WO2000075327A1 - Methods and compositions for inhibiting neoplastic cell growth - Google Patents

Methods and compositions for inhibiting neoplastic cell growth Download PDF

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Publication number
WO2000075327A1
WO2000075327A1 PCT/US2000/004914 US0004914W WO0075327A1 WO 2000075327 A1 WO2000075327 A1 WO 2000075327A1 US 0004914 W US0004914 W US 0004914W WO 0075327 A1 WO0075327 A1 WO 0075327A1
Authority
WO
WIPO (PCT)
Prior art keywords
pro
polypeptide
prol
acid sequence
amino acid
Prior art date
Application number
PCT/US2000/004914
Other languages
French (fr)
Inventor
Avi J. Ashkenazi
Kenneth J. Hillan
Mary A. Napier
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/US1999/012252 external-priority patent/WO1999063088A2/en
Priority claimed from PCT/US2000/000219 external-priority patent/WO2000053753A2/en
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to AU32461/00A priority Critical patent/AU3246100A/en
Priority to AT00916675T priority patent/ATE478145T1/en
Priority to CA002383254A priority patent/CA2383254A1/en
Priority to EP00916675A priority patent/EP1210418B1/en
Priority to JP2001500766A priority patent/JP2004522402A/en
Priority to PCT/US2000/008439 priority patent/WO2000073454A1/en
Priority to AU37743/00A priority patent/AU3774300A/en
Priority to PCT/US2000/015264 priority patent/WO2000073452A2/en
Priority to EP06000588A priority patent/EP1690873A3/en
Priority to EP06000582A priority patent/EP1666495A1/en
Priority to CA002492070A priority patent/CA2492070A1/en
Priority to CA002492049A priority patent/CA2492049A1/en
Priority to CA002491433A priority patent/CA2491433A1/en
Priority to CA2709291A priority patent/CA2709291A1/en
Priority to EP05025102A priority patent/EP1672070A3/en
Priority to EP06000585A priority patent/EP1661996A1/en
Priority to EP10005292A priority patent/EP2228446A1/en
Priority to EP00983846A priority patent/EP1250426A2/en
Priority to CA002391455A priority patent/CA2391455A1/en
Priority to CA002490853A priority patent/CA2490853A1/en
Priority to EP06000584A priority patent/EP1669371A3/en
Priority to EP06000587A priority patent/EP1690872A3/en
Priority to JP2001542531A priority patent/JP2004522404A/en
Priority to EP06000586A priority patent/EP1688497A1/en
Priority to CA002491258A priority patent/CA2491258A1/en
Priority to CA002490909A priority patent/CA2490909A1/en
Priority to CA002496312A priority patent/CA2496312A1/en
Priority to EP06000581A priority patent/EP1666494A1/en
Priority to CA002491610A priority patent/CA2491610A1/en
Priority to PCT/US2000/032678 priority patent/WO2001040466A2/en
Priority to EP06000589A priority patent/EP1661997A1/en
Priority to CA002494705A priority patent/CA2494705A1/en
Priority to AU20554/01A priority patent/AU2055401A/en
Priority to EP06000583A priority patent/EP1686134A3/en
Publication of WO2000075327A1 publication Critical patent/WO2000075327A1/en
Priority to US09/941,992 priority patent/US20030082546A1/en
Priority to US09/993,583 priority patent/US7074897B2/en
Priority to US09/990,427 priority patent/US20030073809A1/en
Priority to US09/990,436 priority patent/US20020198148A1/en
Priority to US09/991,073 priority patent/US20020127576A1/en
Priority to US09/993,687 priority patent/US20020198149A1/en
Priority to US09/990,442 priority patent/US20020132252A1/en
Priority to US09/990,711 priority patent/US20030032023A1/en
Priority to US09/990,562 priority patent/US20030027985A1/en
Priority to US09/990,443 priority patent/US20030054987A1/en
Priority to US09/990,726 priority patent/US20030054359A1/en
Priority to US09/990,444 priority patent/US6930170B2/en
Priority to US09/993,469 priority patent/US20030068623A1/en
Priority to US09/991,854 priority patent/US20030059780A1/en
Priority to US09/991,163 priority patent/US20020132253A1/en
Priority to US09/992,598 priority patent/US6956108B2/en
Priority to US09/990,440 priority patent/US20030060407A1/en
Priority to US09/993,748 priority patent/US20030069403A1/en
Priority to US09/993,667 priority patent/US20030022187A1/en
Priority to US09/992,521 priority patent/US20030083461A1/en
Priority to US09/990,456 priority patent/US20020137890A1/en
Priority to US09/990,438 priority patent/US20030027754A1/en
Priority to US09/997,601 priority patent/US7189814B2/en
Priority to US09/997,614 priority patent/US20030124531A1/en
Priority to US09/997,585 priority patent/US20030119055A1/en
Priority to US09/997,641 priority patent/US20030224358A1/en
Priority to US09/997,683 priority patent/US20030059783A1/en
Priority to US09/997,529 priority patent/US7309761B2/en
Priority to US09/997,666 priority patent/US20030027163A1/en
Priority to US09/997,857 priority patent/US20030064375A1/en
Priority to US09/997,333 priority patent/US6953836B2/en
Priority to US09/997,666 priority patent/US7244816B2/en
Priority to US09/997,641 priority patent/US7112656B2/en
Priority to US09/997,529 priority patent/US20030134284A1/en
Priority to US09/997,542 priority patent/US20030068647A1/en
Priority to US09/997,585 priority patent/US7166282B2/en
Priority to US09/997,573 priority patent/US20030049682A1/en
Priority to US09/997,559 priority patent/US20030054403A1/en
Priority to US09/997,514 priority patent/US7019116B2/en
Priority to US09/998,041 priority patent/US7309775B2/en
Priority to US09/997,653 priority patent/US7034122B2/en
Priority to US09/997,349 priority patent/US7034106B2/en
Priority to US09/998,156 priority patent/US20030044806A1/en
Priority to US09/997,384 priority patent/US20030087305A1/en
Priority to US09/998,041 priority patent/US20030119001A1/en
Priority to US09/997,428 priority patent/US20030027162A1/en
Priority to US09/997,628 priority patent/US20030059782A1/en
Priority to US09/997,601 priority patent/US20030054404A1/en
Priority to US09/997,440 priority patent/US20030059833A1/en
Priority to US09/991,150 priority patent/US20030194760A1/en
Priority to US09/991,172 priority patent/US20030050457A1/en
Priority to US09/990,441 priority patent/US7041804B2/en
Priority to US09/990,437 priority patent/US20030045463A1/en
Priority to US09/991,181 priority patent/US6913919B2/en
Priority to US09/991,157 priority patent/US7101687B2/en
Priority to US09/989,862 priority patent/US20030130182A1/en
Priority to US09/989,279 priority patent/US7083978B2/en
Priority to US09/989,723 priority patent/US20020072092A1/en
Priority to US09/989,722 priority patent/US20020072067A1/en
Priority to US09/989,727 priority patent/US20020072497A1/en
Priority to US09/989,729 priority patent/US20030059831A1/en
Priority to US09/989,735 priority patent/US6972185B2/en
Priority to US09/989,721 priority patent/US20020142961A1/en
Priority to US09/989,732 priority patent/US7037679B2/en
Priority to US09/989,734 priority patent/US7491529B2/en
Priority to US09/989,328 priority patent/US7056736B2/en
Priority to US09/989,726 priority patent/US7018811B2/en
Priority to US09/989,725 priority patent/US20030139329A1/en
Priority to US09/989,728 priority patent/US7029873B2/en
Priority to US09/989,731 priority patent/US20020103125A1/en
Priority to US09/989,730 priority patent/US7157247B2/en
Priority to US09/989,724 priority patent/US7060812B2/en
Priority to US09/989,293 priority patent/US7034136B2/en
Priority to US10/027,603 priority patent/US7264801B2/en
Priority to US10/028,072 priority patent/US20030004311A1/en
Priority to US10/081,056 priority patent/US20040043927A1/en
Priority to US10/119,480 priority patent/US20040087769A1/en
Priority to US10/121,047 priority patent/US20030077778A1/en
Priority to US10/121,044 priority patent/US20030190717A1/en
Priority to US10/121,059 priority patent/US20030190721A1/en
Priority to US10/121,042 priority patent/US20030096386A1/en
Priority to US10/121,046 priority patent/US20030194791A1/en
Priority to US10/121,045 priority patent/US20030073210A1/en
Priority to US10/121,040 priority patent/US20030082759A1/en
Priority to US10/121,041 priority patent/US20030077776A1/en
Priority to US10/121,051 priority patent/US20030092147A1/en
Priority to US10/121,052 priority patent/US20030199052A1/en
Priority to US10/121,048 priority patent/US20030199051A1/en
Priority to US10/121,043 priority patent/US7220831B2/en
Priority to US10/121,062 priority patent/US20030077779A1/en
Priority to US10/121,061 priority patent/US20030082761A1/en
Priority to US10/121,057 priority patent/US20030190719A1/en
Priority to US10/121,063 priority patent/US20030199055A1/en
Priority to US10/121,060 priority patent/US20030190722A1/en
Priority to US10/121,055 priority patent/US20030190718A1/en
Priority to US10/121,056 priority patent/US20030082760A1/en
Priority to US10/121,054 priority patent/US20030199054A1/en
Priority to US10/121,058 priority patent/US20030190720A1/en
Priority to US10/121,050 priority patent/US20030054516A1/en
Priority to US10/121,049 priority patent/US20030022239A1/en
Priority to US10/121,053 priority patent/US20030199053A1/en
Priority to US10/123,771 priority patent/US20030199060A1/en
Priority to US10/123,214 priority patent/US7343721B2/en
Priority to US10/123,236 priority patent/US20030068795A1/en
Priority to US10/123,212 priority patent/US7276577B2/en
Priority to US10/123,215 priority patent/US7291329B2/en
Priority to US10/123,108 priority patent/US7635478B2/en
Priority to US10/123,322 priority patent/US20030199059A1/en
Priority to US10/123,157 priority patent/US20030190725A1/en
Priority to US10/123,213 priority patent/US20030199057A1/en
Priority to US10/123,291 priority patent/US20030199058A1/en
Priority to US10/123,154 priority patent/US20030190724A1/en
Priority to US10/123,261 priority patent/US20030068796A1/en
Priority to US10/123,109 priority patent/US20030190723A1/en
Priority to US10/123,235 priority patent/US20030082762A1/en
Priority to US10/123,155 priority patent/US20030068794A1/en
Priority to US10/123,292 priority patent/US20030073211A1/en
Priority to US10/123,213 priority patent/US7193048B2/en
Priority to US10/123,262 priority patent/US20030049816A1/en
Priority to US10/123,156 priority patent/US20030194792A1/en
Priority to US10/123,905 priority patent/US20030087344A1/en
Priority to US10/123,905 priority patent/US7285625B2/en
Priority to US10/123,902 priority patent/US20030077781A1/en
Priority to US10/123,904 priority patent/US20030022328A1/en
Priority to US10/123,909 priority patent/US7193049B2/en
Priority to US10/123,911 priority patent/US7408032B2/en
Priority to US10/123,903 priority patent/US20030073212A1/en
Priority to US10/123,908 priority patent/US7335728B2/en
Priority to US10/123,907 priority patent/US7084258B2/en
Priority to US10/123,913 priority patent/US20030203462A1/en
Priority to US10/123,910 priority patent/US7329404B2/en
Priority to US10/123,912 priority patent/US20030100087A1/en
Priority to US10/123,906 priority patent/US20030190726A1/en
Priority to US10/124,823 priority patent/US20030199062A1/en
Priority to US10/124,822 priority patent/US7109305B2/en
Priority to US10/124,814 priority patent/US7105335B2/en
Priority to US10/124,824 priority patent/US20030077659A1/en
Priority to US10/124,819 priority patent/US7285626B2/en
Priority to US10/124,820 priority patent/US20030190729A1/en
Priority to US10/124,817 priority patent/US20030077786A1/en
Priority to US10/124,813 priority patent/US7312307B2/en
Priority to US10/125,795 priority patent/US7304131B2/en
Priority to US10/125,704 priority patent/US7357926B2/en
Priority to US10/124,818 priority patent/US20030082763A1/en
Priority to US10/125,805 priority patent/US20030194794A1/en
Priority to US10/124,816 priority patent/US20030190728A1/en
Priority to US10/124,821 priority patent/US20030199023A1/en
Priority to US10/125,922 priority patent/US7309762B2/en
Priority to US10/125,924 priority patent/US7342097B2/en
Priority to US10/125,927 priority patent/US20030190731A1/en
Priority to US10/125,932 priority patent/US7317079B2/en
Priority to US10/125,931 priority patent/US20030199063A1/en
Priority to US10/127,831 priority patent/US20030082689A1/en
Priority to US10/128,689 priority patent/US20030087365A1/en
Priority to US10/131,825 priority patent/US7282566B2/en
Priority to US10/131,823 priority patent/US7304132B2/en
Priority to US10/131,817 priority patent/US7291701B2/en
Priority to US10/137,867 priority patent/US20030207349A1/en
Priority to US10/137,865 priority patent/US20030032155A1/en
Priority to US10/137,868 priority patent/US20030082764A1/en
Priority to US10/140,474 priority patent/US20030032156A1/en
Priority to US10/140,020 priority patent/US20030207415A1/en
Priority to US10/140,470 priority patent/US20030022331A1/en
Priority to US10/140,024 priority patent/US20040058424A1/en
Priority to US10/139,963 priority patent/US7288625B2/en
Priority to US10/139,980 priority patent/US7247710B2/en
Priority to US10/140,023 priority patent/US20030207416A1/en
Priority to US10/140,864 priority patent/US20030207419A1/en
Priority to US10/140,921 priority patent/US7317080B2/en
Priority to US10/140,809 priority patent/US20030207418A1/en
Priority to US10/140,925 priority patent/US20030073215A1/en
Priority to US10/140,808 priority patent/US7425621B2/en
Priority to US10/140,865 priority patent/US20030207420A1/en
Priority to US10/140,928 priority patent/US20030068798A1/en
Priority to US10/140,860 priority patent/US7307151B2/en
Priority to US10/140,805 priority patent/US20030207417A1/en
Priority to US10/141,755 priority patent/US7297764B2/en
Priority to US10/141,754 priority patent/US7361732B2/en
Priority to US10/141,701 priority patent/US20030207421A1/en
Priority to US10/141,760 priority patent/US7342104B2/en
Priority to US10/141,756 priority patent/US7488586B2/en
Priority to US10/143,114 priority patent/US20030036180A1/en
Priority to US10/142,430 priority patent/US7309766B2/en
Priority to US10/143,113 priority patent/US7329730B2/en
Priority to US10/142,425 priority patent/US20030207424A1/en
Priority to US10/142,417 priority patent/US7304133B2/en
Priority to US10/142,419 priority patent/US7153941B2/en
Priority to US10/143,032 priority patent/US7408033B2/en
Priority to US10/142,423 priority patent/US20030049817A1/en
Priority to US10/142,431 priority patent/US7285629B2/en
Priority to US10/146,730 priority patent/US20030207427A1/en
Priority to US10/146,792 priority patent/US20030207428A1/en
Priority to US10/147,528 priority patent/US20030219885A1/en
Priority to US10/147,536 priority patent/US20040077064A1/en
Priority to US10/147,492 priority patent/US20030082765A1/en
Priority to US10/147,519 priority patent/US20030077791A1/en
Priority to US10/152,395 priority patent/US7189534B2/en
Priority to US10/153,934 priority patent/US20030129695A1/en
Priority to US10/156,843 priority patent/US20030207805A1/en
Priority to US10/157,786 priority patent/US20030208055A1/en
Priority to US10/157,782 priority patent/US20030077792A1/en
Priority to US10/158,791 priority patent/US20030207429A1/en
Priority to US10/158,782 priority patent/US20030082766A1/en
Priority to US10/160,498 priority patent/US20030073216A1/en
Priority to US10/187,885 priority patent/US20030032138A1/en
Priority to JP2002341509A priority patent/JP4017507B2/en
Priority to US10/692,299 priority patent/US7446168B2/en
Priority to US11/100,159 priority patent/US7425613B2/en
Priority to JP2005264293A priority patent/JP2006068016A/en
Priority to US11/341,175 priority patent/US7468427B2/en
Priority to US11/537,382 priority patent/US7727536B2/en
Priority to US11/537,472 priority patent/US7736645B2/en
Priority to JP2007172879A priority patent/JP5512073B2/en
Priority to JP2007326613A priority patent/JP2008161190A/en
Priority to JP2007326424A priority patent/JP2008167749A/en
Priority to JP2007325484A priority patent/JP2008148699A/en
Priority to JP2007326609A priority patent/JP2008148701A/en
Priority to US12/724,964 priority patent/US8557238B2/en
Priority to US12/760,354 priority patent/US7960531B2/en
Priority to US13/102,517 priority patent/US20130189273A1/en
Priority to JP2011138276A priority patent/JP2011236224A/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention concerns methods and compositions for inhibiting neoplastic cell growth
  • the present invention concerns antitumor compositions and methods for the treatment of tumors
  • the invention further concerns screening methods for identifying growth inhibitory e g antitumor compounds
  • Malignant tumors are the second leading cause of death in the United States after heart disease (Boring et al , CA Cancel J C n . 43 7 (1993))
  • Cancer is characterized by the 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
  • the present invention relates to methods and compositions for inhibiting neoplastic cell growth More particularly, the invention concerns methods and compositions for the treatment of tumors, including cancers, such as breast, prostate, colon, lung, ovarian, renal and CNS cancers, leukemia, melanoma, etc , in mammalian patients, preferably humans
  • the present invention concerns compositions of matter useful for the inhibition of neoplastic cell growth comprising an effective amount of a PRO 1 186 or PRO] 84 polypeptide as herein defined or an agonist thereof, in admixture with a pharmaceutically acceptable carrier
  • the composition of matter comprises a growth inhibitory amount of a PROl 186 or PRO 184 polypeptide, or an agonist thereof
  • the composition comp ⁇ ses a cytotoxic amount of a PROl 186 or PRO 184 polypeptide, or an agonist thereof
  • the compositions of matter may contain one or more additional growth inhibitory and or cytotoxic and/or other chemotherapeutic agents.
  • the present invention concerns compositions of matter useful for the treatment of a tumor in a mammal comprising a therapeutically effective amount of a PRO 1186 or PRO 184 polypeptide as herein defined, or an agonist thereof.
  • the tumor is preferably a cancer.
  • the invention concerns a method for inhibiting the growth of a tumor cell comprising exposing the cell to an effective amount of a PROl 186 or PROl 84 polypeptide as herein defined, or an agonist thereof.
  • the agonist is an anti-PRO 1186 or anti-PRO 184 agonist antibody.
  • the agonist is a small molecule that mimics the biological activity of a PROl 186 or PRO 184 polypeptide.
  • the method may be performed in vitro or in vivo.
  • the invention concerns an article of manufacture comprising:
  • composition comprising an active agent contained within the container; wherein the composition is effective for inhibiting the neoplastic cell growth, e.g., growth of tumor cells, and the active agent in the composition is a PROl 186 or PROl 84 polypeptide as herein defined, or an agonist thereof; and (c) a label affixed to said container, or a package insert included in said container referring to the use of said PROl 186 or PRO 184 polypeptide or agonist thereof, for the inhibition of neoplastic cell growth, wherein the agonist may be an antibody which binds to the PROl 186 or PROl 84 polypeptide. In a particular embodiment, the agonist is an anti-PROH86 or anti-PR0184 agonist antibody.
  • the agonist is a small molecule that mimics the biological activity of a PROl 186 or PRO 184 polypeptide.
  • Similar articles of manufacture comprising a PRO 1186 or PRO 184 polypeptide as herein defined, or an agonist thereof in an amount that is therapeutically effective for the treatment of tumor are also within the scope of the present invention.
  • articles of manufacture comprising a PRO 1186 or PRO 184 polypeptide as herein defined, or an agonist thereof, and a further growth inhibitory agent, cytotoxic agent or chemotherapeutic agent.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PROl 186 or PR0184 polypeptide.
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic
  • the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about
  • Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PROl 186 or PROl 84 polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide are disclosed herein. Therefore, soluble extracellular domains of the herein described PROl 186 or PRO 184 polypeptides are contemplated.
  • Another embodiment is directed to fragments of a PRO 1186 or PRO 184 polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PROl 186 or PRO 184 polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PROl 186 or anti-PR0184 antibody or as antisense oligonucleotide probes.
  • nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 110 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternat vely at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternat: vely at least about 160 nucleotides in length, alternatively at least about 170 nucleotides in length, alternat: vely at least about 180 nucleotides in
  • novel fragments of a PRO 1186 or PRO 184 polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO 1186 or PRO 184 polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PROl 186 or PROl 84 polypeptide-encoding nucleotide sequence fragment(s) are novel.
  • PROl 186 or PROl 84 polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PROl 186 or PROl 84 polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PROl 186 or PRO 184 polypeptide fragments that comprise a binding site for an anti-PROl 186 or anti-PRO 184 antibody. In another embodiment, the invention provides isolated PRO 1186 or PRO 184 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
  • the invention concerns an isolated PROl 186 or PROl 84 polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98%
  • the invention concerns an isolated PROl 186 or PROl 84 polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino
  • the invention concerns an isolated PROl 186 or PRO 184 polypeptide comprising an amino acid sequence scoring at least about 80% positives, alternatively at least about 81% positives, alternatively at least about 82% positives, alternatively at least about 83% positives, alternatively at least about 84% positives, alternatively at least about 85% positives, alternatively at least about 86% positives, alternatively at least about 87% positives, alternatively at least about 88% positives, alternatively at least about 89% positives, alternatively at least about 90% positives, alternatively at least about 91% positives, alternatively at least about 92% positives, alternatively at least about 93% positives, alternatively at least about 94% positives, alternatively at least about 95% positives, alternatively at least about 96% positives, alternatively at least about 97% positives, alternatively at least about 98% positives and alternatively at least about 99% positives when compared with the amino acid sequence of a PRO 1186 or PRO 184 polypeptide having a full-length amino acid sequence
  • the invention provides an isolated PROl 186 or PRO 184 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 PROl 186 or PROl 84 polypeptide and recovering the PROl 186 or PROl 84 polypeptide from the cell culture.
  • the invention provides an isolated PROl 186 or PRO 184 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 PROl 186 or PROl 84 polypeptide and recovering the PROl 186 or PROl 84 polypeptide from the cell culture.
  • the invention concerns agonists of a native PRO 1 186 or PRO 184 polypeptide as defined herein.
  • the agonist is an anti-PROl 186 or anti-PROl 84 antibody or a small molecule.
  • the invention concerns a method of identifying agonists to a PRO 1186 or PRO 184 polypeptide which comprise contacting the PRO 1186 or PRO 184 polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO 1186 or PRO 184 polypeptide.
  • the PRO 1186 or PRO 184 polypeptide is a native PRO 1186 or PRO 184 polypeptide.
  • the invention concerns a composition of matter comprising a PROl 186 or PRO 184 polypeptide, or an agonist of a PRO 1186 or PRO 184 polypeptide as herein described, or an anti-PRO 1186 or anti-PRO 184 antibody, in combination with a carrier.
  • the carrier is a pharmaceutically acceptable carrier.
  • Another embodiment of the present invention is directed to the use of a PRO 1186 or PRO 184 polypeptide, or an agonist thereof as hereinbefore described, or an anti-PRO 1 186 or anti-PRO 184 antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO 1 186 or PRO 184 polypeptide, an agonist thereof or an anti-PROl 186 or anti-PROl 84 antibody.
  • 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, __.. coli, yeast, or Baculovirus-infected insect cells.
  • a process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
  • the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence.
  • Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin
  • the invention provides an antibody which specifically binds to any of the above or below described polypeptides
  • the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody
  • the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences
  • Figure 1 shows a nucleotide sequence (SEQ ID NO 1) of a native sequence PROl 186 cDNA, wherein SEQ ID NO 1 is a clone designated herein as "DNA60621-1516"
  • Figure 2 shows the amino acid sequence (SEQ ID NO 2) derived from the coding sequence of SEQ ID NO 1 shown in Figure 1
  • Figure 3 shows a nucleotide sequence (SEQ ID NO 3) of a native sequence PRO 184 cDNA, wherein SEQ ID NO 3 is a clone designated herein as "DNA28500”
  • Figure 4 shows the amino acid sequence (SEQ ID NO 4) derived from the coding sequence of SEQ ID NO
  • PRO polypeptide and “PRO” as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (i 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
  • native sequence PRO polypeptide specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO
  • 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
  • the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionine residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionine 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/or cytoplasmic domains and preferably,
  • PRO polypeptide variant means an active PRO polypeptide as defined above or below having at least about 80% ammo acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein
  • Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence
  • a PRO polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid
  • Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program
  • 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 hypotheUcal 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 sequence identity
  • filel and file2 are two dna or two protein sequences.
  • Max file length is 65535 (limited by unsigned short x in the jmp struct)
  • a sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
  • the program may create a tmp file in /tmp to hold info about traceback.
  • static nm matches in core — for checking */ static lmax; /* lengths of stripped file names */ static UP]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static ni[2]; /* current ele number — for gapping */ static siz[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */ static char out[2][P_LINE]; /* output line */ static char star[P LINE]; /* set by stars() *//
  • *ps[ ⁇ ] toupper(*ps[ ⁇ ]), po[ ⁇ ] + + , ps[ ⁇ ] + + ,
  • *py+ + *px, else if ( ⁇ slower(*px))
  • *py++ toupper(*px), if ( ⁇ ndex("ATGCU",*(py-l))) natgc+ + , ⁇ ⁇
  • Page2ofnwsubr.c ...readjmps if 0 ⁇ 0 && dx[dmax] . offset && fj) ⁇
  • Percent (%) amino acid sequence identit with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the 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 amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or 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
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows
  • a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (.
  • 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
  • the am o acid sequence B is the amino acid sequence of the PRO polypeptide of interest
  • PRO variant polynucleotide' or "PRO variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Ordinarily, a PRO variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least
  • PRO variant polynucleotides are at least about 30 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively 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 or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measu ⁇ ng alignment, including any algorithms needed to achiev e 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
  • % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows:
  • % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 ( Altschul et al. , Nucleic Acids Res., 25:3389-3402 (1997)).
  • NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov, or otherwise obtained from the National Institute of Health, Bethesda, MD.
  • % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows:
  • a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide-encoding nucleic acid and the comparison nucleic acid molecule of interest (.
  • 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: 0
  • PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide
  • Amino acid residues that score a positive value to an amino acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in
  • the % value of positives of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows
  • isolated when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a component of its natural environment Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes
  • the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stam Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PROl 186 or PR0184 natural environment will not be present Ordinarily, however, isolated polypeptide will be prepared by at least one pur
  • 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 PROl 186 or PRO 184 polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase
  • enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice
  • antibody is used in the broadest sense and specifically covers, for example, single anti- PROl 186 or anti-PRO 184 and anti-PRO 1186 or anti-PRO 184 monoclonal antibodies (including agonist antibodies), anti-PROl 186 and anti-PRO 184 antibody compositions with polyepitopic specificity, single chain anti- PRO 1186 and anti-PRO 184 antibodies, and fragments of anti-PRO 1186 and anti-PRO 184 antibodies (see below)
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, J e , the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures Hybridization generally depends on the ability of denatured DNA
  • “Stringent conditions or 'high-stringency conditions' may be identified by those that (1 ) employ low ionic strength and high temperature for washing, for example, 0 015 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, for example, 50% (v/v) formamide with 0 1 % bovine serum album ⁇ n/0 1% F ⁇ coll/0 1% polyv ⁇ nylpyrrol ⁇ done/50mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C, or (3) employ 50% formamide, 5 x SSC (0 75 M NaCl, 0 075 M sodium citrate), 50 mM sodium phosphate (pH 6 8), 0 1% sodium pyrophosphate, 5 x Denhardt's solution, sonic
  • formamide for example
  • 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 of washing solution and hybridization conditions (e g , temperature, ionic strength, and % SDS) less stringent than those described above
  • An example of moderately stringent conditions is overnight incubation at 37 °C in a solution comprising 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7 6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37 C -50°C
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc as necessary to accommodate factors such as probe length and the like
  • epitope tagged when used herein refers to a chimeric polypeptide comprising a PROl 186 or PRO 184 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)
  • the term “lmmunoadhesin” designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin' ) with the effector functions of lmmunoglobulin constant domains Structurally, the lmmunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (.
  • the adhesin part of an lmmunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand
  • the lmmunoglobulin constant domain sequence m the lmmunoadhesin may be obtained from any lmmunoglobulin, such as IgG- 1 , IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA 2), IgE, IgD or IgM
  • Activity refers to form(s) of PROl 186 or PROl 84 which retain a biological and or an immunological activity of native or naturally-occurring PROl 186 or PROl 84, wherein "biological " activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally
  • Bio activity in the context of an antibody or another agonist that can be identified by the screening assays disclosed herein ⁇ e.g., an organic or inorganic small molecule, peptide, etc. is used to refer to the ability of such molecules to invoke one or more of the effects listed herein in connection with the definition of a "therapeutically effective amount.”
  • biological activity is the ability to inhibit neoplastic cell growth or proliferation.
  • a preferred biological activity is inhibition, including slowing or complete stopping, of the growth of a target tumor (e.g., cancer) cell.
  • Another preferred biological activity is cytotoxic activity resulting in the death of the target tumor (e.g. , cancer) cell.
  • Yet another preferred biological activity is the induction of apoptosis of a target tumor (e.g., cancer) cell.
  • immunological activity means immunological cross-reactivity with at least one epitope of a PROl 186 or PRO 184 polypeptide.
  • Immunological cross-reactivity means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO 1186 or PRO 184 polypeptide having this activity with polyclonal antisera raised against the known active PROl 186 or PROl 84 polypeptide.
  • antisera are prepared in conventional fashion by injecting goats or rabbits, for example, subcutaneously with the known active analogue in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freunds.
  • the immunological cross-reactivity preferably is "specific", which means that the binding affinity of the immunologically cross-reactive molecule (e.g., antibody) identified, to the corresponding PROl 186 or PROl 84 polypeptide is significantly higher (preferably at least about 2-times, more preferably at least about 4-times, even more preferably at least about 6-times, most preferably at least about 8-times higher) than the binding affinity of that molecule to any other known native polypeptide.
  • the immunologically cross-reactive molecule e.g., antibody
  • 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. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented
  • a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e g , radiation and/or chemotherapy
  • the "pathology” of cancer includes all phenomena that compromise the well-being of the patient This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, etc
  • an “effective amount” of a polypeptide disclosed herein or an agonist thereof, in reference to inhibition of neoplastic cell growth is an amount capable of inhibiting, to some extent, the growth of target cells
  • the term includes an amount capable of invoking a growth inhibitory, cytostatic and/or cytotoxic effect and/or apoptosis of the target cells
  • An "effective amount" of a PRO 1186 or PRO 184 polypeptide or an agonist thereof for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
  • a “therapeutically effective amount”, in reference to the treatment of tumor, refers to an amount capable of invoking one or more of the following effects (1) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest, (2) reduction in the number of tumor cells, (3) reduction in tumor size,
  • a "therapeutically effective amount" of a PRO 1186 or PRO 184 polypeptide or an agonist thereof for purposes of treatment of tumor may be determined empirically and in a routine manner
  • a “growth inhibitory amount” of a PROl 186 or PROl 84 polypeptide or an agonist thereof is an amount capable of inhibiting the growth of a cell, especially tumor, e g , cancer cell, either in vitro or in vivo
  • a “growth inhibitory amount” of a PRO 1 186 or PRO 184 polypeptide or an agonist thereof for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
  • a "cytotoxic amount” of a PROl 186 or PRO 184 polypeptide or an agonist thereof is an amount capable of causing the destruction of a cell, especially tumor, e g , cancer cell, either in vitro or in vivo
  • a "cytotoxic amount" of a PROl 186 or PRO 184 polypeptide or an agonist thereof for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
  • 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 ⁇ , I 125 , Y 90 and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof
  • chemotherapeutic agent is a chemical compound useful in the treatment of tumor, e g , cancer
  • chemotherapeutic agents include ad ⁇ amycin, doxorubicin. epirubicin, 5-fluorourac ⁇ l, cytosme arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e g , pac taxel (Taxol, Bristol- Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rh ⁇ ne-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide.
  • ad ⁇ amycin doxorubicin. epirubicin, 5-fluorourac ⁇ l, cytosme arabinoside ("Ara-C"), cyclophosphamide, thiotepa,
  • mitomycin C mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide. daunomycin, carminomycin, aminopterin, dactinomycin. mitomycins, esperamicins (see, U.S. Patent No. 4,675,187), melphalan and other related nitrogen mustards. Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapristone.
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo.
  • the growth inhibitory agent is one which significantly reduces the percentage of the target cells in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M- phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine. mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds.. Chapter 1, entitled "Cell cycle regulation, oncogens, and antineoplastic drugs” by Murakami et al., (WB Saunders: Philadelphia, 1995), especially p. 13.
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor- ⁇ and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet- growth factor;
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions. 14, pp. 375-382, 615th Meeting Harbor (1986) and Stella et al. , “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al, (ed.), pp. 247-267, Humana Press (1985).
  • the prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, glycosylated prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above
  • agonist' is used in the broadest sense and includes any molecule that mimics a biological activity of a native PROl 186 or PROl 84 polypeptide disclosed herein Suitable agonist molecules specifically include agonist antibodies or antibody fragments fragments or amino acid sequence variants of native PRO 1186 or PRO 184 polypeptides, peptides, small organic molecules, etc.
  • Methods for identifying agonists of a PROl 186 or PROl 84 polypeptide may comprise contacting a tumor cell with a candidate agonist molecule and measuring the inhibition of tumor cell growth
  • Chronic' administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time
  • Intermittent administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc
  • the mammal is human Administration "in combination with" one or more further therapeutic agents includes simultaneous
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed Often the physiologically acceptable carrier is an aqueous pH buffered solution
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptide, proteins, such as serum albumin, gelatin, or lmmunoglobulins, hydrophilic polymers such as polyvinylpyrro done, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccha ⁇ des, and other carbohydrates including glucose, mannose, or dext ⁇ ns, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming countenons such as sodium, and/or nonionic surfactants such
  • “Native antibodies” and “native lmmunoglobulins” 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 lmmunoglobulin isotypes Each heavy and light chain also has regularly spaced mtrachain disulfide bridges Each heavy chain has at one end a ⁇ arable domain (V H ) 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 refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen
  • CDRs complementarity-determining regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, Kabat etal , NIH Publ No 91-3242, Vol I, pages 647-669 ( 1991 ))
  • the constant domains are not involved directly
  • Antibody fragments comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody
  • antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments, diabodies, linear antibodies (Zapata et al , Protein Eng . 8(10) 1057-1062 [1995]), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, a designation reflecting the 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 configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer Collectively, the six CDRs confer antigen-binding specificity to the antibody However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower 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 fragments differ from Fab' fragments by the addition of a few 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') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known
  • the ' light chains of antibodies (lmmunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains
  • lmmunoglobulins can be assigned to different classes There are five major classes of lmmunoglobulins IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e g , IgGl IgG2, IgG3, IgG4, IgA, and IgA2
  • the 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 antigenic site Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hyb ⁇ doma culture, uncontaminated by other lmmunoglobulins The modifier ' monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hy
  • Humanized' forms of non-human (e g , munne) antibodies are chimeric lmmunoglobulins, lmmunoglobulin 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 lmmunoglobulin
  • humanized antibodies are human lmmunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity
  • Fv FR residues of the human lmmunoglobulin are replaced by corresponding non-human residues
  • humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences
  • the humanized antibody includes a PRIM ATIZEDTMant ⁇ body wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest
  • Single-chain Fv' or “sFv' antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding
  • 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 93/11161 , 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 matenals which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present Ordinarily, however, isolated antibody will be prepared by at least one purification step
  • label when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody
  • the label may be detectable by itself
  • the label may also be a non-detectable entity such as a toxin
  • solid phase is meant a non-aqueous matrix to which the antibody of the present invention can adhere
  • solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysaccharides (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and si cones
  • the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275,149
  • a " posome” is a small vesicle composed of various types of pids, phosphohpids and/or surfactant which is useful for delivery of a drug (such as a PRO 1 186 or PRO 184 polypeptide or antibody thereto) to a mammal
  • a drug such as a PRO 1 186 or PRO 184 polypeptide or antibody thereto
  • the components of the hposo e are commonly arranged in a bilayer formation, similar to the hpid arrangement of biological membranes
  • a "small molecule” is defined herein to have a molecular weight below about 500 Daltons
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROl 186
  • cDNAs encoding the PROl 186 polypeptide has been identified and isolated, as disclosed in further detail in the Examples below
  • cDNA clones encoding PROl 186 polypeptide has been deposited with the ATCC
  • the actual nucleotide sequences of the clones can readily be determined by the skilled artisan by sequencing of the deposited clones using routine methods in the art
  • the predicted amino acid sequences can be determined from the nucleotide sequences using routine skill For the PROl 186 and PRO 184 polypeptides and encoding nucleic acids described herein, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time
  • PROl 186 and PRO 184 variants can be prepared by introducing approp ⁇ ate nucleotide changes into the PROl 186 or PRO 184 DNA, and/or by synthesis of the desired PRO 1186 or PRO 184 polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PROl 186 or PRO 184 polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchonng characteristics
  • Variations in the native full-length sequence PRO 1186 or PRO 184 or in various domains of the PRO 1186 or PROl 84 described herein can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U S Patent No 5,364,934 Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO 1186 or PRO 184 that results in a change in the amino acid sequence of the PROl 186 or PRO 184 as compared with the native sequence PROl 186 or PROl 84
  • 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 PROl 186 or PRO 184
  • Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PROl 186 or PRO 184 with that of homologous known protein molecules and minimizing the number of ammo acid sequence changes made in regions of high homology Amin
  • Insertions or deletions may optionally be in the range of about 1 to 5 amino acids The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence
  • PRO 1 186 and PRO 184 polypeptide fragments are provided herein Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein Certain fragments lack amino acid residues that are not essential for a desired biological activity of the PRO 1186 or PRO 184 polypeptide
  • PROl 186 and PRO 184 fragments may be prepared by any of a number of conventional techniques Desired peptide fragments may be chemically synthesized
  • An alternative approach involves generating PROl 186 and PRO 184 fragments by enzymatic digestion, e g , by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment
  • Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR) Ohgonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR
  • PROl 186 and PROl 84 polypeptide fragments share at least one biological and/or immunological activity with the native PRO 1186 or PRO 184 polypeptide shown in Figure 2 (SEQ ID NO 2) and Figure 4 (SEQ ID NO 4), respectively
  • 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
  • Substantial modifications in function or immunological identity of the PRO 1186 or PRO 184 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
  • the variations can be made using methods known in the art such as oligonucleotide-mediated (site- directed) mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis [Carter et al., Nucl. Acids Res.. B:4331 (1986); Zoller et al, Nucl. Acids Res.. 10:6487 (1987)].
  • cassette mutagenesis [Wells et al, Gene, 34:315 (1985)], restriction selection mutagenesis [Wells etal. Philos. Trans. R. Soc. London SerA, 317:415 ( 1986)] or other known techniques can be performed on the cloned DNA to produce the PROl 186 or PR0184 variant DNA
  • Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence
  • preferred scanning ammo acids are relatively small, neutral ammo acids
  • amino acids include alanine, glycme, se ⁇ ne, and cysteine
  • Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science, 244 1081 -1085 (1989)]
  • Alanine is also typically preferred because it is the most common amino acid Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins. (W H Freeman & Co , N Y ), Chothia, J Mol Biol . 150 1 (1976)] If alanine substitution does not yield adequate amounts of variant, an lsote ⁇ c ammo acid can be used
  • Covalent modifications of PRO 1186 and PRO 184 are included within the scope of this invention
  • One type of covalent modification includes reacting targeted amino acid residues of a PRO 1186 or PRO 184 polypeptide with an organic de ⁇ vatizmg agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PROl 186 or PRO 184
  • Denvatization with bifunctional agents is useful, for instance, for crosslinking PRO 1186 or PRO 184 to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO 1186 or anti-PRO 184 antibodies, and vice- versa
  • Commonly used crosslinking agents include, e g , 1,1- b ⁇ s(d ⁇ azoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4- azidosahcylic acid, homobifunctional lmidoesters, including disuccinimidyl esters such as 3,
  • Another type of covalent modification of the PRO 1 186 or PRO 184 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PROl 186 or PROl 84 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PROl 186 or PROl 84
  • the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present
  • Addition of glycosylation sites to the PRO 1186 or PROl 84 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 se ⁇ ne or threonine residue
  • Removal of carbohydrate moieties present on the PRO 1186 or PRO 184 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation
  • Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al , Arch Biochem Biophvs , 259 52 (1987) and by Edge et al , Anal Biochem .
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al , Meth Enzvmol , 138 350 (1987)
  • PRO 1186 or PROl 84 comprises linking the PROl 186 or PROl 84 polypeptide to one of a variety of nonproteinaceous polymers, e g , polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U S Patent Nos 4,640,835, 4,496,689, 4,301 ,144, 4,670,417, 4,791,192 or 4,179,337
  • PRO 1186 or PRO 184 polypeptide of the present invention may also be modified in a way to form a chimeric molecule comprising PROl 186 or PRO 184 fused to another, heterologous polypeptide or amino acid sequence
  • such a chimeric molecule comprises a fusion of the PRO 1186 or PRO 184 polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind
  • the epitope tag is generally placed at the amino- or carboxyl- terminus of the PRO 1186 or PRO 184 polypeptide
  • the presence of such epitope-tagged forms of the PROl 186 or PRO 184 polypeptide can be detected using an antibody against the tag polypeptide
  • provision of the epitope tag enables the PRO 1186 or PRO 184 polypeptide to be readily punfied 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 Bio
  • tag polypeptides include the Flag-peptide [Hopp etal , BioTechnology.6 1204-1210 (1988)], the KT3 epitope peptide [Martin et al , Science, 255 192-194 (1992)], an ⁇ -tubuhn epitope peptide [Skinner et al , J Biol Chem . 266 15163-15166 (1991)], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl Acad Sci USA 87 6393-6397 (1990)]
  • the chimeric molecule may comprise a fusion of the PRO 1186 or PRO 184 polypeptide with an lmmunoglobulin or a particular region of an lmmunoglobulin
  • a bivalent form of the chimeric molecule also referred to as an "lmmunoadhesin )
  • the Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO 1186 or PROl 84 polypeptide in place of at least one variable region within an Ig molecule
  • the lmmunoglobulin fusion includes the hinge, CH2 and CH3 or the hinge, CHI , CH2 and CH3 regions of an IgG 1 molecule
  • PROl 186 or PROl 84 by culturing cells transformed or transfected with a vector containing PROl 186 or PR0184 nucleic acid It is of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PROl 186 or PRO 184
  • the PRO 1186 or PRO 184 polypeptide 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.
  • DNA encoding PRO 1186 or PRO 184 may be obtained from a cDNA library prepared from tissue believed to possess the PRO 1 186 or PRO 184 mRNA and to express it at a detectable level Accordingly, human PRO 1186 or human PROl 84 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as descnbed in the Examples
  • the PRO 1186- or PRO 184-encod ⁇ ng gene may also be obtained from a genomic library or by known synthetic procedures (e g , automated nucleic acid synthesis)
  • Probes such as antibodies to the PRO 1 186 or PRO 184 or ohgonucleotides of at least about 20-80 bases
  • Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al , Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1989)
  • An alternative means to isolate the gene encoding PRO 1186 or PROl 84 is to use PCR methodology [Sambrook et al , supra, Dieffenbach et al , PCR Primer A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)]
  • the oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized
  • the oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened Methods of labeling are ell known
  • Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases
  • Sequence identity at either the amino acid or nucleotide level within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein
  • Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced am o acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al , supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA
  • Host cells are transfected or transformed with expression or cloning vectors described herein for PRO 1 186 or PRO 184 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
  • principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology a Practical Approach, M Butler, ed (IRL Press, 1991 ) and Sambrook et al supra
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells
  • Suitable prokaryotes include but are not limited to eubacte ⁇ a, such as Gram-negative or Gram-positive organisms, for example, Enterobactenaceae such as E coli
  • Various E coli strains are publicly available, such as £ coli YLU strain MM294 (ATCC 31,
  • Strain W31 10 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes.
  • strain W3110 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 W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA El 5 (argF-lac)169 degP ompTkan';
  • E. coli W31 10 strain 37D6 which has the complete genotype tonA pti phoA E15 (argF-lac)]69 degP ompT rbs7 ilvG kan r ;
  • E. coli W31 10 strain 40B4 which is strain 37D6 with a non- kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990.
  • in vitro methods of cloning e.g., PCR or other nucleic acid polymerase reactions, are suitable.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO 1186- or PRO 184-encoding vectors.
  • Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism.
  • Others include Schizosaccharomyces po be (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 etal, J. Bacteriol., 737 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), /-. wickeramii (ATCC 24, 178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg etal, Bio/Technolo gy , 8: 135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna etal, J.
  • 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.
  • Suitable host cells for the expression of glycosylated PRO 1 186 or PRO 184 are derived from multicellular organisms.
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.
  • useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV 1 line transformed by S V40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J. Gen.
  • the nucleic acid (e g cDNA or genomic DNA) encoding PROl 186 or PRO 184 may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression
  • a replicable vector for cloning (amplification of the DNA) or for expression
  • the vector may, for example, be 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
  • DNA is inserted into an appropriate restriction endonuclease s ⁇ te(s) using techniques known in the art
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan
  • the PROl 186 or PROl 84 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 PROl 186- or PROl 84-encod ⁇ ng DNA that is inserted into the vector
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicilhnase, lpp, or heat-stable enterotoxin II leaders
  • the signal sequence may be, e g , the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces ⁇ -factor leaders, the latter described in U S PatentNo 5,010,182), oracid phosphatas
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells Such sequences are well known for a variety of bacteria, yeast, and viruses
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells
  • Selection genes will typically contain a selection gene, also termed a selectable marker
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e g , ampicil n, neomycin, methotrexate, or tetracyc ne, (b) complement auxotrophic deficiencies, or (c) supply critical nutnents not available from complex media, e g , the gene encoding D-alamne racemase for Bacilli
  • selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PROl 186- or PR0184-encod ⁇ ng 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 Uriaub et al , Proc Natl Acad Sci USA.
  • a suitable selection gene for use in yeast is the trpl gene present the yeast plasmid YRp7 [Stinchcomb etal , Nature, 282 39 (1979), Kingsman et al , Gene, 7 141 (1979), Tsche per et al , Gene. JO 157 (1980)]
  • the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No 44076 or PEP4-1 [Jones, Genetics, 85 12 (1977)]
  • Expression and cloning vectors usually contain a promoter operably linked to the PROl 186- or PROl 84- encoding nucleic acid sequence to direct mRNA synthesis.
  • Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems [Chang et al, Nature, 275:615 (1978); Goeddel et al, Nature, 28J_: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 PR01 186 or PR0184.
  • S.D. Shine-Dalgarno
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al, J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al, J. Adv.
  • Enzyme Reg. 2:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde- 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phos ⁇ hoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • PROl 186 or PROl 84 transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (S V40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalo
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription.
  • Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus.
  • Examples include the S V40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the PRO 1186 or PRO 184 coding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs . These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PROl 186 or PR0184. Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PROl 186 or PROl 84 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 of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • Gene expression may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence PROl 186 or PRO 184 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO 1186 or PRO 184 DNA and encoding a specific antibody epitope.
  • PROl 186 or PROl 84 may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells employed in expression of PROl 186 or PROl 84 can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • PROl 186 or PRO 184 may be desired to purify PROl 186 or PRO 184 from recombinant cell proteins or polypeptides.
  • the following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the PROl 186 or PROl 84.
  • Antibodies Some drug candidates for use in the compositions and methods of the present invention are antibodies and antibody fragments which mimic the biological activity of a PROl 186 or PROl 84 polypeptide
  • 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 PROl 186 or PRO 184 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 hemocyamn, serum albumin, bovine thyroglobuhn, and soybean trypsin inhibitor Examples of adjuvants which may be employed include Freund's complete adj uvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate)
  • the immunization protocol may be selected by one or more injections of an immunizing agent and,
  • the antibodies may, alternatively, be monoclonal antibodies
  • Monoclonal antibodies may be prepared using hybndoma methods, such as those described by Kohler and Milstein, Nature.256 495 (1975)
  • a hybndoma method a mouse, hamster, or other approp ⁇ ate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent
  • the lymphocytes may be immunized in vitro
  • the immunizing agent will typically include the PROl 186 or PRO 184 polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hyb ⁇ doma cell fGoding. Monoclonal Antibodies Principles and Practice.
  • 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 hybndoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells
  • the culture medium for the hyb ⁇ domas typically will include hypoxanthme, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium More preferred immortalized cell lines are u ⁇ ne myeloma lines,
  • 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 hybndoma 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 lmmunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Patent No 4,816,567
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of mu ⁇ ne antibodies)
  • the hybndoma cells of the invention serve as a preferred source of such DNA
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce lmmunoglobulin 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 mu ⁇ ne sequences [U S)
  • Non-immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobuhn polypeptide
  • a non-immunoglobuhn 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 lmmunoglobulin light chain and modified heavy chain
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking
  • the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking
  • the antibodies of the invention may further compnse humanized antibodies or human antibodies
  • Humanized forms of non-human (e g , mu ⁇ ne) antibodies are chimeric lmmunoglobulins, lmmunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two. variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.. 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non- human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain.
  • Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239: 1534- 1536 ( 1988)] , by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol Biol, 227:381 (1991); Marks et al., J. Mol Biol. 222:581 (1991)].
  • human antibodies can be made by the introducing of human immunoglobulin loci into transgenic animals, e.g. , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
  • 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 PROl 186 or PRO 184, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature. 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker etal, EMBO J., 10:3655-3659 (1991).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy- chain constant domain, comprising at least part of the hinge. CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.
  • CHI first heavy-chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. , Science.229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • Fab' fragments may be directly recovered from __.. coli and chemically coupled to form bispecific antibodies.
  • Shalaby et al, J. Exp. Med bend 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab') 2 molecule.
  • Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147:60 (1991).
  • Exemplary bispecific antibodies may bind to two different epitopes on a given PROl 186 or PRO 184 polypeptide herein.
  • an anti-PROl 186 or anti-PROl 84 polypeptide arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. , CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular PROl 186 or PROl 84 polypeptide.
  • a triggering molecule such as a T-cell receptor molecule (e.g. , CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PROl 186 or PRO 184 polypeptide. These antibodies possess a PROl 186- or PR0184-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the PROl 186 or PR0184 polypeptide and further binds tissue factor (TF).
  • TF tissue factor
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089].
  • the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676.980.
  • cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See, Caron et al, J. Exp. Med., 176: 1191-1195 (1992) and Shopes, J. Immunol, 148: 2918-2922 (1992).
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research, 53: 2560-2565 (1993).
  • an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See, Stevenson et al, Anti-Cancer Drug Design, 3: 219-230 (1989).
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (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 radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin. enomycin, and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 2,2 Bi, 131 I, I31 In, 90 Y, and 186 Re.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldi thiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis- active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidy
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science. 238: 1098(1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, W094/11026.
  • 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) that is conjugated to a cytotoxic agent (e.g., a radionucleotide).
  • a "receptor” such as streptavidin
  • a ligand e.g., avidin
  • cytotoxic agent e.g., a radionucleotide
  • the antibodies disclosed herein may also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl Acad. Sci. USA. 82: 3688 (1985); Hwang et al, Proc. Natl Acad. Sci. USA. 77: 4030 (1980): and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG- PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin etal, J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.
  • a chemotherapeutic agent such as Doxorubicin is optionally contained within the liposome. See, Gabizon et al, J. National Cancer Inst.. 81(19): 1484 (1989).
  • the proteins disclosed in the present application have been assayed in a panel of 60 tumor cell lines currently used in the investigational, disease-oriented, in vitro drug-discovery screen of the National Cancer Institute (NCI).
  • NCI National Cancer Institute
  • the purpose of this screen is to identify molecules that have cytotoxic and/or cytostatic activity against different types of tumors.
  • NCI screens more than 10,000 new molecules per year (Monks et al., J. Natl. Cancer Inst., 83:757-766 (1991); Boyd, Cancer: Princ. Pract. Oncol Update, 3(10):1-12 ([1989]).
  • the tumor cell lines employed in this study have been described in Monks et al., supra.
  • the cell lines the growth of which has been significantly inhibited by the proteins of the present application are specified in the Examples.
  • cell-based assays and animal models for tumors can also be used to verify the findings of the NCI cancer screen, and to further understand the relationship between the protein identified herein and the development and pathogenesis of neoplastic cell growth.
  • primary cultures derived from tumors in transgenic animals as described below can be used in the cell-based assays herein, although stable cell lines are preferred. Techniques to derive continuous cell lines from transgenic animals are well known in the art (see, e.g., Small et al, Mol Cell Biol.. 5:642-648 [1985]).
  • Animal Models A variety of well known animal models can be used to further understand the role of the molecules identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies, and other agonists of the native polypeptides, including small molecule agonists. The in vivo nature of such models makes them particularly predictive of responses in human patients.
  • Animal models of tumors and cancers e.g., breast cancer, colon cancer, prostate cancer, lung cancer, etc.
  • Non-recombinant animal models include, for example, rodent, e.g., murine models.
  • Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g., colon cancer cells implanted in colonic tissue.
  • standard techniques e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g., colon cancer cells implanted in colonic tissue.
  • mice Probably the most often used animal species in oncological studies are immunodeficient mice and, in particular, nude mice.
  • the autosomal recessive nu gene has been introduced into a very large number of distinct congenic strains of nude mouse, including, for example, ASW, A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL.
  • the cells introduced into such animals can be derived from known tumor/cancer cell lines, such as. any of the above-listed tumor cell lines, and, for example, the B 104- 1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene); ra_--transfected NIH-3T3 cells; Caco-2 (ATCC HTB-37); a moderately well- differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or from tumors and cancers.
  • Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions, involving freezing and storing in liquid nitrogen (Karmali et al., Br. J. Cancer, 48:689-696 [1983]).
  • Tumor cells can be introduced into animals, such as nude mice, by a variety of procedures.
  • the subcutaneous (s.c.) space in mice is very suitable for tumor implantation.
  • Tumors can be transplanted s.c. as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions.
  • tumor tissue fragments of suitable size are introduced into the s.c. space.
  • Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously.
  • Tumor cells can also be injected as subdermal implants. In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s.c. tissue. Boven and Winograd (1991), supra.
  • Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogen was initially isolated), or neu- transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al., Proc. Natl. Acad. Sci. USA. 83:9129-9133 (1986).
  • animal models of colon cancer can be generated by passaging colon cancer cells in animals, e.g. , nude mice, leading to the appearance of tumors in these animals.
  • An orthotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang et al. , Cancer Research, 54:4726-4728 ( 1994) and Too et al, Cancer Research, 55:681-684 (1995). This model is based on the so-called "METAMOUSETM” sold by AntiCancer, Inc., (San Diego, California).
  • Tumors that arise in animals can be removed and cultured in vitro. Cells from the in vitro cultures can then be passaged to animals. Such tumors can serve as targets for further testing or drug screening. Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest. Such passaging techniques can be performed with any known tumor or cancer cell lines.
  • Meth A, CMS4, CMS5, CMS21, and WEHI-164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et al, J. Exp. Med.. 146:720 [1977]), which provide a highly controllable model system for studying the anti-tumor activities of various agents (Palladino et al, J. Immunol, 138:4023-4032 [1987]). Briefly, tumor cells are propagated in vitro in cell culture. Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about 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 investigational tumor model. Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small cell carcinoma of the lung (SCCL).
  • SCCL small cell carcinoma of the lung
  • This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture (Zupi et al, Br. J. Cancer. 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, Hae ostasis. 16:300-320 [1986]).
  • One way of evaluating the efficacy of a test compound in an animal model on an implanted tumor is to measure the size of the tumor before and after treatment.
  • the size of implanted tumors has been measured with a slide caliper in two or three dimensions.
  • the measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted into the corresponding volume by using a mathematical formula.
  • the measurement of tumor size is very inaccurate.
  • the therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay.
  • Another important variable in the description of tumor growth is the tumor volume doubling time.
  • Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc. 6th Int.
  • Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals.
  • Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g., baboons, chimpanzees and monkeys.
  • Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and Wanger, U.S. Patent No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g., Van der Putten et al, Proc. Natl Acad. Sci. USA.
  • transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals").
  • the transgene can be integrated either as a single transgene, or in concatamers, e.g. , head-to-head or head-to-tail tandems. Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al. , Proc. Natl Acad. Sci. USA. 89:6232- 636 (1992).
  • the expression of the transgene in transgenic animals can be monitored by standard techniques. For example. Southern blot analysis or PCR amplification can be used to verify the integration of the transgene.
  • the level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or imrnunocytochemistry. The animals are further examined for signs of tumor or cancer development.
  • SCC feline oral squamous cell carcinoma
  • Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy of cats, accounting for over 60% of the oral tumors reported in this species. It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor.
  • These tumors are usually not amenable to surgery, primarily because of the anatomy of the feline oral cavity. At present, there is no effective treatment for this tumor.
  • each cat Prior to entry into the study, each cat undergoes complete clinical examination, biopsy, and is scanned by computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors are excluded from the study. The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor, the animals may not be able to feed themselves.
  • CT computed tomography
  • Each cat is treated repeatedly, over a longer period of time. Photographs of the tumors will be taken daily during the treatment period, and at each subsequent recheck.
  • CT scans and thoracic radiograms are evaluated every 8 weeks thereafter. The data are evaluated for differences in survival, response and toxicity as compared to control groups. Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span.
  • fibrosarcoma adenocarcinoma
  • lymphoma adenocarcinoma
  • chrondroma adenocarcinoma of dogs, cats
  • baboons a preferred model as its appearance and behavior are very similar to those in humans.
  • the use of this model is limited by the rare occurrence of this type of tumor in animals.
  • Screening assays for drug candidates are designed to identify compounds that competitively bind or complex with the receptor(s) of the polypeptides identified herein, or otherwise signal through such receptor(s).
  • 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)peptide- immunoglobulin fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.
  • a receptor of a polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments .
  • Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the polypeptide and drying.
  • an immobilized antibody e.g., a monoclonal antibody, specific for the polypeptide to be immobilized can be used to anchor it to a solid surface.
  • the assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component.
  • the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected.
  • the detection of label immobilized on the surface indicates that complexing occurred.
  • complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
  • the candidate compound interacts with but does not bind to a particular receptor, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions.
  • assays include traditional approaches, such as, cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns.
  • protein-protein interactions can be monitored by using a yeast-based genetic systemdescribed by Fields and co-workers [Fields and Song, Nature (London). 340:245-246 (1989); Chien etal, Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)] as disclosed by Chevray and Nathans [Proc. Natl Acad. Sci. USA.
  • yeast GAL4 Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain.
  • 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-/ ⁇ cZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction.
  • Colonies containing interacting polypeptides are detected with a chromogenic substrate for ⁇ -galactosidase.
  • a complete kit (MATCHMAKERTM) for identifying protein-protein interactions between two specific proteins using the two- hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
  • compositions The polypeptides of the present invention, agonist antibodies specifically binding proteins identified herein, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions.
  • the smallest inhibitory fragment which specifically binds to the binding domain of the target protein is preferred.
  • peptide molecules can be designed which retain the ability to bind the target protein sequence.
  • Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e.g., Marasco et al. Proc. Natl. Acad. Sci. USA. 90:7889-7893 [1993]).
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • an agent that enhances its function such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • Therapeutic formulations of the polypeptides identified herein, or agonists thereof are prepared for storage by mixing the active ingredient having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences. 16th edition, Osol, A. ed. [1980]), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent.
  • cytotoxic agent cytokine or growth inhibitory agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
  • compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • the polypeptides of the present invention and their agonists may be used to treat various tumors, e.g., cancers.
  • exemplary conditions or disorders to be treated include benign or malignant tumors (e.g., renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas; sarcomas; glioblastomas; and various head and neck tumors); leukemias and lymphoid malignancies; other disorders such as neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders.
  • benign or malignant tumors e.g., renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas; sarcom
  • the anti-tumor agents of the present invention are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intracerobrospinal, intraocular, intraarterial, intralesional, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • 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 & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may precede, or follow administration of the anti-tumor agent of the present invention, or may be given simultaneously therewith.
  • the anti-cancer agents of the present invention may be combined with an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
  • the anti-cancer agents herein are co-administered with a growth inhibitory agent.
  • the growth inhibitory agent may be administered first, followed by the administration of an anti-cancer agent of the present invention.
  • simultaneous administration or administration of the anti-cancer agent of the present invention first is also contemplated. Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the antibody herein.
  • an anti-tumor agent for the prevention or treatment of disease, 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. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell W. "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development. Yacobi et al., eds., Pergamon Press, New York 1989, pp. 42-96.
  • an antitumor agent is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays. Guidance as to particular dosages and methods of delivery is provided in the literature; see, for example, U.S. Pat. Nos.
  • 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 an anti-tumor agent of the present invention.
  • 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.
  • 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.
  • DNA60621-1516 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 methionine codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under consideration.
  • the nucleotides following the first ATG must code for at least 35 unambiguous amino acids without any stop codons.
  • the candidate sequence is not scored.
  • the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals.
  • Incyte EST cluster sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homologies.
  • EST expressed sequence tag
  • the homology search was performed using the computer program BLAST or BLAST2 (Altshul et al , Methods in Enzymology. 266:460-480 (1996)).
  • Clone DN A60621-1516 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 91-93 and ending at the stop codon at nucleotide positions 406-408 (Figure 1)
  • the predicted polypeptide precursor is 105 amino acids long ( Figure 2, SEQ ID NO 2)
  • the full-length PROl 186 protein shown in Figure 2 has an estimated molecular weight of about 1 1,715 daltons and a pi of about 9 05
  • Analysis of the full-length PRO 1186 sequence shown in Figure 2 (SEQ ID NO 2) evidences the presence of important polypeptide domains as shown in Figure 2, wherein the locations given for those important polypeptide domains are approximate as described above
  • Analysis of the full-length PROl 186 sequence evidences the presence of a signal peptide from about amino acid 1 to about ammo acid 19, a tyrosine kinase phosphorylation site from about amino acid 88 to about amino acid 95, and N-my ⁇ stoylation sites from about am o
  • 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
  • the tubes were incubated at 37°C for one hour. A total of 1.0 ⁇ l RQ1 DNase was added, followed by incubation at 37°C for 15 minutes. A total of 90 ⁇ l TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) was added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a MICROCON-50TM ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, a total of 100 ⁇ l TE was added, then 1 ⁇ l of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR IITM.
  • the probe was run on a TBE/urea gel. A total of 1-3 ⁇ l of the probe or 5 ⁇ l of RNA Mrk III was added to 3 ⁇ l of loading buffer. After heating on a 95 °C heat block for three minutes, the gel was immediately placed on ice. The wells of gel were flushed, and the sample was loaded and run at 180-250 volts for 45 minutes. The gel was wrapped in plastic wrap (SARANTM brand) and exposed to XAR film with an intensifying screen in a -70 °C freezer one hour to overnight.
  • SARANTM brand plastic wrap
  • the slides were removed from the freezer, placed on aluminum trays, and thawed at room temperature for 5 minutes. The trays were placed in a 55 °C incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H 2 0). After deproteination in 0.5 ⁇ g/ml proteinase K for 10 minutes at 37°C (12.5 ⁇ l of 10 mg/ml stock in 250 ml prewarmed RNAse-free RNAse buffer), the sections were washed in 0.5 x SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%, and 100% ethanol, 2 minutes each.
  • the slides were deparaffinized, placed in SQ H 2 0, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time.
  • the sections were deproteinated in 20 ⁇ g/ml proteinase K (500 ⁇ l of 10 mg/ml in 250 ml
  • RNase-free RNase buffer 37 °C, 15 minutes
  • 8 x proteinase K 100 ⁇ l in 250 ml Rnase buffer, 37 °C, 30 minutes
  • Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above.
  • DNA60621-15I6 Black Mamba Homolog expression in 2 year old chimp ovary
  • the most intense signal was seen over granulosa cells, especially those nearest to the oocyte, in primary follicles in both the chimp and cyno monkey ovary.
  • a weaker signal was seen over granulosa cells in primordial follicles and in the cumulus oophorus.
  • a significant signal was observed in the ovarian cortex, especially around small, involuted corpora lutea (cyno monkey sample) and in poorly defined clusters of cells at the vascular pole of the ovary. No signal was seen over fallopian tubes or uterine tissue.
  • EXAMPLE 3 Use of PROl 186 or PROl 84 as a Hybridization Probe The following method describes use of a nucleotide sequence encoding PROl 186 or PROl 84 as a hybridization probe.
  • DNA comprising the coding sequence of full-length or mature PRO 1186 or PRO 184 (as shown in Figure 1 and 3, respectively, SEQ ID NOS : 1 and 3, respectively) or a fragment thereof is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PROl 186 or PROl 84) in human tissue cDNA libraries or human tissue genomic libraries.
  • Hybridization and washing of filters containing either library DNAs is performed under the following high- stringency conditions .
  • Hybridization of radiolabeled probe derived from the gene encoding a PRO 1186 or PRO 184 polypeptide to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42°C for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1 x SSC and 0.1 % SDS at 42°C.
  • DNAs having a desired sequence identity with the DNA encoding full-length native sequence can then be identified using standard techniques known in the art.
  • EXAMPLE 4 Expression of PROl 186 or PROl 84 in E. coli This example illustrates preparation of an unglycosylated form of PROl 186 or PRO 184 by recombinant expression in E. coli.
  • the DNA sequence encoding PROl 186 or PROl 84 is initially amplified using selected PCR primers.
  • the primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector.
  • a variety of expression vectors may be employed.
  • An example of a suitable vector is pBR322 (derived from E. coli: see Bolivar et al, Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance.
  • the vector is digested with restriction enzyme and dephosphorylated.
  • the PCR amplified sequences are then ligated into the vector.
  • the vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a poly-His leader (including the first six STTI codons, poly-His sequence, and enterokinase cleavage site), the PRO 1186 or PRO 184 coding region, lambda transcriptional terminator, and an argU gene.
  • the ligation mixture is then used to transform a selected E. coli strain using the methods described in
  • 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.
  • the cells After culturing the cells for several more hours, the cells can be harvested by centrifugation.
  • the cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized PROl 186 or PROl 84 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
  • PRO 1186 or PRO 184 may be expressed in E. coli in a poly-His tagged form, using the following procedure.
  • the DNA encoding PROl 186 or PROl 84 is initially amplified using selected PCR primers.
  • the primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase.
  • the PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E.
  • Transformants are first grown in LB containing 50 mg/ml carbenicillin at 30 °C with shaking until an OD 600 of 3-5 is reached.
  • Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH 4 ) 2 S0 4 , 0.71 g sodium citrate»2H 2 0, 1.07 g KC1, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 1 10 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgS0 4 ) and grown for approximately 20-30 hours at 30°C with shaking. Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.
  • CRAP media prepared by mixing 3.57 g (NH 4 ) 2 S0 4 , 0.71 g sodium citrate»2H 2 0, 1.07 g KC1, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 1 10 mM M
  • E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer.
  • Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1M and 0.02 M, respectively, and the solution is stirred overnight at 4°C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization.
  • the solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min.
  • the supernatant is 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 is loaded onto a 5 ml Qiagen Ni 2+ -NTA metal chelate column equilibrated in the metal chelate column buffer.
  • the column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4.
  • the protein is eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein are pooled and stored at 4°C. Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.
  • the proteins are refolded by diluting the 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 are chosen so that the final protein concentration is between 50 to 100 micrograms/ml.
  • the refolding solution is stirred gently at 4°C for 12-36 hours.
  • the refolding reaction is quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3).
  • the solution is filtered through a 0.22 micron filter and acetonitrile is added to 2-10% final concentration.
  • the refolded protein is chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A 280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.
  • Fractions containing the desired folded PROl 186 or PRO 184 polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered. PROl 186 and PRO 184 were successfully expressed in E. coli in a poly-His tagged form by the above procedure.
  • EXAMPLE 5 Expression of PROl 186 or PROl 84 in mammalian cells This example illustrates preparation of a potentially glycosylated form of PROl 186 or PROl 84 by recombinant expression in mammalian cells
  • the vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector
  • the PRO 1186 or PRO 184 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PRO 1186 or PRO 184 DNA using ligation methods such as described in Sambrook et al supra
  • the resulting vector is called pRK5-PR01186 or pRK5-PR0184
  • 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 ⁇ g pRK5-PRO 1186 or pRK5-PRO 184 DNA is mixed with about 1 ⁇ g DNA encoding the VA RNA gene [Thimmappaya etal , Cell 31 543 (1982)] and dissolved in 500 ⁇ l of 1 mM T ⁇ s-HCl, 0 1 mM EDTA, 0 227 M CaCl 2
  • 500 ⁇ l of 50 mM HEPES pH 7 35
  • 280 mM NaCl 1 5 mM NaP0 4
  • the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ⁇ Ci/ml 35 S-cyste ⁇ ne and 200 ⁇ Ci/ml 35 S-meth ⁇ on ⁇ ne
  • the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel
  • the processed gel may be dned and exposed to film for a selected period of time to reveal the presence of the PROl 186 or PRO 184 polypeptide
  • the cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays
  • PROl 186 or PRO 184 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac etal , Proc Natl Acad Sci .12 7575 (1981) 293 cells are grown to maximal density in a spinner flask and 700 ⁇ g pRK5-PRO 1186 or pRK5-PRO 184 DNA is added The cells are first concentrated from the spinner flask by centrifugation and washed with PBS The DNA-dextran precipitate is incubated on the cell pellet for four hours The cells are treated with 20% glycerol for 90 seconds washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 ⁇ g/ml bovine insulin and 0 1 ⁇ g/ml bovine transfernn After about four days, the conditioned media is centrifuged and filtered to remove cells and debris The sample containing expressed PRO 1186 or PRO 184 can then be concentrated and pun
  • PROl 186 or PROl 84 can be expressed in CHO cells
  • the pRK5-PR01186 or ⁇ RK5-PR0184 can be transfected into CHO cells using known reagents such as CaP0 4 or DEAE-dextran
  • the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35 S-meth ⁇ on ⁇ ne
  • the culture medium may be replaced with serum free medium
  • the cultures are incubated for about 6 days, and then the conditioned medium is harvested
  • the medium containing the expressed PRO 1186 or PRO 184 polypeptide can then be concentrated and purified by any selected method
  • Epitope-tagged PRO 1186 or PRO 184 may also be expressed in host CHO cells
  • the PRO 1186 or PRO 184 may be subcloned out of the ⁇ RK5 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 PRO 1186 or PRO 184 insert can then be subcloned into a S V40 driven vector containing a selection marker such as DHFR for selection of stable clones
  • the CHO cells can be transfected (as described above) with the SV40 driven vector Labeling may be performed, as described above, to venfy expression
  • the culture medium containing the expressed poly-His tagged PRO 1186 or PRO 184 can then be concentrated and purified by any selected method, such as by N ⁇ ,+ -chelate affinity chromatography
  • PRO 1186 or PRO 184 may also be expressed in CHO and/or COS cells by
  • the proteins are expressed as an IgG construct (lmmunoadhesin), in which the coding sequences for the soluble forms (e g , extracellular domains) of the respective proteins are fused to an IgGl constant region sequence containing the hinge, CH2 and CH2 domains and/or as a poly-His tagged form
  • CHO expression vectors are constructed to have compatible restriction sites 5 and 3' of the DNA of interest to allow the convenient shuttling of cDNA's
  • the vector used in 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
  • the ampules containing the plasmid DNA are thawed by placement into a water bath and mixed by vortexing
  • the contents are pipetted into a centrifuge tube containing 10 ml of media and centrifuged at 1000 rpm for 5 minutes
  • the supernatant is aspirated and the cells are resuspended in 10 ml of selective media (0 2 ⁇ m filtered PS20 with 5% 0 2 ⁇ m diafiltered fetal bovine serum)
  • the cells are then aliquoted into a 100 ml spinner containing 90 ml of selective media After 1-2 days, the cells are 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 are seeded with 3 x 10 5 cells/ml
  • the cell media is exchanged with fresh media by centrifugation and resuspension in production medium
  • the pH is adjusted as necessary to keep it at around 7.2.
  • the cell culture is harvested by centrifugation and filtering through a 0.22 ⁇ m filter.
  • the filtrate is either stored at 4 C C or immediately loaded onto columns for purification.
  • the proteins are purified using a Ni 2+ -NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni 2+ -NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole.
  • the highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
  • lmmunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows.
  • the conditioned medium 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 ⁇ l 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 is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
  • yeast expression vectors are constructed for intracellular production or secretion of PRO 1186 or PRO 184 from the ADH2/GAPDH promoter.
  • DNA encoding PROl 186 or PRO 184 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PROl 186 or PRO 184.
  • DNA encoding PROl 186 or PRO 184 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PROl 186 or PRO 184 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 PROl 186 or PROl 84.
  • yeast cells such as yeast strain AB 110
  • yeast cells 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 PRO 1186 or PRO 184 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 PROl 186 or PROl 84 may further be purified using selected column chromatography resins.
  • EXAMPLE 7 Expression of PROl 186 or PROl 84 in Baculovirus-Infected Insect Cells The following method describes recombinant expression in Baculovirus-infected insect cells. The sequence coding for PROl 186 or PRO 184 is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG) .
  • plasmids may be employed, including plasmids derived from commercially available plasmids such as pVLl 393 (Novagen). Briefly, the sequence encoding PROl 186 or PR0184 or the desired portion of the coding sequence of PROl 186 or PRO 184 (such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular) is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.
  • pVLl 393 Novagen
  • Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA
  • Expressed poly-His tagged PRO 1186 or PRO 184 can then be purified, for example, by Ni 2+ -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 2 ; 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
  • sonication buffer 25 ml Hepes, pH 7.9; 12.5 mM MgCl 2 ; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40;
  • a Ni 2+ -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 A 280 with loading buffer, at which point fraction collection is started.
  • 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.
  • a secondary wash buffer 50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0
  • 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 Ni 2+ -NTA-conjugated to alkaline phosphatase
  • Fractions containing the eluted His )0 -tagged PRO 1186 or PRO 184, respectively, are pooled and dialyzed against loading buffer.
  • purification of the IgG tagged (or Fc tagged) PRO 1186 or PRO 184 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.
  • 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 Spodopterafrugiperda ("Sf9”) cells (ATCC CRL 1711), using Lipofectin (Gibco BRL).
  • Sf9 Spodopterafrugiperda
  • pb.PH.IgG and pb.PH.His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences.
  • the cells are grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). Cells are incubated for 5 days at 28 °C. The supernatant is harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Cells are incubated for 3 days at 28°C.
  • MOI multiplicity of infection
  • the supernatant is harvested and the expression of the constructs in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml of Ni 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 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 C. The supernatant is harvested and filtered. Batch binding and SDS-PAGE analysis is 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.
  • the protein construct is purified using a Ni 2+ -NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM.
  • the conditioned media is pumped onto a 6 ml Ni 2+ -NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml min. at 4 C C.
  • 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.
  • lmmunoadhesin (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.
  • the column 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.
  • PEG polyacrylamide gel
  • N-terminal amino acid sequencing by Edman degradation.
  • a modified baculovirus procedure may be used incorporating high-5 cells.
  • the DNA encoding the desired sequence is amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector.
  • suitable systems such as Pfu (Stratagene)
  • epitope tags include poly- His tags and immunoglobulin tags (like Fc regions of IgG).
  • a variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIE 1 - 1 (Novagen) .
  • the pIE 1 - 1 and pIE 1 -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus iel promoter in stably- transformed insect cells (1 ).
  • 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.
  • the desired sequence or the desired portion of the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions.
  • the 5' primer may incorporate flanking (selected) restriction enzyme sites.
  • the product is then digested with those selected restriction enzymes and subcloned into the expression vector.
  • derivatives of pIE 1 - 1 can include the Fc region of human IgG (pb.PH.IgG) or an 8 histidine (pb.PH.His) tag downstream (3'-of) the desired sequence.
  • the vector construct is sequenced for confirmation.
  • High-5 cells are grown to a confluency of 50% under the conditions of, 27 °C, no C0 2 , NO pen/strep.
  • 30 ⁇ g of pIE based vector containing the sequence is mixed with 1 ml Ex-Cell medium (Media: Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401-78P (note: this media is light sensitive)), and in a separate tube, 100 ⁇ l ofCellFectin (CellFEC ⁇ N (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.
  • Ex- Cell media 8 ml of Ex- Cell media is added to the 2 ml of DNA/CellFECTIN mix and this is layered on high-5 cells that have been washed once with Ex-Cell media. The plate is then incubated in darkness for 1 hour at room temperature. The DNA/CellFECTIN mix is then aspirated, and the cells are washed once with Ex-Cell to remove excess CellFECTIN, 30 ml of fresh Ex-Cell media is added and the cells are incubated for 3 days at 28°C.
  • the supernatant is harvested and the expression of the sequence in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml of Ni 2+ -NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
  • the conditioned media from the transfected cells (0.5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0.22 micron filters.
  • the protein comprising the sequence is purified using a Ni 2+ -NTA column (Qiagen).
  • imidazole is added to the conditioned media to a concentration of 5 mM.
  • the conditioned media is pumped onto a 6 ml Ni 2+ -NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 48°C.
  • 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.
  • lmmunoadhesin (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 had 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.
  • PROl 186 was expressed using the above baculovirus procedure employing high-5 cells.
  • EXAMPLE 8 Preparation of Antibodies that Bind PROl 186 or PROl 84 This example illustrates preparation of monoclonal antibodies which can specifically bind PROl 186 or PROl 84. Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding. supra. Immunogens that may be employed include purified PROl 186 or PRO 184, fusion proteins containing PROl 186 or PROl 84, and cells expressing recombinant PROl 186 or PRO 184 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 PROl 186 or PRO 184 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms.
  • the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads.
  • MPL-TDM adjuvant Ribi Immunochemical Research, Hamilton, MT
  • the immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-PROl 186 or anti-PROl 84 antibodies.
  • the animals "positive" for antibodies can be injected with a final intravenous injection of PROl 186 or PR0184.
  • the mice are sacrificed and the spleen cells are harvested.
  • the spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.l, available from ATCC, No. CRL 1597.
  • the fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
  • HAT hyperxanthine, aminopterin, and thymidine
  • the hybridoma cells will be screened in an ELISA for reactivity against PROl 186 or PRO 184. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PROl 186 or PRO 184 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-PROl 186 or anti-PR0184 monoclonal antibodies.
  • 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.
  • PROl 186 or PROl 84 Polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, pro-PR01186 or pro-PR0184 polypeptide, mature PROl 186 or mature PRO 184 polypeptide, or pre-PR01186 or pre-PR0184 polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PROl 186 or PR0184 polypeptide of interest. In general, an immunoaffinity column is constructed by covalently coupling the anti-PROl 186 or anti-PRO 184 polypeptide antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared frommouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSETM (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • a chromatographic resin such as CnBr-activated SEPHAROSETM (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • Such an immunoaffinity column is utilized in the purification of the PRO 1186 or PRO 184 polypeptide by preparing a fraction from cells containing the PROl 186 or PROl 84 polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble PROl 186 or PR0184 polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
  • a soluble PROl 186 or PRO 184 polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of the PROl 186 or PR0184 polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/PROl 186 or antibody/PR0184 polypeptide binding (e.g., a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and the PROl 186 or PRO 184 polypeptide is collected.
  • a low pH buffer such as approximately pH 2-3
  • a chaotrope such as urea or thiocyanate ion
  • This invention is particularly useful for screening compounds by using PRO 1186 or PRO 184 polypeptides or a binding fragment thereof in any of a variety of drug screening techniques.
  • the PROl 186 or PRO 184 polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO 1186 or PRO 184 polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • the present invention provides methods of screening for drugs or any other agents which can affect a PROl 186 or PRO 184 polypeptide-associated disease or disorder. These methods comprise contacting such an agent with a PRO 1186 or PRO 184 polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the PROl 186 or PRO 184 polypeptide or fragment, or (ii) for the presence of a complex between the PROl 186 or PROl 84 polypeptide or fragment and the cell, by methods well known in the art. In such competitive binding assays, the PROl 186 or PR0184 polypeptide or fragment is typically labeled.
  • the free PROl 186 or PROl 84 polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to the PRO 1186 or PRO 184 polypeptide or to interfere with the PRO 1186 or PRO 184 polypeptide/cell complex.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on September 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to a PROl 186 or PROl 84 polypeptide, the peptide test compounds are reacted with the PROl 186 or PROl 84 polypeptide and washed. Bound PROl 186 or PROl 84 polypeptide is detected by methods well known in the art. Purified PROl 186 or PROl 84 polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding a PRO 1186 or PRO 184 polypeptide specifically compete with a test compound for binding to the PROl 186 or PROl 84 polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with a PRO 1186 or PRO 184 polypeptide.
  • EXAMPLE 11 Rational Drug Design The goal of rational drug design is to produce structural analogs of a biologically active polypeptide of interest (i.e., a PROl 186 or PR0184 polypeptide) or of small molecules with which they interact, e.g., agonists, antagonists, or inhibitors. Any of these examples can be used to fashion drugs which are more active or stable forms of the PROl 186 or PRO 184 polypeptide or which enhance or interfere with the function of the PROl 186 or PRO 184 polypeptide in vivo (c.f., Hodgson, Bio/Technology, 9: 19-21 (1991)).
  • the three-dimensional structure of the PRO 1186 or PRO 184 polypeptide, or of a PRO 1186 or PROl 84 polypeptide-inhibitor complex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the PROl 186 or PRO 184 polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of the PROl 186 or PRO 184 polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous PROl 186 or PROl 84 polypeptide-like molecules or to identify efficient inhibitors.
  • Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton and Wells, Biochemistry.31 :7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda et al. J. Biochem.. 113:742-746 (1993).
  • a target-specific antibody selected by functional assay, as described above, and then to solve its crystal structure.
  • This approach in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
  • anti-ids anti-idiotypic antibodies
  • PROl 186 or PROl 84 polypeptide may be made available to perform such analytical studies as X-ray crystallography.
  • knowledge of the PRO 1 186 or PRO 184 polypeptide amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.
  • EXAMPLE 12 In Vitro Antitumor Assay The antiproliferative activity of the PROl 186 and PRO 184 polypeptides was determined in the investigational, disease-oriented in vitro anti-cancer drug discovery assay of the National Cancer Institute (NCI), using a sulforhodamine B (SRB) dye binding assay essentially as described by Skehan et al, J. Natl. Cancer Inst., 82:1 107-1112 (1990). The 60 tumor cell lines employed in this study ("the NCI panel"), as well as conditions for their maintenance and culture in vitro have been described by Monks etal, J. Natl. Cancer Inst., 83 :757-766 ( 1991 ).
  • NCI National Cancer Institute
  • SRB sulforhodamine B
  • This screen is to initially evaluate the cytotoxic and/or cytostatic activity of the test compounds against different types of tumors (Monks et al, supra; Boyd, Cancer: Princ. Pract. Oncol Update, 3(101:1-12 [1989]).
  • Cells from approximately 60 human tumor cell lines were harvested with trypsin/EDTA (Gibco), washed once, resuspended in IMEM and their viability was determined.
  • the cell suspensions were added by pipet (100 ⁇ l volume) into separate 96-well microtiter plates. The cell density for the 6-day incubation was less than for the 2-day incubation to prevent overgrowth. Inoculates were allowed a preincubation period of 24 hours at 37 °C for stabilization. Dilutions at twice the intended test concentration were added at time zero in 100 ⁇ l aliquots to the microtiter plate wells (1 :2 dilution). Test compounds were evaluated at five half-log dilutions (1000 to 100,000- fold). Incubations took place for two days and six days in a 5% C0 2 atmosphere and 100% humidity.
  • a test sample is considered positive if it shows at least 40% growth inhibitory effect at one or more concentrations.
  • PROl 186 95 nM 2 NSCL NCI-H226
  • PROl 186 95 nM 2 Colon Colo205
  • PROl 186 8.1 nM 6 Leukemia K-562 PROl 186 8.1 nM 6 NSCL HOP-62
  • PROl 186 8.1 nM 6 Colon Colo205 HCC-2998
  • PRO 184 0.65 nM 6 NSCL A549/ATCC; EKVX PRO 184 0.65 nM 6 Colon HT29 PRO 184 0.65 nM 6 CNS SF-268; SF-295; SNB-19 PRO 184 0.65 nM 6 Melanoma MALME-3M; Ml 4; SK-MEL-28; UACC-25 * cytotoxic (% growth less than 0)

Abstract

The present invention concerns methods and compositions for inhibiting neoplastic cell growth. In particular, the present invention concerns antitumor compositions and methods for the treatment of tumors. The invention further concerns screening methods for identifying growth inhibitory, e.g., antitumor compounds. 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

METHODS AND COMPOSITIONS FOR INHIBITING NEOPLASTIC CELL
GROWTH
FIELD OF THE INVENTION
The present invention concerns methods and compositions for inhibiting neoplastic cell growth In particular, the present invention concerns antitumor compositions and methods for the treatment of tumors The invention further concerns screening methods for identifying growth inhibitory e g antitumor compounds
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 C n . 43 7 (1993))
Cancer is characterized by the 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
Despite recent advances in cancer therapy, there is a great need for new therapeutic agents capable of inhibiting neoplastic cell growth Accordingly, it is the objective of the present invention to identify compounds capable of inhibiting the growth of neoplastic cells such as cancer cells
SUMMARY OF THE INVENTION
A Embodiments
The present invention relates to methods and compositions for inhibiting neoplastic cell growth More particularly, the invention concerns methods and compositions for the treatment of tumors, including cancers, such as breast, prostate, colon, lung, ovarian, renal and CNS cancers, leukemia, melanoma, etc , in mammalian patients, preferably humans
In one aspect, the present invention concerns compositions of matter useful for the inhibition of neoplastic cell growth comprising an effective amount of a PRO 1 186 or PRO] 84 polypeptide as herein defined or an agonist thereof, in admixture with a pharmaceutically acceptable carrier In a preferred embodiment the composition of matter comprises a growth inhibitory amount of a PROl 186 or PRO 184 polypeptide, or an agonist thereof In another preferred embodiment, the composition compπses a cytotoxic amount of a PROl 186 or PRO 184 polypeptide, or an agonist thereof Optionally, the compositions of matter may contain one or more additional growth inhibitory and or cytotoxic and/or other chemotherapeutic agents.
In a further aspect, the present invention concerns compositions of matter useful for the treatment of a tumor in a mammal comprising a therapeutically effective amount of a PRO 1186 or PRO 184 polypeptide as herein defined, or an agonist thereof. The tumor is preferably a cancer. In another aspect, the invention concerns a method for inhibiting the growth of a tumor cell comprising exposing the cell to an effective amount of a PROl 186 or PROl 84 polypeptide as herein defined, or an agonist thereof. In a particular embodiment, the agonist is an anti-PRO 1186 or anti-PRO 184 agonist antibody. In another embodiment, the agonist is a small molecule that mimics the biological activity of a PROl 186 or PRO 184 polypeptide. The method may be performed in vitro or in vivo. In a still further embodiment, the invention concerns an article of manufacture comprising:
(a) a container;
(b) a composition comprising an active agent contained within the container; wherein the composition is effective for inhibiting the neoplastic cell growth, e.g., growth of tumor cells, and the active agent in the composition is a PROl 186 or PROl 84 polypeptide as herein defined, or an agonist thereof; and (c) a label affixed to said container, or a package insert included in said container referring to the use of said PROl 186 or PRO 184 polypeptide or agonist thereof, for the inhibition of neoplastic cell growth, wherein the agonist may be an antibody which binds to the PROl 186 or PROl 84 polypeptide. In a particular embodiment, the agonist is an anti-PROH86 or anti-PR0184 agonist antibody. In another embodiment, the agonist is a small molecule that mimics the biological activity of a PROl 186 or PRO 184 polypeptide. Similar articles of manufacture comprising a PRO 1186 or PRO 184 polypeptide as herein defined, or an agonist thereof in an amount that is therapeutically effective for the treatment of tumor are also within the scope of the present invention. Also within the scope of the invention are articles of manufacture comprising a PRO 1186 or PRO 184 polypeptide as herein defined, or an agonist thereof, and a further growth inhibitory agent, cytotoxic agent or chemotherapeutic agent.
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 PROl 186 or PR0184 polypeptide.
In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule encoding a PROl 186 or PROl 84 polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).
In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length PROl 186 or PRO 184 polypeptide cDNA as disclosed herein, the coding sequence of a PRO 1186 or PRO 184 polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PROl 186 or PROl 84 polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).
In a further aspect, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a).
Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PROl 186 or PROl 84 polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide are disclosed herein. Therefore, soluble extracellular domains of the herein described PROl 186 or PRO 184 polypeptides are contemplated.
Another embodiment is directed to fragments of a PRO 1186 or PRO 184 polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PROl 186 or PRO 184 polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PROl 186 or anti-PR0184 antibody or as antisense oligonucleotide probes. Such nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 110 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternat vely at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternat: vely at least about 160 nucleotides in length, alternatively at least about 170 nucleotides in length, alternat: vely at least about 180 nucleotides in length, alternatively at least about 190 nucleotides in length, alternat; vely at least about 200 nucleotides in length alternatively at least about 250 nucleotides in length, alternat vely at least about 300 nucleotides in length, alternatively at least about 350 nucleotides in length, alternat: vely at least about 400 nucleotides in length, alternatively at least about 450 nucleotides in length, alternat; vely at least about 500 nucleotides in length, alternatively at least about 600 nucleotides in length, alternat: vely at least about 700 nucleotides in length. alternatively at least about 800 nucleotides in length, alternatively at least about 900 nucleotides in length and alternatively at least about 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length. It is noted that novel fragments of a PRO 1186 or PRO 184 polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO 1186 or PRO 184 polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PROl 186 or PROl 84 polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such PROl 186 or PROl 84 polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PROl 186 or PROl 84 polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PROl 186 or PRO 184 polypeptide fragments that comprise a binding site for an anti-PROl 186 or anti-PRO 184 antibody. In another embodiment, the invention provides isolated PRO 1186 or PRO 184 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
In a certain aspect, the invention concerns an isolated PROl 186 or PROl 84 polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a PROl 186 or PRO 184 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 PROl 186 or PROl 84 polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid 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 PROl 186 or PRO 184 polypeptide comprising an amino acid sequence scoring at least about 80% positives, alternatively at least about 81% positives, alternatively at least about 82% positives, alternatively at least about 83% positives, alternatively at least about 84% positives, alternatively at least about 85% positives, alternatively at least about 86% positives, alternatively at least about 87% positives, alternatively at least about 88% positives, alternatively at least about 89% positives, alternatively at least about 90% positives, alternatively at least about 91% positives, alternatively at least about 92% positives, alternatively at least about 93% positives, alternatively at least about 94% positives, alternatively at least about 95% positives, alternatively at least about 96% positives, alternatively at least about 97% positives, alternatively at least about 98% positives and alternatively at least about 99% positives when compared with the amino acid sequence of a PRO 1186 or PRO 184 polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein.
In a specific aspect, the invention provides an isolated PROl 186 or PRO 184 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 PROl 186 or PROl 84 polypeptide and recovering the PROl 186 or PROl 84 polypeptide from the cell culture. Another aspect the invention provides an isolated PROl 186 or PRO 184 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 PROl 186 or PROl 84 polypeptide and recovering the PROl 186 or PROl 84 polypeptide from the cell culture. In yet another embodiment, the invention concerns agonists of a native PRO 1 186 or PRO 184 polypeptide as defined herein. In a particular embodiment, the agonist is an anti-PROl 186 or anti-PROl 84 antibody or a small molecule.
In a further embodiment, the invention concerns a method of identifying agonists to a PRO 1186 or PRO 184 polypeptide which comprise contacting the PRO 1186 or PRO 184 polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO 1186 or PRO 184 polypeptide. Preferably, the PRO 1186 or PRO 184 polypeptide is a native PRO 1186 or PRO 184 polypeptide.
In a still further embodiment, the invention concerns a composition of matter comprising a PROl 186 or PRO 184 polypeptide, or an agonist of a PRO 1186 or PRO 184 polypeptide as herein described, or an anti-PRO 1186 or anti-PRO 184 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 PRO 1186 or PRO 184 polypeptide, or an agonist thereof as hereinbefore described, or an anti-PRO 1 186 or anti-PRO 184 antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO 1 186 or PRO 184 polypeptide, an agonist thereof or an anti-PROl 186 or anti-PROl 84 antibody. In additional 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, __.. coli, yeast, or Baculovirus-infected insect cells. A process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
In other embodiments, the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence. Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin
In yet another embodiment, the invention provides an antibody which specifically binds to any of the above or below described polypeptides Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody In yet other embodiments, the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a nucleotide sequence (SEQ ID NO 1) of a native sequence PROl 186 cDNA, wherein SEQ ID NO 1 is a clone designated herein as "DNA60621-1516"
Figure 2 shows the amino acid sequence (SEQ ID NO 2) derived from the coding sequence of SEQ ID NO 1 shown in Figure 1
Figure 3 shows a nucleotide sequence (SEQ ID NO 3) of a native sequence PRO 184 cDNA, wherein SEQ ID NO 3 is a clone designated herein as "DNA28500" Figure 4 shows the amino acid sequence (SEQ ID NO 4) derived from the coding sequence of SEQ ID
NO 3 shown in Figure 3
DETAILED DESCRIPTION OF THE INVENTION
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 (i 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 allelic 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 methionine residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionine 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/or 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 likely by no more than about 5 amino acids at either end of the domain as initially identified herein Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain extracellular domain boundary as identified 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 of 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 may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e g , Nielsen et al , Prot Eng . 10 1-6 (1997) and von Heinje et al , Nucl Acids Res . 144683-4690 (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% ammo acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence Ordinarily, a PRO polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herein Ordinarily, PRO variant polypeptides are at least about 10 ammo acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 ammo acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 300 amino acids in length, or more
As shown below, Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program In addition, Tables 2A-2D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid sequence identity (Tables 2C-2D) using the ALIGN-2 sequence comparison computer program, wherein "PRO" represents the amino acid sequence of a hypothetical 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 hypotheUcal 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 -8 /* value of a match with a stop */ int day [26] [26] = {
/* A "B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/ /* A*/ (2, 0, -2, 0,0,-4, 1,-1,-1, 0,-1,-2, -1, 0,_M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3, 0}, /*B */ {0, 3, -4, 3,2,-5,0, 1,-2,0,0,-3,- 2, 2,_M,-1, 1, 0, 0, 0, 0,-2,-5, 0,-3, 1}, /*C */ {-2, -4, 15 ,,-5,-5,-4,-3,-3,-2, 0,-5,-6 ,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0, 0,-5}, /*D */ {0, 3, -5, 4, 3,-6, 1, 1,-2, 0, 0,-4,- 3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 2},
/*E*/ (0, 2, -5, 3, 4,-5, 0, 1,-2, 0, 0,-3,- 2, 1,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 3}, /* p */ {-4, -5, -4, -6,-5,9,-5,-2, 1,0,-5,2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5},
/*G*/ { i. 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*/ {-i. 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 {-i, -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},
/*] */ {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}, ι*κ*ι {-i. 0, -5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1,_M,-1, 1, 3, 0, 0, 0,-2,-3, 0,-4, 0}, l*L*l {-2, -3 -6, -4,-3, 2,-4,-2, 2, 0,-3, 6 4,-3,_M,-3,-2,-3,-3,-l, 0, 2,-2, 0,-1,-2}, l*M*l {-i. -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},
/*0*/ { 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*/ I 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 */ { 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},
/*u*/ {0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/* v */ (0, -2 -2, -2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2,_M,-l ,-2,-2,-1,0,0,4,-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},
1*1*1 {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 8 /* penalty for a gap */ .define DINS1 1 /* penalty per base */
#define PINSO 8 /* penalty for a gap */
#define PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP], /* size of jmp (neg for dely) */ unsigned short x[MAXJMP]; / '* base no. of jmp in seq x */
}; / '* limits seq to 2A16 -1 struct diag { int score, /* score at last jmp */ long offset, /* offset of prev block */ short ljmp; /* current jmp index */ struct jmp JP; /* list of jmps */
}; struct path { int spc; /* number of leading spaces */ short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (last elem before gap) */
}; char *ofιle; /* output file name */ char *namex[2]; seq names: getseqs() */ char *prog; prog name for err msgs */ char *seqx[2]; /* seqs: getseqs() */ int dmax; best diag: nw() */ int dmaxO; final diag */ int dna; /* set if dna: maιn() */ int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* total gaps in seqs */ int lenO, lenl ; /* seq lens */ int ngapx, ngapy; /* total size of gaps */ int smax; /* max score: nw() */ int *xbm; /* bitmap for matching */ long offset; /* current offset in jmp file */ struct diag *dx, /* holds diagonals */ struct path pp[2]; /* holds path for seqs */ char *calloc(), *malloc(., *mdex(), *strcpy(); char *getseq(). *g_calloc();
Page 1 of nw.h /* Needleman-Wunsch alignment program
*
* usage: progs filel file2
* where filel and file2 are two dna or two protein sequences.
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with ',', '>' or ' < ' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align.out"
*
* The program may create a tmp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 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'))|(1<<('Q'-'A'))
}; m
Figure imgf000013_0001
ain int ac; char *av[];
{ prog = = av[0]; if (ac ! = 3){ fpπntf(stderr. usage: %s filel file2\n", prog); fpπntf(stderr. where filel and file2 are two dna or two protein sequencesΛn"); fpnntf(stderr,"The sequences can be in upper- or lower-case\n"); fpπntf(stderr,"Any lines beginning with ';' or ' < ' are ignored. n"); fpπntf(stderr, "Output is in the file \"ahgn.out\"\n"); exιt(l);
} namex[0] = av[l]; namex[l] = av[2]; seqx[0] = getseq(namex[0], &len0); seqx[l] = getseq(namex[l], &lenl); xb = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ ofile = "align.out"; /* output file */ nw(), /* fill in the matrix, get the possible jmps */ readjmpsO; /* get the actual jmps */ printQ, /* print stats, alignment */ cleanup(O), /* unlink any tmp files */
Page 1 of nw.c /* do the alignment, return best score- mainO
* 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() n 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, msl, /* insertion penalties */ register id, /* diagonal index */ register •J. /* 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("_o get colO", lenl + 1, sizeof(int)); coll = (int *)g_calloc("to get coll ", lenl + 1, sizeof(int)); insO = (dna)? DINSO : PINSO; msl = (dna)? DINS1 : PINS1; smax = -10000; if (endgaps) { for (col0[0] = dely[0] = -msO, yy = 1; yy < = lenl; yy+ +) { colOfyy] = dely[yy] = col0[yy-l] - msl; ndelyfyy] = yy,
} col0[0] = 0, /* Waterman Bull Math Biol 84 */
} else for (yy = 1 ; yy < = lenl; yy+ +) delyfyy] = -insO;
/* fill in match matrix */ for (px = seqx[0], xx = 1; xx < = lenO; px+ + , xx+ +) { /* initialize first entry in col */ if (endgaps) { if (xx = = 1) coll[0] = delx = -(msO + insl); else coll[0] = delx = col0[0] - msl , ndelx = xx;
} else { coll[0] = 0; delx = -msO ndelx = 0;
Page 2 of nw.c ...nw for (p> = seqx[l], yy = 1; yy < = lenl, py + + , yy + +) {
Figure imgf000015_0001
mis + = (xbm[*px-'A']&xbm[*py 'A'])'' DMAT DMIS, else mis + = _day[*px-'A'][*py-'A'],
/* update penalty for del in x seq,
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps */ if (endgaps 1 1 ndely[yy] < MAXGAP) { if (colO[yy] - insO > = dely[yy]) { dely[yy] = colO[yy] - (lnsO + msl), ndely [yy] = 1, } else { dely[yy] -= msl, ndely [yy] + + ,
} } else { if (colO[yy] - (insO + insI) > = dely[yy]) { dely[yy] = colO[yy] - (insO + insI), ndely[yy] = 1 , } else ndely[yy] + +,
}
/* update penalty for del in y seq;
* favor new del over ongong del */ if (endgaps | | ndelx < MAXGAP) { if (coll[yy-l] - insO > = delx) { delx = coll[yy-l] - (insO+insI), ndelx = 1 ,
Figure imgf000015_0002
ndelx+ + , }
} else { if (coll[yy-l] - (insO+insI) > = delx) { delx = coll [yy-l] - (insO+insI), ndelx = 1,
} else ndelx+ + ,
}
/* pick the maximum score; we're favoring * mis over any del and delx over dely
*/
Page 3 of nw.c ...nw id = xx - yy + lenl 1 if (mis > = delx && mis > = dely[yy])
Figure imgf000016_0001
else if (delx > = dely[yy]) { coll[yy] = delx IJ = dx[ιd] ljmp if (dx[ιd] jp n[0] && ('dna | | (ndelx > = MAXJMP && xx > dx[ιd] jp x[ιj] +MX) | | mis > dx[ιd] score+DINSO)) { dx[ιd] ιjmp+ + , ιf (+ + ιj > = MAXJMP) { wπtejmps(ιd)
Figure imgf000016_0002
dx[ιd] offset = offset offset + = sιzeof(struct jmp) + sιzeof(offset),
} } dx[ιd] jp n[ιj] = ndelx, dx[ιd] jp x[ιj] = xx, dx[ιd] score = delx,
} else { coll [yy] = dely[yy],
Figure imgf000016_0003
if (dx[ιd] jp n[0] && ('dna | | (ndely[yy] > = MAXJMP
&& xx > dx[ιd] jp x[ιj] + MX) | | mis > dx[ιd] score+DINSO)) { dx[ιd] ιjmp+ + , ιf (+ +ιj > = MAXJMP) { wπtejmps(ιd), ij = dx[ιd] ljmp = 0 dx[ιd] offset = offset, offset + = sιzeof(struct jmp) + sιzeof(offset),
}
} dxfid] jp n[ιj] = -ndely [yy], dx[ιd] jp x[ιj] = xx, dx[ιd] score = dely[yy],
} if (xx = = lenO && yy < lenl) {
/* last col */ if (endgaps) coll [yy] -= insO+insI *(lenl-yy) if (col 1 [yy] > smax) { smax = coll[yy], dmax = id, } } } if (endgaps && xx < lenO) coll [yy 1] -= ιnsO+ιnsl*(lenO-xx), if (coll [yy 1] > smax) { smax = coll[yy-l], dmax = id,
} tmp = colO, colO = coll, coll = tmp,
}
(void) free((char *)ndely) (void) free((char *)dely), (void) free((char *)colO), (void) free((char *)coll),
Page 4 of nw.c * pπnt() — only routine visible outside this module
*
* static-
* getmat() — trace back best path, count matches: pπnt()
* pr_ahgn() - print alignment of described in array p[]: pπnt()
* dumpblockO — dump a block of lines with numbers, stars- pr_alιgn()
* nums() — put out a number line dumpblockO
* putline() — 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, " < fιrst sequence: %s (length = %d)\n", namex[0], lenO); φπntf(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 */ pp[0].spc = firstgap = lenl - dmax - 1 : ly -= pp[0].spc;
} else if (dmax > lenl - 1) { /* leading gap in y */ pp[l].spc = firstgap = dmax - (lenl - 1); lx -= pp[l].spc;
} if (dmaxO < lenO - 1) { /* trailing gap in x */ lastgap = lenO - dmaxO -1 ; lx -= lastgap;
} else if (dmaxO > lenO - 1) { /* trailing gap in y */ lastgap = dmaxO - (lenO - 1); ly -= lastgap;
} getmat(lx, ly, firstgap, lastgap); pr_ahgn();
Page 1 of nwprint.c * trace back the best path, count matches */ static getmatøx, ly, firstgap, lastgap) getmat int lx, 1> , /* "core" (minus endgaps) */ int firstgap, lastgap, /* leading trailing overlap */ int nm, lO, ii , sizO, sizl, char outx[32], double pet, register nO, nl ; register char *p0, *pl ;
/* get total matches, score */
Figure imgf000018_0001
pO = seqx[0] + pp[l].spc, pl = seqx[l] + pp[0].spc; nO = pp[l].spc + 1, nl = pp[0].spc + 1 ; nm = 0, while ( *p0 && *pl ) {
Figure imgf000018_0002
else { if (xbm[*pO-'A']&xbm[*pl-'A']) nm+ + ; if (nO+ + = = pp[0].x[ιO])
Figure imgf000018_0003
if (nl + + -= = pp[l].x[ιl]) sizl = pp[l].n[ιl + +], p0+ + , pi + + ;
}
/* pet homology
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core */ if (endgaps) lx = (lenO < lenl)? lenO : lenl ; else lx = (lx < ly)? lx : ly; pet = 100.*(double)nm/(double)lx, fpnntf(fx, "\n"), fpπntf(fx, " < %d match%s in an overlap of %d % 2f percent similarity \n" nm, (nm = = 1)? " " : "es", lx, pet),
Page 2 of nwprint.c fprintf(fx, " < gaps in first sequence: %d", gapx); ...getmat if (gapx) {
(void) sprintf(outx, " (%d %s%s)" , ngapx, (dna)? "base" : "residue" , (ngapx = = 1)? "s"); fprintf(fx, " %s", outx); fprintf(fx, " , gaps in second sequence: %d", gapy); if (gapy) {
(void) sprintf(outx, " (%d %s%s)", ngapy, (dna)? "base" : "residue", (ngapy 1)? ": "s");
_printf(fx, " s", outx);
} if (dna) fprintf(fx,
"\n < score: %d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n" smax, DMAT, DMIS, DINSO, DINS1); else fprintf(fx,
"\n< score: %d (Davhoff PAM 250 matrix, gap penalty = %d + %d per residue)\n", smax, PINSO, PINS.); if (endgaps) fprintf(fx,
" < endgaps penalized, left endgap: %d %s%s, right endgap: %d %s%s\n" . firstgap, (dna)? "base" : "residue", (firstgap = = 1)? " " : "s", lastgap, (dna)? "base" : "residue" , (lastgap = = 1)? " " : "s"); else fprintf(fx, " < endgaps not penalized\n");
static nm; /* matches in core — for checking */ static lmax; /* lengths of stripped file names */ static UP]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static ni[2]; /* current ele number — for gapping */ static siz[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */ static char out[2][P_LINE]; /* output line */ static char star[P LINE]; /* set by stars() */
/*
* print alignment of described in struct path ppQ • */ static pr_align() pr align
{ int nn; /* char count */ int more; register i; for (i = 0, lmax = 0; i < 2: i+ +) { nn = stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1 ; siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i];
Page 3 of nwprint.c for (nn = nm = 0, more = 1 , more, ) { ...pr align for (l = more = 0, l < 2, ι + +) {
/*
* do we have more of this sequence' */ if C*ps[ι])
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[.] x[ιj[ι]]) { /*
* we need to merge all gaps
* at this location */ sιz[ι] = pp[ι] n[ιj[ι] + +], while (nι[ι] = = pp[ι] x[ιj[ι]]) sιz[ι] + = pp[ι] n[ιj[ι] + +]
} nι[ι] + + ,
}
} 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()
*/ static dumpblockO dumpblock
{ register i, for (ι = 0, l < 2, ι + +) *po[ι]- = \0 ,
Page 4 of nwprmt. c .dumpblock
(void) putc('\n'. , fx), for (1 = 0, l < 2,ι++){ if (*out[ι] && (*out[ι] ' = ' || *(po[ι]) ' = ' ')){ if (i = = 0) nums(ι), if (l == 0&&*out[l]) stars(), putlιne(ι), if (I = = 0 && *out[l]) fpπntf(fx, star); if(ι == 1) nums(ι),
}
}
}
/*
* put out a number line dumpblockO
*/ static nums IX) nums int IX, /* index in out[] holding seq line */
{ char nlιne[P_LINE], register ι,j; register char *pn, *px, *py, for (pn = nline , l = 0; l < lmax+P SPC; ι+ + ,pn++)
*pn = = ' ', for (1 = nc[ιx], py = out[ιx], *py; py+ + , pn++) { if(*py == " || *py = = '-')
*pn = ' ', else { if (ι%10 = = 0 I I (l = = 1 && nc[ιx] ' = 1)) { j = (ι < 0)9 -i - i; for (px = pn, j, j /= 10, px-)
*px =j%10 + '0', if (l < 0)
*px = '-',
} else
*pn ι+ + ;
} }
*pn = '\0', nc[ιx] = l, for (pn = nline; *pn, pn+ +) (void) putc(*pn, fx), (void) putc('\n', fx), }
/*
* put out a line (name, [num], seq, [num]) dumpblockO
*/ static putime(ιx) putline
Figure imgf000021_0001
Page5ofnwprint.c ...putline int i; register char *px; for (px = namexfix], i = 0; *px && *px != ':'; px+ + , i++)
(void) putc(*px, fx); for (: i < lmax + P_SPC; i + +)
(void) putc(' ', fx);
/* these count from 1 :
* niQ is current element (from 1)
* ncQ is number at start of current line */ for (px = out[ix]; *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 starso stars
{ int i; register char *p0, *pl, ex, *px; if (!*out[0] 11 (*out[0] == ' '&& *(po[0]) == ' ') 11 !*out[l] 11 (*out[l] == " '&& *(po[l]) == ' ')) return; px = star; for (i = lmax+P SPC; i; i-) *px++ = ' '; for (pO = out[0], pl = out[l]; *p0 && *pl; p0+ + , pl + +) { if (isalpha(*pO) && isalpha(*pl)) { if (xbm[*pO-'A']&xbm[*pl-'A']) { ex = '*'; nm++;
} else if (!dna && _day[*pO-'A'][*pl-'A'] > 0) ex = '.'; else ex = ' ';
} else ex = ' '; *px+ + = ex;
}
*px++ = '\n'; *px = '\0';
Page 6 of nwprint.c /*
* strip path or prefix from pn, return len: pr_alιgn()
*/ static stπpname(pn) Stripname char *pn; /* file name (may be path) */
{ register char *px, *py; py = 0; for (px = pn; *px; px+ +) if (*px = = V) py = px + 1 ; if (PY)
(void) strcpy(pn, py); return(strlen(pn)) ;
Page 7 of nwprint.c * cleanupO - cleanup any tmp file
* getseqO — read in seq, set dna, len, maxlen
* g_calloc() — callocO with error checkm
* readjmpsO — get the good jmps, from tmp file if necessary
* wπtejmpsO — write a filled array of jmps to a tmp file: nw() */
^include "nw.h" ^include < sys/file.h > char *jname = "/tmp/homgX /* tmp file for jmps */
FILE *fj; int cleanupO; /* cleanup tmp file */ long lseek(); z*
* remove any tmp file if we blow
*/ cleanup(ι) cleanup int { if (fj)
(void) unlιnk(jname), exit(ι);
/*
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with ' ; ' , ' < ' , or ' > '
* seq in upper or lower case */ char * getseq(fιle, len) getseq char *file; /* file name */ int *len; /* seq len */ char lme[1024], *pseq register char *px, *py, int natgc, tlen;
FILE *fp; if ((fp = fopen(file, "r")) = = 0) { fpπntf(stderr, " %s: can't read %s\n", prog, file); exιt(l);
} tlen = natgc = 0; while (fgets(lιne, 1024, fp)) { if (*lme = = ' ;' 1 1 *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 bytes for 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 (*Ime == ',' || *lme == '< | | *hne == '>') continue, for (px = line, *px ' = '\n', px+ +) { if (ιsupper(*px))
*py+ + = *px, else if (ιslower(*px))
*py++ = toupper(*px), if (ιndex("ATGCU",*(py-l))) natgc+ + , } }
*py++ = '\0', *py = '\0', (void) fclose(fp), dna = natgc > (tlen3), return(pseq+4),
} char * g_calloc(msg, nx, sz) g_CallθC char *msg, /* program, calling routine */ int nx, sz, /* number and size of elements */
{ char *px, *calloc(), if ((px = calloc((unsigned)nx, (unsigned)sz)) = = 0) { if (*msg) { fpπntf(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 imgf000025_0001
register I, j, xx; if(fj){
(void) fclose(fj), if ((fd = open(jname, 0_RDONLY, 0)) < 0) { fpπntf(stderr, "%s- can't open() %s\n", prog, j name), cleanup(l), } } for (I = lO = ii = 0, dmaxO = dmax, xx = lenO, , ι + +) { while (1) { for (j = dx[dmax] ijmp, j > = 0 && dx[dmax] jp.xD] > = xx, J--)
Page2ofnwsubr.c ...readjmps if 0 < 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].ιjmp = MAXJMP- 1,
} else break;
} if (l > = JMPS) { fpπntf(stderr, " %s: too many gaps in ahgnmentW, prog); cleanup(l); } if 0 > = 0) {
Figure imgf000026_0001
if (siz < 0) { /* gap m second seq */
Figure imgf000026_0002
/* 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)? -siz : MAXGAP; ιl + + ;
} else if (siz > 0) { /* gap in first seq */
Figure imgf000026_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, ι0~; j < lO; j + + , ι0~) { i = pp[0].n[)]; pp[0].nU] = ρp[0].n[ι0]; pp[0] .n[ιO] = i; i = pp[0].xD]; pp[0].xD] = pp[0].x[ι0], pp[0].x[ι0] = i; } for (j = 0, ιl-; j < ιl ; j + + , il-) { i = pp[l].n[j]; pp[l].nh] = pp[l].n[ιl]; pp[l].n[ιl] = i; i = pp[i].χD]; pp[i].χDl = pp[i].χ[ιi]; PP-UAii] = i;
} if (fd > = 0)
(void) close(fd); if (fj) {
(void) unlinkfjname); fj = 0; offset = 0;
}
Page 3 of nwsubr.c /*
* write a filled jmp struct offset of the prev one (if any): nw()
*/ wπtejmps(ιx) writejmps int IX;
{ char *mktemp(), if (!fj) { if (mktemp(jname) < 0) { fpπntf(stderr, " %s. can't mktempO s\n", prog, jname); cleanup(l);
} if ((fj = fopen(jname, "w")) = = 0) { fpπnt stderr, " %s: can't write %s\n", prog, jname), exιt(l); } }
(void) fwπte((char *)&dx[ιx].jp, sizeof(structjmp), 1 , fj); (void) fwπte((char *)&dx[ιx]. offset, sizeof(dx[ιx] . offset), 1 , fj),
Page4 ofnwsubr.c Table 2A
PRO xxxxxxxxxxxxxxx (Length = 15 ammo acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 ammo acids)
% ammo acid sequence identity =
(the number of identically matchmg amino 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 15 = 33.3%
Table 2B
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 ammo 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 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%
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 identit " with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the 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 amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or 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 ammo acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % am o acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A As examples of % amino acid sequence identity calculations, Tables 2A-2B demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein" to the amino acid sequence designated "PRO" Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program However, % amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al , Nucleic Acids Res . 25 3389-3402 (1997)) The NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov, or otherwise obtained from the National Institute of Health, Bethesda, MD NCBI- BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi- pass e value -= 0 01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62
In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A
In addition, % amino acid sequence identity may also be determined using the WU-BLAST-2 computer program (Altschul et al , Methods in Enzvmology, 266 460-480 ( 1996)) Most of the WU-BLAST-2 search parameters are set to the default values Those not set to default values, ( 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 amino acids residues between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (. e , the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest For example, in the statement "a polypeptide comprising an amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B", the amino acid sequence A is the comparison amino acid sequence of interest and the am o acid sequence B is the amino acid sequence of the PRO polypeptide of interest
"PRO variant polynucleotide' or "PRO variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Ordinarily, a PRO variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity with a 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, alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively 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 or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuπng alignment, including any algorithms needed to achiev e maximal alignment over the full-length of the sequences being compared For purposes herein, however, % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc , and the source code shown in Table 1 has been filed with user documentation in the U S Copyright Office, Washington D C , 20559, where it is registered under U S Copyright Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech, Inc , South San Francisco, California or may be compiled from the source code provided in Table 1 The ALIGN 2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary
For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program" s alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C. As examples of % nucleic acid sequence identity calculations, Tables 2C-2D demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO- DNA". Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 ( Altschul et al. , Nucleic Acids Res., 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov, or otherwise obtained from the National Institute of Health, Bethesda, MD. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix - BLOSUM62.
In situations where NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI- BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
In addition, % nucleic acid sequence identity values may also be generated using the WU-BLAST- 2 computer program (Altschul et al.. Methods in Enzvmology, 266:460-480 ( 1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e. , the adjustable parameters, are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. For purposes herein, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide-encoding nucleic acid and the comparison nucleic acid molecule of interest (. 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) and Figure
4 (SEQ ID NO 4), 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 amino acid residue of interest or are a preferred substitution (as defined in
Table 3 below) of the amino acid residue of interest
For purposes herein, the % value of positives of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % positives to, with, or against a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of amino acid residues scoring a positive value by the sequence alignment program ALIGN- 2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % positives of A to B will not equal the % positives of B to A
"Isolated", when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a component of its natural environment Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes In preferred embodiments the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stam Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PROl 186 or PR0184 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 1 186 or PRO 184 polypeptide or an "isolated' nucleic acid molecule encoding an anti-PROl 186 or anti-PROl 84 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 PROl 186- or PROl 84-encodιng nucleic acid or the natural source of the anti-PROl 186- or anti- PRO 184 encoding nucleic acid Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated An isolated PROl 186- or PROl 84-encodιng nucleic acid molecule or an isolated anti-PROl 186- or anti-PROl 84-encodιng 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 1186- or PRO 184- encoding nucleic acid molecule or from the anti-PROl 186- or anti-PRO 184-encodιng nucleic acid molecule as it exists in natural cells However, an isolated nucleic acid molecule encoding a PROl 186 or PR0184 polypeptide or an isolated nucleic acid molecule encoding an anti-PROl 186 or anti-PROl 84 antibody includes PROl 186 or PR0184-nucleιc acid molecules or anti-PROl 186- or anti-PRO 184-nucleιc acid molecules contained in cells that ordinarily express PROl 186 or PRO 184 polypeptides or anti-PRO 1186 or anti-PROl 84 antibodies where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells The ter ' control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence For example, DNA for a presequence or secretory leader is operably linked to DNA for a PROl 186 or PRO 184 polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice
The term "antibody" is used in the broadest sense and specifically covers, for example, single anti- PROl 186 or anti-PRO 184 and anti-PRO 1186 or anti-PRO 184 monoclonal antibodies (including agonist antibodies), anti-PROl 186 and anti-PRO 184 antibody compositions with polyepitopic specificity, single chain anti- PRO 1186 and anti-PRO 184 antibodies, and fragments of anti-PRO 1186 and anti-PRO 184 antibodies (see below) The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, J e , the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature The higher the degree of desired homology between the probe and hybπdizable sequence the higher the relative temperature that can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so For additional details and explanation of stringency of hybridization reactions, see, Ausubel et al Current Protocols in Molecular Biology (Wiley Interscience Publishers. 1995)
"Stringent conditions or 'high-stringency conditions' , as defined herein, may be identified by those that (1 ) employ low ionic strength and high temperature for washing, for example, 0 015 M sodium chloπde/0 0015 M sodium cιtrate/0 1 % sodium dodecyl sulfate at 50 °C, (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0 1 % bovine serum albumιn/0 1% Fιcoll/0 1% polyvιnylpyrrolιdone/50mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C, or (3) employ 50% formamide, 5 x SSC (0 75 M NaCl, 0 075 M sodium citrate), 50 mM sodium phosphate (pH 6 8), 0 1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ ml), 0 1 % SDS, 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 CC, 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 of washing solution and hybridization conditions (e g , temperature, ionic strength, and % SDS) less stringent than those described above An example of moderately stringent conditions is overnight incubation at 37 °C in a solution comprising 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7 6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37C-50°C The skilled artisan will recognize how to adjust the temperature, ionic strength, etc as necessary to accommodate factors such as probe length and the like
The term "epitope tagged' when used herein refers to a chimeric polypeptide comprising a PROl 186 or PRO 184 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)
As used herein, the term "lmmunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin' ) with the effector functions of lmmunoglobulin constant domains Structurally, the lmmunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (. e , is "heterologous"), and an lmmunoglobulin constant domain sequence The adhesin part of an lmmunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand The lmmunoglobulin constant domain sequence m the lmmunoadhesin may be obtained from any lmmunoglobulin, such as IgG- 1 , IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA 2), IgE, IgD or IgM "Active" or "activity" for the purposes herein refers to form(s) of PROl 186 or PROl 84 which retain a biological and or an immunological activity of native or naturally-occurring PROl 186 or PROl 84, wherein "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally- occurring PRO 1 186 or PRO 184 other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO 1 186 or PRO 184 and an "immunological" activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally- occurring PROl 186 or PROl 84.
"Biological activity" in the context of an antibody or another agonist that can be identified by the screening assays disclosed herein {e.g., an organic or inorganic small molecule, peptide, etc.) is used to refer to the ability of such molecules to invoke one or more of the effects listed herein in connection with the definition of a "therapeutically effective amount." In a specific embodiment, "biological activity" is the ability to inhibit neoplastic cell growth or proliferation. A preferred biological activity is inhibition, including slowing or complete stopping, of the growth of a target tumor (e.g., cancer) cell. Another preferred biological activity is cytotoxic activity resulting in the death of the target tumor (e.g. , cancer) cell. Yet another preferred biological activity is the induction of apoptosis of a target tumor (e.g., cancer) cell.
The phrase "immunological activity" means immunological cross-reactivity with at least one epitope of a PROl 186 or PRO 184 polypeptide.
"Immunological cross-reactivity" as used herein means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO 1186 or PRO 184 polypeptide having this activity with polyclonal antisera raised against the known active PROl 186 or PROl 84 polypeptide. Such antisera are prepared in conventional fashion by injecting goats or rabbits, for example, subcutaneously with the known active analogue in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freunds. The immunological cross-reactivity preferably is "specific", which means that the binding affinity of the immunologically cross-reactive molecule (e.g., antibody) identified, to the corresponding PROl 186 or PROl 84 polypeptide is significantly higher (preferably at least about 2-times, more preferably at least about 4-times, even more preferably at least about 6-times, most preferably at least about 8-times higher) than the binding affinity of that molecule to any other known native polypeptide.
"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, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
"Treatment" is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented In tumor (e g , cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e g , radiation and/or chemotherapy The "pathology" of cancer includes all phenomena that compromise the well-being of the patient This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, etc
An "effective amount" of a polypeptide disclosed herein or an agonist thereof, in reference to inhibition of neoplastic cell growth, is an amount capable of inhibiting, to some extent, the growth of target cells The term includes an amount capable of invoking a growth inhibitory, cytostatic and/or cytotoxic effect and/or apoptosis of the target cells An "effective amount" of a PRO 1186 or PRO 184 polypeptide or an agonist thereof for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
A "therapeutically effective amount", in reference to the treatment of tumor, refers to an amount capable of invoking one or more of the following effects (1) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest, (2) reduction in the number of tumor cells, (3) reduction in tumor size,
(4) inhibition (. e , reduction, slowing down or complete stopping) of tumor cell infiltration into peripheral organs,
(5) inhibition (. e , reduction, slowing down or complete stopping) of metastasis, (6) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor, and/or (7) relief, to some extent, of one or more symptoms associated with the disorder A "therapeutically effective amount" of a PRO 1186 or PRO 184 polypeptide or an agonist thereof for purposes of treatment of tumor may be determined empirically and in a routine manner
A "growth inhibitory amount" of a PROl 186 or PROl 84 polypeptide or an agonist thereof is an amount capable of inhibiting the growth of a cell, especially tumor, e g , cancer cell, either in vitro or in vivo A "growth inhibitory amount" of a PRO 1 186 or PRO 184 polypeptide or an agonist thereof for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
A "cytotoxic amount" of a PROl 186 or PRO 184 polypeptide or an agonist thereof is an amount capable of causing the destruction of a cell, especially tumor, e g , cancer cell, either in vitro or in vivo A "cytotoxic amount" of a PROl 186 or PRO 184 polypeptide or an agonist thereof for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
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 , Iπι, I125, Y90 and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof A "chemotherapeutic agent" is a chemical compound useful in the treatment of tumor, e g , cancer
Examples of chemotherapeutic agents include adπamycin, doxorubicin. epirubicin, 5-fluorouracιl, cytosme arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e g , pac taxel (Taxol, Bristol- Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rhόne-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide. mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide. daunomycin, carminomycin, aminopterin, dactinomycin. mitomycins, esperamicins (see, U.S. Patent No. 4,675,187), melphalan and other related nitrogen mustards. Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapristone.
A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. Thus, the growth inhibitory agent is one which significantly reduces the percentage of the target cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M- phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine. mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds.. Chapter 1, entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami et al., (WB Saunders: Philadelphia, 1995), especially p. 13.
The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet- growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G- CSF); interleukins (ILs) such as IL-1, IL-l α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11 , IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions. 14, pp. 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. 247-267, Humana Press (1985). The prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, glycosylated prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above
The term agonist' is used in the broadest sense and includes any molecule that mimics a biological activity of a native PROl 186 or PROl 84 polypeptide disclosed herein Suitable agonist molecules specifically include agonist antibodies or antibody fragments fragments or amino acid sequence variants of native PRO 1186 or PRO 184 polypeptides, peptides, small organic molecules, etc Methods for identifying agonists of a PROl 186 or PROl 84 polypeptide may comprise contacting a tumor cell with a candidate agonist molecule and measuring the inhibition of tumor cell growth
"Chronic' administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature
"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc Preferably, the mammal is human Administration "in combination with" one or more further therapeutic agents includes simultaneous
(concurrent) and consecutive administration in any order
"Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed Often the physiologically acceptable carrier is an aqueous pH buffered solution Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptide, proteins, such as serum albumin, gelatin, or lmmunoglobulins, hydrophilic polymers such as polyvinylpyrro done, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disacchaπdes, and other carbohydrates including glucose, mannose, or dextπns, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming countenons such as sodium, and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™
"Native antibodies" and "native lmmunoglobulins" 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 lmmunoglobulin isotypes Each heavy and light chain also has regularly spaced mtrachain disulfide bridges Each heavy chain has at one end a \ arable domain (VH) followed by a number of constant domains Each light chain has a variable domain at one end (VL) and a constant domain at its other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains
The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen However, the variability is not evenly distributed throughout the variable domains of antibodies It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervanable regions both in the light-chain and the heavy-chain variable domains The more highly conserved portions of variable domains are called the framework regions (FR) The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, Kabat etal , NIH Publ No 91-3242, Vol I, pages 647-669 ( 1991 )) The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity The term "hypervanable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-bind g The hypervanable region comprises amino acid residues from a "complementarity determining region or "CDR" (i e , residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain, Kabat et al , Sequences of Proteins of Immunological Interest. 5th Ed Public Health Service, National Institute of Health, Bethesda, MD [1991 ]) and/or those residues from a "hypervanable loop" (i e , residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI ), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain, Clothia and Lesk, J Mol Biol , 196 901-917 [1987]) "Framework" or "FR" residues are those variable domain residues other than the hypervanable region residues as herein defined
"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies (Zapata et al , Protein Eng . 8(10) 1057-1062 [1995]), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments
Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the 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 configuration 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 comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH 1 ) of the heavy chain Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain 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')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known The ' light chains of antibodies (lmmunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains
Depending on the amino acid sequence of the constant domain of their heavy chains, lmmunoglobulins can be assigned to different classes There are five major classes of lmmunoglobulins IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e g , IgGl IgG2, IgG3, IgG4, IgA, and IgA2
The 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 antigenic site Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybπdoma culture, uncontaminated by other lmmunoglobulins The modifier ' monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybπdoma method first described by Kohler etal , Nature, 256495 [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 descnbed 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 (lmmunoglobulins) 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 et al , Proc Natl Acad Sci USA. 81 6851-6855 [1984])
"Humanized' forms of non-human (e g , munne) antibodies are chimeric lmmunoglobulins, lmmunoglobulin 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 lmmunoglobulin For the most part, humanized antibodies are human lmmunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity In some instances, Fv FR residues of the human lmmunoglobulin are replaced by corresponding non-human residues Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences These modifications are made to further refine and maximize antibody performance In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human lmmunoglobulin and all or substantially all of the FR regions are those of a human lmmunoglobulin sequence The humanized antibody optimally also will comprise at least a portion of an lmmunoglobulin constant region (Fc), typically that of a human lmmunoglobulin For further details, see Jones et al , Nature, 321 522 525 (1986). Reichmann et al , Nature, 332 323-329 [1988], and Presta, Curr Op Struct Biol , 2 593-596 (1992) The humanized antibody includes a PRIM ATIZED™antιbody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest
"Single-chain Fv' or "sFv' antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding For a review of sFv, see, Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 1 13, Rosenburg and Moore eds , Springer- Verlag, New York, pp 269-315 ( 1994)
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites Diabodies are described more fully in, for example, EP 404,097, WO 93/11161 , 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 matenals which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present Ordinarily, however, isolated antibody will be prepared by at least one purification step
The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody The label may be detectable by itself
(e g , radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable The label may also be a non-detectable entity such as a toxin
By " solid phase' is meant a non-aqueous matrix to which the antibody of the present invention can adhere Examples of solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysaccharides (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and si cones In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275,149
A " posome" is a small vesicle composed of various types of pids, phosphohpids and/or surfactant which is useful for delivery of a drug (such as a PRO 1 186 or PRO 184 polypeptide or antibody thereto) to a mammal The components of the hposo e are commonly arranged in a bilayer formation, similar to the hpid arrangement of biological membranes
A "small molecule" is defined herein to have a molecular weight below about 500 Daltons
II Compositions and Methods of the Invention
A Full-length PRO 1 186 and PRO 184 Polypeptides
The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROl 186 In particular, cDNAs encoding the PROl 186 polypeptide has been identified and isolated, as disclosed in further detail in the Examples below As disclosed in the Examples below, cDNA clones encoding PROl 186 polypeptide has been deposited with the ATCC The actual nucleotide sequences of the clones can readily be determined by the skilled artisan by sequencing of the deposited clones using routine methods in the art The predicted amino acid sequences can be determined from the nucleotide sequences using routine skill For the PROl 186 and PRO 184 polypeptides and encoding nucleic acids described herein, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time
B PROl 186 and PRO 184 Variants
In addition to the full-length native sequence PRO 1186 and PRO 184 polypeptides described herein, it is contemplated that PROl 186 and PRO 184 variants can be prepared PRO 1186 and PRO 184 variants can be prepared by introducing appropπate nucleotide changes into the PROl 186 or PRO 184 DNA, and/or by synthesis of the desired PRO 1186 or PRO 184 polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PROl 186 or PRO 184 polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchonng characteristics
Variations in the native full-length sequence PRO 1186 or PRO 184 or in various domains of the PRO 1186 or PROl 84 described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U S Patent No 5,364,934 Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO 1186 or PRO 184 that results in a change in the amino acid sequence of the PROl 186 or PRO 184 as compared with the native sequence PROl 186 or PROl 84 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 PROl 186 or PRO 184 Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PROl 186 or PRO 184 with that of homologous known protein molecules and minimizing the number of ammo acid sequence changes made in regions of high homology Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a senne, . e conservative amino acid replacements Insertions or deletions may optionally be in the range of about 1 to 5 amino acids The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence
PRO 1 186 and PRO 184 polypeptide fragments are provided herein Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein Certain fragments lack amino acid residues that are not essential for a desired biological activity of the PRO 1186 or PRO 184 polypeptide
PROl 186 and PRO 184 fragments may be prepared by any of a number of conventional techniques Desired peptide fragments may be chemically synthesized An alternative approach involves generating PROl 186 and PRO 184 fragments by enzymatic digestion, e g , by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR) Ohgonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR Preferably, PROl 186 and PROl 84 polypeptide fragments share at least one biological and/or immunological activity with the native PRO 1186 or PRO 184 polypeptide shown in Figure 2 (SEQ ID NO 2) and Figure 4 (SEQ ID NO 4), respectively
In particular embodiments, conservative substitutions of interest are shown in Table 3 under the heading of preferred substitutions If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 3, or as further described below in reference to amino acid classes, are introduced and the products screened
Table 3
Original Exemplary Preferred Residue Substitutions Substitutions
Ala (A) val; leu; ile val Arg (R) lys; gin; asn lys Asn (N) gin; his; lys; arg gin Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gin; lys; arg arg He (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile
Lys (K) arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu
Substantial modifications in function or immunological identity of the PRO 1186 or PRO 184 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as oligonucleotide-mediated (site- directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res.. B:4331 (1986); Zoller et al, Nucl. Acids Res.. 10:6487 (1987)]. cassette mutagenesis [Wells et al, Gene, 34:315 (1985)], restriction selection mutagenesis [Wells etal. Philos. Trans. R. Soc. London SerA, 317:415 ( 1986)] or other known techniques can be performed on the cloned DNA to produce the PROl 186 or PR0184 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 ammo acids Such amino acids include alanine, glycme, seπne, and cysteine Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science, 244 1081 -1085 (1989)] Alanine is also typically preferred because it is the most common amino acid Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins. (W H Freeman & Co , N Y ), Chothia, J Mol Biol . 150 1 (1976)] If alanine substitution does not yield adequate amounts of variant, an lsoteπc ammo acid can be used
C Modifications of PROl 186 and PROl 84
Covalent modifications of PRO 1186 and PRO 184 are included within the scope of this invention One type of covalent modification includes reacting targeted amino acid residues of a PRO 1186 or PRO 184 polypeptide with an organic deπvatizmg agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PROl 186 or PRO 184 Denvatization with bifunctional agents is useful, for instance, for crosslinking PRO 1186 or PRO 184 to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO 1186 or anti-PRO 184 antibodies, and vice- versa Commonly used crosslinking agents include, e g , 1,1- bιs(dιazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4- azidosahcylic acid, homobifunctional lmidoesters, including disuccinimidyl esters such as 3,3'- dιthιobιs(succιnιmιdylpropιonate), bifunctional maleimides such as bιs-N-maleιmιdo-1 ,8-octane and agents such as methyl-3-[(p-azιdophenyl)dιthιo]propιoιmιdate
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of pro ne and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains [T E Creighton, Proteins Structure and Molecular Properties. W H Freeman & Co , San Francisco, pp 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group
Another type of covalent modification of the PRO 1 186 or PRO 184 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PROl 186 or PROl 84 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PROl 186 or PROl 84 In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present Addition of glycosylation sites to the PRO 1186 or PROl 84 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 seπne or threonine residues to the native sequence PROl 186 or PROl 84 (for O-hnked glycosylation sites) The PROl 186 or PRO 184 ammo acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PROl 186 or PROl 84 polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids
Another means of increasing the number of carbohydrate moieties on the PROl 186 or PROl 84 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide Such methods are described in the art, _ , in WO 87/05330 published 1 1 September 1987, and in Aplin and Wπston, CRC Cπt Rev Biochem , pp 259-306 (1981 )
Removal of carbohydrate moieties present on the PRO 1186 or PRO 184 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al , Arch Biochem Biophvs , 259 52 (1987) and by Edge et al , Anal Biochem . 118 131 (1981) Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al , Meth Enzvmol , 138 350 (1987)
Another type of covalent modification of PRO 1186 or PROl 84 comprises linking the PROl 186 or PROl 84 polypeptide to one of a variety of nonproteinaceous polymers, e g , polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U S Patent Nos 4,640,835, 4,496,689, 4,301 ,144, 4,670,417, 4,791,192 or 4,179,337
The PRO 1186 or PRO 184 polypeptide of the present invention may also be modified in a way to form a chimeric molecule comprising PROl 186 or PRO 184 fused to another, heterologous polypeptide or amino acid sequence
In one embodiment, such a chimeric molecule comprises a fusion of the PRO 1186 or PRO 184 polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind The epitope tag is generally placed at the amino- or carboxyl- terminus of the PRO 1186 or PRO 184 polypeptide The presence of such epitope-tagged forms of the PROl 186 or PRO 184 polypeptide can be detected using an antibody against the tag polypeptide Also, provision of the epitope tag enables the PRO 1186 or PRO 184 polypeptide to be readily punfied by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag Various tag polypeptides and their respective antibodies are well known in the art Examples include poly- histidine (poly-His) or poly-histidine-glycine (poly-His-gly) tags, the flu HA tag polypeptide and its antibody 12CA5 [Field etal , Mol Cell Biol , 8 2159-2165 (1988)], the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and9E10 antibodies thereto [Evan et al , Molecular and Cellular Biology.5 3610-3616 ( 1985)] , and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al , Protein Engineering. 3(6) 547-553 (1990)] Other tag polypeptides include the Flag-peptide [Hopp etal , BioTechnology.6 1204-1210 (1988)], the KT3 epitope peptide [Martin et al , Science, 255 192-194 (1992)], an α-tubuhn epitope peptide [Skinner et al , J Biol Chem . 266 15163-15166 (1991)], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl Acad Sci USA 87 6393-6397 (1990)]
In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO 1186 or PRO 184 polypeptide with an lmmunoglobulin or a particular region of an lmmunoglobulin For a bivalent form of the chimeric molecule (also referred to as an "lmmunoadhesin ), 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 PRO 1186 or PROl 84 polypeptide in place of at least one variable region within an Ig molecule In a particularly preferred embodiment, the lmmunoglobulin fusion includes the hinge, CH2 and CH3 or the hinge, CHI , CH2 and CH3 regions of an IgG 1 molecule For the production of lmmunoglobulin fusions see also, US Patent No 5,428, 130 issued June 27, 1995
D Preparation of PROl 186 and PROl 84
The description below relates primarily to production of PROl 186 or PROl 84 by culturing cells transformed or transfected with a vector containing PROl 186 or PR0184 nucleic acid It is of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PROl 186 or PRO 184 For instance, the PRO 1186 or PRO 184 polypeptide 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), Merπfield, J Am Chem Soc , 85 2149-2154 (1963)] In vitro protein synthesis may be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer s instructions Various portions of the PRO 1186 or PRO 184 polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PROl 186 or PRO 184 polypeptide
1 Isolation of DNA Encoding PROl 186 or PROl 84
DNA encoding PRO 1186 or PRO 184 may be obtained from a cDNA library prepared from tissue believed to possess the PRO 1 186 or PRO 184 mRNA and to express it at a detectable level Accordingly, human PRO 1186 or human PROl 84 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as descnbed in the Examples The PRO 1186- or PRO 184-encodιng gene may also be obtained from a genomic library or by known synthetic procedures (e g , automated nucleic acid synthesis)
Libraries can be screened with probes (such as antibodies to the PRO 1 186 or PRO 184 or ohgonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al , Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1989) An alternative means to isolate the gene encoding PRO 1186 or PROl 84 is to use PCR methodology [Sambrook et al , supra, Dieffenbach et al , PCR Primer A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)] The Examples below describe techniques for screening a cDNA library The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened Methods of labeling are ell known in the art and include the use of radiolabels like ^P-labeled ATP, biotinylation or enzyme labeling H) bπdization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supra
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein
Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced am o acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al , supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA
2 Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors described herein for PRO 1 186 or PRO 184 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 , supra, or electroporation is generally used for prokaryotes Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as descnbed 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 efa- . J Bact , 130 946 (1977) and Hsiao etal . Proc Natl Acad Sci (USA).76 3829 (1979) However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e g , polybrene, polyornithine, may also be used For various techniques for transforming mammalian cells, see, Keown etal , Methods in Enzvmology, 185 527-537 (1990) and Mansour et al , Nature, 336 348-352 (1988) Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells Suitable prokaryotes include but are not limited to eubacteπa, such as Gram-negative or Gram-positive organisms, for example, Enterobactenaceae such as E coli Various E coli strains are publicly available, such as £ coli YLU strain MM294 (ATCC 31,446), E coh Xlll (ATCC 31,537), E coli strain W3110 (ATCC 27,325) and K5772 (ATCC 53,635) Other suitable prokaryotic host cells include Enterobactenaceae such as Escherichia, e g , E coli, Entewbacter, Erwinia, Klebsiella, Proteus, Salmonella, e g , Salmonella typhimunum, Serratia, e g , Serratia marcescans, and Shigella, as well as Bacilli such as B subtihs and B hcheniformis (e g , B hcheniformis 41P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W31 10 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 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; __.. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA El 5 (argF-lac)169 degP ompTkan'; E. coli W31 10 strain 37D6, which has the complete genotype tonA pti phoA E15 (argF-lac)]69 degP ompT rbs7 ilvG kanr; E. coli W31 10 strain 40B4, which is strain 37D6 with a non- kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO 1186- or PRO 184-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces po be (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 etal, J. Bacteriol., 737 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), /-. wickeramii (ATCC 24, 178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg etal, Bio/Technology, 8: 135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna etal, J. Basic Microbiol, 28:265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al, Proc. Natl. Acad. Sci. USA, 76:5259-5263 [ 1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance et al, Biochem. Biophvs. Res. Commun.. 112:284-289 [1983]; Tilburn et al, Gene, 26:205-221 [1983]; Yelton et al, Proc. Natl. Acad. Sci. USA. 81: 1470-1474 [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 PRO 1 186 or PRO 184 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 1 line transformed by S V40 (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, Uriaub and Chasin, Proc. Nat Acad. Sci. USA.77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol Reprod.. 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51 ). The selection of the appropriate host cell is deemed to be within the skill in the art. 3 Selection and Use of a Replicable Vector
The nucleic acid (e g cDNA or genomic DNA) encoding PROl 186 or PRO 184 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 ligation techniques which are known to the skilled artisan
The PROl 186 or PROl 84 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 PROl 186- or PROl 84-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, penicilhnase, lpp, or heat-stable enterotoxin II leaders For yeast secretion the signal sequence may be, e g , the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces α-factor leaders, the latter described in U S PatentNo 5,010,182), oracid phosphatase leader, the C alb wans glucoamylase leader (EP 362, 179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990 In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders
Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells Such sequences are well known for a variety of bacteria, yeast, and viruses The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e g , ampicil n, neomycin, methotrexate, or tetracyc ne, (b) complement auxotrophic deficiencies, or (c) supply critical nutnents not available from complex media, e g , the gene encoding D-alamne 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 PROl 186- or PR0184-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 Uriaub et al , Proc Natl Acad Sci USA. 77 4216 (1980) A suitable selection gene for use in yeast is the trpl gene present the yeast plasmid YRp7 [Stinchcomb etal , Nature, 282 39 (1979), Kingsman et al , Gene, 7 141 (1979), Tsche per et al , Gene. JO 157 (1980)] The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No 44076 or PEP4-1 [Jones, Genetics, 85 12 (1977)] Expression and cloning vectors usually contain a promoter operably linked to the PROl 186- or PROl 84- encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al, Nature, 275:615 (1978); Goeddel et al, Nature, 28J_: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 PR01 186 or PR0184.
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al, J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al, J. Adv. Enzyme Reg., 2:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde- 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosρhoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. PROl 186 or PROl 84 transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (S V40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
Transcription of a DNA encoding the PROl 186 or PROl 84 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 S V40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the PRO 1186 or PRO 184 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 PROl 186 or PR0184. Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PROl 186 or PROl 84 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.
4. Detecting Gene Amplification Expression Gene amplification and/or expression may be measured in a sample directly, for example, by conventional
Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence PROl 186 or PRO 184 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO 1186 or PRO 184 DNA and encoding a specific antibody epitope.
5. Purification of Polypeptide Forms of PROl 186 or PROl 84 may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells employed in expression of PROl 186 or PROl 84 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 PROl 186 or PRO 184 from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the PROl 186 or PROl 84. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzvmology, 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 PROl 186 or PROl 84 produced.
E. Antibodies Some drug candidates for use in the compositions and methods of the present invention are antibodies and antibody fragments which mimic the biological activity of a PROl 186 or PROl 84 polypeptide
1 Polyclonal Antibodies
Methods of preparing polyclonal antibodies are known to the skilled artisan Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections The immunizing agent may include the PROl 186 or PRO 184 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 hemocyamn, serum albumin, bovine thyroglobuhn, and soybean trypsin inhibitor Examples of adjuvants which may be employed include Freund's complete adj uvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate) The immunization protocol may be selected by one skilled in the art without undue experimentation
2 Monoclonal Antibodies
The antibodies may, alternatively, be monoclonal antibodies Monoclonal antibodies may be prepared using hybndoma methods, such as those described by Kohler and Milstein, Nature.256 495 (1975) In a hybndoma method, a mouse, hamster, or other appropπate 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 PROl 186 or PRO 184 polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybπdoma cell fGoding. Monoclonal Antibodies Principles and Practice. Academic Press, (1986) pp 59-103] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin Usually, rat or mouse myeloma cell lines are employed The hybndoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells For example, if the parental cells lack the enzyme hypoxanthme guanine phosphoπbosyl transferase (HGPRT or HPRT), the culture medium for the hybπdomas typically will include hypoxanthme, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium More preferred immortalized cell lines are uπne myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J Immunol , 133 3001 (1984), Brodeur et al , Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc , New York, (1987) pp 51-63] The culture medium in which the hybndoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PROl 186 or PROl 84 Preferably, the binding specificity of monoclonal antibodies produced by the hybndoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme linked lmmunoabsorbent assay (ELIS A) 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 hybndoma 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 hybndoma 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 lmmunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Patent No 4,816,567 DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of muπne antibodies) The hybndoma cells of the invention serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce lmmunoglobulin 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 muπne sequences [U S
Patent No 4,816,567, Mornson et al , supra] or by covalently joining to the lmmunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobuhn polypeptide Such a non-immunoglobuhn 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 lmmunoglobulin light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking
In vitro methods are also suitable for preparing monovalent antibodies Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art
3 Human and Humanized Antibodies The antibodies of the invention may further compnse humanized antibodies or human antibodies
Humanized forms of non-human (e g , muπne) antibodies are chimeric lmmunoglobulins, lmmunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two. variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.. 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non- human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239: 1534- 1536 ( 1988)] , by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol Biol, 227:381 (1991); Marks et al., J. Mol Biol. 222:581 (1991)].
The techniques of Cole et al, and Boerner et al., are also available for the preparation of human monoclonal antibodies (Cole etal, Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, p. 77 (1985) and Boerner etal,
J. Immunol. 147(l):86-95 (1991)]. Similarly, human antibodies can be made by the introducing of human immunoglobulin loci into transgenic animals, e.g. , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5.569,825; 5,625,126;
5,633,425; 5,661,016, and in the following scientific publications: Marks et al, Bio/Technology. 10: 779-783
(1992); Lonber e. α ., Nature.368: 856-859 (1994); Morrison. Nature, 368 : 812- 13 (1994); Fishwild e a/., Nature Biotechnology. 14:845-51 (1996): Neuberger. Nature Biotechnology. 14: 826 (1996); Lonberg and Huszar, Intern.
Rev. Immunol. U :65-93 (1995). 4. 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 PROl 186 or PRO 184, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature. 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker etal, EMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy- chain constant domain, comprising at least part of the hinge. CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh etal, Methods in Enzymology. 121:210 (1986). According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. , Science.229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Fab' fragments may be directly recovered from __.. coli and chemically coupled to form bispecific antibodies. Shalaby et al, J. Exp. Med„ 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny etal, J. Immunol, 148(51: 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al , Proc. Natl Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al, J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies may bind to two different epitopes on a given PROl 186 or PRO 184 polypeptide herein. Alternatively, an anti-PROl 186 or anti-PROl 84 polypeptide arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. , CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular PROl 186 or PROl 84 polypeptide. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PROl 186 or PRO 184 polypeptide. These antibodies possess a PROl 186- or PR0184-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the PROl 186 or PR0184 polypeptide and further binds tissue factor (TF).
5. Heteroconiugate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676.980.
6. Effector Function Engineering
It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See, Caron et al, J. Exp. Med., 176: 1191-1195 (1992) and Shopes, J. Immunol, 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See, Stevenson et al, Anti-Cancer Drug Design, 3: 219-230 (1989).
7. Immunoconiugates
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin. enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 2,2Bi, 131I, I31In, 90Y, and 186Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldi thiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis- active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science. 238: 1098(1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, W094/11026.
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) that is conjugated to a cytotoxic agent (e.g., a radionucleotide). 8. 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. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG- PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin etal, J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See, Gabizon et al, J. National Cancer Inst.. 81(19): 1484 (1989).
F. Identification of Proteins Capable of Inhibiting Neoplastic Cell Growth or Proliferation
The proteins disclosed in the present application have been assayed in a panel of 60 tumor cell lines currently used in the investigational, disease-oriented, in vitro drug-discovery screen of the National Cancer Institute (NCI). The purpose of this screen is to identify molecules that have cytotoxic and/or cytostatic activity against different types of tumors. NCI screens more than 10,000 new molecules per year (Monks et al., J. Natl. Cancer Inst., 83:757-766 (1991); Boyd, Cancer: Princ. Pract. Oncol Update, 3(10):1-12 ([1989]). The tumor cell lines employed in this study have been described in Monks et al., supra. The cell lines the growth of which has been significantly inhibited by the proteins of the present application are specified in the Examples.
The results have shown that the proteins tested show cytostatic and, in some instances and concentrations, cytotoxic activities in a variety of cancer cell lines, and therefore are useful candidates for tumor therapy.
Other cell-based assays and animal models for tumors (e.g. , cancers) can also be used to verify the findings of the NCI cancer screen, and to further understand the relationship between the protein identified herein and the development and pathogenesis of neoplastic cell growth. For example, primary cultures derived from tumors in transgenic animals (as described below) can be used in the cell-based assays herein, although stable cell lines are preferred. Techniques to derive continuous cell lines from transgenic animals are well known in the art (see, e.g., Small et al, Mol Cell Biol.. 5:642-648 [1985]).
G. Animal Models A variety of well known animal models can be used to further understand the role of the molecules identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies, and other agonists of the native polypeptides, including small molecule agonists. The in vivo nature of such models makes them particularly predictive of responses in human patients. Animal models of tumors and cancers (e.g., breast cancer, colon cancer, prostate cancer, lung cancer, etc.) include both non- recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodent, e.g., murine models. Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g., colon cancer cells implanted in colonic tissue. (See, e.g., PCT publication No. WO 97/33551, published September 18, 1997).
Probably the most often used animal species in oncological studies are immunodeficient mice and, in particular, nude mice. The observation that the nude mouse with hypo/aplasia could successfully act as a host for human tumor xenografts has lead to its widespread use for this purpose. The autosomal recessive nu gene has been introduced into a very large number of distinct congenic strains of nude mouse, including, for example, ASW, A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL. In addition, a wide variety of other animals with inherited immunological defects other than the nude mouse have been bred and used as recipients of tumor xenografts. For further details see, e.g. , The Nude Mouse in Oncology Research. E. Boven and B. Winograd, eds., CRC Press, Inc., 1991.
The cells introduced into such animals can be derived from known tumor/cancer cell lines, such as. any of the above-listed tumor cell lines, and, for example, the B 104- 1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene); ra_--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 neu- transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al., Proc. Natl. Acad. Sci. 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 μl of the cell suspension, allowing one to three weeks for a tumor to appear. In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the most thoroughly studied experimental tumors, can be used as an investigational tumor model. Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small cell carcinoma of the lung (SCCL). This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture (Zupi et al, Br. J. Cancer. 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, Hae ostasis. 16:300-320 [1986]).
One way of evaluating the efficacy of a test compound in an animal model on an implanted tumor is to measure the size of the tumor before and after treatment. Traditionally, the size of implanted tumors has been measured with a slide caliper in two or three dimensions. The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted into the corresponding volume by using a mathematical formula. However, the measurement of tumor size is very inaccurate. The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay. Another important variable in the description of tumor growth is the tumor volume doubling time. Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc. 6th Int. Workshop on Immune-Deficient Animals, Wu and Sheng eds., Basel, 1989, 301. It is noted, however, that necrosis and inflammatory responses following treatment may actually result in an increase in tumor size, at least initially. Therefore, these changes need to be carefully monitored, by a combination of a morphometric method and flow cytometric analysis. Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals. Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g., baboons, chimpanzees and monkeys. Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and Wanger, U.S. Patent No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g., Van der Putten et al, Proc. Natl Acad. Sci. USA. 82:6148-615 [1985]); gene targeting in embryonic stem cells (Thompson etal, Cell, 56:313-321 [1989]); electroporation of embryos (Lo, Mol. Cell. Biol, 3:1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al, Cell, 57:717-73 [1989]). For review, see, for example, U.S. Patent No. 4,736,866.
For the purpose of the present invention, transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals"). The transgene can be integrated either as a single transgene, or in concatamers, e.g. , head-to-head or head-to-tail tandems. Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al. , Proc. Natl Acad. Sci. USA. 89:6232- 636 (1992). The expression of the transgene in transgenic animals can be monitored by standard techniques. For example. Southern blot analysis or PCR amplification can be used to verify the integration of the transgene. The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or imrnunocytochemistry. The animals are further examined for signs of tumor or cancer development.
The efficacy of antibodies specifically binding the polypeptides identified herein and other drug candidates, can be tested also in the treatment of spontaneous animal tumors. A suitable target for such studies is the feline oral squamous cell carcinoma (SCC). Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy of cats, accounting for over 60% of the oral tumors reported in this species. It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor. These tumors are usually not amenable to surgery, primarily because of the anatomy of the feline oral cavity. At present, there is no effective treatment for this tumor. Prior to entry into the study, each cat undergoes complete clinical examination, biopsy, and is scanned by computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors are excluded from the study. The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor, the animals may not be able to feed themselves. Each cat is treated repeatedly, over a longer period of time. Photographs of the tumors will be taken daily during the treatment period, and at each subsequent recheck. After treatment, each cat undergoes another CT scan. CT scans and thoracic radiograms are evaluated every 8 weeks thereafter. The data are evaluated for differences in survival, response and toxicity as compared to control groups. Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span.
In addition, other spontaneous animal tumors, such as fibrosarcoma, adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma of dogs, cats, and baboons can also be tested. Of these mammary adenocarcinoma in dogs and cats is a preferred model as its appearance and behavior are very similar to those in humans. However, the use of this model is limited by the rare occurrence of this type of tumor in animals.
H. Screening Assays for Drug Candidates
Screening assays for drug candidates are designed to identify compounds that competitively bind or complex with the receptor(s) of the polypeptides identified herein, or otherwise signal through such receptor(s). 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)peptide- immunoglobulin fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.
In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, a receptor of a polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments . Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the polypeptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non- immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
If the candidate compound interacts with but does not bind to a particular receptor, 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 systemdescribed by Fields and co-workers [Fields and Song, Nature (London). 340:245-246 (1989); Chien etal, Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)] as disclosed by Chevray and Nathans [Proc. Natl Acad. Sci. USA. 89:5789-5793 (1991)]. Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl-/αcZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two- hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
I. Pharmaceutical Compositions The polypeptides of the present invention, agonist antibodies specifically binding proteins identified herein, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions.
Where antibody fragments are used, the smallest inhibitory fragment which specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable region sequences of an antibody, peptide molecules can be designed which retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e.g., Marasco et al. Proc. Natl. Acad. Sci. USA. 90:7889-7893 [1993]). 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 an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
Therapeutic formulations of the polypeptides identified herein, or agonists thereof are prepared for storage by mixing the active ingredient having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences. 16th edition, Osol, A. ed. [1980]), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980).
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
Therapeutic compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
J. Methods of Treatment
It is contemplated that the polypeptides of the present invention and their agonists, including antibodies, peptides, and small molecule agonists, may be used to treat various tumors, e.g., cancers. Exemplary conditions or disorders to be treated include benign or malignant tumors (e.g., renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas; sarcomas; glioblastomas; and various head and neck tumors); leukemias and lymphoid malignancies; other disorders such as neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders. The anti-tumor agents of the present invention (including the polypeptides disclosed herein and agonists which mimic their activity, e.g., antibodies, peptides and small organic molecules), 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, or by intramuscular, intraperitoneal, intracerobrospinal, intraocular, intraarterial, intralesional, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
Other therapeutic regimens may be combined with the administration of the anti-cancer agents of the instant invention. For example, the patient to be treated with such anti-cancer agents may also receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may precede, or follow administration of the anti-tumor agent of the present invention, or may be given simultaneously therewith. The anti-cancer agents of the present invention may be combined with an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
It may be desirable to also administer antibodies against tumor associated antigens, such as antibodies which bind to the ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, or in addition, two or more antibodies binding the same or two or more different cancer-associated antigens 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 anti-cancer agents herein are co-administered with a growth inhibitory agent. For example, the growth inhibitory agent may be administered first, followed by the administration of an anti-cancer agent of the present invention. However, simultaneous administration or administration of the anti-cancer agent of the present invention first is also contemplated. Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the antibody herein.
For the prevention or treatment of disease, the appropriate dosage of an anti-tumor agent 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. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell W. "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development. Yacobi et al., eds., Pergamon Press, New York 1989, pp. 42-96.
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 an antitumor agent is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays. Guidance as to particular dosages and methods of delivery is provided in the literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue.
K. 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 an anti-tumor agent of the present invention. 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. 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, Manassas, VA.
EXAMPLE 1 Isolation of cDNA clones encoding PROl 186
DNA60621-1516 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 methionine codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under consideration. The nucleotides following the first ATG must code for at least 35 unambiguous amino acids without any stop codons. If the first ATG has the required amino acids, the second is not examined. If neither meets the requirement, the candidate sequence is not scored. In order to determine whether the EST sequence contains an authentic signal sequence, the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals.
Use of the signal sequence algorithm described above allowed identification of an EST cluster sequence from the Incyte database. This Incyte EST cluster sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homologies. The homology search was performed using the computer program BLAST or BLAST2 (Altshul et al , Methods in Enzymology. 266:460-480 (1996)). Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into a consensus DNA sequence with the program "phrap" (Phil Green, University of Washington, Seattle, Washington). The consensus sequence obtained therefrom is herein designated DNA56748.
In light of an observed sequence homology between the DNA56748 consensus sequence and an Incyte EST sequence no. 3476792 encompassed within a clone (from a library constructed from ovarian tissue) including this Incyte EST, identified from the Incyte database, the Incyte clone including Incyte EST no. 3476792 was purchased and the cDNA insert was obtained and sequenced. It was found herein that that insert encoded a full- length protein. The sequence of this cDNA insert is shown in Figure 1 (SEQ ID NO:l) and is herein designated DNA60621-1516. Clone DN A60621-1516 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 91-93 and ending at the stop codon at nucleotide positions 406-408 (Figure 1) The predicted polypeptide precursor is 105 amino acids long (Figure 2, SEQ ID NO 2) The full-length PROl 186 protein shown in Figure 2 has an estimated molecular weight of about 1 1,715 daltons and a pi of about 9 05 Analysis of the full-length PRO 1186 sequence shown in Figure 2 (SEQ ID NO 2) evidences the presence of important polypeptide domains as shown in Figure 2, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PROl 186 sequence evidences the presence of a signal peptide from about amino acid 1 to about ammo acid 19, a tyrosine kinase phosphorylation site from about amino acid 88 to about amino acid 95, and N-myπstoylation sites from about am o acid 33 to about amino acid 39, from about amino acid 35 to about ammo acid 41 , and from about amino acid 46 to about amino acid 52 Clone DNA60621-1516 was deposited with the ATCC on August 4, 1998, and is assigned ATCC deposit no 203091
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 2 (SEQ ID NO 2), evidenced sequence identity between the PRO 1186 amino acid sequence and the following Dayhoff sequences VPRA_DENPO, LFE4_CHICK, AF034208_1, AF030433_1, A55035, COL_RABIT, CELB0507_9, S67826_l, S34665 and CRU73817_1
EXAMPLE 2 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), usιngPCR-generated33P-labeledrιboprobes Bπefly, 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 πboprobe was generated from a PCR product and hybndized at 55 °C overnight The slides were dipped in Kodak NTB2™ nuclear track emulsion and exposed for 4 weeks — P-Riboprobe synthesis 6 0 μl (125 mCi) of 33P-UTP (AmershamBF 1002, SA<2000 Ci/mmol) were speed- vacuum dried To each tube containing dried 33P-UTP, the following ingredients were added 2 0 μl 5x transcription buffer
1 0 μl DTT (100 mM)
2 0 μl NTP mix (2 5 mM 10 μl each of 10 mM GTP, CTP & ATP + 10 μl H-O) 1 0 μl UTP (50 μM)
1 0 μl RNAsin
1 0 μl DNA template (1 μg) 1.0 μl H,O
1.0 μl RNA polymerase (for PCR products T3 = AS, T7 = S, usually)
The tubes were incubated at 37°C for one hour. A total of 1.0 μl RQ1 DNase was added, followed by incubation at 37°C for 15 minutes. A total of 90 μl TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) was added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a MICROCON-50™ ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, a total of 100 μl TE was added, then 1 μl of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR II™.
The probe was run on a TBE/urea gel. A total of 1-3 μl of the probe or 5 μl of RNA Mrk III was added to 3 μl of loading buffer. After heating on a 95 °C heat block for three minutes, the gel was immediately placed on ice. The wells of gel were flushed, and the sample was loaded and run at 180-250 volts for 45 minutes. The gel was wrapped in plastic wrap (SARAN™ brand) and exposed to XAR film with an intensifying screen in a -70 °C freezer one hour to overnight.
— P-Hybridization A. Pretreatment of frozen sections
The slides were removed from the freezer, placed on aluminum trays, and thawed at room temperature for 5 minutes. The trays were placed in a 55 °C incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H20). After deproteination in 0.5 μg/ml proteinase K for 10 minutes at 37°C (12.5 μl of 10 mg/ml stock in 250 ml prewarmed RNAse-free RNAse buffer), the sections were washed in 0.5 x SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%, and 100% ethanol, 2 minutes each.
B. Pretreatment of paraffin-embedded sections
The slides were deparaffinized, placed in SQ H20, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time. The sections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml
RNase-free RNase buffer; 37 °C, 15 minutes) for human embryo tissue, or 8 x proteinase K (100 μl in 250 ml Rnase buffer, 37 °C, 30 minutes) for formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above.
C. Prehybridization The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper. The tissue was covered with 50 μl of hybridization buffer (3.75 g dextran sulfate + 6 ml SQ H20), vortexed, and heated in the microwave for 2 minutes with the cap loosened. After cooling on ice, 18.75 ml formamide, 3.75 ml 20 x SSC, and 9 ml SQ H20 were added, and the tissue was vortexed well and incubated at 42 °C for 1 -4 hours.
D. Hybridization 1.0 x IO6 cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heated at 95°C for 3 minutes. The slides were cooled on ice, and 48 μl hybridization buffer was added per slide. After vortexing, 50 μl 33P mix was added to 50 μl prehybridization on the slide. The slides were incubated overnight at 55 °C. E. Washes
Washing was done for 2x10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml
0.25 M EDTA, V 4L), followed by RNAseA treatment at 37 °C for 30 minutes (500 μl of 10 mg/ml in 250 ml
Rnase buffer = 20 μg/ml), The slides were washed 2 10 minutes with 2 x SSC, EDTA at room temperature. The stringency wash conditions were as follows: 2 hours at 55 °C, 0.1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA,
V=4L).
F. Oligonucleotides
In situ analysis was performed on one of the DNA sequences disclosed herein. The oligonucleotides employed for these analyses are as follows: (1) DNA60621-1516 (PROl 186) (Black Ma ba snake venom (VPRA DENPO) homolog pl :
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC GCT TCG AGG GCT GCG GAT GT-3' (SEQ ID NO:5) p2: 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA TGC CTT GGG GTG ACT GTC TGC-3' (SEQ ID NO:6)
G. Results
In situ analysis was performed on the above DNA60621-1516 sequence disclosed herein. The results from this analysis are as follows:
DNA60621-15I6 (Black Mamba Homolog) expression in 2 year old chimp ovary
The most intense signal was seen over granulosa cells, especially those nearest to the oocyte, in primary follicles in both the chimp and cyno monkey ovary. A weaker signal was seen over granulosa cells in primordial follicles and in the cumulus oophorus. A significant signal was observed in the ovarian cortex, especially around small, involuted corpora lutea (cyno monkey sample) and in poorly defined clusters of cells at the vascular pole of the ovary. No signal was seen over fallopian tubes or uterine tissue.
Expression in frozen and paraffin-embedded human ovaries There was diffuse moderate expression in ovarian cortical stroma; an intense signal was seen over the externa around corpus hemorrhagicum In testes, a moderate to strong signal was seen over Leydig cells. Fibrous cysts and ovarian surface epithelium were negative.
EXAMPLE 3 Use of PROl 186 or PROl 84 as a Hybridization Probe The following method describes use of a nucleotide sequence encoding PROl 186 or PROl 84 as a hybridization probe.
DNA comprising the coding sequence of full-length or mature PRO 1186 or PRO 184 (as shown in Figure 1 and 3, respectively, SEQ ID NOS : 1 and 3, respectively) or a fragment thereof is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PROl 186 or PROl 84) in human tissue cDNA libraries or human tissue genomic libraries.
Hybridization and washing of filters containing either library DNAs is performed under the following high- stringency conditions . Hybridization of radiolabeled probe derived from the gene encoding a PRO 1186 or PRO 184 polypeptide to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42°C for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1 x SSC and 0.1 % SDS at 42°C. DNAs having a desired sequence identity with the DNA encoding full-length native sequence can then be identified using standard techniques known in the art.
EXAMPLE 4 Expression of PROl 186 or PROl 84 in E. coli This example illustrates preparation of an unglycosylated form of PROl 186 or PRO 184 by recombinant expression in E. coli.
The DNA sequence encoding PROl 186 or PROl 84 is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli: see Bolivar et al, Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a poly-His leader (including the first six STTI codons, poly-His sequence, and enterokinase cleavage site), the PRO 1186 or PRO 184 coding region, lambda transcriptional terminator, and an argU gene. The ligation mixture is then used to transform a selected E. coli strain using the methods described in
Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.
Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.
After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized PROl 186 or PROl 84 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
PRO 1186 or PRO 184 may be expressed in E. coli in a poly-His tagged form, using the following procedure. The DNA encoding PROl 186 or PROl 84 is initially amplified using selected PCR primers. The primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E. coli host based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(ladq). Transformants are first grown in LB containing 50 mg/ml carbenicillin at 30 °C with shaking until an OD600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH4)2S04, 0.71 g sodium citrate»2H20, 1.07 g KC1, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 1 10 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgS04) and grown for approximately 20-30 hours at 30°C with shaking. Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.
E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1M and 0.02 M, respectively, and the solution is stirred overnight at 4°C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant is 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 is loaded onto a 5 ml Qiagen Ni 2+-NTA metal chelate column equilibrated in the metal chelate column buffer. The column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein are pooled and stored at 4°C. Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.
The proteins are refolded by diluting the 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 are chosen so that the final protein concentration is between 50 to 100 micrograms/ml. The refolding solution is stirred gently at 4°C for 12-36 hours. The refolding reaction is 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 is filtered through a 0.22 micron filter and acetonitrile is added to 2-10% final concentration. The refolded protein is chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.
Fractions containing the desired folded PROl 186 or PRO 184 polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered. PROl 186 and PRO 184 were successfully expressed in E. coli in a poly-His tagged form by the above procedure. EXAMPLE 5 Expression of PROl 186 or PROl 84 in mammalian cells This example illustrates preparation of a potentially glycosylated form of PROl 186 or PROl 84 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 PRO 1186 or PRO 184 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PRO 1186 or PRO 184 DNA using ligation methods such as described in Sambrook et al supra The resulting vector is called pRK5-PR01186 or pRK5-PR0184
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-PRO 1186 or pRK5-PRO 184 DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya etal , Cell 31 543 (1982)] and dissolved in 500 μl of 1 mM Tπs-HCl, 0 1 mM EDTA, 0 227 M CaCl2 To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7 35), 280 mM NaCl, 1 5 mM NaP04, and a precipitate is allowed to form for 10 minutes at 25 °C The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days
Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml 35S-cysteιne and 200 μCi/ml 35S-methιonιne 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 dned and exposed to film for a selected period of time to reveal the presence of the PROl 186 or PRO 184 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, PROl 186 or PRO 184 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac etal , Proc Natl Acad Sci .12 7575 (1981) 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-PRO 1186 or pRK5-PRO 184 DNA is added The cells are first concentrated from the spinner flask by centrifugation and washed with PBS The DNA-dextran precipitate is incubated on the cell pellet for four hours The cells are treated with 20% glycerol for 90 seconds washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0 1 μg/ml bovine transfernn After about four days, the conditioned media is centrifuged and filtered to remove cells and debris The sample containing expressed PRO 1186 or PRO 184 can then be concentrated and punfied by any selected method, such as dialysis and/or column chromatography
In another embodiment, PROl 186 or PROl 84 can be expressed in CHO cells The pRK5-PR01186 or ρRK5-PR0184 can be transfected into CHO cells using known reagents such as CaP04 or DEAE-dextran As descnbed above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35S-methιonιne After determining the presence of a PRO 1186 or PRO 184 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 PRO 1186 or PRO 184 polypeptide can then be concentrated and purified by any selected method
Epitope-tagged PRO 1186 or PRO 184 may also be expressed in host CHO cells The PRO 1186 or PRO 184 may be subcloned out of the ρRK5 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 PRO 1186 or PRO 184 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 venfy expression The culture medium containing the expressed poly-His tagged PRO 1186 or PRO 184 can then be concentrated and purified by any selected method, such as by Nι,+-chelate affinity chromatography PRO 1186 or PRO 184 may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure
Stable expression in CHO cells is performed using the following procedure The proteins are expressed as an IgG construct (lmmunoadhesin), in which the coding sequences for the soluble forms (e g , extracellular domains) of the respective proteins are fused to an IgGl constant region sequence containing the hinge, CH2 and CH2 domains and/or as a poly-His tagged form
Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al , Current Protocols of Molecular Biology. Unit 3 16, John Wiley and Sons ( 1997) CHO expression vectors are constructed to have compatible restriction sites 5 and 3' of the DNA of interest to allow the convenient shuttling of cDNA's The vector used in 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 is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Qiagen), Dosper® or Fugene® (Boehπnger Mannheim) The cells are grown as described in Lucas et al , supra Approximately 3 x 107 cells are frozen in an ampule for further growth and production as described below
The ampules containing the plasmid DNA are thawed by placement into a water bath and mixed by vortexing The contents are pipetted into a centrifuge tube containing 10 ml of media and centrifuged at 1000 rpm for 5 minutes The supernatant is aspirated and the cells are resuspended in 10 ml of selective media (0 2 μm filtered PS20 with 5% 0 2 μm diafiltered fetal bovine serum) The cells are then aliquoted into a 100 ml spinner containing 90 ml of selective media After 1-2 days, the cells are 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 are seeded with 3 x 105 cells/ml The cell media is exchanged with fresh media by centrifugation and resuspension in production medium Although anv suitable CHO media may be employed, a production medium described in U S Patent No 5,122,469, issued June 16 1992 may actually be used A 3L production spinner is seeded at 1 2 x 106 cells/ml On day 0, the cell number and pH is determined On day 1 , the spinner is sampled and sparging with filtered air is commenced On day 2 the spinner is 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) taken. Throughout the production, the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability drops below 70%, the cell culture is harvested by centrifugation and filtering through a 0.22 μm filter. The filtrate is either stored at 4CC or immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins are purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C. lmmunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium 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 μl 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 is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
EXAMPLE 6 Expression of PROl 186 or PROl 84 in Yeast
The following method describes recombinant expression of PROl 186 or PRO 184 in yeast. First, yeast expression vectors are constructed for intracellular production or secretion of PRO 1186 or PRO 184 from the ADH2/GAPDH promoter. DNA encoding PROl 186 or PRO 184 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PROl 186 or PRO 184. For secretion, DNA encoding PROl 186 or PRO 184 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PROl 186 or PRO 184 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 PROl 186 or PROl 84.
Yeast cells, such as yeast strain AB 110, 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 PRO 1186 or PRO 184 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 PROl 186 or PROl 84 may further be purified using selected column chromatography resins. EXAMPLE 7 Expression of PROl 186 or PROl 84 in Baculovirus-Infected Insect Cells The following method describes recombinant expression in Baculovirus-infected insect cells. The sequence coding for PROl 186 or PRO 184 is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG) . A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVLl 393 (Novagen). Briefly, the sequence encoding PROl 186 or PR0184 or the desired portion of the coding sequence of PROl 186 or PRO 184 (such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular) is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold™ virus DNA
(Pharmingen) into Spodopterafrugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28°C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual Oxford: Oxford University Press (1994).
Expressed poly-His tagged PRO 1186 or PRO 184 can then be purified, for example, by Ni2+-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert 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 MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KC1), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer
(50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 mm filter. A Ni2+-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 ml, washed with 25 ml of water and equilibrated with 25 ml of loading buffer. The filtered cell extract is loaded onto the column at 0.5 ml per minute. The column is washed to baseline A280 with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 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 Ni2+-NTA-conjugated to alkaline phosphatase
(Qiagen). Fractions containing the eluted His)0-tagged PRO 1186 or PRO 184, respectively, are pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PRO 1186 or PRO 184 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography. 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 Spodopterafrugiperda ("Sf9") cells (ATCC CRL 1711), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences. The cells are grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). Cells are incubated for 5 days at 28 °C. The supernatant is harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Cells are incubated for 3 days at 28°C. The supernatant is harvested and the expression of the constructs in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml of Ni 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 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 CC. The supernatant is harvested and filtered. Batch binding and SDS-PAGE analysis is repeated, as necessary, until expression of the spinner culture is confirmed.
The conditioned medium from the transfected cells (0.5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein construct is purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml min. at 4CC. 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. lmmunoadhesin (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 fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector. Such epitope tags include poly- His tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIE 1 - 1 (Novagen) . The pIE 1 - 1 and pIE 1 -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus iel promoter in stably- transformed insect cells (1 ). The plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for iel -mediated gene expression in uninfected insect cells as well as the hr5 enhancer element. pIEl - 1 and pIEl -2 include the translation initiation site and can be used to produce fusion proteins. Briefly, the desired sequence or the desired portion of the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector. For example, derivatives of pIE 1 - 1 can include the Fc region of human IgG (pb.PH.IgG) or an 8 histidine (pb.PH.His) tag downstream (3'-of) the desired sequence. Preferably, the vector construct is sequenced for confirmation.
High-5 cells are grown to a confluency of 50% under the conditions of, 27 °C, no C02, NO pen/strep. For each 150 mm plate, 30 μg of pIE based vector containing the sequence is mixed with 1 ml Ex-Cell medium (Media: Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401-78P (note: this media is light sensitive)), and in a separate tube, 100μl ofCellFectin (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/CellFECTIN mix is then aspirated, and the cells are washed once with Ex-Cell to remove excess CellFECTIN, 30 ml of fresh Ex-Cell media is added and the cells are incubated for 3 days at 28°C. The supernatant is harvested and the expression of the sequence in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml of Ni 2+-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining. The conditioned media from the transfected cells (0.5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein comprising the sequence is purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 48°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is then subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C. lmmunoadhesin (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 had 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.
PROl 186 was expressed using the above baculovirus procedure employing high-5 cells. EXAMPLE 8 Preparation of Antibodies that Bind PROl 186 or PROl 84 This example illustrates preparation of monoclonal antibodies which can specifically bind PROl 186 or PROl 84. Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding. supra. Immunogens that may be employed include purified PROl 186 or PRO 184, fusion proteins containing PROl 186 or PROl 84, and cells expressing recombinant PROl 186 or PRO 184 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 PROl 186 or PRO 184 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-PROl 186 or anti-PROl 84 antibodies.
After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PROl 186 or PR0184. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.l, available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
The hybridoma cells will be screened in an ELISA for reactivity against PROl 186 or PRO 184. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PROl 186 or PRO 184 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-PROl 186 or anti-PR0184 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.
EXAMPLE 9
Purification of PROl 186 or PROl 84 Polypeptides Using Specific Antibodies Native or recombinant PROl 186 or PRO 184 polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, pro-PR01186 or pro-PR0184 polypeptide, mature PROl 186 or mature PRO 184 polypeptide, or pre-PR01186 or pre-PR0184 polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PROl 186 or PR0184 polypeptide of interest. In general, an immunoaffinity column is constructed by covalently coupling the anti-PROl 186 or anti-PRO 184 polypeptide antibody to an activated chromatographic resin. Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared frommouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
Such an immunoaffinity column is utilized in the purification of the PRO 1186 or PRO 184 polypeptide by preparing a fraction from cells containing the PROl 186 or PROl 84 polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble PROl 186 or PR0184 polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
A soluble PROl 186 or PRO 184 polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of the PROl 186 or PR0184 polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/PROl 186 or antibody/PR0184 polypeptide binding (e.g., a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and the PROl 186 or PRO 184 polypeptide is collected.
EXAMPLE 10 Drug Screening
This invention is particularly useful for screening compounds by using PRO 1186 or PRO 184 polypeptides or a binding fragment thereof in any of a variety of drug screening techniques. The PROl 186 or PRO 184 polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO 1186 or PRO 184 polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between a PROl 186 or PRO 184 polypeptide or a fragment and the agent being tested. Alternatively, one can examine the diminution in complex formation between the PRO 1186 or PRO 184 polypeptide and its target cell or target receptors caused by the agent being tested.
Thus, the present invention provides methods of screening for drugs or any other agents which can affect a PROl 186 or PRO 184 polypeptide-associated disease or disorder. These methods comprise contacting such an agent with a PRO 1186 or PRO 184 polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the PROl 186 or PRO 184 polypeptide or fragment, or (ii) for the presence of a complex between the PROl 186 or PROl 84 polypeptide or fragment and the cell, by methods well known in the art. In such competitive binding assays, the PROl 186 or PR0184 polypeptide or fragment is typically labeled. After suitable incubation, the free PROl 186 or PROl 84 polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to the PRO 1186 or PRO 184 polypeptide or to interfere with the PRO 1186 or PRO 184 polypeptide/cell complex.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on September 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to a PROl 186 or PROl 84 polypeptide, the peptide test compounds are reacted with the PROl 186 or PROl 84 polypeptide and washed. Bound PROl 186 or PROl 84 polypeptide is detected by methods well known in the art. Purified PROl 186 or PROl 84 polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding a PRO 1186 or PRO 184 polypeptide specifically compete with a test compound for binding to the PROl 186 or PROl 84 polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with a PRO 1186 or PRO 184 polypeptide.
EXAMPLE 11 Rational Drug Design The goal of rational drug design is to produce structural analogs of a biologically active polypeptide of interest (i.e., a PROl 186 or PR0184 polypeptide) or of small molecules with which they interact, e.g., agonists, antagonists, or inhibitors. Any of these examples can be used to fashion drugs which are more active or stable forms of the PROl 186 or PRO 184 polypeptide or which enhance or interfere with the function of the PROl 186 or PRO 184 polypeptide in vivo (c.f., Hodgson, Bio/Technology, 9: 19-21 (1991)).
In one approach, the three-dimensional structure of the PRO 1186 or PRO 184 polypeptide, or of a PRO 1186 or PROl 84 polypeptide-inhibitor complex, is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the PROl 186 or PRO 184 polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of the PROl 186 or PRO 184 polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous PROl 186 or PROl 84 polypeptide-like molecules or to identify efficient inhibitors. Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton and Wells, Biochemistry.31 :7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda et al. J. Biochem.. 113:742-746 (1993).
It is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
By virtue of the present invention, sufficient amounts of the PROl 186 or PROl 84 polypeptide may be made available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the PRO 1 186 or PRO 184 polypeptide amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.
EXAMPLE 12 In Vitro Antitumor Assay The antiproliferative activity of the PROl 186 and PRO 184 polypeptides was determined in the investigational, disease-oriented in vitro anti-cancer drug discovery assay of the National Cancer Institute (NCI), using a sulforhodamine B (SRB) dye binding assay essentially as described by Skehan et al, J. Natl. Cancer Inst., 82:1 107-1112 (1990). The 60 tumor cell lines employed in this study ("the NCI panel"), as well as conditions for their maintenance and culture in vitro have been described by Monks etal, J. Natl. Cancer Inst., 83 :757-766 ( 1991 ). The purpose of this screen is to initially evaluate the cytotoxic and/or cytostatic activity of the test compounds against different types of tumors (Monks et al, supra; Boyd, Cancer: Princ. Pract. Oncol Update, 3(101:1-12 [1989]).
Cells from approximately 60 human tumor cell lines were harvested with trypsin/EDTA (Gibco), washed once, resuspended in IMEM and their viability was determined. The cell suspensions were added by pipet (100 μl volume) into separate 96-well microtiter plates. The cell density for the 6-day incubation was less than for the 2-day incubation to prevent overgrowth. Inoculates were allowed a preincubation period of 24 hours at 37 °C for stabilization. Dilutions at twice the intended test concentration were added at time zero in 100 μl aliquots to the microtiter plate wells (1 :2 dilution). Test compounds were evaluated at five half-log dilutions (1000 to 100,000- fold). Incubations took place for two days and six days in a 5% C02 atmosphere and 100% humidity.
After incubation, the medium was removed and the cells were fixed in 0.1 ml of 10% trichloroacetic acid at 40CC. The plates were rinsed five times with deionized water, dried, stained for 30 minutes with 0.1 ml of 0.4% sulforhodamine B dye (Sigma) dissolved in 1% acetic acid, rinsed four times with 1% acetic acid to remove unbound dye, dried, and the stain was extracted for five minutes with 0.1 ml of 10 mM Tris base [tris(hydroxymethyl)aminomethane], pH 10.5. The absorbance (OD) of sulforhodamine B at 492 nm was measured using a computer-interfaced, 96-well microtiter plate reader. A test sample is considered positive if it shows at least 40% growth inhibitory effect at one or more concentrations. The results are shown in the following Table 4, where the tumor cell type abbreviations are as follows: NSCL = non-small cell lung carcinoma; CNS = central nervous system Table 4
Test compound Concentration Days Tumor Cell Line Type Cell Line Designation
PROl 186 95 nM 2 NSCL NCI-H226 PROl 186 95 nM 2 Colon Colo205
PROl 186 2.2 nM 6 Breast MDA-N
PROl 186 114 nM 2 NSCL NCI-H322M PROl 186 1 14 nM 2 CNS SF-268; SF-539 PROl 186 1 14 nM 2 Ovarian IGFOV1 PRO 1 186 1 14 nM 2 Renal 786-0; SN12C; TK-10*
PROl 186 1 14 nM 6 Leukemia MOLT-4*; RPMI-8226* PROl 186 1 14 nM 6 Melanoma LOX IMVI PROl 186 114 nM 6 Ovarian OVCAR-4; SK-OV-3 PRO 1 186 1 14 nM 6 Breast MDA-MB-435; T-47D
PROl 186 8.1 nM 6 Leukemia K-562 PROl 186 8.1 nM 6 NSCL HOP-62 PROl 186 8.1 nM 6 Colon Colo205; HCC-2998 PROl 186 8.1 nM 6 Breast T-47D
PROl 186 15.4 nM 6 Leukemia K-562
PROl 186 3.6 nM 2 Ovarian OVCAR-3
PRO 1186 3.6 nM 6 NSCL HOP-62
PRO 184 0.65 nM 6 NSCL A549/ATCC; EKVX PRO 184 0.65 nM 6 Colon HT29 PRO 184 0.65 nM 6 CNS SF-268; SF-295; SNB-19 PRO 184 0.65 nM 6 Melanoma MALME-3M; Ml 4; SK-MEL-28; UACC-25 * = cytotoxic (% growth less than 0)
Table 4 Continued
Test compound Concentration Days Tumor Cell Line Type Cell Line Designation
PRO 184 0.65 nM 6 Renal 786-0; RXF393
PRO 184 0.65 nM 6 Breast MDA-N
PROl 84 0.65 nM Renal CAKI-1
PRO 184 0.65 nM 6 Leukemia K562 PRO 184 0.65 nM 6 NSCL NCI-H460 PRO 184 0.65 nM 6 CNS SNB-75; U251 PRO 184 0.65 nM 6 Melanoma SK-MEL-2 PRO 184 0.65 nM 6 Ovarian OVCAR-5; OVCAR-8 PRO 184 0.65 nM 6 Renal SN12C PRO 184 0.65 nM 6 Prostate DU-145 PROl 84 0.65 nM 6 Breast MCF7
PRO 184 53.3 nM 2 Leukemia HL-60 (TB)* PRO 184 53.3 nM 2 NSCL HOP-92* PRO 184 53.3 nM 2 Colon HCC-2998* PRO 184 53.3 nM 2 CNS U251 * PRO 184 53.3 nM 2 Melanoma MALME-3M PROl 84 53.3 nM 2 Ovarian IGROV1 *; OVCAR-5*; OVCAR-8 PRO 184 53.3 nM 2 Renal TK-10* PRO 184 53.3 nM 2 Prostate PC-3* PROl 84 53.3 nM 2 Breast MCF7; T-47D
PRO 184 53.3 nM 6 Leukemia HL-60 (TB)*; MOLT-4*; RPMI-8226*; SR PRO 184 53.3 nM 6 NSCL NCI-H226 PRO 184 53.3 nM 6 Colon HCC-2998* PRO 184 53.3 nM 6 CNS U251 * PRO 184 53.3 nM 6 Ovarian IGROV1 * PRO 184 53.3 nM 6 Renal TK-10*
= cytotoxic (% growth less than 0)
Table 4 Continued
Test compound Concentration Days Tumor Cell Line Type Cell Line Designation
PRO 184 53.3 nM 6 Prostate DU-145
PRO 184 53.3 nM 6 Breast T-47D
* = cytotoxic (% growth less than 0)
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 Pep. No. Deposit Date DNA60621 -1516 203091 August 4, 1998
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 maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposits 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 U.S.C. § 122 and the Commissioner's rules pursuant thereto (including 37 CFR § 1.14 with particular reference to 886 OG 638).
The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS
1 A composition of matter useful for the inhibition of neoplastic cell growth, said composition comprising an effective amount of a PROl 186 or PROl 84 polypeptide, or an agonist thereof, in admixture with a pharmaceutically acceptable carrier
2 The composition of matter of Claim 1 comprising a growth inhibitory amount of a PRO 1 186 or PROl 84 polypeptide, or an agonist thereof
3 The composition of matter of Claim 1 comprising a cytotoxic amount of a PRO 1186 or PRO 184 polypeptide, or an agonist thereof
4 The composition of matter of Claim 1 additionally comprising a further growth inhibitory agent, cytotoxic agent or chemotherapeutic agent
5 A composition of matter useful for the treatment of a tumor in a mammal, said composition compnsing a therapeutically effective amount of a PROl 186 or PROl 84 polypeptide, or an agonist thereof
6 The composition of matter of Claim 5, wherein said tumor is a cancer
7 The composition of matter of Claim 6, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, renal cancer, colorectal cancer, uterine cancer, prostate cancer, lung cancer, bladder cancer, central nervous system cancer, melanoma and leukemia
8 A method for inhibiting the growth of a tumor cell comprising exposing said tumor cell to an effective amount of a PROl 186 or PRO 184 polypeptide, or an agonist thereof
9 The method of Claim 8, wherein said agonist is an anti-PROl 186 or anti-PROl 84 agonist antibody
10 The method of Claim 8, wherein said agonist is a small molecule mimicking the biological activity of a PROl 186 or PROl 84 polypeptide
11 The method of Claim 8, wherein said step of exposing occurs in vitro
12 The method of Claim 8, wherein said step of exposing occurs in vivo
13 An article of manufacture comprising a container, and a composition comprising an active agent contained within the container; wherein said active agent in the composition is a PROl 186 or PROl 84 polypeptide, or an agonist therof.
14. The article of manufacture of Claim 13, further comprising a label affixed to said container, or a package insert included in said container, referring to the use of said composition for the inhibition of neoplastic cell growth.
15. The article of manufacture of Claim 13 , wherein said agonist is an anti-PRO 1186 or anti-PRO 184 agonist antibody.
16. The article of manufacture of Claim 13, wherein said agonist is a small molecule mimicking the biological activity of a PROl 186 or PRO 184 polypeptide.
17. The article of manufacture of Claim 13, wherein said active agent is present in an amount that is effective for the treatment of tumor in a mammal.
18. The article of manufacture of Claim 13 , wherein said composition additionally comprises a further growth inhibitory agent, cytotoxic agent or chemotherapeutic agent.
19. 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), and Figure 4 (SEQ ID NO:4).
20. 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:l) and Figure 3 (SEQ ID NO:3).
21. 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) and Figure 3 (SEQ ID NO:3).
22. 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 203091.
23. A vector comprising the nucleic acid of any one of Claims 19 to 22.
24. The vector of Claim 23 operably linked to control sequences recognized by a host cell transformed with the vector.
25. A host cell comprising the vector of Claim 23.
26. The host cell of Claim 25, wherein said cell is a CHO cell
27. The host cell of Claim 25, wherein said cell is an E. coli.
28. The host cell of Claim 25, wherein said cell is a yeast cell.
29. The host cell of Claim 25, wherein said cell is a Baculovirus-infected insect cell.
30. A process for producing a PRO 1186 or PRO 184 polypeptide comprising culturing the host cell of Claim 25 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.
31. 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) and Figure 4 (SEQ ID NO:4).
32. An isolated polypeptide scoring at least 80% positives when compared to an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2) and Figure 4 (SEQ ID NO:4).
33. 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 203091.
34. A chimeric molecule comprising a polypeptide according to any one of Claims 31 to 33 fused to a heterologous amino acid sequence.
35. The chimeric molecule of Claim 34, wherein said heterologous amino acid sequence is an epitope tag sequence.
36. The chimeric molecule of Claim 34, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin.
37. An antibody which specifically binds to a polypeptide according to any one of Claims 31 to 33.
38. The antibody of Claim 37, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody
39 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) or Figure 4 (SEQ ID NO 4), lacking its associated signal peptide,
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO 2) or Figure 4 (SEQ ID NO 4), 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) or Figure 4 (SEQ ID NO 4), lacking its associated signal peptide
40 An isolated polypeptide having at least 80% amino acid sequence identity to
(a) the polypeptide shown in Figure 2 (SEQ ID NO 2) or Figure 4 (SEQ ID NO 4), lacking its associated signal peptide,
(b) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO 2) or Figure 4 (SEQ ID NO 4), with its associated signal peptide, or
(c) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO 2) or Figure 4 (SEQ ID NO 4), lacking its associated signal peptide
PCT/US2000/004914 1996-11-06 2000-02-24 Methods and compositions for inhibiting neoplastic cell growth WO2000075327A1 (en)

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Application Number Priority Date Filing Date Title
AU32461/00A AU3246100A (en) 1999-06-02 2000-02-24 Methods and compositions for inhibiting neoplastic cell growth
AT00916675T ATE478145T1 (en) 1999-06-02 2000-03-30 SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC ACIDS CODING THEREFOR
CA002383254A CA2383254A1 (en) 1999-06-02 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP00916675A EP1210418B1 (en) 1999-06-02 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2001500766A JP2004522402A (en) 1999-06-02 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/008439 WO2000073454A1 (en) 1999-06-02 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU37743/00A AU3774300A (en) 1999-06-02 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/015264 WO2000073452A2 (en) 1999-06-02 2000-06-02 Compositions and methods for the treatment of immune related diseases
EP06000588A EP1690873A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
EP06000582A EP1666495A1 (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
CA002492049A CA2492049A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002491433A CA2491433A1 (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
EP05025102A EP1672070A3 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000585A EP1661996A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP10005292A EP2228446A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptieds and nucleic acids encoding the same
EP00983846A EP1250426A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding same
CA002391455A CA2391455A1 (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
EP06000584A EP1669371A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
EP06000587A EP1690872A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
JP2001542531A JP2004522404A (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding them
EP06000586A EP1688497A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002491258A CA2491258A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002490909A CA2490909A1 (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
EP06000581A EP1666494A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002491610A CA2491610A1 (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
EP06000589A EP1661997A1 (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
AU20554/01A AU2055401A (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
US09/941,992 US20030082546A1 (en) 1996-11-06 2001-08-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,583 US7074897B2 (en) 1997-06-16 2001-11-14 Pro943 polypeptides
US09/990,427 US20030073809A1 (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
US09/991,073 US20020127576A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,687 US20020198149A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,442 US20020132252A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,711 US20030032023A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,562 US20030027985A1 (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
US09/990,726 US20030054359A1 (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
US09/993,469 US20030068623A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,854 US20030059780A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,163 US20020132253A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/992,598 US6956108B2 (en) 1997-06-16 2001-11-14 PRO1184 antibodies
US09/990,440 US20030060407A1 (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
US09/993,667 US20030022187A1 (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
US09/990,456 US20020137890A1 (en) 1997-03-31 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,438 US20030027754A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,601 US7189814B2 (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
US09/997,585 US20030119055A1 (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
US09/997,683 US20030059783A1 (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
US09/997,666 US20030027163A1 (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
US09/997,333 US6953836B2 (en) 1997-06-16 2001-11-15 PRO844 polypeptides
US09/997,666 US7244816B2 (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
US09/997,529 US20030134284A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,542 US20030068647A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,585 US7166282B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,573 US20030049682A1 (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
US09/997,514 US7019116B2 (en) 1997-06-16 2001-11-15 PRO 1387 polypeptides
US09/998,041 US7309775B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,653 US7034122B2 (en) 1997-06-16 2001-11-15 Antibodies to PRO1159 polypeptides
US09/997,349 US7034106B2 (en) 1997-06-16 2001-11-15 Pro1159 polypeptides
US09/998,156 US20030044806A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,384 US20030087305A1 (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
US09/997,428 US20030027162A1 (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
US09/997,601 US20030054404A1 (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
US09/991,150 US20030194760A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,172 US20030050457A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,441 US7041804B2 (en) 1997-06-16 2001-11-16 Antibodies to PRO1387 polypeptides
US09/990,437 US20030045463A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,181 US6913919B2 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,157 US7101687B2 (en) 1997-06-16 2001-11-16 Nucleic acids encoding PRO943
US09/989,862 US20030130182A1 (en) 1997-11-05 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,279 US7083978B2 (en) 1997-06-16 2001-11-19 Nucleic acid encoding PRO1111 polypeptides
US09/989,723 US20020072092A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,722 US20020072067A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,727 US20020072497A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,729 US20030059831A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,735 US6972185B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO844 polypeptides
US09/989,721 US20020142961A1 (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
US09/989,734 US7491529B2 (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
US09/989,726 US7018811B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO189 polypeptides
US09/989,725 US20030139329A1 (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
US09/989,731 US20020103125A1 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,730 US7157247B2 (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
US09/989,293 US7034136B2 (en) 1997-06-16 2001-11-20 Nucleic acids encoding PRO1159 polypeptides
US10/027,603 US7264801B2 (en) 1998-08-11 2001-12-19 EG-VEGF nucleic acids and polypeptides and method of use
US10/028,072 US20030004311A1 (en) 1997-06-18 2001-12-19 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
US10/121,047 US20030077778A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,044 US20030190717A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,059 US20030190721A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,042 US20030096386A1 (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
US10/121,045 US20030073210A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,040 US20030082759A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,041 US20030077776A1 (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
US10/121,052 US20030199052A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,048 US20030199051A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,043 US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/121,062 US20030077779A1 (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
US10/121,057 US20030190719A1 (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
US10/121,060 US20030190722A1 (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
US10/121,056 US20030082760A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,054 US20030199054A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,058 US20030190720A1 (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
US10/121,049 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
US10/123,771 US20030199060A1 (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
US10/123,236 US20030068795A1 (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
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,108 US7635478B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,322 US20030199059A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,157 US20030190725A1 (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
US10/123,291 US20030199058A1 (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
US10/123,261 US20030068796A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,109 US20030190723A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,235 US20030082762A1 (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
US10/123,292 US20030073211A1 (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
US10/123,262 US20030049816A1 (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
US10/123,905 US20030087344A1 (en) 1997-06-18 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
US10/123,902 US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,904 US20030022328A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,911 US7408032B2 (en) 1997-03-31 2002-04-16 PRO1188 polypeptides
US10/123,903 US20030073212A1 (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
US10/123,907 US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,913 US20030203462A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,910 US7329404B2 (en) 1997-03-31 2002-04-16 Antibodies against PRO1310
US10/123,912 US20030100087A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,906 US20030190726A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,823 US20030199062A1 (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
US10/124,814 US7105335B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,824 US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,819 US7285626B2 (en) 1997-03-31 2002-04-17 PRO1076 polypeptides
US10/124,820 US20030190729A1 (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
US10/124,813 US7312307B2 (en) 1997-03-31 2002-04-17 PRO1056 polypeptides
US10/125,795 US7304131B2 (en) 1997-03-31 2002-04-17 PRO1483 polypeptides
US10/125,704 US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
US10/124,818 US20030082763A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,805 US20030194794A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,816 US20030190728A1 (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
US10/125,922 US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/125,924 US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/125,927 US20030190731A1 (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
US10/125,931 US20030199063A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,831 US20030082689A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,689 US20030087365A1 (en) 1997-03-31 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,825 US7282566B2 (en) 1997-03-31 2002-04-24 PRO1779 polypeptide
US10/131,823 US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 polypeptides
US10/131,817 US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
US10/137,867 US20030207349A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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
US10/140,474 US20030032156A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,020 US20030207415A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,470 US20030022331A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,024 US20040058424A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,963 US7288625B2 (en) 1997-03-31 2002-05-06 PRO4395 polypeptides
US10/139,980 US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/140,023 US20030207416A1 (en) 1997-03-31 2002-05-06 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
US10/140,921 US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
US10/140,809 US20030207418A1 (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
US10/140,808 US7425621B2 (en) 1997-03-31 2002-05-07 Antibodies against the PRO4401 polypeptide
US10/140,865 US20030207420A1 (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
US10/140,860 US7307151B2 (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
US10/141,755 US7297764B2 (en) 1997-03-31 2002-05-08 PRO4318 polypeptides
US10/141,754 US7361732B2 (en) 1997-03-31 2002-05-08 PRO4400 polypeptides
US10/141,701 US20030207421A1 (en) 1997-03-31 2002-05-08 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
US10/143,114 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
US10/142,425 US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,417 US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/142,419 US7153941B2 (en) 1997-03-31 2002-05-10 Antibodies that bind PRO4994 polypeptides
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 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
US10/146,730 US20030207427A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,792 US20030207428A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,528 US20030219885A1 (en) 1997-03-31 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,536 US20040077064A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,492 US20030082765A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,519 US20030077791A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,395 US7189534B2 (en) 1997-03-31 2002-05-21 PRO4320 polynucleotide
US10/153,934 US20030129695A1 (en) 1997-03-31 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,843 US20030207805A1 (en) 1997-06-18 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,786 US20030208055A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,782 US20030077792A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,791 US20030207429A1 (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
US10/160,498 US20030073216A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,885 US20030032138A1 (en) 1998-06-24 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2002341509A JP4017507B2 (en) 1999-06-02 2002-09-25 Medicament containing polypeptide
US10/692,299 US7446168B2 (en) 1998-08-11 2003-10-22 EG-VEGF nucleic acids and polypeptides and methods of use
US11/100,159 US7425613B2 (en) 1997-11-05 2005-04-05 PRO1375 polypeptides
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
US11/537,382 US7727536B2 (en) 1998-08-11 2006-09-29 EG-VEGF nucleic acids and polypeptides and methods of use
US11/537,472 US7736645B2 (en) 1998-08-11 2006-09-29 EG-VEGF nucleic acids and polypeptides and methods of use
JP2007172879A JP5512073B2 (en) 1999-06-02 2007-06-29 Medicament containing polypeptide
JP2007326613A JP2008161190A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptide and nucleic acid encoding the same
JP2007326424A JP2008167749A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptide and nucleic acid encoding the same
JP2007325484A JP2008148699A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007326609A JP2008148701A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US12/724,964 US8557238B2 (en) 1998-08-11 2010-03-16 EG-VEGF nucleic acids and polypeptides and methods of use
US12/760,354 US7960531B2 (en) 1998-08-11 2010-04-14 EG-VEGF nucleic acids and polypeptides and methods of use
US13/102,517 US20130189273A1 (en) 1998-08-11 2011-05-06 Eg-vegf nucleic acids and polypeptides and methods of use
JP2011138276A JP2011236224A (en) 1999-06-02 2011-06-22 Medicine containing polypeptide

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
USPCT/US99/12252 1999-06-02
PCT/US1999/012252 WO1999063088A2 (en) 1998-06-02 1999-06-02 Membrane-bound proteins and nucleic acids encoding the same
US14569899P 1999-07-26 1999-07-26
US60/145,698 1999-07-26
PCT/US2000/000219 WO2000053753A2 (en) 1999-03-08 2000-01-05 Promotion or inhibition of angiogenesis and cardiovascularization
USPCT/US00/00219 2000-01-05

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/000219 Continuation-In-Part WO2000053753A2 (en) 1994-09-08 2000-01-05 Promotion or inhibition of angiogenesis and cardiovascularization

Related Child Applications (6)

Application Number Title Priority Date Filing Date
PCT/US2000/008439 Continuation-In-Part WO2000073454A1 (en) 1994-09-08 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/032678 Continuation-In-Part WO2001040466A2 (en) 1996-11-06 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/027,603 Continuation-In-Part US7264801B2 (en) 1998-08-11 2001-12-19 EG-VEGF nucleic acids and polypeptides and method of use
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
US10/692,299 Continuation-In-Part US7446168B2 (en) 1998-08-11 2003-10-22 EG-VEGF nucleic acids and polypeptides and methods of use

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WO2001072958A3 (en) * 2000-03-28 2002-03-21 Beth Israel Hospital Bladder cancer-specific peptides for diagnosis and therapy
WO2002000711A3 (en) * 2000-06-23 2002-06-20 Genentech Inc Ev-vegf nucleic acids and polypeptides and methods of use
WO2002062944A2 (en) * 2001-02-02 2002-08-15 Takeda Chemical Industries, Ltd. Novel physiologically active peptide and use thereof
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GB2378183A (en) * 2001-04-25 2003-02-05 Smithkline Beecham Corp Assays of the interaction between AXOR8 or AXOR52 and BV-8 ligand
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EP1386615A1 (en) * 2002-06-26 2004-02-04 Schering AG EG-VEGF/Prokineticin 2 receptor antagonists
US7052674B2 (en) 2000-11-03 2006-05-30 The Regents Of The University Of California Prokineticin polypeptides, related compositions and methods
US7223727B2 (en) 1998-04-09 2007-05-29 Serono Genetics Institute S.A. GSSP4 polynucleotides and polypeptides and uses thereof
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US9212228B2 (en) 2005-11-24 2015-12-15 Ganymed Pharmaceuticals Ag Monoclonal antibodies against claudin-18 for treatment of cancer
US9512232B2 (en) 2012-05-09 2016-12-06 Ganymed Pharmaceuticals Ag Antibodies against Claudin 18.2 useful in cancer diagnosis
US9775785B2 (en) 2004-05-18 2017-10-03 Ganymed Pharmaceuticals Ag Antibody to genetic products differentially expressed in tumors and the use thereof
US10414824B2 (en) 2002-11-22 2019-09-17 Ganymed Pharmaceuticals Ag Genetic products differentially expressed in tumors and the use thereof

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US7727536B2 (en) 1998-08-11 2010-06-01 Genentech, Inc. EG-VEGF nucleic acids and polypeptides and methods of use
US7960531B2 (en) 1998-08-11 2011-06-14 Genetech, Inc. EG-VEGF nucleic acids and polypeptides and methods of use
US7736645B2 (en) 1998-08-11 2010-06-15 Genentech, Inc. EG-VEGF nucleic acids and polypeptides and methods of use
US8557238B2 (en) 1998-08-11 2013-10-15 Genentech, Inc. EG-VEGF nucleic acids and polypeptides and methods of use
US7264801B2 (en) 1998-08-11 2007-09-04 Genentech, Inc. EG-VEGF nucleic acids and polypeptides and method of use
US7446168B2 (en) 1998-08-11 2008-11-04 Genentech, Inc. EG-VEGF nucleic acids and polypeptides and methods of use
WO2001072958A3 (en) * 2000-03-28 2002-03-21 Beth Israel Hospital Bladder cancer-specific peptides for diagnosis and therapy
WO2002000711A3 (en) * 2000-06-23 2002-06-20 Genentech Inc Ev-vegf nucleic acids and polypeptides and methods of use
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WO2002069689A3 (en) * 2001-02-09 2003-02-06 Genset Sa Gssp4 polynucleotides and polypeptides and uses thereof
WO2002069689A2 (en) * 2001-02-09 2002-09-12 Genset S.A. Gssp4 polynucleotides and polypeptides and uses thereof
GB2378183A (en) * 2001-04-25 2003-02-05 Smithkline Beecham Corp Assays of the interaction between AXOR8 or AXOR52 and BV-8 ligand
WO2003029427A3 (en) * 2001-10-03 2003-12-18 Univ Rochester Human glandular kallikrein (hk2)-specific monoclonal antibodies that enhance or inhibit the enzymatic activity of hk2
WO2003029427A2 (en) * 2001-10-03 2003-04-10 University Of Rochester Human glandular kallikrein (hk2)-specific monoclonal antibodies that enhance or inhibit the enzymatic activity of hk2
EP1386615A1 (en) * 2002-06-26 2004-02-04 Schering AG EG-VEGF/Prokineticin 2 receptor antagonists
US10414824B2 (en) 2002-11-22 2019-09-17 Ganymed Pharmaceuticals Ag Genetic products differentially expressed in tumors and the use thereof
US7632810B2 (en) 2003-03-12 2009-12-15 Genentech, Inc. Compositions with hematopoietic and immune activity
US8858936B2 (en) 2003-03-12 2014-10-14 Genentech, Inc. Compositions with hematopoietic and immune activity
US8257702B2 (en) 2003-03-12 2012-09-04 Genentech, Inc. Compositions with hematopoietic and immune activity
US9775785B2 (en) 2004-05-18 2017-10-03 Ganymed Pharmaceuticals Ag Antibody to genetic products differentially expressed in tumors and the use thereof
US9212228B2 (en) 2005-11-24 2015-12-15 Ganymed Pharmaceuticals Ag Monoclonal antibodies against claudin-18 for treatment of cancer
US9499609B2 (en) 2005-11-24 2016-11-22 Ganymed Pharmaceuticals Ag Monoclonal antibodies against claudin-18 for treatment of cancer
US9751934B2 (en) 2005-11-24 2017-09-05 Ganymed Pharmaceuticals Ag Monoclonal antibodies against claudin-18 for treatment of cancer
US10017564B2 (en) 2005-11-24 2018-07-10 Ganymed Pharmaceuticals Gmbh Monoclonal antibodies against claudin-18 for treatment of cancer
US10174104B2 (en) 2005-11-24 2019-01-08 Ganymed Pharmaceuticals Gmbh Monoclonal antibodies against claudin-18 for treatment of cancer
US10738108B2 (en) 2005-11-24 2020-08-11 Astellas Pharma Inc. Monoclonal antibodies against claudin-18 for treatment of cancer
US11739139B2 (en) 2005-11-24 2023-08-29 Astellas Pharma Inc. Monoclonal antibodies against Claudin-18 for treatment of cancer
US9512232B2 (en) 2012-05-09 2016-12-06 Ganymed Pharmaceuticals Ag Antibodies against Claudin 18.2 useful in cancer diagnosis
US10053512B2 (en) 2012-05-09 2018-08-21 Ganymed Pharmaceuticals Ag Antibodies against claudin 18.2 useful in cancer diagnosis

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