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WO2000053751A1 - 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
WO2000053751A1
WO2000053751A1 PCT/US1999/031243 US9931243W WO0053751A1 WO 2000053751 A1 WO2000053751 A1 WO 2000053751A1 US 9931243 W US9931243 W US 9931243W WO 0053751 A1 WO0053751 A1 WO 0053751A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
acid sequence
amino acid
seq
prol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1999/031243
Other languages
French (fr)
Inventor
Avi J. Ashkenazi
Kevin P. Baker
Audrey Goddard
Austin L. Gurney
Mary A. Napier
William I. Wood
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genentech Inc
Original Assignee
Genentech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US1999/005028 external-priority patent/WO1999046281A2/en
Priority claimed from PCT/US1999/020594 external-priority patent/WO2000015666A2/en
Priority claimed from PCT/US1999/030999 external-priority patent/WO2001005836A1/en
Application filed by Genentech Inc filed Critical Genentech Inc
Priority to AU23993/00A priority Critical patent/AU2399300A/en
Priority to PCT/US2000/000277 priority patent/WO2000053754A1/en
Priority to AU22248/00A priority patent/AU2224800A/en
Priority to EP00907314A priority patent/EP1263948A2/en
Priority to PCT/US2000/004341 priority patent/WO2000053756A2/en
Priority to CA002361840A priority patent/CA2361840A1/en
Priority to JP2000603377A priority patent/JP2004513602A/en
Priority to KR1020017011399A priority patent/KR20030002292A/en
Publication of WO2000053751A1 publication Critical patent/WO2000053751A1/en
Priority to CA2709291A priority patent/CA2709291A1/en
Priority to JP2001542531A priority patent/JP2004522404A/en
Priority to EP06000586A priority patent/EP1688497A1/en
Priority to CA002491258A priority patent/CA2491258A1/en
Priority to CA002494705A priority patent/CA2494705A1/en
Priority to CA002491433A priority patent/CA2491433A1/en
Priority to CA002491610A priority patent/CA2491610A1/en
Priority to CA002490909A priority patent/CA2490909A1/en
Priority to CA002492049A priority patent/CA2492049A1/en
Priority to EP06000587A priority patent/EP1690872A3/en
Priority to EP06000582A priority patent/EP1666495A1/en
Priority to CA002492070A priority patent/CA2492070A1/en
Priority to EP06000584A priority patent/EP1669371A3/en
Priority to AU20554/01A priority patent/AU2055401A/en
Priority to CA002496312A priority patent/CA2496312A1/en
Priority to EP05025102A priority patent/EP1672070A3/en
Priority to EP06000588A priority patent/EP1690873A3/en
Priority to EP10005292A priority patent/EP2228446A1/en
Priority to EP06000583A priority patent/EP1686134A3/en
Priority to EP06000585A priority patent/EP1661996A1/en
Priority to EP06000581A priority patent/EP1666494A1/en
Priority to CA002391455A priority patent/CA2391455A1/en
Priority to PCT/US2000/032678 priority patent/WO2001040466A2/en
Priority to EP06000589A priority patent/EP1661997A1/en
Priority to EP00983846A priority patent/EP1250426A2/en
Priority to CA002490853A priority patent/CA2490853A1/en
Priority to US09/918,585 priority patent/US20030060406A1/en
Anticipated expiration legal-status Critical
Priority to US09/978,295 priority patent/US20020156006A1/en
Priority to US09/978,189 priority patent/US6972325B2/en
Priority to US09/978,298 priority patent/US20030134785A1/en
Priority to US09/978,191 priority patent/US20030050239A1/en
Priority to US09/978,188 priority patent/US20030139328A1/en
Priority to US09/978,192 priority patent/US20020177553A1/en
Priority to US09/978,299 priority patent/US20030199435A1/en
Priority to US09/978,194 priority patent/US20030195333A1/en
Priority to US09/978,193 priority patent/US20030073624A1/en
Priority to US09/978,403 priority patent/US20030050240A1/en
Priority to US09/978,585 priority patent/US20030049633A1/en
Priority to US09/978,375 priority patent/US7196165B2/en
Priority to US09/978,681 priority patent/US20030195148A1/en
Priority to US09/978,564 priority patent/US7195760B2/en
Priority to US09/978,665 priority patent/US7294700B2/en
Priority to US09/981,915 priority patent/US20030054986A1/en
Priority to US09/978,423 priority patent/US20030069178A1/en
Priority to US09/978,643 priority patent/US20030104998A1/en
Priority to US09/978,824 priority patent/US20050124789A9/en
Priority to US09/978,697 priority patent/US20020169284A1/en
Priority to US09/978,757 priority patent/US20030083248A1/en
Priority to US09/978,802 priority patent/US20030199674A1/en
Priority to US09/978,544 priority patent/US20030199436A1/en
Priority to US09/981,915 priority patent/US7285623B2/en
Priority to US09/978,824 priority patent/US20030055216A1/en
Priority to US10/152,388 priority patent/US20040223964A1/en
Priority to US10/210,028 priority patent/US20030203446A1/en
Priority to US10/145,089 priority patent/US7208575B2/en
Priority to US10/145,017 priority patent/US20030186365A1/en
Priority to US10/164,749 priority patent/US20040029218A1/en
Priority to US10/165,067 priority patent/US7279553B2/en
Priority to US10/166,709 priority patent/US20030104536A1/en
Priority to US10/164,829 priority patent/US20030194780A1/en
Priority to US10/165,247 priority patent/US7112657B2/en
Priority to US10/143,029 priority patent/US7105640B2/en
Priority to US10/164,728 priority patent/US20030186368A1/en
Priority to US10/162,521 priority patent/US7067628B2/en
Priority to US10/145,124 priority patent/US20030190701A1/en
Priority to US10/160,502 priority patent/US7220835B2/en
Priority to US10/164,929 priority patent/US20030194781A1/en
Priority to US10/165,036 priority patent/US20050227342A1/en
Priority to US10/013,922 priority patent/US20030195345A1/en
Priority to US10/002,967 priority patent/US20030148373A1/en
Priority to US09/999,830 priority patent/US20030077700A1/en
Priority to US09/999,832 priority patent/US20020192706A1/en
Priority to US09/999,833 priority patent/US6916648B2/en
Priority to US10/017,086 priority patent/US7122375B2/en
Priority to US10/017,081 priority patent/US20030049684A1/en
Priority to US09/999,834 priority patent/US20030064407A1/en
Priority to US10/020,445 priority patent/US20030198994A1/en
Priority to US10/017,084 priority patent/US20030203402A1/en
Priority to US10/017,083 priority patent/US20030148376A1/en
Priority to US09/999,832 priority patent/US7132283B2/en
Priority to US10/017,085 priority patent/US6974696B2/en
Priority to US09/999,829 priority patent/US20030195344A1/en
Priority to US10/013,921 priority patent/US20030068648A1/en
Priority to US10/013,927 priority patent/US7189529B2/en
Priority to US10/013,928 priority patent/US20030215905A1/en
Priority to US10/013,923 priority patent/US7169912B2/en
Priority to US10/013,925 priority patent/US7037710B2/en
Priority to US10/016,177 priority patent/US20030073131A1/en
Priority to US10/013,920 priority patent/US20040006219A1/en
Priority to US10/013,918 priority patent/US20030211091A1/en
Priority to US10/013,917 priority patent/US7029874B2/en
Priority to US10/013,929 priority patent/US7019124B2/en
Priority to US10/013,926 priority patent/US7074593B2/en
Priority to US09/990,456 priority patent/US20020137890A1/en
Priority to US10/028,072 priority patent/US20030004311A1/en
Priority to US09/978,187 priority patent/US20030096744A1/en
Priority to US10/081,056 priority patent/US20040043927A1/en
Priority to US10/119,480 priority patent/US20040087769A1/en
Priority to US10/121,044 priority patent/US20030190717A1/en
Priority to US10/121,047 priority patent/US20030077778A1/en
Priority to US10/121,045 priority patent/US20030073210A1/en
Priority to US10/121,042 priority patent/US20030096386A1/en
Priority to US10/121,059 priority patent/US20030190721A1/en
Priority to US10/121,046 priority patent/US20030194791A1/en
Priority to US10/121,051 priority patent/US20030092147A1/en
Priority to US10/121,040 priority patent/US20030082759A1/en
Priority to US10/121,041 priority patent/US20030077776A1/en
Priority to US10/121,056 priority patent/US20030082760A1/en
Priority to US10/121,053 priority patent/US20030199053A1/en
Priority to US10/121,060 priority patent/US20030190722A1/en
Priority to US10/121,043 priority patent/US7220831B2/en
Priority to US10/121,048 priority patent/US20030199051A1/en
Priority to US10/121,062 priority patent/US20030077779A1/en
Priority to US10/121,063 priority patent/US20030199055A1/en
Priority to US10/121,049 priority patent/US20030022239A1/en
Priority to US10/121,052 priority patent/US20030199052A1/en
Priority to US10/121,054 priority patent/US20030199054A1/en
Priority to US10/121,061 priority patent/US20030082761A1/en
Priority to US10/121,057 priority patent/US20030190719A1/en
Priority to US10/121,058 priority patent/US20030190720A1/en
Priority to US10/121,050 priority patent/US20030054516A1/en
Priority to US10/121,055 priority patent/US20030190718A1/en
Priority to US10/123,213 priority patent/US20030199057A1/en
Priority to US10/123,261 priority patent/US20030068796A1/en
Priority to US10/123,156 priority patent/US20030194792A1/en
Priority to US10/123,157 priority patent/US20030190725A1/en
Priority to US10/123,236 priority patent/US20030068795A1/en
Priority to US10/123,109 priority patent/US20030190723A1/en
Priority to US10/123,212 priority patent/US7276577B2/en
Priority to US10/123,213 priority patent/US7193048B2/en
Priority to US10/123,771 priority patent/US20030199060A1/en
Priority to US10/123,108 priority patent/US7635478B2/en
Priority to US10/123,235 priority patent/US20030082762A1/en
Priority to US10/123,214 priority patent/US7343721B2/en
Priority to US10/123,215 priority patent/US7291329B2/en
Priority to US10/123,322 priority patent/US20030199059A1/en
Priority to US10/123,155 priority patent/US20030068794A1/en
Priority to US10/123,154 priority patent/US20030190724A1/en
Priority to US10/123,292 priority patent/US20030073211A1/en
Priority to US10/123,262 priority patent/US20030049816A1/en
Priority to US10/123,291 priority patent/US20030199058A1/en
Priority to US10/123,905 priority patent/US7285625B2/en
Priority to US10/123,911 priority patent/US7408032B2/en
Priority to US10/123,913 priority patent/US20030203462A1/en
Priority to US10/123,904 priority patent/US20030022328A1/en
Priority to US10/123,907 priority patent/US7084258B2/en
Priority to US10/123,906 priority patent/US20030190726A1/en
Priority to US10/123,910 priority patent/US7329404B2/en
Priority to US10/123,912 priority patent/US20030100087A1/en
Priority to US10/123,908 priority patent/US7335728B2/en
Priority to US10/123,909 priority patent/US7193049B2/en
Priority to US10/123,905 priority patent/US20030087344A1/en
Priority to US10/123,902 priority patent/US20030077781A1/en
Priority to US10/123,903 priority patent/US20030073212A1/en
Priority to US10/124,817 priority patent/US20030077786A1/en
Priority to US10/124,814 priority patent/US7105335B2/en
Priority to US10/124,820 priority patent/US20030190729A1/en
Priority to US10/124,813 priority patent/US7312307B2/en
Priority to US10/124,819 priority patent/US7285626B2/en
Priority to US10/124,818 priority patent/US20030082763A1/en
Priority to US10/125,704 priority patent/US7357926B2/en
Priority to US10/124,822 priority patent/US7109305B2/en
Priority to US10/124,821 priority patent/US20030199023A1/en
Priority to US10/124,823 priority patent/US20030199062A1/en
Priority to US10/124,816 priority patent/US20030190728A1/en
Priority to US10/125,805 priority patent/US20030194794A1/en
Priority to US10/125,795 priority patent/US7304131B2/en
Priority to US10/124,824 priority patent/US20030077659A1/en
Priority to US10/125,927 priority patent/US20030190731A1/en
Priority to US10/125,924 priority patent/US7342097B2/en
Priority to US10/125,922 priority patent/US7309762B2/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 US10/131,823 priority patent/US7304132B2/en
Priority to US10/131,817 priority patent/US7291701B2/en
Priority to US10/131,825 priority patent/US7282566B2/en
Priority to US10/137,867 priority patent/US20030207349A1/en
Priority to US10/137,868 priority patent/US20030082764A1/en
Priority to US10/137,865 priority patent/US20030032155A1/en
Priority to US10/139,980 priority patent/US7247710B2/en
Priority to US10/140,024 priority patent/US20040058424A1/en
Priority to US10/140,023 priority patent/US20030207416A1/en
Priority to US10/140,470 priority patent/US20030022331A1/en
Priority to US10/140,020 priority patent/US20030207415A1/en
Priority to US10/140,474 priority patent/US20030032156A1/en
Priority to US10/139,963 priority patent/US7288625B2/en
Priority to US10/140,809 priority patent/US20030207418A1/en
Priority to US10/140,925 priority patent/US20030073215A1/en
Priority to US10/140,928 priority patent/US20030068798A1/en
Priority to US10/140,921 priority patent/US7317080B2/en
Priority to US10/140,864 priority patent/US20030207419A1/en
Priority to US10/140,808 priority patent/US7425621B2/en
Priority to US10/140,805 priority patent/US20030207417A1/en
Priority to US10/140,860 priority patent/US7307151B2/en
Priority to US10/140,865 priority patent/US20030207420A1/en
Priority to US10/141,755 priority patent/US7297764B2/en
Priority to US10/141,701 priority patent/US20030207421A1/en
Priority to US10/141,756 priority patent/US7488586B2/en
Priority to US10/141,760 priority patent/US7342104B2/en
Priority to US10/141,754 priority patent/US7361732B2/en
Priority to US10/142,417 priority patent/US7304133B2/en
Priority to US10/142,425 priority patent/US20030207424A1/en
Priority to US10/143,114 priority patent/US20030036180A1/en
Priority to US10/143,113 priority patent/US7329730B2/en
Priority to US10/142,430 priority patent/US7309766B2/en
Priority to US10/143,032 priority patent/US7408033B2/en
Priority to US10/142,419 priority patent/US7153941B2/en
Priority to US10/142,431 priority patent/US7285629B2/en
Priority to US10/142,423 priority patent/US20030049817A1/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 US10/157,782 priority patent/US20030077792A1/en
Priority to US10/157,786 priority patent/US20030208055A1/en
Priority to US10/158,791 priority patent/US20030207429A1/en
Priority to US10/160,498 priority patent/US20030073216A1/en
Priority to US10/158,782 priority patent/US20030082766A1/en
Priority to US10/176,913 priority patent/US20030022298A1/en
Priority to JP2005264293A priority patent/JP2006068016A/en
Priority to US11/341,175 priority patent/US7468427B2/en
Priority to US11/786,466 priority patent/US20080182275A1/en
Priority to JP2007325484A priority patent/JP2008148699A/en
Priority to JP2007326609A priority patent/JP2008148701A/en
Priority to JP2007326424A priority patent/JP2008167749A/en
Priority to JP2007326613A priority patent/JP2008161190A/en
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • 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
  • cancers Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al , CA Cancel J Clin . 43 7 (1993))
  • Cancer is characterized by 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 piesent invention relates to methods and compositions for inhibiting neoplastic cell growth More particularly, the invention concerns methods and compositions for the treatment ot 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 ot matter useful for the inhibition oi neoplastic cell growth comprising an effective amount ot a PRO 181 or PR0237 polypeptide as herein defined, or an agonist thereof in admixture with a pharmaceutically acceptable carrier
  • the composition of matter comprises a growth mhibitoiy amount of a PR0181 or PR0237 polypeptide, or an agonist thereof
  • the composition compnses a cytotoxic amount of a PR0181 or PR0237 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 PROl 81 or PR0237 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 81 or PR0237 polypeptide as herein defined, or an agonist thereof
  • the agonist is an anti-PROl 81 or ant ⁇ -PR0237 agonist antibody
  • the agonist is a small molecule that mimics the biological activity of a PROl 81 or PR0237 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 81 or PR0237 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 PRO 181 or PR0237 polypeptide or agonist thereof, for the inhibition of neoplastic cell growth, wherein the agonist may be an antibody which binds to the PROl 81 or PR0237 polypeptide
  • the agonist is an ant ⁇ -PR0181 or ant ⁇ -PR0237 agonist antibody
  • the agonist is a small molecule that mimics the biological activity of a PROl 81 or PR0237 polypeptide
  • Similar articles of manufacture comprising a PROl 81 or PR0237 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 181 or PR0237 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 81 or PR0237 polypeptide
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity , yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more prefei ably at least about 86% sequence identity, yet moie preferably at least about 87% sequence identit) , yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identit) , yet more preferably at least about 91 % sequence identity vet moi e prefei ably at least about 92% sequence identity yet more preferably at least about 93% sequence identity, yet more prefeiably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity yet
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99%
  • the invention concerns an isolated nucleic acid molecule comp ⁇ sing a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity yet mote preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet moi e preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet moi e preferably at least about 94% sequence identity, yet more preferably at least about 95%- sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98%
  • nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 60 nucleotides in length, yet more preferably at least about 70 nucleotides in length, yet more preferably at least about 80 nucleotides in length, yet more preferably at least about 90 nucleotides in length, yet more preferably at least about 100 nucleo
  • the invention provides isolated PROl 81 or PR0237 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified
  • the l ention concerns an isolated PROl 81 oi PR0237 polypeptide, comp ⁇ sing an amino acid sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more prefeiably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 9
  • the invention concerns an isolated PRO 181 or PR0237 polypeptide comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 81 % positives, more preferably at least about 82% positnes, yet more preferably at least about 83% positives yet more preferably at least about
  • the invention provides an isolated PRO l 81 oi PR0237 polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded bv 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 81 or PR0237 polypeptide and recovering the PROl 81 or PR0237 polypeptide from the cell culture
  • Another aspect of the invention provides an isolated PROl 81 or PR0237 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 81 or PR0237 polypeptide
  • the invention concerns a method of identifying agonists to a PRO 181 or PR0237 polypeptide which comprise contacting the PROl 81 or PR0237 polypeptide with a candidate molecule and monitoring a biological activity mediated by said PR0181 or PR0237 polypeptide
  • the PR0181 or PR0237 polypeptide is a native PROl 81 or PR0237 polypeptide
  • the invention concerns a composition of matter comprising a PROl 81 or PR0237 polypeptide, or an agonist of a PROl 81 or PR0237 polypeptide as herein described, or an anti-PROl 81 or ant ⁇ -PR0237 agonist 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 181 or PR0237 polypeptide, or an agonist thereof as hereinbefore described, or an anti-PRO l 81 or ant ⁇ -PR0237 agonist antibody for the preparation of a medicament useful in the treatment of a condition which is responsive to the PROl 81 or PR0237 polypeptide, an agonist thereof or an anti-PROl 81 or ant ⁇ -PR0237 agonist antibody
  • the invention provides vectors comp ⁇ sing DNA encoding any of the herein described polypeptides
  • Host cell comprising any such vector are also ⁇ ded
  • the host cells may be CHO cells, E toll, yeast or Baculovirus-intected insect cells
  • a process for producing any ot the herein described polypeptides is further provided and comprises culturing host cells undei conditions suitable for expression of the desired polypeptide and recovering the desued 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 chi enr molecules comprise any of the herein described polypeptides fused to an epitope tag sequence oi a Fc region of an immunoglobulin
  • the invention provides an antibody which specifically binds to any ot the above or below described polypeptides
  • the antibody is a monoclonal antibody, humanized antibody antibody fragment or single-chain antibody
  • the invention provides ohgonucleotide probes useful tor 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 PRO 181 cDNA, wherein SEQ ID NO 1 is a clone designated herein as "DNA23330-1390"
  • 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 5) of a native sequence PR0237 cDNA, wherein SEQ ID NO 5 is a clone designated herein as "DNA34353-1428"
  • Figure 4 shows the amino acid sequence (SEQ ID NO 6) derived from the coding sequence of SEQ ID NO
  • PROl 81 or “PR0237” polypeptide or protein when used herein encompass native sequence PR0181 orPR0237 andPR0181 or PR0237 variants (which are further defined herein)
  • ThePROl ⁇ l orPR0237 polypeptide may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant and/or synthetic methods
  • a “native sequence PR0181 " or “native sequence PR0237” comprises a polypeptide having the same amino acid sequence as the PRO 181 or PR0237 polypeptide as derived from nature Such native sequence PRO 181 or PR0237 polypeptide can be isolated from nature or can be produced by recombinant and/or synthetic means
  • the term "native sequence” PRO 181 or PR0237 specifically encompasses naturally-occurring truncated or secreted forms (e g , an extracellular domain sequence), naturally-occurring variant forms (e g , alternatively spliced forms) and naturally-occurring alle c variants of the PROl 81 and PR0237 polypeptides
  • the native sequence PRO 181 or PR0237 polypeptide is a mature or full-length nati ⁇ e sequence PRO 181 or PR0237 polypeptide as shown in Figure 2 (SEQ ID NO 2) or Figure 4 (SEQ ID NO 6) respectively
  • a polypeptide ECD will have less than about 1 % of such transmembrane and/or cytoplasmic domains and preferably will have less than about 0 57c of such domains It will be understood that any transmembrane domam(s) identified tor the polypeptides of the present invention are identified pursuant to criteria routinely employed in the art tor identifying that type ot hydrophobic domain The exact boundaries ot a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end ot the domain as initially identified and as shown in the appended figures As such, in one embodiment of the present invention, the extracellular domain of a polypeptide of the present invention comprises amino acids 1 to X of the mature amino acid sequence, wherein X is any amino acid within 5 amino acids on either side of the extracellular domain/transmembrane domain boundary
  • PROl 81 variant polypeptide means an active PROl 81 polypeptide (other than a native sequence PRO 181 polypeptide) as defined below, having at least about 807c amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 21 to 144 of the PRO 181 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) X to 144 of the PROl 81 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any amino acid residue from 16 to 25 of Figure 2 (SEQ ID NO 2), (c) 1 or about 21 to X of Figure 2 (SEQ ID NO 2), wherein X is any amino acid from amino acid 27 to amino acid 36 of Figure 2 (SEQ ID NO 2) or (d) another specifically derived fragment of the am o acid sequence shown in Figure 2 (SEQ ID NO 2)
  • PR0237 variant polypeptide means an active PR0237 polypeptide (other than a native sequence PR0237 polypeptide) as defined below, having at least about 807c amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 24 to 328 of the PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), (b) X to 328 of the PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), wherein X is any amino acid residue from 19 to 28 of Figure 4 (SEQ ID NO 6), (c) 1 or about 24 to X of Figure 4 (SEQ ID NO 6), wherein X is any amino acid from amino acid 172 to amino acid 181 ofF ⁇ gure4 (SEQ ID NO 6) or (d) anothe ⁇ specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 6)
  • PROl 81 and PR0237 variants include, for instance, PROl 81 and PR0237 polypeptides wherein one or more amino acid residues are added, or deleted, at the N or C-termmus, as well as within one or more internal domains of the native sequence
  • a PROl 81 variant will have at least about 807c amino acid sequence identity, more preferably at least about 81 % amino acid sequence identity, more preferably at least about 827c amino acid sequence identity, more preferably at least about 837c amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence ⁇ dent ⁇ t ⁇ , more preferably at least about 86% amino acid sequence identm more preferably at least about 877c amino acid sequence identity more preferably at least about 887c amino acid sequence identity more preferably at least about 897c amino acid sequence identity, moi e prefei ably at least about 90% amino acid sequence identity, more preferably at least about 91 % amino acid sequence identity, more preferably at least about 927r amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least
  • PROl 81 and PR0237 variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 ammo acids in length, more often at least about 50 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 amino acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, oie often at least about 250 amino acids in length, more often at least about 300 amino acids in length, or more
  • Table 1 provides the complete source code tor the ALIGN-2 sequence comparison computer program This source code may be routinely compiled toi use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program
  • Tables 2A-2D show hypothetical exemplifications tor using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and 7c 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 PR0181 or PR0237 polypeptide of interest, “Comparison Protein” represents the amino acid sequence of a polypeptide against which the "PRO” polypeptide of interest is being compared, "PRO-DNA” represents a hypothetical PROl 81 or PR0237-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.
  • a sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
  • the program may create a tmp file m /tmp to hold info about traceback
  • *ps[ ⁇ ] toupper(*ps[ ⁇ ]), po[ ⁇ ] + +, ps[ ⁇ ] + + ,
  • *py + + *px; else if ( ⁇ slower(*px))
  • *py+ + toupper(*px), if ( ⁇ ndex("ATGCU",*(py-l))) natgc + + , ⁇ ⁇
  • Page 3 of nwsubr.c write a filled jmp struct offset of the prev one (if any)- nw()
  • Percent (%) amino acid sequence identity' with respect to the PRO 181 and PR0237polypept ⁇ de 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 PROl 81 or PR0237 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
  • % 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 (; 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 WU-BLAST
  • PR0237 variant polynucleotide or "PR0237 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PR0237 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 24 to 328 of the PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), (b) a nucleic acid sequence which encodes amino acids X to 328 of the PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), wherein X is any amino acid residue from 19 to 28 of Figure 4 (SEQ ID NO 6), (c) a nucleic acid sequence which encodes amino acids 1 or about 24 to X of Figure 4 (SEQ ID NO 6), wherein X is any amino acid from amino acid 172 to amino acid 181 of Figure 4 (SEQ ID NO 6) or (d) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4
  • PRO 181 and PR0237 variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in length, more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more
  • Percent (%) nucleic acid sequence identity with respect to the PROl ⁇ l and PR0237 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 PROl 81 or PR0237 polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Ine , 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 TXU 10087
  • the ALIGN-2 program is publicly available through Genentech, Ine , South San Francisco, California or may be compiled fio the source code
  • the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows
  • % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul etal , Nucleic Acids Res , _25 3389-3402 (1997))
  • NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov
  • the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucle
  • % nucleic acid sequence identity values may also be generated using the WU-BLAST-2 computer program (Altschul et al , Methods in Enzymology, 266460-480 ( 1996)) Most of the WU-BLAST-2 search parametei s aie set to the default values Those not set to default values / e .
  • a % nucleic acid sequence identity value is determined by div iding (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 ot interest (i e the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST 2 by (b) the total number ot nucleotides of the PRO polypeptide- encoding nucleic acid molecule of interest
  • an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic
  • 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
  • the % value of positives of a given amino acid sequence A to, with, or against a given am o 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
  • Isolated, 1 when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non proteinaceous solutes
  • the polypeptide will be purified ( 1 ) to a degree sufficient to obtain at least 15 residues of N-termmal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-i educing or reducing conditions using Coomassie blue or, preferably, silver stain Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component ot the PROl 81 or PR0237 natural environment will not be present Ordinarily, however, isolated polypep
  • An isolated nucleic acid molecule encoding a PROl 81 or PR0237 polypeptide or an isolated nucleic acid molecule encoding an anti-PROl 81 or ant ⁇ -PR0237 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 natuial source of the PRO 181 - or PR0237-encod ⁇ ng nucleic acid or the anti PRO 181 - or ant ⁇ -PR0237-encod ⁇ ng nucleic acid Preferably the isolated nucleic acid is tree of association with all components with which it is naturally associated An isolated PRO 181 - or PR0237-encod ⁇ ng nucleic acid molecule or an isolated anti-PRO 181 - or anti- PR0237-encod ⁇ ng 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 PROl 81- or PR0237-
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence
  • a ⁇ bosome 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 ohgonucleotide 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 8 land ant ⁇ -PR0237 monoclonal antibodies (including agonist antibodies), anti-PROl 81 and ant ⁇ -PR0237 antibody compositions with polyepitopic specificity, single chain anti-PROl 81 and ant ⁇ -PR0237 antibodies, and fragments of anti-PROl 81 and ant ⁇ -PR0237 antibodies (see below)
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, ; e the individual antibodies comprising the population are identical except tor possible naturally-occurring mutations that may be present in minor amounts "Stringency" of hybridization reactions is readily determinable by one ot ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperatuie, and salt concentration In general, longer probes require higher temperatures for proper annealing, while shorter probes need lowei temperatures Hybridization generally depends on the ability
  • Modely stringent conditions may be identified as described by Sambrook et al , Molecular Cloning A Laboratory Manual. New York Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e g , temperature, ionic strength and % SDS) less stringent that those described above
  • An example of moderately stringent conditions is overnight incubation at 37 °C in a solution comprising 20% formamide, 5 x SSC (150 mM NaCl, 15 mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH 7 6), 5 x Denhardt' s solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc as necessary to accommodate factors such as probe length and the like
  • epitope tagged when used herein refers to a chimeric polypeptide comprising a PROl 81 or PR0237 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)
  • immunoadhesin designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site ot an antibody (/ e is "heterologous"), and an immunoglobulin constant domain sequence
  • the adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand
  • the immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin. such as IgG 1 , IgG-2, IgG-3, or IgG 4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM
  • Activity or ' activity refers to torm(s) of PR0181 oi PR0237 which retain a biological and/or an immunological activity of native oi naturally-occurring PROl 81 or PR0237, wherein
  • biological activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally occurring PRO 181 or PR0237 other than the ability to induce the production ot an antibody against an antigemc epitope possessed by a native or naturally occurring PROl 81 or PR0237 and an ' immunological activity refers to the ability to induce the production of an antibody against an antigemc epitope possessed by a native or naturally- occurring PROl 81 or PR0237
  • 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 cross-reactivity means immunological cross-reactivity with at least one epitope of a PROl 81 or PR0237 polypeptide
  • Immunological cross-reactivity means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO 181 or PR0237 polypeptide having this activity with polyclonal antisera raised against the known active PROl 81 or PR0237 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 lntrape ⁇ toneal or subcutaneous injection in incomplete Freunds
  • the immunological cross-reactivity preferably is "specific", which means that the binding affinity of the lmmunologically cross-reactive molecule (e g , antibody) identified, to the corresponding PROl 81 or PR0237 polypeptide is significantly higher (preferably at least about 2-t ⁇ mes, more preferably at least about 4-t ⁇ mes, even more preferably at least about 6-t ⁇ mes, most preferably at least about 8-t ⁇ mes higher) than the binding affinity of that molecule to any other known native polypeptide
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth
  • cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, ghoblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer endomet ⁇ al carcinoma, salivary gland carcinoma, kidney cancer vulval cancer, thyroid cancer, hepatic carcinoma and vanous types of head and neck cancer
  • Treatment is an intervention performed with the intention ot preventing the development or i ermg the pathology of a disorder
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures
  • Those in need ot 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 lender 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 ot 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 PROl 81 or PR0237 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 PROl 81 or PR0237 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 PR0181 or PR0237 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 181 or PR0237 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 81 or PR0237 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 81 or PR0237 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 p ⁇ Y 9 " and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof
  • chemotherapeutic agent' is a chemical compound useful in the treatment of tumor, e g , cancer
  • chemotherapeutic agents include ad ⁇ amycin, doxorubicin, epiiubicin, 5-fluorourac ⁇ l, cytosine arabinoside ( Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e g , pachtaxel (Taxol Bristol- Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxoteie Rhone Poulenc Rorer, Antony.
  • Rnace) toxotere methotrexate cisplatin, melphalan, vinblastine, bleomycin, etoposide, lfosfamide, mitomycin C mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin carminomycin, aminopterin dactinomycin, mitomycins, esperamicins (see, U S Patent No 4,675, 187).
  • 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
  • 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, daunorubicm, etoposide, and bleomycin
  • DNA alkylating agents such as tamoxifen, prednis
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators
  • lymphokines include lymphokines, monokines, and traditional polypeptide hormones Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsuhn, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteimzing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor- ⁇ and - ⁇ , mulle ⁇ an-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothe al growth factor, integrin, thrombopoietin (TPO), nerve growth factors such as NGF- ⁇ , platelet- growth factor
  • growth hormone
  • 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-contaimng prodrugs, glycosylated prodrugs or optionally substituted phenylacetamide containing prodrugs, 5-fluorocytos ⁇ ne and other 5-fluorou ⁇ d ⁇ ne prodrugs which can be deriva
  • agonist is used in the broadest sense and includes any molecule that mimics a biological activity of a native PROl 81 or PR0237 polypeptide disclosed herein Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments oi amino acid sequence variants of native PRO 181 or PR0237 polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists of a PROl 81 or PR0237 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 polyvinylpyrrohdone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccha ⁇ des, 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 noniom
  • “Native antibodies” and “native immunoglobulins” 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 v a ⁇ es among the heavy chains of different immunoglobulin isotypes Each heavy and light chain also has regularly spaced mtrachain disulfide bridges Each heavy chain has at one end a variable 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 ot 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 tor its particulai antigen
  • CDRs complementarity-determining regions
  • FR framework regions
  • the variable domains ot native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, Kabat et al , NIH Publ No 91-3242, Vol I, pages 647-669
  • 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 En , 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-bindmg 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
  • the six CDRs confer antigen-binding specificity to the antibody
  • a single variable domain or half of an Fv comprising only three CDRs specific for an antigen
  • 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 ot a few residues at the carboxy terminus of the heavy chain CH 1 domain including one or more cysteines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine 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 w Inch have hinge cysteines between them Other chemical couplings of antibody fragments are also known
  • the "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on the amino acid sequences of their constant domains
  • immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e g , IgG 1 , 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, J 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
  • monoclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody is directed against a single determinant on the antigen
  • the monoclonal antibodies is directed against a single determinant on the antigen
  • the monoclonal antibodies
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the cha ⁇ n(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U S Patent No 4.816,567, Morrison etal , Proc Natl Acad Sci USA. 81 6851-6855 [1984])
  • humanized forms of non-human (e , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') . or other antigen-bindmg subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donoi antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity
  • Fv FR residues of the human immunoglobulin are replaced by corresponding non-human residues
  • humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences
  • the humanized antibody includes a PRIMATIZEDTMant ⁇ body wherein the antigen-binding region of the antibody is de ⁇ v ed from an antibody produced by immunizing macaque monkeys w ith 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 93/1 1 161 , and Hollmger et al , Proc Natl Acad Sci USA. 90 6444-6448 (1993)
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes
  • the antibody will be purified (1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-termmal 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 ot the present invention can adhere
  • solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysaccha ⁇ des (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and sihcones
  • the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275 149
  • a “liposome' is a small vesicle composed of various types of hpids. phospholipids and/or surfactant which is useful for delivery of a drug (such as a PROl 81 or PR0237 polypeptide or antibody thereto) to a mammal
  • a drug such as a PROl 81 or PR0237 polypeptide or antibody thereto
  • the components of the liposome are commonly arranged in a bilayer formation similai to the lipid arrangement of biological membranes
  • a "small molecule” is defined herein to have a moleculai weight below about 500 Daltons II Compositions and Methods of the Invention
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO 181 and PR0237
  • cDNAs encoding PROl 81 and PR0237 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below
  • cDNA clones encoding PRO 181 and PR0237 polypeptides have 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 8 land PR0237 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 81 and PR0237 variants can be prepared PRO 181 and PR0237 variants can be prepared by introducing appropriate nucleotide changes into the PRO 181 or PR0237 DNA, and/or by synthesis of the desired PROl 81 or PR0237 polypeptide Those skilled in the art will appreciate that amino acid changes may alter post- translational processes of the PROl 81 or PRO 237 polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics
  • Variations in the native full-length sequence PROl 81 or PR0237 or in various domains ot the PROl 81 or PR0237 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 181 or PR0237 that results in a change in the amino acid sequence of the PRO 181 or PR0237 as compared with the native sequence PRO 181 or PR0237
  • the variation is by substitution of at least one amino acid with any other amino acid in one or more ot the domains of the PROl 81 or PR0237
  • 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 81 or PR0237 with that of homologous known protein molecules and minimizing the number ot ammo acid sequence changes made in regions of high homo
  • PROl 81 and PR0237 polypeptide fragments are prov ided herein Such fragments may be ti uncated at the N-terminus or C-terminus, or may lack internal residues tor example, when compared with a full length native protein Certain fragments lack amino acid residues that are not essential tor a desned biological activ ity of the PRO 181 or PR0237 polypeptide
  • PRO 181 and PR0237 fragments may be prepared by any of a number of con ventional techniques Desired peptide fragments may be chemically synthesized
  • An alternative approach involves generating PROl 81 and PR0237 fragments by enzymatic digestion, e g , by treating the protein with an enzyme known to cleave proteins at sites defined by particular ammo 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
  • Substantial modifications in function or immunological identity of the PRO l 81 or PR0237 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, tor 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
  • Non-conservative substitutions will entail exchanging a member ot one ot these classes for another class
  • Such substituted residues also may be introduced into the conseivative substitution sites or more preferably, into the remaining (non-conserved) sites
  • the variations can be made using methods known in the art such as ohgonucleotide-mediated (site directed) mutagenesis, alanine scanning and PCR mutagenesis Site-directed utagenesis [Carter et al , Nucl Acids Res . H 4331 (1986), Zoller et al , Nucl Acids Res , H) 6487 ( 1987)], cassette mutagenesis [Wells et al , Gene, 34 315 (1985)], restriction selection mutagenesis [Wells etal .
  • Covalent modifications of PROl 81 andPR0237 are included within the scope of this invention
  • One type of covalent modification includes reacting targeted amino acid residues of a PROl 81 or PR0237 polypeptide with an organic de ⁇ vatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO 181 or PR0237
  • Den vatization with bifunctional agents is useful, for instance, for crosshnking PRO 181 or PR0237 to a water-insoluble support matrix or surface for use in the method for purifying anti-PROl 81 or anti- PR0237 antibodies, and vice-versa
  • Commonly used crosshnking agents include, e g , l ,l-b ⁇ s(d ⁇ azoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-az ⁇ dosal ⁇ cyl ⁇ c acid, homobifunctional lmidoesters, including disuccin
  • Another type of covalent modification of the PRO 181 or PR0237 polypeptide included within the scope of this invention comprises altering the native glycosylation pattem of the polypeptide "Altering the native glycosylation pattern" is intended toi purposes herein to mean deleting one or moie carbohydrate moieties found in native sequence PROl 81 or PR0237 (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 81 or PR0237
  • the phrase includes qualitative changes in the glycosylation of the native proteins involving a change in the natuie and piopoitions ot the various carbohydrate moieties present
  • Addition of glycosylation sites to the PRO 181 or PR0237 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 senne or threonine residues to the native sequence PROl 81 or PR0237 (for O-linked glycosylation sites)
  • the PROl 81 or PR0237 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PROl 81 or PR0237 polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids
  • Another type of covalent modification of PRO 181 or PR0237 comprises linking the PRO 181 or PR0237 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, 4,670,4 P. 4,791 ,192 or 4, 179,337
  • the PROl 81 or PR0237 polypeptide of the present invention may also be modified in a way to form a chimeric molecule comprising PROl 81 or PR0237 fused to another, heterologous polypeptide or amino acid sequence
  • such a chimeric molecule comprises a fusion of the PRO 181 or PR0237 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 PROl 81 or PR0237 polypeptide
  • the presence of such epitope-tagged forms of the PRO 181 or PR0237 polypeptide can be detected using an antibody against the tag polypeptide
  • provision of the epitope tag enables the PRO l 81 or PR0237 polypeptide to be ieadily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag
  • Various tag polypeptides and their respective antibodies are well known in the art Examples include poly histidine (poly-His) or poly-histidme-glycine (poly-His-gly) tags the flu HA tag polypeptide and its antibody 12CA5 [Field e
  • the chimeiic molecule may comprise a fusion of the PRO l 81 or PR0237 polypeptide with an immunoglobulin or a particular region of an immunoglobulin
  • an immunoglobulin or a particular region of an immunoglobulin
  • the immunoglobulin fusion preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PROl 81 or PR0237 polypeptide in place of at least one variable region within an Ig molecule
  • the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH 1 , CH2 and CH3 regions of an I-gGl molecule
  • PROl 81 or PR0237 The description below relates primarily to production of PROl 81 or PR0237 by culturing cells transformed or transfected with a vector containing PRO 181 or PR0237 nucleic acid It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PROl 81 or PR0237
  • the PROl 81 or PR0237 polypeptide sequence, or portions thereof may be produced by direct peptide synthesis using solid-phase techniques [see, e g , Stewart et al .
  • DNA encoding PROl 81 or PR0237 may be obtained from a cDNA library prepared from tissue believed to possess the PROl 81 or PR0237 mRNA and to express it at a detectable level Accordingly, human PROl 81 or human PR0237 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples
  • the PROl 81 - or PR0237-encod ⁇ ng 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 PROl 81 or PR0237 or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures such as described in Sambiook et al , Moleculai Cloning A Laboratoi v Manual (New York Cold).
  • the o gonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positiv es are minimized
  • the ohgonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the hbi ary being screened
  • Methods of labeling are well known in the art. and include the use of radiolabels like P-labeled ATP, biotinylation or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supia
  • 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 ammo acid sequence disclosed herein for the first time, and if necessary, using conventional primer extension procedures as described in Sambrook et al , supia, 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 181 or PR0237 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 heie include prokaryote, yeast, oi higher eukaryote cells Suitable prokaryotes include but aie not limited to eubacte ⁇ a.
  • Enterobacte ⁇ aceae such as E coli Vanous E coli strains are publicly available, such as E coli Kl 2 stram MM294 (ATCC 31 446), E coli X 1776 (ATCC 1 537), E coli strain W 1 10 (ATCC 27,325) and K5772 (ATCC 53,635)
  • Other suitable prokaryotic host cells include Enterobacte ⁇ aceae such & Eschench ⁇ a, e g , E coli. Entewbai.
  • Strain W31 10 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations
  • the host cell secretes minimal amounts of proteolytic enzymes
  • strain W31 10 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E colt W3110 strain 1 A2, which
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO 181 - or PR0237 encoding vectors Sacchai om ces cerevisiae is a commonly used lower eukaryotic host microorganism
  • Others include po be (Beach and Nu ⁇ se, Na e, 290 140 [1981], EP 139,383 published 2 May 1985), Kluvveiomyces hosts (U S Patent No 4,943,529, Fleer et ⁇ l , Bio/Technology, 9 968-975 ( 1991 )) such as, e g , K l ⁇ ctis (MW98-8C, CBS683, CBS4574, Louvencourt et ⁇ l , J_ Bacte ⁇ ol .737 [ 1983] ), K fr ⁇ gihs (ATCC 12,424), K bulg ⁇ i icus (ATCC 16,045), K wicker ⁇ mu (ATCC 24,
  • K ther otolei ⁇ , and K m ⁇ txi ⁇ nus, y ⁇ nowi ⁇ EP 402,226)
  • Piclu ⁇ p ⁇ stoi is (EP 183,070, Sreeknshna et ⁇ l , J_ Basic Microbiol , 28 265-278 [1988])
  • Candida Ti ichoderma teesia EP 244,234
  • Neuiospoia ctassa (Case et al .
  • 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 Kloeckeia Pichia Sacchai om y ces 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 181 or PR0237 are derived from multicellulai 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 hamstei 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 Vnol 36 59 (1977)), Chinese hamster ovary cellsADHFR (CHO, Urlaub and Chasm, Pioc Natl Acad Sci USA, 77 4216 (1980)), mouse sertoh cells (TM4 Mathei , Biol Reprod , 23 243 251 ( 1980)) human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), and mouse
  • the nucleic acid (e g , cDNA or genomic DNA) encoding PROl 81 or PR0237 may be inserted into a rephcable 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
  • 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 vectois containing one or more of these components employs standard ligation techniques which are known to the skilled artisan
  • the PROl 81 or PR0237 may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide
  • the signal sequence may be a component of the vector, or it may be a part of the PRO 181 - or PR0237-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, penicillmase, lpp, or heat-stable enterotoxin II leaders
  • the signal sequence may be, e g , the yeast invertase leader, alpha factor leader (including Sacchai omyces and Kluyveiomyces oc-factor leaders, the latter described in U S Patent No 5,010,182).
  • 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 w ell known tor a variety of bacteria, yeast, and viruses
  • the origin of replication from the plasmid pBR322 is suitable tor most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable tor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful tor 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) contei resistance to antibiotics or other toxins, e (> , ampicillin neomycin, methotrexate, or tetracychne, (b) complement auxotrophic deficiencies, or (c) supply critic. 1 1 nutrients not available from complex media, e g , the gene encoding D-alanine racemase tor Bacilli
  • suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PR0181 - or PR0237-encod ⁇ ng nucleic acid, such as DHFR or thymidine kinase
  • An appropnate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity prepared and propagated as described by Urlaub et al Proc Natl Acad Sci USA. 77 4216 ( 1980)
  • a suitable selection gene tor use in yeast is the tip 1 gene present in the yeast plasmid YRp7 [Stinchcomb et al , Nature.
  • 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 81- or PR0237 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 61 (1978), Goeddel et al , Nature, 281 544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res .
  • Promoters for use in bacterial systems also will contain a Shine Dalgarno (S D ) sequence operably linked to the DNA encoding PROl 81 or PR0237
  • suitable promoting sequences for use with yeast hosts include the promoters for 3 phosphoglycerate kinase [Hitzeman et al , J Biol Chem , 255 2073 (1980)] or other glycolytic enzymes [Hess et al , J Adv Enzyme Reg .7 149 (1968), Holland, Biochemistry, J_7 4900 ( 1978)], such as enolase, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 phosphoglycerate mutase, pyruvate kinase, t ⁇ osephosphate isomerase, phosphoglucose isomerase, and glucokinase
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metal lothione , 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 81 or PR0237 transcription from vectors in mammalian host cells is controlled for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 1 504 published 5 July 1989) adenovirus (such as Adenovirus 2) bovine papilloma virus avian sarcoma virus cvtomegalovirus a retrovirus, hepatitis-B virus and Simian Virus 40 (S V40) from heterologous mammalian promoters, e % the actin promoter or an immunoglobulin promoter, and from heat shock promoters, provided such promoters are compatible with the host cell systems
  • viruses such as polyoma virus, fowlpox virus (UK 2,21 1 504 published 5 July 1989) adenovirus (such as Adenovirus 2) bovine papilloma virus avian sarcoma virus cvtomegalovirus a retrovirus, hepatit
  • 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 w ill use an enhancer from a eukaryotic cell vn us Examples include the S V40 enhancer on the late side of the replication oi lgin (bp 100 270) the cvtomegalovirus early promoter enhancer the polyoma enhancer on the late side ot the replication origin and adenovn us enhancers
  • the enhancer may be spliced into the vector at a position 5 oi 3 to the PRO 181 or PR0237 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 tor the termination ot 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 81 or PR0237
  • 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 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 immunohisto
  • PROl 81 or PR0237 may be recovered from culture medium or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (e g , T ⁇ ton-X 100) or by enzymatic cleavage Cells employed in expression of PROl 81 or PR0237 can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents
  • PROl 81 or PR0237 may be desired to purify PROl 81 or PR0237 from recombinant cell proteins or polypeptides
  • the following procedures are exemplary of suitable purification procedures bv tractionation 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, to.
  • 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 81 or PR0237 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 81 or PR0237 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 ot such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobuhn, and soybean trypsin inhibitor Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adj uvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate) The imm
  • the antibodies may, alternatively, be monoclonal antibodies
  • Monoclonal antibodies may be prepared using hyb ⁇ doma methods, such as those described by Kohler and Milstein, Nature, 256495 ( 1975)
  • a hyb ⁇ doma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent
  • the lymphocytes may be immunized in viti o
  • the immunizing agent will typically include the PROl 81 or PR0237 polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs”) are used it cells of human origin aie desired, or spleen cells or lymph node cells are used if non human mammalian souices are desired The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as pol vethvlene glycol to form a hyb ⁇ doma cell [Goding.
  • PBLs peripheral blood lymphocytes
  • a suitable fusing agent such as pol vethvlene glycol
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells ot rodent, bovine and human origin Usually, rat or mouse myeloma cell lines are employed
  • the hyb ⁇ doma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the giowth or survival ot the unfused immortalized cells
  • a suitable culture medium that preferably contains one or more substances that inhibit the giowth or survival ot the unfused immortalized cells
  • HGPRT or HPRT the enzyme hy poxanthine guamne phospho ⁇ bosyl transferase
  • HGPRT or HPRT the culture medium tor the hybndomas typically will include hypoxanthine aminopterin, and thymidine ("HAT medium' ), which substances prevent the growth of HGPRT-deficient cells
  • Preferred immortalized cell lines are those that fuse efficiently, suppott stable high level expression of antibody by the selected antibody -producing cells and aie sensitive to a medium such as HAT medium
  • Moie preferred immortalized cell lines are murine myeloma lines, which can be obtained, tor instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia Human myeloma and mouse-human hetei omyeloma cell lines also hav e 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, Ine , New York, (1987) pp 51-63]
  • the culture medium in which the hyb ⁇ doma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PROl 81 or PR0237
  • 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 immunoabsorbent assay (ELIS A)
  • RIA radioimmunoassay
  • enzyme-linked immunoabsorbent assay enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal Biochem , 107 220 (1980)
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra] Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium 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 immunoglobulin purification procedures such as, for example, protein A Sepharose hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Patent No 4,816,567
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using ohgonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine 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 immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U S Patent No 4 816 567 Morrison
  • the antibodies may be monovalent antibodies
  • Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prev ent heavy chain crosshnking Alternatively, the relevant cysteine residues are substituted with another amino acid iesidue or are deleted so as to prevent crosshnking
  • the antibodies of the invention may fuither comprise humanized antibodies or human antibodies
  • Humanized forms of non-human (e g , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv , Fab, Fab', F(ab ) .
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit
  • 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 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.
  • 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 222 381 ( 1991 ). Marks et al , J Mol Biol 222 581 ( 1991 )] The techniques ot Cole et al , and Boerner et al aie also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R L ⁇ ss, p 77 (1985) and Boerner etal , J Immunol 147( 1 ) 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, whit h 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
  • 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 81 or PR0237, 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/hght-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 hybndomas (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 et al , EMBO J .
  • 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, comp ⁇ sing 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, it desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism
  • CHI first heavy-chain constant region
  • Bispecific antibodies can be prepared as full length antibodies oi antibody fragments (e g , F(ab') ., bispecific antibodies) Techniques for generating bispecific antibodies from antibody fragments have been described in the literature For example, bispecific antibodies can be prepaied using chemical linkage Brennan et al , Science, 229 81 (1985) describe a procedure wherein intact antibodies aie proteoly tically 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 pi event intermolecular disulfide formation The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives One of the Fab'-TNB derivativ es 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
  • Fab' fragments may be dnectly recovered from E coli and chemically coupled to form bispecific antibodies
  • Shalaby et al , J Exp Med , 175 217 225 (1992) describe the production of a fully humanized bispecific antibody F(ab')-, molecule
  • Each Fab' fragment was separately secreted from £ coli and subjected to directed chemical coupling in viti o 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 Kostelny ef ⁇ / , J Immunol , 148(5) 1547-1553 (1992)
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers
  • This method can also be utilized for the production of antibody homodimers
  • the "diabody' technology described by Holhnger et al , Proc Natl Acad Sci USA, 90 6444-6448 ( 1993) has provided an alternative mechanism for making bispecific antibody fragments
  • the fragments comprise a heavy-chain variable domain (V 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
  • t ⁇ specific antibodies can be prepared Tutt et al , J Immunol , 147 60 (1991 )
  • bispecific antibodies may bind to two different epitopes on a given PRO! 81 or PR0237 polypeptide herein
  • an anti PROl 81 or anti PR0237 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 particulai PROl 81 or PR0237 polypeptide
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which expiess a particular PROl 81 or PR0237 polypeptide
  • These antibodies possess a PROl 81- or PR0237-b ⁇ nd ⁇ ng arm and an arm which binds a cytotoxic agent or a radionuchde chelator, such
  • Heteroconjugate antibodies are also within the scope of the present inv ention Heteroconjugate antibodies are composed of two covalently pined antibodies Such antibodies have tor example, been proposed to target immune system cells to unwanted cells [U S Patent No 4,676 9801 and for treatment of HIV infection [WO 91 /00360, WO 92/200373 EP 03089 ] It is contemplated that the antibodies may be prepared m viti o using know n methods in synthetic protein chemistry, including those inv olv ing crosshnking agents For example, immunotoxins may be constructed using a disulfide exchange reaction oi by toi ming a thioether bond Examples of suitable reagents for this purpose include lminothiolate and methy l 4 mercaptobutv ⁇ midate and those disclosed, for example, in U S Patent No 4,676,980
  • cysteine res ⁇ due(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region
  • the homodime ⁇ c antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC)
  • ADCC antibody-dependent cellular cytotoxicity
  • Homodime ⁇ c 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.
  • the invention also pertains to immunoconjugates compi ising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (- e , a radioconjugate)
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (- e , a radioconjugate)
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeiuginosa), ⁇ cin A chain, ab ⁇ n A chain, modeccm A chain, alpha-sarcin, Aleuritesfoidu proteins dianthin proteins, Plntolaca amencana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin.
  • radioconjugated antibodies examples include : B ⁇ , ⁇ I, ' ''In, 9 "Y, and l 6 Re
  • Conjugates of the antibody and cytotoxic agent are made using a variety ot bifunctional protein-coupling agents such as N-succ ⁇ mm ⁇ dyl-3-(2-py ⁇ dyld ⁇ th ⁇ ol) propionate (SPDP).
  • SPDP N-succ ⁇ mm ⁇ dyl-3-(2-py ⁇ dyld ⁇ th ⁇ ol) propionate
  • lminothiolane (IT) bifunctional derivatives of lmidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate).
  • aldehydes such as glutareldehyde
  • bis-azido compounds such as bis (p-azidobenzoyl) hexanediamine
  • bis-diazomum derivatives such as b ⁇ s-(p-d ⁇ azon ⁇ umbenzoyl)-ethylened ⁇ am ⁇ ne
  • diisocyanates such as tolyene 2,6-dnsocyanate
  • bis- active fluorine compounds such as 1 ,5-d ⁇ fluoro-2,4-d ⁇ n ⁇ trobenzene
  • a ⁇ cin lmmunotoxin can be prepared as described in Vitetta et al .
  • MX-DTPA Carbon- 14-labeled 1 - ⁇ soth ⁇ ocyanatobenzyl-3 methyldiethylene t ⁇ aminepentaacetic acid
  • the antibody may be conjugated to a ' receptor' (such as streptavidin) toi 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 e a radionucleotide) 8 Immunoliposomes
  • a ' receptor' such as streptavidin
  • the antibodies disclosed herein may also be formulated as immunoliposomes Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein etal , 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 phosphatidylchohne, cholesterol, and PEG-de ⁇ vatized 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 .
  • chemotherapeutic agent such as Doxorubic
  • a chemotherapeutic agent is optionally contained within the liposome See, Gabizon et al , J National Cancer Inst , 81 (19) 1484 (1989)
  • NCI National Cancer Institute
  • cell-based assays and animal models tor 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 aie 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 ot tumors, and to test the efficacy ot candidate therapeutic agents, including antibodies, and other agonists of the nativ e polypeptides, including small molecule agonists.
  • the in i ⁇ o 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 cancel , etc
  • Non-recombinant animal models include toi example rodent, e g .
  • Such models can be generated by intioducing tumor cells into syngeneic mice using standard techniques, e g , subcutaneous injection, tail vein injection, spleen implantation, lntrape ⁇ toneal 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)
  • nude 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, BIO LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and S JL
  • 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
  • e g The Nude Mouse in Oncology Research, E Boven and B Winograd, e
  • 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), / -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 (Karmah 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 tumois 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), supia
  • 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 Aca
  • 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, Ine , (San Diego, California)
  • Tumors that arise in animals can be removed and cultured in ⁇ itio Cells from the in viti o cultures can then be passaged to animals Such tumors can serve as targets tor 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 oi more rounds of passage analyzed toi differential expression of genes of interest Such passaging techniques can be performed with any known tumor oi cancer cell lines
  • 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
  • tumor cells are propagated in vitio in cell culture Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about l Oxl O 6 to 10x10' 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 This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture (Zupi et al , Br J Cancer, 41 , suppl 4 309 [1980]), and evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells survive For further information about this tumor model see, Zacharski, Haemostasis, 16 300-320 [1986])
  • transgenic animals include those that carry the transgene only in part of their cells (' mosaic animals”).
  • the transgene can be integrated eithei as a single transgene, oi in concatamers, e g , head-to-head or head-to-tail tandems
  • Selective introduction ot a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al Pioc Natl Acad Sci USA.
  • transgenic animals can be monitored by standard techniques For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunocytochemistry The animals are further examined for signs of tumor or cancer development
  • Screening assays for drug candidates are designed to identity 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, ( ⁇ oly)pept ⁇ de immunoglobulin fusions, and, in particular, antibodies including without limitation, poly- and antibodies and antibody fiagments, single-chain antibodies, anti ldiotypic antibodies, and chimeric oi humanized versions ot such antibodies oi fragments, as well as human antibodies and antibody fragments
  • the assay s can be performed in a variety of formats, including protein protein binding assays biochemical screening assay s immunoassays and cell based assays, which are well characterized in the ai t
  • a receptor ot a polypeptide encoded by the gene identified heiein 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 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
  • protem-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and Song, Nature (London). 340 245-246 (1989), Chien et ⁇ l , Proc Natl Acad Sci USA, 88 9578-9582 ( 1991 )] as disclosed by Chevray and Nathans [Proc Natl Acad Sci USA.
  • yeast GAL4 89 5789-5793 ( 1991 )]
  • Many transcriptional activators such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-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 ot GAL4, and another, in which candidate activating proteins are fused to the activation domain
  • the expression of a GAL1 -l ⁇ cZ reporter gene under control of a GAL4-act ⁇ vated promoter depends on reconstitution of GAL4 activity via protein-protein interaction Colonies containing interacting polypeptides are detected with a chromoge c substrate for ⁇ -galactosidase
  • MATCHMAKERTM complete kit for identifying protein-protein interactions between two specific proteins using the two- hybrid technique is commercially
  • compositions The polypeptides of the present invention, agonist antibodies specifically binding proteins identified herein, as well as other molecules identified by the scieening assay s disclosed herein, can be administered tor the treatment of tumors, including cancers, in the form of phaimaceutical compositions
  • peptide molecules can be designed which ietain 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 ⁇ l . 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
  • 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 hei ein, 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 lyophihzed formulations or aqueous solutions
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethomum chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cycl
  • compositions such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrohdone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccha ⁇ des, disaccha ⁇ des, and other carbohydrates including glucose, mannose, or dext ⁇ ns, 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 noniomc surfactants such as TWEENTM, PLURONICSTM 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 inhibitor
  • the active ingredients may also be entrapped in microcapsules prepared for example by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose oi gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery svstems (for example, liposomes. albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macioemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A ed ( 1980)
  • 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 lyophihzation and reconstitution
  • compositions herein generally are placed into a container hav ing a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a h podermic injection needle
  • Sustained-release preparations may be prepared Suitable examples ot sustained-release preparations include semipermeable matrices ot solid hydrophobic polymers containing the antibody which matrices are in the form of shaped articles, e g , films, or microcapsules Examples ot sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate).
  • poly(v ⁇ nylalcohol)) polylactides U S Pat No 3,773,919
  • copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycohc acid copolymers such as the LUPRON DEPOTTM (miectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate)
  • LUPRON DEPOTTM miectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate
  • polymers such as ethylene-vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days
  • certain hydrogels release proteins for shorter time periods
  • When encapsulated antibodies remain in the body for a long time they may denature
  • 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, ghoblastomas, and various head and neck tumors), leukemias and lymphoid malignancies, other disorders such as neuronal, ghal, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoehc 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 agonists which mimic their activity, e g , antibodies, peptide
  • chemotherapeutic agents may be administered to the patient Prepaiation and dosing schedules tor 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-piogesterone such as onap ⁇ stone (see, EP 616812) in dosages known foi such molecules
  • the anti cancer agents herein are co administered w ith 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, 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
  • 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 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
  • an article of manufactuie containing materials useful for the diagnosis or treatment of the disorders described abov e is prov ided
  • 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 stoppei pierceable by a hypodermic injection needle)
  • the active agent in the composition is an anti-tumor agent of the present invention
  • the label on oi associated w ith, 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 buff er, such as phosphate buffered saline Ringers solution and dextrose solution It may further include
  • the yeast methods were divided into three categories ( 1 ) Transformation of yeast with the plasmid/cDNA combined vector, (2) Detection and isolation of yeast clones secreting amylase, and (3) PCR amplification of the insert directly from the yeast colony and purification of the DNA for sequencing and further analysis
  • yeast strain used was HD56-5A (ATCC-90785) This strain has the following genotype MAT alpha, ura3-52, leu2-3, leu2-l 12, h ⁇ s3 1 1, h ⁇ s3-15, MAL + , SUC + , GAL +
  • yeast mutants can be employed that have deficient post-translational pathways Such mutants may have translocation deficient alleles in seel 1 , secll, sec62, with truncated secl being most preferred
  • antagonists including antisense nucleotides and/or ligands which interfere with the normal operation of these genes, other proteins implicated in this post translation pathway (e g , SEC ⁇ lp, SEC72p, SEC62p, SEC63p, TDJlp or SSAlp-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast
  • the cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and cent ⁇ fuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centnfuge The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tns-HCl, 1 mM EDTA pH 7 5 100 mM Li-OOCCH,), and resuspended into LiAc/TE (2 5 ml)
  • Transformation took place by mixing the prepared cells (100 ⁇ l) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs Gaithersburg, MD) and transforming DNA ( 1 ⁇ g vol ⁇ l O ⁇ l) in microtuge tubes The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 ⁇ l 40% polyethylene glycol-4000, 10 mM T ⁇ s HCl, 1 M EDTA, 100 mM Li .OOCCH,, pH 7 5) was added This mixture was gently mixed and incubated at 30°C while agitating tor 30 minutes The cells were then heat shocked at 42°C tor 1 minutes, and the reaction vessel cent ⁇ fuged in a microfuge at 12,000 rpm for 5-10 seconds decanted and resuspended into TE (500 ⁇ l, lO mM Tns-HCl, 1 mM EDTA pH 7 5) followed by recenti if ugation The cells were then diluted into TE (1 m
  • the transformation was pei formed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly
  • the selective media used w s a synthetic complete dextrose agar lacking uracil (SCD Ura) prepared as described in Kaiser et al , Methods in Yeast Genetics, Cold Spring Harbor Pi ess Cold Spring Harbor, NY, p 208 210 (1994) Transformants were grown at 30°C for 2-3 days
  • the detection of colonies secreting amylase was performed by including red starch in the selective growth media Starch was coupled to the red dye (Reactive Red-120, Sigma) as per the procedure described by Biely et al , Anal Biochem , 172 176-179 (1988)
  • the coupled starch was incorporated into the SCD-Ura agar plates at a final concentration of 0 15% (w/v), and was buffered with potassium phosphate to a pH of 7 0 (50-100 mM final concentration)
  • the underlined regions of the oligonucleotides annealed to the ADH promoter region and the amylase region, respectively and amplified a 307 bp region from vectoi pSST-AMY 0 when no insert was present
  • the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers
  • the total product of the PCR reaction tiom an empty vector was 343 bp Howevei .
  • cDNA sequence isolated in the above screen was found, by BLAST and FastA sequence alignment, to have sequence homology to a nucleotide sequence encoding the cornichon protein
  • This cDNA sequence is herein designated DNA13242 and DNA25383
  • probes were generated from the sequence of the DNA13242 molecule and used to screen a human placenta (LIB89) library prepared as described in paragraph 1 above
  • the cloning vector was pRK5B (pRK5B is a precursor of pRK5D that does not contain the Sfi site, see, Holmes et al , Science, 253 1278-1280 (1991 )), and the cDNA size cut was less than 2800 bp
  • PRO 181 may be a novel cornichon homolog More specifically, an analysis of the Dayhott database (version 35 45 SwissProt 35) evidenced significant homology between the PROl 81 amino acid sequence and the following Dayhott sequences AF02281 1 CET09E8_3, S64058, YGF4_YEAST, YB60_YEAST EBU89455 SIU36383_3 AND PH1 71
  • the extracellular domain (ECD) sequences (including the secretion signal sequence, it any) from about 950 known secreted proteins from the Swiss Prot public database were used to search EST databases
  • the EST databases included public EST databases (e g , GenBank) and a piop ⁇ etary EST database (LIFESEQ 8 Incyte Pharmaceuticals Palo Alto, CA)
  • the search was performed using the computer program BLAST or BLAST2 [Altschul et al , Methods in Enzymology, 266 460 480 ( 1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins weie clustered and assembled into consensus DNA sequences with the program ' phrap" (Phil Green University of Washington, Seattle Washington)
  • a consensus DNA sequence was assembled relative to other EST sequences using phrap as descnbed above This consensus sequence is herein designated Consen0514 and DNA30905
  • the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above
  • oligonucleotides were synthesized 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0237
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length
  • the probe sequences are typically 40-55 bp in length
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1 5 kbp In order to screen several libraries foi a full-length clon
  • PCR primers (forward and reverse) were synthesized forward PCR primer (30905 f )
  • a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA30905 sequence which had the following nucleotide sequence hybridization probe (30905 p)
  • RNA for construction of the cDNA libraries was isolated from human fetal brain tissue
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with ohgo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursoi of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278- 1280 ( 1991 )) in the unique Xhol and Notl sites
  • DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PR0237 polypeptide (designated
  • the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 586-588 and a stop signal at nucleotide positions 1570- 1 72 (Figuie 3 SEQ ID NO 5)
  • the predicted polypeptide precursor is 328 amino acids long, and has a calculated molecular weight of approximately 36,238 daltons and a pi ot about 9 90 Analysis ot the full-length PR0237 sequence shown in Figure
  • PRO 181 or PRQ237 as a Hybridization Probe The following method describes use of a nucleotide sequence encoding PROl 81 or PR0237 as a hybridization probe
  • DNA comprising the coding sequence of full-length or mature PRO 181 or PR0237 (as shown in Figure 1 and 3, respectively, SEQ ID NOS 1 and 5, respectively) or a fragment thereof is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PROl 81 or PR0237) 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 181 or PR0237 polypeptide to the filters is performed in a solution of 50% formamide, 5 ⁇ SSC 0 1 % SDS, 0 1 % sodium pyrophosphate, 50 mM sodium phosphate, pH 6 8, 2x Denhardt's solution, and 109c dextran sulfate at 42"C for 20 hours
  • Washing ot 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
  • the DNA sequence encoding PROl 81 or PR0237 is initially amplified using selected PCR pi lmers
  • the primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vectoi
  • a variety of expression vectors may be employed An example ot a suitable vector is pBR322 (derived from £ coli, see Bolivar et al , Gene, 2 95 (1977)) which contains genes for ampicillin and tetracychne resistance
  • the vector is digested with restriction enzyme and dephosphorylated
  • the PCR amplified sequences are then ligated into the vector
  • the vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a poly His leader (including the first six STII codons, poly His sequence, and enterokinase cleavage site), the PROl 81 or PR0237 coding region, lambda transcriptional terminator, and an argU gene
  • the ligation mixture is then used to transform a selected £ coli strain using the methods described in Sambrook et al , supia Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected Plasmid DNA can be isolated and confirmed by 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
  • 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 PRO 181 or PR0237 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein
  • PRO 181 or PR0237 may be expressed in £ coli in a poly-His tagged form, using the following procedure
  • the DNA encoding PRO 181 or PR0237 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 £ coli host based on strain 52 (W31 10 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(lacIq) Transformants are first grown in LB containing 50 mg/ml carbenicilhn at 30°C with shaking until an OD 6IK , of 3 5 is reached Cultures are then diluted 50 100 fold into CRAP media (
  • the proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of 20 mM T ⁇ s, 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 acetonitnle 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 acetonitnle from 10 to 80% Aliquots of fractions
  • 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 81 and PR0237 were successfully expressed in £ coli in a poly-His tagged form by the above procedure
  • EXAMPLE 4 Expression of PROl 81 or PRQ237 in mammalian cells This example illustrates preparation of a potentially glycosylated form of PROl 81 or PR0237 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 181 or PR0237 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PROl 81 or PR0237 DNA using ligation methods such as described in Sambrook etal , supia
  • the resulting vector is called pRK5-PRO 181 or pRK5-PR0237
  • the selected host cells may be 293 cells Human 293 cells (ATCC CCI 1573) aie 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 181 or pRK5-PR0237 DNA is mixed with about 1 ⁇ g DNA encoding the VA RNA gene [Thimmappav ef al . Cell.
  • the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ⁇ Ci/ml and 200 ⁇ Ci/ml 15 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 dried and exposed to film for a selected period of time to reveal the presence of the PROl 81 or PR0237 polypeptide
  • the cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays
  • PROl 81 or PR0237 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-PR0181 or ⁇ RK5-PR0237 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 transfer ⁇ n After about four days, the conditioned media is cent ⁇ fuged and filtered to remove cells and debris The sample containing expressed PROl 81 or PR0237 can then be
  • PROl 81 or PR0237 can be expressed in CHO cells
  • the pRK5-PR0181 or pRK5-PR0237 can be transfected into CHO cells using known reagents such as CaP0 or DEAE-dextran
  • the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as
  • 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 PROl 81 or PR0237 polypeptide can then be concentrated and purified by any selected method
  • Epitope-tagged PROl 81 or PR0237 may also be expressed in host CHO cells
  • the PROl 81 or PR0237 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
  • PROl 81 orPR0237 may also be expressed in CHO and/or COS cells by a tiansient expression procedure or in CHO cells by another stable expression procedure Stable expression in CHO cells is performed using the following procedure
  • the proteins aie expressed as an IgG construct immunoadhesin
  • the coding sequences for the soluble forms (e g extiacellular 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
  • the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al , Cunent 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
  • 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 mis of media and cent ⁇ fuged 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
  • 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
  • the proteins are purified using a Ni ⁇ NTA column (Qiagen) Before purification, lmidazole is added to the conditioned media to a concentration of 5 mM The conditioned media is pumped onto a 6 ml Ni + -NTA column equilibrated in 20 mM Hepes, pH 7 4, buftei containing 0 3 M NaCl and 5 mM lmidazole at a flow rate ot 4-5 ml/min at 4°C After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration butter containing 0 25 M lmidazole The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80' C
  • Immunoadhesin (Fc-containing) constructs 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
  • yeast expression vectors are constructed for intracellular production or secretion of PRO 181 or PR0237 from the ADH2/GAPDH promoter DNA encoding PROl 81 or PR0237 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PROl 81 or PR0237
  • DNA encoding PR0181 or PR0237 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PROl 81 or PR0237 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 81 or PR0237
  • yeast cells such as yeast strain AB110
  • 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% t ⁇ chloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain
  • Recombinant PRO 181 or PR0237 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 81 or PR0237 may further be purified using selected column chromatography resins
  • PROl 81 or PRQ237 in Baculovirus-Intected Insect Cells
  • the sequence coding for PROl 81 or PR0237 is fused upstream ot an epitope tag contained within a baculovirus expression vector
  • epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG)
  • plasmids may be employed, including plasmids derived ti om commercially available plasmids such as pVLl 393 (Novagen)
  • the sequence encoding PRO 181 or PR0237 or the desired portion of the coding sequence of PROl 81 or PR0237 is amplified by PCR with primers complementary to the 5' and 3' regions
  • Recombinant baculovirus is generated by co-transtecting the abov e plasmid and BaculoGold 1 virus DNA (Pharmingen) into Spodopteiafiugipei da (“Sf9") cells (ATCC CRL 171 1 ) using hpotectin (commercially available from GIBCO-BRL) After 4 - 5 days of incubation at 28"C. the leleased viruses are harvested and used for furthei amplifications Vnal 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 PROl 81 orPR0237 can then be purified, for example, by Nf + -chelate affinity chromatography as follows Extracts are prepared from recombinant virus-inf ected Sf 9 cells as described by Rupert et al , Natuie, 362 175 179 (1993) Briefly, Sf9 cells are washed, resuspended in so cation 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 butter (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7 8) and filtered through a 0 45 mm filter A N ⁇ 2+ -
  • purification of the IgG tagged (or Fc tagged) PROl 81 or PR0237 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography
  • baculovirus expression vector pb PH IgG for IgG fusions and pb PH His c for poly-His tagged proteins
  • vector and Baculogold® baculovirus DNA are co-transfected into 105 Spodoptera fi ugiperda ("Sf9") cells (ATCC CRL 171 1 ), using Lipofectin (Gibco BRL)
  • pb PH IgG and pb PH His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polyhnker regions to include the His or Fc tag sequences
  • the cells are grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone) Cells are incubated for 5 days at 28 °C The supernatant is harvested and subsequently used for the first viral amplification by infecting S
  • the first viral amplification supernatant is used to infect a spinner cultuie (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an appioximate MOI of 0 1 Cells are incubated for 3 day s at 28 °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 lemove the cells and tilteied through 0 22 micron filters
  • the protein construct is purified using a Ni 1+ -NTA column (Qiagen) Before purification lmidazole is added to the conditioned media to a concentration of 5 mM
  • the conditioned media is pumped onto a 6 ml Ni ,+ -NTA column equilibrated in 20 mM Hepes, pH 7 4, buffer containing 0 3 M NaCl and 5 mM lmidazole at a flow rate of 4-5 ml/min at 4°C
  • the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M lmidazole
  • the highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mann
  • Immunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows
  • the conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3 5
  • the eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M T ⁇ s 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
  • a modified baculovirus procedure may be used incorporating h ⁇ gh-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
  • 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 pIEl -1 (Novagen)
  • the pIEl -1 and pIEl -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus lei promoter in stably- transformed insect cells (1)
  • the plasmids diftei only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for lei -mediated gene expression in uninfected insect cells as well as the hr5
  • H ⁇ gh-5 cells are grown to a confluency of 50% under the conditions of, 27 °C, no CO ⁇ NO pen/strep
  • 30 ⁇ g ofpIE 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, lOO ⁇ l of CellFectin (CellFEC ⁇ N (G ⁇ bcoBRL# 10362-010) (vortexed to mix)) is mixed with 1 ml of Ex-Cell medium
  • the two solutions are combined and allow ed to incubate at room temperature foi 15 minutes 8 ml of Ex Cell media is added to the 2 ml of DNA/CellFECTIN mix and this is layered on h ⁇ gh-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 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) 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
  • 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% mannito
  • Immunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows
  • the conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8
  • 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 T ⁇ s 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
  • PR0237 was expressed using the above baculovirus procedure employing h ⁇ gh-5 cells
  • supia Immunogens that may be employed include purified PROl 81 or PR0237.
  • fusion proteins containing PROl 81 or PR0237, and cells expressing recombinant PROl 81 or PR0237 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 81 or PR0237 immunogen emulsified in complete
  • mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant Thereattei , tor several weeks, the mice may also be boosted with additional immunization injections Serum samples may be pei iodicallv obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-PRO l 81 or ant ⁇ -PR0237 antibodies
  • 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 1 , available from ATCC, No CRL 1597
  • the fusions generate hybndoma 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 hybndoma cells will be screened in an ELISA for reactivity against PROl 81 or PR0237 Determination of positive' hybndoma cells secreting the desired monoclonal antibodies against PROl 81 or PR0237 is within the skill in the art
  • the positive hybndoma cells can be injected lntrape ⁇ toneally into syngeneic Balb/c mice to produce ascites containing the anti-PROl 81 or anti PR0237 monoclonal antibodies
  • the hybndoma 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
  • Native or recombinant PROl 81 or PR0237 polypeptides may be purified by a variety of standard techniques in the art of protein purification
  • pro-PR0181 or pro PR0237 polypeptide, mature PRO 181 or mature PR0237 polypeptide, or pre PRO 181 or pre-PR0237 polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PROl 81 or PR0237 polypeptide of interest
  • an immunoaffinity column is constructed by covalently coupling the anti-PROl 81 or ant ⁇ -PR0237 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 from mouse ascites fluid by ammonium sultate 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
  • Such an immunoaffinity column is utilized in the purification of the PROl 81 or PR0237 polypeptide by preparing a fraction from cells containing the PROl 81 or PR0237 polypeptide in a soluble form This preparation is derived by solubihzation of the whole cell or of a subcellular fraction obtained v la differential centrifugation by the addition of detergent or by other methods well known in the art Alternatively soluble PROl 81 or PR0237 polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown
  • a soluble PROl 81 or PR0237 polypeptide containing pieparation is passed over the lmmunoatfmitv column, and the column is washed under conditions that allow the preferential absorbance of the PROl 81 or
  • PR0237 polypeptide (e g , high ionic strength buffers in thespectnce ot deumbleent) Then, the column is eluted under conditions that disrupt antibody/PROl 81 or ant ⁇ body/PR0237 polypeptide binding (e ? , 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 81 or PR0237 polypeptide is collected
  • EXAMPLE 9 Drug Screening This invention is particularly useful for screening compounds by using PRO 181 or PR0237 polypeptides or a binding fragment thereof in any of a variety of drug screening techniques
  • the PROl 81 or PR0237 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 PROl 81 or PR0237 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 81 or PR0237 polypeptide or a fragment and the agent being tested Alternatively, one can examine the diminution in complex formation between the PROl 81 or PR0237 polypeptide and its target cell or target
  • Another technique for drug screening provides high throughput screening tor 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 81 or PR0237 polypeptide, the peptide test compounds are reacted with the PROl 81 or PR0237 polypeptide and washed Bound PROl 81 or PR0237 polypeptide is detected by methods well known in the art Purified PRO 181 or PR0237 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 PROl 81 or PR0237 polypeptide specifically compete with a test compound for binding to the PROl 81 or PR0237 polypeptide or fragments thereof In this manner, the antibodies can be used to detect the presence ot any peptide which shares one oi more antigenic detei minants with a PRO 181 oi PR0237 polypeptide
  • 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 81 or PR0237 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 81 or PR0237 polypeptide or which enhance or interfere with the function of the PROl 81 or PR0237 polypeptide in vivo (cf, Hodgson, Bio/
  • the three-dimensional structure of the PRO 181 or PR0237 polypeptide. or of a PROl 81 or PR0237 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 81 or PR0237 polypeptide must be ascertained to elucidate the structure and to determine active s ⁇ te(s) of the molecule
  • useful information regarding the structure of the PRO 181 or PR0237 polypeptide may be gained by modeling based on the structure of homologous proteins In both cases, relevant structural information is used to design analogous PRO 181 or PR0237 polypeptide-hke 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
  • the antiprohferative activity of the PR0181 and PR0237 polypeptides was determined in the investigational, disease oriented in vitio anti-cancer drug discoveiy 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 .
  • NCI National Cancer Institute
  • SRB sulforhodamine B
  • 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.

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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 Clin . 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 obiective 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 piesent invention relates to methods and compositions for inhibiting neoplastic cell growth More particularly, the invention concerns methods and compositions for the treatment ot 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 ot matter useful for the inhibition oi neoplastic cell growth comprising an effective amount ot a PRO 181 or PR0237 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 mhibitoiy amount of a PR0181 or PR0237 polypeptide, or an agonist thereof In another preferred embodiment, the composition compnses a cytotoxic amount of a PR0181 or PR0237 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 PROl 81 or PR0237 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 81 or PR0237 polypeptide as herein defined, or an agonist thereof In a particular embodiment, the agonist is an anti-PROl 81 or antι-PR0237 agonist antibody In another embodiment, the agonist is a small molecule that mimics the biological activity of a PROl 81 or PR0237 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 81 or PR0237 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 PRO 181 or PR0237 polypeptide or agonist thereof, for the inhibition of neoplastic cell growth, wherein the agonist may be an antibody which binds to the PROl 81 or PR0237 polypeptide
In a particular embodiment, the agonist is an antι-PR0181 or antι-PR0237 agonist antibody In another embodiment, the agonist is a small molecule that mimics the biological activity of a PROl 81 or PR0237 polypeptide Similar articles of manufacture comprising a PROl 81 or PR0237 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 181 or PR0237 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 81 or PR0237 polypeptide
In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity , yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more prefei ably at least about 86% sequence identity, yet moie preferably at least about 87% sequence identit) , yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identit) , yet more preferably at least about 91 % sequence identity vet moi e prefei ably at least about 92% sequence identity yet more preferably at least about 93% sequence identity, yet more prefeiably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to (a) a DNA molecule encoding a PROl 81 or PR0237 polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a)
In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length PROl 81 or PR0237 polypeptide cDNA as disclosed herein, the coding sequence of a PRO 181 or PR0237 polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PRO 181 or PR0237 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 compπsing a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity yet mote preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet moi e preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet moi e preferably at least about 94% sequence identity, yet more preferably at least about 95%- sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 997c sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule ot (a) Another aspect of the invention provides an isolated nucleic acid molecule compi ising a nucleotide sequence encoding a PROl 81 oi PR0237 polypeptide which is eithei transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembi ane domaιn(s) of such polypeptide are disclosed herein Therefore, soluble extracellular domains of the herein described PROl 81 or PR0237 polypeptides are contemplated
Another embodiment is directed to fragments of a PROl 81 or PR0237 polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PROl 81 or PR0237 polypeptide that may optionally encode a polypeptide comprising a binding site for an antι-PR0181 or antι-PR0237 antibody or as antisense ohgonucleotide probes Such nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 60 nucleotides in length, yet more preferably at least about 70 nucleotides in length, yet more preferably at least about 80 nucleotides in length, yet more preferably at least about 90 nucleotides in length, yet more preferably at least about 100 nucleotides in length, yet more preferably at least about 110 nucleotides in length, yet more preferably at least about 120 nucleotides in length, yet more preferably at least about 130 nucleotides in length, yet more preferably at least about 140 nucleotides in length yet more preferably at least about 150 nucleotides in length, yet more preferably at least about 160 nucleotides in length, yet more preferably at least about 170 nucleotides in length, yet more preferably at least about 180 nucleotides in length, yet more preferably at least about 190 nucleotides in length, yet more preferably at least about 200 nucleotides in length, yet more preferably at least about 250 nucleotides in length, yet more preferably at least about 300 nucleotides in length, yet more preferably at least about 350 nucleotides in length, yet more preferably at least about 400 nucleotides in length, yet more preferably at least about 450 nucleotides in length, yet more preferably at least about 500 nucleotides m length, yet more preferably at least about 600 nucleotides in length, yet more preferably at least about 700 nucleotides in length, yet more preferably at least about 800 nucleotides in length yet more preferably at least about 900 nucleotides in length and yet more preferably at least about 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length It is noted that novel fragments of a PROl 81 or PR0237 polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PROl 81 or PR0237 polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a numbei of well know n sequence alignment programs and determining which PRO 181 or PR0237 polypeptide-encoding nucleotide sequence fιagment(s) are novel All of such PRO 181 or PR0237 polypeptide-encoding nucleotide sequences are contemplated herein Also contemplated are the PROl 81 or PR0237 polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO 181 or PR0237 polypeptide fragments that comprise a binding site tor an anti-PRO 181 or antι-PR0237 antibody
In another embodiment, the invention provides isolated PROl 81 or PR0237 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified
In a certain aspect, the l ention concerns an isolated PROl 81 oi PR0237 polypeptide, compπsing an amino acid sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more prefeiably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to a PROl 81 or PR0237 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 81 or PR0237 polypeptide compπsing an amino acid sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein
In a further aspect, the invention concerns an isolated PRO 181 or PR0237 polypeptide comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 81 % positives, more preferably at least about 82% positnes, yet more preferably at least about 83% positives yet more preferably at least about
84% positives, yet more preferably at least about 85% positnes \et more preferably at least about 86% positives, yet more preferably at least about 87% positives, yet more preferably at least about 88% positives, yet more preferably at least about 89% positives yet more preferably at least about 90% positives, yet more preferably at least about 91 % positives, yet more preferably at least about 92% positives, yet more preferably at least about 93% positives, yet more preferably at least about 94% positives yet more preferably at least about 95% positn es, yet more preferably at least about 96% positives, yet more preferably at least about 97% positives, yet more prefei ably at least about 98% positives and yet more preferably at least about 99% positives when compared with the amino acid sequence of a PRO 181 or PR0237 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 oi any other specifically defined fragment ot the full-length amino acid sequence as disclosed herein
In a specific aspect the invention provides an isolated PRO l 81 oi PR0237 polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded bv 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 81 or PR0237 polypeptide and recovering the PROl 81 or PR0237 polypeptide from the cell culture Another aspect of the invention provides an isolated PROl 81 or PR0237 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 81 or PR0237 polypeptide and recovering the PROl 81 or PR0237 polypeptide from the cell culture In yet another embodiment, the invention concerns agonists of a native PRO 181 or PR0237 polypeptide as defined herein In a particular embodiment, the agonist is an anti-PROl 81 or antι-PR0237 agonist antibody or a small molecule
In a further embodiment, the invention concerns a method of identifying agonists to a PRO 181 or PR0237 polypeptide which comprise contacting the PROl 81 or PR0237 polypeptide with a candidate molecule and monitoring a biological activity mediated by said PR0181 or PR0237 polypeptide Preferably, the PR0181 or PR0237 polypeptide is a native PROl 81 or PR0237 polypeptide
In a still further embodiment, the invention concerns a composition of matter comprising a PROl 81 or PR0237 polypeptide, or an agonist of a PROl 81 or PR0237 polypeptide as herein described, or an anti-PROl 81 or antι-PR0237 agonist 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 181 or PR0237 polypeptide, or an agonist thereof as hereinbefore described, or an anti-PRO l 81 or antι-PR0237 agonist antibody for the preparation of a medicament useful in the treatment of a condition which is responsive to the PROl 81 or PR0237 polypeptide, an agonist thereof or an anti-PROl 81 or antι-PR0237 agonist antibody In other embodiments of the present invention, the invention provides vectors compπsing DNA encoding any of the herein described polypeptides Host cell comprising any such vector are also
Figure imgf000008_0001
ιded By way ot example, the host cells may be CHO cells, E toll, yeast or Baculovirus-intected insect cells A process for producing any ot the herein described polypeptides is further provided and comprises culturing host cells undei conditions suitable for expression of the desired polypeptide and recovering the desued 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 chi enr molecules comprise any of the herein described polypeptides fused to an epitope tag sequence oi a Fc region of an immunoglobulin In another embodiment, the invention provides an antibody which specifically binds to any ot the above or below described polypeptides Optionally, the antibody is a monoclonal antibody, humanized antibody antibody fragment or single-chain antibody
In yet other embodiments, the invention provides ohgonucleotide probes useful tor 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 PRO 181 cDNA, wherein SEQ ID NO 1 is a clone designated herein as "DNA23330-1390"
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 5) of a native sequence PR0237 cDNA, wherein SEQ ID NO 5 is a clone designated herein as "DNA34353-1428" Figure 4 shows the amino acid sequence (SEQ ID NO 6) derived from the coding sequence of SEQ ID
NO 5 shown in Figure 3
DETAILED DESCRIPTION OF THE INVENTION
The terms "PROl 81 " or "PR0237" polypeptide or protein when used herein encompass native sequence PR0181 orPR0237 andPR0181 or PR0237 variants (which are further defined herein) ThePROl δl orPR0237 polypeptide may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant and/or synthetic methods
A "native sequence PR0181 " or "native sequence PR0237" comprises a polypeptide having the same amino acid sequence as the PRO 181 or PR0237 polypeptide as derived from nature Such native sequence PRO 181 or PR0237 polypeptide can be isolated from nature or can be produced by recombinant and/or synthetic means The term "native sequence" PRO 181 or PR0237 specifically encompasses naturally-occurring truncated or secreted forms (e g , an extracellular domain sequence), naturally-occurring variant forms (e g , alternatively spliced forms) and naturally-occurring alle c variants of the PROl 81 and PR0237 polypeptides In one embodiment of the invention, the native sequence PRO 181 or PR0237 polypeptide is a mature or full-length nati \ e sequence PRO 181 or PR0237 polypeptide as shown in Figure 2 (SEQ ID NO 2) or Figure 4 (SEQ ID NO 6) respectively Also, while the PR0181 and PR0237 polypeptides disclosed in Figure 2 (SEQ ID NO 2) oi Figuie 4 (SEQ ID NO 6), respectively, are shown to begin with the methionine residue designated therein as amino acid position 1 , it is conceivable and possible that another methionine residue located either upstream or downstream from amino acid position 1 in Figure 2 (SEQ ID NO 2) or Figure 4 (SEQ ID NO 6), respectively, ma\ be employed as the starting amino acid residue for the PRO 181 or PR0237 polypeptide The "extracellular domain ' or "ECD" ot a polypeptide disclosed herein retei s to a form of the nolypeptide which is essentially free of the transmembrane and cytoplasmic domains Oidinaπly . a polypeptide ECD will have less than about 1 % of such transmembrane and/or cytoplasmic domains and preferably will have less than about 0 57c of such domains It will be understood that any transmembrane domam(s) identified tor the polypeptides of the present invention are identified pursuant to criteria routinely employed in the art tor identifying that type ot hydrophobic domain The exact boundaries ot a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end ot the domain as initially identified and as shown in the appended figures As such, in one embodiment of the present invention, the extracellular domain of a polypeptide of the present invention comprises amino acids 1 to X of the mature amino acid sequence, wherein X is any amino acid within 5 amino acids on either side of the extracellular domain/transmembrane domain boundary
The approximate location of the "signal peptides" of the various PRO polypeptides disclosed herein are shown in the accompanying figures It is noted, however, that the C terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e g , Nielsen et al , Prot Eng , JO 1 6 (1997) and von Heinje et al , Nucl Acids Res , ]4 4683-4690 (1986)) Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention
"PROl 81 variant polypeptide" means an active PROl 81 polypeptide (other than a native sequence PRO 181 polypeptide) as defined below, having at least about 807c amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 21 to 144 of the PRO 181 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) X to 144 of the PROl 81 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any amino acid residue from 16 to 25 of Figure 2 (SEQ ID NO 2), (c) 1 or about 21 to X of Figure 2 (SEQ ID NO 2), wherein X is any amino acid from amino acid 27 to amino acid 36 of Figure 2 (SEQ ID NO 2) or (d) another specifically derived fragment of the am o acid sequence shown in Figure 2 (SEQ ID NO 2)
"PR0237 variant polypeptide" means an active PR0237 polypeptide (other than a native sequence PR0237 polypeptide) as defined below, having at least about 807c amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 24 to 328 of the PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), (b) X to 328 of the PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), wherein X is any amino acid residue from 19 to 28 of Figure 4 (SEQ ID NO 6), (c) 1 or about 24 to X of Figure 4 (SEQ ID NO 6), wherein X is any amino acid from amino acid 172 to amino acid 181 ofFιgure4 (SEQ ID NO 6) or (d) anotheι specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 6)
Such PROl 81 and PR0237 variants include, for instance, PROl 81 and PR0237 polypeptides wherein one or more amino acid residues are added, or deleted, at the N or C-termmus, as well as within one or more internal domains of the native sequence
Ordinarily, a PROl 81 variant will have at least about 807c amino acid sequence identity, more preferably at least about 81 % amino acid sequence identity, more preferably at least about 827c amino acid sequence identity, more preferably at least about 837c amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence ιdentιt\ , more preferably at least about 86% amino acid sequence identm more preferably at least about 877c amino acid sequence identity more preferably at least about 887c amino acid sequence identity more preferably at least about 897c amino acid sequence identity, moi e prefei ably at least about 90% amino acid sequence identity, more preferably at least about 91 % amino acid sequence identity, more preferably at least about 927r amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 997c amino acid sequence identity with (a) residues 1 or about 21 to 144 of the PRO 181 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) X to 144 of the PRO 181 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any amino acid residue from 16 to 25 of Figure 2 (SEQ ID NO 2), (c) 1 or about 21 to X of Figure 2 (SEQ ID NO 2), wherein X is any amino acid from amino acid 27 to amino acid 36 of Figure 2 (SEQ ID NO 2) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2) Ordinarily, a PR0237 variant will have at least about 807c amino acid sequence identity, more preferably at least about 81 % amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91 % amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 937c amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 957c amino acid sequence identity, more preferably at least about 967o amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 997c ammo acid sequence identity with (a) residues 1 or about 24 to 328 of the PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), (b) X to 328 of the PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), wherein X is any amino acid residue from 19 to 28 of Figure 4 (SEQ ID NO 6), (c) 1 or about 24 to X of Figure 4 (SEQ ID NO 6), wherein X is any amino acid from amino acid 172 to amino acid 181 of Figure 4 (SEQ ID NO 6) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 6)
Ordinarily , PROl 81 and PR0237 variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 ammo acids in length, more often at least about 50 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 amino acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, oie often at least about 250 amino acids in length, more often at least about 300 amino acids in length, or more
As shown below, Table 1 provides the complete source code tor the ALIGN-2 sequence comparison computer program This source code may be routinely compiled toi use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program
In addition Tables 2A-2D show hypothetical exemplifications tor using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and 7c 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 PR0181 or PR0237 polypeptide of interest, "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "PRO" polypeptide of interest is being compared, "PRO-DNA" represents a hypothetical PROl 81 or PR0237-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}, l*C*l ■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},
/*¥*/ 4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5}, l*G*l 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,_M,-l,-l,-3, 1, 0, 0,-1,-7, 0,-5, 0},
/*H*/ 1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2,_M, 0, 3, 2,-1,-1, 0,-2,-3, 0, 0, 2},
1*1*1 -1,-2,-2,-2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2,_M,-2,-2,-2,-l, 0, 0, 4,-5, 0,-1,-2},
/*J */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/*K*/ -1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1,_M,-1, 1, 3, 0, 0, 0,-2,-3, 0,-4, 0},
/*L*/ -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},
/*M */ -1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2,_M,-2,-l, 0,-2,-1, 0, 2,-4, 0,-2,-1},
/*N*/ 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2,_M,-1, 1, 0, 1, 0, 0,-2,-4, 0,-2, 1}, ι*o*ι _M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M, 0,_M,_M,Jvl,_M,_M,_M,_M,_M,_M,_M,_M},
/* p */ 1, -1, -3, -1, -1. ,-5, ,-ι. , 0, ,-2. , 0. ,-ι. ,-3. ,-2 ,-1. , M ,6. , o, , 0, 1,0,0,-1,-6,0,-5,0},
/*Q*/ 0, 1, -5, 2, 2, -5, -1, 3, -2, 0, 1, -2, -1, 1, M, 0, 4, 1,-1,-1,0,-2,-5,0,-4,3}, /* R*/ -2, 0, -4, -1, -1, ,-4, ,-3. ,2. ,-2. , 0. ,3, ,-3, , 0, , 0, M, 0, 1, ,6,0,-1,0,-2,2,0,-4,0},
/*S*/ 1, 0, 0, 0, 0, -3, 1, -1, -1, 0, 0, -3, -2, 1, M, 1, -1.0,2, 1,0,-1,-2,0,-3,0}, /* T */ 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, o, 0, 0, 0, 0, 0, 0,' M, 0, 0, 10, 0, 0, 0, 0, 0, 0, 0, 0},
/* V */ 0, -2, -2, -2, -2, -i, A, ,-2: ,4, ,0, ,-2.. , 2, ,2, -2, ~M, -1, ,-2,,-2,-1,0,0,4,-6,0,-2,-2},
/*w */ ■6, -5, -8, -7, -7, ,0, -7, ,-3, ,-5. , 0. ,-3. ,-2. ,-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,ι 0, 0, 0, 0, 0, 0, 0, 0, 0},
/* Y */ ■3, -3, 0, -4, -4, 7, -5, 0, -1, 0, -4, -1, -2, ,-2~ , ,-5, ,-4,,-4,-3,-3,0,-2,0,0,10,-4},
/*z */ 0, 1, -5, 2, 3, -5, 0, 2, -2, 0, 0, -2, -1, 1, M, 0, 3, 0, 0, 0, 0,-2,-6, 0,-4, 4} };
Page 1 of day. h /* */
#include < stdio h > #include < 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 nfMAXJMP], /* size of jmp (neg for dely) */ unsigned short xfMAXJMP], /* base no of jmp in seq x */
}, /* limits seq to 2Λ16 -l */ 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 tor 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() *ιndex(), *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 m /tmp to hold info about traceback
* Original version developed under BSD 43 on a vax 8650 */
^include "nw.h" ^include "day h" static _dbval[26] = {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
>. static _pbval[26] = {
1,2|(1<<('D'-'A'))|(K<('N'-'A')), 4, 8, 16, 32,64,
128, 256, OxFFFFFFF, 1< < 10, 1< < 11, 1< < 12, 1< < 13, 1< < 14,
1< < 15, 1< < 16, 1< < 17, 1< < 18, 1< < 19, 1< <20, 1< <21, 1< <22,
1< <23, 1< <24, 1< <25|(1< <('E'-'A*))|(1< <('Q'-'A )) }, maιn(ac, av) main int ac, char *av[],
{ prog = av[0], if (ac ' = 3) { fpπntf(stderr, "usage %s filel file2\n", prog), fpπntf(stderr, "where filel and file2 are two dna or two protein sequences \n"), fpπntf(stderr, "The sequences can be in upper or lower-case\n"), fpπntf(stderr, " Any lines beginning with ',' or ' < are ιgnored\n"), fpπntf(stderr, "Output is in the file V align out\"\n' ) exιt(l),
} namex[0J = av[l], namex[l] = av[2], seqx[0] = getseq(namex[0], &len0), seqx[l] = getseq(namex[l], &lenl), xbm = (dna) 7 dbval _pbval, endgaps = 0, I* I to penalize endgaps "I ofile = "align out", /* output file */ nw(), /* fill in the matrix, get the possible jmps */ readjmpsO, /* get the actual jmps */ pπnt(), /* print stats, alignment */ cleanup(O) /* unlink any tmp files */
Page 1 of nw.c /* do the alignment, return best score maιn()
* dna values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y
*/ nw() nw
{ char *px, *py, /* seqs and ptrs */ int *ndely, *dely, /* keep track of dely */ int ndelx, delx, /* keep track of delx */ int *tmp, /* for swapping rowO, rowl */ int mis, /* score for each type */ int insO, insl , /* insertion penalties */ register id, /* diagonal index */ register •J. /* jmp index */ register *col0, *coll , /* score for curr, last row */ register x , 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, sιzeof(int)), colO = (int *)g_calloc("to get coIO" lenl + 1 , sιzeof(int)), coll = (int *)g_calloc("to get coll " lenl + 1, sizeof(int)), insO = (dna)'' DINSO PINSO, insl = (dna)1' DINS 1 • PINS1 , smax = -10000, if (endgaps) { for (col0[0] = dely[0] = -insO, yy = 1 , > > < = lenl , yy+ +) { col0[yy] = dely[yy] = col0[yy 1] - insl, ndely [yy] = yy,
} col0[0] = 0, /* Waterman Bull Math Biol 84 */
} else for (yy = 1 , yy < = lenl , yy + +)
Figure imgf000016_0001
/* 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 = -(ιns0 + ιnsl), else col 1 [0J = delx = col0[0] - insl , ndelx = xx,
} else { coll[0] = 0,
Figure imgf000016_0002
ndelx = 0,
Pasje 2 of nw.c
4 ...nw for (py = seqx[l], yy = 1 ; yy < = lenl ; py + + , yy+ +) {
Figure imgf000017_0001
mis + = (xbm[*px-' --']&xbm[;i-py-'A']y, DMAT DMIS; else mis + = _day[*px-'A'][*py-'A'];
/* update penalty for del in x seq;
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps */ if (endgaps | | ndely[yy] < MAXGAP) { if (col0[yy] - insO > = delyfyy]) { dely[yy] = col0[yy] - (msO + insl); ndely[yy] = 1; } else { delyfyy] - = insl; ndely[yy] + + ;
} } else { if (col0[yy] - (msO + insl) > = dely[yy]) { dely[yy] = col0[yy] - (msO + insl), ndely [yy] = 1; } else ndely[yy] + + , }
/* update penalty for del in y seq;
* favor new del over ongong del */ if (endgaps 1 1 ndelx < MAXGAP) { if (coll [yy-l] - insO > = delx) { delx = coll [yy-l] - (msO + insl); ndelx = 1 , } else { delx -= insl , ndelx+ + , }
} else { if (coll[yy-l] - (msO + insl) > = delx) { delx = col l [yy-l] - (msO + insl), ndelx = 1 ,
} else ndelx+ + ,
/* pick the maximum score; we're favoring * mis over any del and delx over dely
*/
Page 3 of nw.c ...n id = xx - yy + lenl 1 , if (mis > = delx && mis > = dely[yy]) coll [yy] = mis, else if (delx > = dely[yy]) { col l [yy] = delx, IJ = dx[ιd] ljmp, if (dx[ιd] jp n[0] && ('dna | | (ndelx > = MAXJMP && xx > dx[ιd] jp x[ιj] + MX) 1 1 mis > dx[ιd] score + DINSO)) { dx[ιd] ιjmp+ + , if (+ +ιj > = MAXJMP) { wπtejmps(ιd),
Figure imgf000018_0001
dx[ιd] offset = offset, offset + = sizeof (struct jmp) + sizeof(offset),
}
} dx[ιd] jp n[ιj] = ndelx. dx[ιd] jp x['j] = XX, dx[ιd] score = delx,
} else { coll [yy] = dely[yy], ij = dx[ιd] ljmp, 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+ + , if (+ +ιj > = MAXJMP) { wπtejmps(ιd), ij = dx[ιd] ljmp = 0, dx[ιd] offset = offset, offset + = sizeof (struct jmp) + sizeof (offset),
} } dx[ιd] jp n[ιj] = -ndely [yy], dx[ιd] jp x[ιj] = xx, dx[ιd] score = delyfyy],
} if (xx = = lenO && >y < lenl) { /* last col */ if (endgaps) coll fyy] -= ιnsO + ιnsl*(lenl-y>), if (col l [yy] > smax) { smax = coll[yy] dmax = id, } } } if (endgaps && xx < lenO) coll [yy-l] -= ιnsO + ιnsl*(lenO xx) if (coll [yy-1] > smax) { smax = col l[yy-l] , dmax = id
} tmp = colO, colO = coll , col l = tmp,
}
(void) free((char *)ndely), (void) tree((char *)dely), (void) free((char *)col0), (void) tree((char ^)col 1)
Page 4 of nw.c /* *
* pπntO — only routine visible outside this module
*
* static:
* getmat() — trace back best path, count matches: pπnt()
* pr_align() — print alignment of described in array p[]: pπnt()
* dumpblockO — dump a block of lines with numbers, stars: pr_ahgn()
* nums() — put out a number line: dumpblockO
* puthne() - 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(ofιle, "w")) = = 0) { fpπntf(stderr, " %s: can't write %s\n" , prog, ofile), cleanup(l);
} fpπntf(tλ, " < first sequence: %s (length = %d)\n", namex[0], lenO). fpπntf(fx, " < second sequence- s (length = %d)\n", namex[l], lenl), olen = 60, lx = lenO, ly = lenl , firstgap = lastgap = 0, if (dmax < lenl - 1) { /* leading gap in x */ 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 - d axO -1 ; lx -= lastgap,
} else if (dmaxO > lenO - 1) { trailing gap in y */ lastgap = dmaxO - (lenO - 1), ly - = lastgap;
} getmat(lx, ly, firstgap, lastgap), pr_alιgn(),
Page 1 of nwprint.c * trace back the best path, count matches */ static getmat(lx, ly, firstgap, lastgap) getmat int lx, ly, /* "core" (minus endgaps) */ int firstgap, lastgap; /* leading trailing overlap */
{ int nm, lO, ii , sizO, sizl , char outx[32]; double pet; register nO, nl ; register char *p0, *pl ;
/* get total matches, score */
Figure imgf000020_0001
pO = seqx[0] + pp[l].spc, pi = seqx[l] + pp[0].spc; nO = pp[l].spc + 1; nl = pp[0].spc + 1 ; nm = 0, while ( *p0 && *pl ) {
Figure imgf000020_0002
} else { if (xbm[*pO-'A']&xbm[*pl-'A']) nm + + , if (n0+ + = = pp[OJ.x[ιO])
Figure imgf000020_0003
/* 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 < \y)f lx . ly; pet = 100 *(double)nm/(double)lx; fpπntf(fx, "\n"), fpπntf(fx, " < ?( d match%s in an overlap ot %d. 7 2t percent sιmιlaπty\n" nm, (nm = = l) " " "es" , lx, pet),
Page2 ofnwprint.c fpπntf(fx, " < gaps in first sequence- %d", gapx), .getmat if (gapx) {
(void) spπntf(outx, " (%d %s%s)", ngapx, (dna)9 "base": "residue", (ngapx = = 1)? "s"); fpπntf(fx, " %s", outx); fpπntf(fx, ", gaps in second sequence: %d", gapy); if (gapy) {
(void) spπntf(outx, " (%d %s%s)", ngapy, (dna)9 "base" : "residue", (ngapy = = 1)? " " : "s"); fpπntf(fx, " % s " , outx) ;
} if (dna) fpπntf(fx,
"\n< score: %d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n" smax, DMAT, DMIS, DINSO, DINS1); else fpπntf(fx,
"\n < score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %d per resιdue)\n", smax, PINSO, PINS1), if (endgaps) fpπntf(fx,
" < endgaps penalized, left endgap: %d %s%s, right endgap: %d %s%s\n". firstgap, (dna)? "base" . "residue", (firstgap = = 1)? " " : "s" , lastgap, (dna)? "base" "residue", (lastgap = = l)9 "" • "s"); else fpπntf(fx, " < endgaps not penalιzed\n");
static nm; /* matches in core — for checking */ static lmax; /* lengths of stripped file names */ static ιj[2], /* jmp index for a path */ static nc[2] , /* number at start of current line */ static m[2], /* current elem number — for gapping */ static sιz[2], static char *ps[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 pp[] */ static pr align() pr align
{ int nn, /* char count */ int more, register 1, for (l = 0, lmax = 0, l < 2; ι+ +) { nn = stπpname(namex[ι] , if (nn > lmax) lmax = nn. nc[ι] = 1, m[ι] = 1 , sιz[ι] = ιj[ι] = 0; ps[ι] = seqx[ι]; po[ι] = out[ι] ,
Page 3 of nwprint.c for (nn = nm = 0, more = 1, more, ) { .pr align for (i = more = 0, I < 2, ι++) { do we have more of this sequence9
*/ if 0*ps[ι]) continue, more++, if (pp[ι] spc) { /* leading space */ *po[ι] + + = ' ', pp[ι] spc- ,
} else if (sιz[ι]) { /* in a gap */
*po[ι] + + = '-', sιz[ι] ,
} else { /* we're putting a seq element */ *po[ι] = *ps[ι], if (ιslower(*ps[ι]))
*ps[ι] = toupper(*ps[ι]), po[ι] + +, ps[ι] + + ,
/* * are we at next gap for this seq9
*/ if („,[,] ==pP[.] x[lJ [!]]){
/*
* we need to merge all gaps
* at this location */ sιz[ι] = pp[ι] n[ιj[ι] + +], while (m[ι] == pp[ι] x[ιj[ι]]) sιz[ι] += pp[ι] n[ιj[ι] + +]
} m[ι] + + ,
} if (+ +nn = = olen 11 'more && nn) { dumpblockO, for(ι = 0, l < 2, ι++) po[ι] = out[ι], nn = 0, }
* dump a block of lines, including numbers, stars pr_alιgn() */ static dumpblockO dumpblock
{ register I, for (I 0, l < 2 1++) *po[ι) = \0 ,
Page 4 of nwprmt.c ...dumpblock
(void) putc('\n', fx), for(ι = 0, i < 2, ι + +){ if (*out[ι] && (*out[ι] '= ' ' || *(po[ι]) ' = ' )) { if(. ==0) nums(ι), if (l == 0&&*out[l]) stars(), puthne(ι), if == 0&&*out[l]) fpπntf(fx, star), if(ι == 1) nums(t),
}
}
/* * put out a number line dumpblockO
*/ nums(ιx) nums int lx, /* index in out[
{ char nhne[P_LINE], register LJ; register char *pn, *px, *py, for (pn = nhne, l = 0; I < lmax+P_SPC, ι + + , pn++)
*pn = ' ', for (i = nc[ιx], py = out[ιx]. *py, py++, pn++) { if (*py == ' ' 11 *py == '-')
*pn = ' ', else { if 0%10 ==0 || (l == 1 &&nc[ιx] '= 1)){ j = (l < 0)> l l, for (px = pn, j, j /= 10, ρx~)
*px =j 10 + '0 if (i < 0)
} else
*pn = ι + + ,
}
*pn = '\0 ncfix] = l, for (pn = nhne, *pn, pn+ +) (void) putc(-pn, fx), (void) putc( \n', fx),
/*
* put out a line (name, [num], seq, [num]) dumpblockO
*/ static puthne(ιx) putline
21 Page 5 of nwpπnt.c ...putline int i; register char *px; for (px = namex[ix], i = 0; *px && *px != ':'; px++, ι++)
(void) putc(-px, fx); for(; i < lmax+P SPC; i++)
(void) putcO ', fx);
/* these count from 1 :
* ni[] is current element (from 1)
* nc[] is number at start of current line */ for (px = 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 stars() stars
{ int i; register char *p0, *pl, ex, *px; if (!*out[0] I I (*out[0] == ' '&& *(ρo[0]) == ' ') 11 !*out[l] I I (*out[l] == ' '&& *(po[l]) = = ' ')) return; px = star; for (i = lmax+P SPC; i; ι~)
*px++ = ' '; for(p0 = outl0],pl = out[l]; *p0 && *pl; p0+ + , pi + +) { if (ιsalpha(*pO) && isalpha(*pl)) { if (xbm[*pO-'A']&xbm[*pl-'A']) { ex = '*'; nm+ + ;
} else if (!dna && _day[*pO-'A'][*pl-'A'] > 0) ex = '.'; else
} else ex =
*px + + = ex;
}
*px+ + = '\n';
*px = ' \0';
Pageόofnwprint.c /*
* strip path or prefix from pn, return len: pr_align()
*/ static stripname(pn) stripname char *pn; /* file name (may be path) */
{ register char *px, *py; py = 0; for (px = pn; *px; px+ +) if (*px = = V) py = px + 1; if (py)
(void) strcpy(pn, py); return(strlen(pn)) ;
Page 7 ofnwprint.c * cleanupO - cleanup any tmp file
* getseqO — read in seq, set dna, len, maxlen
* g_calloc() — calloc() with error checkin
* readjmpsO — get the good jmps, from tmp file if necessary
* wntejmpsO ~ write a filled array of jmps to a tmp file: nw() */
#include "nw.h" #inchιde < sys/file.h > char *jname = "/tmp/homgXXXXXX"; I* tmp file for jmps */ FILE *fj; int cleanupO; /* cleanup tmp file */ long lseek(),
/*
* remove any tmp file if we blow */ cleanup(ι) cleanup int { if (fj)
(void) unhnk(jname); exιt(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 hne[1024] , *pseq; register char *px, *py, int natgc, tlen.
FILE *fp. if ((fp = topen(file, "r")) = = 0) { fpπntf(stderr," %s: can't read s\n", prog, file), exιt(l),
} tlen = natgc = 0, while (fgets(lιne, 1024, fp)) { if (*hne = = ' , ' | | *hne = = ' < ' | | *lme = = ' > ') continue, for (px = line; *px ' = '\n' ; px+ +) if (ιsupper(*px) | | islower0-px)) tlen+ + ,
} if ((pseq = malloc((unsigned)(tlen+6))) = = 0) { tpπntt(stderr," %s malloc() failed to get d bytes tor s\n" , prog, tlen + 6, file), exιt(l),
} pseq[0] = pseq[l] = pseq[2] = pseq[3] = '\0' ,
Page 1 of nwsubr.c ...getseq py = pseq + 4, *len = tlen, rewιnd(fp), while (fgets(hne, 1024, fp)) { if (*hne = = ' , ' 1 1 *lιne = = ' < ' | | *lιne = = ' > ') 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 > (tlen/3), return(pseq+4),
} char * g_calloc(msg, nx, sz) g_callθC char *msg, /* program, calling routine */ int nx, sz, /* number and size of elements */
{ char *px, *calloc(), if ((px = calloc((unsigned)nx, (unsigned)sz)) = = 0) { if (*msg) { 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 , register l, j, xx, if (fj) {
(void) fclose(fj), if ((td = open(jname, O 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 x[j] > = xx, j— )
Page 2 ofnwsubr.c ...readjmps if 0 < 0 && dxfdmax]. offset && fj) {
(void) lseek(fd, dx[dmax] offset, 0),
(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)),
(void) read(fd, (char *)&dx[dmax] offset, sizeof (dx [dmax] offset)), dx[dmax] ijmp = MAXJMP- 1,
} else break,
} if (l > = JMPS) { fpπntf(stderr, "%s too many gaps in ahgnment\n", prog), cleanup(l), } if 0 >=0){
Figure imgf000028_0001
xx = dxfdmax] jpx[)],
Figure imgf000028_0002
if (siz < 0) { /* gap in second seq */
Figure imgf000028_0003
/* id = xx - y> + lenl - 1 */ pp[l].x[ιl] = xx - dmax + lenl - 1, gapy+ + , ngapy -= siz, /* ignore MAXGAP when doing endgaps */ siz = (-siz < MAXGAP | | endgaps)9 -siz . MAXGAP, ιl + + ,
} else if (siz > 0) { /* gap in first seq */
Figure imgf000028_0004
gapx + + , ngapx + = siz, /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP | | endgaps)' siz MAXGAP, ι0+ + , } } else break, }
/* reverse the order of jmps */ for 0 =0, lO--, j < lO, j+ +, ι0~) { i = PP[0] n[j], pp[0] nDJ = ppfO] nfiO], pp[0] n[ι0] = i, i = PP[0] x[j], pp[0].x[)l = pp[0) x[ι0], ppfO] x[ι0] = l, } forO = 0, ιl-,j < ιl,j++, ιl- ){ i = pp[l] n[)l, PP[1] n[|] = pp[l] n[ιl], pp[l] nlil] = i, i = pp[l] x|j], pp[l] xU| = pp[l] x[ιl], pp[l] x[ιl] = i,
} if (fd > = 0)
(void) close(fd),
(void) unhnkOname), fj = o, 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 (mktempOname) < 0) { fpπntf(stderr, " %s: can't mktempO %s\n", prog, j name), cleanup(l);
} if ((fj = fopenOname, "w")) = = 0) { fpπntf(stderr, "%s: can't write %s\n", prog, jname), exιt(l); } }
(void) fwπte((char *)&dx[ιx] jp, sizeof (struct jmp), 1, fj), (void) fwπte((char *)&dx[ιx]. offset, sizeof (dx[ιx]. offset), 1, fj),
Pase 4 ofnwsubr.c Table 2A
PRO XXXXXXXXXXXXXXX (Length = 15 ammo acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% amino acid sequence identity =
(the number of identically matching ammo acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) =
5 divided by 15 = 33.3 %
Table 2B
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) =
5 divided by 10 = 50%
Table 2C
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =
6 divided by 14 = 42.9%
Table 2D
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides)
Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =
4 divided by 12 = 33.3%
"Percent (%) amino acid sequence identity' with respect to the PRO 181 and PR0237polypeptιde 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 PROl 81 or PR0237 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, Ine , 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, Ine , South San Francisco, California or may be compiled from the source code provided in Table 1 The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary
For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to. with, or against a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program s alignment of A and B, and where Y is the total number ot 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 w ill not equal the ck ammo acid sequence identity of B to A As examples ot % amino acid sequence identity calculations, Tables 2A-2B demonstrate how to calculate the % amino acid sequence identity ot 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
Figure imgf000034_0001
obtained as described above using the ALIGN-2 sequence comparison computer pi ogram
Figure imgf000034_0002
, % amino acid sequence identity may also be determined using the sequence comparison progiam NCBI-BLAST2 (Altschul et al , Nucleic Acids Res , 25 3389-3402 (1997)) The NCBI-BLAST2 sequence comparison piogiam may be downloaded from http //www ncbi nlm nih gov NCBI-BLAST2 uses several search paiameteis wheiein all of those search parameters are set to default values including, tor example unmask = yes, strand = all. expected occurrences = 10, minimum low complexity length = 1 /5 multi-pass e- value = 0 01 , constant tor 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 am o 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 ammo acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A
In addition, % amino acid sequence identity may also be determined using the WU-BLAST-2 computer program (Altschul et al , Methods in Enzymology, 266 460 480 (1996)) Most of the WU BLAST-2 search parameters are set to the default values Those not set to default values, ; e , the adjustable parameters, are set with the following values overlap span = 1 , overlap fraction = 0 125, word threshold (T) = 1 1 , and scoring matrix = BLOSUM62 For purposes herein, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical 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 ammo acid sequence of interest and the amino acid sequence B is the amino acid sequence ot the PRO polypeptide of interest "PROl 81 variant polynucleotide ' or "PROl 81 variant nucleic acid sequence ' means a nucleic acid molecule which encodes an active PRO 181 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence hich encodes residues I or about 21 to 144 ot the PROl 81 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) a nucleic acid sequence which encodes amino acids X to 144 of the PRO 181 polypeptide shown in Figure 2 (SEQ ID NO 2) wherein X is any amino acid residue from 16 to 25 of Figure 2 (SEQ ID NO 2), (c) a nucleic acid sequence which encodes amino acids 1 or aboui 1 to X of Figure 2 (SEQ ID NO 2) wherein X is any amino acid from amino acid 27 to amino acid 36 of Figure 2 (SEQ ID NO 2) or (d) a nucleic acid sequence which encodes another specifically derived tiagment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2) Ordinarily, a PRO 181 \ aπant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81 % nucleic acid sequence identity moie preferably at least about 82% nucleic acid sequence identity moie pieterably at least about 83% nucleic acid sequence identity, moie preferably at least about 84% nucleic acid sequence identity, more prefeiably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91 % nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 21 to 144 of the PR0181 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) a nucleic acid sequence which encodes ammo acids X to 144 of the PRO 181 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any amino acid residue from 16 to 25 of Figure 2 (SEQ ID NO 2), (c) a nucleic acid sequence which encodes amino acids 1 or about 21 to X of Figure 2 (SEQ ID NO 2), wherein X is any amino acid from amino acid 27 to amino acid 36 of Figure 2 (SEQ ID NO 2) or (d) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2) PROl 81 polynucleotide variants do not encompass the native PROl 81 nucleotide sequence
"PR0237 variant polynucleotide" or "PR0237 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PR0237 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 24 to 328 of the PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), (b) a nucleic acid sequence which encodes amino acids X to 328 of the PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), wherein X is any amino acid residue from 19 to 28 of Figure 4 (SEQ ID NO 6), (c) a nucleic acid sequence which encodes amino acids 1 or about 24 to X of Figure 4 (SEQ ID NO 6), wherein X is any amino acid from amino acid 172 to amino acid 181 of Figure 4 (SEQ ID NO 6) or (d) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 6) Ordinal ily a PR0237 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81 % nucleic acid sequence identitv, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more pieterably at least about 84% nucleic acid sequence identity, more preterablv at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity more preferably at least about 87% nucleic acid sequence identitv moie preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preterabl at least about 90% nucleic acid sequence identity, more preferably at least about 91 % nucleic acid sequence identitv more preferably at least about 92% nucleic acid sequence identitv more preferably at least about 93% nucleic acid sequence identity more preferably at least about 94% nucleic acid sequence identity, more preterablv at least about 95% nucleic acid sequence identity more preferably at least about 96% nucleic acid sequence identity more preferably at least about 97% nucleic acid sequence identity moie preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 24 to 328 ot the PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), (b) a nucleic acid sequence which encodes amino acids X to 328 of the PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), wherein X is any amino acid residue from 19 to 28 of Figure 4 (SEQ ID NO 6), (c) a nucleic acid sequence which encodes amino acids 1 or about 24 to X of Figure 4 (SEQ ID NO 6), wherein X is any amino acid from amino acid 172 to amino acid 181 of Figure 4 (SEQ ID NO 6) or (d) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 6) PR0237 polynucleotide variants do not encompass the native PR0237 nucleotide sequence
Ordinarily, PRO 181 and PR0237 variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in length, more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more
"Percent (%) nucleic acid sequence identity" with respect to the PROlδl and PR0237 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 PROl 81 or PR0237 polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 The ALIGN-2 sequence comparison computer program was authored by Genentech, Ine , 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 TXU 10087 The ALIGN-2 program is publicly available through Genentech, Ine , South San Francisco, California or may be compiled fio the source code provided inTable 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 pui poses 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 giv en nucleic acid sequence D) is calculated as follows
100 times the traction 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 etal , Nucleic Acids Res , _25 3389-3402 (1997)) The NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0 01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62 In situations where NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI- BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C
In addition, % nucleic acid sequence identity values may also be generated using the WU-BLAST-2 computer program (Altschul et al , Methods in Enzymology, 266460-480 ( 1996)) Most of the WU-BLAST-2 search parametei s aie 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 % nucleic acid sequence identity value is determined by div iding (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 ot interest (i e the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST 2 by (b) the total number ot 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 ot 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, PROl 81 and PR0237 variant polynucleotides are nucleic acid molecules that encode an active PRO 181 or PR0237 polypeptide, respectively, and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PROl 81 polypeptide shown in Figure 2 (SEQ ID NO 2) or to nucleotide sequences encoding the full-length PR0237 polypeptide shown in Figure 4 (SEQ ID NO 6), respectively PROl 81 and PR0237 variant polypeptides may be those that are encoded by a PROl 81 or PR0237 variant polynucleotide
The term "positives", in the context of the ammo 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 am o acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % positives to, with, or against a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of amino acid residues scoring a positive value as defined above by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % positives of A to B will not equal the % positives of B to A
"Isolated,1 when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non proteinaceous solutes In preferred embodiments, the polypeptide will be purified ( 1 ) to a degree sufficient to obtain at least 15 residues of N-termmal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-i educing or reducing conditions using Coomassie blue or, preferably, silver stain Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component ot the PROl 81 or PR0237 natural environment will not be present Ordinarily, however, isolated polypep'ule will be prepared by at least one purification step
An isolated nucleic acid molecule encoding a PROl 81 or PR0237 polypeptide or an isolated nucleic acid molecule encoding an anti-PROl 81 or antι-PR0237 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 natuial source of the PRO 181 - or PR0237-encodιng nucleic acid or the anti PRO 181 - or antι-PR0237-encodιng nucleic acid Preferably the isolated nucleic acid is tree of association with all components with which it is naturally associated An isolated PRO 181 - or PR0237-encodιng nucleic acid molecule or an isolated anti-PRO 181 - or anti- PR0237-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 PROl 81- or PR0237-encodιng nucleic acid molecule or from the anti-PROl 81- or antι-PR0237-encodιng nucleic acid molecule as it exists in natural cells However, an isolated nucleic acid molecule encoding a PROl 81 or PR0237 polypeptide or an isolated nucleic acid molecule encoding an anti-PRO 181 or antι-PR0237 antibody includes PRO 181 - or PR0237-nucleιc acid molecules or anti- PR0181- or antι-PR0237-nucleιc acid molecules contained in cells that ordinarily express PROl 81 or PR0237 polypeptides or anti-PROl 81 or antι-PR0237 antibodies where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a πbosome binding site Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a πbosome 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 ohgonucleotide 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 8 land antι-PR0237 monoclonal antibodies (including agonist antibodies), anti-PROl 81 and antι-PR0237 antibody compositions with polyepitopic specificity, single chain anti-PROl 81 and antι-PR0237 antibodies, and fragments of anti-PROl 81 and antι-PR0237 antibodies (see below) 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 tor possible naturally-occurring mutations that may be present in minor amounts "Stringency" of hybridization reactions is readily determinable by one ot ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperatuie, and salt concentration In general, longer probes require higher temperatures for proper annealing, while shorter probes need lowei 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 which can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent while lower temperatures less so For additional details and explanation ot stringency of hybridization reactions see Ausubel et al , Current Protocols in Molecular Biology, Wiley Interscience Pubhsheis, (1995) "Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that (1) employ low ionic strength and high temperature for washing, for example 0 015 M sodium chloride/0 0015 M sodium 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 μg/ml), 0 1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0 2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55 °C, followed by a high-stringency wash consisting of 0 1 x SSC containing EDTA at 55°C
"Moderately stringent conditions" may be identified as described by Sambrook et al , Molecular Cloning A Laboratory Manual. New York Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e g , temperature, ionic strength and % SDS) less stringent that those described above An example of moderately stringent conditions is overnight incubation at 37 °C in a solution comprising 20% formamide, 5 x SSC (150 mM NaCl, 15 mM tπsodium citrate), 50 mM sodium phosphate (pH 7 6), 5 x Denhardt' s solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C The skilled artisan will recognize how to adjust the temperature, ionic strength, etc as 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 81 or PR0237 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 "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site ot an antibody (/ e is "heterologous"), and an immunoglobulin constant domain sequence The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin. such as IgG 1 , IgG-2, IgG-3, or IgG 4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM
"Active" or ' activity" for the purposes herein refers to torm(s) of PR0181 oi PR0237 which retain a biological and/or an immunological activity of native oi naturally-occurring PROl 81 or PR0237, wherein
"biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally occurring PRO 181 or PR0237 other than the ability to induce the production ot an antibody against an antigemc epitope possessed by a native or naturally occurring PROl 81 or PR0237 and an ' immunological activity refers to the ability to induce the production of an antibody against an antigemc epitope possessed by a native or naturally- occurring PROl 81 or PR0237
"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 81 or PR0237 polypeptide
"Immunological cross-reactivity" as used herein means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO 181 or PR0237 polypeptide having this activity with polyclonal antisera raised against the known active PROl 81 or PR0237 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 lntrapeπtoneal or subcutaneous injection in incomplete Freunds The immunological cross-reactivity preferably is "specific", which means that the binding affinity of the lmmunologically cross-reactive molecule (e g , antibody) identified, to the corresponding PROl 81 or PR0237 polypeptide is significantly higher (preferably at least about 2-tιmes, more preferably at least about 4-tιmes, even more preferably at least about 6-tιmes, most preferably at least about 8-tιmes 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, ghoblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer endometπal carcinoma, salivary gland carcinoma, kidney cancer vulval cancer, thyroid cancer, hepatic carcinoma and vanous types of head and neck cancer
"Treatment" is an intervention performed with the intention ot preventing the development or i ermg the pathology of a disorder Accordingly "treatment" refers to both therapeutic treatment and prophylactic or preventative measures Those in need ot 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 lender 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 ot 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 PROl 81 or PR0237 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 PROl 81 or PR0237 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 PR0181 or PR0237 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 181 or PR0237 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 81 or PR0237 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 81 or PR0237 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 I ι, Ip\ Y9" and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof A "chemotherapeutic agent' is a chemical compound useful in the treatment of tumor, e g , cancer
Examples of chemotherapeutic agents include adπamycin, doxorubicin, epiiubicin, 5-fluorouracιl, cytosine arabinoside ( Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e g , pachtaxel (Taxol Bristol- Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxoteie Rhone Poulenc Rorer, Antony. Rnace) toxotere, methotrexate cisplatin, melphalan, vinblastine, bleomycin, etoposide, lfosfamide, mitomycin C mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin carminomycin, aminopterin dactinomycin, mitomycins, esperamicins (see, U S Patent No 4,675, 187). melphalan and othei 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 onapπstone 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, daunorubicm, etoposide, and bleomycin Those agents that arrest Gl also spill over into S-phase arrest, tor example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracιl, 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, proinsuhn, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteimzing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α and -β, mulleπan-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothe al 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-contaimng prodrugs, glycosylated prodrugs or optionally substituted phenylacetamide containing prodrugs, 5-fluorocytosιne and other 5-fluorouπdιne prodrugs which can be derivatized into a prodrug form for use in this invention include but aie 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 81 or PR0237 polypeptide disclosed herein Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments oi amino acid sequence variants of native PRO 181 or PR0237 polypeptides, peptides, small organic molecules, etc Methods for identifying agonists of a PROl 81 or PR0237 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 polyvinylpyrrohdone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosacchaπdes, 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 noniomc surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™
"Native antibodies" and "native immunoglobulins" 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 v aπes among the heavy chains of different immunoglobulin isotypes Each heavy and light chain also has regularly spaced mtrachain disulfide bridges Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains Each light chain has a variable domain at one end (VL) and a constant domain at its other end, the constant domain of the light chain is aligned with the first constant domain ot 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 tor its particulai antigen However, the variability is not evenly distributed throughout the variable domains ot antibodies It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervaπable regions both in the light-chain and the heavy-chain variable domains The more highly conserved portions of v ariable domains are called the framework regions (FR) The variable domains ot native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, Kabat et al , NIH Publ No 91-3242, Vol I, pages 647-669 ( 1991 )) The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity The term "hypervaπable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding The hypervaπable 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 "hypervaπable loop" (t 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 hypervaπable 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 En , 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-bindmg 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 ot a few residues at the carboxy terminus of the heavy chain CH 1 domain including one or more cysteines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine resιdue(s) of the constant domains bear a free thiol group F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments w Inch have hinge cysteines between them Other chemical couplings of antibody fragments are also known
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on the amino acid sequences of their constant domains
Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e g , IgG 1 , 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, J 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 immunoglobulins The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybπdoma method first described by Kohler et al , Nature, 256 495 [1975], or may be made by recombinant DNA methods (see, e g , U S Patent No 4,816,567) The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al , Nature. 352 624-628 [1991 ] and Marks et al , J Mol Biol , 222 581-597 (1991 ), for example The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chaιn(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U S Patent No 4.816,567, Morrison etal , Proc Natl Acad Sci USA. 81 6851-6855 [1984])
"Humanized" forms of non-human (e , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') . or other antigen-bindmg subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donoi antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity In some instances, Fv FR residues of the human immunoglobulin are replaced by corresponding non-human residues Furthermore humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences These modifications are made to further refine and maximize antibody performance In general, the humanized antibody will comprise substantially all of at least one, and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all oi substantially all ot the FR regions are those of a human immunoglobulin sequence The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that ot a human immunoglobulin For further details see, Jones et al Nature, 321 522 525 ( 1986), Reichmann et al , Nature. 332 323 329 [ 1988], and Presta Cuπ Op Struct Biol , 2 593 596 (1992) The humanized antibody includes a PRIMATIZED™antιbody wherein the antigen-binding region of the antibody is deπv ed from an antibody produced by immunizing macaque monkeys w ith the antigen of interest "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding For a review of sFv, see, Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 1 13, Rosenburg and Moore eds , Springer- Verlag, New York, pp 269-315 (1994)
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites Diabodies are described more fully in, for example, EP 404,097, WO 93/1 1 161 , and Hollmger et al , Proc Natl Acad Sci USA. 90 6444-6448 (1993)
An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes In preferred embodiments, the antibody will be purified (1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-termmal 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 ot the present invention can adhere Examples of solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysacchaπdes (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and sihcones In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275 149
A "liposome' is a small vesicle composed of various types of hpids. phospholipids and/or surfactant which is useful for delivery of a drug (such as a PROl 81 or PR0237 polypeptide or antibody thereto) to a mammal The components of the liposome are commonly arranged in a bilayer formation similai to the lipid arrangement of biological membranes
A "small molecule" is defined herein to have a moleculai weight below about 500 Daltons II Compositions and Methods of the Invention
A Full-length PROl 81 and PRQ237 Polypeptides
The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO 181 and PR0237 In particular, cDNAs encoding PROl 81 and PR0237 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below
As disclosed in the Examples below, cDNA clones encoding PRO 181 and PR0237 polypeptides have 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 8 land PR0237 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 81 and PRQ237 Variants
In addition to the full-length native sequence PROl 81 and PR0237 polypeptides described herein, it is contemplated that PROl 81 and PR0237 variants can be prepared PRO 181 and PR0237 variants can be prepared by introducing appropriate nucleotide changes into the PRO 181 or PR0237 DNA, and/or by synthesis of the desired PROl 81 or PR0237 polypeptide Those skilled in the art will appreciate that amino acid changes may alter post- translational processes of the PROl 81 or PRO 237 polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics
Variations in the native full-length sequence PROl 81 or PR0237 or in various domains ot the PROl 81 or PR0237 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 181 or PR0237 that results in a change in the amino acid sequence of the PRO 181 or PR0237 as compared with the native sequence PRO 181 or PR0237 Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more ot the domains of the PROl 81 or PR0237 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 81 or PR0237 with that of homologous known protein molecules and minimizing the number ot ammo acid sequence changes made in regions of high homology Am o acid substitutions can be the result ot replacing one ammo acid with another amino acid having similar structural and/or chemical properties, such as the replacement ot a leucine with a seπne, i e , conservative amino acid replacements Insertions or deletions may optionally be in the range of about 1 to 5 ammo acids The variation allowed may be determined by systematically making insertions, deletions or substitutions of ammo acids in the sequence and testing the resulting variants toi activity exhibited by the full-length or mature native sequence
PROl 81 and PR0237 polypeptide fragments are prov ided herein Such fragments may be ti uncated at the N-terminus or C-terminus, or may lack internal residues tor example, when compared with a full length native protein Certain fragments lack amino acid residues that are not essential tor a desned biological activ ity of the PRO 181 or PR0237 polypeptide PRO 181 and PR0237 fragments may be prepared by any of a number of con ventional techniques Desired peptide fragments may be chemically synthesized An alternative approach involves generating PROl 81 and PR0237 fragments by enzymatic digestion, e g , by treating the protein with an enzyme known to cleave proteins at sites defined by particular ammo acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR) Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR Preferably PROl 81 and PR0237 polypeptide fragments share at least one biological and/or immunological activity with the native PRO 181 or PR0237 polypeptide shown in Figure 2 (SEQ ID NO 2) and Figure 4 (SEQ ID NO 6), 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
Figure imgf000051_0001
Arg (R) lys, gin, asn lys Asn (N) gin, his, lys, arg gin Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro, ala ala His (H) asn, gin, lys, arg arg He (I) leu, val, met, ala, phe, norleucine leu Leu (L) norleucine, lie, val, met, ala, phe lie
Lys (K) arg, gin, asn arg Met (M) leu, phe, lie leu Phe (F) leu, val, lie, ala, tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser
Figure imgf000051_0002
Tyr (Y) trp, phe, thr, ser phe Val (V) lie, leu, met, phe, ala, norleucine leu
Substantial modifications in function or immunological identity of the PRO l 81 or PR0237 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, tor 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, lie,
(2) neutral hydrophilic cys, ser, thr
(3) acidic asp, glu, (4) basic asn, gin, his, lys, arg,
(5) residues that influence chain orientation gly, pro, and
(6) aromatic trp, tyr, phe
Non-conservative substitutions will entail exchanging a member ot one ot these classes for another class Such substituted residues also may be introduced into the conseivative substitution sites or more preferably, into the remaining (non-conserved) sites
The variations can be made using methods known in the art such as ohgonucleotide-mediated (site directed) mutagenesis, alanine scanning and PCR mutagenesis Site-directed utagenesis [Carter et al , Nucl Acids Res . H 4331 (1986), Zoller et al , Nucl Acids Res , H) 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 81 or PR0237 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 am o acids are relatively small, neutral amino acids Such amino acids include alamne, glycine, 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 amino acid can be used
C Modifications of PROl 81 and PRQ237
Covalent modifications of PROl 81 andPR0237 are included within the scope of this invention One type of covalent modification includes reacting targeted amino acid residues of a PROl 81 or PR0237 polypeptide with an organic deπvatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO 181 or PR0237 Den vatization with bifunctional agents is useful, for instance, for crosshnking PRO 181 or PR0237 to a water-insoluble support matrix or surface for use in the method for purifying anti-PROl 81 or anti- PR0237 antibodies, and vice-versa Commonly used crosshnking agents include, e g , l ,l-bιs(dιazoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azιdosalιcylιc acid, homobifunctional lmidoesters, including disuccinimidyl esters such as 3 3 -dιthιobιs(succιnιmιdylproριonate), bifunctional maleimides such as bιs-N-maleιmιdo-l ,8-octane and agents such as methyl-3-[(p- azιdophenyl)dιthιo]propιoιmιdate
Other modifications include deamidation of glutaminyl and asparaginyl residues to the coπesponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histid e side chains [T E Creighton, Proteins Structuie 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 181 or PR0237 polypeptide included within the scope of this invention comprises altering the native glycosylation pattem of the polypeptide "Altering the native glycosylation pattern" is intended toi purposes herein to mean deleting one or moie carbohydrate moieties found in native sequence PROl 81 or PR0237 (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 81 or PR0237 In addition the phrase includes qualitative changes in the glycosylation of the native proteins involving a change in the natuie and piopoitions ot the various carbohydrate moieties present
Addition of glycosylation sites to the PRO 181 or PR0237 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 senne or threonine residues to the native sequence PROl 81 or PR0237 (for O-linked glycosylation sites) The PROl 81 or PR0237 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PROl 81 or PR0237 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 PRO 181 or PR0237 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide Such methods are described in the art, e g , in WO 87/05330 published 1 1 September 1987, and in Aphn and Wnston, CRC Cπt Rev Biochem , pp 259-306 (1981 ) Removal of carbohydrate moieties present on the PRO 181 or PR0237 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al , Arch Biochem Biophys , 259 52 (1987) and by Edge et al , Anal Biochem , 1 18 131 (1981 ) Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al , Meth Enzvmol , 138 350 (1987)
Another type of covalent modification of PRO 181 or PR0237 comprises linking the PRO 181 or PR0237 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, 4,670,4 P. 4,791 ,192 or 4, 179,337 The PROl 81 or PR0237 polypeptide of the present invention may also be modified in a way to form a chimeric molecule comprising PROl 81 or PR0237 fused to another, heterologous polypeptide or amino acid sequence
In one embodiment, such a chimeric molecule comprises a fusion of the PRO 181 or PR0237 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 PROl 81 or PR0237 polypeptide The presence of such epitope-tagged forms of the PRO 181 or PR0237 polypeptide can be detected using an antibody against the tag polypeptide Also, provision of the epitope tag enables the PRO l 81 or PR0237 polypeptide to be ieadily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag Various tag polypeptides and their respective antibodies are well known in the art Examples include poly histidine (poly-His) or poly-histidme-glycine (poly-His-gly) tags the flu HA tag polypeptide and its antibody 12CA5 [Field etal . Mol Cell Biol . 8 2159-2165 (1988)1. 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 v uus glycoprotein D (gD) tag and its antibody [Paborsky et al , Protein Engineering, 3(6) 547-553 ( 1990)] Other tag polypeptides include the Flag-peptide [Hopp et al BioTechnologv, 6 1204- 1210 ( 1988) J , the KT3 epitope peptide [Martin et al , Science, 255 192-194 ( 1992)], an -tubulin epitope peptide [Skinner et al J Biol Chem 266 15163-15166 ( 1991 )], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al . Pioc Natl Acad Sci USA. 87 6393-6397 (1990)]
In an alternative embodiment, the chimeiic molecule may comprise a fusion of the PRO l 81 or PR0237 polypeptide with an immunoglobulin or a particular region of an immunoglobulin For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PROl 81 or PR0237 polypeptide in place of at least one variable region within an Ig molecule In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH 1 , CH2 and CH3 regions of an I-gGl molecule For the production of immunoglobulin fusions see also, US Patent No 5,428,130 issued June 27, 1995
D Preparation of PROl 81 and PRQ237
The description below relates primarily to production of PROl 81 or PR0237 by culturing cells transformed or transfected with a vector containing PRO 181 or PR0237 nucleic acid It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PROl 81 or PR0237 For instance, the PROl 81 or PR0237 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 PROl 81 or PR0237 polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PROl 81 or PR0237 polypeptide
1 Isolation of DNA Encoding PROl 81 or PRQ237 DNA encoding PROl 81 or PR0237 may be obtained from a cDNA library prepared from tissue believed to possess the PROl 81 or PR0237 mRNA and to express it at a detectable level Accordingly, human PROl 81 or human PR0237 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples The PROl 81 - or PR0237-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 PROl 81 or PR0237 or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures such as described in Sambiook et al , Moleculai Cloning A Laboratoi v Manual (New York Cold Spring Harbor Laboratory Press, 1989) An alternative means to isolate the gene encoding PROl 81 or PR0237 is to use PCR methodology [Sambrook et al , supia, Dieffenbach et al , PCR Primer A Laboratory Manual (Cold Spring Harbor I aboratory Press, 1995)]
The Examples below describe techniques tor screening a cDNA library The o gonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positiv es are minimized The ohgonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the hbi ary being screened Methods of labeling are well known in the art. and include the use of radiolabels like P-labeled ATP, biotinylation or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supia
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 ammo acid sequence disclosed herein for the first time, and if necessary, using conventional primer extension procedures as described in Sambrook et al , supia, 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 181 or PR0237 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 electioporation Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells The calcium treatment employing calcium chloride, as described in Sambrook et al , supia, or electroporation is generally used for prokaryotes Infection with Agiobacteiium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al , Gene, 23 315 (1983) and WO 89/05859 published 29 June 1989 For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and v an 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 er α/ . 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 microiniection 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 Enzv mology, 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 heie include prokaryote, yeast, oi higher eukaryote cells Suitable prokaryotes include but aie not limited to eubacteπa. such as Gram-negative oi Gram-positive organisms, for example, Enterobacteπaceae such as E coli Vanous E coli strains are publicly available, such as E coli Kl 2 stram MM294 (ATCC 31 446), E coli X 1776 (ATCC 1 537), E coli strain W 1 10 (ATCC 27,325) and K5772 (ATCC 53,635) Other suitable prokaryotic host cells include Enterobacteπaceae such & Eschenchιa, e g , E coli. Entewbai. tei Emuua, Klebs-iella, Pi oteus, Salmonella e -> Salmonella nphimiii ium, Seiratia, e g , Serratia marcescans, and Shigella, as well as Bacilli such as B subtihs and B licheniformis (e g , B /«c/?«n/orw(i 41 P disclosed in DD 266J10 published 12 Apπl 1989), Pseudomonas such as P aeruginosa, and Streptomvces 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 W31 10 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E colt W3110 strain 1 A2, which has the complete genotype tonA , E coli W31 10 strain 9E4, which has the complete genotype tonA ptr3, E coli W31 10 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA El 5 (argF-lac)169 degP OmpTkan' ', E colt W31 10 strain 37D6, which has the complete genotype tonA ptr3 phoA £75 (argF-lac)169 degP ompT rbs7 ύvG la , E colt W31 10 strain 40B4, which is strain 37D6 with a non- kanamycin resistant degP deletion mutation, and an £ coli strain having mutant peπplasmic protease disclosed in U S Patent No 4,946,783 issued 7 August 1990 Alternatively, in vitio 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 181 - or PR0237 encoding vectors Sacchai om ces cerevisiae is a commonly used lower eukaryotic host microorganism Others include
Figure imgf000056_0001
po be (Beach and Nuτse, Na e, 290 140 [1981], EP 139,383 published 2 May 1985), Kluvveiomyces hosts (U S Patent No 4,943,529, Fleer et αl , Bio/Technology, 9 968-975 ( 1991 )) such as, e g , K lαctis (MW98-8C, CBS683, CBS4574, Louvencourt et αl , J_ Bacteπol .737 [ 1983] ), K frαgihs (ATCC 12,424), K bulgαi icus (ATCC 16,045), K wickerαmu (ATCC 24, 178), K wαltu (ATCC 56 500), K drosophύαrum (ATCC 36,906, Van den Berg et αl , Bio/Technology. 8 135 (1990)), K ther otoleiα , and K mαtxiαnus, yαnowiα (EP 402,226), Picluα pαstoi is (EP 183,070, Sreeknshna et αl , J_ Basic Microbiol , 28 265-278 [1988]), Candida Ti ichoderma teesia (EP 244,234), Neuiospoia ctassa (Case et al . Proc Natl Acad Sci USA, 76 5259-5263 [\979]), Schwannioimces such as Schwanmonnces occidentals (EP 394,538 published 31 October 1990), and filamentous fungi such as e g , Neuiospoia Penicillium, Tohψocladium (WO 91/00357 published 10 January 1991 ), and Aspeigύlus hosts such as A mdulans (Ballance et al , B ochejjL Biophvs Res Commun . 1 12 284-289 1 1983], Tilburn et al , Gene 26 205-221 [ 19831. Yelton et al . Proc Natl Acad Sci USA, 8i 1470 1474 [1984]) and A nigei (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 Kloeckeia Pichia Sacchai omyces 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 181 or PR0237 are derived from multicellulai 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 hamstei 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 Vnol 36 59 (1977)), Chinese hamster ovary cellsADHFR (CHO, Urlaub and Chasm, Pioc Natl Acad Sci USA, 77 4216 (1980)), mouse sertoh cells (TM4 Mathei , Biol Reprod , 23 243 251 ( 1980)) human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), and mouse mammaiy 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 Rephcable Vector
The nucleic acid (e g , cDNA or genomic DNA) encoding PROl 81 or PR0237 may be inserted into a rephcable 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 vectois containing one or more of these components employs standard ligation techniques which are known to the skilled artisan
The PROl 81 or PR0237 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 PRO 181 - or PR0237-encodιng DNA that is inserted into the vector The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillmase, lpp, or heat-stable enterotoxin II leaders For yeast secretion the signal sequence may be, e g , the yeast invertase leader, alpha factor leader (including Sacchai omyces and Kluyveiomyces oc-factor leaders, the latter described in U S Patent No 5,010,182). or acid phosphatase leader, the C albicans glucoamylase leader (EP 362, 179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990 In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders
Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells Such sequences are w ell known tor a variety of bacteria, yeast, and viruses The origin of replication from the plasmid pBR322 is suitable tor most Gram-negative bacteria, the 2μ plasmid origin is suitable tor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful tor 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) contei resistance to antibiotics or other toxins, e (> , ampicillin neomycin, methotrexate, or tetracychne, (b) complement auxotrophic deficiencies, or (c) supply critic.11 nutrients not available from complex media, e g , the gene encoding D-alanine racemase tor Bacilli
An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PR0181 - or PR0237-encodιng nucleic acid, such as DHFR or thymidine kinase An appropnate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity prepared and propagated as described by Urlaub et al Proc Natl Acad Sci USA. 77 4216 ( 1980) A suitable selection gene tor use in yeast is the tip 1 gene present in the yeast plasmid YRp7 [Stinchcomb et al , Nature. 282 39 (1979), Kmgsman et al , Gene, 7 141 (1979), Tsche per et al , Gene. K) 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 81- or PR0237 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 61 (1978), Goeddel et al , Nature, 281 544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res . 8 4057 (1980), EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al , Proc Natl Acad Sci USA, 80 21-25 (1983)] Promoters for use in bacterial systems also will contain a Shine Dalgarno (S D ) sequence operably linked to the DNA encoding PROl 81 or PR0237
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3 phosphoglycerate kinase [Hitzeman et al , J Biol Chem , 255 2073 (1980)] or other glycolytic enzymes [Hess et al , J Adv Enzyme Reg .7 149 (1968), Holland, Biochemistry, J_7 4900 ( 1978)], such as enolase, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 phosphoglycerate mutase, pyruvate kinase, tπosephosphate isomerase, phosphoglucose isomerase, and glucokinase
Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metal lothione , 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 81 or PR0237 transcription from vectors in mammalian host cells is controlled for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 1 504 published 5 July 1989) adenovirus (such as Adenovirus 2) bovine papilloma virus avian sarcoma virus cvtomegalovirus a retrovirus, hepatitis-B virus and Simian Virus 40 (S V40) from heterologous mammalian promoters, e % 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 81 or PR0237 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, fetoprotein and insulin) Typically, however one w ill use an enhancer from a eukaryotic cell vn us Examples include the S V40 enhancer on the late side of the replication oi lgin (bp 100 270) the cvtomegalovirus early promoter enhancer the polyoma enhancer on the late side ot the replication origin and adenovn us enhancers The enhancer may be spliced into the vector at a position 5 oi 3 to the PRO 181 or PR0237 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 oi nucleated cells from other multicellular organisms) will also contain sequences necessary tor the termination ot 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 81 or PR0237
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PROl 81 or PR0237 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 81 or PR0237 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PROl 81 or PR0237 DNA and encoding a specific antibody epitope
5 Purification of Polypeptide
Forms of PROl 81 or PR0237 may be recovered from culture medium or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (e g , Tπton-X 100) or by enzymatic cleavage Cells employed in expression of PROl 81 or PR0237 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 81 or PR0237 from recombinant cell proteins or polypeptides The following procedures are exemplary of suitable purification procedures bv tractionation 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, to. example, Sephadex G 75, protein A Sepharose columns to remove contaminants such as IgG and metal chelating columns to bind epitope-tagged forms ot the PRO 181 or PR0237 Various methods of protein purification may be employed and such methods aie known in the art and described for example in Deutscher, Methods in Enzymology, 182 ( 1990), Scopes, Protein Purification Principles and Practice, Springer-Vei lag New York ( 1982) The purification step(s) selected will depend, tor example on the nature of the production process used and the particular PROl 81 or PR0237 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 81 or PR0237 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 81 or PR0237 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 ot such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobuhn, and soybean trypsin inhibitor Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adj uvant (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 hybπdoma methods, such as those described by Kohler and Milstein, Nature, 256495 ( 1975) In a hybπdoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent Alternatively , the lymphocytes may be immunized in viti o
The immunizing agent will typically include the PROl 81 or PR0237 polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs") are used it cells of human origin aie desired, or spleen cells or lymph node cells are used if non human mammalian souices are desired The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as pol vethvlene glycol to form a hybπdoma cell [Goding. Monoclonal Antibodies Principles and Practice, Academic Press ( 1986) pp 59-103] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells ot rodent, bovine and human origin Usually, rat or mouse myeloma cell lines are employed The hybπdoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the giowth or survival ot the unfused immortalized cells For example, if the parental cells lack the enzyme hy poxanthine guamne phosphoπbosyl transferase (HGPRT or HPRT) the culture medium tor the hybndomas typically will include hypoxanthine aminopterin, and thymidine ("HAT medium' ), which substances prevent the growth of HGPRT-deficient cells
Preferred immortalized cell lines are those that fuse efficiently, suppott stable high level expression of antibody by the selected antibody -producing cells and aie sensitive to a medium such as HAT medium Moie preferred immortalized cell lines are murine myeloma lines, which can be obtained, tor instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia Human myeloma and mouse-human hetei omyeloma cell lines also hav e 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, Ine , New York, (1987) pp 51-63]
The culture medium in which the hybπdoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PROl 81 or PR0237 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 immunoabsorbent 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 hybπdoma 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 immunoglobulin purification procedures such as, for example, protein A Sepharose hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Patent No 4,816,567 DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using ohgonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine 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 immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U S Patent No 4 816 567 Morrison et al , supia] or by covalently loining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobuhn polypeptide Such a non immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention or can be substituted foi the v ariable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody
The antibodies may be monovalent antibodies Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prev ent heavy chain crosshnking Alternatively, the relevant cysteine residues are substituted with another amino acid iesidue or are deleted so as to prevent crosshnking
In \ iti o methods are also suitable for preparing monovalent antibodies Digestion of antibodies to pi oduce fragments thereof particularly, Fab fragments, can be accomplished using routine techniques known in the ait
3 Human and Humanized Antibodies
The antibodies of the invention may fuither comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv , Fab, Fab', F(ab ) . 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 222 381 ( 1991 ). Marks et al , J Mol Biol 222 581 ( 1991 )] The techniques ot Cole et al , and Boerner et al aie also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R Lιss, p 77 (1985) and Boerner etal , J Immunol 147( 1 ) 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, whit h 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 JJ3 779 783 (1992) Lonberge/α/ Nature, 368 856 859 ( 1994) Morπson, Nature, 68 812-13 (1994) Fishwild er-;/ Nature Biotechnology. 14 845 51 ( 1996). Neubergei . Nature Biotechnology, 14 826 ( 1996). Lonberg and Huszar. Intern Rev Immunol . J_3 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 81 or PR0237, 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/hght-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 hybndomas (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 et al , EMBO J . JO 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, compπsing 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, it 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 Enzvmology, 121 210(1986) According to another approach described in WO 96/2701 1 , the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture The preferred interface comprises at least a part of the CH3 region of an antibody constant domain In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e g , tyrosine or tryptophan) Compensatory "cavities of identical or similar size to the large side chaιn(s) are created on the interface of the second antibody molecule bv replacing large amino acid side chains with smaller ones (e g , alanine or threomne) This provides a mechanism tor increasing the yield of the heterodimer over other unwanted end-products such as homodimers
Bispecific antibodies can be prepared as full length antibodies oi antibody fragments (e g , F(ab') ., bispecific antibodies) Techniques for generating bispecific antibodies from antibody fragments have been described in the literature For example, bispecific antibodies can be prepaied using chemical linkage Brennan et al , Science, 229 81 (1985) describe a procedure wherein intact antibodies aie proteoly tically 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 pi event intermolecular disulfide formation The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives One of the Fab'-TNB derivativ es 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 dnectly recovered from E coli and chemically coupled to form bispecific antibodies Shalaby et al , J Exp Med , 175 217 225 (1992) describe the production of a fully humanized bispecific antibody F(ab')-, molecule Each Fab' fragment was separately secreted from £ coli and subjected to directed chemical coupling in viti o 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 ef α/ , J Immunol , 148(5) 1547-1553 (1992) The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers This method can also be utilized for the production of antibody homodimers The "diabody' technology described by Holhnger et al , Proc Natl Acad Sci USA, 90 6444-6448 ( 1993) has provided an alternative mechanism for making bispecific antibody fragments The fragments comprise a heavy-chain variable domain (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) di ers has also been reported See, Gruber et al , J Immunol 152 5368 (1994)
Antibodies with more than two valencies are contemplated For example, tπspecific antibodies can be prepared Tutt et al , J Immunol , 147 60 (1991 )
Exemplary bispecific antibodies may bind to two different epitopes on a given PRO! 81 or PR0237 polypeptide herein Alternatively, an anti PROl 81 or anti PR0237 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 particulai PROl 81 or PR0237 polypeptide Bispecific antibodies may also be used to localize cytotoxic agents to cells which expiess a particular PROl 81 or PR0237 polypeptide These antibodies possess a PROl 81- or PR0237-bιndιng arm and an arm which binds a cytotoxic agent or a radionuchde chelator, such as EOTUBE, DPTA, DOTA, or TETA Another bispecific antibody of interest binds the PROl 81 or PR0237 polypeptide and further binds tissue factor (TF)
5 Heteroco ugate Antibodies
Heteroconjugate antibodies are also within the scope of the present inv ention Heteroconjugate antibodies are composed of two covalently pined antibodies Such antibodies have tor example, been proposed to target immune system cells to unwanted cells [U S Patent No 4,676 9801 and for treatment of HIV infection [WO 91 /00360, WO 92/200373 EP 03089 ] It is contemplated that the antibodies may be prepared m viti o using know n methods in synthetic protein chemistry, including those inv olv ing crosshnking agents For example, immunotoxins may be constructed using a disulfide exchange reaction oi by toi ming a thioether bond Examples of suitable reagents for this purpose include lminothiolate and methy l 4 mercaptobutvπmidate 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 resιdue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region The homodimeπc antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See, Caron et al , J Exp Med , 176 1 191-1 195 (1992) and Shopes, J Immunol , J48 2918-2922 (1992) Homodimeπc 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 Immunocomugates
The invention also pertains to immunoconjugates compi ising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (- e , a radioconjugate)
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 aeiuginosa), πcin A chain, abπn A chain, modeccm A chain, alpha-sarcin, Aleuritesfoidu proteins dianthin proteins, Plntolaca amencana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin. crotin, sapaonaπa officinahs inhibitor, gelonin, mitogelhn, restπctocin, phenomycin, enomycin, and the tπcothecenes A variety ot radionuchdes are available tor the production of radioconjugated antibodies Examples include : Bι, πιI, ' ''In, 9"Y, and l 6Re
Conjugates of the antibody and cytotoxic agent are made using a variety ot bifunctional protein-coupling agents such as N-succιmmιdyl-3-(2-pyπdyldιthιol) propionate (SPDP). lminothiolane (IT), bifunctional derivatives of lmidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate). aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazomum derivatives (such as bιs-(p-dιazonιumbenzoyl)-ethylenedιamιne), diisocyanates (such as tolyene 2,6-dnsocyanate) and bis- active fluorine compounds (such as 1 ,5-dιfluoro-2,4-dιnιtrobenzene) For example, a πcin lmmunotoxin can be prepared as described in Vitetta et al . Science, 238 1098(1987) Carbon- 14-labeled 1 -ιsothιocyanatobenzyl-3 methyldiethylene tπaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody See, W094/1 1026
In another embodiment, the antibody may be conjugated to a ' receptor' (such as streptavidin) toi 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 e 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 etal , 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 phosphatidylchohne, cholesterol, and PEG-deπvatized 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 Doxorubic ) 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 in vestigational, disease-oriented, in viti o 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 Pπnc Pract Oncol Update, 3(10) 1 -12 ([1989]) The tumor cell lines employed in this study have been described in Monks et al , supia 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 tor 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 aie 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 ot tumors, and to test the efficacy ot candidate therapeutic agents, including antibodies, and other agonists of the nativ e polypeptides, including small molecule agonists The in i ι\ o 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 cancel , etc ) include both non recombinant and recombinant (transgenic) animals Non-recombinant animal models include toi example rodent, e g . murine models Such models can be generated by intioducing tumor cells into syngeneic mice using standard techniques, e g , subcutaneous injection, tail vein injection, spleen implantation, lntrapeπtoneal 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, BIO LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and S JL 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, Ine , 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), / -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 (Karmah 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 tumois 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), supia 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, Ine , (San Diego, California)
Tumors that arise in animals can be removed and cultured in \ itio Cells from the in viti o cultures can then be passaged to animals Such tumors can serve as targets tor 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 oi more rounds of passage analyzed toi differential expression of genes of interest Such passaging techniques can be performed with any known tumor oi 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 vitio in cell culture Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about l Oxl O6 to 10x10' 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, Haemostasis, 16 300-320 [1986])
One way of evaluating the efficacy of a test compound 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 calmer 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 morphometπc 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 f oi 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 et al , 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 puipose 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 eithei as a single transgene, oi in concatamers, e g , head-to-head or head-to-tail tandems Selective introduction ot a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al Pioc Natl Acad Sci USA. 89 6232- 636 (1992) The expression of the transgene in transgenic animals can be monitored by standard techniques For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunocytochemistry The animals are further examined for signs of tumor or cancer development
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 surgeiy, primarily because of the anatomy of the feline oral cavity At present, there is no effective treatment for this tumor Prior to entry into the study, each cat undergoes complete clinical examination, biopsy, and is scanned by computed tomography (CT) Cats diagnosed with subhngual oral squamous cell tumors are excluded from the study The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor, the animals may not be able to feed themselves Each cat is treated repeatedly, over a longer period of time Photographs ot 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, chrondioma leiomyosarcoma of dogs, cats, and baboons can also be tested Ot 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 identity 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, (ρoly)peptιde immunoglobulin fusions, and, in particular, antibodies including without limitation, poly- and
Figure imgf000069_0001
antibodies and antibody fiagments, single-chain antibodies, anti ldiotypic antibodies, and chimeric oi humanized versions ot such antibodies oi fragments, as well as human antibodies and antibody fragments The assay s can be performed in a variety of formats, including protein protein binding assays biochemical screening assay s immunoassays and cell based assays, which are well characterized in the ai t
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 ot a polypeptide encoded by the gene identified heiein 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- lmmobihzed 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, protem-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and Song, Nature (London). 340 245-246 (1989), Chien et αl , 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 ot GAL4, and another, in which candidate activating proteins are fused to the activation domain The expression of a GAL1 -lαcZ reporter gene under control of a GAL4-actιvated promoter depends on reconstitution of GAL4 activity via protein-protein interaction Colonies containing interacting polypeptides are detected with a chromoge c 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 scieening assay s disclosed herein, can be administered tor the treatment of tumors, including cancers, in the form of phaimaceutical 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 ot an antibody, peptide molecules can be designed which ietain 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 αl . 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 hei ein, 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 lyophihzed formulations or aqueous solutions Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethomum 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 polyvinylpyrrohdone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosacchaπdes, disacchaπdes, and other carbohydrates including glucose, mannose, or dextπns, 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 noniomc 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 tor 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 oi gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery svstems (for example, liposomes. albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macioemulsions 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 lyophihzation and reconstitution
Therapeutic compositions herein generally are placed into a container hav ing a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a h podermic injection needle
Sustained-release preparations may be prepared Suitable examples ot sustained-release preparations include semipermeable matrices ot solid hydrophobic polymers containing the antibody which matrices are in the form of shaped articles, e g , films, or microcapsules Examples ot sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate). or poly(vιnylalcohol)) 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-glycohc acid copolymers such as the LUPRON DEPOT™ (miectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate), and ρoly-D-(-)-3-hydroxybutyπc acid While polymers such as ethylene-vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated antibodies 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, ghoblastomas, and various head and neck tumors), leukemias and lymphoid malignancies, other disorders such as neuronal, ghal, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoehc 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, lntrapeπtoneal, intracerobrospinal, intraocular, lntraarteπal, intralesional, subcutaneous, intraarticular, intrasynovial intrathecal, oral, topical, or inhalation routes
Other therapeutic regimens may be combined with the administration ot 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 oi in addition, a chemotherapeutic agent may be administered to the patient Prepaiation and dosing schedules tor 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-piogesterone such as onapπstone (see, EP 616812) in dosages known foi such molecules
It may be desirable to also administer antibodies against tumor associated antigens such as antibodies which bind to the ErbB2 EGFR EιbB3, Eι bB4, or vasculai endothehal factor (VEGF) Alternatively, oi in addition, two or more antibodies binding the same or two or moi e 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 w ith a growth inhibitory agent Foi 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 manufactuie containing materials useful for the diagnosis or treatment of the disorders described abov e is prov ided 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 stoppei pierceable by a hypodermic injection needle) The active agent in the composition is an anti-tumor agent of the present invention The label on oi associated w ith, 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 buff er, such as phosphate buffered saline Ringers solution and dextrose solution It may further include other materials desirable from a commercial and user standpoint including other butters, diluents, filters needles syringes and package inserts with instructions tor 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 refeiences 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 81 and PRQ237
(A) PROl 81
1 Preparation of ohgo dT primed cDNA library mRNA was isolated from human placenta tissue using reagents and protocols from Invitrogen, San Diego CA (Fast Track 2) This RNA was used to generate an ohgo dT primed cDNA library in the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script Plasmid System) In this procedure, the double stranded cDNA was sized to greater than 1000 bp and the Sall/Notl hnkered cDNA was cloned into Xhol/Notl cleaved vector pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an Sfil restriction enzyme site preceding the Xhol/Notl cDNA cloning sites
2 Preparation of random primed cDNA library A secondary cDNA library was generated in order to preferentially represent the 5 ends of the primary cDNA clones Sp6 RNA was generated from the primary library (described above), and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY 0 using leagents and protocols from Life Technologies (Super Script Plasmid System, referenced above) In this procedure the double stranded cDNA was sized to 500- 1000 bp hnkered with blunt to Notl adaptors, cleaved with Sfil, and cloned into Sfil/Notl cleaved vector pSST-AMY 0 is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cDNA cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminatoi after the cloning sites Thus, cDNAs cloned into this v ector that aie fused in frame with amylase sequence will lead to the secretion of amylase from appropriately transfected yeast colonies
3 Transformation and Detection DNA from the library described in paragraph 2 above was chilled on ice to w hich was added electrocompetent DH10B bacteria (Life Technologies 20 ml) The bacteria and vectoi mixture was then electropoi ated as recommended by the manufacturei Subsequently, SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37 °C for 30 minutes The transformants were then plated onto 20 standard 150 mm LB plates containing ampicillin and incubated for 16 hours (37 °C) Positive colonies were scraped off the plates and the DNA was isolated from the bacterial pellet using standard protocols, e g , CsCl-gradient The purified DNA was then carried on to the yeast protocols below
The yeast methods were divided into three categories ( 1 ) Transformation of yeast with the plasmid/cDNA combined vector, (2) Detection and isolation of yeast clones secreting amylase, and (3) PCR amplification of the insert directly from the yeast colony and purification of the DNA for sequencing and further analysis
The yeast strain used was HD56-5A (ATCC-90785) This strain has the following genotype MAT alpha, ura3-52, leu2-3, leu2-l 12, hιs3 1 1, hιs3-15, MAL+, SUC+, GAL+ Preferably, yeast mutants can be employed that have deficient post-translational pathways Such mutants may have translocation deficient alleles in seel 1 , secll, sec62, with truncated secl being most preferred Alternatively, antagonists (including antisense nucleotides and/or ligands) which interfere with the normal operation of these genes, other proteins implicated in this post translation pathway (e g , SECόlp, SEC72p, SEC62p, SEC63p, TDJlp or SSAlp-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast
Transformation was performed based on the protocol outlined by Gietz et al , Nucl Acid Res , 20 1425 (1992) Transformed cells were then inoculated from agar into YEPD complex media broth (100 ml) and grown overnight at 30°C The YEPD broth was prepared as described in Kaiser et al , Methods in Yeast Genetics. Cold Spring Harbor Press, Cold Spring Harbor, NY, p 207 (1994) The overnight culture was then diluted to about 2 x I O6 cells/ml (approx OD^^O 1 ) into fresh YEPD broth (500 ml) and regrown to 1 x 10" cells/ml (approx 00^=0 4-0 5)
The cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centπfuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centnfuge The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tns-HCl, 1 mM EDTA pH 7 5 100 mM Li-OOCCH,), and resuspended into LiAc/TE (2 5 ml)
Transformation took place by mixing the prepared cells (100 μl) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs Gaithersburg, MD) and transforming DNA ( 1 μg vol < l O μl) in microtuge tubes The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 μl 40% polyethylene glycol-4000, 10 mM Tπs HCl, 1 M EDTA, 100 mM Li .OOCCH,, pH 7 5) was added This mixture was gently mixed and incubated at 30°C while agitating tor 30 minutes The cells were then heat shocked at 42°C tor 1 minutes, and the reaction vessel centπfuged in a microfuge at 12,000 rpm for 5-10 seconds decanted and resuspended into TE (500 μl, lO mM Tns-HCl, 1 mM EDTA pH 7 5) followed by recenti if ugation The cells were then diluted into TE (1 ml) and aliquots (200 μl) were spread onto the selective media previously prepared in 150 mm growth plates (VWR)
Alternatively, instead of multiple small reactions the transformation was pei formed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly The selective media used w s a synthetic complete dextrose agar lacking uracil (SCD Ura) prepared as described in Kaiser et al , Methods in Yeast Genetics, Cold Spring Harbor Pi ess Cold Spring Harbor, NY, p 208 210 (1994) Transformants were grown at 30°C for 2-3 days The detection of colonies secreting amylase was performed by including red starch in the selective growth media Starch was coupled to the red dye (Reactive Red-120, Sigma) as per the procedure described by Biely et al , Anal Biochem , 172 176-179 (1988) The coupled starch was incorporated into the SCD-Ura agar plates at a final concentration of 0 15% (w/v), and was buffered with potassium phosphate to a pH of 7 0 (50-100 mM final concentration)
The positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies Well isolated single colonies positive for amylase secretion were detected by direct incorporation of red starch into buffered SCD-Ura agar Positive colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly
4 Isolation of DNA by PCR Amplification
When a positive colony was isolated, a portion of it was picked by a toothpick and diluted into sterile water (30 μl) in a 96 well plate At this time, the positive colonies were either frozen and stored for subsequent analysis or immediately amplified An aliquot of cells (5 μl) was used as a template for the PCR reaction in a 25 μl volume containing 0 5 μl Klentaq (Clontech, Palo Alto, CA), 4 0 μl 10 mM dNTP's (Perkm Elmer-Cetus), 2 5 μl Kentaq buffer (Clontech), 0 25 μl forward ohgo 1 , 0 25 μl reverse ohgo 2 12 5 μl distilled water The sequence of the forward ohgonucleotide 1 was
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID NO 3) The sequence of reverse ohgonucleotide 2 was
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID NO 4)
PCR was then performed as follows
Denature 92 °C, 5 minutes
3 cycles of Denature 92 °C 30 seconds
Anneal 59 °C 30 seconds
Extend 72 °C 60 seconds
3 cycles of Denature 92 °C 30 seconds
Anneal 57 CC 30 seconds
Extend 72 °C 60 seconds
25 cycles of Denature 92 CC 30 seconds
Anneal 55 °C 30 seconds
Extend 72 °C. 60 seconds
Hold 4°C
The underlined regions of the oligonucleotides annealed to the ADH promoter region and the amylase region, respectively and amplified a 307 bp region from vectoi pSST-AMY 0 when no insert was present Typically, the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers Thus, the total product of the PCR reaction tiom an empty vector was 343 bp Howevei . signal sequence-fused cDNA resulted in considerably longer nucleotide sequences Following the PCR an aliquot of the reaction (5 μl) was examined by agarose gel electrophoresis in a 1 % agarose gel using a Tπs-Borate-EDTA (TBE) buffering system as described by Sambrook et al , supia Clones resulting in a single strong PCR product larger than 400 bp were furthei analyzed by DNA sequencing after purification with a 96 Qiaquick PCR clean up column (Qiagen Ine , Chatsworth, CA)
5 Identification of Full-length Clone
A cDNA sequence isolated in the above screen was found, by BLAST and FastA sequence alignment, to have sequence homology to a nucleotide sequence encoding the cornichon protein This cDNA sequence is herein designated DNA13242 and DNA25383 Based on the sequence homology, probes were generated from the sequence of the DNA13242 molecule and used to screen a human placenta (LIB89) library prepared as described in paragraph 1 above The cloning vector was pRK5B (pRK5B is a precursor of pRK5D that does not contain the Sfi site, see, Holmes et al , Science, 253 1278-1280 (1991 )), and the cDNA size cut was less than 2800 bp
A full length clone was identified that contained a single open reading frame with an apparent translational initiation site at nucleotide positions 14-16 and ending at the stop codon found at nucleotide positions 446 448 (Figure 1 , SEQ ID NO 1 ) The predicted polypeptide precursor is 144 amino acids long, has a calculated molecular weight of approximately 16,699 daltons and an estimated pi of approximately 5 6 Analysis of the full-length PR0181 sequence shown in Figure 2 (SEQ ID NO 2) evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 20, a putative type II transmembrane domain from about amino acid 1 1 to about amino acid 31 and other ti ansmembrane domains from about amino acid 57 to about amino acid 77 and from about amino acid 123 to about amino acid 143, and a glycosaminoglycan attachment site from about amino acid 96 to about amino acid 100 Clone UNQ155 (DNA23330 1390) has been deposited with ATCC on April 14, 1998 and is assigned ATCC deposit no 209775
Analysis of the ammo acid sequence of the full-length PROl 81 poypeptide suggests that it possesses significant sequence similarity to the cornichon protein, thereby indicating that PRO 181 may be a novel cornichon homolog More specifically, an analysis of the Dayhott database (version 35 45 SwissProt 35) evidenced significant homology between the PROl 81 amino acid sequence and the following Dayhott sequences AF02281 1 CET09E8_3, S64058, YGF4_YEAST, YB60_YEAST EBU89455 SIU36383_3 AND PH1 71
(B) PRQ237
The extracellular domain (ECD) sequences (including the secretion signal sequence, it any) from about 950 known secreted proteins from the Swiss Prot public database were used to search EST databases The EST databases included public EST databases (e g , GenBank) and a piopπetary EST database (LIFESEQ 8 Incyte Pharmaceuticals Palo Alto, CA) The search was performed using the computer program BLAST or BLAST2 [Altschul et al , Methods in Enzymology, 266 460 480 ( 1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins weie clustered and assembled into consensus DNA sequences with the program ' phrap" (Phil Green University of Washington, Seattle Washington)
A consensus DNA sequence was assembled relative to other EST sequences using phrap as descnbed above This consensus sequence is herein designated Consen0514 and DNA30905 In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above Based on the DNA30905 consensus sequence oligonucleotides were synthesized 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0237 Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length The probe sequences are typically 40-55 bp in length In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1 5 kbp In order to screen several libraries foi a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel etal , Current Protocols in Molecular Biology, supia, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe ohgonucleotide and one of the primer pairs
PCR primers (forward and reverse) were synthesized forward PCR primer (30905 f )
5'-TCTGCTGAGGTGCAGCTCATTCAC-3' (SEQ ID NO 7) reverse PCR primer (30905 r) 5'-GAGGCTCTGGAAGATCTGAGATGG-3' (SEQ ID NO 8)
Additionally , a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA30905 sequence which had the following nucleotide sequence hybridization probe (30905 p)
5'-GCCTCTTTGTCAACGTTGCCAGTACCTCTAACCCATTCCTCAGTCGCCTC-3' (SEQ ID NO 9)
RNA for construction of the cDNA libraries was isolated from human fetal brain tissue The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with ohgo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursoi of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278- 1280 ( 1991 )) in the unique Xhol and Notl sites DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PR0237 polypeptide (designated herein as DNA34353- 1428 [Figure 3, SEQ ID NO 5]) and the derived protein sequence for that PR0237 polypeptide
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 586-588 and a stop signal at nucleotide positions 1570- 1 72 (Figuie 3 SEQ ID NO 5) The predicted polypeptide precursor is 328 amino acids long, and has a calculated molecular weight of approximately 36,238 daltons and a pi ot about 9 90 Analysis ot the full-length PR0237 sequence shown in Figure
4 (SEQ ID NO 6) evidences the presence ot a variety ot important polypeptide domains w herein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PR0237 sequence evidenced the following a signal peptide from about amino acid 1 to about amino acid 23, a transmembrane domain from about amino acid 177 to about amino acid 199, potential N-glycosylation sites from about amino acid 1 18 to about ammo acid 122, from about amino acid 170 to about amino acid 174, and from about amino acid 260 to about amino acid 264, and eukaryotic-type carbonic anhydrase sequence homology blocks from about ammo acid 222 to about amino acid 271 , from about amino acid 128 to about amino acid 165 and fiom about amino acid 45 to about amino acid 93
Clone DNA34353-1428 has been deposited with ATCC on May 12, 1998 and is assigned ATCC deposit no 209855 Analysis of the amino acid sequence of the full-length PR0237 polypeptide suggests that it possesses significant sequence similarity to the carbonic anhydrase protein More specifically, an analysis of the Dayhoff database (version 35 45 SwissProt 35) evidenced significant homology between the PR0237 amino acid sequence and the following Dayhoff sequences AFO50106_l , OACALP_l , CELD1022_8, CAH2_HUMAN, 1CAC, CAH5_HUMAN, CAHP_HUMAN, CAH3_HUMAN, CAH1_HUMAN and 2CAB
EXAMPLE 2
Use of PRO 181 or PRQ237 as a Hybridization Probe The following method describes use of a nucleotide sequence encoding PROl 81 or PR0237 as a hybridization probe
DNA comprising the coding sequence of full-length or mature PRO 181 or PR0237 (as shown in Figure 1 and 3, respectively, SEQ ID NOS 1 and 5, respectively) or a fragment thereof is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PROl 81 or PR0237) 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 181 or PR0237 polypeptide to the filters is performed in a solution of 50% formamide, 5\ SSC 0 1 % SDS, 0 1 % sodium pyrophosphate, 50 mM sodium phosphate, pH 6 8, 2x Denhardt's solution, and 109c dextran sulfate at 42"C for 20 hours Washing ot 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 3
Expression of PROl 81 oi PRQ237 in £ c oli This example illustrates preparation of an unglycosylated form ot PROl 81 or PR0237 by recombinant expression in £ coli
The DNA sequence encoding PROl 81 or PR0237 is initially amplified using selected PCR pi lmers The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vectoi A variety of expression vectors may be employed An example ot a suitable vector is pBR322 (derived from £ coli, see Bolivar et al , Gene, 2 95 (1977)) which contains genes for ampicillin and tetracychne resistance The vector is digested with restriction enzyme and dephosphorylated The PCR amplified sequences are then ligated into the vector The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a poly His leader (including the first six STII codons, poly His sequence, and enterokinase cleavage site), the PROl 81 or PR0237 coding region, lambda transcriptional terminator, and an argU gene
The ligation mixture is then used to transform a selected £ coli strain using the methods described in Sambrook et al , supia Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected Plasmid DNA can be isolated and confirmed by 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 PRO 181 or PR0237 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein
PRO 181 or PR0237 may be expressed in £ coli in a poly-His tagged form, using the following procedure The DNA encoding PRO 181 or PR0237 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 £ coli host based on strain 52 (W31 10 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(lacIq) Transformants are first grown in LB containing 50 mg/ml carbenicilhn at 30°C with shaking until an OD6IK, of 3 5 is reached Cultures are then diluted 50 100 fold into CRAP media (prepared by mixing 3 57 g (NH4) ,S04, 0 71 g sodium cιtrate»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 M 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 centπf uged to pellet the cells Cell pellets are frozen until purification and refolding
£ 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 Tπs, pH 8 buffer Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0 I M and 0 02 M, respectively, and the solution is stiπed overnight at 4°C This step results in a denatured protein with all cvsteine residues blocked by sulfitohzation The solution is centπfuged at 40 000 rpm in a Beckman Ultracentifuge for 30 mm The supernatant is diluted with 3-5 volumes ot metal chelate column buffer (6 M guanidine 20 mM Ti is, pH 7 4) and filteied through 0 22 micron filters to clarity The clai if led extract is loaded onto a 5 ml Qiagen Ni -NTA metal chelate column equilibrated in the metal chelate column buffei The column is washed ith additional buttei containing 50 mM lmidazole (Calbiochem Utrol grade), pH 7 4 The protein is eluted with buffer containing 250 mM lmidazole 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 Tπs, 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 acetonitnle 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 acetonitnle from 10 to 80% Aliquots of fractions with A28„ 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 acetonitnle since those species are the most compact with then hydrophobic interiors shielded from interaction with the reversed phase resin Aggregated species are usually eluted at higher acetonitnle 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 81 or PR0237 polypeptide are pooled and the acetonitnle 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 81 and PR0237 were successfully expressed in £ coli in a poly-His tagged form by the above procedure
EXAMPLE 4 Expression of PROl 81 or PRQ237 in mammalian cells This example illustrates preparation of a potentially glycosylated form of PROl 81 or PR0237 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 181 or PR0237 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PROl 81 or PR0237 DNA using ligation methods such as described in Sambrook etal , supia The resulting vector is called pRK5-PRO 181 or pRK5-PR0237
In one embodiment, the selected host cells may be 293 cells Human 293 cells (ATCC CCI 1573) aie 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 181 or pRK5-PR0237 DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappav ef al . Cell. 31 543 ( 1982)] and dissolved in 500 μl of 1 mM Tπs-HCI, 0 1 mM EDTA, 0 227 M CaCl 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 houis 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
Figure imgf000082_0001
and 200 μCi/ml 15S-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 dried and exposed to film for a selected period of time to reveal the presence of the PROl 81 or PR0237 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 81 or PR0237 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-PR0181 or ρRK5-PR0237 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 transferπn After about four days, the conditioned media is centπfuged and filtered to remove cells and debris The sample containing expressed PROl 81 or PR0237 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography
In another embodiment, PROl 81 or PR0237 can be expressed in CHO cells The pRK5-PR0181 or pRK5-PR0237 can be transfected into CHO cells using known reagents such as CaP0 or DEAE-dextran As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as
Figure imgf000082_0002
After determining the presence of a PROl 81 or PR0237 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 PROl 81 or PR0237 polypeptide can then be concentrated and purified by any selected method Epitope-tagged PROl 81 or PR0237 may also be expressed in host CHO cells The PROl 81 or PR0237 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 181 or PR0237 insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector Labeling may be performed, as described above, to verify expression The culture medium containing the expressed poly His tagged PRO 181 or PR0237 can then be concentrated and purified by any selected method such as by Nι1+- chelate affinity chromatography
PROl 81 orPR0237 may also be expressed in CHO and/or COS cells by a tiansient expression procedure or in CHO cells by another stable expression procedure Stable expression in CHO cells is performed using the following procedure The proteins aie expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e g extiacellular 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 , Cunent 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® (Boehringer Mannheim) The cells are grown as described in Lucas et al , supia Approximately 3 x 107 cells are frozen in an ampule for further growth and production as described below
The ampules containing the plasmid DNA are thawed by placement into a water bath and mixed by vortexing The contents are pipetted into a centrifuge tube containing 10 mis of media and centπfuged 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 any 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 4°C or immediately loaded onto columns for purification
For the poly-His tagged constructs, the proteins are purified using a Ni ^ NTA column (Qiagen) Before purification, lmidazole is added to the conditioned media to a concentration of 5 mM The conditioned media is pumped onto a 6 ml Ni +-NTA column equilibrated in 20 mM Hepes, pH 7 4, buftei containing 0 3 M NaCl and 5 mM lmidazole at a flow rate ot 4-5 ml/min at 4°C After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration butter containing 0 25 M lmidazole The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80' C
Immunoadhesin (Fc-containing) constructs 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 ith equilibration buifer 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 ot 1 M Tπs 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-termmal amino acid sequencing by Edman degradation
EXAMPLE 5 Expression of PRO! 81 or PRQ237 in Yeast
The following method describes recombinant expression of PROl 81 or PR0237 in yeast First, yeast expression vectors are constructed for intracellular production or secretion of PRO 181 or PR0237 from the ADH2/GAPDH promoter DNA encoding PROl 81 or PR0237 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PROl 81 or PR0237 For secretion, DNA encoding PR0181 or PR0237 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PROl 81 or PR0237 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 81 or PR0237
Yeast cells, such as yeast strain AB110, 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% tπchloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain
Recombinant PRO 181 or PR0237 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 81 or PR0237 may further be purified using selected column chromatography resins
EXAMPLE 6 Expression of PROl 81 or PRQ237 in Baculovirus-Intected Insect Cells The following method describes recombinant expression in Baculov lrus-mfected insect cells The sequence coding for PROl 81 or PR0237 is fused upstream ot 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 ti om commercially available plasmids such as pVLl 393 (Novagen) Briefly, the sequence encoding PRO 181 or PR0237 or the desired portion of the coding sequence of PROl 81 or PR0237 (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 sues The product is then digested with those selected restriction enzymes and subcloned into the expression vector
Recombinant baculovirus is generated by co-transtecting the abov e plasmid and BaculoGold1 virus DNA (Pharmingen) into Spodopteiafiugipei da ("Sf9") cells (ATCC CRL 171 1 ) using hpotectin (commercially available from GIBCO-BRL) After 4 - 5 days of incubation at 28"C. the leleased viruses are harvested and used for furthei amplifications Vnal 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 PROl 81 orPR0237 can then be purified, for example, by Nf +-chelate affinity chromatography as follows Extracts are prepared from recombinant virus-inf ected Sf 9 cells as described by Rupert et al , Natuie, 362 175 179 (1993) Briefly, Sf9 cells are washed, resuspended in so cation 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 butter (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7 8) and filtered through a 0 45 mm filter A Nι2+-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 A28„ 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 60), which elutes nonspecifically bound protein After reaching A2M baseline again, the column is developed with a 0 to 500 mM lmidazole gradient in the secondary wash buffer One ml fractions are collected and analyzed by SDS PAGE and silver staining or Western blot with Nι2+-NTA-conjugated to alkaline phosphatase (Qiagen) Fractions containing the eluted HisHI-tagged PROl 81 or PR0237, respectively, are pooled and dialyzed against loading buffer
Alternatively, purification of the IgG tagged (or Fc tagged) PROl 81 or PR0237 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 Spodoptera fi ugiperda ("Sf9") cells (ATCC CRL 171 1 ), using Lipofectin (Gibco BRL) pb PH IgG and pb PH His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polyhnker regions to include the His or Fc tag sequences The cells are grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone) Cells are incubated for 5 days at 28 °C The supernatant is harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10 Cells are incubated for 3 days at 28 °C The supernatant is harvested and the expression ot the constructs in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml of Ni 1*-NTA beads (QIAGEN) for histidine tagged proteins or Piotem-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 cultuie (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an appioximate MOI of 0 1 Cells are incubated for 3 day s at 28 °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 lemove the cells and tilteied through 0 22 micron filters For the poly-His tagged constructs, the protein construct is purified using a Ni 1+-NTA column (Qiagen) Before purification lmidazole is added to the conditioned media to a concentration of 5 mM The conditioned media is pumped onto a 6 ml Ni ,+-NTA column equilibrated in 20 mM Hepes, pH 7 4, buffer containing 0 3 M NaCl and 5 mM lmidazole 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 lmidazole The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C
Immunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3 5 The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tπs 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 hιgh-5 cells In this procedure, the DNA encoding the desired sequence is amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector Such epitope tags include poly- His tags and immunoglobulin tags (like Fc regions of IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIEl -1 (Novagen) The pIEl -1 and pIEl -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus lei promoter in stably- transformed insect cells (1) The plasmids diftei only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for lei -mediated gene expression in uninfected insect cells as well as the hr5 enhancer element pIE 1 - 1 and pIE 1 -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 pIEl - 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
Hιgh-5 cells are grown to a confluency of 50% under the conditions of, 27 °C, no CO^ NO pen/strep For each 150 mm plate, 30 μg ofpIE 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, lOO μl of CellFectin (CellFECΗN (GιbcoBRL# 10362-010) (vortexed to mix)) is mixed with 1 ml of Ex-Cell medium The two solutions are combined and allow ed to incubate at room temperature foi 15 minutes 8 ml of Ex Cell media is added to the 2 ml of DNA/CellFECTIN mix and this is layered on hιgh-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 CellFECΗN, 30 ml of fresh Ex-Cell media is added and the cells are incubated for 3 days at 28"C The supernatant is harvested and the expression of the sequence in the baculovirus expression vector is determined by batch binding of 1 ml of supernatent to 25 ml of Ni 2+-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining
The conditioned media from the transfected cells (0 5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0 22 micron filters For the poly-His tagged constructs, the protein comprising the sequence is purified using a Ni 2+-NTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration of 5 mM The conditioned media is pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 48°C After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein is then subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C
Immunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which 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 Tπs 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
PR0237 was expressed using the above baculovirus procedure employing hιgh-5 cells
EXAMPLE 7 Preparation of Antibodies that Bind PRO! 81 or PRQ237 This example illustrates preparation of monoclonal antibodies which can specifically bind PROl 81 or PR0237
Techniques for producing the monoclonal antibodies are known in the ait and aie described, foi instance in Goding, supia Immunogens that may be employed include purified PROl 81 or PR0237. fusion proteins containing PROl 81 or PR0237, and cells expressing recombinant PROl 81 or PR0237 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 81 or PR0237 immunogen emulsified in complete
Freund's adjuvant and injected subcutaneously or lntrapeπtoneally in an amount from 1 -100 miuograms Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Im unochemical Research, Hamilton MT) and ιn]ected 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 Thereattei , tor several weeks, the mice may also be boosted with additional immunization injections Serum samples may be pei iodicallv obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-PRO l 81 or antι-PR0237 antibodies
After a suitable antibody titer has been detected the animals "positiv e toi antibodies can be injected with a final intravenous injection of PROl 81 or PR0237 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 1 , available from ATCC, No CRL 1597 The fusions generate hybndoma 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 hybndoma cells will be screened in an ELISA for reactivity against PROl 81 or PR0237 Determination of positive' hybndoma cells secreting the desired monoclonal antibodies against PROl 81 or PR0237 is within the skill in the art
The positive hybndoma cells can be injected lntrapeπtoneally into syngeneic Balb/c mice to produce ascites containing the anti-PROl 81 or anti PR0237 monoclonal antibodies Alternatively, the hybndoma 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 8 Purification of PROl 81 or PRQ237 Polypeptides Using Specific Antibodies
Native or recombinant PROl 81 or PR0237 polypeptides may be purified by a variety of standard techniques in the art of protein purification For example, pro-PR0181 or pro PR0237 polypeptide, mature PRO 181 or mature PR0237 polypeptide, or pre PRO 181 or pre-PR0237 polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PROl 81 or PR0237 polypeptide of interest In general, an immunoaffinity column is constructed by covalently coupling the anti-PROl 81 or antι-PR0237 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 from mouse ascites fluid by ammonium sultate 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 PROl 81 or PR0237 polypeptide by preparing a fraction from cells containing the PROl 81 or PR0237 polypeptide in a soluble form This preparation is derived by solubihzation of the whole cell or of a subcellular fraction obtained v la differential centrifugation by the addition of detergent or by other methods well known in the art Alternatively soluble PROl 81 or PR0237 polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown
A soluble PROl 81 or PR0237 polypeptide containing pieparation is passed over the lmmunoatfmitv column, and the column is washed under conditions that allow the preferential absorbance of the PROl 81 or
PR0237 polypeptide (e g , high ionic strength buffers in the piesence ot deteigent) Then, the column is eluted under conditions that disrupt antibody/PROl 81 or antιbody/PR0237 polypeptide binding (e ? , 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 81 or PR0237 polypeptide is collected
EXAMPLE 9 Drug Screening This invention is particularly useful for screening compounds by using PRO 181 or PR0237 polypeptides or a binding fragment thereof in any of a variety of drug screening techniques The PROl 81 or PR0237 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 PROl 81 or PR0237 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 81 or PR0237 polypeptide or a fragment and the agent being tested Alternatively, one can examine the diminution in complex formation between the PROl 81 or PR0237 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 81 or PR0237 polypeptide-associated disease or disorder These methods comprise contacting such an agent with a PROl 81 or PR0237 polypeptide or fragment thereof and assaying (I) for the presence of a complex between the agent and the PROl 81 or PR0237 polypeptide or fragment, or (π) for the presence of a complex between the PROl 81 or PR0237 polypeptide or fragment and the cell, by methods well known in the art In such competitive binding assays, the PROl 81 or PR0237 polypeptide or fragment is typically labeled After suitable incubation, the free PRO 181 or PR0237 polypeptide or fragment is separated from that present in bound form and the amount of tree or uncomplexed label is a measure of the ability of the particular agent to bind to the PROl 81 or PR0237 polypeptide or to interfere with the PROl 81 or PR0237 polypeptide/cell complex
Another technique for drug screening provides high throughput screening tor 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 81 or PR0237 polypeptide, the peptide test compounds are reacted with the PROl 81 or PR0237 polypeptide and washed Bound PROl 81 or PR0237 polypeptide is detected by methods well known in the art Purified PRO 181 or PR0237 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 PROl 81 or PR0237 polypeptide specifically compete with a test compound for binding to the PROl 81 or PR0237 polypeptide or fragments thereof In this manner, the antibodies can be used to detect the presence ot any peptide which shares one oi more antigenic detei minants with a PRO 181 oi PR0237 polypeptide EXAMPLE 10 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 81 or PR0237 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 81 or PR0237 polypeptide or which enhance or interfere with the function of the PROl 81 or PR0237 polypeptide in vivo (cf, Hodgson, Bio/Technology, 9 19-21 (1991 ))
In one approach, the three-dimensional structure of the PRO 181 or PR0237 polypeptide. or of a PROl 81 or PR0237 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 81 or PR0237 polypeptide must be ascertained to elucidate the structure and to determine active sιte(s) of the molecule Less often, useful information regarding the structure of the PRO 181 or PR0237 polypeptide may be gained by modeling based on the structure of homologous proteins In both cases, relevant structural information is used to design analogous PRO 181 or PR0237 polypeptide-hke 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 . 1 13 742-746 (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 minor 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 PR0181 or PR0237 polypeptide may be made available to perform such analytical studies as X-ray crystallography In addition, knowledge ot the PROl 81 or PR0237 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 1 1 In Vitio Antitumor Assay
The antiprohferative activity of the PR0181 and PR0237 polypeptides was determined in the investigational, disease oriented in vitio anti-cancer drug discoveiy 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 1 107-1 1 12 (1990) The 60 tumoi cell lines employed in this study ( 'the NCI panel ), as well as conditions tor their maintenance and culture in viti o have been described by Monks etal J Natl Cancel Inst 83 757 766 ( 1991 ) The purpose of this screen is to initially evaluate the cvtotoxic and/or cytostatic activ ity ot the test compounds against different types ot tumors (Monks et al , supi Boyd, Cancer Pπnc Pract Oncol L pdate. 3( 10) 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 40°C. 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 Davs Tumor Cell Line Type Cell Line Designation
PRO I 81 122 nM 2 Leukemia RPMI-8226
PR0181 122 nM 2 NSCL NCI-H226; NCI-H522
PRO l 81 122 nM 2 Melanoma MALME-3M; SK-MEL-5
PR01 I 122 nM 2 Ovarian OVCAR-4
PRO l 81 122 nM 2 Breast NCI/ADR-RES
PR0181 122 nM 6 Leukemia MOLT-4
PRO l 81 122 nM 6 NSCL NCI-H226*
PR0181 122 nM 6 CNS SNB- 19
PR0181 122 nM 6 Ovarian OVCAR-3; OVCAR-8
PR0181 122 nM 6 Renal A498
PR0181 122 nM 6 Breast MDA-MB-231 /ATCC; MDA-N
PR0181 122 nM 6 Melanoma LOX IMVI
PRO l 81 165 nM 6 Leukemia CCRF-CEM; RPMI-8226*
PRO 181 165 nM 6 NSCL HOP-62
PR0181 165 nM 6 Leukemia HL-60 (TB)
PR0237 0.2 nM 6 Leukemia K-562
PR0237 0.2 nM 6 NSCL NCI-H322M
PR0237 0.2 nM 6 Colon HCC-2998; HCT-15
PR0237 0.02 nM 6 Colon KM I 2
PR0237 0.02 nM 6 Prostate DU- 145
PR0237 0.02 nM 6 Breast MDA-N
cytotoxic
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 DNA23330-1390 209775 April 14, 1998
DNA34353-1428 209855 May 12, 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, Ine , 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 gi anted 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 inv ention The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice ot 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 tiom the foregoing description and fall ithin 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 81 or PR0237 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 181 or PR0237 polypeptide, or an agonist thereof
3 The composition of matter of Claim 1 compπsing a cytotoxic amount of a PRO 181 or PR0237 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 comprising a therapeutically effective amount of a PROl 81 or PR0237 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 ot a tumor cell comprising exposing said tumor cell to an effective amount of a PRO 181 or PR0237 polypeptide or an agonist thereof
9 The method of Claim 8 wherein said agonist is an anti PRO 181 or antι-PR0237 agonist antibody
10 The method of Claim 8, wherein said agonist is a small molecule mimicking the biological activity of a PROl 81 or PR0237 polypeptide
11 The method of Claim 8, wherein sa d step of exposing occurs in viti o
12 The method of Claim 8, wherein said step of exposing occurs in vivo
13 An article ot manufacture comprising a container, and a composition comprising an active agent contained within the container, wherein said activ e agent in the composition is a PROl 81 or PR0237 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 PR0181 or antι-PR0237 agonist antibody
16 The article of manufacture of Claim 13, wherein said agonist is a small molecule mimicking the biological activity of a PROl 81 or PR0237 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 6)
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 1 ) and Figure 3 (SEQ ID NO 5)
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 1 ) and Figure 3 (SEQ ID NO 5)
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 209775 oi 209855
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 £ 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 PROl 81 or PR0237 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 6)
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 6)
33 An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence encoded by the full-length coding sequence ot the DNA deposited under ATCC accession number 209775 or 209855
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 ot 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:6), 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:6), 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:6), 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:6), 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:6), 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:6), lacking its associated signal peptide.
PCT/US1999/031243 1997-03-31 1999-12-30 Methods and compositions for inhibiting neoplastic cell growth Ceased WO2000053751A1 (en)

Priority Applications (241)

Application Number Priority Date Filing Date Title
AU23993/00A AU2399300A (en) 1999-03-08 1999-12-30 Methods and compositions for inhibiting neoplastic cell growth
PCT/US2000/000277 WO2000053754A1 (en) 1999-03-08 2000-01-06 Compositions and methods for the treatment of tumor
AU22248/00A AU2224800A (en) 1999-03-08 2000-01-06 Compositions and methods for the treatment of tumor
EP00907314A EP1263948A2 (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/004341 WO2000053756A2 (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002361840A CA2361840A1 (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2000603377A JP2004513602A (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding them
KR1020017011399A KR20030002292A (en) 1999-03-08 2000-02-18 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
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
CA002494705A CA2494705A1 (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
CA002491610A CA2491610A1 (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
CA002492049A CA2492049A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000587A EP1690872A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
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
EP06000584A EP1669371A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
AU20554/01A AU2055401A (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
EP05025102A EP1672070A3 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000588A EP1690873A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
EP10005292A EP2228446A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptieds and nucleic acids encoding the same
EP06000583A EP1686134A3 (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
EP06000581A EP1666494A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002391455A CA2391455A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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
EP00983846A EP1250426A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding same
CA002490853A CA2490853A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/918,585 US20030060406A1 (en) 1997-10-17 2001-07-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,295 US20020156006A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,189 US6972325B2 (en) 1997-10-17 2001-10-15 PRO273 polypeptides
US09/978,298 US20030134785A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,191 US20030050239A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,188 US20030139328A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,192 US20020177553A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,299 US20030199435A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,194 US20030195333A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,193 US20030073624A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,403 US20030050240A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,585 US20030049633A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,375 US7196165B2 (en) 1997-10-17 2001-10-16 PRO363 polypeptides
US09/978,681 US20030195148A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,564 US7195760B2 (en) 1997-10-17 2001-10-16 Anti-pro363 antibodies
US09/978,665 US7294700B2 (en) 1997-10-17 2001-10-16 Anti-PRO846 antibodies
US09/981,915 US20030054986A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,423 US20030069178A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,643 US20030104998A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,824 US20050124789A9 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,697 US20020169284A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,757 US20030083248A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,802 US20030199674A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,544 US20030199436A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/981,915 US7285623B2 (en) 1997-10-17 2001-10-16 PRO337 polypeptides
US09/978,824 US20030055216A1 (en) 1997-10-17 2001-10-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,388 US20040223964A1 (en) 1998-03-17 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/210,028 US20030203446A1 (en) 1998-10-07 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,089 US7208575B2 (en) 1998-10-07 2001-10-19 PRO531 polypeptides
US10/145,017 US20030186365A1 (en) 1998-03-26 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,749 US20040029218A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,067 US7279553B2 (en) 1998-05-13 2001-10-19 PRO1083 polypeptides
US10/166,709 US20030104536A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,829 US20030194780A1 (en) 1998-04-29 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,247 US7112657B2 (en) 1998-10-07 2001-10-19 PRO697 polypeptides
US10/143,029 US7105640B2 (en) 1997-10-17 2001-10-19 Anti-pro792 antibodies
US10/164,728 US20030186368A1 (en) 1998-05-13 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/162,521 US7067628B2 (en) 1998-03-17 2001-10-19 PRO788 polypeptides
US10/145,124 US20030190701A1 (en) 1998-04-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,502 US7220835B2 (en) 1998-07-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,929 US20030194781A1 (en) 1998-03-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,036 US20050227342A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,922 US20030195345A1 (en) 1997-10-17 2001-10-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/002,967 US20030148373A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,830 US20030077700A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,832 US20020192706A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,833 US6916648B2 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,086 US7122375B2 (en) 1997-10-17 2001-10-24 PRO274 nucleic acids
US10/017,081 US20030049684A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,834 US20030064407A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/020,445 US20030198994A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,084 US20030203402A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,083 US20030148376A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,832 US7132283B2 (en) 1997-10-17 2001-10-24 PRO273 polypeptides
US10/017,085 US6974696B2 (en) 1997-10-17 2001-10-24 PRO853 nucleic acids
US09/999,829 US20030195344A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,921 US20030068648A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,927 US7189529B2 (en) 1997-10-17 2001-10-25 PRO792 nucleic acids
US10/013,928 US20030215905A1 (en) 1998-10-07 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,923 US7169912B2 (en) 1997-10-17 2001-10-25 PRO1017 nucleic acids
US10/013,925 US7037710B2 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/016,177 US20030073131A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,920 US20040006219A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,918 US20030211091A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,917 US7029874B2 (en) 1998-03-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,929 US7019124B2 (en) 1997-10-17 2001-10-25 PRO788 nucleic acids
US10/013,926 US7074593B2 (en) 1998-04-01 2001-10-25 PRO 703 nucleic acids
US09/990,456 US20020137890A1 (en) 1997-03-31 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/028,072 US20030004311A1 (en) 1997-06-18 2001-12-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,187 US20030096744A1 (en) 1997-10-17 2002-01-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/081,056 US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/119,480 US20040087769A1 (en) 1998-09-10 2002-04-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,044 US20030190717A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,047 US20030077778A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,045 US20030073210A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,042 US20030096386A1 (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
US10/121,046 US20030194791A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,051 US20030092147A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,040 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
US10/121,043 US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/121,053 US20030199053A1 (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
US10/121,056 US20030082760A1 (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
US10/121,062 US20030077779A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,063 US20030199055A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,049 US20030022239A1 (en) 1997-06-18 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,052 US20030199052A1 (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
US10/121,061 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
US10/121,058 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
US10/121,055 US20030190718A1 (en) 1997-03-31 2002-04-12 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
US10/123,261 US20030068796A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,156 US20030194792A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,157 US20030190725A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,236 US20030068795A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,109 US20030190723A1 (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
US10/123,213 US7193048B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,771 US20030199060A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,108 US7635478B2 (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
US10/123,214 US7343721B2 (en) 1997-03-31 2002-04-15 PRO4406 polypeptide
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,322 US20030199059A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,155 US20030068794A1 (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
US10/123,292 US20030073211A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,262 US20030049816A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,291 US20030199058A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,905 US7285625B2 (en) 1997-06-18 2002-04-16 PRO536 polypeptides
US10/123,911 US7408032B2 (en) 1997-03-31 2002-04-16 PRO1188 polypeptides
US10/123,913 US20030203462A1 (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
US10/123,907 US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,906 US20030190726A1 (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
US10/123,908 US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,905 US20030087344A1 (en) 1997-06-18 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,902 US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,903 US20030073212A1 (en) 1997-03-31 2002-04-16 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
US10/124,814 US7105335B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,820 US20030190729A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,813 US7312307B2 (en) 1997-03-31 2002-04-17 PRO1056 polypeptides
US10/124,819 US7285626B2 (en) 1997-03-31 2002-04-17 PRO1076 polypeptides
US10/124,818 US20030082763A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,704 US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
US10/124,822 US7109305B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,821 US20030199023A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,823 US20030199062A1 (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
US10/125,805 US20030194794A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,795 US7304131B2 (en) 1997-03-31 2002-04-17 PRO1483 polypeptides
US10/124,824 US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,922 US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/125,924 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
US10/131,823 US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 polypeptides
US10/131,817 US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
US10/131,825 US7282566B2 (en) 1997-03-31 2002-04-24 PRO1779 polypeptide
US10/137,867 US20030207349A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,868 US20030082764A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,865 US20030032155A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,980 US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/140,024 US20040058424A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,023 US20030207416A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,470 US20030022331A1 (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
US10/140,474 US20030032156A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,963 US7288625B2 (en) 1997-03-31 2002-05-06 PRO4395 polypeptides
US10/140,809 US20030207418A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,925 US20030073215A1 (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
US10/140,921 US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
US10/140,864 US20030207419A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,808 US7425621B2 (en) 1997-03-31 2002-05-07 Antibodies against the PRO4401 polypeptide
US10/140,805 US20030207417A1 (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
US10/140,865 US20030207420A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,754 US7361732B2 (en) 1997-03-31 2002-05-08 PRO4400 polypeptides
US10/141,701 US20030207421A1 (en) 1997-03-31 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,756 US7488586B2 (en) 1997-03-31 2002-05-08 PRO4409 polypeptides
US10/141,760 US7342104B2 (en) 1997-03-31 2002-05-08 Antibodies against the PRO4320 polypeptide
US10/141,755 US7297764B2 (en) 1997-03-31 2002-05-08 PRO4318 polypeptides
US10/142,417 US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/142,425 US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,114 US20030036180A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,113 US7329730B2 (en) 1997-03-31 2002-05-09 PRO4348 polypeptides
US10/142,430 US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 polypeptides
US10/142,419 US7153941B2 (en) 1997-03-31 2002-05-10 Antibodies that bind PRO4994 polypeptides
US10/142,431 US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/142,423 US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,730 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
US10/147,519 US20030077791A1 (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
US10/147,536 US20040077064A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,395 US7189534B2 (en) 1997-03-31 2002-05-21 PRO4320 polynucleotide
US10/153,934 US20030129695A1 (en) 1997-03-31 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,843 US20030207805A1 (en) 1997-06-18 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,782 US20030077792A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,786 US20030208055A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,498 US20030073216A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,791 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
US10/176,913 US20030022298A1 (en) 1997-09-15 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2005264293A JP2006068016A (en) 1999-12-01 2005-08-15 Secreted and transmembrane polypeptides and nucleic acids encoding them
US11/341,175 US7468427B2 (en) 1997-03-31 2006-01-27 Antibodies to PRO1275 polypeptide
US11/786,466 US20080182275A1 (en) 1998-12-22 2007-04-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007325484A JP2008148699A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids encoding them
JP2007326609A JP2008148701A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids encoding them
JP2007326424A JP2008167749A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids encoding them
JP2007326613A JP2008161190A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids encoding them

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
USPCT/US99/05028 1999-03-08
PCT/US1999/005028 WO1999046281A2 (en) 1998-03-10 1999-03-08 Novel polypeptides and nucleic acids encoding the same
US12677399P 1999-03-29 1999-03-29
US60/126,773 1999-03-29
US14475899P 1999-07-20 1999-07-20
US60/144,758 1999-07-20
PCT/US1999/020594 WO2000015666A2 (en) 1998-09-10 1999-09-08 Compositions and methods for the treatment of tumors
USPCT/US99/20594 1999-09-08
USPCT/US99/30999 1999-12-20
PCT/US1999/030999 WO2001005836A1 (en) 1999-07-20 1999-12-20 Polypeptidic compositions and methods for the treatment of tumors

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/030095 Continuation-In-Part WO2000037640A2 (en) 1994-09-08 1999-12-16 Compositions and methods for the treatment of tumor

Related Child Applications (3)

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
US10/081,056 Continuation US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/119,480 Continuation US20040087769A1 (en) 1998-09-10 2002-04-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same

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WO2002008284A3 (en) * 2000-07-20 2003-03-13 Genentech Inc Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
WO2002000690A3 (en) * 2000-06-23 2003-03-13 Genentech Inc Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis

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WO2002008284A3 (en) * 2000-07-20 2003-03-13 Genentech Inc Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis

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