WO2000055319A1 - 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
WO2000055319A1
WO2000055319A1 PCT/US1999/028564 US9928564W WO0055319A1 WO 2000055319 A1 WO2000055319 A1 WO 2000055319A1 US 9928564 W US9928564 W US 9928564W WO 0055319 A1 WO0055319 A1 WO 0055319A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
polypeptide
sequence identity
Prior art date
Application number
PCT/US1999/028564
Other languages
French (fr)
Inventor
Avi J. Ashkenazi
Audrey Goddard
Austin L. Gurney
Robert D. Klein
Mary A. Napier
William I. Wood
Jean Yuan
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/US1999/020594 external-priority patent/WO2000015666A2/en
Priority claimed from PCT/US1999/021090 external-priority patent/WO2000015796A2/en
Priority claimed from PCT/US1999/023089 external-priority patent/WO2000021996A2/en
Priority claimed from PCT/US1999/028313 external-priority patent/WO2000032221A2/en
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to AU17498/00A priority Critical patent/AU1749800A/en
Priority to AU23907/00A priority patent/AU2390700A/en
Priority to PCT/US1999/030999 priority patent/WO2001005836A1/en
Priority to AU25967/00A priority patent/AU2596700A/en
Priority to PCT/US2000/000219 priority patent/WO2000053753A2/en
Priority to AU22248/00A priority patent/AU2224800A/en
Priority to PCT/US2000/000277 priority patent/WO2000053754A1/en
Priority to PCT/US2000/004414 priority patent/WO2001004311A1/en
Priority to AU28839/00A priority patent/AU2883900A/en
Priority to PCT/US2000/005841 priority patent/WO2000053758A2/en
Priority to CA002362427A priority patent/CA2362427A1/en
Priority to AU35144/00A priority patent/AU3514400A/en
Priority to EP00913764A priority patent/EP1220905A2/en
Priority to KR1020017011406A priority patent/KR20010103046A/en
Priority to JP2000603379A priority patent/JP2004516227A/en
Publication of WO2000055319A1 publication Critical patent/WO2000055319A1/en
Priority to EP06000587A priority patent/EP1690872A3/en
Priority to CA002491610A priority patent/CA2491610A1/en
Priority to EP05025102A priority patent/EP1672070A3/en
Priority to EP06000588A priority patent/EP1690873A3/en
Priority to CA002491258A priority patent/CA2491258A1/en
Priority to AU20554/01A priority patent/AU2055401A/en
Priority to EP10005292A priority patent/EP2228446A1/en
Priority to CA002490909A priority patent/CA2490909A1/en
Priority to PCT/US2000/032678 priority patent/WO2001040466A2/en
Priority to EP06000582A priority patent/EP1666495A1/en
Priority to JP2001542531A priority patent/JP2004522404A/en
Priority to EP00983846A priority patent/EP1250426A2/en
Priority to EP06000583A priority patent/EP1686134A3/en
Priority to EP06000584A priority patent/EP1669371A3/en
Priority to CA002492049A priority patent/CA2492049A1/en
Priority to EP06000581A priority patent/EP1666494A1/en
Priority to CA2709291A priority patent/CA2709291A1/en
Priority to EP06000585A priority patent/EP1661996A1/en
Priority to CA002494705A priority patent/CA2494705A1/en
Priority to EP06000586A priority patent/EP1688497A1/en
Priority to CA002496312A priority patent/CA2496312A1/en
Priority to CA002391455A priority patent/CA2391455A1/en
Priority to CA002491433A priority patent/CA2491433A1/en
Priority to CA002490853A priority patent/CA2490853A1/en
Priority to EP06000589A priority patent/EP1661997A1/en
Priority to CA002492070A priority patent/CA2492070A1/en
Priority to US09/828,366 priority patent/US20020010137A1/en
Priority to US09/902,634 priority patent/US20030082540A1/en
Priority to US09/902,692 priority patent/US20030054400A1/en
Priority to US09/902,979 priority patent/US20030113718A1/en
Priority to US09/902,572 priority patent/US20030108983A1/en
Priority to US09/902,615 priority patent/US20030092002A1/en
Priority to US09/902,903 priority patent/US20030044839A1/en
Priority to US09/902,713 priority patent/US20030082541A1/en
Priority to US09/902,736 priority patent/US20030049676A1/en
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Priority to US09/903,786 priority patent/US20030044793A1/en
Priority to US09/903,603 priority patent/US20030148419A1/en
Priority to US09/904,011 priority patent/US20030003530A1/en
Priority to US09/903,806 priority patent/US20030130489A1/en
Priority to US09/904,119 priority patent/US20030049621A1/en
Priority to US09/903,823 priority patent/US20030104381A1/en
Priority to US09/903,562 priority patent/US6965015B2/en
Priority to US09/903,520 priority patent/US20030054401A1/en
Priority to US09/903,749 priority patent/US7147853B2/en
Priority to US09/903,640 priority patent/US7208308B2/en
Priority to US09/903,943 priority patent/US20030054349A1/en
Priority to US09/903,925 priority patent/US20030096233A1/en
Priority to US09/904,766 priority patent/US20030152999A1/en
Priority to US09/904,877 priority patent/US20030186358A1/en
Priority to US09/904,859 priority patent/US20030036060A1/en
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Priority to US09/905,088 priority patent/US20030073077A1/en
Priority to US09/905,291 priority patent/US20020160374A1/en
Priority to US09/905,125 priority patent/US6664376B2/en
Priority to US09/905,056 priority patent/US20030054441A1/en
Priority to US09/904,992 priority patent/US20030135025A1/en
Priority to US09/904,786 priority patent/US7557192B2/en
Priority to US09/904,532 priority patent/US7151160B2/en
Priority to US09/904,462 priority patent/US6878807B2/en
Priority to US09/904,485 priority patent/US20030064367A1/en
Priority to US09/904,553 priority patent/US20030059828A1/en
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Priority to US09/904,820 priority patent/US20030036094A1/en
Priority to US09/904,838 priority patent/US20030148370A1/en
Priority to US09/905,075 priority patent/US7169906B2/en
Priority to US09/904,920 priority patent/US6806352B2/en
Priority to US09/905,381 priority patent/US6818746B2/en
Priority to US09/905,449 priority patent/US6965011B2/en
Priority to US09/904,956 priority patent/US20030049622A1/en
Priority to US09/906,760 priority patent/US20030096340A1/en
Priority to US09/906,646 priority patent/US6852848B2/en
Priority to US09/906,722 priority patent/US6946262B2/en
Priority to US09/906,700 priority patent/US6723535B2/en
Priority to US09/906,742 priority patent/US20030023054A1/en
Priority to US09/906,815 priority patent/US7094567B2/en
Priority to US09/906,838 priority patent/US7070979B2/en
Priority to US09/906,777 priority patent/US20030148371A1/en
Priority to US09/906,618 priority patent/US6828146B2/en
Priority to US09/907,824 priority patent/US20020197671A1/en
Priority to US09/907,794 priority patent/US6635468B2/en
Priority to US09/907,942 priority patent/US7087738B2/en
Priority to US09/907,613 priority patent/US20030027145A1/en
Priority to US09/907,728 priority patent/US20030190611A1/en
Priority to US09/908,093 priority patent/US20030017498A1/en
Priority to US09/907,575 priority patent/US20030073079A1/en
Priority to US09/907,979 priority patent/US20030082542A1/en
Priority to US09/907,925 priority patent/US20030054352A1/en
Priority to US09/907,652 priority patent/US20030104469A1/en
Priority to US09/907,841 priority patent/US7033825B2/en
Priority to US09/909,088 priority patent/US20020146709A1/en
Priority to US09/909,064 priority patent/US6818449B2/en
Priority to US09/908,576 priority patent/US20040005553A1/en
Priority to US09/909,204 priority patent/US20030036061A1/en
Priority to US09/909,320 priority patent/US7074592B2/en
Priority to US09/990,456 priority patent/US20020137890A1/en
Priority to US10/028,072 priority patent/US20030004311A1/en
Priority to US10/081,056 priority patent/US20040043927A1/en
Priority to US10/121,044 priority patent/US20030190717A1/en
Priority to US10/121,047 priority patent/US20030077778A1/en
Priority to US10/121,059 priority patent/US20030190721A1/en
Priority to US10/121,041 priority patent/US20030077776A1/en
Priority to US10/121,040 priority patent/US20030082759A1/en
Priority to US10/121,045 priority patent/US20030073210A1/en
Priority to US10/121,042 priority patent/US20030096386A1/en
Priority to US10/121,051 priority patent/US20030092147A1/en
Priority to US10/121,046 priority patent/US20030194791A1/en
Priority to US10/121,054 priority patent/US20030199054A1/en
Priority to US10/121,061 priority patent/US20030082761A1/en
Priority to US10/121,049 priority patent/US20030022239A1/en
Priority to US10/121,062 priority patent/US20030077779A1/en
Priority to US10/121,058 priority patent/US20030190720A1/en
Priority to US10/121,055 priority patent/US20030190718A1/en
Priority to US10/121,060 priority patent/US20030190722A1/en
Priority to US10/121,063 priority patent/US20030199055A1/en
Priority to US10/121,056 priority patent/US20030082760A1/en
Priority to US10/121,048 priority patent/US20030199051A1/en
Priority to US10/121,052 priority patent/US20030199052A1/en
Priority to US10/121,050 priority patent/US20030054516A1/en
Priority to US10/121,053 priority patent/US20030199053A1/en
Priority to US10/121,043 priority patent/US7220831B2/en
Priority to US10/121,057 priority patent/US20030190719A1/en
Priority to US10/123,155 priority patent/US20030068794A1/en
Priority to US10/123,292 priority patent/US20030073211A1/en
Priority to US10/123,215 priority patent/US7291329B2/en
Priority to US10/123,157 priority patent/US20030190725A1/en
Priority to US10/123,291 priority patent/US20030199058A1/en
Priority to US10/123,156 priority patent/US20030194792A1/en
Priority to US10/123,236 priority patent/US20030068795A1/en
Priority to US10/123,213 priority patent/US20030199057A1/en
Priority to US10/123,212 priority patent/US7276577B2/en
Priority to US10/123,108 priority patent/US7635478B2/en
Priority to US10/123,322 priority patent/US20030199059A1/en
Priority to US10/123,109 priority patent/US20030190723A1/en
Priority to US10/123,213 priority patent/US7193048B2/en
Priority to US10/123,261 priority patent/US20030068796A1/en
Priority to US10/123,235 priority patent/US20030082762A1/en
Priority to US10/123,262 priority patent/US20030049816A1/en
Priority to US10/123,771 priority patent/US20030199060A1/en
Priority to US10/123,214 priority patent/US7343721B2/en
Priority to US10/123,154 priority patent/US20030190724A1/en
Priority to US10/123,909 priority patent/US7193049B2/en
Priority to US10/123,906 priority patent/US20030190726A1/en
Priority to US10/123,912 priority patent/US20030100087A1/en
Priority to US10/123,907 priority patent/US7084258B2/en
Priority to US10/123,913 priority patent/US20030203462A1/en
Priority to US10/123,905 priority patent/US20030087344A1/en
Priority to US10/123,905 priority patent/US7285625B2/en
Priority to US10/123,902 priority patent/US20030077781A1/en
Priority to US10/123,910 priority patent/US7329404B2/en
Priority to US10/123,908 priority patent/US7335728B2/en
Priority to US10/123,903 priority patent/US20030073212A1/en
Priority to US10/123,904 priority patent/US20030022328A1/en
Priority to US10/123,911 priority patent/US7408032B2/en
Priority to US10/124,824 priority patent/US20030077659A1/en
Priority to US10/124,823 priority patent/US20030199062A1/en
Priority to US10/124,816 priority patent/US20030190728A1/en
Priority to US10/125,704 priority patent/US7357926B2/en
Priority to US10/124,818 priority patent/US20030082763A1/en
Priority to US10/124,822 priority patent/US7109305B2/en
Priority to US10/124,814 priority patent/US7105335B2/en
Priority to US10/125,805 priority patent/US20030194794A1/en
Priority to US10/124,820 priority patent/US20030190729A1/en
Priority to US10/124,817 priority patent/US20030077786A1/en
Priority to US10/124,819 priority patent/US7285626B2/en
Priority to US10/124,821 priority patent/US20030199023A1/en
Priority to US10/124,813 priority patent/US7312307B2/en
Priority to US10/125,795 priority patent/US7304131B2/en
Priority to US10/125,931 priority patent/US20030199063A1/en
Priority to US10/125,932 priority patent/US7317079B2/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 US10/127,831 priority patent/US20030082689A1/en
Priority to US10/128,689 priority patent/US20030087365A1/en
Priority to US10/131,825 priority patent/US7282566B2/en
Priority to US10/131,823 priority patent/US7304132B2/en
Priority to US10/131,817 priority patent/US7291701B2/en
Priority to US10/137,868 priority patent/US20030082764A1/en
Priority to US10/137,865 priority patent/US20030032155A1/en
Priority to US10/137,867 priority patent/US20030207349A1/en
Priority to US10/140,024 priority patent/US20040058424A1/en
Priority to US10/140,023 priority patent/US20030207416A1/en
Priority to US10/140,020 priority patent/US20030207415A1/en
Priority to US10/140,470 priority patent/US20030022331A1/en
Priority to US10/140,474 priority patent/US20030032156A1/en
Priority to US10/139,980 priority patent/US7247710B2/en
Priority to US10/139,963 priority patent/US7288625B2/en
Priority to US10/140,808 priority patent/US7425621B2/en
Priority to US10/140,805 priority patent/US20030207417A1/en
Priority to US10/140,921 priority patent/US7317080B2/en
Priority to US10/140,860 priority patent/US7307151B2/en
Priority to US10/140,864 priority patent/US20030207419A1/en
Priority to US10/140,928 priority patent/US20030068798A1/en
Priority to US10/140,865 priority patent/US20030207420A1/en
Priority to US10/140,809 priority patent/US20030207418A1/en
Priority to US10/140,925 priority patent/US20030073215A1/en
Priority to US10/141,754 priority patent/US7361732B2/en
Priority to US10/141,756 priority patent/US7488586B2/en
Priority to US10/141,760 priority patent/US7342104B2/en
Priority to US10/141,701 priority patent/US20030207421A1/en
Priority to US10/141,755 priority patent/US7297764B2/en
Priority to US10/142,417 priority patent/US7304133B2/en
Priority to US10/142,425 priority patent/US20030207424A1/en
Priority to US10/143,113 priority patent/US7329730B2/en
Priority to US10/142,430 priority patent/US7309766B2/en
Priority to US10/143,114 priority patent/US20030036180A1/en
Priority to US10/142,431 priority patent/US7285629B2/en
Priority to US10/142,419 priority patent/US7153941B2/en
Priority to US10/142,423 priority patent/US20030049817A1/en
Priority to US10/143,032 priority patent/US7408033B2/en
Priority to US10/146,792 priority patent/US20030207428A1/en
Priority to US10/146,730 priority patent/US20030207427A1/en
Priority to US10/147,528 priority patent/US20030219885A1/en
Priority to US10/147,519 priority patent/US20030077791A1/en
Priority to US10/147,536 priority patent/US20040077064A1/en
Priority to US10/147,492 priority patent/US20030082765A1/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 US10/158,782 priority patent/US20030082766A1/en
Priority to US10/160,498 priority patent/US20030073216A1/en
Priority to US10/158,791 priority patent/US20030207429A1/en
Priority to US10/176,913 priority patent/US20030022298A1/en
Priority to US10/771,187 priority patent/US7355002B2/en
Priority to US10/970,823 priority patent/US7307152B2/en
Priority to JP2005264293A priority patent/JP2006068016A/en
Priority to US11/341,175 priority patent/US7468427B2/en
Priority to US11/518,609 priority patent/US20070077623A1/en
Priority to JP2007326424A priority patent/JP2008167749A/en
Priority to JP2007326609A priority patent/JP2008148701A/en
Priority to JP2007325484A priority patent/JP2008148699A/en
Priority to JP2007326613A priority patent/JP2008161190A/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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.
  • Malignant tumors are the second leading cause of death in the United States, after heart disease (Boring et al, CA Cancel J. Clin., 43:7 (1993)).
  • Cancer is characterized by 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.
  • the present invention relates to methods and compositions for inhibiting neoplastic cell growth. More particularly, the invention concerns methods and compositions for the treatment of tumors, including cancers, such as breast, prostate, colon, lung, ovarian, renal and CNS cancers, leukemia, melanoma, etc., in mammalian patients, preferably humans.
  • cancers such as breast, prostate, colon, lung, ovarian, renal and CNS cancers, leukemia, melanoma, etc.
  • the present invention concerns compositions of matter useful for the inhibition of neoplastic cell growth comprising an effective amount of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide as herein defined, or an agonist thereof, in admixture with a pharmaceutically acceptable carrier.
  • the composition of matter comprises a growth inhibitory amount of a PR021 1 , PR0228, PR0538,
  • 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 m a mammal comprising a therapeutically effective amount of a PR021 1 , PR0228, PR0538, PR0172 or PRO 182 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide as herein defined, or an agonist thereof
  • the agonist is an ant ⁇ -PR021 1 , ant ⁇ -PR0228, ant ⁇ -PR0538, ant ⁇ -PR0172 or ant ⁇ -PR0182 agonist antibody
  • the agonist is a small molecule that mimics the biological activity of a PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide
  • the method may be performed in vitro or in vivo
  • 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 m the composition is a PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide as herein defined, or an agonist thereof, and
  • the agonist is an ant ⁇ -PR021 1 , ant ⁇ -PR0228, ant ⁇ -PR0538, anti-PROl 72 or anti-PROl 82 agonist antibody
  • the agonist is a small molecule that mimics the biological activity of a PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide
  • Similar articles of manufacture compnsmg a PR0211 , PR0228, PR0538, PRO 172 or PRO 182 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.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide
  • the isolated nucleic acid molecule comp ⁇ ses 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 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
  • the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at
  • Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide are disclosed herein. Therefore, soluble extracellular domains ofthe herein described PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptides are contemplated.
  • Another embodiment is directed to fragments of a PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PR021 1 , anti-PR0228, anti-PR0538, anti-PROl 72 or anti-PROl 82 antibody or as antisense oligonucleotide probes.
  • nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 60 nucleotides in length, yet more preferably at least about 70 nucleotides in length, yet more preferably at least about 80 nucleotides in length, yet more preferably at least about 90 nucleotides in length, yet more preferably at least about 100 nucleotides in length, yet more preferably at least about 1 10 nucleotides in length, yet more preferably at least about 120 nucleotides in length, yet more preferably at least about 130 nucleotides in length, yet more preferably at least about 140 nucleotides in length, yet more preferably at least about 150 nucleotides in length, yet more preferably at least about 160 nucleotides in length, yet more preferably at least about 170 nucleo
  • novel fragments of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide-encoding nucleotide sequence fragment(s) are novel.
  • PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PR0211 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide fragments that comprise a binding site for an anti-PR021 1, anti-PR0228, anti-PR0538, anti-PR0172 or anti-PR0182 antibody.
  • the invention provides isolated PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide encoded by any ofthe isolated nucleic acid sequences hereinabove identified.
  • the invention concerns an isolated PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide, comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93%o 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
  • the invention concerns an isolated PR021 1 , PR0228, PR0538, PROl 72 or PRO 182 polypeptide comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85%) sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87%o 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%)
  • the invention concerns an isolated PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 81 % positives, more preferably at least about 82% positives, yet more preferably at least about 83% positives, yet more preferably at least about 84% positives, yet more preferably at least about 85% positives, yet more preferably at least about 86% positives, yet more preferably at least about 87% positives, yet more preferably at least about 88% positives, yet more preferably at least about 89%> positives, yet more preferably at least about 90%> positives, yet more preferably at least about 91 % positives, yet more preferably at least about 92% positives, yet more preferably at least about 93% positives, yet more preferably at least about 94% positives, yet more preferably at least about 95% positives, yet more preferably at least about 96% positives, yet more preferably at least about 97% positives
  • the invention provides an isolated PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described Processes for producing the same are also herein described, wherein those processes comprise cultu ⁇ ng a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide and recovering the PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 polypeptide from the cell culture
  • Another aspect ofthe invention provides an isolated PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated Processes foi producing the same are also herein described, wherein those processes comprise cultu ⁇ ng a host cell comprising a vector which comprises the approp ⁇ ate encoding nucleic acid molecule under conditions suitable for expression of the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide and recovering the PR021 1 PR0228, PR0538, PROl 72 or PROl 82 polypeptide from the cell culture
  • the invention concerns agonists of a native PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide as defined herein
  • the agonist is an ant ⁇ -PR021 1 , ant ⁇ -PR0228, ant ⁇ -PR0538, anti-PROl 72 or anti-PROl 82 antibody or a small molecule
  • the invention concerns a method of identifying agonists to a PR021 1 , PR0228
  • PR0538, PRO 172 or PRO 182 polypeptide which comprise contacting the PR021 1, PR0228, PR0538, PRO 172 or PROl 82 polypeptide with a candidate molecule and monitoring a biological activity mediated by said PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide
  • the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide is a native PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide
  • the invention concerns a composition of matter comprising a PR021 1 ,
  • PR0228, PR0538, PRO 172 or PRO 182 polypeptide or an agonist of a PR021 1 , PR0228, PR0538, PRO 172 or PR0182 polypeptide as herein described, or an ant ⁇ -PR021 1 , ant ⁇ -PR0228, ant ⁇ -PR0538, ant ⁇ -PRO! 72 or anti- PROl 82 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 PR021 1, PR0228, PR0538,
  • PRO 172 or PRO 182 polypeptide or an agomstthereofas hereinbefore described, or an ant ⁇ -PR021 1 , ant ⁇ -PR0228, ant ⁇ -PR0538, anti-PROl 72 or anti-PROl 82 antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide, an agonist thereof or an ant ⁇ -PR021 1, ant ⁇ -PR0228, ant ⁇ -PR0538, ant ⁇ -PR0172 or ant ⁇ -PR0182 antibody
  • the invention provides vectors comprising DNA encoding any of the herein described polypeptides
  • Host cell comprising any such vector are also provided
  • the host cells may be CHO cells, E colt, yeast, or Baculovirus-infected insect cells
  • a process for producing any ofthe herein described polypeptides is further provided and comp ⁇ ses culturing host cells under conditions suitable for expression ofthe desired polypeptide and recovering the desired polypeptide from the cell culture
  • the invention provides chimenc molecules comprising any ofthe herein described polypeptides fused to a heterologous polypeptide or amino acid sequence
  • Example of such chimenc molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin
  • the invention provides an antibody which specifically binds to any ofthe above or below described polypeptides
  • the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody
  • the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any ofthe above or below described nucleotide sequences
  • Figure 1 shows a nucleotide sequence (SEQ ID NO 1 ) of a native sequence PR021 1 cDN A, wherein SEQ ID NO 1 is a clone designated herein as "DN A32292- 1 131 "
  • 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
  • Figures 3A-3B show a nucleotide sequence (SEQ ID NO 6) of a native sequence PR0228 cDNA, wherem SEQ ID NO 6 is a clone designated herein as "DNA33092-1202"
  • Figure 4 shows the amino acid sequence (SEQ ID NO 7) derived from the coding sequence of SEQ ID NO 6
  • Figure 5 shows a nucleotide sequence (SEQ ID NO 15) of a native sequence PR0538 cDNA, wherein SEQ ID NO 15 is a clone designated herein as "DNA48613-1268"
  • Figure 6 shows the amino acid sequence (SEQ ID NO 16) derived from the coding sequence of SEQ ID NO 15 shown in Figure 5
  • Figures 7A-7B show a nucleotide sequence (SEQ ID NO 20) of a native sequence PRO 172 cDNA, wherein SEQ ID NO 20 is a clone designated herein as "DNA35916-1 161 "
  • Figure 8 shows the amino acid sequence (SEQ ID NO 21) derived from the coding sequence of SEQ ID NO 20 shown in Figures 7A-7B
  • Figure 9 shows a nucleotide sequence (SEQ ID NO 25) of a native sequence PRO 182 cDNA, wherein SEQ ID NO 25 is a clone designated herein as "DN A27865- 1091 "
  • Figure 10 shows the amino acid sequence (SEQ ID NO 26) derived from the coding sequence of SEQ ID NO 25 shown in Figure 9
  • PR0211 refers to native sequence PR021 1, PR0228, PR0538, PRO 172 and PROl 82 variants (which are further defined herein)
  • PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1 ", “native sequence PR0228”, “native sequence PR0538” “native sequence PRO 172" or “native sequence PROl 82” comprises a polypeptide having the same amino acid sequence as the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide as derived from nature Such native sequence PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide can be isolated from nature or can be produced by recombinant and/or synthetic means
  • the term “native sequence” PR021 1, PR0228, PR0538, PR0172 or PROl 82 specifically encompasses naturally-occurring truncated or secreted forms (e g , an extracellular domain sequence), naturally-occurring variant forms (e g , alternatively spliced forms) and naturally-occurring allelic variants of the PR021 1, PR0228, PR0538, PR0172 and PR0182 polypeptides
  • the ' extracellular domain" or "ECD" of a polypeptide disclosed herein refers to a form ofthe polypeptide which is essentially tree ofthe transmembrane and cytoplasmic domains
  • ECD extracellular domain
  • a polypeptide ECD will have less than about 1% of such transmembrane and/or cytoplasmic domains and preferably, will have less than about 0 5% of such domains
  • any transmembrane doma ⁇ n(s) identified for the polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain.
  • the extracellular domain of a polypeptide ofthe present invention comprises amino acids 1 to X ofthe mature amino acid sequence, wherein X is any amino acid within 5 amino acids on either side ofthe extracellular domain/transmembrane domain boundary.
  • 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.
  • PR021 1 variant polypeptide means an active PR021 1 polypeptide (other than a native sequence PR021 1 polypeptide) as defined below, having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 25 to 353 ofthe PR021 1 polypeptide shown in Figure 2 (SEQ ID NO:2), (b) X to 353 ofthe PR021 1 polypeptide shown in Figure 2 (SEQ ID NO:2), wherein X is any amino acid residue from 20 to 29 of Figure 2 (SEQ ID NO:2) or (c) another specifically derived fragment ofthe amino acid sequence shown in Figure 2 (SEQ ID NO:2).
  • PR0228 variant polypeptide means an active PR0228 polypeptide (other than a native sequence PR0228 polypeptide) as defined below, having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 20 to 690 ofthe PR0228 polypeptide shown in Figure 4 (SEQ ID NO:7), (b) X to 690 ofthe PR0228 polypeptide shown in Figure 4 (SEQ ID NO:7), wherein X is any amino acid residue from 15 to 24 of Figure 4 (SEQ ID NO:7), (c) 1 or about 20 to X of Figure 4 (SEQ ID NO:7), wherein X is any amino acid from amino acid 425 to amino acid 434 of Figure 4 (SEQ ID NO:7) or (d) another specifically derived fragment ofthe amino acid sequence shown in Figure 4 (SEQ ID NO:7).
  • PR0538 variant polypeptide means an active PR0538 polypeptide (other than a native sequence PR0538 polypeptide) as defined below, having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 27 to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO: 16), (b) X to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO: 16), wherein X is any amino acid residue from 22 to 31 of Figure 6 (SEQ ID NO: 16), (c) 1 or about 27 to X of Figure 6 (SEQ ID NO: 16), wherein X is any amino acid from amino acid 374 to amino acid 383 of Figure 6 (SEQ ID NO: 16) or (d) another specifically derived fragment ofthe amino acid sequence shown in Figure 6 (SEQ ID NO: 16).
  • PRO 172 variant polypeptide means an active PRO 172 polypeptide (other than a native sequence PR0172 polypeptide) as defined below, having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 22 to 723 ofthe PROl 72 polypeptide shown in Figure 8 (SEQ ID NO 21), (b) X to 723 ofthe PRO 172 polypeptide shown in Figure 8 (SEQ ID NO 21 ), wherein X is any amino acid residue from 17 to 26 of Figure 8 (SEQ ID NO 21), (c) 1 or about 22 to X of Figure 8 (SEQ ID NO 21), wherein X is any am o acid from amino acid 543 to amino acid 552 of Figure 8 (SEQ ID NO 21 ) or (d) another specifically derived fragment ofthe amino acid sequence shown in Figure 8 (SEQ ID NO 21 )
  • PRO 182 variant polypeptide means an active PRO 182 polypeptide (other than a native sequence PROl 82 polypeptide) as defined below, having at least about 80% ammo acid sequence identity with the amino acid sequence of (a) residues 1 or about 19 to 135 ofthe PRO 182 polypeptide shown in Figure 10 (SEQ ID NO 26), (b) X to 135 ofthe PROl 82 polypeptide shown in Figure 10 (SEQ ID NO 26), wherein X is any ammo acid residue from 14 to 23 of Figure 10 (SEQ ID NO 26) or (c) another specifically derived fragment ofthe amino acid sequence shown in Figure 10 (SEQ ID NO 26)
  • PR021 1 , PR0228, PR0538, PRO 172 and PRO 182 variants include, for instance, PR021 1 , PR0228, PR0538, PR0172 and PR0182 polypeptides wherein one or more am o acid residues are added, or deleted, at the N- or C-terminus, as well as within one or more internal domains ofthe native sequence
  • a PR021 1 variant will have at least about 80%> amino acid sequence identity, more preferably at least about 81 % amino acid sequence identity, more preferably at least about 82%o amino acid sequence identity, more preferably at least about 83%o 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%o 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
  • a PR0228 variant will have at least about 80% 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% ammo acid sequence identity, more preferably at least about 84% ammo acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% ammo acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% ammo 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%> am o acid sequence identity, more preferably at least about 92%> amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% ammo acid sequence
  • a PR0538 variant will have at least about 80% am o 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%.
  • am o 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%> am o acid sequence identity, more preferably at least about 88%> amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91%) amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% am o acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% ammo 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 99% amino acid sequence identity with (a) residues 1 or about 27 to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO 16),
  • a PROl 72 variant will have at least about 80% 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 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% ammo acid sequence identity, more preferably at least about 9
  • a PROl 82 variant will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% am o 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% am o 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% ammo 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% ammo acid sequence identity, more preferably at least about 97% amino acid sequence identity
  • PR021 1 , PR0228, PR0538, PR0172 and PR0182 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 am o acids in length, more often at least about 40 amino acids in length, more often at least about 50 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 ammo acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, more often at least about 250 ammo acids in length, more often at least about 300 amino acids in length, or more As shown below, Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program
  • Tables 2A-2D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid sequence identity (Tables 2C-2D) using the ALIGN-2 sequence comparison computer program, wherein "PRO” represents the amino acid sequence of a hypothetical PEACH 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 PROXXX- or PROXXX-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.
  • Max file length is 65535 (limited by unsigned short x in the jmp struct)
  • a sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
  • the program may create a tmp file in /t p to hold info about traceback
  • static nm, /* matches in core — for checking */ static lmax; /* lengths of stripped file names */ static ⁇ j[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static n.[2]; /* current elem number — for gapping */ static s ⁇ z[2]; static char *ps[2]; /* ptr to current element */ static char *po[2] /* ptr to next output char slot */ static char oouutt[[22]][[P_LINE]; /* output line */ static char starfP E]; /* set by stars() *//
  • */ *po[ ⁇ ] *ps[ ⁇ ] if ( ⁇ slower(*ps[ ⁇ ]))
  • *ps[ ⁇ ] toupper(*ps[ ⁇ ]), po[ ⁇ ] + + , ps[ ⁇ ] + + ,
  • *py++ *px; else if ( ⁇ slower(*px))
  • *py++ toupper(*px), if ( ⁇ ndex("ATGCU",*(py-l))) natgc + + ;
  • Percent (%) amino acid sequence identity with respect to the PR021 1 , PR0228, PR0538, PROl 72 and PROl 82 polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PR021 1 , PR0228, PR0538, PR0172 or PR0182 sequence, after a gningthe sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part ofthe sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill m the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) software Those skilled m the art can determine approp ⁇ ate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length ofthe sequences being compared For purposes herein, however, % amino acid sequence identity values are obtained
  • 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
  • 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
  • a %> amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence ofthe PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (t 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 ofthe PRO polypeptide of interest For example,
  • PR021 1 variant polynucleotide or "PR021 1 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PR021 1 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 25 to 353 ofthe PR021 1 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) a nucleic acid sequence which encodes amino acids X to 353 ofthe PR021 1 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any ammo acid residue from 20 to 29 of Figure 2 (SEQ ID NO 2) or(c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2)
  • a PR021 1 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81 % nucleic acid sequence identity
  • PR0228 variant polynucleotide or "PR0228 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PR0228 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 20 to 690 ofthe PR0228 polypeptide shown in Figure 4 (SEQ ID NO:7), (b) a nucleic acid sequence which encodes amino acids X to 690 ofthe PR0228 polypeptide shown in Figure 4 (SEQ ID NO:7), wherein X is any amino acid residue from 15 to 24 of Figure 4 (SEQ ID NO:7), (c) a nucleic acid sequence which encodes amino acids 1 or about 20 to X of Figure 4 (SEQ ID NO:7), wherein X is any amino acid from amino acid 425 to amino acid 434 of Figure 4 (SEQ ID NO:7) or (d) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence
  • a PR0228 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81%> nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84%> nucleic acid sequence identity, more preferably at least about 85%o nucleic acid sequence identity, more preferably at least about 86%o 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
  • PR0538 variant polynucleotide or "PR0538 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PR0538 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 27 to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO: 16), (b) a nucleic acid sequence which encodes amino acids X to 400 of the PR0538 polypeptide shown in Figure 6 (SEQ ID NO: 16), wherein X is any amino acid residue from 22 to 31 of Figure 6 (SEQ ID NO: 16), (c) a nucleic acid sequence which encodes amino acids 1 or about 27 to X of Figure 6 (SEQ ID NO:16), wherein X is any amino acid from amino acid 374 to amino acid 383 of Figure 6 (SEQ ID NO: 16) or (d) a nucleic acid sequence which encodes another specifically derived fragment ofthe amino acid sequence shown
  • a PR0538 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81 % nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86%o 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 is 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 27 to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO:16), (b) a nucleic acid sequence which encodes amino acids X to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO: 16), wherein X is any amino acid residue from 22 to 31 of Figure 6 (SEQ ID NO: 16), (c) a nucleic acid sequence which encodes amino acids 1 or about 27 to X of Figure 6 (SEQ ID NO: 16), wherein
  • PR0172 variant polynucleotide or "PR0172 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PRO 172 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 22 to 723 ofthe PROl 72 polypeptide shown in Figure 8 (SEQ ID NO 21), (b) a nucleic acid sequence which encodes amino acids X to 723 ofthe PRO 172 polypeptide shown in Figure 8 (SEQ ID NO 21), wherem X is any amino acid residue from 17 to 26 of Figure 8 (SEQ ID NO 21), (c) a nucleic acid sequence which encodes amino acids 1 or about 22 to X of Figure 8 (SEQ ID NO 21), wherein X is any ammo acid from ammo acid 543 to am o acid 552 of Figure 8 (SEQ ID NO 21 ) or (d) a nucleic acid sequence which encodes another specifically derived fragment ofthe
  • PROl 82 variant polynucleotide or "PRO 182 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PROl 82 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 19 to 135 of the PROl 82 polypeptide shown in Figure 10 (SEQ ID NO 26), (b) a nucleic acid sequence which encodes amino acids X to 135 ofthe PR0182 polypeptide shown in Figure 10 (SEQ ID NO 26), wherein X is any amino acid residue from 14 to 23 of Figure 10 (SEQ ID NO 26) or (c) a nucleic acid sequence which encodes another specifically derived fragment ofthe amino acid sequence shown in Figure 10 (SEQ ID NO 26)
  • a PRO 182 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at
  • 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 PR0211 , PR0228, PR0538, PRO 172 and PROl 82 polypeptide-encod g nucleic acid sequences identified herein is defined as the percentage of nucleotides m a candidate sequence that are identical with the nucleotides in a PR021 1 , PR0228, PR0538, PR0172 or PRO 182 polypeptide-encodmg nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % nucle
  • % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows:
  • % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, %> nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al, Nucleic Acids Res., _25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov.
  • % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows:
  • a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide- encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide- encodmg nucleic acid and the comparison nucleic acid molecule of interest (/ e , the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant
  • PR0211, PR0228, PR0538, PRO 172 and PRO 182 variant polynucleotides are nucleic acid molecules that encode an active PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide, respectively, and which are capable of hybridizing, preferably under stringent hybridization and wash conditions to nucleotide sequences encoding the full-length PR0211 polypeptide shown in Figure 2 (SEQ ID NO 2), to nucleotide sequences encoding the full-length PR0228 polypeptide shown in Figure 4 (SEQ ID NO 7), to nucleotide sequences encoding the full-length PR0538 polypeptide shown in Figure 6 (SEQ ID NO 16), to nucleotide sequences encoding the full-length PRO 172 polypeptide shown in Figure 8 (SEQ ID NO 21), to nucleotide sequences encoding the full-length PRO 182 polypeptide shown in Figure 10 (SEQ ID NO 26), respectively PR021 1 , PR
  • ammo acid residues in the context of the amino acid sequence identity comparisons performed as described above, includes ammo acid residues m 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 ammo acid residue of interest
  • the % value of positives of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows
  • 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
  • Y is the total number of amino acid residues in B
  • Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified ( 1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
  • An "isolated" nucleic acid molecule encoding a PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide or an "isolated" nucleic acid molecule encoding an anti-PR021 1, anti- PR0228, anti-PR0538, anti- PROl 72 or anti-PROl 82 antibody is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the PR021 1-, PR0228-, PR0538-, PRO 172- or PR0182-encoding nucleic acid or the anti-PR021 1-, anti- PR0228-, anti- PR0538-, anti-PROl 72- or anti-PROl 82-encoding nucleic acid.
  • the isolated nucleic acid is free of association with all components with which it is naturally associated.
  • An isolated PR021 1-, PR0228-, PR0538-, PRO 172- or PRO 182-encoding nucleic acid molecule or an isolated anti-PR021 1 -, anti- PR0228-, anti-PR0538-, anti-PROl 72- or anti-PROl 82-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature.
  • Isolated nucleic acid molecules therefore are distinguished from the PR021 1-, PR0228-, PR0538, -PR0172- or PROl 82-encoding nucleic acid molecule or from the anti-PR021 l-,anti- PR0228-, anti- PR0538-, anti-PROl 72- or anti-PROl 82-encoding nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule encoding a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide or an isolated nucleic acid molecule encoding an anti-PR021 l ,anti- PR0228, anti-PR0538, anti-PR0172 or anti- PRO 182 antibody includes PR021 1 -, PR0228-, PR0538-, PRO 172- or PRO 182-nucleic acid molecules or anti- PR021 1- , anti- PR0228-, anti-PR0538-, anti-PRO 172- or anti-PRO 182-nucleic acid molecules contained in cells that ordinarily express PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptides or anti-PR021 l ,anti- PR0228, anti-PR0538, anti-PRO 172 or anti-PRO 182 antibodies where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • control sequences refers to D A sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription ofthe sequence, or 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 m reading phase
  • enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice
  • antibody is used in the broadest sense and specifically covers, for example, single anti- PR021 1, ant ⁇ -PR0228, ant ⁇ -PR0538, ant ⁇ -PR0172 and ant ⁇ -PR0182 monoclonal antibodies (including agonist antibodies), ant ⁇ -PR021 1 , ant ⁇ -PR0228, ant ⁇ -PR0538, anti-PRO 172and anti-PRO 182ant ⁇ body compositions with polyepitopic specificity, single chain ant ⁇ -PR0211, ant ⁇ -PR0228, ant ⁇ -PR0538, ant ⁇ -PR0172 and ant ⁇ -PR0182 antibodies, and fragments of ant ⁇ -PR021 1 , ant ⁇ -PR0228, ant ⁇ -PR0538, anti-PRO 172 and anti-PRO 182 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
  • 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 hybndizable sequence, the higher the relative temperature which can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so
  • stringency of hybridization reactions see Ausubel et al , Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995)
  • “Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that (1) employ low ionic strength and high temperature for washing, for example 0 015 M sodium chlo ⁇ de/0 0015 M sodium c ⁇ trate/0 1% sodium dodecyl sulfate at 50°C, (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0 1 % bovine serum album ⁇ n/0 1% F ⁇ coll/0 1 % polyv ⁇ nylpyrrol ⁇ done/50mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C, or (3) employ 50% fo ⁇ namide, 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 sper
  • Modely stringent conditions may be identified as described by Sambrook etal , Molecular Cloning A Laboratory Manual, New York Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e g , temperature, ionic strength and % SDS) less stringent that those described above
  • An example of moderately stringent conditions is overnight incubation at 37 C 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
  • the term "epitope tagged" when used herein refers to a chimen
  • 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 ofthe polypeptide to which it is ftised
  • 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 ammo acid residues)
  • the term “lmmunoadhesin” designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains Structurally, the immunoadhesms comprise a ftision of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (t e .
  • the adhesin part of an lmmunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a hgand
  • the immunoglobulin constant domain sequence in the lmmunoadhesin 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 form(s) of PR021 1, PR0228, PR0538, PR0172 or PROl 82 which retain a biological and or an immunological activity of native or naturally-occurring PR021 1 , PR0228, PR0538, PRO 172 or PRO 182, wherein "biological" activity refers to a biological function (either inhibitory or stimulatory) caused
  • 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 ofthe 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 that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide having this activity with polyclonal antisera raised against the known active PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide
  • antisera are prepared in conventional fashion by injecting goats or rabbits, for example, subcutaneously with the known active analogue in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freunds
  • the immunological cross-reactivity preferably is "specific", which means that the binding affinity ofthe immunologically cross-reactive molecule (e g , antibody) identified, to the corresponding PR021 1 , PR0228, PR
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth
  • cancer include but are not limited to, carcinoma lymphoma, blastoma, sarcoma, and leukemia More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, 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 various types of head and neck cancer
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology of a disorder
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented
  • a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e g radiation and/or chemotherapy
  • the "pathology" of cancer includes all phenomena that compromise the well-being of the patient This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, etc
  • an “effective amount” of a polypeptide disclosed herein or an agonist thereof, in reference to inhibition of neoplastic cell growth is an amount capable of inhibiting, to some extent, the growth of target cells
  • the term includes an amount capable of invoking a growth inhibitory, cytostatic and/or cytotoxic effect and/or apoptosis of the target cells
  • An "effective amount" of a PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 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 PR021 1 , PR0228, PR0538, PR0172 or PROl 82 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1, PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 131 , 1 125 , Y 90 and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof
  • chemotherapeutic agent is a chemical compound useful in the treatment of tumor, e g , cancer
  • chemotherapeutic agents include ad ⁇ amycm, doxorubicin, epirubicin, 5-fluorourac ⁇ l, cytosine arabmoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxm, taxoids, e g , pachtaxel (Taxol, B ⁇ stol- Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rh ⁇ ne-PoulencRorer, Antony, Rnace), toxotere, methotrexate, cisplatm, melphalan vmblastine, bleomycin, etoposide, lfosfamide, mitomycin C, mitoxantrone, vinc ⁇ stine, vinorelbme, carb
  • 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 m vivo
  • the growth inhibitory agent is one which significantly reduces the percentage ofthe 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 G 1 arrest and M- phase arrest
  • Classical M-phase blockers include the vincas (vinc ⁇ stine and vinblastme), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin
  • Those agents that arrest G I also spill over into S-phase arrest, for example, D
  • D A alkylat g agents such as tamoxifen, prednisone, dacarbazine, mechlorethamme, cisplatin, methotrexate, 5-fluorourac
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators
  • cytokines are lymphokmes, monokines, and traditional polypeptide hormones Included among the cytokines are growth hormone such as human growth hormone, N- methionyl human growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxm, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor- ⁇ and - ⁇ , mulle ⁇ an-inhibiting substance, mouse gonadotrop - associated peptide, inhibin, activin, vascular endothelial growth factor, lnteg ⁇ n, thrombopoietin (TPO), nerve growth factors such as NGF- ⁇ , platelet
  • Chronic administration refers to administration ofthe 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 (concurrent) and consecutive administration m 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
  • 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 immunoglobulins, hydrophihc polymers such as polyvinylpyrrohdone, amino acids such as glycine, glutamine, asparagme, 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 counte ⁇ ons such as sodium, and/or nonionic surfactants
  • “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 varies among the heavy chains of different immunoglobulin isotypes Each heavy and light chain also has regularly spaced lntracha 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 ofthe light chain is aligned with the first constant domain ofthe heavy chain, and the light-chain variable domain is aligned with the variable domain ofthe heavy chain Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen
  • CDRs complementarity-determining regions
  • hyperva ⁇ able regions both in the light-chain and the heavy-chain variable domains
  • the more highly conserved portions of variable domains are called the framework (FR)
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation ofthe antigen-b ding site of antibodies (see, Kabat et al , NIH Publ No 91-3242, Vol I, pages 647-669 ( 1991 ))
  • the constant domains are
  • hyperva ⁇ able region when used herein refers to the ammo acid residues of an antibody which are responsible for antigen-binding
  • the hyperva ⁇ able region comprises amino acid residues from a "complementarity determining region" or "CDR" (/ 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 "hyperva ⁇ able loop” (i e , residues 26-32 (L 1 ), 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,
  • 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')chor and Fv fragments, diabodies, linear antibodies (Zapata et al , Protein Eng . 8(10) 1057-1062 [1995]), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments each with a single antigen-binding site, and a residual "Fc” fragment, a designation reflecting the ability to crystallize readily Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and -binding site This region consists of a dimer of one heavy- and one light-cham variable domain in tight, non-covalent association It is m this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer Collectively, the six CDRs confer antigen-binding specificity to the antibody However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site
  • the Fab fragment also contains the constant domain ofthe light chain and the first constant domain (CH 1 ) of the heavy chain Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus ofthe 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 cyste
  • immunoglobulins 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 am o 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, /. 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 againstdifferentdeterminants(epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character ofthe antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et at. 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 ofthe 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 ofthe chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA, 8 6851 - 6855 [1984]).
  • chimeric antibodies immunoglobulins in which a portion ofthe 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 ofthe chain(s) is identical with or homologous to corresponding sequences
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • Fv FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the humanized antibody includes a PRIMATIZEDTMantibody wherein the antigen-binding region ofthe antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
  • Single-chain Fv or “sFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present m a single polypeptide chain
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding
  • 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 1161 , 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%o by weight of antibody as determined by the Lowry method, and most preferably more than 99%> by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal ammo 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
  • radioisotope labels or fluorescent labels 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
  • solid phase is meant a non-aqueous matrix to which the antibody ofthe 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 silicones
  • the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275, 149
  • a “liposome” is a small vesicle composed of various types of hpids, phosphohpids and/or surfactant which is useful for delivery of a drug (such as a PR021 1 , PR0228 PR0538, PRO 172 or PRO 182 polypeptide or antibody thereto) to a mammal
  • a drug such as a PR021 1 , PR0228 PR0538, PRO 172 or PRO 182 polypeptide or antibody thereto
  • the components ofthe liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes
  • a "small molecule” is defined herein to have a molecular weight below about 500 Daltons II.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0211 , PR0228, PR0538, PROl 72 and PROl 82.
  • PR0211 polypeptides referred to in the present application as PR0211 , PR0228, PR0538, PROl 72 and PROl 82.
  • cDNAs encoding PR0211, PR0228, PR0538, PR0172 and PROl 82 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below.
  • cDNA clones encoding PR021 1 , PR0228, PR0538, PR0172 and PROl 82 polypeptides have been deposited with the ATCC.
  • the actual nucleotide sequences of the clones can readily be determined by the skilled artisan by sequencing ofthe deposited clones using routine methods in the art.
  • the predicted amino acid sequences can be determined from the nucleotide sequences using routine skill.
  • PR021 1 , PRQ228, PRQ538, PRQ172 and PRQ182 Variants In addition to the full-length native sequence PR021 1, PR0228, PR0538, PR0172 and PR0182 polypeptides described herein, it is contemplated that PR021 1 , PR0228, PR0538, PROl 72 and PROl 82 variants can be prepared. PR021 1, PR0228, PR0538, PR0172 and PR0182 variants can be prepared by introducing appropriate nucleotide changes into the PR021 1 , PR0228, PR0538, PR0172 or PR0182 DNA, and/or by synthesis of the desired PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide.
  • amino acid changes may alter post-translational processes ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
  • Variations in the native full-length sequence PR021 1 , PR0228, PR0538, PR0172 or PR0182 or in various domains of the PR021 1 , PR0228, PR0538, PR0172 or PR0182 described herein, can be made, for example, using any ofthe 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 PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 that results in a change in the amino acid sequence ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 as compared with the native sequence PR021 1 , PR0228, PR0538, PROl 72 or PROl 82.
  • the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the PR021 1 , PR0228, PR0538, PR0172 or PR0182.
  • 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 ofthe PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements.
  • Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full- length or mature native sequence.
  • PR021 1, PR0228, PR0538, PRO 172 and PRO 182 polypeptide fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity ofthe PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide.
  • PR021 1, PR0228, PR0538, PR0172 and PR0182 fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized.
  • An alternative approach involves generating PR021 1, PR0228, PR0538, PR0172 and PR0182 fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment.
  • Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR).
  • Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR.
  • PR021 1 , PR0228, PR0538, PR0172 and PR0182 polypeptide fragments share at least one biological and/or immunological activity with the native PR021 1 , PR0228, PR0538, PR0172 orPR0182 polypeptide shown in Figure 2 (SEQ IDNO:2), Figure 4 (SEQ IDN0:7), Figure 6 (SEQ ID NO: 16), Figure 8 (SEQ ID NO:21) and Figure 10 (SEQ ID NO:26), respectively.
  • conservative substitutions of interest are shown in Table 3 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 3, or as further described below in reference to amino acid classes, are introduced and the products screened.
  • PRO 172 or PRO 182 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
  • the variations can be made using methods known in the art such as oligonucleotide-mediated (site- directed) mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis [Carter et al, Nucl. Acids Res., L3:4331 (1986); Zoller et al, Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al, Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al, Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PR021 1 , PR0228, PR0538, PR0172 or PR0182 variant DNA.
  • Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence.
  • preferred scanning amino acids are relatively small, neutral amino acids.
  • amino acids include alanine, glycine, serine, and cysteine.
  • Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main- chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)].
  • Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins. (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150: 1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
  • PRQ21 1 , PRQ228, PRQ538, PRQ172 and PRQ182 Covalent modifications of PR021 1, PR0228, PR0538, PR0172 and PR0182 are included within the scope of this invention.
  • One type of covalent modification includes reacting targeted amino acid residues of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues ofthe PR021 1 , PR0228, PR0538, PRO 172 or PROl 82.
  • Derivatization with bifunctional agents is useful, for instance, for crosslinking PR021 1, PR0228, PR0538, PR0172 or PR0182 to a water-insoluble support matrix or surface for use in the method for purifying anti-PR021 1, anti-PR0228, anti-PR0538, anti-PRO 172 or anti-PRO 182 antibodies, and vice-versa.
  • crosslinking agents include, e.g., l ,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l ,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
  • esters with 4-azidosalicylic acid homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate
  • bifunctional maleimides such as bis-N-maleimido-l ,8-octan
  • Another type of covalent modification of the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern ofthe polypeptide.
  • "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 (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 PR021 1 , PR0228, PR0538, PR0172 or PR0182.
  • the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
  • Addition of glycosylation sites to the PR0211 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide may be accomplished by altering the amino acid sequence.
  • the alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PR021 1 , PR0228, PR0538, PR0172 or PR0182 (for O-linked glycosylation sites).
  • the PR0211, PR0228, PR0538, PR0172 or PR0182 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PR0211, PR0228, PR0538, PRO 172 or PRO 182 polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • PROl 72 or PROl 82 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 1 1 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
  • Removal of carbohydrate moieties present on the PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation.
  • Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al, Arch. Biochem. Biophvs.. 259:52 (1987) and by Edge et al, Anal. Biochem., 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. Enzymol.. 138:350 (1987).
  • PR021 1 , PR0228, PR0538, PR0172 or PR0182 comprises linking the PR021 1 , PR0228, PR0538, PR0172 or PROl 82 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • PEG polyethylene glycol
  • PROl 82 polypeptide e.g., polypropylene glycol, or polyoxyalkylenes
  • the PR021 1, PR0228, PR0538, PRO 172 or PROl 82 polypeptide ofthe present invention may also be modified in a way to form a chimeric molecule comprising PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 fused to another, heterologous polypeptide or amino acid sequence.
  • such a chimeric molecule comprises a fusion of the PR021 1 , PR0228, PR0538, PR0172 or PR0182 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 PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide.
  • the presence of such epitope-tagged forms ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide can be detected using an antibody against the tag polypeptide .
  • the epitope tag enables the PR021 1 , PR0228, PR0538, PROl 72 or PRO 182 polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-His) or poly-histidine-glycine (poly-His-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al, Mol. Cell. Biol..
  • Tag polypeptides include the Flag-peptide [Hopp et al , BioTechnology, 6 1204-1210 (1988)], the KT3 epitope peptide [Martin et al , Science, 255 192- 194 ( 1992)] , an ⁇ -tubuhn epitope peptide [Skinner et al , J Biol Chem .266 15163 - 15166 ( 1991 )], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl
  • the chimenc molecule may comprise a fusion of the PR021 1, PR0228, PR0538, PR0172 or PROl 82 polypeptide with an immunoglobulin or a particular region of an immunoglobulin
  • an immunoglobulin or a particular region of an immunoglobulin
  • the Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 IgG 1 molecule
  • PRQ21 1. PRQ228. PRQ538. PRQ172 and PRQ182
  • PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 by cultu ⁇ ng cells transformed or transfected with a vector containing PR021 1 , PR0228, PR0538, PR0172 or PROl 82 nucleic acid
  • alternative methods which are well known in the art, may be employed to prepare PR0211, PR0228, PR0538, PR0172 or PR0182
  • the PR021 1, PR0228, PR0538, PRO 172 or PRO 182 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), Mernfield, 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
  • DNA encoding PR021 1, PR0228, PR0538, PRO 172 or PROl 82 may be obtained from a cDNA library prepared from tissue believed to possess the PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 mRNA and to express it at a detectable level Accordingly, human PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples
  • the PR021 1-, PR0228-, PR0538-, PR0172- or PR0182-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 PR021 1 , PR0228, PR0538, PR0172 or PRO 182 or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al, Molecular Cloning: A Laboratory Manual (New York: Cold
  • the Examples below describe techniques for screening a cDNA library.
  • the oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized.
  • the oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened.
  • Methods of labeling are well known in the art, and include the use of radiolabels like 32 P-labeled ATP, biotinylation or enzyme labeling.
  • Hybridization conditions including moderate stringency and high stringency, are provided in Sambrook et al, supra.
  • Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions ofthe molecule or across the full-length sequence can be determined using methods known in the art and as described herein.
  • Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al, supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
  • Host cells are transfected or transformed with expression or cloning vectors described herein for PR021 1 , PR0228, PR0538, PR0172 or PR0182 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, CaCh, CaP0 4 , liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride, as described in Sambrook et al, supra, or electroporation is generally used for prokaryotes.
  • Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al, Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells
  • Suitable prokaryotes include but are not limited to eubacte ⁇ a, such as Gram-negative or Gram-positive organisms, for example, Enterobacte ⁇ aceae such as E coli
  • Various £ colt strains are publicly available, such as £ colt K12 strain MM294 (ATCC 31 ,446), £ colt XI 776 (ATCC 31,537), £ co strain W31 10 (ATCC 27,325) and K5772 (ATCC 53,635)
  • Other suitable prokaryotic host cells include Enterobacte ⁇ aceae such as Escherichia, e g , E coli, Enterobacter, Erwinta, Klebsiella, Proteus, Salmonella, e g , Salmonella typhtmurium, Serratia, e g , Serratia
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PR021 1-, PR0228-, PR0538-, PR0172- or PR0182-encod ⁇ ng vectors Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290 140 [1981], EP 139,383 pubhshed 2 May 1985), Kluyveromyces hosts O S Patent No 4,943,529, Fleer et al , Bio/Technology, 9 968-975 (1991 )) such as, e g , K lactts (MW98-8C, CBS683, CBS4574, Louvencourt e/-./ , J Bactenol , 737119831), K fragilis (ATCC 12,424) K bul gar tcus (ATCC 16,045), K wicker
  • thermotolerans, and K maixianus yarrowia EP 402,226), Pichia pastoris (EP 183.070, Sreek ⁇ shna et al . J Basic Microbiol , 28 265-278 119881), Candida Trichoderma reesia (EP 244,234) Neurospora crassa (Case et al , Proc Natl Acad Sci USA, 76 5259-5263 [1979]), Schwanniomyces such as Schwanniomyces occidentals (EP 394,538 published 31 October 1990), and filamentous fungi such as, e g , Neurospora Penicilhum, Tolypocladmm (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A mdulans (Ballance et al , Biochem Biophys Res Commun .
  • Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsts, 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 PR021 1, PR0228, PR0538, PR0172 or PR0182 are derived from multicellular organisms
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells
  • useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham etal , J Gen Virol , 36 59 (1977)), Chinese hamster ovary cellsADHFR (CHO, Urlaub and Chasm, Proc Natl Acad Sci USA.
  • mice sertoh cells TM4, Mather, Biol Reprod , 23 243- 251 (1980)
  • human lung cells W138, ATCC CCL 75
  • human liver cells Hep G2, HB 8065
  • mouse mammary tumor MMT 060562, ATCC CCL51
  • the selection ofthe appropriate host cell is deemed to be within the skill in the art
  • the nucleic acid (e g , cDNA or genomic DNA) encoding PR021 1 , PR0228, PR0538, PR0172 or PRO 182 may be inserted into a rephcable vector for cloning (amplification ofthe DNA) or for expression
  • a rephcable vector for cloning (amplification ofthe DNA) or for expression
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures
  • DNA is inserted into an appropriate restriction endonuclease s ⁇ te(s) using techniques known in the art
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled
  • the PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 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 ofthe mature protein or polypeptide
  • the signal sequence may be a component of the vector, or it may be a part of the PR021 1 -, PR0228-, PR0538-, PRO 172- or PR0182-encodmg DNA that is inserted into the vector
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group ofthe 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 Saccharomyces and Kluyveromyces ⁇ -
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
  • Selection genes will typically contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PR021 1 -, PR0228-, PR0538-, PRO 172- or PRO 182-encoding nucleic acid, such as DHFR or thymidine kinase.
  • An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al, Proc. Natl. Acad. Sci. USA, 77:4216 (1980).
  • a suitable selection gene for use in yeast is the trp ⁇ gene present in the yeast plasmid YRp7 [Stinchcomb et al, Nature, 282:39 (1979); Kingsman et al, Gene. 7: 141 (1979); Tschemper et al, Gene, JO: 157 (1980)].
  • the trp ⁇ gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85.: 12 (1977)].
  • Expression and cloning vectors usually contain a promoter operably linked to the PR0211-, PR0228-,
  • Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems [Chang et al, Nature, 275:615 (1978); Goeddel et al, Nature. 281 :544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al, Proc. Natl.
  • Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PR021 1 , PR0228, PR0538, PR0172 or PR0182.
  • S.D. Shine-Dalgarno
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al, J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al, J. Adv.
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • PR021 1 , PR0228, PR0538, PR0172 or PR0182 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
  • adenovirus such as Adenovirus 2
  • bovine papilloma virus avian sarcoma virus
  • cytomegalovirus a retrovirus
  • hepatitis-B virus a retrovirus
  • Simian Virus 40 SV40
  • heterologous mammalian promoters e.g., the actin promoter or an immunoglobulin promoter
  • heat-shock promoters provided such promoters are compatible with the host cell systems.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription.
  • Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein,and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus.
  • Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side ofthe replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the PR021 1 , PR0228, PR0538, PR0172 or PR0182 coding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PR021 1, PR0228, PR0538,
  • PR0172 or PR0182 are examples of PR0172 or PR0182.
  • PR0538, PR0172 or PR0182 in recombinant vertebrate cell culture are described in Gething et al, Nature, 293:620-625 (1981 : Mantei et al. Nature, 281 :40-46 (1979): EP 1 17,060; and EP 1 17,058.
  • Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
  • the antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • Gene expression 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.
  • the antibodies may be prepared against a native sequence PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PR021 1, PR0228, PR0538, PRO 172 or PRO 182 DNA and encoding a specific antibody epitope.
  • PR021 1, PR0228, PR0538, PR0172 or PR0182 may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells employed in expression of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • PR0211, PR0228, PR0538, PR0172 or PR0182 may be desired to purify PR0211, PR0228, PR0538, PR0172 or PR0182 from recombinant cell proteins or polypeptides.
  • the following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182.
  • Some drug candidates for use in the compositions and methods ofthe present invention are antibodies and antibody fragments which mimic the biological activity of a PR021 1, PR0228, PR0538, PR0172 or PR0182 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.
  • the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include the PR021 1 , PR0228, PR0538, PR0172 or PROl 82 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.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the immunization protocol may be selected by one skilled in the art without undue experimentation.
  • the antibodies may, alternatively, be monoclonal antibodies.
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature.256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include the PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59- 103].
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PR0211 , PR0228, PR0538, PRO 172 or PRO 182.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 ( 1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Patent No 4,816,567
  • DNA encoding the monoclonal antibodies ofthe invention can be readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies)
  • the hybridoma cells of the invention serve as a preferred source of such DNA
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place ofthe homologous murine sequences [U S Patent No 4,816,567, Morrison et
  • the antibodies may be monovalent antibodies
  • Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking
  • the relevant cysteine residues are substituted with another ammo acid residue or are deleted so as to prevent crosslinking
  • the antibodies of the invention may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , murine) antibodies are chimenc 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) ofthe 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 having the desired specificity, affinity and capacity
  • 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
  • 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 chimenc 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
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J Mol Biol , 227 381 (1991), Marks et al , J Mol Biol , 222 581 (1991)] The techniques of Cole et al , and Boerner et al , are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy.
  • human antibodies can be made by the introducing of human immunoglobulin loci into transgenic animals, e g , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire
  • transgenic animals e g , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated
  • human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire
  • This approach is described, for example, in U S Patent Nos 5,545,807, 5,545,806, 5,569,825, 5,625,126 5,633,425, 5,661 ,016, and in the following scientific publications Marks et al Bio/Technology, 10 779-783 (1992), Lonberg e.
  • 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182
  • the other one is for any other antigen, and preferably for a cell- surface protein or receptor or receptor subunit
  • Methods for making bispecific antibodies are known in the art Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-cham pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305 537-539 (1983)] Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure The purification ofthe correct molecule is usually accomplished by affinity chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences
  • the fusion preferably is with an immunoglobulin heavy- chain constant domain, comprising at least part ofthe hmge, CH2, and CH3 regions It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism
  • CHI first heavy-chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture
  • the preferred interface comprises at least a part ofthe CH3 region of an antibody constant domain
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e g , tyrosine or tryptophan)
  • Compensatory "cavities" of identical or similar size to the large side cha ⁇ n(s) are created on the interface ofthe second antibody molecule by replacing large ammo acid side chains with smaller ones (e g , alanine or threomne)
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e g , F(ab'), bispecific antibodies) Techniques for generating bispecific antibodies from antibody fragments have been described in the literature For example, bispecific antibodies can be prepared using chemical linkage Brennan et al , Science, 229 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab'), fragments These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation The Fab' fragments generated are then converted to thiomtrobenzoate (TNB) derivatives One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody
  • the bispecific antibodies produced can be used as agents for the selective immobil
  • Fab' fragments may be directly recovered from £ coli and chemically coupled to form bispecific antibodies
  • Shalaby et al , J Exp Med , 175 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')-, molecule
  • Each Fab' fragment was separately secreted from £ coli and subjected to directed chemical coupling in vitro to form the bispecific antibody
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets
  • bispecific antibodies have been produced using leucine zippers Kostelnv et al A 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 Hollmger 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-cham variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain Accordingly, the V H and
  • t ⁇ specific antibodies can be prepared Tutt et al , J Immunol , 147 60 (1991) Exemplary bispecific antibodies may bind to two different epitopes on a given PR021 1, PR0228,
  • an ant ⁇ -PR0211 , ant ⁇ -PR0228, ant ⁇ -PR0538, anti-PRO 172 or anti-PRO 182 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 , CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular PR0211 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PR021 1, PR0228, PR0538, PROl 72 or PROl 82 polypeptide These antibodies possess a PR021 1 -, PR0228-, PR0538-
  • Heteroconjugate antibodies are also within the scope ofthe present invention Heteroconjugate antibodies are composed of two covalently joined antibodies Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U S Patent No 4,676,980], and for treatment of HIV infection [WO 91 /00360, WO 92/200373 , EP 03089] It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents For example, lmmunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond Examples of suitable reagents for this purpose include lminothiolate and methyl-4-mercaptobuty ⁇ m ⁇ date and those disclosed, for example, in U S Patent No 4,676,980 6 Effector Function Engineering
  • the homodime ⁇ c antibody thus generated may have improved lnteraahzation capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See, Caron et al , J Exp Med , j_76 1 191 - 1195 ( 1992) and Shopes, J Immunol , 148 2918-2922 ( 1992) Homodime ⁇ c antibodies with enhanced antitumor 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
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (/ e , a radioconjugate)
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (/ e , a radioconjugate)
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbmding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aerugmosd), ⁇ cin A chain, ab ⁇ n A chain, modeccin A chain, alpha-sarcin, Aleurites fordn proteins, dianth proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcm, crotm, sapaona ⁇ a officinahs inhibitor, gelonin, mitogelhn, rest ⁇ ctocin, phenomycin, enomycin, and the t ⁇ cothecenes
  • radionuchdes are available for the production of radioconjugated antibodies Examples include 212 B ⁇ , lj l I, 131 In, 90 Y, and
  • Conjugates ofthe antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such asN-succ ⁇ n ⁇ m ⁇ dyl-3-(2-py ⁇ dyld ⁇ th ⁇ ol)prop ⁇ onate (SPDP), lminothiolane (IT), bifunctional derivatives of ⁇ m ⁇ doesters(such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium de ⁇ vativ es (such as b ⁇ s-(p-d ⁇ azon ⁇ umbenzoyl)-ethylened ⁇ am ⁇ ne), dnsocyanates (such as tolyene 2,6-dnsocyanate), and bis- active fluorine compounds (such as l ,5-d ⁇ fluor
  • the antibody may be conjugated to a "receptor” (such as streptavidin) for utilization in tumor pretargetmg wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e g , avidin) that is conjugated to a cytotoxic agent (e g , a radionucleotide) 8.
  • a "receptor” such as streptavidin
  • the antibodies disclosed herein may also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. Sci. USA. 82: 3688 (1985); Hwang et al, Proc. Natl. Acad. Sci. USA. 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG- PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments ofthe antibody ofthe present invention can be conjugated to the liposomes as described in Martin et al, J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.
  • a chemotherapeutic agent such as Doxorubicin is optionally contained within the liposome. See, Gabizon et al, J. National Cancer Inst.. 81(19): 1484 (1989).
  • the proteins disclosed in the present application have been assayed in a panel of 60 tumor cell lines currently used in the investigational, disease-oriented, in vitro drug-discovery screen of the National Cancer Institute (NCI).
  • NCI National Cancer Institute
  • the purpose of this screen is to identify molecules that have cytotoxic and/or cytostatic activity against different types of tumors.
  • NCI screens more than 10,000 new molecules per year (Monks et al, J. Natl. Cancer Inst., 83:757-766 (1991); Boyd, Cancer: Princ. Pract. Oncol. Update, 3(10 ⁇ : 1-12 ([1989]).
  • the tumor cell lines employed in this study have been described in Monks et al, supra.
  • the cell lines the growth of which has been significantly inhibited by the proteins ofthe present application are specified in the Examples.
  • cell-based assays and animal models for tumors can also be used to verify the findings ofthe NCI cancer screen, and to further understand the relationship between the protein identified herein and the development and pathogenesis of neoplastic cell growth.
  • primary cultures derived from tumors in transgenic animals as described below can be used in the cell-based assays herein, although stable cell lines are preferred. Techniques to derive continuous cell lines from transgenic animals are well known in the art (see, e.g., Small et al, Mol. Cell. Biol., 5:642-648 [1985]).
  • Animal Models A variety of well known animal models can be used to further understand the role of the molecules identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies, and other agonists ofthe native polypeptides, including small molecule agonists. The in vivo nature of such models makes them particularly predictive of responses in human patients.
  • Animal models of tumors and cancers e.g., breast cancer, colon cancer, prostate cancer, lung cancer, etc.
  • Non-recombinant animal models include, for example, rodent, e.g. , murine models.
  • Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g. , colon cancer cells implanted in colonic tissue.
  • standard techniques e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g. , colon cancer cells implanted in colonic tissue.
  • orthopin implantation e.g., colon cancer cells implanted in colonic tissue.
  • the autosomal recessive nu gene has been introduced into a very large number of distinct congenic strains of nude mouse, including, for example, ASW, A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL.
  • a wide variety of other animals with inherited immunological defects other than the nude mouse have been bred and used as recipients of tumor xenografts. For further details see, e.g. , The Nude Mouse in Oncology Research, E. Boven and B. Winograd, eds., CRC Press, Inc., 1991.
  • the cells introduced into such animals can be derived from known tumor/cancer cell lines, such as, any ofthe above-listed tumor cell lines, and, for example, the B104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene); ras-transfected NIH-3T3 cells; Caco-2 (ATCC HTB-37); a moderately well- differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or from tumors and cancers.
  • Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions, involving freezing and storing in liquid nitrogen (Karmali et al, Br. J. Cancer, 48:689-696 [1983]).
  • Tumor cells can be introduced into animals, such as nude mice, by a variety of procedures.
  • the subcutaneous (s.c.) space in mice is very suitable for tumor implantation.
  • Tumors can be transplanted s.c. as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions.
  • tumor tissue fragments of suitable size are introduced into the s.c. space.
  • Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously.
  • Tumor cells can also be injected as subdermal implants. In this location, the inoculum is deposited between the lower part ofthe dermal connective tissue and the s.c. tissue. Boven and Winograd ( 1991 ), supra.
  • Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogen was initially isolated), or ne - transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al, Proc. Natl. Acad. Sci. USA, 83:9129-9133 (1986).
  • animal models of colon cancer can be generated by passaging colon cancer cells in animals, e g , nude mice, leading to the appearance of tumors in these animals.
  • An orthotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang et at. Cancer Research, 54:4726-4728 (1994) and Too et al, Cancer Research. 55:681-684 (1995). This model is based on the so-called "METAMOUSE” sold by AntiCancer, Inc., (San Diego, California).
  • Tumors that arise in animals can be removed and cultured in vitro. Cells from the in vitro cultures can then be passaged to animals. Such tumors can serve as targets for further testing or drug screening. Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest. Such passaging techniques can be performed with any known tumor or cancer cell lines
  • Meth A, CMS4, CMS5, CMS21, and WEHI-164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et al , J Exp Med , 146 720 [1977]), which provide a highly controllable model system for studying the anti-tumor activities of various agents (Palladmo et al , J Immunol , 138 4023-4032 [1987]) Briefly, tumor cells are propagated in vitro in cell culture Prior to injection into the animals, the cell lines are washed and suspended m buffer, at a cell density of about lOxlO 6 to lOxlO 7 cells/ml The animals are then infected subcutaneously with 10 to 100 ⁇ ofthe 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
  • 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 morphomet ⁇ c method and flow cytomet ⁇ c analysis
  • Recombinant (transgenic) animal models can be engineered by introducing the coding portion ofthe genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals
  • Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e g , baboons, chimpanzees and monkeys
  • Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and Wanger, U S Patent No 4,873,191), retrovirus-mediated gene transfer into germ lines (e g , Van der Putten et al , Proc Natl Acad Sci USA, 82 6148-615 [1985]), gene targeting in embryonic stem cells (Thompson et al , Cell, 56 313-321 [1989]), electroporation of embryos (Lo, Mol Cell Biol . 3 18
  • transgenic animals include those that carry the transgene only in part of their cells (“mosaic animals”).
  • the transgene can be integrated either as a single transgene, or in concatamers, e g , head-to-head or head-to-tail tandems
  • Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al , Proc Natl Acad Sci USA, 89 6232- 636 (1992)
  • transgenic animals can be monitored by standard techniques For example, Southern blot analysis or PCR amplification can be used to verify the integration ofthe 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
  • fibrosarcoma adenocarcinoma
  • lymphoma adenocarcinoma
  • chrondroma adenocarcinoma of dogs, cats
  • baboons a preferred model as its appearance and behavior are very similar to those in humans
  • the use of this model is limited by the rare occurrence of this type of tumor in animals
  • Screening assays for drug candidates are designed to identify compounds that competitively bind or complex with the receptor(s) ofthe polypeptides identified herein, or otherwise signal through such receptor(s)
  • Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates
  • Small molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides, (poly)pept ⁇ de- lmmunoglobuhn fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimenc or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.
  • a receptor of a polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments.
  • Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the polypeptide and drying.
  • an immobilized antibody e.g., a monoclonal antibody, specific for the polypeptide to be immobilized can be used to anchor it to a solid surface.
  • the assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component.
  • the non-reacted components are removed, e.g. , by washing, and complexes anchored on the solid surface are detected.
  • the detection of label immobilized on the surface indicates that complexing occurred.
  • complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
  • the candidate compound interacts with but does not bind to a particular receptor, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions.
  • assays include traditional approaches, such as, cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns.
  • protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and Song, Nature (London), 340:245-246 ( 1989); Chien etal, Proc. Natl. Acad. Sci. USA. 88:9578-9582 (1991)] as disclosed by Chevray and Nathans fProc. Natl. Acad. Sci. USA.
  • yeast GAL4 Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain.
  • the yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain.
  • the expression of a GALl-/ ⁇ _.Z reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction.
  • Colonies containing interacting polypeptides are detected with a chromogenic substrate for ⁇ -galactosidase.
  • a complete kit (MATCHMAKERTM) for identifying protein-protein interactions between two specific proteins using the two- hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
  • polypeptides of the present invention agonist antibodies specifically binding proteins identified herein, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions.
  • antibody fragments the smallest inhibitory fragment which specifically binds to the binding domain of the target protein is preferred
  • peptide molecules can be designed which retain the ability to bind the target protein sequence
  • Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e g , Marasco et at . Proc Natl Acad Sci USA, 90 7889-7893 119931)
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other
  • the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent
  • cytotoxic agent 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 ofthe polypeptides identified herein, or agonists thereof are prepared for storage by mixing the active ingredient having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences. 16th edition, Osol, A ed [1980]), in the form of lyophilized formulations or aqueous solutions
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other orgamcacids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzy 1 ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethomum chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohe
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by mterfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A ed (1980)
  • 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 having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle
  • Sustained-release preparations may be prepared Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e g , films, or microcapsules
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(v ylalcohol)), polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vmyl acetate, degradable lactic acid-glycohc acid copolymers such as the LUPRON DEPOTTM (injectable microspheres
  • the polypeptides ofthe present invention and their agonists may be used to treat various tumors, e g , cancers
  • Exemplary conditions or disorders to be treated include benign or malignant tumors (e g renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas, sarcomas, glioblastomas, and various head and neck tumors), leukemias and lymphoid malignancies, other disorders such as neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoehc disorders, and inflammatory, angiogenic and lmmunologic disorders
  • the anti-tumor agents ofthe present invention including the polypeptides disclosed herein and agonists which mimic their activity, e g , antibodies, peptide
  • chemotherapeutic agents may be administered to the patient Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, ed , M C Perry, Williams & Wilkins, Baltimore, MD (1992)
  • the chemotherapeutic agent may precede, or follow administration ofthe anti-tumor agent ofthe present invention, or may be given simultaneously therewith
  • the anti-cancer agents ofthe present invention may be combined with an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onap ⁇ stone (see, EP 616812) in dosages known for such molecules
  • the anti-cancer agents herein are co-administered with a growth inhibitory agent
  • the growth inhibitory agent may be administered first, followed by the administration of an anti-cancer agent ofthe present invention
  • simultaneous administration or administration ofthe anti-cancer agent of the present invention is also contemplated Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) ofthe 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 ofthe 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 ofthe 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 Toxicokmetics 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 manufacture containing materials useful for the diagnosis or treatment of the disorders described above comprises a container and a label Suitable containers include, for example, bottles, vials, syringes, and test tubes
  • the containers may be formed from a variety of materials such as glass or plastic
  • the container holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle)
  • the active agent in the composition is an anti-tumor agent of the present invention
  • the label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice
  • the article of manufacture may further comprise a second container comprising a pharmaceutical ly- acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution It may further include other materials desirable from a commercial and user standpoint, including other buffers, dilu,
  • PRQ21 1 Isolation of cDNA clones Encoding PRQ21 1 , PRQ228. PRQ538, PRQ172 and PRQ182 (A) PRQ21 1
  • ECD extracellular domain sequences (including the secretion signal sequence, if any) from about
  • EST databases included public EST databases (e g , GenBank), and a proprietary EST database (LIFESEQ®, 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 ofthe ECD protein sequences to a 6 frame translation of the EST sequences Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington)
  • DNA28730 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA28730 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
  • 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 ofthe full-length coding sequence for PR021 1
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length
  • the probe sequences are typically 40-55 bp m length
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1 5 kbp
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one ofthe primer pairs
  • PCR primers (forward and reverse) were synthesized forward PCR primer 5'-AGAGTGTATCTCTGGCTACGC-3' (SEQ ID NO 3) reverse PCR primer
  • a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28730 sequence which had the following nucleotide sequence hybridization probe 5'-AGGGAGCACGGACAGTGTGCAGATGTGGACGAGTGCTCACTAGCA-3' (SEQ ID NO 5)
  • RNA for construction of the cDNA libraries was isolated from human fetal lung tissue
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278- 1280 ( 1991 )) m 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 PR021 1 polypeptide (designated herein as DNA32292- 1 131 [ Figure 1 , SEQ ID NO 1 ]) and the derived protein sequence for that PR021 1 polypeptide
  • the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 65-67 and a stop signal at nucleotide positions 1 124- 1 126 ( Figure 1 , SEQ ID NO 1)
  • the predicted polypeptide precursor is 353 amino acids long, has a calculated molecular weight of approximately 38,190 daltons
  • Analysis ofthe full-length PR021 1 sequence shown in Figure 2 evidences the presence of a va ⁇ ety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above
  • Analysis ofthe full-length PR021 1 sequence evidenced the following a signal peptide from about amino acid 1 to about amino acid 24, N-glycosylation sites from about amino acid 190 to about amino acid 194 and from about amino acid 251 to about amino acid 255, glycosaminoglycan attachment sites from about amino acid 149 to about amino acid 153 and from about amino acid 155 to about amino acid 159, a
  • the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases
  • the EST databases included public EST databases (e g , GenBank), and a proprietary EST database (LIFESEQ®, Incyte 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 ofthe ECD protein sequences to a 6 frame translation of the EST sequences Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap” (Phil Green, University of Washington, Seattle, Washington)
  • DNA28758 An EST proprietary to Genentech, Inc , designated herein as DNA21951, was employed in the consensus assembly. 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
  • 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 ofthe full-length coding sequence for PR0228
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length
  • the probe sequences are typically 40-55 bp in length
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about 1 -1 5 kbp
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one ofthe p ⁇ mer pairs PCR primers
  • oligonucleotide hybridization probe was constructed from the consensus DNA28758 sequence which had the following nucleotide sequence hybridization probe
  • RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278- 1280 ( 1991 )) in the unique Xhol and Notl sites
  • the predicted polypeptide precursor is 690 amino acids long
  • Analysis of the full-length PR0228 sequence shown in Figure 4 evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above
  • Analysis ofthe full-length PR0228 sequence evidenced the following a signal peptide from about amino acid 1 to about amino acid 19; transmembrane domains from about amino acid 430 to about amino acid 450, from about amino acid 465 to about amino acid 486, from about amino acid 499 to about amino acid 513, from about amino acid 535 to about amino acid 549, from about amino acid 573 to about amino acid 593, from about amino acid 619 to about amino acid 636, and from about amino acid 648 to about amino acid 664; N-glycosylation sites from about amino acid 15 to about amino acid 19, from about amino acid 21 to about amino acid 25, from about amino acid 64 to about amino acid 68, from about amino acid 74 to about amino acid 78, from about amino
  • RNA for construction of cDNA libraries was then isolated from human fetal lung tissue
  • the cDNA libraries used to isolate the cDNA clones encoding human PR0538 were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation mto a suitable cloning vector (such as pRKB or pRKD, ⁇ RK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278-1280 (1991)) in the unique Xhol and Notl
  • Oligonucleotides probes based upon the above described EST sequence were then 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 ofthe full-length coding sequence for PR0538
  • 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
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one ofthe primer pairs
  • oligonucleotide probes employed were as follows forward PCR primer
  • DNA48613-1268 encodes a protein designated as human GFR ⁇ 3, and DNA48614- 1268 encodes its splice variant
  • GFR ⁇ 3 hGFR ⁇ 3 versus hGFR ⁇ 1 34% hGFR ⁇ 3 versus hGFR ⁇ 2 34% hGFR ⁇ 1 versus hGFR ⁇ 2 48% From the sequence comparisons it can be seen that human GFR ⁇ 3 (PR0538) is less related to its rodent homolog than is either GFR ⁇ l or GFR ⁇ 2 In addition, GFR ⁇ 3 (PR0538) appears to be more distantly related to GFR ⁇ l and GFR ⁇ 2 than GFR l and GFR ⁇ 2 are to each other
  • the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases
  • the EST databases included public EST databases (e g , GenBank), and a proprietary EST database (LIFESEQ®, Incyte 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 ofthe ECD protein sequences to a 6 frame translation of the EST sequences Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap” (Phil Green, University of Washington, Seattle, Washington)
  • DNA28765 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA28765 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
  • 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 ofthe full-length coding sequence for PRO 172
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length
  • the probe sequences are typically 40-55 bp in length
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1 5 kbp
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one ofthe primer pairs
  • PCR primers (forward and reverse) were synthesized forward PCR primer
  • RNA for construction of the cDN A libraries was isolated from human fetal kidney tissue
  • the cDN A libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see,
  • a signal peptide from about amino acid 1 to about amino acid 21 ; a transmembrane domain from about amino acid 548 to about amino acid 568; an N-glycosylation site from about amino acid 477 to about amino acid 481 ; a cAMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 660 to about amino acid 664; casein kinase II phosphorylation sites from about amino acid 93 to about amino acid 97, from about amino acid 131 to about amino acid 135, from about amino acid 154 to about amino acid 158, from about amino acid 203 to about amino acid 207, from about amino acid 342 to about amino acid 346, from about amino acid 344 to about amino acid 348, from about amino acid 369 to about amino acid 373, from about amino acid 457 to about amino acid 461 , from about amino acid 483 to about amino acid 487, from about amino acid 495 to about amino acid 499
  • RNA for construction of cDNA libraries was then isolated from human uterine tissue
  • the cDN A libraries used to isolate the cDNA clones encoding human PRO 182 were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278-1280 (1991)) in the unique Xhol and Notl
  • Oligonucleotides probes based upon the above described EST sequence were then 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 ofthe full-length coding sequence for PROl 82
  • 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
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one ofthe p ⁇ mer pairs
  • oligonucleotide probes employed were as follows 5'-CACATTCAGTCCTCAGCAAAATGAA-3' (SEQ ID NO 27) 5'-GAGAATAAAAACAGAGTGAAAATGGAGCCCTTCATTTTGC-3' (SEQ ID NO 28) 5'-CTCAGCTTGCTGAGCTTGAGGGA-3' (SEQ ID NO 29)
  • the DNA sequence encoding PR021 1, PR0228, PR0538, PR0172 or PR0182 is initially amplified using selected PCR primers
  • the primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector
  • a variety of expression vectors may be employed
  • An example of a suitable vector is ⁇ BR322 (derived from £ coh, see Bolivar et al , Gene, 2 95 (1977)) which contains genes for ampicillin and tetracycline resistance
  • the vector is digested with restriction enzyme and dephosphorylated
  • the PCR amplified sequences are then hgated 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 enterokmase cleavage site), the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 coding region, lambda transcriptional terminator
  • the ligation mixture is then used to transform a selected £ co strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics The overnight culture may subsequently be used to inoculate a larger scale culture The cells are then grown to a desired optical density, during which the expression promoter is turned on
  • 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 solubi zed using various agents known in the art, and the solubihzed PR021 1, PR0228, PR0538, PRO 172 or PRO 182 protein can then be purified using a metal chelating column under conditions that allow tight binding ofthe protein
  • PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 may be expressed in £ co in a poly-His tagged form, using the following procedure
  • the DNA encoding PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 enterok ase
  • the PCR-amplified, poly-His tagged sequences are then hgated into an expression vector, which is used to transform an £ coh host based on strain 52 (W31 10 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(ladq) Transformants are first grown in LB containing 50 mg/ml carbenicillm at 30°C with shaking until an
  • 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 A 280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitnle since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin Aggregated species are usually eluted at higher acetonitnle concentrations In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endot
  • PRQ538, PRO 172 or PRO 182 in mammalian cells This example illustrates preparation of a potentially glycosylated form of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 by recombinant expression in mammalian cells.
  • the vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector.
  • the PR021 1 , PR0228, PR0538, PR0172 or PROl 82 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 DNA using ligation methods such as described in Sambrook et al, supra.
  • the resulting vector is called pRK5-PR021 1, pRK5- PR0228, pRK5-PR0538, pRK5-PR0172 or pRK5-PR0182.
  • the selected host cells may be 293 cells.
  • Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics.
  • DMEM fetal calf serum
  • About 10 ⁇ g pRK5-PRO21 l , pRK5-PR0228, pRK5-PR0538, pRK5-PR0172 or pRK5-PR0182 DNA is mixed with about 1 ⁇ g DNA encoding the VA RNA gene [Thimmappaya et al, Ceh, 31:543 (1982)] and dissolved in 500 ⁇ l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl 2 .
  • the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ⁇ Ci/ml 35 S-cysteine and 200 ⁇ Ci/ml 35 S-methionine.
  • 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 ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide.
  • the cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
  • PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al, Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 ⁇ g pRK5-PR021 1 , pRK5-PR0228, pRK5-PR0538, pRK5-PR0172 or pRK5-PRO 182 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 A-g/ml bovine insulin and 0.1 /.g/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris.
  • the sample containing expressed PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
  • PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 can be expressed in CHO cells.
  • the pRK5-PR021 1 , pRK5-PR0228, pRK5-PR0538, pRK5-PR0172 or pRK-5PR0182 can be transfected into CHO cells using known reagents such as CaP0 4 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 -"S- methionme After determining the presence of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide can then be concentrated and
  • Epitope-tagged PR021 1 , PR0228, PR0538, PR0172 or PR0182 may also be expressed in host CHO cells
  • the PR021 1, PR0228, PR0538, PR0172 or PR0182 may be subcloned out of the pRK5 vector
  • the subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-His tag into a Baculovirus expression vector
  • the poly-His tagged PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones
  • the CHO cells can be transfected (as described above) with the SV40 driven vector Labeling may be performed, as described above, to verify expression
  • the culture medium containing the expressed poly-His tagged PR021 1, PR0228, PR0538, PR0172 or PR0182 can then
  • PR021 1 , PR0228, PR0538, PR0172 or PR0182 may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure
  • the proteins are expressed as an IgG construct (lmmunoadhesin), in which the coding sequences for the soluble forms (e g , extracellular domains) ofthe respective proteins are fused to an IgG 1 constant region sequence containing the hinge, CH2 and CH2 domains and/or as a poly-His tagged form
  • CHO expression vectors are constructed to have compatible restriction sites 5' and 3' ofthe 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 ofthe cDNA of interest and dihydrofolate reductase (DHFR) DHFR expression permits selection for stable maintenance ofthe plasmid following transfection
  • the ampules containing the plasmid DNA are thawed by placement mto a water bath and mixed b> vortexing
  • the contents are pipetted into a centrifuge tube containing 10 mis of media and centrifuged at 1000 rpm for 5 minutes
  • the supernatant is aspirated and the cells are resuspended in 10 ml of selective media (02 ⁇ m filtered PS20 with 5%o 0 2 ⁇ m diafiltered fetal bovine serum)
  • the cells are then aliquoted into a 100 ml spinner containing 90 ml of selective media
  • the cells are transferred into a 250 ml spinner filled with 150 ml selective growth medium and incubated at 37°C.
  • spinners 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.
  • 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 10 6 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.
  • the spinner 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 10%, the cell culture is harvested by centrifugation and filtering through a 0.22 ⁇ m filter. The filtrate is either stored at 4°C or immediately loaded onto columns for purification.
  • 10% antifoam e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion
  • the proteins are purified using a Ni 2+ -NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni 2+ -NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole.
  • the highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
  • lmmunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows.
  • the conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5.
  • the eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ⁇ l of 1 M Tris buffer, pH 9.
  • the highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins.
  • the homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
  • PR021 1 , PRO 172 and PRO 182 were stably expressed in CHO cells by the above described method.
  • PRO 172 was expressed in CHO cells by the transient expression procedure.
  • EXAMPLE 4 Expression of PRQ21 1 , PRQ228, PRQ538.
  • PROl 72 or PROl 82 in Yeast The following method describes recombinant expression of PR021 1 , PR0228, PR0538, PR0172 or PRO 182 in yeast.
  • yeast expression vectors are constructed for intracellular production or secretion of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 from the ADH2/GAPDH promoter.
  • DNA encoding PR021 1 , PR0228, PR0538, PR0172 or PR0182 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182.
  • DNA encoding PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or lnvertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182
  • yeast cells such as yeast strain ABI 10
  • yeast supematants can then be transformed with the expression plasmids described above and cultured in selected fermentation media
  • the transformed yeast supematants can be analyzed by precipitation with 10%> t ⁇ chloroacetic acid and separation by SDS-PAGE, followed by staining ofthe gels with Coomassie Blue stain
  • Recombinant PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 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 PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 may further be purified using selected column chromatography resins
  • EXAMPLE 5 Expression of PRQ21 1, PRQ228, PRQ538, PRQ172 or PRQ182 in Baculovirus-Infected Insect Cells
  • the sequence coding for PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 is fused upstream of an epitope tag contained within a baculovirus expression vector
  • 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 pVL 1393 (Novagen) Briefly, the sequence encoding PR021 1 , PR0228, PR0538, PR0172 or PR0182 or the desired portion of the coding sequence of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 (such as the sequence encoding the extracellular domain of a trans
  • Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGoldTM virus DNA
  • Expressed poly-His tagged PR021 1 , PR0228, PR0538, PR0172 or PR0182 can then be purified, for example, by N ⁇ 2+ -chelate affinity chromatography as follows Extracts are prepared from recombinant virus- infected Sf9 cells as described by Rupert et al , Nature, 362 175-179 (1993) Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes, pH 7 9, 12 5 mM MgCh, 0 1 mM EDTA, 10% glycerol, 0 1% NP- 40, 0 4 M KCI), and sonicated twice for 20 seconds on ice The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7 8) and filtered through a 0 45 mm filter A
  • the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.
  • a secondary wash buffer 50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0
  • the column is developed with a 0 to 500 mM imidazole gradient in the secondary wash buffer.
  • One ml fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni 2+ -NTA- conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His, --tagged PR021 1 , PR0228, PR0538, PR0172 or PROl 82, respectively, are pooled and dialyzed against loading buffer.
  • purification of the IgG tagged (or Fc tagged) PR0211 , PR0228, PR0538, PR0172 or PROl 82 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
  • Baculogold® baculovirus DNA Puringen
  • Sf9 Spodoptera frugiperda
  • pb.PH.IgG and pb.PH.His are modifications ofthe commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences.
  • the cells are grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). Cells are incubated for 5 days at 28 C C. The supernatant is harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10%> FBS at an approximate multiplicity of infection (MOI) of 10. Cells are incubated for 3 days at 28 °C.
  • MOI multiplicity of infection
  • the supernatant is harvested and the expression of the constructs in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml of Ni 2+ -NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
  • the first viral amplification supernatant is used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells are incubated for 3 days at 28 °C. The supernatant is harvested and filtered. Batch binding and SDS-PAGE analysis is repeated, as necessary, until expression ofthe spinner culture is confirmed.
  • the conditioned medium from the transfected cells (0.5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0.22 micron filters.
  • the protein construct is purified using a Ni 2+ -NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM.
  • the conditioned media is pumped onto a 6 ml Ni 2+ -NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C.
  • the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole.
  • the highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
  • lmmunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows.
  • the conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3 5
  • the eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9
  • the highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins The homogeneity ofthe proteins is verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation
  • PR0228, PR0538 and PR0172 were expressed in baculovirus infected Sf9 insect cells
  • 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)
  • a variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIE 1 - 1 (Novagen)
  • the pIE 1 - 1 and pIE 1 -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus ⁇ el promoter in stably- transformed insect cells (1)
  • the plasmids differ only in the orientation ofthe 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
  • 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, 100 ⁇ l ofCellFect ⁇ n (CellFECTIN (G ⁇ bcoBRL #10362-010) (vortexed to m ⁇ x)) ⁇ s m ⁇ xed w ⁇ th 1 ml of Ex-Cell medium
  • the two solutions are combined and allowed to incubate at room temperature for 15 minutes 8 ml of Ex-Cell media is added to the 2 ml of DNA/CellFECTIN mix and this is layered on h ⁇ gh-5 cells that have been washed once with Ex-Cell media The plate is then incubate
  • PR0182 fusion proteins containing PR021 1 , PR0228, PR0538, PR0172 or PR0182, and cells expressing recombinant PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 on the cell surface
  • Selection ofthe immunogen can be made by the skilled artisan without undue experimentation
  • mice such as Balb/c are immunized with the PR021 1 , PR0228, PR0538, PR0172 or PR0182 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneal ly in an amount from 1-100 micrograms
  • the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads
  • the immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant Thereafter, for several weeks, the mice may also be boosted with additional immunization injections
  • Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect ant ⁇ -PR021 1 , ant ⁇ -PR0228, ant ⁇ -PR0538, ant ⁇ -PR0172 or ant ⁇ -PR0182 antibodies
  • the animals "positive" for antibodies can be injected with a final intravenous injection of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182
  • 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 hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids
  • HAT hyperxanthine, aminopterin, and thymidine
  • PR0172 or PR0182 Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PR0211 , PR0228, PR0538, PR0172 or PR0182 is within the skill in the art
  • the positive hybridoma cells can be injected lntrape ⁇ toneally into syngeneic Balb/c mice to produce ascites containmg the ant ⁇ -PR021 1 , ant ⁇ -PR0228, ant ⁇ -PR0538, ant ⁇ -PR0172 or ant ⁇ -PR0182 monoclonal antibodies
  • the hybridoma cells can be grown in tissue culture flasks or roller bottles Purification ofthe 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 PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptides may be purified by a variety of standard techniques in the art of protein purification
  • pro-PR021 1 , pro-PR0228, pro- PR0538, pro-PR0172 or pro-PR0182 polypeptide, mature PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide, or pre-PR021 1 , pre-PR0228, pre-PR0538, pre-PR0172 or pre-PR0182 polypeptide is purified by immunoaffimty chromatography using antibodiesspecificforthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide of interest
  • an immunoaffimty column is constructed by covalently coupling the ant ⁇ -PR021 1, ant ⁇ -PR0228, ant ⁇ -PR0538, ant ⁇ -PR0172 or ant ⁇ -PR0182 polypeptide antibody to an activated
  • 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 monoclonalantibodiesare prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSETM (Pharmacia LKB Biotechnology) The antibody is coupled to the resm, the resm is blocked, and the derivative resin is washed according to the manufacturer's instructions Such an immunoaffimty column is utilized in the purification ofthe PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 polypeptide by preparing a fraction from cells containing the PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide in a
  • a soluble PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide-contai ng preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/PR021 1 , antibody/PR0228, antibody/PR0538, antibody /PRO 172 or antibody/PRO 182 polypeptide binding (e.g.
  • a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion
  • a chaotrope such as urea or thiocyanate ion
  • EXAMPLE 8 Drug Screening This invention is particularly useful for screening compounds by using PR021 1 , PR0228, PR0538, PR0172 or PROl 82 polypeptides or a binding fragment thereof in any of a variety of drug screening techniques.
  • the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide or fragment.
  • Drugs are screened against such transformed cells in competitive binding assays.
  • Such cells can be used for standard binding assays.
  • One may measure, for example, the formation of complexes between a PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide or a fragment and the agent being tested.
  • one can examine the diminution in complex formation between the PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide and its target cell or target receptors caused by the agent being tested.
  • the present invention provides methods of screening for drugs or any other agents which can affect a PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 polypeptide-associated disease or disorder.
  • These methods comprise contacting such an agent with a PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide or fragment, or (ii) for the presence of a complex between the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide or fragment and the cell, by methods well known in the art.
  • the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide or fragment is typically labeled.
  • the free PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure ofthe ability ofthe particular agent to bind to the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide or to interfere with the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide/cell complex.
  • peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • a solid substrate such as plastic pins or some other surface.
  • the peptide test compounds are reacted with the PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide and washed.
  • Bound PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide is detected by methods well known in the art Purified PR021 1 , PR0228, PR0538, PR0172 or PR0182 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 sohd ⁇ upport
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide specifically compete with a test compound for binding to the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide or fragments thereof In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide
  • the goal of rational drug design is to produce structural analogs of a biologically active polypeptide of interest ( ⁇ e , a PR021 1, PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide or which enhance or interfere with the function ofthe PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide in vivo (cf Hodgson, Bio/Technology, 9 19-21 (1991))
  • the three-dimensional structure ofthe PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 polypeptide, or of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide-inhibitorcomplex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination ofthe two approaches Both the shape and charges ofthe PR0211, PR0228, PR0538, PR0172 or PR0182 polypeptide must be ascertained to elucidate the structure and to determine active s ⁇ te(s) ofthe molecule Less often, useful information regarding the structure of the PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide may be gained by modeling based on the structure of homologous proteins In both cases, relevant structural information is used to design analogous PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide-hke molecules or to identify efficient inhibitors Useful examples
  • PRO 182 polypeptide may be made available to perform such analytical studies as X-ray crystallography In addition, knowledge ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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
  • the antiprohferative activity ofthe PR021 1 , PR0228, PR0538, PRO 172 and PRO 182 polypeptides was determined in the investigational, disease-oriented in vitro anti-cancer drug discovery assay ofthe National Cancer Institute (NCI), using a sulforhodamme B (SRB) dye binding assay essentially as described by Skehan et al , J_ Natl Cancer Inst , 82 1 107-1 1 12 (1990)
  • SRB sulforhodamme B
  • a test sample is considered positive if it shows at least 40% growth inhibitory effect at one or more concentrations
  • PRO 172 1.25 nM 6 Leukemia CCRF-CEM

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 objective ofthe present invention to identify compounds capable of inhibiting the growth of neoplastic cells, such as cancer cells.
SUMMARY OF THE INVENTION
A. Embodiments
The present invention relates to methods and compositions for inhibiting neoplastic cell growth. More particularly, the invention concerns methods and compositions for the treatment of tumors, including cancers, such as breast, prostate, colon, lung, ovarian, renal and CNS cancers, leukemia, melanoma, etc., in mammalian patients, preferably humans.
In one aspect, the present invention concerns compositions of matter useful for the inhibition of neoplastic cell growth comprising an effective amount of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide as herein defined, or an agonist thereof, in admixture with a pharmaceutically acceptable carrier. In a preferred embodiment, the composition of matter comprises a growth inhibitory amount of a PR021 1 , PR0228, PR0538,
PRO 172 or PRO 182 polypeptide, or an agonist thereof. In another preferred embodiment, the composition compπses a cytotoxic amount of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 m a mammal comprising a therapeutically effective amount of a PR021 1 , PR0228, PR0538, PR0172 or PRO 182 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide as herein defined, or an agonist thereof In a particular embodiment, the agonist is an antι-PR021 1 , antι-PR0228, antι-PR0538, antι-PR0172 or antι-PR0182 agonist antibody In another embodiment, the agonist is a small molecule that mimics the biological activity of a PR021 1 , PR0228, PR0538, PR0172 or PR0182 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 m the composition is a PR021 1, PR0228, PR0538, PR0172 or PR0182 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 PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide or agonist thereof, for the inhibition of neoplastic cell growth, wherein the agonist may be an antibody which binds to the PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide
In a particular embodiment, the agonist is an antι-PR021 1 , antι-PR0228, antι-PR0538, anti-PROl 72 or anti-PROl 82 agonist antibody In another embodiment, the agonist is a small molecule that mimics the biological activity of a PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide Similar articles of manufacture compnsmg a PR0211 , PR0228, PR0538, PRO 172 or PRO 182 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 compπsing a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide
In one aspect, the isolated nucleic acid molecule compπses a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to (a) a DNA molecule encoding a PR021 1 , PR0228, PR0538, PR0172 or PR0182 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 atransmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment ofthe full-length amino acid sequence as disclosed herein, or (b) the complement ofthe 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide cDNA as disclosed herein, the coding sequence of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 ofthe DNA molecule of (a).
In a further aspect, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any ofthe human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement ofthe DNA molecule of (a).
Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide are disclosed herein. Therefore, soluble extracellular domains ofthe herein described PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptides are contemplated.
Another embodiment is directed to fragments of a PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PR021 1 , anti-PR0228, anti-PR0538, anti-PROl 72 or anti-PROl 82 antibody or as antisense oligonucleotide probes. Such nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 60 nucleotides in length, yet more preferably at least about 70 nucleotides in length, yet more preferably at least about 80 nucleotides in length, yet more preferably at least about 90 nucleotides in length, yet more preferably at least about 100 nucleotides in length, yet more preferably at least about 1 10 nucleotides in length, yet more preferably at least about 120 nucleotides in length, yet more preferably at least about 130 nucleotides in length, yet more preferably at least about 140 nucleotides in length, yet more preferably at least about 150 nucleotides in length, yet more preferably at least about 160 nucleotides in length, yet more preferably at least about 170 nucleotides in length, yet more preferably at least about 180 nucleotides in length, yet more preferably at least about 190 nucleotides in length, yet more preferably at least about 200 nucleotides in length, yet more preferably at least about 250 nucleotides in length, yet more preferably at least about 300 nucleotides in length, yet more preferably at least about 350 nucleotides in length, yet more preferably at least about 400 nucleotides in length, yet more preferably at least about 450 nucleotides in length, yet more preferably at least about 500 nucleotides in length, yet more preferably at least about 600 nucleotides in length, yet more preferably at least about 700 nucleotides in length, yet more preferably at least about 800 nucleotides in length, yet more preferably at least about 900 nucleotides in length and yet more preferably at least about 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length. It is noted that novel fragments of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PR0211 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide fragments that comprise a binding site for an anti-PR021 1, anti-PR0228, anti-PR0538, anti-PR0172 or anti-PR0182 antibody.
In another embodiment, the invention provides isolated PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide encoded by any ofthe isolated nucleic acid sequences hereinabove identified.
In a certain aspect, the invention concerns an isolated PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide, comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93%o 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%ι sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 ofthe full-length amino acid sequence as disclosed herein.
In a further aspect, the invention concerns an isolated PR021 1 , PR0228, PR0538, PROl 72 or PRO 182 polypeptide comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85%) sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87%o 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 PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 81 % positives, more preferably at least about 82% positives, yet more preferably at least about 83% positives, yet more preferably at least about 84% positives, yet more preferably at least about 85% positives, yet more preferably at least about 86% positives, yet more preferably at least about 87% positives, yet more preferably at least about 88% positives, yet more preferably at least about 89%> positives, yet more preferably at least about 90%> positives, yet more preferably at least about 91 % positives, yet more preferably at least about 92% positives, yet more preferably at least about 93% positives, yet more preferably at least about 94% positives, yet more preferably at least about 95% positives, yet more preferably at least about 96% positives, yet more preferably at least about 97% positives, yet more preferably at least about 98%) positives and yet more preferably at least about 99%> positives when compared with the amino acid sequence of a PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide having a full-length amino acid sequence as disclosed herein, an am o acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein
In a specific aspect, the invention provides an isolated PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described Processes for producing the same are also herein described, wherein those processes comprise cultuπng a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide and recovering the PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 polypeptide from the cell culture
Another aspect ofthe invention provides an isolated PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated Processes foi producing the same are also herein described, wherein those processes comprise cultuπng a host cell comprising a vector which comprises the appropπate encoding nucleic acid molecule under conditions suitable for expression of the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide and recovering the PR021 1 PR0228, PR0538, PROl 72 or PROl 82 polypeptide from the cell culture
In yet another embodiment, the invention concerns agonists of a native PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide as defined herein In a particular embodiment, the agonist is an antι-PR021 1 , antι-PR0228, antι-PR0538, anti-PROl 72 or anti-PROl 82 antibody or a small molecule In a further embodiment, the invention concerns a method of identifying agonists to a PR021 1 , PR0228
PR0538, PRO 172 or PRO 182 polypeptide which comprise contacting the PR021 1, PR0228, PR0538, PRO 172 or PROl 82 polypeptide with a candidate molecule and monitoring a biological activity mediated by said PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide Preferably, the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide is a native PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide In a still further embodiment, the invention concerns a composition of matter comprising a PR021 1 ,
PR0228, PR0538, PRO 172 or PRO 182 polypeptide, or an agonist of a PR021 1 , PR0228, PR0538, PRO 172 or PR0182 polypeptide as herein described, or an antι-PR021 1 , antι-PR0228, antι-PR0538, antι-PRO! 72 or anti- PROl 82 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 PR021 1, PR0228, PR0538,
PRO 172 or PRO 182 polypeptide, or an agomstthereofas hereinbefore described, or an antι-PR021 1 , antι-PR0228, antι-PR0538, anti-PROl 72 or anti-PROl 82 antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide, an agonist thereof or an antι-PR021 1, antι-PR0228, antι-PR0538, antι-PR0172 or antι-PR0182 antibody
In other embodiments ofthe present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides Host cell comprising any such vector are also provided By way of example, the host cells may be CHO cells, E colt, yeast, or Baculovirus-infected insect cells A process for producing any ofthe herein described polypeptides is further provided and compπses culturing host cells under conditions suitable for expression ofthe desired polypeptide and recovering the desired polypeptide from the cell culture
In other embodiments, the invention provides chimenc molecules comprising any ofthe herein described polypeptides fused to a heterologous polypeptide or amino acid sequence Example of such chimenc molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin
In another embodiment, the invention provides an antibody which specifically binds to any ofthe above or below described polypeptides Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody In yet other embodiments, the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any ofthe 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 PR021 1 cDN A, wherein SEQ ID NO 1 is a clone designated herein as "DN A32292- 1 131 "
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
Figures 3A-3B show a nucleotide sequence (SEQ ID NO 6) of a native sequence PR0228 cDNA, wherem SEQ ID NO 6 is a clone designated herein as "DNA33092-1202" Figure 4 shows the amino acid sequence (SEQ ID NO 7) derived from the coding sequence of SEQ ID
NO 6 shown in Figures 3A-3B
Figure 5 shows a nucleotide sequence (SEQ ID NO 15) of a native sequence PR0538 cDNA, wherein SEQ ID NO 15 is a clone designated herein as "DNA48613-1268"
Figure 6 shows the amino acid sequence (SEQ ID NO 16) derived from the coding sequence of SEQ ID NO 15 shown in Figure 5
Figures 7A-7B show a nucleotide sequence (SEQ ID NO 20) of a native sequence PRO 172 cDNA, wherein SEQ ID NO 20 is a clone designated herein as "DNA35916-1 161 "
Figure 8 shows the amino acid sequence (SEQ ID NO 21) derived from the coding sequence of SEQ ID NO 20 shown in Figures 7A-7B
Figure 9 shows a nucleotide sequence (SEQ ID NO 25) of a native sequence PRO 182 cDNA, wherein SEQ ID NO 25 is a clone designated herein as "DN A27865- 1091 "
Figure 10 shows the amino acid sequence (SEQ ID NO 26) derived from the coding sequence of SEQ ID NO 25 shown in Figure 9
DETAILED DESCRIPTION OF THE INVENTION
The terms "PR0211 ", "PR0228", " PR0538", "PRO 172" or "PROl 82" polypeptide orprotein when used herein encompass native sequence PR021 1, PR0228, PR0538, PRO 172 and PROl 82 variants (which are further defined herein) The PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1 ", "native sequence PR0228", "native sequence PR0538" "native sequence PRO 172" or "native sequence PROl 82" comprises a polypeptide having the same amino acid sequence as the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide as derived from nature Such native sequence PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide can be isolated from nature or can be produced by recombinant and/or synthetic means The term "native sequence" PR021 1, PR0228, PR0538, PR0172 or PROl 82 specifically encompasses naturally-occurring truncated or secreted forms (e g , an extracellular domain sequence), naturally-occurring variant forms (e g , alternatively spliced forms) and naturally-occurring allelic variants of the PR021 1, PR0228, PR0538, PR0172 and PR0182 polypeptides In one embodiment of the invention, the native sequence PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide is a mature or full- length native sequence PR0211 , PR0228, PR0538, PR0172 or PR0182 polypeptide as shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7), Figure 6 (SEQ ID NO 16) Figure 8 (SEQ ID NO 21 ) or Figure 10 (SEQ ID NO 26), respectively Also, while the PR021 1 , PR0228, PR0538, PRO 172 and PRO 182 polypeptides disclosed in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7), Figure 6 (SEQ ID NO 16), Figure 8 (SEQ ID NO 21 ) and Figure 10 (SEQ ID NO 26), respectively, are shown to begin with the methionine residue designated therein as amino acid position 1 , it is conceivable and possible that another methionine residue located either upstream or downstream from amino acid position 1 in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7), Figure 6 (SEQ ID NO 16), Figure 8 (SEQ ID NO 21 ) or Figure 10 (SEQ ID NO 26), respectively, may be employed as the starting ammo acid residue for the PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide
The ' extracellular domain" or "ECD" of a polypeptide disclosed herein refers to a form ofthe polypeptide which is essentially tree ofthe transmembrane and cytoplasmic domains Ordinarily, a polypeptide ECD will have less than about 1% of such transmembrane and/or cytoplasmic domains and preferably, will have less than about 0 5% of such domains It will be understood that any transmembrane domaιn(s) identified for the polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified and as shown in the appended figures. As such, in one embodiment ofthe present invention, the extracellular domain of a polypeptide ofthe present invention comprises amino acids 1 to X ofthe mature amino acid sequence, wherein X is any amino acid within 5 amino acids on either side ofthe extracellular domain/transmembrane domain boundary.
The approximate location ofthe "signal peptides" ofthe 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 ofthe signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary ofthe signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g. , Nielsen et al, Prot. Eng.. 10:1-6 (1997) and von Heinje et al, Nucl. Acids. Res., 14:4683-4690 (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.
"PR021 1 variant polypeptide" means an active PR021 1 polypeptide (other than a native sequence PR021 1 polypeptide) as defined below, having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 25 to 353 ofthe PR021 1 polypeptide shown in Figure 2 (SEQ ID NO:2), (b) X to 353 ofthe PR021 1 polypeptide shown in Figure 2 (SEQ ID NO:2), wherein X is any amino acid residue from 20 to 29 of Figure 2 (SEQ ID NO:2) or (c) another specifically derived fragment ofthe amino acid sequence shown in Figure 2 (SEQ ID NO:2).
"PR0228 variant polypeptide" means an active PR0228 polypeptide (other than a native sequence PR0228 polypeptide) as defined below, having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 20 to 690 ofthe PR0228 polypeptide shown in Figure 4 (SEQ ID NO:7), (b) X to 690 ofthe PR0228 polypeptide shown in Figure 4 (SEQ ID NO:7), wherein X is any amino acid residue from 15 to 24 of Figure 4 (SEQ ID NO:7), (c) 1 or about 20 to X of Figure 4 (SEQ ID NO:7), wherein X is any amino acid from amino acid 425 to amino acid 434 of Figure 4 (SEQ ID NO:7) or (d) another specifically derived fragment ofthe amino acid sequence shown in Figure 4 (SEQ ID NO:7).
"PR0538 variant polypeptide" means an active PR0538 polypeptide (other than a native sequence PR0538 polypeptide) as defined below, having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 27 to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO: 16), (b) X to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO: 16), wherein X is any amino acid residue from 22 to 31 of Figure 6 (SEQ ID NO: 16), (c) 1 or about 27 to X of Figure 6 (SEQ ID NO: 16), wherein X is any amino acid from amino acid 374 to amino acid 383 of Figure 6 (SEQ ID NO: 16) or (d) another specifically derived fragment ofthe amino acid sequence shown in Figure 6 (SEQ ID NO: 16).
"PRO 172 variant polypeptide" means an active PRO 172 polypeptide (other than a native sequence PR0172 polypeptide) as defined below, having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 22 to 723 ofthe PROl 72 polypeptide shown in Figure 8 (SEQ ID NO 21), (b) X to 723 ofthe PRO 172 polypeptide shown in Figure 8 (SEQ ID NO 21 ), wherein X is any amino acid residue from 17 to 26 of Figure 8 (SEQ ID NO 21), (c) 1 or about 22 to X of Figure 8 (SEQ ID NO 21), wherein X is any am o acid from amino acid 543 to amino acid 552 of Figure 8 (SEQ ID NO 21 ) or (d) another specifically derived fragment ofthe amino acid sequence shown in Figure 8 (SEQ ID NO 21 )
"PRO 182 variant polypeptide" means an active PRO 182 polypeptide (other than a native sequence PROl 82 polypeptide) as defined below, having at least about 80% ammo acid sequence identity with the amino acid sequence of (a) residues 1 or about 19 to 135 ofthe PRO 182 polypeptide shown in Figure 10 (SEQ ID NO 26), (b) X to 135 ofthe PROl 82 polypeptide shown in Figure 10 (SEQ ID NO 26), wherein X is any ammo acid residue from 14 to 23 of Figure 10 (SEQ ID NO 26) or (c) another specifically derived fragment ofthe amino acid sequence shown in Figure 10 (SEQ ID NO 26)
Such PR021 1 , PR0228, PR0538, PRO 172 and PRO 182 variants include, for instance, PR021 1 , PR0228, PR0538, PR0172 and PR0182 polypeptides wherein one or more am o acid residues are added, or deleted, at the N- or C-terminus, as well as within one or more internal domains ofthe native sequence Ordinarily, a PR021 1 variant will have at least about 80%> amino acid sequence identity, more preferably at least about 81 % amino acid sequence identity, more preferably at least about 82%o amino acid sequence identity, more preferably at least about 83%o 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%o amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91%) amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93%> amino acid sequence identity, more preferably at least about 94%> amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 or about 25 to 353 of the PR021 1 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) X to 353 ofthe PR021 1 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any amino acid residue from 20 to 29 of Figure 2 (SEQ ID NO 2) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2)
Ordinarily, a PR0228 variant will have at least about 80% 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% ammo acid sequence identity, more preferably at least about 84% ammo acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% ammo acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% ammo 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%> am o acid sequence identity, more preferably at least about 92%> amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% ammo acid sequence identity, more preferably at least about 98%. amino acid sequence identity and yet more preferably at least about 99%> amino acid sequence identity with (a) residues 1 or about 20 to 690 ofthe PR0228 polypeptide shown in Figure 4 (SEQ ID NO 7), (b) X to 690 ofthe PR0228 polypeptide shown in Figure 4 (SEQ ID NO 7), wherein X is any amino acid residue from 15 to 24 of Figure 4 (SEQ ID NO 7), (c) 1 or about 20 to X of Figure 4 (SEQ ID NO 7), wherein X is any am o acid from ammo acid 425 to ammo acid 434 of Figure 4 (SEQ ID NO 7) or (d) another specifically derived fragment ofthe am o acid sequence shown in Figure 4 (SEQ ID NO 7) Ordinarily, a PR0538 variant will have at least about 80% am o 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%. am o 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%> am o acid sequence identity, more preferably at least about 88%> amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91%) amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% am o acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% ammo 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 99% amino acid sequence identity with (a) residues 1 or about 27 to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO 16), (b) X to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO 16), wherein X is any amino acid residue from 22 to 31 of Figure 6 (SEQ ID NO 16), (c) 1 or about 27 to X of Figure 6 (SEQ ID NO 16), wherein X is any amino acid from amino acid 374 to amino acid 383 of Figure 6 (SEQ ID NO 16) or (d) another specifically derived fragment ofthe amino acid sequence shown in Figure 6 (SEQ ID NO 16)
Ordinarily, a PROl 72 variant will have at least about 80% 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 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% ammo acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 or about 22 to 723 of the PRO 172 polypeptide shown in Figure 8 (SEQ ID NO 21 ), (b) X to 723 ofthe PRO 172 polypeptide shown in Figure 8 (SEQ ID NO 21 ), wherein X is any amino acid residue from 17 to 26 of Figure 8 (SEQ ID NO 21 ), (c) 1 or about 22 to X of Figure 8 (SEQ ID NO 21 ), wherein X is any amino acid from amino acid 543 to amino acid 552 of Figure 8 (SEQ ID NO 21 ) or (d) another specifically derived fragment ofthe amino acid sequence shown in Figure 8 (SEQ ID NO 21 )
Ordinarily, a PROl 82 variant will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% am o 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% am o 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%ι amino acid sequence identity, more preferably at least about 92% ammo 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% ammo 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 99% amino acid sequence identity with (a) residues 1 or about 19 to 135 ofthe PRO 182 polypeptide shown in Figure 10 (SEQ ID NO 26), (b) X to 135 ofthe PR0182 polypeptide shown in Figure 10 (SEQ ID NO 26), wherein X is any ammo acid residue from 14 to 23 of Figure 10 (SEQ ID NO 26) or (c) another specifically derived fragment ofthe amino acid sequence shown in Figure 10 (SEQ ID NO 26)
Ordinarily, PR021 1 , PR0228, PR0538, PR0172 and PR0182 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 am o acids in length, more often at least about 40 amino acids in length, more often at least about 50 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 ammo acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, more often at least about 250 ammo acids in length, more often at least about 300 amino acids in length, or more As shown below, Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program
In addition, Tables 2A-2D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid sequence identity (Tables 2C-2D) using the ALIGN-2 sequence comparison computer program, wherein "PRO" represents the amino acid sequence of a hypothetical PEACH 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 PROXXX- or PROXXX-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,-l, 1,0,0,0,0,-2,-5,0,-3, 1}, /*C */ [-2,-4,15,-5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4 ,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0, 0,-5}, /*D */ 0,3,-5,4,3,-6, 1, 1,-2,0,0,-4,-3,2, _M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 2}, /*E */ 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1, M.-l, 2,-1, 0, 0, 0,-2,-7, 0,-4, 3}, /* p */ -4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4," Vl,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5},
/*G */ 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,' _M,-l,-l,-3, 1,0,0,-1,-7,0,-5,0},
/*H */ -1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2.-2, 2,[ _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},
/*] */ 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},
/*0*/ M,_M, M, M, M,_M, M, M, M " M,_M, M, M, M,
0,_M,_M M,_M,_M,_M,_M,_M,_M,_M,_M},
I* p */ 1,-1,-3,-1,-1,-5,-1, 0,-2, 0,-l,-3,-2.-l,_M, 6, 0,0, 1,0,0,-1,-6,0,-5,0}, /*Q */ 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1,_M, 0, 4, 1,-1,-1, 0,-2,-5,0,-4, 3}, /*R*/ -2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0, 0,_M, 0, 1,6,0,-1,0,-2,2,0,-4,0}, /*S */ 1, 0, 0, 0, 0,-3, 1,-1,-1, 0, 0,-3,-2, 1,_M, 1,- 1,0,2, 1,0,-1,-2,0,-3,0}, /* 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, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* v */ 0,-2,-2,-2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2,_M,-l, ,-2,-2,-1,0, 0,4,-6,0,-2,-2}, /* W */ -6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3,-2,-4.-4._M,-6 5, 2,-2,-5,0.-6,17,0, 0,-6},
/*x */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/* Y *ι -3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-4,-1, -2.-2,_M,-5 ,-4,-4,-3,-3,0,-2,0,0,10,-4},
/*z *l 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}
/* */
^include < stdιo.h > #include < ctype.h >
#define MAXJMP 16 /* max jumps in a diag */
-. define MAXGAP 24 /* don't continue to penalize gaps larger than this */
#define JMPS 1024 /* max jmps in an path */
#define MX 4 /* save if there's at least MX-1 bases since last jmp */
#define DMAT 3 /* value of matching bases */
#define DMIS 0 /* penalty for mismatched bases */
#define DINSO 8 /* penalty for a gap */
#define DINS1 1 /* penalty per base */
#define PINSO 8 /* penalty for a gap */
#define PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP]; /* size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. of jmp in seq x */
}; /* limits seq to 2A16 -1 */ struct diag { int score; /* score at last jmp */ long offset; /* offset ot prev block */ short ljmp; /* current jmp index */ struct jmp jp; /* list of jmps */
}; struct path { int spc; /* number of leading spaces */ short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (last elem before gap) */
}; char *ofιle; /* output file name */ char *namex[2]; /* seq names: getseqs() */ char *prog; /* prog name for err msgs */ char *seqx[2]; /* seqs: getseqs() */ int dmax; /* best diag- nw() */ int dmaxO, /* final diag */ int dna, /* set if dna: maιn() */ int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* total gaps in seqs */ int lenO, lenl ; /* seq lens */ int ngapx, ngapy, /* total size of gaps */ int smax; /* max score: nw() */ int *xbm, /* bitmap for matching */ long offset; /* current offset in jmp file */ struct diag *dx; /* holds diagonals */ struct path PP12]; /* holds path for seqs */ char *calloc(), *malloc(), *mdex(), *strcpy(), char *getseq(), *g_calloc(). /* Needleman-Wunsch alignment program
*
* usage- progs filel fιle2
* where filel and fιle2 are two dna or two protein sequences
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with ',', ' > ' or ' < ' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align. out"
* The program may create a tmp file in /t p to hold info about traceback
* Original version developed under BSD 43 on a vax 8650 */
#include "nw h" ^include "day h" static _dbval[26] = {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
static _pbval[26] = {
1,2|(1<<('D'-,A'))|(1< <('N'-'A,)),4, 8, 16, 32, 64,
128, 256, OxFFFFFFF, 1< < 10, 1< < 11, 1< < 12, 1< < 13, 1< < 14,
1< < 15, 1< < 16, 1< < 17, 1< < 18, 1< < 19, 1< <20, 1< <21, 1< <22,
1<<23, 1<<24, 1<<25|(1<<('E'-'A'))|(1<<(,Q'-'A'))
Figure imgf000018_0001
int ac, char *av[], prog = av[0], if (ac »= 3) {
.pπntf(stderr, "usage %s filel fιle2\n", prog), fpπntf(stderr, "where filel and fιle2 are two dna or two protein sequences \n"), fpπntf(stderr, "The sequences can be in upper- or lower case\n"), fpπntf(stderr, "Any lines beginning with ,' or < ' are ιgnored\n"), fpπntf(stderr, "Output is in the file \ align outV \n ) exιt(l),
} namex[0] = av[l], namex[l] = av[2], seqx[0] = getseq(namex[0], &len0), seqx[l] = getseq(namex[l], &lenl), xbm = (dna)9 dbval _pbval endgaps = 0, /* 1 to penalize endgaps *l 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 tiles */ /* 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, I* score for each type */ int insO, msl , /* insertion penalties */ register id, /* diagonal index */ register 'J> /* jmp index */ register *col0, *coll , /* score for curr, last row */ register χχ ; yy, /* index into seqs */ dx = (struct diag *)g_calloc("to get diags", lenO+lenl + 1 , sizeof(struct diag)), ndely = (int *)g_calloc("to get ndely" , lenl + 1 , sizeof(int)), dely = (int *)g_calloc(' to get dely", lenl + 1 , sizeof(int)), colO = (int *)g_calloc("to get colO" , lenl + 1 , sizeof(int)) , coll = (int *)g_calloc("to get col l " , lenl + 1 , sizeof(int)), msO = (dna)9 DINSO PINSO, msl = (dna)9 DINS 1 • PINS1 , smax = -10000, if (endgaps) { for (col0[0] = dely[0] = -insO, yy = 1, yy < = lenl , yy+ +) { col0[yy] = delyhyy] = col0[yy-l] - msl ndely [yy] = yy,
} col0[0] = 0, /* Waterman Bull Math Biol 84 */
} else for (yy = 1 , yy < = lenl , yy + +) delylyy] = -insO,
/* fill in match matrix */ for (px = seqx[0], xx = 1 , xx < = lenO, px+ + , xx+ +) { /* initialize first entry m col
*/ if (endgaps) { if (xx = = 1) coll [0] = = delx = = -(insO + insI), else col l [0] = = delx = = col0[0) - msl ndelx = xx
} else { col l [0J = 0,
Figure imgf000019_0001
ndelx = 0 ...nw for (py = seqx[l], yy = 1 ; yy < = lenl ; py + + , yy + +) { mis = col0[yy-l]; if (dna) mis + = (xbm[*px-*A']&xbm[*py-'A'])? DMAT : DMIS; else mis + = _day[*px-'A'][*py-'A'];
/* update penalty for del in x seq;
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps */ if (endgaps | | ndely[yy] < MAXGAP) { if (colO /y] - insO > = dely[yy]) { delylyy] = col0[yy] - (insO + insI); ndely [yy] = 1 ; } else { dely[yy] -= msl ; ndely [yy] + + ;
} } else { if (colOfyy] - (msO+ insl) > = delylyy]) { delylyy] = colOfyy] - (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 -= msl ; ndelx + + ;
} } else { if (coll [yy-l] - (msO + insl) > = delx) { delx = coll [yy-l ] - (msO + insl). ndelx = 1 , } else ndelx + + , }
/* pick the maximum score; we're favoring
* mis over any del and delx over dely
*/ ...n id = xx - yy + lenl - 1 if (mis > = delx && mis > = dely[yy]) collfyy] = mis, else if (delx > = dely[yy]) { collfyy] = delx, IJ = dxfid] ljmp, if (dxfid] jp n[0] && ('dna | | (ndelx > = MAXJMP && xx > dxfid] jp x[ιj]+MX) | | mis > dxfid] score+DINSO)) { dxfid] πmp+ + , if (++ιj >= MAXJMP) { wπtejmps(ιd), ij = dxfid] lj p = 0, dxfid] offset = offset, offset += sizeof (struct jmp) + sizeof (off set), } } dxfid] jp nfij] = ndelx, dxfid] jp xfij] = XX. dxfid] score = delx,
} else { collfyy] = delyfyy], ij = dxfid] ljmp, if (dxfid] jp n[0] && ('dna 11 (ndelyfyy] > = MAXJMP
&& xx > dxfid] jp x[ιj]+MX) | | mis > dxfid] score+DINSO)) { dxfid] ιjmp+ + , if (++ιj >= MAXJMP) { wπtejmps(ιd), ij = dxfid] ljmp = 0, dxfid] offset = offset, offset + = sizeof (struct jmp) + sizeof (off set), } } dxfid] jp nfij] = -ndelyfyy], dxfid] jp xfij] = xx dxfid] score = delyfyy],
} if (xx = = lenO && yy < lenl) { /* last col */ if (endgaps) collfyy] -= ιnsO+ιnsl*(lenl-yy) if (collfyy] > s ax) { smax = collfyy] dmax = id,
} } } if (endgaps && xx < lenO) collfyy 1] -= ιnsO + ιnsl*(lenO-xx), if (collfyy 1] > smax) { smax = coll[yy-l] dmax = id,
} tmp = colO, colO = coll, coll = tmp
}
(void) free((char *)ndely), (void) free((char *)dely), (void) free((char *)col0) (void) free((char *)coll), /* *
* pπnt() — only routine visible outside this module
*
* static:
* getmat() — trace back best path, count matches: pπnt()
* pr_alιgn() — print alignment of described in array pf]: pπnt()
* dumpblockO — dump a block of lines with numbers, stars. pr_alιgn()
* nums() - put out a number line: dumpblockO
* putlmeO — put out a line (name, [num], seq, [num]). dumpblockO
* stars() - -put a line of stars: dumpblockO
* stripnameO ~ strip any path and prefix from a seqname */
/ include "nw h"
#define SPC 3
^define P LINE 256 /* maximum output line */
^define P SPC 3 /* space between name or num and seq */ extern day [26] [26] ; int olen, /* set output line length */
FILE *fx, /* output file */ pnnto 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(fx, " < first sequence: %s (length = %d)\n", namex[0], lenO), fpπntf(fx, " < second sequence- s (length = %d)\n' , namexfl], lenl), olen = 60, lx = lenO, ly = lenl ; firstgap = lastgap = 0, if (dmax < lenl - 1) { /* leading gap in x */ pp[0].spc = firstgap = lenl - dmax - 1 , ly -= pp[0] .spc,
} else if (dmax > lenl - 1) { /* leading gap in y */ pp[l].spc = firstgap = dmax - (lenl - 1), lx -= pp[l] .spc;
} if (dmaxO < lenO - 1) { /* trailing gap in x */ lastgap = lenO - dmaxO -1 ; lx - = lastgap,
} else if (dmaxO > lenO - 1) { /* trailing gap in y */ lastgap = dmaxO - (lenO - 1), ly - = lastgap,
} getmat(lx, ly, firstgap, lastgap). pr_alιgn(), * trace back the best path, count matches
*/ static getmat(l; ., ly, first gap, lastgap) getmat int lx, iy. /* "core" (minus endgaps) */ int firstgap, lastgap, /* leading trailing overlap */
{ int nm, lO, il sizO, sizl , char outx[32], double pet, register nO, nl , register char *p0, *pl ,
/* get total matches, score */
Figure imgf000023_0001
pO = seqx[0] + pp[l] spc, pi = seqx[l] + pp[0] spc, nO = ppfl] spc + 1 , nl = pp[0].spc + 1 ,
= o, lie (
Figure imgf000023_0002
} else if i (sizl) { pθ+ + , n0+ + ,
Figure imgf000023_0003
} else { if (xbm[*pO-'A']&xbm[*pl-'A']) nm+ + ,
Figure imgf000023_0004
}
/* pet homology
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core */ if (endgaps) lx = (lenO < lenl)9 lenO lenl , else lx = (lx < ly)9 lx ly, pet = 100 *(double)nm/(double)lx, fpπntf(fx, "\n' ), fpπntf(fx, " < %d match%s in an overlap of %d % 2f percent similarity \n" nm, (nm = = l)9 ' es", lx, pet), fpπntf(fx, " < gaps in first sequence: %d", gapx), .getmat if (gapx) {
(void) spπntf(outx, " (%d %s%s)", ngapx, (dna)? "base" : "residue", (ngapx = = 1)? :"s"); fpπntf(fx, " %s", outx); fprintf(fx, ", gaps in second sequence: %d" , gapy); if (gapy) {
(void) spπntf(outx, " (%d %s%s)" , ngapy, (dna)? "base" : "resιdue", (ngapy = = l)9 : "s"); fpπntf(fx," %s" , outx);
} if (dna) fpπntf(fx,
"\n < core: %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 n.[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 oouutt[[22]][[P_LINE]; /* output line */ static char starfP E]; /* set by stars() */
/*
* print alignment of described in struct path ppf]
*/ static pr ahgn() pr align
{ int nn; /* char count */ int more, register 1 , for (l = 0, l ax = 0; l < 2; ι + +) { nn = stπpname(namex[ι]), if (nn > lmax) lmax = nn;
Figure imgf000024_0001
sizfi] = ijfi] = 0; psfi] = seqxfi]; pofi] = outfi] , for (nn = nm = 0, more = 1 , more, ) { ...pr align for (l = more = 0, l < 2, ι+ +) { /* * do we have more of this sequence9
*/ if ('*ps[ι]) continue, more+ + , if (ppfl] spc) { /* leading space */ *po[ι] + + = ' , pp[ι] spc- ,
} else if (sιz[ι]) { /* in a gap */ *po[ι] + + = '-' , sizfi] -,
} else { /* we're putting a seq element
*/ *po[ι] = *ps[ι] if (ιslower(*ps[ι]))
*ps[ι] = toupper(*ps[ι]), po[ι] + + , ps[ι] + + ,
/*
* are we at next gap for this seq9 */ if (m[ι] = = ppfi] xfij [.]]) {
* we need to merge all gaps
* at this location */ sizfi] = ppfi] n[ιj[ι] + +] while (nι[ι] = = ppfi] xfijfi]]) sizfi] + = ppfi] n[ιj[ι] + +]
} nι[ι] + + ,
}
} if (+ +nn = = olen | | 'more && nn) { dumpblockO for (ι = 0, l < 2, ι + +) pofi] = outfi] , nn = 0, }
}
/*
* dump a block of lines, including numbers, stars pr_alιgn()
*/ static dumpblockO dumpblock
{ register 1, for (ι = 0, l < 2, ι + +) *po[ι]- = '\0 ...dumpblock
(void) putc('\n', fx), for(ι = 0, l < 2, ι++) { if (*out[ι] && (*out[ι] ' = *(pofι]) ')){ if (i == 0) nums(ι), if (l == 0 && *out[l]) stars(), putlιne(ι), if (l = = 0 && *out[l]) fpπntf(fx, star), if(ι == 1) nums(ι),
}
/*
* put out a number line. dumpblockO */ static nums(ιx) nums int lx, /* index in out|
{ char nlmefP LINE] register i.J. register char *pn, *px, *py, for (pn = nlme, l = 0, 1 < lmax+P SPC, ι++, pn++)
*pn = ' ', for (I = ncfix], py = outfix], *py, py+ + , pn++) { if (*py == ' ' I I *py == '-')
*pn = ' ', else { if (ι%10 == 0 I I (l == 1 &&nc[ιx] '= 1)) { j = (. < 0)9 -i i, for (px = pn, j j /= 10, px-)
*px = j%10 + '0 , if (l < 0)
*px = -
} else
*pn ι++,
}
}
*pn = '\0', ncfix] = l, for (pn = nline, *pn, pn+ +) (void) putc(*pn, tx), (void) putc('\n'. fx),
}
/*
* put out a line (name, fnum], seq, [num]) dumpblockO */ static puthne(ιx) putline int
{ .putline
Figure imgf000027_0001
register char *px, for (px = namexfix], I = 0, *px && *px ', px+ + , ι++)
(void) putc(*px ,fχ), for (, l < lmax+P SPC, 1++)
(void) putc(' ', fx),
/* these count from 1
* nif] is current element (from 1)
* ncf] is number at start of current line */ for (px = outfix], *px, px+ +)
(void) putc(*px&0x7F, fx), (void) putc('\n', fx),
/*
* put a line of stars (seqs always in outfO], outfi]) dumpblockO
*/ static stars() stars int l, register char *p0, *pl, ex, *px, if 0*out[0] I I (*out[0] == ' '&& *(po[0]) == ' ') I | '*out[l] 11 (*out[l] == • '&& *(po[l]) == ' ')) return, px = star, for (I = lmax+P SPC, l, ι~)
*px++ = ' , for(p0 = outfO], pi = out[l], *p0&&*pl, p0+ + ,pl + +) { if (ιsalpha(*pO) && ιsalpha(*pl)) { if (xbm[*pO-'A']&xbm[*pl-'A ]) { ex = '*', nm+ + ,
} else if ('dna && _day[*pO-'A'][*pl A'] > 0) ex = ' ', else ex
} else ex = *px+ + = ex,
}
*px++ = '\n', *px = '\0', /*
* strip path or prefix from pn, return len: pr_align()
*/ static stripname(pn) Stripnamβ 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));
/*
* cleanupO — cleanup any tmp file
* getseqO — read in seq, set dna, len, maxlen
* g_calloc() — calloc() with error checkm
* readjmpsO — get the good jmps, from tmp file if necessary
* writejmpsO — write a filled array of jmps to a tmp file nw() */
#include "nw h" #include < sys/file h > char *jname = "/tmp/homgXXXXXX" , /* tmp file for jmps */
FILE *fj, int cleanupO, /* cleanup tmp file */ long lseek(),
/*
* remove any tmp file if we blow cleanup(ι) cleanup int { if (fj)
(void) unlιnk(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(file, 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) { fpπntt(stderr, " %s can't read %s\n" , prog, file) exιt(l),
} tlen = natgc = 0, while (fgets(lιne, 1024, fp)) { if (*hne = = ' , ' | | *lιne = = ' < ' | | *lιne = = > ) continue, for (px = line, *px ' = '\n' , px+ +) if (ιsupper(*px) | | ιslower(*px)) tlen+ + ,
} if ((pseq = malloc((unsigned)(tlen + 6))) = = 0) { fpπntf(stderr, " %s malloc() failed to get %d bytes for %s\n' , prog, tlen + 6, file), exιt(l),
} pseqfO] = pseqfl] = pseq[2] = pseq[3] = '\0' , .getseq py = pseq + 4; *len = tlen; rewmd(fp); while (fgets(lιne, 1024, fp)){ if (*lιne == ' ;' |1 *lme == '<' | | *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_calloc char *msg; /* program, calling routine */ int nx, sz; /* number and size of elements */
{ char *px, *calloc(); if ((px = calloc((unsigned)nx, (unsigned)sz)) = = 0) { if (*msg) { fpπntf(stderr, "%s: g_calloc() failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz): exιt(l);
} } return(px),
/* * get final jmps from dxf] or tmp file, set ppf], reset dmax: maιn()
*/ readjmpsO readjmps
{ int fd = -1;
Figure imgf000030_0001
register I, j, xx, if(f)){
(void) fclose(fj): if ((fd = openOna e. O RDONLY, 0)) < 0) { fpπntf(stderr, "%s. can't open() %s\n". prog, jname), cleanup(l), } } for (I = .0 = il = 0, dmaxO = dmax, xx = lenO, ; ι++) { while (1) { for = dx[dmax].ιjmp, j > = 0 && dx[dmax].jp.x[j] > = xx; j~) ...readjmps if 0 < 0 && dxfdmax]. offset && fj) {
(void) lseek(fd, dxfdmax]. offset, 0),
(void) read(fd, (char *)&dx[dmax] jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax]. offset, sizeof (dxfdmax]. offset)); dxfdmax]. ljmp = MAXJMP- 1 ,
} else break;
} if (l > = JMPS) { fpπntf(stderr, " %s: too many gaps in alιgnment\n", prog); cleanupO );
} if 0 > = 0) { siz = dxfdmax] .jp.nfj]; xx = dx[dmax].jp.x[j],
Figure imgf000031_0001
if (siz < 0) { /* gap in second seq */
Figure imgf000031_0002
/* id = xx - yy + lenl - 1 */ pp[l] .x[ιl] = xx - dmax + lenl - 1 , gapy + + ; ngapy -= siz; /* ignore MAXGAP when doing endgaps */ siz = (-siz < MAXGAP | | endgaps)? -siz : MAXGAP; ιl + + ;
} else if (siz > 0) { /* gap in first seq */
Figure imgf000031_0003
gapx + + , ngapx + = siz, /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP | | endgaps)9 siz : MAXGAP, ι0+ + ; } } else break, }
/* reverse the order of jmps */ for 0 = 0, ι0~; j < ι0; j + + , ι0~) { i = pp[0].n[|], PPf0].n[j] = pp[0].n[ι0]; pp[0].n[ι0] = i;
. = pp[0].x[)]; pp[0].xU] = PPl0].x[i0]; pp[0] .x[ι0] = .,
} for (j = 0, il — , j < ιl ; j + + , il-) { i = pp[l].n ]; pp[l].n l = pp[l].n[il]; pp[l].n[ιl] = i, i = pp[i] .χ[j]; pp[i].χDl = PPM χ['U; pp[i] .χ[-i] = >,
} if (fd > = 0)
(void) close(fd), if (f)) {
(void) unlinkt name), fj = 0; offset = 0.
} /*
* write a filled jmp struct offset of the prev one (if any) nw() * *// wπtejmps(ιχ) writejmps
char *mktemp(), if ('fj) { if (mktemρ(jname) < 0) { fpπntf(stderr, " %s can't mktempO %s\n , prog, jname), cleanup(l),
} if ((fj = fopen(jname, "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(dxfιx] offset), 1 , fj),
Table 2A
PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide)
5 divided by 15 = 33.3%
Table 2B
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) =
5 divided by 10 = 50%
Table 2C
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =
6 divided by 14 = 42.9%
Table 2D
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =
4 divided by 12 = 33.3%
"Percent (%) amino acid sequence identity" with respect to the PR021 1 , PR0228, PR0538, PROl 72 and PROl 82 polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PR021 1 , PR0228, PR0538, PR0172 or PR0182 sequence, after a gningthe sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part ofthe sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill m the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) software Those skilled m the art can determine appropπate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length ofthe sequences being compared For purposes herein, however, % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc , and the source code shown in Table 1 has been filed with user documentation in the U S Copyright Office, Washington D C , 20559, where it is registered under U S Copyright Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech, Inc , South San Francisco, California or may be compiled from the source code provided in Table 1 The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary
For purposes herein, the %> amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of ammo acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the %> ammo acid sequence identity of B to A As examples of % amino acid sequence identity calculations, Tables 2A-2B demonstrate how to calculate the % amino acid sequence identity ofthe amino acid sequence designated "Comparison Protein" to the amino acid sequence designated "PRO"
Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program However, %> am mo acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul el al , Nucleic
Acids Res , 25 3389-3402 (1997)) The NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov NCB1-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0 01 , constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62
In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the %> amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given ammo acid sequence A that has or comprises a certain % ammo 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 ammo 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 ammo acid residues m B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the %> amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A
In addition, %> ammo 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, t 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 ofthe PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (t 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 ofthe PRO polypeptide of interest For example, in the statement 'a polypeptide comprising an amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B", the amino acid sequence A is the comparison amino acid sequence of interest and the amino acid sequence B is the am o acid sequence of the PRO polypeptide of interest
"PR021 1 variant polynucleotide" or "PR021 1 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PR021 1 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 25 to 353 ofthe PR021 1 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) a nucleic acid sequence which encodes amino acids X to 353 ofthe PR021 1 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any ammo acid residue from 20 to 29 of Figure 2 (SEQ ID NO 2) or(c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2) Ordinarily, a PR021 1 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81 % nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88%) nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90%> nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95%) nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98%o 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 25 to 353 ofthe PR021 1 polypeptide shown in Figure 2 (SEQ ID NO:2), (b) a nucleic acid sequence which encodes amino acids X to 353 of the PR021 1 polypeptide shown in Figure 2 (SEQ ID NO:2), wherein X is any amino acid residue from 20 to 29 of Figure 2 (SEQ ID NO:2) or (c) a nucleic acid sequence which encodes another specifically derived fragment ofthe amino acid sequence shown in Figure 2 (SEQ ID NO:2). PR021 1 polynucleotide variants do not encompass the native PR021 1 nucleotide sequence.
"PR0228 variant polynucleotide" or "PR0228 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PR0228 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 20 to 690 ofthe PR0228 polypeptide shown in Figure 4 (SEQ ID NO:7), (b) a nucleic acid sequence which encodes amino acids X to 690 ofthe PR0228 polypeptide shown in Figure 4 (SEQ ID NO:7), wherein X is any amino acid residue from 15 to 24 of Figure 4 (SEQ ID NO:7), (c) a nucleic acid sequence which encodes amino acids 1 or about 20 to X of Figure 4 (SEQ ID NO:7), wherein X is any amino acid from amino acid 425 to amino acid 434 of Figure 4 (SEQ ID NO:7) or (d) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO:7). Ordinarily, a PR0228 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81%> nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84%> nucleic acid sequence identity, more preferably at least about 85%o nucleic acid sequence identity, more preferably at least about 86%o 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%ι 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 20 to 690 ofthe PR0228 polypeptide shown in Figure 4 (SEQ ID NO:7), (b) a nucleic acid sequence which encodes amino acids X to 690 ofthe PR0228 polypeptide shown in Figure 4 (SEQ ID NO:7), wherein X is any amino acid residue from 15 to 24 of Figure 4 (SEQ ID NO:7), (c) a nucleic acid sequence which encodes amino acids 1 or about 20 to X of Figure 4 (SEQ ID NO:7), wherein X is any amino acid from amino acid 425 to amino acid 434 of Figure 4 (SEQ ID NO:7) or (d) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO:7). PR0228 polynucleotide variants do not encompass the native PR0228 nucleotide sequence.
"PR0538 variant polynucleotide" or "PR0538 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PR0538 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 27 to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO: 16), (b) a nucleic acid sequence which encodes amino acids X to 400 of the PR0538 polypeptide shown in Figure 6 (SEQ ID NO: 16), wherein X is any amino acid residue from 22 to 31 of Figure 6 (SEQ ID NO: 16), (c) a nucleic acid sequence which encodes amino acids 1 or about 27 to X of Figure 6 (SEQ ID NO:16), wherein X is any amino acid from amino acid 374 to amino acid 383 of Figure 6 (SEQ ID NO: 16) or (d) a nucleic acid sequence which encodes another specifically derived fragment ofthe amino acid sequence shown in Figure 6 (SEQ ID NO: 16). Ordinarily, a PR0538 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81 % nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86%o 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 27 to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO:16), (b) a nucleic acid sequence which encodes amino acids X to 400 ofthe PR0538 polypeptide shown in Figure 6 (SEQ ID NO: 16), wherein X is any amino acid residue from 22 to 31 of Figure 6 (SEQ ID NO: 16), (c) a nucleic acid sequence which encodes amino acids 1 or about 27 to X of Figure 6 (SEQ ID NO: 16), wherein X is any amino acid from amino acid 374 to amino acid 383 of Figure 6 (SEQ ID NO: 16) or (d) a nucleic acid sequence which encodes another specifically derived fragment ofthe amino acid sequence shown in Figure 6 (SEQ ID NO: 16). PR0538 polynucleotide variants do not encompass the native PR0538 nucleotide sequence.
"PR0172 variant polynucleotide" or "PR0172 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO 172 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 22 to 723 ofthe PROl 72 polypeptide shown in Figure 8 (SEQ ID NO 21), (b) a nucleic acid sequence which encodes amino acids X to 723 ofthe PRO 172 polypeptide shown in Figure 8 (SEQ ID NO 21), wherem X is any amino acid residue from 17 to 26 of Figure 8 (SEQ ID NO 21), (c) a nucleic acid sequence which encodes amino acids 1 or about 22 to X of Figure 8 (SEQ ID NO 21), wherein X is any ammo acid from ammo acid 543 to am o acid 552 of Figure 8 (SEQ ID NO 21 ) or (d) a nucleic acid sequence which encodes another specifically derived fragment ofthe ammo acid sequence shown in Figure 8 (SEQ ID NO 21 ) Ordinarily, a PRO 172 variant polynucleotide will have at least about 80%) nucleic acid sequence identity, more preferably at least about 81 % nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83%> nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86%> nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90%) nucleic acid sequence identity, more preferably at least about 91%> nucleic acid sequence identity, more preferably at least about 92%> nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94%> nucleic acid sequence identity, more preferably at least about 95%) nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98%> nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 22 to 723 ofthe PRO 173 polypeptide shown in Figure 8 (SEQ ID NO 21), (b) a nucleic acid sequence which encodes amino acids X to 723 ofthe PRO 172 polypeptide shown in Figure 8 (SEQ ID NO 21), wherein X is any amino acid residue from 17 to 26 of Figure 8 (SEQ ID NO 21), (c) a nucleic acid sequence which encodes amino acids 1 or about 22 to X of Figure 8 (SEQ ID NO 21), wherein X is any amino acid from amino acid 543 to amino acid 552 of Figure 8 (SEQ ID NO 21) or (d) a nucleic acid sequence which encodes another specifically derived fragment ofthe amino acid sequence shown in Figure 8 (SEQ ID NO 21 ) PRO 172 polynucleotide variants do not encompass the native PRO 172 nucleotide sequence
"PROl 82 variant polynucleotide" or "PRO 182 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PROl 82 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 19 to 135 of the PROl 82 polypeptide shown in Figure 10 (SEQ ID NO 26), (b) a nucleic acid sequence which encodes amino acids X to 135 ofthe PR0182 polypeptide shown in Figure 10 (SEQ ID NO 26), wherein X is any amino acid residue from 14 to 23 of Figure 10 (SEQ ID NO 26) or (c) a nucleic acid sequence which encodes another specifically derived fragment ofthe amino acid sequence shown in Figure 10 (SEQ ID NO 26) Ordinarily, a PRO 182 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85%o 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%o 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 19 to 135 of the PRO 182 polypeptide shown in Figure 10 (SEQ ID NO 26), (b) a nucleic acid sequence which encodes amino acids X to 135 ofthe PR0182 polypeptide shown in Figure 10 (SEQ ID NO 26), wherein X is any amino acid residue from 14 to 23 of Figure 10 (SEQ ID NO 26) or (c) a nucleic acid sequence which encodes another specifically derived fragment of the ammo acid sequence shown in Figure 10 (SEQ ID NO 26) PRO 182 polynucleotide variants do not encompass the native PRO 182 nucleotide sequence Ordinarily, PR0211 , PR0228, PR0538, PROl 72 and PROl 82 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 PR0211 , PR0228, PR0538, PRO 172 and PROl 82 polypeptide-encod g nucleic acid sequences identified herein is defined as the percentage of nucleotides m a candidate sequence that are identical with the nucleotides in a PR021 1 , PR0228, PR0538, PR0172 or PRO 182 polypeptide-encodmg nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc , and the source code shown in Table 1 has been filed with user documentation in the U S Copyright Office, Washington D C , 20559, where it is registered under U S Copyright Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech, Inc , South San Francisco, California or may be compiled from the source code provided 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 purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the %> nucleic acid sequence identity of D to C. As examples of % nucleic acid sequence identity calculations,Tables 2C-2D demonstrate how to calculate the %> nucleic acid sequence identity ofthe nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO- DNA". Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, %> nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al, Nucleic Acids Res., _25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01 , constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI- BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
In addition, % nucleic acid sequence identity values may also be generated using the WU-BLAST-2 computer program (Altschul et al, Methods in Enzymology. 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values Those not set to default values, / e , the adjustable parameters, are set with the following values overlap span = I , overlap fraction = 0 125, word threshold (T) = 1 1, and scoring matrix = BLOSUM62 For purposes herein, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide- encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide- encodmg nucleic acid and the comparison nucleic acid molecule of interest (/ e , the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides ofthe PRO polypeptide- encoding nucleic acid molecule of interest For example, in the statement "an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B", the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence ofthe PRO polypeptide-encoding nucleic acid molecule of interest
In other embodiments, PR0211, PR0228, PR0538, PRO 172 and PRO 182 variant polynucleotides are nucleic acid molecules that encode an active PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide, respectively, and which are capable of hybridizing, preferably under stringent hybridization and wash conditions to nucleotide sequences encoding the full-length PR0211 polypeptide shown in Figure 2 (SEQ ID NO 2), to nucleotide sequences encoding the full-length PR0228 polypeptide shown in Figure 4 (SEQ ID NO 7), to nucleotide sequences encoding the full-length PR0538 polypeptide shown in Figure 6 (SEQ ID NO 16), to nucleotide sequences encoding the full-length PRO 172 polypeptide shown in Figure 8 (SEQ ID NO 21), to nucleotide sequences encoding the full-length PRO 182 polypeptide shown in Figure 10 (SEQ ID NO 26), respectively PR021 1 , PR0228, PR0538, PR0172 and PR0182 variant polypeptides may be those that are encoded by a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 variant polynucleotide
The term "positives", in the context of the amino acid sequence identity comparisons performed as described above, includes ammo acid residues m 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 ammo acid residue of interest
For purposes herein, the % value of positives of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain %> positives to, with, or against a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of amino acid residues scoring a positive value as defined above by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of ammo acid sequence A is not equal to the length of amino acid sequence B, the % positives of A to B will not equal the % positives of B to A "Isolated," when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified ( 1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
An "isolated" nucleic acid molecule encoding a PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide or an "isolated" nucleic acid molecule encoding an anti-PR021 1, anti- PR0228, anti-PR0538, anti- PROl 72 or anti-PROl 82 antibody is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the PR021 1-, PR0228-, PR0538-, PRO 172- or PR0182-encoding nucleic acid or the anti-PR021 1-, anti- PR0228-, anti- PR0538-, anti-PROl 72- or anti-PROl 82-encoding nucleic acid. Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated. An isolated PR021 1-, PR0228-, PR0538-, PRO 172- or PRO 182-encoding nucleic acid molecule or an isolated anti-PR021 1 -, anti- PR0228-, anti-PR0538-, anti-PROl 72- or anti-PROl 82-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the PR021 1-, PR0228-, PR0538, -PR0172- or PROl 82-encoding nucleic acid molecule or from the anti-PR021 l-,anti- PR0228-, anti- PR0538-, anti-PROl 72- or anti-PROl 82-encoding nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule encoding a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide or an isolated nucleic acid molecule encoding an anti-PR021 l ,anti- PR0228, anti-PR0538, anti-PR0172 or anti- PRO 182 antibody includes PR021 1 -, PR0228-, PR0538-, PRO 172- or PRO 182-nucleic acid molecules or anti- PR021 1- , anti- PR0228-, anti-PR0538-, anti-PRO 172- or anti-PRO 182-nucleic acid molecules contained in cells that ordinarily express PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptides or anti-PR021 l ,anti- PR0228, anti-PR0538, anti-PRO 172 or anti-PRO 182 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 D A sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription ofthe 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 m reading phase However, enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice
The term "antibody" is used in the broadest sense and specifically covers, for example, single anti- PR021 1, antι-PR0228, antι-PR0538, antι-PR0172 and antι-PR0182 monoclonal antibodies (including agonist antibodies), antι-PR021 1 , antι-PR0228, antι-PR0538, anti-PRO 172and anti-PRO 182antιbody compositions with polyepitopic specificity, single chain antι-PR0211, antι-PR0228, antι-PR0538, antι-PR0172 and antι-PR0182 antibodies, and fragments of antι-PR021 1 , antι-PR0228, antι-PR0538, anti-PRO 172 and anti-PRO 182 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 for possible naturally-occurring mutations that may be present in minor amounts "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art. and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature The higher the degree of desired homology between the probe and hybndizable sequence, the higher the relative temperature which can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so For additional details and explanation of stringency of hybridization reactions, see Ausubel et al , Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995)
"Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that (1) employ low ionic strength and high temperature for washing, for example 0 015 M sodium chloπde/0 0015 M sodium cιtrate/0 1% sodium dodecyl sulfate at 50°C, (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0 1 % bovine serum albumιn/0 1% Fιcoll/0 1 % polyvιnylpyrrolιdone/50mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C, or (3) employ 50% foπnamide, 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 etal , Molecular Cloning A Laboratory Manual, New York Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e g , temperature, ionic strength and % SDS) less stringent that those described above An example of moderately stringent conditions is overnight incubation at 37CC 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 chimenc polypeptide comprising a PR021 1 ,
PR0228, PR0538, PRO 172 or PRO 182 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 ofthe polypeptide to which it is ftised 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 ammo acid residues)
As used herein, the term "lmmunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains Structurally, the immunoadhesms comprise a ftision of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (t e . is "heterologous"), and an immunoglobulin constant domain sequence The adhesin part of an lmmunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a hgand The immunoglobulin constant domain sequence in the lmmunoadhesin 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 form(s) of PR021 1, PR0228, PR0538, PR0172 or PROl 82 which retain a biological and or an immunological activity of native or naturally-occurring PR021 1 , PR0228, PR0538, PRO 172 or PRO 182, wherein "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring PR021 1 , PR0228, PR0538, PROl 72 or PRO 182 other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 and an "immunological" activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PR021 1 , PR0228, PR0538, PRO 172 or PRO 182
"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 ofthe 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 PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide "Immunological cross-reactivity" as used herein means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide having this activity with polyclonal antisera raised against the known active PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide Such antisera are prepared in conventional fashion by injecting goats or rabbits, for example, subcutaneously with the known active analogue in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freunds The immunological cross-reactivity preferably is "specific", which means that the binding affinity ofthe immunologically cross-reactive molecule (e g , antibody) identified, to the corresponding PR021 1 , PR0228, PR0538, PR0172 or PR0182 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 various types of head and neck cancer
"Treatment" is an intervention performed with the intention of preventing the development or altering the pathology of a disorder Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented In tumor (e g , cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e g radiation and/or chemotherapy
The "pathology" of cancer includes all phenomena that compromise the well-being of the patient This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, etc
An "effective amount" of a polypeptide disclosed herein or an agonist thereof, in reference to inhibition of neoplastic cell growth, is an amount capable of inhibiting, to some extent, the growth of target cells The term includes an amount capable of invoking a growth inhibitory, cytostatic and/or cytotoxic effect and/or apoptosis of the target cells An "effective amount" of a PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 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 PR021 1 , PR0228, PR0538, PR0172 or PROl 82 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1, PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 , I131, 1125, Y90 and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof
A "chemotherapeutic agent" is a chemical compound useful in the treatment of tumor, e g , cancer Examples of chemotherapeutic agents include adπamycm, doxorubicin, epirubicin, 5-fluorouracιl, cytosine arabmoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxm, taxoids, e g , pachtaxel (Taxol, Bπstol- Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rhόne-PoulencRorer, Antony, Rnace), toxotere, methotrexate, cisplatm, melphalan vmblastine, bleomycin, etoposide, lfosfamide, mitomycin C, mitoxantrone, vincπstine, vinorelbme, carboplatin, teniposide, daunomycin, carminomycin, aminopteπn, dactinomycin, mitomycins, esperamicms (see, U S Patent No 4 675, 187), melphalan and other related nitrogen mustards Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and 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 m vivo Thus, the growth inhibitory agent is one which significantly reduces the percentage ofthe 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 G 1 arrest and M- phase arrest Classical M-phase blockers include the vincas (vincπstine and vinblastme), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin Those agents that arrest G I also spill over into S-phase arrest, for example, D A alkylat g agents such as tamoxifen, prednisone, dacarbazine, mechlorethamme, 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 lymphokmes, monokines, and traditional polypeptide hormones Included among the cytokines are growth hormone such as human growth hormone, N- methionyl human growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxm, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α and -β, mulleπan-inhibiting substance, mouse gonadotrop - associated peptide, inhibin, activin, vascular endothelial growth factor, lntegπn, 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, erythropoietm (EPO), osteoinductive factors, mterferons such as ιnterferon-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-1 1, IL- 12, a tumor necrosis factor such as TNF-α or TNF-β, and other polypeptide factors including LIF and kit hgand (KL) As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form See, e g , Wilman, "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions, 14, pp 375-382, 615th Meeting Belfast (1986) and Stella et al , "Prodrugs A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al , (ed ), pp 247-267, Humana Press (1985) The prodrugs of this invention include, but are not limited to phosphate-containing prodrugs, thiophosphate-containing prodrugs, glycosylated prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosιne and other 5-fluorouπdιne prodrugs which can be deπvatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above The term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide disclosed herein Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptides, peptides, small organic molecules, etc Methods for identifying agonists of a PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide may comprise contacting a tumor cell with a candidate agonist and measuring the inhibition of tumor cell growth
"Chronic" administration refers to administration ofthe 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 m 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 immunoglobulins, hydrophihc polymers such as polyvinylpyrrohdone, amino acids such as glycine, glutamine, asparagme, 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 counteπons such as sodium, and/or nonionic 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 varies among the heavy chains of different immunoglobulin isotypes Each heavy and light chain also has regularly spaced lntracha 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 ofthe light chain is aligned with the first constant domain ofthe heavy chain, and the light-chain variable domain is aligned with the variable domain ofthe heavy chain Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains
The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen However, the variability is not evenly distributed throughout the variable domains of antibodies It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervaπable regions both in the light-chain and the heavy-chain variable domains The more highly conserved portions of variable domains are called the framework (FR) The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation ofthe antigen-b ding 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 ofthe antibody in antibody-dependent cellular toxicity
The term "hypervaπable region" when used herein refers to the ammo 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" (/ 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" (i e , residues 26-32 (L 1 ), 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 , F96 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')„ and Fv fragments, diabodies, linear antibodies (Zapata et al , Protein Eng . 8(10) 1057-1062 [1995]), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen
"Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site This region consists of a dimer of one heavy- and one light-cham variable domain in tight, non-covalent association It is m 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 ofthe light chain and the first constant domain (CH 1 ) of the heavy chain Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH 1 domain including one or more cysteines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteme resιdue(s) ofthe constant domains bear a free thiol group F(ab'), antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains Depending on the am o 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, /. 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 againstdifferentdeterminants(epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character ofthe antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et at. 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 ofthe 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 ofthe chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA, 8 6851 - 6855 [1984]).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, Jones et al, Nature, 321 :522-525 (1986); Reichmann et al, Nature, 332:323-329 [1988]; and Presta, Curr. Op. Struct. Biol.. 2:593-596 (1992). The humanized antibody includes a PRIMATIZED™antibody wherein the antigen-binding region ofthe antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest. "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present m 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 1161 , 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%o by weight of antibody as determined by the Lowry method, and most preferably more than 99%> by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal ammo 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 ofthe 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 silicones In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275, 149
A "liposome" is a small vesicle composed of various types of hpids, phosphohpids and/or surfactant which is useful for delivery of a drug (such as a PR021 1 , PR0228 PR0538, PRO 172 or PRO 182 polypeptide or antibody thereto) to a mammal The components ofthe liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes
A "small molecule" is defined herein to have a molecular weight below about 500 Daltons II. Compositions and Methods ofthe Invention
A. Full-length PRQ21 1, PRQ228. PRQ538, PRQ172 and PRQ182 Polypeptides
The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0211 , PR0228, PR0538, PROl 72 and PROl 82. In particular, cDNAs encoding PR0211, PR0228, PR0538, PR0172 and PROl 82 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below.
As disclosed in the Examples below, cDNA clones encoding PR021 1 , PR0228, PR0538, PR0172 and PROl 82 polypeptides have been deposited with the ATCC. The actual nucleotide sequences of the clones can readily be determined by the skilled artisan by sequencing ofthe 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 PR0211, PR0228, PR0538, PR0172 and PR0182 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. PRQ21 1 , PRQ228, PRQ538, PRQ172 and PRQ182 Variants In addition to the full-length native sequence PR021 1, PR0228, PR0538, PR0172 and PR0182 polypeptides described herein, it is contemplated that PR021 1 , PR0228, PR0538, PROl 72 and PROl 82 variants can be prepared. PR021 1, PR0228, PR0538, PR0172 and PR0182 variants can be prepared by introducing appropriate nucleotide changes into the PR021 1 , PR0228, PR0538, PR0172 or PR0182 DNA, and/or by synthesis of the desired PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence PR021 1 , PR0228, PR0538, PR0172 or PR0182 or in various domains of the PR021 1 , PR0228, PR0538, PR0172 or PR0182 described herein, can be made, for example, using any ofthe 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 PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 that results in a change in the amino acid sequence ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 as compared with the native sequence PR021 1 , PR0228, PR0538, PROl 72 or PROl 82. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the PR021 1 , PR0228, PR0538, PR0172 or PR0182. 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 ofthe PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full- length or mature native sequence.
PR021 1, PR0228, PR0538, PRO 172 and PRO 182 polypeptide fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity ofthe PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide.
PR021 1, PR0228, PR0538, PR0172 and PR0182 fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating PR021 1, PR0228, PR0538, PR0172 and PR0182 fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, PR021 1 , PR0228, PR0538, PR0172 and PR0182 polypeptide fragments share at least one biological and/or immunological activity with the native PR021 1 , PR0228, PR0538, PR0172 orPR0182 polypeptide shown in Figure 2 (SEQ IDNO:2), Figure 4 (SEQ IDN0:7), Figure 6 (SEQ ID NO: 16), Figure 8 (SEQ ID NO:21) and Figure 10 (SEQ ID NO:26), respectively.
In particular embodiments, conservative substitutions of interest are shown in Table 3 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 3, or as further described below in reference to amino acid classes, are introduced and the products screened.
Table 3
Original Exemplary Preferred Residue Substitutions Substitutions
Ala (A) val; leu; ile val Arg (R) lys; gin; asn lys Asn (N) gin; his; lys; arg gin Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gin; lys; arg arg He (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile
Lys (K) arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu
Substantial modifications in function or immunological identity of the PR021 1, PR0228, PR0538,
PRO 172 or PRO 182 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as oligonucleotide-mediated (site- directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al, Nucl. Acids Res., L3:4331 (1986); Zoller et al, Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al, Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al, Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PR021 1 , PR0228, PR0538, PR0172 or PR0182 variant DNA.
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main- chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins. (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150: 1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
C. Modifications of PRQ21 1 , PRQ228, PRQ538, PRQ172 and PRQ182 Covalent modifications of PR021 1, PR0228, PR0538, PR0172 and PR0182 are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues ofthe PR021 1 , PR0228, PR0538, PRO 172 or PROl 82. Derivatization with bifunctional agents is useful, for instance, for crosslinking PR021 1, PR0228, PR0538, PR0172 or PR0182 to a water-insoluble support matrix or surface for use in the method for purifying anti-PR021 1, anti-PR0228, anti-PR0538, anti-PRO 172 or anti-PRO 182 antibodies, and vice-versa. Commonly used crosslinking agents include, e.g., l ,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l ,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation ofthe α-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation ofthe N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern ofthe polypeptide. "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 (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 PR021 1 , PR0228, PR0538, PR0172 or PR0182. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
Addition of glycosylation sites to the PR0211 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PR021 1 , PR0228, PR0538, PR0172 or PR0182 (for O-linked glycosylation sites). The PR0211, PR0228, PR0538, PR0172 or PR0182 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PR0211, PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1, PR0228, PR0538,
PROl 72 or PROl 82 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 1 1 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
Removal of carbohydrate moieties present on the PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al, Arch. Biochem. Biophvs.. 259:52 (1987) and by Edge et al, Anal. Biochem., 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. Enzymol.. 138:350 (1987).
Another type of covalent modification of PR021 1 , PR0228, PR0538, PR0172 or PR0182 comprises linking the PR021 1 , PR0228, PR0538, PR0172 or PROl 82 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The PR021 1, PR0228, PR0538, PRO 172 or PROl 82 polypeptide ofthe present invention may also be modified in a way to form a chimeric molecule comprising PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PR021 1 , PR0228, PR0538, PR0172 or PR0182 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 PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide. The presence of such epitope-tagged forms ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide can be detected using an antibody against the tag polypeptide . Also, provision ofthe epitope tag enables the PR021 1 , PR0228, PR0538, PROl 72 or PRO 182 polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-His) or poly-histidine-glycine (poly-His-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al, Mol. Cell. Biol.. 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E 10 antibodies thereto [Evan et al , Molecular and Cellular Biology. 5 3610-3616 (1985)], and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al , Protein Engineering, 3(6) 547-553 ( 1990)] Other tag polypeptides include the Flag-peptide [Hopp et al , BioTechnology, 6 1204-1210 (1988)], the KT3 epitope peptide [Martin et al , Science, 255 192- 194 ( 1992)] , an α-tubuhn epitope peptide [Skinner et al , J Biol Chem .266 15163 - 15166 ( 1991 )], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl Acad Sci USA, 87 6393-6397 (1990)]
In an alternative embodiment, the chimenc molecule may comprise a fusion of the PR021 1, PR0228, PR0538, PR0172 or PROl 82 polypeptide with an immunoglobulin or a particular region of an immunoglobulin For a bivalent form ofthe chimenc molecule (also referred to as an "lmmunoadhesin"), such a fusion could be to the Fc region of an IgG molecule The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 IgG 1 molecule For the production of immunoglobulin fusions see also, US Patent No 5,428,130 issued June 27, 1995
D Preparation of PRQ21 1. PRQ228. PRQ538. PRQ172 and PRQ182
The description below relates primarily to production of PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 by cultuπng cells transformed or transfected with a vector containing PR021 1 , PR0228, PR0538, PR0172 or PROl 82 nucleic acid It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PR0211, PR0228, PR0538, PR0172 or PR0182 For instance, the PR021 1, PR0228, PR0538, PRO 172 or PRO 182 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), Mernfield, 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 PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PR021 1 , PR0228 PR0538, PR0172 or PR0182 polypeptide
3 Isolation of DNA Encoding PRQ21 1. PRQ228, PRQ538, PRO 172 or PRO 182
DNA encoding PR021 1, PR0228, PR0538, PRO 172 or PROl 82 may be obtained from a cDNA library prepared from tissue believed to possess the PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 mRNA and to express it at a detectable level Accordingly, human PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples
The PR021 1-, PR0228-, PR0538-, PR0172- or PR0182-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 PR021 1 , PR0228, PR0538, PR0172 or PRO 182 or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al, Molecular Cloning: A Laboratory Manual (New York: Cold
Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding PR0211 , PR0228, PR0538, PRO 172 or PRO 182 is to use PCR methodology [Sambrook etal, supra; Dieffenbach et al, PCR Primer:
A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
The Examples below describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized.
The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al, supra.
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions ofthe molecule or across the full-length sequence can be determined using methods known in the art and as described herein.
Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al, supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
2. Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors described herein for PR021 1 , PR0228, PR0538, PR0172 or PR0182 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, CaCh, CaP04, liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al, supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al, Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456- 457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in U S Patent No 4,399,216 Transformations into yeast are typically carried out according to the method of Van Sohngen et al , J Bact . L30 946 ( 1977) and Hsiao et al , Proc Natl Acad Sci (USA), 76 3829 ( 1979) However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e g , polybrene, polyormthine, may also be used For various techniques for transforming mammalian cells, see, Keown etal , Methods in Enzymology, 185 527-537 ( 1990) and Mansour et al , Nature, 336 348-352 (1988)
Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells Suitable prokaryotes include but are not limited to eubacteπa, such as Gram-negative or Gram-positive organisms, for example, Enterobacteπaceae such as E coli Various £ colt strains are publicly available, such as £ colt K12 strain MM294 (ATCC 31 ,446), £ colt XI 776 (ATCC 31,537), £ co strain W31 10 (ATCC 27,325) and K5772 (ATCC 53,635) Other suitable prokaryotic host cells include Enterobacteπaceae such as Escherichia, e g , E coli, Enterobacter, Erwinta, Klebsiella, Proteus, Salmonella, e g , Salmonella typhtmurium, Serratia, e g , Serratia marcescans, and Shigella, as well as Bacilli such as B subtihs and B licheniformis (e g , B licheniformis 41 P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P aeruginosa, and Streptomyces These examples are illustrative rather than limiting Strain 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 £ coh W31 10 strain 1A2, which has the complete genotype tonA , £ colt W31 10 strain 9E4, which has the complete genotype tonA ptr3, E co// W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonAptriphoA El 5 (argF-lac) 169 degP ompTka , E coli W31 10 strain 37D6, which has the complete genotype tonA ptr3 phoA El 5 (argF-lac) 169 degP ompT rbs7 ilvG karf, E coli W31 10 strain 40B4, which is strain 37D6 with a non- kanamycm 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 vitro methods of cloning, e g , PCR or other nucleic acid polymerase reactions, are suitable
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PR021 1-, PR0228-, PR0538-, PR0172- or PR0182-encodιng vectors Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290 140 [1981], EP 139,383 pubhshed 2 May 1985), Kluyveromyces hosts O S Patent No 4,943,529, Fleer et al , Bio/Technology, 9 968-975 (1991 )) such as, e g , K lactts (MW98-8C, CBS683, CBS4574, Louvencourt e/-./ , J Bactenol , 737119831), K fragilis (ATCC 12,424) K bul gar tcus (ATCC 16,045), K wickeramu (ATCC 24,178), K waltu (ATCC 56,500), K drosophilarum (ATCC 36,906, Van den Berg et al Bio/Technology, 8 135 (1990)), A. thermotolerans, and K maixianus yarrowia (EP 402,226), Pichia pastoris (EP 183.070, Sreekπshna et al . J Basic Microbiol , 28 265-278 119881), Candida Trichoderma reesia (EP 244,234) Neurospora crassa (Case et al , Proc Natl Acad Sci USA, 76 5259-5263 [1979]), Schwanniomyces such as Schwanniomyces occidentals (EP 394,538 published 31 October 1990), and filamentous fungi such as, e g , Neurospora Penicilhum, Tolypocladmm (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A mdulans (Ballance et al , Biochem Biophys Res Commun . 1 12 284-289 [1983], Tilburn et al , Gene, 26 205-221 [1983], Yelton et al , Proc Natl Acad Sci USA, 8i 1470-1474 [1984]) and A ntger (Kelly and Hynes, EMBO J , 4 475-479 [1985]) Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsts, 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 PR021 1, PR0228, PR0538, PR0172 or PR0182 are derived from multicellular organisms Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham etal , J Gen Virol , 36 59 (1977)), Chinese hamster ovary cellsADHFR (CHO, Urlaub and Chasm, Proc Natl Acad Sci USA. 77 4216 (1980)), mouse sertoh cells (TM4, Mather, Biol Reprod , 23 243- 251 (1980)), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), and mouse mammary tumor (MMT 060562, ATCC CCL51 ) The selection ofthe 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 PR021 1 , PR0228, PR0538, PR0172 or PRO 182 may be inserted into a rephcable vector for cloning (amplification ofthe DNA) or for expression Various vectors are publicly available The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures In general, DNA is inserted into an appropriate restriction endonuclease sιte(s) using techniques known in the art Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan
The PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 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 ofthe mature protein or polypeptide In general, the signal sequence may be a component of the vector, or it may be a part of the PR021 1 -, PR0228-, PR0538-, PRO 172- or PR0182-encodmg DNA that is inserted into the vector The signal sequence may be a prokaryotic signal sequence selected, for example, from the group ofthe 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 Saccharomyces and Kluyveromyces α-factor leaders, the latter described in U S Patent No 5 ,010, 182), or acid phosphatase leader, the C albicans glucoamy lase 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 ofthe protein, such as signal sequences from secreted polypeptides ofthe same or related species, as well as viral secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PR021 1 -, PR0228-, PR0538-, PRO 172- or PRO 182-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al, Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is the trp\ gene present in the yeast plasmid YRp7 [Stinchcomb et al, Nature, 282:39 (1979); Kingsman et al, Gene. 7: 141 (1979); Tschemper et al, Gene, JO: 157 (1980)]. The trp\ gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85.: 12 (1977)]. Expression and cloning vectors usually contain a promoter operably linked to the PR0211-, PR0228-,
PR0538-, PROl 72- or PROl 82-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al, Nature, 275:615 (1978); Goeddel et al, Nature. 281 :544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al, Proc. Natl. Acad. Sci. USA. 80_:21 -25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PR021 1 , PR0228, PR0538, PR0172 or PR0182.
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al, J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al, J. Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde- 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
PR021 1 , PR0228, PR0538, PR0172 or PR0182 transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus
(UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the 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 PR021 1, PR0228, PR0538, PR0172 or PR0182 by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein,and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side ofthe replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the PR021 1 , PR0228, PR0538, PR0172 or PR0182 coding sequence, but is preferably located at a site 5' from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PR021 1, PR0228, PR0538,
PR0172 or PR0182.
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PR021 1 , PR0228,
PR0538, PR0172 or PR0182 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 PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PR021 1, PR0228, PR0538, PRO 172 or PRO 182 DNA and encoding a specific antibody epitope.
5. Purification of Polypeptide
Forms of PR021 1, PR0228, PR0538, PR0172 or PR0182 may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells employed in expression of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR0211, PR0228, PR0538, PR0172 or PR0182 from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182.
Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology. 182 (1990); Scopes, Protein Purification: Principles and Practice. Springer- Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular PR021 1 , PR0228, PR0538, PR0172 or PR0182 produced.
E. Antibodies
Some drug candidates for use in the compositions and methods ofthe present invention are antibodies and antibody fragments which mimic the biological activity of a PR021 1, PR0228, PR0538, PR0172 or PR0182 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 PR021 1 , PR0228, PR0538, PR0172 or PROl 82 polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
2. Monoclonal Antibodies The antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature.256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include the PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59- 103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PR0211 , PR0228, PR0538, PRO 172 or PRO 182. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 ( 1980). After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Patent No 4,816,567 DNA encoding the monoclonal antibodies ofthe invention can be readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) The hybridoma cells of the invention serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place ofthe homologous murine sequences [U S Patent No 4,816,567, Morrison et al , supra] or by covalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulm polypeptide Such a non-immunoglobu n polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimenc bivalent antibody
The antibodies may be monovalent antibodies Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking Alternatively, the relevant cysteine residues are substituted with another ammo acid residue or are deleted so as to prevent crosslinking
In vitro methods are also suitable for preparing monovalent antibodies Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art
3 Human and Humanized Antibodies
The antibodies of the invention may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , murine) antibodies are chimenc 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) ofthe 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 ofthe CDR regions correspond to those of a non-human immunoglobulin and all or substantially all ofthe 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 chimenc antibodies (U S Patent No 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J Mol Biol , 227 381 (1991), Marks et al , J Mol Biol , 222 581 (1991)] The techniques of Cole et al , and Boerner et al , are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy. Alan R Liss, p 77 (1985) and Boerner et al , J Immunol , 147(1) 86-95 (1991)] Similarly, human antibodies can be made by the introducing of human immunoglobulin loci into transgenic animals, e g , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire This approach is described, for example, in U S Patent Nos 5,545,807, 5,545,806, 5,569,825, 5,625,126 5,633,425, 5,661 ,016, and in the following scientific publications Marks et al Bio/Technology, 10 779-783 (1992), Lonberg e. _./ Nature, 368 856-859 (1994), Morrison, Nature, 368 812-13 (1994), Fishwild e. α/ Nature Biotechnology, 14 845-51 ( 1996), Neuberger, Nature Biotechnology, 14 826 ( 1996), Lonberg and Huszar. Intern Rev Immunol , 13 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182, the other one is for any other antigen, and preferably for a cell- surface protein or receptor or receptor subunit Methods for making bispecific antibodies are known in the art Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-cham pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305 537-539 (1983)] Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure The purification ofthe correct molecule is usually accomplished by affinity chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker etal , EMBO J . K) 3655-3659 (1991 ) Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences The fusion preferably is with an immunoglobulin heavy- chain constant domain, comprising at least part ofthe hmge, CH2, and CH3 regions It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism For further details of generating bispecific antibodies see, for example, Suresh et al , Methods in Enzymology, 121 210 (1986)
According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture The preferred interface comprises at least a part ofthe 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 ofthe second antibody molecule by replacing large ammo acid side chains with smaller ones (e g , alanine or threomne) This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e g , F(ab'), bispecific antibodies) Techniques for generating bispecific antibodies from antibody fragments have been described in the literature For example, bispecific antibodies can be prepared using chemical linkage Brennan et al , Science, 229 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab'), fragments These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation The Fab' fragments generated are then converted to thiomtrobenzoate (TNB) derivatives One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes
Fab' fragments may be directly recovered from £ coli and chemically coupled to form bispecific antibodies Shalaby et al , J Exp Med , 175 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')-, molecule Each Fab' fragment was separately secreted from £ coli and subjected to directed chemical coupling in vitro to form the bispecific antibody The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described For example, bispecific antibodies have been produced using leucine zippers Kostelnv et al A 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 Hollmger 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-cham 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-b ding sites Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported See, Gruber et al , J Immunol , 152 5368 (1994)
Antibodies with more than two valencies are contemplated For example, 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 PR021 1, PR0228,
PR0538, PRO 172 or PRO 182 polypeptide herein Alternatively, an antι-PR0211 , antι-PR0228, antι-PR0538, anti-PRO 172 or anti-PRO 182 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 , CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular PR0211 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PR021 1, PR0228, PR0538, PROl 72 or PROl 82 polypeptide These antibodies possess a PR021 1 -, PR0228-, PR0538-, PRO 172- or PRO 182-bιndmg arm and an arm which binds a cytotoxic agent or a radionuc de chelator, such as EOTUBE, DPTA, DOTA, or TETA Another bispecific antibody of interest binds the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide and further binds tissue factor (TF)
5 Heteroconiugate Antibodies
Heteroconjugate antibodies are also within the scope ofthe present invention Heteroconjugate antibodies are composed of two covalently joined antibodies Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U S Patent No 4,676,980], and for treatment of HIV infection [WO 91 /00360, WO 92/200373 , EP 03089] It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents For example, lmmunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond Examples of suitable reagents for this purpose include lminothiolate and methyl-4-mercaptobutyπmιdate 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 lnteraahzation capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See, Caron et al , J Exp Med , j_76 1 191 - 1195 ( 1992) and Shopes, J Immunol , 148 2918-2922 ( 1992) Homodimeπc antibodies with enhanced antitumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al , Cancer Research, 53 2560-2565 (1993) Alternatively, an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities See, Stevenson et al , Anti-Cancer Drug Design. 3. 219-230 (1989)
7 Immunoconiugates
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (/ 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, nonbmding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aerugmosd), πcin A chain, abπn A chain, modeccin A chain, alpha-sarcin, Aleurites fordn proteins, dianth proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcm, crotm, sapaonaπa officinahs inhibitor, gelonin, mitogelhn, restπctocin, phenomycin, enomycin, and the tπcothecenes A variety of radionuchdes are available for the production of radioconjugated antibodies Examples include 212Bι, lj lI, 131In, 90Y, and l86Re
Conjugates ofthe antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such asN-succιnιmιdyl-3-(2-pyπdyldιthιol)propιonate (SPDP), lminothiolane (IT), bifunctional derivatives of ιmιdoesters(such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium deπvativ es (such as bιs-(p-dιazonιumbenzoyl)-ethylenedιamιne), dnsocyanates (such as tolyene 2,6-dnsocyanate), and bis- active fluorine compounds (such as l ,5-dιfluoro-2,4-dιnιtrobenzene) For example, a πcin i munotoxin can be prepared as described in Vitetta et al , Science, 238 1098(1987) Carbon- 14-labeled l-ιsothιocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody See, W094/1 1026
In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargetmg wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e g , avidin) that is conjugated to a cytotoxic agent (e g , a radionucleotide) 8. Immunoliposomes
The antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. Sci. USA. 82: 3688 (1985); Hwang et al, Proc. Natl. Acad. Sci. USA. 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG- PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments ofthe antibody ofthe present invention can be conjugated to the liposomes as described in Martin et al, J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See, Gabizon et al, J. National Cancer Inst.. 81(19): 1484 (1989).
F. Identification of Proteins Capable of Inhibiting Neoplastic Cell Growth or Proliferation
The proteins disclosed in the present application have been assayed in a panel of 60 tumor cell lines currently used in the investigational, disease-oriented, in vitro drug-discovery screen of the National Cancer Institute (NCI). The purpose of this screen is to identify molecules that have cytotoxic and/or cytostatic activity against different types of tumors. NCI screens more than 10,000 new molecules per year (Monks et al, J. Natl. Cancer Inst., 83:757-766 (1991); Boyd, Cancer: Princ. Pract. Oncol. Update, 3(10}: 1-12 ([1989]). The tumor cell lines employed in this study have been described in Monks et al, supra. The cell lines the growth of which has been significantly inhibited by the proteins ofthe present application are specified in the Examples.
The results have shown that the proteins tested show cytostatic and, in some instances and concentrations, cytotoxic activities in a variety of cancer cell lines, and therefore are useful candidates for tumor therapy.
Other cell-based assays and animal models for tumors (e.g. , cancers) can also be used to verify the findings ofthe NCI cancer screen, and to further understand the relationship between the protein identified herein and the development and pathogenesis of neoplastic cell growth. For example, primary cultures derived from tumors in transgenic animals (as described below) can be used in the cell-based assays herein, although stable cell lines are preferred. Techniques to derive continuous cell lines from transgenic animals are well known in the art (see, e.g., Small et al, Mol. Cell. Biol., 5:642-648 [1985]).
G. Animal Models A variety of well known animal models can be used to further understand the role of the molecules identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies, and other agonists ofthe native polypeptides, including small molecule agonists. The in vivo nature of such models makes them particularly predictive of responses in human patients. Animal models of tumors and cancers (e.g., breast cancer, colon cancer, prostate cancer, lung cancer, etc.) include both non- recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodent, e.g. , murine models. Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g. , colon cancer cells implanted in colonic tissue. (See, e.g., PCT publication No. WO 97/33551, published September 18, 1997). Probably the most often used animal species in oncological studies are immunodeficient mice and, in particular, nude mice. The observation that the nude mouse with hypo/aplasia could successfully act as a host for human tumor xenografts has lead to its widespread use for this purpose. The autosomal recessive nu gene has been introduced into a very large number of distinct congenic strains of nude mouse, including, for example, ASW, A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL. In addition, a wide variety of other animals with inherited immunological defects other than the nude mouse have been bred and used as recipients of tumor xenografts. For further details see, e.g. , The Nude Mouse in Oncology Research, E. Boven and B. Winograd, eds., CRC Press, Inc., 1991.
The cells introduced into such animals can be derived from known tumor/cancer cell lines, such as, any ofthe above-listed tumor cell lines, and, for example, the B104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene); ras-transfected NIH-3T3 cells; Caco-2 (ATCC HTB-37); a moderately well- differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or from tumors and cancers. Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions, involving freezing and storing in liquid nitrogen (Karmali et al, Br. J. Cancer, 48:689-696 [1983]).
Tumor cells can be introduced into animals, such as nude mice, by a variety of procedures. The subcutaneous (s.c.) space in mice is very suitable for tumor implantation. Tumors can be transplanted s.c. as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions. For solid block or trochar implantation, tumor tissue fragments of suitable size are introduced into the s.c. space. Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously. Tumor cells can also be injected as subdermal implants. In this location, the inoculum is deposited between the lower part ofthe dermal connective tissue and the s.c. tissue. Boven and Winograd ( 1991 ), supra. Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogen was initially isolated), or ne - 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 at. Cancer Research, 54:4726-4728 (1994) and Too et al, Cancer Research. 55:681-684 (1995). This model is based on the so-called "METAMOUSE" sold by AntiCancer, Inc., (San Diego, California).
Tumors that arise in animals can be removed and cultured in vitro. Cells from the in vitro cultures can then be passaged to animals. Such tumors can serve as targets for further testing or drug screening. Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest. Such passaging techniques can be performed with any known tumor or cancer cell lines
For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et al , J Exp Med , 146 720 [1977]), which provide a highly controllable model system for studying the anti-tumor activities of various agents (Palladmo et al , J Immunol , 138 4023-4032 [1987]) Briefly, tumor cells are propagated in vitro in cell culture Prior to injection into the animals, the cell lines are washed and suspended m buffer, at a cell density of about lOxlO6 to lOxlO7 cells/ml The animals are then infected subcutaneously with 10 to 100 μ\ ofthe 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 ofthe lung (SCCL) This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture (Zupi et al , Br J Cancer, 41 , suppl 4 309 [1980]), and evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells survive For further information about this tumor model see, Zacharski, Haemostasis. 16.300-320 [1986]) One way of evaluating the efficacy of a test compound in an animal model on an implanted tumor is to measure the size of the tumor before and after treatment Traditionally, the size of implanted tumors has been measured with a slide cahper in two or three dimensions The measure limited to two dimensions does not accurately reflect the size ofthe 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 cytometπc analysis
Recombinant (transgenic) animal models can be engineered by introducing the coding portion ofthe genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e g , baboons, chimpanzees and monkeys Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and Wanger, U S Patent No 4,873,191), retrovirus-mediated gene transfer into germ lines (e g , Van der Putten et al , Proc Natl Acad Sci USA, 82 6148-615 [1985]), gene targeting in embryonic stem cells (Thompson 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 , CeU, 57 717-73 [1989]) For review, see, for example, U S Patent No 4,736,866
For the purpose ofthe present invention, transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals") The transgene can be integrated either as a single transgene, or in concatamers, e g , head-to-head or head-to-tail tandems Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al , Proc Natl Acad Sci USA, 89 6232- 636 (1992)
The expression of the transgene in transgenic animals can be monitored by standard techniques For example, Southern blot analysis or PCR amplification can be used to verify the integration ofthe 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%) ofthe oral tumors reported in this species It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor These tumors are usually not amenable to surgery, primarily because ofthe anatomy ofthe 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 ofthe tumors will be taken daily during the treatment period, and at each subsequent recheck After treatment, each cat undergoes another CT scan CT scans and thoracic radiograms are evaluated every 8 weeks thereafter The data are evaluated for differences in survival, response and toxicity as compared to control groups Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span
In addition, other spontaneous animal tumors, such as fibrosarcoma, adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma of dogs, cats, and baboons can also be tested Of these mammary adenocarcinoma in dogs and cats is a preferred model as its appearance and behavior are very similar to those in humans However, the use of this model is limited by the rare occurrence of this type of tumor in animals
H Screening Assays for Drug Candidates
Screening assays for drug candidates are designed to identify compounds that competitively bind or complex with the receptor(s) ofthe polypeptides identified herein, or otherwise signal through such receptor(s) Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates Small molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides, (poly)peptιde- lmmunoglobuhn fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimenc or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.
In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, a receptor of a polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the polypeptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g. , by washing, and complexes anchored on the solid surface are detected. When the originally non- immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex. If the candidate compound interacts with but does not bind to a particular receptor, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and Song, Nature (London), 340:245-246 ( 1989); Chien etal, Proc. Natl. Acad. Sci. USA. 88:9578-9582 (1991)] as disclosed by Chevray and Nathans fProc. Natl. Acad. Sci. USA. 89:5789-5793 (1991)]. Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl-/α_.Z reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two- hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
I. Pharmaceutical Compositions
The polypeptides of the present invention, agonist antibodies specifically binding proteins identified herein, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions. Where antibody fragments are used, the smallest inhibitory fragment which specifically binds to the binding domain of the target protein is preferred For example, based upon the variable region sequences of an antibody, peptide molecules can be designed which retain the ability to bind the target protein sequence Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e g , Marasco et at . Proc Natl Acad Sci USA, 90 7889-7893 119931)
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 ofthe polypeptides identified herein, or agonists thereof are prepared for storage by mixing the active ingredient having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences. 16th edition, Osol, A ed [1980]), in the form of lyophilized formulations or aqueous solutions Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other orgamcacids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzy 1 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, hydrophihc polymers such as polyvinylpyrrolidone, 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 non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG) The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by mterfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A ed (1980)
The formulations to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophihzation and reconstitution
Therapeutic compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle Sustained-release preparations may be prepared Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e g , films, or microcapsules Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(v ylalcohol)), polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vmyl acetate, degradable lactic acid-glycohc acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate), and poly-D-(-)-3-hydroxybutyπc acid While polymers such as ethylene-vmyl 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 lntermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophihzing 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 ofthe present invention and their agonists, including antibodies, peptides, and small molecule agonists, may be used to treat various tumors, e g , cancers Exemplary conditions or disorders to be treated include benign or malignant tumors (e g renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas, sarcomas, glioblastomas, and various head and neck tumors), leukemias and lymphoid malignancies, other disorders such as neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoehc disorders, and inflammatory, angiogenic and lmmunologic disorders The anti-tumor agents ofthe present invention (including the polypeptides disclosed herein and agonists which mimic their activity, e g , antibodies, peptides and small organic molecules), are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, ιntracerobrospιnal, ιntraocular,ιntraarteπal,ιntralesιonal,subcutaneous,ιntraartιcular,ιntrasynovιal, intrathecal, oral, topical, or inhalation routes
Other therapeutic regimens may be combined with the administration of the anti-cancer agents of the instant invention For example, the patient to be treated with such anti-cancer agents may also receive radiation therapy Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, ed , M C Perry, Williams & Wilkins, Baltimore, MD (1992) The chemotherapeutic agent may precede, or follow administration ofthe anti-tumor agent ofthe present invention, or may be given simultaneously therewith The anti-cancer agents ofthe present invention may be combined with an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onapπstone (see, EP 616812) in dosages known for such molecules
It may be desirable to also administer antibodies against tumor associated antigens, such as antibodies which bind to the ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF) Alternatively, or in addition, two or more antibodies binding the same or two or more different cancer-associated antigens may be co- admmistered to the patient Sometimes, it may be beneficial to also administer one or more cytokines to the patient In a preferred embodiment, the anti-cancer agents herein are co-administered with a growth inhibitory agent For example, the growth inhibitory agent may be administered first, followed by the administration of an anti-cancer agent ofthe present invention However, simultaneous administration or administration ofthe 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) ofthe 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 ofthe 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 ofthe 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 Toxicokmetics and New Drug Development, Yacobi et al , eds , Pergamon Press, New York 1989, pp 42-96
For example, depending on the type and severity ofthe disease, about 1 g/kg to 15 mg/kg (e g , 0 1-20 mg/kg) of an antitumor agent is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs However, other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays Guidance as to particular dosages and methods of delivery is provided in the literature, see, for example, U S Pat Nos 4,657,760, 5,206,344, or 5,225,212 It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue
K Articles of Manufacture
In another embodiment of the invention, an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above is provided The article of manufacture comprises a container and a label Suitable containers include, for example, bottles, vials, syringes, and test tubes The containers may be formed from a variety of materials such as glass or plastic The container holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) The active agent in the composition is an anti-tumor agent of the present invention The label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice The article of manufacture may further comprise a second container comprising a pharmaceutical ly- acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use
The following examples are offered for illustrative purposes only, and are not intended to limit the scope ofthe present invention in any way
All patent and literature references cited the present specification are hereby incorporated by reference m 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 CultureCollection, Manassas, VA
EXAMPLE 1
Isolation of cDNA clones Encoding PRQ21 1 , PRQ228. PRQ538, PRQ172 and PRQ182 (A) PRQ21 1 The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about
950 known secreted proteins from the Swiss-Prot public database were used to search EST databases The EST databases included public EST databases (e g , GenBank), and a proprietary EST database (LIFESEQ®, Incyte 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 ofthe ECD protein sequences to a 6 frame translation of the EST sequences Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington)
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA28730 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 DNA28730 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 ofthe full-length coding sequence for PR021 1 Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length The probe sequences are typically 40-55 bp m length In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1 5 kbp In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one ofthe primer pairs
PCR primers (forward and reverse) were synthesized forward PCR primer 5'-AGAGTGTATCTCTGGCTACGC-3' (SEQ ID NO 3) reverse PCR primer
5'-TAAGTCCGGCACATTACAGGTC-3' (SEQ ID NO 4)
Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28730 sequence which had the following nucleotide sequence hybridization probe 5'-AGGGAGCACGGACAGTGTGCAGATGTGGACGAGTGCTCACTAGCA-3' (SEQ ID NO 5)
RNA for construction of the cDNA libraries was isolated from human fetal lung tissue The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278- 1280 ( 1991 )) m 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 PR021 1 polypeptide (designated herein as DNA32292- 1 131 [Figure 1 , SEQ ID NO 1 ]) and the derived protein sequence for that PR021 1 polypeptide
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 65-67 and a stop signal at nucleotide positions 1 124- 1 126 (Figure 1 , SEQ ID NO 1) The predicted polypeptide precursor is 353 amino acids long, has a calculated molecular weight of approximately 38,190 daltons Analysis ofthe full-length PR021 1 sequence shown in Figure 2 (SEQ ID NO 2) evidences the presence of a vaπety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above Analysis ofthe full-length PR021 1 sequence evidenced the following a signal peptide from about amino acid 1 to about amino acid 24, N-glycosylation sites from about amino acid 190 to about amino acid 194 and from about amino acid 251 to about amino acid 255, glycosaminoglycan attachment sites from about amino acid 149 to about amino acid 153 and from about amino acid 155 to about amino acid 159, a cAMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 26 to about amino acid 30, casein kinase II phosphorylation sites from about amino acid 58 to about amino acid 62, from about amino acid 66 to about amino acid 70, from about amino acid 86 to about amino acid 90, from about ammo acid 197 to about amino acid 201 , from about amino acid 210 to about amino acid 214, from about amino acid 255 to about amino acid 259, from about am o acid 295 to about amino acid 299, from about am o acid 339 to about amino acid 343, and from about amino acid 349 to about ammo acid 353, a tyrosine kinase phosphorylation site from about amino acid 303 to about ammo acid 310, N-myπstoylation sites from about amino acid 44 to about ammo acid 50, from about amino acid 54 to about amino acid 60, from about ammo acid 55 to about amino acid 61, from about amino acid 81 to about ammo acid 87, from about ammo acid 150 to about amino acid 156, from about amino acid 158 to about amino acid 164, from about amino acid 164 to about amino acid 170, from about amino acid 252 to about amino acid 258, and from about amino acid 313 to about am o acid 319, an aspartic acid and asparagine hydroxylation site from about amino acid 308 to about amino acid 320, an EGF-like domain cysteine pattern signature from about amino acid 166 to about amino acid 178, and a leucine zipper pattern from about amino acid 94 to about amino acid 1 16
Clone DNA32292-1 131 has been deposited with ATCC on September 16, 1997 and is assigned ATCC deposit no 209258
An analysis of the Dayhoff database (version 35 45 SwissProt 35), using the WU-BLAST2 sequence alignment analysis of the full-length sequence shown in Figure 2 (SEQ ID NO 2), evidenced sequence identity between the PR0211 ammo acid sequence and human EGF
(B) PRQ228
The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases The EST databases included public EST databases (e g , GenBank), and a proprietary EST database (LIFESEQ®, Incyte 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 ofthe ECD protein sequences to a 6 frame translation of the EST sequences Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington)
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA28758 An EST proprietary to Genentech, Inc , designated herein as DNA21951, was employed in the consensus assembly 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 DNA28758 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 ofthe full-length coding sequence for PR0228 Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length The probe sequences are typically 40-55 bp in length In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1 -1 5 kbp In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one ofthe pπmer pairs PCR primers (forward and reverse) were synthesized forward PCR primer 1
5'-GGTAATGAGCTCCATTACAG-3' (SEQ ID NO 8) forward PCR primer 2
5'-GGAGTAGAAAGCGCATGG-3' (SEQ ID NO 9) forward PCR primer 3
5'-CACCTGATACCATGAATGGCAG-3' (SEQ ID NO 10) reverse PCR primer 1
5'-CGAGCTCGAATTAATTCG-3' (SEQ ID NO 1 1 ) reverse PCR primer 2 5'-GGATCTCCTGAGCTCAGG-3' (SEQ ID NO 12) reverse PCR primer 3
5'-CCTAGTTGAGTGATCCTTGTAAG-3' (SEQ ID NO 13)
Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28758 sequence which had the following nucleotide sequence hybridization probe
5'-ATGAGACCCACACCTCATGCCGCTGTAATCACCTGACACATTTTGCAATT-3' (SEQ ID NO 14)
RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278- 1280 ( 1991 )) in the unique Xhol and Notl sites
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PR0228 polypeptide (designated herein as DN A33092- 1202 [Figures 3 A-B, SEQ ID NO 6]) and the derived protein sequence for that PR0228 polypeptide
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 24-26 and a stop signal at nucleotide positions 2094-2096 (Figures 3 A-B, SEQ
ID NO 6) The predicted polypeptide precursor is 690 amino acids long Analysis of the full-length PR0228 sequence shown in Figure 4 (SEQ ID NO 7) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above Analysis ofthe full-length PR0228 sequence evidenced the following a signal peptide from about amino acid 1 to about amino acid 19; transmembrane domains from about amino acid 430 to about amino acid 450, from about amino acid 465 to about amino acid 486, from about amino acid 499 to about amino acid 513, from about amino acid 535 to about amino acid 549, from about amino acid 573 to about amino acid 593, from about amino acid 619 to about amino acid 636, and from about amino acid 648 to about amino acid 664; N-glycosylation sites from about amino acid 15 to about amino acid 19, from about amino acid 21 to about amino acid 25, from about amino acid 64 to about amino acid 68, from about amino acid 74 to about amino acid 78, from about amino acid 127 to about amino acid 131, from about amino acid 177 to about amino acid 181 , from about amino acid 188 to about amino acid 192, from about amino acid 249 to about amino acid 253, from about amino acid 381 to about amino acid 385, and from about amino acid 395 to about amino acid 399; a glycosaminoglycan attachment site from about amino acid 49 to about amino acid 53; a c-AMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 360 to about amino acid 364; casein kinase II phosphorylation sites from about amino acid 54 to about amino acid 58, from about amino acid 68 to about amino acid 72, from about amino acid 76 to about amino acid 80, from about amino acid 94 to about amino acid 98, from about amino acid 135 to about amino acid 139, from about amino acid 150 to about amino acid 154, from about amino acid 155 to about amino acid 159, from about amino acid 161 to about amino acid 165, from about amino acid 181 to about amino acid 185, from about amino acid 190 to about amino acid 194, from about amino acid 244 to about amino acid 248, from about amino acid 310 to about amino acid 314, from about amino acid 325 to about amino acid 329, from about amino acid 346 to about amino acid 350, and from about amino acid 608 to about amino acid 612; tyrosine kinase phosphorylation sites from about amino acid 36 to about amino acid 44 and from about amino acid 670 to about amino acid 677; N-myristoylation sites from about amino acid 38 to about amino acid 44, from about amino acid 50 to about amino acid 56, from about amino acid 52 to about amino acid 58, from about amino acid 80 to about amino acid 86, from about amino acid 382 to about amino acid 388, from about amino acid 388 to about amino acid 394, from about amino acid 434 to about amino acid 440, from about amino acid 480 to about amino acid 486, and from about amino acid 521 to about amino acid 527; and an aspartic acid and asparagine hydroxylation site from about amino acid 75 to about amino acid 87.
Clone DNA33092-1202 has been deposited with ATCC on October 28, 1997 and is assigned ATCC deposit no. 209420.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using the WU-BLAST2 sequence alignment analysis ofthe full-length sequence shown in Figure 4 (SEQ ID NO:7), evidenced significant sequence identity between the PR0228 amino acid sequence and the secretin related proteins CD97 and EMR1 as well as the secretin member, latrophilin, thereby indicating that PR0228 may be a new member of the secretin related proteins.
(C) PRQ538
An expressed sequence tag (EST) DNA database and a proprietary EST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) was searched and an Incyte EST (INC3574209) was identified which had 61% sequence identity to murine GFRcβ. RNA for construction of cDNA libraries was then isolated from human fetal lung tissue The cDNA libraries used to isolate the cDNA clones encoding human PR0538 were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation mto a suitable cloning vector (such as pRKB or pRKD, ρRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278-1280 (1991)) in the unique Xhol and Notl
Oligonucleotides probes based upon the above described EST sequence were then 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 ofthe full-length coding sequence for PR0538 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 order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one ofthe primer pairs
The oligonucleotide probes employed were as follows forward PCR primer
5'-GCCTCTCGCAGCCGGAGACC-3' (SEQ ID NO 17) reverse PCR primer 5'-CAGGTGGGATCAGCCTGGCAC-3' (SEQ ID NO 18) hybridization probe 5'-TCTCGCAGCCGGAGACCCCCTTCCCACAGAAAGCCGACTCA-3' (SEQ ID NO 19)
Pure positive clones were obtained after colony purification and secondary screening Five positive clones were identified Two ofthe isolated clones were sequenced These cDNA sequences were designated DN A48613- 1268 and DNA48614-1268 A full length clone for DNA48613- 1268 was identified that contained a single open reading frame with an apparent translational initiation site at nucleotide positions 38-40 and a stop signal at nucleotide positions 1238-1240 (Figure 5, SEQ ID NO 15) The predicted polypeptide precursor is 400 amino acids long, has a calculated molecular weight of approximately 44,51 1 daltons and an estimated pi of approximately 8 15 A comparison of the amino acid sequence of DNA48614-1268 to the amino acid sequence of DNA48613-1268 (Figure 5, SEQ ID NO 15), revealed it to be an alternatively spliced form of DNA48613-1268, with a 30 ammo acid deletion (amino acids 127-157, counting from the initiation methionine)
Analysis ofthe full-length PR0538 sequence shown in Figure 6 (SEQ ID NO 16) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PR0538 sequence evidenced the following a signal peptide from about amino acid 1 to about amino acid 26, a transmembrane domain from about amino acid 379 to about amino acid 395, N-glycosylation sites from about amino acid 95 to about amino acid 99, from about ammo acid 148 to about amino acid 152, and from about amino acid 309 to about amino acid 313, a cAMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 231 to about amino acid 235, casein kinase II phosphorylation sites from about amino acid 134 to about am o acid 138, from about amino acid 170 to about amino acid 174, and from about amino acid 202 to about amino acid 206, N-myπstoylation sites from about ammo acid 279 to about ammo acid 285 and from about amino acid 294 to about amino acid 300, and prokaryotic membrane hpoprotein lipid attachment sites from about amino acid 306 to about amino acid 317 and from about amino acid 379 to about amino acid 390
Clone DNA48613-1268 has been deposited with ATCC on April 7, 1998 and is assigned ATCC deposit no 209752
As discussed below, a sequence comparison of the full-length sequence shown in Figure 6 (SEQ ID NO 16) encoded by DNA48613-1268 to the sequences of human GFRα 1 and GFRα2 indicated that the human protein is a new member ofthe GFRα receptor family, and is a human homolog of murine GFRα3 Accordingly,
DNA48613-1268 encodes a protein designated as human GFRα3, and DNA48614- 1268 encodes its splice variant
An analysis of the Dayhoff database (version 35 45 SwissProt 35), using the BLAST-2 and FastA sequence alignment analysis ofthe full-length sequence of PR0538 shown in Figure 6 (SEQ ID NO 16) and other GFRα family members is provided in Table 4
Table 4 Sequence Identity Between Members ofthe GFRα Family
Proteins Compared Percent Identity rGFRα 1 versus hGFRα 1 92% rGFRα2 versus hGFRα2 94% mGFRα3 versus hGFRα3 77%
hGFRα3 versus hGFRα 1 34% hGFRα3 versus hGFRα2 34% hGFRα 1 versus hGFRα2 48% From the sequence comparisons it can be seen that human GFRα3 (PR0538) is less related to its rodent homolog than is either GFRαl or GFRα2 In addition, GFRα3 (PR0538) appears to be more distantly related to GFRαl and GFRα2 than GFR l and GFRα2 are to each other
(D) PRO 172
The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases The EST databases included public EST databases (e g , GenBank), and a proprietary EST database (LIFESEQ®, Incyte 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 ofthe ECD protein sequences to a 6 frame translation of the EST sequences Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington)
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA28765 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 DNA28765 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 ofthe full-length coding sequence for PRO 172 Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length The probe sequences are typically 40-55 bp in length In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1 5 kbp In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one ofthe primer pairs
PCR primers (forward and reverse) were synthesized forward PCR primer
5'-GGATCTCGAGAACAGCTACTCC-3" (SEQ ID NO 22) reverse PCR primer 5'-TCGTCCACGTTGTCGTCACATG-3' (SEQ ID NO 23)
Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28765 sequence which had the following nucleotide sequence hybridization probe 5'-AAATCTGTGAATTGAGTGCCATGGACCTGTTGCGGACGGCCCTTGCTT-3' (SEQ ID NO 24) RNA for construction of the cDN A libraries was isolated from human fetal kidney tissue The cDN A libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278- 1280 ( 1991 )) in the unique Xhol and Notl sites
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PRO 172 polypeptide (designated herein as DNA35916-1 161 [Figures 7 A-B, SEQ ID NO 20]) and the derived protein sequence for that PRO 172 polypeptide The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 38-40 and a stop signal at nucleotide positions 2207-2209 (Figures 7 A-B, SEQ ID NO 20) The predicted polypeptide precursor is 723 amino acids long Analysis of the full-length PRO 172 sequence shown in Figure 8 (SEQ ID NO:21 ) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis ofthe full-length PROl 72 sequence evidenced the following: a signal peptide from about amino acid 1 to about amino acid 21 ; a transmembrane domain from about amino acid 548 to about amino acid 568; an N-glycosylation site from about amino acid 477 to about amino acid 481 ; a cAMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 660 to about amino acid 664; casein kinase II phosphorylation sites from about amino acid 93 to about amino acid 97, from about amino acid 131 to about amino acid 135, from about amino acid 154 to about amino acid 158, from about amino acid 203 to about amino acid 207, from about amino acid 342 to about amino acid 346, from about amino acid 344 to about amino acid 348, from about amino acid 369 to about amino acid 373, from about amino acid 457 to about amino acid 461 , from about amino acid 483 to about amino acid 487, from about amino acid 495 to about amino acid 499, from about amino acid 659 to about amino acid 663, from about amino acid 670 to about amino acid 674, from about amino acid 671 to about amino acid 675, and from about amino acid 698 to about amino acid 702; tyrosine kinase phosphorylation sites from about amino acid 176 to about amino acid 185 and from about amino acid 252 to about amino acid 261 ; N-myristoylation sites from about amino acid 2 to about amino acid 8, from about amino acid 37 to about amino acid 43, from about amino acid 40 to about amino acid 46, from about amino acid 98 to about amino acid 104, from about amino acid 99 to about amino acid 105, from about amino acid 262 to about amino acid 268, from about amino acid 281 to about amino acid 287, from about amino acid 282 to about amino acid 288, from about amino acid 301 to about amino acid 307, from about amino acid 310 to about amino acid 316, from about amino acid 328 to about amino acid 334, from about amino acid 340 to about amino acid 346, from about amino acid 378 to about amino acid 384, from about amino acid 387 to about amino acid 393, from about amino acid 512 to about amino acid 518, from about amino acid 676 to about amino acid 682, from about amino acid 683 to about amino acid 689, and from about amino acid 695 to about amino acid 701 ; aspartic acid and asparagine hydroxylation sites from about amino acid 343 to about amino acid 355, from about amino acid 420 to about amino acid 432, and from about amino acid 458 to about amino acid 480; a prokaryotic membrane lipoprotein lipid attachment site from about amino acid 552 to about amino acid 563; and EGF-like domain cysteine pattern signatures from about amino acid 243 to about amino acid 255, from about amino acid 274 to about amino acid 286, from about amino acid 314 to about amino acid 326, from about amino acid 352 to aout amino acid 364, from about amino acid 391 to about amino acid 403, from about amino acid 429 to about amino acid 441, from about amino acid 467 to about amino acid 479, and from about amino acid 505 to about amino acid 517.
Clone DNA35916-1 161 has been deposited with ATCC on October 28, 1997 and is assigned ATCC deposit no. 209419.
An analysis ofthe Dayhoff database (version 35.45 SwissProt 35), using the BLAST and FastAsequence alignment analysis of the full-length sequence shown in Figure 8 (SEQ ID NO:21 ), evidenced 89% sequence identity between the PR0172 amino acid sequence and delta-1 mouse protein. (E) PROl 82
An expressed sequence tag (EST) DNA database and a proprietary EST database (LIFESEQ®, Incyte
Pharmaceuticals, Palo Alto, CA) was searched and two EST sequences were identified (Incyte EST INC2328985 and Incyte EST INC778319), each having approximately 40% homology to a region of the relaxin nucleic acid sequence, and representing sequences within a gene of an insulin-like polypeptide The EST corresponding to
INC778319 was used to clone the full-length PRO 182 gene
RNA for construction of cDNA libraries was then isolated from human uterine tissue The cDN A libraries used to isolate the cDNA clones encoding human PRO 182 were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278-1280 (1991)) in the unique Xhol and Notl
Oligonucleotides probes based upon the above described EST sequence were then 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 ofthe full-length coding sequence for PROl 82 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 order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one ofthe pπmer pairs
The oligonucleotide probes employed were as follows 5'-CACATTCAGTCCTCAGCAAAATGAA-3' (SEQ ID NO 27) 5'-GAGAATAAAAACAGAGTGAAAATGGAGCCCTTCATTTTGC-3' (SEQ ID NO 28) 5'-CTCAGCTTGCTGAGCTTGAGGGA-3' (SEQ ID NO 29)
A full length clone for DNA27865-1091 was identified that contained a single open reading frame with an apparent translational initiation site at nucleotide positions 39-41 and a stop signal at nucleotide positions 444- 446 (Figure 9, SEQ ID NO 25) The predicted polypeptide precursor is 135 amino acids long
Analysis ofthe full-length PRO 182 sequence shown in Figure 10 (SEQ ID NO 26) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PROl 82 sequence evidenced the following a signal peptide from about amino acid 1 to about amino acid 18, a cAMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 107 to about amino acid 1 1 1 , casein kinase II phosphorylation sites from about amino acid 88 to about amino acid 92, from about amino acid 1 13 to about amino acid 1 17, and from about amino acid 127 to about am o acid 131, N-myπstoylation sites from about amino acid 3 to about am o acid 9, from about amino acid 52 to about amino acid 58, from about amino acid 96 to about ammo acid 102, and from about am o acid 125 to about amino acid 131 , and an insulin family signature from about amino acid 121 to about amino acid 136
Clone DNA27865-1091 has been deposited with ATCC on September 23, 1997 and is assigned ATCC deposit no 209296
An analysis of the Dayhoff database (version 35 45 SwissProt 35), using the WU-BLAST2 sequence alignment analysis ofthe full-length sequence shown in Figure 10 (SEQ ID NO 26) evidenced sequence identity between the PR0182 amino acid sequence and a human msuhn-hke polypeptide, thus indicating that PR0182 is a novel human msuhn-hke protein
EXAMPLE 2 Expression of PRQ21 1. PRQ228, PRQ538, PRO 172 or PROl 82 in £ co This example illustrates preparation of an unglycosylated form of PR021 1 , PR0228, PR0538, PR0172 or PRO 182 by recombinant expression in £ co
The DNA sequence encoding PR021 1, PR0228, PR0538, PR0172 or PR0182 is initially amplified using selected PCR primers The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector A variety of expression vectors may be employed An example of a suitable vector is ρBR322 (derived from £ coh, see Bolivar et al , Gene, 2 95 (1977)) which contains genes for ampicillin and tetracycline resistance The vector is digested with restriction enzyme and dephosphorylated The PCR amplified sequences are then hgated 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 enterokmase cleavage site), the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 coding region, lambda transcriptional terminator, and an argU gene
The ligation mixture is then used to transform a selected £ co strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics The overnight culture may subsequently be used to inoculate a larger scale culture The cells are then grown to a desired optical density, during which the expression promoter is turned on
After culturing the cells for several more hours, the cells can be harvested by centrifugation The cell pellet obtained by the centrifugation can be solubi zed using various agents known in the art, and the solubihzed PR021 1, PR0228, PR0538, PRO 172 or PRO 182 protein can then be purified using a metal chelating column under conditions that allow tight binding ofthe protein
PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 may be expressed in £ co in a poly-His tagged form, using the following procedure The DNA encoding PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 enterok ase The PCR-amplified, poly-His tagged sequences are then hgated into an expression vector, which is used to transform an £ coh host based on strain 52 (W31 10 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(ladq) Transformants are first grown in LB containing 50 mg/ml carbenicillm at 30°C with shaking until an OD600 of 3-5 is reached Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3 57 g (NH4)2S04, 0 71 g sodium cιtrate«2H20, 1 07 g KCI, 5 36 g Difco yeast extract, 5 36 g Sheffield hycase SF in 500 ml water, as well as 1 10 mM MPOS, pH 7 3, 0 55% (w/v) glucose and 7 mM MgS04) and grown for approximately 20-30 hours at 30 °C with shaking Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells Cell pellets are frozen until purification and refolding
£ coh paste from 0 5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) m 7 M guanidme, 20 mM Tris, pH 8 buffer Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0 IM and 0 02 M, respectively, and the solution is stirred overnight at 4°C This step results in a denatured protein with all cysteine residues blocked by sulfitolization The solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 mm The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidme, 20 mM Tris, pH 7 4) and filtered through 0 22 micron filters to clarify The clarified extract is loaded onto a 5 ml Qiagen Ni 2+-NTA metal chelate column equilibrated in the metal chelate column buffer The column is washed with additional buffer containing 50 mM 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 Tris, pH 8 6, 0 3 M NaCl, 2 5 M urea, 5 mM cysteine, 20 mM glycme 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 A280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitnle since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin Aggregated species are usually eluted at higher 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 EXAMPLE 3 Expression of PRQ21 1. PRQ228. PRQ538, PRO 172 or PRO 182 in mammalian cells This example illustrates preparation of a potentially glycosylated form of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1 , PR0228, PR0538, PR0172 or PROl 82 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 DNA using ligation methods such as described in Sambrook et al, supra. The resulting vector is called pRK5-PR021 1, pRK5- PR0228, pRK5-PR0538, pRK5-PR0172 or pRK5-PR0182. In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 μg pRK5-PRO21 l , pRK5-PR0228, pRK5-PR0538, pRK5-PR0172 or pRK5-PR0182 DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al, Ceh, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500 μ\ of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaP04, and a precipitate is allowed to form for 10 minutes at 25 °C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C. The culture medium is aspirated off and 2 ml of 20%> glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days. Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml 35S-cysteine and 200 μCi/ml 35S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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, PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al, Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-PR021 1 , pRK5-PR0228, pRK5-PR0538, pRK5-PR0172 or pRK5-PRO 182 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 A-g/ml bovine insulin and 0.1 /.g/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
In another embodiment, PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 can be expressed in CHO cells. The pRK5-PR021 1 , pRK5-PR0228, pRK5-PR0538, pRK5-PR0172 or pRK-5PR0182 can be transfected into CHO cells using known reagents such as CaP04 or DEAE-dextran As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as -"S- methionme After determining the presence of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide can then be concentrated and purified by any selected method
Epitope-tagged PR021 1 , PR0228, PR0538, PR0172 or PR0182 may also be expressed in host CHO cells The PR021 1, PR0228, PR0538, PR0172 or PR0182 may be subcloned out of the pRK5 vector The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-His tag into a Baculovirus expression vector The poly-His tagged PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 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 PR021 1, PR0228, PR0538, PR0172 or PR0182 can then be concentrated and purified by any selected method, such as by Nι2+-chelate affinity chromatography
PR021 1 , PR0228, PR0538, PR0172 or PR0182 may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure
Stable expression in CHO cells is performed using the following procedure The proteins are expressed as an IgG construct (lmmunoadhesin), in which the coding sequences for the soluble forms (e g , extracellular domains) ofthe respective proteins are fused to an IgG 1 constant region sequence containing the hinge, CH2 and CH2 domains and/or as a poly-His tagged form
Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al , Current Protocols of Molecular Biology, Unit 3 16, John Wiley and Sons (1997) CHO expression vectors are constructed to have compatible restriction sites 5' and 3' ofthe 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 ofthe cDNA of interest and dihydrofolate reductase (DHFR) DHFR expression permits selection for stable maintenance ofthe plasmid following transfection
Twelve micrograms ofthe desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Quiagen), Dosper® or Fugene® (Boehringer Mannheim) The cells are grown as described in Lucas et al , supra Approximately 3 x 10 7 cells are frozen in an ampule for further growth and production as described below
The ampules containing the plasmid DNA are thawed by placement mto a water bath and mixed b> vortexing The contents are pipetted into a centrifuge tube containing 10 mis of media and centrifuged at 1000 rpm for 5 minutes The supernatant is aspirated and the cells are resuspended in 10 ml of selective media (02 μm filtered PS20 with 5%o 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 10%, 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 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C. lmmunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 μl of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation. PR021 1 , PRO 172 and PRO 182 were stably expressed in CHO cells by the above described method. In addition, PRO 172 was expressed in CHO cells by the transient expression procedure.
EXAMPLE 4 Expression of PRQ21 1 , PRQ228, PRQ538. PROl 72 or PROl 82 in Yeast The following method describes recombinant expression of PR021 1 , PR0228, PR0538, PR0172 or PRO 182 in yeast.
First, yeast expression vectors are constructed for intracellular production or secretion of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 from the ADH2/GAPDH promoter. DNA encoding PR021 1 , PR0228, PR0538, PR0172 or PR0182 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182. For secretion, DNA encoding PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or lnvertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182
Yeast cells, such as yeast strain ABI 10, can then be transformed with the expression plasmids described above and cultured in selected fermentation media The transformed yeast supematants can be analyzed by precipitation with 10%> tπchloroacetic acid and separation by SDS-PAGE, followed by staining ofthe gels with Coomassie Blue stain
Recombinant PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 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 PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 may further be purified using selected column chromatography resins
EXAMPLE 5 Expression of PRQ21 1, PRQ228, PRQ538, PRQ172 or PRQ182 in Baculovirus-Infected Insect Cells The following method describes recombinant expression in Baculovirus-infected insect cells The sequence coding for PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 is fused upstream of an epitope tag contained within a baculovirus expression vector Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL 1393 (Novagen) Briefly, the sequence encoding PR021 1 , PR0228, PR0538, PR0172 or PR0182 or the desired portion of the coding sequence of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 (such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular) is amplified by PCR with primers complementary to the 5' and 3' regions The 5' primer may incorporate flanking (selected) restriction enzyme sites The product is then digested with those selected restriction enzymes and subcloned into the expression vector
Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA
(Pharmingen) into Spodopterafrugφerda ("Sf9") cells (ATCC CRL 171 1 ) using lιpofectιn(commercιally available from GIBCO-BRL) After 4 - 5 days of incubation at 28°C, the released viruses are harvested and used for further amplifications Viral infection and protein expression are performed as described by O'Reilley et al , Baculovirus expression vectors A Laboratory Manual Oxford Oxford University Press (1994)
Expressed poly-His tagged PR021 1 , PR0228, PR0538, PR0172 or PR0182 can then be purified, for example, by Nι2+-chelate affinity chromatography as follows Extracts are prepared from recombinant virus- infected Sf9 cells as described by Rupert et al , Nature, 362 175-179 (1993) Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes, pH 7 9, 12 5 mM MgCh, 0 1 mM EDTA, 10% glycerol, 0 1% NP- 40, 0 4 M KCI), and sonicated twice for 20 seconds on ice The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7 8) and filtered through a 0 45 mm filter A Nr+-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 ml, washed with 25 ml of water and equilibrated with 25 ml of loading buffer The filtered cell extract is loaded onto the column at 0 5 ml per minute The column is washed to baseline A280 with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein. After reaching A280 baseline again, the column is developed with a 0 to 500 mM imidazole gradient in the secondary wash buffer. One ml fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni2+-NTA- conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His, --tagged PR021 1 , PR0228, PR0538, PR0172 or PROl 82, respectively, are pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PR0211 , PR0228, PR0538, PR0172 or PROl 82 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography. Following PCR amplification, the respectivecodingsequencesare subcloned into a baculovirus expression vector (pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged proteins), and the vector and Baculogold® baculovirus DNA (Pharmingen) are co-transfected into 105 Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are modifications ofthe commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences. The cells are grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). Cells are incubated for 5 days at 28 CC. The supernatant is harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10%> FBS at an approximate multiplicity of infection (MOI) of 10. Cells are incubated for 3 days at 28 °C. The supernatant is harvested and the expression of the constructs in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml of Ni 2+-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
The first viral amplification supernatant is used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells are incubated for 3 days at 28 °C. The supernatant is harvested and filtered. Batch binding and SDS-PAGE analysis is repeated, as necessary, until expression ofthe spinner culture is confirmed.
The conditioned medium from the transfected cells (0.5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein construct is purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C. lmmunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows. The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3 5 The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9 The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins The homogeneity ofthe proteins is verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation
PR0228, PR0538 and PR0172 were expressed in baculovirus infected Sf9 insect cells
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 pIE 1 - 1 (Novagen) The pIE 1 - 1 and pIE 1 -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus ιel promoter in stably- transformed insect cells (1) The plasmids differ only in the orientation ofthe 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-l 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, 100 μl ofCellFectιn (CellFECTIN (GιbcoBRL #10362-010) (vortexed to mιx)) ιs mιxed wιth 1 ml of Ex-Cell medium The two solutions are combined and allowed to incubate at room temperature for 15 minutes 8 ml of Ex-Cell media is added to the 2 ml of DNA/CellFECTIN mix and this is layered on 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 CellFECTIN, 30 ml of fresh Ex-Cell media is added and the cells are incubated for 3 days at 28°C The supernatant is harvested and the expression ofthe sequence in the baculovirus expression vector is determined by batch binding of 1 ml of supernatent to 25 ml of Ni ,+-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining The 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 ,+-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 1 O mM Hepes, 0 14 M aCl and 4%> mannιtol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C lmmunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3 5 The eluted protein is immediately neutralized by collecting 1 ml fractions mto tubes containing 275 ml of 1 M Tris buffer, pH 9 The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins The homogeneity ofthe 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 PR021 1 , PR0228, PR0538, PRO 172 and PRO 182 were expressed using the above baculovirus procedure employing hιgh-5 cells
EXAMPLE 6 Preparation of Antibodies that Bind PRQ21 1 , PRQ228, PRQ538, PRQ172 or PRQ182 This example illustrates preparation of monoclonal antibodies which can specifically bind PR021 1 , PR0228, PR0538, PR0172 or PROl 82
Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, m Goding, supra Immunogens that may be employed include purified PR021 1, PR0228, PR0538. PRO 172 or
PR0182, fusion proteins containing PR021 1 , PR0228, PR0538, PR0172 or PR0182, and cells expressing recombinant PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 on the cell surface Selection ofthe immunogen can be made by the skilled artisan without undue experimentation
Mice, such as Balb/c, are immunized with the PR021 1 , PR0228, PR0538, PR0172 or PR0182 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneal ly in an amount from 1-100 micrograms Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant Thereafter, for several weeks, the mice may also be boosted with additional immunization injections Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect antι-PR021 1 , antι-PR0228, antι-PR0538, antι-PR0172 or antι-PR0182 antibodies
After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids The hybridoma cells will be screened in an ELISA for reactivity against PR0211, PR0228, PR0538,
PR0172 or PR0182 Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PR0211 , PR0228, PR0538, PR0172 or PR0182 is within the skill in the art
The positive hybridoma cells can be injected lntrapeπtoneally into syngeneic Balb/c mice to produce ascites containmg the antι-PR021 1 , antι-PR0228, antι-PR0538, antι-PR0172 or antι-PR0182 monoclonal antibodies Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles Purification ofthe 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 7 Purification of PRQ21 1 , PRQ228, PRQ538, PRO 172 or PRO 182 Polypeptides Using Specific Antibodies
Native or recombinant PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptides may be purified by a variety of standard techniques in the art of protein purification For example, pro-PR021 1 , pro-PR0228, pro- PR0538, pro-PR0172 or pro-PR0182 polypeptide, mature PR021 1, PR0228, PR0538, PR0172 or PR0182 polypeptide, or pre-PR021 1 , pre-PR0228, pre-PR0538, pre-PR0172 or pre-PR0182 polypeptide is purified by immunoaffimty chromatography using antibodiesspecificforthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide of interest In general, an immunoaffimty column is constructed by covalently coupling the antι-PR021 1, antι-PR0228, antι-PR0538, antι-PR0172 or antι-PR0182 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 monoclonalantibodiesare prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKB Biotechnology) The antibody is coupled to the resm, the resm is blocked, and the derivative resin is washed according to the manufacturer's instructions Such an immunoaffimty column is utilized in the purification ofthe PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 polypeptide by preparing a fraction from cells containing the PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide in a soluble form This preparation is derived by solubihzation ofthe whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art Alternatively, soluble PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown
A soluble PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide-contai ng preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/PR021 1 , antibody/PR0228, antibody/PR0538, antibody /PRO 172 or antibody/PRO 182 polypeptide binding (e.g. , a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide is collected.
EXAMPLE 8 Drug Screening This invention is particularly useful for screening compounds by using PR021 1 , PR0228, PR0538, PR0172 or PROl 82 polypeptides or a binding fragment thereof in any of a variety of drug screening techniques. The PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide or a fragment and the agent being tested. Alternatively, one can examine the diminution in complex formation between the PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 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 PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 polypeptide-associated disease or disorder. These methods comprise contacting such an agent with a PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide or fragment, or (ii) for the presence of a complex between the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide or fragment and the cell, by methods well known in the art. In such competitive binding assays, the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide or fragment is typically labeled. After suitable incubation, the free PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure ofthe ability ofthe particular agent to bind to the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide or to interfere with the PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide/cell complex.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on September 13, 1984.
Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to a PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide, the peptide test compounds are reacted with the PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide and washed. Bound PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide is detected by methods well known in the art Purified PR021 1 , PR0228, PR0538, PR0172 or PR0182 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 sohd^upport
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide specifically compete with a test compound for binding to the PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide or fragments thereof In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide
EXAMPLE 9 Rational Drug Design
The goal of rational drug design is to produce structural analogs of a biologically active polypeptide of interest (ι e , a PR021 1, PR0228, PR0538, PRO 172 or PRO 182 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 PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide or which enhance or interfere with the function ofthe PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide in vivo (cf Hodgson, Bio/Technology, 9 19-21 (1991))
In one approach, the three-dimensional structure ofthe PR021 1 , PR0228, PR0538, PRO 172 or PROl 82 polypeptide, or of a PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide-inhibitorcomplex, is determined by x-ray crystallography, by computer modeling or, most typically, by a combination ofthe two approaches Both the shape and charges ofthe PR0211, PR0228, PR0538, PR0172 or PR0182 polypeptide must be ascertained to elucidate the structure and to determine active sιte(s) ofthe molecule Less often, useful information regarding the structure of the PR021 1, PR0228, PR0538, PRO 172 or PRO 182 polypeptide may be gained by modeling based on the structure of homologous proteins In both cases, relevant structural information is used to design analogous PR021 1 , PR0228, PR0538, PR0172 or PR0182 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 αl , 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 mirror image, the binding site ofthe 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 PR021 1 , PR0228, PR0538, PROl 72 or
PRO 182 polypeptide may be made available to perform such analytical studies as X-ray crystallography In addition, knowledge ofthe PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 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 10 In Vitro Antitumor Assay
The antiprohferative activity ofthe PR021 1 , PR0228, PR0538, PRO 172 and PRO 182 polypeptides was determined in the investigational, disease-oriented in vitro anti-cancer drug discovery assay ofthe National Cancer Institute (NCI), using a sulforhodamme 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 tumor cell lines employed in this study ("the NCI panel"), as well as conditions for their maintenance and culture in vitro have been described by Monks et al , J Natl Cancer Inst , 83 757-766 (1991 ) The purpose of this screen is to initially evaluate the cytotoxic and/or cytostatic activity ofthe test compounds against different types of tumors (Monks et al , supra, Boyd, Cancer Pπnc Pract Oncol Update, 3(10) 1-I2 fl9891)
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% CO, atmosphere and 100% humidity
After incubation, the medium was removed and the cells were fixed in 0 1 ml of 10% tπchloroacetic 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% sulforhodamme 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 [tπs(hydroxymethyl)amιnomethane],pH 10 5 The absorbance (OD) of sulforhodamme B at 492 nm was measured using a computer-interfaced, 96-well microtiter plate readei
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 Tables 5-9, where the tumor cell type abbreviations are as follows NSCL = non-small cell lung carcinoma, CNS = central nervous system Table 5
Compound Concentration Days Tumor Cell Type Designation
PR0211 0.65 nM 6 NSCL HOP62
PR02U 6.50 nM 6 Leukemia RPMI-8226
PR0211 6.50 nM 6 Leukemia HL-60 (TB)
PR0211 6.50 nM 6 NSCL NCI-H522
PR0211 6.50 nM 6 CNS SF-539
PR0211 6.50 nM 6 Melanoma LOX IMVI
PR0211 6.50 nM 6 Breast MDA-MB-435
PR0211 3.90 nM 6 Leukemia MOLT-4
PR0211 3.90 nM 6 CNS U251
PR0211 3.90 nM 6 Breast MCF7
PR0211 39.00 nM 6 Leukemia HT-60 (TB)
PR0211 39.00 nM 6 Leukemia MOLT-4
PR0211 39.00 nM 6 NSCL EKVX
PR0211 39.00 nM 6 NSCL NCI-H23
PR0211 39.00 nM 6 NSCL NCI-H322M
PR0211 39.00 nM 6 NSCL NCI-H460
PR0211 39.00 nM 6 Colon HCT-116
PR0211 39.00 nM 6 Colon HT29
PR0211 39.00 nM 6 CNS SF-268
PR0211 39.00 nM 6 CNS SF-295
PR0211 39.00 nM 6 CNS SNB-19
PR0211 39.00 nM 6 CNS U251
PR0211 39.00 nM 6 Melanoma LOX IMVI
PR0211 39.00 nM 6 Melanoma SK-MEL-5
PR0211 39.00 nM 6 Melanoma UACC-257
PR0211 39.00 nM 6 Melanoma UACC-62
PR0211 39.00 nM 6 Ovarian OVCAR-8
PR0211 39.00 nM 6 Renal RXF393
PR0211 39.00 nM 6 Breast MCF7
PR0211 39.00 nM 6 Breast NCI/ADR-REHS 578T
PR0211 39.00 nM 6 Breast T-47D
PR0211 39.00 nM 2 Leukemia HL-60 (TB)
PR0211 39.00 nM 2 Leukemia SR
PR0211 39.00 nM 2 NSCL NCI-H23
PR0211 39.00 nM 2 Colon HCT-116
PR0211 39.00 nM 2 Melanoma LOX-IMVI
PR0211 39.00 nM 2 Melanoma SK-MEL-5
PR0211 39.00 nM 2 Breast T-47D
Table 6
Compound Concentration Days Tumor Cell Type Designation
PR0228 0.77 nM 6 Leukemia MOLT-4
PR0228 0.77 nM 6 NSCL EKVX
PR0228 0.77 nM 6 Colon KM12
PR0228 0.77 nM 6 Melanoma UACC-62
PR0228 0.77 nM 6 Ovarian OVCAR-3
PR0228 0.77 nM 6 Renal TK10
PR0228 0.77 nM 6 Renal SN12C
PR0228 0.77 nM 6 Breast MCF7
PR0228 7.77 nM 6 Leukemia CCRF-CEM
PR0228 7.77 nM 6 Leukemia HL-60 (TB)
PR0228 7.77 nM 6 Colon COLO 205
PR0228 7.77 nM 6 Colon HCT-15
PR0228 7.77 nM 6 Colon KM12
PR0228 7.77 nM 6 CNS SF-268
PR0228 7.77 nM 6 CNS SNB-75
PR0228 7.77 nM 6 Melanoma LOX-IMVI
PR0228 7.77 nM 6 Melanoma SK-MEL2
PR0228 7.77 nM 6 Melanoma UACC-257
PR0228 • 7.77 nM 6 Ovarian IGROV1
PR0228 7.77 nM 6 Ovarian OVCAR-4
PR0228 7.77 nM 6 Ovarian OVCAR-5
PR0228 7.77 nM 6 Ovarian OVCAR-8
PR0228 7.77 nM 6 Renal 786-0
PR0228 7.77 nM 6 Renal CAKI-1
PR0228 7.77 nM 6 Renal RXF 393
PR0228 7.77 nM 6 Renal TK-10
PR0228 7.77 nM 6 Renal UO-31
PR0228 7.77 nM 6 Prostate PC-3
PR0228 7.77 nM 6 Prostate DU-145
PR0228 7.77 nM 6 Breast MCF7
PR0228 7.77 nM 6 Breast NCI/ADR-REHS 578T
PR0228 7.77 nM 6 Breast MDA-MB-435MDA-N
PR0228 7.77 nM 6 Breast T-47D
Table 7
Compound Concentration Dav Tumor Cell Tvoe Designation
PR0538 2 Leukemia SR
PR0538 2 CNS SF-539
PR0538 2 Renal RXF 393
PR0538 6 Leukemia HL-60 (TB)
PR0538 6 NSCL EKVX
PR0538 6 NSCL HOP*
PR0538 6 NSCL NCI-H23*
PR0538 6 NSCL NCI-H322M
PR0538 6 NSCL NCI-H460*
PR0538 6 Colon HCC-2998
PR0538 6 Colon HCT-1 16
PR0538 6 Colon HT29
PR0538 6 CNS SF-268*
PR0538 6 CNS SF-295
PR0538 6 CNS SNB-19
PR0538 6 CNS U251
PR0538 6 Melanoma LOX IMVI
PR0538 6 Melanoma SK-MEL-2
PR0538 6 Melanoma SK-MEL-28
PR0538 6 Melanoma SK-MEL-5
PR0538 6 Melanoma UACC-25*
PR0538 6 Melanoma UACC-62
PR0538 6 Ovarian OVCAR-5*
PR0538 6 Ovarian OVCAR-8*
PR0538 6 Renal 768-0
PR0538 6 Renal ACHN
PR0538 6 Renal CAKI- 1 **
PR0538 6 Renal RXF 393*
PR0538 6 Renal SN 12C
PR0538 6 Renal TK-10
PR0538 6 Prostate PC-3
PR0538 6 Prostate DU- 145*
PR0538 6 Breast MDA-MB-23
PR0538 6 Breast HS 578T*
PR0538 6 Breast ST-549*
PR0538 6 Breast T-47D
* cytotoxic effect ** cytostatic effect
Table 8
Compound Concentration Days Tumor Cell Type Designation
PRO 172 1.25 nM 2 Breast T-470
PRO 172 1.25 nM 6 NSCL NCI-H460
PRO 172 1.25 nM 6 Colon KM12
PRO 172 1.25 nM 6 CNS SF-295
PRO 172 1.25 nM 6 Melanoma UACC-62
PRO 172 1.25 nM 2 Breast MDA-MB-231/ATCC
PRO 172 1.25 nM 6 Leukemia CCRF-CEM
PRO 172 1.25 nM 6 Leukemia MOLT4
PRO 172 1.25 nM 6 NSCL NCI-H460
PRO 172 1.25 nM 6 Colon HCT-1 16
PRO 172 1.25 nM 6 Colon HT29
PRO 172 1.25 nM 6 CNS SF-295
PRO 172 1.25 nM 6 CNS U251
PRO 172 1.25 nM 6 Melanoma LOX IMVI
PRO 172 1.25 nM 6 Melanoma UACC-62
PRO 172 1.25 nM 6 Ovarian OVCAR-8
PRO 172 1.25 nM 6 Renal RXF 393
PRO 172 1.25 nM 6 Breast T-470
Table 9
Compound Concentration Days Tumor Cell Type Designation
PRO 182 0.85 nM 2 Leukemia K-562
PRO 182 0.85 nM 6 Leukemia HL-60 (TB)
PRO 182 6.70 nM 6 Ovarian OVCAR-5
PRO 182 6.70 nM 6 Leukemia HL-60 (TB)
PRO 182 6.70 nM 6 Colon COLO205
PRO 182 6.70 nM 6 Melanoma LOX IMVI
PRO 182 67.0 nM 2 NSCL EKVX
PRO 182 67.0 nM 2 NSCL NCI-H226
PRO 182 67.0 nM 2 Ovarian IGROV1
PRO 182 67.0 nM 2 Ovarian OVCAR3
PRO 182 67.0 nM 2 Breast HS378T
PRO 182 67.0 nM 2 Breast T47D
PRO 182 67.0 nM 6 Leukemia CCRF-CEM
PRO 182 67.0 nM 6 Leukemia HL-60 (TB)
PRO 182 67.0 nM 6 Leukemia MOLT4
PRO 182 67.0 nM 6 Leukemia SR
PRO 182 67.0 nM 6 NSCL NCI-H23
PRO 182 67.0 nM 6 NSCL NCI-H460
PRO 182 67.0 nM 6 CNS U251
PRO 182 67.0 nM 6 Melanoma UACC-257
PRO 182 67.0 nM 6 Melanoma UACC-62
PRO 182 67.0 nM 6 Renal RXF-393
PRO 182 42.0 nM 6 Leukemia MOLT4
PRO 182 42.0 nM 6 Leukemia SR
PRO 182 42.0 nM 6 NSCL A549/ATCC
PRO 182 42.0 nM 6 NSCL NCI/H322M
PRO 182 42.0 nM 6 Colon HCT-18
PRO 182 42.0 nM 6 Melanoma UACC-257
PRO 182 42.0 nM 6 Melanoma USCC-62
PRO 182 42.0 nM 2 Renal RXF 393
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
DNA32292-1 131 209258 September 16, 1997
DNA33092-1202 209420 October 28, 1997
DNA48613-1268 209752 April 7, 1998 DNA35916-1 161 209419 October 28, 1997
DNA27865-1091 209296 September 23, 1997
These deposits were made under the provisions ofthe Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposits will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc., and ATCC, which assures permanent and unrestricted availability ofthe progeny ofthe culture ofthe deposit to the public upon issuance ofthe 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 ofthe present application has agreed that if a culture ofthe 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 ofthe same. Availability ofthe deposited material is not to be construed as a license to practice the invention in contravention ofthe rights granted under the authority of any government in accordance with its patent laws.
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect ofthe invention, including the best mode thereof, nor is it to be construed as limiting the scope ofthe claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope ofthe 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 PR021 1 , PR0228, PR0538, PR0172 or PROl 82 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide, or an agonist thereof
3 The composition of matter of Claim 1 comprising a cytotoxic amount of a PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 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 PR021 1 , PR0228, PR0538, PRO 172 or PRO 182 polypeptide, or an agonist thereof
6 The composition of matter of Claim 5, wherein said tumor is a cancer
7 The composition of matter of Claim 6, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, renal cancer, colorectal cancer, uterine cancer, prostate cancer, lung cancer, bladder cancer, central nervous system cancer, melanoma and leukemia
8 A method for inhibiting the growth of a tumor cell comprising exposing said tumor cell to an effective amount of a PR021 1 , PR0228, PR0538, PROl 72 or PROl 82 polypeptide, or an agonist thereof
9 The method of Claim 8, wherein said agonist is an antι-PR021 1 , antι-PR0228, antι-PR0538, anti-PRO 172 or anti-PRO 182 agonist antibody
10 The method of Claim 8, wherein said agonist is a small molecule mimicking the biological activity of a PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide
1 1 The method of Claim 8, wherein said step of exposing occurs in vitro
12 The method of Claim 8, wherein said step of exposing occurs in vivo
13 An article of manufacture comprising a container, and a composition comprising an active agent contained within the container, wherein said active agent in the composition is a PR021 1 , PR0228, PR0538, PR0172 or PR0182 polypeptide, or an agonist therof
14 The article of manufacture of Claim 13, further compnsmg 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 antι-PR021 1 , antι-PR0228, antι-PR0538, antι-PR0172 or anti-PROl 82 agonist antibody
16 The article of manufacture of Claim 13, wherein said agonist is a small molecule mimicking the biological activity of a PR021 1, PR0228, PR0538, PR0172 or PR0182 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), Figure 4 (SEQ ID NO 7), Figure 6 (SEQ ID NO 16), Figure 8 (SEQ ID NO 21), and Figure 10 (SEQ ID NO 26)
20 Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting ofthe nucleotide sequence shown in Figure 1 (SEQ ID NO 1), Figures 3A-3B (SEQ ID NO 6), Figure 5 (SEQ ID NO 15), Figures 7A-7B (SEQ ID NO 20), and Figure 9 (SEQ ID NO 25)
21 Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting ofthe full-length coding sequence ofthe nucleotide sequence shown in Figure 1 (SEQ ID NO 1 ), Figures 3A-3B (SEQ ID NO 6), Figure 5 (SEQ ID NO 15), Figures 7A-7B (SEQ ID NO 20) and Figure 9 (SEQ ID NO 25)
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 209258, 209420, 209752, 209419, or 209296
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, wherem said cell is a CHO cell
27 The host cell of Claim 25, wherein said cell is an £ coh
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 PR021 1 , PR0228, PR0538, PR0172 or PR0182 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 ofthe amino acid sequence shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7), Figure 6 (SEQ ID NO 16), Figure 8 (SEQ ID NO 21 ), and Figure 10 (SEQ ID NO 26)
32 An isolated polypeptide scoring at least 80% positives when compared to an amino acid sequence selected from the group consisting ofthe amino acid sequence shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7), Figure 6 (SEQ ID NO 16), Figure 8 (SEQ ID NO 21 ), and Figure 10 (SEQ ID NO 26)
33 An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence encoded by the full-length coding sequence of the DNA deposited under ATCC accession number 209258, 209420, 209752, 209419, or 209296
34 A chimenc molecule comprising a polypeptide according to any one of Claims 31 to 33 fused to a heterologous amino acid sequence
35 The chimenc molecule of Claim 34, wherein said heterologous amino acid sequence is an epitope tag sequence
36. The chimeric molecule of Claim 34, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin.
37. An antibody which specifically binds to a polypeptide according to any one of Claims 31 to 33.
38. The antibody of Claim 37, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody.
39. Isolated nucleic acid having at least 80% nucleic acid sequence identity to:
(a) a nucleotide sequence encoding the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO: 16), Figure 8 (SEQ ID NO:21 ), or Figure 10 (SEQ ID NO:26), lacking its associated signal peptide;
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO: 16), Figure 8 (SEQ ID NO:21), or Figure 10 (SEQ ID NO:26), with its associated signal peptide; or
(c) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO: 16), Figure 8 (SEQ ID NO:21), or Figure 10 (SEQ ID NO:26), 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), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO: 16), Figure 8 (SEQ ID NO:21), or Figure 10 (SEQ ID NO:26), lacking its associated signal peptide;
(b) an extracellular domain ofthe polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO: 16), Figure 8 (SEQ ID NO:21), or Figure 10 (SEQ ID NO:26), with its associated signal peptide; or
(c) an extracellular domain ofthe polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO: 16), Figure 8 (SEQ ID NO:21 ), or Figure 10 (SEQ ID NO:26), lacking its associated signal peptide.
I l l
PCT/US1999/028564 1994-09-08 1999-12-02 Methods and compositions for inhibiting neoplastic cell growth WO2000055319A1 (en)

Priority Applications (251)

Application Number Priority Date Filing Date Title
AU17498/00A AU1749800A (en) 1999-03-12 1999-12-02 Methods and compositions for inhibiting neoplastic cell growth
AU23907/00A AU2390700A (en) 1999-07-20 1999-12-20 Polypeptidic compositions and methods for the treatment of tumors
PCT/US1999/030999 WO2001005836A1 (en) 1999-07-20 1999-12-20 Polypeptidic compositions and methods for the treatment of tumors
AU25967/00A AU2596700A (en) 1999-03-08 1999-12-30 Promotion or inhibition of angiogenesis and cardiovascularization
PCT/US2000/000219 WO2000053753A2 (en) 1999-03-08 2000-01-05 Promotion or inhibition of angiogenesis and cardiovascularization
AU22248/00A AU2224800A (en) 1999-03-08 2000-01-06 Compositions and methods for the treatment of tumor
PCT/US2000/000277 WO2000053754A1 (en) 1999-03-08 2000-01-06 Compositions and methods for the treatment of tumor
PCT/US2000/004414 WO2001004311A1 (en) 1999-07-07 2000-02-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU28839/00A AU2883900A (en) 1999-07-07 2000-02-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/005841 WO2000053758A2 (en) 1999-03-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
CA002362427A CA2362427A1 (en) 1999-03-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
AU35144/00A AU3514400A (en) 1999-03-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
EP00913764A EP1220905A2 (en) 1999-03-08 2000-03-02 Composition and methods for the treatment of immune related diseases
KR1020017011406A KR20010103046A (en) 1999-03-08 2000-03-02 Compositions and Methods for the Treatment of Immune Related Diseases
JP2000603379A JP2004516227A (en) 1999-03-08 2000-03-02 Compositions and methods for treating immune-related diseases
EP06000587A EP1690872A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
CA002491610A CA2491610A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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
CA002491258A CA2491258A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU20554/01A AU2055401A (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP10005292A EP2228446A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptieds 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
PCT/US2000/032678 WO2001040466A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000582A EP1666495A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2001542531A JP2004522404A (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding them
EP00983846A EP1250426A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding same
EP06000583A EP1686134A3 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000584A EP1669371A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
CA002492049A CA2492049A1 (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
CA2709291A CA2709291A1 (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
CA002494705A CA2494705A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000586A EP1688497A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002496312A CA2496312A1 (en) 1999-12-01 2000-12-01 Colon tumour marker pro4799 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
CA002491433A CA2491433A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002490853A CA2490853A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000589A EP1661997A1 (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
US09/828,366 US20020010137A1 (en) 1997-09-18 2001-04-05 Methods and compositions for inhibiting neoplastic cell growth
US09/902,634 US20030082540A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,692 US20030054400A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,979 US20030113718A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,572 US20030108983A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,615 US20030092002A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,903 US20030044839A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,713 US20030082541A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,736 US20030049676A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,853 US20020192659A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,759 US20030077654A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,786 US20030044793A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,603 US20030148419A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,011 US20030003530A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,806 US20030130489A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,119 US20030049621A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids enconding the same
US09/903,823 US20030104381A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,562 US6965015B2 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,520 US20030054401A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,749 US7147853B2 (en) 1997-09-17 2001-07-11 Anti-pro211 polypeptide antibodies
US09/903,640 US7208308B2 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,943 US20030054349A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,925 US20030096233A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,766 US20030152999A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,877 US20030186358A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,859 US20030036060A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,938 US20030211569A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,088 US20030073077A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,291 US20020160374A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,125 US6664376B2 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,056 US20030054441A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,992 US20030135025A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,786 US7557192B2 (en) 1997-09-17 2001-07-12 Anti-PRO335 antibodies
US09/904,532 US7151160B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,462 US6878807B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,485 US20030064367A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,553 US20030059828A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,348 US20030064923A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,820 US20030036094A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,838 US20030148370A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,075 US7169906B2 (en) 1997-09-17 2001-07-13 PRO211 polypeptides
US09/904,920 US6806352B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,381 US6818746B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,449 US6965011B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,956 US20030049622A1 (en) 1995-12-01 2001-07-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,760 US20030096340A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,646 US6852848B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,722 US6946262B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,700 US6723535B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,742 US20030023054A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,815 US7094567B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,838 US7070979B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,777 US20030148371A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,618 US6828146B2 (en) 1997-09-17 2001-07-16 Nucleic acid encoding PRO229 polypeptides
US09/907,824 US20020197671A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,794 US6635468B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,942 US7087738B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,613 US20030027145A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,728 US20030190611A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/908,093 US20030017498A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,575 US20030073079A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,979 US20030082542A1 (en) 1994-09-08 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,925 US20030054352A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,652 US20030104469A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,841 US7033825B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,088 US20020146709A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,064 US6818449B2 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/908,576 US20040005553A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,204 US20030036061A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,320 US7074592B2 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides nucleic acid encoding
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
US10/081,056 US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/121,046 US20030194791A1 (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
US10/121,059 US20030190721A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,041 US20030077776A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,040 US20030082759A1 (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
US10/121,051 US20030092147A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,044 US20030190717A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,054 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
US10/121,049 US20030022239A1 (en) 1997-06-18 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
US10/121,058 US20030190720A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,055 US20030190718A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,060 US20030190722A1 (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
US10/121,056 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
US10/121,052 US20030199052A1 (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
US10/121,053 US20030199053A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,043 US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/121,057 US20030190719A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,155 US20030068794A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,292 US20030073211A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,157 US20030190725A1 (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
US10/123,156 US20030194792A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,236 US20030068795A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,213 US20030199057A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,212 US7276577B2 (en) 1997-03-31 2002-04-15 PRO1866 polypeptides
US10/123,108 US7635478B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,322 US20030199059A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,109 US20030190723A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,213 US7193048B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,261 US20030068796A1 (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
US10/123,262 US20030049816A1 (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
US10/123,214 US7343721B2 (en) 1997-03-31 2002-04-15 PRO4406 polypeptide
US10/123,154 US20030190724A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,906 US20030190726A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,912 US20030100087A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,907 US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,913 US20030203462A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,905 US20030087344A1 (en) 1997-06-18 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,905 US7285625B2 (en) 1997-06-18 2002-04-16 PRO536 polypeptides
US10/123,902 US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,910 US7329404B2 (en) 1997-03-31 2002-04-16 Antibodies against PRO1310
US10/123,908 US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,903 US20030073212A1 (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
US10/123,911 US7408032B2 (en) 1997-03-31 2002-04-16 PRO1188 polypeptides
US10/125,795 US7304131B2 (en) 1997-03-31 2002-04-17 PRO1483 polypeptides
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
US10/125,704 US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
US10/124,818 US20030082763A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,822 US7109305B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,814 US7105335B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,805 US20030194794A1 (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
US10/124,817 US20030077786A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,819 US7285626B2 (en) 1997-03-31 2002-04-17 PRO1076 polypeptides
US10/124,821 US20030199023A1 (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
US10/124,824 US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,931 US20030199063A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,932 US7317079B2 (en) 1997-03-31 2002-04-19 PRO812 polypeptides
US10/125,927 US20030190731A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,924 US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/125,922 US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/127,831 US20030082689A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,689 US20030087365A1 (en) 1997-03-31 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,825 US7282566B2 (en) 1997-03-31 2002-04-24 PRO1779 polypeptide
US10/131,823 US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 polypeptides
US10/131,817 US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
US10/137,868 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
US10/137,867 US20030207349A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,024 US20040058424A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,023 US20030207416A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,020 US20030207415A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,470 US20030022331A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,474 US20030032156A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,980 US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/139,963 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
US10/140,805 US20030207417A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,921 US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
US10/140,860 US7307151B2 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,864 US20030207419A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,928 US20030068798A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,865 US20030207420A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,808 US7425621B2 (en) 1997-03-31 2002-05-07 Antibodies against the PRO4401 polypeptide
US10/140,925 US20030073215A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,754 US7361732B2 (en) 1997-03-31 2002-05-08 PRO4400 polypeptides
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
US10/141,701 US20030207421A1 (en) 1997-03-31 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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
US10/143,113 US7329730B2 (en) 1997-03-31 2002-05-09 PRO4348 polypeptides
US10/142,430 US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/143,114 US20030036180A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,431 US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/142,419 US7153941B2 (en) 1997-03-31 2002-05-10 Antibodies that bind PRO4994 polypeptides
US10/142,423 US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 polypeptides
US10/146,792 US20030207428A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,730 US20030207427A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,528 US20030219885A1 (en) 1997-03-31 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,519 US20030077791A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,536 US20040077064A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,492 US20030082765A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,395 US7189534B2 (en) 1997-03-31 2002-05-21 PRO4320 polynucleotide
US10/153,934 US20030129695A1 (en) 1997-03-31 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,843 US20030207805A1 (en) 1997-06-18 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,782 US20030077792A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,786 US20030208055A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,782 US20030082766A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,498 US20030073216A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,791 US20030207429A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,913 US20030022298A1 (en) 1997-09-15 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/771,187 US7355002B2 (en) 1997-11-12 2004-02-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/970,823 US7307152B2 (en) 1997-10-17 2004-10-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2005264293A JP2006068016A (en) 1999-12-01 2005-08-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/341,175 US7468427B2 (en) 1997-03-31 2006-01-27 Antibodies to PRO1275 polypeptide
US11/518,609 US20070077623A1 (en) 1997-09-17 2006-09-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007326424A JP2008167749A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptide and nucleic acid encoding the same
JP2007326609A JP2008148701A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007325484A JP2008148699A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007326613A JP2008161190A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptide and nucleic acid encoding the same

Applications Claiming Priority (16)

Application Number Priority Date Filing Date Title
US12395799P 1999-03-12 1999-03-12
US60/123,957 1999-03-12
US13144599P 1999-04-28 1999-04-28
US60/131,445 1999-04-28
US14475899P 1999-07-20 1999-07-20
US60/144,758 1999-07-20
US14569899P 1999-07-26 1999-07-26
US60/145,698 1999-07-26
USPCT/US99/20594 1999-09-08
PCT/US1999/020594 WO2000015666A2 (en) 1998-09-10 1999-09-08 Compositions and methods for the treatment of tumors
PCT/US1999/021090 WO2000015796A2 (en) 1998-09-16 1999-09-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
USPCT/US99/21090 1999-09-15
USPCT/US99/23089 1999-10-05
PCT/US1999/023089 WO2000021996A2 (en) 1998-10-13 1999-10-05 Methods and compositions for inhibiting neoplastic cell growth
USPCT/US99/28313 1999-11-30
PCT/US1999/028313 WO2000032221A2 (en) 1998-12-01 1999-11-30 Promotion or inhibition of angiogenesis and cardiovascularization

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/028301 Continuation-In-Part WO2000032776A2 (en) 1994-09-08 1999-12-01 Secreted amd transmembrane polypeptides and nucleic acids encoding the same

Related Child Applications (3)

Application Number Title Priority Date Filing Date
PCT/US1999/030911 Continuation-In-Part WO2000075316A1 (en) 1994-09-08 1999-12-20 Methods and compositions for inhibiting neoplastic cell growth
US09/903,749 Continuation US7147853B2 (en) 1997-09-17 2001-07-11 Anti-pro211 polypeptide antibodies
US10/081,056 Continuation US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis

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WO2000055319A1 true WO2000055319A1 (en) 2000-09-21

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WO (1) WO2000055319A1 (en)

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US7169906B2 (en) 1997-09-17 2007-01-30 Genentech, Inc. PRO211 polypeptides
US7449551B2 (en) 1997-09-18 2008-11-11 Genentech, Inc. PRO211 polypeptides

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US7169906B2 (en) 1997-09-17 2007-01-30 Genentech, Inc. PRO211 polypeptides
US7449551B2 (en) 1997-09-18 2008-11-11 Genentech, Inc. PRO211 polypeptides

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