US 2005O175581A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0175581A1 Haupts et al. (43) Pub. Date: Aug. 11, 2005 (54) BIOLOGICAL ENTITIES AND THE (60) Provisional application No. 60/524.960, filed on Nov. PHARMACEUTICAL AND DIAGNOSTIC USE 25, 2003. Provisional application No. 60/543,518, THEREOF filed on Feb. 11, 2004. (76) Inventors: Ulrich Haupts, Koln (DE); Andre (30) Foreign Application Priority Data Koltermann, Koln (DE); Andreas Scheidig, Koln (DE); Christian Jun. 18, 2003 (EP)........................................... O3O13819 Votsmeier, Koln (DE); Ulrich Kettling, Nov. 10, 2003 (EP). ... O3O25851 Koln (DE); Wayne Michael Coco, Nov. 11, 2003 (EP). ... O3O25871 Koln (DE) Feb. 11, 2004 (EP)........................................... O4003058 Correspondence Address: Publication Classification NEEDLE & ROSENBERG, PC. SUTE 1000 (51) Int. Cl." .......................... A61K 38/19; A61K 38/48 999 PEACHTREE STREET (52) U.S. Cl. ........................................ 424/85.1; 424/94.63 ATLANTA, GA 30309-3915 (US) (57) ABSTRACT (21) Appl. No.: 11/021,951 The present invention provides method for the treatment of a disease by applying a medicament comprising a protease (22) Filed: Dec. 22, 2004 with a defined Specificity is capable to hydrolyze specific Related U.S. Application Data peptide bonds within a target Substrate related to Such disease. The proteases with Such a defined specificity can (63) Continuation-in-part of application No. 10/872,198, further be used for related therapeutic or diagnostic pur filed on Jun. 18, 2004. pOSes. Patent Application Publication Aug. 11, 2005 Sheet 1 of 18 US 2005/0175581 A1 Fig. 1 Patent Application Publication Aug. 11, 2005 Sheet 2 of 18 US 2005/0175581 A1 Trypsin IVGGYNCEENSVPYQVSL---NSGYHF- CGGSLINEQWVVSAGHCY - - - - a-Thrombin IVEGSDAEIGMSPWQVMLFRKSPQELL - CGASLISDRWVLTAAHCLLYPP Enteropeptidase WGGSNAKEGAWPWVVGL - - - YYGGRLLCGASLVSSDWLVSAAHCWYGRN t Trypsin - ar. KSRIOVRLGEH---NIEVLEGN-EOFINAAKIIRHPQYD-RKTL a-Thrombin WDKNFTENDLLVRIGKH---SRTRYERNIEKISMLEKIYIHPRYNWRENL Enteropeptidase LE- - - - PSKWTAILGLHMKSNLTSPOTV-PRLID. --EIVINPHYN-RRRK - - - - - Trypsin NNDIMLIKLSSRAVINARVSTISLPTA- - - - PPAT- - - - - GTKCISGWG a-Thrombin DRDIALMKLKKPVAFSDYIHPVCLPDR- - - - ETAASLLOAGYKGRVTGWG Enteropeptidase DNDIAMMHLEFKWNYTDYIOPICLPEENOVFPP- - - - - - - GRNCSIAGWG - - - - - - 2 - - - - - - Trypsin N- - - - - TASSGADYPDELOCLDAPVLSOAKCEASYPG-KITSNMFCWGFL a-Thrombin NLKETWTANVGKGOPSVLOVVNLPIVERPVCKDSTRI-RITDNMFCAGYK Enteropeptidase T- - - - - VVYOGTT-ANILOEADVPLLSNERCOOOMPEYNITENIMICAGYE - -3 - - Trypsin -EGGK--DSCOGDSGGPWVCNGO- - - - LQ- - - - - - GVVSWGDGCAQKNKP a-Thrombin PDEGKRGDACEGDSGGPFWMKSP- - - - FNNRWYQMGIWSWGEGCDRDGKY Enteropeptidase -EGGI- - DSCOGDSGGPLMCOENNRWFLA- - - - - - GVTSFGYKCALPNRP . 4 - - - - - Trypsin GVYTKWYNYWKWIKNTIAANS a-Thrombin GFYTHVFRLKKWIOKVIDOFGE Enteropeptidase GVYARWSRFTEWIQSFLH- - - - Fig. 2 Patent Application Publication Aug. 11, 2005 Sheet 3 of 18 US 2005/0175581 A1 Patent Application Publication Aug. 11, 2005 Sheet 4 of 18 US 2005/0175581 A1 sub IAHEYAOSV-- PY- - - - - - - - - - - - - - - GISO--IKAPALHSQGY - - - - - - - - - - - - - - - furin VAKRRAKRD-- VYOEPTDPKFPQQWYLSGVTQRDLNVKEAWAQGF- - - - - - - - - - - - - - - PC SK1 EKERSKRSALRDS- - - - - - - - - - - - - - - ALNL--FNDPMWNOOWYLODTRMTAALPKLDL PC SK5 NTHPCO- - - - - - - - - - - - - - - - - - - - - - - - SD--MNEGAWKRGY - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 - - sub - - - - - - - - - - TGSNWKVAWIDSGDSSHPDL. --NVRGGAS- - FWPSETN- - - - - - - P- - - furin - - - - - - - - - - TGHGIWWSILDDGIEKNHPDLAGNYDPGAS--FDVNDOD- - - - - - - PDPO PC SK1 HVIPVWQKGITGKGVVITVLDDGLEWNHTDIYANYDPEASYDFNDNDHD- - - - - - - P- - - PC SK5 - - - - - - - - - - TGKNIWVTILDDGIERTHPDL. --MONYDA- -LASCDVNGNDLDPMP- - - as a or a lar a k - - - - - -3 - - - - - - - - - - - - - - - - - sub - - - - YO- - - - - DGSS- - -HGTHVAGTIA- -A-NNSIGWGVSPSASLYAVKVLDS - - - - furin PRYTOM- - - - - NDNR - - - HGTRCAGEVA- -AWANNGWCGVGWAYNARGGVRMLD - - - - - PC SK1 - - - - FPRYDPTNENK---HGTRCAGEIAMOAN-NHKCGV-GVAYNSKVGGIRMLDG - - - - PC SK5 - - - -RY - - - - - DASNENKHGTRCAGEVA- -AAANNSHCTWGIAFNAKIGGWRMLDGDWTD 4 - - - - - - - - - - - r a r r - air sub -TGSGOYSWIINGIE-WAISNNMDVINMSLG - - - - - - - - - GP - - GSTA - - - - - - LKT- - furin - - - - GEVTDAWEARS-LGLNPNHIHIYSASW - - - - - - - - - GPEDDGKVDGPARIAEE - - PC SK1 - IVTDAIEASSIGFN- - - - PGHVDIYSASWGPNDDGKTVEGP- --GRIA - - - - - - OKAFE PC SK5 MVEAKSVSFNPOHVHIYSASWGPDDDGKTVD- - - - - - - - - GPA - - PLT- - - - - - - - RQ- - - S - - - - - - - - - - - - 6- - - - - - - - - - - - - - 7- - - - - - - - - - - - - - - - - - - 8 - sub - -WWDKAWSSG - - - - - WVAAAAGNEGSS - - - - - - - GSTSTWGYPAKYPSTIAVGAV- - - furin --AFFRGVSOGRGGLGSIFVWASGNGGREHDSCNCDGYTNSI-YTLSISSATOFGNV- - - PC SK1 YGVKOGROGKG - - - - - SIFVWASGNGGRO - - - - - - - GDNCDCD- - - GYTDSIYTISI - - - PC SK5 --AFENGWRMGRRGLGSVFWWASGNGGRSKDHCSCDGYTNSI-YTISSSTAESGKKPWY - - - - 8-- - - - - - - - - - - - - - - - - - - - - 9 - - - - - - - - Sub --N- - - - - - SSNOR- - - - - - - - - - - - - - ASFSSAG-SELDVMAPGVSIOSTLPGGTYGAY furin --PWYSEACSSTLA- - - - - - - - - - - - - - TTYSSGNONEKOIVTTDLROKCT- - - - - - ESH PC SKl --S- - - - - - SASQOGLSPWYAEKCSSTLATSYSSG-DYTDQRITSADLHNDCT- - - - ETH PC SK5 LEE- - - - - - CSSTL - - - - - - - - - - - - - - ATTYSSG-ESYDKKI - - - - ITTDLRQRCTDNH - - - - - - - - - - O - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 11 - - - sub NGTSMATPHVAGAAALIL--SKHP- - TWTNAOVRDRLESTATY--LG-NSFYYGKGLINV furin TGTSASAPLAAGIIALTLEANKNL--TWRDMOHLVVOTSKPAH--LN-ADDWATNGWGRK PC SKl TGTSASAPLAAGIFALAL- -EANP--NLTWRDMQHLVVWTSEYDPLA-NNPGWKKNGAGL PC SK5 TGTSASAPMAAGIIALAL- -EANPFLTWRDVOHVIVRTSRAGH--LNANDWKTNAAGFKV - - - r Fig. 4 Patent Application Publication Aug. 11, 2005 Sheet 5 of 18 US 2005/0175581 A1 Fig.5 Patent Application Publication Aug. 11, 2005 Sheet 6 of 18 US 2005/0175581 A1 Peps. TLVDEOP- - - - LENYLDMEYFGTIGIGTPAODFTWWFDTGSSNLWVPSVYCSSL--ACTN Secr. EMWDN - - - - - - LRGKSGOGYYVEMTVGSPPQTLNILVDTGSSNFAVGAAPHPFL- - - - - - Cath. PAVTEGPIPEVLKNYMDAQYYGEIGIGTPPQCFTVVFDTGSSNLWVPSIHCKLLDIACWI t - - - - 1 a a - - - - - 2- - Peps. HNRFNPEDSSTYQSTSETVSITYGTGSMTGILGYDTVOV- - - - - - - - - - - G - - - GISDTN Secr. HRYYOROLSSTYRDLRKGVYVPYTOGKWEGELGTDLVSI - - - - - - - - - - - PHGPNWTVRA Cath. HHKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSODTVSWPCOSASSASALG---GVKVER - - - - - 3 - - - - - - - - - - - - - 4. - - - - - - - - - w x Peps. QIFGLSETEPGSFLYYAPFDGILGLAYPSIS--SSGATPVFDNIWNOGLVSQDLFSVYLS Sec. NIAAITESDK-FFINGSNWEGILGLAYAEIARPDDSLEPFFDSLVKQTHVP-NLFSLQLC Cath. OVFGEATKOPGITFIAAKFDGILGMAYPRIS--VNNVLPVFDNLMQQKLVDONIFSFYLS - - - - 5 - - - - it it - - - - - - 6 - Peps. ADD- - - - - - - - - - KS-- GSWWIFGGIDSSYYTGSLNWVPVTVEGYWOITVDSITMNGETI Secr. GAGFPLNOSEVLASV-- GGSMIIGGIDHSLYTGSLWYTPIRREWYYEWIIWRVEINGODL Cath. RDP- - - - - - - - - - DAOPGGEL.MLGGTDSKYYKGSLSYLNVTRKAYWOVHLDOVEVASGLT - - - - - - - - 7- - - - - - - - - - - - - - 8 - - - - - Peps. A- - CAEGC--OAIVDTGTSLLTGPTSPIANIOSDIGASENSD- - - - - - - - GDMVVSCSAI Secr. KMDCKEYNYDKSIVDSGTTNLRLPKKWFEAAVKSIKAASSTEKFPDGFWLGEOLV-CWOA Cath. L--CKEGC--EAIVDTGTSLMWGPWDEVRELOKAIGAVPLIO- - - - - - - - GEYMPCEKV - - - - - - - 9 - - - - - - t Peps. SSLPDIWFT - - - - - - - - - - - NGVCYPVPPSAYILOSEGS - - - - CISGFOGMNVP-TESG Sec. GTTPWNIFPVISLYLMGEWTNOSFRETILPOOYLRPVEDV - - - -ATSODDCYKFAISQSS Cath. STLPATLK - - - - - - - - - - - GGKGYKLSPEDYTLKVSOAGKTLCLSGFMGMDIP-PPSG - - - - - - - O - - - - s F - - - - - - - - - - - - - - 1. O - - Peps. ELWILGDWFIROYFTVFDRANNOVGLAPVA Secr. TGTWMGAVIMEGFYWWFDRARKRIGFAVSA Cath. PLWILGDVFIGRYYTVFDRDNNRWGFAEAA s Fig. 6 Patent Application Publication Aug. 11, 2005 Sheet 7 of 18 US 2005/0175581 A1 Fig. 7 Patent Application Publication Aug. 11, 2005 Sheet 8 of 18 US 2005/0175581 A1 Ol MLEADDQGCI EEOGVEDSAN EDSWDAKPDR SSFVPSLFSK KKKNWTMRSI KTTRDRVPTY 61 OYNMNFEKLG KCIIINNKNF DKVTGMGWRN GTDKDAEALF KCFRSLGFDV IWYNDCSCAK - - - - - - - - - - - - - - - 121 MODLLKKASE EDHTNAACFA CILLSHGEEN VIYGKDGVTP IKDLTAHFRG DRSKTLLEKP a - - - - 2- - - - - - - - - 181 KLFFIOACRG TELDDGIOAD SGPINDTDAN PRYKIPWEAD FLFAYSTVPG YYSWRSPGRG - - - - - 3 - - - - - - - - - - - - - - - - - - - 4 - - - - 241 SWFVQALCSI LEEHGKDLEI MOILTRVNDR VARHFESQSD DPHFHEKKQI PCVVSMLTKE - - - - - - - - - - - - - 5- - - - - - - - - - - - 301 LYFSQ Fig. 8 Patent Application Publication Aug. 11, 2005 Sheet 9 of 18 US 2005/0175581 A1 Protein Scaffod Protease A Protease B 1 2 3 Candidate SDR insertion sites Insertion of random SDRs into protein scaffold H- - - N-N-1 SpecificitySelection for increased NBE with intended specificity SDR1 SDR2 SDR3 Fig. 9 Patent Application Publication Aug. 11, 2005 Sheet 10 of 18 US 2005/0175581 A1 54 kDa 48 kDa 35 kDa 24 kDa Fig. 10 Patent Application Publication Aug. 11, 2005 Sheet 11 of 18 US 2005/0175581 A1 -- expressing cells - A - negative control O 1O 20 30 40 50 60 time (min) Fig. 11 Patent Application Publication Aug. 11, 2005 Sheet 13 of 18 US 2005/0175581 A1 trypsin SDR2 SDR1 & SDR2 Fig. 13 Patent Application Publication Aug. 11, 2005 Sheet 14 of 18 US 2005/0175581 A1 Fig. 14 Patent Application Publication Aug. 11, 2005 Sheet 15 of 18 US 2005/0175581 A1 4. O 3 5 22 O5 1 O live Cells dead Cells Varianti variantii variant iii Fig. 15 Patent Application Publication Aug. 11, 2005 Sheet 16 of 18 US 2005/0175581 A1 CNCN LOO 15 10 5 O 19 16 Patent Application Publication Aug. 11, 2005 Sheet 17 of 18 US 2005/0175581 A1 proteolytic digestion of TNF -ol-variantx -O- variantxi -A-variantxii -v-Trypsin