WO2000053760A2 - Method of preventing the death of retinal neurons and treating ocular diseases - Google Patents

Method of preventing the death of retinal neurons and treating ocular diseases Download PDF

Info

Publication number
WO2000053760A2
WO2000053760A2 PCT/US2000/006319 US0006319W WO0053760A2 WO 2000053760 A2 WO2000053760 A2 WO 2000053760A2 US 0006319 W US0006319 W US 0006319W WO 0053760 A2 WO0053760 A2 WO 0053760A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
acid sequence
pro
polypeptide
amino acid
Prior art date
Application number
PCT/US2000/006319
Other languages
French (fr)
Other versions
WO2000053760A3 (en
Inventor
Napoleone Ferrara
Audrey Goddard
Austin L. Gurney
Caroline Hebert
William J. Henzel
Rhona C. Kabakoff
Robert D. Klein
Ivar J. Kljavin
Sophia S. Kuo
Monique La Fleur
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
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to AU37375/00A priority Critical patent/AU3737500A/en
Priority to AT00939307T priority patent/ATE393825T1/en
Priority to ES00939307T priority patent/ES2307515T3/en
Priority to EP00939307A priority patent/EP1212417B1/en
Priority to PT00939307T priority patent/PT1212417E/en
Priority to PCT/US2000/013705 priority patent/WO2000073445A2/en
Priority to DK00939307T priority patent/DK1212417T3/en
Priority to CA002376116A priority patent/CA2376116A1/en
Priority to JP2001500757A priority patent/JP4297317B2/en
Priority to JP2001500672A priority patent/JP2003524406A/en
Priority to CA002373915A priority patent/CA2373915A1/en
Priority to EP07005021A priority patent/EP1873244A3/en
Priority to PCT/US2000/014941 priority patent/WO2000073348A2/en
Priority to DK00941164T priority patent/DK1185648T3/en
Priority to EP07005022A priority patent/EP1870464A3/en
Priority to AT00941164T priority patent/ATE357518T1/en
Priority to EP07004855A priority patent/EP1820860A3/en
Priority to EP07005023A priority patent/EP1867719A3/en
Priority to ES00941164T priority patent/ES2287020T3/en
Priority to EP00941164A priority patent/EP1185648B1/en
Publication of WO2000053760A2 publication Critical patent/WO2000053760A2/en
Priority to AU20554/01A priority patent/AU2055401A/en
Priority to EP00983846A priority patent/EP1250426A2/en
Priority to JP2001542531A priority patent/JP2004522404A/en
Priority to EP06000586A priority patent/EP1688497A1/en
Priority to CA002496312A priority patent/CA2496312A1/en
Priority to CA002491610A priority patent/CA2491610A1/en
Priority to EP06000581A priority patent/EP1666494A1/en
Priority to EP06000587A priority patent/EP1690872A3/en
Priority to CA002490909A priority patent/CA2490909A1/en
Priority to EP05025102A priority patent/EP1672070A3/en
Priority to EP06000583A priority patent/EP1686134A3/en
Priority to CA002494705A priority patent/CA2494705A1/en
Priority to CA002491433A priority patent/CA2491433A1/en
Priority to CA002492070A priority patent/CA2492070A1/en
Priority to EP10005292A priority patent/EP2228446A1/en
Priority to CA002492049A priority patent/CA2492049A1/en
Priority to EP06000584A priority patent/EP1669371A3/en
Priority to EP06000585A priority patent/EP1661996A1/en
Priority to CA002490853A priority patent/CA2490853A1/en
Priority to PCT/US2000/032678 priority patent/WO2001040466A2/en
Priority to EP06000589A priority patent/EP1661997A1/en
Priority to CA2709291A priority patent/CA2709291A1/en
Priority to EP06000588A priority patent/EP1690873A3/en
Priority to EP06000582A priority patent/EP1666495A1/en
Priority to CA002491258A priority patent/CA2491258A1/en
Priority to CA002391455A priority patent/CA2391455A1/en
Publication of WO2000053760A3 publication Critical patent/WO2000053760A3/en
Priority to US09/828,366 priority patent/US20020010137A1/en
Priority to US09/918,585 priority patent/US20030060406A1/en
Priority to US09/941,992 priority patent/US20030082546A1/en
Priority to US09/978,295 priority patent/US20020156006A1/en
Priority to US09/978,298 priority patent/US20030134785A1/en
Priority to US09/978,189 priority patent/US6972325B2/en
Priority to US09/978,299 priority patent/US20030199435A1/en
Priority to US09/978,192 priority patent/US20020177553A1/en
Priority to US09/978,188 priority patent/US20030139328A1/en
Priority to US09/978,191 priority patent/US20030050239A1/en
Priority to US09/978,193 priority patent/US20030073624A1/en
Priority to US09/978,194 priority patent/US20030195333A1/en
Priority to US09/978,665 priority patent/US7294700B2/en
Priority to US09/978,802 priority patent/US20030199674A1/en
Priority to US09/978,375 priority patent/US7196165B2/en
Priority to US09/978,564 priority patent/US7195760B2/en
Priority to US09/978,697 priority patent/US20020169284A1/en
Priority to US09/978,757 priority patent/US20030083248A1/en
Priority to US09/978,643 priority patent/US20030104998A1/en
Priority to US09/978,544 priority patent/US20030199436A1/en
Priority to US09/981,915 priority patent/US20030054986A1/en
Priority to US09/978,585 priority patent/US20030049633A1/en
Priority to US09/978,681 priority patent/US20030195148A1/en
Priority to US09/978,403 priority patent/US20030050240A1/en
Priority to US09/981,915 priority patent/US7285623B2/en
Priority to US09/978,423 priority patent/US20030069178A1/en
Priority to US09/978,824 priority patent/US20050124789A9/en
Priority to US09/978,824 priority patent/US20030055216A1/en
Priority to US10/152,388 priority patent/US20040223964A1/en
Priority to US10/210,028 priority patent/US20030203446A1/en
Priority to US10/162,521 priority patent/US7067628B2/en
Priority to US10/166,709 priority patent/US20030104536A1/en
Priority to US10/145,124 priority patent/US20030190701A1/en
Priority to US10/145,089 priority patent/US7208575B2/en
Priority to US10/160,502 priority patent/US7220835B2/en
Priority to US10/164,829 priority patent/US20030194780A1/en
Priority to US10/165,067 priority patent/US7279553B2/en
Priority to US10/165,247 priority patent/US7112657B2/en
Priority to US10/164,728 priority patent/US20030186368A1/en
Priority to US10/165,036 priority patent/US20050227342A1/en
Priority to US10/164,749 priority patent/US20040029218A1/en
Priority to US10/143,029 priority patent/US7105640B2/en
Priority to US10/145,017 priority patent/US20030186365A1/en
Priority to US10/164,929 priority patent/US20030194781A1/en
Priority to US10/013,922 priority patent/US20030195345A1/en
Priority to US09/999,829 priority patent/US20030195344A1/en
Priority to US09/999,833 priority patent/US6916648B2/en
Priority to US09/999,830 priority patent/US20030077700A1/en
Priority to US10/017,085 priority patent/US6974696B2/en
Priority to US09/999,832 priority patent/US20020192706A1/en
Priority to US10/020,445 priority patent/US20030198994A1/en
Priority to US10/017,084 priority patent/US20030203402A1/en
Priority to US10/017,081 priority patent/US20030049684A1/en
Priority to US09/999,832 priority patent/US7132283B2/en
Priority to US10/017,086 priority patent/US7122375B2/en
Priority to US10/002,967 priority patent/US20030148373A1/en
Priority to US09/999,834 priority patent/US20030064407A1/en
Priority to US10/017,083 priority patent/US20030148376A1/en
Priority to US10/013,925 priority patent/US7037710B2/en
Priority to US10/013,927 priority patent/US7189529B2/en
Priority to US10/013,921 priority patent/US20030068648A1/en
Priority to US10/013,928 priority patent/US20030215905A1/en
Priority to US10/013,917 priority patent/US7029874B2/en
Priority to US10/013,918 priority patent/US20030211091A1/en
Priority to US10/013,923 priority patent/US7169912B2/en
Priority to US10/013,929 priority patent/US7019124B2/en
Priority to US10/016,177 priority patent/US20030073131A1/en
Priority to US10/013,920 priority patent/US20040006219A1/en
Priority to US10/013,926 priority patent/US7074593B2/en
Priority to US09/993,748 priority patent/US20030069403A1/en
Priority to US09/993,687 priority patent/US20020198149A1/en
Priority to US09/990,456 priority patent/US20020137890A1/en
Priority to US09/991,163 priority patent/US20020132253A1/en
Priority to US09/991,073 priority patent/US20020127576A1/en
Priority to US09/992,521 priority patent/US20030083461A1/en
Priority to US09/990,444 priority patent/US6930170B2/en
Priority to US09/991,854 priority patent/US20030059780A1/en
Priority to US09/990,726 priority patent/US20030054359A1/en
Priority to US09/993,667 priority patent/US20030022187A1/en
Priority to US09/990,438 priority patent/US20030027754A1/en
Priority to US09/990,562 priority patent/US20030027985A1/en
Priority to US09/990,427 priority patent/US20030073809A1/en
Priority to US09/990,443 priority patent/US20030054987A1/en
Priority to US09/992,598 priority patent/US6956108B2/en
Priority to US09/993,469 priority patent/US20030068623A1/en
Priority to US09/990,440 priority patent/US20030060407A1/en
Priority to US09/990,442 priority patent/US20020132252A1/en
Priority to US09/990,711 priority patent/US20030032023A1/en
Priority to US09/990,436 priority patent/US20020198148A1/en
Priority to US09/993,583 priority patent/US7074897B2/en
Priority to US09/997,333 priority patent/US6953836B2/en
Priority to US09/997,428 priority patent/US20030027162A1/en
Priority to US09/998,156 priority patent/US20030044806A1/en
Priority to US09/997,529 priority patent/US7309761B2/en
Priority to US09/997,614 priority patent/US20030124531A1/en
Priority to US09/997,384 priority patent/US20030087305A1/en
Priority to US09/997,514 priority patent/US7019116B2/en
Priority to US09/997,349 priority patent/US7034106B2/en
Priority to US09/997,641 priority patent/US7112656B2/en
Priority to US09/998,041 priority patent/US20030119001A1/en
Priority to US09/997,666 priority patent/US20030027163A1/en
Priority to US09/997,440 priority patent/US20030059833A1/en
Priority to US09/997,601 priority patent/US7189814B2/en
Priority to US09/997,641 priority patent/US20030224358A1/en
Priority to US09/998,041 priority patent/US7309775B2/en
Priority to US09/997,585 priority patent/US20030119055A1/en
Priority to US09/997,384 priority patent/US7119177B2/en
Priority to US09/997,559 priority patent/US20030054403A1/en
Priority to US09/997,601 priority patent/US20030054404A1/en
Priority to US09/997,542 priority patent/US20030068647A1/en
Priority to US09/997,857 priority patent/US20030064375A1/en
Priority to US09/997,666 priority patent/US7244816B2/en
Priority to US09/997,529 priority patent/US20030134284A1/en
Priority to US09/997,683 priority patent/US20030059783A1/en
Priority to US09/997,628 priority patent/US20030059782A1/en
Priority to US09/997,653 priority patent/US7034122B2/en
Priority to US09/997,573 priority patent/US20030049682A1/en
Priority to US09/997,585 priority patent/US7166282B2/en
Priority to US09/991,157 priority patent/US7101687B2/en
Priority to US09/990,441 priority patent/US7041804B2/en
Priority to US09/990,437 priority patent/US20030045463A1/en
Priority to US09/991,181 priority patent/US6913919B2/en
Priority to US09/991,150 priority patent/US20030194760A1/en
Priority to US09/991,172 priority patent/US20030050457A1/en
Priority to US09/989,726 priority patent/US7018811B2/en
Priority to US09/989,328 priority patent/US7056736B2/en
Priority to US09/989,734 priority patent/US7491529B2/en
Priority to US09/989,279 priority patent/US7083978B2/en
Priority to US09/989,722 priority patent/US20020072067A1/en
Priority to US09/989,735 priority patent/US6972185B2/en
Priority to US09/989,727 priority patent/US20020072497A1/en
Priority to US09/989,729 priority patent/US20030059831A1/en
Priority to US09/989,723 priority patent/US20020072092A1/en
Priority to US09/989,721 priority patent/US20020142961A1/en
Priority to US09/989,732 priority patent/US7037679B2/en
Priority to US09/989,862 priority patent/US20030130182A1/en
Priority to US09/989,293 priority patent/US7034136B2/en
Priority to US09/989,725 priority patent/US20030139329A1/en
Priority to US09/989,731 priority patent/US20020103125A1/en
Priority to US09/989,728 priority patent/US7029873B2/en
Priority to US09/989,730 priority patent/US7157247B2/en
Priority to US09/978,187 priority patent/US20030096744A1/en
Priority to US10/081,056 priority patent/US20040043927A1/en
Priority to US10/121,047 priority patent/US20030077778A1/en
Priority to US10/121,045 priority patent/US20030073210A1/en
Priority to US10/121,059 priority patent/US20030190721A1/en
Priority to US10/121,051 priority patent/US20030092147A1/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 US10/121,040 priority patent/US20030082759A1/en
Priority to US10/121,046 priority patent/US20030194791A1/en
Priority to US10/121,043 priority patent/US7220831B2/en
Priority to US10/121,061 priority patent/US20030082761A1/en
Priority to US10/121,053 priority patent/US20030199053A1/en
Priority to US10/121,062 priority patent/US20030077779A1/en
Priority to US10/121,054 priority patent/US20030199054A1/en
Priority to US10/121,048 priority patent/US20030199051A1/en
Priority to US10/121,052 priority patent/US20030199052A1/en
Priority to US10/121,050 priority patent/US20030054516A1/en
Priority to US10/121,057 priority patent/US20030190719A1/en
Priority to US10/121,056 priority patent/US20030082760A1/en
Priority to US10/121,060 priority patent/US20030190722A1/en
Priority to US10/121,055 priority patent/US20030190718A1/en
Priority to US10/121,058 priority patent/US20030190720A1/en
Priority to US10/121,063 priority patent/US20030199055A1/en
Priority to US10/123,261 priority patent/US20030068796A1/en
Priority to US10/123,154 priority patent/US20030190724A1/en
Priority to US10/123,291 priority patent/US20030199058A1/en
Priority to US10/123,155 priority patent/US20030068794A1/en
Priority to US10/123,157 priority patent/US20030190725A1/en
Priority to US10/123,109 priority patent/US20030190723A1/en
Priority to US10/123,215 priority patent/US7291329B2/en
Priority to US10/123,262 priority patent/US20030049816A1/en
Priority to US10/123,236 priority patent/US20030068795A1/en
Priority to US10/123,156 priority patent/US20030194792A1/en
Priority to US10/123,322 priority patent/US20030199059A1/en
Priority to US10/123,213 priority patent/US20030199057A1/en
Priority to US10/123,108 priority patent/US7635478B2/en
Priority to US10/123,212 priority patent/US7276577B2/en
Priority to US10/123,771 priority patent/US20030199060A1/en
Priority to US10/123,214 priority patent/US7343721B2/en
Priority to US10/123,213 priority patent/US7193048B2/en
Priority to US10/123,292 priority patent/US20030073211A1/en
Priority to US10/123,235 priority patent/US20030082762A1/en
Priority to US10/123,908 priority patent/US7335728B2/en
Priority to US10/123,905 priority patent/US7285625B2/en
Priority to US10/123,907 priority patent/US7084258B2/en
Priority to US10/123,912 priority patent/US20030100087A1/en
Priority to US10/123,906 priority patent/US20030190726A1/en
Priority to US10/123,905 priority patent/US20030087344A1/en
Priority to US10/123,911 priority patent/US7408032B2/en
Priority to US10/123,913 priority patent/US20030203462A1/en
Priority to US10/123,910 priority patent/US7329404B2/en
Priority to US10/123,903 priority patent/US20030073212A1/en
Priority to US10/123,909 priority patent/US7193049B2/en
Priority to US10/123,902 priority patent/US20030077781A1/en
Priority to US10/123,904 priority patent/US20030022328A1/en
Priority to US10/124,820 priority patent/US20030190729A1/en
Priority to US10/124,814 priority patent/US7105335B2/en
Priority to US10/124,813 priority patent/US7312307B2/en
Priority to US10/124,823 priority patent/US20030199062A1/en
Priority to US10/124,821 priority patent/US20030199023A1/en
Priority to US10/124,818 priority patent/US20030082763A1/en
Priority to US10/124,824 priority patent/US20030077659A1/en
Priority to US10/125,795 priority patent/US7304131B2/en
Priority to US10/124,817 priority patent/US20030077786A1/en
Priority to US10/125,805 priority patent/US20030194794A1/en
Priority to US10/124,819 priority patent/US7285626B2/en
Priority to US10/124,816 priority patent/US20030190728A1/en
Priority to US10/125,704 priority patent/US7357926B2/en
Priority to US10/124,822 priority patent/US7109305B2/en
Priority to US10/125,922 priority patent/US7309762B2/en
Priority to US10/125,931 priority patent/US20030199063A1/en
Priority to US10/125,924 priority patent/US7342097B2/en
Priority to US10/125,927 priority patent/US20030190731A1/en
Priority to US10/125,932 priority patent/US7317079B2/en
Priority to US10/127,831 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 US10/137,867 priority patent/US20030207349A1/en
Priority to US10/137,865 priority patent/US20030032155A1/en
Priority to US10/137,868 priority patent/US20030082764A1/en
Priority to US10/140,024 priority patent/US20040058424A1/en
Priority to US10/140,020 priority patent/US20030207415A1/en
Priority to US10/140,470 priority patent/US20030022331A1/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 US10/139,980 priority patent/US7247710B2/en
Priority to US10/140,928 priority patent/US20030068798A1/en
Priority to US10/140,860 priority patent/US7307151B2/en
Priority to US10/140,809 priority patent/US20030207418A1/en
Priority to US10/140,805 priority patent/US20030207417A1/en
Priority to US10/140,921 priority patent/US7317080B2/en
Priority to US10/140,864 priority patent/US20030207419A1/en
Priority to US10/140,865 priority patent/US20030207420A1/en
Priority to US10/140,925 priority patent/US20030073215A1/en
Priority to US10/140,808 priority patent/US7425621B2/en
Priority to US10/141,701 priority patent/US20030207421A1/en
Priority to US10/141,755 priority patent/US7297764B2/en
Priority to US10/141,754 priority patent/US7361732B2/en
Priority to US10/141,760 priority patent/US7342104B2/en
Priority to US10/141,756 priority patent/US7488586B2/en
Priority to US10/142,425 priority patent/US20030207424A1/en
Priority to US10/143,113 priority patent/US7329730B2/en
Priority to US10/142,430 priority patent/US7309766B2/en
Priority to US10/142,417 priority patent/US7304133B2/en
Priority to US10/143,114 priority patent/US20030036180A1/en
Priority to US10/142,419 priority patent/US7153941B2/en
Priority to US10/143,032 priority patent/US7408033B2/en
Priority to US10/142,423 priority patent/US20030049817A1/en
Priority to US10/142,431 priority patent/US7285629B2/en
Priority to US10/146,730 priority patent/US20030207427A1/en
Priority to US10/146,792 priority patent/US20030207428A1/en
Priority to US10/147,528 priority patent/US20030219885A1/en
Priority to US10/147,519 priority patent/US20030077791A1/en
Priority to US10/147,492 priority patent/US20030082765A1/en
Priority to US10/147,536 priority patent/US20040077064A1/en
Priority to US10/152,395 priority patent/US7189534B2/en
Priority to US10/153,934 priority patent/US20030129695A1/en
Priority to US10/156,843 priority patent/US20030207805A1/en
Priority to US10/157,786 priority patent/US20030208055A1/en
Priority to US10/157,782 priority patent/US20030077792A1/en
Priority to US10/158,791 priority patent/US20030207429A1/en
Priority to US10/160,498 priority patent/US20030073216A1/en
Priority to US10/158,782 priority patent/US20030082766A1/en
Priority to US10/176,913 priority patent/US20030022298A1/en
Priority to US10/176,492 priority patent/US20030027272A1/en
Priority to US10/187,885 priority patent/US20030032138A1/en
Priority to US10/197,942 priority patent/US20030175882A1/en
Priority to US11/100,159 priority patent/US7425613B2/en
Priority to JP2005229454A priority patent/JP4145314B2/en
Priority to JP2005264293A priority patent/JP2006068016A/en
Priority to US11/341,175 priority patent/US7468427B2/en
Priority to JP2006225770A priority patent/JP4072181B2/en
Priority to JP2006225772A priority patent/JP2007029098A/en
Priority to JP2006225771A priority patent/JP4074645B2/en
Priority to US11/786,466 priority patent/US20080182275A1/en
Priority to JP2007326424A priority patent/JP2008167749A/en
Priority to JP2007326609A priority patent/JP2008148701A/en
Priority to JP2007325484A priority patent/JP2008148699A/en
Priority to JP2007326613A priority patent/JP2008161190A/en
Priority to JP2008145176A priority patent/JP2009019032A/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention relates to a method of promoting retinal neuion surv ⁇ al as well as preventing photoneuron degradation and to the treatment of diseases or conditions w hich are characterized by photoneuron death
  • the retina is the light-sensitive portion of the eye
  • the ietina contains the cones and rods
  • the rods contain rhodops .
  • the rod photopigment and the cones contain 3 distinct photopigments, which respond to light and transmit signals through successive neurons to ultimately trigger a neural discharge in the output cells of the retina, the ganglion cells
  • the signal is carried by the optic nerve to the visual cortex where it is registered as a visual stimulus
  • the macula lutea In the center of the retina is the macula lutea, which is about 1/3 to 1/2 cm in diameter
  • the macula provides detailed vision, particularly in the center (the fovea), because the cones are higher in density Blood vessels, ganglion cells, inner nuclear layer and cells, and the plexiform layers are all displaced to one side (rather than resting above the ones), thereby allowing light a more direct path to the cones
  • the retinal pigment epithelial (RPE) cells which make up the PE, produce, store and transport a variety of factors that are responsible for the normal function and survival of photoreceptors RPE arc multifunctional cells that transport metabolites to the photoreceptoi s from their blood suppK the cho ⁇ o capillaries of the eye
  • the RPE cells also function to recycle vitamin A as it moves between the photoreceptors and the RPE during light and dark adaptation
  • RPE cells also function as macrophages phagocytizing the rhythmically-shed tips of the outer segments of rods and cones, which are produced in the normal course of cell physiology Various ions, proteins and water move between the RPE cells and the interphotoreceptor space,
  • the Muller cell is the most prominent glial cell within the retina, and could also be important foi maintaining the viability ol visual cells Muller cells traverse the entire retina in a iadial dnection from the ganglion cells to the external limiting membrane, a photoreceptor-photoreceptor and Muller cell photoreceptor contact point
  • Muller cells regulate the control ol ionic concentralions, degradation of neurotransmitter, removal of certain metabolites and may be a source of important factors that promote the normal differentiation of photoreceptoi cells [Kl'avin and Re ( 1991 ), J Neuioscience JJ .
  • photoreceptor cells are particularly susceptible to injury since they are often the first cells to degenerate or suffer damage as a result of a traumatizing event or condition.
  • Hereditary defects in specific photoreceptor genes, retinal detachment, circulatory disorders, overexposure to light, toxic effects to drugs and nutritional deficiencies are among the wide array of causes that can result in the death of photoreceptor cells.
  • retinitis pigmentosa a genetic based progressive disease is characterized by incremental loss of peripheral vision and night blindness, which is due in large part to the loss of photoreceptor cells.
  • RP retinitis pigmentosa
  • Total blindness is the usual outcome in more progressive stages of this disease.
  • Macular degeneration another major cause of blindness, is a complex group of disorders that affects the central or predominantly cone portion of the retina. Cones are primarily responsible for acute vision.
  • Diabetic retinopathy a frequent complication in individuals with diabetes mellitus, is estimated to be the fifth leading cause of new blindness. However, it is the second leading cause of blindness among individuals of 45-74 years of age. Moreover, these problems are only expected to get worse as the general population ages.
  • Photoreceptor degeneration may also occur as a result of overexposure to light, various environmental trauma or by any pathological condition characterized by death or injury of retinal neurons or photoreceptors.
  • Photoreceptor loss may also be influenced by cellular or extracellular retinal components.
  • the primary example of extracellular stimulus is related to the close association between the pigment epithelium (PE) and the photoreceptor cells.
  • the PE transports metabolites to and from the photoreceptors as well as removes discarded cellular material.
  • Retinal detachment which involves the separation of the neural retina from the PE leads to photoreceptor death.
  • the degree of cell loss is dependent upon the duration of the separation [Gouras et al. ( 1991 ) 10VS 32: 3167-3174].
  • a delay of photoreceptor death is obtained through the proximal placement of normal PE cells both in experimental chimeras, Mullen & LaVail. supra and by transplantation of PE from healthy animals [Li & Turner ( 1988). E.xp. Eye Res. 47: 91 1 -917: Sheedlo et al. ( 1992). Int. Rev. Cylol. Y__: 1 -49: Lavail et al. ( 1992). E.xp. Eye Res. 55: 555-562: Lavail et al. ( 1992). PNAS 89: I 1249- 1 1253].
  • the "'rescue *' extended beyond the boundaries of the normal PE cells, and suggests the presence of difussible trophic factor(s) produced by the PE cells.
  • Another useful animal model is the albino rat. In this animal, normal illumination levels of light, if continuous, can cause complete degeneration of photoreceptors. Results obtained using such rats as a model to identify survival enhancing factors appear to correlate well with data obtained using RCS rats. Moreover. different factors can be compared and complications can be assessed more quickly in the light damage model than can be assessed by testing factors in models which are based on the slowly evolving dystrophy of the RCS rat.
  • Vitamin A has been observed to halt the decline of retinal function by ov er 20% as administered over the course of 4 6 years in the progression of patients with letmttis pigmentosa (RP) [E L Berson et al Aich Ophthalmol 1 1 1 761 772 ( 1993)] While this study did indicate a potential lengthening of yeais of useful vision, several criticisms of vitamin A therapv exist ( 1 ) the mechanism by which vitamin A (and even vitamin E) alter the progression of RP is unknown, (2) it is not known whethei or not patients with different genetic forms of RP will respond to vitamin A therapy, (3) it is not apparent whether or not quantifiable measurements of visual function (l e pe ⁇ metry and v isual acuity) revealed any significant benefit from vitamin A therapy and (4) long term ingestion of vitamin A may have detrimental side effects in other organ systems
  • a number of agents when administered systemically can be used to ameliorate retinal cell death or injury caused by exposure to light
  • exposure to light generates oxygen free radicals and lipid peroxidation products
  • genetically detective photoreceptors are abnormally sensitive to photooxidation from light levels as encountered normally in the environment
  • PE were secreting a putative survival promoting factor.
  • FGF acidic and basic fibroblast growth factors
  • aFGF and bFGF acidic and basic fibroblast growth factors
  • FGF can be detected through immunohistochemical, biochemical or molecular approaches on a variety of retinal cells including PE, photoreceptor cells and the interphotoreceptor cell matrix (IPM), and a collection of extracellular matrix molecules surrounding photoreceptor cells [Jacquemin et al, Neurosci. Lett. ir. 23-28. ( 1990); Caruelle et al, J. Cell Biol. 39: 1 17- 128 ( 1989): Hageman et al., PNAS __: 6706-6710 ( 1991 ); Connolly et al..
  • TGF- ⁇ TGF- ⁇
  • BDNF brain derived neurotrophic factors
  • IGF insulin-like giowth iactoi II
  • TGF- ⁇ Transforming Growth Factoi beta
  • TNF- ⁇ Transforming Growth Factoi beta
  • TNF- ⁇ tumoi necrosis factois alpha and beta
  • NGF tumoi necrosis factois alpha and beta
  • bFGF pio ote detiimental side effects
  • injections of bFGF results in an increased incidence of macrophages and cataracts
  • bFGF is mitogenic for PE, Muller cells and retinal v ascular cells Faktorovich et al . supra . La Vail et al . supra
  • suitable growth factors which will not only promote the survival of photoreceptor cells, but lack undesired side effects have yet to be discovered
  • the present invention relates to a method of delaying, preventing or rescuing photoreceptor cells from in j ury or death by the administration of a therapeutically effective amount of a PRO polypeptide
  • the present invention relates to the use of PRO polypeptides to delay, prevent or rescue other retinal cells or supportive cells (e g Muller cells or RPE cells) fiom injury and death
  • retinal neurons include, but are not limited to retinal ganglion cells, displaced retinal ganglion cells.
  • the invention relates to the use of PRO to stimulate the regeneration of such cells
  • the PRO polypeptide is an active polypeptide which is at least 90% homologous to a native sequence PRO molecule
  • the PRO polypeptide is an active PRO polypeptide encoded by an isolated nucleic acid comprising DNA encoding a PRO 200 (VEGF-E).
  • the isolated nucleic acid comprises DNA encoding a ( 1 ) PRO200 polypeptide having ammo acid residues 1 to 345 (alternatively 15 to 345) ot SEQ ID NO 2.
  • PRO540 polypeptide having amino acid residues I to 412 alternatively 29 to
  • the present invention relates to the use of PRO polypeptides to treat any condition which results in injury or death of photoreceptor or other retinal cells
  • ot conditions include retinal detachment, age-related and other maculopathies.
  • photic retinopathies surgery-induced retinopathies (either mechanically oi light-induced), toxic retinopathies including those resulting from foreign bodies in the eye.
  • ischemic retinopathies due to venous or arterial occlusion or other ascular disoi der retinopathies due to trauma or penetrating lesions of the eye, peripheral Mtreoretinopathy .
  • inherited retinal degenerations Exemplary letinal degenerations include e g hereditary spastic paraplegia w ith retinal degeneration ( Kiellin and Barnard Schol/ syndromes) l erinitis pigmentosa Stargardt disease Ushei sy ndiome (letimtis pigmentosa w ith congenital hearing loss), and Retsum syndrome (/ et itis pigmentosa hereditary hearing loss and polv neuiopathy ) Additional disordeis which result in death ot retinal neurons include retinal teais.
  • ARN degenerative myopia acute ietinal necrosis syndrome
  • Purtscher's Retinopath degenerative myopia acute ietinal necrosis syndrome
  • the present invention provides to a method of delay ing, preventing or rescuing retinal neuions ( g photoreceptors) or other retinal cells fiom injury or death resulting from disease or injury comprising the administration of a composition of PRO polypeptide and a pharmaceutically -acceptable carrier
  • the composition comprises a therapeutically effective amount of PRO polypeptide
  • the composition comp ⁇ ses a turther activ e ingredient, which may for example, be a further neuronal survival agent
  • the composition is sterile
  • the present invention provides articles of manufacture and kits that include PRO polypeptide
  • the articles of manufacture and kits include a container an instruction on the container, and a composition contained within the container
  • the instruction on the container indicates that the composition can be used to delay, prevent or rescue retinal neurons or other retinal cells from injury or death
  • the composition contains an active agent, and the active agent comprises PRO
  • 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, or yeast
  • a process tor producing any of the herein described polypeptides is further provided and comprises cultu ⁇ ng 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 ammo acid sequence
  • chime ⁇ c molecules compi ise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin
  • the invention provides an antibody which specifically binds to any of the above or below described polypeptides
  • the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody
  • the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense piobes. wheiein those probes may be derived from any of the above oi below described nucleotide sequences
  • 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% nucleic acid sequence identity , alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity , alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity , alternatively at leas! about 87% nucleic acid sequence identity , alternatively at lea**.!
  • nucleic acid 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 othei specifically defined fragment o
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity. alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about
  • nucleic acid sequence identity alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about
  • nucleic acid sequence identity alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about
  • nucleic acid sequence identity alternatively at least about 96% nucleic acid sequence identity. alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length 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).
  • the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity.
  • nucleic acid sequence identity alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity alternatively at least about 92% nucleic acid sequence identity alternatively at least about 93% nucleic acid sequence identity du elv at least about 94% nucleic acid sequence identity, du elv at least about 95% nucleic acid sequence identity du ely at least about 96% nucleic acid sequence identity , alternatively at least about 97% nucleic acid sequence identity .
  • nucleic acid sequence identity alternatively at least about 98% nucleic acid sequence identity and du ely at least about 99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same mature poly peptide encoded by any ot the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a)
  • Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is eithei transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane doma ⁇ n(s) of such polypeptide are disclosed herein Therefore, soluble extracellular domains of the herein desc ⁇ bed PRO polypeptides are contemplated
  • Anothei 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
  • nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 1 10 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleo
  • 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% amino acid sequence identity , altei natively at least about 81 % amino acid sequence identity , alternatively at least about 82% ammo acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity. alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about
  • amino acid sequence identity alternatively at least about 88% amino acid sequence identity , alternatively at least about 89% amino acid sequence identity. alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % ammo acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about
  • amino acid sequence identity alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about
  • 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
  • the invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% ammo acid sequence identity. alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about
  • amino acid sequence identity alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about
  • amino acid sequence identity alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% * amino acid sequence identity, alternatively at least about
  • the invention concerns an isolated PRO polypeptide comprising an ammo acid sequence scoring at least about 80% positives, alternatively at least about 81 % positives, alternatively at least about 82% positives, alternatively at least about 83% positives, alternatively at least about 84% positives, alternatively at least about 85% positives, alternatively at least about 86% positives, alternatively at least about 87% positives, alternatively at least about 88% positives, alternatively at least about 89% positives, alternatively at least about 90% positives, alternatively at least about 91 % positives, alternatively at least about 92% positives, alternatively at least about 93% positives, alternatively at least about 94% positives, alternatively at least about 95% positives, alternatively at least about 96% positives, alternatively at least about 97% positives, alternatively at least about 98% positives and alternatively at least about 99% positives when compared with the amino acid sequence of a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acids, alternatively at
  • the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described.
  • Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.
  • Another aspect 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 culturing 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 sequence 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 carrier 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.
  • Figure 1 show s DNA29101 1272 (SEQ ID NO 1 ) a DNA sequence encoding a PRO200 (VEGF- E) (SEQ ID NO 1 )
  • Figure 2 show s the native sequence PRO200 (SEQ ID NO 2)
  • Figure 3 shows DNA44189- 1 22 (SEQ ID NO 3 ).
  • a DNA sequence encoding PRO 40 SEQ ID NO 3
  • Figure 4 shows the native sequence PRO540 (SEQ ID NO 4)
  • Figure 5 shows DNA39631 (SEQ ID NO 5). a consensus nucleotide used in the preparation ot DNA44189
  • Figure 6 shows DNA44196-1353 (SEQ ID NO 12). a DNA sequence encoding the native sequence
  • Figuie 7 shows the native sequence PR0846 (SEQ ID NO 13)
  • Figure 8 shows a single-stranded (sense) consensus nucleotide sequence used in the isolation of DNA44196 ( Figure 6. SEQ ID NO 12), designated in the present application as DNA39949 (SEQ ID NO 14)
  • Figure 9 shows DNA48309-1280 (SEQ ID NO 18). a DNA sequence encoding a native sequence PR0617 polypeptide (SEQ ID NO.19)
  • Figure 10 shows the native sequence PR0617 (SEQ ID NO 19)
  • Figure 1 1 shows the single stranded nucleotide sequence (sense strand) DNA42798 consensus DNA (SEQ ID NO. 20) which was used in the isolation of DNA48309 (SEQ ID NO 18)
  • Figure 12 shows DNA48613-1268 (SEQ ID NO 24), a DNA sequence encoding a native sequence
  • Figure 13 shows DNA48614-1268 (SEQ ID NO.25), a DNA sequence encoding a native sequence PR03664 polypeptide (SEQ ID NO.27)
  • Figure 14 shows the native sequence PR0538 (SEQ ID NO 26)
  • Figure 15 shows the native sequence PR03664 (SEQ ID NO 27)
  • Figures 16A and B show a comparison between a native sequence PR0538 (SEQ ID NO 26) and PR03664 (SEQ ID NO 27) encoded by DNA48613 (SEQ ID NO 24) and DNA48614 (SEQ ID NO 25), respectively, resulting in a 92 25% similarity (% value generated by the Align program using PAM250 matrix and a gap penalty of 8 + 4 per residue)
  • Figure 17 shows the EST sequence (Incyte INC3574209)(SEQ ID NO 28) sequence which was used in the cloning of the full length PR0538 (GFR ⁇ 3) and PR03664 (GFR ⁇ 3) sequences DNA48613 (SEQ ID NO 24) and DNA48614 (SEQ ID NO 25). respectively
  • Figure 18 shows the native sequence PRO770 (hFIZZ- 1 )(SEQ ID NO 33)
  • Figure 19 shows DNA54228-1366 (SEQ ID NO 34). a DNA sequence encoding a native sequence
  • PRO770 polypeptide SEQ ID NO 33
  • Figure 20 shows DNA53517 (SEQ ID NO 35).
  • Figure 21 show s AA524300 (SEQ ID NO 36) an ESI w hich was identified as hav ing homology to murine TIZZ as described in Example 14
  • Figure 22 show s Incy te EST 13025 16 an ESI exhibiting homology to human VEGF. and was used in the isolation of DNA29101 (SEQ ID NO 1 ) described in Example 8
  • polypeptide aie intended to be used interchangeably They refer to a chain of two (2) or more amino acids which are linked together w ith peptide or amide bonds, regardless of post-translational modification (e g , glycosylation or phosphorylation)
  • the polypeptides of this invention may comprise more than one subunit. where each subunit is encoded by a separate DNA sequence
  • 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 provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein) The
  • PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
  • PRO polypeptide refers to each individual PRO/number polypeptide disclosed herein All disclosures in this specification which refer to the "PRO polypeptide” refer to each of the polypeptides individually (j e , PRO200 (VEGF-E), PRO540. PR0846, PR0617. PR0538 (GFR 3), PR03664 (GFRo ⁇ ) or PRO770
  • PRO polypeptide also includes variants ot the PRO/number polypeptides disclosed herein
  • a "native sequence PRO polypeptide” comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature Such nativ e 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 foims (e g , alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide
  • the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comp ⁇ sing 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
  • PRO polypeptide disclosed in the accompanying figures are show n to begin with methionine residues designated herein as amino acid position 1 in the figures it is conceivable and possible that other methionine residues located either upstream oi dow nstream from the amino acid position 1 in the figures may be employed as the starting amino acid residue toi the PRO pol peptides
  • the nativ e-sequence ( 1 ) PRO200 (VEGF-E) polypeptide is a mature or full-length nativ e sequence PRO200 (VEGF-E) polypeptide comprising amino acids 1 though 345 as depicted in Figure 2.
  • PRO540 polypeptide is a mature or full-length native sequence PRO540 polypeptide comprising amino acids 1 to 412 of Figuie 4.
  • PR0846 poly peptide is a mature or full-length native sequence PR0846 polypeptide comprising amino acids 1 to 332 of Figure 7.
  • PR0617 polypeptide is a mature or full-length native sequence PR0617 polypeptide comprising amino acids 1 to 67 of Figure 10.
  • PR0538 (GFR ⁇ 3) polypeptide is a mature or full-length native sequence PR0538
  • PR03664 (GFR ⁇ 3) polypeptide is a mature oi full-length native sequence PR03664 (GFRc ⁇ ) comprising amino acids I to 369 of Figure 14, or (7)
  • PRO770 (hFIZZ- 1 ) polypeptide is a matuie or full-length native sequence PRO770 (hFIZZ-1 ) comprising ammo acids 1 to 1 1 1 of Figure 16. respectively
  • the PRO polypeptide "extracellular domain " or "ECD” refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains Ordinarily, a PRO polypeptide
  • ECD will have less than 1 % of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0 5% of such domains It will be understood that any transmembrane domains identified for the
  • PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain
  • the exact boundaries ot a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein
  • 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.
  • a PR0846 polypeptide ECD can comprise amino acid residues 1 or about 18 to 247 of Figure 7 (SEQ ID NO 13), while a PR0846 polypeptide ECD may optionally comprise amino acids 1 or about 18 to X of Figure 7
  • 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 tor identifying that type of amino acid sequence element (e g , Nielsen et al , Piot Eng JO 1 -6 ( 1997) and von Heinje et al , Nucl Acids Res J4 4683-4690 ( 1986)) Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secieted polypeptide is not entnely uniform, resulting in more than one secreted species These mature polypeptides, where the signal peptide is cleav ed within no more than about 5 amino acids on either side of the C-terminal boundary ot the signal peptide as identified herein, and the polynucleotides encoding them, are
  • a PRO polypeptide variant will have at least about 80% amino acid sequence identity , more preferably at least about 90% am o acid sequence identity , and even more preferablv at least about 95% ammo acid sequence identity with a full-length native sequence PRO polypeptide identified herein Foi example, a biologically active
  • PRO200 (VEGF-E) PRO540. PR0846 PR0617. PR0538 (GFRc ⁇ ). PR03664 (GFR ⁇ 3) oi PRO770 (hFIZZ- 1 ) variants can be functional fiag ents or analogs of native-sequence
  • PRO200 (VEGF-E).
  • PRO540 PRO540, PR0846.
  • PR0617 PR0538 GFRc ⁇
  • PR03664 GFRc ⁇
  • PRO770 PRO770
  • PR0846, PR0617 PR0538 (GFRc ⁇ ), PR03664 (GFRo ⁇ ) or PRO770 (hFIZZ- 1 ) variant is an isolated nucleic acid which hybridizes under moderate, and optionally under high stringency conditions, and remains stably bound thereto, the compliment of the coding DNA tor a.
  • PR0617 polypeptide having amino acid residues 1 to 67 of SEQ ID NO 19 (5) PR0538 (GFR ⁇ 3) polypeptide having amino acid residues 1 to 400 of SEQ ID NO 26, (6) PR03664 polypeptide having amino acid residues 1 to 369 of SEQ ID NO 27, (7)
  • PRO770 (hFIZZ- 1 ) polypeptide having amino acid residues 1 to 1 1 1 of SEQ ID NO 33, respectively As used herein. "PRO200. PRO540, PR0846. PR0617. PR0538 (GFRc ⁇ ), PR03664 (GFRc ⁇ ) or PRO770
  • 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 extracellulai domain of a PRO polypeptide, with or without the signal peptide. as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Such PRO polypeptide variants include, for instance.
  • PRO polypeptides wheiein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native ammo acid sequence Ordinarily, a PRO polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81 % ammo acid sequence identity, alternatively at least about 82% ammo acid sequence identity altei natively at least about 83% amino acid sequence identity , alternatively at least about 84% amino acid sequence identity , altei natively at least about 85% amino acid sequence identity , alternatively at least about 86% amino acid sequence identity, du ely at least about 87% ammo acid sequence identity, alternatively al least about 88% amino acid sequence identity , alternatively at least about 89% amino acid sequence identity ,
  • ely at least about 90% ammo acid sequence identity Converse ely at least about 91 % amino acid sequence identity , alternatively at least about 92% amino acid sequence identity . alternatively at least about 93% amino acid sequence identity , alternatively at least about
  • PRO variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 ammo acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 250 amino acids in length, alternatively at least about 300 amino acids in length, alternatively at least about 350 amino acids in length, alternatively at least about 400 amino acids in length, alternatively at least about 450 amino acids in length or more
  • Percent (%) amino acid sequence identity with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the ammo acid residues in the specific PRO polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in various way s that are within the skill in the art, tor instance, using publicly available computei software such as BLAST. BLAST-2.
  • ALIGN-2 ALIGN or Megalign (DNASTAR) software
  • ALIGN-2 sequence comparison computer program
  • Table 1 the complete source code for the ALIGN-2 program is provided in Table 1 below
  • the ALIGN-2 sequence comparison computer program was authored by Genentech. Inc and the source code shown in Table 1 below 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 av ailable through Genentech.
  • ALIGN-2 piogram 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 progiam and do not vary In situations w heie ALIGN 2 is emploved for amino acid sequence comparisons the % amino acid sequence identitv ot a giv en amino acid sequence A to w ith oi against a giv en amino acid sequence B ⁇ hich can alternativelv be phiased as a given amino acid sequence A that has oi comprises a certain % amino acid sequence identitv to with or against a giv en amino acid sequence B ) is calculated as follows
  • WU BLAST 2 is employed a % amino acid sequence identity v alue is determined by dividing (a) the number ot matching identical amino acid 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 (l e the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU BLAST 2 by (b) the total number of amino acid residues of the PRO polypeptide ot interest For example in the statement ' a polypeptide comprising an the amino acid
  • Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI BLAST2 (Altschul et al Nucleic Acids R s 25 3389 3402 ( 1997))
  • NCBI BLAST2 sequence companson program may be downloaded fiom http //www ncbi nlm mil gov oi otherwise obtained fiom the National Institute of Health Bethesda MD
  • % amino acid sequence identity of a giv en amino acid sequence A to with, or against a given amino acid sequence B (which can alternatively be phrased as a giv en amino acid sequence A that has or compnses a certain % amino acid sequence identity to with, or against a given ammo acid sequence B) is calculated as follows
  • Polypeptide variants may come in different foims "Substitutional ' variants are those that have at least one amino acid residue in a native sequence removed and a different amino acid inserted in its place at the same position The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more am o acids have been substituted in the same molecule "Insertional” variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native sequence Immediately adjacent to an amino acid means connected to either the ⁇ -carboxyl or ⁇ -amino functional group of the amino acid "Deletional” variants are those with one or more amino acids in the native amino acid sequence removed Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the molecule Polypeptide variants also include covalent modifications to residues in addition to epitope-tagged heterogeneous PRO
  • PRO variant polynucleotide or “PRO variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide.
  • a PRO variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity.
  • nucleic acid sequence identity alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity alternatively at least about 93% nucleic acid sequence identity , alternatively at least about 94% nucleic acid sequence identity, alternativelv at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identitv, .
  • PRO variant polynucleotides are at least about 30 nucleotides in length alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length,
  • ALIGN-2 sequence comparison computer program
  • Table 1 the complete source code for the ALIGN-2 program is provided in Table 1 below
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc and the source code shown in Table 1 below has been filed with user documentation in the U S Copyright Office, Washington D C , 20559.
  • 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 below
  • 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
  • the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows 100 times the ti action W/Z
  • PRO-DNA' represents a hypothetical PRO-encoding nucleic acid sequence of interest.
  • Comparison DNA represents the nucleotide sequence ot a nucleic acid molecule against which the "PRO-DNA” nucleic acid molecule ot interest is being compared, and "N", “L' and “V” each represent different hypothetical nucleotides
  • % nucleic acid sequence identity values may also be obtained as described below by using the
  • a % nucleic acid sequence identity value is determined by dividing (a) the number ot 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
  • WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide-encoding nucleic acid molecule of interest
  • nucleic acid sequence A is the comparison nucleic acid molecule of interest
  • nucleic acid sequence B is the nucleic acid sequence ot the PRO polypeptide-encoding nucleic acid molecule ot interest
  • Percent nucleic acid sequence identity may also be determined using the sequence comparison program NCB1-BLAST2 (Altschul et al , Nucleic Acids Res 25 3389-3402 (1997))
  • NCBI-BLAST2 sequence comparison program may be downloaded from "http //www ncbi nlm nih gov” or otherwise obtained tiom the National Institute of Health, Bethesda.
  • NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a giv en nucleic acid sequence C to. w ith, oi against a giv en nucleic acid sequence D (which can Var ely be phrased as a given nucleic acid sequence C that has oi compnses a DCtam % nucleic acid sequence identity to. w ith, or against a given nucleic acid sequence D) is calculated as folk lows
  • 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 a full-length PRO polypeptide as disclosed herein
  • PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide
  • identity values used herein can be generated by the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively
  • the % value of positives is determined by dividing (a) the number of amino acid residues scoring a positive value between the PRO polypeptide amino acid sequence of interest having a sequence derived from the native PRO polypeptide sequence and the comparison amino acid sequence of interest (/ e , the amino acid sequence against which the PRO polypeptide sequence is being compared) as determined in the BLOSUM62 matrix of WU- BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest
  • the % value of positives is calculated as described in the immediately preceding paragraph
  • Am o 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 prefened substitution (as defined in Table 6 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:
  • X is the number of amino acid residues scoring a positive value as defined above by the sequence alignment program ALIGN-2 or 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 % positives of A to B will not equal the % positives of B to A.
  • Isolated when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide. and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified ( 1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
  • An "isolated" PRO polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid 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 polypeptide-encoding nucleic acid.
  • An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells.
  • an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of 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 for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ⁇ bosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation
  • Generallv opeiably linked' means that the DNA sequences being linked are contiguous and in the case ot a secretory leadei contiguous and in reading phase Howev ei enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites It such sites do not exist the synthetic oligonucleotide adaptors or linkers aie used in accordance w ith conventional practice
  • the term ' antibody ' is used in the broadest sense and specifically covers, tor example single anti PRO monoclonal antibodies (including agonist antagonist and neutiahzing antibodies).
  • anti-PRO antibodv compositions with polyepitopic specificity single chain anti-PRO antibodies, and fragments of anti-PRO antibodies (see below)
  • the term ' monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, l e the indiv idual antibodies comprising the population are identical except for possible naturally-occurung mutations that may be present in minor amounts
  • “Stringent conditions” or “high stringency conditions” are exemplified by reaction conditions characterized by ( 1 ) employing low ionic strength and high temperature for washing, foi example 0 015 M sodium chloride/00015 M sodium c ⁇ trate/0 1 % sodium dodecyl sulfate at 50°C. (2) employing during hybridization a denaturing agent, such as formamide. tor example. 50% (vol/vol) formamide with 0 1 % bovine serum album ⁇ n/0 1 % F ⁇ coll/0 1 % polyv ⁇ nylpyrrol ⁇ done/50 mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride.
  • a denaturing agent such as formamide. tor example.
  • 75 mM sodium citrate at 42°C, oi (3) employ 50%) formamide, 5 x SSC (0 75 M NaCl, 0 075 M sodium citrate), 50 mM sodium phosphate (pH 6 8). 0 1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0 1 % SDS, and 10% dextran sulfate at
  • Modely stringent conditions may be identified as descnbed by Sambrook et al Moleculai Cloning A Lahoiaton Manual. New York Cold Spnng Harbor Press 1989. and include the use of washing solution and hybridization conditions (e.g.. temperature, ionic strength and %SDS) less stringent that those described above.
  • An example of moderately stringent conditions is overnight incubation at 37 C C in a solution comprising: 20% formamide. 5 x SSC ( 150 mM NaCl. 15 mM trisodium citrate). 50 mM sodium phosphate (pH 7.6). 5 x Denhardt ' s solution. 10% dextran sulfate.
  • 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 amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
  • immunoadhesin designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains.
  • the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is “heterologous"), and an immunoglobulin constant domain sequence.
  • the adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand.
  • the immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1 , IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE. IgD or IgM.
  • immunoglobulin such as IgG-1 , IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE. IgD or IgM.
  • Activity refers to form(s) of a PRO polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring PRO
  • biological activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally- occurring PRO other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO
  • an '"immunological” activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally- occurring PRO.
  • biological activity of a PRO means delaying, preventing or rescuing retinal neurons, e.g., photoreceptor cells from injury, degradation or death.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the objective is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • Chronic administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
  • Intermittent administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
  • Delaying, prev enting or rescuing retinal cells fiom injury or death as a result ot the method ot the invention refers to the abilitv to keep such retinal cells viable or aliv e tor a period of time greater than is observed without application ot said method
  • Retinal cell death can iesult from injury , disease or even aging Retinal cell injury can also result in degradated cells 01 those hav ing a limited capacity tor normal physiological operation The effect can be measured either in ⁇ ⁇ t ⁇ o w ith isolated retinal cells or ;// o w ith subjects having compromised retinal cells due to injury or disease
  • “Mammal” tor purposes ot treatment refers to anv animal classified as a mammal, including humans, domestic and from animals, and zoo. sports, or pet animals, such as dogs, horses, cats, sheep, pigs. cattle, etc
  • the mammal is human
  • a "disorder" is any condition that would benefit from tieatment w ith PRO polypeptides This includes both chronic and acute disorders, as well as those pathological conditions which predispose the mammal to the disorder in question
  • disorders to be treated herein include any condition which results in injury or death of photoreceptor or other retinal cells Examples of conditions include retinal detachment, age-related and other maculopathies, photic retinopathies.
  • retinopathy of prematurity viral retinopathies such as CMV or HIV retinopathy related to AIDS, uveitis, ischemic retinopathies due to venous or arterial occlusion or other vascular disorder, retinopathies due to trauma or penetrating lesions of the eye, peripheral vitreoretinopathy, and inherited retinal degenerations
  • retinopathy of prematurity viral retinopathies such as CMV or HIV retinopathy related to AIDS, uveitis, ischemic retinopathies due to venous or arterial occlusion or other vascular disorder, retinopathies due to trauma or penetrating lesions of the eye, peripheral vitreoretinopathy, and inherited retinal degenerations
  • Exemplaiy retinal degenerations include e g .
  • hereditary spastic paraplegia with retinal degeneration Kerman and Barnard-Scholz syndromes
  • retinitis pigmentosa Hereditary spastic paraplegia with retinal degeneration
  • Stargardt disease Hereditary spastic paraplegia with retinal degeneration
  • Usher syndrome retinitis pigmentosa with congenital hearing loss
  • Refsum syndrome retinitis pigmentosa, hereditary hearing loss, and polyneuropathy
  • Additional disorders which result in death of retinal neurons include, retinal tears, detachment of the retina and pigment epithelium, degenerative myopia, acute retinal necrosis syndiome (ARN). traumatic chorioretinopathies or contusion (Purtscher's Retinopathy) and edema
  • a therapeutically effective amount is an amount of active PRO which is lequired to achieve measurable delay, rescue or prevention ot damage to retinal neurons
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients. or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed Often the physiologically acceptable carrier is an aqueous pH buffered solution
  • physiologically acceptable carriers include butters such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptide. proteins, such as serum albumin gelatin or immunoglobulins, hydrophihc polymers such as polyvinylpyrrolidone. amino acids such as glycine, glutamine. asparagine.
  • argimne or lysine monosaccha ⁇ des, disaccha ⁇ des, and other carbohydrates including glucose, mannose. or dext ⁇ ns, chelatmg agents such as EDTA, sugar alcohols such as mannitol or sorbitol. salt-forming counte ⁇ ons such as sodium, and/or no onic surfactants such as TWEEN 1 .
  • Antibody fragments' comprise a portion ot an intact antibody , preterablv the antigen binding oi v ariable region ot the intact antibody Examples ot antibody fragments include Fab Fab F(ab') 2 . and Fv fragments, diabodies.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and - binding site This region consists of a dimer ot 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 Vn-V dimer Collectivelv the six CDRs confer antigen- binding specificity to the antibody However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH I ) of the heavy chain Fab fragments diffei from Fab' fragments by the addition of a tew residues at the carboxy terminus of the heavy chain CH I domain including one or more cysteines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine res ⁇ due(s) of the constant domains bear a free thiol group F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known
  • immunoglobulins The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD. IgE. IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g , IgG l , IgG2, IgG3, IgG4. IgA. and IgA2
  • Single-chain Fv” oi “sFv” antibody fragments comprise the V** and V, domains of antibody, wherein these domains are present in a single polypeptide chain
  • the Fv polypeptide further comprises a polypeptide linker between the V and V* domains which enables the sFv to form the desired structure for antigen binding
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V ) in the same polypeptide chain (V -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 tullv in. foi example, EP 404.097, WO 93/1 1 161 , and Holhnger et al , Pioc Natl Acad Sci USA.
  • an "isolated antibody is one which has been identified and sepaiated and/or recovered from a component ot its natural env nonment Contaminant components ot its natural env ironment are materials which would mterfeie w ith diagnostic or therajDeutic uses for the antibody , and may include enzymes hormones, and other proteinaceous or nonproteinaceous solutes
  • the antibody will be purified ( 1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably moie than 99% by weight.
  • silver stain Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present Ordinarily, however, isolated antibody will be prepared by at least one purification step
  • label when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody
  • the label may be detectable by itself (e g radioisotope labels or fluorescent labels) or. in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable
  • solid phase is meant a non-aqueous matrix to which the antibody of the present invention can adhere
  • solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccha ⁇ des (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and sihcones
  • the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275,149
  • a “liposome” is a small vesicle composed of various types of hpids, phospholipids and/or surfactant which is useful tor delivery of a drug (such as a PRO polypeptide or antibody thereto) to a mammal
  • a drug such as a PRO polypeptide or antibody thereto
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement ot biological membranes
  • a "small molecule” is defined heiein to have a molecular weight below about 500 Daltons
  • Max file length is 65535 (limited bv unsigned short x in the jmp struct)
  • the program may create a tmp file in /tmp to hold info about traceback
  • ij dx[ ⁇ d] lj p, if (dx[ ⁇ d].jp n[0] && Cdna
  • (ndelx > MAXJMP && xx > dx[ ⁇ d] jp x[ ⁇ j] + MX)
  • dumpbiockO dump a block of lines with numbers, stars: pr_ahgn() * nums() - put out a number line. dumpbiockO
  • *ps[i] toupper(*ps[i]); po[i]+ + ; ps[i] + + ;
  • *py++ toupper(*px), if (mdex( ATGCU ,*(py-l))) natgc + + , ⁇ ⁇
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides.
  • cDNAs encoding various PRO 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.
  • PRO/number the protein encoded by the full length native nucleic acid molecules disclosed herein as well as all further native homologues and variants included in the foregoing definition of PRO. will be referred to as "PRO/number " . regardless of their origin or mode of preparation.
  • PRO variants can be prepared.
  • PRO variants can be prepared by introducing appropriate nucleotide changes into the PRO DNA, and/or by synthesis of the desired PRO polypeptide.
  • amino acid changes may alter post-translational processes of the PRO, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
  • Variations in the native full-length sequence PRO or in various domains of the PRO described herein can be made, for example, using any of the techniques and guidelines for conservative and non- conservative mutations set forth, for instance, in U.S. Patent No. 5.364.934.
  • Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO that results in a change in the amino acid sequence of the PRO as compared with the native sequence PRO.
  • 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 PRO.
  • Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e.. conservative amino acid replacements.
  • Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
  • PRO polypeptide fragments are provided herein. Such fragments may be truncated at the N- terminus or C-terminus. or may lack internal residues, for example, when compared with a full length nativ e protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the PRO polypeptide.
  • PRO fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized.
  • An alternative approach involves generating PRO fragments by enzymatic digestion, e.g.. by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment.
  • Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, PRO polypeptide fragments share at least one biological and/or immunological activity with the native PRO polypeptide disclosed herein.
  • PCR polymerase chain reaction
  • conservative substitutions of interest are shown in Table 6 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 6, or as further described below in reference to amino acid classes, are introduced and the products screened.
  • Substantial modifications in function or immunological identity ot the PRO polypeptide are accomplished by selecting substitutions that ditfei significantly in then effect on maintaining (a) the structure ot the polypeptide backbone in the area ot the substitution, for example, as a sheet or helical conformation, (b) the charge or hydiophobicity ot the molecule at the target site, or (c) the bulk of the side chain Naturally occurring residues are divided into groups based on common side-chain properties ( I ) hydrophobic norleucine. met. ala. val. leu. lie. (2) neutral hydrophihc cys ser. thr.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class Such substituted residues also may be introduced into the conservative substitution sites or. more preferably, into the remaining (non-conserved) sites
  • Scanning amino acid analysis can also be employed to identify one or more ammo acids along a contiguous sequence
  • preferred scanning amino acids are relatively small, neutral amino acids
  • Such am o acids include alanine, glycine, se ⁇ ne. and cysteine
  • Alanine is typically a piefe ⁇ ed scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to altei 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 Pioteins (W H Freeman & Co .
  • amino acid sequence variants of native PRO polypeptides and functional fragments theieof may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant PRO or by in vitio synthesis of the desired polypeptide
  • There are two principal variables in the construction of amino acid sequence variants ( 1 ) the location ot the mutation site and. (2) the nature of the mutation With the exception of naturally-occurring alleles. which do not require the manipulation ot the DNA sequence encoding the PRO.
  • the amino acid sequence variants of PRO are preferably constructed bv mutating PRO. either to arriv e at an allele oi an ammo acid sequence v ariant that does not occur in nature
  • cysteine residues can be mutated in order to assist in the assembly and refolding
  • one or more cysteine residues can be mutated to serine to assist in refolding of the protein subsequent to expression in E coli or a similar piokaryote
  • Covalent modifications ot PRO are included within the scope of this invention
  • One type ot covalent modification includes reacting targeted amino acid residues of a PRO polypeptide with an organic de ⁇ vatizing agent that is capable ot reacting with selected side chains or the N- or C- terminal residues of the PRO De ⁇ vatization with bifunctional agents is useful, tor instance, for crosslinking PRO to a water- msoluble support matrix or surface for use in the method for purifying anti-PRO antibodies, and vice-versa
  • Commonly used crosslinking agents include, e g , l . l -b ⁇ s(d ⁇ azoacetyl)-2-phenylethane. glutaraldehyde.
  • N- hydroxysuccinimide esters for example, esters with 4-az ⁇ dosal ⁇ cyl ⁇ c acid, homobifunctional lmidoesters, including disuccinimidyl esters such as 3,3'-d ⁇ th ⁇ ob ⁇ s (succinimidylpropionate), bifunctional maleimides such as b ⁇ s-N- ⁇ nale ⁇ m ⁇ do- 1 ,8-octane and agents such as methyl-3-[(p-az ⁇ dophenyl)d ⁇ th ⁇ o]prop ⁇ o ⁇ m ⁇ date
  • Another type of covalent modification ot the PRO polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO
  • the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydiate moieties present Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence The alteration may be made, for example, by the addition of.
  • the PRO amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons are generated that w ill translate into the desired amino acids
  • Another means of increasing the number ot carbohydrate moieties on the PRO polypeptide is by chemical or enzymatic coupling ot glycosides to the polypeptide Such methods dre described in the art e.g , in WO 87/05330 published 1 1 September 1987 and in Aplm and W ⁇ ston. CRC Cut Biochem . pp. 259-306 ( 1981 )
  • Removal ot carbohydiate moieties present on the PRO polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation
  • Chemical deglycosy lation techniques are know n in the art and described. for instance, by Hakimuddin, et al Aich Biochem B ⁇ oph ⁇ s_. 259 52 ( 1987) and by Edge et al , Anal
  • Enzymatic cleavage of carbohydrate moieties on poly peptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al , Meth 138 350 ( 1987)
  • PRO comp ⁇ ses linking the PRO polypeptide to one of a variety of nonproteinaceous polymers, e g , polyethylene glycol (PEG) polypropylene glycol. or polyoxyalkylenes, in the manner set forth in U S Patent Nos 4,640.835. 4.496.689. 4.301.144. 4,670,417 4,791 ,192 or 4, 179,337
  • the PRO of the present invention may also be modified in a way to form a chime ⁇ c molecule comprising PRO fused to another, heterologous polypeptide or amino acid sequence
  • a chime ⁇ c molecule comprises a fusion of the PRO with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind
  • the epitope tag is generally placed at the amino- or carboxyl- terminus of the PRO
  • the presence of such epitope-tagged forms of the PRO can be detected using an antibody against the tag polypeptide
  • provision of the epitope tag enables the PRO to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag
  • Various tag polypeptides and their respective antibodies are well known in the art Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags, the flu HA tag polypeptide and its antibody 12CA5 [Field e
  • the chime ⁇ c molecule may comprise a fusion of the PRO with an immunoglobulin or a particular region of an immunoglobulin For a biv alent form of the chime ⁇ c molecule (also referred to as an "immunoadhesin").
  • the lg fusions preferably include the substitution oi a soluble (transmembrane domain deleted or inactivated) form of a PRO polypeptide in place of at least one v ariable region within an lg molecule
  • the immunoglobulin fusion includes the lunge. CH2 and CH ⁇ or the hinge.
  • DNA encoding PRO may be obtained from a cDNA library prepared from tissue believed to possess the PRO mRNA and to express it at a detectable level Accordingly, human PRO DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples
  • the PRO-encoding gene may also be obtained from a genomic library or by known synthetic procedures ( g . automated nucleic acid synthesis)
  • Probes such as antibodies to the PRO or oligonucleotides ot at least about 20-80 bases
  • Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al , Moleculai Cloning A Laboiaton Manual (New York Cold Spring Harbor Laboratory Pi ess, 1989)
  • An alternative means to isolate the gene encoding PRO is to use PCR methodology [Sambiook et al , supia. Dieftenbach et al , PCR Pi i ei A Laboiaton Manual (Cold Spring Harbor Laboratory Press. 1995)]
  • 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 tull-length sequence can be determined using methods know n in the art and as described herein
  • Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time and if necessary, using conventional primer extension procedures as described in Sambrook et al , supia. to detect precursors and processing intermediates ot mRNA that may not hav e been reverse-transcribed into cDNA 2 Selection and Transformation of Host Cells
  • Host cells are transfected or transformed with expression or cloning vectors described herein for PRO production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants. or amplifying the genes encoding the desired sequences
  • the culture conditions such as media, temperature. pH and the like, can be selected by the skilled artisan without undue experimentation
  • principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology a Practical Approach. M Butler, ed (IRL Press, 1991 ) and Sambrook et al supra
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells
  • Suitable prokaryotes include but are not limited to eubacteria. such as Gram-negative or Gram-positive organisms, for example. Enterobacte ⁇ aceae such as E.
  • E colt strains are publicly available, such as E coli KI2 strain MM294 (ATCC 31.446), E coli X1776 (ATCC 31 ,537), E coli strain W 1 10 (ATCC 27,325) and K5 772 (ATCC 53,635)
  • Other suitable prokaryotic host cells include Enterobacte ⁇ aceae such as Escherichia, e g , E coli, Enterobacter, Erwtnia. Klebsiella, Proteus. Salmonella, e g . Salmonella tvphimurium. Serratia, e g , Serratia marcescans. and Shigella. as well as Bacilli such as B subtilis and B licheniformis (e g . ? lichenifonms 41 P disclosed in
  • Strain W31 1 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations
  • the host cell secretes minimal amounts of proteolytic enzymes
  • strain W3 ] 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 1A2 which has the complete genotype tonA E coli W31 10 strain 9E4 which has the complete genoty pe tonA pt ⁇ 3.
  • E coli W31 10 strain 27C7 (ATCC 55.244) which has the complete genotype tonA ptr3 phoA El 5 (argF lac)169 degP ompT kan' ' .
  • E coli W31 10 strain 37D6 which has the complete genotype tonA pti 1 phoA El 5 (a ⁇ gF-lac)169 degP ompT ⁇ bs7 G kan .
  • E coli W31 10 strain 40B4 which is strain 37D6 with a non-kanamycin resistant degP deletion mutation, and an £ coli strain having mutant pe ⁇ plasmic protease disclosed in U S Patent No 4 946,783 issued 7 August 1990
  • Other suitable strains have impaired heat shock response in combination with protease deletions and mutations
  • the E Coli W31 10 strain 44C6 which has the complete genotype fhuA ⁇ (tonA ⁇ ) lon ⁇ galE rpoHts (htpHts) ⁇ cIpP
  • Other potential cloning hosts are E coli 294 (ATCC 31 ,446), E coli B and E coli XI 776 (ATCC 31 ,537)
  • in vitro methods of cloning e g . PCR or other nucleic acid polymerase reactions, are suitable
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts tor PRO-encoding vectors Saccharom ces DCevisiae is a commonly used lower eukaryotic host microorganism Others include Schi osacchai oun ces pombe (Beach and Nurse
  • K lactis MW98-8C, CBS683 CBS4574, Louvencourt et al , J Bactenol , 154(2) 737-742 [ 1983]
  • K fragihs ATCC 12,424), K bulgaricus (ATCC 16,045), K wickeramu (ATCC 24,178), K waltu (ATCC 56,500), K diosophilarum (ATCC 36,906, Van den Berg et al , Bw Technolog), 8 135 (1990)), K thermotolerans, and K m ⁇ xianus yarrowta (EP 402,226), Pichia pastons (EP 183,070.
  • Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckeia Pichia Saccharomyces, Toiulopsis, and Rhodotorula A list of specific species that are exemplary of this class of yeasts may be found in C Anthony, The Bwchemistn of Methylotrophs, 269 ( 1982)
  • Suitable host cells for the expression of glycosylated PRO are derived from multicellular organisms
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9 as well as plant cells
  • useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidney CV1 line transformed by SV40 (COS 7
  • the nucleic acid (e g . cDNA oi genomic DNA) encoding PRO may be inseited into a replicable vector foi cloning (amplification ot the DNA) oi tor expression
  • Various v ectors are publicly available
  • the vector may, for example, be in the form of a plasmid. cosmid. v iral particle, or phage
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety ot proceduies In general. DNA is inserted into an appropnate restriction endonuclease s ⁇ te(s) using techniques known in the art
  • Vector components generally include, but are not limited to. one or more of a signal sequence, an origin of replication, one or more mai ker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan
  • the PRO may be pioduced 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 matuie protein or polypeptide
  • the signal sequence may be a component ot the vector, or it may be a part of the PRO-encoding DNA that is inserted into the vector
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group ot the alkaline phosphatase. penicilhnase, 1pp. or heat-stable enterotoxin II leaders.
  • yeast secretion the signal sequence may be, e.g..
  • yeast invertase leader alpha tactor leader (including Saccharomyces and Kluyveronnces ⁇ - factor leaders, the latter described in U.S. Patent No 5,010,182), or acid phosphatase leader, the C albicans glucoamylase leader (EP 362, 179 published 4 April 1990). or the signal described in WO 90/13646 published 15 November 1990
  • mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences fiom 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 moie selected host cells Such sequences are well known for a vanety ot bacteria, yeast, and viruses
  • the origin ot 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
  • Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin.
  • methotrexate. or tetracyclme (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available fiom complex media, e.g . the gene encoding D-alanine racemase for Bacilli
  • suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO-encoding nucleic acid, such as DHFR or thvmidine kinase
  • An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al , Pioc . Natl Acad.
  • a suitable selection gene for use in yeast is the tip] gene present in the yeast plasmid YRp7 [Stinchcomb et al , Natuie. 282 39 ( 1979). Kmgsman et al.. Gene. 7 141 ( 1979). Tschemper el al . Gene. 10 157 ( 1980)]
  • the t ⁇ p ⁇ gene prov ides a selection marker tor a mutant strain ot v east lacking the ability to giow in try ptophan. tor example. ATCC No 44076 oi PEP4- 1 [Jones. Genetic s 85 12 ( 1977)]
  • Expression and cloning vectors usually contain a promotei operably linked to the PRO-encoding nucleic acid sequence to dnect mRNA sy nthesis Promoters iecogmzed by a variety of potential host cells are well known Promotei s suitable foi use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems [Chang et al . Natitie. 275 615 ( 1978). Goeddel et al . Natuie. 2_ ⁇ 544 (1979)]. alkaline phosphatase. a tryptophan (trp) promoter system [Goeddel. Nucleic Acids Res . 8 4057 ( 1980). EP 36.776].
  • Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S D ) sequence operably linked to the DNA encoding PRO
  • suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al , J Biol Chem , 255 2073 ( 1980)] or other glycolytic enzymes [Hess et al . J Enzyme Reg , 7 149 ( 1968), Holland, Biocheimstn , JJ 4900 ( 1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase. hexokinase, pyruvate decarboxylase. phosphofructokinase, glucose-6-phosphate isomerase. 3-phosphoglycerate mutase, pyruvate kinase. t ⁇ osephosphate isomerase. phosphoglucose isomerase, and glucokinase
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, 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.
  • PRO 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 bovine papilloma virus
  • avian sarcoma virus cytomegalovirus
  • a retrovirus hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e g , the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems
  • Enhancers are cis-acting elements of DNA. usually about from 10 to 300 bp, that act on a promotei 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 vnus examples include the SV40 enhancei on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancei .
  • the enhancer may be spliced into the vector at a position 5' or 3' to the PRO coding sequence, but is prefeiably located at a site 5' from the promoter
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary tor the termination of transcription and foi stabilizing the mRNA Such sequences are commonly available from the 5' and. occasionally 3. untranslated regions of eukaryotic or vnal DNAs oi cDNAs These regions contain nucleotide segments transcnbed as polv aden lated fragments in the untranslated portion of the mRNA encoding PRO
  • Prokaryotic cells used to produce the PRO polypeptides of this invention may be cultured in suitable media as described generally in Sambrook et al supia and Ausubel et al , supia Briefly, the transformed cells are grown at 30°C or 37°C until the optical density (measured at 550 nm) reaches about 2
  • the culture is diluted into a production medium, regrown with aeration, and 3- ⁇ -Indole acrylic acid (I A A) is added Growth is continued with aeration for about another 1 hours after hich time the cells are harvested by cent ⁇ tugation When refolding is necessary the proceduie outlined below may be employed
  • a 10 liter fermentation may be carried out as follows
  • the Kirtor is first sterilized with a sterilization solution of about 5-6 5 liters of deionized water to which is added ammonium sullate (50 0 g).
  • ammonium sullate 50 0 g
  • sodium phosphate sodium phosphate
  • monobasic dihydrate 30 0 g
  • sodium citrate dihydrate ( 10 0 g).
  • the growth media after inoculation has a volume typically of about 8 5 liters
  • the media components are comprised of 50% glucose solution (15 mL), IM magnesium sulfate (70 mL), 20%e Hycase solution (250 mL), 20%> yeast extract solution (250 mL), 2 mg/mL ampicillin (250 mL) and trace metals (5 mL)
  • a typical IL trace metal solution is composed ot the following HCI (100 mL), Ferric chloride hexahydrate (27 g), Zinc sulphate heptahydrate (8 g).
  • Gene amplification and/or expression may be measured in ⁇ sample directly, for example, by conventional Southern blotting, northern blotting to quantitate the transcription of mRNA [Thomas, Pioc Natl Acad Sci USA 77 5201 5205 ( 1980)].
  • Vanous labels may be employed most commonly radioisotopes, particularly " P Howevei other techniques may also be employed, such as using biotin-modified nucleotides tor introduction into a polynucleotide
  • the biotin then serves as the site for binding to av idin oi antibodies which mav be labeled with a wise variety of labels such as radionuchdes.
  • fiuorescers enzv mes. oi the like Alternativ ely antibodies mav be emploved that can recognize specific duplexes including DNA duplexes.
  • RNA duplexes and DNA-RNA hvb ⁇ d duplexes or DNA-protein duplexes
  • the antibodies in tui n may be labeled and the assay mav be earned out w here the duplex is bound to a surface so that upon the formation of duplex on the surface thefilmnce of antibody bound to the duplex can be detected
  • Gene expression alternatively may be measured by immunological methods, such as lmmunohistochemical staining ot tissue sections and assay of cell culture or body fluids to quantitate directly the expression of gene product
  • Antibodies useful tor lmmunohistochemical staining and/or assaying of sample fluids may be eithei 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.
  • Labels are preferably visually detectable, such as enzymatic labels, fluorescent labels luminescent labels, and the like Foi example, see Hsu et al , Am J Clin Path 75 734-738 ( 1980)
  • Forms of PRO may be recovered from culture medium as a secreted polypeptide or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (e g .
  • Cells employed in expression of PRO polypeptides can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents
  • the PRO polypeptide can be produced intracellularly, in the pe ⁇ plasmic space, or directly secreted into the medium If the PRO is produced intracellularly. It will usually be necessary to PRO, respectively from other recombinant cell proteins or polypeptides to obtain preparations that are substantially homogenous to the respective PRO
  • the culture medium or lysate is centrifuged to remove the particulate debris, e g host cells or lysed fragments
  • the procedure is described in Carter et al , Bio/Technology J_0 163- 167 ( 1992) for isolating proteins which are secreted to the pe ⁇ plasmic space of E coli Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) ovei about 30 minutes Cell debris can be removed by centrifugation The following procedures are examplary ot other suitable purification procedures by fractionation
  • a microorganism such as E c oli which is fermented undei conditions suitable tor the expression of PRO (but not secretion) is deposited by the microorganism in insoluble "refiactile bodies", and is recoverable by a lysing procedure
  • cells are first washed in a cell disruption buffer Foi example, about 100 g of cells are resuspended in about 10 volumes of a cell disruption buffer (e g 10 mM
  • Tris 5 mM EDTA. pH 8) with, for example, a Polytron homogemzer. followed by centrifugation at 5000 x g tor 30 minutes Cells are then lysed using any conventional technique such as tonic shock, sonication. pressure cycling, chemical or enzymatic methods For example, the washed cell pellet above may be resuspended in another 10 volumes of a cell disruption buffer with a homogemzer and the cell suspension is passed through an LH Cell Disrupter (LH Inceltech.
  • LH Cell Disrupter LH Inceltech.
  • the particulate matter containing PRO is then separated form the liquid phase and optionally washed with any suitable liquid
  • a suspension of cell lysate may be centrifuged at 5,000 x g for 30 minutes, iesuspended and optionally centrifuged a second time to make a washed refractile body pellet
  • the washed pellet may be used immediately or optionally stored frozen (at e g -70°C)
  • Insoluble PRO polypeptide in the refractile body fiom the procedure above is solubihzed with a solubilizing buffer
  • the solubilizing buffer contains a chaotropic agent and is usually buffered at a basic pH and contains a reducing agent to improve the yield of monome ⁇ c PRO
  • Representative chaotropic agents include urea. guanidine-HCl, and sodium thiocyanate
  • a preferred chaotropic agent is guamdine-HCl
  • concentration ot chaotropic agent is usually 4-9 M. preferably 6-8 M
  • the pH of the solubilizing buffer is maintained by any suitable buffer in a pH range of from about 7 5-9 5, preferably 8 0-9.0.
  • the solubilizing buffer also contains a leducing agent to aid formation ot the monome ⁇ c form of PRO
  • Suitable reducing agents include organic compounds containing a free thiol (RDH)
  • Representative reducing agents include dithiothreitol (DTT), dithioeryth ⁇ tol (DTE), mercaptoethanol, glutathione (GSH), cysteamine and cysteine
  • DTT dithiothreitol
  • the solubilizing buffer may contain a mild oxidizing agent (e g molecular oxygen) and a sulfite salt to form monoine ⁇ c mutant PRO via sulfitolysis
  • the resulting PRO-S- sultonate is later refolded in the presence of redox buffer (e g . GSH/GSSG) to form the properly folded PRO
  • the PRO protein is usually further purified using, foi example, centnfugation, gel filtration chromatography and reversed phase column chromatography
  • the refractile body pellet may be resuspended in about 5 volumes by weight of the solubilizing buffer (20 mM Tris. pH 8. with 6-8 M guanidine and 25 mM DTT) and stirred for 1 -3 hr.. or overnight at 4°C to effect solubilization of the mutant PRO protein.
  • High concentrations of urea (6-8M) are also useful but generally result in somewhat lower yields compared to guanidine.
  • the solution is centrifuged at 30.000 x g for 30 min. to produce a clear supernatant containing denatured, monomeric PRO. The supernatant is then chromatographed on a Superdex ® 200 gel filtration column
  • the purified reduced protein elutes at approximately 50% acetonitrile. This material is used for refolding to obtain biologically active.
  • Triton ® X-100 Triton ® X-100, dodecyl-beta-maltoside, CHAPS, CHAPSO, SDS, sarkosyl, Tween ® 20 and Tween ® 80, Zwittergent 3-14 and others may be used to produce at least minimal folding.
  • the most preferred detergents are of the CHAPS family (CHAPS and CHAPSO) which appear to work best in refolding and limit protein aggregation and improper disulfide formation. Levels of CHAPS greater than about 1 % are most preferred. To optimize yields, it is preferred to have sodium chloride present (0.1 M-0.5M).
  • EDTA 1 -5 mM
  • EDTA 1 -5 mM
  • At least 15% glycerol is further preferred in order to reach optimal refolding conditions.
  • the redox buffer have both an oxidized and reduced organic thiol
  • Suitable redox pairs include mercaptoethanol, glutathione (GSH). cysteamine, cysteine and their corresponding oxidized forms. Preferred redox are glutathione (GSH):oxidized glutathione (GSSG) or cysteinexystine. The most preferred redox pair is glutathione (GSH):oxidized glutathione (GSSG).
  • Refolding yields of 40-60% are typical for preparations of the respective PRO that have been purified through the first C4 step.
  • Active material can be obtained when less pure preparations (e.g. directly after the Superdex"" 200 column or after the initial refractile body extraction) although the yields can be less due to precipitation and interference of non-PRO proteins during the respective PRO refolding process.
  • Nucleotide sequences (or their complement) encoding PRO have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA.
  • PRO nucleic acid will also be useful for the preparation of PRO polypeptides by the recombinant techniques described herein.
  • the full-length native sequence PRO gene, or portions thereof may be used as hybridization probes for a cDNA library to isolate the full-length PRO cDNA or to isolate still other cDNAs (for instance, those encoding naturally-occurring variants of PRO or PRO from other species) which have a desired sequence identity to the native PRO sequence disclosed herein.
  • the length of the probes will be about 20 to about 50 bases.
  • the hybridization probes may be derived from at least partially novel regions of the full length native nucleotide sequence wherein those regions may be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and introns of native sequence PRO.
  • a screening method will comprise isolating the coding region of the PRO gene using the known DNA sequence to synthesize a selected probe of about 40 bases.
  • Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 2 P or " 3 S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems.
  • Labeled probes having a sequence complementary to that of the PRO gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below.
  • antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target PRO mRNA (sense) or PRO DNA (antisense) sequences.
  • Antisense or sense oligonucleotides comprise a fragment of the coding region of PRO DNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.
  • Stein and Cohen Cancer Res. 48:2659.
  • binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means. including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means.
  • the antisense oligonucleotides thus may be used to block expression of PRO proteins.
  • Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar- phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases.
  • Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences.
  • Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10048, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine).
  • intercalating agents such as ellipticine. and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
  • Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaP0 4 -mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Ban * virus.
  • an antisense or sense oligonucleotide is inserted into a suitable retroviral vector.
  • a cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo.
  • Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
  • Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO
  • Suitable ligand binding molecules include, but are not limited to. cell surface receptors, growth factors, other cytokines. or other ligands that bind to cell surface receptors.
  • conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
  • a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448.
  • the sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
  • Antisense or sense 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
  • the probes may also be employed in PCR techniques to generate a pool of sequences tor identification ot closely related PRO coding sequences
  • Nucleotide sequences encoding a PRO can also be used to onsti uct hy bridization probes foi mapping the gene which encodes that PRO and for the genetic analysis of individuals with genetic disorders
  • the nucleotide sequences prov ided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hyb ⁇ dization. linkage analysis against known chromosomal markers, and hybridization screening with libraries
  • the coding sequences for PRO encode a protein which binds to another protein (example.
  • the PRO can be used in assays to identify the othei proteins or molecules involved in the binding interaction
  • inhibitors of the receptor/hgand binding interaction can be identified
  • Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists ot the binding interaction
  • the receptor PRO can be used to isolate correlative l ⁇ gand(s) Screening assays can be designed to find lead compounds that mimic the biological activity of ⁇ native PRO or a receptor for PRO Such screening assays will include assays amenable to high-throughput screening of chemical libia ⁇ es.
  • Small molecules contemplated include synthetic organic or inorganic compounds
  • 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.
  • Nucleic acids which encode PRO or its modified forms can also be used to generate either transgemc animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents
  • a transgemc animal e g , a mouse or rat
  • a transgemc animal is an animal having cells that contain a transgene. which tr ⁇ nsgene was introduced into the animal or an ancestor of the animal at a prenatal, e g .
  • transgene is a DNA which is integrated into the genome of a cell from which a transgemc animal develops
  • cDNA encoding PRO can be used to clone genomic DNA encoding PRO in accordance with established techniques and the genomic sequences used to generate transgemc animals that contain cells which express DNA encoding PRO
  • Methods for generating transgemc animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U S Patent Nos 4,736,866 and 4,870.009
  • particular cells would be targeted for PRO transgene incorporation with tissue-specific enhancers
  • Transgemc animals that include a copy of a transgene encoding PRO introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expiession of DNA encoding PRO
  • Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression
  • an animal is treated with the following embryonic embryonic stage
  • cDNA encoding PRO can be used to clone genomic DNA encoding PRO in accordance with established techniques A portion of the genomic DNA encoding PRO can be deleted or replaced with another gene, such as a gene encoding a selectable markei which can be used to monitor integration
  • another gene such as a gene encoding a selectable markei which can be used to monitor integration
  • aie included in the vector [see e g Thomas and Capecchi. Cell. 5J_ 503 (1987) for a description of homologous recombination vectors]
  • the vector is introduced into an embryonic stem cell line (e g .
  • Embryonic Stem Cells A Piaciical Appioach. E J Robertson ed (IRL Oxford 1987), pp 1 13- 152]
  • a chime ⁇ c 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.
  • Nucleic acid encoding the PRO polypeptides may also be used in gene therapy In gene therapy applications, genes are introduced into cells in order to achieve in vn o synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene "Gene therapy” includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in ⁇ ⁇ vo It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite then low intr ⁇ cellular concentrations caused by their restricted uptake by the cell membrane (Zamecnik et al , Pioc Natl Acad Sci USA 83 4143-4146 [ 19861)
  • the oligonucleotides can be modified to enhance their uptake, e g by substituting their negatively charged phosphodiester groups by uncharged groups
  • nucleic acid there are a variety of techniques available for intioducing nucleic acids into viable cells The techniques vary depending upon whether the nucleic acid is transfened into cultured cells in vitio, or m vivo in the cells of the intended host Techniques suitable tor the transfer of nucleic acid into mammalian cells in vitro include the use ot liposomes. electroporation, microinjection. cell tusion. DEAE-dextran.
  • the currently preferred m ⁇ ⁇ vo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transtection (Dzau et al , Tiends in Biotechnology J_[, 205-210 [ 1993])
  • viral typically retroviral
  • viral coat protein-liposome mediated transtection Dzau et al , Tiends in Biotechnology J_[, 205-210 [ 1993]
  • an agent that targets the target cells such as an antibody specific for a cell surface membrane piotein or the target cell, a ligand for a receptor on the target cell, etc
  • proteins which 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 which undergo intemalization in cycling, proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example, by Wu et al.. J. Biol. Chem. 262. 4429-4432 ( 1987): and Wagner et al.. Proc. Natl.
  • PRO polypeptides described herein may also be employed as molecular weight markers for protein electrophoresis purposes and the isolated nucleic acid sequences may be used for recombinantly expressing those markers.
  • nucleic acid molecules encoding the PRO polypeptides or fragments thereof described herein are useful for chromosome identification.
  • there exists an ongoing need to identify new chromosome markers since relatively few chromosome marking reagents, based upon actual sequence data are presently available.
  • Each PRO nucleic acid molecule of the present invention can be used as a chromosome marker.
  • PRO polypeptides and nucleic acid molecules of the present invention may also be used for tissue typing, wherein the PRO polypeptides of the present invention may be differentially expressed in one tissue as compared to another.
  • PRO nucleic acid molecules will find use for generating probes for PCR, Northern analysis, Southern analysis and Western analysis.
  • This invention encompasses methods of screening compounds to identify those that mimic the PRO polypeptide (agonists) or prevent the effect of the PRO polypeptide (antagonists).
  • Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PRO polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins.
  • Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
  • 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 PRO 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 PRO 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 PRO polypeptide and drying.
  • an immobilized antibody e.g.
  • a monoclonal antibody, specific for the PRO polypeptide to be immobilized can be used to anchor it to a solid surlace
  • the assav is performed bv adding the non immobilized component which ma's be labeled by a detectable label to the immobilized component e g , tht coated surface containing the anchored component
  • the non reacted components are removed, e g , by w ashing and complexes anchored on the solid surface are detected
  • complexing can be detected for example, by using a labeled antibody specifically binding the immobilized complex
  • the candidate compound interacts with but does not bind to a particular PRO polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protem-protem 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 USA 88 9578 9582 (1991)) as disclosed by Chevray and Nathans, Proc Natl
  • yeast GAL4 a transcriptional activator
  • 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-tocZ 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 chromogemc substrate for ⁇ galactosidase
  • 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 tor these interactions
  • a reaction mixture is prepared containing the product ot the gene and the intra or extracellular component under conditions and for a time allowing for the interaction and binding of the two products
  • 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 tormation ot a complex in the control react ⁇ on(s) but not in the reaction mixture containing the test compound indicates that the test compound interteres with the interaction of the test compound and its reaction partner
  • the PRO polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of
  • 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. Coligan et al., Current Protocols in Immun. , j_(2): Chapter 5 (1991).
  • 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 labeled PRO polypeptide.
  • the PRO polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and re-trans ected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
  • labeled PRO polypeptide can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule.
  • Cross-linked material is resolved by PAGE and exposed to X-ray film.
  • the labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing.
  • the amino acid sequence obtained from micro- sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.
  • mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured.
  • potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with 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 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 polvnucleotide sequence, which encodes the mature PRO polypeptides herein, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length
  • a DNA oligonucleotide is designed to be complementary to a region ot the gene involved in transcription (triple hehx - see Lee et al , Nucl Acids Res , 6 3073 ( 1979), Cooney et al , Science.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in and blocks translation ot 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 /// vivo to inhibit production of the PRO polypeptide
  • antisense DNA oligodeoxy ⁇ bonucleotides derived trom the translation-initiation site, e g , between about -10 and + 10 positions of the target gene nucleotide sequence, are preferred
  • 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 ot 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 ot catalyzing the specific cleavage of RNA Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific ⁇ bozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g , Rossi, Current Biology, 4 469-471 (1994), and PCT publication No WO 97/33551 (published September 18, 1997)
  • Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single- stranded and composed of deoxynucleotides
  • the base composition ot these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand ot a duplex
  • Hoogsteen base-pairing rules which generally require sizeable stretches of purines or pyrimidines on one strand ot a duplex
  • the present invention further provides anti-PRO antibodies
  • Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies 1 Polyclonal Antibodies
  • the anti-PRO antibodies may comprise polyclonal antibodies Methods ot 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 ad)uvant T ypically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or lntrapentoneal injections
  • the immunizing agent may include the PRO polypeptide or a fusion protein thereof It mav be useful to conjugate the immunizing agent to ⁇ protein know n to be lmmunogenic in the mammal being immunized Examples of such lmmunogenic proteins include but are not limited to keyhole limpet hemocyanin.
  • adiuv ants which may be employed include Fieund s complete ad
  • the immunization piotocol may be selected by one skilled in the art without undue experimentation
  • the anti-PRO antibodies may. alternatively be monoclonal antibodies Monoclonal antibodies may be prepared using hyb ⁇ doma methods, such as those described by Kohler and Milstein Nature.
  • a mouse, hamster, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable ot producing antibodies that will specifically bind to the immuni/ing agent Alternatively the lymphocytes may be immunized in vitro
  • the immunizing agent will typically include the PRO polypeptide or a fusion protein thereof
  • peripheral blood lymphocytes PBLs
  • spleen cells or lymph node cells it non-human mammalian sources are desired
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hyb ⁇ doma cell [Goding, Monoclonal Antibodies Piinciples and Practice.
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin Usually, rat or mouse myeloma cell lines are employed
  • the hyb ⁇ doma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused. immortalized cells
  • the parental cells lack the enzyme hypoxanthine guanine phospho ⁇ bosyl transferase (HGPRT or HPRT).
  • HGPRT or HPRT hypoxanthine guanine phospho ⁇ bosyl transferase
  • the culture medium for the hyb ⁇ domas typically will include hypoxanthine. aminopte ⁇ n and thvmidine ("HAT medium”), which substances prevent the growth of HGPRT-deticient cells
  • Prete ⁇ ed immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium
  • More preferred immortalized cell lines are murine myeloma lines which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego. California and the American Type Culture Collection
  • the culture medium in which the hyb ⁇ doma cells aie cultured can then be assayed for the presence ot monoclonal antibodies directed against PRO
  • the binding specificity ot monoclonal antibodies pioduced by the hyb ⁇ doma cells is determined by immunoprecipitation or by an in vitio binding assay , such as ladioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA)
  • RIA ladioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can. for example be determined by the Scatchaid anal sis ot Munson and Pollard Anal Bioc hem 107 220 ( 1980)
  • telomeres may be subcloned by limiting dilution procedures and grow n by standard methods [Goding supia] Suitable cultuie media foi this purpose include, tor example. Dulbecco's Modified Eagle's Medium and RPMI 1640 medium Alternatively the hybndoma cells may be grown in o as ascites in a mammal
  • the monoclonal antibodies secieted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as. for example, protein A-Sepharose hydroxylapatite chromatography. gel electrophoresis dialy sis, or affinity chromatography
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Patent No 4.816,567
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies)
  • the hybndoma cells of the invention serve as a preferred source ot such DNA
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U S Patent No 4,816,567, Morrison et al , supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobuhn polypeptide
  • a non-immunoglobuhn polypeptide can be substituted tor the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimenc bivalent antibody
  • the antibodies may be monovalent antibodies Methods tor preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain
  • the anti-PRO antibodies of the invention may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , murine) antibodies arc chimenc immunoglobulins, immunoglobulin chains oi fragments thereof (such as Fv, Fab, Fab', F( ⁇ b') 2 oi other antigen-binding subsequences ot antibodies) which contain minimal sequence denved from non-human immunoglobulin Humanized antibodies include human immunoglobulins (recipient antibody ) in which residues from a complementary determining region (CDR) of the recipient are replaced by lesidues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • non-human antibodies arc chimenc immunoglobulins, immunoglobulin chains oi fragments thereof (such as Fv, Fab, Fab', F( ⁇ b') 2 oi other antigen-binding subsequences
  • Fv framew ork residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one. and typically two. variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature. 321 :522-525 ( 1986): Riechmann et a!., Nature, 332:323-329 ( 1988); and Presta. Curr. Op. Struct. Biol., 2:593-596 ( 1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones el al.. Nature, 32J . :522-525 ( 1986): Riechmann el al., Nature, 332:323-327 ( 1988);
  • 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.,
  • human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent
  • the antibodies may also be affinity matured using known selection and/or mutagenesis methods as described above.
  • Preferred affinity matured antibodies have an affinity which is five times, more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (generally murine, humanized or human) from which the matured antibody is prepared.
  • 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 PRO.
  • the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light- chain pairs, where the two heavy chains have different specificities [Milstein and Cuello. Nature. 305:537- 539 ( 1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829. published 13 May 1993. and in Traunecker et al., EMBO J., J0:3655-3659 (1991 ).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge. CH2. and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI ) containing the site necessary for light- chain binding present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared can be prepared using chemical linkage. Brennan et al., Science 229:81 ( 1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab ' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • bispecific antibodies pioduced can be used as agents for the selectiv e immobilization ot enzv mes
  • Fab ' fragments may be directlv recov ered from E coli and chemically coupled to form bispecific antibodies Shalaby et al . J Exp Med 175 217-225 ( 1992) descnbe the production of a fully humanized bispecific antibody F(ab ' ); molecule Each Fab ' tragment w as sepaiately secreted from E coli and subjected to directed chemical coupling in vitio to torm the bispecific antibody The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic acti ity of human cy totoxic lymphocytes against human breast tumor targets
  • bispecific antibodies have been produced using leucine zippers Kostelny et al , J Immunol 148(5) 1547- 1553 ( 1992)
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab ' portions of two different antibodies by gene fusion
  • the antibody homodimers were reduced at the hinge region to foi m monomei and then te oxidized to form the antibody heterodimers
  • This method can also be utilized for the production of antibody homodimers
  • Exemplary bispecific antibodies may bind to two different epitopes on a given PRO polypeptide herein
  • an anti-PRO polypeptide arm may be combined with an arm w hich binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e g CD2, CD3, CD28.
  • a triggering molecule on a leukocyte such as a T-cell receptor molecule (e g CD2, CD3, CD28.
  • Fc receptors foi IgG Fc ⁇ R
  • Fc ⁇ R Fc ⁇ RI
  • CD64 Fc ⁇ RII
  • CD32 Fc ⁇ RII
  • Fc ⁇ RIII CD 16
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PRO polypeptide
  • These antibodies possess a PRO-binding arm and an arm which binds a cytotoxic agent or a radionuchde chelator, such as
  • EOTUBE DPTA.
  • DOTA DOTA
  • TETA TETA
  • Another bispecific antibody of inteiest binds the PRO polypeptide and further binds tissue factor (TF)
  • Ugate antibodies are also within the scope of the present invention
  • Heteroconjugate antibodies are composed of two covalently joined antibodies
  • Such antibodies have for example, been proposed to target immune system cells to unwanted cells [U S Patent No 4.676 980], and for treatment of HIV infection [WO 91/00360. WO 92/200373.
  • the antibodies may be prepared in ⁇ ⁇ tw using known methods in synthetic protein chemistry, including those involving ciosslmkmg agents
  • immunotoxms may be constructed using ⁇ disulfide exchange reaction or by forming a thioether bond
  • suitable reagents for this purpose include lminothiolate and methy l-4-mercaptobutv ⁇ m ⁇ date and those disclosed for example, in U S Patent No 4.676.980
  • cysteine res ⁇ due(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region
  • the homodime ⁇ c antibody thus generated may have improv ed intemalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See Caron et al . J Exp Med . JJ6 1 191 - 1 195 ( 1992) and Shopes, J Immunol .
  • Homodime ⁇ c antibodies with enhanced anti-tumor activity may also be prep ⁇ ied using heterobifunction ⁇ l cross-linkers as described in Wolff et al , Cancel Reseaich. 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-Cancei Di ug Design, 3 219-230 ( 1989)
  • the invention also pertains to lmmunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (/ e , a radioconjugate)
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof)
  • toxin e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof
  • radioactive isotope / e , a radioconjugate
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeiuginosa), ⁇ cin A chain, ab ⁇ n A chain, modeccin A chain, alpha-sarcin, Aleuriles foi du proteins, dianthin proteins, Phytolaca amencana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonana officinahs inhibitor, gelonin. mitogellin.
  • radioconjugated antibodies examples include 2l2 B ⁇ , ⁇ I, ⁇ 'ln, 9I, Y, and ' 8 ⁇ >
  • Re Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succ ⁇ mm ⁇ dyl-3-(2-py ⁇ dyld ⁇ th ⁇ ol) propionate (SPDP), lminothiolane (IT), bifunctional derivatives of lmidoesters (such as dimethyl adipimidate HCL).
  • SPDP N-succ ⁇ mm ⁇ dyl-3-(2-py ⁇ dyld ⁇ th ⁇ ol) propionate
  • I lminothiolane
  • bifunctional derivatives of lmidoesters such as dimethyl adipimidate HCL.
  • active esters such as disuccinimidyl suberate
  • aldehydes such as glutaraldehyde
  • bis-azido compounds such as bis (p-azidobenzoyl) hexanediamine
  • bis-diazomum derivatives such as b ⁇ s-(p-d ⁇ azon ⁇ umbenzoyl)- ethylenediamine
  • dnsocyanates such as tolyene 2.6-dnsocyanate.
  • a ⁇ cin lmmunotoxin can be prepared as desc ⁇ bed in Vitetta et al , Science, 238 1098 (1987) Carbon- 14-labeled l - ⁇ soth ⁇ ocyanatobenzyl-3-methyld ⁇ ethylene tnaminepentaacetic 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 streptavidin) tor utilization in tumoi pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound con
  • T he antibodies disclosed herein may also be toimulated as immunoliposomes
  • Liposomes containing the antibody are prepared by methods know n in the ait. such as desc ⁇ bed in Epstein el al Pioc Natl Acad Sci USA. 82 3688 ( 1985). Hwang et al Pioc 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
  • Particularly useful liposomes can be generated by the reverse-phase ev aporation method with a lipid composition comprising phosphatidy lchohne. cholesterol, and PEG-de ⁇ v atized 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 Che . 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 Cancel lust , 81( 19) 1484 ( 1989)
  • chemotherapeutic agent such as Doxorubicin
  • compositions of Antibodies specifically binding a PRO polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders in the form of pharmaceutical compositions
  • the PRO polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred
  • 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 et al , Pioc Nail 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, pieferably those with complementary activities that do not adversely affect each other
  • the composition may comprise an agent that enhances its function, such as, tor example, a cytotoxic agent, cytokine, chemotherapeutic agent,
  • anti-PRO antibodies of the invention have various utilities
  • anti-PRO antibodies may be used in diagnostic assays for PRO, e g , detecting its expression in specific cells, tissues, or serum
  • diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola. Monoclonal Antibodies A Manual of Techniques. CRC Press. Inc ( 1987) pp
  • the antibodies used in the diagnostic assays can be labeled with a detectable moiety
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal
  • the detectable moiety may be a radioisotope. such as ⁇ H, l4 C. ,_ P, 1-> S, or l 2" 'I.
  • a fluorescent or chemilummescent compound such as fluoiescein isothiocyan ⁇ te. rhodamine.
  • Anti-PRO antibodies also are useful foi the affinity purification of PRO from recombinant cell culture or natural sources
  • the antibodies against PRO are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art
  • the immobilized antibody then is contacted w ith a sample containing the PRO to be purified and theieatter the support is washed with a suitable solvent that w ill remove substantially all the material in the sample except the PRO which is bound to the immobilized antibody
  • the support is washed w ith another suitable solvent that will release the PRO from the antibody H Therapeutic Indications
  • Retinal tears is a condition characterized by where the retina tears or separates from the underlying choroid, which can sometimes result in rupturing of the choroid Retinal tears may occur for a wide variety of reasons Particularly troublesome are macular holes which produce blurred central vision or metamorphopsia
  • macular holes While the direct cause of most macular holes is unknown, they have been associated with trauma. cystic degeneration and vitreoretinal traction Also, full thickness macular holes have appeared following myopic degeneration, laser photocoagulation, lightning strike and pilocarpine administration Macular holes are also present in high frequency after cataract extraction A particular form of acute macular holes is ldiopathic senile macular hole, which involves a full thickness hole through the macula surrounded by annular retinal detachment It is believed that macular holes begin with central or toveolar detachment. which then eventually develops into a full-depth macular hole [Gass et al (1988) Aich Ophthalmol 106
  • edema ischemic conditions
  • uveitis Macular and retinal edema are often associated with metabolic illnesses such as diabetes mellitus Retinal edema is found in a large percentage of individuals who have undergone cataract extraction and other surgical procedures upon the eye Edema is also found with accelerated or malignant hypertension Macular edema is a common complication of prolonged inflammation due to uveitis. Eales disease, or other diseases Local edema is associated with multiple cystoid bodies ("cotton bodies”) as a result of AIDS
  • Retinal ischemia can occur from either choroidal or retinal vascular diseases, such as central or branch retinal vision occlusion collagen vascular diseases and thrombocytopemc purpura Retinal vascuhtis and occlusion is seen with Eales disease and systemic lupus erythematosus
  • Age-related macular degeneration is the ma
  • AMD patients hav e a build up of deposits w ithin and under the retinal pigment epithelium in the macular region resulting in atiophy of the retina and the retinal pigment epithelium
  • the retinal pigment scav enge for photoieceptor discs from the tods and cones tor years and accumulate intracellular w astes
  • the incompletely digested residues reduce cytoplasmic space and
  • Exudative AMD is characterized by the growth of blood vessels from the cho ⁇ ocapill ⁇ s through detects in Bruch ' s membrane, and in some cases the underlying retinal pigment epithelium (RPE)
  • RPE retinal pigment epithelium
  • the accumulation of serous oi hemorrhagic exudates escaping from these vessels results in fibrous scarring of the macular region with attendant degeneration of the neuroretina and permanent loss of central vision
  • Exudative AMD has also been associated with choroidal neovascul ⁇ zation, detachment and tears of the retinal pigment epithelium The cascade retinal events is responsible for more than 80% of cases of significant visual loss in patients with AMD
  • the retinal neuron survival agents of the present invention are promising candidates for the treatment of retinal tears, degenerative myopia, acute retinal necrosis syndrome (ARN). and traumatic chorioretinopathies or contusion (including Purtscher's retinopathy). macular holes, macular degeneration
  • ischemic conditions e g central or branch retinal vision occlusion, collagen vacuolar diseases, thrombocytopenic purpura
  • the PRO polypeptides of the present invention can be delivered to the eye through a variety of routes Methods of introduction include any mode of administration known in the art, including but not limited to intravenously, lntraarte ⁇ ally. intrathecally, subcutaneously. intradermally.
  • biodegradable polymers or proteinaceous material.
  • the factors may be administered prior to the onset of the condition, to prevent its occurrence, for example, during surger on the eye. or immediately after the onset of the pathological condition or during the occurrence of an acute or protracted condition.
  • Intravitreal injection of potential retinal neuron survival promoting factors has several advantages over systemic applications.
  • the amount of any specific agent that reaches the retina can be more accurately determined, since the eye is a round, relatively contained structure and the agent is injected directly into it.
  • the amount of agent that needs to be injected is minuscule compared to systemic injections. For example, a single microliter in volume (about 1 microgram of agent) is used for intravitreal injection, as compared to one to several milliliters (ten to several hundred milligrams of agent) necessary for systemic injections.
  • the intravitreal route of administration avoids the potentially toxic effect of some agents.
  • compositions of the invention may be administered locally to the area in need of treatment, this may be achieved by. for example, local infusion during surgery, by injection, by means of a catheter, or by means of an implant, wherein such implant can be of a porous, non- porous, or gelatinous material, including membranes, such as silastic membranes or fibers.
  • the factors of the present invention may be modified to enhance their ability to penetrate the blood-retinal barrier.
  • Such modification may include increasing their lipophilicity by, for example, glycosylation, or increasing their net charge by methods known in the art.
  • the factors may be delivered alone or in combination, and may be delivered along with a pharmaceutically acceptable vehicle. Ideally, such a vehicle would enhance the stability and/or delivery properties.
  • the invention also provides for pharmaceutical compositions containing the active factor or fragment or derivative thereof, which can be administered using a suitable vehicle such as liposomes, microparticles or microcapsules. In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the active component. J. Pharmaceutical Compositions and Dosages
  • Therapeutic formulations of the PRO polypeptide are prepared for storage as lyophilized formulations or aqueous solutions by mixing the polypeptide having the desired degree of purity with optional "pharmaceutically-acceptable” or “physiologically-acceptable” carriers, excipients or stabilizers typically employed in the art (all of which are termed “excipients”).
  • excipients typically employed in the art
  • buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and other miscellaneous additives See Remington 's Pharmaceutical Sciences. 16th edition, A. Osol. Ed. ( 1980)).
  • Such additives must be nontoxic to the recipients at the dosages and concentrations employed.
  • Buffering agents help to maintain the pH in the range which approximates physiological conditions. They are preferably present at concentration ranging from about 2mM to about 50 mM. Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof such as citrate, succinate, tartrate, fumarate, gluconate, oxalate. lactate, acetate, phosphate, and histidine buffers. Additionally, there may be mentioned phosphate buffers, histidine buffers and trimethylamine salts such as Tris.
  • Preservativ es are added to retaid microbial grow th and aie added in amounts ranging from 0 2% - 1%- (w/v )
  • Suitable preservatives tor use w ith the present inv ention include phenol, benzyl alcohol, meta cresol. methy l paiaben. propy l paraben. octadecv ldimethv lbenzy I ammonium chloride, benzalconium halides (e g . chloride, bromide, iodide), hexamethonium chloride, alky l parabens such as methv l or propy l paraben. catechol. resorcinol cyc ohexanol. and 3-pentanol
  • Isotonicifiers sometimes known as “stabilizers" are present to ensure isotonicity of liquid compositions of the present invention and include polyhyd ⁇ c sugar alcohols, preferably t ⁇ hyd ⁇ c or higher sugar alcohols, such as glyce ⁇ n. eryth ⁇ tol. arabitol. xylitol. sorbitol and manmtol Polyhyd ⁇ c alcohols can be present in an amount between 0 1 % to 25%- by weight preteiably 1 % to 5% taking into account the relative amounts of the other ingredients
  • Stabilizers refer to a broad category of excipients which can lange in function from a bulking agent to an additive which solubihzes the therapeutic agent or helps to prevent denaturation oi adherence to the container wall
  • Typical stabilizers can be polyhyd ⁇ c sugai alcohols (enumerated above), amino acids such as argimne, lysine. glycine, glutamme. aspar ⁇ gine. histidine. alanine. ormthine, L-leucine, 2-phenylalan ⁇ ne. glutamic acid, threonine, etc , organic sugars or sugar alcohols, such as lactose, trehalose. stachyose, mannitol. sorbitol.
  • xylitol ⁇ bitol, myoimsitol, galactitol, glycerol and the like, including cychtols such as mositol, polyethylene glycol, amino acid polymers, sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, ⁇ -monothioglycerol and sodium thio sulfate, low molecular weight polypeptides (I e ⁇ 10 residues), proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins, hydrophihc polymers, such as polyvinylpyrrohdone monosaccha ⁇ des.
  • cychtols such as mositol, polyethylene glycol, amino acid polymers
  • sulfur containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thi
  • polysaccha ⁇ des such as dextran Stabilizers can be present in the range from 0 1 to 10,000 weights per part of weight active protein
  • Non-ionic surfactants or detergents are present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein
  • Suitable non-ionic surfactants include polysorbates (20. 80, etc ), polyoxamers ( 184, 188 etc ), Pluronic 0 polyols, polyoxyethylene sorbitan monoethers (Tween ® -20, Tween ® -80. etc )
  • Non-ionic surfactants are present in a range of about 0 05 mg/ml to about 1 0 mg/ml, preferably about 0 07 mg/ml to about 0 2 mg/ml
  • Additional miscellaneous excipients include bulking agents, (e g starch), chelating agents (e g EDTA), antioxidants (e g . ascorbic acid, methionine, vitamin E), and cosolvents
  • 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
  • an immunosuppressive 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 microcapsule prepared, for example, by coascervation techniques oi by interfacial polymerization, foi example, hydioxymethylcellulose or gelatin- microcapsule and polv-(methv lmethacv late) microcapsule respectiv elv in colloidal drug dehverv sv stems (tor example liposomes albumin microspheres mac oemulsions nano particles and nanocapsules) or in macroemulsions Such techniques are disclosed in R mington s Phaimac eutical Sc i nces 16th edition A Osal Ed ( 1980) Sustained release preparations mav be prepared Suitable examples ot sustained lelease preparations include semi permeable matrices of solid hydrophobic polymers containing the antibody mutant which matrices are in the form of shaped articles e g films oi microcapsules Examples of sustained-release matrices include polyesters hydrogels (for example poly(2
  • the formulations to be used for ;/; vivo administration must be sterile This is readily accomplished, for example, by filtration through sterile filtration membranes
  • the amount of PRO polypeptide which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques Where possible it is desirable to determine the dose response curve and the pharmaceutical compositions of the invention first ;// ⁇ ⁇ t ⁇ o and then in useful animal model systems prior to testing in humans
  • Interspecies scaling of effective doses can be performed following the principles laid down by Moienti J and Chappell W , "The use of interspecies scaling in toxicokmetics", Toxicokinetics and New Diug De ⁇ elopment, Tacobi et al ,
  • a pharmaceutical composition effective in promoting the survival of sensory neurons may provide a local therapeutic agent concentration of between about 5 and 20 ng/ml and preferably between about 10 and 20 ng/ml
  • a pharmaceutical composition effective in promoting the growth and survival of retinal neurons may prov ide a local therapeutic agent concentration of between about 10 ng/ml and 100 ng/ml Additional guidance to particular dosages and methods of delivery is provided in the literature
  • an aqueous solution of PRO polypeptide is administered by subcutaneous injection
  • Each dose may range from about 0 5 ug to about 50 ug per kilogram of body weight or more preferably, from about 3 ug to about 30 ⁇ g per kilogram body weight
  • the dosing schedule for subcutaneous administration may v aiy form once a week to daily depending on a number of clinical factois. including the tv pe of disease, sev enty of disease and the subject s sensitiv ity to the theiapeutic agent Additional guidance to particulai dosages and methods of delivery is provided in the hteratuie - see. for example. U S Pat Nos 4.657.769.
  • the amount of PRO protein which will be effectiv e in the tieatment ot a particular disoidei oi condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques Where possible it is desirable to determine the dose-response curve and the pharmaceutical compositions of the inv ention first ;; ⁇ ⁇ t ⁇ o, and then in useful animal model systems prior to testing in humans Howevei . based on common knowledge of the art.
  • a pharmaceutical composition effective in promoting the survival of sensory neurons may provide a local PRO protein concentiation of between about 10 and 1000 ng/ml.
  • a pharmaceutical composition effective in promoting the growth and survival ot retinal neurons may provide a local PRO protein concentration ot between about 10 ng/ml and 1000 ng/ml
  • the dosing schedule for subvitreous administration of PRO may vary from once a week to daily depending on a number of clinical factors, including the type of disease, severity of disease, and the subject's sensitivity to PRO. respectively
  • Nonhmiting examples of dosing schedules are 3 ⁇ g/kg administered twice a week, thiee times a week or daily, a dose of 7 ug/kg twice a week, three times a week or daily, a dose of 10 ⁇ g/kg twice a week, three times a week or daily
  • neurotrophic factors include nerve growth factor (NGF), aGF. ciliary neurotrophic factoi (CNTF). bovine- de ⁇ ved neurotrophic factor (BDNF). neurotroph ⁇ n-3 (NT-3), neurotroph ⁇ n-4 (NT-4), aFGF, IL- 1 ⁇ , TNF ⁇ , Insulin-like growth factor (IGF- 1.
  • NGF nerve growth factor
  • aGF aGF. ciliary neurotrophic factoi
  • BDNF bovine- de ⁇ ved neurotrophic factor
  • NT-3 neurotroph ⁇ n-3
  • NT-4 neurotroph ⁇ n-4
  • aFGF IL- 1 ⁇
  • TNF ⁇ Insulin-like growth factor
  • IGF-2 transforming growth factor beta
  • TGF- ⁇ transforming growth factor beta
  • skeletal muscle extract may be administered in any sterile biocompatible pharmaceutical carrier, including, but not limited to. saline, buffered saline, dextrose, and water
  • certain factors such as bFGF.
  • CNTF or IL- l ⁇ should be employed cautiously, as these agents may cause undesirable retinal complications such as macrophage proliferation, disorganization oi the retinal structure, cell proliferation or inflammation
  • One or more additional drugs may be administered in combination with PRO to alleviate such undesired side effects, for example, an anti-pyretic, anti-inflammatory or analgesic agent III.
  • neuial letinas ⁇ ie removed from pigment epithelium and dissociated into a single cell suspension using 0 25% try psin in Ca ⁇ + .
  • Mg "+ -free PBS The cells are then plated out in 96-welI plates at 100.000 cells per well in DMEM/F12 supplemented w ith N2 After 2-3 day in culture, the cells are fixed and stained Since death typically occuis upon detachment of neuial retinal cells from the underlying pigment epithelium the relative survival enhancing effect of the tested agent can be readily detei mined by comparison with the untreated control wells The procedure is described in greater detail in the examples
  • ALD Age-related macular degeneration
  • D. Light-induced photoreceptor injury In this assay, an albino rat is maintained in first a cyclic light environment followed by exposure to a constant light source with and without administration of the tested photoreceptor survival agent The intravitreal administiation of factors into the eyes of albino rats enables the assessment of both the ability of the factors to rescue photoreceptors from degeneration as well as the side ettects. such as incidence of macrophages.
  • rats are given intraocular injections prior to constant light exposure and compared to control animals who received sham and no injections Subsequent to constant light exposure, the eyes are removed, embedded in epoxy resin and sectioned along the vertical meridian The degree ot light-induced retinal degeneration can be measured first by examining the outer nuclear layer thickness and second by a subjective score assigned to the relative integrity of the retina E.
  • Light ablation :
  • the degree of photoreceptor rescue is measured in female Spi ⁇ gue-Daw ley rats in a modification ot the procedure described in Reme et al . Degcn Dis Retina. Ch 3. Ed R E Anderson et al . Plenum Press. New Yoik ( 1995) Briefly , animals are fust acclimated to cyclical lighting, followed by immersion in total darkness Animals are injected w ith test factor prior to intermittent light exposuie The degree of retinal degeneration or survival promoting activ ity of the tested factors is reported as the thickness ot the photoreceptor cell layer or number of TUNEL labeled photoreceptor cell nuclei
  • bovine adrenal cortex-derived cells are grown and maintained in culture in the presence of low glucose DMEM. the test factor is administered and test cultures vs controls are measuied
  • Apoptosis appears to be a tightly controlled "shutdown" process or self-selecting cell suicide which by preventing the leakage of destructive enzymes, allows healthy neighboring cells to continue their normal functioning [Wong, F , Aich Ophthalmol 1 13 1245-47 ( 1995)] During this process, the cell's outer membrane remains intact as the cell undergoes nuclear condensation, cytoplasmic shrinkage, membrane blebbing.
  • Apoptosis is now believed to play a key role in degenerative diseases of the eye, such as letimtis pigmentosa RP is believed to be caused by mutations in the rhodopsin gene [Dryja. TP, Nature, 343 364- 366 ( 1990)]
  • letimtis pigmentosa RP is believed to be caused by mutations in the rhodopsin gene [Dryja. TP, Nature, 343 364- 366 ( 1990)]
  • other photoreceptor-specific genetic mutations have been uncovered which induce RP. among them the mutants known as retinal degeneration (id) [McLaughlin ME, et al Nat Genet .
  • Rhodopsin mutations are know n to be the basis of autosomal recessive RP in some families as well
  • a patient pool with v ary ing stages of macular holes (i e . 2. 3 or 4) ot varying ages are chosen and the presence of macular holes is confirmed
  • the pool is selected so as to exclude patients w ith histories of cystoid macula edema, diabetic retinopathy or exudative age-related macular degeneration Vision is examined in each subject to determine the best Snellen visual acuity and analyzed by intraocular pressure, fundus photographs, and fluorescein angiography
  • Each macular hole is graded according to the criteria described by Gass.
  • Stage 3 is characterized by a full-thickness hole with overlying operculum Macular holes are classified as Stage 4 when a posterior vitreous detachment is present Treatment is scheduled within 2 weeks of the baseline examination Under the criteria, patients should be excluded if they had greater than 2+ nuclear sclerotic or posterior subcapsular lens changes Patients are followed for 6- 10 months, with mean follow-up of 8 months Doses are determined at a level below therapeutic effectiveness, in the middle of the effective range, and at a level well above the minimal effective range Eyes are randomly chosen for the indicated level of PRO In addition, some eyes may separately receive 100 ⁇ l of intravitreal hyaluromc acid at the time of installation of PRO in an attempt to delay clearance of PRO from the area of the macular hole Surgical procedure
  • a standard three-port vitrectomy may be performed In eyes with Stage 2 and Stage 3 macular holes, a core vitrectomy is performed In Stage 4 macular hole, ⁇ complete pat s plana vitrectomy is performed
  • an epiretinal membrane may be peeled from the surface of the retina and removed from the eye
  • any fluid which migrates posteriorly is also aspirated
  • a tapered, bent-tipped cannula is then connected to a 1 cc syringe containing a solution of PRO
  • the reconstituted formulation contains the desired concentration of PRO aftei dilution Eyes are randomly assigned a dose of PRO About 0 1 cc of PRO solution is gently infused into the macular hole The same volume of hyaluiomc acid may also be administered
  • the patient After surgery, the patient should he in a supine position for the first 24 hours following surgery Thereafter, each patient should remain in a face-down position as much as possible for a 2 week period Patients are examined at 1 d ⁇ 2 weeks 4 6 weeks and monthly post surgerv Fluoiescein dngiographv is pei toimed at 4 to 6 weeks 3 months and 6 months Best corrected Snellen v isual acuitv intraocular pressure lens status bubble size status ot macular hole and occurrence of adv erse effects are determined at each examination
  • Albino rats (F344 of Sprague Dawley) of 2 5 months of age are maintained in a cyclic light environment ( 12 hours on followed by 12 hours off from an in cage illuminance of less than 25 ft c) for 9 days or more days before exposure to a constant light source
  • the constant light source is maintained at an illuminance level of 1 15 200 ft c
  • 2 40 watt white reflector fluorescent bulbs suspended 60 cm above the floor of a transparent polycarbonate cage with stainless steel wire-bar covers
  • the rats Two days before the constant light exposure the rats are anesthetized with a ketamme xylazine mixture which is administered intravitreally with 1 ul of the tested factor dissolved in phosphate buffered saline (PBS) at a concentration ot 50 1000 ng/ul
  • PBS phosphate buffered saline
  • the injections weie made with the insertion of a 32 gauge needle through the sclera choroid and retina approximately midway between the ora serrat ⁇ and equator of the eye
  • the factor injected animals are compared to eithet unmjected httermates of those that receive control injections as well as to control animals who are not exposed to constant light Controls should include an injection of PBS alone, or a sham injection (insertion of needle with no injection) In all cases, the injections are made into the superior hemisphere of the eye
  • the rats are killed by any suitable means, c g carbon dioxide anesthetization followed by
  • the 3 measurements are made at defined points separated from one another by 75 um. In all. 54 measurements are taken in the two hemispheres which sample representative regions of almost the entire retinal section.
  • the second method of assessing the degree of photoreceptor rescue is through a subjective evaluation by an examining pathologist on a scale of 0-4- ⁇ wherein 4+ is maximal rescue and nearly normal retinal integrity.
  • the degree of photoreceptor rescue in each section based in comparison to the control eye in the same rat. is scored by four individuals. This method not only takes into account the ONL thickness. but also more subtle degenerative changes to the photoreceptor inner and outer segments, as well as degenerative gradients within the eye.
  • the intravitreal administration of various factors into the eyes of albino rats can enable the rapid assessment of both the ability of the factors to rescue photoreceptors from degeneration and the side effects, such as incidence of macrophages, associated with each factor.
  • the model described herein is the albino rat, the eyes of other albino mammals, such as mice and rabbits, are also useful for this purpose.
  • Sprague Dawley rat pups at postnatal day 7 are killed by decapitation following C0 2 anesthesia and the eyes are removed under sterile conditions.
  • the neural retina is dissected away from the pigment epithelium and other ocular tissue and then dissociated into a single cell suspension using 0.25% trypsin in Ca ⁇ + , Mg" + -free PBS.
  • the retinas are incubated at 37°C for 7-10 minutes after which the trypsin is inactivated by adding 1 ml soybean trypsin inhibitor.
  • the cells are plated at 100,000 cells per well in 96 well plates in DMEM/F12 supplemented with N2.
  • Sprague Dawley rat pups at 7 day postnatal are killed by decapitation following CO-> anesthesis and the eyes are removed under sterile conditions
  • the neural retina is dissected away form the pigment epithelium and other ocular tissue and then dissociated into a single cell suspension using 0 25% trypsin in Ca 2+ , Mg 2+ free PBS
  • the retinas are incubated at 37°C foi
  • retina degeneration can be induced by exposure to strong light This light ablation model permits a quantitative comparison ot photoreceptor survival promoting activity of a tested substance
  • Test factors employed are 0 5 - 1 0 ⁇ g/ ⁇ l of PRO polypeptide and phosphate buffeted saline with and without bovine sei um albumin (0 1 %) controls Tdt-mediated dUTP nick-end labeling (TUNEL) (Gav ⁇ eli, Y et al J Cell Biol JJ9 493-501
  • the degree of retina! degeneiation or survival promoting activ ity of the tested tactoi s is reported as the thickness of the photoreceptor cell layer or number of TUNEL labeled photoieceptoi cell nuclei
  • Three transverse sections through the cential retina (approx 10 um intervals) are used toi the analysis
  • the entire retinal surface area is digitized using a cooled CCD camera and NIH image software (Macintosh) to derive the quantitative data
  • Sprague-Dawley iats 250 g. male are maintained in plastic carriers under darkened conditions 24 hours prior to treatment and then anesthetized
  • Each amm ⁇ l s eyes are gently proptosed and secured in place with nontraumatic forceps (BRI- 1725) Using a No 15 blade (Baid-Parker).
  • a 1 5 mm incision is made approximately 1 mm from the center of the cornea into the stroma. but not through it
  • ASSI ST 80017] is then inserted under the lip of the incision and gently blunt-dissected through the stroma toward the outer canthus of the eye
  • the final distance between the base of the pocket and the hmbus should be at least 1 mm
  • Pellets are prepared by mixing together tested growth factor ( 100 ng), sucralfate (50 ⁇ g, BM Research. Denmark) and Hydron (Interferon Sciences, New Brunswick. N J , Lot # 90005) in a 500 1 ratio of growth factor to sucralfate and Hydron (4 ⁇ l)
  • the sucralfate is present to stabilize the molecule by interacting with the hepa ⁇ n-binding region
  • the control pellet is composed of Hydron and sucralfate vehicles only
  • Three treatment groups tested are composed of I ) bovine bFGF (SEQ ID NO 4) (Calbiochem, 10 ug/50 ul) PBS + sucralfate (6 animals).
  • sucralfate control (3 animals), and 3) PRO polypeptide + sucralfate (e g , at least 6 animals)
  • a Hydron pellet (2 x 2 mm) prepared as described in the previous paragraph is inserted into the base of incision whereupon the pocket should reseal spontaneously The eyes are coated with artificial tears ointment and then the animals are returned to their plastic carriers, then permitted to awaken and returned to their cages
  • the assay is terminated on day 5 At time of sacrifice the animals are perfused with FITC dextran (2 x 10 f ' m w ) and cornea! whole mounts prepared by careful dissection of the cornea from the eyes followed by strategic placement of 2-3 cuts to permit permitting the cornea to he flat, followed by placement under a coverslip
  • the image can be captured through a l x objective mounted on a Nikon inverted fluorescent scope Image-Pro ® sottw are-edge detection routine can be used to evaluate growth areas
  • Mitogenic assays on vascular endothelial cells were initiallv dev eloped in order to monitor the purification ot bFGF growth factor Howev er they are also a useful measure to determine the presence ot mitogenicity in the tested substance Materials and Methods
  • Bovine adrenal cortex derived capillai y endothelial (ACE) cells are established according to know n procedures as described by Ferrara et al J98 391 -405 ( 1991 )
  • Stock plates of ACE cells are were maintained in 10 cm tissue culture dishes in thecetnce of low glucose DMEM supplemented w ith 10% calf serum 2 mM glutamine and penicillin G ( 1000 Units/mL) and streptomycin (1000 ug/mL) and basic FGF at a final concentration of 1 ng/ml and weekly passaged at a split ratio of 1 10
  • Mitogenic controls are prepared by adding basic FGF at final concentrations of 1 ng/ml and 5 ng/ml and culturing for 5-6 days
  • ACE cells can be passaged 10 12 times before showing signs of senescence
  • the stock cultures are trypsinized resuspended in growth media and seeded at a density of 1 0 x 10 cells/well in 6 well plates (Costar Cambridge MA
  • Example 8 Identification of clones encoding a PRO200 (VEGF-E) related protein Probes based on an expressed sequence tag (EST) identified from a private EST database provided by Incyte Pharmaceuticals (LileSeq ® ) which exhibited homology with VEGF (v ascular epithelial growth factor) were used to screen a cDNA librarv derived from the human glioma cell line G61 In particular
  • Incyte clone "INC 1302516 (SEQ ID NO 41 , Figure 22) was used to generate the following tour probes 5'-AC rTCTCAGTGTCCATAAGGG 3 (SEQ ID NO 6)
  • the VEGF-E-IgE plasmid was co-transtected with BaculgoldTM baculovu us NDA (Pharmingen) using LipotectinTM (GibcoBRL) into 10 s Sf9 cells grown in Hink's TM TNM-FH medium (JRH Biosciences) supplemented with 10% fetal bovine serum Cells were incubated tor 5 days at 28°C The supernatant was harvested and subsequently used for the first viral amplification by infecting Sf9 cells at an approximate multiplicity of infection (MOI) of 10 Cells were incubated for 3 days, then supernatant was harvested, and expression of the recombinant plasmid was determined by binding of 1 ml of supernatant to 30 ml of Protein-A SepharoseTM CL-4B beads (Pharmacia) followed by subsequent SDS-PAGE analysis The first amplification supernatant was used to infect a 500 ml spinner culture of Sf9 cells grown in
  • DNA sequencing of the clones isolated as described above gave the full-length DNA sequence PRO200 [herein designated UNQ174, VEGF-E, DNA29101 -1276 (SEQ ID NO 1 )] Clone DNA29101 contains a single open reading frame with an apparent translation initiation site at nucleotide positions 285- 287 and ending at the stop codon at nucleotide positions 1320- 1322 ( Figure 1 )
  • the predicted polypeptide precursor is 345 amino acids in length ( Figure 2.
  • SEQ ID NO 2 The full-length PRO200 polypeptide shown in Figure 2 has an estimated molecular weight ot about 39,029 daltons and a pi of about 6 06 Important regions of amino acid sequence of PRO200 include a signal sequence at about residues 1 - 14.
  • N- glycosylation sites at about residues 25-29, 55-59, 254-258.
  • N-my ⁇ stoylation site at about residues 15-21 , 1 17- 123, 127- 133, 281 -287, 282-288 and 319-325 and an amidation site at about residues 229-233 Clone
  • ECD extracellular domain sequences (including the secretion signal, if any ) ot from about 950 known secreted proteins from the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases
  • the EST databases included public EST databases (e GenBank) and a proprietary EST DNA database (LIFESEQ f .
  • phrap Phil Green. Umveisity ot Washington. Seattle. Washington edu/phrap docs/phrap html
  • DNA39631 SEQ ID NO 5
  • oligonucleotides were synthesized 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) foi use as probes to isolate a clone of the full-length coding sequence for PRO540
  • 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 , Curient Piotocols in Moleculai Biolog ⁇ . with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest by an
  • Forward and reverse PCR primers were synthesized forward PCR primer (39631 fl )
  • 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 was ohgo dT containing a Notl site, linked w ith blunt to Sail hemikin ⁇ sed adaptors, cleaved with Notl, sized appropriately by gel electrophoresis. and cloned in a defined orientation into a suitable cloning vector (such as pRKB or PRKD. pRK5B is a precursoi of pRK5D that does not contain the Sfil site. see. Holmes el al ,
  • EST databases included public EST databases (e g , GenBank)
  • the search was performed using the computer program BLAST or BLAST-2 [Altschul et al , Methods in Enzymologv 266 460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequence Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap” (Phil Green, University of Washington. Seattle, WA)
  • DNA39949 ( Figure 8. SEQ ID NO 14) Based on the DNA39949 (SEQ ID NO 14) shown in Figure 8.
  • oligonucleotide probes (primers) were synthesized I ) to identity by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0846
  • 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 screen by PCR amplification, as per Ausubel et al , Cuiient
  • a synthetic oligonucleotide hy bridization probe was constructed from the consensus DNA39949 sequence w hich had the follow ing nucleotide sequence hybridization probe (39949 pi ) 5'-GGTGCAGGAAGGGTGGGATCC TCTTCTCTCGCTGCTCTGGCCACATC-3 (SEQ ID NO 17)
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with ohgo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved w ith Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is ⁇ 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
  • Clone UNQ422 (DNA44196-1353) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 25-27 and ending at the stop codon at nucleotide positions 1021 - 1023 ( Figure 6) (SEQ ID NO 13)
  • the full-length PR0846 protein shown in Figure 7 (SEQ ID NO 13) is 332 amino acids in length, has an estimated molecular weight ot about 36,143 daltons and a pi of about 5 89 Important regions of the amino acid PR0846 (and approximate locations) include the signal peptide
  • the extiacellular domain (ECD) sequences (including the secretion signal, if any) of about 950 know n secreted proteins trom the Swiss-Piot public protein database weie used to search expressed sequence tag (EST) databases
  • the EST databases included the public EST databases (c g . GenBank) and a proprietary EST DNA database (LIFESEQ ® , Inycte Pharmaceuticals, Palo Alto. CA)
  • the search was performed using the computei program BLAST or BLAST-2 [Altschul et al , Methods in 266 460-480 ( I996)
  • 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 tor PR0617
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are otten 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 form the libraries was screen 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
  • a pair of PCR primers (forward and reverse) were synthesized forward PCR primer
  • RNA for construction of the cDNA libraries was isolated form human fetal kidney tissue
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego. CA
  • the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor ot pRK5D that does not contain the Sfil site, see. Holmes et al , Science 253 1278- 1280 ( 1991 )) in the unique Xhol and Notl sites
  • DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for
  • PR0617 [herein designated as UNQ353 (DNA48309- 1280)](SEQ ID NO 18) is shown in Figure 9
  • Clone UNQ353 (DNA48309) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 723-725 and ending at the stop codon at nucleotide positions 924-926 ( Figure 9)
  • the predicted polypeptide precursor is 67 amino acids long ( Figure 10)
  • the tull-length PR0617 protein shown in Figure I 1 (SEQ ID NO 19) has an estimated moleculai weight of about 6 981 daltons and a pi of about 7 47
  • Analy sis ot the PR0617 amino acid sequence also ev idences the existence of a putative signal peptide trom about amino acid 1 to about amino acid 27 and a putative piotein kinase C phosphorylation site from about amino acid 41 to about amino acid 43
  • Clone UNQ353 (DNA48309 1230
  • Example 13 Isolation of cDNA clones encoding human PR0538 (GFRc ⁇ ) and PR03664 (GFR ⁇ 3) A proprietary expressed sequence tag (EST) DNA database (LIFESEQ* Incyte Pharmaceuticals.
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al Curient Protocols in Moleculai Biology ( 1995), with the PCR primer pair A strong PCR product was identified in all libraries analyzed (fetal lung, fetal kidney and placenta)
  • a human fetal lung pRK5 vector library was selected and enriched for positive cDNA clones by extension of single stranded DNA from plasmid libraries grown in dug-/bung- hosts using the newa3 R (SEQ ID NO 31 ) primer RNA for construction of the cDNA libraries was isolated trom human fetal lung tissue
  • the cDNA library used to isolate the cDNA clones was constructed by standard methods using commercially available reagents (e g .
  • 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 (pRKB or pRKD, pRK5B is a precursor ot pRK5D that does not contain the Sfil site, see.
  • pRKB or pRKD is a precursor ot pRK5D that does not contain the Sfil site, see.
  • the primer extension reaction contained 10 ⁇ l of lOx PCR buffer (Perkin Elmer. USA). 1 ⁇ l dNTP (20 mM). I ⁇ l library DNA
  • DNA48613 SEQ ID NO 24. Figure 12
  • DNA48614 SEQ ID NO 25. Figure 13
  • Clone DNA48613- 1268 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 38-40 and ending at the stop codon at nucleotide positions 1238- 1240 ( Figure 12)
  • the predicted native sequence polypeptide precursor PR0538 is 400 ammo acids in length, has an approximate molecular weight of 44.51 1 daltons and a pi of 8 15 Further analysis of the PR0538 sequence of Figure 14 (SEQ ID NO.26) reveals a signal sequence at about amino acid residues 1 - 16.
  • N-my ⁇ stolation sites at residues 279-285 and 294-300 and prokaryotic membrane hpoprotein lipid attachment site are residues 306- 17 and 379-390
  • Clone DNA48614-1268 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 142-144 and ending at the stop codon at nucleotide positions 1249- 1251 ( Figure 13)
  • the predicted native sequence PR03664 polypeptide precursor is 369 ammo acids long, with a calculated molecular weight of approximately 40,930 Da and a pi of 8 16
  • Further analysis of the PR03664 sequence of Figure 15 (SEQ ID NO.27) reveals a signal sequence at about amino acid residues 1 -26, N-glycosylation sites at about residues 95-99 and 278-282, a cAMP- and cGMP- dependent protein kinase phosphorylation site at about residues 200-204.
  • Example 14 Isolation of DNA encoding PRO770 (hFIZZ-1)
  • EST public expressed sequence tag
  • PRO770 protein shown in Figuie 18 has an estimated molecular w eight of 1 1 730 daltons and a pi of 7 82 Further analysis reveals a signal sequence at about amino acid residues 1 -28 a cAMP and cGMP-dependent protein kinase phosphorylation site at residues 51 -55 N mv ⁇ stolation sites at residues 21 27, 70 76 and 75 81 and a prenvl group binding site (CAAX) at about residues 108-1 13 Clone DNA54228-1366 has been deposited with the ATCC on April 23 1998 and is assigned deposit number
  • EXAMPLE 15 Identification and cloning of m-FIZZ-1 (DNA53517) Mous asthma model
  • the asthmatic group was immunized lntrape ⁇ toneally with 10 ⁇ g ovalbumin + 1 mg alum while the control group was not
  • Two weeks later mice were exposed daily to an aerosol of 10 mg/ml ov albumin in PBS aerosolized with a UltraNebTM nebulizer (DeVilbiss) at the rate of 2 ml/min tor 30 mm each day, for 7 consecutive days
  • a UltraNebTM nebulizer UltraNebTM nebulizer
  • the following oligos were used as RT-PCR primers in combination with 5 and 3 amplifiers
  • PRO as a hybridization probe
  • the following method describes use ol a nucleotide sequence encoding PRO as a hybridization probe.
  • DNA comprising the coding sequence of full-length or mature PRO as disclosed herein is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO) m 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 ot radiolabeled PRO-de ⁇ ved probe to the filters is performed in a solution of 50% tormamide, 5x SSC, 0 1 % SDS, 0 1 % sodium pyrophosphate, 50 mM sodium phosphate, pH 6 8, 2x Denhardt's solution, and 10% dextran sultate at 42°C for 20 hours
  • Washing of the filters is performed in an aqueous solution ot 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
  • EXAMPLE 17 Expression of PRO in E. coli This example illustrates preparation of an unglycosylated form of PRO by recombinant expression
  • the DNA sequence encoding PRO is initially amplified using selected PCR primers
  • the primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector
  • a variety of expression vectors may be employed
  • An example of a suitable vector is pBR322 (derived from E coli, see Bolivar et al , Gene, 2 95 ( 1977)) which contains genes tor ampicillin and tetracycline resistance
  • the vector is digested with restriction enzyme and dephosphorylated
  • the PCR amplified sequences are then ligated into the vector
  • the vector will preferably include sequences which encode tor an antibiotic resistance gene, a trp promotei , a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the PRO coding region, lambda transcriptional terminator, and an argU gene
  • the ligation mixture is then used to transform a selected E coli strain using the methods described in Sambrook et al , supia Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics The overnight culture may subsequently be used to inoculate a larger scale culture The cells are then grown to a desired optical density, during which the expression promoter is turned on
  • the cells After culturing the cells for several more hours, the cells can be harvested by centrifugation
  • the cell pellet obtained by the cent ⁇ fugation can be solubihzed using various agents known in the ait. and the solubi zed PRO protein can then be purified using a metal chelating column under conditions that allow tight binding ot the protein
  • PRO mav be expiessed in E oli in a polv His tagged form using the follo ing pioceduie
  • the DNA encoding PRO is initially amplified using selected PCR p ⁇ mei s
  • the primers will contain restriction enzyme sites w hich correspond to the restriction enzv me sites on the selected expression vector and othei useful sequences prov iding for efficient and reliable translation initiation rapid pot ification on a metal chelation column and proteolv tic removal with enterokin ⁇ se
  • the PCR amplified poly His tagged sequences are then ligated into an expression v ector which is used to transform an £ coli host based on strain 52 (W31 10 futiA (tonA) Ion galE rpoHts (htpRts) clpP (laclq)
  • Transformants are first gro n in LB containing 50 mg/ml carbenicilhn at 30°C with shaking until an O D
  • E coli paste trom 0 5 to 1 L fermentations (6 10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine 20 mM Tris, pH 8 buffer Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0 I M and 0 02 M respectively and the solution is stirred overnight at 4* C This step results in a denatured protein with all cysteine residues blocked by sulfitolization
  • the solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min
  • the supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine 20 mM Tris, pH 7 4) and filtered through 0 22 micron filters to clarify
  • the clarified extract is loaded onto a 5 ml Qiagen Ni NTA metal chelate column equilibrated in the metal chelate column buffer The column is washed with additional buffer containing 50
  • the proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of 20 mM Tris pH 8 6, 0 3 M NaCl, 2 5 M urea, 5 mM cysteine 20 mM glycine and 1 mM EDTA Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml
  • the refolding solution is stirred gently at 4°C for 12-36 hours
  • the refolding reaction is quenched by the addition ot TFA to a final concentration of 0 4% (pH of approximately 3)
  • the solution is filtered through a 0 22 micron filter and acetonitrile is added to 2 10% final concentration I he refolded protein is chromatographed on a Poros R l/H reversed phase column using a mobile buftei of 0 1 % TFA with elution with a gradient of acetonitrile trom 10 to 80%
  • Pioteins Fractions containing the desued folded PRO polypeptide are pooled and the acetonitrile remov ed using a gentle siteam of nitiogen directed at the solution Pioteins are formulated into 20 mM Hepes pH 6 8 with 0 14 M sodium chloride and 4% m ⁇ nnitol by d ⁇ al ⁇ s ⁇ s or by gel filtration using G25 Superfine
  • EXAMPLE 18 Expression of PRO in mammalian cells This example illustrates preparation of a potentially glycosylated form ot PRO by recombinant expression in mammalian cells
  • the vector, pRK5 (see EP 307 247 published March 15 1989) is emploved as the expression vector
  • the PRO DNA is ligated into pRK5 with selected restriction enzy es to allow insertion of the PRO DNA using ligation methods such as described in Sambrook et al supia
  • the resulting vector is called pRK5-PRO
  • the selected host cells may be 293 cells
  • Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics
  • About 10 ⁇ g pRK5 PRO DNA is mixed with about 1 ⁇ g DNA encoding the VA RNA gene [Thimmappaya et al Cell, 3J_ 543 ( 1982)] and dissolved in 500 ⁇ l of 1 mM Tris-HCl, 0 1 mM EDTA, 0 227 M CaCl 2 To this mixture is added dropwise, 500 ul of
  • the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 uCi/ml ⁇ S cysteine and 200 uO/ml 1S 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 PRO polypeptide
  • the cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays
  • PRO may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al Pioc Natl Acad Sci , ⁇ 2 7575 ( 1981 ) 293 cells are grown to maximal density in a spinner flask and 700 ug pRK5 PRO DNA is added The cells are first concentrated from the spinner flask by centrifugation and washed with PBS 1 he 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 ug/ml bovine insulin and 0 1 ⁇ g/ml bovine transter ⁇ n After about tour days the conditioned media is centrifuged and filtered to remov e cells and debris The sample containing expressed PRO can then be concentrated and purified by anv selected method such as dialv sis and/oi column chiomatography In another embodiment
  • PRO can be expressed in CHO cells
  • the pRK5-PRO can be transtected into CHO cells using kno n reagents such as CaP0 4 or DEAE-dextran As described abov e, the cell cultuies can be incubated, and the medium replaced w ith culture medium (alone ) or medium containing a radiolabei such as After determining theêtnce of PRO polypeptide.
  • the culture medium may be replaced with serum tree medium
  • the cultures are incubated for about 6 days, and then the conditioned medium is harvested
  • the medium containing the expressed PRO can then be concentrated and purified by any selected method
  • Epitope-tagged PRO may also be expiessed in host CHO cells
  • the PRO may be subcloned out ot the pRK5 vector
  • the subclone insert can undergo PCR to tuse in frame with a selected epitope tag such as a poly-his tag into ⁇
  • Baculovirus expression vector 1 he poly-his tagged PRO insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection ot stable clones
  • the CHO cells can be transtected (as described above) with the SV40 driven vector Labeling may be performed, as described above, to verify expression
  • His tagged PRO can then be concentrated and purified by any selected method, such as by N ⁇ 2+ -chelate affinity chromatography
  • PRO may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure Stable expression in CHO cells is performed using the following procedure
  • the proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins are fused to an IgG l constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form
  • CHO expression vectors are consti ucted to hav e compatible restriction sites 5' and 3' ot the DNA of interest to allow the convenient shuttling of cDNA's
  • the vector used expression in CHO cells is as described in Lucas et al , Nucl Acids Res 24 9 ( 1774- 1779 ( 1996), and uses the SV40 early promoter/enhancer to drive expression ot the cDNA of interest and dihydrotolate reductase (DHFR) DHFR expression permits selection tor stable maintenance of the plasmid following transfection
  • the cells are giown as described in Lucas et al , supia Approximately 3 x 10 cells are tiozen in an ampule for further growth and production as described below
  • the ampules containing the plasmid DNA are thawed by placement into water bath and mixed by vortexing
  • the contents are pipetted into a centrifuge tube containing 10 niLs of media and centrifuged at 1000 rpm foi 5 minutes
  • the supernatant is aspirated and the cells are resuspended in 1 mL ot selective media (0 2 ⁇ m filtered PS20 with 5% 0 2 ⁇ m diafiltered fetal bovine serum)
  • the cells are then aliquoted into a 100 mL spinner containing 90
  • the proteins are purified using a Ni NTA column (Qiagen) Before purification lmidazole is added to the conditioned media to a concentration of 5 mM The conditioned media is pumped onto a 6 ml Ni NTA column equilibrated in 20 mM Hepes pH 7 4 buffei containing 0 3 M NaCl and 5 mM lmidazole at a flow rate ot 4 5 ml/min at 4 C After loading the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M lmidazole The highly purified protein is subsequentlv desalted into a storage buffer containing 10 mM Hepes 0 14 M NaCl and 4% man tol pH 6 8 with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C Immunoadhesin (Fc containing) constructs aie pur
  • the conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading the column is washed extensively with equilibration buffer before elution with 100 mM citric acid pH 3 5
  • the eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ⁇ L of 1 M Tris buffer pH 9
  • the highly purified protein is subsequently desalted into storage buffer as described above for the poly His tagged proteins The homogeneity is assessed by SDS polyacrylamide gels and bv N-terminal amino acid sequencing by Edman degradation
  • yeast expression vectors are constructed for intracellular production or secretion ol PRO from the ADH2/GAPDH promoter DNA encoding PRO and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO
  • DNA encoding PRO can be cloned into the selected plasmid, together with DNA encoding the
  • ADH2/GAPDH promoter a native PRO signal peptide or other mammalian signal peptide, or tor example, a yeast alpha-factoi or invertase secretory signal/leader sequence, and linker sequences (if needed) tor expression of PRO Yeast cells such as ⁇ east strain AB1 10 can then be transtormed ith the expression plasmids desc ⁇ bed above and cultured in selected Voltation media
  • the transtormed yeast supernatants can be analyzed by precipitation w ith 10% t ⁇ chloroacetic acid and separation by SDS PAGE tollovved by staining ot the gels w ith Coomassie Blue stam Recombinant PRO can subsequently be isolated and purified by removing the yeast cells trom the Krutation medium bv centrifugation and then concentrating the medium using selected cartridge filters
  • the concentrate containing PRO may further be purified using selected column chromatography resms
  • the tollowing method describes recombinant expression of insulin or insulin v ariant in Baculovirus infected insect cells
  • sequence coding for insulin or insulin v ⁇ ant is fused upstream of an epitope tag contained within a baculovirus expression vector
  • epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG)
  • a variety ot plasmids may be employed including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly the sequence encoding insulin or insulin variant or the desired portion of the coding sequence of this polypeptide [such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular] is amplified by PCR with primers complementary to the 5 and 3 regions The 5' primer may incorporate flanking (selected) restriction enzyme sites The product is then digested with those selected restriction enzymes and subcloned into the expression vector
  • Recombinant baculovirus is generated by co transfecting the above plasmid and BaculoGold ® virus DNA (Pharmingen) into Spodoptera frugipeida (' Sf9 ) cells (ATCC CRL 171 1 ) 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 Bac uloMi us expiession ⁇ ectoi s A Laboiaton Manual Oxford Oxford University Press (1994)
  • Expressed poly-his tagged insulin or insulin variant can then be purified, for example by N ⁇ " + chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al Natuie, 362 175-179 ( 1993) Briefly Sf9 cells are washed resuspended in so cation buffei (25 mL Hepes pH 7 9, 12 5 mM MgCl 2 0 1 mM EDTA 10%.
  • the insulin or insulin variant molecules ot the invention may be expressed using a modified baculovirus procedure employing H ⁇ -5 cells
  • the DNA encoding the desired sequence was amplified w ith suitable systems such as Pfu (Stratagene).
  • plasmids may be employed, including plasmids derived from commercially available plasmids such as pIE- 1 (Novagen)
  • the pIE l -1 and pIEl -2 vectors are designed tor constitutive expression of recombinant proteins trom the baculovirus le i 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 le i -mediated gene expression in u nfected insect cells as well as the hr5 enhancer element pIE l -1 and pIEl -2 include the lei 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 en
  • the vector construct is sequenced for confirmation H ⁇ 5 cells are grown to a confluency of 50% under the conditions ot 27°C, no C0 . no pen/strep
  • the conditioned media trom the transtected cells (0 5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0 22 cron filters
  • the protein comp ⁇ sing the sequence is purified using a Ni-NTA column (Qiagen ) Before purification, imida/ole at ⁇ flow rate of 4-5 ml/mm at 48°C Attei loading, the column is washed with additional equilibrium butter and the protein eluted with equilibrium butter containing 0 25M imidazole The highly purified protein was then subsequently desalted into a stoiage butter 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
  • Immunoadhesion (Fc-containing) constructs may also be purified from the conditioned media as follow s
  • the conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which had been previously equilibrated in 20 mM sodium phosphate buffei , pH 6 8 After loading, the column is washed extensively with equilibrium buffer before elution with 100 mM citric acid, pH 3 5
  • the eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ⁇ l of 1 M Tris buffer, pH 9
  • the highly purified protein is subsequently desalted into storage butter as described above for the poly-His tagged proteins
  • the homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation
  • EXAMPLE 21 Preparation of Antibodies that Bind PRO This example illustrates preparation of monoclonal antibodies which can specifically bind PRO Techniques for producing the monoclonal antibodies are known in the art and are desc ⁇ bed, for instance, in Goding, supra Immunogens that may be employed include purified PRO, fusion proteins containing PRO, and cells expressing recombinant PRO 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 PRO immunogen emulsified in complete Freund's adjuvant and miected subcutaneously or lntrape ⁇ toneally in an amount from 1 - 100 micrograms
  • the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research.
  • 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-PRO antibodies
  • the animals "positive" for antibodies can be injected with a final intravenous injection ot PRO
  • the mice are sacrificed and the spleen cells are harvested The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU 1.
  • HAT hypoxanthine. aminopte ⁇ n. and thymidine
  • the hybndoma cells will be screened in an ELISA for reactiv ity against PRO Determination of "positive" hybndoma cells secreting the desired monoclonal antibodies against PRO is within the skill in the art
  • the positive hvb ⁇ dom ⁇ cells can be injected liurape ⁇ toneally into sy ngeneic Balb/c mice to produce ascites containing the anti-PRO monoclonal antibodies Alternativ ely the hy b ⁇ doma 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 bv gel exclusion chromatography Alternatively , affinity chromatography based upon binding of antibody to protein A or protein G can be employed
  • PRO polypeptides may be pu ⁇ fied by a variety of standard techniques in the art of protein purification For example, pro-PRO polypeptide. mature PRO polypeptide. or pie-PRO polypeptide is pu ⁇ fied by lmmunoaffinity chromatography using antibodies specific for the PRO polypeptide of interest In geneial, an lmmunoaffinity column is constructed by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resin Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway.
  • monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSETM (Pharmacia LKB Biotechnology) The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions
  • Such an lmmunoaffinity column is utilized in the purification of PRO polypeptide by preparing a fraction from cells containing PRO polypeptide in a soluble form This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art Alternatively, soluble PRO polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown
  • a soluble PRO polypeptide-containing preparation is passed over the lmmunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PRO polypeptide (e g , high ionic strength buffers in the presence of detergent) Then, the column is eluted under conditions that disrupt antibody/PRO polypeptide binding (e g . ⁇ low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and PRO polypeptide is collected
  • This invention is particularly useful for screening compounds by using PRO polypeptides or binding fragment thereof in any of a variety of drug screening techniques
  • the PRO polypeptide oi fragment employed in such a test may either be tree in solution, affixed to a solid support, borne on a cell surface, or located intracellularly
  • One method of di ug screening utilizes eukarvotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • One may measure, for example, the formation of complexes between PRO polypeptide or a fragment and the agent being tested. Alternatively, one can examine the diminution in complex formation between the PRO polypeptide and its target cell or target receptors caused by the agent being tested.
  • the present invention provides methods of screening for drugs or any other agents which can affect a PRO polypeptide-associated disease or disorder. These methods comprise contacting such an agent with an PRO polypeptide or fragment thereof and assaying (I) for the presence of a complex between the agent and the PRO polypeptide or fragment, or (ii) for the presence of a complex between the PRO polypeptide or fragment and the cell, by methods well known in the art. In such competitive binding assays.
  • the PRO polypeptide or fragment is typically labeled. After suitable incubation, free PRO polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to PRO polypeptide or to interfere with the PRO polypeptide/cell complex.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on September 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to a PRO polypeptide, the peptide test compounds are reacted with PRO polypeptide and washed. Bound PRO polypeptide is detected by methods well known in the art. Purified PRO polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding PRO polypeptide specifically compete with a test compound for binding to PRO polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with PRO polypeptide.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptide of interest (i.e.. a PRO polypeptide) or of small molecules with which they interact, e.g., agonists, antagonists, or inhibitors. Any of these examples can be used to fashion drugs which are more active or stable forms of the PRO polypeptide or which enhance or interfere with the function of the PRO polypeptide in vivo (c.f, Hodgson. Bio/Technology, 9: 19-21 (1991 )).
  • the three-dimensional structure of the PRO polypeptide. or of an PRO polypeptide-inhibitor complex is determined by x-ray crystallography, by computer modeling or. most typically, by a combination of the two approaches. Both the shape and charges of the PRO polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous PRO polypeptide-like molecules or to identify efficient inhibitors. Useful examples of rational drug design may include molecules which have improved activity or stability as show n by Bra.xton and Wells. Biochemistry. 3J_:7796-7801 ( 1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda et al., J. Biochem.. 1 13:742-746 ( 1993).
  • a target-specific antibody selected by functional assay, as described above, and then to solve its crystal structure.
  • This approach in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
  • anti-ids anti-idiotypic antibodies
  • PRO polypeptide may be made available to perform such analytical studies as X-ray crystallography.
  • knowledge of the PRO polypeptide amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.

Abstract

The present invention relates to the use of PRO200 (VEGF-E), PRO540, PRO846, PRO617, PRO538 (GFRα3), PRO3664 (GFRα3) or PRO770 (hFIZZ-1) polypeptides to delay, prevent or rescue retinal neurons, including photoreceptors, other retinal cells or supportive cells (e.g Müller cells or RPE cells) from injury and degradation. Conditions comprehended by treatment of the present PRO200 (VEGF-E), PRO540, PRO846, PRO617, PRO538 (GFRα3), PRO3664 (GFRα3) or PRO770 (hFIZZ-1) polypeptides (including variants), antibodies, compositions and articles of manufacture include: retinal detachment, age-related and other maculopathies, photic retinopathies, surgery-induced retinopathies (either mechanically or light-induced), toxic retinopathies including those resulting from foreign bodies in the eye, diabetic retinpathies, retinpathy of prematurity, viral retinopathies such as CMV or HIV retinopathy related to AIDS, uveitis, ischemic retinopathies due to venous or arterial occlusion or other vascular disorder, retinopathies due to trauma or penetrating lesions of the eye, peripheral vitreoretinopathy, and inherited retinal degenerations. Exemplary retinal degenerations include e.g., hereditary spastic paraplegia with retinal degeneration (Kjellin and Barnard-Scholz syndromes), retinitis pigmentosa, Stargardt disease, Usher syndrome (retinitis pigmentosa with congenital hearing loss), and Refsum syndrome (retinitis pigmentosa, hereditary hearing loss, and polyneuropathy). Additional disorders which result in death of retinal neurons include, retinal tears, detachment of the retina and pigment epithelium, degenerative myopia, acute retinal necrosis syndrome (ARN), traumatic chorioretinopathies or contusion (Purtscher's Retinopathy) and edema.

Description

METHOD OF PREVENTING THE DEATH OF RETINAL NEURONS AND TREATING OCULAR DISEASES
BACKGROUND
The present invention relates to a method of promoting retinal neuion survι\al as well as preventing photoneuron degradation and to the treatment of diseases or conditions w hich are characterized by photoneuron death
The retina is the light-sensitive portion of the eye The ietina contains the cones and rods
(photoreceptors), the photosensitive cells The rods contain rhodops . the rod photopigment and the cones contain 3 distinct photopigments, which respond to light and transmit signals through successive neurons to ultimately trigger a neural discharge in the output cells of the retina, the ganglion cells The signal is carried by the optic nerve to the visual cortex where it is registered as a visual stimulus
In the center of the retina is the macula lutea, which is about 1/3 to 1/2 cm in diameter The macula provides detailed vision, particularly in the center (the fovea), because the cones are higher in density Blood vessels, ganglion cells, inner nuclear layer and cells, and the plexiform layers are all displaced to one side (rather than resting above the ones), thereby allowing light a more direct path to the cones
Under the retina is the choroid, a collection of blood vessels embedded within a fibrous tissue, and the pigmented epithelium (PE), which overlays the choroid layer The choroidal blood vessels provide nutrition to the retina (particularly its visual cells) The choroid and PE are found at the posterior of the eye The retinal pigment epithelial (RPE) cells, which make up the PE, produce, store and transport a variety of factors that are responsible for the normal function and survival of photoreceptors RPE arc multifunctional cells that transport metabolites to the photoreceptoi s from their blood suppK the choπo capillaries of the eye The RPE cells also function to recycle vitamin A as it moves between the photoreceptors and the RPE during light and dark adaptation RPE cells also function as macrophages phagocytizing the rhythmically-shed tips of the outer segments of rods and cones, which are produced in the normal course of cell physiology Various ions, proteins and water move between the RPE cells and the interphotoreceptor space, and these molecules ultimately effect the metabolism and v iability of the photoreceptors
The Muller cell is the most prominent glial cell within the retina, and could also be important foi maintaining the viability ol visual cells Muller cells traverse the entire retina in a iadial dnection from the ganglion cells to the external limiting membrane, a photoreceptor-photoreceptor and Muller cell photoreceptor contact point In addition to providing structuial suppoit Muller cells regulate the control ol ionic concentralions, degradation of neurotransmitter, removal of certain metabolites and may be a source of important factors that promote the normal differentiation of photoreceptoi cells [Kl'avin and Re ( 1991 ), J Neuioscience JJ. 2985 2994] Although a search lor defects in Muller cells has not specifically been examined, any disease or injury affecting their normal function most likely would have a dramatic influence on the health of rods and cones. Finally, the death of rod photoreceptors may influence the viability of cones. One common feature in degenerations involving mutations in rod specific genes (i.e.. rhodopsin) is that cones also eventually die. The reason for the loss of cones has not been determined, although it has been suggested that dying rods may release endotoxins [Bird ( 1992 ). Opthal. Pediati c. Genet. Y_: 57-661.
Diseases or injury to the retina can lead to blindness if retinal cells are injured or killed. The photoreceptor cells are particularly susceptible to injury since they are often the first cells to degenerate or suffer damage as a result of a traumatizing event or condition. Hereditary defects in specific photoreceptor genes, retinal detachment, circulatory disorders, overexposure to light, toxic effects to drugs and nutritional deficiencies are among the wide array of causes that can result in the death of photoreceptor cells.
Developmental and hereditary diseases of the retina account for around 20 percent of all legal blindness in the United States [Report of the Retinal and Choroidal Panel: Vision Research - A National Plan 1983-
1987, vol. 2, part I, summary page 2], For example, retinitis pigmentosa (RP), a genetic based progressive disease is characterized by incremental loss of peripheral vision and night blindness, which is due in large part to the loss of photoreceptor cells. RP is a group of hereditary diseases and presently afflicts about one in 3000 persons worldwide [Wong. F. ( 1995) Arch. Ophthal wl. J 3 1245-47]. Total blindness is the usual outcome in more progressive stages of this disease. Macular degeneration, another major cause of blindness, is a complex group of disorders that affects the central or predominantly cone portion of the retina. Cones are primarily responsible for acute vision. Diabetic retinopathy, a frequent complication in individuals with diabetes mellitus, is estimated to be the fifth leading cause of new blindness. However, it is the second leading cause of blindness among individuals of 45-74 years of age. Moreover, these problems are only expected to get worse as the general population ages.
Photoreceptor degeneration may also occur as a result of overexposure to light, various environmental trauma or by any pathological condition characterized by death or injury of retinal neurons or photoreceptors.
Photoreceptor loss may also be influenced by cellular or extracellular retinal components. The primary example of extracellular stimulus is related to the close association between the pigment epithelium (PE) and the photoreceptor cells. As mentioned previously, the PE transports metabolites to and from the photoreceptors as well as removes discarded cellular material. Retinal detachment, which involves the separation of the neural retina from the PE leads to photoreceptor death. Furthermore, the degree of cell loss is dependent upon the duration of the separation [Gouras et al. ( 1991 ) 10VS 32: 3167-3174].
Additionally, diseases of the PE can lead to photoreceptor cell loss. The primary example of this is the Royal College of Surgeons (RCS) rat. which has an inherited retinal dystrophy due to a defect in the PE. resulting in photoreceptor cell death during the normal course of the animal's life [Mullen & LaVail ( 1 76), Science 192: 799-801 ]. In this animal, the PE are unable to phagocytize outersegment debris which accumulates between the photoreceptor cells and the PE, and as a result, provide a useful model system to study the role of trophic factors on the retina. A delay of photoreceptor death is obtained through the proximal placement of normal PE cells both in experimental chimeras, Mullen & LaVail. supra and by transplantation of PE from healthy animals [Li & Turner ( 1988). E.xp. Eye Res. 47: 91 1 -917: Sheedlo et al. ( 1992). Int. Rev. Cylol. Y__: 1 -49: Lavail et al. ( 1992). E.xp. Eye Res. 55: 555-562: Lavail et al. ( 1992). PNAS 89: I 1249- 1 1253]. In all of these experiments, the "'rescue*' extended beyond the boundaries of the normal PE cells, and suggests the presence of difussible trophic factor(s) produced by the PE cells. Another useful animal model is the albino rat. In this animal, normal illumination levels of light, if continuous, can cause complete degeneration of photoreceptors. Results obtained using such rats as a model to identify survival enhancing factors appear to correlate well with data obtained using RCS rats. Moreover. different factors can be compared and complications can be assessed more quickly in the light damage model than can be assessed by testing factors in models which are based on the slowly evolving dystrophy of the RCS rat.
Using albino rats, it has been determined that a number of agents, when administered systemically
(intraperitoneally) can be used to ameliorate retinal cell death or injury caused by exposure to light. In general, exposure to light generates oxygen free radicals and lipid peroxidation products. Accordingly, compounds that act as antioxidants or as scavengers of oxygen free radicals reduce photoreceptor degeneration. Agents such as ascorbate [Organisciak et al. ( 1985). Invest. Opt al. & Vis. Sci. 26: 1580-
1588], flunarizine [Edward et al. ( 1991 ), Arch. Ophthalmoi. J09: 554-562], and dimethylthiourea, [Lam et al. (1990). Arch. Opthal. 108: 1751 -1757] have been used to ameliorate the damaging effects of constant light. There is no evidence, however, that these compounds will act to ameliorate other forms of photoreceptor degeneration and their administration can potentiate harmful side effects. Further, these studies are limited because they utilize systemic delivery, which does not provide an adequate means of assessing the effectiveness of a particular factor. As a result, it is nearly impossible to assess the amount of agent that actually reaches the retina. A large amount of agent must be injected to attain a sufficient concentration at the site of the retina. In addition, systemic toxic effects may result from the injection of certain agents. Traditional approaches to treating the loss of vision due to photoreceptor cell death has taken at least two routes: ( 1 ) replacing the defective cells by physical transplantation: and (2) slowing, arresting or preventing the process of degeneration. The transplantation of healthy pigment epithelium cells into a degenerating retina or one which has defective epithelium cells can rescue photoreceptor cells from dying
[Sheedlo et al.. Int. Rev. Cytol. J38: 1 -49 (1992)); Lavail et al,, Exp. Eye Res. 55: 555-562 ( 1992); and Lavail et al., PNAS 89: 1 1249-1 1253 ( 1992)]. PE transplants in humans have been attempted, but the results have been less than satisfactory [Peyman et al., Opthal. Surg. 22: 102- 108 ( 1991 )]. More promising, but as yet unproven is the transfer of embryonic retina containing mostly undifferentiated progenitor cells, which can differentiate in response to environmental cues into appropriate missing cell types [Cepko, Ann.
Rev. Neurosci. _2: 47-65 ( 1989)]. In conclusion, therapy via functional integration of transplanted retinal cells into a human host retinas remain unproven.
Other strategies have focused on "rescuing" or slowing the loss of visual cells. These techniques include corrective gene therapy, limiting the exposure to normal light during disease, vitamin A supplemented diets and the administration of growth factors to damaged or degenerating eyes. However, these treatment schemes have sev eral limitations Foi example gene therapv or the insertion ot a replacement allele into the cells carry ing the know n mutation av piov e problematic [Mila Cui t Opm _ 797 804 ( 1993)] Since rods and cones aie somew hat inaccessible it might be difficult to delivei replacement genes to them Moreov er the use ot retroviral vectors foi insertion of replacement genes is limited to dividing cells such as cultured PE w hereas post mitotic neurons e g photoreceptors will require other viral vectors such as HSV (Herpes simplex v irus) for effectiv e deliverv Finally gene replacement may not correct a disease where the mutant gene product is deleterious to the cell but ma) be more useful for correcting defects due to the loss of function of a gene product as is found in most recessive disorders Limiting light exposure a low technology conventional approach to attenuating vision loss typically using such approaches as eye patches dark goggles, etc is impractical since the practical effect of the treatment is the same as the disease itself blindness and inability to detect light
Vitamin A has been observed to halt the decline of retinal function by ov er 20% as administered over the course of 4 6 years in the progression of patients with letmttis pigmentosa (RP) [E L Berson et al Aich Ophthalmol 1 1 1 761 772 ( 1993)] While this study did indicate a potential lengthening of yeais of useful vision, several criticisms of vitamin A therapv exist ( 1 ) the mechanism by which vitamin A (and even vitamin E) alter the progression of RP is unknown, (2) it is not known whethei or not patients with different genetic forms of RP will respond to vitamin A therapy, (3) it is not apparent whether or not quantifiable measurements of visual function (l e peπmetry and v isual acuity) revealed any significant benefit from vitamin A therapy and (4) long term ingestion of vitamin A may have detrimental side effects in other organ systems
A number of agents when administered systemically (intraperitoneally) can be used to ameliorate retinal cell death or injury caused by exposure to light In general, exposure to light generates oxygen free radicals and lipid peroxidation products It has been suggested that genetically detective photoreceptors are abnormally sensitive to photooxidation from light levels as encountered normally in the environment
[Hargrave, PA &. O'Brien PJ Retinal Degeneiations Anderson RE et al eds Boca Raton FL CRC Press, p 17 528 (1991 )] Compounds that act as antioxidants or as scavengers of oxygen tree radicals reduce photoreceptor degeneration Anti oxidants or calcium overload blockers (c g flunaπzine) have been reported to prevent degeneration of normal photoreceptors after exposure to high light levels [Rosner et al Arch Ophthalmol 1 10 857-861 (1992) Li et al Exp EM Res 56 71 78 (1993)] Additional success in reducing photoreceptor degeneration has been observed through administration of ascorbate [Orgamsciak et al fin est Ophthal & Vιs Sci 26 1580 1588 ( 1985)] flunaπzine [Edward al Aic Ophthalmol J09 554 562 (1991 )] and dimethylthiourea [Lam et al Aich Ophthal 108 1751 1757 ( 1990)] However there is no evidence that administration of these compounds will reduce photoreceptor degeneration induced by other than intense light exposure Moreover, there is great concern that their administration can generate potentially harmful side eflects As a result the search continues for factors which can somehow protect photoreceptors or even promote their regeneration aftei light induced damage A particular area of interest is the administration of grow th factors. Growth factors have been found to participate in diverse roles such as neuronal differentiation, transmitter specificity, regulation of programmed cell death, and neurite growth in several regions of the central nervous system. However, only recently has their role been studied during retinal development and disease. An early study indicating that diffusible growth factors can rescue photoreceptor cells from dying was based on a chimeric rat constructed to contain both normal and RCS pigment epithelial cells. The animals were produced by fusing blastula from both normal and RCS rat embryos. Mullen and LaVail. supra. In the retina of these chimeras, photoreceptor cells adjacent to RCS PE showed degeneration, and those that were lying next to normal PE were healthy. However, photoreceptor cells that were lying just beyond the immediate contact site of normal PE also appeared healthy, suggesting that photoreceptor-PE contact was not needed, and that normal
PE were secreting a putative survival promoting factor.
Among the best characterized growth factors in the retina are the acidic and basic fibroblast growth factors (aFGF and bFGF). FGF can be detected through immunohistochemical, biochemical or molecular approaches on a variety of retinal cells including PE, photoreceptor cells and the interphotoreceptor cell matrix (IPM), and a collection of extracellular matrix molecules surrounding photoreceptor cells [Jacquemin et al, Neurosci. Lett. ir. 23-28. ( 1990); Caruelle et al, J. Cell Biol. 39: 1 17- 128 ( 1989): Hageman et al., PNAS __: 6706-6710 ( 1991 ); Connolly et al.. IOVS 32 (suppl.): 754 ( 1991 )]. Intravitreal injection of basic fibroblast growth factor (bFGF) in the RCS rat or rats with light damaged retina prevents photoreceptor cell degeneration for several month, even as outersegment debris accumulates [Faktorovich et al, Nature 347: 83-86. (1990)]. Similar results have been seen when bFGF is injected into the subretinal space, the area between the photoreceptors and the PE. However, even sham operations, or injections of phosphate buffered saline (PBS) in both the RCS rat and light damaged retina can delay photoreceptor cell death. However, the rescue effect is small and localized to the needle track, and differs quantitatively from the effect obtained from bFGF [Faktorovich et al., supra: Silverman and Hughes, Curr. Eye Res. 9: 183- 191 ( 1990): Sheedlo H.J. et al.. Int. Rev. Cyto. 138: 1-49 ( 1992)]. In these experiments it is likely that various growth factors derived from damaged retinal tissues or macrophages present in the damaged area were locally released. Sheedlo et al., supra.; Silverman and Hughes, supra. Macrophages themselves are known to produce many different growth factors or cytokines, some of which could have photoreceptor survival activity [Rappolee and Werb, Curr. Top. Microbiol. Immunol. 181 : 87- 140 ( 1992)]. Various agents disclosed to have survival-enhancing and/or growth activity on retinal neurons are described in certain issued patents and pending patent applications. These include Transforming Growth
Factor-β (TGF-β) [WO 94/01 124], brain derived neurotrophic factors (BDNF) [U.S.P 5.180.820] [U.S.P.
5,438, 121 ] and [WO 91/03568], neurotrophin-4 (NT-4) [WO 93/25684], and insulin-like growth factors
(IGF) [WO 93/08826]. Other experiments have shown that intravitreal injections of human subretinal fluid as well as other growth factors can rescue dying photoreceptor cells. For example, one recent study tested eight different factors injected into the retina of rats exposed to constant high intensity light, all showing the ability to delay the degeneration of photoreceptor cells. These include FGF (both acidic and basic forms), brain derived neurotrophic factor ( BDNF). ciliary neurotrophic factor (CNTF). and interleukin 1 (IL- I ) Neurotiophin 3 (NT 3). insulin-like giowth iactoi II (IGF II). Transforming Growth Factoi beta (TGF-β ) and the tumoi necrosis factois alpha and beta (TNF-σ. TNF-β) also show ed surv iv al activ ity , but to a much lessei degiee than the other tactoi s NGF has been reported to reduce the incidence of apoptosis in diabetic rats in addition to minimizing peπcyte loss and acellular occluded capillaπes conditions associated with diabetic retinopathy [Hammes. HP et al Moleculai Med 1 (5 ) 527-534 ( 1995)] How evei . while it does appear that growth factors can enhance surv iv al of photoreceptors. some of these factors may pio ote detiimental side effects For example, injections of bFGF results in an increased incidence of macrophages and cataracts In addition. bFGF is mitogenic for PE, Muller cells and retinal v ascular cells Faktorovich et al . supra . La Vail et al . supra As a result, suitable growth factors which will not only promote the survival of photoreceptor cells, but lack undesired side effects have yet to be discovered
SUMMARY The present invention relates to a method of delaying, preventing or rescuing photoreceptor cells from injury or death by the administration of a therapeutically effective amount of a PRO polypeptide In another aspect, the present invention relates to the use of PRO polypeptides to delay, prevent or rescue other retinal cells or supportive cells (e g Muller cells or RPE cells) fiom injury and death Other retinal neurons include, but are not limited to retinal ganglion cells, displaced retinal ganglion cells. amacπne cells, displaced amacπne cells, horizontal and bipolar neurons Additionally, the invention relates to the use of PRO to stimulate the regeneration of such cells In one aspect, the PRO polypeptide is an active polypeptide which is at least 90% homologous to a native sequence PRO molecule
In yet another aspect, the PRO polypeptide is an active PRO polypeptide encoded by an isolated nucleic acid comprising DNA encoding a PRO 200 (VEGF-E). PRO540. PR0846. PR0617, PR0538 (GFRα3), PR03664 (GFRα3) or PRO770 (hFIZZ-1 ) polypeptide In one aspect, the isolated nucleic acid comprises DNA encoding a ( 1 ) PRO200 polypeptide having ammo acid residues 1 to 345 (alternatively 15 to 345) ot SEQ ID NO 2. (2) PRO540 polypeptide having amino acid residues I to 412 (alternatively 29 to
412) of SEQ ID NO 4, (3) PR0846 polypeptide having amino acid residues 1 to 332 (alternatively 18 to 332) of SEQ ID NO 13; (4) PR0617 polypeptide having amino acid residues 1 to 67 (alternatively 16 to 67) of SEQ ID NO 19, (5) PR0538 polypeptide having amino acid residues I to 400 (alternatively 27 to 400) of SEQ ID NO 26, (6) PR03664 polypeptide having amino acid residues 1 to 369 (alternatively 27 to 369) of SEQ ID NO 27, (7) PRO770 polypeptide having amino acid residues 1 to 1 1 1 (alternatively 29 to
1 1 1 ) of SEQ ID NO 33 or hybridizes under moderate, and optionally under high stringency conditions to the compliment of such encoding nucleic acid sequence and remains stably bound thereto
In yet another aspect, the present invention relates to the use of PRO polypeptides to treat any condition which results in injury or death of photoreceptor or other retinal cells Examples ot conditions include retinal detachment, age-related and other maculopathies. photic retinopathies, surgery-induced retinopathies (either mechanically oi light-induced), toxic retinopathies including those resulting from foreign bodies in the eye. diabetic retinopathies. retinopathy of prematui it> , viral retinopathies such as CMV or HIV retinopathy related to AIDS, uveitis. ischemic retinopathies due to venous or arterial occlusion or other ascular disoi der, retinopathies due to trauma or penetrating lesions of the eye, peripheral Mtreoretinopathy . and inherited retinal degenerations Exemplary letinal degenerations include e g hereditary spastic paraplegia w ith retinal degeneration ( Kiellin and Barnard Schol/ syndromes) l erinitis pigmentosa Stargardt disease Ushei sy ndiome (letimtis pigmentosa w ith congenital hearing loss), and Retsum syndrome (/ et itis pigmentosa hereditary hearing loss and polv neuiopathy ) Additional disordeis which result in death ot retinal neurons include retinal teais. detachment ot the retina and pigment epithelium, degenerative myopia acute ietinal necrosis syndrome (ARN) traumatic choπoretinopathies or contusion (Purtscher's Retinopath ) and edema
In yet another aspect, the present invention prov ides to a method of delay ing, preventing or rescuing retinal neuions ( g photoreceptors) or other retinal cells fiom injury or death resulting from disease or injury comprising the administration of a composition of PRO polypeptide and a pharmaceutically -acceptable carrier In one part, the composition comprises a therapeutically effective amount of PRO polypeptide In another, the composition compπses a turther activ e ingredient, which may for example, be a further neuronal survival agent Preferably the composition is sterile
In still yet another aspect, the present invention provides articles of manufacture and kits that include PRO polypeptide The articles of manufacture and kits include a container an instruction on the container, and a composition contained within the container The instruction on the container indicates that the composition can be used to delay, prevent or rescue retinal neurons or other retinal cells from injury or death The composition contains an active agent, and the active agent comprises PRO In other embodiments ot 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, or yeast A process tor producing any of the herein described polypeptides is further provided and comprises cultuπng 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 ammo acid sequence Example of such chimeπc molecules compi ise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin
In another embodiment, the invention provides an antibody which specifically binds to any of the above or below described polypeptides Optionally the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody
In yet other embodiments, the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense piobes. wheiein those probes may be derived from any of the above oi below described nucleotide sequences
In other embodiments, 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% nucleic acid sequence identity , alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity , alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity , alternatively at leas! about 87% nucleic acid sequence identity , alternatively at lea**.! about 88% nucleic acid sequence identity , alternatively at least about 89% nucleic acid sequence identity, alternativ ely at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, altei natively at least about 94% nucleic acid sequence identity , alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule encoding a 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 othei 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% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity. alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about
85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about
90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about
95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity. alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length 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 turthei aspect, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity. alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity alternatively at least about 92% nucleic acid sequence identity alternatively at least about 93% nucleic acid sequence identity alternativ elv at least about 94% nucleic acid sequence identity, alternativ elv at least about 95% nucleic acid sequence identity alternativ ely at least about 96% nucleic acid sequence identity , alternatively at least about 97% nucleic acid sequence identity . alternatively at least about 98% nucleic acid sequence identity and alternativ ely at least about 99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same mature poly peptide encoded by any ot the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a)
Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is eithei transmembrane domain-deleted or transmembrane 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 of the herein descπbed PRO polypeptides are contemplated
Anothei 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 tor an anti-PRO antibody or as antisense oligonucleotide probes Such nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 1 10 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleotides in length, alternatively at least about 170 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 190 nucleotides in length, alternatively at least about 200 nucleotides in length, alternatively at least about 250 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 350 nucleotides in length, alternatively at least about 400 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 500 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 700 nucleotides in length, alternatively at least about 800 nucleotides in length, alternatively at least about 900 nucleotides in length and alternatively at least about 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length It is noted that novel fragments of a PRO 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 tragment(s) are nov el All of such PRO polypeptide-encoding nucleotide sequences aie contemplated heiein Also contemplated are the PRO polypeptide fragments encoded by these nucleotide molecule fragments, pieferably 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% amino acid sequence identity , altei natively at least about 81 % amino acid sequence identity , alternatively at least about 82% ammo acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity. alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about
88% amino acid sequence identity , alternatively at least about 89% amino acid sequence identity. alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % ammo acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about
93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about
98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a
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
In a further aspect, the invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% ammo acid sequence identity. alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about
88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about
93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97%* amino acid sequence identity, alternatively at least about
98% amino acid sequence identity and alternatively at least about 99% ammo acid sequence identity to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein
In a further aspect, the invention concerns an isolated PRO polypeptide comprising an ammo acid sequence scoring at least about 80% positives, alternatively at least about 81 % positives, alternatively at least about 82% positives, alternatively at least about 83% positives, alternatively at least about 84% positives, alternatively at least about 85% positives, alternatively at least about 86% positives, alternatively at least about 87% positives, alternatively at least about 88% positives, alternatively at least about 89% positives, alternatively at least about 90% positives, alternatively at least about 91 % positives, alternatively at least about 92% positives, alternatively at least about 93% positives, alternatively at least about 94% positives, alternatively at least about 95% positives, alternatively at least about 96% positives, alternatively at least about 97% positives, alternatively at least about 98% positives and alternatively at least about 99% positives when compared with the amino acid sequence of a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein.
In a specific aspect, the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.
Another aspect 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 culturing 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 sequence 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 carrier 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.
Other aspects of the invention will become apparent from the following detailed description and the claims. Brief Description of the Drawings
Figure 1 show s DNA29101 1272 (SEQ ID NO 1 ) a DNA sequence encoding a PRO200 (VEGF- E) (SEQ ID NO 1 )
Figure 2 show s the native sequence PRO200 (SEQ ID NO 2) Figure 3 shows DNA44189- 1 22 (SEQ ID NO 3 ). a DNA sequence encoding PRO 40 (SEQ ID
NO 4)
Figure 4 shows the native sequence PRO540 (SEQ ID NO 4)
Figure 5 shows DNA39631 (SEQ ID NO 5). a consensus nucleotide used in the preparation ot DNA44189 Figure 6 shows DNA44196-1353 (SEQ ID NO 12). a DNA sequence encoding the native sequence
PR0846 (SEQ ID NO 13)
Figuie 7 shows the native sequence PR0846 (SEQ ID NO 13)
Figure 8 shows a single-stranded (sense) consensus nucleotide sequence used in the isolation of DNA44196 (Figure 6. SEQ ID NO 12), designated in the present application as DNA39949 (SEQ ID NO 14)
Figure 9 shows DNA48309-1280 (SEQ ID NO 18). a DNA sequence encoding a native sequence PR0617 polypeptide (SEQ ID NO.19)
Figure 10 shows the native sequence PR0617 (SEQ ID NO 19)
Figure 1 1 shows the single stranded nucleotide sequence (sense strand) DNA42798 consensus DNA (SEQ ID NO. 20) which was used in the isolation of DNA48309 (SEQ ID NO 18)
Figure 12 shows DNA48613-1268 (SEQ ID NO 24), a DNA sequence encoding a native sequence
PR0538 polypeptide (SEQ ID NO 26)
Figure 13 shows DNA48614-1268 (SEQ ID NO.25), a DNA sequence encoding a native sequence PR03664 polypeptide (SEQ ID NO.27) Figure 14 shows the native sequence PR0538 (SEQ ID NO 26)
Figure 15 shows the native sequence PR03664 (SEQ ID NO 27)
Figures 16A and B show a comparison between a native sequence PR0538 (SEQ ID NO 26) and PR03664 (SEQ ID NO 27) encoded by DNA48613 (SEQ ID NO 24) and DNA48614 (SEQ ID NO 25), respectively, resulting in a 92 25% similarity (% value generated by the Align program using PAM250 matrix and a gap penalty of 8 + 4 per residue)
Figure 17 shows the EST sequence (Incyte INC3574209)(SEQ ID NO 28) sequence which was used in the cloning of the full length PR0538 (GFRα3) and PR03664 (GFRσ3) sequences DNA48613 (SEQ ID NO 24) and DNA48614 (SEQ ID NO 25). respectively
Figure 18 shows the native sequence PRO770 (hFIZZ- 1 )(SEQ ID NO 33) Figure 19 shows DNA54228-1366 (SEQ ID NO 34). a DNA sequence encoding a native sequence
PRO770 polypeptide (SEQ ID NO 33)
Figure 20 shows DNA53517 (SEQ ID NO 35). a murine hFIZZ- 1 sequence which has used in the isolation of DNA54228 (SEQ ID NO 34) as described in Examples 14 and 15 Figure 21 show s AA524300 (SEQ ID NO 36) an ESI w hich was identified as hav ing homology to murine TIZZ as described in Example 14
Figure 22 show s Incy te EST 13025 16 an ESI exhibiting homology to human VEGF. and was used in the isolation of DNA29101 (SEQ ID NO 1 ) described in Example 8
Detailed Description of the Im ention 1. Definitions:
The terms employed throughout this application are to be construed with the normal meaning to those of ordinary skill in the art Howev er, applicants desire that the following terms be consti ued with the particular definitions as described All references mentioned in this application should be interpreted and read as being incorporated by reference
The terms "protein" oi " polypeptide" aie intended to be used interchangeably They refer to a chain of two (2) or more amino acids which are linked together w ith peptide or amide bonds, regardless of post-translational modification (e g , glycosylation or phosphorylation) The polypeptides of this invention may comprise more than one subunit. where each subunit is encoded by a separate DNA sequence
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 provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein) The
PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods The term "PRO polypeptide" refers to each individual PRO/number polypeptide disclosed herein All disclosures in this specification which refer to the "PRO polypeptide" refer to each of the polypeptides individually (j e , PRO200 (VEGF-E), PRO540. PR0846, PR0617. PR0538 (GFR 3), PR03664 (GFRoβ) or PRO770
(hFIZZ- 1 )) as well as jointly - depending upon the context Foi example, descriptions of the preparation ol, purification of, derivation of, formation of antibodies to oi against, administration of. compositions containing, treatment of a disease with, etc . pertain to each polypeptide of the invention individually The term "PRO polypeptide" also includes variants ot the PRO/number polypeptides disclosed herein A "native sequence PRO polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature Such nativ e 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 foims (e g , alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide In various embodiments of the invention, the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides compπsing 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 show n to begin with methionine residues designated herein as amino acid position 1 in the figures it is conceivable and possible that other methionine residues located either upstream oi dow nstream from the amino acid position 1 in the figures may be employed as the starting amino acid residue toi the PRO pol peptides
In one embodiment of the invention, the nativ e-sequence ( 1 ) PRO200 (VEGF-E) polypeptide is a mature or full-length nativ e sequence PRO200 (VEGF-E) polypeptide comprising amino acids 1 though 345 as depicted in Figure 2. (2) PRO540 polypeptide is a mature or full-length native sequence PRO540 polypeptide comprising amino acids 1 to 412 of Figuie 4. (3) PR0846 poly peptide is a mature or full-length native sequence PR0846 polypeptide comprising amino acids 1 to 332 of Figure 7. (4) PR0617 polypeptide is a mature or full-length native sequence PR0617 polypeptide comprising amino acids 1 to 67 of Figure 10. (5) PR0538 (GFRα3) polypeptide is a mature or full-length native sequence PR0538
(GFRcβ) comprising amino acids 1 to 400 of Figure 13, (6) PR03664 (GFRα3) polypeptide is a mature oi full-length native sequence PR03664 (GFRcβ) comprising amino acids I to 369 of Figure 14, or (7)
PRO770 (hFIZZ- 1 ) polypeptide is a matuie or full-length native sequence PRO770 (hFIZZ-1 ) comprising ammo acids 1 to 1 1 1 of Figure 16. respectively The PRO polypeptide "extracellular domain" or "ECD" refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains Ordinarily, a PRO polypeptide
ECD will have less than 1 % of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0 5% of such domains It will be understood that any transmembrane domains identified for the
PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain The exact boundaries ot a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides. with or without the associated signal peptide. and nucleic acid encoding them, are contemplated by the present invention For example, a PR0846 polypeptide ECD can comprise amino acid residues 1 or about 18 to 247 of Figure 7 (SEQ ID NO 13), while a PR0846 polypeptide ECD may optionally comprise amino acids 1 or about 18 to X of Figure 7
(SEQ ID NO 13), wherein X is any one of amino acid residues 243 to 252 of Figure 7 (SEQ ID NO 13)
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 tor identifying that type of amino acid sequence element (e g , Nielsen et al , Piot Eng JO 1 -6 ( 1997) and von Heinje et al , Nucl Acids Res J4 4683-4690 ( 1986)) Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secieted polypeptide is not entnely uniform, resulting in more than one secreted species These mature polypeptides, where the signal peptide is cleav ed within no more than about 5 amino acids on either side of the C-terminal boundary ot the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention The term "PRO v ariant" can mean an activ e PRO polypeptide as defined below hav ing at least about 80% ammo acid sequence identity w ith the PRO hav ing the deduced amino acid sequence identity shown in the Figures heiein toi the full-length nativ e-sequence PRO polypeptides Such PRO poly peptide variants include foi instance PRO poly peptides w herein one or more ammo acid residues are added deleted, or substituted at the N- or C-terminus or w ithin the sequence Oidinaπly . a PRO polypeptide variant will have at least about 80% amino acid sequence identity , more preferably at least about 90% am o acid sequence identity , and even more preferablv at least about 95% ammo acid sequence identity with a full-length native sequence PRO polypeptide identified herein Foi example, a biologically active
Alternatively. "PRO200 (VEGF-E) PRO540. PR0846 PR0617. PR0538 (GFRcβ). PR03664 (GFRα3) oi PRO770 (hFIZZ- 1 ) variants can be functional fiag ents or analogs of native-sequence
PRO200 (VEGF-E). PRO540, PR0846. PR0617 PR0538 (GFRcβ), PR03664 (GFRcβ) or PRO770
(hFIZZ- 1 ) hav ing qualitative biological activity in common with the full-length PRO200 (VEGF-E).
PRO540. PR0846. PR0617. PR0538 (GFRcβ). PR03664 (GFRoβ) or PRO770 (hFIZZ-1 ) polypeptide. including variants from other species, but excludes a native-sequence PRO200 (VEGF-E) PRO540 PR0846. PR0617. PR0538 (GFRcβ). PR03664 (GFRcβ) or PRO770 (hFIZZ-1 )polypeptιde Yet another alternative for a PRO200 (VEGF-E). PRO540. PR0846, PR0617 PR0538 (GFRcβ), PR03664 (GFRoβ) or PRO770 (hFIZZ- 1 ) variant is an isolated nucleic acid which hybridizes under moderate, and optionally under high stringency conditions, and remains stably bound thereto, the compliment of the coding DNA tor a. (1 ) PRO200 polypeptide having amino acid residues 1 to 345 of SEQ ID NO 2. (2) PRO540 polypeptide having amino acid residues 1 to 412 (preferably 29 to 412) of SEQ ID NO 3. (3) PR0846 polypeptide having amino acid residues 1 to 332 of SEQ ID NO 13. (4) PR0617 polypeptide having amino acid residues 1 to 67 of SEQ ID NO 19, (5) PR0538 (GFRα3) polypeptide having amino acid residues 1 to 400 of SEQ ID NO 26, (6) PR03664 polypeptide having amino acid residues 1 to 369 of SEQ ID NO 27, (7)
PRO770 (hFIZZ- 1 ) polypeptide having amino acid residues 1 to 1 1 1 of SEQ ID NO 33, respectively As used herein. "PRO200. PRO540, PR0846. PR0617. PR0538 (GFRcβ), PR03664 (GFRcβ) or PRO770
(hFIZZ- 1 ) variant' expressly excludes "nativ e-sequence" PRO200. PRO540. PR0846, PR0617. PR0538
(GFRoβ), PR03664 (GFRα3) oi PRO770 (hFIZZ- 1 )" polypeptides
Alternatively still, "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 extracellulai domain of a PRO polypeptide, with or without the signal peptide. as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Such PRO polypeptide variants include, for instance. PRO polypeptides wheiein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native ammo acid sequence Ordinarily, a PRO polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81 % ammo acid sequence identity, alternatively at least about 82% ammo acid sequence identity altei natively at least about 83% amino acid sequence identity , alternatively at least about 84% amino acid sequence identity , altei natively at least about 85% amino acid sequence identity , alternatively at least about 86% amino acid sequence identity, alternativ ely at least about 87% ammo acid sequence identity, alternatively al least about 88% amino acid sequence identity , alternatively at least about 89% amino acid sequence identity , alternativ ely at least about 90% ammo acid sequence identity , alternativ ely at least about 91 % amino acid sequence identity , alternatively at least about 92% amino acid sequence identity . alternatively at least about 93% amino acid sequence identity , alternativ ely at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity alternatively at least about
96% am o acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide. with or without the signal peptide. as disclosed herein or any other specifically defined fragment ot a full-length PRO polypeptide sequence as disclosed herein Ordinal Uy. PRO variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 ammo acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 250 amino acids in length, alternatively at least about 300 amino acids in length, alternatively at least about 350 amino acids in length, alternatively at least about 400 amino acids in length, alternatively at least about 450 amino acids in length or more
"Percent (%) amino acid sequence identity" with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the ammo acid residues in the specific PRO polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in various way s that are within the skill in the art, tor instance, using publicly available computei software such as BLAST. BLAST-2. ALIGN or Megalign (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below The ALIGN-2 sequence comparison computer program was authored by Genentech. Inc and the source code shown in Table 1 below 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 av ailable through Genentech.
Inc . South San Francisco, California or may be compiled from the source code provided in Table 1 below The ALIGN-2 piogram 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 progiam and do not vary In situations w heie ALIGN 2 is emploved for amino acid sequence comparisons the % amino acid sequence identitv ot a giv en amino acid sequence A to w ith oi against a giv en amino acid sequence B < hich can alternativelv be phiased as a given amino acid sequence A that has oi comprises a certain % amino acid sequence identitv to with or against a giv en amino acid sequence B ) is calculated as follows
100 times the fraction XΛ
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN 2 in that program s alignment of A and B and w here Y is the total number ol am o acid residues in B It will be appreciated that where the length ot amino acid sequence A is not equal to the length of am o acid sequence B the % amino acid sequence identitv of A to B will not equal the % amino acid sequence identity ot B to A As examples of % amino acid sequence identity calculations using this method Tables 2 and 3 demonstrate how to calculate the % amino acid sequence identitv of the amino acid sequence designated Comparison Protein' to the am o acid sequence designated "PRO 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 and "X Y and ' Z each represent different hypothetical amino acid residues
Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN 2 computer program However, % amino acid sequence identity values may also be obtained as described below by using the
WU BLAST 2 computer program (Altschul et al Methods in
Figure imgf000019_0001
266 460 480 ( 1996)) Most of the WU-BLAST 2 search parameters are set to the default values Those not set to default values, I e , the adjustable parameters are set with the following values overlap span = 1 overlap fraction = 0 125, word threshold (T) = 1 1 , and scoring matrix = BLOSUM62 When WU BLAST 2 is employed a % amino acid sequence identity v alue is determined by dividing (a) the number ot matching identical amino acid 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 (l e the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU BLAST 2 by (b) the total number of amino acid residues of the PRO polypeptide ot interest For example in the statement ' a polypeptide comprising an the amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B the amino acid sequence A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest
Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI BLAST2 (Altschul et al Nucleic Acids R s 25 3389 3402 ( 1997)) The NCBI BLAST2 sequence companson program may be downloaded fiom http //www ncbi nlm mil gov oi otherwise obtained fiom the National Institute of Health Bethesda MD NCBI BLAST2 uses several search parametei s wherein all of those seaich parameters are set to default values including for example unmask = yes strand = all expected occurrences = 10 minimum low complexity length •= 1 /5 multi pass e value = 001. constant tor multi-pass = 25. diopotf for final gapped alignment = 25 and scoring matrix = BLOSUM62
In situations w here NCBI-BLAST2 is emploved tor am o acid sequence comparisons, the % amino acid sequence identity of a giv en amino acid sequence A to with, or against a given amino acid sequence B (which can alternatively be phrased as a giv en amino acid sequence A that has or compnses a certain % amino acid sequence identity to with, or against a given ammo acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program 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 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
Polypeptide variants may come in different foims "Substitutional ' variants are those that have at least one amino acid residue in a native sequence removed and a different amino acid inserted in its place at the same position The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more am o acids have been substituted in the same molecule "Insertional" variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native sequence Immediately adjacent to an amino acid means connected to either the α-carboxyl or α-amino functional group of the amino acid "Deletional" variants are those with one or more amino acids in the native amino acid sequence removed Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the molecule Polypeptide variants also include covalent modifications to residues in addition to epitope-tagged heterogeneous PRO
"PRO variant polynucleotide" or "PRO variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide. as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Ordinarily , a PRO variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity. alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity alternatively at least about 93% nucleic acid sequence identity , alternatively at least about 94% nucleic acid sequence identity, alternativelv at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identitv, . alternatively at least about 97% nucleic acid sequence identity alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity with a nucleic acid sequence encoding a full- length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein an extracellular domain of a PRO polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Variants do not encompass the native nucleotide sequence Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in length alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 500 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 700 nucleotides in length, alternatively at least about 800 nucleotides in length, alternatively at least about 900 nucleotides in length, alternatively at least about 1000 nucleotides in length, alternatively at least about 1 100 nucleotides in length, alternatively at least about 1200 nucleotides in length, or more "Percent (%) nucleic acid sequence identity" with respect to PRO-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the PRO nucleic acid sequence of interest, 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 or
Megalign (DNASTAR) software For purposes herein, however, % nucleic acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc and the source code shown in Table 1 below 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 below 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
In situations where ALIGN-2 is employed for nucleic acid sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to. with, or against a given nucleic acid sequence D) is calculated as follows 100 times the ti action W/Z
where W is the number ot nucleotides scoied as identical matches by the sequence alignment program ALIGN-2 in that piogram's alignment of C and D and wheie Z is the total number ot nucleotides in D It will be appreciated that where the length of nucleic acid sequence C is not equal to the length ot 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 ot % nucleic acid sequence identity calculations. Tables 4 and 5. demonstrate how to calculate the % nucleic acid sequence identity ot the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO-DNA". wherein "PRO-DNA' represents a hypothetical PRO-encoding nucleic acid sequence of interest. "Comparison DNA" represents the nucleotide sequence ot a nucleic acid molecule against which the "PRO-DNA" nucleic acid molecule ot interest is being compared, and "N", "L' and "V" each represent different hypothetical nucleotides
Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program
However, % nucleic acid sequence identity values may also be obtained as described below by using the
WU-BLAST-2 computer program (Altschul et al , Methods in Enzvmologs 266 460-480 ( 1996)) Most of the WU-BLAST-2 search parameters are set to the default values Those not set to default values, I e., the adjustable parameters, are set with the following values overlap span = 1 , overlap fraction = 0 125, word threshold (T) = 1 1 , and scoring matrix = BLOSUM62 When WU-BLAST-2 is employed, a % nucleic acid sequence identity value is determined by dividing (a) the number ot 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, in the statement "an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B". the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence ot the PRO polypeptide-encoding nucleic acid molecule ot interest
Percent nucleic acid sequence identity may also be determined using the sequence comparison program NCB1-BLAST2 (Altschul et al , Nucleic Acids Res 25 3389-3402 (1997)) The NCBI-BLAST2 sequence comparison program may be downloaded from "http //www ncbi nlm nih gov" or otherwise obtained tiom the National Institute of Health, Bethesda. MD NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, tor 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 w here NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a giv en nucleic acid sequence C to. w ith, oi against a giv en nucleic acid sequence D (which can alternativ ely be phrased as a given nucleic acid sequence C that has oi compnses a ceitam % nucleic acid sequence identity to. w ith, or against a given nucleic acid sequence D) is calculated as folk lows
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment piogram NCBI-BLAST2 in that progiam'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 ot D to C
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 a full-length PRO polypeptide as disclosed herein PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide
For example, it will be appreciated that particular fragments or subregions of two sequences may have a greater or lesser degree of homology than a comparison between the entire fragments themselves The identity values used herein can be generated by the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively
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 example, it will be appreciated that particular fragments or subregions of two sequences may have a greater or lesser degree of homology than a comparison between the entire fragments themselves The term "positives", in the context ot sequence comparison performed as descπbed above, includes residues in the sequences compared that are not identical but have similar properties (e.g as a result of conservative substitutions, see Table 6 below) For purposes herein, the % value of positives is determined by dividing (a) the number of amino acid residues scoring a positive value between the PRO polypeptide amino acid sequence of interest having a sequence derived from the native PRO polypeptide sequence and the comparison amino acid sequence of interest (/ e , the amino acid sequence against which the PRO polypeptide sequence is being compared) as determined in the BLOSUM62 matrix of WU- BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest
Unless specifically stated otherwise, the % value of positives is calculated as described in the immediately preceding paragraph However, in the context of the amino acid sequence identity comparisons performed as described tor ALIGN-2 and NCBI-BLAST-2 above, includes amino acid residues in the sequences compared that are not only identical, but also those that have similar properties Am o 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 prefened substitution (as defined in Table 6 below ) of the amino acid residue of interest For amino acid sequence comparisons using ALIGN-2 or NCBI-BLAST2. the % value of positives of a given amino acid sequence A to. with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % positives to. with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scoring a positive value as defined above by the sequence alignment program ALIGN-2 or 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 % positives of A to B will not equal the % positives of B to A.
"Isolated," when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide. and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified ( 1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
An "isolated" PRO polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid 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 polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example. DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a πbosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation Generallv opeiably linked' means that the DNA sequences being linked are contiguous and in the case ot a secretory leadei contiguous and in reading phase Howev ei enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites It such sites do not exist the synthetic oligonucleotide adaptors or linkers aie used in accordance w ith conventional practice
The term ' antibody ' is used in the broadest sense and specifically covers, tor example single anti PRO monoclonal antibodies (including agonist antagonist and neutiahzing antibodies). anti-PRO antibodv compositions with polyepitopic specificity , single chain anti-PRO antibodies, and fragments of anti-PRO antibodies (see below) The term ' monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, l e the indiv idual antibodies comprising the population are identical except for possible naturally-occurung mutations that may be present in minor amounts
' Stringency " of hybridization reactions is readily deteπninable 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 tempeiatures Hybridization generally depends upon the ability of denatured DNA to reanneal when complementary strands are present in an environment near but below their T'" (melting temperature) The higher the degree ol desired homology between the probe and hybπdizable sequence, the higher the relative temperature which can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so Moreover, stringency is also inversely proportional to salt concentrations For additional details and explanation of stringency of hybridization reactions, see Ausubel et al , Protocols in Moleculai BiologΛ (1995)
"Stringent conditions" or "high stringency conditions" are exemplified by reaction conditions characterized by ( 1 ) employing low ionic strength and high temperature for washing, foi example 0 015 M sodium chloride/00015 M sodium cιtrate/0 1 % sodium dodecyl sulfate at 50°C. (2) employing during hybridization a denaturing agent, such as formamide. tor example. 50% (vol/vol) formamide with 0 1 % bovine serum albumιn/0 1 % Fιcoll/0 1 % polyvιnylpyrrolιdone/50 mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride. 75 mM sodium citrate at 42°C, oi (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 0 1 % SDS Yet another example of high stringency conditions is hybridization using a buffer of 10% dextran sulphate. 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 The skilled artisan w ill recognize how to adjust the temperatuie, ionic strength, etc as necessary to accommodate factors such as piobe length and the like
Other conditions previously described and well known can be used to arrive at high, low or moderate stringencies
"Moderately stringent conditions" may be identified as descnbed by Sambrook et al Moleculai Cloning A Lahoiaton Manual. New York Cold Spnng Harbor Press 1989. and include the use of washing solution and hybridization conditions (e.g.. temperature, ionic strength and %SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37CC in a solution comprising: 20% formamide. 5 x SSC ( 150 mM NaCl. 15 mM trisodium citrate). 50 mM sodium phosphate (pH 7.6). 5 x Denhardt's solution. 10% dextran sulfate. and 20 μg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50:C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
The term "epitope tagged" when used herein refers to a 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 amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues). As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1 , IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE. IgD or IgM.
"Active" or "activity" for the purposes herein refers to form(s) of a PRO polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring PRO, wherein "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally- occurring PRO other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO and an '"immunological" activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally- occurring PRO. Preferably, biological activity of a PRO means delaying, preventing or rescuing retinal neurons, e.g., photoreceptor cells from injury, degradation or death.
"Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the objective is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
"Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature. "Delaying, prev enting or rescuing retinal cells fiom injury or death" as a result ot the method ot the invention refers to the abilitv to keep such retinal cells viable or aliv e tor a period of time greater than is observed without application ot said method Retinal cell death can iesult from injury , disease or even aging Retinal cell injury can also result in degradated cells 01 those hav ing a limited capacity tor normal physiological operation The effect can be measured either in \ ιtιo w ith isolated retinal cells or ;//
Figure imgf000027_0001
o w ith subjects having compromised retinal cells due to injury or disease
"Mammal" tor purposes ot treatment refers to anv animal classified as a mammal, including humans, domestic and from animals, and zoo. sports, or pet animals, such as dogs, horses, cats, sheep, pigs. cattle, etc Preferably, the mammal is human A "disorder" is any condition that would benefit from tieatment w ith PRO polypeptides This includes both chronic and acute disorders, as well as those pathological conditions which predispose the mammal to the disorder in question Non-limiting examples of disorders to be treated herein include any condition which results in injury or death of photoreceptor or other retinal cells Examples of conditions include retinal detachment, age-related and other maculopathies, photic retinopathies. surgery-induced retinopathies (either mechanically or light-induced), toxic retinopathies including those resulting from foreign bodies in the eye. diabetic retinopathies. retinopathy of prematurity, viral retinopathies such as CMV or HIV retinopathy related to AIDS, uveitis, ischemic retinopathies due to venous or arterial occlusion or other vascular disorder, retinopathies due to trauma or penetrating lesions of the eye, peripheral vitreoretinopathy, and inherited retinal degenerations Exemplaiy retinal degenerations include e g . hereditary spastic paraplegia with retinal degeneration (Kjellin and Barnard-Scholz syndromes), retinitis pigmentosa, Stargardt disease, Usher syndrome (retinitis pigmentosa with congenital hearing loss), and
Refsum syndrome (retinitis pigmentosa, hereditary hearing loss, and polyneuropathy ) Additional disorders which result in death of retinal neurons include, retinal tears, detachment of the retina and pigment epithelium, degenerative myopia, acute retinal necrosis syndiome (ARN). traumatic chorioretinopathies or contusion (Purtscher's Retinopathy) and edema
"A therapeutically effective amount" is an amount of active PRO which is lequired to achieve measurable delay, rescue or prevention ot damage to retinal neurons
Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order "Carriers" as used herein include pharmaceutically acceptable carriers, excipients. or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed Often the physiologically acceptable carrier is an aqueous pH buffered solution Examples of physiologically acceptable carriers include butters such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptide. proteins, such as serum albumin gelatin or immunoglobulins, hydrophihc polymers such as polyvinylpyrrolidone. amino acids such as glycine, glutamine. asparagine. argimne or lysine. monosacchaπdes, disacchaπdes, and other carbohydrates including glucose, mannose. or dextπns, chelatmg agents such as EDTA, sugar alcohols such as mannitol or sorbitol. salt-forming counteπons such as sodium, and/or no onic surfactants such as TWEEN 1 . polyethylene glycol (PEG), and PLURONICS1 M ' Antibody fragments' comprise a portion ot an intact antibody , preterablv the antigen binding oi v ariable region ot the intact antibody Examples ot antibody fragments include Fab Fab F(ab')2. and Fv fragments, diabodies. lineal antibodies (Zapata et al Piotem Em; 8( 10) 1 57- 1062 [ 1995] ) single-chain antibody molecules, and multispecific antibodies formed from antibody fragments Papain digestion of antibodies produces two identical antigen-binding fiagments. called "Fab" fragments, each with a single antigen-binding site, and a residual ' Fc" fragment, a designation reflecting the ability to crystallize readily Pepsin tieatment yields an F(ab'); fragment that has two antigen-combining sites and is still capable of cioss- nking antigen
"Fv " is the minimum antibody fragment which contains a complete antigen-recognition and - binding site This region consists of a dimer ot 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 Vn-V dimer Collectivelv the six CDRs confer antigen- binding specificity to the antibody However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH I ) of the heavy chain Fab fragments diffei from Fab' fragments by the addition of a tew residues at the carboxy terminus of the heavy chain CH I domain including one or more cysteines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine resιdue(s) of the constant domains bear a free thiol group F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD. IgE. IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g , IgG l , IgG2, IgG3, IgG4. IgA. and IgA2
"Single-chain Fv" oi "sFv" antibody fragments comprise the V** and V, domains of antibody, wherein these domains are present in a single polypeptide chain Preferably, the Fv polypeptide further comprises a polypeptide linker between the V and V* domains which enables the sFv to form the desired structure for antigen binding For a review ot sFv, see Pluckthun in The Phaimacologs of Monoclonal
Antibodies, vol 1 13, Rosenburg and Moore eds . Spπngei -Verlag. New York, pp 269-315 ( 1994)
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (V ) in the same polypeptide chain (V -VL) By using a linker that is too short to allow painng 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 tullv in. foi example, EP 404.097, WO 93/1 1 161 , and Holhnger et al , Pioc Natl Acad Sci USA. 90 6444-6448 ( 1993) An "isolated antibody is one which has been identified and sepaiated and/or recovered from a component ot its natural env nonment Contaminant components ot its natural env ironment are materials which would mterfeie w ith diagnostic or therajDeutic uses for the antibody , and may include enzymes hormones, and other proteinaceous or nonproteinaceous solutes In preferred embodiments, the antibody will be purified ( 1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably moie than 99% by weight. (2) to a degree sufficient to obtain at least 15 residues of N- terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under ieducing or nonreducing conditions using Coomassie blue or. preferably, silver stain Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present Ordinarily, however, isolated antibody will be prepared by at least one purification step
The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody The label may be detectable by itself (e g radioisotope labels or fluorescent labels) or. in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable
By ''solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysacchaπdes (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and sihcones In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275,149
A "liposome" is a small vesicle composed of various types of hpids, phospholipids and/or surfactant which is useful tor delivery of a drug (such as a PRO polypeptide or antibody thereto) to a mammal The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement ot biological membranes
A "small molecule" is defined heiein to have a molecular weight below about 500 Daltons
Table 1
* C-C increased from 12 to 15
* Z is average of EQ
* B is average of ND
* match with stop is _M; stop-stop = 0; J (joker) match = 0 */
#define _M -8 /*■* value of a match with a stop */ int day[26][26] = {
/* A~ B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/
/* A */ 2, 0,-2.0, 0,-4, 1 -1,-1, 0,-1,-2,-1, 0,_M.1.0,-2, 1, 1, 0, 0,-6.0.-3, 0}, /*B */ 0, 3,-4, 3, 2,-5, 0. 1,-2.0, 0,-3,-2, 2,_M,-1, 1, 0, 0, 0, 0,-2,-5.0.-3, 1}, /*C */ ■2,-4,15,-5,-5,-4,-3 -3,-2, 0,-5,-6,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8.0.0,-5}, /* D ■**/ 0, 3,-5.4, 3,-6, 1. 1,-2, 0, 0.-4,-3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0.-4.2}, /* E */ 0, 2,-5, 3, 4,-5.0, 1,-2, 0, 0,-3,-2, 1,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0.-4.3}, /*F*/ -4,-5,-4.-6,-5, 9,-5, ■2, 1, 0,-5, 2, 0,-4._M,-5,-5,-4,-3,-3, 0,-1, 0.0, 7,-5}, /*G */ 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,-2,-2,-2,-2, 1,-3 ,-2, 5, 0,-2, 2.2,-2,_M,-2,-2,-2,-l, 0, 0, 4,-5.0.-1,-2},
/* J */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0.0}, /*K */ -1,0,-5,0,0,-5,-2, 0,-2, 0, 5,-3.0, 1,_M,-1, 1, 3, 0, 0, 0,-2,-3, 0.-4, 0}, /*L*/ -2,-3,-6.-4,-3, 2,-4, 2, 2, 0,-3.6, 4,-3._ ,-3,-2,-3,-3,-l, 0, 2,-2.0.-1,-2}, /*M */ -1,-2,-5,-3,-2,0,-3, -2, 2, 0, 0, 4, 6,-2,_M,-2,-l, 0,-2,-1, 0, 2,-4, 0.-2,-1}, /*N*/ 0,2,-4,2, 1,-4,0.2,-2, 0, 1,-3,-2, 2,_M,-1, 1, 0, 1, 0, 0,-2,-4, 0.-2, 1},
/*o*/ M, M, M, M, M M ,_M ,_M ,_M ,_M ,_M ,_M ,_M ,_M , 0,_M ,_M ,_M ._M ,_M ,_M ,_M ,_M ,_M , Λ ,_M } ,
/* p */ 1, -1, .-3, -1. ,-L ,-5, ,-1 , o ,-2. , 0, ,-1 ,-3 ,-2 ,-1 . M , 6 ,0,0, 1,0,0,-1,-6,0,-5,0},
/*Q*/ 0, 1, -5, 2, 2, -5, -1, 3, -2, 0, 1, -2, -1, 1, M, 0, 4, 1,-1,-1,0,-2,-5, 0,-4, 3}, /*R*/ -2, 0, -4, -1, ,-1, ,-4, ,-3. , 2. ,-2, , 0, ,3. ,-3. , 0, 0, M, 0, 1, 6,0,-1,0,-2,2,0,-4,0}, /*S*/ 1, 0, 0, 0, 0, -3, 1, -1, -1, 0, 0, -3, -2, 1, M, 1, 1,0,2, 1,0,-1,-2, 0.-3,0}, 1*1*1 1, 0, -2, 0, 0, -3, o, -1, 0, 0, 0, -1, -1, 0, M, 0, 1,-1, 1,3,0,0,-5,0.-3,0}, ι*υ*ι 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/* v */ 0, -2, -2, -2, -2, -1, -L ,-2. ,4, , 0, rl. ,2, ,2, -2, M, ■-1, ,-2,-2,-1,0,0,4,-6,0.-2,-2},
/* w*/ -6, -5, -8, -7, ,-1: ,0, -1. ,-3, ,-5. , 0, ,-3. ,-2. ,-4. ,-4. , M ,-6 5,2,-2,-5,0,-6,17,0, 0,-6}, /*x */ 0, 0, 0, 0, 0, 0, o, 0, 0, 0, 0, 0, 0, 0, M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/* Y */ -3, -3, 0, -4, -4, 7, -5, 0, -1, 0, -4, -i, -2, -2, M, ,-5, ,-4,-4,-3,-3,0,-2,0.0,10,-4},
/*z */ 0, 1, -5, 2, 3, -5, 0, 2, -2, 0, 0, -2, -1, 1, M, 0, 3, 0, 0, 0, 0,-2,-6, 0,-4, 4} };
Page 1 of day. h Table 1 (conf)
/*
*/
-^include < stdιo.h >
-^include <ctype h>
^define MAXJMP 16 /* max jumps in a diag */
#define MAXGAP 24 /* don't continue to penalize gaps larger than this */
#define JMPS 1024 /* max jmps in an path */
^define MX 4 /•*■ save it 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 DINS0 8 /* penalty tor a gap */
-^define DINS1 1 /* penalty per base */
•^define PINS0 8 /* penalty for a gap */
#defϊne PINS1 4 /* penalty per residue */ struct jmp { short nfMAXJMP], /* size of jmp (neg for dely) */ unsigned short x[MAXJMP], /* base no. of jmp in seq x */
}; /* limits seq to 2"16 -1 */ struct diag { int score, /* score at last jmp */ long offset, /* offset of prev block */ short ijmp, /* current jmp index */ struct jmp JP, /* list ot jmps */
}; struct path { int spc; /* number of leading spaces */ short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (last elem before gap) */
}; char *ofιle; /* output file name */ char "■namex[2], /* seq names. getseqs() */ char *prog, /* prog name for err msgs */ char *seqx[2], /* seqs getseqs() */ int dmax, /* best diag: nw() */ int dmaxO, /* final diag */ int dna; /* set if dna maιn() */ int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* total gaps in seqs */ int lenO, lenl ; /*•* seq lens */ int ngapx, ngapy; /* total size of gaps */ int smax; /* max score: nw() */ int *xbm; /* bitmap for matching */ long offset; /* current offset in jmp file */ struct diag *dx; /* holds diagonals */ struct path PPPJ; /* holds path for seqs */ char *calloc(), *malloc(), *ιndex(), •■strcpyO, char *getseq(), *g_calloc(),
Page 1 of nw h Table 1 (cont')
/* Needieman-Wunsch alignment program
* usage progs fϊlel fιle2 * 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 bv unsigned short x in the jmp struct)
* A sequence with 1/3 or more ot its elements ACGTU is assumed to be DNA * Output is in the file ' align out'
*
* The program may create a tmp file in /tmp to hold info about traceback
* Original version developed under BSD 43 on a v ax 8650 */ #ιnclude "nw h"
static _dbvalI26] = {
1,14,2,13,0,0,4,11,00,12,0,3,15,0,0,0,5,6.: ,8,7,9,0,10,0
static _pbvalf26] = {
1, 2|(1< <('D A'))|(l < <('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< <(Ε'-Α'))|(1< <('Q'-Α'))
maιn(ac, av) main int ac, char *av[], prog = av[0], if(ac '= 3){ fpπntf(stderr, "usage %s filel fιle2\n", prog), fpπntf(stderr, "where filel and file2 are two dna or two protein sequences \n"), fpπntf(stderr, "The sequences can be in upper- or lower case\n"), fpπntf(stderr,"Any lines beginning with ',' or ' < ' are ιgnored\n"), fpπntf(stderr, "Output is in the file V'align out\"\n"), exιt(l),
} namex[0] = av|l], namex[l] = av[2], seqxfO] = getseq(namex[0J, &len0), seqx[l] = getseq(namex[l], &lenl), xb = (dna)'' dbval pbval, endgaps = 0, /* 1 to penalize endgaps */ ofile = "align out' /* output file */ nw(), /* fill in the matrix, get the possible jmps */ readjmps(); /* get the actual jmps */ pπntO, /* print stats, alignment */ cleanup(O), /* unlink any tmp files */
Page 1 ol nw c Table 1 (cont')
I* 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
Figure imgf000033_0001
nw() nw
{ char •*px, *p ; /* seqs and ptrs */ int •••ndely . *del> ; /* keep track of dely */ int ndelx, delx; /* keep track of delx */ int *tmp; /* for swapping rowO, ro l int mis; /* score for each type */ int insO, insi ; I* insertion penalties */ register id, /* diagonal index */ register ij ; /* jmp index */ register *col0, *coll , /*•• score tor 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 *i-)g_calloc("to get colO" , lenl + 1 , sizeof(int)); col l = (int *)g_calloc("to get coll " , lenl + 1 , sizeof(int)); insO = (dna)? DINS0 : PINS0, insi = (dnaϊ.' DINSl : PINS1 ; smax = -10000; if (endgaps) { for (colOfO] = dely[0J = -insO, yy = 1 ; yy < = lenl ; yy + +) { col0[yy] = dely[yy] = col0[yy-l] - msl ; ndely [yy] = yy;
} co!0[0] = 0; /* Waterman Bull Math Biol 84 */
} else for (yy = 1 ; yy < = lenl ; yy -t- +) delylyy] = -insO,
/* fill in match matrix */ for (px = seqxfO]. , xx = 1 ; xx < = lenO; px + + , xx+ +) { /* initial lze first entry in col
/ if (endgaps) { if (xx = = 1) coll [0] = delx = -(ιns0 + ιnsl); else coll fO] = delx = colO[0] - msl , ndelx = xx;
} else { coll [0] = 0; delx = -insO; ndelx = 0;
Pace 2 of nw.c Table 1 (conf)
...nw for (pv = seqx[l].yy = 1; vy < = lenl; py+ + , yy+ +) {
Figure imgf000034_0001
mis += (xbm[*px-'A']&xbml*py-'A'])> DMAT : DMIS; else mis + = _day|*px-'A'][*py-'A'];
10 /* update penalty for del in x seq;
* favor new del over ongong del
•>* ignore MAXGAP if weighting endgaps */" if (endgaps | | ndely[yy] < MAXGAP) { 15 if (colOlyy] - insO > = dely[yy]) { delylyy] = col0[yy] - (msO + insl); ndely[yy] = 1; } else { dely[yy] -= insi; 20 ndely[yy]++;
} } else { if (colOlyy] - (msO+insl) > = delylyy]) { delylyy] = colOlyy] - (msO + insl); 25 ndely lyy] = 1;
} else ndelylyy] + + ; }
30 /* update penalty for del in y seq;
* favor new del over ongong del */ if (endgaps | | ndelx < MAXGAP) { if (coll[yy-l] - insO > = delx) { 35 delx = coll[yy-l] - (insO + insl); ndelx = 1; } else { delx -= msl; ndelx + +; 40 }
} else { if (coll[yy-lj -(insO + insl) >= delx) { delx = coll[yy-l] - (insO + insl); ndelx = 1; 45 } else ndelx + + ;
}
/*•* pick the maximum score; we're favoring 50 * mis over any del and delx over dely
*/
55
60
Page 3 of nw.c Table 1 (conf)
Figure imgf000035_0001
if (mis > = delx && mis > = delylyy]) colllyy] = mis else if (delx >= del lyy]) { coll[yy] = delx. ij = dx[ιd] lj p, if (dx[ιd].jp n[0] && Cdna | | (ndelx > = MAXJMP && xx > dx[ιd] jp x[ιj] + MX) | | mis > dx[ιd].score + DINSO)) { dx[ιd] ιjmp+ + , if ( + +ιj >= MAXJMP) { wπtejmps(ιd), IJ = dxlιd].i)mp = 0, dx[ιd]. offset = offset, offset += sizeof(struct jmp) - sizeof (offset), } } dx[ιdj.jp.nlιj] = ndelx, dx[ιd] jp x[ιj] = xx, dxjid] score = delx,
} else { colllyy] = delylyy], il = dx[ιd].ιjmp, if (dx[id].jp.nl0] && (Idna | | (ndelylyy] > = MAXJMP
&& xx > dx[ιd] jp.x[ιj] + MX) | | mis > dx[ιd].score + DINS0)) {
Figure imgf000035_0002
if (++ιj >= MAXJMP) { wπtejmps(ιd), ij = dx[ιd].ιjmp = 0; dx[ιdj. offset = offset; offset += sizeof(struct jmp) + sizeof (offset);
dx[ιd].jp.n[ιj] = -ndelylyy]; dxfιd].jp.x[ιj] = xx; dxjid]. score = delylyy]; if (xx = -- = len0&&yy < lenl) { /* last col */ if (endgaps) colllyy] -= ιns0 + ιnsl*(lenl -yy) if (coll[yy] > smax) { smax = coll[yy]; dmax = id, }
} if (endgaps && xx < lenO) coil |yy-l] -= ins0 + insl*(len0-xx), if (colllyy- 1] > : smax) { smax = col 1 Lyy-1 j; dmax = : id,
} t p = i -olO; colO = coll; coll = tmp,
}
(void) free((char *)ndely);
(void) free((char *)dely),
(void) free((char *)col0),
(void) tree((char *)coll).
Page 4 of nw c Table 1 (conf)
/*
*
* pπntO - only routine visible outside this module *
* static:
* getmatO — trace back best path, count matches: pπnt()
*** pr_ahgn() — print alignment of described in array p[] : pπnt()
* dumpbiockO — dump a block of lines with numbers, stars: pr_ahgn() * nums() - put out a number line. dumpbiockO
* puthneO - put out a line (name, [num], seq. [num])* dumpbiockO
* stars() - -put a line ot stars. dumpbiockO
* stπpnameO -- strip any path and prefix from a seqname */
^include "nw.h"
^define SPC 3
#defιne P LINE 256 /* maximum output line */ #define P SPC 3 /* space between name or nu and seq */ extern _day[26][26]; int olen; /* set output line length */
FILE *fx; /* output file */ pπntO print
{ int lx, ly, firstgap, lastgap; /* overlap */ if ((fx = fopen(ofile, "w")) = = 0) { fpπntf(stderr, " %s: can't write %s\n", prog, ofile); cleanup(l);
} fprintf(fx, " < first sequence: %s (length = %d)\n", namex[0], lenO); fprιntf(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[01.spc;
} else if (dmax > lenl - 1) { I* leading gap in y */ pp[l].spc = firstgap = dmax - (lenl - 1); lx -= ppf lj.spc;
} if (dmaxO < lenO - 1) { I* trailing gap in x */ lastgap = lenO - dmaxO -1 ; lx - = lastgap;
} else if (dmaxO > lenO - 1 ) { /* trailing gap in y */ lastgap = dmaxO - (lenO - 1); ly -= lastgap; } getmat(lx, ly, firstgap, lastgap); pr_alιgn(); }
Page 1 ol nwpπnt.c Table 1 (conf)
/*
* trace back the best path, count matches
*/ static getmatOx. ly, firstgap, lastgap) getmat int lx, ly; I* "core" (minus endgaps) "I int firstgap, lastgap. I* leading trailing overlap */ int nm. lO, il, sizO, sizl, char outx[32]; double pet; register nO, nl; register char *p0, *pl;
/* get total matches, score
Figure imgf000037_0001
pO = seqx[0] + ppllj.spc, pi = seqx[l| + pp]0].spc; nO = pp|l].spc + 1; nl = ppfO].spc + 1; nm = 0; while (*p0&& *pl ){
Figure imgf000037_0002
sizl- } else { if (xbm[*pO-'A']&xbm[*pl-'A']) nm+ + ; if (n0++ == pp[0].x[ιO])
Figure imgf000037_0003
}
/* pet homology:
* it penalizing endgaps, base is the shorter seq * else, knock otf overhangs and take shorter core
*/ if (endgaps) lx = (lenO < lenlV'lenO : lenl; else lx = (lx < ly)? lx : ly; pet = 100.***(double)nm/(double)lx; fpπntf(tx, "\n"); tpπntf(tx, " < 7ιά match' s in an overlap of %d: %.2t percent sιmιlaπty\n' , n , (nm = = 1)'' "" * "es", lx, pet);
Page 2 ot'nwprint.c Table 1 (cont1) tpπnttdx. < gaps first sequence id , gapx), .getmat if (gapx) {
(void) sprintftoutx. " (7<d ts9£s)", ngapx, (dna).' "base": "residue", (ngapx ιy>""."s"), fpnntf(fx,"9(s", outx), fpπntf(f\. ", gaps in second sequence* ?d", gapy), if (gapy) {
(void) spπntft utx, " (7cd 9?s%s)", ngapy, (dna)'' "base":"resιdue", (ngapy == l)'' "":"s"); fpπntt(fx,"%s", outx);
} if (dna) tpπntf(ix,
"\n< score: %d (match = Sid, mismatch = S?d, gap penalty = STd + 7cd per base)\n" smax, DMAT, DMIS, DINS0, DINS1); else fprιntf(fx,
"\n<score: 7<d (Dayhoff PAM 250 matrix, gap penalty = 7cd + 7ιd per resιdue)\n", smax, PINS0, PINS1); if (endgaps) tpπntf(lx,
"<endgaps penalized, left endgap: %d Ss%s, right endgap: 7ιd %sS?s\n", firstgap, (dna)? "base" : "residue", (firstgap == 1)'' "" : "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 UP]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static ni[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 ]; I* set by stars() */
/*
* print alignment of described in struct path pp[] */ static pr_ahgn() pr align
{ int nn; /* char count */ int more; register I; for (l = 0, Imax = 0; i < 2; 1++) nn = stπpname(namex[ι]); if (nn > Imax) Imax = nn nc[ι] = l; nι|ι] = = 1; sizii] = ijlil = 0; psfil = seqx[ι]; p |ι| = ouφj,
Page 3 ot nwpπnt. Table 1 (conf) for (nn = nm = 0, more = 1 ; more; ) { .pr align for (i = more = 0; i < 2; i + +) { /*
* do we have more of this sequence? */ if(!*ps[i]) continue; more-f +; if (pp[i].spc) { /* leading space */ *po[i] + + = ' '; pp[i].spc-;
} else if (siz[i]) { /* in a gap */
*po[i] + + = '-'; siz[i]-;
} else { /* we're putting a seq element
*/ *po[i] = *ps[i]; if (islower(*ps[i]))
*ps[i] = toupper(*ps[i]); po[i]+ + ; ps[i] + + ;
/*
* are we at next gap for this seq?
*/ if(ni[i] ==pp[i].x[ij[i]]){ /*
* we need to merge all gaps
* at this location */ siz[i] = pp[i].n[ij[i] + +]; while (ni[i] == pp[i].x[ij[i]]) sizfi] +=pp[i].n[ij[i] + +];
} ni[i] + + ;
if (+ +nn = = olen | | Imore && nn){ dumpbiockO; for(i = 0; i < 2; i++) po[i] = out[i]; nn = 0;
}
}
}
/*
* dump a block of lines, including numbers, stars: pr_ align()
*/ static dumpbiockO dumpblock
{ register i; for (i = 0;i < 2;i++) *po[i] - = '\0';
Page 4 of nwprint.c Table 1 (cont')
...dumpblock
(void) ρutc( \n . fx) for(ι =0, i < 2 ! + +){ if (*out[ι] && (*out[ι] '= || *(po[ι]) '= ' ')){ if (. = = 0) nums(ι), if 0 == 0&& *out[l]) starsO, puthne(ι), if (l == 0&& *out[l]) fpπntf(tx, star), if(ι •= = 1) nums(ι),
}
}
* put out a number line dumpbiockO */ static nu s(ιx) nums int lx, /* index in out[] holding seq line */
{ char nhne[P_LINE], register I, j, register char *pn, *px, *py, for (pn = nhne, 1 = 0, 1 < lmax + P SPC, ι + + , pn++)
*pn = ' ', for (i = nc[ιx], py = out[ιx], *py, py++, pn++) { if(*py == ' ' || *py == '.')
*pn = ' '; else { if (ι%10 == 0 || (l == 1 &&nc[ιx] ' = !)){
J = 0 < 0)'-ι l, for (px = pn, j, j /= 10, px-)
**px = j%10 + '0', if (l < 0)
*px = '-',
} else
*pn = ' ',
1++,
}
}
*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]) dumpbiockO
*/ static putlιne(ιx) putline int IX,
Page 5 of nwpπnt c Table 1 (cont')
...putline register char *px, for (px = namex[ιx|, i = 0, *px && ***px '= . p\+ +, ι+ + )
(void) putc(*px, fx), for(, l < lma\ + P_SPC, ι++)
Figure imgf000041_0001
/* these count trom 1
* nι[] is current element (from 1)
* nc[] is number at start of current line */ for (px = out[ιx] *px. px+ +)
(void) putc(*px&0x7F, tx), (void) putc('\n'. fx),
}
/* *** put a line of stars (seqs always in out[0], out[l]) dumpbiockO
*/ static stars() stars { int l, register char *p0, *pl, ex, *px, if C*out[0] I I (*outf0] == • '&& *(po[0]) == ' ') I <*out[l] I I (*out[l] == ' '&& *(p [l]) == ' ')) return, px = star, for (l = lmax + P_SPC; I; ι~)
*px ++ = ' ', for(p0 = out[0], pi = out[l], *p0 && *pl, p0+ + , pl + +) { if (isalpha(*p0) && ιsalpha(*pl)) { if (xbm[*p0 'A']&xbm[-**pl-'A']) { ex = '*', nm+ + ,
} else if (!dna && _day[*p0-'A'][*pl-'A'] > 0) ex = ' ', else ex = ' ',
} else ex = ' ', *px+ + = CX,
}
*px+ + = '\ll , *px = '\0';
Page 6 of nwpπnt c Table 1 (cont')
/*
* strip path or prefix from pn. return len: pr_align()
*/ static stripname(pn) stripname char *pn; /* file name (may be path) */ register char *px, *py; py = 0; for (px = pn; *px; px + + ) if (*px = = V) py = px + l ; if (py)
(void) strcpy(pn, py); return(strlen(pn));
Page 7 of nwprint.c Table 1 (cont')
/*
* cleanupO -- cleanup any tmp file
* getseqO — read in seq set dna, len maxlen
* g_callocO - callocO with error checkin
* readjmpsO -- get the good jmps, from tmp file if necessary
* vvritejmpsO — write a filled array of jmps to a tmp file nw() */
#include "nw h" ^include < sy s/file h > char *jname = "/tmp/homgXXXXXX' /* tmp file tor jmps */ FILE *t], int cleanupO, /*•* cleanup tmp file */ long lseek(),
/*
* remove any tmp file if we blow */ cleanupO) cleanup int { if(fj)
(void) unhnk(jname), exιt(ι),
/*
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with ',', '<', or '>'
* seq in upper or lower case
*/ char * getseq(file, len) getseq char ♦file, /* file name */ int *len, 1* seq len */
{ char hne[1024], *pseq, register char *px, *py, int natgc, tlen,
FILE *fp; if ((fp = fopen(file,"r")) == 0) { fpπntf(stderr,"%s can't read %s\n", prog, file), exιt(l);
} tlen = natgc = 0, while (tgets(hne, 1024, fp)) { if (*lιne == ',' | | *hne == '<' | | *hne == '>') continue, for (px = line, *px ' = '\n', px++) if (ιsupper(*px) | | ιslower(*px)) tlen+ + ,
} if ((pseq = malloc((unsιgned)(tlen + 6))) = = 0) { tpπntf(stdeπ,"%s mallocO failed to get Sd bytes tor S?s\n , prog, tlen+6, file), exιt(l),
} pseq[0] - pseq[l] = pseq[2] = pseq|3] = '\0',
Page 1 ot nwsubr c Table 1 (cont')
...getseq pv = pseq + 4
*len = tlen, rev ιnd(fp) while (tgets(lιne 1024, fp)) { if(*Iιne = = , || *lιne == < | | *hne = = > ) continue, for (p\ = line, *px '= \n , px++) { if (ιsupper(*px))
*P> + + = *pχ, else if (ιslo\ver(*px))
*py++ = toupper(*px), if (mdex( 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((unsιgned)nx, (unsιgned)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 imgf000044_0001
register l, j, xx, if(fj){
(void) fclose(fj), if ((fd = open(jname, O RDONLY, 0)) < 0) { fprmtf(stderr, ' %s can't open() S?s\n , prog, jname), 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 Table 1 (cont')
...readjmps if (j < 0 && dx[dmax] offset && tj) {
(void) lseek(td, dx[dmax] offset, 0), (void) read(fd, (char *)&dx[dmax] jp sizeofistruct jmp))
(void) read(td, (char *)&dx|dmax] oftset sιzeof(dx[dmax] offset)), dxldmax] ijmp = MAXJMP 1,
} else break,
} if (i >= JMPS){ fpπntt(stderr, S?s too many gaps in alιgnment\n , prog) cleanupO ), } if 0 > = 0){ siz = dxfdmax] jp n ], xx = dx[dmax] jp x[j],
Figure imgf000045_0001
if (siz < 0) { /* gap in second seq */
Figure imgf000045_0002
/* id = xx - yy + lenl - 1 */ PPfl] 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 imgf000045_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 = PPfO] nUI, PPlO] nl,] = pp[0] n[ι0j, pp[0] n[ι0] = i, i = PPlO] xUI, PPlOJ xQ] = pp[01 x[ι0], pp[0] x[ι0] = i, } for = 0, il- ,j < ιl,j + + , ιl--){
. = pp|l] nb], pp[l] n[|] = ppll] n[ιll, pp[l] n[ιl] = i, i = PPHJ χϋl, PPIU xUI = PPEIJ x[»H. PPHl hi] = L
} if (fd > = 0)
(void) close(fd), if(fj){
(void) unlιnk(|name), tj =0, ottset = 0,
} }
Page 3 ot nwsubr c Table 1 (cont')
* w rite a filled jmp sti uct ottset ot the prev one (if anv ) nw()
*/ wπtejmps(ιx) ritejmps
{ char *mktemp(), if CO) { if (mktemp(jname) < 0) { fpπntf(stderr, " ? s can t mktempO Sl \n , prog, jname), cleanupO),
} if ((fj = topenfjname, "w")) = = 0) { fpπntf(stderr, ' %s can t write %s\n , prog, jname), exιt(l), }
}
(void) twπte((char *)&dx[ιx] ιp, sizeofistruct jmp), 1 , fj),
(void) twπte((char *)&dx[ιx] offset, sizeof(dx[ιx] oftset), 1 , fj),
Page 4 of nwsubr c Table 2
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 3
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 4
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 5
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 % II. Compositions and Methods of the Invention
A. Full-Len th PRO Polypeptides
The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides. In particular. cDNAs encoding various PRO 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 the full length native nucleic acid molecules disclosed herein as well as all further native homologues and variants included in the foregoing definition of PRO. will be referred to as "PRO/number". regardless of their origin or mode of preparation.
As disclosed in the Examples below, various cDNA clones have been deposited with the ATCC. The actual nucleotide sequences of those clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods in the art. The predicted amino acid sequence can be determined from the nucleotide sequence using routine skill. For the PRO polypeptides and encoding nucleic acids described herein, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time. B. PRO Polypeptide Variants
In addition to the full-length native sequence PRO polypeptides described herein, it is contemplated that PRO variants can be prepared. PRO variants can be prepared by introducing appropriate nucleotide changes into the PRO DNA, and/or by synthesis of the desired PRO polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence PRO or in various domains of the PRO described herein, can be made, for example, using any of the techniques and guidelines for conservative and non- conservative mutations set forth, for instance, in U.S. Patent No. 5.364.934. Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO that results in a change in the amino acid sequence of the PRO as compared with the native sequence PRO. 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 PRO. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e.. conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence. PRO polypeptide fragments are provided herein. Such fragments may be truncated at the N- terminus or C-terminus. or may lack internal residues, for example, when compared with a full length nativ e protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the PRO polypeptide. PRO fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating PRO fragments by enzymatic digestion, e.g.. by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, PRO polypeptide fragments share at least one biological and/or immunological activity with the native PRO polypeptide disclosed herein.
In particular embodiments, conservative substitutions of interest are shown in Table 6 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 6, or as further described below in reference to amino acid classes, are introduced and the products screened.
Table 6
Original Exemplary Preferred Residue Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gin; asn lys
Asn (N) gin; his; lys; arg gin
Asp (D) glu glu Cys (C) ser ser
Gin (Q) asn asn
Glu (E) asp asp
Gly (G) pro; ala ala
His (H) asn; gin; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leu
Leu (L) norleucine; ile; val; met: ala; phe ile
Lys (K) arg; gin; asn arg Met (M) leu: phe; ile leu
Phe (F) leu; val: ile; ala; tyr leu
Pro (P) ala ala
Ser (S) thr thr
Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp. phe. thr. sei phe
Val (V) ile leu. met. phe ala. norleucine leu
Substantial modifications in function or immunological identity ot the PRO polypeptide are accomplished by selecting substitutions that ditfei significantly in then effect on maintaining (a) the structure ot the polypeptide backbone in the area ot the substitution, for example, as a sheet or helical conformation, (b) the charge or hydiophobicity ot the molecule at the target site, or (c) the bulk of the side chain Naturally occurring residues are divided into groups based on common side-chain properties ( I ) hydrophobic norleucine. met. ala. val. leu. lie. (2) neutral hydrophihc cys ser. thr.
(3) acidic asp, glu.
(4) basic asn. gin. his. lys. arg,
(5) residues that influence chain orientation gly , pro. and
(6) aromatic trp, tyr. phe Non-conservative substitutions will entail exchanging a member of one of these classes for another class Such substituted residues also may be introduced into the conservative substitution sites or. more preferably, into the remaining (non-conserved) sites
The variations can be made using methods known in the art such as ohgonucleotide-mediated (site- directed) mutagenesis, alanine scanning, and PCR mutagenesis Site-directed mutagenesis [Carter et al , Nucl Acids Res., 13.4331 (1986). Zoller et al , Nucl Acids Res . JO 6487 ( 1987)], cassette mutagenesis
[Wells et al , Gene. 34 315 (1985)], restriction selection mutagenesis [Wells et al , Philos Tians R Soc London SerA, 317.415 ( 1986)] oi other known techniques can be performed on the cloned DNA to produce the PRO variant DNA
Scanning amino acid analysis can also be employed to identify one or more ammo acids along a contiguous sequence Among the preferred scanning amino acids are relatively small, neutral amino acids
Such am o acids include alanine, glycine, seπne. and cysteine Alanine is typically a piefeπed scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to altei 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 Pioteins (W H Freeman & Co .
N Y ). Chothia. J Mol Biol , 150 1 ( 1976)] If alanine substitution does not y ield adequate amounts of variant, an lsoteπc amino acid can be used
Alternatively, amino acid sequence variants of native PRO polypeptides and functional fragments theieof may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant PRO or by in vitio synthesis of the desired polypeptide There are two principal variables in the construction of amino acid sequence variants ( 1 ) the location ot the mutation site and. (2) the nature of the mutation With the exception of naturally-occurring alleles. which do not require the manipulation ot the DNA sequence encoding the PRO. the amino acid sequence variants of PRO are preferably constructed bv mutating PRO. either to arriv e at an allele oi an ammo acid sequence v ariant that does not occur in nature
Sites ot mutations w ill ty pically be modified in series, e g . by ( 1 ) substituting first with conservative choices, and then with more radical selections depending upon the results achieved (2) deleting the target residue or residues, oi (3) inserting iesidues ot the same or ditterent class ad|acent to the located site, or combinations ot options ( 1 )-(3)
Depending upon the protein, certain residues can be mutated in order to assist in the assembly and refolding For example, in a pi otein having an odd nuinbei of cysteines, wheiein only an ev en number are joined in mtramoleculai disulfide bonds, one or more cysteine residues can be mutated to serine to assist in refolding of the protein subsequent to expression in E coli or a similar piokaryote
C Modifications of PRO
Covalent modifications ot PRO are included within the scope of this invention One type ot covalent modification includes reacting targeted amino acid residues of a PRO polypeptide with an organic deπvatizing agent that is capable ot reacting with selected side chains or the N- or C- terminal residues of the PRO Deπvatization with bifunctional agents is useful, tor instance, for crosslinking PRO to a water- msoluble support matrix or surface for use in the method for purifying anti-PRO antibodies, and vice-versa Commonly used crosslinking agents include, e g , l . l -bιs(dιazoacetyl)-2-phenylethane. glutaraldehyde. N- hydroxysuccinimide esters, for example, esters with 4-azιdosalιcylιc acid, homobifunctional lmidoesters, including disuccinimidyl esters such as 3,3'-dιthιobιs (succinimidylpropionate), bifunctional maleimides such as bιs-N-ιnaleιmιdo- 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 prohne and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains [T E Creighton, Proteins Stiuctuie and Moleculai Pioperties, W.H Freeman & Co . San Francisco, pp 79-86 ( 1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group
Another type of covalent modification ot the PRO polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydiate moieties present Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence The alteration may be made, for example, by the addition of. oi substitution by, one or more serine or threonine residues to the native sequence PRO (for O-linked glycosylation sites) The PRO amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons are generated that w ill translate into the desired amino acids
Another means of increasing the number ot carbohydrate moieties on the PRO polypeptide is by chemical or enzymatic coupling ot glycosides to the polypeptide Such methods dre described in the art e.g , in WO 87/05330 published 1 1 September 1987 and in Aplm and Wπston. CRC Cut
Figure imgf000053_0001
Biochem . pp. 259-306 ( 1981 )
Removal ot carbohydiate moieties present on the PRO polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation Chemical deglycosy lation techniques are know n in the art and described. for instance, by Hakimuddin, et al Aich Biochem Bιoph\ s_. 259 52 ( 1987) and by Edge et al , Anal
Biochem . 1 18 131 (1981 ) Enzymatic cleavage of carbohydrate moieties on poly peptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al , Meth
Figure imgf000053_0002
138 350 ( 1987)
Another type of covalent modification of PRO compπses linking the PRO polypeptide to one of a variety of nonproteinaceous polymers, e g , polyethylene glycol (PEG) polypropylene glycol. or polyoxyalkylenes, in the manner set forth in U S Patent Nos 4,640.835. 4.496.689. 4.301.144. 4,670,417 4,791 ,192 or 4, 179,337
The PRO of the present invention may also be modified in a way to form a chimeπc molecule comprising PRO fused to another, heterologous polypeptide or amino acid sequence In one embodiment, such a chimeπc molecule comprises a fusion of the PRO with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind The epitope tag is generally placed at the amino- or carboxyl- terminus of the PRO The presence of such epitope-tagged forms of the PRO can be detected using an antibody against the tag polypeptide Also, provision of the epitope tag enables the PRO to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag Various tag polypeptides and their respective antibodies are well known in the art Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags, the flu HA tag polypeptide and its antibody 12CA5 [Field et al , Mol Cell Bιol_, 8.2159-2165 ( 1988)], the c-myc tag and the 8F9, 3C7. 6E 10, G4. B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellulai
Figure imgf000053_0003
, 5 3610-3616 ( 1985)], and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody fPaborsky et al . Protein Engineering. 3(6) 547-553 ( 1990)] Other tag polypeptides include the Flag-peptide [Hopp et al ,
Figure imgf000053_0004
, 6 1204- 1210 ( 1988)|. the KT3 epitope peptide [Martin et al , Science. 255 192- 194 ( 1992)]. an cc-tubuhn epitope peptide [Skinner et al , J Biol Chem , 266 15163- 15166 ( 1991 )]. and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al . Pioc Natl Acad S t USA, 87 6393-6397 (1990)] In an alternative embodiment, the chimeπc molecule may comprise a fusion of the PRO with an immunoglobulin or a particular region of an immunoglobulin For a biv alent 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 lg fusions preferably include the substitution oi a soluble (transmembrane domain deleted or inactivated) form of a PRO polypeptide in place of at least one v ariable region within an lg molecule In a particularly preferred embodiment the immunoglobulin fusion includes the lunge. CH2 and CH^ or the hinge. CH I CH2 and CH3 regions ot an IgG 1 molecule Foi the pioduction ot immunoglobulin tusιon-> see also US Patent No 5.428.1 0 issued June 27 1995 D Preparation of PRO The description below relates primarily to production of PRO by culturing cells transformed oi transfected with a vector containing PRO nucleic acid It is of course, contemplated that alternative methods which aie well known in the art. may be employed to prepare PRO For instance, the PRO sequence, or portions theieot. may be produced by direct peptide synthesis using solid-phase techniques [see. g.. Stewart et al , Solid-Phase Peptide S\nthests, W H Freeman Co . San Francisco, CA ( 1969), Merπfield, J Am Chem Soc . 85 2149-2154 ( 1963)] In \ ιtιo protein synthesis mav be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City. CA) using manufacturer's instructions Various portions of the PRO may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO 1 Isolation of DNA Encoding PRO
DNA encoding PRO may be obtained from a cDNA library prepared from tissue believed to possess the PRO mRNA and to express it at a detectable level Accordingly, human PRO DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples
The PRO-encoding gene may also be obtained from a genomic library or by known synthetic procedures ( g . automated nucleic acid synthesis)
Libraries can be screened with probes (such as antibodies to the PRO or oligonucleotides ot 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 , Moleculai Cloning A Laboiaton Manual (New York Cold Spring Harbor Laboratory Pi ess, 1989) An alternative means to isolate the gene encoding PRO is to use PCR methodology [Sambiook et al , supia. Dieftenbach et al , PCR Pi i ei A Laboiaton Manual (Cold Spring Harbor Laboratory Press. 1995)]
The Examples below describe techniques for screening a cDNA library The oligonucleotide sequences selected as probes should be ot sufficient length and sufficiently unambiguous that false positives are minimized The oligonucleotide is preferably labeled such that it can be detected upon hybridization to
DNA in the library being screened Methods of labeling are well known in the art, and include the use of radiolabels like P-labeled ATP. biotinylation or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al . supia
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the tull-length sequence can be determined using methods know n in the art and as described herein Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time and if necessary, using conventional primer extension procedures as described in Sambrook et al , supia. to detect precursors and processing intermediates ot mRNA that may not hav e been reverse-transcribed into cDNA 2 Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors described herein for PRO production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants. or amplifying the genes encoding the desired sequences The culture conditions, such as media, temperature. pH and the like, can be selected by the skilled artisan without undue experimentation In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology a Practical Approach. M Butler, ed (IRL Press, 1991 ) and Sambrook et al supra
Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCli, CaP04. liposo e-mediated and electroporation Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells The calcium treatment employing calcium chloride, as described in Sambrook et al , supia. or electroporation is generally used for prokaryotes Infection with Agrobactei nun 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, Vιιolog\ , 52 456-457 (1978) can be employed General aspects of mammalian cell host system transfections have been described in U S Patent No 4,399,216 Transformations into yeast are typically carried out according to the method of Van Solingen et al , J. Bad., 130 946 (1977) and Hsiao et al , Proc Natl Acad Set (USA), 76 3829 (1979) However, other methods for introducing DNA into cells, such as by nuclear microinjection. electroporation, bacterial protoplast fusion with intact cells, or polycations, e g , polybrene, polyornithine. may also be used For various techniques for transforming mammalian cells, see Keown et al , Methods in Enzsmologs . 185 527 537 ( 1990) and Mansour et al ,
Nature, 336 348-352 ( 1988)
Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells Suitable prokaryotes include but are not limited to eubacteria. such as Gram-negative or Gram-positive organisms, for example. Enterobacteπaceae such as E. coli Various E colt strains are publicly available, such as E coli KI2 strain MM294 (ATCC 31.446), E coli X1776 (ATCC 31 ,537), E coli strain W 1 10 (ATCC 27,325) and K5 772 (ATCC 53,635) Other suitable prokaryotic host cells include Enterobacteπaceae such as Escherichia, e g , E coli, Enterobacter, Erwtnia. Klebsiella, Proteus. Salmonella, e g . Salmonella tvphimurium. Serratia, e g , Serratia marcescans. and Shigella. as well as Bacilli such as B subtilis and B licheniformis (e g . ? lichenifonms 41 P disclosed in
DD 266,710 published 12 April 1989). Pseudomonas such as P aeiuginosa. and Stieptotmces These examples are illustrative rather than limiting Strain W31 1 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 W3 ] 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 1A2 which has the complete genotype tonA E coli W31 10 strain 9E4 which has the complete genoty pe tonA ptι3. E coli W31 10 strain 27C7 (ATCC 55.244) which has the complete genotype tonA ptr3 phoA El 5 (argF lac)169 degP ompT kan' '. E coli W31 10 strain 37D6 which has the complete genotype tonA pti 1 phoA El 5 (aιgF-lac)169 degP ompT ιbs7
Figure imgf000056_0001
G kan . E coli W31 10 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation, and an £ coli strain having mutant peπplasmic protease disclosed in U S Patent No 4 946,783 issued 7 August 1990 Other suitable strains have impaired heat shock response in combination with protease deletions and mutations For example, the E Coli W31 10 strain 44C6, which has the complete genotype fhuAΔ (tonAΔ) lonΔ galE rpoHts (htpHts) ΔcIpP Other potential cloning hosts are E coli 294 (ATCC 31 ,446), E coli B and E coli XI 776 (ATCC 31 ,537) 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 tor PRO-encoding vectors Saccharom ces ceievisiae is a commonly used lower eukaryotic host microorganism Others include Schi osacchai oun ces pombe (Beach and Nurse
Nature, 290 140 [ 1981], EP 139,383 published 2 May 1985), Kluweronnces hosts (U S Patent No 4,943,529, Fleer et al , Bιo/Technolog\ , 9 968-975 ( 1991 )) such as, e g , K lactis (MW98-8C, CBS683 CBS4574, Louvencourt et al , J Bactenol , 154(2) 737-742 [ 1983]) K fragihs (ATCC 12,424), K bulgaricus (ATCC 16,045), K wickeramu (ATCC 24,178), K waltu (ATCC 56,500), K diosophilarum (ATCC 36,906, Van den Berg et al , Bw Technolog), 8 135 (1990)), K thermotolerans, and K mωxianus yarrowta (EP 402,226), Pichia pastons (EP 183,070. Sreekπshna et al , J Basic Microbiol , 28 265-278 [1988]), Candida, Tnchoderma reesia (EP 244,234), Neurospora crassa (Case et al , Proc Natl Acad Set USA, 76 5259-5263 [ 1979]), Schwanmomvces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990), and filamentous fungi such as, e g , Neurospoia, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A mdulans (Ballance et al
Biochem Btopfas Res Commun , JJ2 284-289 [ 1983], Tilburn et al , Gen . 26 205-221 [ 1983], Yelton et al , Proc Natl Acad Set USA, _i 1470-1474 [ 1984]) and A gei (Kelly and Hynes, EMBO J , 4 475-479 [1985]) Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckeia Pichia Saccharomyces, Toiulopsis, and Rhodotorula A list of specific species that are exemplary of this class of yeasts may be found in C Anthony, The Bwchemistn of Methylotrophs, 269 ( 1982)
Suitable host cells for the expression of glycosylated PRO are derived from multicellular organisms Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9 as well as plant cells Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidney 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 Vuol , 36 59 ( 1977)), Chinese hamster ovary cellsADHFR (CHO, Urlaub and Chasm. Ptoc Natl Acad Sci USA, 77 4216 ( 1980)), mouse sertoh cells (TM4. Mather Biol Repwd 23 243 251 ( 1980)), human lung cells (W138 ATCC CCL 75) human liver cells (Hep G2, HB 8065). and mouse mammary tumoi (MMT 060562. ATCC CCL5 1 ) The selection of the appropriate host cell is deemed to be ithin the skill in the art
3 Selection and Use ot a Replicable Vectoi
The nucleic acid (e g . cDNA oi genomic DNA) encoding PRO may be inseited into a replicable vector foi cloning (amplification ot the DNA) oi tor expression Various v ectors are publicly available
The vector may, for example, be in the form of a plasmid. cosmid. v iral particle, or phage The appropriate nucleic acid sequence may be inserted into the vector by a variety ot proceduies In general. DNA is inserted into an appropnate restriction endonuclease sιte(s) using techniques known in the art Vector components generally include, but are not limited to. one or more of a signal sequence, an origin of replication, one or more mai ker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan
The PRO may be pioduced 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 matuie protein or polypeptide In general, the signal sequence may be a component ot the vector, or it may be a part of the PRO-encoding DNA that is inserted into the vector The signal sequence may be a prokaryotic signal sequence selected, for example, from the group ot the alkaline phosphatase. penicilhnase, 1pp. or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g.. the yeast invertase leader, alpha tactor leader (including Saccharomyces and Kluyveronnces α- factor leaders, the latter described in U.S. Patent No 5,010,182), or acid phosphatase leader, the C albicans glucoamylase leader (EP 362, 179 published 4 April 1990). or the signal described in WO 90/13646 published 15 November 1990 In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences fiom 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 moie selected host cells Such sequences are well known for a vanety ot bacteria, yeast, and viruses The origin ot replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin. methotrexate. or tetracyclme, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available fiom 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 PRO-encoding nucleic acid, such as DHFR or thvmidine kinase An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al , Pioc . Natl Acad. Set USA, 11 4216 (1980) A suitable selection gene for use in yeast is the tip] gene present in the yeast plasmid YRp7 [Stinchcomb et al , Natuie. 282 39 ( 1979). Kmgsman et al.. Gene. 7 141 ( 1979). Tschemper el al . Gene. 10 157 ( 1980)] The tιp \ gene prov ides a selection marker tor a mutant strain ot v east lacking the ability to giow in try ptophan. tor example. ATCC No 44076 oi PEP4- 1 [Jones. Genetic s 85 12 ( 1977)]
Expression and cloning vectors usually contain a promotei operably linked to the PRO-encoding nucleic acid sequence to dnect mRNA sy nthesis Promoters iecogmzed by a variety of potential host cells are well known Promotei s suitable foi use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al . Natitie. 275 615 ( 1978). Goeddel et al . Natuie. 2_± 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 , Pioc 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 PRO
Examples ot suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al , J Biol Chem , 255 2073 ( 1980)] or other glycolytic enzymes [Hess et al . J
Figure imgf000058_0001
Enzyme Reg , 7 149 ( 1968), Holland, Biocheimstn , JJ 4900 ( 1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase. hexokinase, pyruvate decarboxylase. phosphofructokinase, glucose-6-phosphate isomerase. 3-phosphoglycerate mutase, pyruvate kinase. tπosephosphate isomerase. phosphoglucose isomerase, and glucokinase
Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, 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.
PRO 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, cytomegalovirus, a retrovirus. hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e g , the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems
Transcription of a DNA encoding the PRO 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 promotei to increase its transcription Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) Typically, however, one will use an enhancer from a eukaryotic cell vnus Examples include the SV40 enhancei on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancei . the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers The enhancer may be spliced into the vector at a position 5' or 3' to the PRO coding sequence, but is prefeiably located at a site 5' from the promoter
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, oi nucleated cells fiom other multicellular organisms) will also contain sequences necessary tor the termination of transcription and foi stabilizing the mRNA Such sequences are commonly available from the 5' and. occasionally 3. untranslated regions of eukaryotic or vnal DNAs oi cDNAs These regions contain nucleotide segments transcnbed as polv aden lated fragments in the untranslated portion of the mRNA encoding PRO
Still othei methods vectors and host cells suitable for adaptation to the sy nthesis of PRO in recombinant vertebrate cell culture are described in Gethmg et al , Natuie 293 620 625 ( 1981 ). Mantel et al . Natuie. 281 40-46 ( 1979). EP 1 17 060. and EP 1 17.058
4 Culturing the host cells
Prokaryotic cells used to produce the PRO polypeptides of this invention may be cultured in suitable media as described generally in Sambrook et al supia and Ausubel et al , supia Briefly, the transformed cells are grown at 30°C or 37°C until the optical density (measured at 550 nm) reaches about 2
3. The culture is diluted into a production medium, regrown with aeration, and 3-β-Indole acrylic acid (I A A) is added Growth is continued with aeration for about another 1 hours after hich time the cells are harvested by centπtugation When refolding is necessary the proceduie outlined below may be employed
More specifically, a 10 liter fermentation may be carried out as follows The termentor is first sterilized with a sterilization solution of about 5-6 5 liters of deionized water to which is added ammonium sullate (50 0 g). potassium phosphate, dibasic (60 0 g), sodium phosphate, monobasic dihydrate (30 0 g). sodium citrate, dihydrate ( 10 0 g). 1 -ιsoleucιne (5 g), 25% aq soln of pluro c polyol L-61 (BASF, antifoam) After the fermentor vessel cools down the growth media is added The growth media after inoculation has a volume typically of about 8 5 liters The media components are comprised of 50% glucose solution (15 mL), IM magnesium sulfate (70 mL), 20%e Hycase solution (250 mL), 20%> yeast extract solution (250 mL), 2 mg/mL ampicillin (250 mL) and trace metals (5 mL) A typical IL trace metal solution is composed ot the following HCI (100 mL), Ferric chloride hexahydrate (27 g), Zinc sulphate heptahydrate (8 g). Cobalt Chloride hexahydrate (7 g), Sodium molybdate (7 g), Cupπc sulphate pentahydrate (8 g), boric acid (2 g), Manganese sulphate monohydrate (5 g). distilled water (total volume to 1 L) Inoculation is made with 500 mL of an 18-20 hour LB culture grown in the presence of ampicillin and the termentor is agitated at 750 rpm and aerated at 10 slpm The culture pH is maintained at 7 0 by automatic addition of ammonium hydroxide and the temperature is maintained at 30°C When the initial glucose in the culture is exhausted, a glucose feed is started and maintained at a rate sufficient to sustain growth but not accumulate in the medium Culture growth is monitored by measuring the optical density (O D ) at 550 nm When the culture O D reaches 25-35, 25 mL of a 25 mg/mL solution of IAA is added and the cell paste harvested after 14- 18 hours of centπfugation 5 Detecting Gene Amplification/Expression
Gene amplification and/or expression may be measured in α sample directly, for example, by conventional Southern blotting, northern blotting to quantitate the transcription of mRNA [Thomas, Pioc 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 heiein Vanous labels may be employed most commonly radioisotopes, particularly "P Howevei other techniques may also be employed, such as using biotin-modified nucleotides tor introduction into a polynucleotide The biotin then serves as the site for binding to av idin oi antibodies which mav be labeled with a wise variety of labels such as radionuchdes. fiuorescers enzv mes. oi the like Alternativ ely antibodies mav be emploved that can recognize specific duplexes including DNA duplexes. RNA duplexes, and DNA-RNA hvbπd duplexes or DNA-protein duplexes The antibodies in tui n may be labeled and the assay mav be earned out w here the duplex is bound to a surface so that upon the formation of duplex on the surface the piesence of antibody bound to the duplex can be detected
Gene expression alternatively , may be measured by immunological methods, such as lmmunohistochemical staining ot tissue sections and assay of cell culture or body fluids to quantitate directly the expression of gene product Antibodies useful tor lmmunohistochemical staining and/or assaying of sample fluids may be eithei 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. followed by reaction with labeled antibodies specific for the gene coupled to a detectable label Labels are preferably visually detectable, such as enzymatic labels, fluorescent labels luminescent labels, and the like Foi example, see Hsu et al , Am J Clin Path 75 734-738 ( 1980)
6 Purification of PRO Polypeptides
Forms of PRO may be recovered from culture medium as a secreted polypeptide or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (e g .
Tπton®-X 100) or by enzymatic cleavage Cells employed in expression of PRO polypeptides can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents
When using recombinant techniques, the PRO polypeptide can be produced intracellularly, in the peπplasmic space, or directly secreted into the medium If the PRO is produced intracellularly. it will usually be necessary to PRO, respectively from other recombinant cell proteins or polypeptides to obtain preparations that are substantially homogenous to the respective PRO As a first step, the culture medium or lysate is centrifuged to remove the particulate debris, e g host cells or lysed fragments A procedure is described in Carter et al , Bio/Technology J_0 163- 167 ( 1992) for isolating proteins which are secreted to the peπplasmic space of E coli Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) ovei about 30 minutes Cell debris can be removed by centrifugation The following procedures are examplary ot other suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE. ammonium sulfate precipitation, gel filtration using, for example. Sephadex G-75. protein A Sepharose columns to remove contaminants such as IgG, and metal chelating columns to bind epitope-tagged forms ot the PRO Various method of protein purification may be employed and such methods are known in the ai t and described for example, in Deutscher. Methods in
Figure imgf000060_0001
182 ( 1990). Scopes. Ptotein Puiification Piinciples and Piactic e. 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 PRO produced Many heterogeneous piotems expressed in E coli require refolding in order to impart activ ity When this is necessary , the follow ing piocedure can be used For a geneiαl discussion of piocedures suitable for refolding ot recombinant oi synthetic PRO. include any N- or C-terminal extended forms, the reader is referred to the following patents Builder et al . U S Patent No 4. 1 1.502. Jones et al . U S Patent 4.512.922. Olson. U S Patent No 4.518,526. Builder et al . U S Patent 4.620.948
(a) Reccn en ofnon soluble PRO
A microorganism such as E c oli which is fermented undei conditions suitable tor the expression of PRO (but not secretion) is deposited by the microorganism in insoluble "refiactile bodies", and is recoverable by a lysing procedure Optionally, cells are first washed in a cell disruption buffer Foi example, about 100 g of cells are resuspended in about 10 volumes of a cell disruption buffer (e g 10 mM
Tris. 5 mM EDTA. pH 8) with, for example, a Polytron homogemzer. followed by centrifugation at 5000 x g tor 30 minutes Cells are then lysed using any conventional technique such as tonic shock, sonication. pressure cycling, chemical or enzymatic methods For example, the washed cell pellet above may be resuspended in another 10 volumes of a cell disruption buffer with a homogemzer and the cell suspension is passed through an LH Cell Disrupter (LH Inceltech. Inc ) or through a Microfluidizei® (Microfluidics Int'l) according to the manufacturer's instructions The particulate matter containing PRO is then separated form the liquid phase and optionally washed with any suitable liquid For example, a suspension of cell lysate may be centrifuged at 5,000 x g for 30 minutes, iesuspended and optionally centrifuged a second time to make a washed refractile body pellet The washed pellet may be used immediately or optionally stored frozen (at e g -70°C)
(b) Solubilization and Purification of Monomei ic PRO
Insoluble PRO polypeptide in the refractile body fiom the procedure above is solubihzed with a solubilizing buffer The solubilizing buffer contains a chaotropic agent and is usually buffered at a basic pH and contains a reducing agent to improve the yield of monomeπc PRO Representative chaotropic agents include urea. guanidine-HCl, and sodium thiocyanate A preferred chaotropic agent is guamdine-HCl The concentration ot chaotropic agent is usually 4-9 M. preferably 6-8 M The pH of the solubilizing buffer is maintained by any suitable buffer in a pH range of from about 7 5-9 5, preferably 8 0-9.0. and most preferably 8 0 Preferably, the solubilizing buffer also contains a leducing agent to aid formation ot the monomeπc form of PRO Suitable reducing agents include organic compounds containing a free thiol (RDH) Representative reducing agents include dithiothreitol (DTT), dithioerythπtol (DTE), mercaptoethanol, glutathione (GSH), cysteamine and cysteine A pieferred reducing agent is dithiothreitol (DTT) Optionally, the solubilizing buffer may contain a mild oxidizing agent (e g molecular oxygen) and a sulfite salt to form monoineπc mutant PRO via sulfitolysis In this embodiment, the resulting PRO-S- sultonate is later refolded in the presence of redox buffer (e g . GSH/GSSG) to form the properly folded PRO
The PRO protein is usually further purified using, foi example, centnfugation, gel filtration chromatography and reversed phase column chromatography For example, the refractile body pellet may be resuspended in about 5 volumes by weight of the solubilizing buffer (20 mM Tris. pH 8. with 6-8 M guanidine and 25 mM DTT) and stirred for 1 -3 hr.. or overnight at 4°C to effect solubilization of the mutant PRO protein. High concentrations of urea (6-8M) are also useful but generally result in somewhat lower yields compared to guanidine. After solubilization. the solution is centrifuged at 30.000 x g for 30 min. to produce a clear supernatant containing denatured, monomeric PRO. The supernatant is then chromatographed on a Superdex® 200 gel filtration column
(Pharmacia, 2.6 x 60 cm) at a flow rate of 2 ml/min. and the protein eluted with 20 mM Na phosphate, pH
6.0, with 10 mM DTT. Fractions containing monomeric. denatured PRO eluting between 160 ml and 200 ml are pooled. The PRO protein is further purified on a semi-preparative C4 reversed phase column (2 x 20 cm VYDAC). The sample is applied at 5 ml/min. to a column equilibrated in 0.1 % TFA (trifluoroacetic acid) with 30% acetonitrile. The protein is eluted with a linear gradient of acetonitrile (30-60%c in 60 min.).
The purified reduced protein elutes at approximately 50% acetonitrile. This material is used for refolding to obtain biologically active.
(c) Refolding of PRO to Generate the Biologically Active Form. Following solubilization and further purification of PRO. the biologically active form is obtained by refolding the denatured monomeric PRO in a redox buffer. Depending upon the potency of the PRO. it may be possible to obtain biologically active material utilizing many different buffer, detergent and redox conditions. However, under most conditions, only a small amount of properly folded material (<10%) is obtained. For commercial manufacturing processes, it is desirable to have refolding yields at least 10%, more preferably 30-50 and most preferably >50%c. Many different detergent including Triton® X-100, dodecyl-beta-maltoside, CHAPS, CHAPSO, SDS, sarkosyl, Tween® 20 and Tween® 80, Zwittergent 3-14 and others may be used to produce at least minimal folding. However, the most preferred detergents are of the CHAPS family (CHAPS and CHAPSO) which appear to work best in refolding and limit protein aggregation and improper disulfide formation. Levels of CHAPS greater than about 1 % are most preferred. To optimize yields, it is preferred to have sodium chloride present (0.1 M-0.5M). It is further preferred to have EDTA ( 1 -5 mM) in the redox buffer in order to limit the amount of metal-catalyzed oxidation (and aggregation). At least 15% glycerol is further preferred in order to reach optimal refolding conditions. For maximum yields, it is further preferred that the redox buffer have both an oxidized and reduced organic thiol
(RSH). Suitable redox pairs include mercaptoethanol, glutathione (GSH). cysteamine, cysteine and their corresponding oxidized forms. Preferred redox are glutathione (GSH):oxidized glutathione (GSSG) or cysteinexystine. The most preferred redox pair is glutathione (GSH):oxidized glutathione (GSSG).
Generally higher yields are observed when the mole ratio of oxidized member of the redox pair is equal to or in excess over the reduced member of the redox pair. pH values between 7.5 and about 9 are optimal for refolding of PRO polypeptides. Organic solvents (e.g. ethanol, acetonitrile, methanol) were tolerated at concentrations of 10- 15% or lower. Higher levels of organic solvents increased the amount of improperly folded forms. Tris and phosphate buffers were generally useful. Incubation at 4°C also produced higher levels of properly folded PRO.
Refolding yields of 40-60% (based on the amount of reduced and denatured PRO used in the refolding reaction) are typical for preparations of the respective PRO that have been purified through the first C4 step. Active material can be obtained when less pure preparations (e.g. directly after the Superdex"" 200 column or after the initial refractile body extraction) although the yields can be less due to precipitation and interference of non-PRO proteins during the respective PRO refolding process.
In order to assist in achieving optimal results during refolding, it may become necessary to mutate various cysteine which are not involved in disulphide bonding so as to ensure formation of the disulfide bonds which do occur in the native sequence molecule.
During the initial exploration in determining refolding conditions, different peaks containing the PRO protein can be separated by C4 reverse phase chromatography. Upon testing for the peak with the most significant biological activity, conditions may be optimized to yield preferentially for that version. E. General Uses for PRO
Nucleotide sequences (or their complement) encoding PRO have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA. PRO nucleic acid will also be useful for the preparation of PRO polypeptides by the recombinant techniques described herein. The full-length native sequence PRO gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length PRO cDNA or to isolate still other cDNAs (for instance, those encoding naturally-occurring variants of PRO or PRO from other species) which have a desired sequence identity to the native PRO sequence disclosed herein. Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived from at least partially novel regions of the full length native nucleotide sequence wherein those regions may be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and introns of native sequence PRO. By way of example, a screening method will comprise isolating the coding region of the PRO gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 2P or " 3S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the PRO gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below.
Any EST sequences disclosed in the present application may similarly be employed as probes, using the methods disclosed herein.
Other useful fragments of the PRO nucleic acids include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target PRO mRNA (sense) or PRO DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of PRO DNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example. Stein and Cohen (Cancer Res. 48:2659. 1988) and van der Krol et al. (BioTechniques 6:958, 1988). Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means. including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of PRO proteins. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar- phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences. Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10048, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine. and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaP04-mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Ban* virus. In a preferred procedure, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not limited to. cell surface receptors, growth factors, other cytokines. or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase. Antisense or sense 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
The probes may also be employed in PCR techniques to generate a pool of sequences tor identification ot closely related PRO coding sequences Nucleotide sequences encoding a PRO can also be used to onsti uct hy bridization probes foi mapping the gene which encodes that PRO and for the genetic analysis of individuals with genetic disorders The nucleotide sequences prov ided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybπdization. linkage analysis against known chromosomal markers, and hybridization screening with libraries When the coding sequences for PRO encode a protein which binds to another protein (example. where the PRO is a receptor), the PRO can be used in assays to identify the othei proteins or molecules involved in the binding interaction By such methods, inhibitors of the receptor/hgand binding interaction can be identified Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists ot the binding interaction Also, the receptor PRO can be used to isolate correlative lιgand(s) Screening assays can be designed to find lead compounds that mimic the biological activity of α native PRO or a receptor for PRO Such screening assays will include assays amenable to high-throughput screening of chemical libiaπes. making them particularly suitable for identifying small molecule drug candidates Small molecules contemplated include synthetic organic or inorganic compounds 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.
Nucleic acids which encode PRO or its modified forms can also be used to generate either transgemc animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents A transgemc animal (e g , a mouse or rat) is an animal having cells that contain a transgene. which trαnsgene was introduced into the animal or an ancestor of the animal at a prenatal, e g . an embryonic stage A transgene is a DNA which is integrated into the genome of a cell from which a transgemc animal develops In one embodiment, cDNA encoding PRO can be used to clone genomic DNA encoding PRO in accordance with established techniques and the genomic sequences used to generate transgemc animals that contain cells which express DNA encoding PRO Methods for generating transgemc animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U S Patent Nos 4,736,866 and 4,870.009 Typically, particular cells would be targeted for PRO transgene incorporation with tissue-specific enhancers Transgemc animals that include a copy of a transgene encoding PRO introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expiession of DNA encoding PRO Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention foi the pathological condition Alternatively , non-human homologues of PRO can be used to construct a PRO "knock out animal which has a detectiv e 01 altered gene encoding PRO as a result ot homologous recombination between the endogenous gene encoding PRO and altered genomic DNA encoding PRO introduced into an embryonic stem cell of the animal For example. cDNA encoding PRO can be used to clone genomic DNA encoding PRO in accordance with established techniques A portion of the genomic DNA encoding PRO can be deleted or replaced with another gene, such as a gene encoding a selectable markei which can be used to monitor integration Typically , several kilobases of unaltered flanking DNA (both at the 5' and 3 ends) aie included in the vector [see e g Thomas and Capecchi. Cell. 5J_ 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 15 ( 1992)] The selected cells are then injected into a blastocyst of an animal (e g . a mouse or rat) to foim aggregation chimeras [ see e g Bradley in Teiatocaicinomas and
Embryonic Stem Cells A Piaciical Appioach. E J Robertson ed (IRL Oxford 1987), pp 1 13- 152] A chimeπc 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. for their ability to defend against certain pathological conditions and tor their development of pathological conditions due to absence of the PRO polypeptide Nucleic acid encoding the PRO polypeptides may also be used in gene therapy In gene therapy applications, genes are introduced into cells in order to achieve in vn o synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in \ ιvo It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite then low intrαcellular concentrations caused by their restricted uptake by the cell membrane (Zamecnik et al , Pioc Natl Acad Sci USA 83 4143-4146 [ 19861)
The oligonucleotides can be modified to enhance their uptake, e g by substituting their negatively charged phosphodiester groups by uncharged groups
There are a variety of techniques available for intioducing nucleic acids into viable cells The techniques vary depending upon whether the nucleic acid is transfened into cultured cells in vitio, or m vivo in the cells of the intended host Techniques suitable tor the transfer of nucleic acid into mammalian cells in vitro include the use ot liposomes. electroporation, microinjection. cell tusion. DEAE-dextran. the calcium phosphate precipitation method, etc The currently preferred m \ ιvo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transtection (Dzau et al , Tiends in Biotechnology J_[, 205-210 [ 1993]) 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 piotein or the target cell, a ligand for a receptor on the target cell, etc Where liposomes are employed, proteins which 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 which undergo intemalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al.. J. Biol. Chem. 262. 4429-4432 ( 1987): and Wagner et al.. Proc. Natl.
Acad. Sci. USA 87. 3410-3414 ( 1990). For review of gene marking and gene therapy protocols see Anderson et al.. Science 256. 808-813 ( 1992).
The PRO polypeptides described herein may also be employed as molecular weight markers for protein electrophoresis purposes and the isolated nucleic acid sequences may be used for recombinantly expressing those markers.
The nucleic acid molecules encoding the PRO polypeptides or fragments thereof described herein are useful for chromosome identification. In this regard, there exists an ongoing need to identify new chromosome markers, since relatively few chromosome marking reagents, based upon actual sequence data are presently available. Each PRO nucleic acid molecule of the present invention can be used as a chromosome marker.
The PRO polypeptides and nucleic acid molecules of the present invention may also be used for tissue typing, wherein the PRO polypeptides of the present invention may be differentially expressed in one tissue as compared to another. PRO nucleic acid molecules will find use for generating probes for PCR, Northern analysis, Southern analysis and Western analysis. F. Agonists and Antagonists of PRO Polypeptides
This invention encompasses methods of screening compounds to identify those that mimic the PRO polypeptide (agonists) or prevent the effect of the PRO polypeptide (antagonists). Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PRO polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
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 PRO 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 PRO 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 PRO polypeptide and drying. Alternatively, an immobilized antibody, e.g. , a monoclonal antibody, specific for the PRO polypeptide to be immobilized can be used to anchor it to a solid surlace The assav is performed bv adding the non immobilized component which ma's be labeled by a detectable label to the immobilized component e g , tht coated surface containing the anchored component When the reaction is complete the non reacted components are removed, e g , by w ashing and complexes anchored on the solid surface are detected W hen the originally non immobilized component carries a detectable label, the detection ot label immobilized on the surface indicates that cornplexing 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
It the candidate compound interacts with but does not bind to a particular PRO polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protem-protem 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 Set 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-tocZ 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 chromogemc 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 tor these interactions
Compounds that interfere with the interaction of a gene encoding a PRO polypeptide identified herein and other intra or extracellular components can be tested as follows usually a reaction mixture is prepared containing the product ot 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 ot a candidate compound to inhibit binding, the reaction is run m the absence and in the presence ot 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 tormation ot a complex in the control reactιon(s) but not in the reaction mixture containing the test compound indicates that the test compound interteres with the interaction of the test compound and its reaction partner To assay for antagonists, the PRO polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the 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. Coligan et al., Current Protocols in Immun. , j_(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 labeled PRO polypeptide. The PRO polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and re-trans ected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
As an alternative approach for receptor identification, labeled PRO polypeptide can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule.
Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing. The amino acid sequence obtained from micro- sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor. In another assay for antagonists, mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured.
More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with 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 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 polvnucleotide sequence, which encodes the mature PRO polypeptides herein, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length A DNA oligonucleotide is designed to be complementary to a region ot the gene involved in transcription (triple hehx - see Lee et al , Nucl Acids Res , 6 3073 ( 1979), Cooney et al , Science. 241_ 456 (1988), Dervan et al , Science, 25J_ 1360 (1991)), thereby preventing transcription and the production of the PRO polypeptide The antisense RNA oligonucleotide hybridizes to the mRNA in
Figure imgf000070_0001
and blocks translation ot 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 /// vivo to inhibit production of the PRO polypeptide When antisense DNA is used, oligodeoxyπbonucleotides derived trom the translation-initiation site, e g , between about -10 and + 10 positions of the target gene nucleotide sequence, are preferred
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 ot 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 ot catalyzing the specific cleavage of RNA Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific πbozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g , Rossi, Current Biology, 4 469-471 (1994), and PCT publication No WO 97/33551 (published September 18, 1997)
Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single- stranded and composed of deoxynucleotides The base composition ot these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand ot 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 Uses of the herein disclosed molecules may also be based upon the positive functional assay hits disclosed and described below G Anti-PRO Antibodies
The present invention further provides anti-PRO antibodies Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies 1 Polyclonal Antibodies
The anti-PRO antibodies may comprise polyclonal antibodies Methods ot 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 ad)uvant T ypically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or lntrapentoneal injections The immunizing agent may include the PRO polypeptide or a fusion protein thereof It mav be useful to conjugate the immunizing agent to α protein know n to be lmmunogenic in the mammal being immunized Examples of such lmmunogenic proteins include but are not limited to keyhole limpet hemocyanin. serum albumin, bovine thyioglobulin. and soy bean try psin inhibitor Examples of adiuv ants which may be employed include Fieund s complete ad|uvant and MPL-TDM ad|uvant (monophosphoryl Lipid A. synthetic trehalose dicorynomycolate) The immunization piotocol may be selected by one skilled in the art without undue experimentation
2 Monoclonal Antibodies
The anti-PRO antibodies may. alternatively be monoclonal antibodies Monoclonal antibodies may be prepared using hybπdoma methods, such as those described by Kohler and Milstein Nature.
256 495 ( 1975) In a hybπdoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable ot producing antibodies that will specifically bind to the immuni/ing agent Alternatively the lymphocytes may be immunized in vitro The immunizing agent will typically include the PRO 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 it non-human mammalian sources are desired The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybπdoma cell [Goding, Monoclonal Antibodies Piinciples and Practice. Academic Press, ( 1986) pp 59-103] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin Usually, rat or mouse myeloma cell lines are employed The hybπdoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused. immortalized cells For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoπbosyl transferase (HGPRT or HPRT). the culture medium for the hybπdomas typically will include hypoxanthine. aminopteπn and thvmidine ("HAT medium"), which substances prevent the growth of HGPRT-deticient cells
Preteπed immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium More preferred immortalized cell lines are murine myeloma lines which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego. California and the American Type Culture Collection
Manassas. Virginia Human myeloma and mouse-human heteromyeloma cell lines also have been described for the pioduction of human monoclonal antibodies [Kozbor. J Immunol , 133 3001 ( 1984) Brodeur et al , Monoclonal Antibods Pioduction Techniques and Applications. Marcel Dekker. Inc New York, ( 1987) pp 51 -63] The culture medium in which the hybπdoma cells aie cultured can then be assayed for the presence ot monoclonal antibodies directed against PRO Preferably the binding specificity ot monoclonal antibodies pioduced by the hybπdoma cells is determined by immunoprecipitation or by an in vitio binding assay , such as ladioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) Such techniques and assαvs are know n in the art The binding affinity of the monoclonal antibody can. for example be determined by the Scatchaid anal sis ot Munson and Pollard Anal Bioc hem 107 220 ( 1980)
After the desired hv bπdoma cells are identified the clones may be subcloned by limiting dilution procedures and grow n by standard methods [Goding supia] Suitable cultuie media foi this purpose include, tor example. Dulbecco's Modified Eagle's Medium and RPMI 1640 medium Alternatively the hybndoma cells may be grown in
Figure imgf000072_0001
o as ascites in a mammal
The monoclonal antibodies secieted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as. for example, protein A-Sepharose hydroxylapatite chromatography. gel electrophoresis dialy sis, or affinity chromatography
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Patent No 4.816,567 DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) The hybndoma cells of the invention serve as a preferred source ot such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells. Chinese hamster ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U S Patent No 4,816,567, Morrison et al , supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobuhn polypeptide Such a non-immunoglobuhn polypeptide can be substituted tor the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimenc bivalent antibody The antibodies may be monovalent antibodies Methods tor preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking Alternatively, the relevant cysteine residues are substituted with another amino acid residue or aie deleted so as to prevent crosslinking hi vitio methods are also suitable foi preparing monovalent antibodies Digestion of antibodies to produce fragments thereof, particularly. Fab fragments, can be accomplished using routine techniques known in the art
3 Human and Humanized Antibodies
The anti-PRO antibodies of the invention may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , murine) antibodies arc chimenc immunoglobulins, immunoglobulin chains oi fragments thereof (such as Fv, Fab, Fab', F(αb')2 oi other antigen-binding subsequences ot antibodies) which contain minimal sequence denved from non-human immunoglobulin Humanized antibodies include human immunoglobulins (recipient antibody ) in which residues from a complementary determining region (CDR) of the recipient are replaced by lesidues 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 framew ork residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one. and typically two. variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature. 321 :522-525 ( 1986): Riechmann et a!., Nature, 332:323-329 ( 1988); and Presta. Curr. Op. Struct. Biol., 2:593-596 ( 1992)].
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones el al.. Nature, 32J.:522-525 ( 1986): Riechmann el al., Nature, 332:323-327 ( 1988);
Verhoeyen et al.. Science, 239: 1534-1536 ( 1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4.816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991 ); Marks et al., J. Mol. Biol.,
222:581 (1991 )]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et a!., Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, p. 77
( 1985) and Boerner el al.. J. Immunol., 147( l ):86-95 ( 1991 )]. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent
Nos. 5,545,807; 5.545,806; 5,569,825; 5.625,126; 5.633.425; 5.661.016, and in the following scientific publications: Marks et al., Bio Technology JO, 779-783 ( 1992); Lonberg et al., Nature 368 856-859
(1994); Morrison, Nature 368, 812- 13 ( 1994); Fishwild et a!.. Nature Biotechnology 4, 845-51 ( 1996);
Neuberger, Nature Biotechnology J_4, 826 ( 1996); Lonberg and Huszar, Intern. Rev. Immunol. J_3 65-93 ( 1995).
The antibodies may also be affinity matured using known selection and/or mutagenesis methods as described above. Preferred affinity matured antibodies have an affinity which is five times, more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (generally murine, humanized or human) from which the matured antibody is prepared. 4. Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the PRO. the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light- chain pairs, where the two heavy chains have different specificities [Milstein and Cuello. Nature. 305:537- 539 ( 1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829. published 13 May 1993. and in Traunecker et al., EMBO J., J0:3655-3659 (1991 ).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge. CH2. and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI ) containing the site necessary for light- chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121 :210 (1986).
According to another approach described in WO 96/2701 1 , the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared can be prepared using chemical linkage. Brennan et al., Science 229:81 ( 1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the othei Fab -TNB deriv ative to form the bispecific antibody The bispecific antibodies pioduced can be used as agents for the selectiv e immobilization ot enzv mes
Fab' fragments may be directlv recov ered from E coli and chemically coupled to form bispecific antibodies Shalaby et al . J Exp Med 175 217-225 ( 1992) descnbe the production of a fully humanized bispecific antibody F(ab'); molecule Each Fab' tragment w as sepaiately secreted from E coli and subjected to directed chemical coupling in vitio to torm the bispecific antibody The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic acti ity of human cy totoxic lymphocytes against human breast tumor targets
Various technique for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described For example, bispecific antibodies have been produced using leucine zippers Kostelny et al , J Immunol 148(5) 1547- 1553 ( 1992) The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion The antibody homodimers were reduced at the hinge region to foi m monomei and then te oxidized to form the antibody heterodimers This method can also be utilized for the production of antibody homodimers The "diabody" technology described by Holhnger et al . Pioc Natl Acad Sci USA 90 6444-6448 ( 1993) has provided an alternative mechanism for making bispecific antibody fragments The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain Accordingly, the VH and VL domains of one tragment are forced to pair with the complementary V[ and VH domains ot another fragment, thereby forming two antigen-binding sites Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported See, Gruber et al , J Immunol 152 5368 (1994) Antibodies with more than two valencies are contemplated For example, tπspecific antibodies can be prepared Tutt et al. Immunol J4J 60 ( 1991 )
Exemplary bispecific antibodies may bind to two different epitopes on a given PRO polypeptide herein Alternatively, an anti-PRO polypeptide arm may be combined with an arm w hich binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e g CD2, CD3, CD28. or B7), or Fc receptors foi IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular PRO polypeptide Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PRO polypeptide These antibodies possess a PRO-binding arm and an arm which binds a cytotoxic agent or a radionuchde chelator, such as
EOTUBE, DPTA. DOTA. or TETA Another bispecific antibody of inteiest binds the PRO polypeptide and further binds tissue factor (TF)
5 Heteroconiugate Antibodies
Heterocon|Ugate antibodies are also within the scope of the present invention Heteroconjugate antibodies are composed of two covalently joined antibodies Such antibodies have for example, been proposed to target immune system cells to unwanted cells [U S Patent No 4.676 980], and for treatment of HIV infection [WO 91/00360. WO 92/200373. EP 03089| It is contemplated that the antibodies may be prepared in \ ιtw using known methods in synthetic protein chemistry, including those involving ciosslmkmg agents For example, immunotoxms may be constructed using α disulfide exchange reaction or by forming a thioether bond Examples of suitable reagents for this purpose include lminothiolate and methy l-4-mercaptobutvπmιdate and those disclosed for example, in U S Patent No 4.676.980
6 Effector Function Engineering
It may be desirable to modify the antibody of the invention ith respect to effector function, so as to enhance, g . the effectiveness of the antibodv in treating cancer For example, cysteine resιdue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region The homodimeπc antibody thus generated may have improv ed intemalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See Caron et al . J Exp Med . JJ6 1 191 - 1 195 ( 1992) and Shopes, J Immunol . J48 2918-2922 ( 1992) Homodimeπc antibodies with enhanced anti-tumor activity may also be prepαied using heterobifunctionαl cross-linkers as described in Wolff et al , Cancel Reseaich. 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-Cancei Di ug Design, 3 219-230 ( 1989)
7 Immunoco ugates The invention also pertains to lmmunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (/ e , a radioconjugate)
Chemotherapeutic agents useful in the generation of such lmmunoconjugates have been described above Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeiuginosa), πcin A chain, abπn A chain, modeccin A chain, alpha-sarcin, Aleuriles foi du proteins, dianthin proteins, Phytolaca amencana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonana officinahs inhibitor, gelonin. mitogellin. restπctocin, phenomycin. enomycin, and the tπcothecenes A variety of radionuclides are available for the production of radioconjugated antibodies Examples include 2l2Bι, πιI, π'ln, 9I,Y, and '8<>Re Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succιmmιdyl-3-(2-pyπdyldιthιol) propionate (SPDP), lminothiolane (IT), bifunctional derivatives of lmidoesters (such as dimethyl adipimidate HCL). active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde). bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine). bis-diazomum derivatives (such as bιs-(p-dιazonιumbenzoyl)- ethylenediamine), dnsocyanates (such as tolyene 2.6-dnsocyanate). and bis-active fluorine compounds (such as 1.5-dιfluoro-2.4-dιmtrobenzene) For example, a πcin lmmunotoxin can be prepared as descπbed in Vitetta et al , Science, 238 1098 (1987) Carbon- 14-labeled l -ιsothιocyanatobenzyl-3-methyldιethylene tnaminepentaacetic 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 streptavidin) tor utilization in tumoi pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound con|ugate from the cnculation using a clearing agent and then administration of a "ligand" (e g avidin) that is conjugated to α cytotoxic agent (e g , a radionucleotide) 8 Immunoliposomes
T he antibodies disclosed herein may also be toimulated as immunoliposomes Liposomes containing the antibody are prepared by methods know n in the ait. such as descπbed in Epstein el al Pioc Natl Acad Sci USA. 82 3688 ( 1985). Hwang et al Pioc 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 ev aporation method with a lipid composition comprising phosphatidy lchohne. cholesterol, and PEG-deπv atized 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 Che . 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 Cancel lust , 81( 19) 1484 ( 1989)
9 Pharmaceutical Compositions of Antibodies Antibodies specifically binding a PRO polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders in the form of pharmaceutical compositions
If the PRO 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 et al , Pioc Nail 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, pieferably 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, tor 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 10 Uses for anti-PRO Antibodies
The anti-PRO antibodies of the invention have various utilities For example, anti-PRO antibodies may be used in diagnostic assays for PRO, e g , detecting its expression in specific cells, tissues, or serum Various diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola. Monoclonal Antibodies A Manual of Techniques. CRC Press. Inc ( 1987) pp
147- 158] The antibodies used in the diagnostic assays can be labeled with a detectable moiety The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal For example, the detectable moiety may be a radioisotope. such as λH, l4C. ,_P, 1->S, or l 2"'I. a fluorescent or chemilummescent compound, such as fluoiescein isothiocyanαte. rhodamine. or lucileπn, or an enzyme, such as alkaline phosphatase beta-galactosidase or horseradish peroxidase Anv method known in the art tor conjugating the antibodv to the detectable moiety mav be emploved including those methods described by Hunter et al , Natuie J44 94 ( 1962 ) David al Biochenustn JJ 1014 ( 1974). Pain et al J Immunol Meth .40 219 (1981 ) and Nygren J Hisiochem and Cλtochem 30 407 ( 1982) Anti-PRO antibodies also are useful foi the affinity purification of PRO from recombinant cell culture or natural sources In this process the antibodies against PRO are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art The immobilized antibody then is contacted w ith a sample containing the PRO to be purified and theieatter the support is washed with a suitable solvent that w ill remove substantially all the material in the sample except the PRO which is bound to the immobilized antibody Finally, the support is washed w ith another suitable solvent that will release the PRO from the antibody H Therapeutic Indications
Various disorders can result in death of retinal neurons These include such widely disparate conditions as detachment of the retina and pigment epithelium, degenerative myopia, acute retinal necrosis syndrome (ARN). and traumatic chorioretinopathies or contusion (Purtscher s Retinopathy) Retinal tears is a condition characterized by where the retina tears or separates from the underlying choroid, which can sometimes result in rupturing of the choroid Retinal tears may occur for a wide variety of reasons Particularly troublesome are macular holes which produce blurred central vision or metamorphopsia
While the direct cause of most macular holes is unknown, they have been associated with trauma. cystic degeneration and vitreoretinal traction Also, full thickness macular holes have appeared following myopic degeneration, laser photocoagulation, lightning strike and pilocarpine administration Macular holes are also present in high frequency after cataract extraction A particular form of acute macular holes is ldiopathic senile macular hole, which involves a full thickness hole through the macula surrounded by annular retinal detachment It is believed that macular holes begin with central or toveolar detachment. which then eventually develops into a full-depth macular hole [Gass et al (1988) Aich Ophthalmol 106
629-639] While surgical procedures, such as trans- αra plana vitiectomy may interrupt the progress of macular degeneration to a full blown macular hole, this operation can permanently damage central vision, and typically only improves vision 40% of the time
Other retinal disorders which can result in photoreceptor cell death include edema, ischemic conditions and uveitis Macular and retinal edema are often associated with metabolic illnesses such as diabetes mellitus Retinal edema is found in a large percentage of individuals who have undergone cataract extraction and other surgical procedures upon the eye Edema is also found with accelerated or malignant hypertension Macular edema is a common complication of prolonged inflammation due to uveitis. Eales disease, or other diseases Local edema is associated with multiple cystoid bodies ("cotton bodies") as a result of AIDS
Retinal ischemia can occur from either choroidal or retinal vascular diseases, such as central or branch retinal vision occlusion collagen vascular diseases and thrombocytopemc purpura Retinal vascuhtis and occlusion is seen with Eales disease and systemic lupus erythematosus Age-related macular degeneration (AMD) is the ma|or cause ot seveie v isual loss in United States citizens ov er the age ot 55 AMD may occur eithei in an atrophic or exudativ e form Most AMD patients hav e a build up of deposits w ithin and under the retinal pigment epithelium in the macular region resulting in atiophy of the retina and the retinal pigment epithelium The retinal pigment scav enge for photoieceptor discs from the tods and cones tor years and accumulate intracellular w astes The incompletely digested residues reduce cytoplasmic space and interfere with metabolism [Feeny -Burns, et al . fin est Ophthal Mol Vis Sci . 25 195-200 ( 1984)] As the cell volume available to the organelles diminishes the capacity to digest photoreceptors decreases, which may be the basis for macular degeneration
Exudative AMD is characterized by the growth of blood vessels from the choπocapillαπs through detects in Bruch's membrane, and in some cases the underlying retinal pigment epithelium (RPE) The accumulation of serous oi hemorrhagic exudates escaping from these vessels results in fibrous scarring of the macular region with attendant degeneration of the neuroretina and permanent loss of central vision Exudative AMD has also been associated with choroidal neovasculαπzation, detachment and tears of the retinal pigment epithelium The cascade retinal events is responsible for more than 80% of cases of significant visual loss in patients with AMD
Laser photocoagulation has been attempted in an effort to ameliorate the initial or recurrent neovascular lesions associated with AMD [Aich Ophthalmol , 109 1220 (1991 ). Aich Ophthalmol , J.09 1232 ( 1991 ), Arch Ophthalmol , W9 1242( 1991 )] Unfortunately. AMD patients with subfoveal lesions subjected to laser treatment experienced a severe reduction in visual acuity (mean 3 lines) at 3 months follow-up Moreover, at two years post-treatment treated eyes had only marginally better visual acuity than their untreated counterparts (means of 20/320 and 20/400, respectively) Another drawback of the procedure is that vision immediately after surgery is worse
As a result, the retinal neuron survival agents of the present invention are promising candidates for the treatment of retinal tears, degenerative myopia, acute retinal necrosis syndrome (ARN). and traumatic chorioretinopathies or contusion (including Purtscher's retinopathy). macular holes, macular degeneration
(including age-related macular degeneration or AMD), edema, ischemic conditions (e g central or branch retinal vision occlusion, collagen vacuolar diseases, thrombocytopenic purpura), uveitis and retinal vascuhtis and occlusion associated with Eales disease and systemic lupus erythematosus
I. Administration Methods: The PRO polypeptides of the present invention can be delivered to the eye through a variety of routes Methods of introduction include any mode of administration known in the art, including but not limited to intravenously, lntraarteπally. intrathecally, subcutaneously. intradermally. by injection into involved tissue, intranasally, intramuscularly, lntrapeπtoneally, orally, or via an implanted device They may be delivered intraocularly, by topical application to the eye oi by intraocular injection into, tor example the vitreous or subretinal (interphotoreceptor) space Alternatively, they may be deliveied locally by insertion or inaction into the tissue surrounding the eye They may be delivered systemically through an oral route or by subcutaneous, intravenous or intramuscular injection Alternatively, they may be delivered by means of a catheter or by means of an implant, wherein such an implant is made of a porous, non-porous or gelatinous material, including membranes such as silastic membrane or fibei s. biodegradable polymers. or proteinaceous material. The factors may be administered prior to the onset of the condition, to prevent its occurrence, for example, during surger on the eye. or immediately after the onset of the pathological condition or during the occurrence of an acute or protracted condition.
Intravitreal injection of potential retinal neuron survival promoting factors has several advantages over systemic applications. The amount of any specific agent that reaches the retina can be more accurately determined, since the eye is a round, relatively contained structure and the agent is injected directly into it. Moreover, the amount of agent that needs to be injected is minuscule compared to systemic injections. For example, a single microliter in volume (about 1 microgram of agent) is used for intravitreal injection, as compared to one to several milliliters (ten to several hundred milligrams of agent) necessary for systemic injections. In addition, the intravitreal route of administration avoids the potentially toxic effect of some agents.
Further, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment, this may be achieved by. for example, local infusion during surgery, by injection, by means of a catheter, or by means of an implant, wherein such implant can be of a porous, non- porous, or gelatinous material, including membranes, such as silastic membranes or fibers.
The factors of the present invention may be modified to enhance their ability to penetrate the blood-retinal barrier. Such modification may include increasing their lipophilicity by, for example, glycosylation, or increasing their net charge by methods known in the art.
The factors may be delivered alone or in combination, and may be delivered along with a pharmaceutically acceptable vehicle. Ideally, such a vehicle would enhance the stability and/or delivery properties. The invention also provides for pharmaceutical compositions containing the active factor or fragment or derivative thereof, which can be administered using a suitable vehicle such as liposomes, microparticles or microcapsules. In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the active component. J. Pharmaceutical Compositions and Dosages
Therapeutic formulations of the PRO polypeptide are prepared for storage as lyophilized formulations or aqueous solutions by mixing the polypeptide having the desired degree of purity with optional "pharmaceutically-acceptable" or "physiologically-acceptable" carriers, excipients or stabilizers typically employed in the art (all of which are termed "excipients"). For example, buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and other miscellaneous additives. (See Remington 's Pharmaceutical Sciences. 16th edition, A. Osol. Ed. ( 1980)). Such additives must be nontoxic to the recipients at the dosages and concentrations employed.
Buffering agents help to maintain the pH in the range which approximates physiological conditions. They are preferably present at concentration ranging from about 2mM to about 50 mM. Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof such as citrate, succinate, tartrate, fumarate, gluconate, oxalate. lactate, acetate, phosphate, and histidine buffers. Additionally, there may be mentioned phosphate buffers, histidine buffers and trimethylamine salts such as Tris. Preservativ es are added to retaid microbial grow th and aie added in amounts ranging from 0 2% - 1%- (w/v ) Suitable preservatives tor use w ith the present inv ention include phenol, benzyl alcohol, meta cresol. methy l paiaben. propy l paraben. octadecv ldimethv lbenzy I ammonium chloride, benzalconium halides (e g . chloride, bromide, iodide), hexamethonium chloride, alky l parabens such as methv l or propy l paraben. catechol. resorcinol cyc ohexanol. and 3-pentanol
Isotonicifiers sometimes known as "stabilizers" are present to ensure isotonicity of liquid compositions of the present invention and include polyhydπc sugar alcohols, preferably tπhydπc or higher sugar alcohols, such as glyceπn. erythπtol. arabitol. xylitol. sorbitol and manmtol Polyhydπc alcohols can be present in an amount between 0 1 % to 25%- by weight preteiably 1 % to 5% taking into account the relative amounts of the other ingredients
Stabilizers refer to a broad category of excipients which can lange in function from a bulking agent to an additive which solubihzes the therapeutic agent or helps to prevent denaturation oi adherence to the container wall Typical stabilizers can be polyhydπc sugai alcohols (enumerated above), amino acids such as argimne, lysine. glycine, glutamme. asparαgine. histidine. alanine. ormthine, L-leucine, 2-phenylalanιne. glutamic acid, threonine, etc , organic sugars or sugar alcohols, such as lactose, trehalose. stachyose, mannitol. sorbitol. xylitol. πbitol, myoimsitol, galactitol, glycerol and the like, including cychtols such as mositol, polyethylene glycol, amino acid polymers, sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, σ-monothioglycerol and sodium thio sulfate, low molecular weight polypeptides (I e < 10 residues), proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins, hydrophihc polymers, such as polyvinylpyrrohdone monosacchaπdes. such as xylose, mannose, fructose, glucose, disacchaπdes such as lactose, maltose, sucrose and tπsacchaπdes such as raffinose, polysacchaπdes such as dextran Stabilizers can be present in the range from 0 1 to 10,000 weights per part of weight active protein
Non-ionic surfactants or detergents (also known as "wetting agents") are present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein Suitable non-ionic surfactants include polysorbates (20. 80, etc ), polyoxamers ( 184, 188 etc ), Pluronic0 polyols, polyoxyethylene sorbitan monoethers (Tween®-20, Tween®-80. etc ) Non-ionic surfactants are present in a range of about 0 05 mg/ml to about 1 0 mg/ml, preferably about 0 07 mg/ml to about 0 2 mg/ml
Additional miscellaneous excipients include bulking agents, (e g starch), chelating agents (e g EDTA), antioxidants (e g . ascorbic acid, methionine, vitamin E), and cosolvents
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 For example, it may be desirable to further provide an immunosuppressive 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 microcapsule prepared, for example, by coascervation techniques oi by interfacial polymerization, foi example, hydioxymethylcellulose or gelatin- microcapsule and polv-(methv lmethacv late) microcapsule respectiv elv in colloidal drug dehverv sv stems (tor example liposomes albumin microspheres mici oemulsions nano particles and nanocapsules) or in macroemulsions Such techniques are disclosed in R mington s Phaimac eutical Sc i nces 16th edition A Osal Ed ( 1980) Sustained release preparations mav be prepared Suitable examples ot sustained lelease preparations include semi permeable matrices of solid hydrophobic polymers containing the antibody mutant which matrices are in the form of shaped articles e g films oi microcapsules Examples of sustained-release matrices include polyesters hydrogels (for example poly(2 hvdroxyethyl methacrylate) or poly(vιnylalcohol)), polylactides |U S Pat No 3 773 919] copoly mers of L glutamic acid and ethyl-L- glutamate non degradable ethylene-vin l acetate degradable lactic acid-glycohc acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid gh colic acid copolymer and leuprohde acetate), and poly D-(-)-3-hvdroxybutyπc acid While polymers such as ethylene v inyl acetate and lactic acid-glycohc acid enable release of molecules for ov er 100 days certain hydrogels release proteins for shorter time periods When encapsulated antibodies remain in the bodv for a long time they may denatuie or aggregate as a result of exposure to moisture at 37°C resulting in a loss of biological activity and possible changes in immunogenicity Rational strategies can be devised for stabilization depending on the mechanism involved For example if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues lyophilizing from acidic solutions, controlling moisture content using appropriate additives, and developing specific polymer matrix compositions
The formulations to be used for ;/; vivo administration must be sterile This is readily accomplished, for example, by filtration through sterile filtration membranes The amount of PRO polypeptide which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques Where possible it is desirable to determine the dose response curve and the pharmaceutical compositions of the invention first ;// \ ιtιo and then in useful animal model systems prior to testing in humans Interspecies scaling of effective doses can be performed following the principles laid down by Moienti J and Chappell W , "The use of interspecies scaling in toxicokmetics", Toxicokinetics and New Diug De\ elopment, Tacobi et al ,
Eds Pergamon Press, New York 1989, pp 42-96 However, based on common knowledge of the art a pharmaceutical composition effective in promoting the survival of sensory neurons may provide a local therapeutic agent concentration of between about 5 and 20 ng/ml and preferably between about 10 and 20 ng/ml In an additional specific embodiment of the invention a pharmaceutical composition effective in promoting the growth and survival of retinal neurons may prov ide a local therapeutic agent concentration of between about 10 ng/ml and 100 ng/ml Additional guidance to particular dosages and methods of delivery is provided in the literature
In a prefened embodiment an aqueous solution of PRO polypeptide is administered by subcutaneous injection Each dose may range from about 0 5 ug to about 50 ug per kilogram of body weight or more preferably, from about 3 ug to about 30 μg per kilogram body weight The dosing schedule for subcutaneous administration may v aiy form once a week to daily depending on a number of clinical factois. including the tv pe of disease, sev enty of disease and the subject s sensitiv ity to the theiapeutic agent Additional guidance to particulai dosages and methods of delivery is provided in the hteratuie - see. for example. U S Pat Nos 4.657.769. 5.206.344 or 5.225.212 It is anticipated that different formulations will be effectiv e for different treatment compounds and difterent disorders, that administration targeting one organ or tissue, tor example, may necessitate delivery in α manner different fiom that to another organ or tissue
The amount of PRO protein which will be effectiv e in the tieatment ot a particular disoidei oi condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques Where possible it is desirable to determine the dose-response curve and the pharmaceutical compositions of the inv ention first ;;; \ ιtιo, and then in useful animal model systems prior to testing in humans Howevei . based on common knowledge of the art. a pharmaceutical composition effective in promoting the survival of sensory neurons may provide a local PRO protein concentiation of between about 10 and 1000 ng/ml. preferably between 100 and 800 ng/ml and most preferably between about 200 ng/ml and 600 ng/ml ot PRO In an additional specific embodiment of the invention, a pharmaceutical composition effective in promoting the growth and survival ot retinal neurons may provide a local PRO protein concentration ot between about 10 ng/ml and 1000 ng/ml
The dosing schedule for subvitreous administration of PRO may vary from once a week to daily depending on a number of clinical factors, including the type of disease, severity of disease, and the subject's sensitivity to PRO. respectively Nonhmiting examples of dosing schedules are 3 μg/kg administered twice a week, thiee times a week or daily, a dose of 7 ug/kg twice a week, three times a week or daily, a dose of 10 μg/kg twice a week, three times a week or daily
Effective doses of additional neurotrophic factors administered in combination with PRO, such CNTF are in the same dose ranges as the effective dose of PRO described herein The PRO polypeptide may also optionally be formulated with a second agent, such as a neurotrophic factor Exemplary neurotrophic factors include nerve growth factor (NGF), aGF. ciliary neurotrophic factoi (CNTF). bovine- deπved neurotrophic factor (BDNF). neurotrophιn-3 (NT-3), neurotrophιn-4 (NT-4), aFGF, IL- 1 β, TNFα, Insulin-like growth factor (IGF- 1. IGF-2), transforming growth factor beta (TGF-β, TGF-β l ) or skeletal muscle extract, may be administered in any sterile biocompatible pharmaceutical carrier, including, but not limited to. saline, buffered saline, dextrose, and water However, certain factors such as bFGF. CNTF or IL- l β should be employed cautiously, as these agents may cause undesirable retinal complications such as macrophage proliferation, disorganization oi the retinal structure, cell proliferation or inflammation
If the subject manifests undesired side effects such as temperature elevation, cold or flu-like symptoms, fatigue, etc . it may be desirable to administer a lower dose at more frequent intervals One or more additional drugs may be administered in combination with PRO to alleviate such undesired side effects, for example, an anti-pyretic, anti-inflammatory or analgesic agent III. MODES FOR CARRYING OUT THE INVENTION:
A. Retinal Neuron (including photoreceptor) Surviv al assays:
In these assays neuial letinas αie removed from pigment epithelium and dissociated into a single cell suspension using 0 25% try psin in Ca~+. Mg"+-free PBS The cells are then plated out in 96-welI plates at 100.000 cells per well in DMEM/F12 supplemented w ith N2 After 2-3 day in culture, the cells are fixed and stained Since death typically occuis upon detachment of neuial retinal cells from the underlying pigment epithelium the relative survival enhancing effect of the tested agent can be readily detei mined by comparison with the untreated control wells The procedure is described in greater detail in the examples
B. Age-related macular degeneration (AMD): In this assay, the effectiveness and safety of locally administered PRO (hFIZZ- 1 ) is examined using a procedure substantially similai to that outlined in WO 94/01 124, filed 8 July 1993 which describes subretinal or intravitreal injections of retinal survival promoting therapeutic agents Briefly, patients with visual acuity of 20/160 or better with a recent diagnosis of AMD are examined for change in visual acuity from baseline and stabilization Study parameters should measure best corrected visual acuity for both distance and near vision, intraocular pressure, lens status and refraction The amount of serous and hyperfluorescence from classic/occult neovasculaπzation. total lesion size and foveal involvement are also measured on fluorescein angiography and ICG (indocyanine green) angiography
C. Macular holes:
In this assay, the safety and effectiveness of locally administered PRO is examined using a procedure substantially similar to that outlined in WO 94/01 124, filed 8 July 1993 which describes subretinal or intravitreal injection of retinal survival promoting therapeutic agents Briefly, patients with confirmed macular holes are examined for visual acuity and analyzed by intraocular piessure. fundus photographs, and fluorescein angiography
The rational for treatment is to induce the flattening of the edges of the macular hoes in order to resolve retinal detachment and thickening surrounding the hole It is believed that a reduction in the traction force which elevates the retina around the hole coupled with and induction of the choπoretinal adhesion along the edge ot the hole is necessary for therapeutic effect
The procedure is described in more detail in the examples
D. Light-induced photoreceptor injury: In this assay, an albino rat is maintained in first a cyclic light environment followed by exposure to a constant light source with and without administration of the tested photoreceptor survival agent The intravitreal administiation of factors into the eyes of albino rats enables the assessment of both the ability of the factors to rescue photoreceptors from degeneration as well as the side ettects. such as incidence of macrophages. associated with each factor Briefly, rats are given intraocular injections prior to constant light exposure and compared to control animals who received sham and no injections Subsequent to constant light exposure, the eyes are removed, embedded in epoxy resin and sectioned along the vertical meridian The degree ot light-induced retinal degeneration can be measured first by examining the outer nuclear layer thickness and second by a subjective score assigned to the relative integrity of the retina E. Light ablation:
In this assay, the degree of photoreceptor rescue is measured in female Spi αgue-Daw ley rats in a modification ot the procedure described in Reme et al . Degcn Dis Retina. Ch 3. Ed R E Anderson et al . Plenum Press. New Yoik ( 1995) Briefly , animals are fust acclimated to cyclical lighting, followed by immersion in total darkness Animals are injected w ith test factor prior to intermittent light exposuie The degree of retinal degeneration or survival promoting activ ity of the tested factors is reported as the thickness ot the photoreceptor cell layer or number of TUNEL labeled photoreceptor cell nuclei
F. Corneal Pocket assay:
In this assay, particular agents are tested to determine whether they are angiogemc under a procedure adapted from Polveπni et al . Methods EIIZMIIOI 198 440-450 ( 1991 ) Briefly. Sprague-Dawley are anesthetized, secured and an incision is made in their corneas into which is placed a pellet of the test factor in combination with sucralfate and Hydron
G. Vascular Endothelial Cell Mitogenicity Assay:
This particular assay measures the mitogenicity (e g , angiogenesis) of the test factor on vascular endothelial cells It was developed as a reliable means of measuring the purification of bFGF (SEQ ID
NO.4) as described by Ferrara, et al ,
Figure imgf000085_0001
198 391 -405 ( 1991 ) Briefly, bovine adrenal cortex-derived cells are grown and maintained in culture in the presence of low glucose DMEM. the test factor is administered and test cultures vs controls are measuied
IV. Assay characterizations: Correlation between in vitro assays and in vivo therapeutic effect
Recent studies using agarose gel electrophoresis and terminal dUTP nick-end labeling (TUNEL) indicate that photoreceptor cell death occurs principally by apoptosis [Chang et al . Neuron JJ_ 595-605 ( 1993), Portera-Cailhau, C et al , Pioc. Nat'l Acad Sci USA 9J_ 974-97 ( 1993), Adler R , Curr Top Dev Biol , J_6_ 207-252 ( 1980)] These studies examined mouse models of human retinal degeneration (letimtis pigmentosa) id mice (which have a mutation in the b subunit ot cGMP phosphodiesterase), ids mice (which have a mutation in peπpheπn). and tiansgemc mice, which have a mutation in rhodopsin In all three models there is a substantial increase in apoptosis at the time ot photoreceptor cell death Apoptosis is also known to be prominent in the RCS rat. as well as in the light-damaged rat retina [Tso M. et al , Invest Opththalmol Vis Sci , 35, 2693-2699 (1994). Shahinfar S , et al . Curi Eye Res , J O. 47-59 ( 1991 )] Apoptosis appears to be a tightly controlled "shutdown" process or self-selecting cell suicide which by preventing the leakage of destructive enzymes, allows healthy neighboring cells to continue their normal functioning [Wong, F , Aich Ophthalmol 1 13 1245-47 ( 1995)] During this process, the cell's outer membrane remains intact as the cell undergoes nuclear condensation, cytoplasmic shrinkage, membrane blebbing. formation of apoptotic bodies, and sometimes DNA fragmentation Apoptosis is now believed to play a key role in degenerative diseases of the eye, such as letimtis pigmentosa RP is believed to be caused by mutations in the rhodopsin gene [Dryja. TP, Nature, 343 364- 366 ( 1990)] In addition, other photoreceptor-specific genetic mutations have been uncovered which induce RP. among them the mutants known as retinal degeneration (id) [McLaughlin ME, et al Nat Genet . 4_ 30- 134 ( 1993)], and retinal degeneration slow (ids) [Farrar G J et al Natui e 354 478 80 ( 1991 ) Kajiwara K et al . Natuie 354 480-83 ( 1991 )] It has further been discoveied that the autosomal dominant types of RP mav be caused by any one ot more than 70 mutations of the rhodopsin gene [Humphries. P et al . Science. 256 804-808 (1992) Dryja T P et al , fin est Ophthalmol Vis Sci . 36 1 197- 1200 ( 1995)] Rhodopsin mutations are know n to be the basis of autosomal recessive RP in some families as well
IRosenfeld. P J et al . Nat Genet . J_ 209- 13 (1992) Kumaramanickavel, G et al 8 10 1 1 ( 1994)] As a result, the rhodopsin gene is now considered an archetypal model for the study of RP
The role of apoptosis in RP has been observed in mouse photoreceptors Several lines of transge c mice which express mutant rhodopsin have been created, and as a result can simulate a form of the autosomal dominant RP found in humans These animal models exhibit dying photoreceptors through various chaiacteπstics of apoptosis. including morphological changes and DNA fragmentation [Chang C-G et al Neuwn. U 595-605 ( 1993). Portera-Cailliau C et al, Pioc Natl Acad Sci USA 9J_ 974-978 ( 1994)] Along with other experimental results, these findings have led researches to the conclusion that apoptosis is a major mechanism of murine photoreceptor death, as it is induced not only by mutations in the rhodopsin gene, but also by mutations in the id and rds genes Chang C-G et al . supra. Portera-Cailliau C et al , supra, Lolley R N et al , fin est Ophthalmol Vis Sci , 35 358-362 (1994)
Of great interest is the observation that photoreceptor degeneration occurs through apoptosis in response not only to genetic abnormalities, but also after experimental retina detachment [Cook, BE et al , Invest Ophthalmol Vis Sci , 36 990-996 ( 1995)] Moreover, apoptotic cell death was also observed in acute retinal lesions in the albino rat induced by relatively low light levels and short exposure duration
(1000 & 3000 lux, diffuse, white light for 2 hours) [Reme et al , Degenerative Diseases of the Retina, Anderson R E et al , eds, Plenum Press, pp 19-25 (1995)] This discovery has lead to the search for survival-promoting trophic factors, factors which are believed to become unavailable to photoreceptors when the subretinal space expands and the composition of the interphotoreceptor matrix changes as a consequence ot retinal detachment [Chader G J , fin est Ophthalmol Vis Sci 30_ 7-22 ( 1989). Berman
E R , Biochemistn of the E\e, New York, NY, Plenum Press. Steinbeig R H . Ctii i Opin Neuiobiol 4_ 515-24 ( 1991 )]
The death of photoreceptor cells through apoptosis is indicative that rather than being passive victims of the cumulative effects of mutations, photoreceptors die in genetic disorders such as letimtis pigmentosa by activation of their own "cell-death program" [Adler, R , Aich Ophthalmol 1 14 79-83
(1996)] This implies that there is a role which certain neurotrophic factors and related molecules play in the degeneration ot cones resulting from mutations in rod proteins
The following examples are demonstrative of therapeutic utility because cell death occurs via apoptosis, the same mechanism as has been shown to occur in various retinal degenerativ e disorders The knowledge that known growth factors prevented apoptosis correlated with preserved v ision in animal models is indicative that prospective factors which prevent apoptosis would also hav e therapeutic utility in retinal degenerative disorders The follow ing examples are offered by way of illustration and not by wav of limitation The disclosures of all citations in the specification are expresslv incorporated herein bv reteience
EXAMPLES Example 1
Macular holes
A patient pool with v ary ing stages of macular holes (i e . 2. 3 or 4) ot varying ages are chosen and the presence of macular holes is confirmed The pool is selected so as to exclude patients w ith histories of cystoid macula edema, diabetic retinopathy or exudative age-related macular degeneration Vision is examined in each subject to determine the best Snellen visual acuity and analyzed by intraocular pressure, fundus photographs, and fluorescein angiography Each macular hole is graded according to the criteria described by Gass. Aich Ophthalmol ( 1988), 106 629-39 Eyes with Stage 2 holes have a retinal dehiscence along the margin of the areas of deep retinal cyst formation Stage 3 is characterized by a full-thickness hole with overlying operculum Macular holes are classified as Stage 4 when a posterior vitreous detachment is present Treatment is scheduled within 2 weeks of the baseline examination Under the criteria, patients should be excluded if they had greater than 2+ nuclear sclerotic or posterior subcapsular lens changes Patients are followed for 6- 10 months, with mean follow-up of 8 months Doses are determined at a level below therapeutic effectiveness, in the middle of the effective range, and at a level well above the minimal effective range Eyes are randomly chosen for the indicated level of PRO In addition, some eyes may separately receive 100 μl of intravitreal hyaluromc acid at the time of installation of PRO in an attempt to delay clearance of PRO from the area of the macular hole Surgical procedure
All surgery can be done under local anesthesia with sedation After the eyes are prepped and draped, a standard three-port vitrectomy may be performed In eyes with Stage 2 and Stage 3 macular holes, a core vitrectomy is performed In Stage 4 macular hole, α complete pat s plana vitrectomy is performed
If encountered, an epiretinal membrane may be peeled from the surface of the retina and removed from the eye In other cases, some gelatinous condensation on the inner surface of the retina surrounding the macular hole for about 200-400 am, with a firm adhesion along the margin of the macular hole This was carefully dissected where possible, taking care to limit traction on the edges of the macular hole and damage to the nerves
After allowing for peripheral fluid to drain posteriorly, any fluid which migrates posteriorly is also aspirated A tapered, bent-tipped cannula is then connected to a 1 cc syringe containing a solution of PRO
The reconstituted formulation contains the desired concentration of PRO aftei dilution Eyes are randomly assigned a dose of PRO About 0 1 cc of PRO solution is gently infused into the macular hole The same volume of hyaluiomc acid may also be administered
After surgery, the patient should he in a supine position for the first 24 hours following surgery Thereafter, each patient should remain in a face-down position as much as possible for a 2 week period Patients are examined at 1 dα\ 2 weeks 4 6 weeks and monthly post surgerv Fluoiescein dngiographv is pei toimed at 4 to 6 weeks 3 months and 6 months Best corrected Snellen v isual acuitv intraocular pressure lens status bubble size status ot macular hole and occurrence of adv erse effects are determined at each examination
Discussion
The rational tor treatment in this example is to induce the flattening ot the edges ot the macular hole in order to resolve letinal detachment and thickening surrounding the hole It has been suggested that a reduction in the traction force which elevates the retina around the hole coupled with and induction of the choπoretinal adhesion along the edge of the hole is necessary for therapeutic effect Unlike peripheral retinal holes where surgical techniques can be used to reattach the retina and a small area of destruction is not noticeable macular holes require gentle induction of choπoretinal adhesion to avoid the destruction of adjacent neurosensory tissue and permanent destruction of central vision
Example 2
Light Induced Photoreceptor Injury
Albino rats (F344 of Sprague Dawley) of 2 5 months of age are maintained in a cyclic light environment ( 12 hours on followed by 12 hours off from an in cage illuminance of less than 25 ft c) for 9 days or more days before exposure to a constant light source The constant light source is maintained at an illuminance level of 1 15 200 ft c For example, 2 40 watt white reflector fluorescent bulbs suspended 60 cm above the floor of a transparent polycarbonate cage with stainless steel wire-bar covers
Two days before the constant light exposure the rats are anesthetized with a ketamme xylazine mixture which is administered intravitreally with 1 ul of the tested factor dissolved in phosphate buffered saline (PBS) at a concentration ot 50 1000 ng/ul The injections weie made with the insertion of a 32 gauge needle through the sclera choroid and retina approximately midway between the ora serratα and equator of the eye The factor injected animals are compared to eithet unmjected httermates of those that receive control injections as well as to control animals who are not exposed to constant light Controls should include an injection of PBS alone, or a sham injection (insertion of needle with no injection) In all cases, the injections are made into the superior hemisphere of the eye Immediately following the constant light exposure the rats are killed by any suitable means, c g carbon dioxide anesthetization followed by vascular perfusion of mixed aldehydes The eyes are embedded in epoxv resin and sectioned into 1 um thick sections of the entire retina along the vertical meridian of the eye The degree ot light induced retinal degeneration is then quantified b\ two methods The first is through measurement of the outer nuclear layer (ONL) thickness which is used as an index of photoreceptoi cell loss A mean ONL thickness is obtained from a single section of each animal with the aid of a Bioquant morphometry system In each of the supeπoi and inferior hemispheres ONL thickness is measured in 9 sets of 3 measurements each (total of 27 measurements in each hemisphere) Each set is centered on adjacent 440 um lengths ot retina (the diameter of the microscope field at 400X magnification) The first set of measurements is taken at approximately 440 μm from the optic nerve head, with subsequent sets taken more peripherally. Within each 440-μm length of the retina, the 3 measurements are made at defined points separated from one another by 75 um. In all. 54 measurements are taken in the two hemispheres which sample representative regions of almost the entire retinal section. The second method of assessing the degree of photoreceptor rescue is through a subjective evaluation by an examining pathologist on a scale of 0-4-κ wherein 4+ is maximal rescue and nearly normal retinal integrity. The degree of photoreceptor rescue in each section, based in comparison to the control eye in the same rat. is scored by four individuals. This method not only takes into account the ONL thickness. but also more subtle degenerative changes to the photoreceptor inner and outer segments, as well as degenerative gradients within the eye.
Discussion:
The intravitreal administration of various factors into the eyes of albino rats can enable the rapid assessment of both the ability of the factors to rescue photoreceptors from degeneration and the side effects, such as incidence of macrophages, associated with each factor. Although the model described herein is the albino rat, the eyes of other albino mammals, such as mice and rabbits, are also useful for this purpose.
Example 3 Retinal Neuron Survival
Sprague Dawley rat pups at postnatal day 7 (mixed population: glia and retinal neuronal types) are killed by decapitation following C02 anesthesia and the eyes are removed under sterile conditions. The neural retina is dissected away from the pigment epithelium and other ocular tissue and then dissociated into a single cell suspension using 0.25% trypsin in Ca~+, Mg"+-free PBS. The retinas are incubated at 37°C for 7-10 minutes after which the trypsin is inactivated by adding 1 ml soybean trypsin inhibitor. The cells are plated at 100,000 cells per well in 96 well plates in DMEM/F12 supplemented with N2. Cells for all experiments are grown at 37°C in a water saturated atmosphere of 5% COi. After 2-3 days in culture, cells are stained with calcein AM then fixed using 4% paraformaldehyde and stained with DAP1 for determination of total cell count. The total cells (fluorescent) are quantified at 20X objective magnification using CCD camera and NIH image software for Macintosh. Fields in the well are chosen at random. The effect of various concentration of PRO polypeptides are reported in Table 7.
Table 7 Retinal Neuron Surv ival
Figure imgf000090_0001
Example 4
Rod Photoreceptor Survival
Sprague Dawley rat pups at 7 day postnatal (mixed population glia and retinal neuronal cell types) are killed by decapitation following CO-> anesthesis and the eyes are removed under sterile conditions The neural retina is dissected away form the pigment epithelium and other ocular tissue and then dissociated into a single cell suspension using 0 25% trypsin in Ca2+, Mg2+ free PBS The retinas are incubated at 37°C foi
7- 10 minutes after which the trypsin is inactiv ated by adding 1 ml soybean trypsin inhibitor The cells are plated at 100,000 cells per well in 96 well plates in DMEM/F12 supplemented w ith N2 Cells for all experiments are grown at 37CC in a watei satuiated atmosphere oi 5% COi After 2-3 days in culture cells are fixed using 4% paraformaldehyde and then stained using CellTrackei Green CMFDA Rho 4D2 (ascites or IgG 1 100), a monoclonal antibody directed towards the visual pigment rhodopsin is used to detect rod photoreceptor cells by indirect immunofiuorescence The results are reported as % survival total number of calcein rhodopsin positive cells at 2-3 days in culture, divided by the total number of rhodopsin positive cells at time 2-3 days in culture The total cells (fluorescent) are quantified at 20x objective magnification using a CCD camera and NIH image software for Macintosh Fields in the well are chosen at random
The effect of various concentration of PRO pol peptides are reported in Table 8 Table 8 Rod hotorece tor survi al
Figure imgf000091_0001
Example 5
Light Ablation Study
Introduction
As indicated by Reme C E et al, Degen Dis Retina, Ch 3, Ed R E Anderson et al , Plenum Press, New York (1995), retina degeneration can be induced by exposure to strong light This light ablation model permits a quantitative comparison ot photoreceptor survival promoting activity of a tested substance
Methods
Adult female Sprague Dawley rats are kept in "normal" fluorescent light environment (50 foot candles) for 12 hours on/off until the beginning of the experimental period Light-induced degeneration is initiated through dark adaptation performed by keeping rats in 24 hour total darkness About 5- 10 animals in each treatment group are placed into a 5' x 3 chamber illuminated with 490 580 nm (green) light at 300-
400 toot candles Light exposure is intermittent, 1 hour on, 2 hours off, for a total of eight cycles Both eyes of each animal receive 1 2 ul vitreal injections of test factor two days prior to the light exposure Test factors employed are 0 5 - 1 0 μg/μl of PRO polypeptide and phosphate buffeted saline with and without bovine sei um albumin (0 1 %) controls Tdt-mediated dUTP nick-end labeling (TUNEL) (Gavπeli, Y et al J Cell Biol JJ9 493-501
(1992), is performed with modifications using the ApopTag® In Situ Apoptosis Detection Kit (Oncor, cat no S71 10-KIT) on a 4 um thick paraffin sections The DNA strand breaks (fragments) are labeled with fluorescein while intact DNA aie labeled with DAPI (4',6-dιamιdιno-2-phenv hndole) and visualized with a FITC/DAPI filter on a Vanox AH-3 Olympus microscope Results
The degree of retina! degeneiation or survival promoting activ ity of the tested tactoi s is reported as the thickness of the photoreceptor cell layer or number of TUNEL labeled photoieceptoi cell nuclei Three transverse sections through the cential retina (approx 10 um intervals) are used toi the analysis For each section, the entire retinal surface area is digitized using a cooled CCD camera and NIH image software (Macintosh) to derive the quantitative data
Example 6 Corneal Pocket Assay Introduction
This expeπment is intended to determine whether the tested agent is angiogemc in this rodent m vivo model Sample are formulated and pelleted with a delivery vehicle and stability and then transplanted into the cornea and then observed for angiogemc effect The procedure has been adapted from Polveπm et al . Methods in
Figure imgf000092_0001
J98 440-450 ( 1991 ) Methods
Sprague-Dawley iats (250 g. male) are maintained in plastic carriers under darkened conditions 24 hours prior to treatment and then anesthetized Each ammαl s eyes are gently proptosed and secured in place with nontraumatic forceps (BRI- 1725) Using a No 15 blade (Baid-Parker). a 1 5 mm incision is made approximately 1 mm from the center of the cornea into the stroma. but not through it A curved spatula [2 mm w ide. ASSI ST 80017] is then inserted under the lip of the incision and gently blunt-dissected through the stroma toward the outer canthus of the eye The final distance between the base of the pocket and the hmbus should be at least 1 mm
Pellets are prepared by mixing together tested growth factor ( 100 ng), sucralfate (50 μg, BM Research. Denmark) and Hydron (Interferon Sciences, New Brunswick. N J , Lot # 90005) in a 500 1 ratio of growth factor to sucralfate and Hydron (4 μl) The sucralfate is present to stabilize the molecule by interacting with the hepaπn-binding region The control pellet is composed of Hydron and sucralfate vehicles only Three treatment groups tested are composed of I ) bovine bFGF (SEQ ID NO 4) (Calbiochem, 10 ug/50 ul) PBS + sucralfate (6 animals). 2) sucralfate control (3 animals), and 3) PRO polypeptide + sucralfate (e g , at least 6 animals) A Hydron pellet (2 x 2 mm) prepared as described in the previous paragraph is inserted into the base of incision whereupon the pocket should reseal spontaneously The eyes are coated with artificial tears ointment and then the animals are returned to their plastic carriers, then permitted to awaken and returned to their cages
The assay is terminated on day 5 At time of sacrifice the animals are perfused with FITC dextran (2 x 10f' m w ) and cornea! whole mounts prepared by careful dissection of the cornea from the eyes followed by strategic placement of 2-3 cuts to permit permitting the cornea to he flat, followed by placement under a coverslip The image can be captured through a l x objective mounted on a Nikon inverted fluorescent scope Image-Pro® sottw are-edge detection routine can be used to evaluate growth areas Example 7 \ ascular Endothelial Cell Mitogenecitv Assay
Introduction
Mitogenic assays on vascular endothelial cells were initiallv dev eloped in order to monitor the purification ot bFGF growth factor Howev er they are also a useful measure to determine the presence ot mitogenicity in the tested substance Materials and Methods
Bovine adrenal cortex derived capillai y endothelial (ACE) cells are established according to know n procedures as described by Ferrara et al
Figure imgf000093_0001
J98 391 -405 ( 1991 ) Stock plates of ACE cells are were maintained in 10 cm tissue culture dishes in the piesence of low glucose DMEM supplemented w ith 10% calf serum 2 mM glutamine and penicillin G ( 1000 Units/mL) and streptomycin (1000 ug/mL) and basic FGF at a final concentration of 1 ng/ml and weekly passaged at a split ratio of 1 10 Mitogenic controls are prepared by adding basic FGF at final concentrations of 1 ng/ml and 5 ng/ml and culturing for 5-6 days ACE cells can be passaged 10 12 times before showing signs of senescence For each the test substances the stock cultures are trypsinized resuspended in growth media and seeded at a density of 1 0 x 10 cells/well in 6 well plates (Costar Cambridge MA) at a plating volume of 2 ml PRO samples to be tested are added to duplicate or triplicate wells in 10 ul ahquots shortly after plating After 5 oi 6 days cells are trypsinized and counted in a Coulter Counter (Coulter Electronics Hialeah. FL)
Example 8 Identification of clones encoding a PRO200 (VEGF-E) related protein Probes based on an expressed sequence tag (EST) identified from a private EST database provided by Incyte Pharmaceuticals (LileSeq®) which exhibited homology with VEGF (v ascular epithelial growth factor) were used to screen a cDNA librarv derived from the human glioma cell line G61 In particular
Incyte clone "INC 1302516 (SEQ ID NO 41 , Figure 22) was used to generate the following tour probes 5'-AC rTCTCAGTGTCCATAAGGG 3 (SEQ ID NO 6)
5'-GAACTAAAGAGAACCGATACCATTTTCTGGCCAGGTTGTC-3' (SEQ ID NO 40) 5'-CACCACAGCGTTTAACCAGG-3' (SEQ ID NO 7) 5'-ACAACAGGCACAGTTCCCAC-3' (SEQ ID NO 8)
Nine positives were identified and characterized Three clones contained the full coding region and were identical in sequence Partial clones were also identified from a fetal lung library and were identical in sequence Partial clones were also identified fiom a fetal lung library and were identical with the glioma derived sequence with the exception of one nucleotide change, which did not alter the encoded amino acid Example 9 Expression Constructs for PRO200 (VEGF-E)
For mammalian protein expression, the entire open reading tiαme (ORF) was cloned into a CMV- based expression vectoi An epitope-tag (FLAG Kodak) and Histidine-tag (Hιs8) were inserted between the ORF and stop codon VEGF-E-Hιs8 and VEFG-E-FLAG were transtected into human embryonic kidney 293 cells by SuperFect1 M (Qiagen) and pulse-labeled for 3 hours with ( ^S)-methιomne and XS)- cysteine Both epitope-tagged proteins co-migrate when 20 microliters ot 15-fold concentrated serum-free conditioned medium were electrophoresed on a polyacrylamide gel (Novex) in sodium dodecyl sultate sample buffer (SDS-PAGE) The VEGF-E-IgG expression plasmid was constructed by cloning the ORF in tront ot the human Fc (IgG) sequence
The VEGF-E-IgE plasmid was co-transtected with Baculgold™ baculovu us NDA (Pharmingen) using Lipotectin™ (GibcoBRL) into 10s Sf9 cells grown in Hink's TM TNM-FH medium (JRH Biosciences) supplemented with 10% fetal bovine serum Cells were incubated tor 5 days at 28°C The supernatant was harvested and subsequently used for the first viral amplification by infecting Sf9 cells at an approximate multiplicity of infection (MOI) of 10 Cells were incubated for 3 days, then supernatant was harvested, and expression of the recombinant plasmid was determined by binding of 1 ml of supernatant to 30 ml of Protein-A Sepharose™ CL-4B beads (Pharmacia) followed by subsequent SDS-PAGE analysis The first amplification supernatant was used to infect a 500 ml spinner culture of Sf9 cells grown in ESF- 921 medium (Expression Systems LLC) at an approximate MOI of 0 1 Cells were tieated as above, except harvested supernatant was sterile filtered Specific protein was purified by binding to Protein-A Sepharose
4 Fast Flow™ (Pharmacia) column.
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence PRO200 [herein designated UNQ174, VEGF-E, DNA29101 -1276 (SEQ ID NO 1 )] Clone DNA29101 contains a single open reading frame with an apparent translation initiation site at nucleotide positions 285- 287 and ending at the stop codon at nucleotide positions 1320- 1322 (Figure 1 ) The predicted polypeptide precursor is 345 amino acids in length (Figure 2. SEQ ID NO 2) The full-length PRO200 polypeptide shown in Figure 2 has an estimated molecular weight ot about 39,029 daltons and a pi of about 6 06 Important regions of amino acid sequence of PRO200 include a signal sequence at about residues 1 - 14. N- glycosylation sites at about residues 25-29, 55-59, 254-258. N-myπstoylation site at about residues 15-21 , 1 17- 123, 127- 133, 281 -287, 282-288 and 319-325 and an amidation site at about residues 229-233 Clone
UNQ174 has been deposited with the ATCC on March 5. 1998 and has been assigned deposit number 209653
Example 10 Isolation of cDNA clones encoding human PRO540
The extracellular domain (ECD) sequences (including the secretion signal, if any ) ot from about 950 known secreted proteins from the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases The EST databases included public EST databases (e GenBank) and a proprietary EST DNA database (LIFESEQf . Incv te Pharmaceuticals Palo Alto CA ) The seaich was performed using the computer piogiam BLAST 01 BLAST-2 [Altchul et al Methods in
Figure imgf000095_0001
266 460-480 ( 1996)] as α companson of the ECD protein sequences to a 6 name translation ot the EST sequence Those comparisons resulting in a BLAST score of 70 (oi in some cases 90) oi greater that did not encode know n proteins were clustered an assembled into consensus DNA sequences w ith the piogram
"phrap" (Phil Green. Umveisity ot Washington. Seattle. Washington edu/phrap docs/phrap html)
A consensus DNA sequence was assembled relative to other EST sequences using phrap This consensus sequence is herein designated DNA39631 (SEQ ID NO 5)
Based on the DNA39631 sequences which is show n in Figure 5 (SEQ ID NO 5). oligonucleotides were synthesized 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) foi use as probes to isolate a clone of the full-length coding sequence for PRO540 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 , Curient Piotocols in Moleculai Biologλ . with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest by an in vivo cloning procedure using the probe oligonucleotide and one ot the primer pairs
Forward and reverse PCR primers were synthesized forward PCR primer (39631 fl )
5'-CTGGGGCTACACACGGGGTGAGG-3' (SEQ ID NO 9) reverse PCR primer (39631 rl )
5'-GGTGCCGCTGCAGAAAGTAGAGCG-3' (SEQ ID NO 10) hybridization probe (39631 p i ) 5'-GCCCCAAATGAAAACGGGCCCTACTTCCTGGCCCTCCGCGAGATG-3' (SEQ ID NO 1 1 )
In order to screen several libraries tor a source of a full-length clone. DNA from the libraries was screened by PCR amplification with one of the PCR primer pans identified above A positive library was then used to isolate clones encoding the PRO540 gene using the probe oligonucleotide and one ot the PCR primers RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (L1B227)
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 was ohgo dT containing a Notl site, linked w ith blunt to Sail hemikinαsed adaptors, cleaved with Notl, sized appropriately by gel electrophoresis. and cloned in a defined orientation into a suitable cloning vector (such as pRKB or PRKD. pRK5B is a precursoi of pRK5D that does not contain the Sfil site. see. Holmes el al ,
Science 253 1278- 1280 ( 1991 )) in the unique Xhol and Notl sites
DNA sequencing of the clones isolated as described above gave the tull-length DNA sequence for PRO540 [heiein designated as UNQ341 (DNA44 I 89- 1322)] (SEQ ID NO 3) Clone DNA44189 contains a single open reading frame w ith an appaient translational initiation site at nucleotide positions 21 -23 and ending at the stop codon at nucleotide positions 1257- 1259 (Figure 3) The predicted encoded polypeptide is 412 amino acids long (Figure 4) (SEQ ID NO 4) The full-length PRO540 protein shown in Figure 4 has an estimated moleculai weight of about 46658 daltons and α pi ot about 6 65 Important regions ot the amino acid sequence of PRO540 (including approximate locations) include the signal peptide (residues 1 -
28). potential N-glycosylatιon sites (residues 99-103. 273-277, 289-293 398-402). a potential lipid substrate binding site (residues 147- 164). a sequence typical of hpases and serine proteins (residues 189- 202). tyrosme kinase phosphorylation sites (residues 165- 174 and 178- 186), a beta-transducin family Trp- Asp repeat (residues 353-366) and N-myπstolation sites (residues 200-206. 227-233, 232-238 and 316- 322) Clone UNQ341 (DNA44189- 1322) was deposited with the ATCC on March 26. 1998 and is assigned deposit no 209699
Example 11 Isolation of cDNA clones Encoding Human PR0846 The extracellular domain (ECD) sequences (including the secretion signal it any) of at least about
950 known secreted proteins from the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases The EST databases included public EST databases (e g , GenBank) The search was performed using the computer program BLAST or BLAST-2 [Altschul et al , Methods in Enzymologv 266 460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequence Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington. Seattle, WA)
A consensus DNA sequence was assembled relative to othei EST sequences using phrap This consensus sequence is herein designated DNA39949 (Figure 8. SEQ ID NO 14) Based on the DNA39949 (SEQ ID NO 14) shown in Figure 8. oligonucleotide probes (primers) were synthesized I ) to identity by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0846 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 for a tull-length clone, DNA from the libraries was screen by PCR amplification, as per Ausubel et al , Cuiient Protocols in Moleculai Bιolog\. with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs Forward and reverse PCR primers were synthesized forward PCR primer (39949 11 )
5'-CCCTGCAGTGCACCTACAGGGAAG-3' (SEQ ID NO 15) reverse PCR pnmer (39949 rl ) 5'-CTGTCTTCCCCTGCTTGGCTGTGG-3' (SEQ ID NO 16)
Additionally , a synthetic oligonucleotide hy bridization probe was constructed from the consensus DNA39949 sequence w hich had the follow ing nucleotide sequence hybridization probe (39949 pi ) 5'-GGTGCAGGAAGGGTGGGATCC TCTTCTCTCGCTGCTCTGGCCACATC-3 (SEQ ID NO 17)
In order to screen several libraries for a source of a full-length clone. DNA trom the libraries was screened by PCR amplification with one ot the PCR primer pairs identified abov e A positive library was then used to isolate cones encoding the PR0846 gene using the probe oligonucleotide and one ot the PCR primers RNA for construction of the cDNA libraries was isolated foim human fetal kidney tissue (LIB227)
The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with ohgo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved w ith Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is α precursor of pRK5D that does not contain the Sfil site, see Holmes et al . Science 253 1278-1280 ( 1991 )) in the unique Xhol and Notl sites
DNA sequencing ot the clones isolated as described above gave the full-length DNA sequence for PR0846 [herein designated as UNQ422 (DNA44196- 1 53)](SEQ ID NO 12) and the derived protein sequence for PR0846 The entire nucleotide sequence of UNQ422 (DNA44196- 1353) is shown in Figure 6 (SEQ ID NO
12) Clone UNQ422 (DNA44196-1353) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 25-27 and ending at the stop codon at nucleotide positions 1021 - 1023 (Figure 6) (SEQ ID NO 13) The full-length PR0846 protein shown in Figure 7 (SEQ ID NO 13) is 332 amino acids in length, has an estimated molecular weight ot about 36,143 daltons and a pi of about 5 89 Important regions of the amino acid PR0846 (and approximate locations) include the signal peptide
(residues 1 - 17), the transmembrane domain (residues 248-269). an N-glycosylation site (residues 96- 100), N-myπstolation sites (residues 55-61. 63-69 and 164- 170) α sequence typical of fibrogen beta and gamma chains C-terminal domain (residues 104-1 14), and a sequence typical of lg like V-type domain (residues 13- 128) as shown in Figure 7 Clone UNQ422 (DNA44196- 1353) has been deposited with the ATCC on May 6. 1998 and is assigned ATCC deposit no 209847
Example 12 Isolation of cDNA clones encoding human PR0617
The extiacellular domain (ECD) sequences (including the secretion signal, if any) of about 950 know n secreted proteins trom the Swiss-Piot public protein database weie used to search expressed sequence tag (EST) databases The EST databases included the public EST databases (c g . GenBank) and a proprietary EST DNA database (LIFESEQ®, Inycte Pharmaceuticals, Palo Alto. CA) The search was performed using the computei program BLAST or BLAST-2 [Altschul et al , Methods in 266 460-480 ( I996)| as a comparison of the ECD protein sequences to a 6 frame tianslation of the EST sequence. Those comparisons iesulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clusteied and assembled into consensus DNA sequences w ith the program "phrap" (Phil Gieen. University of Washington. Seattle. Washington) The above analysis resulted in a single hit which is heiein designated DNA42798 (Fig 1 1. SEQ ID
NO. 20). a sequence that corresponds to Washington University /Meick EST RO I 713 Based on the DNA42798 sequence shown in Figure 1 1. 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 tor PR0617 Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are otten designed to give a PCR product of about 100- 1000 bp in length The probe sequences are typically 40-55 bp in length In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1 -1 5 kbp In order to screen several libraries foi a full-length clone, DNA form the libraries was screen 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 oligonucleotide and one of the primer pairs
A pair of PCR primers (forward and reverse) were synthesized forward PCR primer
5'-ACGGGCACACTGGATCCCAAATG-3' (SEQ ID NO. 21 ) reverse PCR primer. 5'-GGTAGAGATGTAGAAGGGCAAGCAAGACC-3' (SEQ ID NO. 22)
Additionally, a synthetic oligonucleotide hybridization probes was constructed from the consensus DNA42798 sequence which had the following sequence- hybridization probe
5'-GCTCCCTACCCGTGCAGGTTTCTTCATTTGTTCCTTTAACCAGTATGCCG-3' (SEQ ID NO. 23) In order to screen several libraries tor 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 PR0617 gene using the probe oligonucleotide and one of the PCR primers
RNA for construction of the cDNA libraries was isolated form human fetal kidney tissue The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego. CA The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor ot pRK5D that does not contain the Sfil site, see. Holmes et al , Science 253 1278- 1280 ( 1991 )) in the unique Xhol and Notl sites DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for
PR0617 [herein designated as UNQ353 (DNA48309- 1280)](SEQ ID NO 18) is shown in Figure 9 Clone UNQ353 (DNA48309) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 723-725 and ending at the stop codon at nucleotide positions 924-926 (Figure 9) The predicted polypeptide precursor is 67 amino acids long (Figure 10) The tull-length PR0617 protein shown in Figure I 1 (SEQ ID NO 19) has an estimated moleculai weight of about 6 981 daltons and a pi of about 7 47 Analy sis ot the PR0617 amino acid sequence also ev idences the existence of a putative signal peptide trom about amino acid 1 to about amino acid 27 and a putative piotein kinase C phosphorylation site from about amino acid 41 to about amino acid 43 Clone UNQ353 (DNA48309 1230) has been deposited on
March 5. 1998 with the ATCC and is assigned deposit numbei 209656
Example 13 Isolation of cDNA clones encoding human PR0538 (GFRcβ) and PR03664 (GFRα3) A proprietary expressed sequence tag (EST) DNA database (LIFESEQ* Incyte Pharmaceuticals.
Palo Alto. CA) was searched with murine GFRcβ (SEQ ID NO 29) and the EST sequence INC3574209 (SEQ ID NO 28) was identified and found to have 61 % sequence identitv in the aligned region between the two sequences
The following primers were created in order to screen for the corresponding full length cDNA newa3 F 5'-GCCTCTCGCAGCCGGAGACC-3' (SEQ ID NO 30) newsa3 R" 5'-CAGGTGGGATCAGCCTGGCAC-3' (SEQ ID NO 31 )
DNA from the libraries was screened by PCR amplification, as per Ausubel et al Curient Protocols in Moleculai Biology ( 1995), with the PCR primer pair A strong PCR product was identified in all libraries analyzed (fetal lung, fetal kidney and placenta) In order to isolate a corresponding cDNA clone encoding a lull length protein, a human fetal lung pRK5 vector library was selected and enriched for positive cDNA clones by extension of single stranded DNA from plasmid libraries grown in dug-/bung- hosts using the newa3 R (SEQ ID NO 31 ) primer RNA for construction of the cDNA libraries was isolated trom human fetal lung tissue The cDNA library used to isolate the cDNA clones was constructed by standard methods using commercially available reagents (e g . Invitrogen. San Diego, CA. Clontech, etc ) 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 (pRKB or pRKD, pRK5B is a precursor ot pRK5D that does not contain the Sfil site, see. Holmes et al Science 253 1278-1280 ( 1991 )) in the unique Xhol and Notl sites To enrich for positive cDNA clones, the primer extension reaction contained 10 μl of lOx PCR buffer (Perkin Elmer. USA). 1 μl dNTP (20 mM). I μl library DNA
(200 ng). I μl primer. 86 5 μl H 0 and 1 μl Amphtaq (Perkin Elmer USA) added after a hot start The reaction was denatured for 1 minute at 95°C. annealed for 1 minute at 60°C, and then extended for 15 minutes at 72°C The DNA was extracted with phenol/chloroform precipitated with ethanol, and then transformed by electroporation into a DH I OHB host bacteria The entire transformation mixture was plated onto 10 plates and colonies were allowed to form Colonies were lilted onto ny lon membranes and screened with the following oligonucleotide probe derived from the original ESI nucleotide sequence newa3 piobe 5'-TCTCGCAGCCGGAGACCCCCTTCCCACAGAAAGCCGACTCA 3' ( SEQ ID NO 32) Filters were hybndi/ed ith the probe overnight at 42°C in 50% toimamide 5xSSC 10x Denhardt s 0 05M sodium phosphate (pH 6 5 ) 0 1 % sodium pyrophosphate and 50 μg/ml of sonicated salmon sperm DNA Filters were then rinsed in 2xSSC. washed in 0 l xSSC, 0 1 % SDS, and then exposed overnight to Kodak X Ray films Fiv e positiv e clones were identified Pure positive clones were obtained after colony purification and secondary screening
Two ot the isolated clones were sequenced These cDNA sequences were designated DNA48613 (SEQ ID NO 24. Figure 12) and DNA48614 (SEQ ID NO 25. Figure 13) Clone DNA48613- 1268 (SEQ ID NO 24) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 38-40 and ending at the stop codon at nucleotide positions 1238- 1240 (Figure 12) The predicted native sequence polypeptide precursor PR0538 is 400 ammo acids in length, has an approximate molecular weight of 44.51 1 daltons and a pi of 8 15 Further analysis of the PR0538 sequence of Figure 14 (SEQ ID NO.26) reveals a signal sequence at about amino acid residues 1 - 16. N-glycosylation sites at residues 95-99. 148- 152 and 309-313. cAMP- and cGMP-dependent protein kinase phosphorylation site at residues 231 - 235. N-myπstolation sites at residues 279-285 and 294-300 and prokaryotic membrane hpoprotein lipid attachment site are residues 306- 17 and 379-390
Clone DNA48614-1268 (SEQ ID NO 25) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 142-144 and ending at the stop codon at nucleotide positions 1249- 1251 (Figure 13) The predicted native sequence PR03664 polypeptide precursor is 369 ammo acids long, with a calculated molecular weight of approximately 40,930 Da and a pi of 8 16 Further analysis of the PR03664 sequence of Figure 15 (SEQ ID NO.27) reveals a signal sequence at about amino acid residues 1 -26, N-glycosylation sites at about residues 95-99 and 278-282, a cAMP- and cGMP- dependent protein kinase phosphorylation site at about residues 200-204. N-myπstoylation sites at about residues 248-254 and 263-269 and prokaryotic membrane hpoprotein lipid attachment sites at about residues 275-286 and 348-359 A comparison of PR03664 (SEQ ID NO 27) and PR0548 (SEQ ID NO 26) reveals the two molecules to be splice variants with α 30 amino acid deletion (amino acid positions 127-157) in PR03664. as shown in Figure 16 Interestingly, none of the cysteines are deleted in PR03664 (SEQ ID NO 27) Clones DNA48613- 1268 and DNA48614- 1268 have been deposited with the ATCC on April 7, 1998 and are assigned ATCC dep nos 209752 and 209751 , respectively
Example 14 Isolation of DNA encoding PRO770 (hFIZZ-1) A public expressed sequence tag (EST) DNA database (Merck/Washington University) was searched with the tull-length murine m-FIZZ- 1 DNA (DNA53517)(SEQ ID NO 35). and an EST. designated AA524300 (SEQ ID NO 36), was identified, which showed homology with the m-FIZZ- 1 DNA
A full length clone corresponding to EST AA524300 (SEQ ID NO 36) was obtained and sequenced in its entirety The full length polv nucleotide sequence corresponding to the AA524300 partial length clone is show n in Figure 21 (SEQ ID NO 34) This full length clone designated DNA54228 1 66 (SEQ ID NO 34) contains a single open reading frame with an apparent translational initiation site at about nucleotide positions 100 102 and ending at the stop codon at nucleotide positions 433 535 (Figure 19 SEQ ID NO 34) The piedicted PRO770 polypeptide precursor is 1 1 1 amino acids long (Figure 18) The full length
PRO770 protein shown in Figuie 18 (SEQ ID NO 33) has an estimated molecular w eight of 1 1 730 daltons and a pi of 7 82 Further analysis reveals a signal sequence at about amino acid residues 1 -28 a cAMP and cGMP-dependent protein kinase phosphorylation site at residues 51 -55 N mvπstolation sites at residues 21 27, 70 76 and 75 81 and a prenvl group binding site (CAAX) at about residues 108-1 13 Clone DNA54228-1366 has been deposited with the ATCC on April 23 1998 and is assigned deposit number
209803
EXAMPLE 15 Identification and cloning of m-FIZZ-1 (DNA53517) Mous asthma model Female Balb/C mice, 6 to 8 weeks of age weie separated into two experimental groups controls and asthmatics The asthmatic group was immunized lntrapeπtoneally with 10 μg ovalbumin + 1 mg alum while the control group was not Two weeks later mice were exposed daily to an aerosol of 10 mg/ml ov albumin in PBS aerosolized with a UltraNeb™ nebulizer (DeVilbiss) at the rate of 2 ml/min tor 30 mm each day, for 7 consecutive days One day after the last aerosol challenge whole blood serum and bronchoalveolar lavage (BAL) samples were collected and the lungs were harvested and preserved for histological examination, immuno histochemistry and m situ hybridization A schematic
Gel electiophoresis of BAL samples Examination ot the BAL samples by gel electrophoresis on a 16% Tπcine gel indicates a low molecular weight protein is expressed in the BAL of asthmatic mice but not in the BAL sample from control mice This low molecular weight protein has been termed mFIZZ 1. and comigrates with a 8300 Dalton marker protein (not shown)
Partial protein sequence The protein of interest was transferred upon a PVDF membrane and sequenced by Edman degradation The first 23 amino acids of the N terminal sequence are DETIEIIVENKVKELLANPANYP (SEQ ID NO 37)
Paitial cDNA sequence Degenerate oligonucleotide PCR primers corresponding to the putative DNA sequence tor the first 7 and the last 7 ammo acids ot SEQ ID NO 37, were made The cDNA prepared from the lungs of normal mice was used a template for the PCR reaction and the result was an 88 bp pioduct which was used to design additional primers which themselves used to obtain a full length FIZZ clone by RT-PCR of mouse lung poly(A)+ RNA Full length cDNA clone The following oligos were used as RT-PCR primers in combination with 5 and 3 amplifiers
(Clontech) and oligo d(T), respectively
5 ACA AAC GCG TGC TGG AGA ATA AGG TCA AGG 3 (SEQ ID NO 38) 5 ACT AAC GCG TAG GCI AAG GAA CTT CTT GCC-3 (SEQ ID NO 39) The complete m-FIZZ- 1 cDNA was been termed DNA53517 (SEQ ID NO 35) and is shown in Figure 20
EXAMPLE 16
Use of PRO as a hybridization probe The following method describes use ol a nucleotide sequence encoding PRO as a hybridization probe.
DNA comprising the coding sequence of full-length or mature PRO as disclosed herein is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO) m 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 ot radiolabeled PRO-deπved probe to the filters is performed in a solution of 50% tormamide, 5x SSC, 0 1 % SDS, 0 1 % sodium pyrophosphate, 50 mM sodium phosphate, pH 6 8, 2x Denhardt's solution, and 10% dextran sultate at 42°C for 20 hours Washing of the filters is performed in an aqueous solution ot 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
PRO can then be identified using standard techniques known the art
EXAMPLE 17 Expression of PRO in E. coli This example illustrates preparation of an unglycosylated form of PRO by recombinant expression
The DNA sequence encoding PRO is initially amplified using selected PCR primers The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector A variety of expression vectors may be employed An example of a suitable vector is pBR322 (derived from E coli, see Bolivar et al , Gene, 2 95 ( 1977)) which contains genes tor ampicillin and tetracycline resistance The vector is digested with restriction enzyme and dephosphorylated The PCR amplified sequences are then ligated into the vector The vector will preferably include sequences which encode tor an antibiotic resistance gene, a trp promotei , a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the PRO coding region, lambda transcriptional terminator, and an argU gene
The ligation mixture is then used to transform a selected E coli strain using the methods described in Sambrook et al , supia Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics The overnight culture may subsequently be used to inoculate a larger scale culture The cells are then grown to a desired optical density, during which the expression promoter is turned on
After culturing the cells for several more hours, the cells can be harvested by centrifugation The cell pellet obtained by the centπfugation can be solubihzed using various agents known in the ait. and the solubi zed PRO protein can then be purified using a metal chelating column under conditions that allow tight binding ot the protein
PRO mav be expiessed in E oli in a polv His tagged form using the follo ing pioceduie The DNA encoding PRO is initially amplified using selected PCR pπmei s The primers will contain restriction enzyme sites w hich correspond to the restriction enzv me sites on the selected expression vector and othei useful sequences prov iding for efficient and reliable translation initiation rapid pui ification on a metal chelation column and proteolv tic removal with enterokinαse The PCR amplified poly His tagged sequences are then ligated into an expression v ector which is used to transform an £ coli host based on strain 52 (W31 10 futiA (tonA) Ion galE rpoHts (htpRts) clpP (laclq) Transformants are first gro n in LB containing 50 mg/ml carbenicilhn at 30°C with shaking until an O D 600 of 3 5 is reached Cultures are then diluted 50 100 told into CRAP media (prepared by mixing 3 57 g (NH )^S0 0 71 g sodium cιtrate-2H20, 1 07 g KC1 5 36 g Difco yeast extract 5 36 g Sheffield hycase SF in 500 mL watei as well as 1 10 mM MPOS pH 7 3 0 55%* (w/v) glucose and 7 mM MgS04) and grow n for approximatelv 20 30 hours at 30°C with shaking Samples are removed to v erify expression by SDS PAGE analv sis and the bulk culture is centrifuged to pellet the cells Cell pellets are frozen until purification and refolding
E coli paste trom 0 5 to 1 L fermentations (6 10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine 20 mM Tris, pH 8 buffer Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0 I M and 0 02 M respectively and the solution is stirred overnight at 4* C This step results in a denatured protein with all cysteine residues blocked by sulfitolization The solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine 20 mM Tris, pH 7 4) and filtered through 0 22 micron filters to clarify The clarified extract is loaded onto a 5 ml Qiagen Ni NTA metal chelate column equilibrated in the metal chelate column buffer The column is washed with additional buffer containing 50 mM lmidazole (Calbiochem, Utrol grade) pH 7 4 The protein is eluted with buffer containing 250 mM lmidazole Fractions containing the desired protein are pooled and stored at 4°C Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence
The proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of 20 mM Tris pH 8 6, 0 3 M NaCl, 2 5 M urea, 5 mM cysteine 20 mM glycine and 1 mM EDTA Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml The refolding solution is stirred gently at 4°C for 12-36 hours The refolding reaction is quenched by the addition ot TFA to a final concentration of 0 4% (pH of approximately 3) Before further purification of the protein the solution is filtered through a 0 22 micron filter and acetonitrile is added to 2 10% final concentration I he refolded protein is chromatographed on a Poros R l/H reversed phase column using a mobile buftei of 0 1 % TFA with elution with a gradient of acetonitrile trom 10 to 80%
Ahquots of fractions with A280 absorbance aie analv zed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled Generally the propei ly refolded species ot most proteins are eluted at the lowest concentrations ot acetonitrile since those species are the most compact with their hydrophobic intenoi s shielded from interaction with the reversed phase resin Aggregated species are usualK eluted at higher acetonitrile concentrations In addition to resolv ing misfolded forms ot proteins trom the desired form the rev ersed phase step also remov es endotoxin trom the samples
Fractions containing the desued folded PRO polypeptide are pooled and the acetonitrile remov ed using a gentle stieam of nitiogen directed at the solution Pioteins are formulated into 20 mM Hepes pH 6 8 with 0 14 M sodium chloride and 4% mαnnitol by dιal\ sιs or by gel filtration using G25 Superfine
(Pharmacia) resins equilibrated in the formulation butter and sterile filtered
EXAMPLE 18 Expression of PRO in mammalian cells This example illustrates preparation of a potentially glycosylated form ot PRO by recombinant expression in mammalian cells
The vector, pRK5 (see EP 307 247 published March 15 1989) is emploved as the expression vector Optionally the PRO DNA is ligated into pRK5 with selected restriction enzy es to allow insertion of the PRO DNA using ligation methods such as described in Sambrook et al supia The resulting vector is called pRK5-PRO
In one embodiment the selected host cells may be 293 cells Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics About 10 μg pRK5 PRO DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al Cell, 3J_ 543 ( 1982)] and dissolved in 500 μl of 1 mM Tris-HCl, 0 1 mM EDTA, 0 227 M CaCl2 To this mixture is added dropwise, 500 ul 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 uCi/ml ^S cysteine and 200 uO/ml 1SS 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 PRO 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 PRO may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al Pioc Natl Acad Sci , \ 2 7575 ( 1981 ) 293 cells are grown to maximal density in a spinner flask and 700 ug pRK5 PRO DNA is added The cells are first concentrated from the spinner flask by centrifugation and washed with PBS 1 he 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 ug/ml bovine insulin and 0 1 μg/ml bovine transterπn After about tour days the conditioned media is centrifuged and filtered to remov e cells and debris The sample containing expressed PRO can then be concentrated and purified by anv selected method such as dialv sis and/oi column chiomatography In another embodiment. PRO can be expressed in CHO cells The pRK5-PRO can be transtected into CHO cells using kno n reagents such as CaP04 or DEAE-dextran As described abov e, the cell cultuies can be incubated, and the medium replaced w ith culture medium (alone ) or medium containing a radiolabei such as
Figure imgf000105_0001
After determining the piesence of PRO polypeptide. the culture medium may be replaced with serum tree medium Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested The medium containing the expressed PRO can then be concentrated and purified by any selected method Epitope-tagged PRO may also be expiessed in host CHO cells The PRO may be subcloned out ot the pRK5 vector The subclone insert can undergo PCR to tuse in frame with a selected epitope tag such as a poly-his tag into α Baculovirus expression vector 1 he poly-his tagged PRO insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection ot stable clones Finally, the CHO cells can be transtected (as described above) with the SV40 driven vector Labeling may be performed, as described above, to verify expression The culture medium containing the expressed poly-
His tagged PRO can then be concentrated and purified by any selected method, such as by Nι2+-chelate affinity chromatography
PRO may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure Stable expression in CHO cells is performed using the following procedure The proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins are fused to an IgG l constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form
Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al , Curient Piotocols of Moleculai Biologs, Unit
3 16, John Wiley and Sons ( 1997) CHO expression vectors are consti ucted to hav e compatible restriction sites 5' and 3' ot the DNA of interest to allow the convenient shuttling of cDNA's The vector used expression in CHO cells is as described in Lucas et al , Nucl Acids Res 24 9 ( 1774- 1779 ( 1996), and uses the SV40 early promoter/enhancer to drive expression ot the cDNA of interest and dihydrotolate reductase (DHFR) DHFR expression permits selection tor stable maintenance of the plasmid following transfection
Twelve micrograms of the desned plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Quiagen). Dosper* or Fugene® (Boehπnger Mannheim) The cells are giown as described in Lucas et al , supia Approximately 3 x 10 cells are tiozen in an ampule for further growth and production as described below The ampules containing the plasmid DNA are thawed by placement into water bath and mixed by vortexing The contents are pipetted into a centrifuge tube containing 10 niLs of media and centrifuged at 1000 rpm foi 5 minutes The supernatant is aspirated and the cells are resuspended in 1 mL ot selective media (0 2 μm filtered PS20 with 5% 0 2 μm diafiltered fetal bovine serum) The cells are then aliquoted into a 100 mL spinner containing 90 mL ot selective media Aftei 1 -2 days, the cells are transterred into a 250 mL spinner tilled ith 150 mL selective growth medium and incubated at 37 C After another 2 3 dav s 250 mL 500 mL and 2000 mL spinners are seeded ith 3 \ 10 cells/mL The cell media is exchanged w ith fresh media bv centrifugation and resuspension in pioduction medium Although anv suitable CHO media may be employed a production medium described in U S Patent No 5 122 469 issued June 16 1992 mav actually be used A 3L production spmnei is seeded at 1 2 x 106 cells/mL On dav 0 the cell number pH is determined On day 1 the spinnei is sampled and sparging w ith filtered air is commenced On day 2 the spinner is sampled the temperature shifted to 33°C and 30 mL of 500 g/L glucose and 0 6 mL of 10% antitoam (e g 35% polvdimethylsiloxane emulsion Dow Corning 365 Medical Grade Emulsion) taken Throughout the production the pH is adjusted as necessary to keep it at around 7 2 After 10 days or until the viability dropped below 70% the cell culture is harvested by centrifugation and filtering through a 0 22 μm filter The filtrate was either stored at 4°C or immediatelv loaded onto columns for purification
For the poly-His tagged constructs the proteins are purified using a Ni NTA column (Qiagen) Before purification lmidazole is added to the conditioned media to a concentration of 5 mM The conditioned media is pumped onto a 6 ml Ni NTA column equilibrated in 20 mM Hepes pH 7 4 buffei containing 0 3 M NaCl and 5 mM lmidazole at a flow rate ot 4 5 ml/min at 4 C After loading the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M lmidazole The highly purified protein is subsequentlv desalted into a storage buffer containing 10 mM Hepes 0 14 M NaCl and 4% man tol pH 6 8 with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C Immunoadhesin (Fc containing) constructs aie purified from the conditioned media as follows
The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading the column is washed extensively with equilibration buffer before elution with 100 mM citric acid pH 3 5 The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 μL of 1 M Tris buffer pH 9 The highly purified protein is subsequently desalted into storage buffer as described above for the poly His tagged proteins The homogeneity is assessed by SDS polyacrylamide gels and bv N-terminal amino acid sequencing by Edman degradation
EXAMPLE 19 Expression of PRO in Yeast
The following method describes recombinant expression of PRO in yeast
First, yeast expression vectors are constructed for intracellular production or secretion ol PRO from the ADH2/GAPDH promoter DNA encoding PRO and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO For secretion, DNA encoding PRO can be cloned into the selected plasmid, together with DNA encoding the
ADH2/GAPDH promoter a native PRO signal peptide or other mammalian signal peptide, or tor example, a yeast alpha-factoi or invertase secretory signal/leader sequence, and linker sequences (if needed) tor expression of PRO Yeast cells such as \east strain AB1 10 can then be transtormed ith the expression plasmids descπbed above and cultured in selected termentation media The transtormed yeast supernatants can be analyzed by precipitation w ith 10% tπchloroacetic acid and separation by SDS PAGE tollovved by staining ot the gels w ith Coomassie Blue stam Recombinant PRO can subsequently be isolated and purified by removing the yeast cells trom the termentation medium bv centrifugation and then concentrating the medium using selected cartridge filters The concentrate containing PRO may further be purified using selected column chromatography resms
EXAMPLE 20 Expression of PRO in Baculov irus-Infected Insect Cells
The tollowing method describes recombinant expression of insulin or insulin v ariant in Baculovirus infected insect cells
The sequence coding for insulin or insulin vαπant is fused upstream of an epitope tag contained within a baculovirus expression vector Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG) A variety ot plasmids may be employed including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly the sequence encoding insulin or insulin variant or the desired portion of the coding sequence of this polypeptide [such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular] is amplified by PCR with primers complementary to the 5 and 3 regions The 5' primer may incorporate flanking (selected) restriction enzyme sites The product is then digested with those selected restriction enzymes and subcloned into the expression vector
Recombinant baculovirus is generated by co transfecting the above plasmid and BaculoGold® virus DNA (Pharmingen) into Spodoptera frugipeida (' Sf9 ) cells (ATCC CRL 171 1 ) 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 Bac uloMi us expiession \ ectoi s A Laboiaton Manual Oxford Oxford University Press (1994)
Expressed poly-his tagged insulin or insulin variant can then be purified, for example by Nι"+ chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al Natuie, 362 175-179 ( 1993) Briefly Sf9 cells are washed resuspended in so cation buffei (25 mL Hepes pH 7 9, 12 5 mM MgCl2 0 1 mM EDTA 10%. glycerol 0 1 % NP-40 0 4 M KC1) and sonicated twice for 20 seconds on ice The sonicates are cleared by centrifugation and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate 300 mM NaCl, 10% glvcerol pH 7 8) and filtered through a 0 45 μm filter A Ni + NTA agarose column (commercially available from Qiagen) is prepared with a bed v olume of 5 mL washed with 25 mL of water and equilibrated with 25 mL of loading buffer The filtered cell extiact is loaded onto the column at 0 5 mL per minute The column is washed to baseline A2W with loading butter t which point fraction collection is started Next the column is washed with a secondarv wash buftei (50 mM phosphate 300 mM NaCl 10% glycerol pH 6 0) which elutes nonspecitically bound protein Atter reaching A78π baseline again the column is developed with a 0 to 500 mM imida/ole gradient in the secondary w ash butter One mL tractions are collected and analyzed by SDS- PAGE and silvei staining or Western blot w ith Nr+-NTA-con|ugated to alkaline phosphatase (Qiagen) Fractions containing the eluted His-n-tagged insulin or insulin variant aie pooled and dialy zed against loading butter Alternatively , purification of the IgG tagged (or Fc tagged) insulin oi insulin variant can be performed using known chromatography techniques, including for instance Protein A 01 Protein G column chromatography
Alternatively still, the insulin or insulin variant molecules ot the invention may be expressed using a modified baculovirus procedure employing Hι-5 cells In this procedure, the DNA encoding the desired sequence was amplified w ith suitable systems such as Pfu (Stratagene). or fused upstream (5'-of) an epitope tag contained within a baculovirus expression vector Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIE- 1 (Novagen) The pIE l -1 and pIEl -2 vectors are designed tor constitutive expression of recombinant proteins trom the baculovirus le i 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 le i -mediated gene expression in u nfected insect cells as well as the hr5 enhancer element pIE l -1 and pIEl -2 include the lei 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 the transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions The 5' primer may incorporate flanking (selected) restriction enzyme sites The product 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 Hι5 cells are grown to a confluency of 50% under the conditions ot 27°C, no C0 . 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)) Separately, 100 μl of Cell Fectin (CellFECTIN. Gibco BRL +10362-010. pre-vortexed) is mixed with 1 ml of Ex-Cell medium The two solutions are combined and incubated at room temperature for 15 minutes 8 ml of Ex-Cell media is added to the 2 ml of DNA/CellFECTIN mix and this is layered on
Hι5 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 Cell FECTIN 30 ml of fresh Ex-Cell media is added and the cells are incubated for 3 days at 28°C The supernatant is harvested and the expression of the sequence in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml ot Ni-NTA beads
(QIAGEN) for histidine tagged proteins of Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged pi oteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining
The conditioned media trom the transtected cells (0 5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0 22 micion filters For the pol -His tagged constructs, the protein compπsing the sequence is purified using a Ni-NTA column (Qiagen ) Before purification, imida/ole at α flow rate of 4-5 ml/mm at 48°C Attei loading, the column is washed with additional equilibrium butter and the protein eluted with equilibrium butter containing 0 25M imidazole The highly purified protein was then subsequently desalted into a stoiage butter 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
Immunoadhesion (Fc-containing) constructs may also be purified from the conditioned media as follow s The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which had been previously equilibrated in 20 mM sodium phosphate buffei , pH 6 8 After loading, the column is washed extensively with equilibrium buffer before elution with 100 mM citric acid, pH 3 5 The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 μl of 1 M Tris buffer, pH 9 The highly purified protein is subsequently desalted into storage butter as described above for the poly-His tagged proteins The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation
EXAMPLE 21 Preparation of Antibodies that Bind PRO This example illustrates preparation of monoclonal antibodies which can specifically bind PRO Techniques for producing the monoclonal antibodies are known in the art and are descπbed, for instance, in Goding, supra Immunogens that may be employed include purified PRO, fusion proteins containing PRO, and cells expressing recombinant PRO 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 PRO immunogen emulsified in complete Freund's adjuvant and miected subcutaneously or lntrapeπtoneally in an amount from 1 - 100 micrograms Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research.
Hamilton. MT) and injected into the animal's hind foot pads The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant Thereafter, for several weeks, the mice may also be boosted with additional immunization injections Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-PRO antibodies After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection ot PRO Three to tour days later, the mice are sacrificed and the spleen cells are harvested The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU 1. available from ATCC. No CRL 1597 The fusions generate hybndoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine. aminopteπn. and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids
The hybndoma cells will be screened in an ELISA for reactiv ity against PRO Determination of "positive" hybndoma cells secreting the desired monoclonal antibodies against PRO is within the skill in the art The positive hvbπdomα cells can be injected liurapeπtoneally into sy ngeneic Balb/c mice to produce ascites containing the anti-PRO monoclonal antibodies Alternativ ely the hy bπdoma 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 bv gel exclusion chromatography Alternatively , affinity chromatography based upon binding of antibody to protein A or protein G can be employed
EXAMPLE 22 Purification of PRO Polypeptides Using Specific Antibodies Native or recombinant PRO polypeptides may be puπfied by a variety of standard techniques in the art of protein purification For example, pro-PRO polypeptide. mature PRO polypeptide. or pie-PRO polypeptide is puπfied by lmmunoaffinity chromatography using antibodies specific for the PRO polypeptide of interest In geneial, an lmmunoaffinity column is constructed by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resin Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway. N J ) Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKB Biotechnology) The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions
Such an lmmunoaffinity column is utilized in the purification of PRO polypeptide by preparing a fraction from cells containing PRO polypeptide in a soluble form This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art Alternatively, soluble PRO polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown
A soluble PRO polypeptide-containing preparation is passed over the lmmunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PRO polypeptide (e g , high ionic strength buffers in the presence of detergent) Then, the column is eluted under conditions that disrupt antibody/PRO polypeptide binding (e g . Ά low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and PRO polypeptide is collected
EXAMPLE 23 Drug Screening
This invention is particularly useful for screening compounds by using PRO polypeptides or binding fragment thereof in any of a variety of drug screening techniques The PRO polypeptide oi fragment employed in such a test may either be tree in solution, affixed to a solid support, borne on a cell surface, or located intracellularly One method of di ug screening utilizes eukarvotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between PRO polypeptide or a fragment and the agent being tested. Alternatively, one can examine the diminution in complex formation between the PRO polypeptide and its target cell or target receptors caused by the agent being tested.
Thus, the present invention provides methods of screening for drugs or any other agents which can affect a PRO polypeptide-associated disease or disorder. These methods comprise contacting such an agent with an PRO polypeptide or fragment thereof and assaying (I) for the presence of a complex between the agent and the PRO polypeptide or fragment, or (ii) for the presence of a complex between the PRO polypeptide or fragment and the cell, by methods well known in the art. In such competitive binding assays. the PRO polypeptide or fragment is typically labeled. After suitable incubation, free PRO polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to PRO polypeptide or to interfere with the PRO polypeptide/cell complex.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on September 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to a PRO polypeptide, the peptide test compounds are reacted with PRO polypeptide and washed. Bound PRO polypeptide is detected by methods well known in the art. Purified PRO polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding PRO polypeptide specifically compete with a test compound for binding to PRO polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with PRO polypeptide.
EXAMPLE 24 Rational Drug Design
The goal of rational drug design is to produce structural analogs of biologically active polypeptide of interest (i.e.. a PRO polypeptide) or of small molecules with which they interact, e.g., agonists, antagonists, or inhibitors. Any of these examples can be used to fashion drugs which are more active or stable forms of the PRO polypeptide or which enhance or interfere with the function of the PRO polypeptide in vivo (c.f, Hodgson. Bio/Technology, 9: 19-21 (1991 )).
In one approach, the three-dimensional structure of the PRO polypeptide. or of an PRO polypeptide-inhibitor complex, is determined by x-ray crystallography, by computer modeling or. most typically, by a combination of the two approaches. Both the shape and charges of the PRO polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous PRO polypeptide-like molecules or to identify efficient inhibitors. Useful examples of rational drug design may include molecules which have improved activity or stability as show n by Bra.xton and Wells. Biochemistry. 3J_:7796-7801 ( 1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda et al., J. Biochem.. 1 13:742-746 ( 1993).
It is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
By virtue of the present invention, sufficient amounts of the PRO polypeptide may be made available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the PRO polypeptide amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.
Deposit of Material
The following material has been deposited with the American Type Culture Collection, 10801 University Blvd.. Manassas, VA 201 10-2209 (USA). Material ATCC Deposit Number Deposit Date DNA2910I - 1272 209653 March 5. 1998
DNA44189- 1322 209699 March 26. 1998
DNA44196- 1353 209847 May 6, 1998
DNA48309-1280 209656 March 5, 1998
DNA48613- 1268 209752 April 7, 1998 DNA48614- 1268 209751 April 7, 1998
DNA54228-1366 209801 April 23, 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 the ATCC under the terms of the Budapest
Treaty, and subject to an agreement between Genentech, Inc. and the ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled theieto according to 35 U S C ij 122 and the Commissioners l ules pursuant theieto ( including 37 C F R • 1 14 w ith particulai refeience to 886 OG 638)
The assignee of the present application has agreed that if a culture of the material on deposit should die or be lost or destroy ed when cultivated under suitable conditions, the material will be promptly replaced on notification w ith another of the same Av ailability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any gov ernment in accordance with its patent laws
The foregoing written specification is considered to be sufficient to enable one of ordinary skill in the art to practice the invention The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent aie within the scope of this invention The deposit of material 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 form the foregoing description and fall within the scope of the appended claims

Claims

WHAT IS CLAIMED IS
1 A method ot delay ing preventing or rescuing retinal cells trom injury or death without causing angiogenesis or mitogenesis comprising the administration of a theiapeutically effective amount ot an active PRO200. PRO540 PR0846. PR0617. PR0538. PR03664 or PRO770 polypeptide
2 The method of claim 1 wherein the active PRO200, PRO540. PR0846 PR0617. PR0538. PR03664 or PRO770 polypeptide is at least 90%o homologous to a native sequence PRO200 (SEQ ID NO.2). PRO540 (SEQ ID NO 4). PR0846 (SEQ ID NO 13). PR0617 (SEQ ID NO 19). PR0538 (SEQ ID NO 26). PR03664 (SEQ ID NO 27) or PRO770 (SEQ ID NO 33) molecule
3 The method of claim 1. where the active PRO200. PRO540. PR0846 PR0617. PR0538. PR03664 or PRO770 polypeptide is selected from the group consisting of SEQ ID NO 2. SEQ ID NO 4, SEQ ID NO 13, SEQ ID NO 19, SEQ ID NO 26. SEQ ID NO 27 and SEQ ID NO 33
4. The method of claim 1 , wherein the active PRO200, PRO540, PR0846, PR0617, PR0538,
PR03664 or PRO770 polypeptide is encoded by a nucleotide sequence which hybridizes under stringent conditions to the complement of a nucleotide encoding a ( 1 ) PRO200 polypeptide comprising residues 15 to 345 of SEQ ID NO 2, (2) PRO540 polypeptide comprising amino acid residues 29 to 412 of SEQ ID NO 4, (3) PR0846 polypeptide comprising amino acid residues 18 to 332 of SEQ ID NO.13; (4) PR0617 polypeptide having amino acid residues 16 to 67 of SEQ ID NO 19, (5) PR0538 polypeptide comprising residues 27 to 400 of SEQ ID NO 26, (6) PR03664 polypeptide comprising residues 27 to 369 of SEQ ID NO 27, (7) PRO770 polypeptide comprising residues 21 to 1 1 1 of SEQ ID N0.33
5 The method of claim I . wherein the retinal cells are selected from the group consisting of retinal neurons and supportive cells
6 The method of claim 5 wherein the retinal neurons are selected from the group consisting of photoreceptors, retinal ganglion cells, displaced retinal ganglion cells, amacπne cells, displaced amacπne cells, horizontal and bipolar neurons
7 The method ot claim 6 wherein the supportive cells are selected from the group consisting of Muller cells and pigment epithelial cells
8 The method of claim 7 wherein the retinal cells are photoreceptors 9 The method of claim 8 wherein the photoreceptoi cell lnjuiy or death is caused by an ocular disease, retinal injury, light or env ironmental trauma
10 The method of claim 9 wherein the photoreceptor cell injury or death is caused by an oculai disease
1 1 The method of claim 9 wherein the administration method is intraocular
12 The method of claim 9 wherein the polypeptide is administered into the vitreous or into the subretinal space
13 The method ot claim 9 wherein the administration method is intravitreal
14 T he method of claim 9 wherein the administration method is by means ot an implant
15 The method of claim 10 wherein the ocular disease is selected from the group consisting of retinitis pigmentosa. macular degeneration, including age-related, retinal detachment, retinal tears, retinopathy, retinal degenerative diseases, macular holes, degenerative myopia, acute retinal necrosis syndrome, traumatic chorioretinopathies or contusion, such as Purtscher's retinopathy, edema, ischemic conditions such as central or branch retinal vision occlusion, collagen vascular diseases, thrombocytopenic purpura, uveitis. retinal vascuhtis and occlusion associated with Eales disease and systemic lupus erythematosus
16 A composition comprising an active PRO200, PRO540, PR0846, PR0617, PR0538. PR03664 or PRO770 polypeptide and a pharmaceutically acceptable carrier for use in a method of delaying, preventing or rescuing retinal neurons from ιn|ury or death without causing angiogenesis or mitogenesis by the administration of a therapeutically effective amount of an PRO200. PRO540, PR0846, PR0617, PR0538, PR03664 or PRO770 polypeptide
17 An article of manufacture, comprising
(a) a container,
(b) a label on said container, and
(c) a composition contained within said container, wherein the composition includes an active agent effective toi promoting the survival of retinal neurons, the label on said container indicates that the composition can be used to delay, prevent or rescue retinal neurons, and the active agent is said composition comprises PRO200, PRO540. PR0846. PR0617, PR0538. PR03664 or PRO770 polypeptide 18 The article ot manufacture of claim 17 further comprising instructions for administering the PRO200 PRO540 PR0846 PR0617 PR0538 PR03664 oi PRO770 polv peptide to a mammal
19 Isolated nucleic acid hav ing at least 80% nucleic acid sequence identitv to a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figuie 2 (SEQ ID NO 2) Figure 4 (SEQ ID NO 4) Figure 7 (SEQ ID NO 13) Figure 10 (SEQ ID NO 19) Figure 14 (SEQ ID NO 26) Figure 15 (SEQ ID NO 27) Figure 18 (SEQ ID NO 33)
20 Isolated nucleic acid having at least 80%; nucleic acid sequence identity to a nucleotide sequence selected trom the group consisting of the nucleotide sequence shown in Figure 2 (SEQ ID NO 2) Figure 4 (SEQ ID NO 4), Figure 7 (SEQ ID NO 13) Figure 10 (SEQ ID NO 19), Figure 14 (SEQ ID NO 26) Figure 1 (SEQ ID NO 27) and Figure 18 (SEQ ID NO 33)
21 Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the full-length coding sequence of the nucleotide sequence shown in Figure 2 (SEQ ID NO 2), Figuie 4 (SEQ ID NO 4) Figure 7 (SEQ ID NO 13), Figure 10 (SEQ ID NO 19), Figure 14 (SEQ ID NO 26), Figure 15 (SEQ ID NO 27) and Figure 18 (SEQ ID NO 33)
22 Isolated nucleic acid having at least 80% nucleic acid sequence identity to the full-length coding sequence of the DNA deposited under ATCC accession number 209653, 209699, 209847, 209656, 209752, 209751 , 209801
23 A vector comprising the nucleic acid of any one of Claims 19 to 22
24 The vector of Claim 23 operably linked to control sequences recognized by a host cell transformed with the vector
25 A host cell comprising the vector of Claim 23
26 The host cell of Claim 25, wherein said cell is a CHO cell
27 The host cell of Claim 25, wherein said cell is an E coli
8 The host cell of Claim 25 wherein said cell is a yeast cell
14
29. A process for producing a PRO polypeptides comprising culturing the host cell of Claim 25 under conditions suitable for expression of said PRO polypeptide and recovering said PRO polypeptide from the cell culture.
30. 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:4). Figure 7 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 19). Figure 14 (SEQ ID NO:26). Figure 15 (SEQ ID NO:27). Figure 18 (SEQ ID NO:33).
31. 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:4). Figure 7 (SEQ ID NO: 13), Figure 10 (SEQ ID NO: 19). Figure 14 (SEQ ID NO:26). Figure 15 (SEQ ID NO:27), Figure 18 (SEQ ID NO:33).
32. 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 209653, 209699, 209847. 209656, 209752, 209751, 209801.
33. A chimeric molecule comprising a polypeptide according to any one of Claims 30 to 32 fused to a heterologous amino acid sequence.
34. The chimeric molecule of Claim 33, wherein said heterologous amino acid sequence is an epitope tag sequence.
35. The chimeric molecule of Claim 33, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin.
36. An antibody which specifically binds to a polypeptide according to any one of Claims 30 to 32.
37. The antibody of Claim 36, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody.
PCT/US2000/006319 1996-11-06 2000-03-10 Method of preventing the death of retinal neurons and treating ocular diseases WO2000053760A2 (en)

Priority Applications (332)

Application Number Priority Date Filing Date Title
AU37375/00A AU3737500A (en) 1999-03-12 2000-03-10 Method of preventing the death of retinal neurons and treating ocular diseases
AT00939307T ATE393825T1 (en) 1999-06-02 2000-05-17 STIMULATION OR INHIBITION OF ANGIOGENESIS AND CARDIAC VASCULARIZATION
ES00939307T ES2307515T3 (en) 1999-06-02 2000-05-17 ACTIVATION OR INHIBITION OF ANGIOGENESIS AND CARDIOVASCULARIZATION.
EP00939307A EP1212417B1 (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and cardiovascularization
PT00939307T PT1212417E (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and cardiovascularization
PCT/US2000/013705 WO2000073445A2 (en) 1999-06-02 2000-05-17 Interleukin-1-receptor associated kinase-3 (irak3)
DK00939307T DK1212417T3 (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and vascularization
CA002376116A CA2376116A1 (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and cardiovascularization
JP2001500757A JP4297317B2 (en) 1999-06-02 2000-05-17 Promotion or inhibition of angiogenesis and cardiovascularization
JP2001500672A JP2003524406A (en) 1999-06-02 2000-05-30 Compositions and methods for inhibiting tumor cell growth
CA002373915A CA2373915A1 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
EP07005021A EP1873244A3 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
PCT/US2000/014941 WO2000073348A2 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
DK00941164T DK1185648T3 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
EP07005022A EP1870464A3 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
AT00941164T ATE357518T1 (en) 1999-06-02 2000-05-30 METHODS AND COMPOSITIONS FOR INHIBITING NEOPLASTIC CELL GROWTH
EP07004855A EP1820860A3 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
EP07005023A EP1867719A3 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
ES00941164T ES2287020T3 (en) 1999-06-02 2000-05-30 PROCEDURE AND COMPOSITIONS TO INHIBIT THE GROWTH OF NEOPLASSIC CELLS.
EP00941164A EP1185648B1 (en) 1999-06-02 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth
AU20554/01A AU2055401A (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
JP2001542531A JP2004522404A (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding them
EP06000586A EP1688497A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002496312A CA2496312A1 (en) 1999-12-01 2000-12-01 Colon tumour marker pro4799 polypeptides and nucleic acids encoding the same
CA002491610A CA2491610A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000581A EP1666494A1 (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
CA002490909A CA2490909A1 (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
EP06000583A EP1686134A3 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002494705A CA2494705A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002491433A CA2491433A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002492070A CA2492070A1 (en) 1999-12-01 2000-12-01 Lung tumor marker pro4329 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
CA002492049A CA2492049A1 (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
EP06000585A EP1661996A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002490853A CA2490853A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/032678 WO2001040466A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000589A EP1661997A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA2709291A CA2709291A1 (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
EP06000582A EP1666495A1 (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
CA002391455A CA2391455A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/828,366 US20020010137A1 (en) 1997-09-18 2001-04-05 Methods and compositions for inhibiting neoplastic cell growth
US09/918,585 US20030060406A1 (en) 1997-10-17 2001-07-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/941,992 US20030082546A1 (en) 1996-11-06 2001-08-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,295 US20020156006A1 (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
US09/978,299 US20030199435A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,192 US20020177553A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,188 US20030139328A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,191 US20030050239A1 (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
US09/978,194 US20030195333A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,665 US7294700B2 (en) 1997-10-17 2001-10-16 Anti-PRO846 antibodies
US09/978,802 US20030199674A1 (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
US09/978,564 US7195760B2 (en) 1997-10-17 2001-10-16 Anti-pro363 antibodies
US09/978,697 US20020169284A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,757 US20030083248A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,643 US20030104998A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,544 US20030199436A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/981,915 US20030054986A1 (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
US09/978,681 US20030195148A1 (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
US09/981,915 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
US09/978,824 US20050124789A9 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,824 US20030055216A1 (en) 1997-10-17 2001-10-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,388 US20040223964A1 (en) 1998-03-17 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/210,028 US20030203446A1 (en) 1998-10-07 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/162,521 US7067628B2 (en) 1998-03-17 2001-10-19 PRO788 polypeptides
US10/166,709 US20030104536A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,124 US20030190701A1 (en) 1998-04-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,089 US7208575B2 (en) 1998-10-07 2001-10-19 PRO531 polypeptides
US10/160,502 US7220835B2 (en) 1998-07-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,829 US20030194780A1 (en) 1998-04-29 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,067 US7279553B2 (en) 1998-05-13 2001-10-19 PRO1083 polypeptides
US10/165,247 US7112657B2 (en) 1998-10-07 2001-10-19 PRO697 polypeptides
US10/164,728 US20030186368A1 (en) 1998-05-13 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
US10/164,749 US20040029218A1 (en) 1998-10-07 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
US10/145,017 US20030186365A1 (en) 1998-03-26 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,929 US20030194781A1 (en) 1998-03-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,922 US20030195345A1 (en) 1997-10-17 2001-10-21 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
US09/999,833 US6916648B2 (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
US10/017,085 US6974696B2 (en) 1997-10-17 2001-10-24 PRO853 nucleic acids
US09/999,832 US20020192706A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/020,445 US20030198994A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,084 US20030203402A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,081 US20030049684A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,832 US7132283B2 (en) 1997-10-17 2001-10-24 PRO273 polypeptides
US10/017,086 US7122375B2 (en) 1997-10-17 2001-10-24 PRO274 nucleic acids
US10/002,967 US20030148373A1 (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
US10/017,083 US20030148376A1 (en) 1997-10-17 2001-10-24 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
US10/013,927 US7189529B2 (en) 1997-10-17 2001-10-25 PRO792 nucleic acids
US10/013,921 US20030068648A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,928 US20030215905A1 (en) 1998-10-07 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
US10/013,918 US20030211091A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,923 US7169912B2 (en) 1997-10-17 2001-10-25 PRO1017 nucleic acids
US10/013,929 US7019124B2 (en) 1997-10-17 2001-10-25 PRO788 nucleic acids
US10/016,177 US20030073131A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,920 US20040006219A1 (en) 1997-10-17 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
US09/993,748 US20030069403A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,687 US20020198149A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,456 US20020137890A1 (en) 1997-03-31 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,163 US20020132253A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,073 US20020127576A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/992,521 US20030083461A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,444 US6930170B2 (en) 1997-06-16 2001-11-14 PRO1184 polypeptides
US09/991,854 US20030059780A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,726 US20030054359A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,667 US20030022187A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,438 US20030027754A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,562 US20030027985A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,427 US20030073809A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,443 US20030054987A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/992,598 US6956108B2 (en) 1997-06-16 2001-11-14 PRO1184 antibodies
US09/993,469 US20030068623A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,440 US20030060407A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,442 US20020132252A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,711 US20030032023A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,436 US20020198148A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,583 US7074897B2 (en) 1997-06-16 2001-11-14 Pro943 polypeptides
US09/997,333 US6953836B2 (en) 1997-06-16 2001-11-15 PRO844 polypeptides
US09/997,428 US20030027162A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/998,156 US20030044806A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,529 US7309761B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,614 US20030124531A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,384 US20030087305A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,514 US7019116B2 (en) 1997-06-16 2001-11-15 PRO 1387 polypeptides
US09/997,349 US7034106B2 (en) 1997-06-16 2001-11-15 Pro1159 polypeptides
US09/997,641 US7112656B2 (en) 1997-06-16 2001-11-15 PRO1312 polypeptides
US09/998,041 US20030119001A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,666 US20030027163A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,440 US20030059833A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,601 US7189814B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,641 US20030224358A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/998,041 US7309775B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,585 US20030119055A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,384 US7119177B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,559 US20030054403A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,601 US20030054404A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,542 US20030068647A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,857 US20030064375A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,666 US7244816B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,529 US20030134284A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,683 US20030059783A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,628 US20030059782A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,653 US7034122B2 (en) 1997-06-16 2001-11-15 Antibodies to PRO1159 polypeptides
US09/997,573 US20030049682A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,585 US7166282B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,157 US7101687B2 (en) 1997-06-16 2001-11-16 Nucleic acids encoding PRO943
US09/990,441 US7041804B2 (en) 1997-06-16 2001-11-16 Antibodies to PRO1387 polypeptides
US09/990,437 US20030045463A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,181 US6913919B2 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,150 US20030194760A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,172 US20030050457A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,726 US7018811B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO189 polypeptides
US09/989,328 US7056736B2 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,734 US7491529B2 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,279 US7083978B2 (en) 1997-06-16 2001-11-19 Nucleic acid encoding PRO1111 polypeptides
US09/989,722 US20020072067A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,735 US6972185B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO844 polypeptides
US09/989,727 US20020072497A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,729 US20030059831A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,723 US20020072092A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,721 US20020142961A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,732 US7037679B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO1184 polypeptides
US09/989,862 US20030130182A1 (en) 1997-11-05 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,293 US7034136B2 (en) 1997-06-16 2001-11-20 Nucleic acids encoding PRO1159 polypeptides
US09/989,725 US20030139329A1 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,731 US20020103125A1 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,728 US7029873B2 (en) 1997-06-16 2001-11-20 Nucleic acids to PRO1387 polypeptides
US09/989,730 US7157247B2 (en) 1997-06-16 2001-11-20 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
US10/121,047 US20030077778A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,045 US20030073210A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,059 US20030190721A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,051 US20030092147A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,044 US20030190717A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,041 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
US10/121,040 US20030082759A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,046 US20030194791A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,043 US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/121,061 US20030082761A1 (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
US10/121,062 US20030077779A1 (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
US10/121,048 US20030199051A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,052 US20030199052A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,050 US20030054516A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,057 US20030190719A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,056 US20030082760A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,060 US20030190722A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,055 US20030190718A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,058 US20030190720A1 (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
US10/123,261 US20030068796A1 (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
US10/123,291 US20030199058A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,155 US20030068794A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,157 US20030190725A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,109 US20030190723A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,262 US20030049816A1 (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
US10/123,156 US20030194792A1 (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
US10/123,213 US20030199057A1 (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
US10/123,212 US7276577B2 (en) 1997-03-31 2002-04-15 PRO1866 polypeptides
US10/123,771 US20030199060A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,214 US7343721B2 (en) 1997-03-31 2002-04-15 PRO4406 polypeptide
US10/123,213 US7193048B2 (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
US10/123,235 US20030082762A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,908 US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,905 US7285625B2 (en) 1997-06-18 2002-04-16 PRO536 polypeptides
US10/123,907 US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,912 US20030100087A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,906 US20030190726A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,905 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
US10/123,913 US20030203462A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,910 US7329404B2 (en) 1997-03-31 2002-04-16 Antibodies against PRO1310
US10/123,903 US20030073212A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,902 US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,904 US20030022328A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,820 US20030190729A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,814 US7105335B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,813 US7312307B2 (en) 1997-03-31 2002-04-17 PRO1056 polypeptides
US10/124,823 US20030199062A1 (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
US10/124,824 US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,795 US7304131B2 (en) 1997-03-31 2002-04-17 PRO1483 polypeptides
US10/124,817 US20030077786A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,805 US20030194794A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,819 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
US10/125,704 US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
US10/124,822 US7109305B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,922 US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/125,931 US20030199063A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,924 US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/125,927 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
US10/137,867 US20030207349A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,865 US20030032155A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,868 US20030082764A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,024 US20040058424A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,020 US20030207415A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,470 US20030022331A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,963 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
US10/139,980 US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/140,928 US20030068798A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,860 US7307151B2 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,809 US20030207418A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,805 US20030207417A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,921 US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
US10/140,864 US20030207419A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,865 US20030207420A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,925 US20030073215A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,808 US7425621B2 (en) 1997-03-31 2002-05-07 Antibodies against the PRO4401 polypeptide
US10/141,701 US20030207421A1 (en) 1997-03-31 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,755 US7297764B2 (en) 1997-03-31 2002-05-08 PRO4318 polypeptides
US10/141,754 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
US10/141,756 US7488586B2 (en) 1997-03-31 2002-05-08 PRO4409 polypeptides
US10/142,425 US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,113 US7329730B2 (en) 1997-03-31 2002-05-09 PRO4348 polypeptides
US10/142,430 US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/142,417 US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/143,114 US20030036180A1 (en) 1997-03-31 2002-05-09 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
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 polypeptides
US10/142,423 US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,431 US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/146,730 US20030207427A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,792 US20030207428A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,528 US20030219885A1 (en) 1997-03-31 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,519 US20030077791A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,492 US20030082765A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,536 US20040077064A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,395 US7189534B2 (en) 1997-03-31 2002-05-21 PRO4320 polynucleotide
US10/153,934 US20030129695A1 (en) 1997-03-31 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,843 US20030207805A1 (en) 1997-06-18 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,786 US20030208055A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,782 US20030077792A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,791 US20030207429A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,498 US20030073216A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,782 US20030082766A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,913 US20030022298A1 (en) 1997-09-15 2002-06-20 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
US10/187,885 US20030032138A1 (en) 1998-06-24 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/197,942 US20030175882A1 (en) 1998-09-10 2002-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/100,159 US7425613B2 (en) 1997-11-05 2005-04-05 PRO1375 polypeptides
JP2005229454A JP4145314B2 (en) 1999-06-02 2005-08-08 Compositions and methods for tumor cell growth inhibition
JP2005264293A JP2006068016A (en) 1999-12-01 2005-08-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/341,175 US7468427B2 (en) 1997-03-31 2006-01-27 Antibodies to PRO1275 polypeptide
JP2006225770A JP4072181B2 (en) 1999-06-02 2006-08-22 Compositions and methods for tumor cell growth inhibition
JP2006225772A JP2007029098A (en) 1999-06-02 2006-08-22 Method and composition for inhibiting neoplastic cell growth
JP2006225771A JP4074645B2 (en) 1999-06-02 2006-08-22 Compositions and methods for tumor cell growth inhibition
US11/786,466 US20080182275A1 (en) 1998-12-22 2007-04-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007326424A JP2008167749A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptide and nucleic acid encoding the same
JP2007326609A JP2008148701A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007325484A JP2008148699A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007326613A JP2008161190A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptide and nucleic acid encoding the same
JP2008145176A JP2009019032A (en) 1999-06-02 2008-06-02 Promotion or inhibition of angiogenesis and cardiovascularization

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US12395799P 1999-03-12 1999-03-12
US60/123,957 1999-03-12

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/005841 Continuation-In-Part WO2000053758A2 (en) 1994-09-08 2000-03-02 Compositions and methods for the treatment of immune related diseases

Related Child Applications (3)

Application Number Title Priority Date Filing Date
PCT/US2000/007532 Continuation-In-Part WO2000070050A1 (en) 1997-03-31 2000-03-21 Compositions and methods for the treatment of immune related diseases
US10/081,056 Continuation US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/197,942 Continuation US20030175882A1 (en) 1998-03-27 2002-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same

Publications (2)

Publication Number Publication Date
WO2000053760A2 true WO2000053760A2 (en) 2000-09-14
WO2000053760A3 WO2000053760A3 (en) 2001-01-18

Family

ID=22411919

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/006319 WO2000053760A2 (en) 1996-11-06 2000-03-10 Method of preventing the death of retinal neurons and treating ocular diseases

Country Status (2)

Country Link
AU (1) AU3737500A (en)
WO (1) WO2000053760A2 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6432673B1 (en) 1998-12-07 2002-08-13 Zymogenetics, Inc. Growth factor homolog ZVEGF3
WO2002079383A2 (en) * 2001-03-30 2002-10-10 Genentech, Inc. Fizz1 for metabolism regulation
WO2004071573A2 (en) * 2003-02-07 2004-08-26 The Johns Hopkins University Hypoxia induced mitogenic factor
WO2013172721A1 (en) * 2012-05-18 2013-11-21 Wise Lyn Marie Combination treatments and compositions for wound healing
KR20150132602A (en) * 2008-06-25 2015-11-25 에스바테크 - 어 노바티스 컴파니 엘엘씨 Stable and soluble antibodies inhibiting vegf

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993008826A1 (en) * 1991-11-08 1993-05-13 Cephalon, Inc. Treating retinal neuronal disorders by the application of insulin-like growth factors and analogs
WO1994001124A1 (en) * 1992-07-08 1994-01-20 Celtrix Pharmaceuticals, Inc. METHOD OF TREATING OPHTHALMIC DISORDERS USING TGF-$g(b)
EP0679716A1 (en) * 1993-11-12 1995-11-02 Kenichi Matsubara Gene signature
WO1999037671A1 (en) * 1998-01-27 1999-07-29 Eli Lilly And Company Vegf related gene and protein
WO1999046281A2 (en) * 1998-03-10 1999-09-16 Genentech, Inc. Novel polypeptides and nucleic acids encoding the same
WO1999047677A2 (en) * 1998-03-17 1999-09-23 Genentech, Inc. Polypeptides homologous to vegf and bmp1
WO2000004183A1 (en) * 1998-07-15 2000-01-27 Human Genome Sciences, Inc. Bone morphogenic protein
WO2000032221A2 (en) * 1998-12-01 2000-06-08 Genentech, Inc. Promotion or inhibition of angiogenesis and cardiovascularization
WO2000034474A2 (en) * 1998-12-07 2000-06-15 Zymogenetics, Inc. Growth factor homolog zvegf3

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993008826A1 (en) * 1991-11-08 1993-05-13 Cephalon, Inc. Treating retinal neuronal disorders by the application of insulin-like growth factors and analogs
WO1994001124A1 (en) * 1992-07-08 1994-01-20 Celtrix Pharmaceuticals, Inc. METHOD OF TREATING OPHTHALMIC DISORDERS USING TGF-$g(b)
EP0679716A1 (en) * 1993-11-12 1995-11-02 Kenichi Matsubara Gene signature
WO1999037671A1 (en) * 1998-01-27 1999-07-29 Eli Lilly And Company Vegf related gene and protein
WO1999046281A2 (en) * 1998-03-10 1999-09-16 Genentech, Inc. Novel polypeptides and nucleic acids encoding the same
WO1999047677A2 (en) * 1998-03-17 1999-09-23 Genentech, Inc. Polypeptides homologous to vegf and bmp1
WO2000004183A1 (en) * 1998-07-15 2000-01-27 Human Genome Sciences, Inc. Bone morphogenic protein
WO2000032221A2 (en) * 1998-12-01 2000-06-08 Genentech, Inc. Promotion or inhibition of angiogenesis and cardiovascularization
WO2000034474A2 (en) * 1998-12-07 2000-06-15 Zymogenetics, Inc. Growth factor homolog zvegf3

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CAYOUETTE M ET AL: "ADENOVIRUS-MEDIATED GENE TRANSFER OF CILIARY NEUROTROPHIC FACTOR CAN PREVENT PHOTORECEPTOR DEGENERATION IN THE RETINAL DEGENERATION (RD) MOUSE" HUMAN GENE THERAPY,XX,XX, vol. 8, no. 4, 1 March 1997 (1997-03-01), pages 423-430, XP000876513 ISSN: 1043-0342 *
DATABASE EMBL - EMEST_HUM1 [Online] Entry/Acc.no. AA631149, 31 October 1997 (1997-10-31) STRAUSBERG, R. ET AL.: "nq75h03.s1 NCI_CGAP_Pr22 Homo sapiens cDNA clone IMAGE:1158197 3', mRNA sequence." XP002113997 *
LAVAIL M M ET AL: "Multiple growth factors, cytokines, and neutrophins rescue photoreceptors from the damaging effects of constant light" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA,US,NATIONAL ACADEMY OF SCIENCE. WASHINGTON, vol. 89, no. 89, December 1992 (1992-12), pages 11249-11253, XP002118631 ISSN: 0027-8424 *
MEYER M ET AL: "A novel vascular endothelial growth factor encoded by orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases" EMBO JOURNAL,GB,OXFORD UNIVERSITY PRESS, SURREY, vol. 18, no. 2, 1999, pages 363-374, XP002110116 ISSN: 0261-4189 *
OGAWA S ET AL: "A novel type of vascular endothelial growth factor, VEGF-E (NZ-7 VEGF), preferentially utilizes KDR/Flk-1 receptor and carries a potent mitotic activity without heparin-binding domain" JOURNAL OF BIOLOGICAL CHEMISTRY,US,AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, vol. 273, no. 47, 20 November 1998 (1998-11-20), pages 31273-31282, XP002110115 ISSN: 0021-9258 *

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7691981B2 (en) 1998-12-07 2010-04-06 Zymogenetics, Inc. Growth factor homolog zvegf3
US6528050B1 (en) 1998-12-07 2003-03-04 Zymogenetics, Inc. Growth factor homolog zvegf3
US6432673B1 (en) 1998-12-07 2002-08-13 Zymogenetics, Inc. Growth factor homolog ZVEGF3
US6814965B2 (en) 1998-12-07 2004-11-09 Zymogenetics, Inc. Methods of decreasing ZVEGF3 activity
US6887982B1 (en) 1998-12-07 2005-05-03 Zymogenetics, Inc. Antibodies reactive to the c-terminal portion of growth factor homolog zvegf3
US7387885B2 (en) 1998-12-07 2008-06-17 Zymogenetics, Inc. Growth factor homolog zvegf3 polynucleotides
US8052976B2 (en) 1998-12-07 2011-11-08 Zymogenetics, Inc. Growth factor homolog ZVEGF3
US7658920B2 (en) 1998-12-07 2010-02-09 Zymogenetics, Inc. Method of inhibiting the activity of growth factor homolog ZVEGF3
WO2002079383A2 (en) * 2001-03-30 2002-10-10 Genentech, Inc. Fizz1 for metabolism regulation
WO2002079383A3 (en) * 2001-03-30 2003-12-04 Genentech Inc Fizz1 for metabolism regulation
US8329177B2 (en) 2003-02-07 2012-12-11 The John Hopkins University Method of treating hypertrophic cardiomyopathy
US7671037B2 (en) 2003-02-07 2010-03-02 The Johns Hopkins University Hypoxia induced mitogenic factor
WO2004071573A3 (en) * 2003-02-07 2008-07-24 Univ Johns Hopkins Hypoxia induced mitogenic factor
US8080533B2 (en) 2003-02-07 2011-12-20 The Johns Hopkins University Hypoxia induced mitogenic factor
WO2004071573A2 (en) * 2003-02-07 2004-08-26 The Johns Hopkins University Hypoxia induced mitogenic factor
US8414891B2 (en) 2003-02-07 2013-04-09 The Johns Hopkins University Method for treating pulmonary hypertension
US9878005B2 (en) 2003-02-07 2018-01-30 The Johns Hopkins University Method of promoting wound healing with hyodxia-induced mitogenic factor
KR101737466B1 (en) 2008-06-25 2017-05-18 에스바테크 - 어 노바티스 컴파니 엘엘씨 Stable and soluble antibodies inhibiting vegf
KR101671886B1 (en) 2008-06-25 2016-11-04 에스바테크 - 어 노바티스 컴파니 엘엘씨 Stable and soluble antibodies inhibiting vegf
KR20150132602A (en) * 2008-06-25 2015-11-25 에스바테크 - 어 노바티스 컴파니 엘엘씨 Stable and soluble antibodies inhibiting vegf
KR101817284B1 (en) 2008-06-25 2018-01-11 에스바테크 - 어 노바티스 컴파니 엘엘씨 Stable and soluble antibodies inhibiting vegf
KR20180098705A (en) * 2008-06-25 2018-09-04 에스바테크 - 어 노바티스 컴파니 엘엘씨 Stable and soluble antibodies inhibiting vegf
KR20190092630A (en) * 2008-06-25 2019-08-07 에스바테크 - 어 노바티스 컴파니 엘엘씨 Stable and soluble antibodies inhibiting vegf
KR102095257B1 (en) 2008-06-25 2020-04-01 노바르티스 아게 Stable and soluble antibodies inhibiting vegf
KR102157097B1 (en) 2008-06-25 2020-09-18 노바르티스 아게 Stable and soluble antibodies inhibiting vegf
KR20200108922A (en) * 2008-06-25 2020-09-21 노바르티스 아게 Stable and soluble antibodies inhibiting vegf
KR102296443B1 (en) 2008-06-25 2021-09-01 노바르티스 아게 Stable and soluble antibodies inhibiting vegf
WO2013172721A1 (en) * 2012-05-18 2013-11-21 Wise Lyn Marie Combination treatments and compositions for wound healing
AU2013263502B2 (en) * 2012-05-18 2018-02-08 Otago Innovation Limited Combination treatments and compositions for wound healing

Also Published As

Publication number Publication date
WO2000053760A3 (en) 2001-01-18
AU3737500A (en) 2000-09-28

Similar Documents

Publication Publication Date Title
CA2503125C (en) Novel composition and methods for the treatment of immune related diseases
CA2586615C (en) Pro87299 (btla), and variants thereof, for treating immune related disorders
WO2000037638A2 (en) Methods and compositions for inhibiting neoplastic cell growth
CA2347835A1 (en) Promotion or inhibition of angiogenesis and cardiovascularization
WO2001009327A2 (en) Method of preventing the injury or death of retinal cells and treating ocular diseases
PT1616878E (en) Secreted and transmembrane polypeptides and nucleic acids encoding the same
WO2000070050A1 (en) Compositions and methods for the treatment of immune related diseases
WO2001005972A1 (en) Compositions and methods for the treatment of immune related diseases
WO2000053752A2 (en) Promotion or inhibition of angiogenesis and cardiovascularization
ZA200103885B (en) Methods and compositions for inhibiting neoplastic cell growth.
WO2000032778A2 (en) Methods and compositions for inhibiting neoplastic cell growth
CA2390786A1 (en) Compositions and methods for the treatment of immune related diseases
ZA200401536B (en) Compositions and methods for the treatment of immune related diseases
WO2000053760A2 (en) Method of preventing the death of retinal neurons and treating ocular diseases
JP2009219492A (en) New polypeptide having sequence similarity to gdnfr and nucleic acid encoding the same
US7642242B2 (en) PRO34128 polypeptides
WO2000015797A2 (en) Compositions and methods for the treatment of immune related diseases
MXPA01011264A (en) Novel chimpanzee erythropoietin (chepo) polypeptides and nucleic acids encoding the same.
WO2000075316A1 (en) Methods and compositions for inhibiting neoplastic cell growth
US20100285016A1 (en) PRO34128 nucleic acids
CA2384055A1 (en) Compositions and methods for the treatment of immune related diseases
CA2450402A1 (en) Methods and compositions for inhibiting cancer cell growth comprising pro224
EP1114154A2 (en) Compositions and methods for the treatment of immune related diseases
KR20020059591A (en) Fibroblast Growth Factor-19 (FGF-19) Nucleic acid and Polypeptides and Methods of Use for the Treatment of Obesity

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase