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WO1999014241A2 - Traitement d'affections liees aux fonctions immunitaires et compositions correspondantes - Google Patents

Traitement d'affections liees aux fonctions immunitaires et compositions correspondantes Download PDF

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Publication number
WO1999014241A2
WO1999014241A2 PCT/US1998/019437 US9819437W WO9914241A2 WO 1999014241 A2 WO1999014241 A2 WO 1999014241A2 US 9819437 W US9819437 W US 9819437W WO 9914241 A2 WO9914241 A2 WO 9914241A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
cells
polypeptide
seq
mammal
Prior art date
Application number
PCT/US1998/019437
Other languages
English (en)
Other versions
WO1999014241A3 (fr
Inventor
Sherman Fong
Audrey Goddard
Austin L. Gurney
Daniel Tumas
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 AU93959/98A priority Critical patent/AU9395998A/en
Priority to ES98959543T priority patent/ES2316175T3/es
Priority to DE69840105T priority patent/DE69840105D1/de
Priority to DE69837897T priority patent/DE69837897T2/de
Priority to EP04010433A priority patent/EP1481990B1/fr
Priority to AU15324/99A priority patent/AU735081B2/en
Priority to DE69839430T priority patent/DE69839430T2/de
Priority to EP98959543A priority patent/EP1032667B1/fr
Priority to ES04010433T priority patent/ES2288649T3/es
Priority to DK04010433T priority patent/DK1481990T3/da
Priority to PT04010433T priority patent/PT1481990E/pt
Priority to ES04010432T priority patent/ES2305608T3/es
Priority to AT98959543T priority patent/ATE410512T1/de
Priority to AT04010433T priority patent/ATE364049T1/de
Priority to PCT/US1998/024855 priority patent/WO1999027098A2/fr
Priority to DK04010432T priority patent/DK1481989T3/da
Priority to AT04010432T priority patent/ATE393786T1/de
Priority to CA002309358A priority patent/CA2309358A1/fr
Priority to JP2000522240A priority patent/JP3497133B2/ja
Priority to EP04010432A priority patent/EP1481989B1/fr
Priority to PT04010432T priority patent/PT1481989E/pt
Priority to EP08017468A priority patent/EP2014677A1/fr
Publication of WO1999014241A2 publication Critical patent/WO1999014241A2/fr
Publication of WO1999014241A3 publication Critical patent/WO1999014241A3/fr
Priority to IL14155199A priority patent/IL141551A0/xx
Priority to KR1020017003430A priority patent/KR20010085816A/ko
Priority to AU64984/99A priority patent/AU6498499A/en
Priority to JP2000570324A priority patent/JP2003524600A/ja
Priority to PCT/US1999/021547 priority patent/WO2000015797A2/fr
Priority to CA002343006A priority patent/CA2343006A1/fr
Priority to US09/902,736 priority patent/US6894148B2/en
Priority to US09/902,903 priority patent/US20030044839A1/en
Priority to US09/902,736 priority patent/US20030049676A1/en
Priority to US09/902,759 priority patent/US20030077654A1/en
Priority to US09/902,572 priority patent/US20030108983A1/en
Priority to US09/902,615 priority patent/US20030092002A1/en
Priority to US09/902,634 priority patent/US20030082540A1/en
Priority to US09/902,979 priority patent/US20030113718A1/en
Priority to US09/902,853 priority patent/US20020192659A1/en
Priority to US09/902,692 priority patent/US20030054400A1/en
Priority to US09/902,713 priority patent/US20030082541A1/en
Priority to US09/904,011 priority patent/US20030003530A1/en
Priority to US09/903,806 priority patent/US20030130489A1/en
Priority to US09/903,603 priority patent/US20030148419A1/en
Priority to US09/903,640 priority patent/US7208308B2/en
Priority to US09/903,562 priority patent/US6965015B2/en
Priority to US09/904,119 priority patent/US20030049621A1/en
Priority to US09/903,786 priority patent/US20030044793A1/en
Priority to US09/903,823 priority patent/US20030104381A1/en
Priority to US09/903,943 priority patent/US20030054349A1/en
Priority to US09/903,749 priority patent/US7147853B2/en
Priority to US09/903,925 priority patent/US20030096233A1/en
Priority to US09/903,520 priority patent/US20030054401A1/en
Priority to US09/904,859 priority patent/US20030036060A1/en
Priority to US09/904,992 priority patent/US20030135025A1/en
Priority to US09/904,877 priority patent/US20030186358A1/en
Priority to US09/905,291 priority patent/US20020160374A1/en
Priority to US09/905,056 priority patent/US20030054441A1/en
Priority to US09/904,938 priority patent/US20030211569A1/en
Priority to US09/905,088 priority patent/US20030073077A1/en
Priority to US09/905,125 priority patent/US6664376B2/en
Priority to US09/904,786 priority patent/US7557192B2/en
Priority to US09/904,766 priority patent/US20030152999A1/en
Priority to US09/905,381 priority patent/US6818746B2/en
Priority to US09/904,838 priority patent/US20030148370A1/en
Priority to US09/904,820 priority patent/US20030036094A1/en
Priority to US09/904,485 priority patent/US20030064367A1/en
Priority to US09/904,920 priority patent/US6806352B2/en
Priority to US09/905,075 priority patent/US7169906B2/en
Priority to US09/905,348 priority patent/US20030064923A1/en
Priority to US09/904,462 priority patent/US6878807B2/en
Priority to US09/904,532 priority patent/US7151160B2/en
Priority to US09/905,449 priority patent/US6965011B2/en
Priority to US09/904,553 priority patent/US20030059828A1/en
Priority to US09/904,956 priority patent/US20030049622A1/en
Priority to US09/906,838 priority patent/US7070979B2/en
Priority to US09/906,700 priority patent/US6723535B2/en
Priority to US09/906,722 priority patent/US6946262B2/en
Priority to US09/906,742 priority patent/US20030023054A1/en
Priority to US09/906,646 priority patent/US6852848B2/en
Priority to US09/906,815 priority patent/US7094567B2/en
Priority to US09/906,618 priority patent/US6828146B2/en
Priority to US09/906,760 priority patent/US20030096340A1/en
Priority to US09/906,777 priority patent/US20030148371A1/en
Priority to US09/907,824 priority patent/US20020197671A1/en
Priority to US09/907,575 priority patent/US20030073079A1/en
Priority to US09/907,728 priority patent/US20030190611A1/en
Priority to US09/907,613 priority patent/US20030027145A1/en
Priority to US09/908,093 priority patent/US20030017498A1/en
Priority to US09/907,942 priority patent/US7087738B2/en
Priority to US09/907,794 priority patent/US6635468B2/en
Priority to US09/907,841 priority patent/US7033825B2/en
Priority to US09/907,979 priority patent/US20030082542A1/en
Priority to US09/907,652 priority patent/US20030104469A1/en
Priority to US09/907,925 priority patent/US20030054352A1/en
Priority to US09/908,576 priority patent/US20040005553A1/en
Priority to US09/909,088 priority patent/US20020146709A1/en
Priority to US09/909,204 priority patent/US20030036061A1/en
Priority to US09/909,320 priority patent/US7074592B2/en
Priority to US09/909,064 priority patent/US6818449B2/en
Priority to US09/941,992 priority patent/US20030082546A1/en
Priority to US09/992,521 priority patent/US20030083461A1/en
Priority to US09/993,469 priority patent/US20030068623A1/en
Priority to US09/990,443 priority patent/US20030054987A1/en
Priority to US09/990,442 priority patent/US20020132252A1/en
Priority to US09/990,456 priority patent/US20020137890A1/en
Priority to US09/991,073 priority patent/US20020127576A1/en
Priority to US09/990,711 priority patent/US20030032023A1/en
Priority to US09/992,598 priority patent/US6956108B2/en
Priority to US09/991,854 priority patent/US20030059780A1/en
Priority to US09/993,604 priority patent/US20020137075A1/en
Priority to US09/993,748 priority patent/US20030069403A1/en
Priority to US09/990,726 priority patent/US20030054359A1/en
Priority to US09/990,427 priority patent/US20030073809A1/en
Priority to US09/990,562 priority patent/US20030027985A1/en
Priority to US09/990,444 priority patent/US6930170B2/en
Priority to US09/993,687 priority patent/US20020198149A1/en
Priority to US09/990,438 priority patent/US20030027754A1/en
Priority to US09/990,440 priority patent/US20030060407A1/en
Priority to US09/993,583 priority patent/US7074897B2/en
Priority to US09/990,436 priority patent/US20020198148A1/en
Priority to US09/993,667 priority patent/US20030022187A1/en
Priority to US09/991,163 priority patent/US20020132253A1/en
Priority to US09/997,666 priority patent/US20030027163A1/en
Priority to US09/997,601 priority patent/US20030054404A1/en
Priority to US10/002,796 priority patent/US20030032057A1/en
Priority to US09/997,683 priority patent/US20030059783A1/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 US09/997,440 priority patent/US20030059833A1/en
Priority to US09/997,601 priority patent/US7189814B2/en
Priority to US09/997,857 priority patent/US20030064375A1/en
Priority to US09/997,641 priority patent/US20030224358A1/en
Priority to US09/997,653 priority patent/US7034122B2/en
Priority to US09/997,585 priority patent/US7166282B2/en
Priority to US09/997,641 priority patent/US7112656B2/en
Priority to US09/997,559 priority patent/US20030054403A1/en
Priority to US09/997,542 priority patent/US20030068647A1/en
Priority to US09/997,428 priority patent/US20030027162A1/en
Priority to US09/997,628 priority patent/US20030059782A1/en
Priority to US09/997,333 priority patent/US6953836B2/en
Priority to US09/997,666 priority patent/US7244816B2/en
Priority to US09/997,585 priority patent/US20030119055A1/en
Priority to US09/997,529 priority patent/US20030134284A1/en
Priority to US09/997,614 priority patent/US20030124531A1/en
Priority to US09/998,041 priority patent/US7309775B2/en
Priority to US09/997,573 priority patent/US20030049682A1/en
Priority to US09/998,041 priority patent/US20030119001A1/en
Priority to US09/998,156 priority patent/US20030044806A1/en
Priority to US09/997,384 priority patent/US7119177B2/en
Priority to US09/997,529 priority patent/US7309761B2/en
Priority to US09/991,181 priority patent/US6913919B2/en
Priority to US09/991,150 priority patent/US20030194760A1/en
Priority to US09/990,437 priority patent/US20030045463A1/en
Priority to US09/991,157 priority patent/US7101687B2/en
Priority to US09/991,172 priority patent/US20030050457A1/en
Priority to US09/990,441 priority patent/US7041804B2/en
Priority to US09/989,727 priority patent/US20020072497A1/en
Priority to US09/989,722 priority patent/US20020072067A1/en
Priority to US09/989,734 priority patent/US7491529B2/en
Priority to US09/989,328 priority patent/US7056736B2/en
Priority to US09/989,726 priority patent/US7018811B2/en
Priority to US09/989,721 priority patent/US20020142961A1/en
Priority to US09/989,732 priority patent/US7037679B2/en
Priority to US09/989,735 priority patent/US6972185B2/en
Priority to US09/989,862 priority patent/US20030130182A1/en
Priority to US09/989,723 priority patent/US20020072092A1/en
Priority to US09/989,729 priority patent/US20030059831A1/en
Priority to US09/989,279 priority patent/US7083978B2/en
Priority to US09/989,725 priority patent/US20030139329A1/en
Priority to US09/989,731 priority patent/US20020103125A1/en
Priority to US09/989,724 priority patent/US7060812B2/en
Priority to US09/989,728 priority patent/US7029873B2/en
Priority to US09/989,293 priority patent/US7034136B2/en
Priority to US09/989,730 priority patent/US7157247B2/en
Priority to US10/028,072 priority patent/US20030004311A1/en
Priority to US10/066,203 priority patent/US20030180796A1/en
Priority to US10/066,494 priority patent/US20030032063A1/en
Priority to US10/066,500 priority patent/US20020177165A1/en
Priority to US10/066,193 priority patent/US20030044902A1/en
Priority to US10/066,273 priority patent/US7317092B2/en
Priority to US10/066,198 priority patent/US20030170721A1/en
Priority to US10/066,211 priority patent/US20030044844A1/en
Priority to US10/066,269 priority patent/US20030040014A1/en
Priority to US10/081,056 priority patent/US20040043927A1/en
Priority to US10/121,040 priority patent/US20030082759A1/en
Priority to US10/121,047 priority patent/US20030077778A1/en
Priority to US10/121,041 priority patent/US20030077776A1/en
Priority to US10/121,042 priority patent/US20030096386A1/en
Priority to US10/121,046 priority patent/US20030194791A1/en
Priority to US10/121,059 priority patent/US20030190721A1/en
Priority to US10/121,045 priority patent/US20030073210A1/en
Priority to US10/121,044 priority patent/US20030190717A1/en
Priority to US10/121,051 priority patent/US20030092147A1/en
Priority to US10/121,063 priority patent/US20030199055A1/en
Priority to US10/121,052 priority patent/US20030199052A1/en
Priority to US10/121,043 priority patent/US7220831B2/en
Priority to US10/121,054 priority patent/US20030199054A1/en
Priority to US10/121,050 priority patent/US20030054516A1/en
Priority to US10/121,056 priority patent/US20030082760A1/en
Priority to US10/121,062 priority patent/US20030077779A1/en
Priority to US10/121,061 priority patent/US20030082761A1/en
Priority to US10/121,060 priority patent/US20030190722A1/en
Priority to US10/121,055 priority patent/US20030190718A1/en
Priority to US10/121,049 priority patent/US20030022239A1/en
Priority to US10/121,053 priority patent/US20030199053A1/en
Priority to US10/121,048 priority patent/US20030199051A1/en
Priority to US10/121,057 priority patent/US20030190719A1/en
Priority to US10/121,058 priority patent/US20030190720A1/en
Priority to US10/123,771 priority patent/US20030199060A1/en
Priority to US10/123,212 priority patent/US7276577B2/en
Priority to US10/123,213 priority patent/US20030199057A1/en
Priority to US10/123,262 priority patent/US20030049816A1/en
Priority to US10/123,109 priority patent/US20030190723A1/en
Priority to US10/123,157 priority patent/US20030190725A1/en
Priority to US10/123,156 priority patent/US20030194792A1/en
Priority to US10/123,236 priority patent/US20030068795A1/en
Priority to US10/123,292 priority patent/US20030073211A1/en
Priority to US10/123,291 priority patent/US20030199058A1/en
Priority to US10/123,214 priority patent/US7343721B2/en
Priority to US10/123,108 priority patent/US7635478B2/en
Priority to US10/123,213 priority patent/US7193048B2/en
Priority to US10/123,215 priority patent/US7291329B2/en
Priority to US10/123,235 priority patent/US20030082762A1/en
Priority to US10/123,155 priority patent/US20030068794A1/en
Priority to US10/123,322 priority patent/US20030199059A1/en
Priority to US10/123,261 priority patent/US20030068796A1/en
Priority to US10/123,154 priority patent/US20030190724A1/en
Priority to US10/123,902 priority patent/US20030077781A1/en
Priority to US10/123,909 priority patent/US7193049B2/en
Priority to US10/123,905 priority patent/US7285625B2/en
Priority to US10/123,907 priority patent/US7084258B2/en
Priority to US10/123,913 priority patent/US20030203462A1/en
Priority to US10/123,904 priority patent/US20030022328A1/en
Priority to US10/123,912 priority patent/US20030100087A1/en
Priority to US10/123,905 priority patent/US20030087344A1/en
Priority to US10/123,906 priority patent/US20030190726A1/en
Priority to US10/123,910 priority patent/US7329404B2/en
Priority to US10/123,903 priority patent/US20030073212A1/en
Priority to US10/123,908 priority patent/US7335728B2/en
Priority to US10/123,911 priority patent/US7408032B2/en
Priority to US10/125,805 priority patent/US20030194794A1/en
Priority to US10/124,814 priority patent/US7105335B2/en
Priority to US10/124,821 priority patent/US20030199023A1/en
Priority to US10/124,818 priority patent/US20030082763A1/en
Priority to US10/124,817 priority patent/US20030077786A1/en
Priority to US10/124,824 priority patent/US20030077659A1/en
Priority to US10/124,822 priority patent/US7109305B2/en
Priority to US10/125,704 priority patent/US7357926B2/en
Priority to US10/124,813 priority patent/US7312307B2/en
Priority to US10/124,823 priority patent/US20030199062A1/en
Priority to US10/124,820 priority patent/US20030190729A1/en
Priority to US10/125,795 priority patent/US7304131B2/en
Priority to US10/124,819 priority patent/US7285626B2/en
Priority to US10/124,816 priority patent/US20030190728A1/en
Priority to US10/125,924 priority patent/US7342097B2/en
Priority to US10/125,927 priority patent/US20030190731A1/en
Priority to US10/125,932 priority patent/US7317079B2/en
Priority to US10/125,922 priority patent/US7309762B2/en
Priority to US10/125,931 priority patent/US20030199063A1/en
Priority to US10/127,831 priority patent/US20030082689A1/en
Priority to US10/128,689 priority patent/US20030087365A1/en
Priority to US10/131,825 priority patent/US7282566B2/en
Priority to US10/131,817 priority patent/US7291701B2/en
Priority to US10/131,823 priority patent/US7304132B2/en
Priority to US10/137,865 priority patent/US20030032155A1/en
Priority to US10/137,868 priority patent/US20030082764A1/en
Priority to US10/137,867 priority patent/US20030207349A1/en
Priority to US10/140,024 priority patent/US20040058424A1/en
Priority to US10/140,020 priority patent/US20030207415A1/en
Priority to US10/140,023 priority patent/US20030207416A1/en
Priority to US10/140,470 priority patent/US20030022331A1/en
Priority to US10/140,474 priority patent/US20030032156A1/en
Priority to US10/139,963 priority patent/US7288625B2/en
Priority to US10/139,980 priority patent/US7247710B2/en
Priority to US10/140,864 priority patent/US20030207419A1/en
Priority to US10/140,809 priority patent/US20030207418A1/en
Priority to US10/140,808 priority patent/US7425621B2/en
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Priority to US10/140,925 priority patent/US20030073215A1/en
Priority to US10/140,805 priority patent/US20030207417A1/en
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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 US10/141,756 priority patent/US7488586B2/en
Priority to US10/142,430 priority patent/US7309766B2/en
Priority to US10/143,114 priority patent/US20030036180A1/en
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Priority to US10/142,425 priority patent/US20030207424A1/en
Priority to US10/142,419 priority patent/US7153941B2/en
Priority to US10/142,431 priority patent/US7285629B2/en
Priority to US10/142,423 priority patent/US20030049817A1/en
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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 US10/147,492 priority patent/US20030082765A1/en
Priority to US10/147,536 priority patent/US20040077064A1/en
Priority to US10/147,519 priority patent/US20030077791A1/en
Priority to US10/152,395 priority patent/US7189534B2/en
Priority to US10/153,934 priority patent/US20030129695A1/en
Priority to US10/156,843 priority patent/US20030207805A1/en
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Priority to US10/157,782 priority patent/US20030077792A1/en
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Priority to US10/160,498 priority patent/US20030073216A1/en
Priority to US10/158,791 priority patent/US20030207429A1/en
Priority to US10/176,913 priority patent/US20030022298A1/en
Priority to JP2003334209A priority patent/JP3950097B2/ja
Priority to JP2003334210A priority patent/JP4040004B2/ja
Priority to US10/767,374 priority patent/US7282565B2/en
Priority to US10/767,904 priority patent/US7211400B2/en
Priority to US10/771,187 priority patent/US7355002B2/en
Priority to US10/785,607 priority patent/US7115713B2/en
Priority to US10/785,433 priority patent/US20040141972A1/en
Priority to US10/785,220 priority patent/US7198917B2/en
Priority to US10/785,221 priority patent/US7273726B2/en
Priority to US10/797,366 priority patent/US7378507B2/en
Priority to US10/964,263 priority patent/US7419663B2/en
Priority to US11/100,159 priority patent/US7425613B2/en
Priority to US11/240,891 priority patent/US20060246540A1/en
Priority to US11/249,825 priority patent/US8007798B2/en
Priority to US11/341,175 priority patent/US7468427B2/en
Priority to US11/518,609 priority patent/US20070077623A1/en
Priority to US11/607,709 priority patent/US20070087007A1/en
Priority to US11/642,816 priority patent/US20070088151A1/en
Priority to JP2007056461A priority patent/JP4382827B2/ja

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—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
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to compositions and methods for the diagnosis and treatment of immune related diseases.
  • Immune related and inflammatory diseases are the manifestation or consequence of fairly complex, often multiple interconnected biological pathways which in normal physiology are critical to respond to insult or injury, initiate repair from insult or injury, and mount innate and acquired defense against foreign organisms. Disease or pathology occurs when these normal physiological pathways cause additional insult or injury either as directly related to the intensity of the response, as a consequence of abnormal regulation or excessive stimulation, as a reaction to self, or as a combination of these.
  • therapeutic intervention can occur by either antagonism of a detrimental process/pathway or stimulation of a beneficial process/pathway.
  • immune-mediated inflammatory diseases include immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, neoplasia, etc.
  • T lymphocytes are an important component of a mammalian immune response. T cells recognise antigens which are associated with a self-molecule encoded by genes within the major histocompatibility complex (MHC). The antigen may be displayed together with MHC molecules on the surface of antigen presenting cells, virus infected cells, cancer cells, grafts, etc. The T cell system eliminates these altered cells which pose a health threat to the host mammal. T cells include helper T cells and cytotoxic
  • Helper T cells proliferate extensively following recognition of an antigen -MHC complex on an antigen presenting cell. Helper T cells also secrete a variety of cytokines, i.e. lymphokines, which play a central role in the activation of B cells, cytotoxic T cells and a variety of other cells which participate in the immune response.
  • cytokines i.e. lymphokines
  • helper T cell activation is initiate by the interaction of the T cell receptor (TCR) - CD3 complex with an antigen-MHC on the surface of an antigen presenting cell. This interaction mediates a cascade of biochemical events that induce the resting helper T cell to enter a cell cycle (the Go to Gl transition) and results in the expression of a high affinity receptor for IL-2 and sometimes IL-4.
  • TCR T cell receptor
  • T cell progresses through the cycle proliferating and differentiating into memory cells or effector cells.
  • activation of T cells involves additional costimulation induced by cytokines released by the antigen presenting cell or through interactions with membrane bound molecules on the antigen presenting cell and the T cell.
  • the cytokines IL-1 and IL-6 have been shown to provide a costimulatory signal.
  • the interaction between the B7 molecule expressed on the surface of an antigen presenting cell and CD28 and CTLA-4 molecules expressed on the T cell surface effect T cell activation.
  • Activated T cells express an increased number of cellular adhesion molecules, such as ICAM- 1. integrins, VLA-4, LFA- 1 , CD56, etc.
  • T-cell proliferation in a mixed lymphocyte culture or mixed lymphocyte reaction is an established indication of the ability of a compound to stimulate the immune system.
  • inflammatory cells infiltrate the site of injury or infection.
  • the migrating cells may be neutrophilic, eosinophilic, monocytic or lymphocytic. Histologic examination of the affected tissues provides evidence of an immune stimulating or inhibiting responseCurrent Protocols in Immunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc. .
  • Immune related diseases can be treated by suppressing the immune response. Using neutralizing antibodies that inhibit molecules having immune stimulatory activity would be beneficial in the treatment of immune-mediated and inflammatory diseases.
  • Molecules which inhibit the immune response can be utilized (proteins directly or via the use of antibody agonists) to inhibit the immune response and thus ameliorate immune related disease. Summary of the Invention
  • the present invention concerns compositions and methods for the diagnosis and treatment of immune related disease in mammals, including humans.
  • the present invention is based on the identification of proteins (including agonist and antagonist antibodies) which either stimulate or inhibit the immune response in mammals.
  • Immune related diseases can be treated by suppressing or enhancing the immune response. Molecules that enhance the immune response stimulate or potentiate the immune response to an antigen. Molecules which stimulate the immune response can be used therapeutically where enhancement of the immune response would be beneficial. Such stimulatory molecules can also be inhibited where suppression of the immune response would be of value.
  • Neutralizing antibodies are examples of molecules that inhibit molecules having immune stimulatory activity and which would be beneficial in the treatment of immune related and inflammatory diseases. Molecules which inhibit the immune response can also be utilized (proteins directly or via the use of antibody agonists) to inhibit the immune response and thus ameliorate immune related disease.
  • the proteins of the invention encoded by the genes of the invention are useful for the diagnosis and/or treatment (including prevention) of immune related diseases.
  • Antibodies which bind to stimulatory proteins are useful to suppress the immune system and the immune response.
  • Antibodies which bind to inhibitory proteins are useful to stimulate the immune system and the immune response.
  • the proteins and antibodies of the invention are also useful to prepare medicines and medicaments for the treatment of immune related and inflammatory diseases.
  • the present invention concerns an isolated antibody which binds a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide.
  • the antibody mimics the activity of a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide (an agonist antibody) or conversely the antibody inhibits or neutralizes the activity of a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide (an antagonist antibody).
  • the antibody is a monoclonal antibody, which preferably has nonhuman complementarity determining region (CDR) residues and human framework region (FR) residues.
  • CDR complementarity determining region
  • FR human framework region
  • the antibody may be labeled and may be immobilized on a solid support.
  • the antibody is an antibody fragment, a single-chain antibody, or an anti-idiotypic antibody.
  • the invention concerns a composition containing a PR0245, PR0217, PRO301. PR0266, PR0335, PR0331 or PR0326 polypeptide or an agonist or antagonist antibody which binds the polypeptide in admixture with a carrier or excipient.
  • the composition contains a therapeutically effective amount of the peptide or antibody.
  • the composition when the composition contains an immune stimulating molecule, the composition is useful for: (a) increasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) stimulating or enhancing an immune response in a mammal in need thereof, or (c) increasing the proliferation of T-lymphocytes in a mammal in need thereof in response to an antigen.
  • the composition when the composition contains an immune inhibiting molecule, the composition is useful for: (a) decreasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) inhibiting or reducing an immune response in a mammal in need thereof, or (c) decreasing the proliferation of T-lymphocytes in a mammal in need thereof in response to an antigen.
  • the composition contains a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent.
  • the composition is sterile.
  • the invention concerns the use of the polypeptides and antibodies of the invention to prepare a composition or medicament which has the uses described above.
  • the invention concerns nucleic acid encoding an anti-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 antibody, and vectors and recombinant host cells comprising such nucleic acid.
  • the invention concerns a method for producing such an antibody by culturing a host cell transformed with nucleic acid encoding the antibody under conditions such that the antibody is expressed, and recovering the antibody from the cell culture.
  • the invention further concerns antagonists and agonists of a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide that inhibit one or more of the functions or activities of the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide.
  • the invention concerns isolated nucleic acid molecules that hybridize to the complement of the nucleic acid molecules encoding the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptides.
  • the nucleic acid preferably is DNA, and hybridization preferably occurs under stringent conditions.
  • Such nucleic acid molecules can act as antisense molecules of the amplified genes identified herein, which, in turn, can find use in the modulation of the respective amplified genes, or as antisense primers in amplification reactions.
  • sequences can be used as part of ribozyme and/or triple helix sequence which, in turn, may be used in regulation of the amplified genes.
  • the invention concerns a method for determining the presence of a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide comprising exposing a cell suspected of containing the polypeptide to an anti-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 antibody and determining binding of the antibody to the cell.
  • the present invention concerns a method of diagnosing an immune related disease in a mammal, comprising detecting the level of expression of a gene encoding a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher expression level in the test sample indicates the presence of immune related disease in the mammal from which the test tissue cells were obtained.
  • the present invention concerns a method of diagnosing an immune disease in a mammal, comprising (a) contacting an anti-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 antibody with a test sample of tissue cells obtained from the mammal , and (b) detecting the formation of a complex between the antibody and the PR0245.
  • PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide in the test sample may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells of the same cell type.
  • a larger quantity of complexes formed in the test sample indicates the presence of tumor in the mammal from which the test tissue cells were obtained.
  • the antibody preferably carries a detectable label. Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art.
  • the test sample is usually obtained from an individual suspected of having a deficiency or abnormality of the immune system.
  • the present invention concerns a diagnostic kit, containing an anti-
  • PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 antibody and a carrier e.g. a buffer
  • the kit preferably contains instructions for using the antibody to detect the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide.
  • the invention concerns an article of manufacture, comprising: a container; a label on the container; and a composition comprising an active agent contained within the container; wherein the composition is effective for stimulating or inhibiting an immune response in a mammal, the label on the container indicates that the composition can be used to treat an immune related disease, and the active agent in the composition is an agent stimulating or inhibiting the expression and/or activity of the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide.
  • the active agent is a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide or an anti-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 antibody.
  • a further embodiment is a method for identifying a compound capable of inhibiting the expression and/or activity of a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide by contacting a candidate compound with a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide under conditions and for a time sufficient to allow these two components to interact.
  • either the candidate compound or the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide is immobilized on a solid support.
  • the non-immobilized component carries a detectable label.
  • Figure 1 shows the nucleotide sequence (SEQ ID N0:1) of a native sequence PR0245 cDNA, wherein the nucleotide sequence is designated herein as "UNQ219" and/or "DNA35638".
  • Figure 2 shows the amino acid sequence (SEQ ID N0:2) derived from the nucleotide sequence shown in Figure 1.
  • Figures 3 A and 3B show an alignment of nucleotide sequences (SEQ ID NOS: 8-11) from a variety of expressed sequence tags as well as a consensus nucleotide sequence derived therefrom designated "DNA30954" (SEQ ID N0:7).
  • Figure 4 shows a BLAST sequence alignment analysis of a portion of the PR0245 amino acid sequence derived from the DNA35638 molecule ("DNA35638”) (SEQ ID NO: 12) with the human c-myb (“HSU22376_2”) (SEQ ID N0:3).
  • Figures 5A, 5B and 5C show the nucleotide sequence comprising a native sequence egf-like homologue cDNA. These are also indicated as SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively.
  • Figures 6A, 6B and 6C show the amino acid sequences encoded by the coding sequences of the nucleotides described in Figures 5A, 5B and 5C. These polypeptide sequences are also identified as SEQ ID NO: 16. SEQ ID NO: 17 and SEQ ID NO: 18 (PR0217), respectively.
  • Figures 7A, 7B and 7C show an alignment comparison between prior art sequences used to create DNA28726 (SEQ ID NO: 19), DNA28730 (SEQ ID NO: 20) and DNA28760 (SEQ ID NO: 21), respectively, virtual sequences which were used in the isolation of the nucleotide sequences of the invention.
  • Figure 7A indicates the alignment between Incyte EST sequences 2305118 (SEQ ID NO: 22), 2544914 (SEQ ID NO: 23), 5 1682522 (SEQ ID NO: 24), 424333 (SEQ ID NO: 25), 640534 (SEQ ID NO: 26), 2211568 (SEQ ID NO: 27), 1436024 (SEQ ID NO: 28), 1600521 (SEQ ID NO: 30), 732577 (SEQ ID NO: 31), 931313 (SEQ ID NO: 33), 045517 (SEQ ID NO: 34), 1557825 (SEQ ID NO: 35), 1555649 (SEQ ID NO: 36), and GenBank sequences W24885 (SEQ ID NO: 29), N95751 (SEQ ID NO: 32).
  • Figure 7B indicates the alignment between Inctye EST sequences 2398238 (SEQ ID NO: 37), 1842628 (SEQ ID NO: 38), 2191592 (SEQ ID NO: 39), 1932631 (SEQ
  • FIG. 10 10 ID NO: 40), 1700782 (SEQ ID NO: 44) and GenBank sequences AA195267 (SEQ ID NO: 41), H99879 (SEQ ID NO: 42), AA 195084 (SEQ ID NO: 43).
  • Figure 7C indicates the alignment between GenBank sequences W27896 (SEQ ID NO: 33), W27851 (SEQ ID NO: 46), W22553 (SEQ ID NO: 47), W23268 (SEQ ID NO: 48), W28670 (SEQ ID NO: 50), W27944 (SEQ ID NO: 51), R55894 (SEQ ID NO: 53), W37154 (SEQ ID NO: 57), W38638 (SEQ ID NO: 59) and Incyte EST sequences 400252 (SEQ ID NO: 49), 399998 (SEQ ID NO: 52),
  • Figure 8 shows oligonucleotide sequences 28726.p (SEQ ID NO: 60), 28726.f (SEQ ID NO: 61) and 28726.r (SEQ ID NO: 62), which were used in the isolation of DNA32279 (SEQ ID NO: 13), also indicated in Figure 5A.
  • Figure 9 shows oligonucleotide sequences 28730.p (SEQ ID NO: 63), 28730.f (SEQ ID NO: 64) and
  • Figure 10 shows oligonucleotide sequences 28760.p (SEQ ID NO: 66), 28760.f (SEQ ID NO: 67) and 28760.r (SEQ ID NO: 68), which were used in the isolation of DNA33094 (SEQ ID NO: 15), also indicated in Figure 5C.
  • DNA32279 (SEQ ID NO: 13), a full-length EGF-like homologue of the invention in comparison with GEN12205 (SEQ ID NO: 69), an epidermal growth factor-like protein Sl-5.
  • Figures 12A and 12B describe the Blast score, match and percent homology alignment between the coding protein of DNA32292 (SEQ ID NO: 14), a full-length EGF-like homologue of the invention in
  • PAC6_RAT SEQ ID NO: 70
  • serine protease pc ⁇ precursor from rattus norvegicus
  • FBLC_MOUSE SEQ ID NO: 71
  • Fibulin-1 isoform c precursor from mus musculus, respectively, each of which contain a cysteine-rich domain which may form EGF-like structures.
  • Figures 13A and 13B describe the Blast score, match and percent homology alignment between the coding protein of DNA33094 (SEQ ID NO: 15), a full-length EGF-like homologue of the invention in
  • Figure 14 shows the derived amino acid sequence of a native sequence PRO301 polypeptide (SEQ ID NO:74). This polypeptide is 299 amino acids long, having signal sequence at residue 1 to 27, an
  • Figure 15 shows the nucleotide sequence of a native sequence DNA40628 cDNA (SEQ ID NO:75).
  • Figure 16 shows the alignment comparison between sequences used to create DNA35936 (SEQ ID NO:76) (from DNA (SEQ ID NOS: 88-91)) from which the consensus sequence used for cloning the cDNA DNA40628 was created.
  • Figure 17 shows the alignment comparison between DNA35936 (SEQ ID N0:76) (from DNA) and further sequences from the LIFESEQTM database (Incyte Pharmaceuticals, Palo Alto, CA) and GenBank (SEQ ID NOS:92-235), which were used to extend the from DNA to obtain a consensus sequence shown in the bottom line of the Figure as "consenOl” (SEQ ID NO:77).
  • Figures 18A-18F show the oligonucleotide sequences OLI2162 (35936.fl) (SEQ ID NO:78); OLI2163 (35936.pl) (SEQ ID NO:79); OLI2164 (35936.f2) (SEQ ID NO:80); OLI2165 (35936.rl) (SEQ ID NO:81); OLI2166 (35936.B) (SEQ ID NO:82); OLI2167 (35936.r2) (SEQ ID NO:83) which were used in the isolation of DNA40628.
  • Figure 19 describes the Blast score, match and percent homology alignment between 2 overlapping fragments of DNA40628 and A33_HUMAN, an human A33 antigen precursor.
  • the first fragment compares the coded residues beginning at nucleotide position 121 to 816 of DNA40628 (SEQ ID NO:84) with nucleotides 17 to 284 of A33 HUMAN (SEQ ID NO:85);
  • the second fragment compares nucleotides 112 to
  • Figures 20A and 20B show a nucleotide sequence (SEQ ID NO:236) containing the nucleotide sequence (SEQ ID NO:237) of a native sequence PR0266 cDNA, wherein the nucleotide sequence (SEQ ID NO:236) is a clone designated herein as "UNQ233" and/or "DNA37150-seq min". Also presented (circled in Figure 20A) is the position of the initiator methionine residue (residues 1-3 of SEQ ID NO: 237: residues 107- 109 of SEQ ID NO: 236).
  • the putative transmembrane domain of the protein is encoded by nucleotides beginning at nucleotide 1843 of SEQ ID NO: 237, underlined in Figure 20B. Also in Figure 20B, the stop codon is circled, immediately after the last nucleotide of SEQ ID NO: 237.
  • Figure 21 shows the amino acid sequence (SEQ ID NO:238) derived from SEQ ID NO:237 shown in Figures 20A and 20B.
  • Figures 22A-22D show a BLAST sequence alignment analysis of portions of the PR0266 amino acid sequence derived from SEQ ID NO: 237 with portions of the SLIT protein precursor from drosophila melanogaster (SEQ ID NOS:239-247).
  • Figures 23A-23D show a BLAST sequence alignment analysis of portions of the PR0266 amino acid sequence derived from SEQ ID NO:237 with portions of the Drosophila SLIT protein involved in axon pathway development (SEQ ID NOS. 248-256).
  • Figure 24 shows an expression sequence tag (SEQ ID NO:257) which was used to form primers herein.
  • Figures 25 A and 25B show the nucleic acid sequence (SEQ ID NO:261) comprising the coding nucleic acid (SEQ ID NO:262) of a native PR0335 polypeptide derived from SEQ ID NO:262.
  • SEQ ID N0:262 begins with at position 65 of SEQ ID NO: 261.
  • the start codon, nucleic acid positions 1-3 of SEQ ID NO:262 is circled.
  • the stop codon is circled, after the last nucleic acid of SEQ ID NO:262, at 3177.
  • Figure 26 shows the amino acid sequence of PR0335 (SEQ ID NO:263).
  • Figures 27A and 27B show an alignment of nucleotide sequences from a variety of expressed sequence tags as well as a consensus nucleotide sequence derived therefrom designated "DNA36685", (SEQ ID NO: 1).
  • FIG. 28A through 28C show the results of a BLAST search against PR0335 and amino acid alignments between portions of PR0335 and portions of LIG-1 (SEQ ID NOS:267-269).
  • Figures 29A and 29B show the amino acid sequence of LIG-1 (SEQ ID NO:270) and the leucine rich repeat domains of LIG-1.
  • Figure 30A through 30C show sequence information related to SEQ ID NO:286 ( Figure 30A).
  • Figure 30B shows the results of a BLAST search using SEQ ID NO:286 and Figure 6A shows primers (SEQ ID NOS:287-289) synthesized based on SEQ ID NO:286.
  • Figure 31 shows primers (SEQ ID NOS:271 -278) related to the identification of SEQ ID NO:261.
  • Figure 32 shows the nucleic acid sequence (SEQ ID NO:279) comprising the coding nucleic acid (SEQ ID NO:280) of a native PR0331 polypeptide derived from SEQ ID NO:280.
  • SEQ ID NO:280 begins with the start codon, nucleic acid positions 1-3 of SEQ ID NO:280, circled. The stop codon is also circled, after the last nucleic acid of SEQ ID NO:280, at 1920.
  • Figure 33 shows the amino acid sequence of PR0331 (SEQ ID NO:281) wherein the signal peptide is shown in parenthesis, and the start of the mature peptide or extracellular domain is shown underlined.
  • the start and end of the leucine rich repeat domains have an X underneath the perspective amino acid.
  • the start of the transmembrane domain is marked with a circle underneath the perspective amino acid.
  • the start of the intracellular domain is marked with a triangle underneath the perspective amino acid.
  • Figures 34A through 34E show the results of a BLAST search against PR0331 and amino acid alignments between portions of PR0331 and portions of LIG-1 (SEQ ID NOS:282-292).
  • Figures 35A and 35B show the results of a BLAST search (Figure 35A) against SEQ ID NO:264 and amino acid alignments between portions of the amino acid sequence for which SEQ ID NO:4 encodes, (SEQ ID NO:310) and portions of LIG-1 (SEQ ID NOS:293 and 294).
  • Figure 36 shows primers (SEQ ID NOS:295-297) related to the identification of SEQ ID NO:280.
  • Figures 37A through 37C show the nucleic acid sequence (SEQ ID NO:298) comprising the coding nucleic acid (SEQ ID NO:299) of a native PR0326 polypeptide derived from SEQ ID NO:299.
  • SEQ ID NO:299 begins with the start codon, nucleic acid positions 1-3 of SEQ ID NO:299, circled. The stop codon is also circled, after the last nucleic acid of SEQ ID NO:299, at position 3357.
  • Figure 38 shows the amino acid sequence of PR0326 (SEQ ID NO:300).
  • Figures 39A through 39D show the results of a BLAST search against PR0326 and amino acid alignments between portions of PR0326 and portions of LIG-1 (SEQ ID NOS:301-303).
  • Figure 40 shows primers (SEQ ID NOS:304-306) related to the identification of SEQ ID NO:299.
  • Figure 41 shows additional primers (SEQ ID NOS:307-308) related to the identification of SEQ ID NO:299.
  • immune related disease means a disease in which a component of the immune system of a mammal causes, mediates or otherwise contributes to a morbidity in the mammal. Also included are diseases in which stimulation or intervention of the immune response has an ameliorative effect on progression of the disease. Included within this term are immune-mediated inflammatory diseases, non- immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, neoplasia, etc.
  • T cell mediated disease means a disease in which T cells directly or indirectly mediate or otherwise contribute to a morbidity in a mammal.
  • the T cell mediated disease may be associated with cell mediated effects, lymphokine mediated effects, etc., and even effects associated with B cells if the B cells are stimulated, for example, by the lymphokines secreted by T cells.
  • immune-related and inflammatory diseases examples include systemic lupus erythematosis, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjsgren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tubulointerstitial
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • a therapeutic agent may directly decrease or increase the magnitude of response of a component of the immune response, or render the disease more susceptible to treatment by other therapeutic agents, e.g. antibiotics, antifungals, anti-inflammatory agents, chemotherapeutics, etc.
  • the "pathology" of an immune related disease includes all phenomena that compromise the well- being of the patient.
  • abnormal or uncontrollable cell growth neutrophililic, eosinophilic, monocytic, lymphocytic cells
  • antibody production auto-antibody production
  • complement production interference with the normal functioning of neighboring cells
  • release of cytokines or other secretory products at abnormal levels suppression or aggravation of any inflammatory or immunological response
  • infiltration of inflammatory cells into cellular spaces, etc.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
  • Administration "in combination with" one or more further therapeutic agents includes simultaneous
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g. I 131 , 1 125 , Y 90 and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
  • a "chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g.
  • paclitaxel Texol, Bristol-Myers Squibb Oncology, Princeton, NJ
  • doxetaxel Tetraxotere, RhDne-Poulenc Rorer, Antony, Rnace
  • toxotere methotrexate
  • cisplatin melphalan
  • vinblastine bleomycin, etoposide
  • ifosfamide mitomycin C
  • mitoxantrone vincristine, vinorelbine
  • carboplatin teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (see U.S. Pat. No. 4,675,187), melphalan and other related nitrogen mustards.
  • hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapristone.
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer cell overexpressing any of the genes identified herein, either in vitro or in vivo.
  • the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • cytokines lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor- ⁇ and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor: integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet-growth factor; transforming growth factors (TGFs) such as TGF- ⁇ and TGF- ⁇ ; insulin-like growth factor-I and
  • cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
  • a "PR0245, PR0217, PRO301, PR0266. PR0335, PR0331 or PR0326 polypeptide" refers to a native sequence PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 having the same amino acid sequence as a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 derived from nature.
  • Such native sequence PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can be isolated from nature or can be produced by recombinant and/or synthetic means.
  • the term specifically encompasses naturally-occurring truncated or secreted forms (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally- occurring allelic variants of the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326.
  • the native sequence PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 is a mature or full-length native sequence PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 comprising amino acids 1-312 of Figure 2 (SEQ ID NO:2), 1-379 of Figure 6C (SEQ ID NO: 18), 1-299 of Figure 14 (SEQ ID NO:74), 1-696 of Figure 21 (SEQ ID NO:238), 1-1059 of Figure 26 (SEQ ID NO:263), 1-640 of Figure 33 (SEQ ID NO:281) or 1-1119 of Figure 38 (SEQ ID NO:300).
  • polypeptide of the invention refers to each individual PR0245, PR0217, PRO301, PR0266. PR0335, PR0331 or PR0326 polypeptide. All disclosures in this specification which refer to the "polypeptide of the invention” or to "the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide” refer to each of the polypeptides individually as well as jointly. For example, descriptions of the preparation of, purification of, derivation of, formation of antibodies to or against, administration of, compositions containing, treatment of a disease with, etc., pertain to each polypeptide of the invention individually.
  • compound of the invention includes the polypeptide of the invention, as well as agonist antibodies for and antagonist antibodies to these polypeptide, peptides or small molecules having agonist or antagonist activity developed from the polypeptide, etc.
  • An "isolated" nucleic acid molecule encoding a polypeptide of the invention 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 nucleic acid molecules therefore are distinguished from the polypeptide-encoding nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule encoding a polypeptide of the invention includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express a polypeptide of the invention 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 ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is
  • SUBST1TUTE SHEET (RULE 26) accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
  • “Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 M sodium chloride, 75 mM sodium citrate at 42C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ug/ml), 0.1% SDS, and 10% dextran sulfate at 42
  • Modely stringent conditions may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above.
  • washing solution and hybridization conditions e.g., temperature, ionic strength and %SDS
  • moderately stringent conditions is overnight incubation at 37C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50C.
  • a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50C.
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length
  • epitope tagged when used herein refers to a chimeric polypeptide comprising a polypeptide of the invention 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).
  • Active or “activity” in the context of variants of the polypeptide of the invention refers to form(s) of proteins of the invention which retain the biologic and/or immunologic activities of a native or naturally- occurring polypeptide of the invention.
  • Bioactivity in the context of an antibody or another molecule that can be identified by the screening assays disclosed herein (e.g. an organic or inorganic small molecule, peptide, etc.) is used to refer to the ability of such molecules to induce or inhibit infiltration of inflammatory cells into a tissue, to stimulate or inhibit T-cell proliferation and to stimulate or inhibit lymphokine release by cells .
  • Another preferred activity is increased vascular permeability or the inhibition thereof.
  • antagonist is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide of the invention disclosed herein.
  • agonist is used in the broadest sense and includes any molecule that mimics a biological activity of a native polypeptide of the invention disclosed herein.
  • Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides of the invention , peptides, small organic molecules, etc.
  • a "small molecule” is defined herein to have a molecular weight below about 600 daltons.
  • Antibodies are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
  • antibody is used in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • “Native antibodies” and “native immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at one end (V ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light- chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy- chain variable domains.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR).
  • CDRs complementarity-determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., NIH Publ. No.91- 3242, Vol. I, pages 647-669 (1991)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody- dependent cellular toxicity.
  • Antibody fragments comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies (Zapata et al. , Protein Eng, 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen- binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose PR0245, PR0217, PRO301. Pro266, pro335, pro331 or pro326 reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V j ⁇ -V ⁇ dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(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 (K) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ counter respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be consfrued as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al.. Nature, 256:495 [1975], or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementarity-determining region
  • humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • the humanized antibody includes a "primatized"antibody where the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest. Antibodies containing residues from Old World monkeys are also possible within the invention. See, for example, U.S. Patent Nos. 5,658,570; 5,693,780; 5,681,722; 5,750,105; and 5,756,096.
  • Single-chain Fv or “sFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V j and V L domains which enables the sFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VJJ) connected to a light-chain variable domain (V j J in the same polypeptide chain (V ⁇ _ - V j J.
  • VJJ heavy-chain variable domain
  • V j J light-chain variable domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • an "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the compound of the invention will be purified (1) to greater than 95% by weight of the compound as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated compound e.g. antibody or polypeptide
  • Isolated compound includes the compound in situ within recombinant cells since at least one component of the compound's natural environment will not be present. Ordinarily, however, isolated compound 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 compound, e.g. antibody or polypeptide, so as to generate a "labelled” compound.
  • 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 compound of the present invention can adhere.
  • solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g, agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.
  • the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149.
  • a “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as the anti-ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent) to a mammal.
  • a drug such as the anti-ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • 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.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0245.
  • PR0266, PR0335, PR0331 or PR0326 (UNQ219, UNQ191, UNQ264, UNQ233, UNQ287V, UNQ292 or UNQ287 respectively).
  • cDNA encoding a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide has 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.
  • PR0245 protein encoded by DNA35638, DNA33094, DNA40628, DNA37150, DNA41388, DNA40981 AND DNA37140 as well as all further native homologues and variants included in the foregoing definition of PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326, will be referred to as PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 or simply as "the polypeptide of the invention", regardless of their origin or mode of preparation.
  • polypeptide sequence or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc, 85:2149-2154 (1963)].
  • In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions. Various portions of the polypeptide of the invention may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length polypeptide. Isolation of DNA Encoding the Polypeptide of the Invention
  • DNA encoding the polypeptide of the invention may be obtained from a cDNA library prepared from tissue believed to possess the polypeptide mRNA and to express it at a detectable level. Accordingly, human DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples.
  • the gene encoding the polypeptide of the invention may also be obtained from a genomic library or by oligonucleotide synthesis.
  • Probes such as antibodies to the polypeptide of the invention or oligonucleotides of at least about 20-80 bases
  • Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).
  • An alternative means to isolate the gene encoding the polypeptide of the invention is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
  • the oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized.
  • the oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32 P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.
  • Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs such as ALIGN, DNAstar, and INHERIT which employ various algorithms to measure homology. Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cD A. ii. Selection and Transformation of Host Cells
  • Host cells are transfected or transformed with expression or cloning vectors described herein for production of the polypeptides of the invention 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.
  • transfection is known to the ordinarily skilled artisan, for example, CaP ⁇ 4 and electroporation.
  • transformation is performed using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers.
  • Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells.
  • Suitable prokaryotes include but are not limited to eubacteria, such as Gram- negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli.
  • Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635).
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding the polypeptides of the invention. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism.
  • Suitable host cells for the expression of glycosylated polypeptides of the invention 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. ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.
  • the nucleic acid (e.g., cDNA or genomic DNA) encoding the polypeptides of the invention may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression.
  • a replicable vector for cloning (amplification of the DNA) or for expression.
  • Various vectors are 0 publicly available.
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, phagemid or phage.
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art.
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination 5 sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
  • the polypeptide of the invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • a heterologous polypeptide which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the signal 0 sequence may be a component of the vector, or it may be a part of the DNA encoding the polypeptide of the invention that is inserted into the vector.
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
  • the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces G-factor leaders, the latter described in U.S. Patent No. 5 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 from secreted polypeptides of the same or related species, as well as viral secretory leaders.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to 0 replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2u plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
  • Selection genes will typically contain a selection gene, also termed a selectable 5 marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • selectable markers for mammalian cells are those that enable the identification of cells competent to take up the nucleic acid encoding the polypeptide of the invention, such as 0 DHFR or thymidine kinase.
  • An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci.
  • a suitable selection gene for use in yeast is the trp ⁇ gene present in the yeast plasmid YRp7 [Stinchcomb et al.. Nature, 282:39 (1979); Kingsman et al.. Gene, 7: 141 (1979); Tschemper et al.. Gene. ⁇ 0: 157 (1980)].
  • the trp I gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
  • Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding the polypeptide of the invention to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the ⁇ - lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 28 544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc.
  • Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the polypeptide of the invention.
  • S.D. Shine-Dalgarno
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 25_5:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv.
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • Transcription of the polypeptide of the invention from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalo
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription.
  • Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein. and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus.
  • Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the coding sequence of the polypeptide of the invention, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and. occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide of the mvention
  • Gene expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc Natl Acad Sci USA, 77 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protem duplexes The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected
  • Gene expression may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence of the mventive polypeptide or against a synthetic peptide based on the DNA sequences provided herein or agamst exogenous sequence fused to DNA encoding the polypeptide of the invention and encoding a specific antibody epitope in Purification of Polypeptide
  • Forms of the polypeptide of the invention may be recovered from culture medium or from host cell lysates If membrane-bound, it can be released from the membrane usmg a suitable detergent solution (e g
  • Cells employed m expression of the polypeptide of the invention can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysmg agents
  • polypeptide of the mvention may be desired to purify from recombinant cell proteins or polypeptides
  • the following procedures are exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, protem A Sepharose columns to remove contaminants such as
  • tissue expressing the polypeptides of the invention can be identified by determining mRNA expression in various human tissues The location of such genes provides information about which tissues are most likely to be affected by the stimulating and inhibiting activities of the polypeptides of the invention. The location of a gene in a specific tissue also provides sample tissue for the activity blocking assays discussed below.
  • gene expression in various tissues may be measured by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 [1980]), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
  • Gene expression in various tissues may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence of a polypeptide of the invention or against a synthetic peptide based on the DNA sequences encoding the polypeptide of the invention or against an exogenous sequence fused to a DNA encoding a polypeptide of the invention and encoding a specific antibody epitope.
  • General techniques for generating antibodies, and special protocols for Northern blotting and in situ hybridization are provided below.
  • the activity of the polypeptides of the invention can be further verified by antibody binding studies, in which the ability of anti-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 antibodies to inhibit the effect of the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptides on tissue cells is tested.
  • Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow.
  • Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).
  • ком ⁇ онентs rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody.
  • the amount of target protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies.
  • the antibodies preferably are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected.
  • the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay).
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example. 4 Cell-Based Assays
  • Cell-based assays and animal models for immune related diseases can be used to further understand the relationship between the genes and polypeptides identified herein and the development and pafhogenesis of immune related disease
  • cells of a cell type known to be involved in a particular immune related disease are transfected with the cDNAs described herein, and the ability of these cDNAs to stimulate or inhibit immune function is analyzed Suitable cells can be transfected with the desired gene, and monitored for immune function activity
  • Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit or stimulate immune function, for example to modulate T-cell proliferation or mflammatory cell infiltration
  • Cells transfected with the coding sequences of the genes identified herein can further be used to identify drug candidates for the treatment of immune related diseases
  • transgenic animals in addition, primary cultures derived from transgenic animals (as described below) can be used in the cell-based assays herem, although stable cell lmes are preferred Techniques to derive continuous cell lines from transgenic animals are well known in the art (see, e g Small et al Mol Cell Biol 5, 642-648 [1985])
  • MLR mixed lymphocyte reaction
  • Polypeptides of the invention as well as other compounds of the invention, which are stimulators (costimulators) of T cell proliferation, as determined by MLR and costimulation assays.for example, are useful m treating immune related diseases characterized by poor, suboptimal or inadequate immune function These diseases are treated by stimulating the proliferation and activation of T cells (and T cell mediated immunity) and enhancing the immune response in a mammal through administration of a stimulaton compound, such as the stimulating polypeptides of the invention.
  • the stimulating polypeptide may be a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide or an agonist antibody therefor.
  • Immunoadjuvant therapy for treatment of tumors is an example of this use of the stimulating compounds of the invention.
  • Antibodies which bind to inhibitory polypeptides function to enhance the immune response by removing the inhibitory effect of the inhibiting polypeptides. This effect is seen in experiments using anti-CTLA-4 antibodies which enhance T cell proliferation, presumably by removal of the inhibitory signal caused by CTLA-4 binding. Walunas, T. L. et al. Immunity (1994) 1 :405. This use is also validated in experiments with 4- IBB glycoprotein, a member of the tumor necrosis factor receptor family which binds to a ligand (4-1BBL) expressed on primed T cells and signals T cell activation and growth. Alderson, M. E.
  • polypeptides of the invention as well as other compounds of the invention, which are inhibitors of T cell proliferation activation and/or lymphokine secretion, can be directly used to suppress the immune response. These compounds are useful to reduce the degree of the immune response and to treat immune related diseases characterized by a hyperactive, superoptimal, or autoimmune response.
  • antibodies which bind to the stimulating polypeptides of the invention and block the stimulating effect of these molecules can be used to suppress the T cell mediated immune response by inhibiting T cell proliferation/activation and/or lymphokine secretion. Blocking the stimulating effect of the polypeptides suppresses the immune response of the mammal. 5. Animal Models
  • the results of the cell based in vitro assays can be further verified using in vivo animal models and assays for T-cell function.
  • a variety of well known animal models can be used to further understand the role of the genes identified herein in the development and pathogenesis of immune related disease, and to test the efficacy of candidate therapeutic agents, including antibodies, and other antagonists of the native polypeptides, including small molecule antagonists.
  • the in vivo nature of such models makes them particularly predictive of responses in human patients.
  • Animal models of immune related diseases include both non-recombinant and recombinant (transgenic) animals.
  • Non-recombinant animal models include, for example, rodent, e.g., murine models.
  • Such models can be generated by introducing cells into syngeneic mice using standard techniques, e.g. subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, etc.
  • Contact hypersensitivity is a simple in vivo assay of cell mediated immune function.
  • epidermal cells are exposed to exogenous haptens which give rise to a delayed type hypersensitivity reaction which is measured and quantitated.
  • Contact sensitivity involves an initial sensitizing phase followed by an elicitation phase.
  • the elicitation phase occurs when the epidermal cells encounter an antigen to which they have had previous contact. Swelling and inflammation occur, making this an excellent model of human allergic contact dermatitis.
  • a suitable procedure is described in detail in Current Protocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek. D. H. Margulies, E. M. Shevach and W.
  • Graft-versus-host disease occurs when immunocompetent cells are transplanted into immunosuppressed or tolerant patients. The donor cells recognize and respond to host antigens. The response can vary from life threatening severe inflammation to mild cases of diarrhea and weight loss. Graft- versus-host disease models provide a means of assessing T cell reactivity against MHC antigens and minor transplant antigens. A suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4.3.
  • An animal model for skin allograft rejection is a means of testing the ability of T cells to mediate in vivo tissue destruction which is indicative of and a measure of their role in anti-viral and tumor immunity.
  • the most common and accepted models use murine tail-skin grafts.
  • Repeated experiments have shown that skin allograft rejection is mediated by T cells, helper T cells and killer-effector T cells, and not antibodies.
  • a suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4.4.
  • transplant rejection models which can be used to test the compounds of the invention are the allogeneic heart transplant models described by Tanabe, M. et al, Transplantation (1994) 58:23 and Tinubu, S. A. et al, J. Immunol. (1994) 4330-4338.
  • Delayed type hypersensitivity reactions are a T cell mediated in vivo immune response characterized by inflammation which does not reach a peak until after a period of time has elapsed after challenge with an antigen. These reactions also occur in tissue specific autoimmune diseases such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE, a model for MS). A suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4.5.
  • MS multiple sclerosis
  • EAE experimental autoimmune encephalomyelitis
  • EAE is a T cell mediated autoimmune disease characterized by T cell and mononuclear cell inflammation and subsequent demyelination of axons in the central nervous system.
  • EAE is generally considered to be a relevant animal model for MS in humans. Bolton, C, Multiple Sclerosis (1995) 1 :143. Both acute and relapsing-remitting models have been developed.
  • the compounds of the invention can be tested for T cell stimulatory or inhibitory activity against immune mediated demyelinating disease using the protocol described in Current Protocols in Immunology, above, units 15.1 and 15.2. See also the models for myelin disease in which ohgodendrocytes or Schwann cells are grafted into the central nervous system as described in Duncan, I. D. et al, Molec. Med. Today (1997) 554-561.
  • An animal model for arthritis is collagen-induced arthritis. This model shares clinical, histological and immunological characteristics of human autoimmune rheumatoid arthritis and is an acceptable model for human autoimmune arthritis.
  • Mouse and rat models are characterized by synovitis, erosion of cartilage and subchondral bone.
  • the compounds of the invention can be tested for activity against autoimmune arthritis using the protocols described in Current Protocols in Immunology, above, units 15.5. See also the model using a monoclonal antibody to CD 18 and VLA-4 integrins described in Issekutz, A. C. et al., Immunology (1996) 88:569.
  • a model of asthma has been described in which antigen-induced airway hyper-reactivity, pulmonary eosinophilia and inflammation are induced by sensitizing an animal with ovalbumin and then challenging the animal with the same protein delivered by aerosol.
  • Several animal models (guinea pig, rat, non-human primate) show symptoms similar to atopic asthma in humans upon challenge with aerosol antigens.
  • Murine models have many of the features of human asthma. Suitable procedures to test the compounds of the invention for activity and effectiveness in the treatment of asthma are described by Wolyniec. W. W. et al, Am. J. Respir. Cell Mol. Biol. (1998) 18:777 and the references cited therein.
  • the compounds of the invention can be tested on animal models for psoriasis like diseases. Evidence suggests a T cell pathogenesis for psoriasis.
  • the compounds of the invention can be tested in the scid/scid mouse model described by Schon, M P et al, Nat Med (1997) 3 183, in which the mice demonstrate histopathologic skin lesions resemblmg psoriasis
  • Another suitable model is the human skin/scid mouse chimera prepared as described by Nickoloff, B J et al, Am J Path (1995) 146 580
  • Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the genes identified herem into the genome of animals of interest, using standard techniques for producing transgenic animals
  • Animals that can serve as a target for transgenic manipulation mclude, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e g baboons, chimpanzees and monkeys
  • Techniques known in the art to introduce a transgene into such animals mclude pronucleic microinjection (Hoppe and Wanger, U S Patent No 4,873,191), retrovirus-mediated gene transfer into germ lines (e g , Van der Putten et al Proc Natl Acad Sci USA 82, 6148-615 [1985]), gene targeting in embryonic stem cells (Thompson et al , Cell 56, 313-321 [1989]), electroporation of embryos (Lo, Mol Cel Biol 3, 1803-18
  • transgenic animals include those that carry the transgene only in part of their cells (“mosaic animals”).
  • the transgene can be integrated either as a single transgene, or in concatamers, e g , head-to-head or head-to-tail tandems
  • Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al , Proc Natl Acad Sci USA 89, 6232-636 (1992)
  • transgene m transgenic animals can be monitored by standard techniques For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or lmmunocytochemistry
  • the animals may be further examined for signs of immune disease pathology, for example by histological examination to determine infiltration of immune cells mto specific tissues
  • Blocking experiments can also be performed in which the transgenic animals are treated with the compounds of the mvention to determine the extent of the T cell proliferation stimulation or inhibition of the compounds
  • blocking antibodies which bind to the polypeptide of the mvention, prepared as described above, are administered to the animal and the effect on immune function is determined
  • "knock out" animals can be constructed which have a defective or altered gene encodmg a polypeptide identified herein, as a result of homologous recombmation between the endogenous gene encodmg the polypeptide and altered genomic DNA encodmg the same polypeptide introduced mto an embryonic cell of the animal
  • cDNA encoding a particular polypeptide can be used to clone genomic DNA encodmg that polypeptide m accordance with established techniques
  • a portion of the genomic DNA encodmg a particular polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor mtegration
  • several kilobases of unaltered flanking DNA are included in the vector [see e g , Thomas and Capecchi, Cell, 5__ 503 (1987) for a description of homologous recombination vectors
  • a chimeric embryo can then be implanted mto 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 for their development of pathological conditions due to absence of the polypeptide.
  • the immunostimulating compounds of the invention can be used in immunoadjuvant therapy for the treatment of tumors (cancer).
  • tumors cancer
  • T cells recognize human tumor specific antigens.
  • DeSmet, C. et al (1996) Proc. Natl. Acad. Sci. USA, 93:7149. It has been shown that costimulation of T cells induces tumor regression and an antitumor response both in vitro and in vivo. Melero, I.
  • the stimulatory compounds of the invention can be administered as adjuvants, alone or together with a growth regulating agent, cytotoxic agent or chemotherapeutic agent , to stimulate T cell proliferation/activation and an antitumor response to tumor antigens.
  • the growth regulating, cytotoxic, or chemotherapeutic agent may be administered in conventional amounts using known administration regimes. Immunostimulating activity by the compounds of the invention allows reduced amounts of the growth regulating, cytotoxic, or chemotherapeutic agents thereby potentially lowering the toxicity to the patient.
  • Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis). In a cancerous state a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
  • Alteration of gene expression is intimately related to the uncontrolled cell growth and de- differentiation which are a common feature of all cancers.
  • the genomes of certain well studied tumors have been found to show decreased expression of recessive genes, usually referred to as tumor suppression genes, which would normally function to prevent malignant cell growth, and/or overexpression of certain dominant genes, such as oncogenes, that act to promote malignant growth.
  • tumor suppression genes which would normally function to prevent malignant cell growth
  • oncogenes such as oncogenes
  • Each of these genetic changes appears to be responsible for importing some of the traits that, in aggregate, represent the full neoplastic phenotype (Hunter, Cell 64, 1129 [1991]; Bishop, Cell 64, 235-248 [1991]).
  • a well known mechanism of gene (e.g. oncogene) overexpression in cancer cells is gene amplification.
  • the process involves unscheduled replication of the region of chromosome comprising the gene, followed by recombination of the replicated segments back into the chromosome (Alitalo et al, Adv. Cancer Res. 47, 235-281 [1986]). It is believed that the overexpression of the gene parallels gene amplification, i.e. is proportionate to the number of copies made.
  • Proto-oncogenes that encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of various human malignancies, including breast cancer.
  • the human ErbB2 gene (erbB2, also known as her2. or c-erbB-2), which encodes a 185- kd transmembrane glycoprotein receptor (pl85 HER2 ; HER2) related to the epidermal growth factor receptor (EGFR)
  • EGFR epidermal growth factor receptor
  • HER2-positive patients responding clinically to treatment with taxanes were greater than three times those of HER2-negative patients (Ibid).
  • a recombinant humanized anti-ErbB2 (anti-HER2) monoclonal antibody (a humanized version of the murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or Herceptin7) has been clinically active in patients with ErbB2-overexpressing metastatic breast cancers that had received extensive prior anticancer therapy. (Baselga et al, J. Clin. Oncol ⁇ :1 1-1AA [1996]).
  • the compounds of the invention may be administered as adjuvants in the treatment of cancers in which one or more genes in cancer cells are amplified.
  • Gene amplification is a quantitative modification, whereby the actual number of complete coding sequence, i.e. a gene, increases, leading to an increased number of available templates for transcription, an increased number of translatable transcripts, and, ultimately, to an increased abundance of the protein encoded by the amplified gene.
  • MTX cytotoxic drug methotrexate
  • DHFR dihydrofolate reductase
  • Gene amplification is most commonly encountered in the development of resistance to cytotoxic drugs (antibiotics for bacteria and chemotherapeutic agents for eukaryotic cells) and neoplastic transformation. Transformation of a eukaryotic cell as a spontaneous event or due to a viral or chemical/environmental insult is typically associated with changes in the genetic material of that cell.
  • One of the most common genetic changes observed in human malignancies are mutations of the p53 protein. p53 controls the transition of cells from the stationary (Gl) to the replicative (S) phase and prevents this transition in the presence of DNA damage.
  • Gl stationary
  • S replicative
  • one of the main consequences of disabling p53 mutations is the accumulation and propagation of DNA damage, i.e. genetic changes.
  • Common types of genetic changes in neoplastic cells are, in addition to point mutations, amplifications and gross, structural alterations, such as translocations.
  • the amplification of DNA sequences may indicate specific functional requirement as illustrated in the DHFR experimental system. Therefore, the amplification of certain oncogenes in malignancies points toward a causative role of these genes in the process of malignant transformation and maintenance of the transformed phenotype.
  • This hypothesis has gained support in recent studies.
  • the bcl-2 protein was found to be amplified in certain types of non-Hodgkin's lymphoma. This protein inhibits apoptosis and leads to the progressive accumulation of neoplastic cells.
  • Members of the gene family of growth factor receptors have been found to be amplified in various types of cancers suggesting that overexpression of these receptors may make neoplastic cells less susceptible to limiting amounts of available growth factor.
  • Examples include the amplification of the androgen receptor in recurrent prostate cancer during androgen deprivation therapy and the amplification of the growth factor receptor homologue ERB2 in breast cancer.
  • genes involved in intracellular signaling and control of cell cycle progression can undergo amplification during malignant transformation. This is illustrated by the amplification of the bcl-I and ras genes in various epithelial and lymphoid neoplasms.
  • CGH comparative genomic hybridization
  • PCR-based assays are most suitable for the final identification of coding sequences, i.e. genes in amplified regions.
  • genes can be identified by quantitative PCR (S. Gelmini et al, Clin. Chem. 43, 752 [1997]), by comparing DNA from a variety of primary tumors, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, etc. tumor, or tumor cell lines, with pooled DNA from healthy donors.
  • Quantitative PCR may be performed using a TaqMan instrument (ABI). Gene-specific primers and fluorogenic probes are designed based upon the coding sequences of the DNAs.
  • the compounds of the invention can be used as immunoadjuvants in the treatment of cancers in which amplified genes have been found in cancer cell lines, such as:
  • SRCC768 Human lung carcinoma cell lines including A549 (SRCC768), Calu-1 (SRCC769), Calu-6 (SRCC770), H157 (SRCC771), H441 (SRCC772), H460 (SRCC773), SKMES-1 (SRCC774) and SW900 (SRCC775), all available from ATCC.
  • Primary human lung tumor cells usually derive from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, non-small cell carcinomas, small cell carcinomas, and broncho alveolar carcinomas, and include, for example, SRCC724 (squamous cell carcinoma abbreviated as ⁇ SqCCaD), SRCC725 (non-small cell carcinoma, abbreviated as "NSCCa”), SRCC726 (adenocarcinoma, abbreviated as "AdenoCa”), SRCC727 (adenocarcinoma), SRCC728 (squamous cell carcinoma), SRCC729 (adenocarcinoma), SRCC730 (adeno/squamous cell carcinoma).
  • SRCC724 squamous cell carcinoma abbreviated as ⁇ SqCCaD
  • SRCC725 non-small cell carcinoma, abbreviated as "NSCCa”
  • SRCC726 adenocar
  • SRCC731 (adenocarcinoma), SRCC732 (squamous cell carcinoma), SRCC733 (adenocarcinoma), SRCC734 (adenocarcinoma), SRCC735 (broncho alveolar carcinoma, abbreviated as "BAC"), SRCC736 (squamous cell carcinoma), SRCC738 (squamous cell carcinoma), SRCC739 (squamous cell carcinoma), SRCC740 (squamous cell carcinoma), SRCC740 (lung cell carcinoma, abbreviated as "LCCa”); Colon cancer cell lines including, for example, ATCC cell lines SW480 (adenocarcinoma,
  • SRCC776 SW620 (lymph node metastasis of colon adenocarcinoma, SRCC777), COLO320 (adenocarcinoma, SRCC778), HT29 (adenocarcinoma, SRCC779), HM7 (carcinoma, SRCC780), CaWiDr (adenocarcinoma, srcc781), HCT116 (carcinoma, SRCC782), SKCOl (adenocarcinoma, SRCC783), SW403 (adenocarcinoma, SRCC784), LS174T (carcinoma, SRCC785), and HM7 (a high mucin producing variant of ATCC colon adenocarcinoma cell line LS 174T, obtained from Dr.
  • Primary colon tumors include colon adenoocarcinomas designated CT2 (SRCC742), CT3 (SRCC743), CT8 (SRCC744), CT10 (SRCC745), CT12 (SRCC746), CT14 (SRCC747), CT15 (SRCC748), CT17 (SRCC750), CT1 (SRCC751), CT4 (SRCC752), CT5 (SRCC753), CT6 (SRCC754), CT7 (SRCC755), CT9 (SRCC756), CT11 (SRCC757), CT18 (SRCC758), and DcR3, BACrev, BACfwd, T160, and T159; and Human breast carcinoma cell lines including, for example, HBL100 (SRCC759), MB435s
  • SRCC760 T47D (SRCC761), MB468(SRCC762), MB175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766), SKBR3 (SRCC767).
  • Screening assays for drug candidates are designed to identify compounds that bind or complex with the polypeptides encoded by the genes identified herein or a biologically active fragment thereof, 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.
  • Small molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides, (poly)peptide- immunoglobulin fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.
  • All assays are common in that they call for contacting the drug candidate with a 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 polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g. on a microtiter plate, by covalent or non-covalent attachments.
  • Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the polypeptide and drying.
  • an immobilized antibody e.g. a monoclonal antibody, specific for the polypeptide to be immobilized can be used to anchor it to a solid surface.
  • the assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g.
  • the coated surface containing the anchored component When the reaction is complete, the non-reacted components are removed, e.g. by washing, and complexes anchored on the solid surface are detected.
  • the detection of label immobilized on the surface indicates that complexing occurred.
  • complexing can be detected, for example, by using a labelled antibody specifically binding the immobilized complex.
  • the candidate compound interacts with but does not bind to a particular protein encoded by a gene identified herein, its interaction with that protein can be assayed by methods well known for detecting protein- protein interactions.
  • assays include traditional approaches, such as, cross-linking, co- immunoprecipitation, and co-purification through gradients or chromatographic columns.
  • protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and Song, Nature (London) 340, 245-246 (1989); Chien et al, Proc. Natl. Acad. Sci. USA 88, 9578-9582 (1991)] as disclosed by Chevray and Nathans [Proc. Natl. Acad. Sci.
  • yeast GAL4 consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain.
  • the yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain.
  • the expression of a GALl-/ ⁇ cZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction.
  • Colonies containing interacting polypeptides are detected with a chromogenic substrate for ⁇ -galactosidase.
  • a complete kit (MATCHMAKERTM) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
  • a reaction mixture is usually prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products.
  • the reaction is run in the absence and in the presence of the test compound.
  • 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 above. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.
  • compositions useful in the treatment of immune related diseases include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple helix molecules, etc. that inhibit or stimulate immune function, for example, T cell proliferation/activation, lymphokine release, or immune cell infiltration.
  • antisense RNA and RNA molecule act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
  • antisense DNA oligodeoxyribonucleotides derived from the translation initiation site, e.g. between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g. Rossi, Current Biology 4, 469-471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997).
  • Nucleic acid molecules in triple helix formation used to inhibit transcription should be single- stranded and composed of deoxynucleotides.
  • the base composition of these oligonucleotides is designed such that it promotes triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
  • Hoogsteen base pairing rules which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
  • Some of the most promising drug candidates according to the present invention are antibodies and antibody fragments which may inhibit (antagonists) or stimulate (agonists) T cell proliferation, eosinophil infiltration, etc. i. Polyclonal Antibodies
  • polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.
  • the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include the polypeptide of the invention or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-
  • TDM adjuvant monophosphoryl Lipid A, synthetic trehalose dicorynomycolate.
  • the immunization protocol may be selected by one skilled in the art without undue experimentation. ii. Monoclonal Antibodies
  • Antibodies which recognize and bind to the polypeptides of the invention or which act as agonist therefor may, alternatively, be monoclonal antibodies.
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include the polypeptide of the invention or a fusion protein thereof.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide of the invention or having similar activity as the polypeptide of the invention.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., ⁇ 07:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as. for example, protein A-Sepharose. hydroxyapatite chromatography, gel electrophoresis. dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein.
  • 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.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies are preferably monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain.
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking.
  • the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. iii. Human and Humanized Antibodies
  • the antibodies of the invention may further comprise humanized antibodies or human antibodies.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances.
  • humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta. Curr. Op. Struct. Biol, 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • Humanization can be essentially performed following the method of Winter and coworkers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • 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 Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss. p. 77 (1985); Boemer et al., J.
  • 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.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities may be for the polypeptide of the invention, 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 coexpression 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., 10:3655-3659 (1991).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.
  • CHI first heavy-chain constant region
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980. vi Effector function engineering
  • the antibody of the invention may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating an immune related disease, for example.
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992).
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al Cancer Research 53:2560-2565 (1993).
  • an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design 3:219-230 (1989). vii Immunoconjugates
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosd), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins.
  • Phytolaca americana proteins PAPI, PAPII, and PAP-S
  • momordica charantia inhibitor curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
  • radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I, 13 , In, 90 Y and 186 Re.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminofhiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl
  • a ricin immunotoxin can be prepared as described in Vitetta et al. , Science 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026.
  • the antibody may be conjugated to a "receptor” (such streptavidin) for utilization in tisue pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g. avidin) which is conjugated to a cytotoxic agent (e.g. a radionucleotide).
  • a "ligand” e.g. avidin
  • cytotoxic agent e.g. a radionucleotide
  • proteins, antibodies, etc. disclosed herein may also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al, Proc. Natl Acad. Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S.
  • Patent No. 5,013,556 is a patent No.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al ., J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange reaction.
  • a chemotherapeutic agent such as doxorubicin
  • active molecules of the invention can be administered for the treatment of immune related diseases, in the form of pharmaceutical compositions.
  • Therapeutic formulations of the active molecule are prepared for storage by mixing the active molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
  • Lipofections or liposomes can also be used to deliver the polypeptide, antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment which specifically binds to the binding domain of the target protein is preferred.
  • peptide molecules can be designed which retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e.g. Marasco et al, Proc. Natl. Acad. Sci. USA 90, 7889-7893 [1993]).
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent.
  • cytotoxic agent cytokine or growth inhibitory agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active molecules may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3- hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37C, 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.
  • polypeptides, antibodies and other active compounds of the present invention may be used to treat various immune related diseases and conditions, such as T cell mediated diseases, including those characterized by infiltration of inflammatory cells into a tissue, stimulation of T-cell proliferation, inhibition of T-cell proliferation, increased or decreased vascular permeability or the inhibition thereof.
  • T cell mediated diseases including those characterized by infiltration of inflammatory cells into a tissue, stimulation of T-cell proliferation, inhibition of T-cell proliferation, increased or decreased vascular permeability or the inhibition thereof.
  • Exemplary conditions or disorders to be treated with the polypeptides, antibodies and other compounds of the invention include, but are not limited to systemic lupus eryfhematosis, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma). idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjsgren's syndrome, systemic vasculitis. sarcoidosis. autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria).
  • autoimmune thrombocytopenia idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia
  • thyroiditis Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis
  • diabetes mellitus idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia
  • thyroiditis Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis
  • immune-mediated renal disease glomerulonephritis, tubulointerstitial nephritis
  • demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy
  • hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis
  • inflammatory and fibrotic lung diseases such as inflammatory bowel disease (ulcerative colitis: Crohn's disease), gluten-sensitive enteropathy, and Whipple's disease
  • autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis, allergic
  • T lymphocytes have not been shown to be directly involved in tissue damage, T lymphocytes are required for the development of auto-reactive antibodies.
  • the genesis of the disease is thus T lymphocyte dependent.
  • Multiple organs and systems are affected clinically including kidney, lung, musculoskeletal system, mucocutaneous, eye, central nervous system, cardiovascular system, gastrointestinal tract, bone marrow and blood.
  • Rheumatoid arthritis is a chronic systemic autoimmune inflammatory disease that mainly involves the synovial membrane of multiple joints with resultant injury to the articular cartilage.
  • the pathogenesis is T lymphocyte dependent and is associated with the production of rheumatoid factors, auto- antibodies directed against self IgG, with the resultant formation of immune complexes that attain high levels in joint fluid and blood.
  • These complexes in the joint may induce the marked infiltrate of lymphocytes and monocytes into the synovium and subsequent marked synovial changes; the joint space/fluid if infiltrated by similar cells with the addition of numerous neutrophils.
  • Tissues affected are primarily the joints, often in symmetrical pattern.
  • extra-articular disease also occurs in two major forms.
  • One form is the development of extra-articular lesions with ongoing progressive joint disease and typical lesions of pulmonary fibrosis, vasculitis, and cutaneous ulcers.
  • the second form of extra-articular disease is the so called Felty's syndrome which occurs late in the RA disease course, sometimes after joint disease has become quiescent, and involves the presence of monropenia, thrombocytopenia and splenomegaly. This can be accompanied by vasculitis in multiple organs with formations of infarcts, skin ulcers and gangrene.
  • RA rheumatoid nodules
  • pericarditis pleuritis
  • coronary arteritis intestitial pneumonitis with pulmonary fibrosis
  • keratoconjunctivitis sicca and rhematoid nodules.
  • Juvenile chronic arthritis is a chronic idiopathic inflammatory disease which begins often at less than
  • SUBST1TUTE SHEET (RULE 26) are classified as juvenile rheumatoid arthritis The disease is sub-classified into three major categories pauciarticular, polyarticular, and systemic The arthritis can be severe and is typically destructive and leads to j oint ankylosis and retarded growth Other manifestations can mclude chronic anterior uveitis and systemic amyloidosis Spondyloarthropathies are a group of disorders with some common clinical features and the common association with the expression of HLA-B27 gene product The disorders include ankylosmg spony tis, Reiter's syndrome (reactive arthritis), arthritis associated with mflammatory bowel disease, spondyhtis associated with psoriasis, juvenile onset spondyloarthropathy and undifferentiated spondyloarthropathy Distinguishing features include sacroileitis with or without spondyhtis, mflammatory asymmetric arthritis, association with HLA-
  • Systemic sclerosis (scleroderma) has an unknown etiology A hallmark of the disease is induration of the skin, likely this is mduced by an active mflammatory process Scleroderma can be localized or systemic, vascular lesions are common and endothelial cell injury in the microvasculature is an early and important event in the development of systemic sclerosis, the vascular injury may be immune mediated An immunologic basis is implied by the presence of mononuclear cell infiltrates in the cutaneous lesions and the presence of anti-nuclear antibodies m many patients ICAM-1 is often upregulated on the cell surface of fibroblasts m skin lesions suggestmg that T cell interaction with these cells may have a role m the pathogenesis of the disease Other organs involved include the gastrointestinal tract smooth muscle atrophy and fibrosis resultmg in abnormal pe ⁇ stalsis/motility, kidney concentric subendofhe al intimal proliferation affectmg small arcuate and interlobular arteries with resultant reduced
  • Idiopathic inflammatory myopathies including dermatomyositis, polymyositis and others are disorders of chronic muscle inflammation of unknown etiology resultmg m muscle weakness Muscle injury/inflammation is often symmetric and progressive Autoantibodies are associated with most forms These myositis-specific autoantibodies are directed agamst and inhibit the function of components, proteins and RNA's, mvolved in protein synthesis
  • Sjsgren's syndrome is due to immune-mediated inflammation and subsequent functional destruction of the tear glands and salivary glands
  • the disease can be associated with or accompanied by inflammatory connective tissue diseases
  • the disease is associated with autoantibody production against Ro and La antigens, both of which are small RNA-protein complexes Lesions result in keratoconjunctivitis sicca, xerostomia, with other manifestations or associations including bilary cirrhosis, peripheral or sensory neuropathy, and palpable purpura
  • Systemic vasculitis are diseases in which the primary lesion is inflammation and subsequent damage to blood vessels which results in ischemia/necrosis/degeneration to tissues supplied by the affected vessels and eventual end-organ dysfunction in some cases
  • Vascu tides can also occur as a secondary lesion or sequelae to other immune-inflammatory mediated diseases such as rheumatoid arthritis, systemic sclerosis, etc., particularly in diseases also associated with the formation of immune complexes.
  • Systemic necrotizing vasculitis poiyarteritis nodosa, allergic angiitis and granulomatosis, polyangiitis; Wegener's granulomatosis; lymphomatoid granulomatosis; and giant cell arteritis.
  • Miscellaneous vasculitides include: mucocutaneous lymph node syndrome (MLNS or Kawasaki's disease), isolated CNS vasculitis, Behet's disease, fhromboangiitis obliterans (Buerger's disease) and cutaneous necrotizing venulitis.
  • MLNS mucocutaneous lymph node syndrome
  • isolated CNS vasculitis Behet's disease
  • fhromboangiitis obliterans Buerger's disease
  • cutaneous necrotizing venulitis The pathogenic mechanism of most of the types of vasculitis listed is believed to be primarily due to the deposition of immunoglobulin complexes in the vessel wall and subsequent in
  • Sarcoidosis is a condition of unknown etiology which is characterized by the presence of epithelioid granulomas in nearly any tissue in the body; involvement of the lung is most common.
  • the pathogenesis involves the persistence of activated macrophages and lymphoid cells at sites of the disease with subsequent chronic sequelae resultant from the release of locally and systemically active products released by these cell types.
  • Autoimmune hemolytic anemia including autoimmune hemolytic anemia, immune pancytopenia, and paroxysmal noctural hemoglobinuria is a result of production of antibodies that react with antigens expressed on the surface of red blood cells (and in some cases other blood cells including platelets as well) and is a reflection of the removal of those antibody coated cells via complement mediated lysis and/or ADCC/Fc- receptor-mediated mechanisms.
  • platelet destruction/removal occurs as a result of either antibody or complement attaching to platelets and subsequent removal by complement lysis, ADCC or FC-receptor mediated mechanisms.
  • Thyroiditis including Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, and atrophic thyroiditis, are the result of an autoimmune response against thyroid antigens with production of antibodies that react with proteins present in and often specific for the thyroid gland.
  • Experimental models exist including spontaneous models: rats (BUF and BB rats) and chickens (obese chicken strain); inducible models: immunization of animals with either thyroglobulin, thyroid microsomal antigen (thyroid peroxidase).
  • Type I diabetes mellitus or insulin-dependent diabetes is the autoimmune destruction of pancreatic islet ⁇ cells; this destruction is mediated by auto-antibodies and auto-reactive T cells.
  • Antibodies to insulin or the insulin receptor can also produce the phenotype of insulin-non-responsiveness.
  • Immune mediated renal diseases including glomerulonephritis and tubulointerstitial nephritis, are the result of antibody or T lymphocyte mediated injury to renal tissue either directly as a result of the production of autoreactive antibodies or T cells against renal antigens or indirectly as a result of the deposition of antibodies and/or immune complexes in the kidney that are reactive against other, non-renal antigens.
  • immune-mediated diseases that result in the formation of immune-complexes can also induce immune mediated renal disease as an indirect sequelae. Both direct and indirect immune mechanisms result in inflammatory response that produces/induces lesion development in renal tissues with resultant organ function impairment and in some cases progression to renal failure.
  • Demyelinating diseases of the central and peripheral nervous systems including Multiple Sclerosis: idiopathic demyelinating polyneuropathy or Guillain-Barr syndrome; and Chronic Inflammatory
  • Demyelinating Polyneuropathy are believed to have an autoimmune basis and result in nerve demyelination as a result of damage caused to ohgodendrocytes or to myelin directly.
  • MS there is evidence to suggest that disease induction and progression is dependent on T lymphocytes.
  • Multiple Sclerosis is a demyelinating disease that is T lymphocyte-dependent and has either a relapsing-remitting course or a chronic progressive course. The etiology is unknown; however, viral infections, genetic predisposition, environment, and autoimmunity all contribute.
  • Lesions contain infiltrates of predominantly T lymphocyte mediated, microglial cells and infiltrating macrophages; CD4+T lymphocytes are the predominant cell type at lesions. The mechanism of oligodendrocyte cell death and subsequent demyelination is not known but is likely T lymphocyte driven.
  • Inflammatory and Fibrotic Lung Disease including Eosinophilic Pneumonias; Idiopathic Pulmonary Fibrosis, and Hypersensitivity Pneumonitis may involve a disregulated immune- inflammatory response. Inhibition of that response would be of therapeutic benefit.
  • Allergic diseases including asthma; allergic rhinitis; atopic dermatitis; food hypersensitivity; and urticaria are T lymphocyte dependent. These diseases are predominantly mediated by T lymphocyte induced inflammation, IgE mediated- inflammation or a combination of both.
  • Transplantation associated diseases including Graft rejection and Graft-Versus-Host-Disease
  • GVHD T lymphocyte-dependent; inhibition of T lymphocyte function is ameliorative.
  • Infectious disease including but not limited to viral infection (including but not limited to AIDS, hepatitis A, B, C, D, E) bacterial infection, fungal infections, and protozoal and parasitic infections (molecules (or derivatives/agonists) which stimulate the MLR can be utilized therapeutically to enhance the immune response to infectious agents), diseases of immunodeficiency (molecules/derivatives/agonists) which stimulate the MLR can be utilized therapeutically to enhance the immune response for conditions of inherited, acquired, infectious induced (as in HIV infection), or iatrogenic (i.e. as from chemotherapy) immunodeficiency), and neoplasia.
  • viral infection including but not limited to AIDS, hepatitis A, B, C, D, E
  • bacterial infection including but not limited to AIDS, hepatitis A, B, C, D, E
  • fungal infections including but not limited to AIDS, hepatitis A, B, C, D, E
  • protozoal and parasitic infections
  • Molecules that inhibit the lymphocyte response in the MLR also function in vivo during neoplasia to suppress the immune response to a neoplasm; such molecules can either be expressed by the neoplastic cells themselves or their expression can be induced by the neoplasm in other cells.
  • Antagonism of such inhibitory molecules enhances immune-mediated tumor rejection.
  • inhibition of molecules with proinflammatory properties may have therapeutic benefit in reperfusion injury; stroke; myocardial infarction; atherosclerosis; acute lung injury; hemorrhagic shock; burn; sepsis/septic shock; acute tubular necrosis; endometriosis; degenerative joint disease and pancreatis.
  • the compounds of the present invention e.g. polypeptides or antibodies, are administered to a subject.
  • 5 mammal preferably a human
  • intravenous administration as a bolus or by continuous infusion over a period of time
  • Intravenous or inhaled administration of polypeptides and antibodies is preferred.
  • the patient to be treated with a the immunoadjuvant of the invention may also receive an anti- cancer agent (chemotherapeutic agent) or radiation therapy.
  • chemotherapeutic agent chemotherapeutic agent
  • Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in
  • chemotherapeutic agent may precede, or follow administration of the immunoadjuvant or may be given simultaneously therewith.
  • an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onapristone (see, EP 616812) may be given in dosages known for such molecules.
  • the polypeptides of the invention are coadministered with a growth inhibitory agent.
  • the growth inhibitory agent may be any growth inhibitory agent.
  • Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the polypeptide of the invention.
  • a compound of the invention will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound, and the discretion of the attending physician.
  • the compound is suitably administered to the patient at one time or over a series of treatments.
  • 35 20mg/kg) of polypeptide or antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 ug/kg to 100 mg kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful.
  • an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above comprises a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port
  • the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the active agent in the composition is usually a polypeptide or an antibody of the invention.
  • the label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. 12. Diagnosis and Prognosis of Immune Related Disease
  • Cell surface proteins such as proteins which are overexpressed in certain immune related diseases, are excellent targets for drug candidates or disease treatment.
  • the same proteins along with secreted proteins encoded by the genes amplified in immune related disease states find additional use in the diagnosis and prognosis of these diseases.
  • antibodies directed against the protein products of genes amplified in multiple sclerosis, rheumatoid arthritis, or another immune related disease can be used as diagnostics or prognostics.
  • antibodies can be used to qualitatively or quantitatively detect the expression of proteins encoded by amplified or overexpressed genes ("marker gene products").
  • the antibody preferably is equipped with a detectable, e.g. fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. These techniques are particularly suitable, if the overexpressed gene encodes a cell surface protein Such binding assays are performed essentially as decribed above.
  • In situ detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy.
  • a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample.
  • This procedure also allows for determining the distribution of the marker gene product in the tissue examined. It will be apparent for those skilled in the art that a wide variety of histological methods are readily available for in situ detection.
  • the extracellular domain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins from the Swiss-Prot public protem database were used to search expressed sequence tag (EST) databases
  • the EST databases included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA)
  • the search was performed using the computer program BLAST or BLAST2 (Altshui et al , Methods m Enzymology 266 460-480 (1996)) as a comparison of the ECD protem sequences to a 6 frame translation of the EST sequence
  • BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled mto consensus DNA sequences with the program Dphrapi; (Phil Green, University of Washington, Seattle, Washmgton)
  • a consensus DNA sequence encodmg PR0245 was assembled relative to the other identified EST sequences, wherein the consensus sequence was designated here as DNA30954 (see Figs 3A-3B) , wherem the polypeptide showed some structural homology to transmembrane protem receptor tyrosine kinase proteins
  • oligonucleotides were synthesized to identify by PCR a cDNA library that contamed the sequence of interest and for use as probes to isolate a clone of the full- length coding sequence for PR0245
  • a pair of PCR primers (forward and reverse) were synthesized forward PCR primer 5'-ATCGTTGTGAAGTTAGTGCCCC-3' (SEQ ID NO 4) reverse PCR primer 5'-ACCTGCGATATCCAACAGAATTG-3' (SEQ ID NO 5)
  • a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30954 sequence which had the following nucleotide sequence hybridization probe 5'-GGAAGAGGATACAGTCACTCTGGAAGTATTAGTGGCTCCAGCAGTTCC-3' (SEQ ID NO 6)
  • SEQ ID NO 6 nucleotide sequence hybridization probe 5'-GGAAGAGGATACAGTCACTCTGGAAGTATTAGTGGCTCCAGCAGTTCC-3'
  • RNA for construction of the cDNA libraries was isolated from human fetal liver 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 NotI site, linked with blunt to Sail hemikinased adaptors cleaved with Notl. sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD,
  • pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science 253 1278- 1280 (1991)) in the unique Xhol and NotI sites
  • DNA sequencmg of the clones isolated as described above gave the full-length DNA sequence for PR0245 [herein designated as UNQ219 (DNA35638)] and the derived protein sequence for PR0245 The entire nucleotide sequence of UNQ219 (DNA35638) is shown in Figure 1 (SEQ ID NO 1)
  • Clone UNQ219 (DNA35638) contains a smgle open readmg frame with an apparent translational initiation site at nucleotide positions 89-91 [Kozak et al , supra] and ending at the stop codon at nucleotide positions 1025-1027 (Fig 1, SEQ ID NO 1)
  • the predicted polypeptide precursor is 312 amino acids long (Fig 2)
  • Clone UNQ219 (DNA35638) has been deposited with ATCC on September 17, 1997 and is assigned ATCC Deposit No 209265
  • EST databases included public databases (e g , Dayhof, GenBank), and proprietary databases (e g LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA)
  • the search was performed usmg the computer program BLAST or BLAST2 (Altschul, SF and Gish (1996), Methods in Enzymology 266 460-80 (1996), http //blast wustl/edu/blast/README html) as a companson of the ECD protein sequences to a 6 frame translation of the EST sequences
  • Blast score 70 (or m some cases 90) or greater that did not encode known protems were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, WA,
  • 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
  • the pair of forward and reverse PCR primers may range from 20 to 30 nucleotides (typically 24), and are designed to give a PCR product of 100-1000 bp in length
  • the probe sequences (notated as * p) are typically 40-55 bp (typically 50) in length
  • additional oligonucleotides are synthesized when
  • RNA for the construction of the cDNA libraries was isolated usmg standard isolation protocols, e g ,
  • the cDNA was primed with oligo dT containing a Noti site, linked with blunt to Sail hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoesis, and cloned in a defined orientation in a suitable cloning vector 5 (pRK5B or pRK5D) in the unique Xhol and NotI sites.
  • a suitable cloning vector 5 pRK5B or pRK5D
  • a cDNA clone was sequenced in its entirety.
  • the entire nucleotide sequence of EGF-like homologues is shown in Figures 5A (SEQ ID NO: 13), 5B (SEQ ID NO: 14) and 5C (SEQ ID NO: 15).
  • the predicted polypeptide is 448, 353, and 379 (PR0217) amino acid in length, respectively, with a molecule weight of approximately 50.15, 38.19 and 41.52 kDa, respectively.
  • the oligonucleotide sequences used in the above procedure were the following:
  • ECD extracellular domain
  • the EST databases included public EST databases (e.g., GenBank), a proprietary EST database (LIFESEQTM,
  • a consensus DNA sequence encoding DNA35936 was assembled using phrap. In some cases, the consensus DNA sequence was extended using repeated cycles of blast and phrap to extend the consensus sequence as far as possible using the three sources of EST sequences listed above. The extended assembly
  • 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.
  • Forward and reverse PCR primers may range from 20 to 30 nucleotides (typically about 24), and are designed to give a PCR product of 100-1000 bp in length.
  • 15 probe sequences are typically 40-55 bp (typically about 50) in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than 1-1.5 kbp.
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al, Current Protocols in Molecular Biology, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest by the in vivo cloning procedure
  • 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 PRO301 gene using the probe oligonucleotide and one of the PCR primers.
  • RNA for construction of the cDNA libraries was isolated from human fetal kidney.
  • cDNA clones 25 libraries used to isolated the cDNA clones were 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 NotI site, linked with blunt to Sail hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al.. Science,
  • FIG. 15 A cDNA clone was sequenced in its entirety.
  • the full length nucleotide sequence of native sequence PRO301 is shown in Figure 15 (SEQ ID NO: 75).
  • Clone DNA40628 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 52-54 [Kozak et al., supra] (Fig. 15; SEQ ID NO: 75).
  • the predicted polypeptide precursor is 299 amino acids long with a predicted molecular weight
  • PRO301 Based on a BLAST and FastA sequence alignment analysis of the full-length sequence, PRO301 shows amino acid sequence identity to A33 antigen precursor (30%) and coxsackie and adenovirus receptor protein (29%).
  • OLI2167 (35936.r2) (SEQ ID NO:83) ACTCAGCAGTGGTAGGAAAG
  • the extracellular domain (ECD) sequences (including the secretion signal, if any) of 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.g., GenBank) and a proprietary EST DNA database (LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequence.
  • oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0266.
  • 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 l-1.5kbp.
  • DNA from the libraries was screened by PCR amplification, as ber 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 by the in vivo clongin procedure using the probe oligonucleotide and one of the primer pairs.
  • PCR primers forward and reverse were synthesized: forward PCR primer 5'-GTTGGATCTGGGCAACAATAAC-3' (SEQ ID NO:258) reverse PCR primer 5'-ATTGTTGTGCAGGCTGAGTTTAAG-3' (SEQ ID NO:259) Additionally, a synthetic oligonucleotide hybridization probe was constructed from SEQ ID NO:257 which had the following nucleotide sequence:
  • 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 PR0266 gene using the probe oligonucleotide and one of the PCR primers.
  • RNA for construction of the cDNA libraries was isolated from human fetal brain tissue.
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA.
  • the cDNA was primed with oligo dT containing a NotI site, linked with blunt to Sail hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science, 253:1278- 1280 (1991)) in the unique Xhol and NotI sites.
  • a suitable cloning vector such as pRKB or pRKD; pRK5B is a precursor of p
  • DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0266 [herein designated as UNQ233 (DNA37150-seq min)] (SEQ ID NO:236) and the derived protein sequence for PR0266.
  • the extracellular domain (ECD) sequences (including the secretion signal, if any) of 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.g., GenBank) and a proprietary EST DNA database (LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequence.
  • oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0335, PR0331 or PR0326.
  • 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 l-1.5kbp.
  • Figure 31 forward SEQ ID NOS:271-274; reverse SEQ ID NOS:275-277) and yet another primer, SEQ ID NO:278 shown in Figure 31 for determination of PR0335.
  • the primers are shown in Figure 36, (forward is SEQ ID NO:295; reverse is SEQ ID NO:296; and the other is SEQ ID NO:297).
  • PR0326 a 5' splice variant of PR0335, the primers used are shown in Figures 40 and Figures 41.
  • RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (PR0335 and PR0326) and human fetal brain (PR0331).
  • 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 NotI site, linked with blunt to Sail hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique Xhol and NotI sites.
  • a suitable cloning vector such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science, 253:1278-1280 (1991)
  • DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0335, PR0331 or PR0326 [herein designated as SEQ ID N0S:261, 279 or 298, and the derived protein sequence for PR0335, PR0331 or PR0326.
  • EXAMPLE 2 Stimulatory Activity in Mixed Lymphocyte Reaction (MLR) Assay This example shows that the polypeptides of the invention are active as a stimulator of the proliferation of stimulated T-lymphocytes.
  • MLR Mixed Lymphocyte Reaction
  • a therapeutic agent may take the form of antagonists of the polypeptide of the invention, for example, murine-human chimeric, humanized or human antibodies against the polypeptide.
  • peripheral blood mononuclear cells are isolated.
  • the cells are frozen in fetal bovine serum and DMSO after isolation. Frozen cells may be thawed overnight in assay media (37 °C, 5%
  • the stimulator PBMCs are prepared by irradiating the cells (about 3000 Rads).
  • the assay is prepared by plating in triplicate wells a mixture of: lOO ⁇ l of test sample diluted to 1% or to 0.1% 50 ⁇ l of irradiated stimulator cells and 50 ⁇ l of responder PBMC cells. 15 100 microliters of cell culture media or 100 microliter of CD4-IgG is used as the control.
  • the wells are then incubated at 37 °C, 5% CO2 for 4 days. On day 5 and each well is pulsed with tritiated thymidine (i.O mC/well; Amersham). After 6 hours the cells are washed 3 times and then the uptake of the label is evaluated.
  • PBMCs are isolated from the spleens of Balb/c mice and C57B6 0 mice.
  • the cells are teased from freshly harvested spleens in assay media (RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential amino acids, 1% pyruvate) and the
  • PBMCs are isolated by overlaying these cells over Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and washing the mononuclear cell layer in assay media and resuspending the cells to lx 10' cells/ml of assay media.
  • Lympholyte M Organic Teknika
  • the assay is then conducted as described above.
  • the results of this 5 assay for compounds of the invention are shown below. Positive increases over control are considered positive with increases of greater than or equal to 180% being preferred. However, any value greater than control indicates a stimulatory effect for the test protein.
  • PRO301 (cont.) 1.0% 127.7
  • This assay shows that certain polypeptides of the invention stimulate an immune response and induce inflammation by inducing mononuclear cell, eosinophil and PMN infiltration at the site of injection of the animal.
  • This skin vascular permeability assay is conducted as follows. Hairless guinea pigs weighing 350 grams or more are anesthetized with ketamine (75-80 mg/Kg) and 5 mg/Kg xylazine intramuscularly (IM). A sample of purified polypeptide of the invention or a conditioned media test sample is injected intradermally onto the backs of the test animals with 100 uL per injection site. It is possible to have about 10-30, preferably about 16-24, injection sites per animal.
  • Evans blue dye 1% in physiologic buffered saline
  • Blemishes at the injection sites are then measured (mm diameter) at lhr and 6 hr post injection. Animals were sacrificed at 6 hrs after injection. Each skin injection site was biopsied and fixed in formalin. The skins were then prepared for histopafhalogic evaluation. Each site was evaluated for inflammatory cell infiltration into the skin. Sites with visible inflammatory cell inflammation were scored as positive. Inflammatory cells can be neutrophilic, eosinophilic, monocytic or lymphocytic. The results of this test for compounds of the invention is shown below.
  • T cell activation requires a costimulatory signal.
  • One costimulatory signal is generated by the interaction of B7 (CD3) with CD28.
  • B7 CD3
  • 96 well plates are coated with CD3 with or without CD28 and then human peripheral blood lymphocytes followed by a test protein, are added. Proliferation of the lymphocytes is determined by tritiated thymidine uptake. A positive assay indicates that the test protein provided a stimulatory signal for lymphocyte proliferation.
  • Material 1) Hyclone D-PBS without Calcium, Magnesium 2) Nunc 96 well certified plates #4-39454
  • Plates are prepared by coating 96 well flat bottom plates with anti-CD3 antibody (Amac) or anti- CD28 antibody (Biodesign) or both in Hyclone D-PBS without calcium and magnesium.
  • Anti -CD3 antibody is used at 10 ng/well (50 ⁇ l of 200 ng/ml) and anti -CD28 antibody at 25 ng/well (50 ⁇ l of 0.5 ⁇ g/ml) in 100 ⁇ l total volume.
  • PBLs are isolated from human donors using standard leukophoresis methods. The cell preparations are frozen in 50% fetal bovine serum and 50% DMSO until the assay is conducted. Cells are prepared by thawing and washing PBLs in media, resuspending PBLs in 25 mis of media and incubating at 37DC, 5% CO2 overnight. In the assay procedure, the coated plate is washed twice with PBS and the PBLs are washed and resuspended to 1 x 10° cells/ml using 100 ⁇ L /well. 100 ul of a test protein or control media are added to the plate making a total volume per well of 200 ⁇ L. The plate is incubated for 72 hours.
  • In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of 5 gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis and aid in chromosome mapping.
  • the probe was run on a TBE/urea gel. 1-3 ⁇ L of the probe or 5 ⁇ L of RNA Mrk III were added to 3 0 ⁇ L of loading buffer. After heating on a 95 ⁇ C heat block for three minutes, the gel was immediately placed on ice. The wells of gel were flushed, the sample loaded, and run at 180-250 volts for 45 minutes. The gel was wrapped in saran wrap and exposed to XAR film with an intensifying screen in -70 °C freezer one hour to overnight.
  • the slides were deparaffinized, placed in SQ H2O, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time.
  • the sections were deproteinated in 20 ⁇ g/ml proteinase K (500 ⁇ L of 10 mg/ml in 250 ml RNase-free RNase buffer; 37C, 15 minutes ) - human embryo, or 8 x proteinase K (100 ⁇ L in 250 ml Rnase buffer, 37°C, 30 minutes) - formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above.
  • Hybridization 1.0 x 10" cpm probe and 1.0 ⁇ L tRNA (50 mg ml stock) per slide were heated at 95 °C for 3 minutes. The slides were cooled on ice, and 48 ⁇ L hybridization buffer were added per slide. After vortexing, 50 ⁇ L 33 P mix were added to 50 ⁇ L prehybridization on slide. The slides were incubated overnight at 55C.
  • Oligo c- 120P (SEQ ID NO:312)
  • PRQ245, PRQ217, PRO301, PRQ266, PRQ335, PRQ331 or PRQ326 as a hybridization probe
  • the following method describes use of a nucleotide sequence encoding PR0245, PR0217,
  • PRO301, PR0266, PR0335, PR0331 or PR0326 as a hybridization probe.
  • PR0266, PR0335, PR0331 or PR0326 (as shown in Figure 1, SEQ ID NO:l; Figure 5C, SEQ ID NO: 15;
  • SEQ ID NO:280 or Figures 37A-C, SEQ ID NO:299) is employed as a probe to screen for homologous
  • DNAs (such as those encoding naturally-occurring variants of PR0245, PR0217, PRO301, PR0266,
  • Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions.
  • PR0335, PR0331 or PR0326-derived probe to the filters is performed in a solution of 50% formamide, 5x
  • SSC 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and
  • DNAs having a desired sequence identity with the DNA encoding full-length native sequence PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can then be identified using standard techniques known in the art.
  • PRQ245, PRQ217, PRO301, PRQ266, PRQ335, PRQ331 or PRQ326 in E. coli
  • This example illustrates preparation of an unglycosylated form of PR0245, PR0217, PRO301,
  • PR0266, PR0335, PR0331 or PR0326 by recombinant expression in E. coli.
  • the primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector.
  • restriction enzyme sites 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,
  • the vector is digested with restriction enzyme and dephosphorylated.
  • the PCR amplified sequences are then ligated into the vector.
  • 35 vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the
  • the ligation mixture is then used to transform a selected E. coli strain using the methods described in
  • Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.
  • liquid culture medium such as LB broth supplemented with antibiotics.
  • the overnight culture may subsequently be used to inoculate a larger scale culture.
  • the cells are then grown to a desired optical density, during which the expression promoter is turned on.
  • the cells After culturing the cells for several more hours, the cells can be harvested by centrifugation.
  • the cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
  • PR0245, PR0217, PRO301 and PR0266 were expressed in E. coli in a poly-His tagged form, using the following procedure.
  • the DNA encoding PR0245, PR0217, PRO301 and PR0266 was initially amplified using selected PCR primers.
  • the primers contained restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase.
  • the PCR-amplified, poly-His tagged sequences were then ligated into an expression vector, which was used to transform an E.
  • Cultures were then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH ) 2 S ⁇ 4, °- 71 8 sodium citrate-2H20, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO_ j ) and grown for approximately 20-30 hours at 30"C with shaking. Samples were removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets were frozen until purification and refolding.
  • CRAP media prepared by mixing 3.57 g (NH ) 2 S ⁇ 4, °- 71 8 sodium citrate-2H20, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH
  • E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) was resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer.
  • Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1M and 0.02 M, respectively, and the solution was stirred overnight at 4°C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization.
  • the solution was centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min.
  • the supernatant was 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. Depending the clarified extract was loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer. The column was washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein was eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4°C. Protein concentration was estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.
  • the proteins were refolded by diluting 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 were chosen so that the final protein concentration was between 50 to 100 micrograms/ml.
  • the refolding solution was stirred gently at 4"C for 12-36 hours.
  • the refolding reaction was quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution was filtered through a 0.22 micron filter and acetonitrile was added to 2-10% final concentration.
  • the refolded protein was chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbance were analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein were pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.
  • This example illustrates preparation of a potentially glycosylated form of PR0245, PR0217, PRO301 , PR0266, PR0335, PR0331 or PR0326 by recombinant expression in mammalian cells.
  • the vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector.
  • the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PR0245, PR0217, PRO301, PR0266,
  • PR0335, PR0331 or PR0326 DNA using ligation methods such as described in Sambrook et al., supra.
  • the resulting vector is called pRK5-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326.
  • 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.
  • medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and or antibiotics.
  • PRO301, PR0266, PR0335, PR0331 or PR0326 DNA is mixed with about 1 ug DNA encoding the VA RNA gene [Thimmappaya et al, Cell, 3L543 (1982)] and dissolved in 500 uL of 1 mM Tris-HCl, 0.1 mM
  • 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.
  • the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 uCi/ml 35 S-cysteine and 200 uCi/ml 35 S- methionine.
  • the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel.
  • the processed gel may be dried and exposed to film for a selected period of time to reveal the presence of PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide.
  • the cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
  • PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci., _12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 ug pRK5-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS.
  • the DNA-dextran precipitate is incubated on the cell pellet for four hours
  • the cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium.
  • the conditioned media is centrifuged and filtered to remove cells and debris
  • the sample containmg expressed PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography
  • PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can be expressed in CHO cells
  • the pRK5-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can be transfected mto CHO cells usmg known reagents such as CaP0 or DEAE-dextran
  • the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35 S-meth ⁇ on ⁇ ne
  • the culture medium may be replaced with serum free medium
  • the cultures are incubated for about 6 days, and then the conditioned medium is harvested
  • the medium contammg the expressed PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can then be concentrated and pur
  • Epitope-tagged PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 may also be expressed in host CHO cells
  • the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 may be subcloned out of the pRK5 vector
  • the subclone insert can undergo PCR to fuse m frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector
  • the poly-his tagged PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 insert can then be subcloned into a SV40 driven vector contammg a selection marker such as DHFR for selection of stable clones
  • the CHO cells can be transfected (as described above) with the SV40 driven vector Labelmg may be performed, as described above, to verify expression
  • PR0245, PR0217 and PRO301 were expressed in CHO cells by both a transient and a stable expression procedure
  • Stable expression m CHO cells was performed usmg the following procedure
  • the proteins were expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e g extracellular domams) of the respective proteins were fused to an IgGl constant region sequence containing the hinge, CH2 and CH2 domams and/or is a poly-His tagged form
  • CHO expression vectors are constructed to have compatible restriction sites 50 and 3D of the DNA of interest to allow the convenient shuttling of cDNADs
  • 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 of the cDNA of mterest and dihydrofolate reductase (DHFR) DHFR expression permits selection for stable maintenance of the plasmid following transfection
  • the cells were grown and described m Lucas et al . supra Approximately 3 x 10 7 cells are frozen in an ampule for further growth and production as described below
  • the ampules containing the plasmid DNA were thawed by placement into water bath and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 mL of selective media (0.2 ⁇ m filtered PS20 with 5% 0.2 ⁇ m diafiltered fetal bovine serum).
  • the cells were then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells were transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37°C. After another 2-3 days, a 250 mL, 500 mL and 2000 mL spinners were seeded with 3 x 10 5 cells/mL. The cell media was exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in US Patent No. 5,122,469, issued June 16, 1992 was actually used. 3L production spinner is seeded at 1.2 x 10 6 cells/mL. On day 0, the cell number pH were determined.
  • the spinner On day 1, the spinner was sampled and sparging with filtered air was commenced. On day 2, the spinner was sampled, the temperature shifted to 33 °C, and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion). Throughout the production, pH was adjusted as necessary to keep at around 7.2. After 10 days, or until viability dropped below 70%, the cell culture was harvested by centrifugtion and filtering through a 0.22 ⁇ m filter. The filtrate was either stored at 4°C or immediately loaded onto columns for purification.
  • 10% antifoam e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion.
  • the proteins were purified using a Ni-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml min. at 4°C. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole.
  • the highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80 °C.
  • Immunoadhesin (Fc containing) constructs of were purified from the conditioned media as follows.
  • the conditioned medium was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ⁇ L of 1 M Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
  • PR0326 was also produced by transient expression in COS cells.
  • PR0245 The following method describes recombinant expression of PR0245. PR0217, PRO301, PR0266, PR0335. PR0331 or PR0326 in yeast.
  • yeast expression vectors are constructed for intracellular production or secretion of PR0245, PR0217. PRO301, PR0266, PR0335, PR0331 or PR0326 from the ADH2/GAPDH promoter.
  • DNA encoding PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PR0245.
  • PR0217. PRO301, PR0266, PR0335, PR0331 or PR0326 for secretion, DNA encoding PR0245,
  • PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PR0245, PR0217, PRO301, PR0266, PR0335,
  • PR0331 or PR0326 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal leader sequence, and linker sequences (if needed) for expression of PR0245,
  • PR0217 PRO301, PR0266, PR0335, PR0331 or PR0326.
  • Yeast cells such as yeast strain ABI 10
  • yeast cells can then be transformed with the expression plasmids described above and cultured in selected fermentation media.
  • the transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
  • Recombinant PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters.
  • PRO301, PR0266, PR0335, PR0331 or PR0326 may further be purified using selected column chromatography resins.
  • the following method describes recombinant expression of PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 in Baculovirus-infected insect cells.
  • the sequence coding for PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 is fused upstream of an epitope tag contained within a baculovirus expression vector.
  • epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG).
  • a variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 or the desired portion of the coding sequence of PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326
  • the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5' and 3' regions.
  • the 5' primer may incorporate flanking (selected) restriction enzyme sites.
  • the product is then digested with those selected restriction enzymes and subcloned into the expression vector.
  • Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus
  • Expressed poly-his tagged PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can then be purified, for example, by Ni 2+ -chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl 2 ; 0.1 mM EDTA;
  • a Ni 2+ -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute.
  • the column is washed to baseline A 28 o with loading buffer, at which point fraction collection is started.
  • the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.
  • a secondary wash buffer 50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0
  • the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer.
  • One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni 2+ -NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His, 0 -tagged PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 are pooled and dialyzed against loading buffer.
  • purification of the IgG tagged (or Fc tagged) PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.
  • PR0245, PRO301 and PR0266 were expressed in baculovirus infected Sf9 insect cells. While the expression was actually performed in a 0.5-2 L scale, it can be readily scaled up for larger (e.g. 8 L) preparations.
  • the proteins were expressed as an IgG construct (immunoadhesin), in which the protein extracellular region was fused to an IgGl constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms.
  • baculovirus expression vector pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged proteins
  • vector and BaculogoldO baculovirus DNA were co-transfected into 105 Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711), using Lipofectin (Gibco BRL).
  • Sf9 Spodoptera frugiperda
  • pb.PH.IgG and pb.PH.His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences.
  • the cells were grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). Cells were incubated for 5 days at 28'C. The supernatant was harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Cells were incubated for 3 days at 28 °C.
  • MOI multiplicity of infection
  • the supernatant was harvested and the expression of the constructs in the baculovirus expression vector was determined by batch binding of 1 ml of supernatant to 25 mL of Ni-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
  • the first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells were incubated for 3 days at 28"C. The supernatant was harvested and filtered. Batch binding and SDS-PAGE analysis was repeated, as necessary, until expression of the spinner culture was confirmed.
  • the conditioned medium from the transfected cells (0.5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0.22 micron filters.
  • the protein construct were purified using a Ni-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media were pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4'C.
  • the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole.
  • the highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
  • Immunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows. The conditioned media were pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8.
  • the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5.
  • the eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 mL of 1 M Tris buffer, pH 9.
  • the highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins.
  • the homogeneity of the proteins was verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.
  • PR0245, PR0217, PRO301, PR0266, PR0331 and PR0326 were also expressed in baculovirus infected High-5 cells using an analogous procedure.
  • Immunogens that may be employed include purified PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326, fusion proteins containing PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326, and cells expressing recombinant PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 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 PR0245, PR0217, PRO301, PR0266, PR0335,
  • PR0331 or PR0326 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms.
  • the immunogen is emulsified in MPL- TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads.
  • 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-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 antibodies. After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.l, available from ATCC, No.
  • a selected murine myeloma cell line such as P3X63AgU.l, available from ATCC, No.
  • the fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
  • HAT hyperxanthine, aminopterin, and thymidine
  • the hybridoma cells will be screened in an ELISA for reactivity against PR0245, PR0217. PRO301,
  • PR0266 PR0335, PR0331 or PR0326. Determination of "positive" hybridoma cells secreting the desired
  • the positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 monoclonal antibodies.
  • the hybridoma cells can be grown in tissue culture flasks or roller bottles.
  • Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.

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Abstract

La présente invention concerne des compositions contenant des protéines récemment découvertes. L'invention concerne également des procédés se rapportant au diagnostic et au traitement d'affections liées aux fonctions immunitaires.
PCT/US1998/019437 1994-09-08 1998-09-17 Traitement d'affections liees aux fonctions immunitaires et compositions correspondantes WO1999014241A2 (fr)

Priority Applications (331)

Application Number Priority Date Filing Date Title
AU93959/98A AU9395998A (en) 1997-09-17 1998-09-17 Compositions and methods for the treatment of immune related diseases
ES98959543T ES2316175T3 (es) 1997-11-21 1998-11-20 Antigenos especificos de plaquetas y sus usos farmacologicos.
DE69840105T DE69840105D1 (de) 1997-11-21 1998-11-20 Plättchen-spezifische antigene und deren pharmazeutische verwendung
DE69837897T DE69837897T2 (de) 1997-11-21 1998-11-20 Mit A33 verwandte Antigene und deren pharmazeutische Verwendungen
EP04010433A EP1481990B1 (fr) 1997-11-21 1998-11-20 Antigènes apparentés à A33 et leurs utilisations pharmacologiques
AU15324/99A AU735081B2 (en) 1997-11-21 1998-11-20 A-33 related antigens and their pharmacological uses
DE69839430T DE69839430T2 (de) 1997-11-21 1998-11-20 Mit A33 verwandte Antigene und deren pharmezeutische Verwendung
EP98959543A EP1032667B1 (fr) 1997-11-21 1998-11-20 Antigenes specifique des plaquettes et leurs utilisations pharmacologiques
ES04010433T ES2288649T3 (es) 1997-11-21 1998-11-20 Antigenos tipo a-33 y sus utilizaciones farmacologicas.
DK04010433T DK1481990T3 (da) 1997-11-21 1998-11-20 A33-beslægtede antigener og deres farmakologiske anvendelser
PT04010433T PT1481990E (pt) 1997-11-21 1998-11-20 Antigénios relacionados com a-33 e suas utilizações farmacológicas
ES04010432T ES2305608T3 (es) 1997-11-21 1998-11-20 Antigenos tipo a-33 y sus utilizaciones farmacologicas.
AT98959543T ATE410512T1 (de) 1997-11-21 1998-11-20 Plättchen-spezifische antigene und deren pharmazeutische verwendung
AT04010433T ATE364049T1 (de) 1997-11-21 1998-11-20 Mit a33 verwandte antigene und deren pharmazeutische verwendungen
PCT/US1998/024855 WO1999027098A2 (fr) 1997-11-21 1998-11-20 Antigenes apparentes a a33 et leurs utilisations pharmacologiques
DK04010432T DK1481989T3 (da) 1997-11-21 1998-11-20 A-33-beslægtede antigener og deres farmakologiske anvendelser
AT04010432T ATE393786T1 (de) 1997-11-21 1998-11-20 Mit a33 vewandte antigene und deren pharmezeutische verwendung
CA002309358A CA2309358A1 (fr) 1997-11-21 1998-11-20 Antigenes apparentes a a33 et leurs utilisations pharmacologiques
JP2000522240A JP3497133B2 (ja) 1997-11-21 1998-11-20 Ａ−３３関連抗原およびそれらの薬理学的使用
EP04010432A EP1481989B1 (fr) 1997-11-21 1998-11-20 Antigènes apparentés à A33 et leurs utilisations pharmacologiques
PT04010432T PT1481989E (pt) 1997-11-21 1998-11-20 Antigénios relacionados com a-33 e suas utilizações farmacológicas
EP08017468A EP2014677A1 (fr) 1997-11-21 1998-11-20 Antigènes apparentés à A33 et leurs utilisations pharmacologiques
IL14155199A IL141551A0 (en) 1998-09-17 1999-09-15 Compositions and methods for the treatment of immune related diseases
KR1020017003430A KR20010085816A (ko) 1998-09-17 1999-09-15 면역 관련 질환 치료용 조성물 및 치료 방법
AU64984/99A AU6498499A (en) 1998-09-17 1999-09-15 Compositions and methods for the treatment of immune related diseases
JP2000570324A JP2003524600A (ja) 1998-09-17 1999-09-15 免疫関連疾患治療のための組成物及び方法
PCT/US1999/021547 WO2000015797A2 (fr) 1998-09-17 1999-09-15 Compositions et methodes de traitement des maladies relatives au syteme immunitaire
CA002343006A CA2343006A1 (fr) 1998-09-17 1999-09-15 Compositions et methodes de traitement des maladies relatives au syteme immunitaire
US09/902,736 US6894148B2 (en) 1997-11-12 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,903 US20030044839A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,736 US20030049676A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,759 US20030077654A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,572 US20030108983A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,615 US20030092002A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,634 US20030082540A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,979 US20030113718A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,853 US20020192659A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,692 US20030054400A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,713 US20030082541A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,011 US20030003530A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,806 US20030130489A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,603 US20030148419A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,640 US7208308B2 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,562 US6965015B2 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,119 US20030049621A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids enconding the same
US09/903,786 US20030044793A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,823 US20030104381A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,943 US20030054349A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,749 US7147853B2 (en) 1997-09-17 2001-07-11 Anti-pro211 polypeptide antibodies
US09/903,925 US20030096233A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,520 US20030054401A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,859 US20030036060A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,992 US20030135025A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,877 US20030186358A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,291 US20020160374A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,056 US20030054441A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,938 US20030211569A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,088 US20030073077A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,125 US6664376B2 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,786 US7557192B2 (en) 1997-09-17 2001-07-12 Anti-PRO335 antibodies
US09/904,766 US20030152999A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,381 US6818746B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,838 US20030148370A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,820 US20030036094A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,485 US20030064367A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,920 US6806352B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,075 US7169906B2 (en) 1997-09-17 2001-07-13 PRO211 polypeptides
US09/905,348 US20030064923A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,462 US6878807B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,532 US7151160B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,449 US6965011B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,553 US20030059828A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,956 US20030049622A1 (en) 1995-12-01 2001-07-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,838 US7070979B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,700 US6723535B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,722 US6946262B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,742 US20030023054A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,646 US6852848B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,815 US7094567B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,618 US6828146B2 (en) 1997-09-17 2001-07-16 Nucleic acid encoding PRO229 polypeptides
US09/906,760 US20030096340A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,777 US20030148371A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,824 US20020197671A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,575 US20030073079A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,728 US20030190611A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,613 US20030027145A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/908,093 US20030017498A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,942 US7087738B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,794 US6635468B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,841 US7033825B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,979 US20030082542A1 (en) 1994-09-08 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,652 US20030104469A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,925 US20030054352A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/908,576 US20040005553A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,088 US20020146709A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,204 US20030036061A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,320 US7074592B2 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides nucleic acid encoding
US09/909,064 US6818449B2 (en) 1997-09-17 2001-07-18 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
US09/992,521 US20030083461A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,469 US20030068623A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,443 US20030054987A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,442 US20020132252A1 (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
US09/991,073 US20020127576A1 (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
US09/992,598 US6956108B2 (en) 1997-06-16 2001-11-14 PRO1184 antibodies
US09/991,854 US20030059780A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,604 US20020137075A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,748 US20030069403A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,726 US20030054359A1 (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
US09/990,562 US20030027985A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,444 US6930170B2 (en) 1997-06-16 2001-11-14 PRO1184 polypeptides
US09/993,687 US20020198149A1 (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
US09/990,440 US20030060407A1 (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
US09/990,436 US20020198148A1 (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
US09/991,163 US20020132253A1 (en) 1997-06-16 2001-11-14 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
US09/997,601 US20030054404A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/002,796 US20030032057A1 (en) 1997-08-26 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
US09/997,384 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
US09/997,440 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
US09/997,857 US20030064375A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,641 US20030224358A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,653 US7034122B2 (en) 1997-06-16 2001-11-15 Antibodies to PRO1159 polypeptides
US09/997,585 US7166282B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,641 US7112656B2 (en) 1997-06-16 2001-11-15 PRO1312 polypeptides
US09/997,559 US20030054403A1 (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
US09/997,428 US20030027162A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,628 US20030059782A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,333 US6953836B2 (en) 1997-06-16 2001-11-15 PRO844 polypeptides
US09/997,666 US7244816B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,585 US20030119055A1 (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
US09/997,614 US20030124531A1 (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
US09/997,573 US20030049682A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/998,041 US20030119001A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/998,156 US20030044806A1 (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
US09/997,529 US7309761B2 (en) 1997-06-16 2001-11-15 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
US09/990,437 US20030045463A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,157 US7101687B2 (en) 1997-06-16 2001-11-16 Nucleic acids encoding PRO943
US09/991,172 US20030050457A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,441 US7041804B2 (en) 1997-06-16 2001-11-16 Antibodies to PRO1387 polypeptides
US09/989,727 US20020072497A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,722 US20020072067A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,734 US7491529B2 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,328 US7056736B2 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,726 US7018811B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO189 polypeptides
US09/989,721 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
US09/989,735 US6972185B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO844 polypeptides
US09/989,862 US20030130182A1 (en) 1997-11-05 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
US09/989,729 US20030059831A1 (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
US09/989,725 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
US09/989,724 US7060812B2 (en) 1997-06-16 2001-11-20 PRO1312 nucleic acids
US09/989,728 US7029873B2 (en) 1997-06-16 2001-11-20 Nucleic acids to PRO1387 polypeptides
US09/989,293 US7034136B2 (en) 1997-06-16 2001-11-20 Nucleic acids encoding PRO1159 polypeptides
US09/989,730 US7157247B2 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/028,072 US20030004311A1 (en) 1997-06-18 2001-12-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,203 US20030180796A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,494 US20030032063A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,500 US20020177165A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding
US10/066,193 US20030044902A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,273 US7317092B2 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,198 US20030170721A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,211 US20030044844A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,269 US20030040014A1 (en) 1997-08-26 2002-02-01 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
US10/121,040 US20030082759A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,047 US20030077778A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,041 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
US10/121,046 US20030194791A1 (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
US10/121,045 US20030073210A1 (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
US10/121,051 US20030092147A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,063 US20030199055A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,052 US20030199052A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,043 US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/121,054 US20030199054A1 (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
US10/121,056 US20030082760A1 (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
US10/121,061 US20030082761A1 (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
US10/121,049 US20030022239A1 (en) 1997-06-18 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,053 US20030199053A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,048 US20030199051A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,057 US20030190719A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,058 US20030190720A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,771 US20030199060A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,212 US7276577B2 (en) 1997-03-31 2002-04-15 PRO1866 polypeptides
US10/123,213 US20030199057A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,262 US20030049816A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,109 US20030190723A1 (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
US10/123,156 US20030194792A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,236 US20030068795A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,292 US20030073211A1 (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
US10/123,214 US7343721B2 (en) 1997-03-31 2002-04-15 PRO4406 polypeptide
US10/123,108 US7635478B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,213 US7193048B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,235 US20030082762A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,155 US20030068794A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,322 US20030199059A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,261 US20030068796A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,154 US20030190724A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,902 US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 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
US10/123,913 US20030203462A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,904 US20030022328A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,912 US20030100087A1 (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
US10/123,906 US20030190726A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,910 US7329404B2 (en) 1997-03-31 2002-04-16 Antibodies against PRO1310
US10/123,903 US20030073212A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,908 US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,911 US7408032B2 (en) 1997-03-31 2002-04-16 PRO1188 polypeptides
US10/125,805 US20030194794A1 (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
US10/124,821 US20030199023A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,818 US20030082763A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,817 US20030077786A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,824 US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,822 US7109305B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,704 US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
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
US10/124,820 US20030190729A1 (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
US10/124,819 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
US10/125,924 US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/125,927 US20030190731A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,932 US7317079B2 (en) 1997-03-31 2002-04-19 PRO812 polypeptides
US10/125,922 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
US10/127,831 US20030082689A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,689 US20030087365A1 (en) 1997-03-31 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,825 US7282566B2 (en) 1997-03-31 2002-04-24 PRO1779 polypeptide
US10/131,817 US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
US10/131,823 US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 polypeptides
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
US10/137,867 US20030207349A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,024 US20040058424A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,020 US20030207415A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,023 US20030207416A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,470 US20030022331A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,474 US20030032156A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,963 US7288625B2 (en) 1997-03-31 2002-05-06 PRO4395 polypeptides
US10/139,980 US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/140,864 US20030207419A1 (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
US10/140,808 US7425621B2 (en) 1997-03-31 2002-05-07 Antibodies against the PRO4401 polypeptide
US10/140,860 US7307151B2 (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
US10/140,805 US20030207417A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,928 US20030068798A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,865 US20030207420A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,921 US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
US10/141,760 US7342104B2 (en) 1997-03-31 2002-05-08 Antibodies against the PRO4320 polypeptide
US10/141,701 US20030207421A1 (en) 1997-03-31 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,755 US7297764B2 (en) 1997-03-31 2002-05-08 PRO4318 polypeptides
US10/141,754 US7361732B2 (en) 1997-03-31 2002-05-08 PRO4400 polypeptides
US10/141,756 US7488586B2 (en) 1997-03-31 2002-05-08 PRO4409 polypeptides
US10/142,430 US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/143,114 US20030036180A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,417 US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/143,113 US7329730B2 (en) 1997-03-31 2002-05-09 PRO4348 polypeptides
US10/142,425 US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,419 US7153941B2 (en) 1997-03-31 2002-05-10 Antibodies that bind PRO4994 polypeptides
US10/142,431 US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/142,423 US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 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
US10/147,492 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
US10/147,519 US20030077791A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,395 US7189534B2 (en) 1997-03-31 2002-05-21 PRO4320 polynucleotide
US10/153,934 US20030129695A1 (en) 1997-03-31 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,843 US20030207805A1 (en) 1997-06-18 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,786 US20030208055A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,782 US20030077792A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,782 US20030082766A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,498 US20030073216A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,791 US20030207429A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,913 US20030022298A1 (en) 1997-09-15 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2003334209A JP3950097B2 (ja) 1997-11-21 2003-09-25 Ａ−３３関連抗原およびそれらの薬理学的使用
JP2003334210A JP4040004B2 (ja) 1997-11-21 2003-09-25 Ａ−３３関連抗原およびそれらの薬理学的使用
US10/767,374 US7282565B2 (en) 1998-03-20 2004-01-29 PRO362 polypeptides
US10/767,904 US7211400B2 (en) 1997-11-21 2004-01-29 Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens
US10/771,187 US7355002B2 (en) 1997-11-12 2004-02-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/785,607 US7115713B2 (en) 1997-11-21 2004-02-24 Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens
US10/785,433 US20040141972A1 (en) 1997-11-21 2004-02-24 Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens
US10/785,220 US7198917B2 (en) 1997-11-21 2004-02-24 Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens
US10/785,221 US7273726B2 (en) 1997-11-21 2004-02-24 Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens
US10/797,366 US7378507B2 (en) 1997-09-18 2004-03-09 PRO217 polypeptides
US10/964,263 US7419663B2 (en) 1998-03-20 2004-10-12 Treatment of complement-associated disorders
US11/100,159 US7425613B2 (en) 1997-11-05 2005-04-05 PRO1375 polypeptides
US11/240,891 US20060246540A1 (en) 1997-08-26 2005-09-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/249,825 US8007798B2 (en) 1997-11-21 2005-10-12 Treatment of complement-associated disorders
US11/341,175 US7468427B2 (en) 1997-03-31 2006-01-27 Antibodies to PRO1275 polypeptide
US11/518,609 US20070077623A1 (en) 1997-09-17 2006-09-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/607,709 US20070087007A1 (en) 1997-11-21 2006-11-30 Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens
US11/642,816 US20070088151A1 (en) 1997-09-18 2006-12-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007056461A JP4382827B2 (ja) 1997-11-21 2007-03-06 Ａ−３３関連抗原およびそれらの薬理学的使用

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US5911997P 1997-09-17 1997-09-17
US5926397P 1997-09-18 1997-09-18
US6355097P 1997-10-28 1997-10-28
US6518697P 1997-11-12 1997-11-12
US6636497P 1997-11-21 1997-11-21
US6677097P 1997-11-24 1997-11-24
US60/063,550 1997-11-25
US60/065,186 1997-11-25
US60/066,770 1997-11-25
US60/059,119 1997-11-25
US60/066,364 1997-11-25
US60/059,263 1997-11-25
US8802698P 1998-06-04 1998-06-04
US60/088,026 1998-06-04

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Related Child Applications (13)

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US15834298A Continuation-In-Part 1997-08-26 1998-09-21
US09/254,465 Continuation-In-Part US6410708B1 (en) 1997-08-26 1998-11-20 Nucleic acids encoding A-33 related antigen polypeptides
PCT/US1998/024855 Continuation-In-Part WO1999027098A2 (fr) 1997-03-31 1998-11-20 Antigenes apparentes a a33 et leurs utilisations pharmacologiques
US09/254,465 A-371-Of-International US6410708B1 (en) 1997-08-26 1998-11-20 Nucleic acids encoding A-33 related antigen polypeptides
PCT/US1998/025108 Continuation-In-Part WO1999028462A2 (fr) 1994-09-08 1998-12-01 Polypeptides et acides nucleiques codant ces derniers
US09254460 A-371-Of-International 1999-03-09
PCT/US1999/012252 Continuation-In-Part WO1999063088A2 (fr) 1996-11-06 1999-06-02 Proteines membranaires et acides nucleiques codant ces proteines
PCT/US2000/004414 Continuation-In-Part WO2001004311A1 (fr) 1994-09-08 2000-02-22 Polypeptides secretes et transmembranaires et acides nucleiques codant pour ces polypeptides
US66535000A Continuation 1994-09-08 2000-09-18
US09/903,749 Continuation US7147853B2 (en) 1997-09-17 2001-07-11 Anti-pro211 polypeptide antibodies
US09/953,499 Continuation US6838554B2 (en) 1997-11-21 2001-09-14 Nucleic acids encoding proteins that stimulate the proliferation of t-lymphocytes
US09/953,499 Continuation-In-Part US6838554B2 (en) 1997-11-21 2001-09-14 Nucleic acids encoding proteins that stimulate the proliferation of t-lymphocytes
US10/081,056 Continuation US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis

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US6403555B1 (en) 1999-12-08 2002-06-11 Xcyte Therapies, Inc. Depsipeptide and congeners thereof for use as immunosuppressants
WO2002102310A2 (fr) * 2001-06-15 2002-12-27 Incyte Genomics, Inc. Proteines associees a la croissance, la differenciation et la mort de cellules
WO2003066881A2 (fr) * 2002-02-08 2003-08-14 Curagen Corporation Polypeptides therapeutiques, acides nucleiques codant ces derniers et leurs procedes d'utilisation
US6828302B1 (en) 1999-12-08 2004-12-07 Xcyte Therapies, Inc. Therapeutic uses of depsipeptides and congeners thereof
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Cited By (26)

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US6878806B2 (en) 1997-03-14 2005-04-12 Human Genome Sciences, Inc. Human secreted protein HTEEB42
US7781176B2 (en) 1997-03-14 2010-08-24 Human Genome Sciences, Inc. Antibodies to HTEEB42 protein
US7169906B2 (en) 1997-09-17 2007-01-30 Genentech, Inc. PRO211 polypeptides
US7147853B2 (en) 1997-09-17 2006-12-12 Genentech, Inc. Anti-pro211 polypeptide antibodies
US7449551B2 (en) 1997-09-18 2008-11-11 Genentech, Inc. PRO211 polypeptides
US7223586B2 (en) 1997-09-18 2007-05-29 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US6974689B1 (en) 1997-09-18 2005-12-13 Genentech, Inc. Nucleic acid encoding PRO211 polypeptides
WO2000015797A3 (fr) * 1998-09-17 2000-08-24 Genentech Inc Compositions et methodes de traitement des maladies relatives au syteme immunitaire
WO2000015797A2 (fr) * 1998-09-17 2000-03-23 Genentech, Inc. Compositions et methodes de traitement des maladies relatives au syteme immunitaire
WO2001019991A1 (fr) * 1999-09-15 2001-03-22 Genentech, Inc. Compositions et techniques permettant de traiter des maladies d'origine immunologique
EP1878794A2 (fr) * 1999-11-30 2008-01-16 Genentech, Inc. Compositions et procédés pour le traitement de maladies liées au système immunitaire
EP1878795A2 (fr) * 1999-11-30 2008-01-16 Genentech, Inc. Compositions et procédés pour le traitement de maladies liées au système immunitaire
EP1878794A3 (fr) * 1999-11-30 2008-01-23 Genentech, Inc. Compositions et procédés pour le traitement de maladies liées au système immunitaire
EP1878795A3 (fr) * 1999-11-30 2008-01-23 Genentech, Inc. Compositions et procédés pour le traitement de maladies liées au système immunitaire
US7041639B2 (en) 1999-12-08 2006-05-09 Xcyte Therapies, Inc. Depsipeptide and congeners thereof for use as immunosuppressants
US6828302B1 (en) 1999-12-08 2004-12-07 Xcyte Therapies, Inc. Therapeutic uses of depsipeptides and congeners thereof
US6548479B1 (en) 1999-12-08 2003-04-15 Xcyte Therapies, Inc. Therapeutic uses of depsipeptides and congeners thereof
US6403555B1 (en) 1999-12-08 2002-06-11 Xcyte Therapies, Inc. Depsipeptide and congeners thereof for use as immunosuppressants
US7601514B2 (en) 2000-01-20 2009-10-13 Genentech, Inc. Nucleic acid encoding PRO10268 polypeptides
WO2002102310A3 (fr) * 2001-06-15 2003-12-04 Incyte Genomics Inc Proteines associees a la croissance, la differenciation et la mort de cellules
WO2002102310A2 (fr) * 2001-06-15 2002-12-27 Incyte Genomics, Inc. Proteines associees a la croissance, la differenciation et la mort de cellules
EP1513867A2 (fr) * 2001-07-16 2005-03-16 Eli Lilly And Company Molecules d'adhesion jonctionnelle extracellulaires
EP1513867A4 (fr) * 2001-07-16 2006-02-08 Lilly Co Eli Molecules d'adhesion jonctionnelle extracellulaires
JP2009159949A (ja) * 2001-10-19 2009-07-23 Genentech Inc 炎症性腸疾患の診断と治療のための組成物と方法
WO2003066881A2 (fr) * 2002-02-08 2003-08-14 Curagen Corporation Polypeptides therapeutiques, acides nucleiques codant ces derniers et leurs procedes d'utilisation
WO2003066881A3 (fr) * 2002-02-08 2005-01-20 Curagen Corp Polypeptides therapeutiques, acides nucleiques codant ces derniers et leurs procedes d'utilisation

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