THI NA MTUUS009758582B2 MTUHULUMTOTTUK ( 12 ) United States Patent ( 10) Patent No. : US 9 ,758 , 582 B2 Govindappa et al. (45 ) Date of Patent: Sep. 12, 2017
( 54 ) ANTI- CTLA4 ANTIBODY - TGF - BRII CO7K 16 /32 (2013 . 01 ) ; GOIN 33/ 6854 TARGETED /IMMUNOMODULATORY ( 2013 . 01 ) ; A61K 38 /00 ( 2013 . 01 ) ; AIK FUSION PROTEINS 2039 /505 (2013 .01 ) ; CO7K 2317 / 14 ( 2013 . 01 ) ; CO7K 2317 /24 ( 2013 . 01 ) ; COZK 2317 / 31 ( 71 ) Applicant: BIOCON LIMITED , Bangalore ( IN ) ( 2013 .01 ) ; CO7K 2317 / 51 ( 2013 .01 ) ; CO7K 2317 /515 ( 2013 .01 ) ; CO7K 2317 / 732 Ê(72 ) Inventors: Nagaraj Govindappa , Karnataka (IN ) ; Kedarnath Sastry , Karnataka ( IN ) ; ( 2013 . 01 ) ; CO7K 2319 /00 ( 2013 .01 ) ; CO7K Maria Melina Soares , Karnatake ( IN ) 2319 /02 ( 2013. 01 ) ; CO7K 2319/ 33 (2013 .01 ) (58 ) Field of Classification Search ( 73 ) Assignee: BIOCON LIMITED ( IN ) None See application file for complete search history . ( * ) Notice : Subject to any disclaimer , the term of this patent is extended or adjusted under 35 U . S . C . 154 (b ) by 0 days . (56 ) References Cited (21 ) Appl . No. : 15 /145 , 135 U .S . PATENT DOCUMENTS ( 22 ) Filed : May 3 , 2016 7 , 141, 651 B2 11/ 2006 Gillies et al. (65 ) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2016 /0237155 A1 Aug. 18 , 2016 WO WO 98/ 33914 8 / 1998 wo WO 2009 /039409 3 /2009 Related U .S . Application Data WO WO 2009 / 114110 9 / 2009 WO WO 2011/ 109789 9 / 2011 (62 ) Division of application No. 14 /458 ,674 , filed on Aug . WO WO 2012033953 3 / 2012 13 , 2014 , now Pat . No . 9 , 340 ,617 , which is a division of application No . 13 /799 ,409 , filed on Mar . 13 , 2013 , OTHER PUBLICATIONS now Pat . No . 8, 815 ,247 . Birch , J . R . et al . “ Antibody production .” Advanced Drug Delivery ( 30 ) Foreign Application Priority Data Reviews , vol. 58 , No. 5 - 6 , Aug. 7 , 2006 . Carton et al. “ Codon engineering for improved antibody expression Apr. 30, 2012 ( IN ) . .. . 1689/ CHE /2012 in mammalian cells . " Protein Expression and Purification , Aca Apr. 30 , 2012 ( IN ) .. . 1690 /CHE / 2012 demic Press, vol. 55 , No. 2 , Sep . 8 , 2007 . Casi, Giulio et al. “ Antibody drug conjugates : Basic concepts , (51 ) Int. CI. examples and future perspectives .” Journal of Controlled Release , CO7K 16 /28 ( 2006 .01 ) vol. 161 , No. 2 , Jan . 10 , 2012 . A61K 38 / 10 ( 2006 . 01 ) Kalwy , S . et al. " Toward more efficient protein expression ." A61K 38 / 18 ( 2006 .01 ) Molecular Biotechnology, Humana Press , Inc. , vol. 34 , No . 2 , Sp . A61K 39 /395 ( 2006 .01 ) Iss. SI, Oct. 1 , 2006 . CO7K 14 /495 ( 2006 . 01 ) Kotsopoulou , E . et al. “ Optimised mammalian expression through CO7K 14 /00 ( 2006 . 01 ) the coupling of codon adaptation with gene amplification ; maxi CO7K 14765 ( 2006 . 01 ) mum yields with minimum effort. ” Journal of Biotechnology, vol . CO7K 14 /705 ( 2006 .01 ) 146 , No. 4 , Apr. 15 , 2010 . CO7K 16 /32 ( 2006 . 01 ) (Continued ) GOIN 33 /68 ( 2006 . 01 ) A61K 38 / 17 ( 2006 .01 ) A61K 45 / 06 ( 2006 .01 ) Primary Examiner - Elly -Gerald Stoica CO7K 7 / 08 ( 2006 .01 ) (74 ) Attorney , Agent, or Firm — Marianne Fuierer; Moore CO7K 14 /71 ( 2006 .01 ) & Van Allen , PLLC COZK 16 /30 ( 2006 . 01 ) A61K 39 /00 ( 2006 .01 ) A61K 38 /00 ( 2006 .01 ) (57 ) ABSTRACT (52 ) U . S . CI. CPC ...... CO7K 16 / 2818 ( 2013 . 01 ) ; A61K 38 / 10 The present invention relates generally to the field of gen (2013 .01 ) ; A61K 38 / 179 (2013 .01 ) ; A61K erating fusion proteins to be used in cancer therapy, and 39 /39558 ( 2013 .01 ) ; A61K 45 /06 ( 2013 .01 ) ; more specifically , to nucleotide sequences encoding the CO7K 7 /08 ( 2013 .01 ) ; CO7K 14 /00 (2013 . 01 ) ; fusion proteins, wherein the chimeric fusion proteins com CO7K 14 /495 ( 2013 . 01 ) ; CO7K 14 /65 prises at least one targeting moiety and at least one immu ( 2013 . 01 ) ; COZK 14 / 70532 ( 2013 .01 ) ; CO7K nomodulatory moiety that counteracts the immune tolerance 14 / 70596 (2013 .01 ) ; C07K 14 / 71 ( 2013 .01 ) ; of cancer cells . CO7K 16 / 2863 ( 2013 . 01 ) ; C07K 16 / 2866 ( 2013 .01 ) ; C07K 16 /2878 (2013 .01 ); CO7K 16 / 2887 ( 2013 .01 ) ; COZK 16 / 30 ( 2013 .01 ) ; 1 Claim , 65 Drawing Sheets US 9, 758 ,582 B2 Page 2
( 56 ) References Cited
OTHER PUBLICATIONS Ortiz - Sanchez, Elizabeth et al. “ Antibody cytokine fusion proteins : applications in cancer therapy. ” Expert Opinion on Biological Therapy, vol . 8 , No . 5 , May 1 , 2008 . Cocco , E . et al . “ hl- con1, a factor for VII- IgGFc chimeric protein targeting tissue factor for immunotherapy of uterine serous papillary carcinoma. ” Br. J . Cancer, 2010 , vol. 3 , is . 6 . pgs . 812 - 819 , the abstract , pp . 813 -818 . Office Action , issued by the Russian Patent Office for corresponding Russian Patent Application No . 2014145158 , issued on Feb . 14 , 2017 . Liu , H . et al. “ Heterogenecity ofmonoclonal antibodies .” Pharma ceutical Sciences , American Pharmaceutical Assoc . , 2007, vol. 97 , No . 7 , pgs. 2426 -447 . Office Action , issued by the European Patent Office for correspond ing European Patent Application No. 1373158 . 2 , issued on Jun . 6 , 2017 . U . S . Patent Sep . 12 , 2017 Sheet 1 of 65 US 9 , 758 ,582 B2
Anti- HER2 / neu - TGFBRII fusion protein at LC constant region Amino acid sequence of Anti- HER2 / neu heavy chain : EVQLVESGGGLVQPGGSLRLSCAASGENIKDTYIHWVRQAPGK GLEWARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSL RAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPS VEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCWWDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG Amino acid sequence of Anti -HER2 /neu light chain fusion protein : DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKA PKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPTFGQGTKVEIKRTVAAPSVEIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGG GSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCK FCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC SSDECNDNIIFSEEYNTSNPD
Figure 1 U . S . Patent Sep. 12 , 2017 Sheet 2 of 65 US 9 , 758, 582 B2
Anti -EGFR1 - TGFBRII fusion protein at LC constant region Amino acid sequence of Anti- EGFR1 heavy chain : QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPG KGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSL QSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG Amino acid sequence of Anti- EGFR1 light chain fusion protein : DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSP RLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQ NNNWPTTFGAGTKLELKRTVAAPSVEIFPPSDEQLKSGTASWC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGS GGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFC DVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITL ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSS DECNDNIIFSEEYNTSNPD Figure 2 U . S . Patent Sep . 12 , 2017 Sheet 3 of 65 US 9 , 758 ,582 B2
Anti- CTLA4 - TGFBRII fusion protein at LC constant region Amino acid sequence of anti- CTLA4 heavy chain : QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQAP GKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSWVTVPSSSLGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCWDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG Amino acid sequence of anti- CTLA4 light chain fusion protein : EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQ APRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY CQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECG GGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQ LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFM CSCSSDECNDNIIFSEEYNTSNPD Figure 3 U . S . Patent Sep. 12 , 2017 Sheet 4 of 65 US 9 ,758 ,582 B2
Anti- HER2 / neu HC - 4 - 1BB and LC - TGFBRII fusion protein : Amino acid sequence of heavy chain - 4 - 1BB fusion protein : EVQLVESGGGLVQPGGSLRLSCAASGENIKDTYIHWVRQAPGKGLEWA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW GGDGFYAMDYWGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCWDVSHEDPEVKENWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGGGGGSGGGGSGGGGSACPWAVSGARASPGSAASPRLREGPE LSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTG GLSYKEDTKELWAKAGVYYVFFQLELRRWAGEGSGSVSLALHLQPLR SAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHT EARARHAWQLTQGATVLGLFRVT PEIPAGLPSPRSE Amino acid sequence of light chain - TGFBRII fusion protein : DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTD NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVW RKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC SSDECNDNIIFSEEYNTSNPD
Figure 4 U . S . Patent Sep. 12 , 2017 Sheet 5 of 65 US 9 ,758 ,582 B2
Anti- EGFR1 HC - 4 - 1BB and LC - TGFBRII fusion protein : Amino acid sequence ofheavy chain - 4 - 1 BB fusion protein : QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWL GVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARA LTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCWWDVSHEDPEVKENWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGSGGGGSGGGGSACPWAVSGARASPGSAASPRLREGPEL SPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGG LSYKEDTKELWAKAGWYVFFQLELRRWAGEGSGSVSLALHLQPLRS AAGAAALALTVOLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTE ARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE Amino acid sequence of light chain - TGFBRIl fusion protein : DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYA SESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAG TKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDN NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR KNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCS SDECNDNIIFSEEYNTSNPD
Figure 5 U . S . Patent Sep. 12 , 2017 Sheet 6 of 65 US 9 ,758 ,582 B2
Anti- CTLA4 HC -4 - 1BB and LC - TGFBRII fusion protein : Amino acid sequence of heavy chain - 4 - 1BB fusion protein : QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEW VTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCA RTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCWDVSHEDPEVKENWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGSGGGGSGGGGSACPWAVSGARASPGSAASPRLREGPEL SPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGG LSYKEDTKELWAKAGWYYVFFQLELRRWAGEGSGSVSLALHLQPLRS AAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTE ARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE Amino acid sequence of light chain - TGFBRII fusion protein : EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIY GAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIV TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMC SCSSDECNDNIIFSEEYNTSNPD
Figure 6 U . S . Patent Sep . 12 , 2017 Sheet 7 of 65 US 9 , 758 ,582 B2
Anti -HER2 / neu HC - PD1 and LC - TGFBRIl fusion protein : Amino acid sequence of heavy chain -PD1 fusion protein : EVQLVESGGGLVQPGGSLRLSCAASGENIKDTYIHWVRQAPGKGLEWA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW GGDGFYAMDYWGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCWDVSHEDPEVKENWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSL SLSPGGGGGSGGGGSGGGGSPGWFLDSPDRPWNPPTFSPALLWTE GDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQD CRFRVTQLPNGRDFHMSWRARRNDSGTYLCGAISLAPKAQIKESL RAELRVTERRAEVPTAHPSPSPRPAGQFQTLV Amino acid sequence of light chain - TGFBRII fusion protein : DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKRTVAAPSVEIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTD NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVW RKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC SSDECNDNIIFSEEYNTSNPD
Figure 7 U . S . Patent Sep. 12 , 2017 Sheet 8 of 65 US 9 ,758 ,582 B2
Anti -EGFR1 HC -PD1 and LC - TGFBRII fusion protein : Amino acid sequence of heavy chain -PD1 fusion protein : QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWL GVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARA LTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCWWDVSHEDPEVKENWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGSGGGGSGGGGSPGWFLDSPDRPWNPPTFSPALLWTEG DNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDC RFRVTQLPNGRDFHMSWRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTERRAEVPTAHPSPSPRPAGQFQTLV Amino acid sequence of light chain - TGFBRII fusion protein : DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYA SESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAG TKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDN NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR KNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCS SDECNDNIIFSEEYNTSNPD
Figure 8 U . S . Patent Sep. 12 , 2017 Sheet 9 of 65 US 9 ,758 ,582 B2
Anti- CTLA4 HC -PD1 and LC - TGFBRII fusion protein : Amino acid sequence of heavy chain -PD1 fusion protein : QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEW VTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCA RTGWLGPFDYWGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCWDVSHEDPEVKENWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLS LSPGGGGGSGGGGSGGGGSPGWFLDSPDRPWNPPTFSPALLWTEG DNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDC RFRVTQLPNGRDFHMSWRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTERRAEVPTAHPSPSPRPAGQFQTLV Amino acid sequence of light chain - TGFBRII fusionprotein : EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIY GAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIV TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMC SCSSDECNDNIIFSEEYNTSNPD
Figure 9 U . S . Patent Sep. 12 , 2017 Sheet 10 of 65 US 9 ,758 ,582 B2
Anti -HER2 /neu HC - TGFBRII - 4 - 1BB fusion protein Amino acid sequence ofheavy chain -TGFBRII -4 - 1BB fusion protein : EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCWWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITL ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDN JIFSEEYNTSNPDEPKSCDKACPWAVSGARASPGSAASPRLREGPELSP DDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLS YKEDTKELVAKAGVYYVFFQLELRRWAGEGSGSVSLALHLQPLRSA AGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEA RARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE Amino acid sequence of light chain : DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKRTVAAPSVEIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC
Figure 10 U . S . Patent Sep. 12 , 2017 Sheet 11 of 65 US 9 ,758 ,582 B2
Anti- EGFR1 HC - TGFBRII- 4 - 1 BB fusion protein Amino acid sequence of heavy chain -TGFBRII - 4 - 1BB fusion protein : QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWL GVIWSGGNTDYNTPFTSRLSINKDNSKSQVEFKMNSLQSNDTAIYYCARA LTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQL CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDN SEEYNTSNPDEPKSCDKACPWAVSGARASPGSAASPRLREGPELSPD DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSY KEDTKELWAKAGVYYVFFQLELRRWAGEGSGSVSLALHLQPLRSAA GAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE Amino acid sequence of light chain : DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYA SESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAG TKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
Figure 11 U . S . Patent Sep. 12 , 2017 Sheet 12 of 65 US 9 ,758 ,582 B2
Anti -CTLA4 HC -TGFBRII -4 -1BB fusion protein Amino acid sequence of heavy chain - TGFBRII - 4 - 1BB fusion protein : QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWYRQAPGKGLEW VTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCA RTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCWDVSHEDPEVKENWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKGGGGSGGGGSGGGGSTIPPHVOKSVNNDMIVTDNNGAVKFPQL CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFEMCSCSSDECNDNUIF SEEYNTSNPDEPKSCDKACPWAVSGARASPGSAASPRLREGPELSPD DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSY KEDTKELWAKAGVYYVFFQLELRRWAGEGSGSVSLALHLQPLRSAA GAAALALTVOLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE Amino acid sequence of light chain : EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIY GAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC
Figure 12 U . S . Patent Sep . 12 , 2017 Sheet 13 of 65 US 9 ,758 ,582 B2
Anti- HER2/ neu HC - TGFBRII -PD1 fusion protein Amino acid sequence ofheavy chain - TGFBRII -PD1 fusion protein : EVQLVESGGGLVQPGGSLRLSCAASGENIKDTYIHWVRQAPGKGLEWVA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCWWDVSHEDPEVKENWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITL ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDN IIFSEEYNTSNPDEPKSCDKPGWFLDSPDRPWNPPTFSPALLWTEGD NATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRF RVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAEL RVTERRAEVPTAHPSPSPRPAGQFQTLV Amino acid sequence of light chain : DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC
Figure 13 U . S . Patent Sep . 12 , 2017 Sheet 14 of 65 US 9, 758 ,582 B2
Anti - EGFR1 HC - TGFBRII - PD1 fusion protein : Amino acid sequence of heavy chain - TGFBRII -PD1 fusion protein : QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWL GVIWSGGNTDYNTPFTSRLSINKDNSKSQVEFKMNSLQSNDTAIYYCARA LTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCWDVSHEDPEVKENWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLS LSPGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQL CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDEPKSCDKPGWFLDSPDRPWNPPTFSPALLWTEGONA TFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV TQLPNGRDFHMSWRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHPSPSPRPAGQFQTLV Amino acid sequence of light chain : DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYA SESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAG TKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
Figure 14 U . S . Patent Sep . 12 , 2017 Sheet 15 of 65 US 9 ,758 ,582 B2
Anti- CTLA4 HC - TGFBRII -PD1 fusion protein Amino acid sequence of heavy chain - TGFBRII -PD1 fusion protein : QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWYROAPGKGLEW VTEISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCA RTGWLGPFDYWGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCWDVSHEDPEVKENWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLS LSPGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQL CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDEPKSCDKPGWFLDSPDRPWNPPTFSPALLWTEGDNA TFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV TQLPNGRDFHMSWRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHPSPSPRPAGQFQTLV Amino acid sequence of light chain : EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIY GAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC
Figure 15 U . S . Patent Sep . 12 , 2017 Sheet 16 of 65 US 9 ,758 ,582 B2
Nucleotide sequence of Anti- HER2 /neu heavy chain constant region with linker : 1 gotagcacca agggcccctc cgtgttccct ctggccccct ccagcaagtc cacctctggc 61 ggcaccgccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 121 tggaactctg gcgctctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 181 ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcca gctctctggg cacccagacc 241 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggaaccc 301 aagtcctgcg acaagaccca cacctgtcccccctgccctg cccctgagct cctgggaggc 361 cctagcgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatctc ccggaccccc 421 gaagtgacct gcgtggtggt ggacgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 481 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacaac 541 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 601 gagtacaagt gcaaggtgtc caacaaggcc ctgcctgccc ccatcgaaaa gaccatctcc 661 aaggccaagg gccagccccg cgagcctcag gtgtacacoc tgccccctag ccgggaagag 721 atgaccaaga accaggtgtc cctgacctgt ctggtcaagg gottctaccc ctccgatatc 781 gccgtggaat gggagtccaa cggccagccc gagaacaact acaagaccac cccccctgtg 841 ctggactccg acggctcatt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 901 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 961 cagaagtccc tgtccctgag cccaggcaaa ggcggaggcg gatctggcgg cggaggatct 1021 ggtggcg gatcc
Nucleotide sequence of TGFBRII ECD : 1 ggatccacca tccccccaca cgtgcagaaa tccgtgaaca acgacatgat cgtgaccgac 61 aacaacggcg ctgtgaagtt cccccagctg tgcaagttct gcgacgtgcg gttctctacc 121 tgcgacaacc agaaatcctg catgtccaac tgctccatca cctccatctg cgagaagccc 181 caggaagtgt gcgtcgccgt ctggcggaag aacgacgaga acatcaccct ggaaaccgtg 241 tgccacgacc ccaagctgcc ctaccacgac ttcatcctgg aagatgccgc ctcccccaag 301 tgcatcatga aggaaaagaa gaagcccggc gagactttct tcatgtgcag ctgctcctcc 361 gacgagtgca acgacaacat catcttctcc gaagagtaca acacctccaa ccccgactga 421 agctt
Figure 16 U . S . Patent Sep . 12 , 2017 Sheet 17 of 65 US 9 ,758 ,582 B2
Nucleotide sequence of Anti- HER2 / neu heavy chain variable region i gcggccgcca tgaacttcgg cctgcggctg atcttcctgg tgctgaccct gaagggcgtg 61 cagtgcgagg tgcagctggt ggaatccggc ggaggcctgg tccagcctgg cggatctctg 121 agactgtcct gcgccgcctc cggcttcaac atcaaggaca cctacatcca ctgggtccga 181 caggcccctg gcaagggcct ggaatgggtg gcccggatct accccaccaa cggctacaco 241 agatacgccg actccgtgaa gggccggttc accatctccg ccgacacctc caagaacacc 301 gcctacctgc agatgaactc cctgcgggcc gaggacaccg ccgtgtacta ctgctccaga 361 tggggaggcg acggcttcta cgccatggac tactggggcc agggcaccct ggtcaccgtg 421 ctccgcta gc
Nucleotide sequence of Anti -HER2 /neu light chain variable region i gcggccgcca tggaatccca gacccaggtg ctgatctccc tgctgttctg ggtgtccggc 61 acctgtggcg acatccagat gacccagtcc ccctccagcc tgtccgcctc tgtgggcgac 121 agagtgacca tcacctgtcg ggcctcccag gacgtgaaca ccgccgtggc ctggtatcag 181 cagaagcccg gcaaggcccc caagctgctg atctactccg cctccttcct gtactccggc 241 gtgccctccc ggttctccgg ctctagatcc ggcaccgact ttaccctgac catctccagc 301 ctgcagcccg aggacttcgc cacctactac tgccagcagc actacaccac cccccccacc 361 tttggccagg gcaccaaggt ggaaatcaag cggaccgtgg ccgctccctc cgtgttcato 421 cccaccct ccgacgagca gctg
Nucleotide sequence of Anti- EGFR1 heavy chain constant region with linker : 1 gctagcacca agggcccctc cgtgtttccc ctggccccct ccagcaagtc cacctctggc 61 ggcaccgccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc A121 tggaactctg gcgctctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 181 ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcca gctctctggg cacccagacc 241 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagcg ggtggaaccc 301 aagtcctgcg acaagaccca cacctgtccc ccctgccctg cccctgaact gctgggaggc 361 ccttccgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatctc coggaccccc 421 gaagtgacct gcgtggtggt ggacgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 481 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacaac 541 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 601 gagtacaagt gcaaggtgtc caacaaggcc ctgcctgccc ccatcgaaaa gaccatctcc 661 aaggccaagg gccagccccg cgagcctcag gtgtacaccc tgcctcccag ccgggacgag 721 ctgaccaaga accaggtgtc cctgacctgt ctggtcaagg gottctaccc ctccgatatc 781 gccgtggaat gggagtccaa cggccagccc gagaacaact acaagaccac cccccctgtg 8418 ctggactccga c acggctcatt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 9019 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa??? ccactacacc 961 cagaagtccc tgtctctgag ccccggcaaa ggcggcggag gatctggcgg tggcggatca 1021 ggcggag gatcc Figure 17 U . S . Patent Sep . 12 , 2017 Sheet 18 of 65 US 9 ,758 ,582 B2
Nucleotide sequence of Anti - EGFR1 heavy chain variable region 1 goggccgcca tgaacttcgg cctgcggctg atcttcctgg tgctgaccct gaagggcgtg 61 cagtgccagg tgcagctgaa gcagtccgga cctggcctgg tgcagccttc ccagtccctg 121 tccatcacct gtaccgtgtc cggcttctcc ctgaccaact acggcgtgca ctgggtccga 181 cagtccccag gcaagggcct ggaatggctg ggagtgattt ggagcggcgg0 caacaccgac
0 0 ???????? ccttcacc t > accaa a c 301 ttcttottcttcaaga tgaactccct10 ctc c gcagtccaac gacaccgccaa totactctactactg cgccagagccc 361 ctgacctact atgactacga gttcgcctac tggggacagg gcaccctggt caccgtgtct 421 cgctagc
Nucleotide sequence of Anti- EGFR1light chain variable region 1 goggccgcca tggaatccca gacccaggtg ctgatctccc tgctgttctg ggtgtccggc ? 61 acctgtggcg acatcctgct gacccagtcc cccgtgatcc tgtccgtgtc tcctggcgag ??121 cgggtgtcct tctcctgccg ggcctcccag tocatcggca ccaacatcca ctggtatcag 181 cagcggacca acggctcccc tcggctgctg attaagtacg cctccgagtc tatctccggc
?241 atcccctccc ggttctccgg ctctggotcc ggcaccgact tcaccctgtc catcaactcc ?????301 gtggaatccg aggatatcgc cgactactac toccagcaga acaacaactg gcccaccacc 361 ttcggcgctg gcaccaagctc ggaactgaag cggaccgtgg ccgctccctcccctc cgtgttcatctcatc ??421 cccaccct ccgacgagca gctg Nucleotide sequence of Anti -CTLA4 heavy chain variable region 1 gcggccgcca tgaacttcgg cctgcggctg atcttcctgg tgctgaccct gaagggcgtg 61 cagtgccagg tgcagctggt ggaatccggc ggaggcgtgg tgcagcctgg cagatccctg 121 agactgtcctD gcgccgcctc cggcttcacc ttctccagct??? acaccatgcaCacc ctgggtccga 181 caggcccctg gcaagggcct ggaatgggtc accttcatca gctacgacgg caacaacaag 241 tactacgccg actccgtgaa gggccggttc accatctcccatoto gggacaactcg a c aactc caagaacaco 301 ctgtacctgc agatgaactc cctgcgggcc gaggacaccg ccatctacta ctgcgcccgg 361 accggctggc tgggcccttt tgattactgg ggccagggca ccctggtcac cgtgtcctcc 421 tagc Nucleotide sequence of Anti- CTLA4light chain variable region 1 geggccgcca tggaatccca gacccaggtg ctgatctccc tgctgttctg ggtgtccggc 61 acctgtggcg agatcgtgct gacccagtcc cccggcaccc tgtctctgag ccctggcgag 121 agagccaccc tgtcctgcag agcctcccag tccgtgggct cctcctacct ggcttggtat 181 cagcagaagc ccggccaggc ccctcggctg ctgatctacg gcgctttctc tcgggccacc 241 ggcatccctg accggttctc tggctccggc tccggcaccg acttcaccct gaccatctcc 301 cggctggaac ccgaggactt cgccgtgtac tactgccagc agtacggctc ctccccctgg 361 acctttggcc agggcaccaa ggtggaaatc aagcggaccg tggccgctcc ctccgtgttc
421N cttcccac cctccgacga gcagctg
Figure 18 U . S . Patent Sep. 12 , 2017 Sheet 19 of 65 US 9 ,758 ,582 B2
Nucleotide sequence of Anti CD20 IgG1 molecule ;
1 gotagcacaa0 agggccctag tgtgtttcct ctggctccct cttccaaatc cacttctggt 61 ggcactgctg0 ctctgggatg cctggtgaag gattactttc ctgaacctgt gactgtctca 121 tggaactctg0 gtgctctgac ttctggtgtc cacactttcc ctgctgtgct gcagtctagt 181 ggactgtact ctctgtcatc tgtggtcact gtgccctctt catctctggg aacccagacc 241 tacatttgta atgtgaacca caaaccatcc aacactaaag tggacaaaaa agccgaaccc
0 the aaats ala 0 CA t t a ct ggaggaa 361 ccttctgtgt ttctgttccc accaaaacca aaagataccc tgatgatctc tagaacccct 421 gaggtgacat gtgtggtggt ggatgtgtct catgaggacc ctgaggtcaa atttaattgg 481 tacgtcgatg gagtggaagt ccacaatgcc aaaaccaagc ctagagagga acagtacaat 541 tcaacctaca gagtcgtcag tgtgctgact gtgctgcatc aggattggct gaatggcaag 601 gaatacaagt gtaaagtctc aaacaaggcc ctgcctgctc caattgagaa aacaatctca 661 aaggccaagg gacagcctag ggaaccccaggaaccc gtctacacccro tgccaccttc0 Cac ??? acgcgacgaa 721 ctgaccaaaaaa acaccaggtgtc cctgacatgc ctggtcaaagcaaag gettctaccctctaccc ttctgacattt 781 gctgtggagtg gggagtcaaart a tggacagcctc gagaacaactcaa acaaaacaacacaac cccccctgtg 841 ctggattctg atggctcttt ctttctgtac tccaaactga ctgtggacaa0 gtctagatgg 901 cagcagggga atgtcttttc ttgctctgtc atgcatgagg ctctgcataa ccactacact 961 cagaaatccc tgtctctgtc tcccgggaaa ggcggcggag gatctggcgg aggcggttct 1021 ggtggtggcg gatec Nucleotide sequence of Anti -CD20 heavy chain variable region 1 goggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 61 cagtgtcagg tgcagctgca gcagcctggt gccgagctcg tgaaacctgg cgcctccgtg 121 aagatgtcct gcaaggcctc cggctacacc ttcaccagct acaacatgca ctgggtcaag 181 cagacccccg gcagaggcct ggaatggatc ggcgctatct accccggcaa cggcgacaco 241 tcctacaacc agaagttcaa gggcaaggcc accctgaccg ccgacaagtc ctcttccacc 301 gcctacatgc agctgtcctc cctgacctcc gaggactccg ccgtgtacta ctgcgcccgg 361 tctacctact acggcggcga ctggtacttc aacgtgtggg gcgctggcac caccgtgacc 421 gtgtctgctg ctagc
Nucleotide sequence of Anti - CD20 light chain variable region
1 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct0 gaaaggcgtc 61 cagtgtcaga togtgctgtc ccagtcccct gccatcctgt ctgctagccc tggcgagaaa 121 gtgacaatgat cctgccgggcO O gggC ctcctcctcc gtgtcctaca tocactggtt ccagcagaag 181 cccggctccaa gccccaagcca ttggatctac gccacctccaa acctggcctc tggcgtgcca 241 gtgcggtttt ccggctctgg ctctggcacc tcctactccc tgaccatctc tcgggtggaa 301 gccgaggatg ccgccacctacta ctactgccag cagtggacca gcaaccccccaccCCCC cacattcacatttggc 361 ggaggcacca agctggaaat caagcggacc gtggcggcgc cctct
Figure 19 U . S . Patent Sep. 12 , 2017 Sheet 20 of 65 US 9 ,758 ,582 B2
Nucleotide sequence of 4 - 1 BB .
formend ggatccgcct gtccttgggc cgtgtccggc gctagagcct ctcctggctc tgccgcctcc 61 cccagactgaro gagagggccc tgagctgtcc cctgacgatc ctgccggcct gctggacctg 121 agacagggca tgtttgccca gctggtggcc cagaacgtgca tgctgatcga cggccccctg 181 tcctggtact ctgatcctgg cctggccggc gtgtccctga ccggcggact ogtcctacaaa 241 gaggacaccaa a aagaactggt ggtggccaag gctggcgtgt actacgtgtt ctttcagctg 301 gaactgcggcc gggtggtggc cggcgagggc tctggatctg tgtccctggc cctgcatctg cagcccctga gatctgccgc tggcgccgct gctctggccc tgacagtgga tctgcctcct 421 gcctcctccg aggcccggaa ctccgcattc gggtttcagg gccggctgct gcacctgtct 481 gctggccaga gactgggagt gcatctgcac accgaggcca gagccagaca cgcctggcag 541 ctgacccagg gcgctaccgt gctgggcctg ttcagagtga cccccgagat cccagccggc 601 ctgcccagcc ctagatccga gtgataagct t
Nucleotide sequence of Anti- ILOR heavy chain :
1 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccctO gaaaggcgtc 61 cagtgtcagg tgcagctgca ggaatctggc cctggactcg tgcggccttc ccaaaccctg 121 tctctgacct gtaccgtgtc cggctactccc atcacctccgc accacgcctggtcttgggtg0 o 181 cgacagcctc ctggcagagg0 cctggaatgg atcggctaca tctcctacto cggcatcacc
241 acctacaacc ccagcctgaa0 gtccagagtg accatgctgc gggacacctc caagaaccag 301 ttctccctgc ggctgtcctc cgtgaccgct gctgataccg ccgtgtacta ctgcgccaga 361 oftctctggcca ggaccaccgc0 catggattac tggggccagg gctccctcgt gaccgtgtcc
0 0 0 421 tctgctagca ccaagggccc ctccgtgttc( cctctggcccLO cttcctctaa atctacctct
481 ggcggcaccg ccgctctggg ctgcctcgtg aaggactact tccccgagcc? cgtgacagtg 541 tcttggaact ctggcgccct gacctccggc gtgcacacct0 ttccagctgt gctgcagtcc 601 tccggcctgt actccctgtc cagcgtcgtg actgtgccctODVP cctcatctct gggcacccag
0 661 acctacatct gcaacgtgaa ccacaagccc tccaacacca0 aggtggacaa gaaggtggaaa 0 0 721 cccaagtcct0 gcgacaagac ccacacctgt0 cccccttgtc ctgcccctga actgctgggc 781 ggaccctctg tgttcctgtt cccaccaaaa ccgaaagaca ccctgatgat ctcccggacc 841 cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 901 tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 961 aactccacct accgggtggt gtccgtgctg accgtgctgc accaggattg gctgaacggc 1021 aaagagtaca agtgcaaggt gtccaacaag gocctgcctgU cccccatcga aaagaccato 1081 tccaaggcca agggccagcc acgggaaccc caggtgtaca0 cactgccccc tagccgcgac 1141 gagctgacca agaatcaggt gtccctgaca tgcctcgtga aaggettcta cccctccgat 1201 r0307atcgccgtgg aatgggagtc caacggccag cctgagaacart actacaagac caccccccct ) 1261 gtgctggactt ccgacggctc attoattcttcctg tottactcaaagc 0 tgacagtgga1. caagtcccggc 1321 tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1381 acccagaagt ccctgtccct gagccccggg aaaggcggcg gaggatctgg cggaggcggt 1441 tctggtggtg goggatec Figure 20 U . S . Patent Sep. 12 , 2017 Sheet 21 of 65 US 9 , 758 ,582 B2
Nucleotide sequence of Anti- ILOR light chain variable region :
1 goggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtcO 61 cagtgtgaca tccagatgac ccagtccccc tccagcctgt ctgcctctgt gggcgacaga 121 gtgaccatca cctgtcgggc0 ctcccaggac atctcctcct acctgaactg gtatcagcag 181 aagcccggca aggcccccaa gctgctgatc tactacacct cccggctgca ctccggcgtg 241 ccctctagatt ttttttccggctc tggctccggc accgactttat t ccttcoccttcaccat cagctccctgc 301 cagcccgagg atatcgccac ctactactgc cagcaaggcaa acaccctgcc ctacaccttt 361 ggccagggca ccaaggtgga aatcaagcgg accgtggcgg cgccc
Nucleotide sequence of Anti- 4 - 1BB heavy chain 1 goggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 61 cagtgtcagg tgcagctgca gcagtgggga gctggactgc tgaagccctc cgagacactg 121 tctctgacct gcgctgtgta0 cggcggctcc ttctccggct actactggtc ctggattcgg 181 cagtcccctg agaagggcct ggaatggatc ggcgagatca accacggcgg ctacgtgacc 241 tacaacccca gcctggaatc cagagtgacc atctccgtgg acacctccaa gaaccagttc 301 tccctgaagc tgtcctccgt gaccgccgct gataccgccg tgtactactg cgccagagac 361 tacggccctg gcaactacga ctggtacttc gacctgtggg gcagaggcac cctcgtgacc 421 gtgtcctctg ctagcaccaa gggcccctcc gtgtttcctc tggccccttg ctcacgctcc 481 acctccgaat ctaccgccgc tctgggctgc ctcgtgaagg actacttccc cgagcccgtg 541 actgtgtctt ggaactctgg cgccctgacc tccggcgtgc acacctttcc agctgtgctg 601 cagtcctccg gcctgtactc cctgtccagc gtcgtgacag tgccctccag ctctctgggc 661 accaagacct acacctgtaaa cgtggaccac aagccctcca acaccaaggt ggacaagcgg 721 gtggaatctac aatacggccc tccctgccct ccttgcccag cccctgaatta tctgggcgga 781 ccttccgtgt tcctgttccccccaaaaccc aaggacacoc tgatgatctc ccggaccccc 841 gaagtgacct gcgtggtggt ggatgtgtcc caggaagatc ccgaggtgca gttcaattgg 901 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ctagagagga acagttcaac 961 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggattggct gaacggcaaa 1021 gagtacaagt gcaaggtgtc caacaagggc ctgcccagct ccatcgaaaa gaccatcago 1081 aaggccaagg gccagccccgggaaccccag gtgtacacac tgcctccaag ccaggaagag 1141 atgaccaagag atcaggtgtc cctgacctgt ctcgtgaaag gettctaccctet ctccgatatct 1201 gccgtggaat gggagtccaa cggccagcct gagaacaact acaagaccac cccccctgtgt 1261 ctggactccg acggcagctt cttcctgtac tctcgcctga ccgtggacaa gtcccggtgg 1321 caggaaggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 1381 cagaagtccc tgtccctgtc tctggggaaa ggcggcggag gatctggcgg aggcggttct 1441 ggtggtggcg gatec Nucleotide sequence of Anti- 4 - 1BB light chain variable region I gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 61 cagtgtgaga tcgtgctgac ccagtctcct gccaccctgt ctctgagccc tggcgagaga 121 gctaccctgt cctgccgtgc ctcccaatcc gtgtcctctt acctggcctg gtatcagcaa 181 aagcccggcc aggctccccg gctgctgatc tacgatgcct ccaatagagc caccggcatc 241 cctgccagat tctccggctc tggctctggc accgacttta ccctgaccat ctcctctctg 301 gaacccgagg acttcgccgt gtactactgc cagcagcggt ccaactggcc tcccgccctg 361 acatttggcg gaggcaccaa ggtggaaatc aagcggaccg tggcggcgcc c
Figure 21 U . S . Patent Sep . 12 ,2017 Sheet 22 of 65 US 9, 758 ,582 B2
AYA Reduced Non -Reduced
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* * www . ww 4SHARAN w . wwwxWX .wasxx USKIEROWA U . S . Patent Sep. 12 , 2017 Sheet 46 of 65 US 9 ,758 ,582 B2
Cantuzumab - TGFBRII fusion protein at LC constant region Amino acid sequence of Cantuzumab heavy chain :
QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWI DTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFD VWGQGTTVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTOKSLSLSPGK Amino acid sequence of Cantuzumab light chain fusion protein : DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYR MSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGTKLE LKRTVAAPSVEIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC GGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCK FCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC SSDECNDNIIFSEEYNTSNPD
Figure 46 U . S . Patent Sep. 12 , 2017 Sheet 47 of 65 US 9 ,758 ,582 B2
Cixutumumab - TGFBRII fusion protein at LC constant region Amino acid sequence of Cixutumumab heavy chain : EVOLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPI FGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLEWSTQ DHYYYYYMDVWGKGTTVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC WDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of Cixutumumab light chain fusion protein : SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWYQQKPGQAPILVIYGENKRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKLTVLG QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECSGGGG SGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCD VRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPD
Figure 47 U . S . Patent Sep. 12 , 2017 Sheet 48 of 65 US 9 ,758 ,582 B2
Clivatuzumab - TGFBRII fusion protein at LC constant region Amino acid sequence of Clivatuzumab heavy chain : QVQLQQSGAEVKKFGASVKVSCEASGYTEPSYVLHWVKQAPGQGLEWIGYINP YNDGTQTNKKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYGFA YNGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVNTEPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTCVVVDVSHEDP EVKENWYVDGVEVHNAKTKPREEQYNSTYRVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVNHEAL HNHYTOKSLSLSPGK Amino acid sequence of Clivatuzumab light chain fusion protein : DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIYSTSNL ASGVPARFSGSGSGTDFTLTISSLOPEDSASYFCHQWNRYPYTFGGGTRLEIKRT VAAPSVFIFPPSDEQLKSGTASWCLLNNFYEAKVQWKVDNALOSGNSQESVTE QDSKDSTYSLSSTLTLSPRKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGG SGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCD VRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPD
Figure 48 U . S . Patent Sep. 12 , 2017 Sheet 49 of 65 US 9 ,758 ,582 B2
Pritumumab - TGFBRII fusion protein at LC constant region Amino acid sequence of Pritumumab heavy chain : EVQLLESGGDLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAITP SGGSTNYADSVKGRFTISRDNSQNTLYLQMNSLRVEDTAVYICGRVPYRSTWYP LYWGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWWDVSH EDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPOVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK Amino acid sequence of Pritumumab light chain fusion protein : DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQKPGKAPKSLIYAASSLH SKVPTQFSGSGSGTDFTLTISSLQPEDFATYYCLQYSTYPITFGGGTKVEIKRTVAA PSVEIFPPSDEQLKSGTASWVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGG GGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN DNIIFSEEYNTSNPD
Figure 49 U . S . Patent Sep. 12 , 2017 Sheet 50 of 65 US 9 ,758 ,582 B2
Cantuzumab HC - 4 -1BB and LC -TGFBRII fusion protein Amino acid sequence of heavy chain - 4 - 1BB fusion protein : QVQLVQSGAEVKKPGETVKISCKASDYTETYYGMNWVKQAPGQGLKWMGWI DTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFD WWGQGTTVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTEPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWWDVSHE DPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSACPWAVSGARASPGS AASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLID GPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQL ELRRWAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPAS SEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT QGATVLGLFRVTPEIPAGLPSPRSE Amino acid sequence of Cantuzumab light chain fusion protein : DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYR MSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGTKLE LKRTVAAPSVEIEPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCK FCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC SSDECNDNJIFSEEYNTSNPD Figure 50 U . S . Patent Sep . 12 , 2017 Sheet 51 of 65 US 9 ,758 ,582 B2
Cixutumumab HC- 4 -1BB and LC -TGFBRII fusion protein Amino acid sequence of heavy chain - 4 - 1BB fusion protein : EVOLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPI FGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLEWSTQ DHYYYYYMDWWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTEPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC WDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VESCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSACPWAVSG ARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVA QNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELWAKAG VYVFFQLELRRWAGEGSGSVSLALHLQPLRSAAGAAALAL TVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARA RHAWQLTQGATVLGLFRVT PEIPAGLPSPRSE Amino acid sequence of Cixutumumab light chain fusion protein : SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWYQQKPGQAPILVIYGENKRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKLTVLG QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECSGGGG SGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCD VRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPD Figure 51 U . S . Patent Sep . 12 , 2017 Sheet 52 of 65 US 9 ,758 ,582 B2
Clivatuzumab HC- 4 - 1BB and LC - TGFBRII fusion protein Amino acid sequence ofheavy chain - 4 - 1BB fusion protein QVQLQQSGAEVKKFGASVKVSCEASGYTEPSYVLHWVKQAPGQGLEWIGYINP YNDGTQTNKKEKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYGFA YNGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVNTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTCVWDVSHEDP EVKENWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVNHEAL HNHYTQKSLSLSPGKGGGGSGGGGSGGGGSACPWAVSGARASPGSAA SPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPL SWYSDPGLAGVSLTGGLSYKEDTKELWAKAGVYYVFFQLELR RWAGEGSGSVSLALHLQPLRSAAGAAALALTVOLPPASSEA RNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQG ATVLGLFRVT PEIPAGLPSPRSE Amino acid sequence of Clivatuzumab light chain fusion protein : DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIYSTSNL ASGVPARFSGSGSGTDFTLTISSLQPEDSASYFCHQWNRYPYTFGGGTRLEIKRT VAAPSVEIFPPSDEQLKSGTASWCLLNNFYEAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSPRKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGG SGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCD VRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPD Figure 52 U . S . Patent Sep. 12 , 2017 Sheet 53 of 65 US 9 ,758 ,582 B2
Pritumumab HC -4 -1BB and LC- TGFBRII fusion protein Amino acid sequence of heavy chain - 4 - 1BB fusion protein : EVQLLESGGDLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAITP SGGSTNYADSVKGRFTISRDNSQNTLYLQMNSLRVEDTAVYICGRVPYRSTWYP LYWGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWDVSH EDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRWYSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSACPWAVSGARASPGS AASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLID GPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQL ELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPAS SEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT QGATVLGLFRVTPEIPAGLPSPRSE Amino acid sequence of Pritumumab light chain fusion protein : DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQKPGKAPKSLIYAASSLH SKVPTQFSGSGSGTDFTLTISSLQPEDFATYYCLQYSTYPITFGGGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGGGGSGG GGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN DNIIFSEEYNTSNPD Figure 53 U . S . Patent Sep . 12 , 2017 Sheet 54 of 65 US 9 ,758 ,582 B2
Cantuzumab - HC- PD1 and LC- TGFBRII fusion protein Amino acid sequence of heavy chain -PD1 fusion protein : QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWI DTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFD WWGQGTTVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTEPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWWDVSHE DPEVKENWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSPGWFLDSPDRPWNPPT FSPALLWTEGONATFTCSFSNTSESFVLNWYRMSPSNQTOKLAAFPE DRSQPGQDCRFRVTQLPNGRDFHMSWRARRNDSGTYLCGAISLA PKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV Amino acid sequence of Cantuzumab light chain fusion protein : DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYR MSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGTKLE LKRTVAAPSVEIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCK FCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC SSDECNDNIIFSEEYNTSNPD
Figure 54 U . S . Patent Sep. 12 , 2017 Sheet 55 of 65 US 9 ,758 ,582 B2
Cixutumumab HC- PD1 and LC - TGFBRII fusion protein Amino acid sequence of heavy chain - PD1 fusion protein : EVOLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPI FGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLEWSTQ DHYYYYYMDVWGKGTTVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC WWDVSHEDPEVKENWYDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSEFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSPGWFLDSPD RPWNPPTFSPALLWTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT DKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSWRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTL
Amino acid sequence of Cixutumumab light chain fusion protein : SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWYQQKPGQAPILVIYGENKRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKLTVLG QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECSGGGG SGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCD VRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPD
Figure 55 U . S . Patent Sep . 12, 2017 Sheet 56 of 65 US 9 ,758 ,582 B2
Clivatuzumab HC -PD1 and LC - TGFBRII - fusion protein Amino acid Amino acid sequence of heavy chain -PD1 fusion protein : QVQLQQSGAEVKKFGASVKVSCEASGYTFPSYVLHWVKQAPGQGLEWIGYINP YNDGTQTNKKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYGFA YNGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVNTEPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTCWWDVSHEDP EVKENWYVDGVEVHNAKTKPREEQYNSTYRVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVNHEAL HNHYTQKSLSLSPGKGGGGSGGGGSGGGGSPGWFLDSPDRPWNPPTFSP ALLWTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRS QPGQDCRFRVTQLPNGRDFHMSWRARRNDSGTYLCGAISLAPKA QIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV Amino acid sequence of Clivatuzumab light chain fusion protein : DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIYSTSNL ASGVPARFSGSGSGTDFTLTISSLQPEDSASYFCHQWNRYPYTFGGGTRLEIKRT VAAPSVEIFPPSDEQLKSGTASWCLLNNFYEAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSPRKADYEKHKVYACEVTHQGLSSPVTKSENRGECGGGG SGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCD VRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFÉMCSCSSD ECNDNIIFSEEYNTSNPD
Figure 56 U . S . Patent Sep . 12 , 2017 Sheet 57 of 65 US 9 ,758 ,582 B2
Pritumumab HC- PD1 and LC - TGEBRII fusion protein Amino acid Amino acid sequence of heavy chain - PD1 fusion protein : EVQLLESGGDLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAITP SGGSTNYADSVKGRFTISRDNSQNTLYLQMNSLRVEDTAVYICGRVPYRSTWYP LYWGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTEPAVLQSSGLYSLSSWVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVVDVSH EDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSPGWFLDSPDRPWNPPT FSPALLWTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPE DRSQPGQDCRFRVTQLPNGRDFHMSVRARRNDSGTYLCGAISLA PKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV Amino acid sequence of Pritumumab light chain fusion protein : .
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQKPGKAPKSLIYAASSLH SKVPTQFSGSGSGTDFTLTISSLOPEDFATYYCLQYSTYPITFGGGTKVEIKRTVAA PSVEIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGG GGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVC HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN DNIIFSEEYNTSNPD Figure 57 U . S . Patent Sep. 12 , 2017 Sheet 58 of 65 US 9 ,758 ,582 B2
Cantuzumab HC- TGFBRII- 4 - 1BB fusion protein Amino acid sequence of heavy chain - TGEBRII- 4 - 1BB fusion protein : QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWI DTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFD VWGQGTTVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWWDVSHE DPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKO KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIV TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQ EVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepkscdkAC PWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMF AQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELV VAKAGVYYVFFQLELRRWAGEGSGSVSLALHLQPLRSAAGA AALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLH TEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE Amino acid sequence of light chain DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYR MSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGTKLE LKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC Figure 58 U . S . Patent Sep . 12 , 2017 Sheet 59 of 65 US 9 ,758 ,582 B2
Cixutumumab HC - TGFBRII - 4 - 1BB fusion protein Amino acid sequence of heavy chain - TGEBRIT - 4 - 1BB fusion protein : EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPI FGTANYAOKFOGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLEWSTO DHYYYYYMDVWGKGTTVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTEPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC WDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI SPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepTSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAA kscdkACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLD LRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELWAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQ RLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSP RSE Amino acid sequence of light chain : SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWYQQKPGQAPILVIYGENKRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKLTVLG QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS Figure 59 U . S . Patent Sep. 12 , 2017 Sheet 60 of 65 US 9 ,758 ,582 B2
Clivatuzumab HC - TGEBRII -4 - 1BB fusion protein Amino acid sequence of heavy chain - TGEBRIT - 4 - 1BB fusion protein : QVQLQQSGAEVKKFGASVKVSCEASGYTFPSYVLHWVKQAPGQGLEWIGYINP YNDGTQTNKKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYGFA YNGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVNTEPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTCVVVDVSHEDP EVKENWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVNHEAL HNHYTOKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTD NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV CVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK KKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepkscdkACPW AVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQ LVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVA KAGVYYVFFQLELRRWAGEGSGSVSLALHLQPLRSAAGAAA LALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTE ARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE Amino acid sequence of light chain : DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIYSTSNL ASGVPARFSGSGSGTDFTLTISSLOPEDSASYFCHQWNRYPYTFGGGTRLEIKRT VAAPSVEIFPPSDEQLKSGTASWCLLNNFYEAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSPRKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Figure 60 U . S . Patent Sep. 12 , 2017 Sheet 61 of 65 US 9 ,758 ,582 B2
Pritumumab HC -TGFBRII - 4 - 1BB fusion protein Amino acid sequence of heavy chain - TGEBRI| - 4 - 1BB fusion protein : EVQLLESGGDLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAITP SGGSTNYADSVKGRFTISRDNSQNTLYLQMNSLRVEDTAVYICGRVPYRSTWYP LYWGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWWVDVSH EDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIV TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQ EVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepkscdkAC PWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMF AQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELV VAKAGVYYVFFQLELRRWAGEGSGSVSLALHLQPLRSAAGA AALALTVOLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLH TEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE Amino acid sequence of light chain : DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQKPGKAPKSLIYAASSLH SKVPTQFSGSGSGTDFTLTISSLQPEDFATYYCLQYSTYPITFGGGTKVEIKRTVAA PSVEIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Figure 61 U . S . Patent Sep. 12 , 2017 Sheet 62 of 65 US 9 ,758 ,582 B2
Cantuzumab HC - TGFBRII- PD1 fusion protein Amino acid sequence of heavy chain - TGFBRII - PD1 fusion protein : QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWI DTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLK?EDTATYFCARRGPYNWYFD WWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTEPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKENWYVDGVEVHNAKTKPREEQYNSTYRWVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIV TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQ EVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepkscdkPG WFLDSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSESFVLNWYR MSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSWRAR RNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRP AGQFQTLV Amino acid sequence of light chain : DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYR MSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGTKLE LKRTVAAPSVEIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC Figure 62 U . S . Patent Sep. 12 , 2017 Sheet 63 of 65 US 9 ,758 ,582 B2
Cixutumumab HC -TGFBRII - PD1 fusion protein Amino acid sequence of heavy chain -TGEBRII - PD1 fusion protein : EVOLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIPI FGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLEWSTQ DHYYYYYMDVWGKGTTVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC WVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAA SPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDep kscdkPGWFLDSPDRPWNPPTFSPALLWTEGONATFTCSFSNTSESF VLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHM SWRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAH PSPSPRPAGQFQTLV Amino acid sequence of light chain : SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWYQQKPGQAPILVIYGENKRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKLTVLG QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS
Figure 63 U . S . Patent Sep. 12 , 2017 Sheet 64 of 65 US 9 ,758 ,582 B2
Clivatuzumab HC - TGFBRII- PD1 fusion protein Amino acid sequence of heavy chain - TGFBRII -PD1 fusion protein : KVSCEASGYTEPSYVLHWVKQAPGQGLEWIGYINP YNDGTQTNKKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYGFA YNGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVNTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTCWVVDVSHEDP EVKENWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVNHEAL HNHYTQKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTD NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV CVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK KKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepkscdkPGWFL DSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSESFVLNWYRMSPS NQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSWRARRND SGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQ FQTLV Amino acid sequence of light chain : DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIYSTSNL ASGVPARFSGSGSGTDFTLTISSLQPEDSASYFCHQWNRYPYTFGGGTRLEIKRT VAAPSVEIFPPSDEQLKSGTASWCLLNNFYEAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSPRKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Figure 64 U . S . Patent Sep. 12 , 2017 Sheet 65 of 65 US 9 ,758 ,582 B2
Pritumumab HC - TGEBRII- PD1 fusion protein Amino acid sequence of heavy chain -TGEBRII -PD1 fusion protein : EVQLLESGGDLVOPGGSLRLSCAASGETFSNYAMSWVRQAPGKGLEWVSAITP SGGSTNYADSVKGRFTISRDN?QNTLYLQMNSLRVEDTAVYICGRVPYRSTWYP LYWGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLOSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWWDVSH EDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIV TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQ EVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepkscdkPG WFLDSPDRPWNPPTFSPALLVTEGDNATFTCSFSNTSESFVLNWYR MSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSWRAR RNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRP AGQFQTLV Amino acid sequence of light chain :
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQKPGKAPKSLIYAASSLH SKVPTQFSGSGSGTDFTLTISSLQPEDFATYYCLQYSTYPITFGGGTKVEIKRTVAA PSVEIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Figure 65 US 9 ,758 , 582 B2 ANTI -CTLA4 ANTIBODY - TGF- BRII moiety that counteracts immune tolerance of cancer cell , TARGETED / IMMUNOMODULATORY wherein the targeting moiety and the immunomodulating FUSION PROTEINS moiety are linked by a amino acid spacer of sufficient length of amino acid residues so that both moieties can successfully CROSS REFERENCE TO RELATED 5 bond to their individual target. In the alternative , the target APPLICATIONS ing moiety and the immunomodulating moiety that coun teract immune tolerance of cancer cell may be bound The application is a divisional application of and claims directly to each other. The chimeric / fusion polypeptides of priority to U . S . patent application Ser. No. 14 /458 ,674 filed the invention are useful for binding to a cancer cell receptor on Aug . 13 , 2014 , now U . S . Pat. No. 9 , 349 ,617 , which in 10 and reducing the ability of cancer cells to avoid an immune turn claims priority to copending U . S . patent application Ser. response . No . 13 /799 .409 filed on Mar. 13 , 2013 , now U . S . Pat. No. The present invention is based on preparing chimeric / 8 ,815 , 247 , which in turn claims priority to Indian Patent fusion proteins by expression of polynucleotides encoding Application No . 1689 /CHE / 2012 filed on Apr. 30 , 2012 and the fusion proteins that counteract or reverse immune tol Indian Patent Application No . 1690 /CHE /2012 filed on Apr. 15 erance of cancer cells . Cancer cells are able to escape 30 , 2012 , the contents of all are hereby incorporated by elimination by chemotherapeutic agents or tumor- targeted reference herein for all purposes . antibodies via specific immunosuppressive mechanisms in the tumor microenvironment and such ability of cancer cells BACKGROUND OF THE INVENTION is recognized as immune tolerance . Such immunosuppres 20 sive mechanisms include immunosuppressive cytokines ( for Technical Field example , Transforming growth factor beta ( TGF- B ) ) and The present invention relates generally to the field of regulatory T cells and /or immunosuppressive myeloid den generating fusion proteins to be used in cancer therapy , and dritic cells (DCs ) . By counteracting tumor - induced immune more specifically , to nucleotide sequences encoding the tolerance , the present invention provides effective compo fusion proteins , wherein the fusion or chimeric polypeptides 25 sitions and methods for cancer treatment, optional in com comprises at least one targeting moiety and at least one bination with another existing cancer treatment. The present immunomodulatory moiety that counteracts the immune invention provides strategies to counteract tumor- induced tolerance of cancer cells . immune tolerance and enhance the antitumor efficacy of Related Art chemotherapy by activating and leveraging T cell -mediated The immune system provides the human body with a 30 adaptive antitumor against resistant or disseminated cancer means to recognize and defend itself against microorgan - cells . isms and substances recognized as foreign or potentially I n another aspect, the present invention provides a mol harmful. While passive immunotherapy of cancer with ecule including at least one targeting moiety fused with at monoclonal antibodies and passive transfer of T cells to least one immunomodulatory moiety . The targeting moiety attack tumor cells have demonstrated clinical efficacy , the 35 specifically binds a target molecule , and the immunomodu goal of active therapeutic vaccination to induce these latory moiety specifically binds one of the following mol immune effectors and establish immunological memory ecules : ( i ) Transforming growth factor- beta ( TGF - B ) : ( ii ) against tumor cells has remained challenging . Several Programmed death - 1 ligand 1 (PD -L1 ) or Programmed tumor -specific and tumor- associated antigens have been death - 1 ligand 2 (PD - L2 ) ; ( iii ) Receptor activator of nuclear identified , yet these antigens are generally weakly immuno - 40 factor- KB (RANK ) ligand (RANKL ) ; ( iv ) Transforming genic and tumors employ diverse mechanisms to create a growth factor- beta receptor ( TGF - PR ) ; ( v ) Programmed tolerogenic environment that allows them to evade immu - death - 1 (PD - 1 ) ; (vi ) 4 - 1BB receptor or ( vii ) Receptor acti nologic attack . Strategies to overcome such immune toler - vator of nuclear factor- KB (RANK ) . ance and activating robust levels of antibody and /or T cell In a further aspect , the targeting moiety includes an responses hold the key to effective cancer immunotherapy. 45 antibody, antibody fragment including the light or heavy More important, the individual proteins and how to create an chains of the antibody , scFv , or Fc -containing polypeptide active chimeric polypeptide with an active tertiary structure that specifically binds a component of a tumor cell , tumor needs to be explored . antigen , tumor vasculature , tumor microenvironment, or tumor - infiltrating immune cell . Preferably , the targeting SUMMARY OF THE INVENTION 50 moiety is an antibody or a fragment thereof having binding affinity for a component on a tumor cell. Notably each of the The present invention provides polynucleotides, as well heavy chain and light chain may individually be linked to a as polypeptides encoded thereby , that are expressed in separate and distinct immunomodulatory moiety . Further, a cancer cells . These polynucleotides and expressed polypep - heavy or light chain of an antibody targeting moiety may be tides are useful in a variety of therapeutic methods for the 55 linked to an immunomodulatory moiety which in turn can be treatment of cancer. The present invention further provides further linked to a second immunomodulatory moiety methods of reducing growth of cancer cells by counteracting wherein there is a linker between the two immunomodula immune tolerance of cancer cells , wherein T cell remain tory moieties . active and inhibit the recruitment of T - regulatory that are In a still further aspect , there is provided a chimeric known to suppress the immune system ' s response to the 60 polypeptide that comprised a tumor targeting moiety and an tumor. Thus the chimeric polypeptides generated by the immunomodulatory moiety comprising a molecule that polynucleotides sequences of the present invention are use - binds transforming growth factor beta ( TGF - B ) , wherein the ful for treating cancer because of the expressed fusion or tumor targeting moiety is an antibody that binds to EGFR1, chimeric polypeptides . where in the antibody can be the full antibody, heavy chain In one aspect, the present invention provides for chimeric 65 or light chain . The tumor targeting moiety may include polypeptides containing at least one targeting moiety to monoclonal antibodies that target a cancer cell , including but target a cancer cell and at least one immunomodulating not limited to cetuximab , trastuzumab , ritubximab , ipilim US 9 , 758 , 582 B2 umab , tremelimumab , muromonab - CD3 , abciximab , dacli In yet another aspect, the immunomodulatory moiety zumab , basiliximab , palivizumab , infliximab . gemtuzumab includes a molecule that specifically binds to and inhibit the ozogamicin , alemtuzumab , ibritumomab tiuxetan , adalim - activity of Programmed death - 1 ligand 1 (PD - L 1 ) or Pro umab , omalizumab , tositumomab , I- 131 tositumomab , efali - grammed death - 1 ligand 2 (PD - L2 ) . In another aspect , the zumab , bevacizumab , panitumumab , pertuzumab , natali - 5 immunomodulatory moiety includes an extracellular ligand zumab , etanercept, IGN101 ( Aphton ) , volociximab (Biogen binding domain or ectodomain of Programmed Death - 1 Idec and PDL BioPharm ) , Anti -CD80 mAb (Biogen Idec ) , (PD - 1 ) . Anti -CD23 mAb ( Biogen Idel ) , CAT- 3888 ( Cambridge In a further aspect, the targeting moiety includes an Antibody Technology ), CDP -791 ( Imclone) , eraptuzumab antibody, antibody fragment, or polypeptide that specifically ( Immunomedics ) , MDX -010 (Medarex and BMS) , MDX - 10 binds to HER2/ neu , EGFR1, CD20 , cytotoxic T -lymphocyte 060 (Medarex ), MDX - 070 (Medarex ), matuzumab (Merck ) , antigen - 4 ( CTLA - 4 ), CD25 ( 1L - 2a receptor ; IL - 2aR ) , or CP -675 , 206 ( Pfizer ) , CAL (Roche ), SGN - 30 ( Seattle Genet - CD4 and wherein , the immunomodulatory moiety includes ics ) , zanolimumab (Serono and Genmab ) , adecatumumab an extracellular ligand -binding domain or ectodomain of ( Sereno ) , oregovomab (United Therapeutics ), nimotuzumab Programmed Death - 1 (PD - 1 ) . ( YM Bioscience ) , ABT- 874 ( Abbott Laboratories ), deno - 15 In a still further aspect , the targeting moiety includes an sumab ( Amgen ) , AM 108 ( Amgen ) , AMG 714 ( Amgen ) , antibody or antibody fragment that specifically binds to fontolizumab (Biogen Idec and PDL BioPharm ) , dacli - CD20 , and the immunomodulatory moiety includes a zumab (Biogent Idec and PDL BioPharm ), golimumab s equence from transforming growth factor- B ( TGF -B ). (Centocor and Schering - Plough ) , CNTO 1275 (Centocor ), In one aspect , the present invention provides for opti ocrelizumab (Genetech and Roche ), HuMax - CD20 (Gen - 20 mized genes encoding for a fusion polypeptide comprising mab ) , belimumab (HGS and GSK ) , epratuzumab ( Immuno - at least one targeting moiety and at least one immunomodu medics ) , MLN1202 (Millennium Pharmaceuticals ), visili - latory moiety for treating cancer in a human subject wherein zumab (PDL BioPharm ) , tocilizumab ( Roche ) , ocrerlizumab the optimized genes have been modified to increase expres (Roche ) , certolizumab pegol (UCB , formerly Celltech ) , ecu - sion in a human subject. preferably the optimized genes lizumab ( Alexion Pharmaceuticals ), pexelizumab (Alexion 25 comprise sequences for encoding a targeting moiety or an Pharmaceuticals and Procter & Gamble ), abciximab (Cen - immunomodulatory moiety selected from SEQ ID NOs : 12 tocor ), ranibizimumab (Genetech ) , mepolizumab (GSK ), to 28 . TNX - 355 (Tanox ), or MYO -029 (Wyeth ) . In another aspect, the present invention provides for a In an another aspect, the tumor targeting moiety is a vector comprising optimized genes for treating cancer in a monoclonal antibody that binds to HER2/ Neu , CD20 , 30 human subject wherein the optimized genes have been CTLA4 , EGFR1 and wherein the antibody can be the full modified to increase CG sequences. Preferably , the vector antibody , heavy chain or light chain . includes sequences for encoding at least one targeting moi In yet another aspect, the targeting moiety is a molecule ety and at least one immunomodulatory moiety selected that specifically binds epidermal growth factor receptor from SEQ ID NOs: 12 to 28 . (EGFR1 , Erb - B 1 ), HER2/ neu ( Erb -B2 ), CD20 , cytotoxic 35 In yet another aspect, the present invention provides for a T - lymphocyte antigen - 4 (CTLA - 4 ) which is essential for method of treating cancer in a subject , the method compris Treg function (CD 152 ) ; H - 1 and Interleukin - 6 ( IL - 6 ) . ing : In a still further aspect , the targeting moiety specifically a . providing at least one recombinant vector comprising binds a component of a regulatory T cell ( treg ), myeloid nucleotide sequences that encode at least one targeting suppressor cell , or dendritic cell . In another aspect , the 40 moiety and at least one immunomodulatory moiety targeting moiety specifically binds one of the following selected from SEO ID NOs: 12 to 28 ; and molecules: ( i) CD4 ; ( ii ) CD25 ( IL - 2ct receptor ; IL -2aR ); b . administering the recombinant vector to the subject ( iii ) Transforming growth factor -beta receptor ( TGF -PR ) ; under conditions such that said nucleotide sequences (vi ) Transforming growth factor- beta ( TGF - B ) : ( vii) Pro are expressed at a level which produces a therapeuti grammed Death - 1 (PD - 1) ; (viii ) Programmed death - 1 ligand 45 cally effective amount of the encoded fusion proteins in (PD - LI or PD - L2 . the subject. In another aspect , the immunomodulatory moiety specifi In an alternative aspect, the present invention provides an cally binds one of the following molecules: ( i) Transforming expression vector comprising polynucleotides of optimized growth factor- beta ( TGF - B ) : ( ii ) Programmed death - 1 ligand genes that encode at least one targeting moiety and at least ( PD - L1 or PD - L2 ) ; or 4 - 1BB receptor. 50 one immunomodulatory moiety selected from SEQ ID NOs: In yet another aspect, the immunomodulatory moiety 12 to 28 . includes a molecule that binds TGF- B and inhibits the In yet another aspect, the present invention provides a function thereof. Specifically the immunomodulatory moi- recombinant host cell transfected with a polynucleotide that ety includes an extracellular ligand -binding domain of encodes a fusion protein peptide of the present invention . Transforming growth factor -beta receptor TGF - BRII , TGF - 55 In a still further aspect, the present invention contem BRIIb , or TGF -BRIII In another aspect the immunomodu - plates a process of preparing a fusion protein of the present latory moiety includes an extracellular ligand -binding invention comprising : domain (ECD ) of TGF -BRII . Still further the immunomodu a . transfecting a host cell with polynucleotide sequences latory moiety may include H - 4 - 1BB ligand which binds to that encode chimeric fusion proteins to produce a the 4 - 1BB receptor to stimulate T - cells to help eradiate 60 transformed host cell , wherein the polynucleotide tumor. sequences encode at least one targeting moiety and at In a still further aspect , the targeting moiety includes an least one immunomodulatory moiety selected from antibody, antibody fragment, or polypeptide that specifically SEQ ID NOs: 12 to 28 ; and binds to HER2 /neu , EGFR1, CD20 , or cytotoxic T -lympho - b . maintaining the transformed host cell under biological cyte antigen -4 (CTLA -4 ) and wherein the immunomodula - 65 conditions sufficient for expression of the peptide . tory moiety includes an extracellular ligand - binding domain In another aspect, the present invention relates to the use of TGF- BRII. of a chimeric fusion protein , as shown in FIGS . 1 to 15 , in US 9 , 758 , 582 B2 the use of a medicament for the treatment of cancer. Pref . In a further aspect, the culture medium can be improved erably , the fusion protein is expressed in a host cell and such by additions to such medium . For example , the culture expressed proteins are administered in a therapeutic amount medium may include a divalent transitional metallic salt to reduce the effects of cancer in a subject in need thereof. which is added to the cell culture either initially or in In a still further aspect, the present invention provides a 5 fed -batch mode to reduce accumulation of lactate during method of preventing or treating a neoplastic disease. The culturing and /or reduce heterogeneity of the fusion proteins. method includes administration to a subject in need thereof A desirable transitional metallic salt includes a zinc ion and one or more fusion proteins of the invention , in various the addition of the metal ion may be carried out during aspects , the subject is administered one or more molecule of different phases of the production . the invention in combination with another anticancer 10 Other features and advantages of the invention will be therapy , in one aspect, the anticancer therapy includes a apparent from the following detailed description , drawings chemotherapeutic molecule , antibody, small molecule and claims. kinase inhibitor, hormonal agent or cytotoxic agent. The anticancer therapy may also include ionizing radiation , 1 BRIEF DESCRIPTION OF THE DRAWINGS ultraviolet radiation , cryoablation , thermal ablation , or radiofrequency ablation . FIG . 1 shows the amino acid sequences of with the amino In yet another aspect, the present invention provides for a acid sequence of Anti- HER2/ neu - TGFBRII fusion protein at method of preparing therapeutically active antibody - peptide LC constant region with the amino acid sequence of anti fusion proteins , the method comprising ; 20 HER2/ neu heavy chain (SEQ ID NO : 1 ) and anti -HER2 / neu a . preparing a codon optimized sequence of the said light chain (SEQ ID NO : 2 ) attached to amino residues for fusion protein ; TGF - BRII ( immunomodulatory moiety ) (SEQ ID NO : 4 ) b . cloning the optimized sequence of said fusion protein identified in bold letters and wherein a linker ( SEQ ID NO : in a host cell capable of transient or continued expres 3 ) is positioned between the anti- HER2/ neu light chain and sion ; 25 TGF -BRII and shown in italics . c . growing the host cell in a media under suitable condi- FIG . 2 shows the amino acid sequences of Anti -EGFR1 tions for growing and allowing the host cell to express TGFBRII fusion protein at LC constant region with amino the cloned protein ; and acid sequence of Anti - EGFR1 heavy chain ( SEQ ID NO : 5 ) d . subjecting the expressed protein to purification and and the amino acid sequence of Anti - EGFR1 light chain optionally checking the bi- specific binding capabilities 30 (SEQ ID NO : 6 ) attached to amino acid residues for TGF of the protein to its targets . BRII (immunomodulatory moiety ) (SEQ ID NO : 4 ) identi In a preferred embodiment the therapeutically active fied in bold letters and wherein a linker ( SEO ID NO : 3 ) is antibody -peptide fusion proteins is a targeting antibody positioned between the Anti -EGFR1 light chain and TGF fused to one or more immunomodulating moiety that coun - BRII and shown in italics. teracts immune tolerance of a cancer cell . In one aspect, the 35 FIG . 3 shows the amino acid sequences of Anti -CTLA4 immunomodulating moiety may be linked by an amino acid TGFBRII fusion protein at LC constant region with amino spacer of sufficient length to allow bi- specific binding of the acid sequence of anti -CTLA4 heavy chain (SEQ ID NO : 7 ) molecule . The immunomodulating moiety may be bound to and amino acid sequence of anti -CTLA4 light chain (SEQ either the C -terminus of the heavy or light chain of the ID NO : 8 ) attached to amino acid residues for TGF - BRII antibody 40 ( immunomodulatory moiety ) (SEO ID NO : 4 ) identified in In a preferred method as described above , the immuno - bold letters and wherein a linker (SEQ ID NO : 3 ) is modulating moiety is ( i ) Transforming growth factor -beta positioned between the anti -CTLA4 light chain and TGF ( TGF - B ) , ( ii ) Programmed death - 1 ( PD - 1 ) , ( iii ) CTLA - 4 or BRII and shown in italics. ( iv ) 4 - 1BB or parts thereof and the targeting antibody binds FIG . 4 shows the amino acid sequences of Anti- HER2 / epidermal growth factor receptor ( EGFR1, Erb - B 1 ) , HER2/ 45 neu HC - 4 - 1BB and LC - TGFBRII fusion protein with amino neune ( Erb - B2 ) , CD20 , CD , CTLA - 4 , Mucin 1 (MUC - 1 ) , acid sequence of Anti -HER2 / neu /HC - 4 - 1BB fusion protein Interleukin - 2 ( IL - 2 ) or Interleukin - 6 ( IL - 6 ) . wherein the amino acid sequence for Anti -HER2 / neu heavy The method of the present invention provides nucleotide chain (SEQ ID NO : 1 ) is attached to a linker ( SEQ ID NO : sequences that encode the therapeutically active antibody . 3 ) shown in italics and the sequence for 4 - 1BB ( immuno peptide fusion proteins and such expression may be con - 50 modulatory moiety ) (SER ID NO : 9 ) is in written text font ducted in a transient cell line or a stable cell line. The and amino acid sequence of anti -HER2 / neu light chain (SEQ transient expression is accomplished by transfecting or ID NO : 2 ) attached to amino residues for TGF - BRII ( immu transforming the host with vectors carrying the fusion pro - nomodulatory moiety ) ( SEQ ID NO : 4 ) identified in bold teins into mammalian host cells letters and wherein a linker ( SEO ID NO : 3 ) is positioned Once the fusion peptides are expressed , they are prefer - 55 between the anti- HER2/ neu light chain and TGF - BRII and ably subjected to purification and in -vitro tests to check its shown in italics . bi- specificity , that being, having the ability to bind to both FIG . 5 shows the amino acid sequence of Anti - EGFR1 the targetmoiety and immunomodulatingmoiety . Such tests HC - 4 - 1BB and LC - TGFBRII fusion protein with amino acid may include in - vitro test such as ELISA or NK / T - cell sequence of Anti - EGFR1 heavy chain - 4 - 1BB fusion protein binding assays to validate bi- functional target binding or 60 wherein the amino acid sequence for Anti -EGFR1 heavy immune cell stimulation . chain (SEQ ID NO : 5 ) is attached to a linker (SEQ ID NO : Notably once the specific fusion peptides demonstrate the 3 ) is shown in italics and the sequence for 4 - 1 BB ( immu desired bi- specificity , such fusion peptides are selected for nomodulatory moiety ) (SEQ ID NO : 9 ) is in written text font sub - cloning into a stable cell line for larger scale expression and amino acid sequence of light chain Anti - EGFR1 (SEO and purification . Such stable cell lines are previously dis - 65 ID NO : 6 ) attached to amino residues for TGF - BRII ( immu closed , such as a mammalian cell line , including but not nomodulatory moiety ) ( SEQ ID NO : 4 ) identified in bold limited to HEK293 , CHO or NSO . letters with a linker ( SEQ ID NO : 3 ) therebetween . US 9 , 758 , 582 B2 FIG . 6 shows the amino acid sequence of Anti -CTLA4 written text font with linker between (SEQ ID NO : 11 ) and HC - 4 - 1BB and LC - TGFBRII fusion protein with amino acid including the amino acid sequence of Anti - EGFR1 light sequence of Anti -CTLA4 heavy chain - 4 -1BB fusion protein chain ( SEQ ID NO : 6 ). wherein the amino acid sequence for Anti -CTLA4 heavy FIG . 12 shows the amino acid sequence of Anti -CTLA4 chain (SEQ ID NO : 7 ) is attached to a linker (SEQ ID NO : 5 HC - TGFBRII- 4 - 1BB fusion protein with amino acid 3 ) is shown in italics and the sequence for 4 -1BB (immu sequence of Anti -CTLA4 heavy chain - TGFBRII -4 - 1BB nomodulatory moiety ) (SEQ ID NO : 9 ) is in written text font fusion protein wherein the amino acid sequence Anti and amino acid sequence of Anti -CTLA4 light chain ( SEQ CTLA4 heavy chain ( SEQ ID NO : 39 ) is attached to a linker ID NO : 8 ) is attached to amino residues for TGF- BRII (SEQ ID NO : 3 ) shown in italics and the sequence for ( immunomodulatory moiety ) ( SEQ ID NO : 4 ) identified in " TGFBRII ( immunomodulatory moiety ) (SEO ID NO : 4 ) is bold letters with a linker (SEO ID NO : 3 ) therebetween identified in bold letters and the amino acid sequence for FIG . 7 shows the amino acid sequence of Anti -HER2 /neu 4 - 1BB ( immunomodulatory moiety ) (SEO ID NO : 9 ) is in HC -PD1 and LC - TGFBRII fusion protein with amino acid written text font with linker between (SEQ ID NO : 11 ) and sequence of Anti -HER2 /neu heavy chain - PD 1 fusion pro - 16 including the amino acid sequence of Anti -CTLA4 light tein wherein the amino acid sequence for the Anti -HER2 / chain (SEQ ID NO : 8 ) . neu heavy chain (SEQ ID NO : 1 ) is attached to a linker (SEQ F IG . 13 shows the amino acid sequence of Anti -HER2 / ID NO : 3 ) is shown in italics and the sequence for PD1 neu HC - TGFBRII- PD1 fusion protein with amino acid ( immunomodulatory moiety ) (SEQ ID NO : 10 ) is in written sequence of Anti- HER2 / neu heavy chain - TGFBRII- PD1 text font and amino acid sequence of Anti - HER2/ neu light 20 fusion protein wherein the amino acid sequence Anti -HER2 / chain ( SEQ ID NO : 2 ) is attached to amino residues for neu heavy chain (SEQ ID NO : 37 ) is attached to a linker TGF -BRII (immunomodulatory moiety ) (SEQ ID NO : 4 ) (SEQ ID NO : 3 ) shown in italics and the sequence for identified in bold letters with a linker ( SEQ ID NO : 3 ) TGFBRII ( immunomodulatory moiety ) (SEQ ID NO : 4 ) is therebetween . identified in bold letters and the amino acid sequence for FIG . 8 shows the amino acid sequence of Anti- EGFR1 25 PD - 1 ( immunomodulatory moiety ) (SEQ ID NO : 10 ) is in HC - PD1 and LC - TGFBRII fusion protein with amino acid written text font with linker between ( SEQ ID No: 11 ) and sequence of Anti - EGFR1 heavy chain -PD1 fusion protein including the amino acid sequence of Anti -HER2 /neu light wherein the amino acid sequence Anti -EGFR1 heavy chain chain ( SEQ ID NO : 2 ). ( SEQ ID NO : 5 ) is attached to a linker ( SEQ ID NO : 3 ) FIG . 14 shows the amino acid sequence of Anti - EGFR1 shown in italics and the sequence for PD1 ( immunomodu - 30 HC - TGFBRII - PD1 fusion protein with amino acid sequence latory moiety ) (SEQ ID NO : 10 ) is in written text font and of Anti- EGFR1 heavy chain - TGFBRII- PD1 fusion protein amino acid sequence of Anti- EGFR1 light chain (SEQ ID wherein the amino acid sequence Anti - EGFR1 heavy chain NO : 6 ) attached to amino residues for TGF - BRII (immuno (SEQ ID NO : 38 ) is attached to a linker (SEQ ID NO : 3 ) modulatory moiety ) (SEQ ID NO : 4 ) identified in bold shown in italics and the sequence for TGFBRII ( immuno letters with a linker (SEQ ID NO : 3 ) therebetween . 35 modulatory moiety ) (SEQ ID NO : 4 ) is identified in bold FIG . 9 shows the amino acid sequence of Anti -CTLA4 letters and the amino acid sequence for PD - 1 ( immuno HC - PD1 and LC - TGFBRII fusion protein with amino acid modulatory moiety ) ( SEQ ID NO : 10 ) is in written text font sequence of Anti -CTLA4 heavy chain - PD1 fusion protein with linker between (SEQ ID No: 11) and including the wherein the amino acid sequence Anti -CTLA4 heavy chain amino acid sequence of Anti - EGFR1 light chain (SEQ ID (SEQ ID NO : 7 ) is attached to a linker (SEQ ID NO : 3 ) 40 NO : 6 ) . shown in italics and the sequence for PD1 ( immunomodu FIG . 15 shows the of Anti- CTLA4 HC - TGFBRII - PD1 latory moiety ) (SEQ ID NO : 10 ) is in written text font and fusion protein with amino acid sequence of Anti -CTLA4 amino acid sequence of Anti- CTLA4 light chain ( SEQ ID heavy chain - TGFBRII - PD1 fusion protein wherein the NO : 8 ) attached to amino residues for TGF -BRII ( immuno - amino acid sequence Anti -CTLA4 heavy chain (SEQ ID modulatory moiety ) (SEQ ID NO : 4 ) identified in bold 45 NO : 39 ) is attached to a linker (SEQ ID NO : 3 ) shown in letters with a linker (SEQ ID NO : 3 ) therebetween . italics and the sequence for TGFBRII ( immunomodulatory FIG . 10 shows the amino acid sequence of Anti -HER2 / moiety ) ( SEQ ID NO : 4 ) is identified in bold letters and the neu HC - TGFBRII- 4 - 1BB fusion protein with amino acid amino acid sequence for PD - 1 ( immunomodulatory moiety ) sequence of Anti- HER2 / neu heavy chain - TGFBRII- 4 - 1BB (SEQ ID NO : 10 ) is in written text font with linker between fusion protein wherein the amino acid sequence for Anti - 50 (SEQ ID NO : 11 ) and including the amino acid sequence of HER2/ neu heavy chain (SEQ ID NO : 37 ) is attached to a Anti - CTLA4 light chain ( SEQ ID NO : 8 ) . linker (SEQ ID NO : 3 ) shown in italics and the sequence for FIG . 16 shows the nucleotide sequence of Anti -HER2 /neu TGFBRII (immunomodulatory moiety ) (SEQ ID NO : 4 ) is heavy chain constant region with linker (SEQ ID NO : 12 ) identified in bold letters and the amino acid sequence for and TGFBRII ECD ( SEQ ID NO : 13 ) that have been codon 4 - 1BB ( immunomodulatory moiety ) (SEQ ID NO : 9 ) is in 55 optimized for expression in CHO cell. written text font with linker between (SEQ ID No : 11 ) and FIG . 17 shows the nucleotide sequence of Anti -HER2 / neu including the amino acid sequence of Anti- HER2 /neu light heavy chain variable region (SEQ ID NO : 14 ), Anti -HER2 / chain (SEQ ID NO : 2 ). neu light chain variable region (SEQ ID NO : 15 ) and FIG . 11 shows the amino acid sequence of Anti - EGFR1 Anti - EGFR1 heavy chain constant region with linker ( SEQ HC - TGFBRII- 4 - 1BB fusion protein with amino acid 60 ID NO : 16 ) that have been codon optimized for expression sequence of Anti - EGFR1 heavy chain - TGFBRII - 4 - 1BB in CHO cell. fusion protein wherein the amino acid sequence for Anti - FIG . 18 shows the nucleotide sequence of Anti- EGFR1 EGFR1 heavy chain (SEQ ID NO : 38 sequence is attached heavy chain variable region ( SEQ ID NO : 17 ) , Anti - EGFR1 to a linker (SEQ ID NO : 3 ) shown in italics and the sequence light chain variable region ( SEQ ID NO : 18 ), Anti- CTLA4 for TGFBRII ( immunomodulatory moiety ) (SEQ ID NO : 4 ) 65 heavy chain variable region (SEQ ID NO : 19 ) and Anti is identified in bold letters and the amino acid sequence for CTLA4 light chain variable region (SEQ ID NO : 20 ) that 4 - 1BB (immunomodulatory moiety ) (SEQ ID NO : 9 ) is in have been codon optimized for expression in CHO cell . US 9 , 758 ,582 B2 10 FIG . 19 shows the nucleotide sequence of Anti CD20 FIGS . 39, 40 and 41 provide lists of expected / observed IgG1 molecule (SEQ ID NO : 21 ) , Anti -CD20 heavy chain tryptic peptide of the light chain , heavy chain and linked variable region (SEQ ID NO : 22 ) and Anti -CD20 light chain motif of the Anti - HER2/ neu - TGFBRII ECD fusion protein , variable region (SEQ ID NO : 23 ) that have been codon respectively . optimized for expression in CHO cell . 5 FIG . 42 A shows the UV Chromatogram of Tryptic FIG . 20 shows the nucleotide sequence of 4 - 1BB (SEQ ID Peptides of Anti- EGFR1- TGFBRII ECD fusion protein and NO : 24 ) and Anti - IL6R heavy chain (SEQ ID NO : 25 ) that B shows the Total Ion Chromatogram ( TIC ) of Tryptic have been codon optimized for expression in CHO cell . Peptides of Anti - EGFR1- TGFBRII ECD fusion protein . FIG . 21 shows the nucleotide sequence of Anti -ILOR light FIG . 43 provides a list of expected /observed tryptic chain variable region (SEQ ID NO : 26 ) , Anti - 4 - 1BB heavy 10 peptide of the light chain of the Anti - EGFR1- TGFBRII ECD chain (SEQ ID NO : 27 ) and Anti - 4 - 1BB light chain variable fusion protein . region ( SEQ ID NO : 28 ) that have been codon optimized for FIG . 44 shows the list of expected /observed tryptic pep expression in CHO cell . tide of the heavy chain of the Anti - EGFR1- TGFBRII ECD FIG . 22 shows the analysis of Protein A purified Anti - fusion protein . HER2/ neu - TGFBRII and Anti- EGFR1- TGFBRII at 12 % 15 FIG . 45 shows the list of expected / observed tryptic pep PAGE tide of the heavy chain of the Anti - EGFR1- TGFBRII ECD FIG . 23 A shows Anti- HER2 /neu - TGFBRII samples ana - fusion protein . lyzed by Protein A / SEC Chromtography and B Anti - FIG . 46 shows the amino acid sequences of Cantuzumab EGFR1 - TGFBRII samples analyzed by Protein A / SEC TGFBRII fusion protein at LC constant region with amino Chromtography . 20 acid sequence of Cantuzumab heavy chain (SEQ ID NO : 29 ) FIG . 24 A shows that Anti -HER2 /neu - TGFBRII and Anti - and amino acid sequence of Cantuzumab light chain (SEQ EGFR1- TGFBRII molecules bind to the TGFB indicating ID NO : 30 ) attached to amino acid residues for TGF - BRII that the fusion protein is functional and B shows that (immunomodulatory moiety ) (SEQ ID NO : 4 ) identified in Anti -HER2 - TGFBRII inhibits the proliferation of BT474 bold letters and wherein a linker (SEQ ID NO : 3 ) is cell line similar to the Bmab200 (Herceptin ) . 25 positioned between the Cantuzumab light chain and TGF FIG . 25 shows that Anti - EGFR1 - TGFBRII- inhibits the BRII and shown in italics. proliferation of A431 cell line similar to the Cetuximab . FIG . 47 shows the amino acid sequences of Cixutu FIG . 26 shows the ADCC activity of Anti -HER2 - mumab - TGFBRII fusion protein at LC constant region with TGFBRII on BT474 cells is similar to that of Bmab200 amino acid sequence of Cixutumumab heavy chain (SEQ ID (Herceptin ) . 30 NO : 31 ) and amino acid sequence of Cixutumumab light FIG . 27 shows the ADCC activity of Anti- EGFR1 - chain (SEQ ID NO : 32 ) attached to amino acid residues for TGFBRII on A431 cells wherein the ADCC activities are TGF- BRII ( immunomodulatory moiety ) (SEQ ID NO : 4 ) similar to that of Cetuximab . identified in bold letters and wherein a linker (SEQ ID NO : FIG . 28 shows the ADCC activity of ADCC activity of 3 ) is positioned between the Cixutumumab light chain and Anti - EGFR1- 4 - 1BB in comparison with Anti - EGFR1 - 35 TGF- BRII and shown in italics. TGFBRII and cetuximab . FIG . 48 shows the amino acid sequences of Clivatu FIG . 29 A shows that the binding activity of Anti- CTLA4 zumab - TGFBRII fusion protein at LC constant region with TGFBRII to TGFB1 is comparable to Anti- EGFR1- TGFBRII amino acid sequence of Clivatuzumab heavy chain (SEQ ID and B shows that the binding activity of Anti -CTLA4 - NO : 33 ) and amino acid sequence of Clivatuzumab light TGFBRII to CTLA4 . 40 chain ( SEQ ID NO : 34 ) attached to amino acid residues for FIG . 30 A shows the binding activity of Anti -CTLA4 - TGF -BRII (immunomodulatory moiety ) (SEQ ID NO : 4 ) TGFBRII to determine the level of PD1- Fc binding and B identified in bold letters and wherein a linker (SEQ ID NO : shows the binding activity of Anti- EGRF1 -4 - 1BB to deter 3 ) is positioned between the Clivatuzumab light chain and mine the binding of 4 - 1BBL . TGF -BRII and shown in italics . FIG . 31 A shows the binding activity of Anti - EGFR1- 4 - 45 FIG . 49 shows the amino acid sequences of Pritumumab 1BB to EGFR and B shows the binding activity of PD1- TGFBRII fusion protein at LC constant region with amino Fc- 4 - 1BB to find out PDL1- Fc. acid sequence of Pritumumab heavy chain (SEQ ID NO : 35 ) FIG . 32 shows the binding activity of Anti - EGFR1- PD1 and amino acid sequence of Pritumumab light chain (SEQ to EGFR and PD1. ID NO : 36 ) attached to amino acid residues for TGF - BRII FIG . 33 shows photographs of expressed proteins and 50 ( immunomodulatory moiety ) ( SEQ ID NO : 4 ) identified in reduction alkylation thereof. bold letters and wherein a linker (SEQ ID NO : 3 ) is FIG . 34 A shows the mass spectrum Mass Spectrum of positioned between the Pritumumab light chain and TGF light chain (LC ) (Reduced ) of Anti -HER2 / neu - TGFBRII BRII and shown in italics. ECD fusion and B shows Deconvoluted Mass Spectrum of FIG . 50 shows the amino acid sequence of Cantuzumab LC (Reduced ) of Anti- HER2 / neu - TGFBRII ECD fusion . 55 HC -4 - 1BB and LC - TGFBRII fusion protein wherein the FIG . 35 shows the Mass Spectrum of heavy chain (HC ) amino acid sequence for the Cantuzumab heavy chain (SEQ ( Reduced ) of Anti- HER2 / neu - TGFBRII ECD fusion . ID NO : 29 ) is attached to a linker ( SEQ ID NO : 3 ) which is FIG . 36 A shows the Mass Spectrum of LC (Reduced ) of shown in italics and the sequence for 4 - 1BB ( immunomodu Anti - EGFR1- TGFBRII ECD and B shows the Deconvoluted latory moiety ) (SEQ ID NO : 9 ) is in written text font and Mass Spectrum of LC (Reduced ) of Anti -EGFR1 - TGFBRII 60 amino acid sequence of Cantuzumab light chain (SEQ ID ECD . NO : 30 ) is attached to amino residues for TGF - BRII ( immu FIG . 37 shows the Mass Spectrum of HC (Reduced ) of nomodulatory moiety ) (SEQ ID NO : 4 ) identified in bold Anti - EGFR1- TGFBRII ECD . letters with a linker (SEQ ID NO : 3 ) therebetween . FIG . 38 A shows the UV Chromatogram of Tryptic FIG . 51 shows the amino acid sequence of Cixutumumab Peptides of Anti -HER2 / neu - TGFBRII ECD fusion protein 65 HC - 4 - 1BB and LC - TGFBRII fusion protein wherein the and B shows the Total Ion Chromatogram ( TIC ) of Tryptic amino acid sequence for the Cixutumumab heavy chain Peptides of Anti - HER2/ neu - TGFBRII ECD fusion protein . (SEQ ID NO : 31 ) is attached to a linker (SEQ ID NO : 3 ) US 9 , 758 , 582 B2 ju 12 shown in italics and the sequence for 4 -1BB ( immunomodu FIG . 58 shows the amino acid sequence of Cantuzumab latory moiety ) ( SEQ ID NO : 9 ) is in written text font and HC - TGFBRII - 4 - 1BB fusion protein wherein the amino acid amino acid sequence of Cixutumumab light chain (SEQ ID sequence for Cantuzumab heavy chain (SEQ ID NO : 29 ) is NO : 32 ) is attached to amino residues for TGF -BRII ( immu - attached to a linker ( SEQ ID NO : 3 ) shown in italics and the nomodulatory moiety ) ( SEO ID NO : 4 ) identified in bold 5 sequence for TGFPRII ( immunomodulatory moiety ) ( SEQ letters with a linker (SEQ ID NO : 3 ) therebetween . ID NO : 4 ) is identified in bold letters and the amino acid FIG . 52 shows the amino acid sequence of Clivatuzumab sequence for 4 - 1BB ( immunomodulatory moiety ) ( SEQ ID NO : 9 ) is in written text font with linker between (SEQ ID HC -4 - 1BB and LC - TGFBRII fusion protein wherein the No : 11 ) and including the amino acid sequence of Cantu amino acid sequence for the Clivatuzumab heavy chain210 zumab light chain (SEQ ID NO : 30 ). (SEQ ID NO : 33 ) is attached to a linker (SEQ ID NO : 3 ) FIG . 59 shows the amino acid sequence of Cixutumumab shown in italics and the sequence for 4 - 1BB ( immunomodu HC - TGFBRII - 4 - 1BB fusion protein wherein the amino acid latory moiety ) (SEQ ID NO : 9 ) is in written text font and sequence for Cixutumumab heavy chain ( SEQ ID NO : 31 ) amino acid sequence of Clivatuzumab light chain (SEQ ID is attached to a linker (SEQ ID NO : 3 ) shown in italics and NO : 34 ) is attached to amino residues for TGF - BRIIhimnu ( immu 15 the sequence for TGFBRII ( immunomodulatory moiety ) nomodulatory moiety ) (SEQ ID NO : 4 ) identified in bold ( SEQ ID NO : 4 ) is identified in bold letters and the amino letters with a linker (SEQ ID NO : 3 ) therebetween . acid sequence for 4 -1BB ( immunomodulatory moiety ) (SEQ FIG . 53 shows the amino acid sequence of Pritumumab ID NO : 9 ) is in written text font with linker between ( SEQ HC - 4 - 1BB and LC - TGFBRII fusion protein wherein the ID No : 11 ) and including the amino acid sequence of amino acid sequence for the Pritumumab heavy chain (SEQ 20 Cixutumumab light chain (SEQ ID NO : 32) . ID NO : 35 ) is attached to a linker ( SEQ ID NO : 3 ) shown FIG . 60 shows the amino acid sequence of Clivatuzumab in italics and the sequence for 4 - 1BB ( immunomodulatory HC - TGFBRII - 4 -1BB fusion protein wherein the amino acid moiety ) (SEQ ID NO : 9 ) is in written text font and amino sequence for Clivatuzumab heavy chain (SEQ ID NO : 33 ) is acid sequence of Pritumumab light chain ( SEQ ID NO : 36 ) attached to a linker ( SEQ ID NO : 3 ) shown in italics and the is attached to amino residues for TGF -BRII ( immunomodu - 25 sequence for TGFBRII ( immunomodulatory moiety ) (SEQ latory moiety ) (SEQ ID NO : 4 ) identified in bold letters with ID NO : 4 ) is identified in bold letters and the amino acid a linker (SEQ ID NO : 3 ) therebetween . sequence for 4 - 1BB (immunomodulatory moiety ) ( SEQ ID FIG . 54 shows the amino acid sequence of Cantuzumab — NO : 9 ) is in written text font with linker between (SEQ ID HC - PD1 and LC - TGFBRII fusion protein wherein the amino No: 11 ) and including the amino acid sequence of Clivatu acid sequence for the Cantuzumab heavy chain (SEQ ID 30 zumab light chain ( SEQ ID NO : 34 ) . NO : 29 ) is attached to a linker (SEQ ID NO : 3 ) shown in FIG . 61 shows the amino acid sequence of Pritumumab italics and the sequence for PD1 ( immunomodulatory moi HC - TGFBRII - 4 - 1BB fusion protein wherein the amino acid ety ) (SEQ ID NO : 10 ) is in written text font and amino acid sequence for Pritumumab heavy chain (SEQ ID NO : 35 ) is sequence of Cantuzumab light chain (SEQ ID NO : 30 ) is attached to a linker ( SEQ ID NO : 3 ) shown in italics and the attached to amino residues for TGF -BRII ( immunomodula - 35 sequence for TGFBRII (immunomodulatory moiety ) (SEQ tory moiety ) ( SEQ ID NO : 4 ) identified in bold letters with ID NO : 4 ) is identified in bold letters and the amino acid a linker (SEQ ID NO : 3 ) therebetween . sequence for 4 - 1BB ( immunomodulatory moiety ) ( SEQ ID FIG . 55 shows the amino acid sequence of Cixutu NO : 9 ) is in written text font with linker between (SEQ ID mumab HC - PD1 and LC - TGFBRII fusion protein wherein No : 11 ) and including the amino acid sequence of Pritu the amino acid sequence for the Cixutumumab heavy chain 40 mumab light chain (SER ID NO : 36 ) . ( SEQ ID NO : 31 ) is attached to a linker (SEQ ID NO : 3 ) FIG . 62 shows the amino acid sequence of Cantuzumab shown in italics and the sequence for PD1 ( immunomodu - HC - TGFBRII -PD1 fusion protein wherein the amino acid latory moiety ) (SEQ ID NO : 10 ) is in written text font and sequence for Cantuzumab heavy chain (SEQ ID NO : 29 ) is amino acid sequence of Cixutumumab light chain (SEQ ID attached to a linker ( SEQ ID NO : 3 ) shown in italics and the NO : 32 ) is attached to amino residues for TGF - BRII ( immu - 45 sequence for TGFBRII ( immunomodulatory moiety ) (SEQ nomodulatory moiety ) (SEQ ID NO : 4 ) identified in bold ID NO : 4 ) is identified in bold letters and the amino acid letters with a linker (SEQ ID NO : 3 ) therebetween . sequence for PD1 ( immunomodulatory moiety ) (SEQ ID FIG . 56 shows the amino acid sequence of Clivatu NO : 10 ) is in written text font with linker between (SEQ ID zumab — HC - PD1 and LC - TGFBRII fusion protein wherein No : 11) and including the amino acid sequence of Cantu the amino acid sequence for the Clivatuzumab heavy chain 50 zumab light chain (SEQ ID NO : 30 ) . (SEQ ID NO : 33 ) is attached to a linker (SEQ ID NO : 3 ) FIG . 63 shows the amino acid sequence of Cixutumumab shown in italics and the sequence for PD1 ( immunomodu - HC - TGFBRII- PD1 fusion protein wherein the amino acid latory moiety ) (SEQ ID NO : 10 ) is in written text font and sequence for Cixutumumab heavy chain (SEQ ID NO : 31) amino acid sequence of Clivatuzumab light chain ( SEQ ID is attached to a linker (SEQ ID NO : 3 ) shown in italics and NO : 34 ) is attached to amino residues for TGF- BRII ( immu- 55 the sequence for TGFBRII ( immunomodulatory moiety ) nomodulatory moiety ) (SEQ ID NO : 4 ) identified in bold ( SEQ ID NO : 4 ) is identified in bold letters and the amino letters with a linker (SEQ ID NO : 3 ) therebetween . acid sequence for PD1 ( immunomodulatory moiety ) ( SEQ FIG . 57 shows the amino acid sequence of Pritumumab — ID NO : 10 ) is in written text font with linker between (SEQ HC - PD1 and LC - TGFBRII fusion protein wherein the amino ID No : 11) and including the amino acid sequence of acid sequence for the Pritumumab heavy chain (SEQ ID 60 Cixutumumab light chain ( SEQ ID NO : 32 ) . NO : 35 ) is attached to a linker (SEQ ID NO : 3 ) shown in FIG . 64 shows the amino acid sequence of Clivatuzumab italics and the sequence for PD1 ( immunomodulatory moi - HC - TGFBRII -PD1 fusion protein wherein the amino acid ety ) (SEQ ID NO : 10 ) is in written text font and amino acid sequence for Clivatuzumab heavy chain (SEQ ID NO : 33 ) is sequence of Pritumumab light chain ( SEQ ID NO : 36 ) is attached to a linker ( SEQ ID NO : 3 ) shown in italics and the attached to amino residues for TGF -BRII ( immunomodula - 65 sequence for TGFBRII ( immunomodulatory moiety ) (SEQ tory moiety ) ( SEQ ID NO : 4 ) identified in bold letters with ID NO : 4 ) is identified in bold letters and the amino acid a linker ( SEQ ID NO : 3 ) therebetween . sequence for PD1 ( immunomodulatory moiety ) (SEQ ID US 9 , 758 , 582 B2 13 14 NO : 10 ) is in written text font with linker between (SEQ ID mammalian cells ; however optimized expression of these No: 11 ) and including the amino acid sequence of Clivatu sequences in other eukaryotic cells is also envisioned herein . zumab light chain (SEQ ID NO : 34 ) . The amino acid sequences encoded by optimized nucleotide FIG . 65 shows the amino acid sequence of Pritumumab sequences are also referred to as optimized . The term HC - TGFBRII -PD1 fusion protein wherein the amino acid 5 “ expression ” as used herein is defined as the transcription sequence for Pritumumab heavy chain ( SEO ID NO : 35 ) is and /or translation of a particular nucleotide sequence driven attached to a linker ( SEO ID NO : 3 ) shown in italics and the by its promoter. sequence for TGFBRII (immunomodulatory moiety ) (SEO The term “ transfection ” of a cell as used herein means that ID NO : 4 ) is identified in bold letters and the amino acid genetic material is introduced into a cell for the purpose of sequence for PD1 ( immunomodulatory moiety ) ( SEO ID 10 genetically modifying the cell . Transfection can be accom NO : 10 ) is in written text font with linker between (SEO ID plished by a variety of means known in the art , such as No : 11 ) and including the amino acid sequence of Pritu transduction or electroporationwotwoneration . mumab light chain ( SEQ ID NO : 36 ) . The term " cancer” as used herein is defined as disease characterized by the rapid and uncontrolled growth of aber DETAILED DESCRIPTION OF THE 15 rant cells . Cancer cells can spread locally or through the INVENTION bloodstream and lymphatic system to other parts of the body . Examples of various cancers include but are not limited to , The practice of the present invention will employ , unless breast cancer , prostate cancer, ovarian cancer, cervical can otherwise indicated , conventional techniques of immunol - cer, skin cancer , ocular cancer, pancreatic cancer, colorectal ogy, molecular biology , microbiology, cell biology and 20 cancer , renal cancer , liver cancer, brain cancer, lymphoma, recombinant DNA , which are within the skill of the art. See , leukemia , lung cancer and the like . e . g . , Sambrook , et al . MOLECULAR CLONING : A LABO The term “ transgene” is used in a broad sense to mean any RATORY MANUAL , 2nd edition ( 1989 ) ; CURRENT PRO - heterologous nucleotide sequence incorporated in a vector TOCOLS IN MOLECULAR BIOLOGY (F . M . Ausubel, et for expression in a target cell and associated expression al . eds. , ( 1987 ) ); the series METHODS IN ENZYMOLOGY 25 control sequences, such as promoters . It is appreciated by ( Academic Press, Inc. ) : PCR 2 : A PRACTICAL those of skill in the art that expression control sequences will APPROACH ( M . J . MacPherson , B . D . Hames and G . R . be selected based on ability to promote expression of the Taylor eds . ( 1995 ) ) , Harlow and Lane , eds. ( 1988 ) ANTI - transgene in the target cell . An example of a transgene is a BODIES, A LABORATORY MANUAL , and ANIMAL nucleic acid encoding a chimeric fusion protein of the CELL CULTURE (R . I. Freshney, ed . ( 1987 ) ). 30 present invention . The term " expression vector” as used herein means a Definitions vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed . Expres Unless otherwise defined , all technical and scientific sion vectors can contain a variety of control sequences, terms used herein have the meaning commonly understood 35 which refer to nucleic acid sequences necessary for the by one of ordinary skill in the art to which this invention transcription and possibly translation of an operatively belongs. The terminology used herein is for the purpose of linked coding sequence in a particular host organism . In describing particular embodiments only and is not intended addition to control sequences that govern transcription and to be limiting of the invention . As used in the description of translation , vectors and expression vectors may contain the invention and the appended claims, the singular forms 40 nucleic acid sequences that serve other functions as well . " a " , " an ” and “ the ” are intended to include the plural forms The term also includes a recombinant plasmid or virus that as well, unless the context clearly indicates otherwise . The comprises a polynucleotide to be delivered into a host cell , following terms have the meanings given : either in vitro or in vivo . Preferably the host cell is a The term “ polynucleotide ” as used herein means a transient cell line or a stable cell line and more preferably a sequence of nucleotides connected by phosphodiester link - 45 mammalian host cell and selected from the group consisting ages . Polynucleotides are presented herein in the direction of HEK293 , CHO and NSO . from the 5 ' to the 3 ' direction . A polynucleotide of the The term “ subject, ” as used herein means a human or present invention can be a deoxyribonucleic acid (DNA ) vertebrate animal including a dog , cat, horse , cow , pig , molecule or ribonucleic acid (RNA ) molecule . Where a sheep , goat, chicken , monkey, rat , and mouse . polynucleotide is a DNA molecule , that molecule can be a 50 The term " therapeutically effective amount” as used gene or a cDNA molecule . Nucleotide bases are indicated herein means the amount of the subject compound that will herein by a single letter code : adenine ( A ), guanine ( G ) , elicit the biological or medical response of a tissue, system , thymine ( T ) , cytosine ( C ) , inosine ( I ) and uracil ( U ) . A animal or human that is being sought by the researcher, polynucleotide of the present invention can be prepared veterinarian , medical doctor or other clinician . using standard techniques well known to one of skill in the 55 The term “ pharmaceutically acceptable ” as used herein art . means the carrier, diluent or excipient must be compatible The term , " optimized ” as used herein means that a nucleo with the other ingredients of the formulation and not del tide sequence has been altered to encode an amino acid e terious to the recipient thereof. sequence using codons that are preferred in the production The term “ recombinant” as used herein means a genetic cell or organism , generally a eukaryotic cell, for example , a 60 entity distinct from that generally found in nature . As cell of Pichia , a cell of Trichoderma, a Chinese Hamster applied to a polynucleotide or gene, this means that the Ovary cell (CHO ) or a human cell . The optimized nucleotide polynucleotide is the product of various combinations of sequence is engineered to retain completely or as much as cloning , restriction and /or ligation steps, and other proce possible the amino acid sequence originally encoded by the dures that result in the production of a construct that is starting nucleotide sequence , which is also known as the 65 distinct from a polynucleotide found in nature . " parental” sequence . The optimized sequences herein have The term “ substantial identity ” or “ substantial similarity , " been engineered to have codons that are preferred in CHO as used herein when referring to a nucleic acid or fragment US 9 , 758 , 582 B2 15 16 thereof, indicates that when optimally aligned with appro antibodies via specific immunosuppressive mechanisms in priate nucleotide insertions or deletions with another nucleic the tumor microenvironment and such ability of cancer cells acid ( or its complementary strand ) , there is nucleotide is recognized as immune tolerance . By counteracting tumor sequence identity in at least about 95 to 99 % of the induced immune tolerance , the present invention provides sequence . 5 effective compositions and methods for cancer treatment, The term “ peptide , " " polypeptide ” and “ protein ” are used optional in combination with another existing cancer treat interchangeably to denote a sequence polymer of at least two ment. amino acids covalently linked by an amide bond . The present invention provides compositions and meth The term " homologous” as used herein and relating to ods for producing fusion proteins that counteract immune peptides refers to amino acid sequence similarity between 10 tolerance in the tumor microenvironment and promote T two peptides . When an amino acid position in both of the cell -mediated adaptive antitumor immunity for maintenance peptides is occupied by identical amino acids , they are of durable long -term protection against recurrent or dissemi homologous at that position . Thus by “ substantially homolo - nated cancers . These fusion proteins are designed to facili gous” means an amino acid sequence that is largely , but not tate effective long term T cell -mediated immune responses entirely , homologous , and which retains most or all of the 15 against tumor cells by at least one of the following : activity as the sequence to which it is homologous . As used a . promoting death of tumor cells via enhancement of herein , " substantially homologous ” as used herein means antibody - dependent cellular cytotoxicity (ADCC ) ; and that a sequence is at least 50 % identical, and preferably at b . increasing activation and proliferation of antitumor CD8 + least 75 % and more preferably 95 % homology to the refer - T cells by negating immune suppression mediated by regu ence peptide. Additional peptide sequence modification are 20 latory T cells and myeloid suppressor cells . These antitumor included , such as minor variations, deletions, substitutions immune responses may be activated in tandem with the or derivitizations of the amino acid sequence of the sensitization of tumor cells to immune effector- mediated sequences disclosed herein , so long as the peptide has cytotoxicity , thereby establishing a positive feedback loop substantially the same activity or function as the unmodified that augments tumor cytoreduction and reinforces adaptive peptides . Notably , a modified peptide will retain activity or 25 antitumor immunity . function associated with the unmodified peptide, the modi- In addition , the fusion proteins of the present invention fied peptide will generally have an amino acid sequence are distinguished from and superior to existing therapeutic , “ substantially homologous ” with the amino acid sequence of molecules in at least one of the following aspects: ( i ) To the unmodified sequence . counteract immune tolerance in the tumor microenviron The term “ administering ” as used herein is defined as the 30 ment and promote T cell -mediated adaptive antitumor actual physical introduction of the composition into or onto immunity for maintenance of long - term protection against ( as appropriate ) the host subject . Any and all methods of recurrent or disseminated cancers ( for prevention or treat introducing the composition into the subject are contem - ment of diverse cancers ) ; ( ii ) To produce immune cell plated according to the present invention ; the method is not compositions for adoptive cellular therapy of diverse can dependent on any particular means of introduction and is not 35 cers ; and ( iii ) To serve as immune adjuvants or vaccines for to be so construed . Means of introduction are well -known to prophylaxis of diverse cancers or infectious diseases . those skilled in the art , and preferably , the composition is The targeted immunostimulatory antibodies and / or fusion administered subcutaneously or intratumorally . One skilled proteins of the invention provide the ability to disrupt in the art will recognize that, although more than one route immunosuppressive networks in the tumor microenviron can be used for administration , a particular route can provide 40 ment. Tumors employ a wide array of regulatory mecha a more immediate and more effective reaction than another nisms to avoid or suppress the immune response . Cancer route . Local or systemic delivery can be accomplished by cells actively promote immune tolerance in the tumor administration comprising application or instillation of the microenvironment via the expression of cytokines and mol immunovaccines into body cavities, inhalation or insuffla - ecules that inhibit the differentiation and maturation of tion of an aerosol, or by parenteral introduction , comprising 45 antigen - presenting dendritic cells (DC ) . The immunosup intramuscular, intravenous , intraportal, intrahepatic , perito - pressive cytokines and ligands produced by tumor cells neal, subcutaneous, or intradermal administration . In the include the following : ( i) Transforming growth factor- beta event that the tumor is in the central nervous system , the ( TGF- B ) ; ( ii ) Programmed death - 1 ligand 1 (PD - L1 ; composition must be administered intratumorally because B7- H1 ) ; (iii ) Vascular endothelial growth factor ( VEGF ) ; there is no priming of the immune system in the central 50 and ( iv ) Interleukin - 10 ( 1L - 10 ) . nervous system . In addition to blocking dendritic cell (DC ) maturation , Although chemotherapeutic agents can induce “ immuno these molecules promote the development of specialized genic " tumor cell death and facilitate cross - presentation of subsets of immunosuppressive CD4 + T cells ( regulatory T antigens by dendritic ceils , tumors create a tolerogenic cells ; Treg cells ) and myeloid - derived suppressor cells environment that allows them to suppress the activation of 55 (MDSC ) . Tregs are a minority sub -population of CD4 + T innate and adaptive immune responses and evade immuno - cells that constitutively express CD25 [ the interleukin - 2 logic attack by immune effector cells . The present invention ( IL - 2 ) receptor cc -chain ) and the forkhead box P3 (FOXP3 ) provides strategies to counteract tumor- induced immune transcription factor . Tregs (CD4 +CD25 +FoxP3 + cells ) tolerance in the tumor microenvironment and can enhance maintain immune tolerance by restraining the activation , the antitumor efficacy of chemotherapy by activating and 60 proliferation , and effector functions of a wide range of leveraging T cell -mediated adaptive antitumor immunity immune cells , including CD4 and CDS T cells , natural killer against disseminated cancer cells . (NK ) and NKT cells , B cells and antigen presenting cells The present invention is based on the discovery that (APCs ) in vitro and in vivo . targeted immunomodulatory antibodies or fusion proteins of The accumulation of Treg cells in the tumor microenvi the present invention can counteract or reverse immune 65 ronment reinforces tumor immune tolerance and facilitates tolerance of cancer cells . Cancer cells are able to escape tumor progression and metastases . The increased expression elimination by chemotherapeutic agents or tumor- targeted of immunosuppressive cytokines ( TGF -B ; PD - L1) and US 9 , 758 , 582 B2 17 18 tumor - infiltrating Tregs is correlated with a reduction of mAb ) ; Girentuximab ( anti- Carbonic anhydrase ix ): or Far survival of patients with diverse types of cancers . The fusion letuzumab (anti - folate receptor ) . Other examples include proteins of the present invention inhibit key immunosup antibodies such as PanorexTM ( 17 - 1 A ) (murine monoclonal pressive molecules expressed by the targeted tumor cell or antibody ) ; Panorex ( @ ( 17 - 1 A ) ( chimeric murine mono tumor - infiltrating Treg cells and myeloid suppressor cells 5 clonal antibody ) ; BEC2 ( ami- idiotypic mAb , mimics the GD (DCs or MDSC ) . As such , they provide the targeted ability epitope ) (with BCG ): Oncolym (Lym - 1 monoclonal anti to inhibit the development or function of Tregs within the body ) ; SMART M 1 95 Ab , humanized 13 ' 1 LYM - 1 tumor microenvironment. (Oncolym ), Ovarex ( B43 .13 , anti- idiotypic mouse mAb ) ; The present invention provides a method of preventing or 3622W94 mAb that binds to EGP40 (17 - 1 A ) pancarcinoma treating a neoplastic disease . The method includes admin - 10 antigen on adenocarcinomas ; Zenapax (SMART Anti - Tac istration to a subject in need thereof one or more fusion (IL - 2 receptor ) ; SMART M1 95 Ab , humanized Ab , human proteins of the present invention in combination with ized ) ; NovoMAb -G2 (pancarcinoma specific Ab ) : TNT (chi another anticancer therapy , wherein the anticancer therapy is meric mAb to histone antigens ) ; TNT ( chimeric mAb to a chemotherapeutic molecule , antibody , small molecule histone antigens ) ; GJiomab - H (Monoclonals — Humanized kinase inhibitor , hormonal agent, cytotoxic agent, targeted 15 Abs ) ; GN1 - 250 Mab ; EMD - 72000 ( chimeric - EGF antago therapeutic agent, anti - angiogenic agent, ionizing radiation , nist ); LymphoCide (humanized IL .L . 2 antibody ) ; and MDX ultraviolet radiation , cryoablation , thermal ablation , or 260 bispecific , targets GD - 2 , ANA Ab , SMART IDIO Ab , radiofrequency ablation . SMART ABL 364 Ab or ImmuRAIT -CEA . As used herein , the term “ antibody ” includes natural or Various methods have been employed to produce anti artificial mono - or polyvalent antibodies including, but not 20 bodies. Hybridoma technology , which refers to a cloned cell limited to , polyclonal, monoclonal, multispecific , human , line that produces a single type of antibody , uses the cells of humanized or chimeric antibodies , single chain antibodies , various species, including mice (murine ) , hamsters , rats , and Fab fragments . F (ab ') fragments , fragments produced by a humans. Another method to prepare an antibody uses Fab expression library , anti- idiotypic ( anti- Id ) antibodies genetic engineering including recombinant DNA tech ( including , e . g . , anti -Id antibodies to antibodies of the inven - 25 niques . For example , antibodies made from these techniques tion ) , and epitope -binding fragments of any of the above . include , among others , chimeric antibodies and humanized The antibody may be from any animal origin including birds antibodies . A chimeric antibody combines DNA encoding and mammals . In one aspect, the antibody is , or derived regions from more than one type of species . For example , a from , a human , murine ( e. g . , mouse and rat ), donkey, sheep , chimeric antibody may derive the variable region from a rabbit , goat , guinea pig , camel, horse , or chicken . Further , 30 mouse and the constant region from a human . A humanized such antibody may be a humanized version of an antibody. antibody comes predominantly from a human , even though The antibody may bemonospecific , bispecific , trispecific , or it contains nonhuman portions . Like a chimeric antibody , a of greater multispecificity . The antibody herein specifically humanized antibody may contain a completely human con include a " chimeric ” antibody in which a portion of the stant region . But unlike a chimeric antibody, the variable heavy and / or light chain is identical with or homologous to 35 region may be partially derived from a human . The nonhu corresponding sequences in antibodies derived from a par - man , synthetic portions of a humanized antibody often come ticular species or belonging to a particular antibody class or from CDRs in murine antibodies . In any event, these regions subclass , while the remainder of the chain ( s ) is identical are crucial to allow the antibody to recognize and bind to a with or homologous to corresponding sequences in antibod - specific antigen . ies derived from another species or belonging to another 40 In one embodiment, a hybridoma can produce a targeted antibody class or subclass , as well as fragments of such fusion protein comprising a targeting moiety and an immu antibodies, so long as they exhibit the desired biological nomodulatory moiety. In one embodiment, a targeting moi activity . ety comprising an antibody , antibody fragment, or polypep Examples of antibodies which can be incorporated into tide is linked or fused to an immunomodulatory moiety compositions and methods disclosed herein include , but are 45 consisting of a polypeptide , with a linker or without a linker. not limited , to antibodies such as trastuzumab ( anti -HER2 The linker can be an amino acid linker. In one embodiment , neu antibody ) ; Pertuzumab ( anti -HER2 mAb ) ; cetuximab a linker is (GGGGS ) n wherein n is 1 , 2 , 3 , 4 , 5 , 6 , 7 , or 8 . ( chimeric monoclonal antibody to epidermal growth factor For example , GGGGSGGGGSGGGGS (SEQ ID NO : 3 ) . In receptor EGFR ) : panitumumab (anti - EGFR antibody ) ; another embodiment, a linker is EPKSCDK (SEO ID NO : nimotuzumab ( anti - EGFR antibody ); Zalutumumab ( anti - 50 11 ). In various aspects , the length of the linker may be EGFR mAb ) ; Necitumumab (anti - EGFR mAb ) ; MDX - 210 modified to optimize binding of the target moiety or the (humanized anti -HER -2 bispecific antibody ); MDX - 210 function of the immunomodulatory moiety . In various (humanized anti -HER - 2 bispecific antibody ) ; MDX - 447 aspects , the immunomodulatory moiety is a polypeptide that (humanized anti - EGF receptor bispecific antibody ) ; Ritux - is fused to the C -terminus of the Fc region of the heavy chain imab ( chimeric murine /human anti -CD20 mAb ) ; Obinutu - 55 of a targeting antibody or Fc -containing fusion protein . In zumab (anti - CD20 mAb ) ; Ofatumumab (anti - CD20 mAb ) ; another aspect, the immunomodulatory moiety is a polypep Tositumumab - 1131 (anti -CD20 mAb ) ; ibritumomab tiux - tide that is fused to the C - terminus of the light chain of a etan (anti - CD20 mAb ) ; Bevacizumab ( anti - VEGF mAb ) ; targeting antibody. Ramucirumab (anti - VEGFR2 mAb ) ; Ranibizumab (anti - An antibody fragment can include a portion of an intact , VEGF mAb ) ; Aflibercept ( extracellular domains of 60 antibody, e . g . including the antigen - binding or variable YEGFR1 and VEGFR2 fused to IgG1 Fc ) : AMG386 ( an - region thereof. Examples of antibody fragments include Fab , giopoietin - 1 and - 2 binding peptide fused to IgG1 Fc ) ; Fab ', F ( ab ') 2 , and Fv fragments ; Fc fragments or Fc - fusion Dalotuzumab ( anti- 1GF - 1R mAb ) : Gemtuzumab ozogami- products ; diabodies ; linear antibodies; single - chain antibody cin (anti -CD33 mAb ); Alemtuzumab (anti - Campath - 1 / molecules ; and multispecific antibodies formed from anti CD52mAb ); Brentuximab vedotin ( anti - CD30 mAb ) ; Catu - 65 body fragment ( s ). An intact antibody is one which includes maxomab (bispecific mAb that targets epithelial cell an antigen - binding variable region as well as a light chain adhesion molecule and CD3) ; Naptumomab ( anti- 5T4 constant domain ( CL ) and heavy chain constant domains, US 9 , 758 , 582 B2 19 20 CHI, CH2 and CH3. The constant domains may be native selection of an appropriate cloning vector is a matter of sequence constant domains ( e . g . , human native sequence choice . The gene can be placed under the control of a constant domains ) or amino acid sequence variant thereof promoter , ribosome binding site ( for bacterial expression ) for any other modified Fc ( e . g . glycosylation or other and , optionally , an operator ( collectively referred to herein engineered Fc ) . 5 as “ control” elements ) , so that the DNA sequence encoding The fusion proteins of the present invention may be the desired polypeptide is transcribed into RNA in the host synthesized by conventional techniques known in the art , for cell transformed by a vector containing this expression example , by chemical synthesis such as solid phase peptide construction . The coding sequence may or may not contain synthesis . Such methods are known to those skilled in the a signal peptide or leader sequence . Heterologous leader art. In general, these methods employ either solid or solution 10 sequences can be added to the coding sequence that causes phase synthesis methods, well known in the art . Specifically, the secretion of the expressed polypeptide from the host the methods comprise the sequential addition of one or more organism . Other regulatory sequences may also be desirable amino acids or suitably protected amino acids to a growing which allow for regulation of expression of the protein peptide chain . Normally, either the amino or carboxyl group sequences relative to the growth of the host cell . Such of the first amino acid is protected by a suitable protecting 15 regulatory sequences are known to those of skill in the art, group . The protected or derivatized amino acid can then be and examples include those which cause the expression of a either attached to an inert solid support or utilized in solution gene to be turned on or off in response to a chemical or by adding the next amino acid in the sequence having the physical stimulus , including the presence of a regulatory complementary ( amino or carboxyl) group suitably pro compound . Other types of regulatory elements may also be tected , under conditions suitable for forming the amide 20 present in the vector, for example , enhancer sequences . linkage . The protecting group is then removed from this The control sequences and other regulatory sequences newly added amino acid residue and the next amino acid may be ligated to the coding sequence prior to insertion into (suitably protected ) is then added , and so forth . After all the a vector, such as the cloning vectors described above . desired amino acids have been linked in the proper Alternatively , the coding sequence can be cloned directly sequence , any remaining protecting groups and any solid 25 into an expression vector which already contains the control support are removed either sequentially or concurrently to sequences and an appropriate restriction site . afford the final polypeptide . By simple modification of this The expression vector may then used to transform an general procedure , it is possible to add more than one amino appropriate host cell . A number ofmammalian cell lines are acid at a time to a growing chain , for example , by coupling known in the art and include immortalized cell lines avail ( under condition that do not racemize chiral centers ) a 30 able from the American Type Culture Collection ( ATCC ) , protected tripeptide with a properly protected dipeptide to such as , but not limited to , Chinese hamster ovary ( CHO ) form , after deprotection , a pentapeptide . cells , HeLa cells , HEK293 , baby hamster kidney (BHK ) Typical protecting groups include t -butyloxycarbonyl cells , monkey kidney cells (COS ) , human hepatocellular (Boc ) , 9 - fluorenylmethoxycarbonyl (Fmoc ) , benxyloxycar - carcinoma cells ( e . g . , Hep G2 ), Madin - Darby bovine kidney bonyl ( Cbz ), p - toluenesulfonyl ( Tos ); 2 ,4 -dinitrophenyl , 35 (“ MDBK ” ) cells , NOS cells derived from carcinoma cells , benzyl (Bzl ) , biphenylisopropyloxy -carboxycarbonyl , such as sarcoma, as well as others. Similarly , bacterial hosts cyclohexyl, isopropyl, acetyl , o -nitrophenylsulfonyl , and the such as E . coli, Bacillus subtilis , and Streptococcus spp ., will like. Of these , Boc and Fmoc are preferred . find use with the present expression constructs . Yeast hosts Typical solid supports are generally cross - linked poly useful in the present invention include inter alia , Saccharo meric materials . These include divinylbenzene cross- linked 40 myces cerevisiae, Candida albicans, Candida maltosa , Han styrene -based polymers , for example , divinylbenzene -hy senula polymorpha , Kluyveromyces fragilis, Kluyveromyces droxymethylstyrene copolymers, divinylbenzene - chlorom - lactis , Pichia guillerimondii , Pichia pastoris , Schizosaccha ethyl styrene copolymers , and divinylbenzene -benzhydry romyces pombe and Yarrowia lipolytica . Insect cells for use laminopolystyrene copolymers . The divinylbenzene with baculovirus expression vectors include , inter alia , benzhydrylaminopolystyrene copolymers , as illustrated 45 Aedes aegypti, Autographa californica , Bombyx mori , herein using p -methyl - benzhydrylamine resin , offers the Drosophila melanogaster, Spodoptera frugiperda , and advantage of directly introducing a terminal amide func - Trichoplusia ni. The proteins may also be expressed in tional group into the peptide chain , which function is Trypanosomes. retained by the chain when the chain is cleaved from the Depending on the expression system and host selected , support. 50 the proteins of the present invention are produced by grow In one method , the polypeptides are prepared by conven - ing host cells transformed by an expression vector described tional solid phase chemical synthesis on , for example, an above under conditions whereby the protein of interest is Applied Biosystems, Inc . (ABI ) 430A peptide synthesizer expressed . The protein is then isolated from the host cells using a resin that permits the synthesis of the amide peptide and purified . If the expression system secretes the protein form and using t - Boc amino acid derivatives (Peninsula 55 into growth media , the protein can be purified directly from Laboratories , Inc . ) with standard solvents and reagents . the media . If the protein is not secreted , it is isolated from Polypeptides containing either L - or D - amino acids may be cell lysates . The selection of the appropriate growth condi synthesized in this manner. Polypeptide composition is tions and recovery methods are within the skill of the art. confirmed by quantitative amino acid analysis and the Once purified , the amino acid sequences of the proteins can specific sequence of each peptide may be determined by 60 be determined , i. e ., by repetitive cycles of Edman degrada sequence analysis . tion , followed by amino acid analysis by HPLC . Other Preferably , the polypeptides can be produced by recom - methods of amino acid sequencing are also known in the art. binant DNA techniques by synthesizing DNA encoding the Once synthesized or otherwise produced , the inhibitory desired polypeptide . Once coding sequences for the desired activity of a candidate polypeptide can be tested by assess polypeptides have been synthesized or isolated , they can be 65 ing the ability of the candidate to inhibit the lipopolysac cloned into any suitable vector for expression . Numerous charide - induced nuclear translocation of NF - kappa . B by , cloning vectors are known to those of skill in the art, and the for example , using murine endothelial cells . US 9 , 758 ,582 B2 21 22 The fusion proteins of the present invention can be TABLE 1 - continued formulated into therapeutic compositions in a variety of dosage forms such as, but not limited to , liquid solutions or Fusion protein Details suspensions , tablets , pills , powders, suppositories, poly PD1 ectodomain - Fc - 4 - 1 BBL meric microcapsules or microvesicles, liposomes , and 5 TGFBRII ECD - Fc -4 - 1BBL Anti - EGFR1 heavy chain + PD1 ectodomain and Anti- EGFR1 injectable or infusible solutions. The preferred form depends light chain upon the mode of administration and the particular cancer Anti- CD20 heavy chain + 4 - 1BBL and Anti - CD20 light chain type targeted . The compositions also preferably include Anti - HER2/ neu heavy chain + PD1 ectodomain and Anti -HER2 / neu light chain pharmaceutically acceptable vehicles, carriers or adjuvants , Anti - IL6Rheavy chain + PD1 ectodomain and Anti -ILOR light well known in the art, such as human serum albumin , ion chain exchangers , alumina , lecithin , buffer substances such as Anti- IL6Rheavy chain + TGFB -RII ECD and Anti- IL6R light phosphates , glycine, sorbic acid , potassium sorbate , and chain salts or electrolytes such as protamine sulfate . Suitable Anti- 4 - 1BB heavy chain + PD1 ectodomain and Anti - 4 - 1BB vehicles are , for example , water, saline , dextrose, glycerol, light chain ethanol, or the like , and combinations thereof . Actual meth ods of preparing such compositions are known, or will be The expressed protein were characterized by using SDS apparent, to those skilled in the art. See , e . g ., Remington ' s PAGE and the expressed fusion proteins Anit -HER2 / neu Pharmaceutical Sciences, Mack Publishing Company, Eas TGFBRII and Anti - EGFR1- TGFBRII were purified from ton , Pa. , 18th edition , 1990 . culture supernatants using ProteinA column and the results The above compositions can be administered using con 20 are shown in FIG . 22 . Notably , Anti - EGFR1 - TGFBRII light ventional modes of delivery including , but not limited to , chain mass is higher and it may be because of the presence intravenous , intraperitoneal, oral , intralymphatic , or subcu - of two glycosylation sites on the variable regions light and taneous administration . Local administration to a tumor in heavy chain . Both the Anti- HER2 / neu - TGFBRII & Anti question , or to a site of inflammation , e . g . , direct injection 25 EGFR1- TGFBRII heavy chains mass are higher because of into an arthritic joint, will also find use with the present the TGFBRII. Also Anti -HER2 / neu - TGFBRII heavy chain invention . has four N - glycosylation sites while Anti- EGFR1- TGFBRII Therapeutically effective doses will be easily determined has five N - glycosylation sites . by one of skill in the art and will depend on the severity and course of the disease , the patient' s health and response to 3020 Example 2 treatment, and the judgment of the treating physician . Protein A / SEC chromatography . The Anti- HER2 /neu EXPERIMENTAL TGFBRII and Anti - EGFR1- TGFBRII samples were ana Below are examples of specific embodiments for carrying lyzed by ProteinA /SEC chromatography and the results are out the present invention . The examples are offered for 35 shown in FIG . 23 . FIG . 23 A shows a sharp peak of elution illustrative purposes only , and are not intended to limit the of Bmab200 (Herceptin ) vs a broader elution peak is scope of the present invention in any way. Efforts have been believed to be a measure of heterogeneity due to presence of made to ensure accuracy with respect to numbers used ( e . g ., glycosylation as there are three additional N - glycosylation amounts , temperatures, etc .) , but some experimental error sites that are present in the TGFBRII region . Notably storage and deviation should , of course , be allowed for. 40 at - 80 C did not causing aggregation . The shift in the position or appearance of the peak early in SEC column Example 1 indicates that the increase in the molecular weight is because of the fusion partner. This once again confirms that the full The Fusion proteins comprising of IgG heavy chain length molecule is being expressed . FIG . 23 B shows a sharp linked to immunomodulator (either suppressor or activator ) * peak of elution of Bmab200 (Herceptin ) vs a broader elution ligands were expressed by codon optimized genes for the peak which is believed to be a measure ofheterogeneity due expression of CHO cells . The codon optimized nucleotide to presence of glycosylation sites as there are three addi sequences defined by SEQ ID NOs: 12 to 28 were expressed tional N -glycosylation sites are present in the TGFBRII in (CHO ) cells and the expressed chimeric / fusion proteins 50 region . Again , storage at - 80 C did not causing aggregation . are shown in The shift in the position or appearance of the peak early in SEC column indicates that the increase in the molecular TABLE 1 weight is because of the fusion partner. This once again Fusion protein Details confirms that the full length molecule is being expressed . 55 Anti- HER2 /neu heavy chain + TGFB -RII ECD and Anti HER2/ neu light chain Example 3 Anti- EGFR1 heavy chain + TGFB -RII ECD and Anti- EGFRI light chain Functional assays for the Fusion proteins . ELISA experi Anti- CTLA4 heavy chain + TGFB -RII ECD and Anti- CTLA4 ment was carried out to check the binding ability of Anti light chain Anti- CTLA4 heavy chain + PD1 ectodomain and Anti- CTLA4 60 HER2/ neu - TGFBRII and Anti - EGFR1 - TGFBRII to TGFB . light chain FIG . 24 A shows that Anti -HER2 /neu - TGFBRII and Anti Anti- HER2 / neu heavy chain + 4 - 1BBL and Anti -HER2 /neu EGFR1- TGFBRII molecules bind to the TGFB indicating light chain Anti- EGFR1 heavy chain + 4 - 1BBL and Anti- EGFR1 that the fusion protein is functional. FIG . 24 B shows that light chain Anti -HER2 - TGFBRII inhibits the proliferation of BT474 Anti- CTLA4 heavy chain + 4 - 1BBL and Anti - CTLA4 65 cell line similar to the Bmab200 (Herceptin ) . FIG . 25 shows light chain that Anti - EGFR1- TGFBRII - inhibits the proliferation of A431 cell line similar to the Cetuximab . US 9 ,758 ,582 B2 23 24 Example 4 was also evaluated . FIG . 38 A shows the UV Chromatogram of Tryptic Peptides of Anti -HER2 / neu - TGFBRII ECD Antibody dependent cellular cytotoxicity ADCC activity fusion protein and B shows the Total Ion Chromatogram for Anti - HER2/ neu - TGFBRII fusion protein was conducted ( TIC ) of Tryptic Peptides of Anti- HER2 /neu - TGFBRII ECD to determine that the protein binds to the target receptors on 5 fusion protein . FIGS. 39 , 40 and 41 provide lists of the cells . The results are shown in FIG . 26 wherein the expected /observed tryptic peptide of the light chain , heavy activity is determined in BT474 cells and it is evident that chain and linked motif of the Anti -HER2 /neu - TGFBRII ADCC activity (% lysis of cells ) ofAnti - HER2 - TGFBRII on ECD fusion protein , respectively . Notably , all the expected BT474 cells is similar to that of Bmab200 (Herceptin ). FIG . peptides of the molecules were identified including the light 27 shows ADCC activity of Anti -EGFR1 - TGFBRII On A431 10 and heavy chain peptides and the peptides of the linked cells wherein the ADCC activities are similar to that of motif ( TGF Cetuximab . FIG . 28 shows the ADCC activity of ADCC FIG . 42 A shows the UV Chromatogram of Tryptic activity of Anti - EGFR1 - 4 - 1BB in comparison with Anti Peptides of Anti - EGFR1 - TGFBRII ECD fusion protein and EGFR1 - TGFBRII and cetuximab . B shows the Total Ion Chromatogram ( TIC ) of Tryptic 15 Peptides of Anti- EGFR1 - TGFBRII ECD fusion protein . Example 5 FIGS. 43 , 44 , and 45 provide lists of expected /observed tryptic peptide of the light chain , heavy chain and linked Binding Activity of the expressed proteins. The aim of motif of the Anti- EGFR1- TGFBRII ECD fusion protein , this assay is to test the functionality of the fusion proteins to respectively. Again all the expected peptides of the mol bind to the target receptors on the cells in a dose dependent 20 ecules were identified including the light and heavy chain manner. FIG . 29 A shows that the binding activity of peptides and the peptides of the linked motif ( TGF BRII) . Anti -CTLA4 - TGFBRII to TGFB1 is comparable to Anti EGFR1 - TGFBRII and B shows that the binding activity of Example 8 Anti -CTLA4 - TGFBRII to CTLA4. FIG . 30 A shows the binding activity of Anti -CTLA4 - TGFBRII to determine the 25 The host cell line used for the expression of recombinant level of PD1 - Fc binding and B shows the binding activity of fusion protein expression is CHO cells or the derivative of Anti - EGRF1 - 4 - 1BB to determine the binding of 4 - 1BBL . the CHO cells . The CHO cells referred here is either FIG . 31 A shows the binding activity of Anti- EGFR1 - 4 - 1BB freedom CHO - S cells ; CHO - S Cells are CHO - derived cells to EGFR and B shows the binding activity of PD1- Fc - 4 - 1BB adapted to high density, serum - free suspension culture in to find out PDL1- Fc. FIG . 32 shows the binding activity of 30 chemically -defined medium that are capable of producing Anti- EGFR1 -PD1 to EGFR and PD1. high levels of secreted , recombinant protein or CHO K1 cells ; having the same as ATCC No. CCL -61 . It is basically Example 6 an adherent cell line . The vectors used for stable cell line : The Freedom pCHO 1. 0 vector, designed by ProBioGen Confirmation of primary structure of molecule . As shown 35 AG , to express one or two genes of interest downstream of in FIG . 33 , the expressed proteins are evaluated to determine the vector ' s two different hybrid CMV promoters . This themolecular weight and the presence of glycosylation . The vectorV contains the dihydrofolate reductase (DHFR ) selec samples were analyzed by reducing and non -reducing SDS tion marker and a puromycin resistance gene , allowing PAGE . The heavy and light chains of the antibody are selection using MTX and Puromycin simultaneously . separated by reduction alkylation so that the reduced struc - 40 The light chain or the light chain fusion protein coding tures can be evaluated . Tryptic digestion of the fusion nucleic acid sequences are cloned into the restriction proteins provides for the identification of the primary enzyme sites Avril and BstZ17 under the control of EF2 / sequence. MS/ MS analysis of the proteins is performed . CMV promoter . The heavy chain or the heavy chain fusion Mass Spectrometry Analysis of Anti -HER2 / neu - TGFBRII protein coding nucleic acid sequences are cloned , in restric and Anti - EGFR1- TGFBRII . The fusion protein shown in 45 tion enzyme sites EcoRV and Pad under the control of FIG . 1 was expressed and tested . FIG . 34 A shows the mass CMV /EF1 promoter. spectrum Mass Spectrum of light chain (LC )( Reduced ) of The construct( s) are transfected into Freedom CHO - S Anti -HER2 / neu - TGFBRII ECD fusion and B showsDecon - cells /CHOK1 cells . The high producer single, clonal cell voluted Mass Spectrum of LC (Reduced ) of Anti- HER2 strain is selected for producing the recombinant fusion neu - TGFBRII ECD fusion . FIG . 35 shows the Mass Spec - 50 protein . Prepare the MCB and characterize for cell viability , trum of heavy chain (HC ) (Reduced ) of Anti- HER2 / neu - productivity , stability and other parameters . The cells are TGFBRII ECD fusion . used for culturing followed by purification . The fusion protein shown in FIG . 2 was expressed and tested . FIG . 36 A shows the Mass Spectrum of LC (Reduced ) Example 9 of Anti - EGFR1 - TGFBRII ECD and B shows the Deconvo - 55 luted Mass Spectrum of LC (Reduced ) of Anti -EGFR1 The cell culture is performed in feed -batch mode. In the TGFBRII ECD . FIG . 37 shows the Mass Spectrum of HC cell culture, the mammalian host cells used is Chinese (Reduced ) of Anti - EGFR1- TGFBRII ECD . Hamster Ovary (CHO ) cells and culture medium are sup plied initially . The CHO cells are genetically engineered to Example 7 60 produce the Antibody -peptide fusion protein . The zinc sul phate hepta hydrate salt is added in the medium at a The fusion proteins having amino acid sequences as concentration of 0. 4 mM . In contrast, there is no addition of described in FIGS . 1 and 2 were inspected using UV any zinc salt in the control medium . The production fer chromatography and providing chromatograms resulting mentation run starts with an initial cell count of 0 . 3 - 0 . 45x from the chromatographic separation of the tryptic digest of 65 10º cells /ml at 37 + 1° C ., the first 3 - 4 days are dedicated to the fusion proteins and tested with UV 218 - 222 nm wave - grow the cells in batch phase . Next step involves lowering length . Total Ion Current ( TIC ) corresponding to UV trace the temperature to 31 + 1° C . and continuing the run till 7th US 9 , 758 , 582 B2 25 26 day. Lactate reduces by almost 10 -40 % throughout the run . with other members of the ErbB family of receptors lead to The produced fusion protein is then collected from the tumor progression . This will involve the binding of growth media using the technique of affinity chromatography. factors associated with the ErbB signaling pathway. In Example 10 addition to this , the tumor creates a milieu wherein the 5 immune system is suppressed by activating TGF B and The cell culture is performed in a feed -batch mode is specific cytokines involved in the subdued immune employed . In the cell cultures the mammalian host cells and culture medium which is Hyclone CDM4Mab are supplied response . A novel molecule is generated wherein Trastu initially . The salts ( zinc ) is also added in the medium ( 0 . 3 zumab (anti ErbB2) is fused with the TGF BRII receptor as mM ). The production fermentation run starts with an initial 10 a fusion protein . While it is hypothesized that Trastuzumab cell count of 0 . 3 - 0 .45x10 cells/ nil at 37 + 1° C ., the first 3 - 4 will act as a targeted molecule homing into the ErbB2 days are dedicated to growing the cells in batch phase . Next overexpressing breast cancer cells , the TGFBRII receptor step involves lowering the temperature to 31 + 1 - 1° C . and will sequester TGFB leading to immune activation . The continuing the run till 7th day . experiment will utilize the growth of Herceptin resistant Example 11 15 ErbB2 expressing cell lines ( selected by growing BT474 cells in the presence of Herceptin ) in the presence of TGFB , Purification of antibody -peptide fusion immunostimula tory molecules using protein A column . Supernatant culture cytotoxic CD8 positive cells and NK cells . While Trastu secreted from recombinant CHO cell line containing the zumab will be ineffective in inducing cytotoxicity Trastu fusion monoclonal antibodies is tested for titer and endo - 20 zumab TGFBRII receptor fusion molecule will sequester the toxins under sterile conditions . The supernatant is subjected TGFB thereby preventing the inhibition of cytotoxic CD8 to affinity chromatography using Mab Select Xtra Protein A and NK cells . This will lead to enhanced cytotoxicity affinity resin , washed and equilibrated with binding buffer. observed in Trastuzumab - TGFBRII receptor fusion treated The pH of the supernatant is adjusted using 0 . 5M phosphate cells over cells treated with Trastuzumab alone . The readout to the same pH as the column ; the supernatant is allowed to 25 for the experiment will use Alamar Blue a resazurin dve bind to the column /pass through the column at the flow rate of 0 . 5 ml/minute to achieve the maximum binding . All the which will get activated directly proportional to live cells Antibody -proteins fusion molecules bind through the Fc present. Another method could be to measure cytotoxicity region while impurities are eliminated as flow through . The by using cytotox glo which measures protease release which column is washed with equilibration buffer and the bound 3n directly corresponds to proportional dead cells . Yet another fusion molecules are eluted using 0 . 1 M glycine at pH 3 . 0 . method could be the use of the flow cytometer directly The pH of the eluted proteins is adjusted to neutral pH or the measuring apoptotic and necrotic cell population by using stable formulation pH and the purified protein are stored at Annexin V and propidium iodide . Results from these mul - 20° C . or at 2 -8° C . tiple experiments will elucidate understanding of the activity Example 12 35 of the conjugate molecule as compared to Trastuzumab alone . Differentiating Trastuzumab from Although the invention has been described with reference Trastuzumab - TGF BRII Receptor Fusion Molecule to the above example , it will be understood that modifica tions and variations are encompassed within the spirit and A breast cancer tumor overexpressing the ErbB2 receptor 40 scope of the invention . Accordingly , the invention is limited will either by constitutive activation or heterodimerization only by the following claims .
SEQUENCE LISTING
< 160 > NUMBER OF SEQ ID NOS : 39
A< 210 > SEQ ID NO 1 A< 211 > LENGTH : 449 A< 212 > TYPE : PRT A 13 > ORGANISM : Artificial Sequence A< 220 > FEATURE : A< 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 1 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 10 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr a 25 Tyr ÅIle His Trp val Arg in Ala Pro Gly Lys Gly Leu Glu Trp Val 40 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr IfyouareAla Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr isthe 75 80 US 9 , 758 , 582 B2
- continued Leu Gin Met Asn Ser Leu Arg ?Ala ?Glu Asp Thr Ala Val Tyr Tyr Cys ET 95
Ser RArg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gin ?? 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr HEEggVal Ser 160 Trp Asn Ser Gly %&REAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys AAsp Thr Leu Met Ile 250 | Ser Arq Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg 300 Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys mmmmm 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr 3501 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 380 gg Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His 430
Glu Ala Leu His Asn His ?Tyr ?Thr Gin Lys Ser Leu Ser Leu Ser Pro ?435 440 mmmmm mmmmm445 Gly g < 210 > SEQ ID NO 2 V< 211 > LENGTH : 214 < 212 > TYPE : PRT < 213 > ORGANISMmmmmmmm : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct US 9 , 758 , 582 B2 30 - continued 1 < 400 > SEQUENCE : 2 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 Asp Arq Val Thr Ile Thr Cys Arq Ala Ser Gin Asp Val Asn Thr Ala 20 30 Val Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 40 Tyr Ser msAla Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin His Tyr Thr Thr Pro Pro 95 Thr Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 120 Thr Ala Ser ammmVal Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 8Ser Gly Asn Ser Gin 145 155 Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser ?Thr Tyr Ser Leu Ser 3? 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 190 . Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 200 ? Phe Asn Arg Gly Glu Cys mmm210 < 210 > SEQ ID No 3 < 211 > LENGTH : 15 | < 212 > TYPE : PRT < 213 > ORGANISM : Artificial sequence < 220 > FEATURE : AAAA< 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 3 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 10 15
< 210 > SEQ ID NO 4 < 211 > LENGTH : 137 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : AAAA< 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 4 Thr Ile Pro Pro His Val Gin Lys Ser Val Asn Asn Asp Met Ile Val
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gin Leu Cys Lys Phe Cys &20 ggg" 25 Asp Val Arg Phe Ser ?Thr Cys Asp Asn Gin Lys Ser Cys Met Ser Asn 35 Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gin Glu Val Cys Val Ala 50 55 ER US 9 , 758 , 582 B2
- continued
Val Trp Arg Lys ?Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His 75 180 Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser 90 g95
&Pro Lys Cys Ile Met Lys Glu Lys Lys Lys &Pro Gly Glu Thr Phe Phe 110 Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 115 ?? 120 125 Glu Glu Tyr Asn Thr Ser Asn Pro Asp 130 ggg 135
< 210 > SEQ ID No 5 < 211 > LENGTH : 448 1 222 1230 ^ TYPE : PRT < 2131 ^> ORGANISM : Artificial Sequence < 2202 ^> FEATURE : AAAAA 2 < 223 > OTHER INFORMATION : Synthetic Construct < 400 > SEQUENCE : 5 Gin Val Gin Leu Lys Gin Ser Gly Pro Gly Leu Val Gin Pro |Ser Gin 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn ETyr Gly Val His Trp Val Arg Gin Ser Pro Gly Lys Gly Leu Glu Trp Leu 40 45 Gly Val Ile Trp Ser Gly ?8Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gin Val Phe Phe $sm 75 &80 Lys Met Asn Ser Leu Gin Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 5 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe REAla Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 155 EggEne& 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gin Thr Tyr Ile Cys Asn Val Asn His Lys Pro mmm 200 205 Ser mammAsn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 255 Arg Thr Pro Glu Val memThr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn mmmmm280 mmmmmmm gmmmm mmmmm 285 US 9 , 758 , 582 B2 34 - continued Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr| Asn Ser Thr Tyr Arg Val 1 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 ESEETyr Lys 38Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 Cys Leu Val Lys Gly Phe Tyr ?Pro Ser Asp Ile Ala Val Glu Trp Glu 375 380
Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly mmmSer Phe Phe Leu Tyr mmmSer Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser cys Ser Val Met His Glu 420 425
Ala Leu His Asn His Tyr Thr ?Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440
< 210 > SEQ ID No 6 < 211 > LENGTH : 214 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 6 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro &Gly
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gin Ser Ile Gly Thr Asn 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser &Pro Arq Leu Leu Ile 40 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly & . Ser Gly Ser 3Gly ?Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 75 Glu Asp Ile Ala Asp Tyr Tyr Cys Gin Gin Asn Asn Asn Trp Pro Thr
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala , 110
Pro Ser Val Phe Ile Phe Pro &Pro Ser Asp Glu Gin Leu Lys Ser Gly 120 Thr Ala Ser Val pgVal ECys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 155 mmmm Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr | 190
? Ala Cys Glu Val Thr His Gin Gly Leu ?Ser ?Ser Pro Val Thr Lys ?Ser 200 ? US 9 , 758 , 582 B2 35 36 - continued Phe Asn Arg Gly Glu Cys 210
< 210 > SED ID No 7 < 211 > LENGTH : 447 < 212 > TYPE : PRT m < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 7 ?Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 25 30 g& Thr Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 70 75 Leu Gln Met Asn Ser Leu Arq Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 90 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gin Gly Thr 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 150 155 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 170 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser mmmm195 200 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 230 235 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 250 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 310 315 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 330 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu mmmm mmmm mm 8mm mmmmmmmmmm 345 350 mmmmm US 9 , 758 , 582 B2 38 - continued Pro Pro Ser Arg Asp Glu Leu ?Thr ?Lys Asn Gin Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 375 380
Asn Gly Gin Pro Glu Asn Asn Tyr Lys ?Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 410 415 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met mmmHis Glu Ala 425 ? Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
< 210 > SEQ ID NO 8 < 211 > LENGTH : 215 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 8 Glu Ile Val Leu Thr Gin Ser Pro Gly ?Thr Leu Ser Leu Ser Pro Gly
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Gly Ser Ser ag& 30 1 Tyr Leu Ala Trp Tyr Gin Gin ?Lys Pro Gly Gin Ala Pro Arg Leu Leu 35
Ile Tyr 3Gly Ala Phe Ser Arg Ala ?Thr 3Gly Ile Pro Asp Arg Phe Ser 855 . Gly Ser Gly Ser Gly Thr Asp Phe ?Thr Leu Thr Ile Ser Arg Leu Glu 70 75 23 3ET&EPro RE&Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arq Thr Val Ala 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser 115 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arq Glu 135 Ala Lys Val Gin Trp emmaLys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser 150 155
Gin Glu Ser Val Thr Glu ?Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 1901 Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215
< 210 > SEQ ID NO 9 < 211 > LENGTH : 205 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223303 > OTHER INFORMATIONsmmm : Synthetic construct < 400 > SEQUENCEammmm : 9 US 9 , 758 , 582 B2 40 - continued
Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro 3Gly Ser Ala
Ala Ser &Pro HArg Leu Arg Glu Gly Pro Glu Leu Ser &Pro Asp Asp Pro 25 30 Ala Gly Leu Leu Asp Leu Arg Gin Gly Met Phe Ala Gin Leu Val Ala Gin Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp
Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val ETyr Tyr Val Phe Phe Gin Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val 105 110 Ser Leu Ala Leu His Leu Gin Pro Leu Arq Ser Ala Ala Gly Ala Ala
Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arq
Asn Ser Ala Phe Gly Phe Gin Gly Arg Leu Leu His Leu Ser Ala Gly
Gin Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala
Trp Gin Leu Thr Gin Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr 185 190 mmmmmm ?
?Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 195
< 210 > SEQ ID NO 10 < 22111 > LENGTH : 150 < 22121 1230 ^ > TYPE : PRT < 22131 ^> ORGANISM : Artificial Sequence AAAAA2 2 ^ FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 10 Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg &Pro Trp Asn Pro Pro Thr ? 15 Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe 25 30
Thr Cys Ser Phe Ser ?Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr
Arg Met Ser Pro Ser Asn Gin Thr Asp Lys Leu Ala Ala Phe Pro Glu 55 60 ?IS&Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gin Leu 2 80
Pro Asn 3Gly IArg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn 95 Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala 105 110 Gln Ile Lys Glu Ser Leu Arq Ala ggsgºGlu Leu Arq Val Thr Glu Arg amArg Ala Glu Val Pro Thr Ala HisHis ?ProPro Ser Propro Serser ProPro ArqArg Pro AlaAla Gly mgm135 140 Gln Phe Gin Thr Leu Val mmmmmm 145 mmmm US 9 ,758 ,582 B2 - continued
< 210 > SEQ ID NO 11 < 211 > LENGTH : 7 < 212 > TYPE : PRT ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 11 Glu Pro Lys Ser Cys Asp Lys 2
< 210 > SEO ID NO 12 < 211 > LENGTH : 1032 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 12 gctagcacca agggcccctc cgtgttccct ctggccccct ccagcaagtc cacctctggc 60 ggcaccgccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 120 tggaactctg gcgctctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 180 ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcca gctctctggg cacccagacc 240 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggaaccc 300 aagtcctgcg acaagaccca cacctgtccc ccctgccctg cccctgagct cctgggaggc 360 cctagcgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatctc ccggaccccc 420 gaagtgacct gcgtggtggt ggacgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 480 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacaac 540 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 600 gagtacaagt gcaaggtgtc caacaaggcc ctgcctgcccccatcgaaaa gaccatctcc 660 aaggccaagg gccagccccg cgagcctcag gtgtacaccc tgccccctag ccgggaagag 720 atgaccaaga accaggtgtc cctgacctgt ctggtcaagg gottctaccc ctccgatato 780 gccgtggaat gggagtccaa cggccagccc gagaacaact acaagaccac cccccctgtg 840 ctggactccg acggctcatt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 900 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 960 cagaagtccc tgtccctgag cccaggcaaa ggcggaggcg gatctggcgg cggaggatot 1020 ggtggcggat CC 1032
< 210 > SEQ ID NO 13 < 211 > LENGTH : 425 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 13 ggatccacca tccccccaca cgtgcagaaa tccgtgaaca acgacatgat cgtgaccgac 60 aacaacggcg ctgtgaagtt cccccagctg tgcaagttct gcgacgtgcg gttctctacc 120 tgcgacaacc agaaatcctg catgtccaac tgctccatca cctccatctg cgagaagccc 180 caggaagtgt gcgtcgccgt ctggcggaag aacgacgaga acatcaccct ggaaaccgtg 240 tgccacgacC ccaagctgcc ctaccacgac ttcatcctgg aagatgccgc ctcccccaag 300 US 9 ,758 ,582 B2 43 44 - continued tgcatcatga aggaaaagaa gaagcccggo gagactttct tcatgtgcag ctgctcctcc 360 gacgagtgca acgacaacat catcttctcc gaagagtaca acacctccaa ccccgactga 420 agott 425
< 210 > SEQ ID NO 14 < 211 > LENGTH : 430 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 14 goggccgcca tgaacttcgg cctgcggctg atcttcctgg tgctgaccct gaagggcgtg 60 cagtgcgagg tgcagctggt ggaatccggc ggaggcctgg tccagcctgg cggatctctg 120 agactgtcct gcgccgcctc cggcttcaac atcaaggaca cctacatcca ctgggtccga 180 caggcccctg gcaagggcct ggaatgggtg gcccggatct accccaccaa cggctacacc 240 agatacgccg actccgtgaa gggccggttc accatctccg ccgacacctc caagaacacc 300 gcctacctgc agatgaactc cctgcgggcc gaggacaccg ccgtgtacta ctgctccaga 360 tggggaggcg acggcttcta cgccatggac tactggggcc agggcaccct ggtcaccgtg 420 ctccgctagc 430
< 210 > SEQ ID NO 15 < 211 > LENGTH : 442 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 15 goggccgcca tggaatccca gacccaggtg ctgatctccc tgctgttctg ggtgtccggc 60 acctgtggcg acatccagat gacccagtcc ccctccagcc tgtccgcctc tgtgggcgac 120 agagtgacca tcacctgtcg ggcctcccag gacgtgaaca ccgccgtggc ctggtatcag 180 cagaagcccg gcaaggcccc caagctgctg atctactccg cctccttcct gtactccggo 240 gtgccctccc ggttctccgg ctctagatcc ggcaccgact ttaccctgac catctccago 300 ctgcagcccg aggacttcgc cacctactac toccagcagc actacaccac cccccccacc 360 tttggccagg gcaccaaggt ggaaatcaag cggaccgtgg ccgctccctc cgtgttcatc 420 cccaccctcc gacgagcagc tg 442
< 210 > SEQ ID NO 16 < 211 > LENGTH : 1032 < 212 > TYPE : DNA < 213M > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 16 gctagcacca agggcccctc cgtgtttccc ctggccccct ccagcaagtc cacctctggc 60 ggcaccgccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 120 tggaactctg gcgctctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 180 ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcca gctctctggg cacccagacc 240 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagcg ggtggaaccc 300 US 9 ,758 ,582 B2 45 46 - continued aagtcctgcg acaagaccca cacctgtccc ccctgccctg cccctgaact gctgggaggc 360 ccttccgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatctc ccggaccccc 420 gaagtgacct gcgtggtggt ggacgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 480 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacaac 540 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 600 gagtacaagt gcaaggtgtc caacaaggcc ctgcctgccc ccatcgaaaa gaccatctcc 660 aaggccaagg gccagccccg cgagcctcag gtgtacaccc tgcctcccag ccgggacgag 720 ctgaccaaga accaggtgtc cctgacctgt ctggtcaagg gottctaccc ctccgatatc 780 gccgtggaat gggagtccaa cggccagccc gagaacaact acaagaccac cccccctgtg 840 ctggactccg acggctcatt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 900 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacaco 960 cagaagtccc tgtctctgag ccccggcaaa ggcggcggag gatctggcgg tggcggatca 1020 ggcggaggat cc 1032
< 210 > SEQ ID NO 17 < 211 > LENGTH : 427 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence 20 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 17 gcggccgcca tgaacttcgg cctgcggctg atcttcctgg tgctgaccct gaagggcgtg 60 cagtgccagg tgcagctgaa gcagtccgga cctggcctgg tgcagccttc ccagtccctg 120 tccatcacct gtaccgtgtc cggcttctcc ctgaccaact acggcgtgca ctgggtccga 180 cagtccccag gcaagggcct ggaatggctg ggagtgattt ggagcggcgg caacaccgac 240 tacaacacoc ccttcacctc ccggctgtcc atcaacaagg acaactccaa gtcccaggtg 300 ttcttcaaga tgaactccct gcagtccaac gacaccgcca tctactactg cgccagagcc 360 ctgacctact atgactacga gttcgcctac tggggacagg gcaccctggt caccgtgtct 420 cgctagc 427
?< 210 > SEQ ID NO 18 ?< 211 > LENGTH : 442 ?< 212 > TYPE : DNA ?< 213 > ORGANISM : Artificial Sequence ?< 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 18 gcggccgcca tggaatccca gacccaggtg ctgatctccc tgctgttctg ggtgtccggc 60 acctgtggcg acatcctgct gacccagtcc cccgtgatcc tgtccgtgtc tcctggcgag 120 cgggtgtcct tctcctgccg ggcctcccag tccatcggca ccaacatcca ctggtatcag 180 cagcggacca acggctcccc tcggctgctg attaagtacg cctccgagtc tatctccggc 240 atcccctccc ggttctccgg ctctggctcc ggcaccgact tcaccctgtc catcaactcc 300 gtggaatccg aggatatcgc cgactactac tgccagcaga acaacaactg gcccaccacc 360 ttcggcgctg gcaccaagct ggaactgaag cggaccgtgg ccgctccctc cgtgttcatc 420 cccaccctcc gacgagcagc tg 442 US 9 ,758 ,582 B2 48 - continued < 210 > SEO ID NO 19 < 211 > LENGTH : 424 < 212 > TYPE : DNA < 2 ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 19 gcggccgcca tgaacttcgg cctgcggctg atcttcctgg tgctgaccct gaagggcgtg 60 cagtgccagg tgcagctggt ggaatccggc ggaggcgtgg tgcagcctgg cagatccctg 120 agactgtcct gcgccgcctc cggcttcacc ttctccagct acaccatgca ctgggtccga 180 caggcccctg gcaagggcct ggaatgggtc accttcatca gctacgacgg caacaacaag 240 tactacgccg actccgtgaa gggccggttc accatctccc gggacaactc caagaacacc 300 ctgtacctgc agatgaactc cctgcgggcc gaggacaccg ccatctacta ctgcgcccgg 360 accggctggc tgggcccttt tgattactgg ggccagggca ccctggtcac cgtgtcctcc 420 tagc 424
< 210 > SEQ ID NO 20 < 211 > LENGTH : 445 < 212 > TYPE : DNA ? 13 > ORGANISM : Artificial Sequence
? 20 > FEATURE : ?< NNN 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 20 goggccgcca tggaatccca gacccaggtg ctgatctccc tgctgttctg ggtgtccggo 60 acctgtggcg agatcgtgct gacccagtcc cccggcaccc tgtctctgag ccctggcgag 120 agagccaccc tgtcctgcag agcctcccag tccgtgggct cctcctacct ggcttggtat 180 cagcagaagc ccggccaggc ccctcggctg ctgatctacg gcgctttctc tcgggccacc 240 ggcatccctg accggttctc tggctccggc tccggcaccg acttcaccct gaccatctcc 300 cggctggaac ccgaggactt cgccgtgtac tactgccagc agtacggctc ctccccctgg 360 acctttggcc agggcaccaa ggtggaaatc aagcggaccg tggccgctcc ctccgtgtto 420 cttcccaccc tccgacgagc agctg 445
< 210 > SEQ ID NO 21 < 211 > LENGTH : 1035 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 21 gctagcacaa agggccctag tgtgtttcct ctggctccct cttccaaatc cacttctggt 60 ggcactgctg ctctgggatg cctggtgaag gattactttc ctgaacctgt gactgtctca 120 tggaactctg gtgctctgac ttctggtgtc cacactttcc ctgctgtgct gcagtctagt 180 ggactgtact ctctgtcatc tgtggtcact gtgccctctt catctctggg aacccagacc 240 tacatttgta atgtgaacca caaaccatcc aacactaaag tggacaaaaa agccgaaccc 300 aaatcctgtg acaaaaccca cacctgcoca ccttgtcctg cccctgaact gctgggagga 360 ccttctgtgt ttctgttccc accaaaacca aaagataccc tgatgatctc tagaacccct 420 gaggtgacat gtgtggtggt ggatgtgtct catgaggacc ctgaggtcaa atttaattgg 480 tacgtcgatg gagtggaagt ccacaatgcc aaaaccaagc ctagagagga acagtacaat 540 US 9 ,758 ,582 B2 49 50 - continued tcaacctaca gagtcgtcag tgtgctgact gtgctgcatc aggattggct gaatggcaag 600 gaatacaagt gtaaagtctc aaacaaggcc ctgcctgctc caattgagaa aacaatctca 660 aaggccaagg gacagcctag ggaaccccag gtctacacoc toccaccttc acgcgacgaa 720 ctgaccaaaa accaggtgtc cctgacatgc ctggtcaaag gcttctaccc ttctgacatt 780 gctgtggagt gggagtcaaa tggacagcct gagaacaact acaaaacaac cccccctgtg 840 ctggattctg atggctcttt ctttctgtac tccaaactga ctgtggacaa gtctagatgg 900 cagcagggga atgtcttttc ttgctctgtc atgcatgagg ctctgcataa ccactacact 960 cagaaatccc tgtctctgtc toccgggaaa ggcggcggag gatctggcgg aggcggttct 1020 ggtggtggcg gatcc 1035
< 210 > SEQ ID NO 22 < 211 > LENGTH : 435 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 22 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtcagg tgcagctgca gcagcctggt gccgagctcg tgaaacctgg cgcctccgtg 120z aagatgtcct gcaaggcctc cggotacacc ttcaccagct acaacatgca ctgggtcaag 180 cagacccccg gcagaggcct ggaatggatc ggcgctatct accccggcaa cggcgacacc 240 tcctacaacc agaagttcaa gggcaaggcc accctgaccg ccgacaagtc ctcttccacc 300 gcctacatge agctgtcctc cctgacctcc gaggactccg ccgtgtacta ctgcgcccgg 360 tctacctact acggcggcga ctggtacttc aacgtgtggg gcgctggcac caccgtgacc 420 gtgtctgctg ctago 435
< 210 > SEQ ID NO 23 < 211 > LENGTH : 405 ?.< 212 > TYPE : DNA ?< 213 > ORGANISM : Artificial Sequence ?< 220 > FEATURE : ?< 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 23 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc cagtgtcaga tcgtgctgtc ccagtcccct gccatcctgt ctgctagccc tggcgagaaa 120 gtgacaatga cctgccgggc ctcctcctcc gtgtcctaca tocactggtt ccagcagaag 180 cccggctcca gccccaagcc ttggatctac gccacctcca acctggcctc tggcgtgcca 240 gtgcggtttt ccggctctgg ctctggcacc tcctactccc tgaccatctc tcgggtggaa 300 gccgaggatg ccgccaccta ctactgccag cagtggacca gcaacccccc cacatttggc 360 ggaggcacca agctggaaat caagcggacc gtggcggcgc cctct 405
< 210 > SEQ ID NO 24 < 211 > LENGTH : 631 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 2 20 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 24 ggatccgcct gtccttgggc cgtgtccggc gctagagcct ctcctggctc tgccgcctcc 60 US 9 ,758 ,582 B2 - continued cccagactga gagagggccc tgagctgtcc cctgacgatc ctgccggcct gctggacctg 120 agacagggca tgtttgccca gctggtggcc cagaacgtgc tgctgatcga cggccccctg 180 tcctggtact ctgatcctgg cctggccggc gtgtccctga ccggcggact gtcctacaaa 240 gaggacacca aagaactggt ggtggccaag gctggcgtgt actacgtgtt ctttcagctg 300 gaactgeggc gggtggtggc cggcgagggc tctggatctg tgtccctggc cctgcatctg 360 cagcccctga gatctgccgc tggcgccgct gctctggccc tgacagtgga tctgcctcct 420 gcctcctccg aggcccggaa ctccgcattc gggtttcagg gccggctgct gcacctgtct 480 gctggccaga gactgggagt gcatctgcac accgaggcca gagccagaca cgcctggcag 540 ctgacccagg gcgctaccgt gctgggcctg ttcagagtga cccccgagat cccagccggc 600 ctgcccagcc ctagatccga gtgataagct t 631
< 210 > SEQ ID NO 25 < 211 > LENGTH : 1458 < 212 > TYPE : DNA V 13 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223NNAPWOWN > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 25 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtcagg tgcagctgca ggaatctggc cctggactcg tgcggccttc ccaaaccctg 120 tctctgacct gtaccgtgtc cggctactcc atcacctccg accacgcctg gtcttgggtg 180 cgacagcctc ctggcagagg cctggaatgg atcggctaca tctcctactc cggcatcacc 240 acctacaacc ccagcctgaa gtccagagtg accatgctgc gggacacctc caagaaccag 300 ttctccctgc ggctgtcctc cgtgaccgct gctgataccg ccgtgtacta ctgcgccaga 360 tctctggcca ggaccaccgc catggattac tggggccagg gctccctcgt gaccgtgtcc 420 tctgctagca ccaagggccc ctccgtgttc cctctggccc cttcctctaa atctacctct 480 ggcggcaccg ccgctctggg ctgcctcgtg aaggactact tccccgagcc cgtgacagtg 540 tcttggaact ctggcgccct gacctccggc gtgcacacct ttccagctgt gctgcagtcc 600 tccggcctgt actccctgtc cagcgtcgtg actgtgccct cctcatctct gggcacccag 660 acctacatct gcaacgtgaa ccacaagccc tccaacacca aggtggacaa gaaggtggaa 720 cccaagtcct gcgacaagac ccacacctgt cccccttgtc ctgcccctga actgctgggc 780 ggaccctctg tgttcctgtt cccaccaaaa ccgaaagaca ccctgatgat ctcccggacc 840 cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 900 tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 960 aactccacct accgggtggt gtccgtgctg accgtgctgc accaggattg gctgaacggc 1020 aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatega aaagaccato 1080 tccaaggcca agggccagcc acgggaaccc caggtgtaca cactgccccc tagccgcgac 1140 gagctgacca agaatcaggt gtccctgaca tgcctcgtga aaggettcta cccctccgat 1200 atcgccgtgg aatgggagtc caacggccag cctgagaaca actacaagac caccccccct 1260 gtgctggact ccgacggctc attcttcctg tactcaaagc tgacagtgga caagtcccgg 1320 tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1380 acccagaagt ccctgtccct gagccccggg aaaggcggcg gaggatctgg cggaggcggt 1440 US 9 ,758 ,582 B2 5454 - continued tctggtggtg goggatcc 1458
< 210 > SEQ ID NO 26 < 211 > LENGTH : 405 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 26 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtgaca tccagatgac ccagtccccc tccagcctgt ctgcctctgt gggcgacaga 120 gtgaccatca cctgtcgggc ctcccaggac atctcctcct acctgaactg gtatcagcag 180 aagcccggca aggcccccaa gctgctgatc tactacacct cccggctgca ctccggcgtg 240 ccctctagat tttccggctc tggctccggc accgacttta ccttcaccat cagctccctg 300 cagcccgagg atatcgccac ctactactgc cagcaaggca acaccctgcc ctacaccttt 360 ggccagggca ccaaggtgga aatcaagcgg accgtggcgg cgccc 405
< 210 > SEQ ID NO 27 < 211 > LENGTH : 1455 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence ? 20 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 27 goggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc cagtgtcagg tgcagctgca gcagtgggga gctggactgc tgaagccctc cgagacactg 120 tctctgacct gcgctgtgta cggcggctcc ttctccggct actactggtc ctggattcgg 180 cagtcccctg agaagggcct ggaatggatc ggcgagatca accacggcgg ctacgtgacc 240 tacaacccca gcctggaatc cagagtgacc atctccgtgg acacctccaa gaaccagttc 300 tccctgaagc tgtcctccgt gaccgccgct gataccgccg tgtactactg cgccagagac 360 tacggccctg gcaactacga ctggtacttc gacctgtggg gcagaggcac cctcgtgacc 420 gtgtcctctg ctagcaccaa gggcccctcc gtgtttcctc tggccccttg ctcacgctcc 480 acctccgaat ctaccgccgc tctgggctgc ctcgtgaagg actacttccc cgagcccgtg 540 actgtgtctt ggaactctgg cgccctgacctccggcgtgc acacctttcc agctgtgctg 600 cagtcctccg gcctgtactc cctgtccagc gtcgtgacag tgccctccag ctctctgggc 660 accaagacct acacctgtaa cgtggaccac aagccctcca acaccaaggt ggacaagcgg 720 gtggaatcta aatacggccc tccctgccct ccttgcccag cccctgaatt tctgggcgga 780 ccttccgtgt tcctgttccc cccaaaaccc aaggacaccc tgatgatctc ccggaccccc 840 gaagtgacct gcgtggtggt ggatgtgtcc caggaagatc ccgaggtgca gttcaattgg 900 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ctagagagga acagttcaac 960 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggattggct gaacggcaaa 1020 gagtacaagt gcaaggtgtc caacaagggc ctgcccagct ccatcgaaaa gaccatcagc 1080 aaggccaagg gccagccccg ggaaccccag gtgtacacac tgcctccaag ccaggaagag 1140 atgaccaaga atcaggtgtc cctgacctgt ctcgtgaaag gottctaccc ctccgatatc 1200 gccgtggaat gggagtccaa cggccagcct gagaacaact acaagaccac cccccctgtg 1260 ctggactccg acggcagctt cttcctgtac tctcgcctga ccgtggacaa gtcccggtgg 1320 US 9 , 758 , 582 B2 55 56 - continued caggaaggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 1380 cagaagtccc tgtccctgtc tctggggaaa ggcggcggag gatctggcgg aggcggttct 1440 ggtggtggcg gatec 1455
< 210 > SEQ ID NO 28 < 211 > LENGTH : 411 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : AAAA< 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 28 goggccgoca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc . | 60 cagtgtgaga tcgtgctgac ccagtctcct gccaccctgt ctctgagccc tggcgagaga 120 gctaccctgt cctgccgtgc ctcccaatcc gtgtcctctt acctggcctg gtatcagcaa 180 aagcccggcc aggctccccg gctgctgatc tacgatgcct ccaatagagc caccggcatc 240 cctgccagat tctccggctc tggctctggc accgacttta ccctgaccat ctcctctctg 300 gaacccgagg acttcgccgt gtactactgc cagcagcggt ccaactggcc tcccgccctg 360 acatttggcg gaggcaccaa ggtggaaatc aagcggaccg tggcggcgccc 411.
< 210 > SEO ID No 29 < 211 > LENGTH : 449 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : 3 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 29 ?Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Asp Tyr Thr Phe Thr Tyr Tyr Gly Met Asn Trp Val Lys Gin Ala Pro Gly Gin Gly Leu Lys Trp Met 35 Gly Trp Ile Asp Thr Thr Thr Gly Glu Pro Thr Tyr Ala Gin Lys Phe
Gin Gly Arg Ile Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gin Ile Lys Ser Leu Lys Ser Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg mmmArg Gly Pro Tyr Asn Trp Tyr Phe Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro ? ? ? 195 am ? US 9 , 758 , 582 B2 57 58 - continued
Ser Asn Thr Lys Val Asp Lys Lys Val Glu &Pro Lys Ser Cys Asp Lys 220
?Thr His Thr RCys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 235 &240 Ser ?3?Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 255 &T RArg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp &260 265 270 23| &Pro Glu Val Lys Phe Asn PERJTrp Tyr Val Asp Gly Val Glu Val His Asn 275 280
EAla Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 300 Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 315 320 Tyr Lys Cys Lys Val Ser Asn &EngLys Ala Leu Pro Ala Pro Ile Glu Lys gggggggg? 335 Thr Ile Ser Lys Ala Lys Gly Gin &Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 mmmm Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 380 Ser Asn EGly Gin Pro Glu Asn Asn Tyr Lys Thr Thr 8mmPro Pro Val Leu 385 mmmm ES 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 415
Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met nHis Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser &Pro Gly 435 mmmmmm 440 Lys
< 210 > SEQ ID NO 30 < 211 > LENGTH : 219 | < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : AAAA< 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 30 | Asp Ile Val ?Met Thr ?Gln Ser Pro Leu Ser Val Pro Val ?Thr Pro Gly ?& a 515 Glu Pro 3Val 8Ser .EpIle Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25
Asn Gly Asn ?Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser g35 45
&Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Val Ser Gly Val Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 1ggAla Phe Thr Leu Arg Ile JRA 70 mm Ser Arg Val Glu EAla Glu Asp Val Gly Val Tyr Tyr Cys Leu Gin His 95 Leu Glu Tyr Pro Phe Thr Phe Gly Pro Gly Thr Lys Leu Glu Leu Lys 100 105 sm US 9 , 758 , 582 B2
- continued Arg ?Thr Val Ala Ala Pro Ser Val Phe ?Ile Phe Pro Pro Ser Asp Glu 120 | 125 |4 Gin Leu Lys Ser Gly Thr Ala Ser Val Val ECys Leu Leu Asn Asn Phe 135 Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 150 am 160 Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser 170 175 4s5 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 E 1 Lys His Lys Val Tyr Ala cys Glu Val Thr His Gin Gly Leu 5Ser Ser 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
< 210 > SEQ ID NO 31 < 211 > LENGTH : 460 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct ?< 400 > SEQUENCE : 31 Glu Val Gin Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly ?Thr ?Phe Ser Ser Tyr on 25 Ala Ile Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 40 . &3? Gly Gly Ile Ile &Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gin Lys Phe
Gin Gly Arg Val Thr Ile Thr Ala Asp Lys Ser ?Thr Ser Thr Ala Tyr 70 Met Glu Leu Ser Ser Leu Arg mamSer Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arq Ala Pro Leu Arq Phe Leu Glu Trp Ser ?Thr Gln Asp His Tyr 105
Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly ?Thr Thr Val Thr Val 120
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala &Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu gVal Lys 150 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu 185
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 200 Gin Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His ?Thr Cys Pro 230 mmmm Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe ? mmmmm mmmmm mmmm ? US 9 , 758 , 582 B2
- continued Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 265 270
Thr ECys Val Val Val Asp Val Ser His Glu Asp &Pro Glu Val Lys Phe 275 280
Asn Trp Tyr Val Asp 3Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 290 ECE 300 Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 305 310 315 320 Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 335
Ser Asn Lys Ala Leu Pro Ala &Pro Ile Glu Lys Thr Ile Ser Lys Ala 345
Lys Gly Gln Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg 355 360 Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly 370 mmm 380 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro 385 390 mmm 395 400 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 415
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin mmmm 425 mmm Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 435 440 Tyr Thr Gin Lys Ser Leu Ser Leu Ser ?Pro Gly Lys 450 ? 460
A< 2101 > SEQ ID NO 32 A< 211 > LENGTH : 213 | A< 212 > TYPE : PRT A< 213 > ORGANISM : Artificial Sequence A< 220 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 32 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln . | 5
Thr 3Val Arg Ile Thr ?Cys 3Gin 3Gly Asp Ser Leu Arg Ser Tyr Tyr Ala o
Thr Trp 2Tyr Gin Gin Lys .Pro Gly 5Gin Ala Pro Ile Leu Val Ile Tyr 35 ?2? Gly Glu Asn Lys Arg &Pro Ser Gly °Ile &Pro Asp Arg Phe Ser Gly Ser *isSer Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gin Ala Glu E2 | 75 TEPE& HER&a Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Gly Ser Gly Gin His
Leu Val Phe Gly Gly Gly ?Thr Lys Leu Thr Val Leu Gly Gin Pro Lys
Ala Ala &Pro Ser Val ?Thr Leu Phe Pro &Pro Ser Ser Glu Glu Leu Gin 115 Ala Asn Lys Ala 3?Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly ? ? Lys Ala Asp ser gggggg155 sam 160 US 9 , 758 , 582 B2 63. 64 - continued Val Glu Thr ?Thr Thr Pro Ser Lys Gin Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gin Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gin |pnVal Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Ala Glu Cys 210
ER< 210 > SEO ID NO 33 < 211 > LENGTH : 449 < 212 > TYPE : PRT < 213 , ORGANISM : Artificial Sequence < 220 > FEATURE : 223 > OTHER INFORMATION : Synthetic construct < 400 > SEQUENCE : 33 Gin Val Gin Leu Gin Gin Ser Gly Ala Glu Val Lys Lys Phe Gly Ala Ser Val Lys Val Ser Cys Glu Ala Ser Gly Tyr Thr Phe Pro Ser Tyr 3 0 Val Leu His Trp Val Lys Gin Ala Pro Gly Gin Gly Leu Glu Trp Ile 35 40 45 m3Gly Tyr Ile Asn Pro Tyr 13?Asn Asp Gly Thr Gin Thr Asn Lys Lys Phe Lys Gly Lys Ala Thr ESLeu Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 Ala Arg Gly Phe Gly Gly Ser Tyr Gly Phe Ala Tyr Asn Gly Gin Gly mm 110 Egg Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser ?mmGly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val Asn Thr Phe Pro Ala Val Leu 165 de& Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro | 195 mammmmm 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu mmmmLeu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Asn Ile Ser 245 ? Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 mm 285 Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val mm mmmm mmmm mmmmmm am 300 US 9 , 758 , 582 B2 65 - continued | Val ?Ser Val Leu ?Thr Val Leu His Gin Asp Trp ?Leu Asn Gly Lys Glu 305 | g310 315 1 320 | Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu &Pro Ala Pro Ile Glu Lys BE 325 335 Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr ?Thr 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu ?Thr 355 360 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 375 Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys ROLeu Thr Val Asp Lys 405 415 Ser Arq Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Asn His Glu ? 425 430 Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 ggggg ? Lys
< 210 > SEQ ID NO 34 < 211 > LENGTH : 215 < 212 > TYPE : PRT < 213 > ORGANISM : Artificial Sequence A 20 > FEATURE : < 223 > OTHER INFORMATION : Synthetic construct < 400 > SEOUENCE : 34 Asp Ile Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
Asp Arq Val Thr Met ?Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser 25 30
ETyr Leu Tyr Trp Tyr 3Gin Gin Lys Pro *Gly Lys Ala Pro Lys Leu Trp E3 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser * 60
Gly Ser Gly Ser Gly Thr Asp Phe ?Thr Leu Thr Ile Ser Ser DLeu Gin 65 170 75 &Pro Glu Asp Ser Ala Ser Tyr Phe Cys His Gin Trp Asn Arg Tyr Pro ?
Tyr Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala 105 | 1101 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Glu Ala Lys 140
g 3Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu 145 150 155 Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
?Thr Leu Thr Leu Ser &Pro Arg Lys Ala Asp Tyr Glu Lys His Lys Val mmm 185 190 Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys ? 205 Ser Phe Asn mmmArg Gly .Glu Cys