US008007813B2

(12) Ulllted States Patent (10) Patent N0.: US 8,007,813 B2 Walczak (45) Date of Patent: Aug. 30, 2011

(54) CD95-FC FUSION W0 WO 98/36768 8/1993 W0 WO 99/50413 10/1999 . - W0 WO 00/18932 4/2000 (75) Inventor: Hennlng Walczak, Heidelberg (DE) W0 W0 01/4 1803 Al 60001 _ _ W0 WO 01/49866 7/2001 (73) Ass1gnees: Apogenix GmbH, Heidelberg (DE); W0 WO 01/49866 A1 7/2001 DeutschesStrftung Des Krebsforschungszentrum Offenthchen Rechts, W0 W0 02/079415 A2 100002 Heldelberg (DE) W0 W0 02/090553 11/2002 W0 W0 02/090553 A2 11/2002 ( ) Not1ce. Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 USC 154(b) by 1099 days. Wells, J .A. (1990). Additivity of mutational effects in proteins. Bio chemistry. 29(37):8509-8517.* (21) Appl. No.: 10/551,004 Ngo et al. (1994). Computational complexity, structure pre diction, and the Levinthal paradox. In MerZ and Le Grand (Eds.) The (22) PCT Filed: Mar. 26, 2004 Protein Folding Problem and Tertiary Structure Prediction. Birkhauser:Boston, pp. 491-495.* Paluet al. (1999). In pursuit of new developments for gene therapy. (86) PCT No.: PCT/EP2004/003239 Journal of Biotechnology. 68:1-13.* § 371 (0)0)’ Phillips, A.J. (2001). The challenge of gene therapy and DNA deliv ery. Journal of Pharmacy and Pharmacology. 53: 1 169-1 174.* (2), (4) Date: Apr. 12, 2007 Wang et al. (1999). Rapid analysis of gene expression (RAGE) facili tates universal expression pro?ling. Nucleic Acds Research. (87) PCT Pub. No.: WO2004/085478 27(23):4609-4618.* PCT Pub. Date: Oct. 7, 2004 Kaufman et al. (1999). Transgenic analysis of a 100-kb human B-globulin cluster-containing DNA fragment propagated as a bacte rial arti?cial . Blood. 94(9):3178-3184.* (65) Prior Publication Data Demjen, D et al., “Neutralization of CD95 ligand promotes regen US 2007/0269449 A1 Nov. 22, 2007 eration and functional recovery after spinal cord injury”, Nat. Med 10(4):389-395, 2004. Kim, Yon Su et al., “Targeting the IL-15 With an Antagonist (30) Foreign Application Priority Data IL-15 Mutant/Fe 2a Protein Blocks Delayed-Type Hypersensitivity”, Jlmmunol 160:5742-5748, 1998. Mar. 26, 2003 (EP) ...... 03006949 Martin-Villalba, A. et al., “Therapeutic neutralization of CD95 ligand and TNF attenuates brain damage in stroke”, Cell Death (51) Int. Cl. Dijj’er. 8(7):679-686, 2001. A61K 38/16 (2006.01) Smith, CA et al., “CD30 antigen, a marker for Hodgkin’s lymphoma, A61K 38/1 7 (2006.01) is a receptor Whose ligand de?nes an emerging family cytokines With C12N 5/10 (2006.01) homology to TNF”, Cell 73(7):1349-1360, 1993. C12N 5/22 (2006.01) Capon, Daniel J. et al., “Designing CD4 immunoadhesins for AIDS C07K 14/705 (2006.01) therapy”, Nature, vol. 337, Feb. 9, 1989, pp. 525-531. C07H 21/04 (2006.01) Haak-Frendscho, M. et al., “Inhibition of interferon-y by an inter feron-y receptor immunoadhesin”, Immunology, 1993 vol. 79, pp. (52) US. Cl...... 424/192.1; 435/697; 435/325; 594-599. 530/350; 536/234; 514/21.2; 514/213 (58) Field of Classi?cation Search ...... None * cited by examiner See application ?le for complete search history. Primary Examiner * Christine J Saoud (56) References Cited Assistant Examiner * Jon M Lockard (74) Attorney, Agent, or Firm * Perkins Coie LLP; Viola T. U.S. PATENT DOCUMENTS Kung 6,015,559 A 1/2000 Lynch et al. (57) ABSTRACT FOREIGN PATENT DOCUMENTS The invention relates to fusion proteins comprising at least a EP 0 526 452 2/1993 ?rst domain and a second domain selected from a constant Fc EP 0 965 637 12/1999 EP 0 992 243 4/2000 immunoglobulin domain. W0 WO 91/08298 A2 6/1991 W0 WO 95/27735 10/1995 14 Claims, 19 Drawing Sheets US. Patent Aug. 30, 2011 Sheet 1 0f 19 US 8,007,813 B2

Figure 1

CD95 >sp]P25445ITNR6_I-IUMAN Tumor necrosis factor receptor superfamily member 6 precursor (FASL receptor) (-mediating surface antigen PAS) antigen) (CD95) - Homo sapiens (Human).

1 6O MLGIW'I‘LLPL VLTSVARLSS KSVNAQV‘I‘DI NSKGLELRK'I‘ VTTVETQNLE GLHHDGQFCH xcPPGERKA RDCTVNGDEP DCVPCQEGKE YTDKAHFSSK CRRCRLCDEG HGLEVEIItgg lt'i‘éNTKCRCK PNFFCNSTVC EHCDPCTKCE HGIIKECTLT SNTKCKEEGS RSNLGWLéEg ItEIi-IPLIVWV K'RKEVQKTCR KHRKENQGSH ESPTLNPETV AINLSDVDLS KYl'l‘TIAgéllod 241 300 'I'LSQVKGFVR KNGVNEAKID EII'CNDNVQDT AEQKVQLLRN WHQLHGKKEA YDTLIKDLKK 301 335 ANLCTLAEKI QTIILKDITS DSENSNFRNE IQSLV AA 1-16 Signal peptide (potential) AA 17-173 extracellular domain (potential) AA 47-83 CRD1 AA 84-127 CRDZ AA 128-166 CRD3 AA 174-190 transmembrane (potential) AA 191-335 cytoplasmic (potential)

US. Patent Aug. 30, 2011 Sheet 4 0f 19 US 8,007,813 B2

Figure 4

3. TRAIL-R1 >splO00220lT10A_HUMAN Tumor necrosis factor receptor superfamily member 10A precursor () (TNF-related apoptosis-inducing ligand receptor 1) (TRAIL receptor-1) (TRAIL-R1) — Homo sapiens (Human) .

1 60 MAPPPARVHL GAFLAVTPNP GSAASGTEAA AATPSKVWGS SAGRIEPRGG GRGALP'I‘SMG 61 120 QHGPSARARA GRAPGPRPAR EASPRLRVHK TFKFWVGVL LQVVPSSAAT IKLHDQSIGT 121 180 QQWEHSPLGE LCPPGSHRSE HPGACNRCTE GVGYTNASNN LFACLPCTAC KSDEEERSPC 181 240 'I'TTRNTACQC KPGTFRNDNS AEMCRKCSRG CPRGMVKVKD CTPWSDIECV HKESGNGHNI 241 300 WVILWTLVV PLLLVAVLIV CCCIGSGCGG DPKCMDRVCF WRLGLLRGPG AEDNAHNEIL 301 360 SNADSLSTFV SEQQMESQEP ADLTGV‘I'VQS PGEAQCLLGP AEAEGSQRRR LLVPANGADP 361 420 TETLMLFFDK FANIVPFDSW DQLMRQLDLT KNEIDVVRAG TAGPGDALYA mwV'NKTG 421 468 RNASIHTLLD ALERI'EERHA KEKIQDLLVD SGKFIYLEDG TGSAVSLE AA 1-23 Signal peptide (potential) AA 24-239 extracellular domain (potential) AA 107-145 CRD1 AA 147-188 CRD2 AA 189-229 CRD3 AA 240-262 transmembrane (potential) AA 263-468 cytoplasmic (potential)

US. Patent Aug. 30, 2011 Sheet 6 0f 19 US 8,007,813 B2

Figure 6

4. TRAIL-R2 (long)

>sp|O14763lT1OB_HUMAN Tumor necrosis factor receptor superfamily member 10B precursor () (‘INF-related apoptosis-inducing ligand receptor 2) (TRAIL receptor-2) (TRAIL-R2) - Homo sapiens (Human) .

1 60 MEQRGQNAPA ASGARKRHGP GPREARGARP GPRVPKTLW WAAvLLLvs AESALITQQD a1 120 LAPQQRAAPQ QKRSSPSEGL CPPGHHISED GRDCISCKYG QDYSTHWNDL LFCLRCTRCD 121 180 SGEVELSPCT TTRN'I'VCQCE EGTFREEDSP EMCRKCRTGC PRGMVKVGDC TPWSDIECVH 181 24o gE_se'rKHsG§ APAVEE'I'V'I'S SPG'I'PASPCS LSGIIIGV'I'V AAVVLIVAVF VCKSLLWKKV 241 300 LPYLKGICSG GGGDPERVDR SSQRPGAEDN VLNEIVSILQ PTQVPEQEME VQEPAEPTGV 301 360 NMLSPGESEH LLEPAEAERS QRRRLLVPAN EGDPTETLRQ CFDDFADLVP FDSWEPLMRK 361 420 LGLM'DNEIKV AKAEAAGHRD TLYTMLIKWV NKTGRDASVH TLLDALETLG ERLAKQKIED 440 HLLSSGKFMY LEGNADSAMS 1-55 Signal peptide 56-210 extracellular domain (potential) 57-94 CRDl 97-137 CRD2 138-178 CRD3 192-206 TAPE 211-231 transmembrane (potential) 232-440 cytoplasmic (potential)

US. Patent Aug. 30, 2011 Sheet 8 0f 19 US 8,007,813 B2

Figure 8 Examples of Trail-R2 (long) -Fc fusion proteins with overlapping amino acids ("repeat" not included) :

Trail R2 (long) extracellular domain huIgGl 171 191 29 120 PWSDIECVH KESGTIG'ISGE A HP KSCDKTHTCP PCPAPELLGG TPWSDIECVH KESGTKHSGjLP KSCDKTHTCP PCPAPELLGG

171 191 99 m 120 PWSDIECVH KESGTil-ISGE A P FIQSCDKTHTCP PCPAPELLGG TPWSDIECVH mass'rgscnm'rcp PCPAPELLGG

1'71 _ 191 99 _ 120 TPWSDIECVH KESGTIG‘IQGE A P KLSCDKTHTCP PCPAPELLGG TPWSDIECVH ICESGTKHECDKTHTCP PCPAPELLGG

171 191 99 120 TPWSDIECVH msef?mser: A P KSCDKE'EHTCP PCPAPELLGG TPWSDIECVH KEsG'gHTcP PCPAPELLGG

171 4 191 9 120 TPWSDIECV'H KEsG'rKg?sGE A P KSCDKTETCP PCPAPELLGG TPWSDIECVH mss'm'g'rcp PCPAPELLGG US. Patent Aug. 30, 2011 Sheet 9 0f 19 US 8,007,813 B2

Figure 9

5. TRAIL-R2 (short) >sp|014763|T10B_HUMAN Tumor necrosis factor receptor superfamily member 10B precursor (Death receptor 5) (TNF-related apoptosis-inducing ligand receptor 2) (TRAIL receptor-2) (TRAIL-R2) — Homo sapiens (Human) .

éEQRGQNAPA ASGARKRHGP GPREARGARP GPRVPKTLVL VVAAVLLLVS AESALITQQD 120 :iPQQRJ-LAPQ QKRSSPSEGL CPPGHHISED GRDCISCKYG QDYSTHWNDL LFCLRCTRCD :ZéVELSPCT TTRNTVCQCE EGTFREEDSP EMCRKCRTGC PRGMVKVGDC TPWSDIECVE 240 :(EGIIIGVT VAAVVLIVAV FVCKSLLWKK VLPYLKGICS GGGGDPERVD RSSQRPGAED 300 Ii'fléNEIVSIL QPTQVPEQEM EVQEPAEPTG VNMLSPGESE HLLEPAEAER SQRRRLLVPA 360 :IgéDPTETLR QCFDDFADLV PFDSWEPLMR KLGLMDNEIK VAKAEAAGHR DTLYTMLIKW 4:11 ialliltTGRDAsv HTLLDALE'I'L GERLAKQKIE DHLLSSGKFM YLEGNADSAM S 1-55 Signal peptide 56-184 extracellular domain (potential) 57-94 CRDl 97-137 CRDZ 138-178 CRD3 213-202 transmembrane (potential) EEEEEEE 203-411 cytoplasmic (potential)

US. Patent Aug. 30, 2011 Sheet 11 0119 US 8,007,813 B2

Figure 11

6. TRAIL-R3

>splO14798|T1OC_HUMAN Tumor necrosis factor receptor superfamily member 10C precursor () (DcRl) (Decoy TRAIL receptor without death domain) (TNF- related apoptosis-inducing ligand receptor 3) (TRAIL receptor-3) (TRAIL-R3) (Trail receptor w

IZARIPKTLKF VWIVAVLLP VLAYSATTAR QEEVPQQTVA PQQQRHSFKG EECPAGSHIEQZ gljilTGACNPCT EGVDYTNASN NEPSCFPC'I'V CKSDQKHKSS CTMTRD'I'VCQ CKEGTFRiIgg :EZMCRKCSR CPSGEVQVSN CTSWDDIQCV EEFGANAEVE IPAAEETDNT SPGTPAP?g é‘i'éNTSPGTP APAAEETMTI‘.‘ SPG'I'PAPAAE ETMTTSPGTP APAAEETMT'I' SPGTPASZfI?’ 241 259 LSCTIVGIIV LIVLLIV'FV 1-23 Signal peptide 24-236 extracellular domain 29-66 CRD1 69-109 CRDZ 110-149 CRD3 162-236 5 x 15 AA tandem tape repeats 55555223 237-259 removed in mature form (potential)

US. Patent Aug. 30, 2011 Sheet 14 0f 19 US 8,007,813 B2

Figure 14

7. TRAIL-R4 >splQ9UBN6| T1OD_HUMAN Tumor necrosis factor receptor superfamily member 10D precursor ( ) (DcRz) (TNF-related apoptosis-inducing ligand receptor 4) (TRAIL receptor-4) (TRAIL-R4) (TRAIL receptor with a truncated death domain) - Homo sapiens

1 6O MGLWGQSVPT ASSARAGRYP GARTASGTRP WLLDPKILKF VVFIVAVLLP VRVDSATIPR 61 120 QDEVPQQTVA PQQQRRSLKE EECPAGSHRS EYTGACN'PCT EGVDYTIASN NLPSCLLCTV 121 180 CKSGQTNKSS CTTTRDTVCQ CEKGSFQDKN SPEMCRTCRT GCPRGMVKVS NCTPRSDIKC 181 240 gwggAAssTs KTPAAEETVLI: IILGMLAS_PY _I-_1_'YLIIIVVLV IILAWWGF SCRKKFISYL 241 300 KGICSGGGGG PERVHRVLFR RRSCPSRVPG AEDNARNETL SNRYLQPTQV SEQEIQGQEL 301 360 AELTGVTVES PEEPQRLLEQ AEAEGCQRRR LLV'PV'NDADS ADIS'ILLDAS ATLEEGHAKE 361 386 TIQDQLVGSE KLFYEEDEAG SATSCL AA 1-55 signal peptide AA 56-211 extracellular domain (potential) AA 58-97 CRD1 AA 98-139 CRDZ AA 140-180 CRD3 AA 212-232 transmembrane (potential) AA 233-386 cytoplasmic (potential) US. Patent Aug. 30, 2011 Sheet 15 0f 19 US 8,007,813 B2

Figure 15

Examples of Trail-Ré-Fc fusion proteins with overlapping amino acids:

Trail-R4 extracellular domain huIgGl

171 _ 211 99 120 NCTPRSDIKC KNESAASSTG KTPAAEETVT TILGMLASEY H 1 KSCDKTHTCP PCPAPELLGG

NCTPRSDIKC KNESAASSTG KTPAAEETVT TILGMLASH KSCDKTHTCP PCPAPELLGG

171 211 99 120 CTPRSDIKC KNESAASSTG K'I‘PAAEETVT TILGMLA‘FSVZPY H P K'STCDKTHTCP PCPAPELLGG NCTPRSDIKC IQIESAASSTG KTPAAEETVT TILGMLAECDKTHTCP PCPAPELLGG

17 l 2 1 1 9 9 12 O CTPRSD IKC KNESAAS STG K'I'PAAEETVT '{?ILGMLASPY H EP KSCDK?gl-ITCP PCPAPELLGG NCTPRSDIKC KNESAASSTG K'I'PAAEETVT EHTCP PCPAPELLGG

171 211 9 120 INCTPRSDIKC KNESAASSTG KTPAAEETVT TILGMLASPY EP KSCDKT'HTTCP PCPAPELLGG NCTPRSDIKC KN‘ESAASSTG KTPAAEETVT TILGMLASPY IEIZTCP PCPAPELLGG US. Patent Aug. 30, 2011 Sheet 16 0119 US 8,007,813 B2

Figure 16

l. TNF-Rl

>splP19438|TR1A__HUMAN Tumor necrosis factor receptor superfamily member 1A precursor (p60) (‘INF-R1) (‘INF-RI) (p55) (CD120a) [Containsz Tumor necrosis factor binding protein 1 (TBPI)] - Homo sapiens (Human) .

IJl-IGLSTV'PDLL LPLVLLELLV GIYPSGVIGL VPHLGDREKR DSVCPQGKYI HPQNNSICgg‘ IiéHKGTYLYN DCPGPGQDTD CRECESGSFT ASENHLRHCL SCSKCRKEMG ovElssc'i'xzzg gévcscm QYRHYWSENL FQCFNCSLCL NGTVHLSCQE KQNTVCTCHA GFFLRENégg égéncxxsm cTKLcLgQIg; nvggeggge; ZVLLPLVIFF GLCLLSLLFI (ELI/121211933112 ggrslvcex STPEKEGELE GTTTKPLAPN PSFSPTPGFT PTLGFSPVPS s'rr'rsss'i'gg ZZIISCPNFAAP RREVAPPYQG ADPILATALA SDPIPNPLQK WEDSAHKPQS LDTDDPA'igg ?gs/Emma WKEFVRRLGL SDHEIDRLEL QNGRCLREAQ YSMLATWRRR TPRREAT?Eg ?gévmpmm. LGCLEDIEEA LCGPAALPPA ps3; 1-21 Signal peptide 22-211 extracellular domain (potential) 43-82 CRDJ. 83-125 CRD2 126-166 CRD3 167-196 CRD4 212-234 transmembrane (potential) EEEEEEEE 235-455 cytoplasmic (potential)

US. Patent Aug. 30, 2011 Sheet 18 0f 19 US 8,007,813 B2

Figure 18

2. INF-R2

>sp|P20333|TR1B_HUMAN Tumor necrosis factor receptor superfamily member 13 precursor (Tumor necrosis factor receptor 2) (p80) (TNF-RZ) (p75) (CD120b) (Etanercept) [Contains: Tumor necrosis factor binding protein 2 (TBPII)] — Homo sapiens (Human).

?APVAVWAAL AvsLELwAAA HALPAQVAFT PYAPEPGSTC RLREYYDQTA QMCCSKCSgg SEAKVFCTKT SDTVCDSCED STYTQLWNWV PECLSCGSRC SSDQVETQAC TREQNRIéZg éiéWYCALSK QEGCRLCAPL RKCRPGFGVA RPGTETSDVV CKPCAPGTFS wrrssrnigg igércuvvAx PGNASMDAVC TSTSPTR§MA gGAvgLgQgv ggngggggpr PEPSTAQZEE gzigmsgggg AgcggspFAr PVGLIVGVTA LGLLIIGVVN CVIMTQVKKK PLCLQREigg :giPADKARG TQGPEQQHLL ITAPSSSSSS LESSASALDR RAPTRNQPQA PGVEASGigg igisressns SPGGHGTQVN VTCIVNVCSS SDHSSQCSSQ ASSTMGDTDS spsespxggg 421 461 VPFSKEECAF RSQLETPETL LGSTEEKPLP LGVPDAGMKP 5 1-22 Signal peptide 23-257 extracellular domain (potential) 39-76 CRDl 77-118 CRD2 119-162 CRD3 163-201 CRD4 258-287 transmembrane (potential) 555%?555 288-461 cytoplasmic (potential)