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,306 US20030170718A1 (en) 1998-09-01 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
US10/017,407 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
US10/017,867 US20030180792A1 (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
US10/028,072 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
US10/081,056 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
US10/121,059 US20030190721A1 (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
US10/121,047 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
US10/121,046 US20030194791A1 (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
US10/121,044 US20030190717A1 (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
US10/121,042 US20030096386A1 (en) 1997-03-31 2002-04-11 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
US10/121,050 US20030054516A1 (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
US10/121,055 US20030190718A1 (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
US10/121,052 US20030199052A1 (en) 1997-03-31 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
US10/121,058 US20030190720A1 (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
US10/121,049 US20030022239A1 (en) 1997-06-18 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
US10/121,043 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
US10/121,056 US20030082760A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,322 US20030199059A1 (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
US10/123,155 US20030068794A1 (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
US10/123,214 US7343721B2 (en) 1997-03-31 2002-04-15 PRO4406 polypeptide
US10/123,157 US20030190725A1 (en) 1997-03-31 2002-04-15 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
US10/123,156 US20030194792A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,212 US7276577B2 (en) 1997-03-31 2002-04-15 PRO1866 polypeptides
US10/123,109 US20030190723A1 (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
US10/123,108 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
US10/123,291 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
US10/123,236 US20030068795A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,213 US20030199057A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,903 US20030073212A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,902 US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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
US10/123,906 US20030190726A1 (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
US10/123,913 US20030203462A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,912 US20030100087A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,911 US7408032B2 (en) 1997-03-31 2002-04-16 PRO1188 polypeptides
US10/123,907 US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,904 US20030022328A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,905 US7285625B2 (en) 1997-06-18 2002-04-16 PRO536 polypeptides
US10/124,823 US20030199062A1 (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
US10/125,805 US20030194794A1 (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
US10/124,818 US20030082763A1 (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
US10/125,704 US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
US10/124,814 US7105335B2 (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
US10/124,822 US7109305B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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
US10/124,824 US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,922 US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/125,927 US20030190731A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,924 US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/125,931 US20030199063A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,932 US7317079B2 (en) 1997-03-31 2002-04-19 PRO812 polypeptides
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
US10/131,817 US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
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
US10/137,865 US20030032155A1 (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
US10/137,867 US20030207349A1 (en) 1997-03-31 2002-05-03 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
US10/140,023 US20030207416A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,980 US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/140,020 US20030207415A1 (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
US10/139,963 US7288625B2 (en) 1997-03-31 2002-05-06 PRO4395 polypeptides
US10/140,809 US20030207418A1 (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
US10/140,865 US20030207420A1 (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
US10/140,805 US20030207417A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,921 US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
US10/140,808 US7425621B2 (en) 1997-03-31 2002-05-07 Antibodies against the PRO4401 polypeptide
US10/140,860 US7307151B2 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,925 US20030073215A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,760 US7342104B2 (en) 1997-03-31 2002-05-08 Antibodies against the PRO4320 polypeptide
US10/141,756 US7488586B2 (en) 1997-03-31 2002-05-08 PRO4409 polypeptides
US10/141,701 US20030207421A1 (en) 1997-03-31 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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
US10/142,425 US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,114 US20030036180A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,430 US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/143,113 US7329730B2 (en) 1997-03-31 2002-05-09 PRO4348 polypeptides
US10/142,417 US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/142,419 US7153941B2 (en) 1997-03-31 2002-05-10 Antibodies that bind PRO4994 polypeptides
US10/142,423 US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,431 US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 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
US10/147,519 US20030077791A1 (en) 1997-03-31 2002-05-17 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
US10/152,395 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
US10/157,782 US20030077792A1 (en) 1997-03-31 2002-05-29 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
US10/160,498 US20030073216A1 (en) 1997-03-31 2002-05-30 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
US10/158,791 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
US10/197,942 US20030175882A1 (en) 1998-09-10 2002-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2002341509A JP4017507B2 (en) 1999-06-02 2002-09-25 Medicament containing polypeptide
US11/021,329 US20050159591A1 (en) 1999-06-02 2004-12-21 Compositions and methods for the diagnosis and treatment of tumor
US11/021,330 US20050159588A1 (en) 1999-06-02 2004-12-21 Compositions and methods for the diagnosis and treatment of tumor
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
US11/341,175 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
JP2006225770A JP4072181B2 (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
JP2007083124A JP2007238619A (en) 2000-01-06 2007-03-27 Composition and method for inhibiting growth of tumorous cell
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
JP2007326424A JP2008167749A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptide and nucleic acid encoding the same
JP2007326609A JP2008148701A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids 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
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 (16)

Application Number Priority Date Filing Date Title
USPCT/US99/05028 1999-03-08
PCT/US1999/005028 WO1999046281A2 (en) 1998-03-10 1999-03-08 Novel polypeptides and nucleic acids encoding the same
PCT/US1999/012252 WO1999063088A2 (en) 1998-06-02 1999-06-02 Membrane-bound proteins and nucleic acids encoding the same
USPCT/US99/12252 1999-06-02
US14103799P 1999-06-23 1999-06-23
US60/141,037 1999-06-23
US14304899P 1999-07-07 1999-07-07
US60/143,048 1999-07-07
US14569899P 1999-07-26 1999-07-26
US60/145,698 1999-07-26
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/US99/30911 1999-12-20
PCT/US1999/030911 WO2000075316A1 (en) 1999-06-02 1999-12-20 Methods and compositions for inhibiting neoplastic cell growth
PCT/US2000/000219 WO2000053753A2 (en) 1999-03-08 2000-01-05 Promotion or inhibition of angiogenesis and cardiovascularization
USPCT/US00/00219 2000-01-05

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PCT/US2000/003565 Continuation-In-Part WO2001053486A1 (en) 1994-09-08 2000-02-11 Compositions and methods for the treatment of tumor
US87203501A Continuation-In-Part 1996-11-06 2001-06-01
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
US10/197,942 Continuation US20030175882A1 (en) 1998-03-27 2002-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same

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US20100015152A1 (en) * 2000-02-25 2010-01-21 Oxford Glycosciences (Uk) Ltd. Diagnostic and therapeutic methods
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US8604162B2 (en) 2000-04-21 2013-12-10 Fuso Pharmaceutical Industries, Ltd. Collectin
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EP1666594A2 (en) * 2000-06-02 2006-06-07 Genentech, Inc. Polypeptide, nucleic acid encoding it, and their use for the diagnosis of cancer
EP1666594A3 (en) * 2000-06-02 2006-06-21 Genentech, Inc. Polypeptide, nucleic acid encoding it, and their use for the diagnosis of cancer
WO2002055533A3 (en) * 2000-11-22 2002-10-03 Immunex Corp Methods of using imxp-888 and imxp-888 antagonists
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US11248021B2 (en) 2004-04-21 2022-02-15 Alexion Pharmaceuticals, Inc. Bone delivery conjugates and method of using same to target proteins to bone
US8546535B2 (en) 2008-01-24 2013-10-01 Esperance Pharmaceuticals, Inc. Lytic domain fusion constructs and methods of making and using same
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US9266939B2 (en) 2010-12-27 2016-02-23 Alexion Pharmaceuticals, Inc. Compositions comprising natriuretic peptides and methods of use thereof
US10052366B2 (en) 2012-05-21 2018-08-21 Alexion Pharmaceuticsl, Inc. Compositions comprising alkaline phosphatase and/or natriuretic peptide and methods of use thereof
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US10822596B2 (en) 2014-07-11 2020-11-03 Alexion Pharmaceuticals, Inc. Compositions and methods for treating craniosynostosis
US11224638B2 (en) 2014-12-05 2022-01-18 Alexion Pharmaceuticals, Inc. Treating seizure with recombinant alkaline phosphatase
US10449236B2 (en) 2014-12-05 2019-10-22 Alexion Pharmaceuticals, Inc. Treating seizure with recombinant alkaline phosphatase
US10603361B2 (en) 2015-01-28 2020-03-31 Alexion Pharmaceuticals, Inc. Methods of treating a subject with an alkaline phosphatase deficiency
US11564978B2 (en) 2015-01-28 2023-01-31 Alexion Pharmaceuticals, Inc. Methods of treating a subject with an alkaline phosphatase deficiency
US11352612B2 (en) 2015-08-17 2022-06-07 Alexion Pharmaceuticals, Inc. Manufacturing of alkaline phosphatases
US11229686B2 (en) 2015-09-28 2022-01-25 Alexion Pharmaceuticals, Inc. Reduced frequency dosage regimens for tissue non-specific alkaline phosphatase (TNSALP)-enzyme replacement therapy of hypophosphatasia
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