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WO2000053752A2 - Promotion or inhibition of angiogenesis and cardiovascularization - Google Patents

Promotion or inhibition of angiogenesis and cardiovascularization Download PDF

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Publication number
WO2000053752A2
WO2000053752A2 PCT/US1999/031274 US9931274W WO0053752A2 WO 2000053752 A2 WO2000053752 A2 WO 2000053752A2 US 9931274 W US9931274 W US 9931274W WO 0053752 A2 WO0053752 A2 WO 0053752A2
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
pro
pro320
antibody
Prior art date
Application number
PCT/US1999/031274
Other languages
French (fr)
Other versions
WO2000053752A3 (en
Inventor
Kevin P. Baker
Napoleone Ferrara
Audrey Goddard
Austin L. Gurney
Kenneth J. Hillan
P. Mickey Williams
William I. Wood
Original Assignee
Genentech, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US1999/005028 external-priority patent/WO1999046281A2/en
Priority claimed from PCT/US1999/028565 external-priority patent/WO2000037638A2/en
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to AU25967/00A priority Critical patent/AU2596700A/en
Priority to AU22248/00A priority patent/AU2224800A/en
Priority to PCT/US2000/000277 priority patent/WO2000053754A1/en
Priority to CA002361840A priority patent/CA2361840A1/en
Priority to EP00907314A priority patent/EP1263948A2/en
Priority to KR1020017011399A priority patent/KR20030002292A/en
Priority to JP2000603377A priority patent/JP2004513602A/en
Priority to PCT/US2000/004341 priority patent/WO2000053756A2/en
Priority to AU35144/00A priority patent/AU3514400A/en
Priority to KR1020017011406A priority patent/KR20010103046A/en
Priority to EP00913764A priority patent/EP1220905A2/en
Priority to JP2000603379A priority patent/JP2004516227A/en
Priority to PCT/US2000/005841 priority patent/WO2000053758A2/en
Priority to CA002362427A priority patent/CA2362427A1/en
Publication of WO2000053752A2 publication Critical patent/WO2000053752A2/en
Priority to CA002491610A priority patent/CA2491610A1/en
Priority to EP10005292A priority patent/EP2228446A1/en
Priority to EP06000586A priority patent/EP1688497A1/en
Priority to CA2709291A priority patent/CA2709291A1/en
Priority to EP00983846A priority patent/EP1250426A2/en
Priority to EP06000589A priority patent/EP1661997A1/en
Priority to CA002492070A priority patent/CA2492070A1/en
Priority to AU20554/01A priority patent/AU2055401A/en
Priority to EP06000587A priority patent/EP1690872A3/en
Priority to EP06000581A priority patent/EP1666494A1/en
Priority to CA002490909A priority patent/CA2490909A1/en
Priority to JP2001542531A priority patent/JP2004522404A/en
Priority to CA002391455A priority patent/CA2391455A1/en
Priority to CA002496312A priority patent/CA2496312A1/en
Priority to CA002492049A priority patent/CA2492049A1/en
Priority to CA002491258A priority patent/CA2491258A1/en
Priority to CA002491433A priority patent/CA2491433A1/en
Priority to EP06000588A priority patent/EP1690873A3/en
Priority to CA002490853A priority patent/CA2490853A1/en
Priority to EP06000583A priority patent/EP1686134A3/en
Priority to EP06000584A priority patent/EP1669371A3/en
Priority to EP06000582A priority patent/EP1666495A1/en
Priority to PCT/US2000/032678 priority patent/WO2001040466A2/en
Priority to EP05025102A priority patent/EP1672070A3/en
Priority to EP06000585A priority patent/EP1661996A1/en
Priority to CA002494705A priority patent/CA2494705A1/en
Priority to AU25909/01A priority patent/AU2590901A/en
Priority to ES00989401T priority patent/ES2333772T3/en
Priority to CA002391374A priority patent/CA2391374A1/en
Priority to EP00989401A priority patent/EP1240325B1/en
Priority to EP07016900A priority patent/EP1897944B1/en
Priority to EP10009491.1A priority patent/EP2258848B1/en
Priority to ES07016899T priority patent/ES2379101T3/en
Priority to DE60043069T priority patent/DE60043069D1/en
Priority to DK07016903.2T priority patent/DK1897947T3/en
Priority to DK00989401.5T priority patent/DK1240325T3/en
Priority to AT07016900T priority patent/ATE519847T1/en
Priority to EP07016899A priority patent/EP1897943B1/en
Priority to AT00989401T priority patent/ATE444361T1/en
Priority to EP10009490A priority patent/EP2290081A3/en
Priority to ES07016901T priority patent/ES2380958T3/en
Priority to DK07016900.8T priority patent/DK1897944T3/en
Priority to EP07016903A priority patent/EP1897947B1/en
Priority to PCT/US2000/034956 priority patent/WO2001046420A2/en
Priority to PT00989401T priority patent/PT1240325E/en
Priority to DK07016901.6T priority patent/DK1897945T3/en
Priority to AT07016899T priority patent/ATE537258T1/en
Priority to ES07016903T priority patent/ES2380812T3/en
Priority to ES09009972.2T priority patent/ES2506665T3/en
Priority to ES07016902T priority patent/ES2388748T3/en
Priority to PT07016901T priority patent/PT1897945E/en
Priority to JP2001546918A priority patent/JP2003527104A/en
Priority to AT07016903T priority patent/ATE541931T1/en
Priority to PT07016900T priority patent/PT1897944E/en
Priority to EP07016902A priority patent/EP1897946B1/en
Priority to US09/747,259 priority patent/US6569645B2/en
Priority to AT07016901T priority patent/ATE541930T1/en
Priority to PT07016903T priority patent/PT1897947E/en
Priority to EP07016901A priority patent/EP1897945B1/en
Priority to ES10009491.1T priority patent/ES2458349T3/en
Priority to EP09009972.2A priority patent/EP2163625B1/en
Publication of WO2000053752A3 publication Critical patent/WO2000053752A3/en
Priority to US09/816,744 priority patent/US6579520B2/en
Priority to US09/874,503 priority patent/US20020177188A1/en
Priority to US09/908,827 priority patent/US20030054442A1/en
Priority to US09/918,585 priority patent/US20030060406A1/en
Priority to US09/978,194 priority patent/US20030195333A1/en
Priority to US09/978,295 priority patent/US20020156006A1/en
Priority to US09/978,192 priority patent/US20020177553A1/en
Priority to US09/978,299 priority patent/US20030199435A1/en
Priority to US09/978,298 priority patent/US20030134785A1/en
Priority to US09/978,189 priority patent/US6972325B2/en
Priority to US09/978,188 priority patent/US20030139328A1/en
Priority to US09/978,193 priority patent/US20030073624A1/en
Priority to US09/978,191 priority patent/US20030050239A1/en
Priority to US09/981,915 priority patent/US20030054986A1/en
Priority to US09/978,403 priority patent/US20030050240A1/en
Priority to US09/978,757 priority patent/US20030083248A1/en
Priority to US09/978,375 priority patent/US7196165B2/en
Priority to US09/978,643 priority patent/US20030104998A1/en
Priority to US09/978,665 priority patent/US7294700B2/en
Priority to US09/981,915 priority patent/US7285623B2/en
Priority to US09/978,423 priority patent/US20030069178A1/en
Priority to US09/978,802 priority patent/US20030199674A1/en
Priority to US09/978,564 priority patent/US7195760B2/en
Priority to US09/978,824 priority patent/US20050124789A9/en
Priority to US09/978,585 priority patent/US20030049633A1/en
Priority to US09/978,608 priority patent/US20030045462A1/en
Priority to US09/978,681 priority patent/US20030195148A1/en
Priority to US09/978,697 priority patent/US20020169284A1/en
Priority to US09/978,544 priority patent/US20030199436A1/en
Priority to US09/978,824 priority patent/US20030055216A1/en
Priority to US10/210,028 priority patent/US20030203446A1/en
Priority to US10/152,388 priority patent/US20040223964A1/en
Priority to US10/165,247 priority patent/US7112657B2/en
Priority to US10/145,124 priority patent/US20030190701A1/en
Priority to US10/143,029 priority patent/US7105640B2/en
Priority to US10/145,089 priority patent/US7208575B2/en
Priority to US10/160,502 priority patent/US7220835B2/en
Priority to US10/164,749 priority patent/US20040029218A1/en
Priority to US10/164,829 priority patent/US20030194780A1/en
Priority to US10/145,017 priority patent/US20030186365A1/en
Priority to US10/165,067 priority patent/US7279553B2/en
Priority to US10/164,929 priority patent/US20030194781A1/en
Priority to US10/166,709 priority patent/US20030104536A1/en
Priority to US10/165,036 priority patent/US20050227342A1/en
Priority to US10/162,521 priority patent/US7067628B2/en
Priority to US10/164,728 priority patent/US20030186368A1/en
Priority to US10/013,922 priority patent/US20030195345A1/en
Priority to US09/999,832 priority patent/US7132283B2/en
Priority to US10/017,085 priority patent/US6974696B2/en
Priority to US10/017,084 priority patent/US20030203402A1/en
Priority to US09/999,832 priority patent/US20020192706A1/en
Priority to US10/002,967 priority patent/US20030148373A1/en
Priority to US09/999,833 priority patent/US6916648B2/en
Priority to US10/020,445 priority patent/US20030198994A1/en
Priority to US10/017,083 priority patent/US20030148376A1/en
Priority to US09/999,830 priority patent/US20030077700A1/en
Priority to US10/017,086 priority patent/US7122375B2/en
Priority to US10/017,081 priority patent/US20030049684A1/en
Priority to US09/999,829 priority patent/US20030195344A1/en
Priority to US09/999,834 priority patent/US20030064407A1/en
Priority to US10/013,927 priority patent/US7189529B2/en
Priority to US10/013,928 priority patent/US20030215905A1/en
Priority to US10/013,918 priority patent/US20030211091A1/en
Priority to US10/016,177 priority patent/US20030073131A1/en
Priority to US10/013,925 priority patent/US7037710B2/en
Priority to US10/013,917 priority patent/US7029874B2/en
Priority to US10/013,926 priority patent/US7074593B2/en
Priority to US10/013,921 priority patent/US20030068648A1/en
Priority to US10/013,929 priority patent/US7019124B2/en
Priority to US10/013,923 priority patent/US7169912B2/en
Priority to US10/013,920 priority patent/US20040006219A1/en
Priority to US10/000,157 priority patent/US20020182673A1/en
Priority to US09/990,456 priority patent/US20020137890A1/en
Priority to US10/028,072 priority patent/US20030004311A1/en
Priority to US10/052,586 priority patent/US20020127584A1/en
Priority to US09/978,187 priority patent/US20030096744A1/en
Priority to US10/081,056 priority patent/US20040043927A1/en
Priority to US10/119,480 priority patent/US20040087769A1/en
Priority to US10/121,046 priority patent/US20030194791A1/en
Priority to US10/121,051 priority patent/US20030092147A1/en
Priority to US10/121,040 priority patent/US20030082759A1/en
Priority to US10/121,047 priority patent/US20030077778A1/en
Priority to US10/121,044 priority patent/US20030190717A1/en
Priority to US10/121,041 priority patent/US20030077776A1/en
Priority to US10/121,042 priority patent/US20030096386A1/en
Priority to US10/121,045 priority patent/US20030073210A1/en
Priority to US10/121,059 priority patent/US20030190721A1/en
Priority to US10/121,055 priority patent/US20030190718A1/en
Priority to US10/121,060 priority patent/US20030190722A1/en
Priority to US10/121,062 priority patent/US20030077779A1/en
Priority to US10/121,052 priority patent/US20030199052A1/en
Priority to US10/121,053 priority patent/US20030199053A1/en
Priority to US10/121,054 priority patent/US20030199054A1/en
Priority to US10/121,056 priority patent/US20030082760A1/en
Priority to US10/121,043 priority patent/US7220831B2/en
Priority to US10/121,057 priority patent/US20030190719A1/en
Priority to US10/121,061 priority patent/US20030082761A1/en
Priority to US10/121,063 priority patent/US20030199055A1/en
Priority to US10/121,058 priority patent/US20030190720A1/en
Priority to US10/121,049 priority patent/US20030022239A1/en
Priority to US10/121,050 priority patent/US20030054516A1/en
Priority to US10/121,048 priority patent/US20030199051A1/en
Priority to US10/123,214 priority patent/US7343721B2/en
Priority to US10/123,155 priority patent/US20030068794A1/en
Priority to US10/123,215 priority patent/US7291329B2/en
Priority to US10/123,291 priority patent/US20030199058A1/en
Priority to US10/123,236 priority patent/US20030068795A1/en
Priority to US10/123,292 priority patent/US20030073211A1/en
Priority to US10/123,262 priority patent/US20030049816A1/en
Priority to US10/123,108 priority patent/US7635478B2/en
Priority to US10/123,771 priority patent/US20030199060A1/en
Priority to US10/123,154 priority patent/US20030190724A1/en
Priority to US10/123,213 priority patent/US7193048B2/en
Priority to US10/123,213 priority patent/US20030199057A1/en
Priority to US10/123,157 priority patent/US20030190725A1/en
Priority to US10/123,261 priority patent/US20030068796A1/en
Priority to US10/123,156 priority patent/US20030194792A1/en
Priority to US10/123,212 priority patent/US7276577B2/en
Priority to US10/123,109 priority patent/US20030190723A1/en
Priority to US10/123,322 priority patent/US20030199059A1/en
Priority to US10/123,235 priority patent/US20030082762A1/en
Priority to US10/123,909 priority patent/US7193049B2/en
Priority to US10/123,908 priority patent/US7335728B2/en
Priority to US10/123,913 priority patent/US20030203462A1/en
Priority to US10/123,912 priority patent/US20030100087A1/en
Priority to US10/123,903 priority patent/US20030073212A1/en
Priority to US10/123,902 priority patent/US20030077781A1/en
Priority to US10/123,906 priority patent/US20030190726A1/en
Priority to US10/123,907 priority patent/US7084258B2/en
Priority to US10/123,910 priority patent/US7329404B2/en
Priority to US10/123,905 priority patent/US20030087344A1/en
Priority to US10/123,911 priority patent/US7408032B2/en
Priority to US10/123,905 priority patent/US7285625B2/en
Priority to US10/123,904 priority patent/US20030022328A1/en
Priority to US10/124,814 priority patent/US7105335B2/en
Priority to US10/124,823 priority patent/US20030199062A1/en
Priority to US10/124,813 priority patent/US7312307B2/en
Priority to US10/124,822 priority patent/US7109305B2/en
Priority to US10/124,819 priority patent/US7285626B2/en
Priority to US10/124,816 priority patent/US20030190728A1/en
Priority to US10/124,821 priority patent/US20030199023A1/en
Priority to US10/124,818 priority patent/US20030082763A1/en
Priority to US10/124,817 priority patent/US20030077786A1/en
Priority to US10/124,820 priority patent/US20030190729A1/en
Priority to US10/124,824 priority patent/US20030077659A1/en
Priority to US10/125,805 priority patent/US20030194794A1/en
Priority to US10/125,795 priority patent/US7304131B2/en
Priority to US10/125,704 priority patent/US7357926B2/en
Priority to US10/125,924 priority patent/US7342097B2/en
Priority to US10/125,931 priority patent/US20030199063A1/en
Priority to US10/125,922 priority patent/US7309762B2/en
Priority to US10/125,927 priority patent/US20030190731A1/en
Priority to US10/125,932 priority patent/US7317079B2/en
Priority to US10/127,831 priority patent/US20030082689A1/en
Priority to US10/128,689 priority patent/US20030087365A1/en
Priority to US10/131,817 priority patent/US7291701B2/en
Priority to US10/131,825 priority patent/US7282566B2/en
Priority to US10/131,823 priority patent/US7304132B2/en
Priority to US10/063,521 priority patent/US20030190669A1/en
Priority to US10/063,519 priority patent/US20030009013A1/en
Priority to US10/063,518 priority patent/US7465785B2/en
Priority to US10/063,517 priority patent/US7232889B2/en
Priority to US10/063,510 priority patent/US7109292B2/en
Priority to US10/063,520 priority patent/US20030187196A1/en
Priority to US10/063,551 priority patent/US20020183494A1/en
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Priority to US10/063,547 priority patent/US20020182638A1/en
Priority to US10/063,526 priority patent/US20030171550A1/en
Priority to US10/063,536 priority patent/US7259238B2/en
Priority to US10/063,569 priority patent/US20030018168A1/en
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Priority to US10/063,564 priority patent/US20030180794A1/en
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Priority to US10/063,532 priority patent/US7202336B2/en
Priority to US10/063,541 priority patent/US20030060601A1/en
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Priority to US10/063,610 priority patent/US7371814B2/en
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Priority to US10/140,024 priority patent/US20040058424A1/en
Priority to US10/140,470 priority patent/US20030022331A1/en
Priority to US10/139,980 priority patent/US7247710B2/en
Priority to US10/139,963 priority patent/US7288625B2/en
Priority to US10/140,023 priority patent/US20030207416A1/en
Priority to US10/140,474 priority patent/US20030032156A1/en
Priority to US10/140,020 priority patent/US20030207415A1/en
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Priority to US10/063,671 priority patent/US20030180840A1/en
Priority to US10/140,921 priority patent/US7317080B2/en
Priority to US10/140,864 priority patent/US20030207419A1/en
Priority to US10/063,675 priority patent/US20030180842A1/en
Priority to US10/063,647 priority patent/US20030187197A1/en
Priority to US10/140,860 priority patent/US7307151B2/en
Priority to US10/063,661 priority patent/US7193047B2/en
Priority to US10/063,642 priority patent/US20030181650A1/en
Priority to US10/063,639 priority patent/US7081520B2/en
Priority to US10/063,659 priority patent/US7186801B2/en
Priority to US10/140,805 priority patent/US20030207417A1/en
Priority to US10/063,670 priority patent/US20030180839A1/en
Priority to US10/063,648 priority patent/US7193062B2/en
Priority to US10/063,660 priority patent/US7189822B2/en
Priority to US10/063,677 priority patent/US20030187242A1/en
Priority to US10/063,652 priority patent/US7230077B2/en
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Priority to US10/063,662 priority patent/US20030180795A1/en
Priority to US10/063,668 priority patent/US20030191290A1/en
Priority to US10/063,674 priority patent/US20030180841A1/en
Priority to US10/140,928 priority patent/US20030068798A1/en
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Priority to US10/140,925 priority patent/US20030073215A1/en
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Priority to JP2005143079A priority patent/JP2005323597A/en
Priority to JP2005171514A priority patent/JP2006006327A/en
Priority to JP2005171206A priority patent/JP4239021B2/en
Priority to JP2005171424A priority patent/JP2006006326A/en
Priority to JP2005171331A priority patent/JP4452659B2/en
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Priority to US11/189,442 priority patent/US20060246465A1/en
Priority to JP2005264293A priority patent/JP2006068016A/en
Priority to US11/311,561 priority patent/US20060088917A1/en
Priority to US11/311,555 priority patent/US7473763B2/en
Priority to US11/323,117 priority patent/US20070092941A1/en
Priority to JP2006000562A priority patent/JP4688682B2/en
Priority to US11/341,175 priority patent/US7468427B2/en
Priority to US11/529,324 priority patent/US8273703B2/en
Priority to US11/786,466 priority patent/US20080182275A1/en
Priority to JP2007114868A priority patent/JP2007291109A/en
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Priority to US13/593,362 priority patent/US20130064827A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • 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
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • the present invention relates to compositions and methods useful for promoting or inhibiting angiogenesis and/or cardiovascularization in mammals in need of such biological effect. This includes the diagnosis and treatment of cardiovascular disorders as well as oncological disorders.
  • Heart failure affects approximately five million Americans, and new cases of heart failure number about 400,000 each year. It is the single most frequent cause of hospitalization for people age 65 and older in the United States. Recent advances in the management of acute cardiac diseases, including acute myocardial infarction, are resulting in an expanding patient population that will eventually develop chronic heart failure. From 1979 to 1995, hospitalizations for congestive heart failure (CHF) rose from 377,000 to 872,000 (a 130 percent increase) and CHF deaths increased 116 percent.
  • CHF congestive heart failure
  • CHF is a syndrome characterized by left ventricular dysfunction, reduced exercise tolerance, impaired quality of life, and markedly shortened life expectancy.
  • the sine qua non of heart failure is an inability of the heart to pump blood at a rate sufficient to meet the metabolic needs of the body's tissues (in other words, there is insufficient cardiac output).
  • At least four major compensatory mechanisms are activated in the setting of heart failure to boost cardiac output, including peripheral vasoconstriction, increased heart rate, increased cardiac contractility, and increased plasma volume. These effects are mediated primarily by the sympathetic nervous system and the renin-angiotensin system. See, Eichhorn, American Journal of Medicine, 104: 163-169 (1998). Increased output from the sympathetic nervous system increases vascular tone, heart rate, and contractility.
  • Angiotensin II elevates blood pressure by 1 ) directly stimulating vascular smooth muscle contraction, 2) promoting plasma volume expansion by stimulating aldosterone and antidiuretic hormone secretion, 3) stimulating sympathetic-mediated vascular tone, and 4) catalyzing the degradation of bradykinin, which has vasodilatory and natriuretic activity. See, review by Brown and Vaughan. Circulation.97: 1411-1420(1998). As noted below, angiotensin II may also have directly deleterious effects on the heart by promoting myocyte necrosis (impairing systolic unction) and intracardiac fibrosis (impairing diastolic and in some cases systolic function). See, Weber, Circulation. 96: 4065-4082 (1998).
  • cardiac hypertrophy an enlargement of the heart that is activated by both mechanical and hormonal stimuli and enables the heart to adapt to demands for increased cardiac output.
  • This hypertrophic response is frequently associated with a variety of distinct pathological conditions such as hypertension, aortic stenosis, myocardial infarction, cardiomyopathy, valvular regurgitation, and intracardiac shunt, all of which result in chronic hemodynamic oveiload
  • Hypertrophy is generally defined as an increase in size of an organ or structuie independent of natural growth that does not involve tumor formation Hypertrophy of the heart is due either to an increase in the mass of the individual cells (myocytes), or to an increase in the number of cells making up the tissue (hyperplasia), or both While the enlargement of an embryonic heart is largely dependent on an increase in myocyte number (which continues until shortly after birth), post-natal cardiac myocytes lose their prohferative capacity Further growth occurs through hypertrophy of the individual cells
  • Myocyte hypertrophy is initially beneficial as a short term response to impaired cardiac function by permitting a decrease in the load on individual muscle fibers With severe, long-standing overload, however, the hypertroph led cells begin to deteriorate and die Katz, "Heart Failure", in Katz A M ed , Physiology of the Heart (New York Raven Press, 1992) pp 638-668 Cardiac hypertrophy is a significant risk factor for both mortality and morbidity in the clinical course of heart failure Katz, Trends Cardiovas
  • non-myocytes are primarily fibroblast/mesenchymal cells, they also include endothehal and smooth muscle cells Indeed, although myocytes make up most of the adult myocardial mass, they represent only about 30% of the total cell numbers present in heart
  • adult ventricular muscle cells can adapt to increased workloads through the activation of a hypertrophic process
  • This response is characterized by an increase in myocyte cell size and contractile protein content of individual cardiac muscle cells, without concomitant cell division and activation of embryonic genes, including the gene for at ⁇ al nat ⁇ uretic peptide (ANP) Chien et al , FASEB J , 5 3037 3046 (1991 ), Chien etal , Annu Rev Phvsiol , 55 77 95 (1993)
  • APP at ⁇ al nat ⁇ uretic peptide
  • CT-1 cardiotrophin 1
  • catecholamines catecholamines
  • adrenocorticostei oids catecholamines
  • angiotensin prostaglandins
  • beta-adrenergic receptor blocking drugs (beta-blockers, e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol. atenolol, etoprolol carvedilol, etc ) and verapamil have been used extensively in the treatment of hypertrophic cardiomyopathy The beneficial effects of beta-blockers on symptoms
  • Antihypertensive drug therapy has been reported to have beneficial effects on cardiac hypertrophy associated with elevated blood pressure
  • examples of drugs used in antihypertensive therapy are calcium antagonists, e g , nitrendipine, adrenergic receptor blocking agents, e g , those listed above, angiotensin converting enzyme (ACE) inhibitors such as quinap ⁇ l, captop ⁇ l, enalap ⁇ l, ramip ⁇ l, benazep ⁇ l, fosinop ⁇ l, and hsinop ⁇ l, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, and mdapamide, and calcium channel blockers, e g , diltiazem, nif
  • ACE inhibitors consistently appear unable to relieve symptoms in more than 60% of heart failure patients and reduce mortality of heart failure only b ⁇ approximately 15 20% For further adverse effects, see Brown and Vaughan, supra
  • thro mbolytic agents e g , streptokmase, uiokinase, and in particular tissue plasminogen activator (t-PA) have significantly increased the survival of patients who suffered myocardial infarction
  • t-PA tissue plasminogen activator
  • t-PA may also be administered as a single bolus, although due to its relatively short half-life, it is better suited for infusion therapy Tebbe et al , Am J Cardiol , 64 448-453 (1989)
  • FGF basic and acidic fibroblast growth factors
  • PD-ECGF platelet-derived endothehal cell growth factor
  • VEGF vascular endothehal growth factor
  • hVEGF human VEGF
  • hVEGF related proteins The 121 -amino acid protein differs from hVEGF by virtue of the deletion of the 44 amino acids between residues 1 16 and 159 in h VEGF
  • the 189-am ⁇ no acid protein differs from hVEGF by virtue of the insertion of 24 amino acids at residue 1 16 in hVEGF, and apparently is identical to human vascular permeability factor (hVPF)
  • hVPF human vascular permeability factor
  • the 206-am ⁇ no acid protein differs from hVEGF by virtue ot an insertion of 41 amino acids at residue 1 16 in h VEGF Houck et al .
  • angiogenesis which involves the formation of new blood vessels from preexisting endothehum is implicated in the pathogenesis of a variety of disorders
  • disorders include solid tumors and metastasis, atheiosclerosis, retiolental fibroplasia, hemangiomas, chronic inflammation, intraocular neovasculai syndromes such as prohferative retinopathies, e g , diabetic retinopathy, age related macular degeneration (AMD) neovascular glaucoma, immune i ejection ot transplanted corneal tissue and other tissues, rheumatoid arthritis, and psoriasis Folkman e/ ⁇ / J Biol Che , 267 10931 -10934 (1992), Klagsbrun ef -./ Annu Rev Physiol , 53 217- 239 (1991), and Garner A , "Vascular diseases", In Pathobiology of Ocular Disease A Dvnamic Approach
  • angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia, and tor pioviding nourishment to the growing solid tumor
  • the neovasculanzation allows the tumor cells to acquire a growth advantage and prohferative autonomy compared to the normal cells
  • a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors Weidner et al , N Engl J Med, 324 1-6 (1991), Horak et al , Lancet, 340 1120-1 124 (1992), Macchia ⁇ ni et al , Lancet, 340 145-146 (1992)
  • the search for positive regulators of angiogenesis has yielded many candidates, including aFGF, bFGF, TGF- ⁇ , TGF- ⁇ , HGF, TNF- ⁇ , angiogenin, IL-8, etc Folkman
  • the negative regulators so far identified include thrombospondin (Good et al , Proc Natl Acad Sci USA , 87 6624- 6628 (1990)), the 16-k ⁇ lodalton N-terminal fragment of prolactin (Clapp et al , Endocrinology, 133 1292-1299 (1993)), angiostatin (O'Reilly et al . Cell. 79 315-328 (1994)), and endostatin O'Reilly et al , CeU, 88 277-285 (1996)
  • VEGF vascular endothehal cell proliferation
  • Ferrara et al vascular permeability and angiogenesis
  • Ferrara et al vascular permeability and angiogenesis
  • VEGF has been shown to be a key mediator of neovasculanzation associated with tumors and intraocular disorders Ferrara et al , Endocr Rev , supra
  • the VEGF mRNA is overexpressed by the majority of human tumors examined Berkman et al , J Clm Invest , 91 153-159 (1993), Brown et al , Human Pathol , 26 86-91 (1995) Brown etal , Cancer Res , 53 4727-4735 (1993), Mattern e.
  • anti-VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of solid tumors and various intraocular neovascular disordei s
  • Such antibodies are described, tor example, in EP 817,648 published January 14, 1998 and in PCT/US 98/06724 filed April 3, 1998
  • CM 61 which is rapidly activated by serum- or platelet-derived growth factor (PDGF) (O'Brien et al , Mol Cell Biol . K) 3569-3577 (1990), human connective tissue growth factor (CTGF) (Bradham et al , J Cell Biol . 1 14 1285- 1294 ( 1991 )), which is secreted by human vascular endothehal cells in high levels after activation with transforming growth factor beta (TGF- ⁇ ).
  • PDGF platelet-derived growth factor
  • CTGF human connective tissue growth factor
  • IGFBPs insulin-like growth factor binding proteins
  • IGF insulin-like growth factor binding proteins
  • IGF insulin-like growth factor binding proteins
  • Inhibitory effects of IGFBP on various IGF actions in vitro include stimulation of glucose transport by adipocytes, sulfate incorporation by chondrocytes, and thymidine incorporation in fibroblast Zapf et al , J Clin Invest , 63 1077 ( 1979)
  • IGFBPs insulin-like growth factor binding proteins
  • the present invention concerns compositions and methods tor promoting or inhibiting angiogenesis and/or cardiovascula ⁇ zation in mammals
  • the piesent invention is based on the identification of proteins that test positive in various cardiovascular assays that test promotion or inhibition of certain biological activities
  • the proteins are believed to be useful drugs for the diagnosis and/or treatment (including prevention) of disoidei s where such effects are desired such as the promotion or inhibition of angiogenesis, inhibition or stimulation ot vascular endothehal cell grow th, stimulation of growth or proliferation of vascular endothehal cells, inhibition of tumor growth, inhibition of angiogenesis dependent tissue growth, stimulation of angiogenesis dependent tissue growth, inhibition of cardiac hypertrophy and stimulation of cardiac hypertrophy , e g , for the treatment of congestive heart failure
  • the present invention provides a composition comprising a PRO polypeptide in admixture with a pharmaceutically acceptable carrier
  • the composition comprises a therapeutically effective amount of the polypeptide
  • the composition comprises a further active ingredient, namely, a cardiovascular, endothehal or angiogemc agent or an angiostatic agent, preferably an angiogemc or angiostatic agent
  • the composition is sterile
  • the PRO polypeptide may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability
  • Preserved liquid pharmaceutical formulations might contain multiple doses of PRO polypeptide, and might, therefore, be suitable for repeated use
  • the present invention provides a method for preparing such a composition useful for the treatment of a cardiovascular, endothehal or angiogemc disorder comprising admixingatherapeutically effective amount of a PRO polypeptide with a pharmaceutically acceptable carrier
  • the present invention provides a composition comprising an agonist or antagonist of a PRO polypeptide in admixture with a pharmaceutically acceptable carrier
  • the composition comprises a therapeutically effective amount of the agonist or antagonist
  • the composition comprises a further active ingredient, namely, a cardiovascular, endothehal or angiogemc agent or an angiostatic agent, preferably an angiogemc or angiostatic agent
  • the composition is sterile
  • the PRO polypeptide agonist or antagonist may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability
  • Preserv ed liquid pharmaceutical formulations might contain multiple doses of a PRO polypeptide agonist or antagonist, and might therefore, be suitable for repeated use
  • the present invention provides a method for preparing such a composition useful for the treatment of a cardiovascular, endothehal or angiogemc disorder comprising admixing a therapeutically effective amount of a PRO polypeptide agonist or antagonist with a pharmaceutically acceptable carrier
  • the present invention concerns a composition comprising an anti PRO antibody in admixture with a pharmaceutically acceptable earner
  • the composition comprises a therapeutically effective amount of the antibody
  • the composition comprises a further active ingredient, namely , a cardiovascular endothehal or angiogemc agent or an angiostatic agent, preferably an angiogemc or angiostatic agent
  • the composition is ste ⁇ le
  • the composition may be administered in the form of a liquid pharmaceutical formulation which may be preserved to achieve extended stoiage stabili Preserv ed liquid pharmaceutical formulations might contain multiple doses of the anti PRO antibody, and might, therefore, be suitable tor lepeated use
  • the antibody is a
  • the present invention provides an article of manufacture comprising (a) a composition of matter comprising a PRO polypeptide or agonist or antagonist thereof,
  • composition may comprise a therapeutically effective amount of the PRO polypeptide or the agonist or antagonist thereof
  • the present invention provides a method for identifying an agonist of a PRO polypeptide comprising
  • the present invention provides a method for identifying an agonist of a PRO polypeptide comprising
  • the invention provides a method for identifying a compound that inhibits the activity of a PRO polypeptide comprising contacting a test compound with a PRO polypeptide under conditions and for a time sufficient to allow the test compound and polypeptide to interact and determining whether the activity of the PRO polypeptide is inhibited
  • either the test compound or the PRO polypeptide is immobilized on a solid support
  • the non-immobilized component carries a detectable label
  • this method comprises the steps of
  • this process comprises the steps of
  • the invention provides a method for identifying a compound that inhibits the expression of a PRO polypeptide in cells that normally expresses the polypeptide, wherein the method comprises contacting the cells with a test compound and determining whether the expression of the PRO polypeptide is inhibited
  • this method comprises the steps of (a) contacting cells and a test compound to be screened under conditions suitable for allowing expression of the PRO polypeptide, and
  • the invention provides a compound that inhibits the expression of a PRO polypeptide, such as a compound that is identified by the methods set forth above
  • a compound that is identified by the methods set forth above Another aspect of the present invention is directed to an agonist or an antagonist of a PRO polypeptide which may optionally be identified by the methods described above
  • the invention provides an isolated antibody that binds a PRO polypeptide
  • the antibody is a monoclonal antibody, which preferably has non-human complementa ⁇ ty-determining-region (CDR) residues and human framework-region (FR) residues
  • CDR non-human complementa ⁇ ty-determining-region
  • FR human framework-region
  • the antibody may be labeled and may be immobilized on a solid support
  • the antibody is an antibody fragment, a single-chain antibody, or a humanized antibody
  • the antibody specifically binds to the polypeptide
  • the present invention provides a method for diagnosing a disease or susceptibility to a disease which is related to a mutation in a PRO polypeptide-encodmg nucleic acid sequence comprising determining the presence or absence of said mutation in the PRO polypeptide nucleic acid sequence, wherein the presence or absence of said mutation is indicative of the presence of said disease or susceptibility to said disease
  • the invention provides a method of diagnosing a cardiovascular, endothehal or angiogemc disorder in a mammal which comprises analyzing the level ot expression of a gene encoding a PRO polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower expression level in the test sample as compared to the control sample is indicative of the presence of a cardiovascular, endothehal or angiogemc disorder in said mammal
  • the expression of a gene encoding a PRO polypeptide may optionally be accomplished by measuring the level of mRNA or the polypeptide in the test sample as compared to the control sample.
  • the present invention provides a method of diagnosing a cardiovascular, endothehal or angiogemc disorder in a mammal which comprises detecting the presence or absence of a PRO polypeptide in a test sample of tissue cells obtained from said mammal
  • the invention provides a method for determining the presence of a PRO polypeptide in a sample comprising exposing a sample suspected of containing the PRO polypeptide to an anti-PRO antibody and determining binding of said antibody to a component of said sample
  • the sample comprises a cell suspected of containing the PRO polypeptide and the antibody binds to the cell
  • the antibody is preferably detectably labeled and/or bound to a solid support
  • the invention provides a cardiovascular, endothehal or angiogemc disorder diagnostic kit comprising an anti-PRO antibody and a earner in suitable packaging
  • kit further comprises instructions for using said antibody to detect the presence of the PRO polypeptide
  • the carrier is a buffer, for example
  • the cardiovascular, endothehal or angiogemc disorder is cancer
  • the present invention provides a method for treating a cardiovascular, endothehal or angiogemc disorder in a mammal comprising administering to the mammal an effective amount of a PRO polypeptide
  • the disorder is cardiac hypertrophy, trauma such as wounds or burns, or a type of cancer
  • the mammal is further exposed to angioplasty or a drug that treats cardiovascular, endothehal or angiogemc disorders such as ACE inhibitors or chemotherapeutic agents if the cardiovascular, endothehal or angiogemc disorder is a type of cancer
  • the mammal is human, preferably one who is at risk of developing cardiac hypertrophy and more preferably has suffered myocardial infarction
  • the cardiac hypertrophy is characterized by the presence of an elevated level of PGF 2 ⁇
  • the cardiac hypertrophy may be induced by myocardial infarction, wherein preferably the administration of the PRO polypeptide is initiated within 48 hours, more preferably within 24 hours, following myocardial infarction
  • the cardiovascular, endothehal or angiogemc disorder is cardiac hypertrophy and said PRO polypeptide is administered together with a cardiovascular, endothehal or angiogemc agent
  • a cardiovascular, endothehal or angiogemc agent for this purpose is selected from the group consisting of an antihypertensive drug, an ACE inhibitor, an endothelin receptor antagonist and a thrombolytic agent If a thrombolytic agent is administered, preferably the PRO polypeptide is administered following administration of such agent More preferably, the thrombolytic agent is recombinant human tissue plasminogen activator
  • the cardiovascular, endothehal or angiogemc disorder is cardiac hypertrophy and the PRO polypeptide is administered following primary angioplasty for the treatment of acute myocardial infarction, preferably wherein the mammal is further exposed to angioplastv or a cardiovascular, endothehal, or angiogemc agent
  • the cardiovascular endothehal or angiogemc disordei is a cancer and the PRO polypeptide is administered in combination with a chemotherapeutic agent, a growth inhibitory agent or a cytotoxic agent
  • the invention concerns a method for treating a cardiovascular, endothehal or angiogemc disorder in a mammal comprising administering to the mammal an effective amount of an agonist of a PRO polypeptide
  • the cardiovascular, endothehal or angiogemc disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration
  • an effective amount of an angiogemc or angiostatic agent is administered in conjunction with the agonist
  • the invention concerns a method for treating a cardiovascular, endothehal or angiogemc disorder m a mammal comprising administering to the mammal an effective amount of an antagonist of a PRO polypeptide
  • the cardiovascular, endothehal or angiogemc disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration
  • the mammal is human, and where an effective amount of an angiogemc or angiostatic agent is administered in conjunction with the antagonist
  • the invention concerns a method for treating a cardiovascular, endothehal or angiogemc disorder in a mammal comprising administering to the mammal an effective amount of an anti-PRO antibody
  • the cardiovascular, endothehal or angiogemc disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration
  • an effective amount of an angiogemc or angiostatic agent is administered in conjunction with the antibody
  • the invention provides a method for treating a cardiovascular, endothehal or angiogemc disorder in a mammal that suffers therefrom comprising administering to the mammal a nucleic acid molecule that codes for either (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide or (c) an antagonist of a PRO polypeptide, wherein said agonist or antagonist may be an anti-PRO antibody
  • the mammal is human
  • the gene is administered via ex vivo gene therapy
  • the gene is comprised within a vector, more preferably an adenoviral, adeno-associated viral, lentiviral, or retroviral vector
  • the invention provides a recombinant retroviral particle comprising a retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO polypeptide, (b) an agonist polypeptide of a PRO polypeptide
  • the invention provides a method for inhibiting endothehal cell giowth in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide or (c) an antagonist of a PRO polypeptide, wherein endothehal cell growth in said mammal is inhibited, and wherein said agonist or antagonist may be an anti-PRO antibody
  • the mammal is human and the endothehal cell growth is associated with a tumor or a retinal disorder
  • the invention provides a method for stimulating endothehal cell growth in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein endothehal cell growth in said mammal is stimulated, and wherein said agonist or antagonist may be an anti-PRO antibody
  • the mammal is human
  • the invention provides a method for inhibiting cardiac hypertrophy in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein cardiac hypertrophy in said mammal is inhibited, and wherein said agonist or antagonist may be an anti-PRO antibody
  • the mammal is human and the cardiac hypertrophy has been induced by myocardial infarction
  • the invention provides a method for stimulating cardiac hypertrophy in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein cardiac hypertrophy in said mammal is stimulated, and wherein said agonist or antagonist may be an anti-PRO antibody
  • the mammal is human who suffers from congestive heart failure
  • the invention provides a method for inhibiting angiogenesis induced by a PRO polypeptide in a mammal comprising administering a therapeutically effective amount of an anti PRO antibody to the mammal
  • the mammal is a human, and more preferably the mammal has a tumor or a retinal disorder
  • the invention provides a method for stimulating angiogenesis induced by a PRO polypeptide in a mammal comprising administering a therapeutically effective amount of a PRO polypeptide to the mammal
  • the mammal is a human, and more preferably angiogeneisis would promote tissue regeneration or wound healing
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more 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, v.
  • sequence identity et more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet moi e 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 moie preferably at least about 989.
  • sequence identity and yet more preferably at least about 99% sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein with or without the signal peptide, as disclosed herein or any other specifically defined fragment ot the full length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a)
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99%
  • the invention concerns an isolated nucleic acid molecule compi ising 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
  • 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
  • the invention provides isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified
  • the invention concerns an isolated PRO 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 pieferably at least about 89% sequence identity, yet more preferablv 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 moie 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 moie preferably at least about 969b 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
  • the invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about
  • the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methio ne and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore descnbed Processes for producing the same are also herein described, wherein those processes comprise cultu ⁇ ng a host cell comprising a v ector which comprises the appropi late encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture
  • Another aspect of the invention provides an isolated PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated Processes for producing the same are also herein described, wherein those processes comprise culturmg a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture
  • the invention concerns agonists and antagonists of a native PRO polypeptide as defined herein
  • the agonist or antagonist is an anti-PRO antibody or a small molecule
  • the invention concerns a method of identifying agonists or antagonists to a PRO polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide
  • the PRO polypeptide is a native PRO polypeptide
  • the invention concerns a composition of matter comprising a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier
  • the earner is a pharmaceutically acceptable carrier
  • Another embodiment of the present invention is directed to the use of a PRO polypeptide, or an agonist or antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO polypeptide, an agonist or antagonist thereof or an anti-PRO 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 coli, yeast, or Baculovirus-infected insect cells
  • a process for producing any of the herein described polypeptides is further provided and comprises culturmg host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture
  • the invention provides chime ⁇ c molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence
  • Example of such chime ⁇ c molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobuhn
  • the invention provides an antibody which specifically binds to any of the above or below described polypeptides
  • the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody
  • the invention provides ohgonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences
  • Figure 1 shows a nucleotide sequence (SEQ ID NO 1 ) of a native sequence PRO320 cDNA, wherein SEQ ID NO 1 is a clone designated herein as ' DNA32284-1307'
  • Figure 2 shows the amino acid sequence (SEQ ID NO 2) derived from the coding sequence of SEQ ID NO 1 shown in Figure 1
  • Figure 3 shows a nucleotide sequence (SEQ ID NO 6) of a native sequence PR0938 cDNA, wherein SEQ ID NO 6 is a clone designated herein as "DNA56433-1406'
  • Figure 4 shows the amino acid sequence (SEQ ID NO 7) derived from the coding sequence of SEQ ID NO 6 shown in Figure 3
  • FIG. 5 shows a nucleotide sequence (SEQ ID NO 1 1 ) of a native sequence PRO 1031 cDNA, wherein SEQ ID NO 1 1 is a clone designated herein as "DNA59294-1381 "
  • Figure 6 shows the amino acid sequence (SEQ ID NO 12) derived from the coding sequence of SEQ ID NO 1 1 shown in Figure 5
  • Figure 7 shows a nucleotide sequence (SEQ ID NO 13) of a native sequence PR0296 cDNA, wherein SEQ ID NO 13 is a clone designated herein as "DNA39979-1213"
  • Figure 8 shows the amino acid sequence (SEQ ID NO 14) derived from the coding sequence of SEQ ID NO 13 shown in Figure 7
  • Figure 9 shows a nucleotide sequence (SEQ ID NO 21 ) of a native sequence PR0213 cDNA, wherein SEQ
  • ID NO 21 is a clone designated herein as "DNA30943-1163"
  • Figure 10 shows the amino acid sequence (SEQ ID NO 22) derived from the coding sequence of SEQ ID NO 21 shown in Figure 9
  • Figure 1 1 shows a nucleotide sequence (SEQ ID NO 23) of a native sequence PRO1330 cDNA, wherein SEQ ID NO 1 1 is a clone designated herein as "DNA64907- 1 163-1 "
  • Figure 12 shows the amino acid sequence (SEQ ID NO 24) derived from the coding sequence of SEQ ID NO 23 shown in Figure 1 1
  • Figure 13 shows a nucleotide sequence (SEQ ID NO 25) ot a native sequence PROl 449 cDNA, wherein SEQ ID NO 13 is a clone designated herein as "DNA64908-1 163 1 "
  • Figure 14 shows the ammo acid sequence (SEQ ID NO 26) derived from the coding sequence of SEQ ID NO
  • cardiovasculai, endothehal and angiogemc dysfunction e.g., cardiovasculai, endothehal and angiogemc dysfunction
  • 'cardiovascular, endothehal or angiogemc disorder and 'cardiovascular, endothehal or angiogemc disfunction are used inteichangeably and refer in part to systemic disorders that affect vessels, such as diabetes melhtus, as well as diseases of the vessels themselves, such as of the arteries, capillaries, veins, and/or lymphatics
  • Such disordei s include for example, arterial disease, such as atherosclerosis, hypertension, inflammatory vascuhtides, Reynaud's disease and Reynaud's phenomenon, aneurysms and arterial restenosis, venous and lymphatic disorders such as thromb
  • Hypertrophy is defined as an increase in mass of an organ or structure independent of natural growth that does not involve tumor formation Hypertrophy of an organ or tissue is due either to an increase in the mass of the individual cells (true hypertrophy), or to an increase in the number of cells making up the tissue (hyperplasia), or both Certain organs, such as the heart, lose the ability to divide shortly after birth Accordingly,
  • cardiac hypertrophy is defined as an increase in mass of the heart, which, in adults, is characterized by an increase in myocyte cell size and contractile protein content without concomitant cell division
  • the character of the stress responsible for inciting the hypertrophy appears to play a critical role in determining the nature of the response
  • the early stage of cardiac hypertrophy is usually characterized morphologically by increases in the size of myofibrils and mitochondria, as well as by enlargement of mitochondria and nuclei At this stage, while muscle cells are larger than normal, cellular organization is largely preserved
  • Cells subjected to long- standing hypertrophy show more obvious disruptions in cellular organization, including markedly enlarged
  • Heart failure refers to an abnormality of cardiac function where the heart does not pump blood at the rate needed for the requirements of metabolizing tissues
  • the heart failure can be caused by a number of factors, including ischemic, congenital, rheumatic, or ldiopathic forms
  • CHF Congestive heart failuie
  • hypotrophic cardiomyopathy Another complex cardiac disease associated with cardiac hypertrophy is "hypertrophic cardiomyopathy" This condition is characterized by a great diversity of morphologic, functional, and clinical features (Maron et al , N Engl J Med . 316 780-789 (1987), Spinto etal , N Engl J Med , 320 749-755 ( 1989), Louie and Edwards, Prog Cardiovasc Pis . 36 275-308 ( 1994), Wigle et al , Circulation, 92 1680- 1692 ( 1995)), the heterogeneity of which is accentuated by the fact that it afflicts patients of all ages Spinto et al , N Engl J Med , 336 775-785 (1997)
  • hypertrophic cardiomyopathy The causative factors of hypertrophic cardiomyopathy are also diverse and little understood In general, mutations in genes encoding sarcome ⁇ c proteins are associated with hypertrophic cardiomyopathy Recent data suggest that ⁇ -myosin heavy chain mutations may account for approximately 30 to 40 percent of cases of familial hypertrophic cardiomyopathy Watkins ef ⁇ / . N Engl j Med , 326 1 108-1 1 14 (1992), Schwartz etal. Circulation, 91 532-540 (1995), Marian and Roberts, Circulation. 92 1336-1347 (1995), Thierfelder et al , Cejl, 77 701 -712 (1994),
  • Supravalvular "aortic stenosis” is an inherited vascular disorder characterized by narrowing of the ascending aorta, but other arteries, including the pulmonary arteries, may also be affected Untreated aortic stenosis may lead to increased intracardiac pressure resulting in myocardial hypertrophy and eventually heart failure and death The pathogenesis of this disorder is not fully understood, but hypertrophy and possibly hyperplasia of medial smooth muscle are prominent features of this disorder It has been reported that molecular variants of the elastin gene are involved in the development and pathogenesis of aortic stenosis U S Patent No 5,650,282 issued July 22, 1997 "Valvular regurgitation" occurs as a result of heart diseases resulting in disorders of the cardiac valves
  • Various diseases can cause the shrinking or pulling apart ot the valve orifice while other diseases may result in endocarditis, an inflammation of the endocardium or lining membrane of the at ⁇ ovent ⁇ culai orifices and operation of the heart Defects such as the narrowing of the valve stenosis or the defectiv e closing of the valve result in an accumulation of blood in the heart cavity or regurgitation of blood past the valve If uncorrected, prolonged valvular stenosis or insufficiency may result in cardiac hypertrophy and associated damage to the heart muscle, which may eventually necessitate valve replacement
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth
  • cancer include but are not limited to, carcinoma including adenocarcmoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, ghoblastoma, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometnal carcinoma, salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, and various
  • 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, l2, 1, 9 "Y, and 186 Re), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof
  • a "chemotherapeutic agent” is a chemical compound useful in the treatment of cancer Examples of chemotherapeutic agents include alkylatmg agents, fohc acid antagonists, anti metabolites of nucleic acid metabolism, antibiotics, py ⁇ midine analogs, 5-fluorourac ⁇ l, cisplatin, punne nucleosides, amines, amino acids, tnazol nucleosides, or corticosteroids Specific examples include Adnamycm, Doxorubicin, 5-Fluorourac ⁇ I, Cytosine arabinoside ("
  • Vmorelbme Carboplatin Temposide, Daunomycin, Carminomycin, Aminopte ⁇ n, Dactinomycin, Mitomycins, Esperamicins (see U S Pat No 4,675,187), Melphalan, and other related nitrogen mustards
  • hormonal agents that act to regulate or inhibit hormone action on tumors, such as tamoxifen and onapnstone
  • a ' growth-inhibitory agent when used herein refers to a compound or composition that inhibits growth of a cell, such as an Wnt-overexpressing cancer cell, either in vitro oi in vix o
  • the growth-inhibitory agent is one which significantly reduces the percentage ot malignant 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 (vincnstine and v inblastme), taxol and topo II inhibitois such
  • TNF tumor necrosis factor
  • HGF hepatocyte growth factoi
  • 4D5 antibody an antibody capable of binding to HER2 receptor (WO 89/06692), such as the 4D5 antibody (and functional equivalents thereof) (e , WO 92/22653)
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology of a cardiovascular, endothehal, and angiogemc disorder
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a cardiovascular, endothehal, and angiogemc disorder such as hypertrophy
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be pi evented
  • the disorder may result from any cause, including ldiopathic, cardiotrophic, or myotrophic causes, or ischemia or lschemic insults, such as myocardial infarction
  • Chronic administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial effect, such as an anti-hypertrophic effect, for an extended period of time
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, pigs, etc Preferably, the mammal is human
  • Administration in combination with one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order
  • cardiovascular agents refers genencally to any drug that acts in treating cardiovascular, endothehal, and angiogemc disordeis
  • cardiovascular agents are those that promote vascular homeostasis by modulating blood pressure, heart rate, heart contractility, and endothehal and smooth muscle biology, all of which factors have a role in cardiovascular disease
  • ang ⁇ otens ⁇ n-II receptor antagonists include ang ⁇ otens ⁇ n-II receptor antagonists, endothelin receptor antagonists such as, for example, BOSENTANTM and MOXONODINTM, interferon-gamma (IFN- ⁇ ), des-aspartate-angiotensin I, thrombolytic agents, e g , streptokmase, urokinase, t-PA, and a t-PA variant specifically designed to have longer half-life and very high fibrin specificity, TNK t-PA (a Tl 03N, N 1 17Q, KHRR(296-299) AAAA
  • inotropic or hypertensive agents such as digoxigenin and ⁇ -adrenergic receptor blocking agents, e g , propranolol. timolol, tertalolol, carteolol nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, and carvedilol.
  • angiotensin converting enzyme (ACE) inhibitors e g , quinapnl, captopnl, enalap ⁇ l ramip ⁇ l, benazep ⁇ l, fosinopnl, and hsinop ⁇ l
  • diuretics e g , chlorothiazide, hydiochlorothiazide hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, and indapamide
  • calcium channel blockers e g , diltiazem, nifedipine, verapamil, mcardipine
  • One prefe ⁇ ed category of this type is a therapeutic agent used for the treatment ot cardiac hypertrophv or of a physiological condition instrumental in the development of cardiac hypei trophy, such as elevated blood pressure, aortic stenosis, or myocardial infarction "Angiogemc
  • an "effective amount" of an active agent such as a PRO polypeptide or agonist or antagonist thereto or an anti-PRO antibody refers to an amount effective for carrying out a stated purpose, wherein such amounts may be determined empirically tor the desired effect
  • PRO polypeptide and "PRO” as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (( e , PRO/number) refers to specific polypeptide sequences as described herein
  • PRO/number polypeptide and “PRO/number” wherein the term “number” is piovided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein)
  • the PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods
  • a “native sequence PRO polypeptide” comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means The term "native sequence PRO polypeptide' specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e g , an extracellular domain sequence), naturally-occurring variant forms (e g , alternatively spliced forms) and naturally-occurring allehc variants of the polypeptide
  • the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures Start and stop codons are shown in bold font and underlined in the figures However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methion e residues designated herein as amino acid position 1 in the figures, it is conceivable
  • the PRO polypeptide ' extracellular domain ' or "ECD" refers to a form of the PRO polypeptide which is essentially free of the tiansmembrane and cytoplasmic domains Ordinarily a PRO polypeptide ECD will have less than 1 % of such transmembiane and/or cytoplasmic domains and pieferably will have less than 0 5% of such domains It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end ot the domain as initially identified herein Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucle
  • PRO polypeptide variant means an active PRO polypeptide as defined above or below having at least about 80% amino acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide with or without the signal peptide, as disclosed herein oi any other fragment of a full-length PRO polypeptide sequence as disclosed herein
  • Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or moie amino acid residues are added, or deleted, at the N or C-terminus of the full-length native amino acid sequence
  • a PRO polypeptide variant will have at least about 80% ammo acid sequence identity, preferably at least about 81 % amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least
  • amino acid sequence identity more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and most preferably at least about 99% ammo acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herein.
  • PRO variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about 50 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 amino acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, more often at least about 300 amino acids in length, or more.
  • Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program. This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program.
  • Tables 2A-2D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid sequence identity (Tables 2C-2D) using the ALIGN-2 sequence comparison computer program, wherein "PRO” represents the amino acid sequence of a hypothetical PRO polypeptide of interest, “Comparison Protein” represents the amino acid sequence of a polypeptide against which the "PRO” polypeptide of interest is being compared, “PRO-DNA” represents a hypothetical PRO-encoding nucleic acid sequence of interest, “Comparison DNA” represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA” nucleic acid molecule of interest is being compared, “X”, “Y”, and “Z” each represent different hypothetical amino acid residues and "N", “L” and “V” each represent different hypothetical nucleotides.
  • filel and file2 are two dna or two protein sequences
  • Max file length is 65535 (limited by unsigned short x in the jmp struct)
  • a sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
  • the program may create a tmp file in /tmp to hold info about traceback.
  • static nm matches in core — for checking */ static Imax; /* lengths of stripped file names */ static U[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static m[2]; /* current elem number — for gapping */ static s ⁇ z[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */ static char oouutt[[22]][[IP_LINE], /* output line */ static char star[P 1 -], /* set by stars() *//
  • *ps[ ⁇ ] toupper(*ps[ ⁇ ]), po[ ⁇ ] + +; ps[i] + +;
  • *py+ + *px, else if ( ⁇ slower(*px))
  • *py++ touppe ⁇ (*px), if ( ⁇ ndex("ATGCU",*(py-l))) natgc+ + , ⁇ ⁇
  • Percent (%) amino acid sequence identity with respect to the PRO polypeptide sequences identified herein is defined as the percentage ot amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO sequence, after aligning the sequences and intioducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in vanous ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 The ALIGN-2 sequence comparison computer program was
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given ammo acid sequence B is calculated as follows
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows
  • a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (; e , the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of ammo acid residues of the PRO polypeptide of interest For example, in
  • PRO variant polynucleotide or "PRO variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extiacellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any othei fragment of a full-length PRO polypeptide sequence as disclosed herein
  • a PRO 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
  • PRO variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in length, more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more
  • Percent (%) nucleic acid sequence identity with respect to the PRO polypeptide-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PRO polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) softwaie 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
  • % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al , Nucleic Acids Res , 25 3389-3402 (1997))
  • NCBI-BLAST2 sequence comparison program may be dow nloaded from http //www ncbi nlm mh 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
  • PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4
  • PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide
  • amino acid residues in the sequences compared that are not only identical, but also those that have similar properties Amino acid residues that score a positive value to an amino acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 3 below) of the amino acid residue of interest
  • 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
  • the % positives of A to B will not equal the % positives of B to A "Isolated", when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a component of its natural environment
  • the isolated polypeptide is fiee of association with all components with which it is naturally associated
  • Contaminant components of its natural environment are materials that would typically mterfeie 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 1 residues of N-terminal or internal amino acids
  • an "isolated" nucleic acid molecule encoding a PRO polypeptide or an "isolated” nucleic acid molecule encoding an anti-PRO antibody is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the PRO-encoding nucleic acid or the natural source of the an ti-PRO-encoding nucleic acid Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated
  • An isolated PRO-encoding nucleic acid molecule or an isolated anti-PRO-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 PRO-encoding nucleic acid molecule or from the anti-PRO-encoding nucleic acid molecule as it exists in natural cells
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism
  • the control sequences that are suitable forprokaryotes include a promoter, optionally an operator sequence, and a nbosome binding site
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence
  • DNA for a presequence or secretory leader is operably linked to DNA for a PRO polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence
  • a nbosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation
  • ' operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase
  • enhancers do not have to be contiguous Linking is accomplished by hgation at convenient restriction sites If such sites do not exist, the synthetic ohgonucleotide adaptors or linkers are used in accordance with conventional practice
  • 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 hyb ⁇ dizable sequence the higher the relative temperature that can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lowei 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 b) those that (1 ) employ low ionic strength and high temperature for washing, tor 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 albumin/ 0 1 % F ⁇ coll/0 1 % polyvinylpyrrolidone/ 50mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 °C, or (3) employ 50% formamide, 5 x SSC (0 75 M NaCl, 0 075 M sodium citrate), 50 mM sodium phosphate (pH 6 8), 0 1 % sodium pyrophosphate, 5 x Denhardt's solution
  • Modely-stringent conditions may be identified as described by Sambrook etal , Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Press, 1989), and include the use of washing solution and hybridization conditions (e g , temperature, ionic strength, and % SDS) less stringent than those described above
  • An example of moderately stringent conditions is overnight incubation at 37 °C in a solution comprising 20% formamide, 5 x SSC (150 mM NaCl, 15 mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH 7 6) 5 x Denhardt ' s solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc as necessary to accommodate factors such as probe length and the like
  • epitope-tagged when used herein refers to a chime ⁇ c polypeptide comprising a PRO polypeptide fused to a "tag polypeptide"
  • the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused
  • the tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes
  • Suitable tag polypeptides generally have at least six ammo acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues)
  • Active or “activity” in the context of PRO variants refers to form(s) of PRO proteins that retain the biologic and/or immunologic activities of a native or naturally-occurring PRO polypeptide
  • Bioactivity in the context of a molecule that antagonizes a PRO polypeptide that can be identified by the screening assays disclosed herein (e g , an organic or inorganic small molecule, peptide, etc ) is used to refer to the ability of such molecules to bind or complex with the PRO polypeptide identified herein or othei wise interfere with the interaction of the PRO polypeptides with other cellular proteins or otherw ise inhibits the transcription or translation of the PRO polypeptide
  • Particularly preferred biological activity includes cardiac hypertrophy, activity that acts on systemic disorders that affect vessels, such as diabetes melhtus, as w ell as diseases of the arteries, capillaries, veins, and/or lymphatics and cancer
  • Antagonist is used in the broadest sense and includes any molecule that partialh or fully blocks, inhibits or neutralizes one or moie of the biological activities of a native PRO polypeptide disclosed herein for example, if applicable, its mitogemc or angiogemc activity
  • Antagonists of a PRO polypeptide may act by interfering with the binding of a PRO pol) peptide to a cellular receptor, by incapacitating or killing cells that have been activated bv a PRO polypeptide, or b> interfering with vascular endothehal cell activation after binding of a PRO polypeptide to a cellular receptor All such points of intervention by a PRO polypeptide antagonist shall be considered equivalent for purposes of this invention
  • the antagonists inhibit the mitogemc, angiogemc, or other biological activity of PRO polypeptides, and thus are useful for the treatment of diseases or disorders characterized by undesirable excessive neovasculanzation, including by way of example tumors, and especially solid malignant tumors, r
  • a "small molecule” is defined herein to have a molecular weight below about 500 daltons
  • PRO polypeptide receptor refers to a cellular receptor for a PRO polypeptide, ordinarily a cell-surface receptor found on vascular endothehal cells, as well as variants thereof that retain the ability to bind a PRO polypeptide
  • Antibodies (Abs) and “immunoglobuhns” are glycoproteins having the same structural characteristics While antibodies exhibit binding specificity to a specific antigen, immunoglobuhns include both antibodies and other antibody-like molecules that lack antigen specificity Polypeptides of the lattei kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas
  • the term ' antibody' is used in the broadest sense and specifically covers without limitation, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e g , bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity
  • “Natne antibodies and ' native immunoglobuhns” are usuallyheterotetramericglycoproteinsof 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 disulf ide bond, while the number of disulf ide linkages varies among the heavy chains of different immunoglobulin isotypes Each heavy and light chain also has regularly spaced intrachain disulfide bridges Each heavy chain has at one end a variable domain (V instruct) followed by a number of constant domains Each light chain has a variable domain at one end (V, ) and a constant domain at its other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains
  • the term "variable” refers to the fact that certain portions of the variable domains differ extensively in
  • 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') ., and Fv fragments, diabodies, linear antibodies (Zapataef ⁇ / , Protein Eng , 8(10) 1057-1062(1995)), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily Pepsin treatment yields an F(ab') 2 fragment that has two an tigen-combimng sites and is still capable of cross-linking antigen
  • Fv is the minimum antibody fragment that contains a complete antigen-recognition and -binding site This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the V H -V L di er
  • the six CDRs confer antigen-binding specificity to the antibody
  • e ⁇ en a single variable domain or half of an Fv comprising only three CDRs specific for an antigen
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI ) of the heavy chain Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH 1 domain including one or more cy steines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteme res ⁇ due(s) of the constant domains bear a free thiol group F(ab')-, antibody fragments originally were produced as pairs of Fab fragments that have hinge cysteines between them Other chemical couplings of antibody fragments are also known
  • immunoglobuhns can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino acid sequences of then constant domains Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobuhns can be assigned to different classes There aie five major classes of immunoglobuhns IgA, IgD, IgE, IgG and IgM, and several ot these may be further divided into subclasses (isotypes), e g IgG 1 , lgG2, IgG3, IgG4, IgA and IgA2 The heavy-chain constant domains that correspond to the different classes ot immunoglobuhns are called ⁇ , e ⁇ , and ⁇ , respectively The subumt structures and three dimensional configurations of different classes of immunoglobuhns are well known
  • the term ' monoclonal antibody' as used herein refers to an antibody obtained from a population ot substantially homogeneous antibodies, . e , the individual antibodies compi ismg the population are identical except for possible naturally-occurring mutations that may be present in minor amounts Monoclonal antibodies are highly specific, being directed against a single antigemc site Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes) each monoclonal antibody is directed against a single determinant on the antigen In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobuhns The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method For example, the monoclonal antibodies to be used in accordance with the present invention may be made
  • the monoclonal antibodies herein specifically include "chime ⁇ c” antibodies (immunoglobuhns) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the cha ⁇ n(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity
  • "Humanized' forms of non-human (e g , unne) antibodies are chime ⁇ c immunoglobuhns, immunoglobulm chains, or fragments thereof (such as Fv, Fab, Fab , F(ab').
  • humanized antibodies are human immunoglobuhns (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
  • Fv FR residues of the human immunoglobulm are replaced by corresponding non-human residues
  • humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H - V L )
  • V H heavy-chain variable domain
  • V L light-chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites
  • Diabodies are described more fully in, for example, EP 404,097, WO 93/1 1 161 , and Holhnger et al , Proc Natl Acad Sci USA, 90 6444-6448 ( 1993 )
  • an “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other protemaceous or nonproteinaceous solutes
  • the antibody will be purified (1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, oi (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 other composition that 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 that is detectable
  • Radionuchdes that can serve as detectable labels include, for example, 1-131 1-123, 1-125, Y-90, Re- 188, At-21 1 , Cu-67, B ⁇ -212, and Pd- 109
  • the label may also be a non- detectable entity such as a toxin
  • solid phase ' is meant a non-aqueous matrix to which an antibody of the present invention can adhere
  • solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysaccha ⁇ des (e c , agarose), polyacrylamides, polystyrene, polyv yl alcohol and si cones
  • the solid phase can comprise the well of an assay plate, in others it is a purification column (e g an affinity chromatography column)
  • This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275, 149
  • a "liposome” is a small vesicle composed of various types of hpids, phosphohpids and/or surfactant that is useful for delivery of a drug (such as the PRO polypeptide or antibodies thereto disclosed herein) to a mammal
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of
  • PRO320, PRO938. PRO1031 PRQ296 PRQ213, PROl 330 and PRO 1449 Variants In addition to the full-length native sequence PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 and
  • PRO 1449 poly peptides described herein it is contemplated that PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO1330 and PR01449 variants can be prepared PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 and PRO 1449 variants can be prepared by introducing appropriate nucleotide changes into the PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PRO 1449 DNA, and/or by synthesis of the desired PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics
  • Variations in the native full-length sequence PRO320, PR0938, PROl 031 , PR0296 PR021 , PROl 330 or PROl 449 or in various domains ot the PRO320, PR0938, PRO 1031 , PR0296, PR0213 PROl 330 or PRO 1449 described herein, can be made, for example, using any of the techniques and guidelines for conservative and non- conservative mutations set forth, for instance, in U S Patent No 5,364,934 Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO1330 or PR01449 that results m a change in the amino acid sequence of the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 as compared with the native sequence PRO320, PR0938, PRO I 031 , PR0296, PR0213, PRO 1330 or PRO 1449
  • the variation is by substitution of at
  • 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.
  • conservative substitutions of interest are shown in Table 3 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 3, or as further described below in reference to amino acid classes, are introduced and the products screened.
  • Substantial modifications in function or lmmunological identitv ofthePRO320 PR0938 PRO1031 , PRO296, PR0213, PRO1330 or PR01449 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain Naturally occurring residues are d ⁇ ided into groups based on common side chain properties
  • Non conservative substitutions will entail exchanging a member of one of these classes for another class
  • Such substituted residues also mav be introduced into the conservative substitution sites or more preferably, into the remaining (non conserved) sites
  • Scanning ammo acid analysis can also be employed to identity one or more ammo acids along a contiguous sequence
  • preferred scanning amino acids are relatively small, neutral amino acids
  • amino acids include alanine, glycine, senne, and cysteme
  • 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 alanme substitution does not yield adequate amounts of variant, an lsotenc amino acid can be used
  • PRO1330 and PRQ1449 Covalent modifications of PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 and PR01449 are included within the scope of this invention
  • One type of covalent modification includes reacting targeted ammo acid residues of a PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 poly peptide with an organic denvatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues ot the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449
  • Denvatization with bifunctional agents is useful, for instance, for crosshnking PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PRO 1449 to a water-insoluble support matrix or surface for use in the method for purifying ant ⁇ -PRO320, anti- PR
  • PRO 1449 polypeptide included within the scope of this invention comprises altering the native giycosylation pattern of the polypeptide ' Altering the native giycosylation pattern is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO320 PR0938, PRO1031 PR0296 PR021 PROl 330 or PRO 1449 (either by removing the underlying giycosylation site or by deleting the giycosylation by chemical and/or enzymatic means), and/or adding one or moie giycosylation sites that are not present in the native sequence PRO320 PR0938 PRO 1031 PR0296 PR0213 PROl 330 or PRO 1449
  • the phrase includes qualitative changes in the ghcosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present
  • Addition of giycosylation sites to the PRO320, PR0938, PRO1031 PR0296, PR0213 PRO1330 or PR01449 polypeptide may be accomplished by altering the amino acid sequence
  • the alteiation may be made, foi example, by the addition of, or substitution by, one or more serine or thieonme residues to the native sequence PRO320, PR0938, PRO1031, PR0296, PR0213, PROl 330 or PRO 1449 (for O-linked giycosylation sites)
  • the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 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 PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide Such methods are described in the art, e g , in WO 87/05330 published 1 1 September 1987, and in Aphn and W ⁇ ston, CRC Cnt Rev Biochem , pp 259-306 (1981 ) Removal of carbohydrate moieties present on the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for giycosylation Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddm, et al , Arch Biochem Biophys , 259 52 (1987) and by Edge e.
  • 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 etal , Meth Enzvmol , 138 350 (1987)
  • Another type of covalent modification of PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PR01449 comprises linking the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 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
  • PRO320, PR0938, PRO 1031 PR0296, PR0213, PRO 1330 or PRO 1449 of the present invention may also be modified in a way to form a chime ⁇ c molecule comprising PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO 1330 or PRO 1449 fused to another, heterologous polypeptide or amino acid sequence
  • a chime ⁇ c molecule comprises a fusion of the PRO320, PR0938, PRO1031 ,
  • the epitope tag is generally placed at the ammo- or carboxyl- terminus ot the PRO320, PR0938.
  • PRO1031 , PR0296 PR0213, PRO1330 or PRO H49 Thecetnce of such epitope-tagged forms of the PRO320, PR0938, PRO 1031 , PR0296.
  • PR0213, PRO 1330 or PRO 1449 can be detected using an antibody against the tag polypeptide
  • provision of the epitope tag enables the PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO 1330 or PRO 1449 to be readily purified by affinity purification using an anti-tag antibody or another t_ ⁇ pe 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 (poIy-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 9E10 antibodies thereto [Evan et al , Molecular and Cellular Biology, 5 3610-3616 (1985)], and the Herpe
  • the chime ⁇ c molecule may comprise a fusion of the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 with an immunoglobulm or a particular region of an immunoglobulm
  • an immunoglobulm or a particular region of an immunoglobulm For a bivalent form of the chime ⁇ c molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule
  • the Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO320, PR0938, PRO1031 , PR0296, PR0213, PROl 330 or PRO 1449 polypeptide in place of at least one variable region within an Ig molecule
  • the immunoglobulm fusion includes the hinge, CH2 and CH3, or the hinge, CHI , CH2 and CH3 regions of an IgGl molecule For the production
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PROl 449
  • cDNAs encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO 1330 or PR01449 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below
  • proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed
  • the protein encoded by DNA32284-1307, DNA56433-1406, DNA59294-1381 , DNA39979-1213, DNA30943-1 163, DNA64907-1 163-1 or DNA64908-1 163 1 as well as all further native homologues and variants included in the foregoing definition of PRO320, PR0938, PROl
  • PRO 1330 or PRO 1449 polypeptides by culturmg cells transformed or transfected with a vector containing nucleic acid encoding PRO320, PR0938, PRO 1031 PR0296, PR0213, PRO 1330 or PRO 1449 polypeptides
  • alternative methods that are well known in the art may be employed to prepare PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO 1330 or PRO 1449
  • the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 oi PRO 1449 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.
  • DNA encoding PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the mRNA encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 and to express it at a detectable level Accordingly, DNAs encoding human PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 can be conveniently obtained from cDNA libraries prepared from human tissues, such as described in the Examples The gene encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide may also be obtained from a genomic library or by ohgonucleotide synthesis Libraries can be screened with probes (such as antibodies to the PRO320, PR0938, PRO1031 , PR0296,
  • PR0213, PRO 1330 or PRO 1449 polypeptide or ohgonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al , supia
  • An alternative means to isolate the gene encoding PRO320, PR0938, PRO10 1 , PR0296, PR0213, PRO1330 or PR01449 is to use PCR methodology Sambrook et al , supia, Dieftenbach et al PCR Primer A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1995)
  • the ohgonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized
  • the ohgonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened
  • Methods of labeling are well known in the art, and include the use of radiolabels like ,2 P-labeled ATP biotmylation, or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supia
  • Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases
  • Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs such as ALIGN, DNAstar, and INHERIT, which employ various algorithms to measure homology
  • Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced ammo acid sequence disclosed herein tor the first time, and, if necessary using conventional primer extension piocedures as described in Sambiook et al , supia to detect precursors and processing intermediates of mRNA that may not have been ieverse transcribed into cDNA li Selection and Transformation of Host Cells
  • Host cells are transfected or transformed with expression or cloning vectors described herein for PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transtormants, 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
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or highei eukaryote cells
  • Suitable prokaryotes include, but are not limited to, eubactena, such as Gram-negative or Gram positive organisms, for example, En terobacte ⁇ aceae such as E coh
  • E coli strains are publicly available, such as - ⁇ coh K12 strain MM294 (ATCC 31 ,446), E coli X1776 (ATCC 31 ,537), £ co strain W31 10 (ATCC 27,325), and K5 772 (ATCC 53,635)
  • Other suitable prokaryotic host cells include Enterobacte ⁇ aceae such as Eschenchia e g , E coli, Enterobacter, Erwima, Klebsiella, Proteus, Salmonella, e g , Salmonella typhimut mm , Serratia, e
  • W31 10 strain 27C7 (ATCC 55,244), which has the complete genotype tonA pti 3 phoA E15 (atgF lac )169 deqP ompTkan
  • E coli W31 10 strain 37D6 which has the complete genotype tonA pt ⁇ 3p hoA El 5 (argF-lac)169 degP ompT rbs7 ilvG kai
  • E. coli W31 10 strain 40B4 which is strain 37D6 with a non- kanamycin resistant degP deletion mutation
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449.
  • Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981 ]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No.
  • K. lactis MW98-8C, CBS683. CBS4574: Louvencourte?-./., J. Bacteriol., 737 [1983]
  • K. frag His ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24, 178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al. , Bio/Technology, 8: 135 (1990)), K . thermotolerans, and K.
  • Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991 ), and Aspergillus hosts such as A. nidulans (Ballance et al. , Biochem. Biophys. Res. Commun., 1 12: 284-289 [1983]; Tilburn etal, Gene. 26: 205-221 [1983]; Yelton etal. Proc. Natl. Acad. Sci. USA. 8J_: 1470-1474 [1984]) and A.
  • Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula,
  • Candida Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula.
  • a list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).
  • PR0213, PROl 330 or PR01449 are derived from multicellular organisms.
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf ' 9, as well as plant cells.
  • useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J. Gen. Virol.. 36: 59 (1977)); Chinese hamster ovary cells/ -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.
  • mice sertoli 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 of the appropriate host cell is deemed to be within the skill in the art.
  • nucleic acid e.g., cDNA or genomic DNA
  • the nucleic acid encoding PRO320, PR0938, PROl 031 , PR0296.
  • PR0213, PRO 1330 or PRO 1449 may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression.
  • Various vectors are publicly available.
  • the vector may, for example, be in the form of a plasmid, cosmid, vn al 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 if the sequence is to be secreted, an origin of replication, one or more markei genes, an enhancer element, a promoter, and a transcription termination sequence Construction of suitable vectois containing one or more of these components employs standard hgation techniques that are known to the skilled artisan
  • the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide
  • the signal sequence may be a component of the vector, or it may be a part of the DNA encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 that is inserted into the vector
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicilhnase, lpp, or heat-stable enterotoxin II leaders For yeast secretion the signal sequence may be, e g , the yeast mvertase leader, alpha factor leader (including Saccharomy
  • 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 , ampicilhn, neomycin, methotrexate, or tetracyc ne, (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 nucleic acid encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PROl 449, such as DHFR or thymidine kinase
  • An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub etal , Proc Natl Acad Sci USA.
  • a suitable selection gene tor use in veast is the t ⁇ p ⁇ gene present in the yeast plasmid YRp7 Stinchcomb et al , Nature, 282 39 (1979), Kings an et al Gene 7 141 ( 1979), Tschemper et al , Gene 10 157 (1980)
  • the tip] 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 promotei operably linked to the nucleic acid sequence encoding PRO320, PR0938, PRO1031 PR0296, PR0213, PROl 330 or PR01449 to direct mRNA synthesis
  • Promoters recognized by a variety of potential host cells are well known Piomoters suitable for use with prokaiyotic 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 .
  • Promoters for use in bacterial systems also will contain a Shine Dalgarno (S D ) sequence operably linked to the DNA encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO1330 or PRO1449
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3 phosphoglycerate kinase (Hitzeman etal , J Biol Chem . 255 2073 (1980)) or other glycol ytic enzymes (Hess et al Adv Enzyme Reg , 7 149 ( 1968). Holland.
  • Othei yeast promoters that 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
  • PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 nucleic acid transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 1 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegaiovirus, a retrovirus, hepatitis B virus, and Simian Virus 40 (S V40), by heterologous mammalian promoters, e g , the actin promoter or an immunoglobulm promoter, and by heat-shock promoters, provided such promoters are compatible ith the host cell systems Transcription of a DNA encoding the PRO320, PR0938, PRO1031 PR0296, PR0213, PRO1330 or
  • PRO 1449 by higher eukaryotes may be increased by inserting an enhancer sequence into the vector
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription
  • Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin)
  • an enhancer from a eukaryotic cell virus examples include the SV40 enhancer on the late side of the replication origin (bp 100 270) the cytomegaiovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers
  • the enhancer may be spliced into the vector at a position 5 or 3' to the sequence coding for PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PRO 1449, but is preferably located at a site 5 from the promotei
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination ot transcription and for stabilizing the mRNA Such sequences are commonly available from the 5 and occasionally 3 , untranslated regions of eukaryotic or viral DNAs or cDNAs These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449
  • PRO1031 , PR0296, PR0213, PRO1330 or PR01449 in recombinant vertebiate cell culture are described in Gething etal . Nature, 293 620-625 (1981 ), Mantel etal , 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 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 may be measured by immunological methods, such as lmmunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product
  • Antibodies useful for lmmunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native-sequence PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to DNA encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 and encoding a specific antibody epitope
  • PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptides 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-XTM 100) or by enzymatic cleavage Cells employed in expression of nucleic acid encoding the PRO320, PR0938, PRO 1031 , PR0296, PR0213 PRO 1330 or PRO 1449 polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, somcation, mechanical disruption, or cell-lysing agents
  • PRO320, PR0938, PRO 1031 , PR0296, PR0213 PRO 1330 or PR01449 polypeptide may be desired to purify the PRO320, PR0938, PRO 1031 , PR0296, PR0213 PRO 1330 or PR01449 polypeptide 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, chromatof ocusing SDS-PAGE, ammonium sulfate precipitation, gel filtration using for example, Sephadex G-75, protein A Sepharose columns to remove contaminants such as IgG, and metal chelating columns to bind epitope tagged forms ot the PRO320, PR0938, PRO 1031 PR0296, PR0213, PR01330 or PRO 1449 polypeptide
  • Various methods ot protein purification may be employed and such methods
  • Assays fortestingforendothelin antagonistactivity include a rat heart ventricle binding assay where the polypeptide is tested for its ability to inhibit lodimzed endothehn-1 binding in a receptor assay, an endothelin receptor binding assay testing for intact cell binding of radiolabeled endothelin- 1 using rabbit renal artery vascular smooth muscle cells, an inositol phosphate accumulation assay where functional activity is determined in Rat-1 cells by measuring intra-cellular levels of second messengers, an arachidomc acid release assay that measures the ability of added compounds to reduce endothehn-stimulated arachidomc acid release in cultured vascular smooth muscles, in vitro (isolated vessel) studies using endothehum from male New Zealand rabbits, and in vivo studies using male Sprague-Dawley rats
  • Assays for tissue generation activity include, without limitation, those described in WO 95/16035 (bone, cartilage, tendon), WO 95/05846 (nerve, neuronal), and WO 91/07491 (skin, endothehum)
  • Assays for wound-healing activity include, for example, those described in Winter, Epidermal Wound Healing. Maibach, HI and Rovee, DT, eds (Year Book Medical Publishers, Inc , Chicago), pp 71 -1 12, as modified by the article of Eaglstein and Mertz, J Invest Dermatol , 7 . 382-384 (1978)
  • An assay to screen for a test molecule relating to a PRO polypeptide that binds an endothelin B, (ETB,) receptor polypeptide and modulates signal transduction activity involves providing a host cell transformed with a DNA encoding endothelin B, receptor polypeptide, exposing the cells to the test candidate, and measuring endothelin B, receptor signal transduction activity, as described, e g , in U S Pat No 5,773,223
  • cardiac hypertrophy assays include induction of spreading of adult rat cardiac myocytes
  • ventricular myocytes are isolated from a single (male Sprague-Dawley) rat, essentially following a modification of the procedure described in detail by Piper et al , "Adult ventricular rat heart muscle cells" in Cell Culture Techniques in Heart and Vessel Research, H M Piper, ed (Berlin Springer- Verlag, 1990), pp 36-60
  • This procedure permits the isolation of adult ventricular myocytes and the long-term culture of these cells in the rod-shaped phenotype Phenyleph ⁇ ne and Prostaglandin F 2 ⁇ (PGF 2c ) have been shown to induce a spreading response in these adult cells
  • PGF 2 ⁇ or PGF 2 ⁇ analogs e g fluprostenol
  • phenyleph ⁇ ne by various potential inhibitors of cardiac hypertrophy
  • an in vivo assay is a test tor inhibiting cardiac hypertrophy induced by fluprostenol /;; vivo This phaimacological model tests the ability of the PRO polypeptide to inhibit cardiac hypertrophy induced in rats
  • an in vivo assay is the pressure-overload cardiac hypertrophy assay
  • rats e g , male Wistar or Sprague Dawley
  • the abdominal aorta of each rat is narrowed down just below the diaphragm Beznak M , Can J Biochem Physiol , 33 985-94 (1955)
  • the aorta is exposed through a surgical incision, and a blunted needle is placed next to the vessel
  • the aorta is constricted with a ligature of silk thread around the needle, which is immediately removed and which reduces the lumen of the aorta to the diameter of the needle
  • This approach is described, for example, in Rossi et al .
  • a variety of well-known animal models can be used to further understand the role of the genes identified herein in the development and pathogenesis of tumors and to test the efficacy of candidate therapeutic agents, including antibodies and other antagonists of the nativ e PRO polypeptides, such as small-molecule antagonists
  • Animal models of tumors and cancers include both non-recombinant and recombinant (transgemc) animals
  • Non-recombinant animal models include, for example, rodent, e g , munne models
  • Such models can be generated by 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 orthopm implantation, e g colon cancer cells implanted in colomc tissue See, e g PCT publication No
  • the cells introduced into such animals can be derived from known tumoi/cancer cell lines such as any of the above-listed tumor cell lines, and, for example, the B 104-1 -1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene), / ⁇ .
  • NIH-3T3 cells Caco-2 (ATCC HTB-37), or a moderately well- differentiated grade II human colon adenocarcmoma 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
  • Tumors can be transplanted s c as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions
  • tumor tissue fragments of suitable size are introduced into the s c space
  • Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously Tumor cells can also be injected as subdermal implants In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s c tissue
  • Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogene was initially isolated), or new-transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al Proc Nat 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 etal , Cancer Research. 54 4726-4728 (1994) and Too et al , Cancer Research.
  • Tumors that arise in animals can be removed and cultured in vitio Cells from the in vitio 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 ot 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 ot
  • mice which provide a highly controllable model system for studying the anti-tumor activities of various agents Palladino et al , J Immunol , 138 4023-4032 (1987) Briefly, tumor cells are propagated in vitio in cell culture Prior to injection into the animals the cell lines are washed and suspended in buffer, at a cell density of about 10x l 0 f to l Oxl O 7 cells/ml The animals are then infected subcutaneously with 10 to 100 ⁇ of the cell suspension, allow ing one to three weeks for a tumor to appeal
  • the Lewis lung (3LL) carcinoma of mice which is one of the most thoioughly studied experimental tumors, can be used as an investigational tumor model Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small-cell carcinoma of the lung (SCCL) This tumor can be introduced in normal mice upon injection of tumor
  • One way of evaluating the efficacy of a test compound in an animal model with an implanted tumor is to measure the size of the tumor before and after treatment Traditionally, the size of implanted tumors has been measured with a slide caliper in two or three dimensions The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted into the corresponding volume by using a mathematical formula However, the measurement of tumor size is very inaccurate The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay Another important variable in the description of tumor growth is the tumor volume doubling time Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen.
  • 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 (transgemc) animal models can be engineered by introducing the coding portion of the PRO gene identified herein into the genome of animals of interest, using standard techniques for producing transgemc animals
  • Animals that can serve as a target for transgemc manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e g , baboons, chimpanzees and monkeys
  • Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (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.
  • transgemc animals include those that carry the transgene only in part of their cells (“mosaic animals”).
  • the transgene can be integrated either as a single transgene, or in concatamers, e g , head-to-head or head-to-tail tandems
  • Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko e. al . Proc Natl Acad Sci USA, 89 6232 636 (1992)
  • transgene expression in transgemc animals can be monitored by standard techniques For example,
  • Southern blot analysis or PCR amplification can be used to verify the integration of the transgene
  • the level ot mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis PCR, or lmmunocvtochemistry
  • the animals are further examined for signs of tumor or cancer development Alternatively, ' knock-out" animals can be constructed that have a defective or altered gene encoding a PRO polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the PRO polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal.
  • cDNA encoding a particular PRO polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques.
  • a portion of the genomic DNA encoding a particular PRO polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration.
  • another gene such as a gene encoding a selectable marker that can be used to monitor integration.
  • several kilobases of unaltered flanking DNA are included in the vector. See, e.g., Thomas and Capecchi, Cell, 51 : 503 (1987) for a description of homologous recombination vectors.
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected. See, e.g., Li et al, Cell, 69: 915 (1992). The selected cells are then injected into a blastocyst of an animal (e.g. , a mouse or rat) to form aggregation chimeras. See, e.g. , Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J. Robertson, ed. (IRL: Oxford, 1987), pp. 1 13-152.
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock-out" animal.
  • Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA.
  • Knockout animals can be characterized, for instance, by their ability to defend against certain pathological conditions and by their development of pathological conditions due to absence of the PRO polypeptide.
  • SCC feline oral squamous cell carcinoma
  • Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy of cats, accounting for over 60% of the oral tumors reported in this species. It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor.
  • These tumors are usually not amenable to surgery, primarily because of the anatomy of the feline oral cavity. At present, there is no effective treatment for this tumor.
  • each cat Prior to entry into the study, each cat undergoes complete clinical examination and biopsy, and is scanned by computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors are excluded from the study. The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor, the animals may not be able to feed themselves.
  • CT computed tomography
  • Each cat is treated repeatedly, over a longer period of time. Photographs of the tumors will be taken daily during the treatment period, and at each subsequent recheck.
  • CT scans and thoracic radiograms are evaluated every 8 weeks thereafter. The data are evaluated for differences in survival, response, and toxicity as compared to control groups. Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span.
  • gene amplification and/or gene expression in various tissues may be measured by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc Natl Acad Sci USA, 77 5201-5205 ( 1980)), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes Gene expression in various tissues, alternatively, may be measured by lmmunological methods, such as lmmunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful for lmmunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently,
  • cardiovascular, endothehal, and angiogemc study can be further verified by antibody binding studies, in which the ability of anti-PRO antibodies to inhibit the effect of the PRO polypeptides on endothehal cells or other cells used in the cardiovascular, endothehal, and angiogemc assays is tested
  • Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow
  • Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays Zola, Monoclonal Antibodies A Manual of Techniques (CRC Press, Inc , 1987), pp 147-158
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different lmmunogemc portion, or epitope, of the protein to be detected
  • the test sample analyte is bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobuhn antibody that is labeled with a detectable moiety (indirect sandwich assay)
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme
  • the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example
  • Cell-based assays and animal models for cardiovascular, endothehal, and angiogemc disorders, such as tumors can be used to verify the findings of a cardiovascular, endothehal, and angiogemc assay herein, and further to understand the relationship between the genes identified herein and the development and pathogenesis of undesirable cardiovascular, endothehal, and angiogemc cell growth
  • the role of gene products identified herein in the development and pathology of undesirable cardiovascular, endothehal, and angiogemc cell growth, e g , tumor cells can be tested by using cells or cells lines that have been identified as being stimulated or inhibited by the PRO polypeptide herein
  • Such cells include, for example, those set forth in the Examples below
  • suitable tumor cells include, for example, stable tumor cells lines such as the B 104-1 -1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and / -w-transfected NIH-3T3 cells, which can be transfected with the desired gene and monitored for tumongemc growth
  • stable tumor cells lines such as the B 104-1 -1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and / -w-transfected NIH-3T3 cells, which can be transfected with the desired gene and monitored for tumongemc growth
  • Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumongemc cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody-dependent cellular cytotoxicity (ADCC) Cells transfected with the coding sequences of
  • PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide herein and polypeptidyl agonists and antagonists may be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as gene therapy
  • nucleic acid (optionally contained in a vector) into the patient's cells in vivo and e ⁇ o
  • the nucleic acid is injected directly into the patient, usually at the sites where the PRO320, PR0938, PRO1031 , PR0296 PR0213, PROl 330 or PR01449 polypeptide is required, i e , the site of synthesis of the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449 polypeptide, if known, and the site (e g , wound) where biological activity of PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PR01449 polypeptide is needed.
  • the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes that are implanted into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283, 187).
  • the techniques vary depending upon whether the nucleic acid is transferred into cultured cells ... vitro, or transferred in vivo in the cells of the intended host.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • Transduction involves the association of a replication-defective, recombinant viral (preferably retroviral) particle with a cellular receptor, followed by introduction of the nucleic acids contained by the particle into the cell.
  • a commonly used vector for ex vivo delivery of the gene is a retrovirus.
  • the currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral vectors (such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV)) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are, for example, DOTM A, DOPE, and DC-Choi ; see, e.g., Tonkinson et al, Cancer Investigation. 14(1 ): 54-65 (1996)).
  • the most preferred vectors for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
  • a viral vector such as a retroviral vector includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • a viral vector such as a retroviral vector includes a nucleic acid molecule that, when transcribed in the presence of a gene encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide, is operably linked thereto and acts as a translation initiation sequence.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used (if these are not already present in the viral vector).
  • such vector typically includes a signal sequence for secretion of the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence, most preferably the native signal sequence for the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide.
  • the vector construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • a signal that directs polyadenylation will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
  • the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • an agent that targets the target cells such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • proteins that bind to a cell-surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins that undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example, by Wu etal. , J. Biol.
  • This invention is also related to the use of the gene encoding the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide as a diagnostic. Detection of a mutated form of the PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide will allow a diagnosis of a cardiovascular, endothehal, and angiogenic disease or a susceptibility to a cardiovascular, endothehal, and angiogenic disease, such as a tumor, since mutations in the PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide may cause tumors.
  • PR0213, PROl 330 or PRO 1449 polypeptide may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy, and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using
  • RNA or cDNA may also be used for the same purpose.
  • PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide can be used to identify and analyze PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PROl 449 polypeptide mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA encoding the PRO320, PR0938,
  • PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide or alternatively, radiolabeled antisense DNA sequences encoding the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide.
  • Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
  • DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamidine gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures. See, e.g. , Myers e. al. Science, 230: 1242 (1985).
  • Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method, for example, Cotton et al, Proc. Natl. Acad. Sci. USA, 85: 4397-4401 (1985).
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing, or the use of restriction enzymes, e.g., restriction fragment length polymorphisms (RFLP), and Southern blotting of genomic DNA.
  • restriction enzymes e.g., restriction fragment length polymorphisms (RFLP), and Southern blotting of genomic DNA.
  • nucleic acid encoding the PRO polypeptide may be linked to vascular disease or neovascularization associated with tumor formation. If the PRO polypeptide has a signal sequence and the mRNA is highly expressed in endothehal cells and to a lesser extent in smooth muscle cells, this indicates that the PRO polypeptide is present in serum. Accordingly, an anti-PRO polypeptide antibody could be used to diagnose vascular disease or neovascularization associated with tumor formation, since an altered level of this PRO polypeptide may be indicative of such disorders.
  • a competition assay may be employed wherein antibodies specific to the PRO polypeptide are attached to a solid support and the labeled PRO polypeptide and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of the PRO polypeptide in the sample.
  • Chromosome Mapping The sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymo ⁇ hisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis for the 3'- untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome- specific cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • FISH requires use of the clones from which the gene encoding the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide was derived, and the longer the better.
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).
  • This invention encompasses methods of screening compounds to identify those that mimic the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide (agonists) or prevent the effect of the
  • screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.
  • All assays for antagonists are common in that they call for contacting the drug candidate with a PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact.
  • the interaction is binding and the complex formed can be isolated or detected in the reaction mixture.
  • the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 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 PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PRO 1449 polypeptide and drying.
  • an immobilized antibody e.g., a monoclonal antibody, specific for the PRO320, PR0938, PRO1031 , PR0296, PR0213, PROl 330 or PR01449 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. If the candidate compound interacts with but does not bind to a particular PRO320, PR0938, PRO1031 ,
  • PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide encoded by a gene identified herein its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions.
  • Such assays include traditional approaches, such as, e.g., 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 et al. , Proc. Natl. Acad. Sci.
  • yeast GAL4 Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, 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.
  • GALl -. ⁇ cZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for ⁇ -galactosidase.
  • a complete kit (MATCHMAKERTM) for identifying protein-protein interactions between two specific proteins using the two- hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
  • the PRO polypeptide has the ability to stimulate the proliferation of endothehal cells in the presence of the co-mitogen ConA
  • a screening method takes advantage of this ability Specifically, in the proliferation assay, human umbilical vein endothehal cells are obtained and cultured in 96-well flat-bottomed culture plates (Costar, Cambridge, MA) and supplemented with a reaction mixture appropriate for facilitating proliferation of the cells, the mixture containing Con-A (Calbiochem, La Jolla, CA) Con-A and the compound to be screened are added and after incubation at 37 °C, cultures are pulsed with H-thymidine and harvested onto glass fiber filters (phD, Cambridge Technology, Watertown, MA) Mean 3 H- thymidine incorporation (cpm) of triplicate cultures is determined using a liquid scintillation counter (Beckman Instruments, Irvine, CA) Significant 3 (H)- thymidine incorporation indicates stimulation of endothehal cell proliferation
  • the assay described above is performed, however, in this assay the PRO polypeptide is added along with the compound to be screened and the ability of the compound to inhibit 3 (H)thym ⁇ d ⁇ ne incorporation in the presence of the PRO polypeptide indicates that the compound is an antagonist to the PRO polypeptide
  • antagonists may be detected by combining the PRO polypeptide and a potential antagonist with membrane-bound PRO polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay
  • the PRO polypeptide can be labeled, such as by radioactivity, such that the number of PRO polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist
  • the gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting Cohgan et al , Current Protocols in Immun , 1(2) Chapter 5 ( 1991 )
  • expression cloning is employed wherein polyadenylated RNA is prepared from
  • the labeled PRO polypeptide can be photoaffimty- hnked with cell membrane or extract preparations that express the receptor molecule Cross-linked material is resolved by PAGE and exposed to X-ray film
  • the labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing
  • the amino acid sequence obtained from micro-sequencing would be used to design a set of degenerate ohgonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor
  • mammalian cells or a membrane preparation expressing the receptor would be incubated with the labeled PRO polypeptide in the presence of the candidate compound The ability of the compound to enhance or block this interaction could then be measured
  • compositions useful in the treatment of cardiovascular, endothehal and angiogenic disorders include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple-helix molecules, etc., that inhibit the expression and/or activity of the target gene product.
  • potential antagonists include an ohgonucleotide that binds to the fusions of immunoglobulin with a PRO polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.
  • a potential antagonist may be a closely related protein, for example, a mutated form of the PRO polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO polypeptide.
  • Another potential PRO polypeptide antagonist or agonist is an antisense RNA or DNA construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
  • Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide sequence, which encodes the mature PRO polypeptides herein is used to design an antisense RNA ohgonucleotide of from about 10 to 40 base pairs in length.
  • a DNA ohgonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see, Lee et al, Nucl. Acids Res., 6:3073 (1979); Cooney etal., Science, 241 : 456 (1988); Dervan et al, Science, 251 : 1360 (1991 )), thereby preventing transcription and the production of the PRO polypeptide.
  • the antisense RNA ohgonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PRO polypeptide (antisense - Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988).
  • the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PRO polypeptide.
  • antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.
  • Antisense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length, about 35 bases in length, about 40 bases in length, about 45 bases in length, about 50 bases in length, about 55 bases in length, about 60 bases in length, about 65 bases in length, about 70 bases in length, about 75 bases in length, about 80 bases in length, about 85 bases in length, about 90 bases in length, about 95 bases in length, about 100 bases in length, or more.
  • Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the PRO polypeptide, thereby blocking the normal biological activity of the PRO polypeptide.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g , Rossi.
  • Nucleic acid molecules in tnple-hehx formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides
  • the base composition of these oligonucleotides is designed such that it promotes tnple-hehx formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of punnes or py ⁇ midines on one strand of a duplex
  • base-pairing rules which generally require sizeable stretches of punnes or py ⁇ midines on one strand of a duplex
  • the PRO polypeptides, or agonists or antagonists thereto, that have activity in the cardiovascular, angiogenic, and endothehal assays described herein, and/or whose gene product has been found to be localized to the cardiovascular system, are likely to have therapeutic uses in a variety of cardiovascular, endothehal, and angiogenic disorders, including systemic disorders that affect vessels, such as diabetes melhtus
  • Their therapeutic utility could include diseases of the arteries, capillaries, veins, and/or lymphatics
  • treatments hereunder include treating muscle wasting disease, treating osteoporosis, aiding in implant fixation to stimulate the growth of cells around the implant and therefore facilitate its attachment to its intended site, increasing IGF stability in tissues or in serum, if applicable, and increasing binding to the IGF receptor (since IGF has been shown in vitro to enhance human marrow erythroid and granulocytic progenitor cell growth)
  • the PRO polypeptides or agonists or antagonists thereto may also be employed to stimulate erythropoiesis or granulopoiesis, to stimulate wound healing or tissue regeneration and associated therapies concerned with re- growth of tissue, such as connective tissue, skin, bone, cartilage, muscle, lung, or kidney, to promote angiogenesis, to stimulate or inhibit migration of endothehal cells, and to proliferate the growth of vascular smooth muscle and endothehal cell production
  • tissue such as connective tissue, skin, bone, cartilage, muscle, lung, or kidney
  • angiogenesis to stimulate or inhibit migration of endothehal cells
  • the increase in angiogenesis mediated by the PRO polypeptide or antagonist would be beneficial to ischemic tissues and to collateral coronary development in the heart subsequent to coronary stenosis Antagonists are used to inhibit the action of such polypeptides, for example, to limit the production of excess connective tissue during wound healing or pulmonary fibrosis if the PRO polypeptide promotes such production
  • the decision of whether to use the molecule itself or an agonist thereof for any particular indication, as opposed to an antagonist to the molecule, would depend mainly on whether the molecule herein promotes cardiovascularization, genesis of endothehal cells, or angiogenesis or inhibits these conditions. For example, if the molecule promotes angiogenesis, an antagonist thereof would be useful for treatment of disorders where it is desired to limit or prevent angiogenesis.
  • vascular tumors such as haemangioma, tumor angiogenesis, neovascularization in the retina, choroid, or cornea, associated with diabetic retinopathy or premature infant retinopathy or macular degeneration and proliferati ve vitreoretinopathy, rheumatoid arthritis, Crohn's disease, atherosclerosis, ovarian hyperstimulation, psoriasis, endometriosis associated with neovascularization, restenosis subsequent to balloon angioplasty, scar tissue overproduction, for example, that seen in a keloid that forms after surgery, fibrosis after myocardial infarction, or fibrotic lesions associated with pulmonary fibrosis.
  • vascular tumors such as haemangioma, tumor angiogenesis, neovascularization in the retina, choroid, or cornea, associated with diabetic retinopathy or premature infant retinopathy or macular degeneration and proliferati ve vitreoretinopathy, rheum
  • the molecule inhibits angiogenesis, it would be expected to be used directly for treatment of the above conditions.
  • the molecule stimulates angiogenesis it would be used itself (or an agonist thereof) for indications where angiogenesis is desired such as peripheral vascular disease, hypertension, inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenon, aneurysms, arterial restenosis, thrombophlebitis, lymphangitis, lymphedema, wound healing and tissue repair, ischemia reperfusion injury, angina, myocardial infarctions such as acute myocardial infarctions, chronic heart conditions, heart failure such as congestive heart failure, and osteoporosis. If, however, the molecule inhibits angiogenesis, an antagonist thereof would be used for treatment of those conditions where angiogenesis is desired.
  • an antagonist thereof would be used for treatment of those conditions where angiogenesis is desired.
  • Atherosclerosis is a disease characterized by accumulation of plaques of intimal thickening in arteries, due to accumulation of lipids, proliferation of smooth muscle cells, and formation of fibrous tissue within the arterial wall.
  • the disease can affect large, medium, and small arteries in any organ. Changes in endothehal and vascular smooth muscle cell function are known to play an important role in modulating the accumulation and regression of these plaques.
  • Hypertension is characterized by raised vascular pressure in the systemic arterial, pulmonary arterial, or portal venous systems. Elevated pressure may result from or result in impaired endothehal function and/or vascular disease.
  • Inflammatory vasculitides include giant cell arteritis, Takayasu's arteritis, polyarteritis nodosa (including the microangiopathic form), Kawasaki's disease, microscopic polyangiitis, Wegener's granulomatosis, and a variety of infectious-related vascular disorders (including Henoch-Schonlein prupura). Altered endothehal cell function has been shown to be important in these diseases.
  • Reynaud's disease and Reynaud's phenomenon are characterized by intermittent abnormal impairment of the circulation through the extremities on exposure to cold. Altered endothehal cell function has been shown to be important in this disease.
  • Aneurysms are saccular or fusiform dilatations of the arterial or venous tree that are associated with altered endothehal cell and/or vascular smooth muscle cells.
  • Arterial restenosis (restenosis of the arterial wall) may occur following angioplasty as a result of alteration in the function and proliferation of endothehal and vascular smooth muscle cells.
  • Thrombophlebitis and lymphangitis are inflammatory disorders of veins and lymphatics, respectively, that may result from, and/or in, altered endothehal cell function.
  • lymphedema is a condition involving impaired lymphatic vessels resulting from endothehal cell function.
  • lymphangiomas are benign tumors of the lymphatic system that are congenital, often cystic, malformations of the lymphatics that usually occur in newborns. Cystic tumors tend to grow into the adjacent tissue. Cystic tumors usually occur in the cervical and axillary region. They can also occur in the soft tissue of the extremities. The main symptoms are dilated, sometimes reticular, structured lymphatics and lymphocysts surrounded by connective tissue. Lymphangiomas are assumed to be caused by improperly connected embryonic lymphatics or their deficiency. The result is impaired local lymph drainage. Griener et al, Lvmphology, 4: 140-144 (1971).
  • tumor angiogenesis involves vascularization of a tumor to enable it to growth and/or metastasize. This process is dependent on the growth of new blood vessels.
  • neoplasms and related conditions that involve tumor angiogenesis include breast carcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervical carcinomas, endometrial carcinoma, endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreas
  • AMD Age-related macular degeneration
  • AMD Age-related macular degeneration
  • the exudative form of AMD is characterized by choroidal neovascularization and retinal pigment epithelial cell detachment. Because choroidal neovascularization is associated with a dramatic worsening in prognosis, the PRO polypeptide or antagonist thereto is expected to be useful in reducing the severity of AMD.
  • a PRO polypeptide or antagonist thereof that induces cartilage and/or bone growth in circumstances where bone is not normally formed has application in the healing of bone fractures and cartilage damage or defects in humans and other animals
  • Such a preparation employing a PRO polypeptide or antagonist thereof may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints
  • De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma-induced, or oncologic, resection-induced craniofacial defects, and also is useful in cosmetic plastic surgery
  • PRO polypeptides or antagonists thereto may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like
  • a PRO polypeptide or antagonist thereto may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, or endothehum), muscle (smooth, skeletal, or cardiac), and vascular (including vascular endothehum) tissue, or for promoting the growth of cells comprising such tissues
  • organs including, for example, pancreas, liver, intestine, kidney, skin, or endothehum
  • muscle smooth, skeletal, or cardiac
  • vascular including vascular endothehum
  • a PRO polypeptide herein or antagonist thereto may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage Also, the PRO polypeptide or antagonist thereto may be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells, or for inhibiting the growth of tissues described above
  • a PRO polypeptide or antagonist thereto may also be used in the treatment of pe ⁇ odontal diseases and in other tooth-repair processes Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells, or induce differentiation of progenitors of bone-forming cells
  • a PRO polypeptide herein or an antagonist thereto may also be useful in the treatment of osteoporosis or osteoarth ⁇ tis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc ) mediated by inflammatory processes, since blood vessels play an important role in the regulation of bone turnover and growth
  • tissue regeneration activity that may be attributable to the PRO polypeptide herein or antagonist thereto is tendon/ligament formation
  • a protein that induces tendon/ ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed has application in the healing of tendon or ligament tears, deformities, and other tendon or ligament defects in humans and other animals
  • Such a preparation may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue
  • De novo tendon/hgament-hke tissue formation induced by a composition of the PRO polypeptide herein or antagonist thereto contributes to the repair ot congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments
  • the compositions herein may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of
  • the PRO polypeptide or its antagonist may also be useful for proliferation ot neui al cells and for 1 egeneration of nerve and brain tissue, . e , for the treatment of central and peripheral nervous system disease and neuropathies, as well as mechanical and traumatic disorders, that involve degeneration, death, or trauma to neural cells or nerve tissue More specifically, a PRO polypeptide or its antagonist may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma, and cerebrovascular diseases such as stroke Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a PRO polypeptide herein or antagonist thereto
  • Ischemia-reperfusion injury is another indication Endothehal cell dysfunction may be important in both the initiation of, and in regulation of the sequelae of events that occur following ischemia-reperfusion injury
  • Rheumatoid arthritis is a further indication
  • Blood vessel growth and targeting of inflammatory cells through the vasculature is an important component in the pathogenesis of rheumatoid and sero-negative forms of arthritis
  • a PRO polypeptide or its antagonist may also be administered prophylactically to patients with cardiac hypertrophy, to prevent the progression of the condition, and avoid sudden death, including death of asymptomatic patients
  • Such preventative therapy is particularly warranted in the case of patients diagnosed with massive left ventricular cardiac hypertrophy (a maximal wall thickness of 35 mm or more in adults, or a comparable value in children), or in instances when the hemodynamic burden on the heart is particularly strong
  • a PRO polypeptide or its antagonist may also be useful in the management of atnal fibrillation, which develops in a substantial portion of patients diagnosed with hypertrophic cardiomyopathy
  • Further indications include angina, myocardial infarctions such as acute myocardial infarctions, and heart failure such as congestive heart failure
  • Additional non-neoplastic conditions include psoriasis, diabetic and other prohferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, nephrotic syndrome, preeclampsia, ascites, pencardial effusion (such as that associated with pericarditis), and pleural effusion
  • PRO polypeptides or agonists or antagonists thereof described herein which are shown to alter or impact endothehal cell function, proliferation, and/or form, are likely to play an important role in the etiology and pathogenesis of many or all ot the disorders noted above, and as such can serve as therapeutic targets to augment or inhibit these processes or for vascular-related drug targeting in these disorders
  • the molecules herein and agonists and antagonists thereto are pharmaceutically useful as a piophylactic and therapeutic agent for various disorders and diseases as set forth above
  • compositions of the PRO polypeptides or agonists or antagonists are prepared for storage by mixing the desired molecule having the appropriate 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 lyophi zed formulations or aqueous solutions
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionme, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethomum chloride, benzalkomum chloride, benzethomum chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexan
  • Such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyce ⁇ de mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium tnsihcate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol
  • Carriers for topical or gel-based forms of antagonist include polysaccha ⁇ des such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrohdone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols
  • conventional depot forms are suitably used Such forms include, for example, microcapsules, nano-capsulin
  • Another formulation comprises incorporating a PRO polypeptide or antagonist thereof into formed articles
  • Such articles can be used in modulating endothehal cell growth and angiogenesis
  • tumor invasion and metastasis may be modulated with these articles
  • PRO polypeptide or antagonist to be used for m vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophihzation and reconstitution
  • PRO polypeptide ordinarily will be stored in lyophihzed form or in solution if administered systemically
  • PRO polypeptide or antagonist thereto is typically formulated in combination with other ingredients for reconstitution with an appropriate diluent at the time for use
  • An example of a liquid formulation of PRO polypeptide or antagonist is a sterile, clear, colorless unpreserved solution filled in a single dose vial for subcutaneous injection
  • Preserved pharmaceutical compositions suitable for repeated use may contain, for example, depending mainly on the indication and type of polypeptide a) PRO polypeptide or
  • non-ionic surfactants permits the formulation to be exposed to shear surface stresses without causing denaturation of the polypeptide
  • surfactant-containing formulations may be employed in aerosol devices such as those used in a pulmonary dosmg, and needleless jet injector guns (see, e g , EP 257,956)
  • An isotonifier may be present to ensure isotonicity of a liquid composition of the PRO polypeptide or antagonist thereto, and includes polyhyd ⁇ c sugar alcohols, preferably t ⁇ hydnc or higher sugar alcohols, such as glycerin, erythntol, arabitol, xyhtol, sorbitol, and mannitol
  • sugar alcohols can be used alone or in combination Alternatively, sodium chloride or other appropriate inorganic salts may be used to render the solutions lsotonic
  • the buffer preferably t ⁇ hydnc or higher sugar alcohols, such as glycerin, erythnto
  • the preservatives phenol, benzyl alcohol and benzetho um hahdes, e g , chloride, are known antimicrobial agents that may be employed
  • Therapeutic PRO polypeptide compositions 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
  • the formulations are preferably administered as repeated intravenous (I v ), subcutaneous (s c ), or intramuscular (l m ) injections, or as aerosol formulations suitable for intranasal or intrapulmonary delivery (for intrapulmonary delivery see, e g , EP 257,956)
  • PRO polypeptide can also be administered in the form of sustained-released preparations Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, e g , films, or microcapsules Examples of sustained
  • Liposomes containing the PRO polypeptide are prepared by methods known/, erse DE 3,218,121 , Epstein etal , Proc Natl Acad Sci USA. 82 3688-3692 (1985), Hwang et al , Proc Natl Acad Sci USA, 77 4030-4034 (1980), EP 52,322, EP 36,676, EP 88,046, EP 143,949, EP 142,641 , Japanese patent application 83-1 18008, U S Patent Nos 4,485,045 and 4,544,545, and EP 102,324 Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol % cholesterol, the selected proportion being adjusted for the optimal therapy
  • the therapeutically effective dose of PRO polypeptide or antagonist thereto will, of course, vary depending on such factors as the pathological condition to be treated (including prevention), the method of administration, the type of compound being used for treatment, any co-therapy involved, the patient's age, weight, general medical condition, medical history, etc , and its determination is well within the skill of a practicing physician Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the maximal therapeutic effect If the PRO polypeptide has a narrow host range, for the treatment of human patients formulations comprising human PRO polypeptide, more preferably native-sequence human PRO polypeptide, are preferred The clinician will administer PRO polypeptide until a dosage is reached that achieves the desired effect for treatment of the condition in question For example, if the objective is the treatment of CHF, the amount would be one that inhibits the progressive cardiac hypertrophy associated with this condition The progress of this therapy is easily monitored by echo cardiography Similarly, in patients with hypertrophic cardiomyopathy, PRO polypeptide can
  • the effective dose generally is within the range of from about 0 001 to about 1 0 mg/kg, more preferably about 0 01 -1 0 mg/kg, most preferably about 0 01 0 1 mg/kg
  • a molecule based on the PRO polypeptide is preferably administered at about 5 mg to 1 g, preferably about 10 to 100 mg, per kg body weight, 1 to 3 times daily
  • endotoxin contamination should be kept minimally at a safe level, for example less than 0 5 ng/mg protein
  • the formulations preferably meet sterility, pyrogemcity, general safety, and purity as required by FDA Office and Biologies standards
  • the dosage regimen of a pharmaceutical composition containing PRO polypeptide to be used in tissue regeneration will be determined by the attending physician considering various factors that modify the action of the polypeptides, e.g.
  • the dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
  • the addition of other known growth factors, such as IGF-I, to the final composition may also affect the dosage.
  • Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomo ⁇ hometric determinations, and tetracycline labeling.
  • the route of PRO polypeptide or antagonist or agonist administration is in accord with known methods, e.g. , by injection or infusion by intravenous, intramuscular, intracerebral, intraperitoneal, intracerobrospinal, subcutaneous, intraocular, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes, or by sustained-release systems as noted below.
  • the PRO polypeptide or antagonists thereof also are suitably administered by intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects.
  • the intraperitoneal route is expected to be particularly useful, for example, in the treatment of ovarian tumors.
  • a peptide or small molecule is employed as an antagonist or agonist, it is preferably administered orally or non-orally in the form of a liquid or solid to mammals.
  • Examples of pharmacologically acceptable salts of molecules that form salts and are useful hereunder include alkali metal salts (e.g., sodium salt, potassium salt), alkaline earth metal salts (e.g., calcium salt, magnesium salt), ammonium salts, organic base salts (e.g., pyridine salt, triethylamine salt), inorganic acid salts (e.g., hydrochloride, sulfate, nitrate), and salts of organic acid (e.g., acetate, oxalate, p-toluenesulfonate).
  • alkali metal salts e.g., sodium salt, potassium salt
  • alkaline earth metal salts e.g., calcium salt, magnesium salt
  • ammonium salts e.g., organic base salts (e.g., pyridine salt, triethylamine salt)
  • organic base salts e.g., pyridine salt, triethylamine salt
  • the therapeutic method includes administering the composition topically, systemically, or locally as an implant or device.
  • the therapeutic composition for use is in a pyrogen-free, physiologically acceptable form.
  • the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage, or tissue damage.
  • Topical administration may be suitable for wound healing and tissue repair.
  • the composition would include a matrix capable of delivering the protein- containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and preferably capable of being resorbed into the body.
  • Such matrices may be formed of materials presently in use for other implanted medical applications.
  • compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid, and polyanhydrides.
  • Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate
  • the bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradabihty
  • One specific embodiment is a 50 50 (mole weight) copolymer of lactic acid and glycohc acid in the form of porous particles having diameters ranging from 150 to 800 microns
  • a sequestering agent such as carboxymethyl cellulose or autologous blood clot, to prevent the polypeptide compositions from disassociating from the matrix
  • sequestering agents include cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydoxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose, one preferred being catio c salts of carboxymethylcellulose (CMC)
  • CMC carboxymethylcellulose
  • Other preferred sequestering agents include hyaluromc acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer, and poly(v ⁇ nyl alcohol)
  • the amount of sequestering agent useful herein is 0 5-20 wt%, preferably 1-10 wt%, based on total formulation weight, which represents the amount necessary to prevent desorption of the polypeptide (or its antagonist) from the polymer mat ⁇ x and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the poly
  • the effectiveness of the PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide or an agonist or antagonist thereof in preventing or treating the disorder in question may be improved by administering the active agent serially or in combination with another agent that is effective for those purposes, either in the same composition or as separate compositions
  • PRO polypeptide therapy can be combined with the administration of inhibitors of known cardiac myocyte hypertrophy factors, e g , inhibitors of ⁇ -adrenergic agonists such as phenyleph ⁇ ne, endothelin- 1 inhibitors such as BOSENTANTM and MOXONODINTM, inhibitors to CT 1 (US Pat No 5,679,545), inhibitors to LIF, ACE inhibitors, des aspartate-angiotensin I inhibitors (U S Pat No 5,773,415), and angiotensin II inhibitors
  • the PRO polypeptide can be administered in combination with ⁇ -adrenergic receptor blocking agents, e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol, ACE inhibitors, e g , quinapnl, captopnl, enalapnl, ramip ⁇ l, benazepnl fosinopnl, or hsinop ⁇ l, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide acetazolamide, or indapamide, and/oi calcium channel blockers
  • Preferred candidates for combination therapy in the treatment of hypertrophic cardiomyopathy are ⁇ - adrenergic-blocking drugs (e.g., propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol), verapamil, difedipine, or diltiazem.
  • ⁇ - adrenergic-blocking drugs e.g., propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol
  • verapamil difedipine
  • diltiazem
  • Treatment of hypertrophy associated with high blood pressure may require the use of antihypertensive drug therapy, using calcium channel blockers, e.g., diltiazem, nifedipine, verapamil, or nicardipine; ⁇ -adrenergic blocking agents; diuretics, e.g., chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlo ⁇ henamide, acetazolamide, or indapamide; and/or ACE-inhibitors, e.g., quinapril, captopril, enalapril, ramipril, benazepril, fosinopril, or lisinopril.
  • calcium channel blockers e.g., diltiazem, nifedipine, verapamil, or nicardipine
  • PRO polypeptides or their antagonists may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.
  • agents include various growth factors such as EGF, PDGF, TGF- ⁇ or TGF- ⁇ , IGF, FGF, and CTGF.
  • PRO polypeptides or their antagonists used to treat cancer may be combined with cytotoxic, chemotherapeutic, or growth-inhibitory agents as identified above.
  • the PRO polypeptide or antagonist thereof is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances.
  • the effective amounts of the therapeutic agents administered in combination with the PRO polypeptide or antagonist thereof will be at the physician's or veterinarian's discretion. Dosage administration and adjustment is done to achieve maximal management of the conditions to be treated. For example, for treating hypertension, these amounts ideally take into account use of diuretics or digitalis, and conditions such as hyper- or hypotension, renal impairment, etc.
  • the dose will additionally depend on such factors as the type of the therapeutic agent to be used and the specific patient being treated. Typically, the amount employed will be the same dose as that used, if the given therapeutic agent is administered without the PRO polypeptide.
  • An article of manufacture such as a kit containing PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PRO 1449 polypeptide or agonists or antagonists thereof useful for the diagnosis or treatment of the disorders described above comprises at least 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 that 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 the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PROl 449 polypeptide or an agonist or antagonist thereto.
  • the label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • the article of manufacture may also comprise a second or third container with another active agent as described above.
  • Antibodies Some of the most promising drug candidates according to the present invention are antibodies and antibody fragments that may inhibit the production or the gene product of the genes identified herein and/or reduce the activity of the gene products.
  • Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.
  • the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include the PRO320, PR0938, PRO 1031 , PR0296, PR0213 , PROl 330 or PRO 1449 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 examples include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A or synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
  • the anti-PRO320, anti-PR0938, anti-PRO 1031 , anti-PR0296, anti-PR0213, anti-PROl 330 or anti-PRO 1449 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 PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide or a fusion protein thereof.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice (New York: 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 parental cells lack the enzyme hypoxanthine guanme phosphonbosyl transferase (HGPRT or HPRT)
  • HGPRT or HPRT hypoxanthine guanme phosphonbosyl transferase
  • the culture medium for the hybndomas typically will include hypoxanthine, aminopte ⁇ n, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells
  • Preferred immortalized cell lines are those that fuse efficiently , support stable high level expression of antibody by the selected antibody producing cells, and are sensitive to a medium such as HAT medium
  • More preferred immortalized cell lines are munne myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, 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 the PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide
  • 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 of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal Biochem . 107 220 (1980)
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods Goding, supra Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulm purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S
  • Patent No 4,816,567 DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using ohgonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of munne antibodies)
  • the hybridoma cells of the invention serve as a preferred source of such DNA
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulm protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light-chain constant domains in place of the homologous munne sequences (U S Patent No 4,816,567, Morrison et al , supia) or by covalently joining to the immunoglobulm coding sequence
  • the ant ⁇ -PRO320, ant ⁇ -PR0938, anti-PRO 1031 , ant ⁇ -PR0296, ant ⁇ -PR0213, anti-PRO 1330 or anti-PRO 1449 antibodies may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , munne) antibodies are chime ⁇ c immunoglobuhns, immunoglobulm chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non- human immunoglobulm Humanized antibodies include human immunoglobuhns (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 framework residues of the human immunoglobulm are replaced by corresponding non-human residues Humanized antibodies may also comprise residue
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human
  • These non-human ammo acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain
  • Humamzation can be essentially performed following the method of Winter and co-workers (Jones et al , Nature, 321 522-525 (1986), Riechmann et al , Nature, 332 323-327 (1988). Verhoeven et al .
  • humanized antibodies are chimeric antibodies (U S Patent No 4,816 567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries Hoogenboom and Winter, J Mol Biol , 227 381 (1991 ). Marks et al , J Mol Biol , 222 581 (1991 ) The techniques of Cole et al and Boerner et al are also available for the preparation of human monoclonal antibodies Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p 77 (1985) and Boerner e.
  • human antibodies can be made by introducing human immunoglobulm loci into transgemc animals, e g , mice in which the endogenous immunoglobulm genes have been partially or completely inactivated Upon challenge, human antibody production is observed that 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, and 5,661 ,016, and in the following scientific publications Marks etal , Bio/Technology.
  • 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 PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PRO 1449 polypeptide, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit
  • bispecific antibodies are known in the art Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulm heavy-cham/hght-chain pairs, where the two heavy chains have different specificities Milstein and Cuello, Nature, 305 537-539 (1983) Because of the random assortment of immunoglobulm heavy and light chains, these hyb ⁇ domas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure The purification of the correct molecule is usually accomplished by affinity chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al , EMBO J , 10 3655-3659 (1991 )
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulm constant-domain sequences
  • the fusion preferably is with an immunoglobulm heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions It is 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 immunoglobulm heavy-chain fusions and, if desired, the immunoglobulm light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism
  • CHI first heavy-chain constant region
  • ugate 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 foi treatment of HIV infection WO 91/00360, WO 92/200373, EP 03089 It is contemplated that the antibodies may be prepared in ⁇ ⁇ t ⁇ o using known methods in synthetic protein chemistry, including those involving crosshnking agents.
  • immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • 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.
  • cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See, Caron et al, J. Exp. Med.. 176: 1191-1 195 (1992) and Shopes, J. Immunol.. 148: 2918-2922 ( 1992).
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al, Cancer Research. 53: 2560-2565 (1993).
  • an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See, Stevenson et al, Anti-Cancer Drug Design. 3: 219-230 (1989).
  • Immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 13I I, 131 In, yo Y, and 186 Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyI-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate
  • a ricin immunotoxin can be prepared as described in Vitetta et al, Science, 238: 1098 (1987).
  • Carbon- 14-labeled l -isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, W094/1 1026.
  • the antibody may be conjugated to a "receptor” (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a radionucleotide).
  • a "receptor” such as streptavidin
  • a ligand e.g., avidin
  • cytotoxic agent e.g., a radionucleotide
  • Immunoliposomes The antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein etal, Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et l, Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG- PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin 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).
  • Antibodies specifically binding a PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders as noted above and below in the form of pharmaceutical compositions. If the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449 polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
  • peptide molecules can be designed that 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 etal, Proc. Natl. Acad. Sci. USA, 90: 7889- 7893 (1993).
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • 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.
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxyme thylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes. albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes. albumin microspheres, microemulsions, nano-particles, and nanocapsules
  • 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(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • PR01449 polypeptide may be used to treat various cardiovascular, endothehal, and angiogenic conditions as noted above.
  • the antibodies are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration of the antibody is preferred.
  • 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 &
  • the chemotherapeutic agent may precede, or follow administration of the antibody, or may be given simultaneously therewith.
  • the antibody may be combined with an anti-estrogen compound such as tamoxifen or EVISTATM or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
  • the antibodies are used for treating cancer, it may be desirable also to administer antibodies against other tumor-associated antigens, such as antibodies that bind to one or more of the ErbB2, EGFR, ErbB3, ErbB4, or VEGF receptor(s). These also include the agents set forth above.
  • the antibody is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances.
  • two or more antibodies binding the same or two or more different antigens disclosed herein may be co-administered to the patient.
  • the antibodies herein are co- administered with a growth-inhibitory agent.
  • the growth-inhibitory agent may be administered first, followed by an antibody of the present invention.
  • simultaneous administration or administration of the antibody of the present invention first is also contemplated.
  • Suitable dosages for the growth-inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth-inhibitory agent and the antibody herein.
  • vascularization of tumors is attacked in combination therapy.
  • the anti-PRO polypeptide antibody and another antibody e.g., anti-VEGF
  • TNF is administered, alone or in combination with an auxiliary agent such as alpha-, beta-, or gamma-interferon, anti-HER2 antibody, heregulin, anti-heregulin antibody, D-factor, interleukin-1 (IL-1 ), interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), or agents that promote microvascular coagulation in tumors, such as anti-protein C antibody, anti-protein S antibody, or C4b binding protein (see, WO 91/01753, published 21 February 1991 ), or heat or radiation.
  • an auxiliary agent such as alpha-, beta-, or gamma-interferon, anti-HER2 antibody, heregulin, anti-heregulin antibody, D-factor, interleukin-1 (IL-1 ), interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), or agents that promote microvascular coagulation in tumors, such as anti-protein C
  • auxiliary agents will vary in their effectiveness, it is desirable to compare their impact on the tumor by matrix screening in conventional fashion.
  • the administration of anti-PRO polypeptide antibody and TNF is repeated until the desired clinical effect is achieved.
  • the anti-PRO polypeptide antibody is administered together with TNF and, optionally, auxiliary agent(s).
  • the therapeutic agents described herein are administered to the isolated tumor or organ.
  • a FGF or PDGF antagonist such as an anti-FGF or an anti-PDGF neutralizing antibody, is administered to the patient in conjunction with the anti-PRO polypeptide antibody.
  • Treatment with anti-PRO polypeptide antibodies preferably may be suspended during periods of wound healing or desirable neovascularization.
  • an antibody for the prevention or treatment of cardiovascular, endothehal, and angiogenic disorder, the appropriate dosage of an antibody herein will depend on the type of disorder to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • g/kg to 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily or weekly dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is repeated or sustained until a desired suppression of disorder symptoms occurs.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic tumor imaging.
  • An article of manufacture containing a container with the antibody and a label is also provided. Such articles are described above, wherein the active agent is an anti-PRO320, anti-PR0938, anti-PRO 1031, anti-PR0296, anti- PR0213, anti-PRO1330 or anti-PR01449.
  • the indication for which the antibodies are used is cancer
  • cell-surface proteins such as growth receptors over expressed in certain tumors
  • growth receptors are excellent targets for drug candidates or tumor (e.g., cancer) treatment
  • the same proteins along with PRO polypeptides find additional use in the diagnosis and prognosis of tumors.
  • antibodies directed against the PRO polypeptides may be used as tumor diagnostics or prognostics.
  • antibodies can be used qualitatively or quantitatively to detect the expression of genes including the gene encoding the PRO polypeptide.
  • the antibody preferably is equipped with a detectable, e.g., fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. Such binding assays are performed essentially as described above.
  • In situ detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy. For this pu ⁇ ose, a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample. This procedure also allows for determining the distribution of the marker gene product in the tissue examined. It will be apparent to those skilled in the art that a wide variety of histological methods are readily available for in situ detection.
  • EXAMPLE 1 Extracellular Domain Homology Screening to Identify Novel Polypeptides and cDNA Encoding Therefor
  • the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases.
  • the EST databases included public databases (e.g., Dayhoff, GenBank), and proprietary databases (e.g. LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST-2 (Altschul et al.
  • consensus DNA sequences were assembled relative to the other identified EST sequences using phrap.
  • consensus DNA sequences obtained were often (but not always) extended using repeated cycles of BLAST or BLAST-2 and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above.
  • oligonucleotides were then synthesized and used to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for a PRO polypeptide.
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length.
  • the probe sequences are typically 40-55 bp in length.
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp.
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al, Current Protocols in Molecular Biology, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe ohgonucleotide and one of the primer pairs.
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA.
  • the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al, Science, 253: 1278-1280 (1991 )) in the unique Xhol and Notl sites.
  • a suitable cloning vector such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al, Science,
  • EXAMPLE 2 Isolation of cDNA Clones Encoding Human PRO320 (a fibulin homolog) A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above This assembled consensus sequence is herein identified as DNA28739 Based on the DNA28739 consensus sequence, oligonucleotides were synthesized 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO320
  • PCR primers (forward and reverse) were synthesized forward PCR primer 5'-CCTCAGTGGCCACATGCTCATG-3' (SEQ ID NO 3) reverse PCR primer 5'-GGCTGCACGTATGGCTATCCATAG-3' (SEQ ID NO 4)
  • a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA28739 sequence which had the following nucleotide sequence hybridization probe
  • DNA32284-1307 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 135 137 and an apparent stop codon at nucleotide positions 1 149-1151
  • the predicted polypeptide precursor is 338 amino acids long
  • Analysis of the full-length PRO320 sequence shown in Figure 2 (SEQ ID NO 2) 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 PRO320 polypeptide shown in Figure 2 evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 21 , a cAMP and cGMP-dependent protein kinase phosphorylation site from about ammo acid 54 to about amino acid 58, N- my ⁇ stoylation sites from about amino acid 18 to about amino acid 24, from about amino acid 21 to about amino acid 27, from about amino acid 30 to
  • DNA49798 consensus sequence oligonucleotides were synthesized: 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0938.
  • PCR primers forward and reverse were synthesized: forward PCR primer 5'-GTCCAGCCCATGACCGCCTCCAAC-3' (SEQ ID NO:8) reverse PCR primer 5'-CTCTCCTCATCCACACCAGCAGCC-3' (SEQ ID NO:9)
  • hybridization probe 5'-GTGGATGCTGAAATTTTACGCCCCATGGTGTCCATCCTGCCAGC-3' SEQ ID NO: 10.
  • RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB227).
  • the predicted polypeptide precursor is 349 amino acids long.
  • Analysis of the full-length PR0938 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 of the full-length PR0938 polypeptide shown in Figure 4 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 22; a transmembrane domain from about amino acid 189 to about amino acid 211 ; an N-glycosylation site from about amino acid 46 to about amino acid 50; and an N-myristoylation site from about amino acid 200 to about amino acid
  • PR0938 may be a novel protein disulfide isomerase.
  • An analysis of the Dayhoff database evidenced significant homology between the PR0938 amino acid sequence and the following Dayhoff sequences P_W03626, P_W03627, P_R70491 , GARP_PLAFF, XLU85970_1 , ACADISPROA_l , IE68_HSVA, KSU52064_1 , U93872_83, P_R97866
  • a secondary cDNA library was generated in order to preferentially represent the 5' ends of the primary cDNA clones.
  • Sp6 RNA was generated from the primary library (described above), and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY.O using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above). In this procedure the double stranded cDNA was sized to 500- 1000 bp, linkered with blunt to Notl adaptors, cleaved with Sfil, and cloned into Sfil/Notl cleaved vector.
  • pSST- AMY.O is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cD ⁇ A cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminator, after the cloning sites.
  • cD ⁇ As cloned into this vector that are fused in frame with amylase sequence will lead to the secretion of amylase from appropriately transfected yeast colonies.
  • D ⁇ A from the library described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and vector mixture was then electroporated as recommended by the manufacturer. Subsequently, SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37°C for 30 minutes. The transformants were then plated onto 20 standard 150 mm LB plates containing ampicillin and incubated for 16 hours (37°C). Positive colonies were scraped off the plates and the D ⁇ A was isolated from the bacterial pellet using standard protocols, e.g., CsCl-gradient. The purified D ⁇ A was then carried on to the yeast protocols below.
  • the yeast methods were divided into three categories: (1) Transformation of yeast with the plasmid/cD ⁇ A combined vector; (2) Detection and isolation of yeast clones secreting amylase; and (3) PCR amplification of the insert directly from the yeast colony and purification of the D ⁇ A for sequencing and further analysis.
  • yeast strain used was HD56-5A (ATCC-90785). This strain has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-l 12, his3-l 1 , his3-15, MAL + , SUC + , GAL + .
  • yeast mutants can be employed that have deficient post-translational pathways. Such mutants may have translocation deficient alleles in seel 1 , sec!2, *ec62, with truncated --ec71 being most preferred.
  • antagonists including antisense nucleotides and/or ligands which interfere with the normal operation of these genes, other proteins implicated in this post translation pathway (e.g., SEC ⁇ lp, SEC72 ⁇ , SEC62p, SEC63p, TDJlp or SSAlp-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast.
  • other proteins implicated in this post translation pathway e.g., SEC ⁇ lp, SEC72 ⁇ , SEC62p, SEC63p, TDJlp or SSAlp-4p
  • the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast.
  • Transformation took place by mixing the prepared cells ( 100 ⁇ l) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1 ⁇ g, vol. ⁇ 10 ⁇ l) in microfuge tubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 ⁇ l, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li,OOCCH 3 , pH 7.5) was added. This mixture was gently mixed and incubated at 30°C while agitating for 30 minutes.
  • PEG/TE 600 ⁇ l, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li,OOCCH 3 , pH 7.5
  • the cells were then heat shocked at 42°C for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000 rp for 5-10 seconds, decanted and resuspended into TE (500 ⁇ l, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followed by recentrifugation. The cells were then diluted into TE ( 1 ml) and aliquots (200 ⁇ l) were spread onto the selective media previously prepared in 150 mm growth plates (VWR).
  • TE 500 ⁇ l, 10 mM Tris-HCl, 1 mM EDTA pH 7.5
  • the transformation was performed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly.
  • the selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser etal, Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 208- 210 (1994). Transformants were grown at 30°C for 2-3 days.
  • the detection of colonies secreting amylase was performed by including red starch in the selective growth media.
  • Starch was coupled to the red dye (Reactive Red-120, Sigma) as per the procedure described by Biely et al., Anal. Biochem., 172:176-179 (1988).
  • the coupled starch was incorporated into the SCD-Ura agar plates at a final concentradon of 0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0 (50-100 mM final concentration).
  • the positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies.
  • Well isolated single colonies positive for amylase secretion were detected by direct incorporation of red starch into buffered SCD-Ura agar. Positive colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly.
  • the underlined regions of the oligonucleotides annealed to the ADH promoter region and the amylase region, respectively, and amplified a 307 bp region from vector pSST-AMY 0 when no insert was present
  • the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers
  • DNA23020 A cDNA sequence isolated in the above screen was found, by BLAST and FastA sequence alignment, to have sequence homology to a nucleotide sequence encoding sarcoma-associated protein SAS This cDNA sequence is herein designated DNA23020
  • EST expressed sequence tag
  • the homology search was performed using the computer program BLAST or BLAST2 ( Altshul etal , Methods in Enzymology, 266 460-480 (1996)) Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into a consensus DNA sequence with the program "phrap'
  • DNA35858 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above An intermediate consensus DNA sequence was extended using repeated cycles of BLAST and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above This consensus sequence is herein designated DNA35858
  • ohgonucleotide probes ere generated and used to scieen a human kidney library (LIB228) prepared as described in paragraph 1 above
  • the cloning vector was pRK5B (pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science, 253: 1278- 1280
  • forward PCR primer 1 5'-ACCCACGTCTGCGTTGCTGCC-3' (SEQ ID NO: 17)
  • forward PCR primer 2 5'-ACCCACGTCTGCGTTGCTGCC-3' (SEQ ID NO: 17)
  • hybridization probe 5'-AGGAATGCACTAGGATTCGCGCGG-3' (SEQ ID NO: 19) Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA35858 sequence which had the following nucleotide sequence: hybridization probe:
  • DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0296 gene using the probe ohgonucleotide and one of the PCR primers.
  • a full-length clone (designated herein as DNA39979- 1213) was identified that contained a single open reading frame with an apparent translational initiation site at nucleotide positions 174-176 and ending at the stop codon found at nucleotide positions 786-788 ( Figure 7; SEQ ID NO: 13).
  • the predicted polypeptide precursor is 204 amino acids long as shown in Figure 8 (SEQ ID NO: 14).
  • the full-length PR0296 protein shown in Figure 8 has acalculated molecular weight of approximately 22, 147 daltons and an estimated pi of approximately 8.37. Analysis of the full-length PR0296 sequence shown in Figure 8 (SEQ ID NO: 14) evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 34; transmembrane domains from about amino acid
  • N-glycosylation sites from about amino acid 1 13 to about amino acid 1 17 and from about amino acid 137 to about amino acid 141 ; N-myristoylation sites from about amino acid 4 to about amino acid 10, from about amino acid 34 to about amino acid 40, from about amino acid 38 to about amino acid 44, from about amino acid 47 to about amino acid 53, from about amino acid 63 to about amino acid 69, from about amino acid
  • PR0296 may be a novel SAS homolog. More specifically, an analysis of the Dayhoff database (version 35.45 SwissProt) evidenced significant homology between the PR0296 amino acid sequence and the following Dayhoff sequences: 158391 ,
  • PROl 330 and PR01449 (human notch 4 homologs) A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in
  • Example 1 above This assembled consensus sequence is herein identified as DNA28735 Based on the DNA28735 consensus sequence, oligonucleotides were synthesized 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0213 , PRO 1330 and or PRO 1449 PCR primers (forward and reverse) were synthesized forward PCR primer
  • DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above A positive library was then used to isolate clones encoding the PR0213, PRO1330 and/or PR01449 gene using the probe ohgonucleotide and one of the PCR primers RNA for construction of the cDNA libraries was isolated from human fetal lung tissue
  • DNA sequencing of the clones isolated as described above gave the full length DNA sequence for PR0213 (herein designated as DNA30943-1 163 [ Figure 9, SEQ ID NO 21 ]), the full-length DNA sequence for PRO1031 (herein designated as DNA64907-1 163-1 [ Figure 1 1 SEQ ID NO 23]), and the full-length DNA sequence for PR01449 (herein designated DNA64908-1 163-1 [ Figure 13, SEQ ID NO 25]), respectively
  • DNA30943-1 163 (SEQ ID NO 21)], Figure 1 1 [DNA64907- 1 163-1 (SEQ ID NO 23)], and Figure 13 [DNA64908- 1163-1 (SEQ ID NO 25)], respectively DNA30943-1 163, DNA64907-1 163-1 and DNA64908 1 163-1 contain single open reading frames with apparent translational initiation sites at nucleotide positions 399-401 , 488 490 and 326 328, respectively, and ending at the stop codon at nucleotide positions 1218-1220, 1307-1309 and 1 145-1 147, respectively ( Figures 9, 1 1 , & 13, respectively)
  • the predicted PR0213 polypeptide precursor is 273 amino acids long as shown in Figure 10 (SEQ ID NO 22), the predicted PROl 330 polypeptide precursor is 273 amino acids long as shown in Figure 12 (SEQ ID NO 24), and the predicted PRO 1449 polypeptide precursor is 273 amino acids long as shown in Figure 14 (
  • amino acid 100 to about amino acid 106 from about amino acid 103 to about amino acid 109, from about amino acid 157 to about amino acid 163, from about amino acid 191 to about amino acid 197, and from about amino acid 265 to about amino acid 271 ; an amidation site from about amino acid 26 to about amino acid 30; an aspartic acid and asparagine hydroxylation site from about amino acid 152 to about amino acid 164; a cell attachment site from about amino acid 130 to about amino acid 133; and an EGF-like domain cysteine pattern signature from about amino acid 123 to about amino acid 135.
  • amino acid 100 to about amino acid 106 from about amino acid 103 to about amino acid 109, from about amino acid 157 to about amino acid 163, from about amino acid 191 to about amino acid 197, and from about amino acid 265 to about amino acid 271 ; an amidation site from about amino acid 26 to about amino acid 30; an aspartic acid and asparagine hydroxylation site from about amino acid 152 to about amino acid 164; a cell attachment site from about amino acid 130 to about amino acid 133; and an EGF-like domain cysteine pattern signature from about amino acid 123 to about amino acid 135.
  • DNA30943- 1 163, DNA64907- 1 163- 1 and DNA64908- 1 163- 1 have been deposited with ATCC on April 21 , 1998, September 9, 1998, and September 9, 1998, respectively, and are assigned ATCC deposit nos. 209791 , 203242, and 203243. respectively.
  • EXAMPLE 7 Inhibition of VEGF-stimulated Endothehal Cell (ACE) Growth (ASSAY #9) The ability of PRO polypeptides to inhibit VEGF stimulated proliferation of endothehal cells was tested. Polypeptides testing positive in this assay are useful for inhibiting endothehal cell growth in mammals where such an effect would be beneficial, e.g., for inhibiting tumor growth.
  • ACE bovine adrenal cortical capillary endothehal cells
  • DMEM 10% calf serum, 2 mM glutamine, and lX penicillin/streptomycin/fungizone.
  • Control wells included the following: (1) no ACE cells added; (2) ACE cells alone; (3) ACE cells plus 5 ng/ml FGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3 ng/ml VEGF plus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGF plus
  • test sample poly-his tagged PRO320 polypeptide (in 100 microliter volumes), was then added to the wells (at dilutions of 1 %, 0.1 % and 0.01 %, respectively). The cell cultures were incubated for 6-7 days at
  • the activity of PRO320 was calculated as the percent inhibition of VEGF (3 ng/ml) stimulated proliferation
  • TGF-beta was employed as an activity reference at 1 ng/ml, TGF-beta blocks 70-90% of VEGF-stimulated ACE cell proliferation. Results of the assay were interpreted as "positive” if the observed inhibition was > 30%.
  • EXAMPLE 8 Induction of c-fos in Endothehal Cells (ASSAY #34) This assay is designed to determine whether PRO polypeptides show the ability to induce c-fos in endothehal cells.
  • PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of conditions or disorders where angiogenesis would be beneficial including, for example, wound healing, and the like (as would agonists of these PRO polypeptides).
  • Antagonists of the PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of cancerous tumors.
  • Human venous umbilical vein endothehal cells (HUVEC, Cell Systems) in growth media (50% Ham's FI 2 w/o GHT: low glucose, and 50% DMEM without glycine: with NaHC03, 1 % glutamine, 10 mM HEPES, 10% FBS, 10 ng/ml bFGF) are plated on 96-well microtiter plates at a cell density of l xl O 4 cells/well.
  • the cells are starved by removing the growth media and treating the cells with 100 ⁇ l/well test samples and controls (positive control: growth media; negative control: 10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8).
  • positive control growth media
  • negative control 10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8
  • the cells are incubated for 30 minutes at 37°C, in 5% CO,.
  • the samples are removed, and the first part of the bDNA kit protocol (Chiron Diagnostics, cat. #6005-037) is followed, where each capitalized reagent/buffer listed below is available from the kit.
  • the amounts of the TM Lysis Buffer and Probes needed for the tests are calculated based on information provided by the manufacturer.
  • the appropriate amounts of thawed Probes are added to the TM Lysis Buffer.
  • the Capture Hybridization Buffer is warmed to room temperature.
  • the bDNA strips are set up in the metal strip holders, and 100 ⁇ l of Capture Hybridization Buffer are added to each b-DNA well needed, followed by incubation for at least 30 minutes.
  • the test plates with the cells are removed from the incubator, and the media are gently removed using the vacuum manifold. 100 ⁇ l of Lysis Hybridization Buffer with Probes are quickly pipetted into each well of the microtiter plates.
  • the plates are then incubated at 55 °C for 15 minutes. Upon removal from the incubator, the plates are placed on the vortex mixer with the microtiter adapter head and vortex on the #2 setting for one minute. 80 ⁇ l of the lysate are removed and added to the bDNA wells containing the Capture Hybridization Buffer, and pipetted up and down to mix. The plates are incubated at 53 °C for at least 16 hours.
  • the second part of the bDNA kit protocol is followed. Specifically, the plates are removed from the incubator and placed on the bench to cool for 10 minutes. The volumes of additions needed are calculated based upon information provided by the manufacturer.
  • An Amplifier Working Solution is prepared by making a 1:100 dilution of the Amplifier Concentrate (20 fm/ ⁇ l) in AL Hybridization Buffer. The hybridization mixture is removed from the plates and washed twice with Wash A. 50 ⁇ l of Amplifier Working Solution are added to each well and the wells are incubated at 53 °C for 30 minutes. The plates are then removed from the incubator and allowed to cool for 10 minutes.
  • the Label Probe Working Solution is prepared by making a 1 : 100 dilution of Label Concentrate (40 pmoles/ ⁇ l) in AL Hybridization Buffer. After the 10-minute cool-down period, the amplifier hybridization mixture is removed and the plates are washed twice with Wash A. 50 ⁇ l of Label Probe Working Solution are added to each well and the wells are incubated at 53°C for 15 minutes. After cooling for 10 minutes, the Substrate is warmed to room temperature. Upon addition of 3 ⁇ l of Substrate Enhancer to each ml of Substrate needed for the assay, the plates are allowed to cool for 10 minutes, the label hybridization mixture is removed, and the plates are washed twice with Wash A and three times with Wash D. 50 ⁇ l of the Substrate Solution with Enhancer are added to each well. The plates are incubated for 30 minutes at 37 °C and RLU is read in an appropriate luminometer.
  • PRO 1031 assayed "positive” as shown in Table 5:
  • In situ Hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis, and aid in chromosome mapping.
  • In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett, Cell Vision, 1 : 169-176 (1994), using PCR-generated 33 P-labeledriboprobes. Briefly, formalin-fixed, paraffin-embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37 °C, and further processed for in situ hybridization as described by Lu and Gillett, supra. A ( 3 -P)UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at 55 °C overnight. The slides were dipped in Kodak NTB2TM nuclear track emulsion and exposed for 4 weeks. — P-Riboprobe synthesis
  • the tubes were incubated at 37 °C for one hour.
  • a total of 1.0 ⁇ l RQ1 DNase was added, followed by incubation at 37 °C for 15 minutes.
  • a total of 90 ⁇ l TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) was added, and the mixture was pipetted onto DE81 paper.
  • the remaining solution was loaded in a MICROCON-50TM ultrafiltration unit, and spun using program 10 (6 minutes).
  • the filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, a total of 100 ⁇ l TE was added, then 1 ⁇ l of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR IITM.
  • the probe was run on a TBE/urea gel. A total of 1-3 ⁇ l of the probe or 5 ⁇ l of RNA Mrk III was added to
  • the slides were removed from the freezer, placed on aluminum trays, and thawed at room temperature for 5 minutes.
  • the trays were placed in a 55 °C incubator for five minutes to reduce condensation.
  • the slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H 2 0).
  • the slides were deparaffinized, placed in SQ H 2 0, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time.
  • the sections were deproteinated in 20 ⁇ g/ml proteinase K (500 ⁇ l of 10 mg/ml in 250 ml
  • RNase-free RNase buffer 37 °C, 15 minutes
  • 8 x proteinase K 100 ⁇ l in 250 ml Rnase buffer, 37 °C, 30 minutes
  • Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above.
  • the slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper.
  • the tissue was covered with 50 ⁇ l of hybridization buffer (3.75 g dextran sulfate + 6 ml SQ H 2 0), vortexed, and heated in the microwave for 2 minutes with the cap loosened. After cooling on ice, 18.75 ml formamide, 3.75 ml 20 x SSC. and 9 ml SQ H 2 0 were added, and the tissue was vortexed well and incubated at 42 °C for 1 -4 hours.
  • DNA39979-1213 (human SAS homolog - sarcoma amplified protein) p1 5'-GGATTCTAATACGACTCACTATAGGGCCCCAAAGGCAAGGACAAAGC-3' (SEQ ID NO:30) p2 5'-CTATGAAATTAACCCTCACTAAAGGGAACAGTGGACAGTGCCCGACAA-3' (SEQ ID NO:31) p3 5'-GGATTCTAATACGACTCACTATAGGGCTGCGCCCTCAACCTGCTTTAC-3' (SEQ ID NO:32) p4 5'-CTATGAAATTAACCCTCACTAAAGGGATGGACAGTGCCCGACAATGAA-3' (SEQ ID NO:33) p5 5'-GGATTCTAATACGACTCACTATAGGGCTGCGCCCTCAACCTGCTTTAC-3' (SEQ ID NO:34) p6 5'-CTATGAAATTAACCCTCACTAAAGGGATGGACAGTGCCCGACAATGAA-3' (SEQ ID NO
  • DNA64907-1 163-1 (PRO1330) (human notch homolog) p7 5'-GGATTCTAATACGACTCACTATAGGGCGCTGCCCCGGCTGGAAGAG-3' (SEQ ID NO:36) p8 5'-CTATGAAATTAACCCTCACTAAAGGGAAGCGCTGGGCAGTCACGAGTC-3' (SEQ ID NO:37) p9 5'-GGATTCTAATACGACTCACTATAGGGCGGATGGCGGGGTGACACTTG-3' (SEQ ID NO:38) plO 5'-CTATGAAATTAACCCTCACTAAAGGGACTTGGGATGCCGTTGGGGTAG-3' (SEQ ID NO:39)
  • DNA39979-1213 PRQ296
  • human SAS homolog - sarcoma amplified protein
  • Human fetal tissues examined included: placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs, great vessels, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis, testis and lower limb.
  • Adult human tissues examined included: kidney (normal and end-stage), adrenal, spleen, lymph node, pancreas, lung, eye (including retina), bladder, liver (normal, cirrhotic, acute failure).
  • Non-human primate tissues examined included: chimp adrenal tissues and cerebral cortex, hippocampus and cerrebellum of rhesus monkey tissues.
  • DNA64908- 1 163- 1 (PRO 1449) - a splice variant of DNA64907-1163- 1 (PRO 1330) with one amino acid difference - may be expected to exhibit similar expression patterns as DNA64907-1 163-1 (PROl 330).
  • PRO320 Use of PRO320. PRQ938. PRO 1031. PRQ296. PRQ213.
  • PROl 330 or PR01449 as a Hybridization Probe The following method describes use of a nucleotide sequence encoding PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO1330 or PR01449 as a hybridization probe. DNA comprising the coding sequence of full-length or mature PRO320, PR0938, PROl 031 , PR0296,
  • PR0213, PROl 330 or PR01449 (as shown in Figures 1 , 3 , 5, 7, 9, 1 1 , and 13, respectively, SEQ ID NOS: 1 , 6. 1 1 , 13, 21 , 23, and 25, respectively) or a fragment thereof is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449) in human tissue cDNA libraries or human tissue genomic libraries. Hybridization and washing of filters containing either library DNAs is performed under the following high- stringency conditions.
  • PR0213, PRO 1330 or PRO 1449 polypeptide to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1 % SDS, 0.1 % sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42"C for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1 x SSC and 0.1 % SDS at 42°C.
  • DNAs having a desired sequence identity with the DNA encoding full-length native sequence can then be identified using standard techniques known in the art.
  • PRO320 PRQ938. PRO1031. PRQ296. PRQ213.
  • PRO 1449 in E. coli This Example illustrates preparation of an unglycosylated form of PRO320, PR0938, PRO1031, PR0296,
  • PR0213, PRO1330 or PR01449 by recombinant expression in E. coli.
  • the DNA sequence encoding PRO320, PR0938. PRO1031, PR0296, PR0213, PRO 1330 or PRO 1449 (SEQ ID NOS: 1 , 6, 11 , 13, 21, 23, and 25, respectively) is initially amplified using selected PCR primers.
  • the primers should contain restriction enzyme sites that correspond to the restriction enzyme sites on the selected expression vector.
  • a variety of expression vectors may be employed.
  • An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al. , Gene. 2: 95 ( 1977)), which contains genes for ampicillin and tetracycline resistance.
  • the vector is digested with restriction enzyme and dephosphorylated.
  • the PCR-amplified sequences are then ligated into the vector.
  • the vector will preferably include sequences that encode an antibiotic-resistance gene, a trp promoter, a poly-His leader (including the first six STII codons, poIy-His sequence, and enterokinase cleavage site), the region encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449, lambda transcriptional terminator, and an argU gene.
  • the ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al. supra. Transformants are identified by their ability to grow on LB plates and antibiotic-resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.
  • Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics.
  • the overnight culture may subsequently be used to inoculate a larger-scale culture.
  • the cells are then grown to a desired optical density, during which the expression promoter is turned on.
  • the cells After culturing the cells for several more hours, the cells can be harvested by centrifugation.
  • the cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide can then be purified using a metal- chelating column under conditions that allow tight binding of the polypeptide.
  • PRO 213, PRO 1330 and PRO 1449 were successfully expressed in E. coli in a poly-His tagged form by the above procedure.
  • EXAMPLE 12 Expression of Nucleic Acid Encoding PRO320, PRQ938. PRO 1031 , PRQ296. PRQ213, PRO1330 or
  • PRQ1449 in Mammalian Cells This Example illustrates preparation of a potentially glycosylated form of PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO1330 or PR01449 by recombinant expression in mammalian cells.
  • the vector, pRK5 (see, EP 307,247, published March 15, 1989), is employed as the expression vector.
  • PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the DNA encoding PRO320, PR0938, PRO 1031 , PR0296,
  • PR0213, PRO 1330 or PRO 1449 using ligation methods such as described in Sambrook et al. , supra.
  • the resulting vector is called pRK5-(DNA encoding PRO320, PR0938, PRO1031, PR0296, PR0213, PRO 1330 or PRO 1449).
  • the selected host cells are 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.
  • medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics.
  • PR0296, PR0213, PRO1330 or PR01449) is mixed with about 1 ⁇ g DNA encoding the VA RNA gene (Thimmappava et al. Cell. 31 : 543 (1982)) and dissolved in 500 ⁇ l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M
  • 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 of the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide.
  • the cultures containing transfected cells may undergo further incubation (in serum-free medium) and the medium is tested in selected bioassays.
  • the gene encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PROl 330 or PRO 1449 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al, Proc. Natl. Acad. Sci.. 2: 7575 (1981 ). 293 cells are grown to maximal density in a spinner flask and 700 ⁇ g pRK5-(DNA encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449) is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS.
  • the DNA-dextran precipitate is incubated on the cell pellet for four hours.
  • the cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 ⁇ g/ml bovine insulin, and 0.1 ⁇ g/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris.
  • the sample containing the expressed gene encoding the PRO320. PR0938, PRO1031. PR0296. PR0213, PRO1330 or PR01449 polypeptide can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
  • the gene encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 can be expressed in CHO cells.
  • the pRK5-(DNA encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449) nucleic acid 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 35 S-methionine.
  • the culture medium may be replaced with serum-free medium.
  • the cultures are incubated for about 6 days, and then the conditioned medium is harvested.
  • the medium containing the expressed PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 can then be concentrated and purified by any selected method.
  • Epitope-tagged gene encoding the PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide may also be expressed in host CHO cells.
  • the gene encoding PRO320, PR0938, PRO 1031, PR0296, PR0213, PRO1330 orPR01449 may be subcloned out of thepRK5 vector.
  • the subclone insert can undergo PCR amplification to fuse in frame with a selected epitope tag such as a poly-His tag into a baculovirus expression vector.
  • the gene insert encoding the poly-His-tagged-PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO 1330 or PR01449 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 gene encoding the poly-His-tagged-PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PRO 1449 can then be concentrated and purified by any selected method, such as by Ni 2+ -chelate affinity chromatography.
  • PRO320 and PROl 031 were stably expressed in CHO cells by the above described method.
  • PR0938 was in expressed in CHO cells by a transient procedure.
  • PRO 1449 in Yeast The following method describes recombinant expression of the gene encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO1330 or PR01449 in yeast.
  • yeast expression vectors are constructed for intracellular production or secretion of PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 from the ADH2/GAPDH promoter.
  • DNA encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of the gene encoding PRO320.
  • PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 are suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of the gene encoding PRO320.
  • DNA encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO 1330 or PRO 1449 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (it needed) for expression of the gene encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449
  • yeast cells such as yeast strain AB 1 10
  • yeast cells can then be transformed with the expression plasmids described above and cultured in selected fermentation media
  • the transformed yeast supernatants can be analyzed by precipitation with 10% t ⁇ chloroacetic acid and separation by SDS PAGE, followed by staining of the gels with Coomassie Blue stain
  • Recombinant PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 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 PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PR01449 may further be purified using selected column-chromatography resins
  • PR01449 in Baculovirus Infected Insect Cells The sequence coding for PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 is fused upstream of an epitope tag contained within a baculovirus expression vector Such epitope tags include poly-His tags and immunoglobulm tags (like Fc regions of IgG)
  • epitope tags include poly-His tags and immunoglobulm tags (like Fc regions of IgG)
  • plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly, the sequence encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 or the desired portion of the coding sequence of PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 [such as the sequence encoding the extracellular domain of a trans
  • Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes pH 7 9, 12 5 mM MgCl. , 0 1 M EDTA, 10% glycerol, 0 1 % NP-40 04 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 thiough a 0 45 ⁇ m filter
  • a Ni 1+ NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 ml, washed with 25 ml of water and equilibrated with 25 ml of loading buffer The filtered cell extract is loaded onto the column at 0 5 ml per minute The column is washed to baseline A, MI with
  • PRO 1330 or PRO 1449 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography
  • PRO 1031 was successfully expressed in baculovirus infected Sf9 insect cells While expression was actually performed in a 0 5 2 L scale, it can be readily scaled up for larger (e g , 8 L) preparations
  • the proteins were expressed as an IgG construct (immunoadhesin), in which the protein extracellular region was fused to an IgGl constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms
  • baculovirus expression vector pb PH IgG for IgG fusions and pb PH His c for poly-His tagged proteins
  • vector and Baculogold® baculovirus DNA were co-transfected into 105 Spodoptera frug ⁇ et da ("Sf9 ) cells (ATCC CRL 171 1), using Lipofectin (Gibco BRL)
  • pb PH IgG and pb PH His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polyhnker regions to include the His or Fc tag sequences
  • the cells were grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone) Cells were incubated foi 5 days at 28 °C The supernatant was harvested and subsequently used for the first viral amplification by infecting
  • ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0 1 Cells were incubated for 3 days at 28 °C The supernatant was harvested and filtered Batch binding and SDS PAGE analysis was repeated, as necessary, until expression of the spinner culture was confirmed
  • the conditioned medium fiom the transfected cells (0 5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0 22 micron filters
  • the protein construct were purified using a Ni ,+ -NTA column (Qiagen) Before purification, lmidazole was added to the conditioned media to a concentration of 5 mM
  • the conditioned media were 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 was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole.
  • the highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
  • Immunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows.
  • the conditioned media were pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5.
  • the eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9.
  • the highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins.
  • the homogeneity of the proteins was verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.
  • a modified baculovirus procedure may be used incorporating high-5 cells.
  • the DNA encoding the desired sequence was amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector.
  • suitable systems such as Pfu (Stratagene)
  • epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG).
  • a variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIEl -1 (Novagen).
  • the pIEl-1 and pIEl -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus iel promoter in stably-transformed insect cells.
  • the plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for iel -mediated gene expression in uninfected insect cells as well as the hr5 enhancer element.
  • pIEl-1 and pIEl-2 include the translation initiation site and can be used to produce fusion proteins. Briefly, the desired sequence or the desired portion of the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) was amplified by PCR with primers complementary to the 5' and 3' regions.
  • the 5' primer may incorporate flanking (selected) restriction enzyme sites.
  • the product was then digested with those selected restriction enzymes and subcloned into the expression vector.
  • derivatives of pIEl - 1 can include the Fc region of human IgG (pb.PH.IgG) or an 8 histidine (pb.PH.His) tag downstream (3'-of) the desired sequence.
  • the vector construct is sequenced for confirmation.
  • High-5 cells are grown to a confluency of 50% under the conditions of, 27 °C, no C02, NO pen/strep.
  • 30 ⁇ g of pIE based vector containing the sequence was 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 of CellFectin (CellFEOTN (GibcoBRL #10362-010) (vortexed to mix)) was mixed with 1 ml of Ex-Cell medium. The two solutions were combined and allowed to incubate at room temperature for 15 minutes.
  • Ex-Cell media 8 ml of Ex-Cell media was added to the 2 ml of DNA/CellFECTIN mix and this is layered on high-5 cells that have been washed once with Ex-Cell media. The plate is then incubated in darkness for 1 hour at room temperature. The DNA/CellFECTIN mix is then aspirated, and the cells are washed once with Ex-Cell to remove excess CellFECTIN, 30 ml of fresh Ex-Cell media was added and the cells are incubated for 3 days at 28 °C.
  • the supernatant was harvested and the expression of the sequence in the baculovirus expression vector was determined by batch binding of 1 ml of supernatant to 25 ml of Ni + -NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
  • the conditioned media from the transfected cells (0.5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0.22 micron filters.
  • the protein comprising the sequence is purified using a Ni 2+ -NTA column (Qiagen).
  • imidazole is added to the conditioned media to a concentration of 5 mM.
  • the conditioned media was pumped onto a 6 ml Ni + -NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 M imidazole at a flow rate of 4-5 ml/min. at 48 °C.
  • the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole.
  • the highly purified protein was then subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80 °C.
  • Immunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows.
  • the conditioned media was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5.
  • the eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9.
  • the highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins.
  • the homogeneity of the sequence was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary.
  • PRO 1031 was successfully expressed by the above modified baculovirus procedure incorporating high-5 cells.
  • PRO1031 PRO1031 , PR0296, PR0213, PRO1330 or PR01449.
  • Immunogens that may be employed include purified PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO1330 or PR01449 fusion proteins containing PRO320, PR0938, PRO1031 , PR0296, PR0213, PROl 330 or PROl 449, and cells expressing the gene encoding PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PRO 1449 on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
  • mice such as Balb/c are immunized with the PRO320.
  • the immunogen is emulsified in MPL-TDM adjuvant (Ribi
  • mice Immunochemical Research, Hamilton, MT
  • the immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-PRO320, anti-PR0938, anti-PRO1031 , anti-PR0296, anti-PR0213, anti-PRO1330 or anti-PR01449 antibodies.
  • the animals "positive" for antibodies can be injected with a final intravenous injection of PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449.
  • 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 P3X63 AgU.1 , available from ATCC, No. CRL 1597.
  • the fusions generate hybridoma cells that can then be plated in 96-well tissue culture plates containing HAT medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
  • hybridoma cells will be screened in an ELISA for reactivity against PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 is within the skill in the art.
  • the positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PRO320, anti-PR0938, anti-PRO1031, anti-PR0296, anti-PR0213, anti-PRO1330 or anti- PRO 1449 monoclonal antibodies.
  • the hybridoma cells can be grown in tissue-culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium- sulfate precipitation, followed by gel-exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.

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Abstract

Compositions and methods are disclosed for stimulating or inhibiting angiogenesis and/or cardiovascularization in mammals, including humans. Pharmaceutical compositions are based on polypeptides or antagonists thereto that have been identified for one or more of these uses. Disorders that can be diagnosed, prevented, or treated by the compositions herein include trauma such as wounds, various cancers, and disorders of the vessels including atherosclerosis and cardiac hypertrophy. In addition, 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

PROMOTION OR INHIBITION OF ANGIOGENESIS AND CARDIOVASCULARIZATION
Background of the Invention Field of the Invention
The present invention relates to compositions and methods useful for promoting or inhibiting angiogenesis and/or cardiovascularization in mammals in need of such biological effect. This includes the diagnosis and treatment of cardiovascular disorders as well as oncological disorders.
Description of Background
A. Cardiac Disorders and Factors
Heart failure affects approximately five million Americans, and new cases of heart failure number about 400,000 each year. It is the single most frequent cause of hospitalization for people age 65 and older in the United States. Recent advances in the management of acute cardiac diseases, including acute myocardial infarction, are resulting in an expanding patient population that will eventually develop chronic heart failure. From 1979 to 1995, hospitalizations for congestive heart failure (CHF) rose from 377,000 to 872,000 (a 130 percent increase) and CHF deaths increased 116 percent.
CHF is a syndrome characterized by left ventricular dysfunction, reduced exercise tolerance, impaired quality of life, and markedly shortened life expectancy. The sine qua non of heart failure is an inability of the heart to pump blood at a rate sufficient to meet the metabolic needs of the body's tissues (in other words, there is insufficient cardiac output).
At least four major compensatory mechanisms are activated in the setting of heart failure to boost cardiac output, including peripheral vasoconstriction, increased heart rate, increased cardiac contractility, and increased plasma volume. These effects are mediated primarily by the sympathetic nervous system and the renin-angiotensin system. See, Eichhorn, American Journal of Medicine, 104: 163-169 (1998). Increased output from the sympathetic nervous system increases vascular tone, heart rate, and contractility. Angiotensin II elevates blood pressure by 1 ) directly stimulating vascular smooth muscle contraction, 2) promoting plasma volume expansion by stimulating aldosterone and antidiuretic hormone secretion, 3) stimulating sympathetic-mediated vascular tone, and 4) catalyzing the degradation of bradykinin, which has vasodilatory and natriuretic activity. See, review by Brown and Vaughan. Circulation.97: 1411-1420(1998). As noted below, angiotensin II may also have directly deleterious effects on the heart by promoting myocyte necrosis (impairing systolic unction) and intracardiac fibrosis (impairing diastolic and in some cases systolic function). See, Weber, Circulation. 96: 4065-4082 (1998).
A consistent feature of congestive heart failure (CHF) is cardiac hypertrophy, an enlargement of the heart that is activated by both mechanical and hormonal stimuli and enables the heart to adapt to demands for increased cardiac output. Morgan and Baker, Circulation. 83: 13-25 (1991). This hypertrophic response is frequently associated with a variety of distinct pathological conditions such as hypertension, aortic stenosis, myocardial infarction, cardiomyopathy, valvular regurgitation, and intracardiac shunt, all of which result in chronic hemodynamic oveiload
Hypertrophy is generally defined as an increase in size of an organ or structuie independent of natural growth that does not involve tumor formation Hypertrophy of the heart is due either to an increase in the mass of the individual cells (myocytes), or to an increase in the number of cells making up the tissue (hyperplasia), or both While the enlargement of an embryonic heart is largely dependent on an increase in myocyte number (which continues until shortly after birth), post-natal cardiac myocytes lose their prohferative capacity Further growth occurs through hypertrophy of the individual cells Adult myocyte hypertrophy is initially beneficial as a short term response to impaired cardiac function by permitting a decrease in the load on individual muscle fibers With severe, long-standing overload, however, the hypertroph led cells begin to deteriorate and die Katz, "Heart Failure", in Katz A M ed , Physiology of the Heart (New York Raven Press, 1992) pp 638-668 Cardiac hypertrophy is a significant risk factor for both mortality and morbidity in the clinical course of heart failure Katz, Trends Cardiovasc Med . 5 37-44 (1995) For further details of the causes and pathology of cardiac hypertrophy see, e g , Heart Disease, A Textbook of Cardiovascular
Medicine, Braunwald, E ed (W B Saunders Co , 1988), Chapter 14, "Pathophysiology of Heart Failure "
On a cellular level, the heart is composed of myocytes and surrounding support cells, geneπcally called non-myocytes While non-myocytes are primarily fibroblast/mesenchymal cells, they also include endothehal and smooth muscle cells Indeed, although myocytes make up most of the adult myocardial mass, they represent only about 30% of the total cell numbers present in heart In response to hormonal, physiological, hemodynamic, and pathological stimuli, adult ventricular muscle cells can adapt to increased workloads through the activation of a hypertrophic process This response is characterized by an increase in myocyte cell size and contractile protein content of individual cardiac muscle cells, without concomitant cell division and activation of embryonic genes, including the gene for atπal natπuretic peptide (ANP) Chien et al , FASEB J , 5 3037 3046 (1991 ), Chien etal , Annu Rev Phvsiol , 55 77 95 (1993) An increment in myocardial mass as a result of an increase in myocyte size that is associated with an accumulation of interstitial collagen within the extracellular matrix and around in tramyocardial coronary arteries has been described in left ventricular hypertrophy secondary to pressure o\ erload in humans Caspaπ et al , Cardiovasc Res 1 1 554-558 (1977), Schwarz et al , Am J Cardiol , 42 895 903 (1978), Hess et al . Circulation, 63 360 371 (1981 ), Pearlman et al . Lab Invest . 46 158 164 (1982) It has also been suggested that paracπne factors produced by non myocyte supporting cells may additionally be involved in the development of cardiac hypertrophy, and various non myocyte derived hypertrophic factors, such as leukocyte inhibitory factor (LIF) and endothehn, have been identified Metcalt, Growth Factors, 2 169 173 (1992). Kurzrock et al , Endocrine Reviews, 12 208-217 (1991 ), Inoue et al , Proc Natl Acad Sci USA 86 2863 2867 (1989), Yanagisawa and Masaki, Trends Pharm Sci . JO 374 378 (1989), U S Patent No 5 573 762 (issued November 12, 1996) Further exemplary factors that have been identified as potential mediatois of cardiac h\pertrophy include cardiotrophin 1 (CT-1 ) (Pennica e. a/ Proc Nat Acad Sci USA, 92 1 142 1 146 ( 1995)) catecholamines, adrenocorticostei oids, angiotensin and prostaglandins
At present, the treatment of cardiac hypertiophy varies depending on the underlying cardiac disease Catecholamines, adrenocorticosteroids, angiotensin, prostaglandins LIF endothelin (including endothelin- 1 , -2, and -3 and big endothelin), and CT-1 are among the factors identified as potential mediators of hypertrophy For example, beta-adrenergic receptor blocking drugs (beta-blockers, e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol. atenolol, etoprolol carvedilol, etc ) and verapamil have been used extensively in the treatment of hypertrophic cardiomyopathy The beneficial effects of beta-blockers on symptoms
(e g , chest pain) and exercise tolerance are largely due to a decrease in the heart rate with a consequent prolongation of diastole and increased passive ventricular filling Thompson etal . Br Heart J , 44 488-98 (1980), Harrison et al , Circulation, 29 84-98 ( 1964) Verapamil has been described to improve ventricular filling and probably reducing myocardial ischemia Bonow et al . Circulation, 72 853-64 (1985) Nifedipine and diltiazem have also been used occasionally in the treatment of hypertrophic cardiomyopathy Lorell etal , Circulation, 65 499-507 (1982), Betocchi e. fl/ . Am J Cardiol .78 451-457 (1996) However, because of its potent vasodilating properties, nifedipine may be harmful, especially in patients with outflow obstruction Disopyramide has been used to relieve symptoms by virtue of its negative inotropic properties Pol ck, N Engl J Med . 307 997-999 (1982) In many patients, however, the initial benefits decrease with time Wiglee. al . Circulation, 92 1680-1692 (1995) Antihypertensive drug therapy has been reported to have beneficial effects on cardiac hypertrophy associated with elevated blood pressure Examples of drugs used in antihypertensive therapy, alone or in combination, are calcium antagonists, e g , nitrendipine, adrenergic receptor blocking agents, e g , those listed above, angiotensin converting enzyme (ACE) inhibitors such as quinapπl, captopπl, enalapπl, ramipπl, benazepπl, fosinopπl, and hsinopπl, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, and mdapamide, and calcium channel blockers, e g , diltiazem, nifedipine, verapamil, and mcardipine
For example, treatment of hypertension with diltiazem and captopπl showed a decrease in left ventricular muscle mass, but the Doppler indices of diastohc function did not normalize Szlachcic et al , Am J Cardiol , 63 198-201 (1989), Shahi et al , Lancet. 336 458 461 ( 1990) These findings were interpreted to indicate that excessive amounts of interstitial collagen may remain after regression of left ventricular hypertrophy Rossi et al
Am Heart J . 124 700-709 (1992) Rossi et al , supra, investigated the effect of captopπl on the prevention and regression of myocardial cell hypertrophy and interstitial fibrosis in pressure overload cardiac hypertrophy, in experimental rats
Agents that increase cardiac contractility directly (lontropic agents) were initially thought to benefit patients with heart failure because they improved cardiac output in the short term However all positive inotropic agents except digoxigemn have been found to result in increased long-term mortality, in spite of short term improvements in cardiac performance Massie, Curr Op in Cardiology 12 209 217 (1997), Reddy etal , Curr Opin Cardiol , 12 233-241 (1997) Beta adrenergic receptor blockers have recently been advocated for use in heart failure Evidence from clinical trials suggests that improvements in cardiac function can be achieved without increased moita ty, though documented improvements patient surv ival have not yet been demonstrated See also
U S Pat Nos 5,935,924, 5,624 806, 5,661 , 122, and 5,610 134 and WO 95/28173 regaiding the use of cardiotro pin 1 oi antagonists thereof, or growth hormone and/or insulin-like growth factor I in the treatment of CHF Another treatment modality is heart transplantation, but this is limited by the availability ot donor hearts Endothelin is a vasoconstπcting peptide comprising 21 ammo acids, isolated from swine arterial endothelial culture supernatant and structurally determined Yanagisawa et al , Nature, 332 41 1 -415 (1988) Endothelin was later found to exhibit various actions and endothelin antibodies as endothelin antagonists have proven effective in the treatment of myocardial infarction renal failure, and other diseases Since endothelin is present in live bodies and exhibits vasoconstπcting action, it is expected to be an endogenous factor involved in the regulation of the circulatory system, and may be associated with hypertension, cardiovascular diseases such as myocardial infarction, and renal diseases such as acute renal failure Endothelin antagonists are described, for example, in U S Pat No 5,773,414, JP Pat Publ 3130299/1991 , EP 457,195, EP 460,679, and EP 552,489 A new endothelin B receptor for identifying endothelin receptor antagonists is described in U S Pat No 5,773,223 Current therapy for heart failure is primarily directed to using angiotensin-converting enzyme (ACE) inhibitors, such as captopnl, and diuretics These drugs improve hemodynamic profile and exercise tolerance and reduce the incidence of morbidity and mortality in patients with CHF Kramer et al , Circulation, 67(4) 807-816 (1983), Captopnl Multicenter Research Group, J A C C . 2(4) 755 763 (1983), The CONSENSUS Trial Study Group, N Engl J Med , 316(23) 1429-1435 (1987), The SOLVD Investigators. N Engl J Med , 325(5) 293-302 (1991 ) Further, they are useful in treating hypertension, left ventricular dysfunction, atherosclerotic vascular disease, and diabetic nephropathy Brown and Vaughan, supra However, despite proven efficacy, response to ACE inhibitors has been limited For example, while prolonging survival in the setting of heart failure, ACE inhibitors appear to slow the progression towards end-stage heart failure, and substantial numbers of patients on ACE inhibitors have functional class III heart failure Moreover, improvement of functional capacity and exercise time is only small and mortality, although reduced, continues to be high The CONSENSUS Trial Study Group, N Engl J Med , 316(23) 1429-1453 (1987), The SOLVD Investigators, N Engl J Med , 325(5) 293 302 (1991 ), Cohn et al , N Engl J Med . 325(5) 303-310 ( 1991 ), The Captopπl-Digoxin Multicenter Research Group, JAMA, 259(4) 539-544 (1988) Hence, ACE inhibitors consistently appear unable to relieve symptoms in more than 60% of heart failure patients and reduce mortality of heart failure only b\ approximately 15 20% For further adverse effects, see Brown and Vaughan, supra
An alternative to ACE inhibitors is represented by specific ATI receptor antagonists Clinical studies are planned to compaie the efficacy of these two modalities in the treatment of cardiovascular and renal disease However, animal model data suggests that the ACE/Ang II pathway, while clearly involved in cardiac hypertrophy , is not the only, or even the primary pathway active in this role Mouse genetic 'knockout' models have been made to test individual components of the pathway In one such model, the primary cardiac receptoi for Ang II, AT sub 1A, has been genetically deleted these mice do not develop hypertrophy when Ang II is given experimentally (confirming the basic success of the model in eliminating hypertrophy secondary to Ang II) However, when the aorta is constricted in these animals (a model of hypertensive cardiac stress), the hearts still become hypertrophic This suggests that altei native signaling pathways not depending on this receptor (AT sub 1 A), aie activated in hypertension ACE inhibitors would presumably not be able to inhibit these pathways See, Harada et al Circulation 97 1952 1959 (1998) See also, Homey, Circulation. 97 1890- 1892 ( 1998) regarding the enigma associated with the process and mechanism of cardiac hypertrophy About 750 000 patients suffer from acute myocardial infarction (AMI) annually, and approximately one-fourth of all deaths in the United States are due to AMI In recent years, thro mbolytic agents, e g , streptokmase, uiokinase, and in particular tissue plasminogen activator (t-PA) have significantly increased the survival of patients who suffered myocardial infarction When administei ed as a continuous intravenous infusion over 1 5 to 4 hours, t-PA produces coronary patency at 90 minutes in 69% to 90% of the treated patients Topol etal , Am J Cardiol , 61., 723-728 (1988), Neuhaus etal , J Am Coll Cardiol . J2 581-587 (1988), Neuhaus et al , J Am Coll Cardiol , 14 1566 1569 ( 1989) The highest patency rates have been reported with high dose or accelerated dosing regimens Topol, J Am Coll Cardiol , 15 922-924 ( 1990) t-PA may also be administered as a single bolus, although due to its relatively short half-life, it is better suited for infusion therapy Tebbe et al , Am J Cardiol , 64 448-453 (1989) A t-PA variant, specifically designed to have longer half-life and very high fibrin specificity, TNK t-PA (a T103N, Nl 17Q, KHRR(296-299)AAAA t-PA variant, Keyt e. g. . Proc Natl Acad Sci USA, 91 3670-3674 ( 1994)) is particularly suitable for bolus administration However, despite all these advances, the long-term prognosis of patient survival depends greatly on the post-infarction monitoring and treatment of the patients, which should include monitoring and treatment of cardiac hypertrophy
B Growth Factors
Various naturally occurring polypeptides reportedly induce the proliferation of endothehal cells Among those polypeptides are the basic and acidic fibroblast growth factors (FGF) (Burgess and Maciag, Annual Rev Biochem , 58 575 (1989)), platelet-derived endothehal cell growth factor (PD-ECGF) (Ishikawa etal , Nature, 338 557 (1989)), and vascular endothehal growth factor (VEGF) Leung etal . Science, 246 1306 (1989), Ferrara and
Henzel, Biochem Biophys Res Commun , 161 851 (1989), Tischer et al . Biochem Biophys Res Commun . 165 1 198 (1989), EP 471 /754B granted July 31 , 1996
Media conditioned by cells transfected with the human VEGF (hVEGF) cDNA promoted the proliferation of capillary endothehal cells, whereas control cells did not Leung et al . Science, 246 1306 (1989) Several additional cDNAs were identified in human cDNA libraries that encode 121 -, 189-, and 206 amino acid isoforms of hVEGF (also collectively referred to as hVEGF related proteins) The 121 -amino acid protein differs from hVEGF by virtue of the deletion of the 44 amino acids between residues 1 16 and 159 in h VEGF The 189-amιno acid protein differs from hVEGF by virtue of the insertion of 24 amino acids at residue 1 16 in hVEGF, and apparently is identical to human vascular permeability factor (hVPF) The 206-amιno acid protein differs from hVEGF by virtue ot an insertion of 41 amino acids at residue 1 16 in h VEGF Houck et al . Mol Endocπn , 5 1806
(1991 ), Ferrara et al , J Cell Biochem , 47 21 1 (1991 ), Ferrara et al Endocrine Reviews 13 18 (1992), Keck etal . Science 246 1309 (1989), Connolly et al , J Biol Chem . 264 20017 (1989), EP 370,989 published May 30, 1990
It is now well established that angiogenesis which involves the formation of new blood vessels from preexisting endothehum is implicated in the pathogenesis of a variety of disorders These include solid tumors and metastasis, atheiosclerosis, retiolental fibroplasia, hemangiomas, chronic inflammation, intraocular neovasculai syndromes such as prohferative retinopathies, e g , diabetic retinopathy, age related macular degeneration (AMD) neovascular glaucoma, immune i ejection ot transplanted corneal tissue and other tissues, rheumatoid arthritis, and psoriasis Folkman e/α/ J Biol Che , 267 10931 -10934 (1992), Klagsbrun ef -./ Annu Rev Physiol , 53 217- 239 (1991), and Garner A , "Vascular diseases", In Pathobiology of Ocular Disease A Dvnamic Approach Garner A , Khntworth GK, eds , 2nd Edition (Marcel Dekker, NY, 1994), pp 1625-1710
In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia, and tor pioviding nourishment to the growing solid tumor Folkman e. α/ , Nature. 339 58 ( 1989) The neovasculanzation allows the tumor cells to acquire a growth advantage and prohferative autonomy compared to the normal cells Accordingly, a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors Weidner et al , N Engl J Med, 324 1-6 (1991), Horak et al , Lancet, 340 1120-1 124 (1992), Macchiaπni et al , Lancet, 340 145-146 (1992) The search for positive regulators of angiogenesis has yielded many candidates, including aFGF, bFGF, TGF- α, TGF-β, HGF, TNF-α, angiogenin, IL-8, etc Folkman et al , J B C , supra, and Klagsbrun et al , supia. The negative regulators so far identified include thrombospondin (Good et al , Proc Natl Acad Sci USA , 87 6624- 6628 (1990)), the 16-kιlodalton N-terminal fragment of prolactin (Clapp et al , Endocrinology, 133 1292-1299 (1993)), angiostatin (O'Reilly et al . Cell. 79 315-328 (1994)), and endostatin O'Reilly et al , CeU, 88 277-285 (1996)
Work done over the last several years has established the key role of VEGF, not only in stimulating vascular endothehal cell proliferation, but also in inducing vascular permeability and angiogenesis Ferrara et al , Endocr Rev . 18 4-25 ( 1997) The finding that the loss of even a single VEGF allele results in embryonic lethality points to an irreplaceable role played by this factor in the development and differentiation of the vascular system Furthermore, VEGF has been shown to be a key mediator of neovasculanzation associated with tumors and intraocular disorders Ferrara et al , Endocr Rev , supra The VEGF mRNA is overexpressed by the majority of human tumors examined Berkman et al , J Clm Invest , 91 153-159 (1993), Brown et al , Human Pathol , 26 86-91 (1995) Brown etal , Cancer Res , 53 4727-4735 (1993), Mattern e. a. , Brit J Cancer, 73 931 -934 (1996), Dvorak et al Am T Pathol , 146 1029-1039 (1995) Also, the concentration levels of VEGF in eye fluids are highly correlated to the presence of active proliferation of blood vessels in patients with diabetic and other ischemia-related retinopathies Aiello e t al , N_ Engl J Med 331 1480-1487 (1994) Furthermore, recent studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients affected by AMD Lopez et al , Invest Ophthalmol Vis Sci , 37 855-868 (1996) Anti-VEGF neutralizing antibodies suppress the growth ot a variety of human tumor cell lines in nude mice
(Kim et al Nature, 362 841 -844 ( 1993), Warren et al J Clin Invest , 95 1789-1797 ( 1995), Borgstrom et al , Cancer Res 56 4032-4039 (1996), Melnvk et al , Cancer Res . 56 921 -924 (1996)) and also inhibit intraocular angiogenesi1. in models of ischemic retinal disordeis Ada is et al Arch Ophthalmol , 1 14 66-71 (1996) Therefore, anti-VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of solid tumors and various intraocular neovascular disordei s Such antibodies are described, tor example, in EP 817,648 published January 14, 1998 and in PCT/US 98/06724 filed April 3, 1998
There exist several other growth factoi s and mitogens, including transforming oncogenes, that are capable of rapidly inducing a complex set of genes to be expressed by cei tain cells Lau and Nathans Moleculai Aspects of Cellular Regulation, 6 165-202 ( 1991 ) These genes, which have been named immediate-early- or early-response genes, are transcnptionally activated within minutes after contact with a growth factor or mitogen, independent of de novo protein synthesis A group of these in termediate-early genes encodes secreted, extracellular proteins that are needed tor coordination of complex biological processes such as differentiation and proliferation, regeneration, and wound healing Rvseck et al , Cell Growth Differ , 2 235-233 (1991 )
Highly-related proteins that belong to this group include cef 10 (Simmons etal , Proc Natl Acad Sci USA, 86 1 178-1 182 (1989)), CM 61, which is rapidly activated by serum- or platelet-derived growth factor (PDGF) (O'Brien et al , Mol Cell Biol . K) 3569-3577 (1990), human connective tissue growth factor (CTGF) (Bradham et al , J Cell Biol . 1 14 1285- 1294 ( 1991 )), which is secreted by human vascular endothehal cells in high levels after activation with transforming growth factor beta (TGF-β). exhibits PDGF-hke biological and immunological activities, and competes with PDGF for a particular cell surface receptor, fisp-12 (Ryseck et al , Cell Growth Differ , 2 235-233 (1991 )), human vascular IBP-hke growth factor (VIGF) (WO 96/17931 ), and nov, normally arrested in adult kidney cells, which was found to be overexpressed in myeloblastosιs-assocιated-vιrus-type-1 - mduced nephroblastomas Joloit et al , Mol Cell Biol . |2 10-21 ( 1992) The expression of these immediate early genes acts as "third messengers" in the cascade of events triggered by growth factors It is also thought that they are needed to integrate and coordinate complex biological processes, such as differentiation and wound healing in which cell proliferation is a common event
As additional mitogens, insulin-like growth factor binding proteins (IGFBPs) have been shown, in complex with sulin-hke growth factor (IGF), to stimulate increased binding of IGF to fibroblast and smooth muscle cell surface receptors Clemmons et al , J Clin Invest . 77 1548 ( 1986) Inhibitory effects of IGFBP on various IGF actions in vitro include stimulation of glucose transport by adipocytes, sulfate incorporation by chondrocytes, and thymidine incorporation in fibroblast Zapf et al , J Clin Invest , 63 1077 ( 1979) In addition, inhibitory effects of IGFBPs on growth factor-mediated mitogen activity in normal cells have been shown
C Need for Further Treatments
In view of the role of vascular endothehal cell growth and angiogenesis in many diseases and disorders, it is desirable to have a means of reducing or inhibiting one or more of the biological effects causing these processes It is also desirable to have a means of assaying for the presence of pathogenic polypeptides in normal and diseased conditions, and especially cancer Further, in a specific aspect, as there is no generally applicable therapy for the treatment of cardiac hypertrophy, the identification of factors that can prevent or reduce cardiac myocyte hypertrophy is of primary importance in the development of new therapeutic strategies to inhibit pathophysiological cardiac growth While there are several treatment modalities for various cardiovascular and oncologic disorders, there is still a need for additional therapeutic approaches
Summary of the Invention
A Embodiments
Accordingly, the present invention concerns compositions and methods tor promoting or inhibiting angiogenesis and/or cardiovasculaπzation in mammals The piesent invention is based on the identification of proteins that test positive in various cardiovascular assays that test promotion or inhibition of certain biological activities Accordingly, the proteins are believed to be useful drugs for the diagnosis and/or treatment (including prevention) of disoidei s where such effects are desired such as the promotion or inhibition of angiogenesis, inhibition or stimulation ot vascular endothehal cell grow th, stimulation of growth or proliferation of vascular endothehal cells, inhibition of tumor growth, inhibition of angiogenesis dependent tissue growth, stimulation of angiogenesis dependent tissue growth, inhibition of cardiac hypertrophy and stimulation of cardiac hypertrophy , e g , for the treatment of congestive heart failure
In one embodiment, the present invention provides a composition comprising a PRO polypeptide in admixture with a pharmaceutically acceptable carrier In one aspect, the composition comprises a therapeutically effective amount of the polypeptide In another aspect, the composition comprises a further active ingredient, namely, a cardiovascular, endothehal or angiogemc agent or an angiostatic agent, preferably an angiogemc or angiostatic agent Preferably, the composition is sterile The PRO polypeptide may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability Preserved liquid pharmaceutical formulations might contain multiple doses of PRO polypeptide, and might, therefore, be suitable for repeated use
In a further embodiment, the present invention provides a method for preparing such a composition useful for the treatment of a cardiovascular, endothehal or angiogemc disorder comprising admixingatherapeutically effective amount of a PRO polypeptide with a pharmaceutically acceptable carrier
In another embodiment, the present invention provides a composition comprising an agonist or antagonist of a PRO polypeptide in admixture with a pharmaceutically acceptable carrier In one aspect, the composition comprises a therapeutically effective amount of the agonist or antagonist In another aspect, the composition comprises a further active ingredient, namely, a cardiovascular, endothehal or angiogemc agent or an angiostatic agent, preferably an angiogemc or angiostatic agent Preferably, the composition is sterile The PRO polypeptide agonist or antagonist may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability Preserv ed liquid pharmaceutical formulations might contain multiple doses of a PRO polypeptide agonist or antagonist, and might therefore, be suitable for repeated use
In a further embodiment, the present invention prov ides a method for preparing such a composition useful for the treatment of a cardiovascular, endothehal or angiogemc disorder comprising admixing a therapeutically effective amount of a PRO polypeptide agonist or antagonist with a pharmaceutically acceptable carrier In yet another embodiment, the present invention concerns a composition comprising an anti PRO antibody in admixture with a pharmaceutically acceptable earner In one aspect the composition comprises a therapeutically effective amount of the antibody In another aspect, the composition comprises a further active ingredient, namely , a cardiovascular endothehal or angiogemc agent or an angiostatic agent, preferably an angiogemc or angiostatic agent Preferably, the composition is steπle The composition may be administered in the form of a liquid pharmaceutical formulation which may be preserved to achieve extended stoiage stabili Preserv ed liquid pharmaceutical formulations might contain multiple doses of the anti PRO antibody, and might, therefore, be suitable tor lepeated use In preteπed embodiments, the antibody is a monoclonal antibody, an antibody fragment a humanized antibody oi a single-chain antibody In a further embodiment, the present invention provides a method for preparing such a composition useful for the treatment of a cardiovascular, endothehal or angiogemc disorder comprising admixing a therapeutically effective amount of an anti PRO antibody with a pharmaceutically acceptable carrier
In a still further aspect, the present invention provides an article of manufacture comprising (a) a composition of matter comprising a PRO polypeptide or agonist or antagonist thereof,
(b) a container containing said composition, and
(c) a label affixed to said container, or a package insert included in said container referring to the use of said PRO polypeptide or agonist or antagonist thereof in the treatment of a cardiovascular, endothehal or angiogemc disorder, wherein the agonist or antagonist may be an antibody which binds to the PRO polypeptide The composition may comprise a therapeutically effective amount of the PRO polypeptide or the agonist or antagonist thereof
In another embodiment, the present invention provides a method for identifying an agonist of a PRO polypeptide comprising
(a) contacting cells and a test compound to be screened under conditions suitable for the induction of a cellular response normally induced by a PRO polypeptide, and
(b) determining the induction of said cellular response to determine if the test compound is an effective agonist, wherein the induction of said cellular response is indicative of said test compound being an effective agonist
In another embodiment, the present invention provides a method for identifying an agonist of a PRO polypeptide comprising
(a) contacting cells and a test compound to be screened under conditions suitable for the stimulation of cell proliferation by a PRO polypeptide, and
(b) measuring the proliferation of said cells to determine it the test compound is an effective agonist wherein the stimulation of cell proliferation is indicative of said test compound being an effective agonist In another embodiment, the invention provides a method for identifying a compound that inhibits the activity of a PRO polypeptide comprising contacting a test compound with a PRO polypeptide under conditions and for a time sufficient to allow the test compound and polypeptide to interact and determining whether the activity of the PRO polypeptide is inhibited In a specific preferred aspect, either the test compound or the PRO polypeptide is immobilized on a solid support In another preferred aspect, the non-immobilized component carries a detectable label In a preferred aspect, this method comprises the steps of
(a) contacting cells and a test compound to be screened in the presence of a PRO pol> peptide undei conditions suitable for the induction of a cellular response normally induced by a PRO polypeptide and
(b) determining the induction of said cellular response to determine if the test compound is an effective antagonist In another pief erred aspect, this process comprises the steps of
(a) contacting cells and a test compound to be screened in the presence of a PRO polvpeptide undei conditions suitable for the stimulation ot cell proliferation by a PRO polypeptide, and
(b) measuring the proliferation of the cells to determine it the test compound is an effective antagonist In another embodiment, the invention provides a method for identifying a compound that inhibits the expression of a PRO polypeptide in cells that normally expresses the polypeptide, wherein the method comprises contacting the cells with a test compound and determining whether the expression of the PRO polypeptide is inhibited In a preferred aspect, this method comprises the steps of (a) contacting cells and a test compound to be screened under conditions suitable for allowing expression of the PRO polypeptide, and
(b) determining the inhibition of expression of said polypeptide
In a still further embodiment, the invention provides a compound that inhibits the expression of a PRO polypeptide, such as a compound that is identified by the methods set forth above Another aspect of the present invention is directed to an agonist or an antagonist of a PRO polypeptide which may optionally be identified by the methods described above
One type of antagonist of a PRO polypeptide that inhibits one or more of the functions or activities of the PRO polypeptide is an antibody Hence, in another aspect, the invention provides an isolated antibody that binds a PRO polypeptide In a preferred aspect, the antibody is a monoclonal antibody, which preferably has non-human complementaπty-determining-region (CDR) residues and human framework-region (FR) residues The antibody may be labeled and may be immobilized on a solid support In a further aspect, the antibody is an antibody fragment, a single-chain antibody, or a humanized antibody Preferably, the antibody specifically binds to the polypeptide
In a still further aspect, the present invention provides a method for diagnosing a disease or susceptibility to a disease which is related to a mutation in a PRO polypeptide-encodmg nucleic acid sequence comprising determining the presence or absence of said mutation in the PRO polypeptide nucleic acid sequence, wherein the presence or absence of said mutation is indicative of the presence of said disease or susceptibility to said disease
In a still further aspect, the invention provides a method of diagnosing a cardiovascular, endothehal or angiogemc disorder in a mammal which comprises analyzing the level ot expression of a gene encoding a PRO polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower expression level in the test sample as compared to the control sample is indicative of the presence of a cardiovascular, endothehal or angiogemc disorder in said mammal The expression of a gene encoding a PRO polypeptide may optionally be accomplished by measuring the level of mRNA or the polypeptide in the test sample as compared to the control sample In a still further aspect, the present invention provides a method of diagnosing a cardiovascular, endothehal or angiogemc disorder in a mammal which comprises detecting the presence or absence of a PRO polypeptide in a test sample of tissue cells obtained from said mammal, wherein the presence or absence of said PRO polypeptide in said test sample is indicative of the presence ot a caidiovascular, endothehal or angiogemc disorder in said mammal In a still further embodiment, the invention provides a method of diagnosing a cardiovasculai , endothehal or angiogemc disorder in a mammal comprising (a) contacting an anti PRO antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the antibody and the PRO polypeptide in the test sample, wherein the formation of said complex is indicative of the presence of a cardiovasculai , endothehal or angiogemc disorder in the mammal The detection may be qualitative 01 quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells of the same cell type A larger or smaller quantity of complexes formed in the test sample indicates the presence of a cardiovascular, endothehal or angiogemc dysfunction in the mammal from which the test tissue cells were obtained The antibody preferably carries a detectable label Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluoπmetry, or other techniques known in the art The test sample is usually obtained from an individual suspected to have a cardiovascular, endothehal or angioge c disorder
In another embodiment, the invention provides a method for determining the presence of a PRO polypeptide in a sample comprising exposing a sample suspected of containing the PRO polypeptide to an anti-PRO antibody and determining binding of said antibody to a component of said sample In a specific aspect, the sample comprises a cell suspected of containing the PRO polypeptide and the antibody binds to the cell The antibody is preferably detectably labeled and/or bound to a solid support
In further aspects, the invention provides a cardiovascular, endothehal or angiogemc disorder diagnostic kit comprising an anti-PRO antibody and a earner in suitable packaging Preferably, such kit further comprises instructions for using said antibody to detect the presence of the PRO polypeptide Preferably, the carrier is a buffer, for example Preferably, the cardiovascular, endothehal or angiogemc disorder is cancer
In yet another embodiment, the present invention provides a method for treating a cardiovascular, endothehal or angiogemc disorder in a mammal comprising administering to the mammal an effective amount of a PRO polypeptide Preferably, the disorder is cardiac hypertrophy, trauma such as wounds or burns, or a type of cancer In a further aspect, the mammal is further exposed to angioplasty or a drug that treats cardiovascular, endothehal or angiogemc disorders such as ACE inhibitors or chemotherapeutic agents if the cardiovascular, endothehal or angiogemc disorder is a type of cancer Preferably, the mammal is human, preferably one who is at risk of developing cardiac hypertrophy and more preferably has suffered myocardial infarction
In another preferred aspect, the cardiac hypertrophy is characterized by the presence of an elevated level of PGF2α Alternatively, the cardiac hypertrophy may be induced by myocardial infarction, wherein preferably the administration of the PRO polypeptide is initiated within 48 hours, more preferably within 24 hours, following myocardial infarction
In another preferred embodiment, the cardiovascular, endothehal or angiogemc disorder is cardiac hypertrophy and said PRO polypeptide is administered together with a cardiovascular, endothehal or angiogemc agent The preferred cardiovascular, endothehal or angiogemc agent for this purpose is selected from the group consisting of an antihypertensive drug, an ACE inhibitor, an endothelin receptor antagonist and a thrombolytic agent If a thrombolytic agent is administered, preferably the PRO polypeptide is administered following administration of such agent More preferably, the thrombolytic agent is recombinant human tissue plasminogen activator
In another prefeired aspect the cardiovascular, endothehal or angiogemc disorder is cardiac hypertrophy and the PRO polypeptide is administered following primary angioplasty for the treatment of acute myocardial infarction, preferably wherein the mammal is further exposed to angioplastv or a cardiovascular, endothehal, or angiogemc agent
In another preferred embodiment, the cardiovascular endothehal or angiogemc disordei is a cancer and the PRO polypeptide is administered in combination with a chemotherapeutic agent, a growth inhibitory agent or a cytotoxic agent
In a further embodiment, the invention concerns a method for treating a cardiovascular, endothehal or angiogemc disorder in a mammal comprising administering to the mammal an effective amount of an agonist of a PRO polypeptide Preferably, the cardiovascular, endothehal or angiogemc disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration Also preferred is where the mammal is human, and where an effective amount of an angiogemc or angiostatic agent is administered in conjunction with the agonist
In a further embodiment, the invention concerns a method for treating a cardiovascular, endothehal or angiogemc disorder m a mammal comprising administering to the mammal an effective amount of an antagonist of a PRO polypeptide Preferably, the cardiovascular, endothehal or angiogemc disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration Also preferred is where the mammal is human, and where an effective amount of an angiogemc or angiostatic agent is administered in conjunction with the antagonist
In a further embodiment, the invention concerns a method for treating a cardiovascular, endothehal or angiogemc disorder in a mammal comprising administering to the mammal an effective amount of an anti-PRO antibody Preferably, the cardiovascular, endothehal or angiogemc disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration Also prefened is where the mammal is human, and where an effective amount of an angiogemc or angiostatic agent is administered in conjunction with the antibody
In still further embodiments, the invention provides a method for treating a cardiovascular, endothehal or angiogemc disorder in a mammal that suffers therefrom comprising administering to the mammal a nucleic acid molecule that codes for either (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide or (c) an antagonist of a PRO polypeptide, wherein said agonist or antagonist may be an anti-PRO antibody In a preferred embodiment, the mammal is human In another preferred embodiment, the gene is administered via ex vivo gene therapy In a further preferred embodiment, the gene is comprised within a vector, more preferably an adenoviral, adeno-associated viral, lentiviral, or retroviral vector In yet another aspect, the invention provides a recombinant retroviral particle comprising a retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO polypeptide, (b) an agonist polypeptide of a PRO polypeptide, or (c) an antagonist polypeptide of a PRO polypeptide, and a signal sequence for cellular secretion of the polypeptide, wherein the retroviral vector is in association with retroviral structural proteins Preferably, the signal sequence is from a mammal, such as from a native PRO polypeptide In a still further embodiment, the invention supplies an e\ vivo producer cell comprising a nucleic acid construct that expresses retroviral structural proteins and also comprises a retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO polypeptide, (b) an agonist polypeptide of a PRO polypeptide or (c) an antagonist polypeptide of a PRO polypeptide, and a signal sequence for cellular secretion of the polypeptide, wherein said producei cell packages the retroviral vector in association with the structural proteins to pioduce recombinant retrovnal particles
In yet another embodiment, the invention provides a method for inhibiting endothehal cell giowth in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide or (c) an antagonist of a PRO polypeptide, wherein endothehal cell growth in said mammal is inhibited, and wherein said agonist or antagonist may be an anti-PRO antibody Preferably, the mammal is human and the endothehal cell growth is associated with a tumor or a retinal disorder
In yet another embodiment, the invention provides a method for stimulating endothehal cell growth in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein endothehal cell growth in said mammal is stimulated, and wherein said agonist or antagonist may be an anti-PRO antibody Preferably, the mammal is human
In yet another embodiment, the invention provides a method for inhibiting cardiac hypertrophy in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein cardiac hypertrophy in said mammal is inhibited, and wherein said agonist or antagonist may be an anti-PRO antibody Preferably, the mammal is human and the cardiac hypertrophy has been induced by myocardial infarction
In yet another embodiment, the invention provides a method for stimulating cardiac hypertrophy in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein cardiac hypertrophy in said mammal is stimulated, and wherein said agonist or antagonist may be an anti-PRO antibody Preferably, the mammal is human who suffers from congestive heart failure
In yet another embodiment, the invention provides a method for inhibiting angiogenesis induced by a PRO polypeptide in a mammal comprising administering a therapeutically effective amount of an anti PRO antibody to the mammal Preferably, the mammal is a human, and more preferably the mammal has a tumor or a retinal disorder
In yet another embodiment, the invention provides a method for stimulating angiogenesis induced by a PRO polypeptide in a mammal comprising administering a therapeutically effective amount of a PRO polypeptide to the mammal Preferably, the mammal is a human, and more preferably angiogeneisis would promote tissue regeneration or wound healing
Additional Embodiments
In other embodiments of the present invention, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide
In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, v. et more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet moi e 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 moie preferably at least about 989. sequence identity and yet more preferably at least about 99% sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein with or without the signal peptide, as disclosed herein or any other specifically defined fragment ot the full length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a)
In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, 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 PRO polypeptide cDNA as disclosed herein, the coding sequence of a PRO polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PRO polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a)
In a furthei aspect, the invention concerns an isolated nucleic acid molecule compi ising 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 ot the human protein cDNAs deposited with the ATCC as disclosed herein, oi (b) the complement ot the DNA molecule of (a) Another aspect ot the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is either transmembrane domain deleted or ti ansmembi ane domain-inactivated, or is complementary to such encoding nucleotide sequence wherein the transmembrane domaιn(s) of such polypeptide are disclosed herein Therefore soluble extracellular domains ot the herein described PRO polypeptides are contemplated
Another embodiment is directed to fragments of a PRO polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PRO polypeptide that may optionally encode a polypeptide comprising a binding site for an anti PRO antibody or as antisense ohgonucleotide probes Such nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 60 nucleotides in length, yet more preferably at least about 70 nucleotides in length, yet more preferably at least about 80 nucleotides in length, yet more preferably at least about 90 nucleotides in length, yet more preferably at least about 100 nucleotides in length, yet more preferably at least about 1 10 nucleotides in length, yet more preferably at least about 120 nucleotides in length, yet more preferably at least about 130 nucleotides in length, yet more preferably at least about 140 nucleotides in length, yet more preferably at least about 150 nucleotides in length, yet more preferably at least about 160 nucleotides in length, yet more preferably at least about 170 nucleotides in length, yet more preferably at least about 180 nucleotides in length, yet more preferably at least about 190 nucleotides 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 PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PRO polypeptide-encoding nucleotide sequence fragment(s) are novel All of such PRO polypeptide-encoding nucleotide sequences are contemplated herein Also contemplated are the PRO polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO polypeptide fragments that comprise a binding site for an anti-PRO antibody
In another embodiment, the invention provides isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified
In a certain aspect, the invention concerns an isolated PRO 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 pieferably at least about 89% sequence identity, yet more preferablv 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 moie 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 moie preferably at least about 969b 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 PRO polypeptide having a full-length amino acid sequence as disclosed heiem, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein
In a furthei aspect, the invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein In a further aspect, the invention concerns an isolated PRO polypeptide comprising an ammo 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 PRO polypeptide having a full-length ammo acid sequence as disclosed herein, an am o acid sequence lacking the signal peptide as disclosed heiein, 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 PRO polypeptide without the N-terminal signal sequence and/or the initiating methio ne and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore descnbed Processes for producing the same are also herein described, wherein those processes comprise cultuπng a host cell comprising a v ector which comprises the appropi late encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture
Another aspect of the invention provides an isolated PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated Processes for producing the same are also herein described, wherein those processes comprise culturmg a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture
In yet another embodiment, the invention concerns agonists and antagonists of a native PRO polypeptide as defined herein In a particular embodiment, the agonist or antagonist is an anti-PRO antibody or a small molecule
In a further embodiment, the invention concerns a method of identifying agonists or antagonists to a PRO polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide Preferably, the PRO polypeptide is a native PRO polypeptide
In a still further embodiment, the invention concerns a composition of matter comprising a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier Optionally, the earner is a pharmaceutically acceptable carrier Another embodiment of the present invention is directed to the use of a PRO polypeptide, or an agonist or antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO polypeptide, an agonist or antagonist thereof or an anti-PRO antibody
In additional embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides Host cell comprising any such vector are also provided By way of example, the host cells may be CHO cells, E coli, yeast, or Baculovirus-infected insect cells A process for producing any of the herein described polypeptides is further provided and comprises culturmg host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture In other embodiments, the invention provides chimeπc molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence Example of such chimeπc molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobuhn
In yet another embodiment, the invention provides an antibody which specifically binds to any of the above or below described polypeptides Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody
In yet other embodiments, the invention provides ohgonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences
Brief Description of the Drawings Figure 1 shows a nucleotide sequence (SEQ ID NO 1 ) of a native sequence PRO320 cDNA, wherein SEQ ID NO 1 is a clone designated herein as ' DNA32284-1307' Figure 2 shows the amino acid sequence (SEQ ID NO 2) derived from the coding sequence of SEQ ID NO 1 shown in Figure 1
Figure 3 shows a nucleotide sequence (SEQ ID NO 6) of a native sequence PR0938 cDNA, wherein SEQ ID NO 6 is a clone designated herein as "DNA56433-1406' Figure 4 shows the amino acid sequence (SEQ ID NO 7) derived from the coding sequence of SEQ ID NO 6 shown in Figure 3
Figure 5 shows a nucleotide sequence (SEQ ID NO 1 1 ) of a native sequence PRO 1031 cDNA, wherein SEQ ID NO 1 1 is a clone designated herein as "DNA59294-1381 "
Figure 6 shows the amino acid sequence (SEQ ID NO 12) derived from the coding sequence of SEQ ID NO 1 1 shown in Figure 5
Figure 7 shows a nucleotide sequence (SEQ ID NO 13) of a native sequence PR0296 cDNA, wherein SEQ ID NO 13 is a clone designated herein as "DNA39979-1213"
Figure 8 shows the amino acid sequence (SEQ ID NO 14) derived from the coding sequence of SEQ ID NO 13 shown in Figure 7 Figure 9 shows a nucleotide sequence (SEQ ID NO 21 ) of a native sequence PR0213 cDNA, wherein SEQ
ID NO 21 is a clone designated herein as "DNA30943-1163"
Figure 10 shows the amino acid sequence (SEQ ID NO 22) derived from the coding sequence of SEQ ID NO 21 shown in Figure 9
Figure 1 1 shows a nucleotide sequence (SEQ ID NO 23) of a native sequence PRO1330 cDNA, wherein SEQ ID NO 1 1 is a clone designated herein as "DNA64907- 1 163-1 "
Figure 12 shows the amino acid sequence (SEQ ID NO 24) derived from the coding sequence of SEQ ID NO 23 shown in Figure 1 1
Figure 13 shows a nucleotide sequence (SEQ ID NO 25) ot a native sequence PROl 449 cDNA, wherein SEQ ID NO 13 is a clone designated herein as "DNA64908-1 163 1 " Figure 14 shows the ammo acid sequence (SEQ ID NO 26) derived from the coding sequence of SEQ ID
NO 25 shown in Figure 13
Detailed Description of the Invention I Definitions The phrases "cardiovascular, endothehal and angiogemc disorder", cardiovasculai, endothehal and angiogemc dysfunction", 'cardiovascular, endothehal or angiogemc disorder and 'cardiovascular, endothehal or angiogemc disfunction" are used inteichangeably and refer in part to systemic disorders that affect vessels, such as diabetes melhtus, as well as diseases of the vessels themselves, such as of the arteries, capillaries, veins, and/or lymphatics This would include indications that stimulate angiogenesis and/or cardiovasculaπzation, and those that inhibit angiogenesis and/or cardiovasculaπzation Such disordei s include for example, arterial disease, such as atherosclerosis, hypertension, inflammatory vascuhtides, Reynaud's disease and Reynaud's phenomenon, aneurysms and arterial restenosis, venous and lymphatic disorders such as thrombophlebitis, lymphangitis and lymphedema and other vascular disoi ders such as periphei al v ascular disease, cancer such as vascular tumors, e g , hemangioma (capillary and cavernous), glomus tumois, telangiectasia, bacillary angiomatosis, hemangioendothehoma, angiosarcoma, haemangiopencytoma, Kaposi's sarcoma, lymphangioma, and lymphangiosarcoma, tumor angiogenesis trauma such as wounds, burns, and other injured tissue, implant fixation scamng, ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular disease renal diseases such as acute renal failuie, and osteoporosis This would also include angina, myocardial infarctions such as acute myocardial infarctions, cardiac hypertrophy, and heart failure such as CHF
"Hypertrophy", as used herein, is defined as an increase in mass of an organ or structure independent of natural growth that does not involve tumor formation Hypertrophy of an organ or tissue is due either to an increase in the mass of the individual cells (true hypertrophy), or to an increase in the number of cells making up the tissue (hyperplasia), or both Certain organs, such as the heart, lose the ability to divide shortly after birth Accordingly,
"cardiac hypertrophy" is defined as an increase in mass of the heart, which, in adults, is characterized by an increase in myocyte cell size and contractile protein content without concomitant cell division The character of the stress responsible for inciting the hypertrophy, (e g , increased preload, increased afterload, loss of myocytes, as in myocardial infarction, or primary depression of contractility), appears to play a critical role in determining the nature of the response The early stage of cardiac hypertrophy is usually characterized morphologically by increases in the size of myofibrils and mitochondria, as well as by enlargement of mitochondria and nuclei At this stage, while muscle cells are larger than normal, cellular organization is largely preserved At a more advanced stage of cardiac hypertrophy, there are preferential increases in the size or number of specific organelles, such as mitochondria, and new contiactile elements are added in localized areas of the cells, in an irregular manner Cells subjected to long- standing hypertrophy show more obvious disruptions in cellular organization, including markedly enlarged nuclei with highly lobulated membranes, which displace adjacent myofibrils and cause breakdown of normal Z-band registration The phrase "cardiac hypertrophy" is used to include all stages of the progression of this condition, characterized by various degrees of structural damage of the heart muscle, regardless of the underlying cardiac disorder Hence, the term also includes physiological conditions instrumental in the development of cardiac hypertrophy, such as elevated blood pressure, aortic stenosis, or myocardial infarction
"Heart failure" refers to an abnormality of cardiac function where the heart does not pump blood at the rate needed for the requirements of metabolizing tissues The heart failure can be caused by a number of factors, including ischemic, congenital, rheumatic, or ldiopathic forms
"Congestive heart failuie" (CHF) is a progressive pathologic state where the heart is increasingly unable to supply adequate cardiac output (the volume of blood pumped by the heart over time) to deliver the oxygenated blood to peripheral tissues As CHF progresses, structural and hemodynamic damages occur While these damages have a variety of manifestations, one characteristic symptom is ventricular hypertrophy CHF is a common end result of a number of various cardiac disorders
"Myocardial infarction geneially results from atheiosclerosis of the coronary arteries often with superimposed coronary thrombosis It may be divided into tw o ma]or types transmui al infarcts, in which myocardial neci osis involves the full thickness of the v entπcular w all and subendocardial (nontransmural) infarcts, in which the necrosis involves the subendocardium the intramural myocardium, or both, without extending all the way through the ventricular wall to the epicardium Mvocardial infarction is known to cause both a change in hemodynamic effects and an alteration in structure in the damaged and healthy zones of the heart Thus, for example, myocardial infarction reduces the maximum cardiac output and the stroke volume of the heart Also associated with myocardial infarction is a stimulation of the DNA synthesis occurring in the interstice as well as an increase in the formation of collagen in the areas of the heart not affected As a result of the increased stress or strain placed on the heart in prolonged hypertension due, for example, to the increased total peripheral resistance, cardiac hypertrophy has long been associated with "hypertension" A characteristic of the ventricle that becomes hypertrophic as a result of chronic pressure overload is an impaired diastohc performance Fo ad etal , J Am Coll Cardiol .4 1500- 1506 (1984) , S mith et al . j Am Coll Cardiol , 5 869-874 ( 1985) A prolonged left ventricular relaxation has been detected in early essential hypertension, in spite of normal or supranormal systolic function Hartford et al , Hypertension, 6 329-338 (1984) However, there is no close parallelism between blood pressure levels and cardiac hypertrophy Although improvement in left ventricular function in response to antihypertensive therapy has been reported in humans, patients variously treated with a diuretic (hydrochlorothiazide), a β-blocker (propranolol), or a calcium channel blocker (diltiazem), have shown reversal of left ventricular hypertrophy, without improvement in diastohc function Inouye et al , Am J Cardiol . 53 1583-7 (1984)
Another complex cardiac disease associated with cardiac hypertrophy is "hypertrophic cardiomyopathy" This condition is characterized by a great diversity of morphologic, functional, and clinical features (Maron et al , N Engl J Med . 316 780-789 (1987), Spinto etal , N Engl J Med , 320 749-755 ( 1989), Louie and Edwards, Prog Cardiovasc Pis . 36 275-308 ( 1994), Wigle et al , Circulation, 92 1680- 1692 ( 1995)), the heterogeneity of which is accentuated by the fact that it afflicts patients of all ages Spinto et al , N Engl J Med , 336 775-785 (1997)
The causative factors of hypertrophic cardiomyopathy are also diverse and little understood In general, mutations in genes encoding sarcomeπc proteins are associated with hypertrophic cardiomyopathy Recent data suggest that β-myosin heavy chain mutations may account for approximately 30 to 40 percent of cases of familial hypertrophic cardiomyopathy Watkins efα/ . N Engl j Med , 326 1 108-1 1 14 (1992), Schwartz etal. Circulation, 91 532-540 (1995), Marian and Roberts, Circulation. 92 1336-1347 (1995), Thierfelder et al , Cejl, 77 701 -712 (1994),
Watkms e. fl/ Nat Gen . 1 1 434-437 (1995) Besides β-myosin heavy chain, other locations of genetic mutations include cardiac tropo n T, alpha topomyosin, cardiac myosin binding protein C, essential myosin light chain, and regulatory myosin light chain See, Malik and Watkms, Cun Opin Cardiol , 12 295-302 ( 1997)
Supravalvular "aortic stenosis" is an inherited vascular disorder characterized by narrowing of the ascending aorta, but other arteries, including the pulmonary arteries, may also be affected Untreated aortic stenosis may lead to increased intracardiac pressure resulting in myocardial hypertrophy and eventually heart failure and death The pathogenesis of this disorder is not fully understood, but hypertrophy and possibly hyperplasia of medial smooth muscle are prominent features of this disorder It has been reported that molecular variants of the elastin gene are involved in the development and pathogenesis of aortic stenosis U S Patent No 5,650,282 issued July 22, 1997 "Valvular regurgitation" occurs as a result of heart diseases resulting in disorders of the cardiac valves
Various diseases, like rheumatic fever, can cause the shrinking or pulling apart ot the valve orifice while other diseases may result in endocarditis, an inflammation of the endocardium or lining membrane of the atπoventπculai orifices and operation of the heart Defects such as the narrowing of the valve stenosis or the defectiv e closing of the valve result in an accumulation of blood in the heart cavity or regurgitation of blood past the valve If uncorrected, prolonged valvular stenosis or insufficiency may result in cardiac hypertrophy and associated damage to the heart muscle, which may eventually necessitate valve replacement
The treatment ot all these, and other cardiovascular, endothehal and angiogemc disorders, which may or may not be accompanied by cardiac hypertrophy, is encompassed by the present invention
The terms "cancer", "cancerous' , and "malignant" 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 including adenocarcmoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, ghoblastoma, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometnal carcinoma, salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, and various types of head and neck cancer The preferred cancers for treatment herein are breast, colon, lung, melanoma, ovarian, and others involving vascular tumors as noted above
The term ' cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells The term is intended to include radioactive isotopes (e g , I, l2,1, 9"Y, and 186Re), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer Examples of chemotherapeutic agents include alkylatmg agents, fohc acid antagonists, anti metabolites of nucleic acid metabolism, antibiotics, pyπmidine analogs, 5-fluorouracιl, cisplatin, punne nucleosides, amines, amino acids, tnazol nucleosides, or corticosteroids Specific examples include Adnamycm, Doxorubicin, 5-FluorouracιI, Cytosine arabinoside ("Ara C"), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin, Melphalan, Vinblastme, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vmcreistme,
Vmorelbme Carboplatin, Temposide, Daunomycin, Carminomycin, Aminopteπn, Dactinomycin, Mitomycins, Esperamicins (see U S Pat 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 onapnstone A ' growth-inhibitory agent" when used herein refers to a compound or composition that inhibits growth of a cell, such as an Wnt-overexpressing cancer cell, either in vitro oi in vix o Thus, the growth-inhibitory agent is one which significantly reduces the percentage ot malignant 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 (vincnstine and v inblastme), taxol and topo II inhibitois such as doxorubicin, daunorubicin, etoposide, and bleomycin Those agents that arrest Gl also spill ov er into S phase arrest, for example DNA alkylatmg agents such as tamoxifen prednisone, dacaibazine, mechlorethamme, cisplatin, methotrexate 5 fluorouracil, and ara-C Further information can be found in The Moleculai Basis of Cancer. Mendelsohn and Israel eds , Chapter 1 , entitled Cell cycle regulation oncogenes, and antineoplastic drugs" by Murakami etal (WB Saunders Philadelphia, 1995), especiallyp 13 Additional examples include tumor necrosis factor (TNF), an antibody capable of inhibiting or neutralizing the angiogemc activity of acidic or basic FGF or hepatocyte growth factoi (HGF), an antibody capable of inhibiting or neutralizing the coagulant activities of tissue factor, protein C, or protein S (see, WO 91/01753, published 21 February 1991 ), or an antibody capable of binding to HER2 receptor (WO 89/06692), such as the 4D5 antibody (and functional equivalents thereof) (e , WO 92/22653)
"Treatment" is an intervention performed with the intention of preventing the development or altering the pathology of a cardiovascular, endothehal, and angiogemc disorder The concept of treatment is used in the broadest sense, and specifically includes the prevention (prophylaxis), moderation, reduction, and curing of cardiovascular, endothehal, and angiogemc disorders of any stage Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a cardiovascular, endothehal, and angiogemc disorder such as hypertrophy Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be pi evented The disorder may result from any cause, including ldiopathic, cardiotrophic, or myotrophic causes, or ischemia or lschemic insults, such as myocardial infarction
"Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial effect, such as an anti-hypertrophic effect, for an extended period of time
"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, pigs, etc Preferably, the mammal is human
Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order
The phrase "cardiovascular, endothehal or angiogemc agents" refers genencally to any drug that acts in treating cardiovascular, endothehal, and angiogemc disordeis Examples of cardiovascular agents are those that promote vascular homeostasis by modulating blood pressure, heart rate, heart contractility, and endothehal and smooth muscle biology, all of which factors have a role in cardiovascular disease Specific examples of these include angιotensιn-II receptor antagonists, endothelin receptor antagonists such as, for example, BOSENTAN™ and MOXONODIN™, interferon-gamma (IFN-γ), des-aspartate-angiotensin I, thrombolytic agents, e g , streptokmase, urokinase, t-PA, and a t-PA variant specifically designed to have longer half-life and very high fibrin specificity, TNK t-PA (a Tl 03N, N 1 17Q, KHRR(296-299) AAAA t-PA variant, Keyt et al , Proc Natl Acad Sci
USA 91 , 3670-3674 (1994)). inotropic or hypertensive agents such as digoxigenin and β-adrenergic receptor blocking agents, e g , propranolol. timolol, tertalolol, carteolol nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, and carvedilol. angiotensin converting enzyme (ACE) inhibitors, e g , quinapnl, captopnl, enalapπl ramipπl, benazepπl, fosinopnl, and hsinopπl, diuretics, e g , chlorothiazide, hydiochlorothiazide hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, and indapamide, and calcium channel blockers, e g , diltiazem, nifedipine, verapamil, mcardipine One prefeπed category of this type is a therapeutic agent used for the treatment ot cardiac hypertrophv or of a physiological condition instrumental in the development of cardiac hypei trophy, such as elevated blood pressure, aortic stenosis, or myocardial infarction "Angiogemc agents" and "endothehal agents" are active agents that promote angiogenesis and/or endothehal cell growth, or, if applicable, vasculogenesis This would include factors that accelerate wound healing, such as growth hormone, msulm-hke growth factor-I (IGF-I), VEGF, VIGF, PDGF, epidermal growth factor (EGF), CTGF and members of its family, FGF, and TGF-α and TGF-β "Angiostatic agents" are active agents that inhibit angiogenesis or vasculogenesis or otherwise inhibit 01 prevent growth of cancer cells Examples include antibodies or other antagonists to angiogemc agents as defined above, such as antibodies to VEGF They additionally include cytotherapeutic agents such as cytotoxic agents, chemotherapeutic agents, growth-inhibitory agents, apoptotic agents, and other agents to treat cancer, such as anti- HER-2, antι-CD20, and other bioactive and organic chemical agents In a pharmacological sense, in the context of the present invention, a "therapeutically effective amount" of an active agent such as a PRO polypeptide or agonist or antagonist thereto or an anti-PRO antibody, refers to an amount effective in the treatment of a cardiovascular, endothehal or angioge c disorder in a mammal and can be determined empirically
As used herein, an "effective amount" of an active agent such as a PRO polypeptide or agonist or antagonist thereto or an anti-PRO antibody, refers to an amount effective for carrying out a stated purpose, wherein such amounts may be determined empirically tor the desired effect
The terms "PRO polypeptide" and "PRO" as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (( e , PRO/number) refers to specific polypeptide sequences as described herein The terms "PRO/number polypeptide" and "PRO/number" wherein the term "number" is piovided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein) The PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods
A "native sequence PRO polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means The term "native sequence PRO polypeptide' specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e g , an extracellular domain sequence), naturally-occurring variant forms (e g , alternatively spliced forms) and naturally-occurring allehc variants of the polypeptide In various embodiments of the invention, the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures Start and stop codons are shown in bold font and underlined in the figures However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methion e residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionme residues located either upstream or downstream from the amino acid position ) in the figures may be employed as the starting amino acid residue for the PRO polypeptides
The PRO polypeptide ' extracellular domain ' or "ECD" refers to a form of the PRO polypeptide which is essentially free of the tiansmembrane and cytoplasmic domains Ordinarily a PRO polypeptide ECD will have less than 1 % of such transmembiane and/or cytoplasmic domains and pieferably will have less than 0 5% of such domains It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end ot the domain as initially identified herein Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are comtemplated by the present invention The approximate location of the "signal peptides" of the various PRO polypeptides disclosed herein are shown in the present specification and/or the accompanying figures It is noted, however, that the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e g Nielsen et al , Prot Eng , 10 1-6 (1997) and von Heinje et al , Nucl Acids Res , J_4 4683-4690 (1986)) Moreovei , 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 withm no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention
"PRO polypeptide variant" means an active PRO polypeptide as defined above or below having at least about 80% amino acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide with or without the signal peptide, as disclosed herein oi any other fragment of a full-length PRO polypeptide sequence as disclosed herein Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or moie amino acid residues are added, or deleted, at the N or C-terminus of the full-length native amino acid sequence Ordinarily, a PRO polypeptide variant will have at least about 80% ammo acid sequence identity, preferably at least about 81 % amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91 % amino acid sequence identity, more preferably at least about 92% amino acid sequence identity more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, moi e preferably at least about 95% amino acid sequence identity more pref ei ably at least about 969. amino acid sequence identity more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and most preferably at least about 99% ammo acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herein. Ordinarily, PRO variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about 50 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 amino acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, more often at least about 300 amino acids in length, or more.
As shown below, Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program. This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program.
In addition, Tables 2A-2D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid sequence identity (Tables 2C-2D) using the ALIGN-2 sequence comparison computer program, wherein "PRO" represents the amino acid sequence of a hypothetical PRO polypeptide of interest, "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "PRO" polypeptide of interest is being compared, "PRO-DNA" represents a hypothetical PRO-encoding nucleic acid sequence of interest, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA" nucleic acid molecule of interest is being compared, "X", "Y", and "Z" each represent different hypothetical amino acid residues and "N", "L" and "V" each represent different hypothetical nucleotides.
Table 1
* C-C increased from 12 to 15 *Zιs average of EQ
* B is average of ND
* match with stop is _M; stop-stop = 0; J (joker) match = 0 */
#define _M -8 /* value of a match with a stop */ int _day[26][26] = {
/* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/
/* A*/ 2, 0,-2, 0, 0,-4, 1,-1,-1, 0,-1,-2,-1, 0,_M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3, 0},
/*B */ 0,3 -4 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2,_M,-1, 1, 0, 0, 0, 0,-2,-5, 0,-3, 1},
/*C*/ -2,-4 15, 5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0, 0,-5},
/*D */ 0,3 -5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 2},
/*E*/ 0,2 -5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 3},
/*¥*/ -4,-5 -4, -6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5}, l*G*l 1,0 -3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,_M,-l,-l,-3, 1, 0, 0,-1,-7, 0,-5, 0},
/*H*/ -1, 1 -3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2,_M, 0, 3, 2,-1,-1, 0,-2,-3, 0, 0, 2},
1*1*1 -1,-2 -2, -2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2,_M,-2,-2,-2,-l, 0, 0, 4,-5, 0,-1,-2},
I*} *! 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}, l*K*i -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 -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,-1, 0,-2,-1, 0, 2,-4, 0,-2,-1},
/*N*/ 0,2 -4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2,_M,-1, 1, 0, 1, 0, 0,-2,-4, 0,-2, 1},
1*0*1 _M,_ M, M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M, 0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M},
/* p */ 1,-1 -3, -1, -1, ,-5, ,-L .0, ,-2. , 0, ,-ι. ,-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, ,- ,-4, .-3, ,2.. ,-2. , o, ,3, ,-3. , 0, . o, M, 0, 1, 6,0,-1, 0,-2,2,0,-4, 0},
/*S*/ 1,0. 0, 0, 0, -3, 1, -1, -1, 0, 0, -3, -2, 1, M, 1,- -1,0,2, 1,0,-1,-2,0,-3,0}, /* Ύ */ 1,0 -2, o, 0, -3, 0, -1, 0, 0, 0, -1, -1, 0," M, 0, -1,-1, 1,3,0, 0,-5,0,-3,0},
/*u*/ 0,0 0, 0, 0, 0, 0, 0, 0, o, 0, 0, 0, o, M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/* v */ 0,-2 -2, -2, -2, .-1, , ,-2. : , 0, .-2, ,2, ,2, -2, M, .-1, ,-2,-2,-1, 0, 0, 4,-6, 0,-2,-2},
/* w */ -6,-5 -1, ,-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. o, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, M, 0, 0,0,0,0,0,0,0,0,0,0},
/* Y */ -3,-3 -4, -4, 7, -5, 0, -1, 0, -4, ,-ι, .-2, ,-ϊ, M, ,-5, ,-4,-4,-3,-3, 0,-2,0,0,10,-4},
/*z*. 0, 1. 2, 3, -5, 0, 2, -2, 0, 0, -2, -1, 1, M, 0, 3, 0, 0, 0, 0,-2,-6, 0,-4, 4} };
Page 1 of day. h /* */
-/include < stdio h > -. include < ctyρe.h >
^define MAXJMP 16 / * max jumps in a diag */
#define MAXGAP 24 I* don't continue to penalize gaps larger than this */
^define JMPS 1024 /* max jmps in an path */
-. define MX 4 /* save if there's at least MX-1 bases since last jmp */
-. define DMAT 3 /* value of matching bases */
#define DMIS 0 /* penalty for mismatched bases */
#define DINSO 8 /* penalty for a gap */
-. define DINS1 1 /* penalty per base */
#define PINSO 8 /* penalty for a gap */
#define PINS1 4 /* penalty per residue */ struct jmp { short nfMAXJMP], /* size of jmp (neg for dely) */ unsigned short xfMAXJMP]; /* base no of jmp in seq x */
}; /* limits seq to 2^16 -1 */ struct diag { int score; /* score at last jmp "7 long offset /* offset of prev block */ short ljπip; /* current jmp index */ struct jmp JP; /* list of jmps */
>; struct path { int spc; /* number of leading spaces */ short n[JMPS]; /* size of mp (gap) */ int x[JMPS]; /* loc of jmp (last elem before gap) */
}; char *ofιle; /* output file name */ char *namex[2]; /* seq names- getseqs() */ char *prog, /* prog name for err msgs */ char *seqx[2], /* seqs. getseqs() */ int dmax; /* best diag. nw() */ int dmaxO, /* final diag */ int dna, /* set if dna: main.) */ int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* total gaps in seqs */ int lenO, lenl; /* seq lens */ int ngapx, ngapy; /* total size of gaps *7 int smax; /* max score: nw() */ int *xbm, /* bitmap for matching */ long offset, /* current offset in jmp file */ struct diag *dx, /* holds diagonals */ struct path PP[2], /* holds path for seqs char *calloc(), *malloc(), *ιndex(), *strcpy(); char *getseq(), *g_calioc(),
Page 1 of nw.h /* Needleman-Wunsch alignment program *
* usage: progs filel file2
* where filel and file2 are two dna or two protein sequences
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with ';', ' > ' or ' < ' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align. out" *
* The program may create a tmp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650 */
^include "nw.h" -/include "day.h" static _dbval[26] = {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
}; static _pbval[26] = {
1,2|(1<<('D'-'A'))|(1<<('N'-'A')), 4, 8, 16, 32,64,
128, 256, OxFFFFFFF, 1< < 10, 1< < 11, 1< < 12, 1< < 13, 1< < 14,
1< < 15, 1< < 16, 1< < 17, 1< < 18, 1< < 19, 1< <20, 1< <21, 1< <22,
1< <23, 1< <24, 1< <25|(1< <('E'-'A'))|(1< <('Q'-'A'))
};
Figure imgf000030_0001
int ac; char *av[];
{ prog = av[0]; if (ac i = 3) { fpπntf(stderr, "usage %s filel file2\n", prog); fprmtf(stderr, "where filel and file2 are two dna or two protein sequences. \n"), fpπntf(stderr,"The sequences can be in upper- or lower-case\n"), fpπntt(stderr,"Any lines beginning with ';' or ' < ' are ιgnored\n"), fpπntf(stdeπ , "Output is in the file \"ahgn.out\"\n"); exιt(l),
} namex[0] = av[l], namex[l] = av[2]; seqx[0] = getseq(namex[0], &len0), seqx[l] = getseq(namex[l], &lenl), xbm = (dna)'' _dbval : _pbval, endgaps = 0, /* 1 to penalize endgaps */ ofile = ' align out", /* output file */ nw(), /* fill in the matrix, get the possible jmps */ readjmps(). /* get the actual jmps */ pπnt() /* print stats, alignment */ cleanup(O), /* unlink any tmp files */
Page 1 of nw.c /* do the alignment, return best score: maιn()
* dna- values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y. */ nw( ) nw { char *px, * .y; /* seqs and ptrs */ int *ndely, *dely; /* keep track of dely */ int ndelx, delx; /* keep track of delx */ int *tmp; /* for swapping rowO, rowl */ int mis; /* score for each type */ int insO, insl; /* insertion penalties */ register id; /* diagonal index */ register u; /* jmp index */ register *col0, *coll ; /* score for curr, last row */ register xx, yy /* index into seqs */ dx = (struct diag *)g_calloc("to get diags", lenO+lenl + 1 , sizeof(struct diag)); ndely = (int *)g_calloc("to get ndely", lenl + 1, sizeof(int)); dely = (int *)g_calloc("to get dely", lenl + 1, sizeof(int)); colO = (int *)g_calloc("to get colO", lenl + 1, sizeof(int)); coll = (int *)g_calIoc("to get coll " , lenl + 1 , sizeof(int)); insO = (dna)? DINSO : PINSO; insl = (dna)? DINSl : PINS1 ; smax = -10000; if (endgaps) { for (col0[0] = dely[0] = -insO, yy = 1 ; yy < = lenl ; yy + +) { col0[yy] = dely[yy] = col0[yy-l] - insl ; ndelyfyy] = yy,
} col0[0] = 0; /* Waterman Bull Math Biol 84 */
} else for (yy = 1 , yy < = lenl ; yy + +) delyfyy] = -insO;
/* fill in match matrix */ for (px = seqx[0], xx = 1 , xx < = lenO; px+ + , xx + +) { /* initialize first entry in col */ if (endgaps) { if (xx = = i) col l [01 = delx = -(msO+insl), else coll [0] = delx = co!0[0] - insl , ndelx = xx,
} else {
Figure imgf000031_0001
ndelx = 0.
Page 2 of nw.c ...nw seqx[l], yy = 1 , yy < = lenl , py + + , yy + +) { mis = col0[yy-lj, if (dna) mis + = (xbm[*px-'A']&xbm[*p> A ])"> DMAT DMIS, else mis + = _day[*px-'A'][*py-'A'],
/* update penalty for del in x seq,
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps */ if (endgaps 1 1 ndely[yy] < MAXGAP) { if (col0[yy] - insO > = dely[yy]) { delyfyyl = colOfyy] - (msO + insl), ndely [yy] = 1, } else { dely[yy] -= insl, ndely[yyl + +,
} } else { if (col0[yy] (msO+insl) > = dely[yy]) { dely[yy] = colOhyy] - (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 Lyy-1] - insO > = delx) { delx = col l [yy 1] - (msO + insl), ndelx = 1 , } else { delx -= msl , ndelx+ + ,
} } else { if (col l [yy 1] - (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
*/
Page 3 of nw c ...nw id = xx - yy + lenl - 1; if (mis > = delx && mis > = dely[yy])
Figure imgf000033_0001
else if (delx > = dely[yy]) { collfyy] = delx; ij = dx[ιd].ιjmp; if (dx[ιd].jp.n[0] && (!dna | [ (ndelx > = MAXJMP &&xx > dx[ιd].jp.x[ιj] + MX) | | mis > dx[ιd].score+DINSO)) { dx[ιd].ιjmp+ + , if (++ιj >= MAXJMP) { wπtejmps(ιd), ij = dx[ιd].ιjmp = 0; dx[ιd]. offset = offset; offset += sizeoftstruct jmp) + sizeof(offset); } } dx[ιd].jp.n[ιj] = ndelx;
Figure imgf000033_0002
dx[ιd]. score = delx;
} else { col 1 [yy] = dely[yy], ij = dx[ιd].ιjmp; if (dx[ιd].jp.n[0] && (!dna | | (ndely[yy] > = MAXJMP
&& xx > dx[ιd].jp.x[ιj] + MX) | | mis > dx[ιd].score + DINSO)) {
Figure imgf000033_0003
if(++ιj >= MAXJMP) { wπtejmps(ιd); ij = dx[ιd].ιjmp = 0; dx[ιd]. offset = offset; offset += sizeof(struct jmp) + sizeof (offset); } } dx[ιd].jp.n[ιj] = -ndelyyy], dx[ιd].jp.x[ιj] = xx; dx[ιd]. score = delyyy];
} if (xx = = lenO && yy < lenl) { /* last col */ if (endgaps) coll[yy] -= ιns0 + msl*(lenl-yy), if (coll [yy] > smax) { smax = coll[yy], dmax = id, } } } if (endgaps && xx < lenO) col 1 [yy- 1 ] -= ιnsO + msl*(lenO-xx), if (collfyy- 1] > smax) { smax = coll [yy-1], dmax = id,
} tmp = colO; colO = coll, coll = tmp,
}
(void) free((char =-)ndeIy), (void) free((char *-)dely), (void) free((char *)co!0), (void) free((char x)coll),
Pace 4 of nw.c /* *
*• pπnt() - onl\ routine visible outside this module *
* static:
* getmatO - trace back best path, count matches: pπnt()
* pr_ahgn() — print alignment of described in array p[]: pπnt()
* dumpblockO - dump a block of lines with numbers, stars. pr_ahgn()
* nums() — put out a number line- dumpblockO
* putline() — put out a line (name, [num], seq, [num]): dumpblockO
* stars() - -put a line of stars: dumpblockO
* stripname() - strip any path and prefix from a seqname */
#include "nw.h"
#define SPC 3
-/define P LINE 256 /* maximum output line */
-/define P SPC 3 /* space between name or num and seq */ extern day [26] [26]; int olen, /* set output line length */
FILE *fx, /* output file */ pπnto print
{ int lx, ly, firstgap, lastgap, /* overlap */ if ((fx = fopentofile, "w")) = = 0) { fpπntf(stderr, " %s: can't write %s\n", prog, ofile), cleanup(l),
} fpπntf(fλ. " < first sequence: %s (length = %d)\n", namex[0], lenO), fpπntf(fx, " < second sequence: %s (length = %d)\n", namex[l], lenl), olen = 60, lx = lenO, ly = lenl , firstgap = lastgap = 0, if (dmax < lenl - 1) { /* leading gap in x */ pp[0] spc = firstgap = lenl - dmax - 1 ; ly - = pp[0].spc;
} else if (dmax > lenl - 1) { /* leading gap in y */ pp[l] .spc = firstgap = dmax - (lenl - 1), lx -= pp[l].spc;
} if (dmaxO < lenO - 1) { /* trailing gap in x *l lastgap = lenO - dmaxO - 1 ; lx -= lastgap;
} else if (dmaxO > lenO - 1) { /* trailing gap in y */ lastgap = dmaxO - (lenO - 1), ly - = lastgap,
} getmat(lλ. ly, firstgap, lastgap), pr_ahgn()
Page 1 of nwprint.c /*
* trace back the best path, count matches
*/ static getmat(lx, ly, firstgap, lastgap) getmat int lx, ly, /* "core" (minus endgaps) */ int firstgap, lastgap, /* leading trailing overlap */
{ int nm, lO, ll , sizO, sizl , char outx[32], double pet, register nO, nl, register char *p0, *ρl ,
/* get total matches, score */
Figure imgf000035_0001
pO = seqx[0] + pp[l].spc, pi = seqx[l] + pp[0].spc, nO = pp[l] spc + 1 , nl = pp[0].spc + 1 , nm = 0, while ( *p0 && *pl ) {
Figure imgf000035_0002
if (xbm[*p0-'A']&xbm[*pl-'A']) nm+ + ,
Figure imgf000035_0003
}
/* pet homology
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core */ if (endgaps) lx = (lenO < lenl)'' lenO lenl, else lx = (lx < ly) ' lx ly, pet = 100 *(double)nm/(double)lx, fpπntf(fx, "\n"), tpπntf(fx, ' < % d match%s in an overlap of %d % 2f percent sιmιlaπty\n nm, (nm = = l)7 " " "es" , lx, pet),
Page 2 of nwprmt.c fpπntt(fx, " < gaps in first sequence: %d", gapx); .getmat if (gapx) {
(void) sprmtf(outx, " (%d %s%s)" , ngapx, (dna) ' ' base' ' residue", (ngapx = = 1 ) ' s"), fpπntf(fx, " %s", outx); fpπntf(fx, ", gaps in second sequence: %d", gapy); if (gapy) {
(void) sprιntf(outx, " (%d %s%s)", ngapy, (dna)? "base": "residue", (ngapy = = 1)? " " : "s"); fpπntf(fx, " %s", outx);
} if (dna) fpπntf(fx,
"\n < score: %d (match = %d, mismatch = %ά, 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) fprιntf(fx,
" < endgaps penalized, left endgap: %d %s%s, right endgap: %d %s%s\n firstgap, (dna)? "base" : "residue", (firstgap = = l)9 " " : "s", lastgap, (dna)? "base" • "residue", (lastgap = = 1)? " " : "s"), else fpπntf(fx, " < endgaps not penahzed\n"),
static nm; /* matches in core — for checking */ static Imax; /* lengths of stripped file names */ static U[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static m[2]; /* current elem number — for gapping */ static sιz[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */ static char oouutt[[22]][[IP_LINE], /* output line */ static char star[P 1 -], /* set by stars() */
/*
* print alignment of described in struct path pp[]
*/ static pr ιhgn() pr align { int nn, /* char count */ int more, register I; for (l = 0, lmax = 0, I < 2; ι + +) { nn = stπpname(namex[ι]), if (nn > lmax) lmax = nn, nc[ι] = 1 , m[ι] = 1 , sιz[ι] = lj[l] = 0, ps[ι] = seqxfi], po[ι] = out[ι],
Page 3 of nwpπnt.c for (nn = nm = 0, more = 1; more; ) { .pr align for (I = more = 0; l < 2; ι + +) {
/
: do we have more of this sequence'' if(!*ps[ι]) continue, more+ + , if (pp[ι]-spc) { /* leading space */ *po[ι] + + = ' ', pp[ι].spc-;
} else if (sιz[i]) { /* in a gap */
*po[ι] + + = '-', sιz[ι]~;
} else { /* we're putting a seq element
*/ *po[ι] = *ps[ι], if (ιslower(*ps[ι]))
*ps[ι] = toupper(*ps[ι]), po[ι] + +; ps[i] + +;
/*
* are we at next gap for this seq?
*/ if(m[ι] ==pp[ι].x[ιj[ι]]){ /*
* we need to merge all gaps
* at this location */ sιz[ι] = pp[ι] n[ιj[ι] + +]; while (nι[ι] == pp[ι].x[ιj[i]]) sιz[ι] +=pp[ι] n[ιj[ι] + +]
} nι[ι] + + ;
}
} if (+ +nn = = olen | | Imore && nn) { dumpblockO; for(ι = 0, l < 2; ι++) po[ι] = out[ι], nn = 0, }
/*
* dump a block of lines, including numbers, stars. pr_ahgn()
*/ static dumpblockO dumpblock
{ register i, for(ι = 0. l < 2. ι + +) *po[ι]~ = '\0
Page 4 of nwprint.c ...dumpblock
(void) putc('\n', fx); for = 0, l < 2, ι + +) { if (*out[ι] && (*out[ι] '= ' ' || (po[ι]) '= ' ')){ if 0 ==0) nums(ι); if (l = = 0 && *out[l]) stars(), puthne(ι); if (l == 0&&*out[l]) fpπntf(fx, star); if(ι == 1) nums(ι);
}
}
/*
* put out a number line: dumpblockO
*/ static nums(ιx) nums int IX; /* index in out[] holding seq line */ char nhnefP LINE]; register ι,j; register char *pn, *px, *py; for (pn = nhne, i = 0; l < lmax+P_SPC; ι++, pn++)
*pn = ' '; for (i = nc[ιx], py = outfix]; *py; py+ + , pn++) { if (*py == ' ' I I *py == '-')
*pn else { if (ι%10 == 0 || (l == 1 &&nc[ιx] '= 1)) { j = (l < 0)? -l : i; for (px = pn; j, j /= 10, px— )
*px =j%10 + '0'; if (l < 0)
} else
*-pn = ι++;
}
}
*pn = '\0', nc[ιx] = l, for (pn = nhne, *pn; pn++)
(void) putc(*pn, fx); (void) putc('\n', fx),
put out a line (name, [num], seq, [num]). dumpblockO static puthne(ιx) putline
Figure imgf000038_0001
Page 5 of nwprint.c ...putline int i; register char "-px; for (px = namexfix], i = 0; *px && *px != ':'; px+ + , i + +)
(void) putc(*px, fx); for(; i < lmax + P_SPC; i++)
(void) putc(' ', fx);
/* these count from 1 :
* nip is current element (from 1)
* nc[] is number at start of current line */ for (px = outfix]; *px; px++) (void) putc(*px&0x7F, fx); (void) ρutc('\n', fx);
* put a line of stars (seqs always in out[0], out[l]): dumpblockO */ static stars() stars
{ int i; register char *p0, *pl, ex, *px; if (!*out[0] 11 (*out[0] == " '&& *(po[0]) == ' ') | | !*out[l] I I (*out[l] == ' '&& *(po[l]) == ' ')) return; px = star; for(i = lmax+P SPC; i; i-)
*px++ = ' '; for(p0 = out[0],pl = out[l]; *p0 && *pl; p0+ +, pl + +){ if (isalpha(*pO) && isalpha(*pl)) { if ,xbm[*pO-'A']&xbm[*pl-'A']) { ex = '*'; nm+ +;
} else if (!dna && _day[*pO-'A'][*pl-'A'] > 0) ex = '.'; else ex = ' ';
} else ex = ' '; *px+ + = ex;
}
*px+ + = '\n'; *px = '\0';
Page 6 of nwprint.c /*
* strip path or prefix from pn, return len: pr_align()
*/ static stripname(pn) stripname char *pn; /* file name (may be path) */
{ register char *px, *py; py = 0; for (px = pn; *px; px+ +) if (*px = = '/') py = px + 1; if (py)
(void) strcpy(pn, py); return(strlen(pn));
Page 7 of nwprint.c /*
* cleanupO — cleanup any tmp file
* getseqO — read in seq, set dna, len, maxien ■* g_calloc0 - cdllocO with error checkin
* readjmpsO ~ get the good jmps, from tmp file if necessary
* writejmpsO — write a filled array of jmps to a tmp file: nw() */
.. include "nw.h" .. include < sys/file h > char *jname = "Λmp/homgXXXXXX"; /* tmp file for jmps */
FILE *fj; int cleanupO; /* cleanup tmp file */ long lseek();
/*
* remove any tmp file if we blow
*/ cieanup(ι) cleanup int l, { if (fj)
(void) unhnk name); exιt(ι), }
/*
* read, return ptr to seq, set dna, len, maxien
* 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 = fopen(file, "r")) = = 0) { fprmtf(stderr, " %s: can't read %s\n", prog, file), exιt(l),
} tlen = natgc = 0, while (tgets(hne, 1024, fp)) { if (*hne = = ' , ' | | *hne = = ' < ' | | *hne = = ' > ') continue; for (px = line; *px ' = '\n' ; px+ +) if (ιsupper(*px) | | ιslower(*px)) tlen+ + ,
} if ((pseq = malloc((unsigned)(tlen+6))) = = 0) { fpπnthstderr, ' %s: malloc() failed to get %d bytes lor %s\n", prog, tlen+6, file), exιt(l).
} pseq[0] = pseq[l] = pseq[2] = pseq[3] = '\0',
Page 1 of nwsubr.c ...getseq py = pseq + 4, *len = tlen, rewιnd(fp) while (fgets(hne, 1024, fp)) { if (*lme == ',' || *hne == '<' | | *hne == '>') continue, for (px = line, *px '= '\n', px++) { if (ιsupper(*px))
*py+ + = *px, else if (ιslower(*px))
*py++ = touppeι(*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 dx[] or tmp file, set pp[], reset dmax maιn()
*/ readjmpsO readjmps { int fd = -1,
Figure imgf000042_0001
register 1, j, xx, if(fj){
(void) fclose(fj), if ((fd = open(jname, 0_RDONLY, 0)) < 0) { fpπntt(stderr, 5? s can t open() %s\n", prog, iname) cleanup(l), } } for (I = lO = il = 0, dmaxO = dmax, xx = lenO, , ι + +) { while (1) { for (j = dx|dmax] ijmp, j > = 0 && dx|dmax] jp x[j] > = xx, j- )
Page 2 of nwsubr.c ...readjmps if 0 < 0 && dx[dmax]. offset && fj) {
(void) lseek(fd, dx[dmax]. offset, 0);
(void) read(fd, (char *)&dx[dmax] jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax] offset sizeof(dx[dmax] offset)), dx[dmax].ιjmp = MAXJMP- 1 ;
} else break,
} if (l > = JMPS) { fpπntf(stderr, " %s: too many gaps in ahgnment\n". prog); cleanup(l);
} if 0 > = 0) { siz = dx[dmax].jp.n[)]; xx = dx[dmax] jp.x[)];
Figure imgf000043_0001
if (siz < 0) { /* gap in second seq "7
Figure imgf000043_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 imgf000043_0003
gapx + + ; ngapx + = siz, /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP | | endgaps)'' siz MAXGAP, ι0+ + , } } else break; }
/* reverse the order of jmps */ for 0 = 0, ι0~; j < lO; j + + , ι0~) { i = pp[0] .n[j]; pp[0] .nD] = pp.0] .n[ι0]; pp[0].n[ι0] = i, i = pp[0].x[)]; pp[0].xD] = pp-0] xfiO]; pp[0J.x[ι0] = i; } for 0 = 0, ιl-; j < ιl ; j + + , il-) { i = pp[l].n[j]; pp[l].n[)] = pp. l].n[ιl]; pp[lj.n[ιl] = i,
■ = pp[i]-χ[j]; PPI I] χϋl = pp[i].χ[ι ij; PPM χ[ι l] = i,
} if (fd > = 0)
(void) close(fd), if (tj) {
(void) unhnk(jname), fj = 0;otfset = 0;}} Page 3 of nwsubr.c /* * write a filled jmp struct offset of the prev one (if any) nw()
*/ wπtejmps(ιx) writejmps
{ char *mktemp(); if (!fj) { if (mktemp(jname) < 0) { fpπntf(stderr, " %s: can't mktempO %s\n", prog, jname); cleanup(l );
} if ((fj = fopen(jname, "w")) = = 0) { fpπntf(stderr, " %s: can't write %s\n" , prog, jna e); exιt(l); } }
(void) fwπte((char *)&dx[ιx].jp, sizeof(struct jmp), 1 , fj); (void) fwπte((char *)&dx[ιx]. offset, sizeof(dx[ιx]. offset), 1, fj);
Page 4 of nwsubr.c Table 2A
PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) =
5 divided by 15 = 33.3%
Table 2B
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) =
5 divided by 10 = 50%
Table 2C
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =
6 divided by 14 = 42.9%
Table 2D
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =
4 divided by 12 = 33.3 %
"Percent (%) amino acid sequence identity" with respect to the PRO polypeptide sequences identified herein is defined as the percentage ot amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO sequence, after aligning the sequences and intioducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in vanous ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc , and the source code shown in Table 1 has been filed with user documentation in the U S Copyright Office, Washington D C , 20559, where it is registered under U S Copyright Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech, Inc , South San Francisco, California or may be compiled from the source code provided in Table 1 The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary
For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given ammo 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 ammo acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A As examples of % amino acid sequence identity calculations, Tables 2A-2B demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein ' to the amino acid sequence designated "PRO" Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described abo\e using the ALIGN-2 sequence comparison computer program Howevei , % am o acid sequence identity ma\ 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 mh gov NCBI-BLAST2 uses several search parameteis wherein all of those search paiameters are set to default values including for example, unmask = yes, strand = all expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0 01 , constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62
In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has oi comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A
In addition, % amino acid sequence identity may also be determined using the WU-BLAST-2 computer program (Altschul et al , Methods in Enzvmology, 266 460-480 (1996)) Most of the WU-BLAST-2 search parameters are set to the default values Those not set to default values, ; e , the adjustable parameters, are set with the following values overlap span = 1 , overlap fraction = 0 125, word threshold (T) = 1 1, and scoring matrix = BLOSUM62 For purposes herein, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (; e , the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of ammo acid residues of the PRO polypeptide of interest For example, in the statement "a polypeptide comprising an amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B", the amino acid sequence A is the comparison amino acid sequence of interest and the ammo acid sequence B is the amino acid sequence of the PRO polypeptide of interest
"PRO variant polynucleotide" or "PRO variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extiacellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any othei fragment of a full-length PRO polypeptide sequence as disclosed herein Ordinarily, a PRO variant polynucleotide will have at least about 80% nucleic acid sequence identity, 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 a nucleic acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Variants do not encompass the native nucleotide sequence
Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in length, more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more
"Percent (%) nucleic acid sequence identity" with respect to the PRO polypeptide-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PRO polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) softwaie Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc , and the source code shown in Table 1 has been filed with user documentation in the U S Copyright Office, Washington D C , 20559, where it is registered under U S Copyright Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech, Inc , South San Francisco California or may be compiled from the source code provided in Table 1 The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C As examples of % nucleic acid sequence identity calculations, Tables 2C-2D demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO- DNA"
Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program However, % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al , Nucleic Acids Res , 25 3389-3402 (1997)) The NCBI-BLAST2 sequence comparison program may be dow nloaded from http //www ncbi nlm mh 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, w ith or against a given nucleic acid sequence D) is calculated as follows
100 times the fi action W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI- BLAST2 in that program's alignment of C and D, and where Z is the total 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 ιdentιt\ of D to C In addition, % nucleic acid sequence identity values may also be generated using the \\ U-BLAST-2 computer program (Altschul et al Methods in Enzymology, 266 460-480 (1996)) Most of the WU-BLAST-2 search parameteis are set to the default values Those not set to default values, t e , the adjustable parameters, aie set with the following \alues overlap span = 1 , overlap fraction = 0 125, word threshold (T) = 1 1 and scoring matrix = BLOSUM62 For purposes herein, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide encoding nucleic acid and the comparison nucleic acid molecule of interest (i e , the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST 2 by (b) the total number of nucleotides of the PRO polypeptide- encoding nucleic acid molecule of interest For example, m the statement "an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B", the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest
In other embodiments, PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4
(SEQ ID NO 7), Figure 6 (SEQ ID NO 12), Figure 8 (SEQ ID NO 14) , Figure 10 (SEQ ID NO 22), Figure 12 (SEQ ID NO 24), and Figure 14 (SEQ ID NO 26), respectively PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide
The term "positives", in the context of the amino acid sequence identity comparisons performed as described above, includes amino acid residues in the sequences compared that are not only identical, but also those that have similar properties Amino acid residues that score a positive value to an amino acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 3 below) of the amino acid residue of interest
For purposes herein, the % value of positives of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given ammo acid sequence A that has or comprises a certain % positives to, with, or against a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of amino acid residues scoring a positive value by the sequence alignment program ALIGN 2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % positives of A to B will not equal the % positives of B to A "Isolated", when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a component of its natural environment Preferably, the isolated polypeptide is fiee of association with all components with which it is naturally associated Contaminant components of its natural environment are materials that would typically mterfeie 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 1 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 PRO natural environment will not be present Ordinarily, howevei, isolated polypeptide will be prepared by at least one purification step
An "isolated" nucleic acid molecule encoding a PRO polypeptide or an "isolated" nucleic acid molecule encoding an anti-PRO antibody is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the PRO-encoding nucleic acid or the natural source of the an ti-PRO-encoding nucleic acid Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated An isolated PRO-encoding nucleic acid molecule or an isolated anti-PRO-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 PRO-encoding nucleic acid molecule or from the anti-PRO-encoding nucleic acid molecule as it exists in natural cells However, an isolated nucleic acid molecule encoding a PRO polypeptide or an isolated nucleic acid molecule encoding an anti- PRO antibody includes PRO-nucleic acid molecules or anti-PRO-nucleic acid molecules contained in cells that ordinarily express PRO polypeptides or anti-PRO antibodies where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells
The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operator sequence, and a nbosome binding site Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence For example, DNA for a presequence or secretory leader is operably linked to DNA for a PRO polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a nbosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation Generally, ' operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by hgation at convenient restriction sites If such sites do not exist, the synthetic ohgonucleotide adaptors or linkers are used in accordance with conventional practice
"Stringency" ofhybπdization reactions is readily determmable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature The higher the degree of desired homology between the probe and hybπdizable sequence the higher the relative temperature that can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lowei temperatures less so For additional details and explanation of stringency of hybridization reactions see, Ausubel et al , Current Protocols in Molecular Biology (Wiley Interscience Publishers, 1995)
"Stringent conditions" or "high-stringency conditions", as defined herein, may be identified b) those that (1 ) employ low ionic strength and high temperature for washing, tor 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 albumin/ 0 1 % Fιcoll/0 1 % polyvinylpyrrolidone/ 50mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 °C, or (3) employ 50% formamide, 5 x SSC (0 75 M NaCl, 0 075 M sodium citrate), 50 mM sodium phosphate (pH 6 8), 0 1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0 1 % SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0 2 x SSC (sodium chloride/ sodium citrate) and 50% formamide at 55 °C, followed by a high-stringency wash consisting of 0 1 x SSC containing EDTA at 55°C
"Moderately-stringent conditions" may be identified as described by Sambrook etal , Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Press, 1989), and include the use of washing solution and hybridization conditions (e g , temperature, ionic strength, and % SDS) less stringent than those described above An example of moderately stringent conditions is overnight incubation at 37 °C in a solution comprising 20% formamide, 5 x SSC (150 mM NaCl, 15 mM 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 modifier "epitope-tagged" when used herein refers to a chimeπc polypeptide comprising a PRO polypeptide fused to a "tag polypeptide" The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes Suitable tag polypeptides generally have at least six ammo acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues)
"Active" or "activity" in the context of PRO variants refers to form(s) of PRO proteins that retain the biologic and/or immunologic activities of a native or naturally-occurring PRO polypeptide
"Biological activity" in the context of a molecule that antagonizes a PRO polypeptide that can be identified by the screening assays disclosed herein (e g , an organic or inorganic small molecule, peptide, etc ) is used to refer to the ability of such molecules to bind or complex with the PRO polypeptide identified herein or othei wise interfere with the interaction of the PRO polypeptides with other cellular proteins or otherw ise inhibits the transcription or translation of the PRO polypeptide Particularly preferred biological activity includes cardiac hypertrophy, activity that acts on systemic disorders that affect vessels, such as diabetes melhtus, as w ell as diseases of the arteries, capillaries, veins, and/or lymphatics and cancer
The term "antagonist" is used in the broadest sense and includes any molecule that partialh or fully blocks, inhibits or neutralizes one or moie of the biological activities of a native PRO polypeptide disclosed herein for example, if applicable, its mitogemc or angiogemc activity Antagonists of a PRO polypeptide may act by interfering with the binding of a PRO pol) peptide to a cellular receptor, by incapacitating or killing cells that have been activated bv a PRO polypeptide, or b> interfering with vascular endothehal cell activation after binding of a PRO polypeptide to a cellular receptor All such points of intervention by a PRO polypeptide antagonist shall be considered equivalent for purposes of this invention The antagonists inhibit the mitogemc, angiogemc, or other biological activity of PRO polypeptides, and thus are useful for the treatment of diseases or disorders characterized by undesirable excessive neovasculanzation, including by way of example tumors, and especially solid malignant tumors, rheumatoid arthritis, psoriasis, atherosclerosis diabetic and other retinopathies, retrolental fibroplasia, age related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, and chronic inflammation The antagonists also are useful tor the treatment of diseases or disorders characterized by undesirable excessive vascular permeability, such as edema associated with brain tumors, ascites associated with malignancies, Meigs' syndrome, lung inflammation, nephrotic syndrome, peπcardial effusion (such as that associated with pericarditis), and pleural effusion In a similar manner, the term ' agonist' is used in the broadest sense and includes any molecule that mimics a biological activity of a native PRO polypeptide disclosed herein Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments, or am o acid sequence variants of native PRO polypeptides, peptides, small organic molecules, etc
A "small molecule" is defined herein to have a molecular weight below about 500 daltons
The term "PRO polypeptide receptor ' as used herein refers to a cellular receptor for a PRO polypeptide, ordinarily a cell-surface receptor found on vascular endothehal cells, as well as variants thereof that retain the ability to bind a PRO polypeptide
"Antibodies (Abs) and "immunoglobuhns" (Igs) are glycoproteins having the same structural characteristics While antibodies exhibit binding specificity to a specific antigen, immunoglobuhns include both antibodies and other antibody-like molecules that lack antigen specificity Polypeptides of the lattei kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas The term ' antibody' is used in the broadest sense and specifically covers without limitation, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e g , bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity
"Natne antibodies and ' native immunoglobuhns" are usuallyheterotetramericglycoproteinsof 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 disulf ide bond, while the number of disulf ide linkages varies among the heavy chains of different immunoglobulin isotypes Each heavy and light chain also has regularly spaced intrachain disulfide bridges Each heavy chain has at one end a variable domain (V„) followed by a number of constant domains Each light chain has a variable domain at one end (V, ) and a constant domain at its other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody to and for its particular antigen Howex er, 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 regions (FR) The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-bindmg site of antibodies See, Kabat et al , NIH Publ No 91 -3242, Vol I, pages 647-669 ( 1991 ) The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity
"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 (Zapataefα/ , Protein Eng , 8(10) 1057-1062(1995)), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments
Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily Pepsin treatment yields an F(ab')2 fragment that has two an tigen-combimng sites and is still capable of cross-linking antigen
"Fv" is the minimum antibody fragment that contains a complete antigen-recognition and -binding site This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL di er Collectively, the six CDRs confer antigen-binding specificity to the antibody However, e\ en a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI ) of the heavy chain Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH 1 domain including one or more cy steines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteme resιdue(s) of the constant domains bear a free thiol group F(ab')-, antibody fragments originally were produced as pairs of Fab fragments that have hinge cysteines between them Other chemical couplings of antibody fragments are also known
The "light chains" of antibodies (immunoglobuhns) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino acid sequences of then constant domains Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobuhns can be assigned to different classes There aie five major classes of immunoglobuhns IgA, IgD, IgE, IgG and IgM, and several ot these may be further divided into subclasses (isotypes), e g IgG 1 , lgG2, IgG3, IgG4, IgA and IgA2 The heavy-chain constant domains that correspond to the different classes ot immunoglobuhns are called δ, e γ, and μ, respectively The subumt structures and three dimensional configurations of different classes of immunoglobuhns are well known
The term ' monoclonal antibody' as used herein refers to an antibody obtained from a population ot substantially homogeneous antibodies, . e , the individual antibodies compi ismg the population are identical except for possible naturally-occurring mutations that may be present in minor amounts Monoclonal antibodies are highly specific, being directed against a single antigemc site Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes) each monoclonal antibody is directed against a single determinant on the antigen In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobuhns The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al Nature, 256 495 ( 1975), or may be made by recombinant DNA methods (see, e g , U S Patent No 4,816,567) The "monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al , Nature, 352 624-628 (1991 ) and Marks et al , J Mol Biol , 222 581-597 (1991 ), for example
The monoclonal antibodies herein specifically include "chimeπc" antibodies (immunoglobuhns) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chaιn(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity U S Patent No 4,816,567, Morrison etal Proc Natl Acad Sci USA, 81 6851 -6855 (1984) "Humanized' forms of non-human (e g , unne) antibodies are chimeπc immunoglobuhns, immunoglobulm chains, or fragments thereof (such as Fv, Fab, Fab , F(ab'). or other antigen-bindmg subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulm For the most part, humanized antibodies are human immunoglobuhns (recipient antibody) in which residues from a CDR 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 immunoglobulm are replaced by corresponding non-human residues Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences These modifications are made to further refme 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 coπespond to those of a non-human immunoglobulm and all or substantially all ot the FR regions are those of a human immunoglobulm sequence The humanized antibody preferably also will comprise at least a portion ot an immunoglobulm constant legion (Fc), typically that of a human immunoglobulm 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 of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest "Single-chain Fv" or "sFv" antibody fragments comprise the VM and VL domains of an antibody wherein these domains are present in a single polypeptide chain Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that 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, 1994), pp 269-315 The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites Diabodies are described more fully in, for example, EP 404,097, WO 93/1 1 161 , and Holhnger et al , Proc Natl Acad Sci USA, 90 6444-6448 ( 1993 )
An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other protemaceous or nonproteinaceous solutes In preferred embodiments, the antibody will be purified (1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, oi (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 other composition that 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 that is detectable Radionuchdes that can serve as detectable labels include, for example, 1-131 1-123, 1-125, Y-90, Re- 188, At-21 1 , Cu-67, Bι-212, and Pd- 109 The label may also be a non- detectable entity such as a toxin
By "solid phase ' is meant a non-aqueous matrix to which an antibody of the present invention can adhere Examples of solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysacchaπdes (e c , agarose), polyacrylamides, polystyrene, polyv yl alcohol and si cones In certain embodiments depending on the context, the solid phase can comprise the well of an assay plate, in others it is a purification column (e g an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275, 149 A "liposome" is a small vesicle composed of various types of hpids, phosphohpids and/or surfactant that is useful for delivery of a drug (such as the PRO polypeptide or antibodies thereto disclosed herein) to a mammal The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes As used herein, the term "lmmunoadhesin" designates antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulm constant domains Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity that is other than the antigen recognition and binding site of an antibody (i e , is "heterologous"), and an immunoglobulm constant domain sequence The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand The immunoglobulm constant domain sequence in the immunoadhesin may be obtained from any immunoglobulm, such as IgG- 1 , IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM
II Compositions and Methods of the Invention
A PRO320, PRO938. PRO1031 PRQ296 PRQ213, PROl 330 and PRO 1449 Variants In addition to the full-length native sequence PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 and
PRO 1449 poly peptides described herein, it is contemplated that PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO1330 and PR01449 variants can be prepared PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 and PRO 1449 variants can be prepared by introducing appropriate nucleotide changes into the PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PRO 1449 DNA, and/or by synthesis of the desired PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics
Variations in the native full-length sequence PRO320, PR0938, PROl 031 , PR0296 PR021 , PROl 330 or PROl 449 or in various domains ot the PRO320, PR0938, PRO 1031 , PR0296, PR0213 PROl 330 or PRO 1449 described herein, can be made, for example, using any of the techniques and guidelines for conservative and non- conservative mutations set forth, for instance, in U S Patent No 5,364,934 Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO1330 or PR01449 that results m a change in the amino acid sequence of the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 as compared with the native sequence PRO320, PR0938, PRO I 031 , PR0296, PR0213, PRO 1330 or PRO 1449 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 PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 oi PRO 1449 Guidance in determining which amino acid residue may be inserted, substituted oi deleted without ad\ ersely affecting the desired actι\ ity may be found by comparing the sequence of the PRO320, PR0938 PRO1031 PR0296, PR0213, PRO 1330 or PR01449 with that of homologous know n protein molecules and minimizing the number ot amino acid sequence changes made in regions of high homology Ammo 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.
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, lie val Arg (R) lys, gin, asn lys Asn (N) gin, his, lys, arg gin Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro, ala ala His (H) asn, gin, lys, arg arg He (I) leu, val, met, ala, phe, norleucine leu Leu (L) norleucme, lie, val,
Figure imgf000062_0001
Phe (F) leu, val, lie, 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) lie, leu, met, phe, ala, norleucine leu
Substantial modifications in function or lmmunological identitv ofthePRO320 PR0938 PRO1031 , PRO296, PR0213, PRO1330 or PR01449 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain Naturally occurring residues are dι\ ided into groups based on common side chain properties
(1 ) hydrophobic norleucine, met, ala, val, leu, lie,
(2) neutral hydrophilic cys, ser, thr, (3) acidic asp, glu,
(4) basic asn, gin, his, lys, arg
(5) residues that influence chain orientation gly, pro, and
(6) aromatic trp, tyr, phe
Non conservative substitutions will entail exchanging a member of one of these classes for another class Such substituted residues also mav 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 ohgonucleotide mediated (site directed) mutagenesis, alanine scanning and PCR mutagenesis Site directed mutagenesis fCarter et al Nucl Acids Res 13 4331 (1986), Zo--er e. ... Nucl Acids Res , 10 6487 ( 1987)1 cassette mutagenesis .Wells et al . Gene. 34 31 (1985)], restriction selection mutagenesis [Wells et al , Philos Trans R Soc London SerA. 317 41 5 (1986)] or other known techniques can be performed on the cloned DNA to produce the PRO320 PR0938, PRO1031 , PR0296, PR0213 PROl 330 or PRO 1449 variant DNA
Scanning ammo acid analysis can also be employed to identity one or more ammo acids along a contiguous sequence Among the preferred scanning amino acids are relatively small, neutral amino acids Such amino acids include alanine, glycine, senne, and cysteme 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 alanme substitution does not yield adequate amounts of variant, an lsotenc amino acid can be used
B Modifications of PRO320 PRQ938 PRO1031 , PRQ296, PRQ213. PRO1330 and PRQ1449 Covalent modifications of PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 and PR01449 are included within the scope of this invention One type of covalent modification includes reacting targeted ammo acid residues of a PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 poly peptide with an organic denvatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues ot the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 Denvatization with bifunctional agents is useful, for instance, for crosshnking PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PRO 1449 to a water-insoluble support matrix or surface for use in the method for purifying antι-PRO320, anti- PR0938, anti-PRO 1031 , antι-PR0296, antι-PR0213, anti-PRO 1330 or anti PROl 449 antibodies, and vice-versa Commonly used crosshnking agents include, e g , 1 , 1 bιs(dιazoacetyl) 2-phenylethane, glutaraldehyde, N-hydroxy- succimmide esters, for example, esters with 4-azιdosalιcyhc acid, homobifunctional lmidoesters, including disuccimmidyl esters such as 3,3'-dιthιobιs(succιmmιdylpropιonate), bifunctional maleimides such as bis N maleιmιdo-1 8-octane and agents such as methyl-3-[(p azιdophenyl)dιthιo]propιoιmιdate Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of prol e and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the -ammo groups of lysine, argimne, and histidine side chains [T E Creighton, Proteins Structure and Molecular Properties, W H Freeman & Co , San Francisco, pp 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal caiboxyl group Another type ofcovalent modification of the PRO320 PR0938 PRO1031. PRO296 PRO213, PRO1 30 oι
PRO 1449 polypeptide included within the scope of this invention comprises altering the native giycosylation pattern of the polypeptide ' Altering the native giycosylation pattern is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO320 PR0938, PRO1031 PR0296 PR021 PROl 330 or PRO 1449 (either by removing the underlying giycosylation site or by deleting the giycosylation by chemical and/or enzymatic means), and/or adding one or moie giycosylation sites that are not present in the native sequence PRO320 PR0938 PRO 1031 PR0296 PR0213 PROl 330 or PRO 1449 In addition, the phrase includes qualitative changes in the ghcosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present
Addition of giycosylation sites to the PRO320, PR0938, PRO1031 PR0296, PR0213 PRO1330 or PR01449 polypeptide may be accomplished by altering the amino acid sequence The alteiation may be made, foi example, by the addition of, or substitution by, one or more serine or thieonme residues to the native sequence PRO320, PR0938, PRO1031, PR0296, PR0213, PROl 330 or PRO 1449 (for O-linked giycosylation sites) The PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 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 PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide Such methods are described in the art, e g , in WO 87/05330 published 1 1 September 1987, and in Aphn and Wπston, CRC Cnt Rev Biochem , pp 259-306 (1981 ) Removal of carbohydrate moieties present on the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for giycosylation Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddm, et al , Arch Biochem Biophys , 259 52 (1987) and by Edge e. α/ 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 etal , Meth Enzvmol , 138 350 (1987)
Another type of covalent modification of PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PR01449 comprises linking the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 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 PRO320, PR0938, PRO 1031 PR0296, PR0213, PRO 1330 or PRO 1449 of the present invention may also be modified in a way to form a chimeπc molecule comprising PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO 1330 or PRO 1449 fused to another, heterologous polypeptide or amino acid sequence In one embodiment, such a chimeπc molecule comprises a fusion of the PRO320, PR0938, PRO1031 ,
PR0296, PR0213, PROl 330 or PRO 1449 with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind The epitope tag is generally placed at the ammo- or carboxyl- terminus ot the PRO320, PR0938. PRO1031 , PR0296 PR0213, PRO1330 or PRO H49 The piesence of such epitope-tagged forms of the PRO320, PR0938, PRO 1031 , PR0296. PR0213, PRO 1330 or PRO 1449 can be detected using an antibody against the tag polypeptide Also, provision of the epitope tag enables the PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO 1330 or PRO 1449 to be readily purified by affinity purification using an anti-tag antibody or another t_\pe 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 (poIy-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 9E10 antibodies thereto [Evan et al , Molecular and Cellular Biology, 5 3610-3616 (1985)], and the Herpes Simplex vnus glycoprotein D (gD) tag and its antibody [Paborsky etal , Protein Engineering, 3(6) 547-553 ( 1990)] Other tag polypeptides include the Flag-peptide [Hopp e_ -./ , BιoTechnologv, 6 1204- 1210 ( 1988)1 , the KT3 epitope peptide [Martin et al . Science, 255 192-194 ( 1992)], an α-tubuhn epitope peptide [Skinner etal , J Biol Che , 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 chimeπc molecule may comprise a fusion of the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 with an immunoglobulm or a particular region of an immunoglobulm For a bivalent form of the chimeπc molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO320, PR0938, PRO1031 , PR0296, PR0213, PROl 330 or PRO 1449 polypeptide in place of at least one variable region within an Ig molecule In a particularly preferred embodiment, the immunoglobulm fusion includes the hinge, CH2 and CH3, or the hinge, CHI , CH2 and CH3 regions of an IgGl molecule For the production of immunoglobulm fusions see also, US Patent No 5,428,130 issued June 27, 1995
C Preparation ot the PRO320. PRQ938. PRO1031 , PRQ296, PRQ213. PRO1330 and PRQ1449
The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PROl 449 In particular, cDNAs encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO 1330 or PR01449 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed However, for sake of simplicity, in the present specification the protein encoded by DNA32284-1307, DNA56433-1406, DNA59294-1381 , DNA39979-1213, DNA30943-1 163, DNA64907-1 163-1 or DNA64908-1 163 1 , as well as all further native homologues and variants included in the foregoing definition of PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO1330 or PR01449, will be referred to as "PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO 1330 or PROl 449", respectively, regardless of their ongin or mode of preparation The description below relates primarily to production of PRO320, PR0938, PRO1031 , PR0296, PR0213,
PRO 1330 or PRO 1449 polypeptides by culturmg cells transformed or transfected with a vector containing nucleic acid encoding PRO320, PR0938, PRO 1031 PR0296, PR0213, PRO 1330 or PRO 1449 polypeptides It is, of course contemplated that alternative methods that are well known in the art may be employed to prepare PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO 1330 or PRO 1449 For instance, the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 oi PRO 1449 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 x itw protein synthesis may be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance with an Applied Biosvstems Peptide Synthesizer (Foster City, CA) using manufacturer s instructions Various portions of PRO320, PR0938, PRO1031 , PR0296, PR0213, PROl 330 or PR01449 may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide
l Isolation of DNA Encoding PRO320, PRQ938 PRO1031 , PRO296. PRO213. PRO1330 or PRO1449
DNA encoding PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the mRNA encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 and to express it at a detectable level Accordingly, DNAs encoding human PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 can be conveniently obtained from cDNA libraries prepared from human tissues, such as described in the Examples The gene encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide may also be obtained from a genomic library or by ohgonucleotide synthesis Libraries can be screened with probes (such as antibodies to the PRO320, PR0938, PRO1031 , PR0296,
PR0213, PRO 1330 or PRO 1449 polypeptide or ohgonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al , supia An alternative means to isolate the gene encoding PRO320, PR0938, PRO10 1 , PR0296, PR0213, PRO1330 or PR01449 is to use PCR methodology Sambrook et al , supia, Dieftenbach et al PCR Primer A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1995)
The Examples below describe techniques for screening a cDNA library The ohgonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized The ohgonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened Methods of labeling are well known in the art, and include the use of radiolabels like ,2P-labeled ATP biotmylation, or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supia
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs such as ALIGN, DNAstar, and INHERIT, which employ various algorithms to measure homology
Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced ammo acid sequence disclosed herein tor the first time, and, if necessary using conventional primer extension piocedures as described in Sambiook et al , supia to detect precursors and processing intermediates of mRNA that may not have been ieverse transcribed into cDNA li Selection and Transformation of Host Cells Host cells are transfected or transformed with expression or cloning vectors described herein for PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transtormants, 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 l , supra
Methods of transfection are known to the ordinarily skilled artisan, for example, CaP04 treatment and electroporation Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells The calcium treatment employing calcium chloride, as described in Sambrook et al , supia, or electroporation is generally used for prokaryotes or other cells that contain substantial cell wall barriers Infection with Agi obacterium 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 transformations 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 , 130 946 (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 or polyormthine, may also be used For various techniques for transforming mammalian cells, see, Keown et al , Methods in Enzvmology, 185 527-537 (1990) and Mansour et al , Nature, 336 348-352 (1988)
Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or highei eukaryote cells Suitable prokaryotes include, but are not limited to, eubactena, such as Gram-negative or Gram positive organisms, for example, En terobacteπaceae such as E coh Various E coli strains are publicly available, such as -± coh K12 strain MM294 (ATCC 31 ,446), E coli X1776 (ATCC 31 ,537), £ co strain W31 10 (ATCC 27,325), and K5 772 (ATCC 53,635) Other suitable prokaryotic host cells include Enterobacteπaceae such as Eschenchia e g , E coli, Enterobacter, Erwima, Klebsiella, Proteus, Salmonella, e g , Salmonella typhimut mm , Serratia, e g , Seπatia marcescans, and Slugella, as well as Bacilli such as B subtilis and -3 lichenifonms (e g , B lichenifoi is W disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P aeιιιgmosa, <mά Streptonn ces These examples are illustrative rather than limiting Strain W31 10 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations Preferably, the host cell secretes minimal amounts of proteolytic enzymes For example, strain W31 10 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host with examples of such hosts including E coli W31 10 strain 1 A2 which has the complete genotype ton A , E coli W31 10 strain 9E4 which has the complete genotype to/i 4 ptι3, E cσ/. W31 10 strain 27C7 (ATCC 55,244), which has the complete genotype tonA pti 3 phoA E15 (atgF lac )169 deqP ompTkan , E coli W31 10 strain 37D6, which has the complete genotype tonA ptι3p hoA El 5 (argF-lac)169 degP ompT rbs7 ilvG kai ; E. coli W31 10 strain 40B4, which is strain 37D6 with a non- kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990. Alternatively, in vitro methods of cloning, e.g.. PCR or other nucleic acid polymerase reactions, are suitable. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981 ]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al, Bio/Technology, 9: 968-975 (1991 )) such as, e.g., K. lactis (MW98-8C, CBS683. CBS4574: Louvencourte?-./., J. Bacteriol., 737 [1983]). K. frag His (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24, 178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al. , Bio/Technology, 8: 135 (1990)), K . thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al, J. Basic Microbiol., 28: 265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al, Proc. Natl. Acad. Sci. USA. 76: 5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991 ), and Aspergillus hosts such as A. nidulans (Ballance et al. , Biochem. Biophys. Res. Commun., 1 12: 284-289 [1983]; Tilburn etal, Gene. 26: 205-221 [1983]; Yelton etal. Proc. Natl. Acad. Sci. USA. 8J_: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J..4: 475-479 [1985]). Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula,
Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).
Suitable host cells for the expression of nucleic acid encoding glycosylated PRO320, PR0938, PRO 1031 ,
PR0296. PR0213, PROl 330 or PR01449 are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf'9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J. Gen. Virol.. 36: 59 (1977)); Chinese hamster ovary cells/ -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4. Mather, Biol. Reprod.. 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51 ). The selection of the appropriate host cell is deemed to be within the skill in the art.
iii. Selection and Use of a Replicable Vector The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO320, PR0938, PROl 031 , PR0296. PR0213, PRO 1330 or PRO 1449 may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, vn al 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 if the sequence is to be secreted, an origin of replication, one or more markei genes, an enhancer element, a promoter, and a transcription termination sequence Construction of suitable vectois containing one or more of these components employs standard hgation techniques that are known to the skilled artisan
The PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide In general, the signal sequence may be a component of the vector, or it may be a part of the DNA encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 that is inserted into the vector The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicilhnase, lpp, or heat-stable enterotoxin II leaders For yeast secretion the signal sequence may be, e g , the yeast mvertase leader, alpha factor leader (including Saccharomyces and Kluvveromyces α factor leaders, the latter described in U S Patent No 5,010, 182), or acid phosphatase leader, the C albicans glucoamylase leader (EP 362, 179 published 4 April 1990), or the signal described m WO 90/13646 published 15 November 1990 In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders
Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells Such sequences are well known for a variety of bacteria, yeast, and viruses The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e g , ampicilhn, neomycin, methotrexate, or tetracyc ne, (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 nucleic acid encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PROl 449, such as DHFR or thymidine kinase An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub etal , Proc Natl Acad Sci USA. 77 4216 (1980) A suitable selection gene tor use in veast is the tιp\ gene present in the yeast plasmid YRp7 Stinchcomb et al , Nature, 282 39 (1979), Kings an et al Gene 7 141 ( 1979), Tschemper et al , Gene 10 157 (1980) The tip] 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 promotei operably linked to the nucleic acid sequence encoding PRO320, PR0938, PRO1031 PR0296, PR0213, PROl 330 or PR01449 to direct mRNA synthesis Promoters recognized by a variety of potential host cells are well known Piomoters suitable for use with prokaiyotic 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 PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO1330 or PRO1449
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3 phosphoglycerate kinase (Hitzeman etal , J Biol Chem . 255 2073 (1980)) or other glycol ytic enzymes (Hess et al 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-ρhosphoglycerate mutase, pyruvate kinase tnosephosphate isomerase, phosphoglucose isomerase, and glucokinase
Othei yeast promoters that 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
PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 nucleic acid transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 1 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegaiovirus, a retrovirus, hepatitis B virus, and Simian Virus 40 (S V40), by heterologous mammalian promoters, e g , the actin promoter or an immunoglobulm promoter, and by heat-shock promoters, provided such promoters are compatible ith the host cell systems Transcription of a DNA encoding the PRO320, PR0938, PRO1031 PR0296, PR0213, PRO1330 or
PRO 1449 by higher eukaryotes may be increased by inserting an enhancer sequence into the vector Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin) Typically, however one will use an enhancer from a eukaryotic cell virus Examples include the SV40 enhancer on the late side of the replication origin (bp 100 270) the cytomegaiovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers The enhancer may be spliced into the vector at a position 5 or 3' to the sequence coding for PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PRO 1449, but is preferably located at a site 5 from the promotei
Expression vectors used in eukaryotic host cells (veast, fungi insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination ot transcription and for stabilizing the mRNA Such sequences are commonly available from the 5 and occasionally 3 , untranslated regions of eukaryotic or viral DNAs or cDNAs These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO320, PR0938,
PRO1031 , PR0296, PR0213, PRO1330 or PR01449 in recombinant vertebiate cell culture are described in Gething etal . Nature, 293 620-625 (1981 ), Mantel etal , Nature. 281 40-46 (1979), EP 1 17,060, and EP 1 17,058
lv 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 lmmunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful for lmmunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native-sequence PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to DNA encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 and encoding a specific antibody epitope
v Purification of Polypeptide Forms of PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptides 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 nucleic acid encoding the PRO320, PR0938, PRO 1031 , PR0296, PR0213 PRO 1330 or PRO 1449 polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, somcation, mechanical disruption, or cell-lysing agents
It may be desired to purify the PRO320, PR0938, PRO 1031 , PR0296, PR0213 PRO 1330 or PR01449 polypeptide 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, chromatof ocusing SDS-PAGE, ammonium sulfate precipitation, gel filtration using for example, Sephadex G-75, protein A Sepharose columns to remove contaminants such as IgG, and metal chelating columns to bind epitope tagged forms ot the PRO320, PR0938, PRO 1031 PR0296, PR0213, PR01330 or PRO 1449 polypeptide Various methods ot 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 PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 produced
D Uses of the PRO320. PRQ938, PRO1031 , PRQ296. PRQ213, PROl 330 or PRQ1449 polypeptides l Assays for Cardiovascular, Endothehal, and Angiogemc Activity Various assays can be used to test the polypeptide herein for cardiovascular, endothehal, and angiogemc activity Such assays include those provided in the Examples below
Assays fortestingforendothelin antagonistactivity. as disclosedin U S Pat No 5,773,414, include a rat heart ventricle binding assay where the polypeptide is tested for its ability to inhibit lodimzed endothehn-1 binding in a receptor assay, an endothelin receptor binding assay testing for intact cell binding of radiolabeled endothelin- 1 using rabbit renal artery vascular smooth muscle cells, an inositol phosphate accumulation assay where functional activity is determined in Rat-1 cells by measuring intra-cellular levels of second messengers, an arachidomc acid release assay that measures the ability of added compounds to reduce endothehn-stimulated arachidomc acid release in cultured vascular smooth muscles, in vitro (isolated vessel) studies using endothehum from male New Zealand rabbits, and in vivo studies using male Sprague-Dawley rats
Assays for tissue generation activity include, without limitation, those described in WO 95/16035 (bone, cartilage, tendon), WO 95/05846 (nerve, neuronal), and WO 91/07491 (skin, endothehum)
Assays for wound-healing activity include, for example, those described in Winter, Epidermal Wound Healing. Maibach, HI and Rovee, DT, eds (Year Book Medical Publishers, Inc , Chicago), pp 71 -1 12, as modified by the article of Eaglstein and Mertz, J Invest Dermatol , 7 . 382-384 (1978)
An assay to screen for a test molecule relating to a PRO polypeptide that binds an endothelin B, (ETB,) receptor polypeptide and modulates signal transduction activity involves providing a host cell transformed with a DNA encoding endothelin B, receptor polypeptide, exposing the cells to the test candidate, and measuring endothelin B, receptor signal transduction activity, as described, e g , in U S Pat No 5,773,223
There are several cardiac hypertrophy assays In vitro assays include induction of spreading of adult rat cardiac myocytes In this assay, ventricular myocytes are isolated from a single (male Sprague-Dawley) rat, essentially following a modification of the procedure described in detail by Piper et al , "Adult ventricular rat heart muscle cells" in Cell Culture Techniques in Heart and Vessel Research, H M Piper, ed (Berlin Springer- Verlag, 1990), pp 36-60 This procedure permits the isolation of adult ventricular myocytes and the long-term culture of these cells in the rod-shaped phenotype Phenylephπne and Prostaglandin F2α (PGF2c) have been shown to induce a spreading response in these adult cells The inhibition of myocyte spreading induced by PGF2α or PGF2π analogs (e g fluprostenol) and phenylephπne by various potential inhibitors of cardiac hypertrophy is then tested
One example of an in vivo assay is a test tor inhibiting cardiac hypertrophy induced by fluprostenol /;; vivo This phaimacological model tests the ability of the PRO polypeptide to inhibit cardiac hypertrophy induced in rats
(e g , male Wistar or Sprague-Dawley) by subcutaneous injection of fluprostenol (an agonist analog of PGF2κ) It is known that rats with pathologic cardiac hypertrophy induced by myocardial infarction have chronically elevated levels of extractable PGF2α in their myocardium Lai et al , Am J Physiol (Heart Circ Physiol ), 271 H2197- H2208 (1996) Accordingly, factors that can inhibit the effects of fluprostenol on myocardial growth in vn o are potentially useful for ti eating cardiac hypertrophy The effects ot the PRO polypeptide on cardiac hypertrophy are determined by measuring the weight of heart, ventricles, and left \ entncle (normalized by body weight) relative to fluprostenol-treated rats not receiving the PRO polypeptide
Another example of an in vivo assay is the pressure-overload cardiac hypertrophy assay For in vivo testing it is common to induce pressure-overload cardiac hypertrophy by constriction of the abdominal aorta of test animals In a typical protocol, rats (e g , male Wistar or Sprague Dawley) are treated under anesthesia, and the abdominal aorta of each rat is narrowed down just below the diaphragm Beznak M , Can J Biochem Physiol , 33 985-94 (1955) The aorta is exposed through a surgical incision, and a blunted needle is placed next to the vessel The aorta is constricted with a ligature of silk thread around the needle, which is immediately removed and which reduces the lumen of the aorta to the diameter of the needle This approach is described, for example, in Rossi et al . Am Heart J , 124 700-709 (1992) and O'Rourke and Reibel P S E M B , 200 95 100 (1992) In yet another in vivo assay, the effect on cardiac hypertrophy following experimentally induced myocardial infarction (MI) is measured Acute MI is induced in rats by left coronary artery hgation and confirmed by electrocardiographic examination A sham-operated group of animals is also prepared as control animals Earlier data have shown that cardiac hypertrophy is present in the group of animals with MI, as evidenced by an 18% increase in heart weight-to-body weight ratio Lai et al supra Treatment of these animals with candidate blockers of cardiac hypertrophy, e g , PRO polypeptide, provides valuable information about the therapeutic potential of the candidates tested One further such assay test for induction of cardiac hypertrophy is disclosed in U S Pat No 5,773,415, using Sprague-Dawley rats
For cancer, a variety of well-known animal models can be used to further understand the role of the genes identified herein in the development and pathogenesis of tumors and to test the efficacy of candidate therapeutic agents, including antibodies and other antagonists of the nativ e PRO polypeptides, such as small-molecule antagonists The in vivo nature of such models makes them particularly predictive of responses in human patients Animal models of tumors and cancers (e g , breast cancer, colon cancer, prostate cancer, lung cancer, etc ) include both non-recombinant and recombinant (transgemc) animals Non-recombinant animal models include, for example, rodent, e g , munne 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 orthopm implantation, e g colon cancer cells implanted in colomc 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 thymic hypo/ aplasia could successfully act as a host tor human tumor xenografts has lead to its widespiead use for this purpose The autosomal recessive nu gene has been introduced into a very large number of distinct congemc stiains of nude mouse, including, for example, ASW, A/He, AKR, BALB/c, B 10 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 tumoi/cancer cell lines such as any of the above-listed tumor cell lines, and, for example, the B 104-1 -1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene), /α. -transfected NIH-3T3 cells, Caco-2 (ATCC HTB-37), or a moderately well- differentiated grade II human colon adenocarcmoma cell line, HT-29 (ATCC HTB-38), or from tumors and cancers Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions involving freezing and storing in liquid nitrogen Karmali et al , Br J Cancer, 48 689-696 (1983) Tumor cells can be introduced into animals such as nude mice by a variety of procedures The subcutaneous
(s c ) space in mice is very suitable for tumor implantation Tumors can be transplanted s c as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions For solid-block or trochar implantation, tumor tissue fragments of suitable size are introduced into the s c space Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously Tumor cells can also be injected as subdermal implants In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s c tissue
Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogene was initially isolated), or new-transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al Proc Nat 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 etal , 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 vitio Cells from the in vitio 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 ot 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 ot
BALB/c female mice (DeLeo et al , J Exp Med , 146 720 (1977)) which provide a highly controllable model system for studying the anti-tumor activities of various agents Palladino et al , J Immunol , 138 4023-4032 (1987) Briefly, tumor cells are propagated in vitio in cell culture Prior to injection into the animals the cell lines are washed and suspended in buffer, at a cell density of about 10x l 0f to l Oxl O7 cells/ml The animals are then infected subcutaneously with 10 to 100 μ\ of the cell suspension, allow ing one to three weeks for a tumor to appeal In addition, the Lewis lung (3LL) carcinoma of mice which is one of the most thoioughly studied experimental tumors, can be used as an investigational tumor model Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small-cell carcinoma of the lung (SCCL) This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture Zupi e. a/ . Bi J Cancer, 41 suppl 4, 30 (1980) 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 with an implanted tumor is to measure the size of the tumor before and after treatment Traditionally, the size of implanted tumors has been measured with a slide caliper in two or three dimensions The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted into the corresponding volume by using a mathematical formula However, the measurement of tumor size is very inaccurate The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay Another important variable in the description of tumor growth is the tumor volume doubling time Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen. Proc 6th Int Workshop on Immune-Deficient Animals, Wu and Sheng eds (Basel, 1989), p 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
Further, recombinant (transgemc) animal models can be engineered by introducing the coding portion of the PRO gene identified herein into the genome of animals of interest, using standard techniques for producing transgemc animals Animals that can serve as a target for transgemc manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e g , baboons, chimpanzees and monkeys Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (U S Patent No 4,873,191 ), retrovirus-mediated gene transfer into germ lines (e g , Van der Putten et al , Proc Natl Acad Sci USA. 82 6148-615 (1985)), gene targeting in embryonic stem cells (Thompson etal , Cdl, 56 313-321 (1989)), electroporation of embryos (Lo, Mol Cell Biol , 3 1803-1814 (1983)), and sperm-mediated gene transfer Lavitrano et al , Cdl, 57 717-73 (1989) For a review, see tor example, U S Patent No 4,736,866
For the purpose of the present invention, transgemc animals include those that carry the transgene only in part of their cells ("mosaic animals") The transgene can be integrated either as a single transgene, or in concatamers, e g , head-to-head or head-to-tail tandems Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko e. al . Proc Natl Acad Sci USA, 89 6232 636 (1992)
The expression of the transgene in transgemc animals can be monitored by standard techniques For example,
Southern blot analysis or PCR amplification can be used to verify the integration of the transgene The level ot mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis PCR, or lmmunocvtochemistry The animals are further examined for signs of tumor or cancer development Alternatively, ' knock-out" animals can be constructed that have a defective or altered gene encoding a PRO polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the PRO polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal. For example, cDNA encoding a particular PRO polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques. A portion of the genomic DNA encoding a particular PRO polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector. See, e.g., Thomas and Capecchi, Cell, 51 : 503 (1987) for a description of homologous recombination vectors. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected. See, e.g., Li et al, Cell, 69: 915 (1992). The selected cells are then injected into a blastocyst of an animal (e.g. , a mouse or rat) to form aggregation chimeras. See, e.g. , Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J. Robertson, ed. (IRL: Oxford, 1987), pp. 1 13-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock-out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized, for instance, by their ability to defend against certain pathological conditions and by their development of pathological conditions due to absence of the PRO polypeptide.
The efficacy of antibodies specifically binding the PRO polypeptides identified herein, and other drug candidates, can be tested also in the treatment of spontaneous animal tumors. A suitable target for such studies is the feline oral squamous cell carcinoma (SCC). Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy of cats, accounting for over 60% of the oral tumors reported in this species. It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor. These tumors are usually not amenable to surgery, primarily because of the anatomy of the feline oral cavity. At present, there is no effective treatment for this tumor. Prior to entry into the study, each cat undergoes complete clinical examination and biopsy, and is scanned by computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors are excluded from the study. The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor, the animals may not be able to feed themselves. Each cat is treated repeatedly, over a longer period of time. Photographs of the tumors will be taken daily during the treatment period, and at each subsequent recheck. After treatment, each cat undergoes another CT scan. CT scans and thoracic radiograms are evaluated every 8 weeks thereafter. The data are evaluated for differences in survival, response, and toxicity as compared to control groups. Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span.
In addition, other spontaneous animal tumors, such as fibrosarcoma, adenocarci noma, lymphoma, chondroma, or leiomyosarcoma of dogs, cats, and baboons can also be tested. Of these, mammary adenocarci noma 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. Other in vitro and in vivo cardiovascular, endothehal, and angiogenic tests known in the art are also suitable herein. π Tissue Distribution
The results of the cardiovascular, endothehal, and angiogemc assays herein can be verified by further studies, such as by determining mRNA expression in various human tissues
As noted befoie, gene amplification and/or gene expression in various tissues may be measured by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc Natl Acad Sci USA, 77 5201-5205 ( 1980)), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes Gene expression in various tissues, alternatively, may be measured by lmmunological methods, such as lmmunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful for lmmunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native-sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO DNA and encoding a specific antibody epitope General techniques for generating antibodies, and special protocols for in situ hybridization are provided hereinbelow
in Antibody Binding Studies
The results of the cardiovascular, endothehal, and angiogemc study can be further verified by antibody binding studies, in which the ability of anti-PRO antibodies to inhibit the effect of the PRO polypeptides on endothehal cells or other cells used in the cardiovascular, endothehal, and angiogemc assays is tested Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow
Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays Zola, Monoclonal Antibodies A Manual of Techniques (CRC Press, Inc , 1987), pp 147-158
Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody The amount of target protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies To facilitate determining the amount of standard that becomes bound, the antibodies preferably are insolubihzed before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte that remain unbound
Sandwich assays involve the use of two antibodies, each capable of binding to a different lmmunogemc portion, or epitope, of the protein to be detected In a sandwich assay, the test sample analyte is bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex See, e g , US Pat No 4,376, 1 10 The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobuhn antibody that is labeled with a detectable moiety (indirect sandwich assay) For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme
For immunohis tochemistry, the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example
lv Cell-Based Tumor Assays
Cell-based assays and animal models for cardiovascular, endothehal, and angiogemc disorders, such as tumors, can be used to verify the findings of a cardiovascular, endothehal, and angiogemc assay herein, and further to understand the relationship between the genes identified herein and the development and pathogenesis of undesirable cardiovascular, endothehal, and angiogemc cell growth The role of gene products identified herein in the development and pathology of undesirable cardiovascular, endothehal, and angiogemc cell growth, e g , tumor cells, can be tested by using cells or cells lines that have been identified as being stimulated or inhibited by the PRO polypeptide herein Such cells include, for example, those set forth in the Examples below
In a different approach, cells of a cell type known to be involved in a particular cardiovascular, endothehal, and angiogemc disorder are transfected with the cDNAs herein, and the ability of these cDNAs to induce excessive growth or inhibit growth is analyzed If the cardiovascular, endothehal, and angiogemc disorder is cancer, suitable tumor cells include, for example, stable tumor cells lines such as the B 104-1 -1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and / -w-transfected NIH-3T3 cells, which can be transfected with the desired gene and monitored for tumongemc growth Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumongemc cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody-dependent cellular cytotoxicity (ADCC) Cells transfected with the coding sequences of the genes identified herein can further be used to identify drug candidates for the treatment of cardiovascular, endothehal, and angiogemc disorders such as cancer
In addition, primary cultures derived from tumors in transgemc animals (as described above) can be used in the cell-based assays herein, although stable cell lines are preferred Techniques to derive continuous cell lines from transgemc animals are well known in the art See, e g , Small et al , Mol Cell Biol . 5 642-648 (1985)
v Gene Therapy The PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide herein and polypeptidyl agonists and antagonists may be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as gene therapy
There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells in vivo and e\
Figure imgf000078_0001
o For in vivo delivery the nucleic acid is injected directly into the patient, usually at the sites where the PRO320, PR0938, PRO1031 , PR0296 PR0213, PROl 330 or PR01449 polypeptide is required, i e , the site of synthesis of the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449 polypeptide, if known, and the site (e g , wound) where biological activity of PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PR01449 polypeptide is needed. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes that are implanted into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283, 187). There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells ... vitro, or transferred in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. Transduction involves the association of a replication-defective, recombinant viral (preferably retroviral) particle with a cellular receptor, followed by introduction of the nucleic acids contained by the particle into the cell. A commonly used vector for ex vivo delivery of the gene is a retrovirus.
The currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral vectors (such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV)) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are, for example, DOTM A, DOPE, and DC-Choi ; see, e.g., Tonkinson et al, Cancer Investigation. 14(1 ): 54-65 (1996)). The most preferred vectors for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. A viral vector such as a retroviral vector includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. In addition, a viral vector such as a retroviral vector includes a nucleic acid molecule that, when transcribed in the presence of a gene encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide, is operably linked thereto and acts as a translation initiation sequence. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used (if these are not already present in the viral vector). In addition, such vector typically includes a signal sequence for secretion of the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence, most preferably the native signal sequence for the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide. Optionally, the vector construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such vectors will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
In some situations, it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins that bind to a cell-surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins that undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu etal. , J. Biol. Chem., 262: 4429-4432 (1987); and Wagner et al. , Proc. Natl. Acad. Sci. USA, 87: 3410-3414 (1990). For a review of the currently known gene marking and gene therapy protocols, see, Anderson et al, Science, 256: 808-813 (1992). See also WO 93/25673 and the references cited therein. Suitable gene therapy and methods for making retroviral particles and structural proteins can be found in, e.g. , U.S. Pat. No. 5,681,746.
vi. Use of Gene as Diagnostic This invention is also related to the use of the gene encoding the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide as a diagnostic. Detection of a mutated form of the PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide will allow a diagnosis of a cardiovascular, endothehal, and angiogenic disease or a susceptibility to a cardiovascular, endothehal, and angiogenic disease, such as a tumor, since mutations in the PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide may cause tumors.
Individuals carrying mutations in the genes encoding a human PRO320, PR0938, PRO1031 , PR0296,
PR0213, PROl 330 or PRO 1449 polypeptide may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy, and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using
PCR (Saiki et al., Nature, 324: 163-166 (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding the PRO320, PR0938,
PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide can be used to identify and analyze PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PROl 449 polypeptide mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA encoding the PRO320, PR0938,
PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide, or alternatively, radiolabeled antisense DNA sequences encoding the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamidine gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures. See, e.g. , Myers e. al. Science, 230: 1242 (1985).
Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method, for example, Cotton et al, Proc. Natl. Acad. Sci. USA, 85: 4397-4401 (1985).
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing, or the use of restriction enzymes, e.g., restriction fragment length polymorphisms (RFLP), and Southern blotting of genomic DNA.
vii. Use to Detect PRO Polypeptide Levels In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
Expression of nucleic acid encoding the PRO polypeptide may be linked to vascular disease or neovascularization associated with tumor formation. If the PRO polypeptide has a signal sequence and the mRNA is highly expressed in endothehal cells and to a lesser extent in smooth muscle cells, this indicates that the PRO polypeptide is present in serum. Accordingly, an anti-PRO polypeptide antibody could be used to diagnose vascular disease or neovascularization associated with tumor formation, since an altered level of this PRO polypeptide may be indicative of such disorders.
A competition assay may be employed wherein antibodies specific to the PRO polypeptide are attached to a solid support and the labeled PRO polypeptide and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of the PRO polypeptide in the sample.
viii. Chromosome Mapping The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymoφhisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis for the 3'- untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for
PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same ohgonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome- specific cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. FISH requires use of the clones from which the gene encoding the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide was derived, and the longer the better. For example, 2,000 bp is good, 4,000 bp is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of this technique, see, Verma et al. , Human Chromosomes: a Manual of Basic Techniques (Pergamon Press, New York, 1988).
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region is then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease. With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).
ix. Screening Assays for Drug Candidates
This invention encompasses methods of screening compounds to identify those that mimic the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide (agonists) or prevent the effect of the
PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide (antagonists). Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PRO320,
PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.
All assays for antagonists are common in that they call for contacting the drug candidate with a PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact.
In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 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 PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PRO 1449 polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the PRO320, PR0938, PRO1031 , PR0296, PR0213, PROl 330 or PR01449 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 PRO320, PR0938, PRO1031 ,
PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., 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 et al. , Proc. Natl. Acad. Sci. USA, 88 : 9578-9582 ( 1991 )) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA. 89: 5789-5793 (1991 ). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription- activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl -.αcZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two- hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
Compounds that interfere with the interaction of a gene encoding a PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PR01449 polypeptide identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner
If the PRO polypeptide has the ability to stimulate the proliferation of endothehal cells in the presence of the co-mitogen ConA, then one example of a screening method takes advantage of this ability Specifically, in the proliferation assay, human umbilical vein endothehal cells are obtained and cultured in 96-well flat-bottomed culture plates (Costar, Cambridge, MA) and supplemented with a reaction mixture appropriate for facilitating proliferation of the cells, the mixture containing Con-A (Calbiochem, La Jolla, CA) Con-A and the compound to be screened are added and after incubation at 37 °C, cultures are pulsed with H-thymidine and harvested onto glass fiber filters (phD, Cambridge Technology, Watertown, MA) Mean 3 H- thymidine incorporation (cpm) of triplicate cultures is determined using a liquid scintillation counter (Beckman Instruments, Irvine, CA) Significant 3 (H)- thymidine incorporation indicates stimulation of endothehal cell proliferation
To assay for antagonists, the assay described above is performed, however, in this assay the PRO polypeptide is added along with the compound to be screened and the ability of the compound to inhibit 3 (H)thymιdιne incorporation in the presence of the PRO polypeptide indicates that the compound is an antagonist to the PRO polypeptide Alternatively, antagonists may be detected by combining the PRO polypeptide and a potential antagonist with membrane-bound PRO polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay The PRO polypeptide can be labeled, such as by radioactivity, such that the number of PRO polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting Cohgan et al , Current Protocols in Immun , 1(2) Chapter 5 ( 1991 ) Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the PRO polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the PRO polypeptide Transfected cells that are grown on glass slides are exposed to the labeled PRO polypeptide The PRO polypeptide can be labeled by a variety of means including lodination or inclusion of a recognition site for a site-specific protein kinase Following fixation and incubation, the slides are subjected to autoradiographic analysis Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor
As an alternative approach for receptor identification, the labeled PRO polypeptide can be photoaffimty- hnked with cell membrane or extract preparations that express the receptor molecule Cross-linked material is resolved by PAGE and exposed to X-ray film The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing The amino acid sequence obtained from micro-sequencing would be used to design a set of degenerate ohgonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor
In another assay for antagonists, mammalian cells or a membrane preparation expressing the receptor would be incubated with the labeled PRO polypeptide in the presence of the candidate compound The ability of the compound to enhance or block this interaction could then be measured
The compositions useful in the treatment of cardiovascular, endothehal and angiogenic disorders include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple-helix molecules, etc., that inhibit the expression and/or activity of the target gene product.
More specific examples of potential antagonists include an ohgonucleotide that binds to the fusions of immunoglobulin with a PRO polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the PRO polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO polypeptide.
Another potential PRO polypeptide antagonist or agonist is an antisense RNA or DNA construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes the mature PRO polypeptides herein, is used to design an antisense RNA ohgonucleotide of from about 10 to 40 base pairs in length. A DNA ohgonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see, Lee et al, Nucl. Acids Res., 6:3073 (1979); Cooney etal., Science, 241 : 456 (1988); Dervan et al, Science, 251 : 1360 (1991 )), thereby preventing transcription and the production of the PRO polypeptide. The antisense RNA ohgonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PRO polypeptide (antisense - Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PRO polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.
Antisense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length, about 35 bases in length, about 40 bases in length, about 45 bases in length, about 50 bases in length, about 55 bases in length, about 60 bases in length, about 65 bases in length, about 70 bases in length, about 75 bases in length, about 80 bases in length, about 85 bases in length, about 90 bases in length, about 95 bases in length, about 100 bases in length, or more.
Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the PRO polypeptide, thereby blocking the normal biological activity of the PRO polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g , Rossi. Current Biology, 4 469-471 (1994), and PCT publication No WO 97/33551 (published September 18, 1997) Nucleic acid molecules in tnple-hehx formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides The base composition of these oligonucleotides is designed such that it promotes tnple-hehx formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of punnes or pyπmidines on one strand of a duplex For further details see, e g , PCT publication No WO 97/33551 , supra These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art
x Types of Cardiovascular. Endothehal. and Angiogenic Disorders to be Treated
The PRO polypeptides, or agonists or antagonists thereto, that have activity in the cardiovascular, angiogenic, and endothehal assays described herein, and/or whose gene product has been found to be localized to the cardiovascular system, are likely to have therapeutic uses in a variety of cardiovascular, endothehal, and angiogenic disorders, including systemic disorders that affect vessels, such as diabetes melhtus Their therapeutic utility could include diseases of the arteries, capillaries, veins, and/or lymphatics Examples of treatments hereunder include treating muscle wasting disease, treating osteoporosis, aiding in implant fixation to stimulate the growth of cells around the implant and therefore facilitate its attachment to its intended site, increasing IGF stability in tissues or in serum, if applicable, and increasing binding to the IGF receptor (since IGF has been shown in vitro to enhance human marrow erythroid and granulocytic progenitor cell growth)
The PRO polypeptides or agonists or antagonists thereto may also be employed to stimulate erythropoiesis or granulopoiesis, to stimulate wound healing or tissue regeneration and associated therapies concerned with re- growth of tissue, such as connective tissue, skin, bone, cartilage, muscle, lung, or kidney, to promote angiogenesis, to stimulate or inhibit migration of endothehal cells, and to proliferate the growth of vascular smooth muscle and endothehal cell production The increase in angiogenesis mediated by the PRO polypeptide or antagonist would be beneficial to ischemic tissues and to collateral coronary development in the heart subsequent to coronary stenosis Antagonists are used to inhibit the action of such polypeptides, for example, to limit the production of excess connective tissue during wound healing or pulmonary fibrosis if the PRO polypeptide promotes such production This would include treatment of acute myocardial infarction and heart failure Moreover, the present invention concerns the treatment of cardiac hypertrophy, regardless of the underlying cause, by administering a therapeutically effective dose of the PRO polypeptide, or agonist or antagonist thereto If the objective is the treatment of human patients, the PRO polypeptide preferably is recombinant human PRO polypeptide (rhPRO polypeptide) The treatment for cardiac hypertrophy can be performed at any of its various stages, which may result from a variety of diverse pathologic conditions, including myocardial infarction, hypertension, hypertrophic cardiomyopathy, and valvular regurgitation The treatment extends to all stages ot the progression of cardiac hypertrophy, with or without structural damage of the heart muscle, regardless of the underlying cardiac disorder.
The decision of whether to use the molecule itself or an agonist thereof for any particular indication, as opposed to an antagonist to the molecule, would depend mainly on whether the molecule herein promotes cardiovascularization, genesis of endothehal cells, or angiogenesis or inhibits these conditions. For example, if the molecule promotes angiogenesis, an antagonist thereof would be useful for treatment of disorders where it is desired to limit or prevent angiogenesis. Examples of such disorders include vascular tumors such as haemangioma, tumor angiogenesis, neovascularization in the retina, choroid, or cornea, associated with diabetic retinopathy or premature infant retinopathy or macular degeneration and proliferati ve vitreoretinopathy, rheumatoid arthritis, Crohn's disease, atherosclerosis, ovarian hyperstimulation, psoriasis, endometriosis associated with neovascularization, restenosis subsequent to balloon angioplasty, scar tissue overproduction, for example, that seen in a keloid that forms after surgery, fibrosis after myocardial infarction, or fibrotic lesions associated with pulmonary fibrosis.
If, however, the molecule inhibits angiogenesis, it would be expected to be used directly for treatment of the above conditions.
On the other hand, if the molecule stimulates angiogenesis it would be used itself (or an agonist thereof) for indications where angiogenesis is desired such as peripheral vascular disease, hypertension, inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenon, aneurysms, arterial restenosis, thrombophlebitis, lymphangitis, lymphedema, wound healing and tissue repair, ischemia reperfusion injury, angina, myocardial infarctions such as acute myocardial infarctions, chronic heart conditions, heart failure such as congestive heart failure, and osteoporosis. If, however, the molecule inhibits angiogenesis, an antagonist thereof would be used for treatment of those conditions where angiogenesis is desired.
Specific types of diseases are described below, where the PRO polypeptide herein or antagonists thereof may serve as useful for vascular-related drug targeting or as therapeutic targets for the treatment or prevention of the disorders. Atherosclerosis is a disease characterized by accumulation of plaques of intimal thickening in arteries, due to accumulation of lipids, proliferation of smooth muscle cells, and formation of fibrous tissue within the arterial wall. The disease can affect large, medium, and small arteries in any organ. Changes in endothehal and vascular smooth muscle cell function are known to play an important role in modulating the accumulation and regression of these plaques.
Hypertension is characterized by raised vascular pressure in the systemic arterial, pulmonary arterial, or portal venous systems. Elevated pressure may result from or result in impaired endothehal function and/or vascular disease.
Inflammatory vasculitides include giant cell arteritis, Takayasu's arteritis, polyarteritis nodosa (including the microangiopathic form), Kawasaki's disease, microscopic polyangiitis, Wegener's granulomatosis, and a variety of infectious-related vascular disorders (including Henoch-Schonlein prupura). Altered endothehal cell function has been shown to be important in these diseases.
Reynaud's disease and Reynaud's phenomenon are characterized by intermittent abnormal impairment of the circulation through the extremities on exposure to cold. Altered endothehal cell function has been shown to be important in this disease.
Aneurysms are saccular or fusiform dilatations of the arterial or venous tree that are associated with altered endothehal cell and/or vascular smooth muscle cells.
Arterial restenosis (restenosis of the arterial wall) may occur following angioplasty as a result of alteration in the function and proliferation of endothehal and vascular smooth muscle cells.
Thrombophlebitis and lymphangitis are inflammatory disorders of veins and lymphatics, respectively, that may result from, and/or in, altered endothehal cell function. Similarly, lymphedema is a condition involving impaired lymphatic vessels resulting from endothehal cell function.
The family of benign and malignant vascular tumors are characterized by abnormal proliferation and growth of cellular elements of the vascular system. For example, lymphangiomas are benign tumors of the lymphatic system that are congenital, often cystic, malformations of the lymphatics that usually occur in newborns. Cystic tumors tend to grow into the adjacent tissue. Cystic tumors usually occur in the cervical and axillary region. They can also occur in the soft tissue of the extremities. The main symptoms are dilated, sometimes reticular, structured lymphatics and lymphocysts surrounded by connective tissue. Lymphangiomas are assumed to be caused by improperly connected embryonic lymphatics or their deficiency. The result is impaired local lymph drainage. Griener et al, Lvmphology, 4: 140-144 (1971).
Another use for the PRO polypeptides herein or antagonists thereto is in the prevention of tumor angiogenesis, which involves vascularization of a tumor to enable it to growth and/or metastasize. This process is dependent on the growth of new blood vessels. Examples of neoplasms and related conditions that involve tumor angiogenesis include breast carcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervical carcinomas, endometrial carcinoma, endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma, glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma, abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
Age-related macular degeneration (AMD) is a leading cause of severe visual loss in the elderly population. The exudative form of AMD is characterized by choroidal neovascularization and retinal pigment epithelial cell detachment. Because choroidal neovascularization is associated with a dramatic worsening in prognosis, the PRO polypeptide or antagonist thereto is expected to be useful in reducing the severity of AMD.
Healing of trauma such as wound healing and tissue repair is also a targeted use for the PRO polypeptides herein or their antagonists. Formation and regression of new blood vessels is essential for tissue healing and repair. This category includes bone, cartilage, tendon, ligament, and/or nerve tissue growth or regeneration, as well as wound healing and tissue repair and replacement, and in the treatment of burns, incisions, and ulcers. A PRO polypeptide or antagonist thereof that induces cartilage and/or bone growth in circumstances where bone is not normally formed has application in the healing of bone fractures and cartilage damage or defects in humans and other animals Such a preparation employing a PRO polypeptide or antagonist thereof may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma-induced, or oncologic, resection-induced craniofacial defects, and also is useful in cosmetic plastic surgery
PRO polypeptides or antagonists thereto may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like
It is expected that a PRO polypeptide or antagonist thereto may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, or endothehum), muscle (smooth, skeletal, or cardiac), and vascular (including vascular endothehum) tissue, or for promoting the growth of cells comprising such tissues Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate
A PRO polypeptide herein or antagonist thereto may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage Also, the PRO polypeptide or antagonist thereto may be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells, or for inhibiting the growth of tissues described above
A PRO polypeptide or antagonist thereto may also be used in the treatment of peπodontal diseases and in other tooth-repair processes Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells, or induce differentiation of progenitors of bone-forming cells A PRO polypeptide herein or an antagonist thereto may also be useful in the treatment of osteoporosis or osteoarthπtis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc ) mediated by inflammatory processes, since blood vessels play an important role in the regulation of bone turnover and growth
Another category of tissue regeneration activity that may be attributable to the PRO polypeptide herein or antagonist thereto is tendon/ligament formation A protein that induces tendon/ ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed has application in the healing of tendon or ligament tears, deformities, and other tendon or ligament defects in humans and other animals Such a preparation may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue De novo tendon/hgament-hke tissue formation induced by a composition of the PRO polypeptide herein or antagonist thereto contributes to the repair ot congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments The compositions herein may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament forming cells, or induce growth of tendon/ligament cells or progenitors ex \ ivo for return in vivo to effect tissue repair The compositions herein may also be useful in the treatment of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art
The PRO polypeptide or its antagonist may also be useful for proliferation ot neui al cells and for 1 egeneration of nerve and brain tissue, . e , for the treatment of central and peripheral nervous system disease and neuropathies, as well as mechanical and traumatic disorders, that involve degeneration, death, or trauma to neural cells or nerve tissue More specifically, a PRO polypeptide or its antagonist may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma, and cerebrovascular diseases such as stroke Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a PRO polypeptide herein or antagonist thereto
Ischemia-reperfusion injury is another indication Endothehal cell dysfunction may be important in both the initiation of, and in regulation of the sequelae of events that occur following ischemia-reperfusion injury
Rheumatoid arthritis is a further indication Blood vessel growth and targeting of inflammatory cells through the vasculature is an important component in the pathogenesis of rheumatoid and sero-negative forms of arthritis
A PRO polypeptide or its antagonist may also be administered prophylactically to patients with cardiac hypertrophy, to prevent the progression of the condition, and avoid sudden death, including death of asymptomatic patients Such preventative therapy is particularly warranted in the case of patients diagnosed with massive left ventricular cardiac hypertrophy (a maximal wall thickness of 35 mm or more in adults, or a comparable value in children), or in instances when the hemodynamic burden on the heart is particularly strong
A PRO polypeptide or its antagonist may also be useful in the management of atnal fibrillation, which develops in a substantial portion of patients diagnosed with hypertrophic cardiomyopathy Further indications include angina, myocardial infarctions such as acute myocardial infarctions, and heart failure such as congestive heart failure Additional non-neoplastic conditions include psoriasis, diabetic and other prohferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, nephrotic syndrome, preeclampsia, ascites, pencardial effusion (such as that associated with pericarditis), and pleural effusion
In view of the above, the PRO polypeptides or agonists or antagonists thereof described herein, which are shown to alter or impact endothehal cell function, proliferation, and/or form, are likely to play an important role in the etiology and pathogenesis of many or all ot the disorders noted above, and as such can serve as therapeutic targets to augment or inhibit these processes or for vascular-related drug targeting in these disorders
xi Administration Protocols, Schedules. Doses, and Formulations
The molecules herein and agonists and antagonists thereto are pharmaceutically useful as a piophylactic and therapeutic agent for various disorders and diseases as set forth above
Therapeutic compositions of the PRO polypeptides or agonists or antagonists are prepared for storage by mixing the desired molecule having the appropriate 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 lyophi zed formulations or aqueous solutions Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionme, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethomum chloride, benzalkomum 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 immunoglobuhns, hydrophihc polymers such as polyvinylpyrrohdone, amino acids such as glycine, glutamine, asparagine, histidine, argimne, or lysine, monosacchaπdes, disacchaπdes, and other carbohydrates including glucose, mannose, or dextnns, chelating agents such as EDTA, sugars such as sucrose, manmtol, trehalose or sorbitol, salt-forming counter-ions such as sodium, metal complexes (e g , Zn-protein complexes), and/or non- ionic sur actants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG)
Additional examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceπde mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium tnsihcate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol Carriers for topical or gel-based forms of antagonist include polysacchaπdes such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrohdone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols For all administrations, conventional depot forms are suitably used Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nose sprays, subhngual tablets, and sustained-release preparations The PRO polypeptides or agonists or antagonists will typically be formulated in such vehicles at a concentration of about 0 1 mg/ml to 100 mg/ml
Another formulation comprises incorporating a PRO polypeptide or antagonist thereof into formed articles Such articles can be used in modulating endothehal cell growth and angiogenesis In addition, tumor invasion and metastasis may be modulated with these articles PRO polypeptide or antagonist to be used for m vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophihzation and reconstitution PRO polypeptide ordinarily will be stored in lyophihzed form or in solution if administered systemically If in lyophihzed form, PRO polypeptide or antagonist thereto is typically formulated in combination with other ingredients for reconstitution with an appropriate diluent at the time for use An example of a liquid formulation of PRO polypeptide or antagonist is a sterile, clear, colorless unpreserved solution filled in a single dose vial for subcutaneous injection Preserved pharmaceutical compositions suitable for repeated use may contain, for example, depending mainly on the indication and type of polypeptide a) PRO polypeptide or agonist or antagonist thereto, b) a buffer capable ot maintaining the pH in a range of maximum stability of the polypeptide or other molecule in solution, preferably about 4-8, c) a detergent/surfactant primarily to stabilize the polypeptide or molecule against agitation-induced aggregation, d) an isotonifier, e) a preservative selected from the group of phenol, benzyl alcohol and a benzethomum hahde, e g , chloride, and f) water If the detergent employed is non-ionic, it may, for example, be polysorbates (e g , POLYSORBATE™
(TWEEN™) 20, 80, etc ) or poloxamers (e g , POLOXAMER™ 188) The use of non-ionic surfactants permits the formulation to be exposed to shear surface stresses without causing denaturation of the polypeptide Further, such surfactant-containing formulations may be employed in aerosol devices such as those used in a pulmonary dosmg, and needleless jet injector guns (see, e g , EP 257,956) An isotonifier may be present to ensure isotonicity of a liquid composition of the PRO polypeptide or antagonist thereto, and includes polyhydπc sugar alcohols, preferably tπhydnc or higher sugar alcohols, such as glycerin, erythntol, arabitol, xyhtol, sorbitol, and mannitol These sugar alcohols can be used alone or in combination Alternatively, sodium chloride or other appropriate inorganic salts may be used to render the solutions lsotonic The buffer may, for example, be an acetate, citrate, succinate, or phosphate buffer depending on the pH desired The pH of one type of liquid formulation of this invention is buffered in the range of about 4 to 8, preferably about physiological pH
The preservatives phenol, benzyl alcohol and benzetho um hahdes, e g , chloride, are known antimicrobial agents that may be employed Therapeutic PRO polypeptide compositions 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 The formulations are preferably administered as repeated intravenous (I v ), subcutaneous (s c ), or intramuscular (l m ) injections, or as aerosol formulations suitable for intranasal or intrapulmonary delivery (for intrapulmonary delivery see, e g , EP 257,956) PRO polypeptide can also be administered in the form of sustained-released preparations Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, e g , films, or microcapsules Examples of sustained- release matrices include polyesters, hydrogels (e g poly(2-hydroxyethyl-methacrylate) as described by Langer et al , J Biomed Mater Res , 15 167-277 ( 1981 ) and Langer, Chem Tech , 12 98- 105 ( 1982) or poly(vιnylalcohol)), polylactides (U S PatentNo 3,773,919, EP 58.481 ) copolymei s of L glutamic acid and gamma ethyl-L-glutamate (Sidman et al , Biopolvmers, 22 547-556 ( 1983)) non-degradable ethylene-vinyl acetate (Langer et al , supia), degradable lactic acid-glycohc acid copolymers such as the Lupron Depot™ (injectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate), and poly-D-(-)-3-hydroxybutync acid (EP 133,988) While polymers such as ethylene-vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated proteins 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 protein stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophihzing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions Sustained-release PRO polypeptide compositions also include hposomally entrapped PRO polypeptides
Liposomes containing the PRO polypeptide are prepared by methods known/, erse DE 3,218,121 , Epstein etal , Proc Natl Acad Sci USA. 82 3688-3692 (1985), Hwang et al , Proc Natl Acad Sci USA, 77 4030-4034 (1980), EP 52,322, EP 36,676, EP 88,046, EP 143,949, EP 142,641 , Japanese patent application 83-1 18008, U S Patent Nos 4,485,045 and 4,544,545, and EP 102,324 Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol % cholesterol, the selected proportion being adjusted for the optimal therapy
The therapeutically effective dose of PRO polypeptide or antagonist thereto will, of course, vary depending on such factors as the pathological condition to be treated (including prevention), the method of administration, the type of compound being used for treatment, any co-therapy involved, the patient's age, weight, general medical condition, medical history, etc , and its determination is well within the skill of a practicing physician Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the maximal therapeutic effect If the PRO polypeptide has a narrow host range, for the treatment of human patients formulations comprising human PRO polypeptide, more preferably native-sequence human PRO polypeptide, are preferred The clinician will administer PRO polypeptide until a dosage is reached that achieves the desired effect for treatment of the condition in question For example, if the objective is the treatment of CHF, the amount would be one that inhibits the progressive cardiac hypertrophy associated with this condition The progress of this therapy is easily monitored by echo cardiography Similarly, in patients with hypertrophic cardiomyopathy, PRO polypeptide can be administered on an empirical basis
With the above guidelines, the effective dose generally is within the range of from about 0 001 to about 1 0 mg/kg, more preferably about 0 01 -1 0 mg/kg, most preferably about 0 01 0 1 mg/kg
For non oral use in treating human adult hypertension, it is advantageous to administer PRO polypeptide in the form of an injection at about 0 01 to 50 mg, preferably about 0 05 to 20 mg, most preferably 1 to 20 mg, per kg body weight, 1 to 3 times daily by intravenous injection For oral administration, a molecule based on the PRO polypeptide is preferably administered at about 5 mg to 1 g, preferably about 10 to 100 mg, per kg body weight, 1 to 3 times daily It should be appreciated that endotoxin contamination should be kept minimally at a safe level, for example less than 0 5 ng/mg protein Moreovei , for human administration, the formulations preferably meet sterility, pyrogemcity, general safety, and purity as required by FDA Office and Biologies standards The dosage regimen of a pharmaceutical composition containing PRO polypeptide to be used in tissue regeneration will be determined by the attending physician considering various factors that modify the action of the polypeptides, e.g. , amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration, and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF-I, to the final composition may also affect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomoφhometric determinations, and tetracycline labeling. The route of PRO polypeptide or antagonist or agonist administration is in accord with known methods, e.g. , by injection or infusion by intravenous, intramuscular, intracerebral, intraperitoneal, intracerobrospinal, subcutaneous, intraocular, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes, or by sustained-release systems as noted below. The PRO polypeptide or antagonists thereof also are suitably administered by intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects. The intraperitoneal route is expected to be particularly useful, for example, in the treatment of ovarian tumors.
If a peptide or small molecule is employed as an antagonist or agonist, it is preferably administered orally or non-orally in the form of a liquid or solid to mammals.
Examples of pharmacologically acceptable salts of molecules that form salts and are useful hereunder include alkali metal salts (e.g., sodium salt, potassium salt), alkaline earth metal salts (e.g., calcium salt, magnesium salt), ammonium salts, organic base salts (e.g., pyridine salt, triethylamine salt), inorganic acid salts (e.g., hydrochloride, sulfate, nitrate), and salts of organic acid (e.g., acetate, oxalate, p-toluenesulfonate).
For compositions herein that are useful for bone, cartilage, tendon, or ligament regeneration, the therapeutic method includes administering the composition topically, systemically, or locally as an implant or device. When administered, the therapeutic composition for use is in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage, or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Preferably, for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein- containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and preferably capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance, and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid, and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradabihty One specific embodiment is a 50 50 (mole weight) copolymer of lactic acid and glycohc acid in the form of porous particles having diameters ranging from 150 to 800 microns In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the polypeptide compositions from disassociating from the matrix
One suitable family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydoxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose, one preferred being catio c salts of carboxymethylcellulose (CMC) Other preferred sequestering agents include hyaluromc acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer, and poly(vιnyl alcohol) The amount of sequestering agent useful herein is 0 5-20 wt%, preferably 1-10 wt%, based on total formulation weight, which represents the amount necessary to prevent desorption of the polypeptide (or its antagonist) from the polymer matπ x and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the polypeptide (or its antagonist) the opportunity to assist the osteogenic activity of the progenitor cells
xn Combination Therapies The effectiveness of the PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide or an agonist or antagonist thereof in preventing or treating the disorder in question may be improved by administering the active agent serially or in combination with another agent that is effective for those purposes, either in the same composition or as separate compositions
For example, for treatment of cardiac hypertrophy, PRO polypeptide therapy can be combined with the administration of inhibitors of known cardiac myocyte hypertrophy factors, e g , inhibitors of α-adrenergic agonists such as phenylephπne, endothelin- 1 inhibitors such as BOSENTAN™ and MOXONODIN™, inhibitors to CT 1 (US Pat No 5,679,545), inhibitors to LIF, ACE inhibitors, des aspartate-angiotensin I inhibitors (U S Pat No 5,773,415), and angiotensin II inhibitors
For treatment of cardiac hypertrophy associated with hypertension, the PRO polypeptide can be administered in combination with β-adrenergic receptor blocking agents, e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol, ACE inhibitors, e g , quinapnl, captopnl, enalapnl, ramipπl, benazepnl fosinopnl, or hsinopπl, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide acetazolamide, or indapamide, and/oi calcium channel blockers, e g diltiazem, nifedipine verapamil or nicardipme Pharmaceutical compositions comprising the therapeutic agents identified herein by their generic names are commercially available, and are to be administered following the manufacturers instructions for dosage, administration, adverse effects, contraindications, etc. See, e.g.. Physicians' Desk Reference (Medical Economics Data Production Co.: Montvale, N.J., 1997), 51th Edition.
Preferred candidates for combination therapy in the treatment of hypertrophic cardiomyopathy are β- adrenergic-blocking drugs (e.g., propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol), verapamil, difedipine, or diltiazem. Treatment of hypertrophy associated with high blood pressure may require the use of antihypertensive drug therapy, using calcium channel blockers, e.g., diltiazem, nifedipine, verapamil, or nicardipine; β-adrenergic blocking agents; diuretics, e.g., chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichloφhenamide, acetazolamide, or indapamide; and/or ACE-inhibitors, e.g., quinapril, captopril, enalapril, ramipril, benazepril, fosinopril, or lisinopril.
For other indications, PRO polypeptides or their antagonists may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as EGF, PDGF, TGF-α or TGF-β, IGF, FGF, and CTGF.
In addition, PRO polypeptides or their antagonists used to treat cancer may be combined with cytotoxic, chemotherapeutic, or growth-inhibitory agents as identified above. Also, for cancer treatment, the PRO polypeptide or antagonist thereof is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances.
The effective amounts of the therapeutic agents administered in combination with the PRO polypeptide or antagonist thereof will be at the physician's or veterinarian's discretion. Dosage administration and adjustment is done to achieve maximal management of the conditions to be treated. For example, for treating hypertension, these amounts ideally take into account use of diuretics or digitalis, and conditions such as hyper- or hypotension, renal impairment, etc. The dose will additionally depend on such factors as the type of the therapeutic agent to be used and the specific patient being treated. Typically, the amount employed will be the same dose as that used, if the given therapeutic agent is administered without the PRO polypeptide.
xiii. Articles of Manufacture
An article of manufacture such as a kit containing PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PRO 1449 polypeptide or agonists or antagonists thereof useful for the diagnosis or treatment of the disorders described above comprises at least 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 that 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 the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PROl 449 polypeptide or an agonist or antagonist thereto. The label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. The article of manufacture may also comprise a second or third container with another active agent as described above.
E. Antibodies Some of the most promising drug candidates according to the present invention are antibodies and antibody fragments that may inhibit the production or the gene product of the genes identified herein and/or reduce the activity of the gene products.
i. 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 PRO320, PR0938, PRO 1031 , PR0296, PR0213 , PROl 330 or PRO 1449 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 that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A or synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
ii. Monoclonal Antibodies The anti-PRO320, anti-PR0938, anti-PRO 1031 , anti-PR0296, anti-PR0213, anti-PROl 330 or anti-PRO 1449 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 PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 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 (New York: 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 guanme phosphonbosyl transferase (HGPRT or HPRT), the culture medium for the hybndomas typically will include hypoxanthine, aminopteπn, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells
Preferred immortalized cell lines are those that fuse efficiently , support stable high level expression of antibody by the selected antibody producing cells, and are sensitive to a medium such as HAT medium More preferred immortalized cell lines are munne myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, 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 the PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) Such techniques and assays are known in the art The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal Biochem . 107 220 (1980)
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods Goding, supra Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulm purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S
Patent No 4,816,567 DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using ohgonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of munne antibodies) The hybridoma cells of the invention serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulm protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light-chain constant domains in place of the homologous munne sequences (U S Patent No 4,816,567, Morrison et al , supia) or by covalently joining to the immunoglobulm coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide Such a non-immunoglobuhn polypeptide can be substituted for the constant domains of an antibody of the inv ention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody The antibodies may be monovalent antibodies Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulm light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy-chain crosshnking Alternatively, the relevant cysteme residues are substituted with another amino acid residue or are deleted so as to prevent crosshnking
In vitro methods are also suitable for preparing monovalent antibodies Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art
in Human and Humanized Antibodies
The antι-PRO320, antι-PR0938, anti-PRO 1031 , antι-PR0296, antι-PR0213, anti-PRO 1330 or anti-PRO 1449 antibodies may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , munne) antibodies are chimeπc immunoglobuhns, immunoglobulm chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non- human immunoglobulm Humanized antibodies include human immunoglobuhns (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 framework residues of the human immunoglobulm are replaced by corresponding non-human residues Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulm, and all or substantially all of the FR regions are those of a human immunoglobulm consensus sequence The humanized antibody preferably also will comprise at least a portion of an immunoglobulm constant region (Fc), typically that of a human immunoglobulm Jones et al , Nature, 321 522-525 (1986), Riechmann et al , Nature. 332 323-329 (1988), 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 that is non-human These non-human ammo acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain Humamzation can be essentially performed following the method of Winter and co-workers (Jones et al , Nature, 321 522-525 (1986), Riechmann et al , Nature, 332 323-327 (1988). Verhoeven et al . Science, 239 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody Accordingly, such "humanized" antibodies are chimeric antibodies (U S Patent No 4,816 567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
Human antibodies can also be produced using various techniques known in the art, including phage display libraries Hoogenboom and Winter, J Mol Biol , 227 381 (1991 ). Marks et al , J Mol Biol , 222 581 (1991 ) The techniques of Cole et al and Boerner et al are also available for the preparation of human monoclonal antibodies Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p 77 (1985) and Boerner e. <- , J Immunol , 147( 1 ) 86-95 (1991 ) Similarly, human antibodies can be made by introducing human immunoglobulm loci into transgemc animals, e g , mice in which the endogenous immunoglobulm genes have been partially or completely inactivated Upon challenge, human antibody production is observed that 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, and 5,661 ,016, and in the following scientific publications Marks etal , Bio/Technology. 10 779-783 ( 1992), Lonberg etal . Nature, 368 856-859 (1994), Morrison, Nature, 368 812-813 (1994), Fishwild . o/ Nature Biotechnology, 14 845-851 (1996), Neuberger, Nature Biotechnology, 14 826(1996). Lonberg and Huszar. Intern Rev Immunol , 13 65-93 (1995)
lv 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 PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PRO 1449 polypeptide, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit
Methods for making bispecific antibodies are known in the art Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulm heavy-cham/hght-chain pairs, where the two heavy chains have different specificities Milstein and Cuello, Nature, 305 537-539 (1983) Because of the random assortment of immunoglobulm heavy and light chains, these hybπdomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure The purification of the correct molecule is usually accomplished by affinity chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al , EMBO J , 10 3655-3659 (1991 )
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulm constant-domain sequences The fusion preferably is with an immunoglobulm heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions It is 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 immunoglobulm heavy-chain fusions and, if desired, the immunoglobulm 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)
v Heteroco ugate Antibodies
Heterocon|ugate 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 foi treatment of HIV infection WO 91/00360, WO 92/200373, EP 03089 It is contemplated that the antibodies may be prepared in \ ιtιo using known methods in synthetic protein chemistry, including those involving crosshnking agents. For example, immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
vi. Effector Function Engineering It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See, Caron et al, J. Exp. Med.. 176: 1191-1 195 (1992) and Shopes, J. Immunol.. 148: 2918-2922 ( 1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff 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).
vii. Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 13II, 131In, yoY, and 186Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyI-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis- active fluorine compounds (such as 1 ,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science, 238: 1098 (1987). Carbon- 14-labeled l -isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, W094/1 1026.
In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a radionucleotide).
viii. Immunoliposomes The antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein etal, Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et l, Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG- PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin 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).
ix. Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders as noted above and below in the form of pharmaceutical compositions. If the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449 polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that 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 that 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 etal, Proc. Natl. Acad. Sci. USA, 90: 7889- 7893 (1993).
The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxyme thylcellulose 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, supra.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. , films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
x. Methods of Treatment using the Antibody
It is contemplated that the antibodies to a PRO320, PR0938, PRO1031, PR0296, PR0213, PRO 1330 or
PR01449 polypeptide may be used to treat various cardiovascular, endothehal, and angiogenic conditions as noted above.
The antibodies are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration of the antibody is preferred.
Other therapeutic regimens may be combined with the administration of the antibodies of the instant invention as noted above. For example, if the antibodies are to treat cancer, the patient to be treated with such antibodies may also receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service. Ed., M.C. Perry (Williams &
Wilkins: Baltimore, MD. 1992). The chemotherapeutic agent may precede, or follow administration of the antibody, or may be given simultaneously therewith. The antibody may be combined with an anti-estrogen compound such as tamoxifen or EVISTA™ or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
If the antibodies are used for treating cancer, it may be desirable also to administer antibodies against other tumor-associated antigens, such as antibodies that bind to one or more of the ErbB2, EGFR, ErbB3, ErbB4, or VEGF receptor(s). These also include the agents set forth above. Also, the antibody is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances. Alternatively, or in addition, two or more antibodies binding the same or two or more different antigens disclosed herein may be co-administered to the patient. Sometimes, it may be beneficial also to administer one or more cytokines to the patient. In a preferred embodiment, the antibodies herein are co- administered with a growth-inhibitory agent. For example, the growth-inhibitory agent may be administered first, followed by an antibody of the present invention. However, simultaneous administration or administration of the antibody of the present invention first is also contemplated. Suitable dosages for the growth-inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth-inhibitory agent and the antibody herein.
In one embodiment, vascularization of tumors is attacked in combination therapy. The anti-PRO polypeptide antibody and another antibody (e.g., anti-VEGF) are administered to tumor-bearing patients at therapeutically effective doses as determined, for example, by observing necrosis of the tumor or its metastatic foci, if any. This therapy is continued until such time as no further beneficial effect is observed or clinical examination shows no trace of the tumor or any metastatic foci. Then TNF is administered, alone or in combination with an auxiliary agent such as alpha-, beta-, or gamma-interferon, anti-HER2 antibody, heregulin, anti-heregulin antibody, D-factor, interleukin-1 (IL-1 ), interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), or agents that promote microvascular coagulation in tumors, such as anti-protein C antibody, anti-protein S antibody, or C4b binding protein (see, WO 91/01753, published 21 February 1991 ), or heat or radiation.
Since the auxiliary agents will vary in their effectiveness, it is desirable to compare their impact on the tumor by matrix screening in conventional fashion. The administration of anti-PRO polypeptide antibody and TNF is repeated until the desired clinical effect is achieved. Alternatively, the anti-PRO polypeptide antibody is administered together with TNF and, optionally, auxiliary agent(s). In instances where solid tumors are found in the limbs or in other locations susceptible to isolation from the general circulation, the therapeutic agents described herein are administered to the isolated tumor or organ. In other embodiments, a FGF or PDGF antagonist, such as an anti-FGF or an anti-PDGF neutralizing antibody, is administered to the patient in conjunction with the anti-PRO polypeptide antibody. Treatment with anti-PRO polypeptide antibodies preferably may be suspended during periods of wound healing or desirable neovascularization.
For the prevention or treatment of cardiovascular, endothehal, and angiogenic disorder, the appropriate dosage of an antibody herein will depend on the type of disorder to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.
For example, depending on the type and severity of the disorder, about 1 g/kg to 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily or weekly 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 repeated or sustained until a desired suppression of disorder symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic tumor imaging.
xi. Articles of Manufacture with Antibodies
An article of manufacture containing a container with the antibody and a label is also provided. Such articles are described above, wherein the active agent is an anti-PRO320, anti-PR0938, anti-PRO 1031, anti-PR0296, anti- PR0213, anti-PRO1330 or anti-PR01449.
xii. Diagnosis and Prognosis of Tumors using Antibodies
If the indication for which the antibodies are used is cancer, while cell-surface proteins, such as growth receptors over expressed in certain tumors, are excellent targets for drug candidates or tumor (e.g., cancer) treatment, the same proteins along with PRO polypeptides find additional use in the diagnosis and prognosis of tumors. For example, antibodies directed against the PRO polypeptides may be used as tumor diagnostics or prognostics.
For example, antibodies, including antibody fragments, can be used qualitatively or quantitatively to detect the expression of genes including the gene encoding the PRO polypeptide. The antibody preferably is equipped with a detectable, e.g., fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. Such binding assays are performed essentially as described above.
In situ detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy. For this puφose, a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample. This procedure also allows for determining the distribution of the marker gene product in the tissue examined. It will be apparent to those skilled in the art that a wide variety of histological methods are readily available for in situ detection.
The following Examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
The disclosures of all patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
EXAMPLES Commercially available reagents referred to in the Examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following Examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, V A. Unless otherwise noted, the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in the following textbooks: Sambrook et al, supra; Ausubel et al, Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, N. Y., 1989); Innis etal, PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc.: N.Y., 1990); Harlow et al, Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold Spring Harbor, 1988); Gait, Ohgonucleotide Synthesis (IRL Press: Oxford, 1984); Freshnev. Animal Cell Culture. 1987; Coligan er-./., Current Protocols in Immunology, 1991.
EXAMPLE 1 Extracellular Domain Homology Screening to Identify Novel Polypeptides and cDNA Encoding Therefor The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST databases included public databases (e.g., Dayhoff, GenBank), and proprietary databases (e.g. LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST-2 (Altschul et al. Methods in Enzymology, 266:460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons with a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington).
Using this extracellular domain homology screen, consensus DNA sequences were assembled relative to the other identified EST sequences using phrap. In addition, the consensus DNA sequences obtained were often (but not always) extended using repeated cycles of BLAST or BLAST-2 and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above.
Based upon the consensus sequences obtained as described above, oligonucleotides were then synthesized and used to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for a PRO polypeptide. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al, Current Protocols in Molecular Biology, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe ohgonucleotide and one of the primer pairs.
The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al, Science, 253: 1278-1280 (1991 )) in the unique Xhol and Notl sites. EXAMPLE 2 Isolation of cDNA Clones Encoding Human PRO320 (a fibulin homolog) A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above This assembled consensus sequence is herein identified as DNA28739 Based on the DNA28739 consensus sequence, oligonucleotides were synthesized 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO320
PCR primers (forward and reverse) were synthesized forward PCR primer 5'-CCTCAGTGGCCACATGCTCATG-3' (SEQ ID NO 3) reverse PCR primer 5'-GGCTGCACGTATGGCTATCCATAG-3' (SEQ ID NO 4)
Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA28739 sequence which had the following nucleotide sequence hybridization probe
5'-GATAAACTGTCAGTACAGCTGTGAAGACACAGAAGAAGGGCCACAGTGCC 3' (SEQ ID NO 5) In order to screen several libraries for a source of a full length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above A positive library was then used to isolate clones encoding the PRO320 gene using the probe ohgonucleotide and one of the PCR primers RNA for construction of the cDNA libraries was isolated from human fetal lung tissue (LIB025)
DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA32284-1307 [Figure 1, SEQ ID NO 1], and the derived protein sequence for PRO320
The entire coding sequence of DNA32284-1307 is included in Figure 1 (SEQ ID NO 1 ) Clone DNA32284- 1307 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 135 137 and an apparent stop codon at nucleotide positions 1 149-1151 The predicted polypeptide precursor is 338 amino acids long Analysis of the full-length PRO320 sequence shown in Figure 2 (SEQ ID NO 2) 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 PRO320 polypeptide shown in Figure 2 evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 21 , a cAMP and cGMP-dependent protein kinase phosphorylation site from about ammo acid 54 to about amino acid 58, N- myπstoylation sites from about amino acid 18 to about amino acid 24, from about amino acid 21 to about amino acid 27, from about amino acid 30 to about amino acid 36, from about amino acid 44 to about ammo acid 50, from about amino acid 59 to about amino acid 65, from about amino acid 68 to about amino acid 74, from about amino acid 1 14 to about amino acid 120, from about amino acid 196 to about ammo acid 202, from about amino acid 241 to about amino acid 247, from about amino acid 255 to about amino acid 261 , from about amino acid 326 to about amino acid 332, and from about amino acid 330 to about amino acid 336, an amidation site from about amino acid 330 to about amino acid 334, aspartic acid and asparagine hydroxylation sites from about amino acid 109 to about amino acid 121, from about amino acid 191 to about ammo acid 203, and from about amino acid 236 to about amino acid 248, and an EGF-hke domain cysteme pattern signature from about amino acid 80 to about amino acid 92 Clone DNA32284- 1307 has been deposited with the ATCC on March 1 1 , 1998 and is assigned ATCC deposit no. 209670. The full-length PRO320 protein shown in Figure 2 has an estimated molecular weight of about 37,143 daltons and a pi of about 8.92.
EXAMPLE 3 Isolation of cDNA Clones Encoding Human PRQ938 (a disulfide isomerase homolog)
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in
Example 1 above. This assembled consensus sequence is herein identified as DNA49798. Based on the
DNA49798 consensus sequence, oligonucleotides were synthesized: 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0938.
PCR primers (forward and reverse) were synthesized: forward PCR primer 5'-GTCCAGCCCATGACCGCCTCCAAC-3' (SEQ ID NO:8) reverse PCR primer 5'-CTCTCCTCATCCACACCAGCAGCC-3' (SEQ ID NO:9)
Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA49798 sequence which had the following nucleotide sequence: hybridization probe 5'-GTGGATGCTGAAATTTTACGCCCCATGGTGTCCATCCTGCCAGC-3' (SEQ ID NO: 10)
In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0938 gene using the probe ohgonucleotide and one of the PCR primers. RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB227).
DNA sequencing of the isolated clones isolated as described above gave the full-length DNA sequence for DNA56433-1406 [Figure 3, SEQ ID NO:6]; and the derived protein sequence for PR0938.
The entire coding sequence of DNA56433-1406 is included in Figure 3 (SEQ ID NO:6). Clone DNA56433- 1406 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 134-
136 and an apparent stop codon at nucleotide positions 1 181-1 183. The predicted polypeptide precursor is 349 amino acids long. Analysis of the full-length PR0938 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 of the full-length PR0938 polypeptide shown in Figure 4 evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 22; a transmembrane domain from about amino acid 189 to about amino acid 211 ; an N-glycosylation site from about amino acid 46 to about amino acid 50; and an N-myristoylation site from about amino acid 200 to about amino acid
206. Clone DNA56433- 1406 has been deposited with the ATCC on May 12, 1998 and is assigned ATCC deposit no. 209857. The full-length PR0938 protein shown in Figure 4 has an estimated molecular weight of about 38,952 daltons and a pi of about 4.34.
Analysis of the amino acid sequence of the full-length PR0938 polypeptide suggests that it possesses significant sequence similarity to protein disulfide isomerase, thereby indicating that PR0938 may be a novel protein disulfide isomerase An analysis of the Dayhoff database (version 35 45 S wissProt) evidenced significant homology between the PR0938 amino acid sequence and the following Dayhoff sequences P_W03626, P_W03627, P_R70491 , GARP_PLAFF, XLU85970_1 , ACADISPROA_l , IE68_HSVA, KSU52064_1 , U93872_83, P_R97866
EXAMPLE 4 Isolation of cDNA Clones Encoding Human PRO1031 (an ιnterleukιn-17 homolog) A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above This assembled consensus sequence is herein identified as DNA47332 Based upon an observed homology between the DNA47332 consensus sequence and the Merck EST clone no W74558 (clone 344649), the Merck EST clone no W74558 (clone 344649) was purchased and the insert obtained and sequenced DNA sequencing of the insert gave the full-length DNA sequence for PRO 1031 (herein designated as DNA59294-1381 [Figure 5, SEQ ID NO 1 1 ]) and the derived protein sequence for PRO 1031 (SEQ ID NO 12)
The entire nucleotide sequence of DNA59294-1381 is shown in Figure 5 (SEQ ID NO 1 1) Clone DNA59294-1381 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 42-44 and ending at the stop codon at nucleotide positions 582-584 (Figure 5) The predicted polypeptide precursor is 180 amino acids long (Figure 6) The full-length PRO 1031 protein shown in Figure 6 has an estimated molecular weight of about 20,437 daltons and a pi of about 9 58 Analysis of the full-length PROl 031 polypeptide sequence shown in Figure 6 evidences the presence of a variety of important polypeptide domains as shown in Figure 6, wherein the locations given for those important polypeptide domains are approximate Analysis of the full- length PRO 1031 polypeptide shown in Figure 6 evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 20, and an N-glycosylation site from about amino acid 75 to about amino acid 79 Clone DNA59294-1381 has been deposited with the ATCC on May 14, 1998 and is assigned ATCC deposit no 209866
EXAMPLE 5
Isolation of cDNA Clones Encoding Human PRQ296 (a sarcoma-amphf led protein SAS homolog) 1 Preparation of ohgo dT primed cDNA library mRNA was isolated from human kidney tissue using reagents and protocols from Invitrogen, San Diego, CA (Fast Track 2) This RNA was used to generate an ohgo dT primed cDNA library in the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script Plasmid System) In this procedure, the double stranded cDNA was sized to greater than 1000 bp and the Sall/Notl nkered cDΝA was cloned into Xhol/Νotl cleaved vector pRK5D is a cloning vector that has an sρ6 transcription initiation site followed by an Sfil restriction enzyme site preceding the Xhol/Νotl cDΝA cloning sites 2. Preparation of random primed cDNA library
A secondary cDNA library was generated in order to preferentially represent the 5' ends of the primary cDNA clones. Sp6 RNA was generated from the primary library (described above), and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY.O using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above). In this procedure the double stranded cDNA was sized to 500- 1000 bp, linkered with blunt to Notl adaptors, cleaved with Sfil, and cloned into Sfil/Notl cleaved vector. pSST- AMY.O is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cDΝA cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminator, after the cloning sites. Thus, cDΝAs cloned into this vector that are fused in frame with amylase sequence will lead to the secretion of amylase from appropriately transfected yeast colonies.
3. Transformation and Detection
DΝA from the library described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and vector mixture was then electroporated as recommended by the manufacturer. Subsequently, SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37°C for 30 minutes. The transformants were then plated onto 20 standard 150 mm LB plates containing ampicillin and incubated for 16 hours (37°C). Positive colonies were scraped off the plates and the DΝA was isolated from the bacterial pellet using standard protocols, e.g., CsCl-gradient. The purified DΝA was then carried on to the yeast protocols below.
The yeast methods were divided into three categories: (1) Transformation of yeast with the plasmid/cDΝA combined vector; (2) Detection and isolation of yeast clones secreting amylase; and (3) PCR amplification of the insert directly from the yeast colony and purification of the DΝA for sequencing and further analysis.
The yeast strain used was HD56-5A (ATCC-90785). This strain has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-l 12, his3-l 1 , his3-15, MAL+, SUC+, GAL+. Preferably, yeast mutants can be employed that have deficient post-translational pathways. Such mutants may have translocation deficient alleles in seel 1 , sec!2, *ec62, with truncated --ec71 being most preferred. Alternatively, antagonists (including antisense nucleotides and/or ligands) which interfere with the normal operation of these genes, other proteins implicated in this post translation pathway (e.g., SECόlp, SEC72ρ, SEC62p, SEC63p, TDJlp or SSAlp-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast.
Transformation was performed based on the protocol outlined by Gietz et al, Νucl. Acid. Res., 20: 1425 (1992). Transformed cells were then inoculated from agar into YEPD complex media broth (100 ml) and grown overnight at 30 °C. The YEPD broth was prepared as described in Kaiser etal, Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, ΝY, p. 207 (1994). The overnight culture was then diluted to about 2 x 106 cells/ml (approx. OD6(K,=0.1 ) into fresh YEPD broth (500 ml) and regrown to 1 x 107 cells/ml (approx. OD6(H)=0.4-0.5). The cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval
GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centrifuged again in 50 ml falcon tubes at 3,500 φra in a Beckman GS-6KR centrifuge. The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li2OOCCH,), and resuspended into LiAc/TE (2.5 ml).
Transformation took place by mixing the prepared cells ( 100 μl) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1 μg, vol. < 10 μl) in microfuge tubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li,OOCCH3, pH 7.5) was added. This mixture was gently mixed and incubated at 30°C while agitating for 30 minutes. The cells were then heat shocked at 42°C for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000 rp for 5-10 seconds, decanted and resuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followed by recentrifugation. The cells were then diluted into TE ( 1 ml) and aliquots (200 μl) were spread onto the selective media previously prepared in 150 mm growth plates (VWR).
Alternatively, instead of multiple small reactions, the transformation was performed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly.
The selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser etal, Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 208- 210 (1994). Transformants were grown at 30°C for 2-3 days.
The detection of colonies secreting amylase was performed by including red starch in the selective growth media. Starch was coupled to the red dye (Reactive Red-120, Sigma) as per the procedure described by Biely et al., Anal. Biochem., 172:176-179 (1988). The coupled starch was incorporated into the SCD-Ura agar plates at a final concentradon of 0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0 (50-100 mM final concentration).
The positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies. Well isolated single colonies positive for amylase secretion were detected by direct incorporation of red starch into buffered SCD-Ura agar. Positive colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly.
4. Isolation of DNA by PCR Amplification
When a positive colony was isolated, a portion of it was picked by a toothpick and diluted into sterile water (30 μl) in a 96 well plate. At this time, the positive colonies were either frozen and stored for subsequent analysis or immediately amplified. An aliquot of cells (5 μl) was used as a template for the PCR reaction in a 25 μl volume containing: 0.5 μl Klentaq (Clontech, Palo Alto, CA); 4.0 μl 10 mM dNTP's (Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clontech); 0.25 μl forward oligo 1 ; 0.25 μl reverse oligo 2; 12.5 μl distilled water. The sequence of the forward ohgonucleotide 1 was:
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID NO: 15) The sequence of reverse ohgonucleotide 2 was: 5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID NO: 16) PCR was then performed as follows
Denature 92°C, 5 minutes b 3 cycles of Denature 92°C, 30 seconds
Anneal 59°C, 30 seconds
Extend 72°C, 60 seconds c 3 cycles of Denature 92°C, 30 seconds
Anneal 57°C, 30 seconds
Extend 72°C, 60 seconds d 25 cycles of Denature 92°C, 30 seconds
Anneal 55°C, 30 seconds
Extend 72°C, 60 seconds
Hold 4°C
The underlined regions of the oligonucleotides annealed to the ADH promoter region and the amylase region, respectively, and amplified a 307 bp region from vector pSST-AMY 0 when no insert was present Typically, the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers
Thus, the total product of the PCR reaction from an empty vector was 343 bp However, signal sequence fused cDNA resulted in considerably longer nucleotide sequences
Following the PCR, an aliquot of the reaction (5 μl) was examined by agarose gel electrophoresis in a 1 % agarose gel using a Tπs-Borate-EDTA (TBE) buffering system as described by Sambrook et al , supra Clones resulting in a single strong PCR product larger than 400 bp were further analyzed by DNA sequencing after purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc , Chatsworth, CA)
5 Identification of Full-length Clone
A cDNA sequence isolated in the above screen was found, by BLAST and FastA sequence alignment, to have sequence homology to a nucleotide sequence encoding sarcoma-associated protein SAS This cDNA sequence is herein designated DNA23020 The DNA23020 sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homologies The homology search was performed using the computer program BLAST or BLAST2 ( Altshul etal , Methods in Enzymology, 266 460-480 (1996)) Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into a consensus DNA sequence with the program "phrap'
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above An intermediate consensus DNA sequence was extended using repeated cycles of BLAST and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above This consensus sequence is herein designated DNA35858
Based on the DNA35858 consensus sequence, ohgonucleotide probes ere generated and used to scieen a human kidney library (LIB228) prepared as described in paragraph 1 above The cloning vector was pRK5B (pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science, 253: 1278- 1280
( 1991 )), and the cDNA size cut was less than 2800 bp.
PCR primers (forward and reverse) were synthesized: forward PCR primer 1 : 5'-ACCCACGTCTGCGTTGCTGCC-3' (SEQ ID NO: 17) forward PCR primer 2:
5'-GAGAATATGCTGGAGAGG-3' (SEQ ID NO: 18) reverse PCR primer:
5'-AGGAATGCACTAGGATTCGCGCGG-3' (SEQ ID NO: 19) Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA35858 sequence which had the following nucleotide sequence: hybridization probe:
5'-GGCCCCAAAGGCAAGGACAAAGCAGCTGTCAGGGAACCTCCGCCG-3' (SEQ ID NO:20)
In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0296 gene using the probe ohgonucleotide and one of the PCR primers.
A full-length clone (designated herein as DNA39979- 1213) was identified that contained a single open reading frame with an apparent translational initiation site at nucleotide positions 174-176 and ending at the stop codon found at nucleotide positions 786-788 (Figure 7; SEQ ID NO: 13). The predicted polypeptide precursor is 204 amino acids long as shown in Figure 8 (SEQ ID NO: 14). The full-length PR0296 protein shown in Figure 8 has acalculated molecular weight of approximately 22, 147 daltons and an estimated pi of approximately 8.37. Analysis of the full-length PR0296 sequence shown in Figure 8 (SEQ ID NO: 14) evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 34; transmembrane domains from about amino acid
47 to about amino acid 63, from about amino acid 72 to about amino acid 95 and from about amino acid 162 to about amino acid 182; N-glycosylation sites from about amino acid 1 13 to about amino acid 1 17 and from about amino acid 137 to about amino acid 141 ; N-myristoylation sites from about amino acid 4 to about amino acid 10, from about amino acid 34 to about amino acid 40, from about amino acid 38 to about amino acid 44, from about amino acid 47 to about amino acid 53, from about amino acid 63 to about amino acid 69, from about amino acid
173 to about amino acid 179, and from about amino acid 183 to about amino acid 189, and prokaryotic membrane lipoprotein lipid attachment sites from about amino acid 84 to about amino acid 95 and from about amino acid 86 to about amino acid 97. Clone DNA39979-1213 has been deposited with ATCC on April 21 , 1998 and is assigned
ATCC deposit no. 209789.
Analysis of the amino acid sequence of the full-length PR0296 polypeptide suggests that it possesses significant sequence similarity to the sarcoma-amplified SAS protein, thereby indicating that PR0296 may be a novel SAS homolog. More specifically, an analysis of the Dayhoff database (version 35.45 SwissProt) evidenced significant homology between the PR0296 amino acid sequence and the following Dayhoff sequences: 158391 ,
GEN1 1061 , SSC2B04J , HSU81031 2, CD63_RAT, CD63_MOUSE, CD63JHUMAN, AF022813 , CD63JRABIT and CO02_HUMAN
EXAMPLE 6
Isolation of cDNA Clones Encoding Human PRQ213 (human giowth arrest specific gene 6 protein homolog,
PROl 330 and PR01449 (human notch 4 homologs) A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in
Example 1 above This assembled consensus sequence is herein identified as DNA28735 Based on the DNA28735 consensus sequence, oligonucleotides were synthesized 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0213 , PRO 1330 and or PRO 1449 PCR primers (forward and reverse) were synthesized forward PCR primer
5'-TGGAGCAGCAATATGCCAAGCC-3' (SEQ ID NO 27) reverse PCR primer
5'-TTTTCCACTCCTGTCGGGTTGG 3' (SEQ ID NO 28) Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA28735 sequence which had the following nucleotide sequence hybridization probe 5'-GGTGACACTTGCCAGTCAGATGTGGATGAATGCAGTGCTAGGAGGG-3' (SEQ ID NO 29)
In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above A positive library was then used to isolate clones encoding the PR0213, PRO1330 and/or PR01449 gene using the probe ohgonucleotide and one of the PCR primers RNA for construction of the cDNA libraries was isolated from human fetal lung tissue
DNA sequencing of the clones isolated as described above gave the full length DNA sequence for PR0213 (herein designated as DNA30943-1 163 [Figure 9, SEQ ID NO 21 ]), the full-length DNA sequence for PRO1031 (herein designated as DNA64907-1 163-1 [Figure 1 1 SEQ ID NO 23]), and the full-length DNA sequence for PR01449 (herein designated DNA64908-1 163-1 [Figure 13, SEQ ID NO 25]), respectively
The entire nucleotide sequences are shown in Figure 9 [DNA30943-1 163 (SEQ ID NO 21)], Figure 1 1 [DNA64907- 1 163-1 (SEQ ID NO 23)], and Figure 13 [DNA64908- 1163-1 (SEQ ID NO 25)], respectively DNA30943-1 163, DNA64907-1 163-1 and DNA64908 1 163-1 contain single open reading frames with apparent translational initiation sites at nucleotide positions 399-401 , 488 490 and 326 328, respectively, and ending at the stop codon at nucleotide positions 1218-1220, 1307-1309 and 1 145-1 147, respectively (Figures 9, 1 1 , & 13, respectively) The predicted PR0213 polypeptide precursor is 273 amino acids long as shown in Figure 10 (SEQ ID NO 22), the predicted PROl 330 polypeptide precursor is 273 amino acids long as shown in Figure 12 (SEQ ID NO 24), and the predicted PRO 1449 polypeptide precursor is 273 amino acids long as shown in Figure 14 (SEQ ID NO 26)
Analysis of the full length PR0213 sequence shown in Figure 10 (SEQ ID NO 22) evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 19; cAMP- and cGMP-dependent protein kinase phosphorylation sites from about amino acid 93 to about amino acid 97 and from about amino acid 270 to about amino acid 274; N-myristoylation sites from about amino acid 19 to about amino acid 25, from about amino acid 78 to about amino acid 84, from about amino acid 97 to about amino acid 103, from about amino acid 100 to about amino acid 106, from about amino acid 103 to about amino acid 109, from about amino acid 157 to about amino acid 163, from about amino acid 191 to about amino acid 197, and from about amino acid 265 to about amino acid 271 ; an amidation site from about amino acid 26 to about amino acid 30; an aspartic acid and asparagine hydroxylation site from about amino acid 152 to about amino acid 164; a cell attachment site from about amino acid 130 to about amino acid 133; and an EGF-like domain cysteine pattern signature from about amino acid 123 to about amino acid 135.
Analysis of the full-length PRO1330 sequence shown in Figure 12 (SEQ ID NO:24) evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 19; cAMP- and cGMP-dependent protein kinase phosphorylation sites from about amino acid 93 to about amino acid 97 and from about amino acid 270 to about amino acid 274; N-myristoylation sites from about amino acid 19 to about amino acid 25, from about amino acid 78 to about amino acid 84, from about amino acid 97 to about amino acid 103. from about amino acid 100 to about amino acid 106, from about amino acid 103 to about amino acid 109, from about amino acid 157 to about amino acid 163, from about amino acid 191 to about amino acid 197, and from about amino acid 265 to about amino acid 271 ; an amidation site from about amino acid 26 to about amino acid 30; an aspartic acid and asparagine hydroxylation site from about amino acid 152 to about amino acid 164; a cell attachment site from about amino acid 130 to about amino acid 133; and an EGF-like domain cysteine pattern signature from about amino acid 123 to about amino acid 135.
Analysis of the full-length PR01449 sequence shown in Figure 14 (SEQ ID NO:26) evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 19; cAMP- and cGMP-dependent protein kinase phosphorylation sites from about amino acid 93 to about amino acid 97 and from about amino acid 270 to about amino acid 274; N-myristoylation sites from about amino acid 19 to about amino acid 25, from about amino acid 78 to about amino acid 84, from about amino acid 97 to about amino acid 103. from about amino acid 100 to about amino acid 106, from about amino acid 103 to about amino acid 109, from about amino acid 157 to about amino acid 163, from about amino acid 191 to about amino acid 197, and from about amino acid 265 to about amino acid 271 ; an amidation site from about amino acid 26 to about amino acid 30; an aspartic acid and asparagine hydroxylation site from about amino acid 152 to about amino acid 164; a cell attachment site from about amino acid 130 to about amino acid 133; and an EGF-like domain cysteine pattern signature from about amino acid 123 to about amino acid 135.
DNA30943- 1 163, DNA64907- 1 163- 1 and DNA64908- 1 163- 1 have been deposited with ATCC on April 21 , 1998, September 9, 1998, and September 9, 1998, respectively, and are assigned ATCC deposit nos. 209791 , 203242, and 203243. respectively. EXAMPLE 7 Inhibition of VEGF-stimulated Endothehal Cell (ACE) Growth (ASSAY #9) The ability of PRO polypeptides to inhibit VEGF stimulated proliferation of endothehal cells was tested. Polypeptides testing positive in this assay are useful for inhibiting endothehal cell growth in mammals where such an effect would be beneficial, e.g., for inhibiting tumor growth.
Specifically, bovine adrenal cortical capillary endothehal cells (ACE) (from primary culture, maximum of 12-
14 passages) were plated in 96-well plates at 500 cells/well per 100 microliter. Assay media included low glucose
DMEM, 10% calf serum, 2 mM glutamine, and lX penicillin/streptomycin/fungizone. Control wells included the following: (1) no ACE cells added; (2) ACE cells alone; (3) ACE cells plus 5 ng/ml FGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3 ng/ml VEGF plus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGF plus
5 ng/ml LIF. The test sample, poly-his tagged PRO320 polypeptide (in 100 microliter volumes), was then added to the wells (at dilutions of 1 %, 0.1 % and 0.01 %, respectively). The cell cultures were incubated for 6-7 days at
37°C/5% C02. After the incubation, the media in the wells was aspirated, and the cells were washed 1 X with PBS.
An acid phosphatase reaction mixture (100 microliter; 0.1M sodium acetate, pH 5.5, 0.1% Triton X-100, 10 mM p-nitrophenyl phosphate) was then added to each well. After a 2 hour incubation at 37 °C, the reaction was stopped by addition of 10 microliters IN NaOH. Optical density (OD) was measured on a microplate reader at 405 nm.
The activity of PRO320 was calculated as the percent inhibition of VEGF (3 ng/ml) stimulated proliferation
(as determined by the acid phosphatase activity at OD 405 nm) relative to the cells without stimulation. TGF-beta was employed as an activity reference at 1 ng/ml, TGF-beta blocks 70-90% of VEGF-stimulated ACE cell proliferation. Results of the assay were interpreted as "positive" if the observed inhibition was > 30%.
PRO320 assayed "positive" as follows: PRO320 at 1%, 0.1 %, and 0.01 % dilutions exhibited 94%, 94% and 63% inhibition, respectively.
EXAMPLE 8 Induction of c-fos in Endothehal Cells (ASSAY #34) This assay is designed to determine whether PRO polypeptides show the ability to induce c-fos in endothehal cells. PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of conditions or disorders where angiogenesis would be beneficial including, for example, wound healing, and the like (as would agonists of these PRO polypeptides). Antagonists of the PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of cancerous tumors. Human venous umbilical vein endothehal cells (HUVEC, Cell Systems) in growth media (50% Ham's FI 2 w/o GHT: low glucose, and 50% DMEM without glycine: with NaHC03, 1 % glutamine, 10 mM HEPES, 10% FBS, 10 ng/ml bFGF) are plated on 96-well microtiter plates at a cell density of l xl O4 cells/well. The day after plating, the cells are starved by removing the growth media and treating the cells with 100 μl/well test samples and controls (positive control: growth media; negative control: 10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8). The cells are incubated for 30 minutes at 37°C, in 5% CO,. The samples are removed, and the first part of the bDNA kit protocol (Chiron Diagnostics, cat. #6005-037) is followed, where each capitalized reagent/buffer listed below is available from the kit.
Briefly, the amounts of the TM Lysis Buffer and Probes needed for the tests are calculated based on information provided by the manufacturer. The appropriate amounts of thawed Probes are added to the TM Lysis Buffer. The Capture Hybridization Buffer is warmed to room temperature. The bDNA strips are set up in the metal strip holders, and 100 μl of Capture Hybridization Buffer are added to each b-DNA well needed, followed by incubation for at least 30 minutes. The test plates with the cells are removed from the incubator, and the media are gently removed using the vacuum manifold. 100 μl of Lysis Hybridization Buffer with Probes are quickly pipetted into each well of the microtiter plates. The plates are then incubated at 55 °C for 15 minutes. Upon removal from the incubator, the plates are placed on the vortex mixer with the microtiter adapter head and vortex on the #2 setting for one minute. 80 μl of the lysate are removed and added to the bDNA wells containing the Capture Hybridization Buffer, and pipetted up and down to mix. The plates are incubated at 53 °C for at least 16 hours.
On the next day, the second part of the bDNA kit protocol is followed. Specifically, the plates are removed from the incubator and placed on the bench to cool for 10 minutes. The volumes of additions needed are calculated based upon information provided by the manufacturer. An Amplifier Working Solution is prepared by making a 1:100 dilution of the Amplifier Concentrate (20 fm/μl) in AL Hybridization Buffer. The hybridization mixture is removed from the plates and washed twice with Wash A. 50 μl of Amplifier Working Solution are added to each well and the wells are incubated at 53 °C for 30 minutes. The plates are then removed from the incubator and allowed to cool for 10 minutes. The Label Probe Working Solution is prepared by making a 1 : 100 dilution of Label Concentrate (40 pmoles/μl) in AL Hybridization Buffer. After the 10-minute cool-down period, the amplifier hybridization mixture is removed and the plates are washed twice with Wash A. 50 μl of Label Probe Working Solution are added to each well and the wells are incubated at 53°C for 15 minutes. After cooling for 10 minutes, the Substrate is warmed to room temperature. Upon addition of 3 μl of Substrate Enhancer to each ml of Substrate needed for the assay, the plates are allowed to cool for 10 minutes, the label hybridization mixture is removed, and the plates are washed twice with Wash A and three times with Wash D. 50 μl of the Substrate Solution with Enhancer are added to each well. The plates are incubated for 30 minutes at 37 °C and RLU is read in an appropriate luminometer.
The replicates are averaged and the coefficient of variation is determined. The measure of activity of the fold increase over the negative control (HEPES buffer described above) value is indicated by chemiluminescence units (RLU). Samples that show an at least two-fold value over the negative control value are considered positive. PR0938 assayed "positive" as shown in Table 4:
Table 4
ASSAY #1
Negative control = 1.0 RLU
Positive control = 10.96 RLU PR0938 at 0.60 nM = 2.02 RLU ASSAY #2
Negative control = 1.0 RLU Positive control = 10.96 RLU PR0938 at 0.60 nM = 2.62 RLU
PRO 1031 assayed "positive" as shown in Table 5:
Table 5
ASSAY #1
Negative control = 1.0 RLU Positive control = 10.96 RLU PRO 1031 at 0.056 nM = 2.22 RLU
ASSAY #2
Negative control = 1.0 RLU Positive control = 10.96 RLU PRO 1031 at 0.56 nM = 2.01 RLU
EXAMPLE 9
In situ Hybridization In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis, and aid in chromosome mapping.
In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett, Cell Vision, 1 : 169-176 (1994), using PCR-generated33P-labeledriboprobes. Briefly, formalin-fixed, paraffin-embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37 °C, and further processed for in situ hybridization as described by Lu and Gillett, supra. A (3 -P)UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at 55 °C overnight. The slides were dipped in Kodak NTB2™ nuclear track emulsion and exposed for 4 weeks. — P-Riboprobe synthesis
6.0 μl (125 mCi) of 33P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed-vacuum dried. To each tube containing dried 33P-UTP, the following ingredients were added: 2.0 μl 5x transcription buffer
LO μl DTT (lOO mM)
2.0 μl NTP mix (2.5 mM: 10 μl each of 10 mM GTP, CTP & ATP + 10 μl H20) 1.0 μl UTP (50 M) l .O μl RNAsin 1.0 μl DNA template (1 μg) 1.0 μl H2O
1.0 μl RNA polymerase (for PCR products T3 = AS, T7 = S, usually) The tubes were incubated at 37 °C for one hour. A total of 1.0 μl RQ1 DNase was added, followed by incubation at 37 °C for 15 minutes. A total of 90 μl TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) was added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a MICROCON-50™ ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, a total of 100 μl TE was added, then 1 μl of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR II™.
The probe was run on a TBE/urea gel. A total of 1-3 μl of the probe or 5 μl of RNA Mrk III was added to
3 μl of loading buffer. After heating on a 95 °C heat block for three minutes, the gel was immediately placed on ice. The wells of gel were flushed, and the sample was loaded and run at 180-250 volts for 45 minutes. The gel was wrapped in plastic wrap (SARAN™ brand) and exposed to XAR film with an intensifying screen in a -70 °C freezer one hour to overnight.
— P-Hvbridization
A. Pretreatment of frozen sections
The slides were removed from the freezer, placed on aluminum trays, and thawed at room temperature for 5 minutes. The trays were placed in a 55 °C incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H20). After deproteination in 0.5 μg/ml proteinase K for 10 minutes at 37 °C (12.5 μl of 10 mg/ml stock in 250 ml prewarmed RNAse-free RNAse buffer), the sections were washed in 0.5 x SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%, and 100% ethanol, 2 minutes each. B. Pretreatment of paraffin-embedded sections
The slides were deparaffinized, placed in SQ H20, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time. The sections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml
RNase-free RNase buffer; 37 °C, 15 minutes) for human embryo tissue, or 8 x proteinase K ( 100 μl in 250 ml Rnase buffer, 37 °C, 30 minutes) for formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above.
C. Prehybridization
The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper. The tissue was covered with 50 μl of hybridization buffer (3.75 g dextran sulfate + 6 ml SQ H20), vortexed, and heated in the microwave for 2 minutes with the cap loosened. After cooling on ice, 18.75 ml formamide, 3.75 ml 20 x SSC. and 9 ml SQ H20 were added, and the tissue was vortexed well and incubated at 42 °C for 1 -4 hours.
D. Hybridization
1.0 x 106 cpm probe and 1.0 μl tRN A (50 mg/ml stock) per slide were heated at 95 °C for 3 minutes. The slides were cooled on ice, and 48 μl hybridization buffer was added per slide. After vortexing, 50 μl 3P mix was added to 50 μl prehybridization on the slide. The slides were incubated overnight at 55 °C.
E. Washes
Washing was done for 2x 10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0.25 M EDTA, Vt=4L), followed by RNAseA treatment at 37 °C for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer = 20 μg/ml), The slides were washed 2 xlO minutes with 2 x SSC, EDTA at room temperature. The stringency wash conditions were as follows: 2 hours at 55 °C, 0.1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA,
VF4L).
F. Oligonucleotides In situ analysis was performed on two of the DNA sequences disclosed herein. The oligonucleotides employed for these analyses are as follows:
(1) DNA39979-1213 (PRQ296) (human SAS homolog - sarcoma amplified protein) p1 5'-GGATTCTAATACGACTCACTATAGGGCCCCAAAGGCAAGGACAAAGC-3' (SEQ ID NO:30) p2 5'-CTATGAAATTAACCCTCACTAAAGGGAACAGTGGACAGTGCCCGACAA-3' (SEQ ID NO:31) p3 5'-GGATTCTAATACGACTCACTATAGGGCTGCGCCCTCAACCTGCTTTAC-3' (SEQ ID NO:32) p4 5'-CTATGAAATTAACCCTCACTAAAGGGATGGACAGTGCCCGACAATGAA-3' (SEQ ID NO:33) p5 5'-GGATTCTAATACGACTCACTATAGGGCTGCGCCCTCAACCTGCTTTAC-3' (SEQ ID NO:34) p6 5'-CTATGAAATTAACCCTCACTAAAGGGATGGACAGTGCCCGACAATGAA-3' (SEQ ID NO:35)
(2) DNA64907-1 163-1 (PRO1330) (human notch homolog) p7 5'-GGATTCTAATACGACTCACTATAGGGCGCTGCCCCGGCTGGAAGAG-3' (SEQ ID NO:36) p8 5'-CTATGAAATTAACCCTCACTAAAGGGAAGCGCTGGGCAGTCACGAGTC-3' (SEQ ID NO:37) p9 5'-GGATTCTAATACGACTCACTATAGGGCGGATGGCGGGGTGACACTTG-3' (SEQ ID NO:38) plO 5'-CTATGAAATTAACCCTCACTAAAGGGACTTGGGATGCCGTTGGGGTAG-3' (SEQ ID NO:39)
G. Results In situ analysis was performed on the above two DNA sequences disclosed herein. The results from these analyses are as follows:
(1 ) DNA39979-1213 (PRQ296) (human SAS homolog - sarcoma amplified protein)
Widespread expression was observed in fetal adult tissues. Expression was found in a variety of adult and fetal epithelia, cardiac and skeletal muscle, developing (including retina) and adult CNS, thymic epithelium, placental villi, hepatocytes in cirrhotic and acetaminophen induced toxicity. High expression was observed in hypertrophic chondrocytes in developing skeletal system.
The overall expression pattern, while not completely overlapping (not expressed in glomeruli, more widely expressed in CNS), is not dissimilar to VEGF. These data are consistent with the potential role of this molecule in angiogenesis.
Human fetal tissues examined (E12-E16 weeks) included: placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs, great vessels, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis, testis and lower limb. Adult human tissues examined included: kidney (normal and end-stage), adrenal, spleen, lymph node, pancreas, lung, eye (including retina), bladder, liver (normal, cirrhotic, acute failure).
Non-human primate tissues examined included: chimp adrenal tissues and cerebral cortex, hippocampus and cerrebellum of rhesus monkey tissues.
(2) DNA64907- 1163-1 (PRO1330) (human notch homolog)
In human fetal tissues there was strong expression over arterial, venous, capillary and sinusoidal endothehum in all tissues examined, except for fetal brain. In normal adult tissues expression was low to absent, but when present appeared expression was confined to the vasculature. Highest expression in adult tissues was observed regionally in vessels running within the white matter of rhesus brain. Elevated expression was observed in vasculature of many inflamed and diseased tissues, including tumor vasculature. In some of these tissues it was unclear if expression was solely confined to vascular endothehum.
In the 15 lung tumors examined no expression was seen over the malignant epithelium, however, vascular expression was observed in many of the tumors and adjacent lung tissue. Moderate, apparently non-specific background, was seen with this probe over hyalinized collagen and sites of tissue necrosis. In the absence of a sense control, however, it is not possible to be absolutely certain that all of this signal is non-specific. Some signal, also thought to be non-specific, was seen over the chimp gastric mucosa, transitional cell epithelium of human adult bladder and fetal retina.
Although not examined, DNA64908- 1 163- 1 (PRO 1449) - a splice variant of DNA64907-1163- 1 (PRO 1330) with one amino acid difference - may be expected to exhibit similar expression patterns as DNA64907-1 163-1 (PROl 330).
EXAMPLE 10 Use of PRO320. PRQ938. PRO 1031. PRQ296. PRQ213. PROl 330 or PR01449 as a Hybridization Probe The following method describes use of a nucleotide sequence encoding PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO1330 or PR01449 as a hybridization probe. DNA comprising the coding sequence of full-length or mature PRO320, PR0938, PROl 031 , PR0296,
PR0213, PROl 330 or PR01449 (as shown in Figures 1 , 3 , 5, 7, 9, 1 1 , and 13, respectively, SEQ ID NOS: 1 , 6. 1 1 , 13, 21 , 23, and 25, respectively) or a fragment thereof is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449) in human tissue cDNA libraries or human tissue genomic libraries. Hybridization and washing of filters containing either library DNAs is performed under the following high- stringency conditions. Hybridization of radiolabeled probe derived from the gene encoding a PRO320, PR0938, PRO 1031 , PR0296. PR0213, PRO 1330 or PRO 1449 polypeptide to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1 % SDS, 0.1 % sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42"C for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1 x SSC and 0.1 % SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length native sequence can then be identified using standard techniques known in the art.
EXAMPLE 1 1
Expression of Nucleic Acid Encoding PRO320. PRQ938. PRO1031. PRQ296. PRQ213. PROl 330 or
PRO 1449 in E. coli This Example illustrates preparation of an unglycosylated form of PRO320, PR0938, PRO1031, PR0296,
PR0213, PRO1330 or PR01449 by recombinant expression in E. coli.
The DNA sequence encoding PRO320, PR0938. PRO1031, PR0296, PR0213, PRO 1330 or PRO 1449 (SEQ ID NOS: 1 , 6, 11 , 13, 21, 23, and 25, respectively) is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites that correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al. , Gene. 2: 95 ( 1977)), which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR-amplified sequences are then ligated into the vector. The vector will preferably include sequences that encode an antibiotic-resistance gene, a trp promoter, a poly-His leader (including the first six STII codons, poIy-His sequence, and enterokinase cleavage site), the region encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449, lambda transcriptional terminator, and an argU gene.
The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al. supra. Transformants are identified by their ability to grow on LB plates and antibiotic-resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.
Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger-scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.
After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide can then be purified using a metal- chelating column under conditions that allow tight binding of the polypeptide.
PRO 213, PRO 1330 and PRO 1449 were successfully expressed in E. coli in a poly-His tagged form by the above procedure. EXAMPLE 12 Expression of Nucleic Acid Encoding PRO320, PRQ938. PRO 1031 , PRQ296. PRQ213, PRO1330 or
PRQ1449 in Mammalian Cells This Example illustrates preparation of a potentially glycosylated form of PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO1330 or PR01449 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 PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the DNA encoding PRO320, PR0938, PRO 1031 , PR0296,
PR0213, PRO 1330 or PRO 1449 using ligation methods such as described in Sambrook et al. , supra. The resulting vector is called pRK5-(DNA encoding PRO320, PR0938, PRO1031, PR0296, PR0213, PRO 1330 or PRO 1449).
In one embodiment, the selected host cells are 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 1 Oμg DNA of ρRK5-(DNA encoding PRO320, PR0938, PRO 1031 ,
PR0296, PR0213, PRO1330 or PR01449) is mixed with about 1 μg DNA encoding the VA RNA gene (Thimmappava et al. Cell. 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 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaP04, and a precipitate is allowed to form for 10 minutes at 25°C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum-free medium, fresh medium is added, and the cells are incubated for about 5 days.
Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml 35S-cysteine and 200 μCi/ml 35S-methionine. After a 12- hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 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, the gene encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PROl 330 or PRO 1449 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al, Proc. Natl. Acad. Sci.. 2: 7575 (1981 ). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-(DNA encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449) is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin, and 0.1 μg/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing the expressed gene encoding the PRO320. PR0938, PRO1031. PR0296. PR0213, PRO1330 or PR01449 polypeptide can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
In another embodiment, the gene encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 can be expressed in CHO cells. The pRK5-(DNA encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449) nucleic acid can be transfected into CHO cells using known reagents such as CaP04 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35S-methionine. After determining the presence of PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 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 PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 can then be concentrated and purified by any selected method.
Epitope-tagged gene encoding the PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide may also be expressed in host CHO cells. The gene encoding PRO320, PR0938, PRO 1031, PR0296, PR0213, PRO1330 orPR01449 may be subcloned out of thepRK5 vector. The subclone insert can undergo PCR amplification to fuse in frame with a selected epitope tag such as a poly-His tag into a baculovirus expression vector. The gene insert encoding the poly-His-tagged-PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO 1330 or PR01449 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 gene encoding the poly-His-tagged-PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PRO 1449 can then be concentrated and purified by any selected method, such as by Ni2+-chelate affinity chromatography.
PRO320 and PROl 031 were stably expressed in CHO cells by the above described method. In addition, PR0938 was in expressed in CHO cells by a transient procedure.
EXAMPLE 13 Expression of Nucleic Acid Encoding PRO320, PRQ938. PRO1031. PRQ296. PRQ213, PRO1330 or
PRO 1449 in Yeast The following method describes recombinant expression of the gene encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO1330 or PR01449 in yeast.
First, yeast expression vectors are constructed for intracellular production or secretion of PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 from the ADH2/GAPDH promoter. DNA encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of the gene encoding PRO320. PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449. For secretion, DNA encoding PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PRO 1449 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO 1330 or PRO 1449 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (it needed) for expression of the gene encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449
Yeast cells, such as yeast strain AB 1 10, can then be transformed with the expression plasmids described above and cultured in selected fermentation media The transformed yeast supernatants can be analyzed by precipitation with 10% tπchloroacetic acid and separation by SDS PAGE, followed by staining of the gels with Coomassie Blue stain
Recombinant PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 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 PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PR01449 may further be purified using selected column-chromatography resins
EXAMPLE 14 Expression of Nucleic Acid Encoding PRO320. PRQ938, PRO1031 , PRQ296, PRQ213, PRO1330 or
PR01449 in Baculovirus Infected Insect Cells The following method describes recombinant expression in Baculovirus infected insect cells The sequence coding for PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 is fused upstream of an epitope tag contained within a baculovirus expression vector Such epitope tags include poly-His tags and immunoglobulm tags (like Fc regions of IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly, the sequence encoding PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 or the desired portion of the coding sequence of PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 [such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular] is amplified by PCR with primers complementary to the 5' and 3' regions The 5 primer may incorporate flanking (selected) restriction enzyme sites The product is then digested with those selected restriction enzymes and subcloned into the expression vector Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA
(Pharmmgen) into Spodopteiafi ugψerda ("Sf9") cells (ATCC CRL 1711) using hpofectin (commercially available from GIBCO-BRL) After 4 - 5 days of incubation at 28 °C, the released viruses are harvested and used for further amplifications Viral infection and protein expression are performed as described by O'Reilley et al , Baculovirus Expression Vectors A Laboratory Manual (Oxford Oxford University Pi ess (1994)) Expressed poly-His tagged-PRO320, PR0938, PRO 1031 , PR0296 PR0213, PROl 330 or PRO 1449 can then be purified, for example, by Ni °+-chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al , Nature. 362 175-179 (1993) Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes pH 7 9, 12 5 mM MgCl. , 0 1 M EDTA, 10% glycerol, 0 1 % NP-40 04 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 thiough a 0 45 μm filter A Ni 1+ NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 ml, washed with 25 ml of water and equilibrated with 25 ml of loading buffer The filtered cell extract is loaded onto the column at 0 5 ml per minute The column is washed to baseline A,MI 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 non-specifically bound protein After reaching A2 „ baseline again, the column is developed with a 0 to 500 mM lmidazole gradient in the secondary wash buffer One ml fractions are collected and analyzed by SDS PAGE and silver staining or Western blot with Ni 2+-NTA-conjugated to alkaline phosphatase (Qiagen) Fractions containing the eluted His,,, tagged- PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449, respectively, are pooled and dialyzed against loading buffer Alternatively, purification of thelgG-tagged (or Fc tagged)-PRO320, PR0938, PRO1031 , PR0296, PR0213,
PRO 1330 or PRO 1449 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography
PRO 1031 was successfully expressed in baculovirus infected Sf9 insect cells While expression was actually performed in a 0 5 2 L scale, it can be readily scaled up for larger (e g , 8 L) preparations The proteins were expressed as an IgG construct (immunoadhesin), in which the protein extracellular region was fused to an IgGl constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms
Following PCR amplification, the respective coding sequences were 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) were co-transfected into 105 Spodoptera frugψet da ("Sf9 ) cells (ATCC CRL 171 1), using Lipofectin (Gibco BRL) pb PH IgG and pb PH His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polyhnker regions to include the His or Fc tag sequences The cells were grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone) Cells were incubated foi 5 days at 28 °C The supernatant was harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hin s TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10 Cells were incubated for 3 days at 28 °C The supernatant was harvested and the expression of the constructs in the baculovirus expression vector was 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 was 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 were incubated for 3 days at 28 °C The supernatant was harvested and filtered Batch binding and SDS PAGE analysis was repeated, as necessary, until expression of the spinner culture was confirmed
The conditioned medium fiom the transfected cells (0 5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0 22 micron filters For the poly-His tagged constructs, the protein construct were purified using a Ni ,+-NTA column (Qiagen) Before purification, lmidazole was added to the conditioned media to a concentration of 5 mM The conditioned media were pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
Immunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows. The conditioned media were pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of the proteins was verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.
Alternatively, a modified baculovirus procedure may be used incorporating high-5 cells. In this procedure, the DNA encoding the desired sequence was amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector. Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIEl -1 (Novagen). The pIEl-1 and pIEl -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus iel promoter in stably-transformed insect cells. The plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for iel -mediated gene expression in uninfected insect cells as well as the hr5 enhancer element. pIEl-1 and pIEl-2 include the translation initiation site and can be used to produce fusion proteins. Briefly, the desired sequence or the desired portion of the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) was amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product was then digested with those selected restriction enzymes and subcloned into the expression vector. For example, derivatives of pIEl - 1 can include the Fc region of human IgG (pb.PH.IgG) or an 8 histidine (pb.PH.His) tag downstream (3'-of) the desired sequence. Preferably, the vector construct is sequenced for confirmation.
High-5 cells are grown to a confluency of 50% under the conditions of, 27 °C, no C02, NO pen/strep. For each 150 mm plate, 30 μg of pIE based vector containing the sequence was 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 of CellFectin (CellFEOTN (GibcoBRL #10362-010) (vortexed to mix)) was mixed with 1 ml of Ex-Cell medium. The two solutions were combined and allowed to incubate at room temperature for 15 minutes. 8 ml of Ex-Cell media was added to the 2 ml of DNA/CellFECTIN mix and this is layered on high-5 cells that have been washed once with Ex-Cell media. The plate is then incubated in darkness for 1 hour at room temperature. The DNA/CellFECTIN mix is then aspirated, and the cells are washed once with Ex-Cell to remove excess CellFECTIN, 30 ml of fresh Ex-Cell media was added and the cells are incubated for 3 days at 28 °C. The supernatant was harvested and the expression of the sequence in the baculovirus expression vector was determined by batch binding of 1 ml of supernatant to 25 ml of Ni +-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining. The conditioned media from the transfected cells (0.5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein comprising the sequence is purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni +-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 M imidazole at a flow rate of 4-5 ml/min. at 48 °C. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was then subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80 °C.
Immunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows. The conditioned media was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of the sequence was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary.
PRO 1031 was successfully expressed by the above modified baculovirus procedure incorporating high-5 cells.
EXAMPLE 15 Preparation of Antibodies that Bind PRO320. PRQ938. PRO1031. PRQ296. PRQ213. PROl 330 or PRQ1449 This Example illustrates preparation of monoclonal antibodies that can specifically bind PRO320, PR0938,
PRO1031 , PR0296, PR0213, PRO1330 or PR01449.
Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO1330 or PR01449 fusion proteins containing PRO320, PR0938, PRO1031 , PR0296, PR0213, PROl 330 or PROl 449, and cells expressing the gene encoding PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PRO 1449 on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the PRO320. PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PRO 1449 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1 to 100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi
Immunochemical Research, Hamilton, MT) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-PRO320, anti-PR0938, anti-PRO1031 , anti-PR0296, anti-PR0213, anti-PRO1330 or anti-PR01449 antibodies. After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449. 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 P3X63 AgU.1 , available from ATCC, No. CRL 1597. The fusions generate hybridoma cells that can then be plated in 96-well tissue culture plates containing HAT 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 PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 is within the skill in the art. The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PRO320, anti-PR0938, anti-PRO1031, anti-PR0296, anti-PR0213, anti-PRO1330 or anti- PRO 1449 monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue-culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium- sulfate precipitation, followed by gel-exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.
Deposit of Material
The following material(s) has/have been deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, VA 201 10-2209, USA (ATCC):
Material ATCC Deo. No. Deposit Date
DNA32284-1307 209670 March 1 1 , 1998
DNA56433-1406 209857 May 12, 1998
DNA59294-1381 209866 May 14, 1998
DNA39979-1213 209789 April 21 , 1998
DNA30943-1 163 209791 April 21, 1998
DNA64907- 1163-1 203242 September 9, 1998
DNA64908- 1163-1 203243 September 9, 1998
This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the
Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty).
This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc., and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC § 122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638).
The assignee of the present application has agreed that if a culture of the material(s) on deposit should die or be lost or destroyed when cultivated under suitable conditions, the material(s) will be promptly replaced on notification with another of the same. Availability of the deposited material(s) is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the cons truct(s) deposited, since the deposited embodiment(s) is/are intended as single illustration(s) of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material(s) herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A composition comprising a PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide or agonist or antagonist thereof, in admixture with a pharmaceutically acceptable carrier.
2. The composition of Claim 1 comprising a therapeutically effective amount of said polypeptide or said agonist or antagonist thereof.
3. The composition of Claim 1 , wherein the agonist is an anti-PRO320, anti-PR0938, anti-PRO 103 l. anti- PR0296, anti-PR0213, anti-PRO1330 or anti-PR01449 antibody.
4. The composition of Claim 1 , wherein the antagonist is an anti-PRO320, anti-PR0938, anti-PRO 1031 , anti-PR0296, anti-PR0213, anti-PRO1330 or anti-PR01449 antibody.
5. The composition of Claim 1 further comprising a cardiovascular, endothehal, angiogenic or angiostatic agent.
6. A method of preparing the composition of Claim 1 comprising admixing a PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide or agonist or antagonist thereof, with a pharmaceutically acceptable carrier.
7. An article of manufacture comprising:
(1 ) a composition comprising (a) a PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide, (b) an agonist of a PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PR01449 polypeptide, or (c) an antagonist of a PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PRO 1449 polypeptide, in admixture with a pharmaceutically acceptable carrier;
(2) a container containing said composition; and
(3) a label affixed to said container, or a package insert included in said container, referring to the use of said composition, in the treatment of a cardiovascular, endothehal, and angiogenic disorder.
8. The article of manufacture of Claim 7, wherein said agonist is an anti-PRO320, anti-PR0938, anti- PRO1031, anti-PR0296, anti-PR0213, anti-PRO1330 or anti-PR01449 antibody.
9. The article of manufacture of Claim 7, wherein said antagonist is an anti-PRO320, anti-PR0938, anti- PRO 1031 , anti-PR0296, anti-PR0213, anti-PRO 1330 or anti-PRO 1449 antibody.
10. The article of manufacture of Claim 7, wherein said composition comprises a therapeutically effective amount of said polypeptide or agonist or antagonist thereof, in admixture with said pharmaceutically acceptable carrier.
1 1. A method for identifying an agonist of a PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide comprising:
(a) contacting cells and a test compound to be screened under conditions suitable for the induction of a cellular response normally induced by a PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PRO 1449 polypeptide; and
(b) determining the induction of said cellular response to determine if the test compound is an effective agonist, wherein the induction of said cellular response is indicative of said test compound being an effective agonist.
12. The method of Claim 11, wherein the cellular response normally induced by said polypeptide is stimulation of cell proliferation.
13. A method for identifying a compound that inhibits an activity of a PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide comprising contacting a test compound with said polypeptide under conditions and for a time sufficient to allow the test compound and polypeptide to interact and determining whether the activity of said polypeptide is inhibited.
14. A method for identifying a compound the inhibits an activity of a PRO320, PR0938, PRO1031, PR0296, PR0213, PRO1330 or PR01449 polypeptide comprising the steps of:
(a) contacting cells and a test compound to be screened in the presence of said polypeptide under conditions suitable for the induction of a cellular response normally induced by said polypeptide; and
(b) determining the induction of said cellular response to determine if the test compound is an effective antagonist.
15. The method of Claim 14, wherein the cellular response normally induced by said polypeptide is stimulation of cell proliferation.
16. A method for identifying a compound that inhibits the expression of a PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PR01449 polypeptide in cells that normally expresses the polypeptide, wherein the method comprises contacting the cells with a test compound under conditions suitable for allowing expression of said polypeptide and determining whether the expression of said polypeptide is inhibited.
17. An agonist of a PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PR01449 polypeptide.
18. An antagonist of a PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide.
19. A compound that inhibits the expression of a PRO320, PR0938, PRO1031 , PR0296, PR0213, PROl 330 or PRO 1449 polypeptide in a mammalian cell which expresses said polypeptide.
20. The compound of Claim 19, wherein said compound is an antisense ohgonucleotide.
21. An isolated antibody that binds to a PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PRO 1449 polypeptide.
22. The antibody of Claim 21 which is a monoclonal antibody.
23. The antibody of Claim 21 which is an antibody fragment.
24. The antibody of Claim 21 which is a single-chain antibody.
25. A method for diagnosing a disease or susceptibility to a disease which is related to a mutation in a PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide-encoding nucleic acid sequence comprising determining the presence or absence of said mutation in said polypeptide-encoding nucleic acid sequence, wherein the presence or absence of said mutation is indicative of the presence of said disease or susceptibility to said disease.
26. A method of diagnosing a cardiovascular, endothehal or angiogenic disorder in a mammal which comprises analyzing the level of expression of a gene encoding a PRO320, PR0938, PRO 1031 , PR0296, PR0213, PROl 330 or PR01449 polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower expression level in the test sample as compared to the control sample is indicative of the presence of a cardiovascular, endothehal or angiogenic disorder in said mammal.
27. A method of diagnosing a cardiovascular, endothehal or angiogenic disorder in a mammal which comprises detecting the presence or absence of a PRO320, PR0938, PROl 031 , PR0296, PR0213, PROl 330 or PRO! 449 polypeptide in a test sample of tissue cells obtained from said mammal, wherein the presence or absence of said polypeptide in said test sample is indicative of the presence of a cardiovascular, endothehal or angiogenic disorder in said mammal.
28. A method of diagnosing a cardiovascular, endothehal or angiogenic disorder in a mammal comprising (a) contacting an anti-PRO320, anti-PR0938, anti-PRO 1031 , anti-PR0296, anti-PR0213, anti-PRO 1330 or anti- PRO 1449 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between said antibody and a PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide in the test sample, wherein the formation of said complex is indicative of the presence of a cardiovascular, endothehal or angiogenic disorder in the mammal.
29. A method for determining the presence of a PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide in a sample comprising contacting a sample suspected of containing said polypeptide with an anti-PRO320, anti-PR0938, anti-PRO1031, anti-PR0296, anti-PR0213, anti-PRO1330 or anti-PR01449 antibody and determining binding of said antibody to a component of said sample.
30. A cardiovascular, endothehal or angiogenic disorder diagnostic kit comprising an anti-PRO320, anti- PR0938, anti-PRO1031, anti-PR0296, anti-PR0213, anti-PRO1330 or anti-PR01449 antibody and a carrier in suitable packaging.
31. A method for treating a cardiovascular, endothehal or angiogenic disorder in a mammal comprising administering to the mammal a therapeutically effective amount of a PRO320, PR0938, PROl 031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide or agonist or antagonist thereof.
32. The method according to Claim 31 , wherein the mammal is human.
33. The method of Claim 32, wherein the human has suffered myocardial infarction.
34. The method of Claim 32, wherein the human has cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration.
35. The method of Claim 34, wherein the cardiac hypertrophy is characterized by the presence of an elevated level of PGF2α.
36. The method of Claim 31 , wherein the PRO320, PR0938, PRO 1031, PR0296, PR0213, PRO 1330 or PR01449 polypeptide is administered together with a cardiovascular, endothehal or angiogenic agent.
37. The method of Claim 34, wherein the PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PRO 1449 polypeptide is administered following primary angioplasty.
38. The method of Claim 31 , wherein the cardiovascular, endothehal or angiogenic disorder is cancer.
39. The method of Claim 38, wherein the PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide is administered in combination with a chemotherapeutic agent, a growth inhibitory agent or a cytotoxic agent.
40. The method of Claim 31 wherein said agonist is an anti-PRO320, anti-PR0938, anti-PRO 1031 , anti- PR0296, anti-PR0213, anti-PRO1330 or anti-PR01449 antibody.
41. The method of Claim 32 wherein said antagonist is an anti-PRO320, anti-PR0938, anti-PRO 1031, anti- PR0296, anti-PR0213, anti-PRO1330 or anti-PR01449 antibody.
42. A method for treating a cardiovascular, endothehal or angiogenic disorder in a mammal comprising administering to the mammal a nucleic acid molecule that encodes a PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide or agonist or antagonist thereof.
43. The method of Claim 42 wherein said agonist is an anti-PRO320, anti-PR0938, anti-PRO1031 , anti- PR0296, anti-PR0213, anti-PRO 1330 or anti-PRO 1449 antibody.
44. The method of Claim 42 wherein said antagonist is an anti-PRO320, anti-PR0938, anti-PRO 1031 , anti- PR0296, anti-PR0213, anti-PRO1330 or anti-PR01449 antibody.
45. The method of Claim 42, wherein the mammal is human.
46. The method of Claim 42, wherein the nucleic acid molecule is administered via ex vivo gene therapy.
47. A recombinant retroviral particle comprising a retroviral vector consisting essentially of ( 1 ) a promoter,
(2) nucleic acid encoding a PRO320, PR0938, PRO 1031 , PR0296, PR0213, PRO 1330 or PROl 449 polypeptide or agonist or antagonist thereof, and (3) a signal sequence for cellular secretion of the polypeptide, wherein the retroviral vector is in association with retroviral structural proteins.
48. An ex vivo producer cell comprising a nucleic acid construct that expresses retroviral structural proteins and also comprises a retroviral vector consisting essentially of a ( 1 ) promoter, (2) nucleic acid encoding a PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide or agonist or antagonist thereof, and
(3) a signal sequence for cellular secretion of the polypeptide, wherein said producer cell packages the retroviral vector in association with the structural proteins to produce recombinant retroviral particles.
49. A method for inhibiting endothehal cell growth in a mammal comprising administering to the mammal a PRO320, PR0938 or PROl 031 polypeptide or agonist thereof, wherein endothehal cell growth in said mammal is inhibited.
50. A method for stimulating endothehal cell growth in a mammal comprising administering to the mammal a PR0296, PR0213, PRO 1330 or PRO 1449 polypeptide or agonist thereof, wherein endothehal cell growth in said mammal is stimulated.
51. A method of inhibiting endothehal cell growth in a mammal comprising administering to the mammal an antagonist of a PR0296, PR0213, PRO1330 or PR01449 polypeptide, wherein endothehal cell growth in said mammal is inhibited.
52. A method of stimulating endothehal cell growth in a mammal comprising administering to the mammal an antagonist of a PRO320, PR0938 or PRO 1031 polypeptide, wherein endothehal cell growth in said mammal is stimulated.
53. A method for inhibiting angiogenesis induced by a PR0296, PR0213, PRO1330 or PR01449 polypeptide in a mammal comprising administering a therapeutically effective amount of an anti-PR0296, anti- PR0213, anti-PRO 1330 or anti-PRO 1449 antibody to the mammal, wherein said angiogenesis is inhibited.
54. A method for stimulating angiogenesis induced by a PR0296, PR0213, PRO1330 or PR01449 polypeptide in a mammal comprising administering a therapeutically effective amount of said polypeptide to the mammal, wherein said angiogenesis is stimulated.
55. 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: 12), Figure 8 (SEQ ID NO: 14), Figure 10 (SEQ ID NO:22), Figure 12 (SEQ ID NO:24) and Figure 14 (SEQ ID NO:26).
56. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1 ), Figure 3 (SEQ ID NO:6), Figure 5 (SEQ ID NO: l 1 ), Figure 7 (SEQ ID NO: 13), Figure 9 (SEQ ID NO:21 ), Figure 1 1 (SEQ ID NO:23) and Figure 13 (SEQ ID NO:25).
57. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the full-length coding sequence of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1 ), Figure 3 (SEQ ID NO:6), Figure 5 (SEQ ID NO: 1 1 ), Figure 7 (SEQ ID NO: 13), Figure 9 (SEQ ID NO:21 ), Figure 1 1 (SEQ ID NO:23) and Figure 13 (SEQ ID NO:25).
58. 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 209670, 209857, 209866, 209789, 209791 , 203242 or 203243.
59. A vector comprising the nucleic acid of any one of Claims 55 to 58.
60. The vector of Claim 59 operably linked to control sequences recognized by a host cell transformed with the vector.
61. A host cell comprising the vector of Claim 59.
62. The host cell of Claim 61 , wherein said cell is a CHO cell.
63. The host cell of Claim 61, wherein said cell is an E. coli.
64. The host cell of Claim 61 , wherein said cell is a yeast cell.
65. The host cell of Claim 61 , wherein said cell is a Baculovirus infected insect cell.
66. A process for producing a PRO320, PR0938, PRO1031 , PR0296, PR0213, PRO1330 or PR01449 polypeptide comprising culturing the host cell of Claim 61 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.
67. An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO: 12), Figure 8 (SEQ ID NO: 14), Figure 10 (SEQ ID NO:22), Figure 12 (SEQ ID NO:24) and Figure 14 (SEQ ID NO:26).
68. An isolated polypeptide scoring at least 80% positives when compared to an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO: 12), Figure 8 (SEQ ID NO: 14), Figure 10 (SEQ ID NO:22), Figure 12 (SEQ ID NO:24) and Figure 14 (SEQ ID NO:26).
69. 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 209670, 209857, 209866, 209789, 209791 , 203242 or 203243.
70. A chimeric molecule comprising a polypeptide according to any one of Claims 67 to 69 fused to a heterologous amino acid sequence.
71. The chimeric molecule of Claim 70, wherein said heterologous amino acid sequence is an epitope tag sequence.
72. The chimeric molecule of Claim 70, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulm.
73. An antibody which specifically binds to a polypeptide according to any one of Claims 67 to 69.
74. The antibody of Claim 73, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody.
75. 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: 12), Figure 8 (SEQ ID NO: 14), Figure 10 (SEQ ID NO:22), Figure 12 (SEQ ID NO:24) or Figure 14 (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: 12), Figure 8 (SEQ ID NO: 14), Figure 10 (SEQ ID NO:22), Figure 12 (SEQ ID NO:24) or Figure 14 (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: 12), Figure 8 (SEQ ID NO: 14), Figure 10 (SEQ ID NO:22), Figure 12 (SEQ ID NO:24) or Figure 14 (SEQ ID NO:26), lacking its associated signal peptide.
76. 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: 12), Figure 8 (SEQ ID NO: 14), Figure 10 (SEQ ID NO:22), Figure 12 (SEQ ID NO:24) or Figure 14 (SEQ ID NO:26), lacking its associated signal peptide;
(b) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO: 12), Figure 8 (SEQ ID NO: 14), Figure 10 (SEQ ID NO:22), Figure 12 (SEQ ID NO:24) or Figure 14 (SEQ ID NO:26), with its associated signal peptide; or
(c) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO: 12), Figure 8 (SEQ ID NO: 14), Figure 10 (SEQ ID NO:22), Figure 12 (SEQ ID NO:24) or Figure 14 (SEQ ID NO:26),lacking its associated signal peptide.
PCT/US1999/031274 1997-03-31 1999-12-30 Promotion or inhibition of angiogenesis and cardiovascularization WO2000053752A2 (en)

Priority Applications (764)

Application Number Priority Date Filing Date Title
AU25967/00A AU2596700A (en) 1999-03-08 1999-12-30 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
CA002361840A CA2361840A1 (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP00907314A EP1263948A2 (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
KR1020017011399A KR20030002292A (en) 1999-03-08 2000-02-18 Secreted and Transmembrane Polypeptides and Nucleic Acids Encoding the Same
JP2000603377A JP2004513602A (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding them
PCT/US2000/004341 WO2000053756A2 (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU35144/00A AU3514400A (en) 1999-03-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
KR1020017011406A KR20010103046A (en) 1999-03-08 2000-03-02 Compositions and Methods for the Treatment of Immune Related Diseases
EP00913764A EP1220905A2 (en) 1999-03-08 2000-03-02 Composition and methods for the treatment of immune related diseases
JP2000603379A JP2004516227A (en) 1999-03-08 2000-03-02 Compositions and methods for treating immune-related diseases
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
CA002491610A CA2491610A1 (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
EP06000586A EP1688497A1 (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
EP00983846A EP1250426A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding 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
AU20554/01A AU2055401A (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000587A EP1690872A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
EP06000581A EP1666494A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002490909A CA2490909A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2001542531A JP2004522404A (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding them
CA002391455A CA2391455A1 (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
CA002492049A CA2492049A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002491258A CA2491258A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002491433A CA2491433A1 (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
CA002490853A CA2490853A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000583A EP1686134A3 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000584A EP1669371A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
EP06000582A EP1666495A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/032678 WO2001040466A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05025102A EP1672070A3 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000585A EP1661996A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002494705A CA2494705A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU25909/01A AU2590901A (en) 1999-12-23 2000-12-20 Il-17 homologous polypeptides and therapeutic uses thereof
ES00989401T ES2333772T3 (en) 1999-12-23 2000-12-20 HOMOLOGICAL POLIPEPTIDES OF IL-17 AND IL-17R AND THEIR THERAPEUTIC USES
CA002391374A CA2391374A1 (en) 1999-12-23 2000-12-20 Il-17 homologous polypeptides and therapeutic uses thereof
EP00989401A EP1240325B1 (en) 1999-12-23 2000-12-20 Il-17 and il-17r homologous polypeptides and therapeutic uses thereof
EP07016900A EP1897944B1 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
EP10009491.1A EP2258848B1 (en) 1999-12-23 2000-12-20 Il-17 homologous polypeptide and therapeutic uses thereof
ES07016899T ES2379101T3 (en) 1999-12-23 2000-12-20 Homologous IL-17 polypeptides and therapeutic uses thereof
DE60043069T DE60043069D1 (en) 1999-12-23 2000-12-20 IL-17 AND IL-17R HOMOLOGOUS POLYPEPTIDES AND THEIR THERAPEUTIC USES
DK07016903.2T DK1897947T3 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and their therapeutic uses
DK00989401.5T DK1240325T3 (en) 1999-12-23 2000-12-20 IL-17 and IL-17R homologous polypeptides and therapeutic use thereof
AT07016900T ATE519847T1 (en) 1999-12-23 2000-12-20 IL-17 HOMOLOGUE POLYPEPTIDES AND THEIR THERAPEUTIC USE
EP07016899A EP1897943B1 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
AT00989401T ATE444361T1 (en) 1999-12-23 2000-12-20 IL-17 AND IL-17R HOMOLOGUE POLYPEPTIDES AND THERAPEUTIC USES
EP10009490A EP2290081A3 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptide and therapeutic uses thereof
ES07016901T ES2380958T3 (en) 1999-12-23 2000-12-20 Homologous IL-17 polypeptides and therapeutic uses thereof
DK07016900.8T DK1897944T3 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and their therapeutic use
EP07016903A EP1897947B1 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
PCT/US2000/034956 WO2001046420A2 (en) 1999-12-23 2000-12-20 Il-17 and il-17r homologous polypeptides and therapeutic uses thereof
PT00989401T PT1240325E (en) 1999-12-23 2000-12-20 Il-17 and il-17r homologous polypeptides and therapeutic uses thereof
DK07016901.6T DK1897945T3 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof.
AT07016899T ATE537258T1 (en) 1999-12-23 2000-12-20 IL-17 HOMOLOGUE POLYPEPTIDES AND THEIR THERAPEUTIC USE
ES07016903T ES2380812T3 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
ES09009972.2T ES2506665T3 (en) 1999-12-23 2000-12-20 Homologous IL-17 and IL-17R polypeptides and therapeutic uses thereof
ES07016902T ES2388748T3 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
PT07016901T PT1897945E (en) 1999-12-23 2000-12-20 Il-17 homologous polypeptides and therapeutic uses thereof
JP2001546918A JP2003527104A (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and their therapeutic uses
AT07016903T ATE541931T1 (en) 1999-12-23 2000-12-20 IL-17 HOMOLOGUE POLYPEPTIDES AND THEIR THERAPEUTIC USE
PT07016900T PT1897944E (en) 1999-12-23 2000-12-20 Il-17 homologous polypeptides and therapeutic uses thereof
EP07016902A EP1897946B1 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
US09/747,259 US6569645B2 (en) 1999-05-14 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
AT07016901T ATE541930T1 (en) 1999-12-23 2000-12-20 IL-17 HOMOLOGUE POLYPEPTIDES AND THEIR THERAPEUTIC USE
PT07016903T PT1897947E (en) 1999-12-23 2000-12-20 Il-17 homologous polypeptides and therapeutic uses thereof
EP07016901A EP1897945B1 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
ES10009491.1T ES2458349T3 (en) 1999-12-23 2000-12-20 Homologous IL-17 polypeptides and therapeutic uses thereof
EP09009972.2A EP2163625B1 (en) 1999-12-23 2000-12-20 IL-17 and IL-17R homologous polypeptides and therapeutic uses thereof
US09/816,744 US6579520B2 (en) 1998-05-15 2001-03-22 IL-17 related mammalian cytokine polypeptides (IL-17E)
US09/874,503 US20020177188A1 (en) 1998-05-15 2001-06-05 IL-17 homologous polypeptides and therapeutic uses thereof
US09/908,827 US20030054442A1 (en) 1998-05-15 2001-07-18 IL-17 homologous polypeptides and therapeutic uses thereof
US09/918,585 US20030060406A1 (en) 1997-10-17 2001-07-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,194 US20030195333A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,295 US20020156006A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,192 US20020177553A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,299 US20030199435A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,298 US20030134785A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,189 US6972325B2 (en) 1997-10-17 2001-10-15 PRO273 polypeptides
US09/978,188 US20030139328A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,193 US20030073624A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,191 US20030050239A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/981,915 US20030054986A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,403 US20030050240A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,757 US20030083248A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,375 US7196165B2 (en) 1997-10-17 2001-10-16 PRO363 polypeptides
US09/978,643 US20030104998A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,665 US7294700B2 (en) 1997-10-17 2001-10-16 Anti-PRO846 antibodies
US09/981,915 US7285623B2 (en) 1997-10-17 2001-10-16 PRO337 polypeptides
US09/978,423 US20030069178A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,802 US20030199674A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,564 US7195760B2 (en) 1997-10-17 2001-10-16 Anti-pro363 antibodies
US09/978,824 US20050124789A9 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,585 US20030049633A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,608 US20030045462A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,681 US20030195148A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,697 US20020169284A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,544 US20030199436A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,824 US20030055216A1 (en) 1997-10-17 2001-10-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/210,028 US20030203446A1 (en) 1998-10-07 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,388 US20040223964A1 (en) 1998-03-17 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,247 US7112657B2 (en) 1998-10-07 2001-10-19 PRO697 polypeptides
US10/145,124 US20030190701A1 (en) 1998-04-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,029 US7105640B2 (en) 1997-10-17 2001-10-19 Anti-pro792 antibodies
US10/145,089 US7208575B2 (en) 1998-10-07 2001-10-19 PRO531 polypeptides
US10/160,502 US7220835B2 (en) 1998-07-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,749 US20040029218A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,829 US20030194780A1 (en) 1998-04-29 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,017 US20030186365A1 (en) 1998-03-26 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,067 US7279553B2 (en) 1998-05-13 2001-10-19 PRO1083 polypeptides
US10/164,929 US20030194781A1 (en) 1998-03-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/166,709 US20030104536A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,036 US20050227342A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/162,521 US7067628B2 (en) 1998-03-17 2001-10-19 PRO788 polypeptides
US10/164,728 US20030186368A1 (en) 1998-05-13 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,922 US20030195345A1 (en) 1997-10-17 2001-10-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,832 US7132283B2 (en) 1997-10-17 2001-10-24 PRO273 polypeptides
US10/017,085 US6974696B2 (en) 1997-10-17 2001-10-24 PRO853 nucleic acids
US10/017,084 US20030203402A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,832 US20020192706A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/002,967 US20030148373A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,833 US6916648B2 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/020,445 US20030198994A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,083 US20030148376A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,830 US20030077700A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,086 US7122375B2 (en) 1997-10-17 2001-10-24 PRO274 nucleic acids
US10/017,081 US20030049684A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,829 US20030195344A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,834 US20030064407A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,927 US7189529B2 (en) 1997-10-17 2001-10-25 PRO792 nucleic acids
US10/013,928 US20030215905A1 (en) 1998-10-07 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,918 US20030211091A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/016,177 US20030073131A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,925 US7037710B2 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,917 US7029874B2 (en) 1998-03-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,926 US7074593B2 (en) 1998-04-01 2001-10-25 PRO 703 nucleic acids
US10/013,921 US20030068648A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,929 US7019124B2 (en) 1997-10-17 2001-10-25 PRO788 nucleic acids
US10/013,923 US7169912B2 (en) 1997-10-17 2001-10-25 PRO1017 nucleic acids
US10/013,920 US20040006219A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/000,157 US20020182673A1 (en) 1998-05-15 2001-10-30 IL-17 homologous polypedies and therapeutic uses thereof
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
US10/052,586 US20020127584A1 (en) 1997-09-18 2002-01-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,187 US20030096744A1 (en) 1997-10-17 2002-01-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/081,056 US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/119,480 US20040087769A1 (en) 1998-09-10 2002-04-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,046 US20030194791A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,051 US20030092147A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,040 US20030082759A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,047 US20030077778A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,044 US20030190717A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,041 US20030077776A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,042 US20030096386A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,045 US20030073210A1 (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
US10/121,055 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
US10/121,062 US20030077779A1 (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
US10/121,053 US20030199053A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,054 US20030199054A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,056 US20030082760A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,043 US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/121,057 US20030190719A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,061 US20030082761A1 (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
US10/121,058 US20030190720A1 (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
US10/121,050 US20030054516A1 (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
US10/123,214 US7343721B2 (en) 1997-03-31 2002-04-15 PRO4406 polypeptide
US10/123,155 US20030068794A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,291 US20030199058A1 (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
US10/123,292 US20030073211A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,262 US20030049816A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,108 US7635478B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,771 US20030199060A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,154 US20030190724A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,213 US7193048B2 (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
US10/123,157 US20030190725A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,261 US20030068796A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,156 US20030194792A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,212 US7276577B2 (en) 1997-03-31 2002-04-15 PRO1866 polypeptides
US10/123,109 US20030190723A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,322 US20030199059A1 (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
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,908 US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,913 US20030203462A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,912 US20030100087A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,903 US20030073212A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,902 US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,906 US20030190726A1 (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
US10/123,910 US7329404B2 (en) 1997-03-31 2002-04-16 Antibodies against PRO1310
US10/123,905 US20030087344A1 (en) 1997-06-18 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,911 US7408032B2 (en) 1997-03-31 2002-04-16 PRO1188 polypeptides
US10/123,905 US7285625B2 (en) 1997-06-18 2002-04-16 PRO536 polypeptides
US10/123,904 US20030022328A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,814 US7105335B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,823 US20030199062A1 (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
US10/124,822 US7109305B2 (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
US10/124,816 US20030190728A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,821 US20030199023A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,818 US20030082763A1 (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
US10/124,820 US20030190729A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,824 US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,805 US20030194794A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,795 US7304131B2 (en) 1997-03-31 2002-04-17 PRO1483 polypeptides
US10/125,704 US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
US10/125,924 US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/125,931 US20030199063A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,922 US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/125,927 US20030190731A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,932 US7317079B2 (en) 1997-03-31 2002-04-19 PRO812 polypeptides
US10/127,831 US20030082689A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,689 US20030087365A1 (en) 1997-03-31 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,817 US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
US10/131,825 US7282566B2 (en) 1997-03-31 2002-04-24 PRO1779 polypeptide
US10/131,823 US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 polypeptides
US10/063,521 US20030190669A1 (en) 1998-12-30 2002-05-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,519 US20030009013A1 (en) 1998-12-30 2002-05-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,518 US7465785B2 (en) 1999-03-08 2002-05-01 Polypeptide encoded by a nucleic acid over-expressed in melanoma
US10/063,517 US7232889B2 (en) 1999-03-08 2002-05-01 PRO300 antibodies
US10/063,510 US7109292B2 (en) 1999-03-08 2002-05-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,520 US20030187196A1 (en) 1998-12-30 2002-05-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,551 US20020183494A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,551 US7214777B2 (en) 1999-03-08 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,547 US20020182638A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,526 US20030171550A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,536 US7259238B2 (en) 1999-03-08 2002-05-02 Polypeptide encoded by a nucleic acid under-expressed in stomach and lung tumor
US10/063,569 US20030018168A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,555 US20030065143A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,545 US20020183505A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,536 US20030181696A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,548 US7232892B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptide encoded by a nucleic acid differentailly expressed in stomach, lung and melanoma tumor
US10/063,569 US7378491B2 (en) 1999-03-08 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,538 US7253255B2 (en) 1999-03-08 2002-05-02 Polypeptide encoded by a nucleic acid underexpressed in esophageal tumor and melanoma
US10/063,544 US20030027212A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,527 US20030181637A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,523 US20030181636A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,564 US20030180794A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,546 US7435798B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptide encoded by a nucleic acid overexpressed in normal stomach, normal skin and kidney tumor
US10/063,528 US20030181666A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,566 US20030073821A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,567 US20030069394A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,540 US7193061B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptide encoded by a nucleic acid underexpressed in esophageal tumor and melanoma
US10/063,563 US20030060602A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,532 US7202336B2 (en) 1999-03-08 2002-05-02 Polypeptide encoded by a nucleic acid overexpressed in kidney tumor and underexpressed in lung tumor
US10/063,541 US20030060601A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,554 US7223841B2 (en) 1999-03-08 2002-05-02 PR0994 antibodies
US10/063,561 US7271247B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptide encoded by a nucleic acid differentially expressed in melanoma
US10/063,547 US7294690B2 (en) 1999-03-08 2002-05-02 Polypeptides encoded by a nucleic acid differentially expressed in lung tumor
US10/063,525 US20030036634A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,524 US7205391B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptide suppressed in stomach tumors
US10/063,553 US7235630B2 (en) 1999-03-08 2002-05-02 PRO994 polypeptides
US10/063,568 US20030181668A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,540 US20030181667A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,523 US7220830B2 (en) 1999-03-08 2002-05-02 Polypeptide underexpressed in stomach tumors
US10/063,570 US7507404B2 (en) 1999-03-08 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,534 US7193060B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptides encoded by a nucleic acid overexpressed in kidney tumor and underexpressed in lung tumor
US10/063,553 US20030045684A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,530 US7193059B2 (en) 1999-03-08 2002-05-02 Antibodies that recognize a polypeptide overexpressed in rectal tumors
US10/063,545 US7256261B2 (en) 1999-03-08 2002-05-02 Polypeptides encoded by a nucleic acid over expressed in normal stomach normal skin and kidney tumor
US10/063,565 US20030180904A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,524 US20030027992A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,560 US7232882B2 (en) 1999-03-08 2002-05-02 Polypeptide uder-expressed in melanoma
US10/063,537 US7276586B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptide encoded by a nucleic acid under-expressed in stomach and lung tumor
US10/063,548 US20030187228A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,562 US20030181697A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,549 US7253256B2 (en) 1999-03-08 2002-05-02 Polypeptides encoded by a nucleic acid are expressed in esophageal and kidney tumor
US10/063,596 US7285624B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,868 US20030082764A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,592 US7220841B2 (en) 1998-10-27 2002-05-03 Antibody to PRO1557 polypeptide encoded by a nucleic acid highly expressed in esophageal and kidney tumors
US10/063,604 US7390876B2 (en) 1999-03-08 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
US10/063,611 US20030181677A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,614 US7393931B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,616 US7358339B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,589 US20030181641A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,591 US7196174B2 (en) 1998-10-27 2002-05-03 PRO1557 polypeptide encoded by a nucleic acid highly expressed in esophageal and kidney tumors
US10/063,606 US20030181675A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,578 US7417125B2 (en) 1999-03-08 2002-05-03 Antibodies that bind a PRO1158 polypeptide
US10/063,610 US7371814B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,579 US20030181638A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,588 US20030130483A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,577 US7423130B2 (en) 1999-03-08 2002-05-03 PRO1158 polypeptides
US10/063,583 US7189804B2 (en) 1999-03-08 2002-05-03 PRO1335 polypeptides
US10/063,582 US7205389B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,610 US20030180907A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,587 US7244428B2 (en) 1998-09-10 2002-05-03 PRO1357 antibodies
US10/063,598 US7223838B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,597 US7227000B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,615 US7405269B2 (en) 1998-10-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,617 US7423119B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,612 US7399828B2 (en) 1998-09-24 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,618 US7468424B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,607 US7345145B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,611 US7378501B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,591 US20030180906A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,609 US7402661B2 (en) 1998-10-06 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
US10/063,592 US20030181672A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,600 US7230076B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,581 US7189803B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,602 US7230082B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,593 US7189805B2 (en) 1999-03-08 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
US10/140,470 US20030022331A1 (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
US10/140,023 US20030207416A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,474 US20030032156A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,020 US20030207415A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,809 US20030207418A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,671 US20030180840A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,921 US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
US10/140,864 US20030207419A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,675 US20030180842A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,647 US20030187197A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,860 US7307151B2 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,661 US7193047B2 (en) 1999-03-08 2002-05-07 Polypeptide encoded by a nucleic acid underexpressed in esophageal tumor
US10/063,642 US20030181650A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,639 US7081520B2 (en) 1998-10-08 2002-05-07 Anti-pro 1570 antibodies
US10/063,659 US7186801B2 (en) 1999-03-08 2002-05-07 Polypeptide encoded by a nucleic acid underexpressed in stomach tumor and lung tumor
US10/140,805 US20030207417A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,670 US20030180839A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,648 US7193062B2 (en) 1998-10-20 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid over expressed in esoprageal and lung tumor, and under expressed in kidney tumor and melanoma
US10/063,660 US7189822B2 (en) 1999-03-08 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid underexpressed in stomach and lung tumor
US10/063,677 US20030187242A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,652 US7230077B2 (en) 1999-03-08 2002-05-07 Polypeptide encoded by a nucleic acid underexpressed in melanoma
US10/063,672 US20030181700A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,662 US20030180795A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,668 US20030191290A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,674 US20030180841A1 (en) 1998-12-30 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
US10/063,651 US7193057B2 (en) 1997-10-29 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid underexpressed in rectal tumor
US10/140,925 US20030073215A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,653 US7238787B2 (en) 1999-03-08 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid underexpressed in melanoma
US10/063,644 US7196167B2 (en) 1998-10-20 2002-05-07 Polypeptide underexpressed in lung tumor
US10/063,646 US7189821B2 (en) 1998-10-20 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid underexpressed in lung tumor
US10/063,644 US20030181651A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,638 US7101970B2 (en) 1999-03-08 2002-05-07 PRO1570 polypeptides
US10/063,676 US20030180843A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,646 US20030181681A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,649 US20030181652A1 (en) 1998-12-30 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
US10/063,673 US20030180908A1 (en) 1998-12-30 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
US10/063,640 US7354997B2 (en) 1999-03-08 2002-05-07 Polypeptide encoded by a nucleic acid underexpressed in stomach tumor
US10/063,669 US20030180838A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,654 US7202337B2 (en) 1999-03-08 2002-05-07 Polypeptide encoded by a nucleic acid overexpressed in esophageal tumor, normal stomach and melanoma
US10/063,643 US20030181680A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,650 US7217787B2 (en) 1999-03-08 2002-05-07 Polypeptide encoded by a nucleic acid underexpressed in rectal tumor
US10/063,664 US7256262B2 (en) 1999-03-08 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,657 US7193063B2 (en) 1999-03-08 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid overexpressed in esophageal tumor, normal stomach and melanoma
US10/063,647 US7193046B2 (en) 1998-10-20 2002-05-07 Polypeptide encoded by a nucleic acid overexpressed in esophageal and lung tumor, and underexpressesd in kidney tumor and melanoma
US10/063,666 US7411037B2 (en) 1999-03-08 2002-05-07 Polypeptides encoded by a nucleic acid underexpressed in melanoma
US10/063,665 US7427664B2 (en) 1999-03-08 2002-05-07 Antibodies to polypeptides that stimulate TNF-α release
US10/063,712 US20030180912A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,715 US20030180914A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,684 US20030186407A1 (en) 1998-12-30 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
US10/063,744 US20030180863A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,707 US20030180853A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,726 US20030180919A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,698 US20030180849A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,711 US20030180911A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,688 US20030186408A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,736 US20030180860A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,721 US20030181702A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,714 US20030180913A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,710 US20030180910A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,754 US7361732B2 (en) 1997-03-31 2002-05-08 PRO4400 polypeptides
US10/141,760 US7342104B2 (en) 1997-03-31 2002-05-08 Antibodies against the PRO4320 polypeptide
US10/063,682 US20030181701A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,705 US7220850B2 (en) 1999-03-08 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,689 US20030180845A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,699 US20030180850A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,728 US20030180920A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,716 US20030180915A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,717 US20030180916A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,718 US20030190698A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,724 US20030180856A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,730 US20030180858A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,756 US7488586B2 (en) 1997-03-31 2002-05-08 PRO4409 polypeptides
US10/063,723 US20030181703A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,731 US20030180921A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,701 US20030207421A1 (en) 1997-03-31 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,720 US20030180917A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,732 US20030180922A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,694 US20030180848A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,713 US20030180855A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,703 US7189563B2 (en) 1999-03-08 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,685 US20030180909A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,727 US20030180857A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,702 US7193074B2 (en) 1999-03-08 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,693 US20030180847A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,735 US20030138882A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,734 US20030180859A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,686 US20030180844A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,722 US20030180918A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,709 US7189564B2 (en) 1999-03-08 2002-05-08 PRO1335 nucleic acids
US10/063,692 US20030180846A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,417 US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/143,113 US7329730B2 (en) 1997-03-31 2002-05-09 PRO4348 polypeptides
US10/142,425 US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,742 US7189532B2 (en) 1999-03-08 2002-05-09 Nucleic acid underexpressed in stomach tumor and lung tumor
US10/063,745 US20040058411A1 (en) 1998-12-30 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,430 US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/063,741 US7118887B2 (en) 1999-03-08 2002-05-09 Nucleic acid overexpressed in esophageal tumor, normal stomach and melanoma
US10/063,743 US20030180862A1 (en) 1998-12-30 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,114 US20030036180A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 polypeptides
US10/142,431 US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/142,423 US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,419 US7153941B2 (en) 1997-03-31 2002-05-10 Antibodies that bind PRO4994 polypeptides
US10/146,730 US20030207427A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,792 US20030207428A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,528 US20030219885A1 (en) 1997-03-31 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,536 US20040077064A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,492 US20030082765A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,519 US20030077791A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,395 US7189534B2 (en) 1997-03-31 2002-05-21 PRO4320 polynucleotide
US10/153,934 US20030129695A1 (en) 1997-03-31 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,843 US20030207805A1 (en) 1997-06-18 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,782 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
US10/158,791 US20030207429A1 (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
US10/173,696 US20030082767A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,695 US20030032101A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,689 US20030166104A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,703 US20030170794A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,701 US20030104538A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,691 US20030166106A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,698 US20030166108A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,707 US20030166110A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,692 US20030166188A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,694 US20030166107A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,706 US20030022293A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,700 US20030027262A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,702 US20030170793A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,704 US20030170795A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,708 US20030040053A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,699 US20030166109A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,705 US20030032103A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,693 US20030073169A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,690 US20030166105A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,697 US20030032102A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,570 US20030211572A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,582 US20030027265A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,576 US7125962B2 (en) 1997-09-18 2002-06-18 Anti-Pro268 antibodies
US10/175,740 US20030027268A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,579 US20030027264A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,578 US20030073170A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,583 US7211645B2 (en) 1997-09-18 2002-06-18 PRO268 polypeptides
US10/174,589 US20030166114A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,591 US20030166115A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,574 US20030170796A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,588 US20030027266A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,581 US7153939B2 (en) 1997-09-18 2002-06-18 PRO354 antibodies
US10/174,572 US20030027263A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,585 US20030032105A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,586 US20030032106A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,587 US20030166113A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,590 US20030008352A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,569 US20030166111A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,735 US20030082715A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,737 US20030013153A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,746 US20030027270A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,739 US20030027267A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,752 US20030022295A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,744 US20030166119A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,736 US20030166117A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,743 US20030027269A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,745 US20030166120A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,751 US20030166122A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,748 US20030166121A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,747 US20030032107A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,754 US20030166123A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,738 US20030022294A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,742 US20030166118A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,741 US20030073171A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,753 US20030077732A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,750 US20030073172A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,749 US20050196832A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,920 US20030166129A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,746 US20030068680A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,482 US20030022296A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,919 US20030032114A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,753 US20030044917A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,491 US20030087373A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,990 US20030036119A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,483 US20030017541A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,989 US20030170803A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,493 US20030032111A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,914 US20030017543A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,981 US20030170800A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,747 US20030027273A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,484 US20030059876A9 (en) 1997-09-18 2002-06-20 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
US10/176,911 US20030032113A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,479 US20030040054A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,988 US20030170802A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,757 US7317082B2 (en) 1997-09-18 2002-06-20 PRO1018 polypeptides
US10/176,754 US7709602B2 (en) 1997-09-18 2002-06-20 PRO1078 polypeptides
US10/176,749 US20030017542A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,759 US20030166128A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,917 US20030044918A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,921 US20030027276A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,993 US20030027280A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,485 US20030032109A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,918 US7495083B2 (en) 1997-09-18 2002-06-20 PRO940 antibodies
US10/176,487 US20030032110A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,490 US20030170798A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,992 US20030027279A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,486 US7354999B2 (en) 1997-09-18 2002-06-21 PRO1481 polypeptides
US10/176,983 US20030170801A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,481 US20030032108A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,987 US20030027278A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,985 US20030027277A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,925 US20030032115A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,488 US20030027271A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,986 US20030073173A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,923 US20030068681A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,751 US20030036117A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,922 US20030166130A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,758 US20030008353A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,755 US20030166127A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,760 US7339033B2 (en) 1998-06-26 2002-06-21 Pro1481
US10/176,978 US20030032116A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,489 US20030166125A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,916 US20030040056A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,492 US20030027272A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,750 US20030027274A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,979 US20030087374A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,480 US20030166124A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,748 US20030040055A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,756 US20030032112A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,752 US20030170799A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,924 US20030166131A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,991 US20030027324A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,915 US20030017544A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,982 US20030044919A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,526 US20030100061A1 (en) 1998-06-26 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,507 US20030040057A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,519 US7339024B2 (en) 1997-09-18 2002-06-24 PRO1772 polypeptides
US10/179,509 US20030207392A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,506 US20030044920A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,525 US20030040060A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,513 US20030044921A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,523 US20030215909A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,516 US20030040058A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,514 US20030044922A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,515 US20030166135A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,517 US20030170805A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,522 US20030044923A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,508 US20030166133A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,520 US20030096353A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,510 US20030032117A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,518 US20030104540A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,511 US20030104539A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,521 US20030170806A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,512 US20030166134A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,552 US7348415B2 (en) 1997-09-18 2002-06-25 PRO1316 antibodies
US10/180,541 US20030036120A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,551 US20030036123A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,554 US20050202526A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,547 US20030032121A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,553 US7365156B2 (en) 1997-09-18 2002-06-25 PRO1316 polypeptides
US10/180,548 US7696319B2 (en) 1997-09-18 2002-06-25 PRO1772 antibodies
US10/180,560 US20030044925A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,557 US20030022301A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,559 US20030032124A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,545 US20030040062A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,549 US20030032122A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,550 US20030064440A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,546 US20030032120A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,543 US20030032118A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,542 US20030036121A1 (en) 1998-06-26 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,555 US20030032123A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,556 US7355000B2 (en) 1997-09-18 2002-06-25 PRO1380 polypeptides
US10/180,544 US20030032119A1 (en) 1998-06-26 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,540 US20030040061A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,998 US7087421B2 (en) 1997-09-18 2002-06-26 Pro1278 polypeptides
US10/183,010 US20030032126A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,011 US20030068682A1 (en) 1998-06-26 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,015 US20030044926A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,002 US20030054454A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,016 US20030082717A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/181,000 US7319137B2 (en) 1997-09-18 2002-06-26 PRO1339 polypeptides
US10/183,018 US20030104541A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,014 US20030064441A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,009 US7339034B2 (en) 1997-09-18 2002-06-26 PRO1305 antibodies
US10/183,003 US20030082716A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,999 US7297767B2 (en) 1997-09-18 2002-06-26 PRO1374 polypeptides
US10/183,005 US7317093B2 (en) 1997-09-18 2002-06-26 PRO1339 antibodies
US10/183,013 US7309769B2 (en) 1997-09-18 2002-06-26 PRO1487 polypeptides
US10/183,017 US20030040065A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,001 US7084255B2 (en) 1997-09-18 2002-06-26 PRO1278 polypeptides
US10/183,006 US7297776B2 (en) 1997-09-18 2002-06-26 PRO1374 antibodies
US10/183,019 US7425605B2 (en) 1997-09-18 2002-06-26 PRO1486 polypeptides
US10/183,012 US7718770B2 (en) 1997-09-18 2002-06-26 PRO1305-polypeptides
US10/183,008 US20030040064A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,627 US7282569B2 (en) 1997-09-18 2002-06-27 PRO1508 antibodies
US10/184,631 US20030036134A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,630 US20030036133A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,638 US20030054456A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,616 US20030036128A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,627 US20030040070A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,628 US7309770B2 (en) 1997-09-18 2002-06-27 PRO1757 polypeptides
US10/184,654 US7378486B2 (en) 1997-09-18 2002-06-27 PRO1482 antibodies
US10/184,651 US7291704B2 (en) 1997-09-18 2002-06-27 PRO1758 polypeptides
US10/184,615 US20030044927A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,641 US20030073174A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,614 US20030032128A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,613 US20030119105A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,633 US20030068683A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,642 US7332573B2 (en) 1997-09-18 2002-06-27 PRO1571 polypeptides
US10/184,612 US20030036127A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,630 US7304143B2 (en) 1997-09-18 2002-06-27 PRO1571 antibodies
US10/184,619 US20030049738A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,618 US7393917B2 (en) 1997-09-18 2002-06-27 PRO1482 polypeptides
US10/184,652 US20030032134A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,640 US7271250B2 (en) 1998-06-26 2002-06-27 PRO1757 antibodies
US10/184,647 US20030032133A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,657 US20030104543A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,650 US20030036138A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,624 US20030104542A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,620 US20030044928A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,646 US20030032132A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,626 US20030040069A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,655 US20030040073A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,658 US20030027281A1 (en) 1998-06-26 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,621 US20030054455A1 (en) 1998-06-26 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,625 US20030040068A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,634 US20030068684A1 (en) 1998-06-26 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,636 US20030036136A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,645 US7291718B2 (en) 1998-06-26 2002-06-28 PRO1758 antibodies
US10/184,629 US20030036132A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,637 US20030032131A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,644 US20030044930A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,617 US20030036129A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,643 US20030044929A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,623 US20030032129A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,632 US20030036135A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,656 US20030044931A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,635 US20030032130A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,622 US20030036130A1 (en) 1997-09-18 2002-06-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,594 US7294335B2 (en) 1998-06-26 2002-07-01 PRO19645 antibodies
US10/187,884 US20030036155A1 (en) 1997-09-18 2002-07-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,597 US20030036141A1 (en) 1997-09-18 2002-07-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,886 US7291708B2 (en) 1997-09-18 2002-07-01 PRO1785 polypeptides
US10/187,601 US7291705B2 (en) 1997-09-18 2002-07-01 PRO19645 polypeptides
US10/187,739 US7291706B2 (en) 1998-06-26 2002-07-01 PRO4352 polypeptides
US10/187,588 US7351795B2 (en) 1998-06-26 2002-07-01 PRO19563 polypeptides
US10/187,598 US20030036142A1 (en) 1997-09-18 2002-07-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,747 US7291707B2 (en) 1997-09-18 2002-07-01 PRO1337 polypeptides
US10/187,887 US7285645B2 (en) 1997-09-18 2002-07-01 PRO4356 antibodies
US10/188,780 US7268217B2 (en) 1998-06-26 2002-07-02 PRO4421 polypeptides
US10/187,743 US20030036148A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,741 US20030036147A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,747 US20030036150A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,596 US20030032136A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,753 US20030036152A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,885 US20030032138A1 (en) 1998-06-24 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,746 US20030036149A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,603 US20030036146A1 (en) 1998-06-26 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,770 US7358340B2 (en) 1997-09-18 2002-07-02 PRO19563 antibodies
US10/188,766 US7351804B2 (en) 1998-06-26 2002-07-02 Antibodies against PRO4421
US10/188,773 US20030036159A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,781 US20030036160A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,774 US20030040074A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,602 US20030036145A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,745 US7250490B2 (en) 1997-09-18 2002-07-02 PRO1480 polypeptides
US10/188,767 US7312310B2 (en) 1997-09-18 2002-07-02 PRO6015 polypeptides
US10/188,775 US20030040075A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,769 US20030036157A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,754 US20030036153A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,751 US20030036151A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,600 US20030036143A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,757 US7276578B2 (en) 1997-09-18 2002-07-02 PRO4334 polypeptides
US10/192,010 US20030044932A1 (en) 1997-09-18 2002-07-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/194,462 US7388073B2 (en) 1998-06-26 2002-07-12 PRO9835 polypeptides
US10/194,461 US20030054459A1 (en) 1998-06-26 2002-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/194,361 US20030036161A1 (en) 1998-06-26 2002-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/194,365 US7381791B2 (en) 1998-06-26 2002-07-12 PRO9739 polypeptides
US10/194,423 US7339025B2 (en) 1998-06-26 2002-07-12 PRO6246 polypeptides
US10/195,888 US20060073545A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,883 US20060073544A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,902 US20030038826A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,894 US20030043176A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,901 US20030036165A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,889 US7534856B2 (en) 1998-06-26 2002-07-15 PRO19624 antibodies
US10/195,892 US7385033B2 (en) 1998-06-26 2002-07-15 PRO12970 polypeptides
US10/195,897 US20030036164A1 (en) 1997-09-18 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,893 US20030206188A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/196,760 US7408034B2 (en) 1998-06-26 2002-07-16 PRO20025 polypeptides
US10/196,759 US20030071835A1 (en) 1998-06-26 2002-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/196,756 US7304145B2 (en) 1998-06-26 2002-07-16 PRO19646 antibodies
US10/196,762 US20030040078A1 (en) 1998-06-26 2002-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/196,745 US7423120B2 (en) 1997-09-18 2002-07-16 PRO19814 polypeptides
US10/196,743 US20030038827A1 (en) 1998-06-26 2002-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/199,462 US20030054468A1 (en) 1998-06-26 2002-07-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/199,316 US20030068726A1 (en) 1998-06-26 2002-07-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/199,464 US20030032140A1 (en) 1997-09-18 2002-07-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/198,768 US20030049756A1 (en) 1998-06-26 2002-07-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/205,904 US20030073813A1 (en) 1998-06-26 2002-07-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2005143079A JP2005323597A (en) 1999-12-23 2005-05-16 Il-17 homologous polypeptide and therapeutic use thereof
JP2005171514A JP2006006327A (en) 1999-12-23 2005-06-10 Il-17-homologous polypeptide and remedial use thereof
JP2005171206A JP4239021B2 (en) 1999-12-23 2005-06-10 IL-17 homologous polypeptide and therapeutic uses thereof
JP2005171424A JP2006006326A (en) 1999-12-23 2005-06-10 Il-17-homologous polypeptide and remedial use thereof
JP2005171331A JP4452659B2 (en) 1999-12-23 2005-06-10 IL-17 homologous polypeptide and therapeutic uses thereof
JP2005171112A JP2005348735A (en) 1999-12-23 2005-06-10 Il-17 homologous polypeptide and its therapeutic use
US11/189,442 US20060246465A1 (en) 1998-06-04 2005-07-25 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
US11/311,561 US20060088917A1 (en) 1999-12-23 2005-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
US11/311,555 US7473763B2 (en) 1999-12-23 2005-12-20 Receptor for IL-17 homologous polypeptides and uses thereof
US11/323,117 US20070092941A1 (en) 1998-09-16 2005-12-29 PRO1298 polypeptides
JP2006000562A JP4688682B2 (en) 1999-12-23 2006-01-05 IL-17 homologous polypeptide and therapeutic uses thereof
US11/341,175 US7468427B2 (en) 1997-03-31 2006-01-27 Antibodies to PRO1275 polypeptide
US11/529,324 US8273703B2 (en) 1998-05-15 2006-09-29 IL-17 receptor-like polypeptides and therapeutic uses thereof
US11/786,466 US20080182275A1 (en) 1998-12-22 2007-04-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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HK08110072.2A HK1114635B (en) 1999-12-23 2008-09-10 Il-17 homologous polypeptides and therapeutic uses thereof
HK08110071.3A HK1114634B (en) 1999-12-23 2008-09-10 Il-17 homologous polypeptides and therapeutic uses thereof
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JP2009171169A JP5258691B2 (en) 1999-12-23 2009-07-22 IL-17 homologous polypeptide and therapeutic uses thereof
JP2010152694A JP2010266454A (en) 1999-12-23 2010-07-05 Il-17 homologous polypeptide and therapeutic use thereof
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US13/593,362 US20130064827A1 (en) 1998-05-15 2012-08-23 Il-17 homologous polypeptides and thereapeutic uses thereof
JP2013038690A JP2013165712A (en) 1999-12-23 2013-02-28 Il-17 homologous polypeptide and therapeutic use thereof
JP2014138440A JP2014239685A (en) 1999-12-23 2014-07-04 Il-17 homologous polypeptides and therapeutic uses thereof
JP2014166220A JP2015007091A (en) 1999-12-23 2014-08-18 Il-17 homologous polypeptides and therapeutic uses thereof
JP2014243369A JP2015077137A (en) 1999-12-23 2014-12-01 Il-17 homologous polypeptides and therapeutic uses thereof
US14/581,971 US20150266953A1 (en) 1998-05-15 2014-12-23 Il-17 homologous polypeptides and therapeutic uses thereof
JP2015187174A JP2016047051A (en) 1999-12-23 2015-09-24 Il-17 homologous polypeptides and therapeutic uses thereof

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
USPCT/US99/05028 1999-03-08
PCT/US1999/005028 WO1999046281A2 (en) 1998-03-10 1999-03-08 Novel polypeptides and nucleic acids encoding the same
US13023299P 1999-04-21 1999-04-21
US60/130,232 1999-04-21
US13102299P 1999-04-26 1999-04-26
US60/131,022 1999-04-26
US13144599P 1999-04-28 1999-04-28
US60/131,445 1999-04-28
US13428799P 1999-05-14 1999-05-14
US60/134,287 1999-05-14
PCT/US1999/028565 WO2000037638A2 (en) 1998-12-22 1999-12-02 Methods and compositions for inhibiting neoplastic cell growth
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PCT/US1999/010733 Continuation-In-Part WO1999060127A2 (en) 1997-03-31 1999-05-14 Il-17 homologous polypeptides and therapeutic uses thereof
US31183299A Continuation-In-Part 1997-09-15 1999-05-14
US38013899A Continuation-In-Part 1996-11-06 1999-08-25
US38014299A Continuation-In-Part 1997-09-15 1999-08-25
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PCT/US2000/004341 Continuation-In-Part WO2000053756A2 (en) 1996-11-06 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/747,259 Continuation-In-Part US6569645B2 (en) 1997-03-31 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
US10/081,056 Continuation US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/119,480 Continuation US20040087769A1 (en) 1998-09-10 2002-04-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same

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US7115398B2 (en) 1998-05-15 2006-10-03 Genentch, Inc. IL-17 homologous polypeptides and therapeutic uses thereof
US7256264B2 (en) 2000-08-24 2007-08-14 Genentech, Inc. Receptor for IL-17 homologous polypeptides and uses thereof
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US7771719B1 (en) 2000-01-11 2010-08-10 Genentech, Inc. Pharmaceutical compositions, kits, and therapeutic uses of antagonist antibodies to IL-17E
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US9650437B2 (en) 2008-05-05 2017-05-16 Novimmune S.A. Nucleic acid encoding and method of producing anti-IL-17A/IL-17F cross-reactive antibodies
US8574576B2 (en) 2009-05-08 2013-11-05 Genentech, Inc. Humanized anti-EGFL7 antibodies and methods using same
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