(12) United States Patent (10) Patent No.: US 8,815,247 B2 Govindappa Et Al

US00881.5247B2

(12) United States Patent (10) Patent No.: US 8,815,247 B2 Govindappa et al. (45) Date of Patent: Aug. 26, 2014

(54) TARGETED/IMMUNOMODULATORY (56) References Cited FUSION PROTEINS FOREIGN PATENT DOCUMENTS (71) Applicant: Biocon Limited, Bangalore (IN) WO WO 98.33914 8, 1998 WO WO 2009/039.409 3, 2009 (72) Inventors: Nagaraj Govindappa, Karnataka (IN); WO WO 2009/114110 9, 2009 Kedarnath Sastry, Karnataka (IN); WO WO 2011, 109789 9, 2011 Maria Melina Soares, Karnatake (IN) OTHER PUBLICATIONS (73) Assignee: Biocon Limited (IN) Birch, J. R. et al. "Antibody production.” Advanced Drug Delivery Reviews, vol. 58, Nos. 5-6, Aug. 7, 2006. Carton et al. "Codon engineering for improved antibody expression (*) Notice: Subject to any disclaimer the term of this in mammalian cells.” Protein Expression and Purification, Academic patent is extended or adjusted under 35 Press, vol. 55, No. 2, Sep. 8, 2007. U.S.C. 154(b) by 0 days. Casi, Giulio et al. "Antibody drug conjugates: Basic concepts, examples and future perspectives.” Journal of Controlled Release, vol. 161, No. 2, Jan. 10, 2012. (21) Appl. No.: 13/799,409 Kalwy, S. etal. “Toward more efficient protein expression.” Molecu lar Biotechnology, Humana Press, Inc., vol.34, No. 2, Sp. Iss. SI, Oct. 22) Filed: Mar 13, 2013 1, 2006. (22) 9 Kotsopoulou, E. et al. "Optimised mammalian expression through O O the coupling of codon adaptation with gene amplification; maximum (65) Prior Publication Data yields with minimum effort.” Journal of Biotechnology, vol. 146, No. 4, Apr. 15, 2010. US 2013/02878O2A1 Oct. 31, 2013 Ortiz-Sanchez, Elizabeth et al. "Antibody cytokine fusion proteins: applications in cancer therapy.” Expert Opinion on Biological (30) Foreign Application Priority Data Therapy, vol. 8, No. 5, May 1, 2008.

Apr. 30, 2012 (IN) 1689/CHFA2012 Primary Examiner — Elly-Gerald Stoica Apr. 30, 2012 (IN) ...... 1690 CHE/2012 (74) Attorney, Agent, or Firm – Marianne Fuierer; Moore & Van Allen, PLLC (51) Int. Cl. (57) ABSTRACT A6 IK39/00 (2006.01) The present invention relates generally to the field of gener (52) U.S. Cl. ating fusion proteins to be used in cancer therapy, and more USPC ...... 424/179.1; 424/1341; 424/185.1 specifically, to nucleotide sequences encoding the fusion pro 424/192.1; 435/69.7:435/328; 530/387.3 teins, wherein the chimeric fusion proteins comprises at least (58) Field of Classification Search one targeting moiety and at least one immunomodulatory None moiety that counteracts the immune tolerance of cancer cells. See application file for complete search history. 1 Claim, 65 Drawing Sheets U.S. Patent Aug. 26, 2014 Sheet 1 of 65 US 8,815,247 B2

Anti-HER2/neu-TGF3Ril fusion protein at LC constant region Amino acid sequence of Anti-HER2/neu heavy chain:

EVOLVESGGGLVOPGGSLRLSCAASGFNIKDTYHWVROAPGK GLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLOMNSL RAEDTAVYYCSRWGGDGFYAMDYWGOGTLWTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWWTVPSSSLGTOTYCNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYN STYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPEKTISKAKG QPREPOVYTLPPSREEMTKNOWSLTCLVKGFYPSDAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOQGNVFSCS VMHEALHNHYTOKSLSLSPG Amino acid sequence of Anti-HER2/neu light chain fusion protein:

DOMTOSPSSLSASVGDRVTITCRASODVNTAVAWYOOKPGKA PKLLYSASFLYSGVPSRFSGSRSGTDFTLTISSLOPEDFATYYC QQHYTTPPTFGOGTKVEKRTVAAPSVFIFPPSDEQLKSGTASW WCLLNNFYPREAKVOWKVDNALOSGNSQESVTECRDSKDSTYS LSSTLTLSKADYEKHKWYACEWTHQGLSSPVTKSFNRGECGGG GSGGGGSGGGGSTIPPHVOKSVNNDMIVTDNNGAVKFPOLCK FCDVRFSTCDNQKSCMSNCSITSICEKPOEVCVAVWRKNDENI TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC SSDECNDNIFSEEYNTSNPD

Figure 1 U.S. Patent Aug. 26, 2014 Sheet 2 of 65 US 8,815,247 B2

Anti-EGFR1-TGFBRI fusion protein at LC constant region Amino acid sequence of Anti-EGFR1 heavy chain:

OVOLKOSGPGLVOPSQSLSITCTVSGFSLTNYGVHWVROSPG KGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSL OSNDTAYYCARALTYYDYEFAYWGOGTLVTVSAASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSWTVPSSSLGTOTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHODWLNGKEYKCKVSNKALPAPEKTISKAKGOP REPORVYTLPPSRDELTKNOWSLTCLVKGFYPSDAVEWESNGO PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOQGNWFSCSVM HEALHNHYTOKSLSLSPG

Amino acid sequence of Anti-EGFR1 light chain fusion protein:

DLLTOSPVILSVSPGERVSFSCRASOSIGTNIHWYOORTNGSP RLLKYASESSGPSRFSGSGSGTDFTLSINSVESEDIADYYCOO NNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEOLKSGTASWC LLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLS STLTLSKADYEKHKWYACEVTHOGLSSPVTKSFNRGECGGGGS GGGGSGGGGSTIPPHVOKSVNNDMVTDNNGAVKFPQLCKFC DVRFSTCDNQKSCMSNCSITSICEKPOEVCVAVWRKNDENITL ETVCHDPKLPYHDFILEDAASPKCMKEKKKPGETFFMCSCSS DECNDNIFSEEYNTSNPD Figure 2 U.S. Patent Aug. 26, 2014 Sheet 3 of 65 US 8,815,247 B2

Anti-CTLA4-TGFBRI fusion protein at LC constant region Amino acid sequence of anti-CTLA4 heavy chain: OVOLVESGGGWOPGRSLRLSCAASGFTFSSYTMHWVROAP GKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLOMN SLRAEDTAIYYCARTGWLGPFDYWGOGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLOSSGLYSLSSWWTVPSSSLGTOTYCNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTP EVTCVVVDVSHEDPEVKFNWYWDGVEVHNAKTKPREEOYNST YRVVSVLTVLHODWLNGKEYKCKVSNKALPAPEKTISKAKGOP REPOVYTLPPSRDELTKNOWSLTCLVKGFYPSDIAVEWESNGO PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVM HEALHNHYTOKSLSLSPG Amino acid sequence of anti-CTLA4 light chain fusion protein:

EIVLTQSPGTLSLSPGERATLSCRASOSVGSSYLAWYOOKPGO APRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY CQQYGSSPWTFGOGTKVEKRTVAAPSVFIFPPSDEOLKSGTA SWCLLNNFYPREAKVOWKVDNALOSGNSOESVTEQDSKDST YSLSSTLTLSKADYEKHKWYACEVTHQGLSSPVTKSFNRGECG GGGSGGGGSGGGGSTIPPHVORKSVNNDMIVTDNNGAVKFPQ LCKFCDVRFSTCDNQKSCMSNCSITSICEKPOEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFM CSCSSDECNDNIFSEEYNTSNPD Figure 3 U.S. Patent Aug. 26, 2014 Sheet 4 of 65 US 8,815,247 B2

Anti-HER2/neu HC-4-1BB and LC-TGFBRI fusion protein: Amino acid sequence of heavy chain-4-1BB fusion protein:

EVOLVESGGGLVOPGGSLRLSCAASGFNKOTYHWVROAPGKGLEWVA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLOMNSLRAEDTAVYYCSRW GGDGFYAMDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLG TOTYCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHCRDWLNGKEYKCKVSNKALPAPEKTISKAKGOP REPOVYTLPPSREEMTKNOWSLTCLVKGFYPSDIAVEWESNGOPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWOQGNVFSCSVMHEALHNHYTORKSL SLSPGGGGGSGGGGSGGGGSACPWAVSGARASPGSAASPRL1REGPE LSp0D?AGLLDLROGMFAQLVAQNVLLIDGPLSWYSD-pGLAGVSLTG GLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHELQ:PLR SAAGAAALALTVOLPPASSEARNSAFGFQGRLLHCLSAGQRLGVHLHCT tARARHAWQLTQGATVLGLFRVTipt IPAGLPSPRSE Amino acid sequence of light chain-TGFBRIl fusion protein:

DOMTOSPSSLSASVGDRVTITCRASODVNTAVAWYOOKPGKAPKLLYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCOOHYTTPPTFGO GTKVEKRTVAAPSVFIFPPSDECQLKSGTASWCLLNNFYPREAKVOWKV DNALOSGNSOESVTEQDSKDSTYSLSSTLTLSKADYEKHKWYACEVTHQ GLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVORKSVNNDMVTD NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSTSICEKPOEVCVAVW RKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC SSDECNDNIFSEEYNTSNPD

Figure 4 U.S. Patent Aug. 26, 2014 Sheet 5 of 65 US 8,815,247 B2

Anti-EGFR1 HC-4-1BB and LC-TGF3RI fusion protein: Amino acid sequence of heavy chain-4-1BB fusion protein:

QVOLKOSGPGLVOPSQSLSITCTVSGFSTNYGVHWVROSPGKGLEWL GVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLOSNDTAIYYCARA LTYYDYEFAYWGOGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCL WKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWWTVPSSSLGT OTYCNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE OYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGOPR EPOVYTLPPSRDELTKNOWSLTCLVKGFYPSDAVEWESNGOPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSLS LSPGGGGGSGGGGSGGGGSACPWAVSGARASPGSAASPRLREGPEL SpoopAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDpGLAGVSLTGG LSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHELQ:PLRS AAGAAALALTVDLPPASSEARNSAFGFQGRLLHELSAGQRLGVHLHETE ARARHAWQLTQGATVLGLFRVTipt IpAGLPSPRSE

Amino acid sequence of light chain-TGFBRIl fusion protein:

DILLTOSPVILSVSPGERVSFSCRASOSIGTNIHWYOORTNGSPRLLIKYA SESSGPSRFSGSGSGTDFTLSNSVESEDIADYYCOONNNWPTTFGAG TKLELKRTVAAPSVFIFPPSDECRLKSGTASWCLLNNFYPREAKVOWKVD NALOSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKWYACEVTHQG LSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVOKSVNNDMIVTDN NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPCREVCVAVWR KNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCS SDECNDNIFSEEYNTSNPD

Figure 5 U.S. Patent Aug. 26, 2014 Sheet 6 of 65 US 8,815,247 B2

Anti-CTLA4 HC-4-1BB and LC-TGFBRI fusion protein: Amino acid sequence of heavy chain-4-1BB fusion protein:

QVOLVESGGGVVOPGRSLRLSCAASGFTFSSYTMHWVROAPGKGLEW WTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAIYYCA RTGWLGPFDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL WKDYFPEPVTVSWNSGALTSGWHTFPAVLOSSGLYSLSSWWTVPSSSLGT QTYCNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE OYNSTYRVVSVLTVLHCRDWLNGKEYKCKVSNKALPAPEKTISKAKGOPR EPOVYTLPPSRDELTKNOWSLTCLVKGFYPSDAVEWESNGOPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWOOGNWFSCSVMHEALHNHYTOKSLS LSPGGGGGSGGGGSGGGGSACPWAVSGARASPGSAASPRLREGPEL SpoDpAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGG LSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHELQpLRS AAGAAALALTVDLPPASSEARNSAFGFQGRLLHELSAGQRLGVHLHETE ARARHAWQLTQGATVLGLFRVTipt IpAGLPSPRSE

Amino acid sequence of light chain-TGFBRI fusion protein:

EVLTQSPGTLSLSPGERATLSCRASCRSVGSSYLAWYOOKPGGRAPRLLY GAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCOOYGSSPWTF GOGTKVEKRTVAAPSVFIFPPSDEOLKSGTASWCLLNNFYPREAKVCRW KVDNALOSGNSOESVTEQDSKDSTYSLSSTLTLSKADYEKHKWYACEVT HQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVORKSVNNDMIV TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSTSCEKPOEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMC SCSSDECNDNIFSEEYNTSNPD

Figure 6 U.S. Patent Aug. 26, 2014 Sheet 7 of 65 US 8,815,247 B2

Anti-HER2/neu HC-PD1 and LC-TGFBRI fusion protein: Amino acid sequence of heavy chain-PD1 fusion protein: EVOLVESGGGLVOPGGSLRLSCAASGFNIKDTYHWVROAPGKGLEWVA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLOMNSLRAEDTAVYYCSRW GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWWTVPSSSLG TOTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYWDGVEVHNAKTKPRE ECRYNSTYRVVSVLTVLHCRDWLNGKEYKCKVSNKALPAPEKTISKAKGOP REPOVYTLPPSREEMTKNOWSLTCLVKGFYPSDIAVEWESNGOPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWOQGNVFSCSVMHEALHNHYTOKSL SLSPGGGGGSGGGGSGGGGSpGMUFLDSPORPMUN22TFSpALLVVTE GDNATFTCSFSNTSESTVLNWYRMSPSNQTDKLAAFPEDRSQpGQD CRFRVTQLPNGRDFHMSWRARRNDSGTYLCGAISLAPKAQIKESL RAELRVTERRAEVPTAHPSPSPRpAGQFQTLV Amino acid sequence of light chain-TGF3RI fusion protein: DIQMTOSPSSLSASVGDRVTITCRASQDVNTAVAWYOOKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLOPEDFATYYCOQHYTTPPTFGO GTKVEKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVOWKV DNALOSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKWYACEVTHQ GLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVOKSVNNDMIVTD NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPCREVCVAVW RKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC SSDECNDNIFSEEYNTSNPD

Figure 7 U.S. Patent Aug. 26, 2014 Sheet 8 of 65 US 8,815,247 B2

Anti-EGFR1 HC-PD1 and LC-TGFBRI fusion protein: Amino acid sequence of heavy chain-PD1 fusion protein:

QVOLKOSGPGLVOPSQSLSTCTVSGFSLTNYGVHWVROSPGKGLEWL GVWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLOSNDTAYYCARA LTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCL WKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE CRYNSTYRVVSVLTVLHCRDWLNGKEYKCKVSNKALPAPEKTISKAKGOPR EPOWYTLPPSRDELTKNOWSLTCLVKGFYPSDAVEWESNGOPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSLS LSPGGGGGSGGGGSGGGGSPGWFLDSPDRPWNPPTFSPALLVVTEG DNATTTCSTSNTSESTVLNWYRMSPSNQTDKLAAFPEDRSQPGQDC rFRVTQLpNGRDFHMSVVRARRNDSGTYLCGAISLApKAQIKESLR AELRVTERRAEVPTAHPSPSPRPAGQFQTLV Amino acid sequence of light chain-TGFBRI fusion protein:

DILLTQSPVILSVSPGERVSFSCRASOSIGTNIHWYOORTNGSPRLLIKYA SESSGPSRFSGSGSGTDFTLSNSVESEDIADYYCOONNNWPTFGAG TKLELKRTVAAPSVFIFPPSDEQLKSGTASVWCLLNNFYPREAKVOWKVD NALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKWYACEVTHQG LSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVOKSVNNDMVTDN NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPOEVCVAVWR KNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCS SDECNDNIFSEEYNTSNPD

Figure 8 U.S. Patent Aug. 26, 2014 Sheet 9 of 65 US 8,815,247 B2

Anti-CTLA4 HC-PD1 and LC-TGFBRI fusion protein: Amino acid sequence of heavy chain-PD1 fusion protein: QVOLVESGGGWOPGRSLRLSCAASGFTFSSYTMHWVROAPGKGLEW VTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAYYCA RTGWLGPFDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL WKDYFPEPVVSWNSGALTSGVHTFPAVLOSSGLYSLSSWTVPSSSLGT QTYCNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGOPR EPOVYTLPPSRDELTKNOWSLTCLVKGFYPSDIAVEWESNGOPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSLS LSPGGGGGSGGGGSGGGGSpGWFLDSPOR:PWNppTFSPALLVVTEG DNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQpGQDC RFRVTQLPNGRDF(MSVVRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTERRAEVPTAH PSPSPRPAGQFQTLV Amino acid sequence of light chain-TGFBRI fusionprotein:

EIVLTOSPGTLSLSPGERATLSCRASOSVGSSYLAWYOOKPGOAPRLLIY GAFSRATGPDRFSGSGSGTDFTLTISRLEPEDFAVYYCOOYGSSPWTF GQGTKVEIKRTVAAPSVFIFPPSDEOLKSGTASWCLLNNFYPREAKVOW KVDNALOSGNSORESVTEQDSKDSTYSLSSTLTLSKADYEKHKWYACEVT HQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVORKSVNNDMIV TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPCREVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCMKEKKKPGETFFMC SCSSDECNDNIFSEEYNTSNPD

Figure 9 U.S. Patent Aug. 26, 2014 Sheet 10 of 65 US 8,815,247 B2

Anti-HER2/neu HC-TGFBRIl-4-1BB fusion protein Amino acid sequence of heavy chain-TGFBRIl-4-1BB fusion protein:

EVOLVESGGGLVOPGGSLRLSCAASGFNIKDTYHWVROAPGKGLEWVA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLORMNSLRAEDTAVYYCSRW GGDGFYAMDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLG TOTYCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPEKTISKAKGOP REPOVYTLPPSREEMTKNOWSLTCLVKGFYPSDIAVEWESNGOPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTORKSL SLSPGKGGGGSGGGGSGGGGSTPPHVOKSVNNDMVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPOEVCVAVWRKNDENITL ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDN IIFSEEYNTSNPDEPKSCDKACPWAVSGARASPGSAASPRLREGPELSP DOpAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDpGLAGVSLTGGLS YKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHELQ:PLRSA AGAAALALTVDLP:PASSEARNSAFGFQGRLLHELSAGQRLGVHLHETEA RARHAWQLTQGATVLGLFRVTipt IPAGLPSPRSE Amino acid sequence of light chain:

DOMTOSPSSLSASVGDRVTITCRASODVNTAVAWYOOKPGKAPKLLYS ASFLYSGVPSRFSGSRSGTDFTLTSSLOPEDFATYYCOQHYTTPPTFGO GTKVEKRTVAAPSVFIFPPSDEOLKSGTASWCLLNNFYPREAKVOWKV DNALOSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKWYACEWTHQ GLSSPVTKSFNRGEC

Figure 10 U.S. Patent Aug. 26, 2014 Sheet 11 of 65 US 8,815,247 B2

Anti-EGFR1 HC-TGFBRIl-4-1BB fusion protein Amino acid sequence of heavy chain-TGFBRIl-4-1BB fusion protein:

OVOLKOSGPGLVCRPSQSLSTCTVSGFSLTNYGVHWVROSPGKGLEWL GVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARA LTYYDYEFAYWGOGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCL WKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGOPR EPOVYTLPPSRDELTKNOWSLTCLVKGFYPSDAVEWESNGOPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWOQGNVFSCSVMHEALHNHYTOKSLS LSPGKGGGGSGGGGSGGGGSTPPHVOKSVNNDMIVTDNNGAVKFPQL CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIF SEEYNTSNPDEPKSCDKACPWAVSGARASPGSAASpRLREG.pELSpid OpAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSD:PGLAGVSLTGGLSY KEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHELQPLRSAA GAAALALTVDLPPASSEARNSAFGFQGRLLHCLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVT pt IpAGLpSPRSE Amino acid sequence of light chain:

DILLTOSPVILSVSPGERVSFSCRASOSGTNIHWYOORTNGSPRLLIKYA SESSGIPSRFSGSGSGTDFTLSINSVESEDIADYYCOONNNWPTTFGAG TKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVOWKVD NALOSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKWYACEVTHQG LSSPVTKSFNRGEC

Figure 11 U.S. Patent Aug. 26, 2014 Sheet 12 of 65 US 8,815,247 B2

Anti-CTLA4 HC-TGFBRIl-4-1BB fusion protein Amino acid sequence of heavy chain-TGFBRIl-4-1BB fusion protein: QVOLVESGGGWOPGRSLRLSCAASGFTFSSYTMHWVROAPGKGLEW WTFISYDGNNKYYADSVKGRFTSRDNSKNTLYLOMNSLRAEDTAIYYCA RTGWLGPFDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL WKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSWWTVPSSSLGT QTYCNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPEKTISKAKGOPR EPOVYTLPPSRDELTKNOWSLTCLVKGFYPSDAVEWESNGOPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLS LSPGKGGGGSGGGGSGGGGSTIPPHVOKSVNNDMVTDNNGAVKFPOL CKFCDVRFSTCDNGRKSCMSNCSITSICEKPCREVCVAVWRKNDENITLET VCHDPKPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIF SEEYNTSNPDEPKSCDKACPWAVSGARASPGSAASpRLREGPELSPD DpaGLLDLROGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSY KEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHELQpLRSAA GAAALALTVDLPPASSEARNSA FGFQGRLLHELSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTipt IPAGLPSPRSE Amino acid sequence of light chain:

EIVLTQSPGTLSLSPGERATLSCRASOSVGSSYLAWYOOKPGOAPRLLY GAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCOQYGSSPWTF GQGTKVEIKRTVAAPSVFIFPPSDEOLKSGTASVWCLLNNFYPREAKVOW KVDNALOSGNSOESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC

Figure 12 U.S. Patent Aug. 26, 2014 Sheet 13 of 65 US 8,815,247 B2

Anti-HER2/neu HC-TGFBRIl-PD1 fusion protein Amino acid sequence of heavy chain-TGFBRI-PD1 fusion protein: EVOLVESGGGLVORPGGSLRLSCAASGFNIKDTYHWVROAPGKGLEWVA RYPTNGYTRYADSVKGRFTISADTSKNTAYLOMNSLRAEDTAVYYCSRW GGDGFYAMDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSWTVPSSSLG TOTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPEKTISKAKGOP REPOVYTLPPSREEMTKNOWSLTCLVKGFYPSDAVEWESNGOPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWOQGNVFSCSVMHEALHNHYTOKSL SLSPGKGGGGSGGGGSGGGGSTIPPHVORKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPOEVCVAVWRKNDENITL ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDN IIFSEEYNTSNPDEPKSCDKPGWFLOSpoRpWNppTTFSPALLVVTEGD NATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQpGQDCRF RVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAEL rVTERRAEVPTAHpSpSpRpAGQFQTLV Amino acid sequence of light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCOOHYTTPPTFGQ GTKVEKRTVAAPSVFIFPPSDEQLKSGTASVWCLLNNFYPREAKVOWKV DNALOSGNSOESVTEQDSKDSTYSLSSTLTLSKADYEKHKWYACEVTHQ GLSSPVTKSFNRGEC

Figure 13 U.S. Patent Aug. 26, 2014 Sheet 14 of 65 US 8,815,247 B2

Anti-EGFR1 HC-TGFBRIl-PD1 fusion protein: Amino acid sequence of heavy chain-TGFBRI-PD1 fusion protein:

QVOLKQSGPGLVOPSQSLSITCTVSGFSLTNYGVHWVROSPGKGLEWL GVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAYYCARA LTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCL WKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSWLTVLHCRDWLNGKEYKCKVSNKALPAPEKTISKAKGOPR EPOVYTLPPSRDELTKNOWSLTCLVKGFYPSDIAVEWESNGOPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLS LSPGKGGGGSGGGGSGGGGSTIPPHVOKSVNNDMIVTDNNGAVKFPQL CKFCDVRFSTCDNQKSCMSNCSTSCEKPQEVCVAVWRKNDENITLE VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIF SEEYNTSNPDEPKSCDKPGWFLDSPORPWNPPTFSPALLVVTEGONA TFTCSFSNTSESFVLNWYRMSPSNQTOKLAAFPEDRSQpGQDCRFRV TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAH(pSpSpRpAGQFQTLV Amino acid sequence of light chain:

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYOORTNGSPRLLKYA SESISGPSRFSGSGSGTDFTLSINSVESEDIADYYCOONNNWPTTFGAG TKLELKRTVAAPSVFIFPPSDEQLKSGTASVWCLLNNFYPREAKVOWKVD NALOSGNSOESVTEQDSKDSTYSLSSTLTLSKADYEKHKWYACEVTHQG LSSPVTKSFNRGEC

Figure 14 U.S. Patent Aug. 26, 2014 Sheet 15 of 65 US 8,815,247 B2

Anti-CTLA4 HC-TGFBRI-PD1 fusion protein Amino acid sequence of heavy chain-TGFBRIl-PD1 fusion protein:

QVOLVESGGGWOPGRSLRLSCAASGFTFSSYTMHWVROAPGKGLEW VTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAIYYCA RTGWLGPFDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL WKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGOPR EPOVYTLPPSRDELTKNOWSLTCLVKGFYPSDAVEWESNGOPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSLS LSPGKGGGGSGGGGSGGGGSTIPPHVORKSVNNDMIVTDNNGAVKFPOL CKFCDVRFSTCDNQKSCMSNCSITSICEKPCREVCVAVWRKNDENITLET VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIF SEEYNTSNPDEPKSCDKPGWFLOSPORpWNppTESPALLWTEGDNA TFTCSFSNTSESTVLNWYRMSPSNQTDKLAAFPEDRSQpGQDCRFRV TQLpNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAH-pSpSpRpAGQFQTLV Amino acid sequence of light chain:

EVLTQSPGTLSLSPGERATLSCRASOSVGSSYLAWYOOKPGOAPRLLY GAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCOQYGSSPWTF GQGTKVEKRTVAAPSVFIFPPSDECRLKSGTASWCLLNNFYPREAKVCRW KVDNALOSGNSOESVTEQDSKDSTYSLSSTLTLSKADYEKHKWYACEVT HQGLSSPVTKSFNRGEC

Figure 15

U.S. Patent Aug. 26, 2014 Sheet 22 of 65 US 8,815,247 B2

Non-Reduced

Molecular wt markers

Herceptin Anti-HER2-TGFBRIA GFR1TGFBRI Herceptin Anti-HER2-TGFBRII Anti-EGFR1-TGFBRI

Heavy chai

Light chain

Figure 22 U.S. Patent Aug. 26, 2014 Sheet 23 of 65 US 8,815,247 B2

U.S. Patent Aug. 26, 2014 Sheet 24 of 65 US 8,815,247 B2

... " -- Brnab 100Awastin) -HTGFBRI-FC s - - Anti-HER2-TGFBRE.2-8°C i --E- Anti-HER2-TGFSRI Refrozen i -a-Anti-FGFR1-TGF3Rf s ----- Anti-EGFR1 -TGFBRI. Refree

O3 O.83 25 2.5 SOC O.O Concentration (giml)

Inhibition of BT474 Proliferation

a BrabOO was Bria 2. was initi-i-Fi: - GFR, see Buffer

. CiscertatioFigi

Figure 24 U.S. Patent Aug. 26, 2014 Sheet 25 Of 65 US 8,815,247 B2

Inhibition of A431 Proliferation

is sesses

in Buffer a Brmab1OOAwastin spa, Cetiximab

. oftetratio.gif

Figure 25

U.S. Patent Aug. 26, 2014 Sheet 27 Of 65 US 8,815,247 B2

sis

s U.S. Patent Aug. 26, 2014 Sheet 28 of 65 US 8,815,247 B2

Anti-EGFR1-4-1BB ADCC ASSAY 2911 2011 E. :: O:

in Bife see Catuxia see Anti-EGFR1-TGFBRII as Anti-EGFR-4-1BB purif

geosy'ssassissists:x:

Canaestratic Irist

Figure 28 U.S. Patent Aug. 26, 2014 Sheet 29 Of 65 US 8,815,247 B2

Anti-CTLA4-TGFBRIELISA 15112011 A (1:120,000 dilm)

in Brnab100 Awastin) as Anti-EGFR1-TGFBRI

ag Anti-As-TGFBRW-3C

again Anti-CTA4-TGFBRFrozen

Conceatristie girl

Anti-CTLA4-TGFBRII./CTLA4 ELISA 09.11.2011

and Bimab100 Awastin

arles Anti-CTLAA-TGFBRI/2-8°C

are Anti-CiA 4-GFR1. Froze

EEEstagi

Figure 29 U.S. Patent Aug. 26, 2014 Sheet 30 of 65 US 8,815,247 B2

A Anti-CTLA-4-PD1 ELISA 1611.2011 4.5 s man Anti-EGFR1-T6FBRII i -secreenessesser-resser re-or 3.5 ------cocoon X&ts:::EEE::: mine PDL1-FC 3. |- A. xwors--war - an Anti-CTLA-4-f6 FBRI/2-8°C s - axia Anti-CTLA-4-TGFBRil Frozen

SY s c S. Concentrating fail

B Anti-EGFR1-4-1BB ELISA 23112011

-e Anti-EGFR1-TGFBRil

as 4-1BBL an Anti-EGFR-4-BB SUP2-8"C sus Anti-EGFR1-4-1BB Purified

Caricetration gal

Figure 30 U.S. Patent Aug. 26, 2014 Sheet 31 of 65 US 8,815,247 B2

A Anti-EGFR1-4-1BBSUPERNATENTEGFRELISA

are Bmab100 Awastin) e Anti-EGFR1-TGFBRI aristi-EGFR-A-BB 2-3'

sai Ai-EGFR1--1BE igger

wnn Anti-EGFR1-TGFBR its 1-c. sis -, -éi-ii: " ... is is E. E.g.: 3 is is

itratics gif

Figure 31 U.S. Patent Aug. 26, 2014 Sheet 32 of 65 US 8,815,247 B2

Anti-EGFR1-PD1 EGFRELISA O4112011.

catassissiekarstatascissassiawikaikaiassausawwuwissisterswissistsFr 388 mm Bimab 100 Awastin an Anti-EGFR1-TGFBRIt seen Anti-EGFR1-PD1 2-8 aul Anti-EGFR1-PD1 frozer

Concentrating.In

Figure 32

U.S. Patent Aug. 26, 2014 Sheet 34 of 65 US 8,815,247 B2

#79eun61– U.S. Patent Aug. 26, 2014 Sheet 35 of 65 US 8,815,247 B2

sae U.S. Patent Aug. 26, 2014 Sheet 36 of 65 US 8,815,247 B2

99eun61– U.S. Patent Aug. 26, 2014 Sheet 37 Of 65 US 8,815,247 B2

GOBII}}{j+9L-THH?3-?quVJO(p?Onp?H 19eun61 JOHJo?ni?b?dsssew U.S. Patent Aug. 26, 2014 Sheet 38 of 65 US 8,815,247 B2

99eun6|-

* 5-0=--~~======——————————————————————————————————————————? U.S. Patent Aug. 26, 2014 Sheet 39 of 65 US 8,815,247 B2

::::::::::::::::::::::: *****************************************naer:::::::::::::::::::: ***********************xw.waer,waewaewaer,*****************:::::::+ ####&$$$$$$$$$$$$$$$::::::::::::::::::::::&##########******* 69?un61–

***************#!*#:############## U.S. Patent Aug. 26, 2014 Sheet 40 of 65 US 8,815,247 B2

07eun6|-

U.S. Patent Aug. 26, 2014 Sheet 41 of 65 US 8,815,247 B2

*********************¿¿.* §§§§;&##################3

|#7eun61–

::::::::&&&&&&&&&#::::::::::::::::::::::::::::::::::::::::::::::::: (CHOMEINE

WONNOIMINNAS

WO3SOSBEWITTE 88N|l U.S. Patent Aug. 26, 2014 Sheet 42 of 65 US 8,815,247 B2

8|9

U.S. Patent Aug. 26, 2014 Sheet 46 of 65 US 8,815,247 B2

Cantuzumab -TGFBRI fusion protein at LC constant region Amino acid sequence of Cantuzumab heavy chain:

DIVMTOSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLORPGOSPOLLIYR MSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLOHLEYPFTFGPGTKLE LKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNS OESVTEODSKDSTYSLSSTLTLSKADYEKHKWYACEVTHOGLSSPVTKSFNRGEC GGGGSGGGGSGGGGSTIPPHVORKSVNNDMVTDNNGAVKFPOLCK FCDVRFSTCDNQKSCMSNCSITSICEKPOEVCVAVWRKNDEN TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC SSDECNDNIFSEEYNTSNPD

Figure 46 U.S. Patent Aug. 26, 2014 Sheet 47 of 65 US 8,815,247 B2

Cixutumumab-TGFBRI fusion protein at LC constant region Amino acid Sequence of Cixutumumab heavy chain:

EVOLVOSGAEVKKPGSSVKVSCKASGGTFSSYAISWVROAPGOGLEWMGGP FGTANYAOKFOGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLEWSTO DHYYYYYMDWWGKGTTWTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODW LNGKEYKCKVSNKALPAPEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCL VKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGN VFSCSVMHEALHNHYTOKSLSLSPGK Amino acid sequence of Cixutumumab light chain fusion protein:

SSELTODPAVSVALGOTVRITCOGDSLRSYYATWYOOKPGOAPILVIYGENKRPS GIPDRFSGSSSGNTASTITGAOAEDEADYYCKSRDGSGOHLVFGGGTKLTVLG OPKAAPSVTLFPPSSEELOANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKOSNNKYAASSYLSLTPEQWKSHRSYSCOVTHEGSTVEKTVAPAECSGGGG SGGGGSGGGGSTIPPHVORKSVNNDMIVTDNNGAVKFPOLCKFCD WRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCMKEKKKPGETFFMCSCSSD ECNDNIFSEEYNTSNPD

Figure 47 U.S. Patent Aug. 26, 2014 Sheet 48 of 65 US 8,815,247 B2

Clivatuzumab -TGF3Ril fusion protein at LC constant region Amino acid sequence of Clivatuzumab heavy chain: OVOLOOSGAEVKKFGASVKVSCEASGYTFPSYWLHWVKOAPGOGLEWIGYINP YNDGTOTNKKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYGFA YNGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVNTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKV SNKALPAPIEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCLVKGFYPSDIAV EWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVNHEAL HNHYTOKSLSLSPGK Amino acid sequence of Clivatuzumab light chain fusion protein: DIOLTOSPSSLSASVGDRVTMTCSASSSVSSSYLYWYOOKPGKAPKLWIYSTSNL ASGVPARFSGSGSGTDFTLTISSLOPEDSASYFCHOWNRYPYTFGGGTRLEIKRT VAAPSVFIFPPSDEOLKSGTASVVCLLNN FYEAKVOWKVDNALOSGNSOESVTE ODSKDSTYSLSSTLTLSPRKADYEKHKWYACEVTHOGLSSPVTKSFNRGECGGGG SGGGGSGGGGSTIPPHVORKSVNNDMVTDNNGAVKFPQLCKFCD WRFSTCDNCRKSCMSNCSTSICEKPQEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIFSEEYNTSNPD

Figure 48 U.S. Patent Aug. 26, 2014 Sheet 49 of 65 US 8,815,247 B2

Pritumumab-TGFBRIl fusion protein at LC constant region Amino acid sequence of Pritumumab heavy chain: EVOLLESGGDLVOPGGSLRLSCAASGFTFSNYAMSWVROAPGKGLEWVSATP SGGSTNYADSVKGRFTISRDNSONTLYLOMNSLRVEDTAVYICGRVPYRSTWYP LYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYCNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYK CKVSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDI AVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMH EALHNHYTOKSLSLSPGK Amino acid sequence of Pritumumab light chain fusion protein: DIOMTOSPSSLSASVGDRVTITCRASODISNYLAWFOOKPGKAPKSLIYAASSLH SKVPTOFSGSGSGTDFTLTISSLOPEDFATYYCLOYSTYPITFGGGTKVEIKRTVAA PSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEO DSKDSTYSLSSTLTLSKADYEKHKWYACEVTHOGLSSPVTKSFNRGECGGGGSGG GGSGGGGSTIPPHVOKSVNNDMIVTDNNGAVKFPQLCKFCDVRF STCDNQKSCMSNCSTSCEKPQEVCVAVWRKNDENITLETWC HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN DNIFSEEYNTSNPD

Figure 49 U.S. Patent Aug. 26, 2014 Sheet 50 Of 65 US 8,815,247 B2

Cantuzumab HC-4-1BB and LC-TGFBRI fusion protein Amino acid sequence of heavy chain-4-1BB fusion protein:

OVOLVOSGAEVKKPGETVKISCKASDYTFTYYGMNWWKOAPGOGLKWMGW DTTTGEPTYAOKFOGRIAFSLETSASTAYLOKSLKSEDTATYFCARRGPYNWYFD WWGOGTTWTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKC KVSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSD AVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSACPWAVSGARAS1PGS AASPRLREGPELSPDOpAGLLOLROGMFAQLVAQNVLLID GPLSWYSDpGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQL ELRRVVAGEGSGSVSLALHELQpLRSAAGAAALALTVDLPPAS SEARNSAFGFQGRLLHCLSAGQRLGVHLHETEARARHAWQLT QGATVLGLFRVTipt IpAGLpSpRSE Amino acid sequence of Cantuzumab light chain fusion protein:

DIVMTOSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLORPGOSPOLLIYR MSNLVSGVPDRFSGSGSGTAFTLRSRVEAEDVGVYYCLOHLEYPFTFGPGTKLE LKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNS OESVTEODSKDSTYSLSSTLTLSKADYEKHKWYACEVTHOGLSSPVTKSFNRGEC GGGGSGGGGSGGGGSTIPPHVORKSVNNDMIVTDNNGAVKFPCRLCK FCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDEN TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC SSDECNDNIFSEEYNTSNPD Figure 50 U.S. Patent Aug. 26, 2014 Sheet 51 of 65 US 8,815,247 B2

Cixutumumab HC-4-1BB and LC-TGFBRI fusion protein Amino acid sequence of heavy chain-4-1BB fusion protein:

EVOLVOSGAEVKKPGSSVKVSCKASGGTFSSYASWVROAPGOGLEWMGGIP FGTANYAOKFOGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLEWSTO DHYYYYYMDWWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODW LNGKEYKCKVSNKALPAPIEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCL VKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGN VFSCSVMHEALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSACPWAVSG ARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVA QNVLLIDGPLSWYSDpGLAGVSLTGGLSYKEDTKELVVAKAG VYYVFFQLELRRVVAGEGSGSVSLALHELQ:PLRSAAGAAALAL TVDLPpASSEARNSAFGFQGRLLHELSAGQRLGVHLHTEARA RHAWQLTQGATVLGLFRVT ptIPAGLpSpRSE Amino acid sequence of Cixutumumab light chain fusion protein:

SSELTODPAVSVALGOTVRITCOG DSLRSYYATWYOOKPGOAPILVIYGENKRPS GIPDRFSGSSSGNTASLTITGAOAEDEADYYCKSRDGSGOHLVFGGGTKLTVLG OPKAAPSVTLFPPSSEELOANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKOSNNKYAASSYLSLTPEOWKSHRSYSCOVTHEGSTVEKTVAPAECSGGGG SGGGGSGGGGSTIPPHVOKSVNNDMIVTDNNGAVKFPQLCKFCD VRFSTCDNQKSCMSNCSTSCEKPOEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIFSEEYNTSNPD Figure 51 U.S. Patent Aug. 26, 2014 Sheet 52 of 65 US 8,815,247 B2

Clivatuzumab HC-4-1BB and LC-TGFBRI fusion protein Amino acid sequence of heavy chain -4-1BB fusion protein OVOLOOSGAEVKKFGASVKVSCEASGYTFPSYWLHWVKOAPGOGLEWIGYINP YNDGTOTNKKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYGFA YNGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVNTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKV SNKALPAPIEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCLVKGFYPSDIAV EWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVNHEAL HNHYTOKSLSLSPGKGGGGSGGGGSGGGGSACPWAVSGARASPGSAA SPRLREGPELSPDDpAGLLDLRQGMFAQLVAQNVLLIDGPL SWYSDpGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR 1RVVAGEGSGSVSLALHELQpLRSAAGAAALALTVDLPPASSEA ?RNSAFGFQGRLLHELSAGQRLGVHLHTEARARHAWQLTQG ATVLGLFRVTPEIPAGLPSPRSE Amino acid sequence of Clivatuzumab light chain fusion protein: DIOLTOSPSSLSASVGDRVTMTCSASSSVSSSYLYWYOOKPGKAPKLWYSTSNL ASGVPARFSGSGSGTDFTLTISSLOPEDSASYFCHOWNRYPYTFGGGTRLEIKRT WAAPSVFIFPPSDEOLKSGTASVWCLLNN FYEAKVOWKVDNALOSGNSOESVTE ODSKDSTYSLSSTLTLSPRKADYEKHKWYACEVTHOGLSSPVTKSFNRGECGGGG SGGGGSGGGGSTIPPHVOKSVNNDMIVTDNNGAVKFPQLCKFCD VRFSTCDNQKSCMSNCSTSCEKPQEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIFSEEYNTSNPD Figure 52 U.S. Patent Aug. 26, 2014 Sheet 53 of 65 US 8,815,247 B2

Pritumumab HC-4-1BB and LC-TGFBRI fusion protein Amino acid sequence of heavy chain-4-1BB fusion protein: EVOLLESGGDLVOPGGSLRLSCAASGFTFSNYAMSWVROAPGKGLEWVSAITP SGGSTNYADSVKGRFTISRDNSONTLYLOMNSLRVEDTAVYICGRVPYRSTWYP LYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYK CKVSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDI AVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSACPWAVSGARAS1PGS AASPRLREGPELSPDDpAGLLDLRQGMFAQLVAQNVLLID GPLSWYSDpGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQL ELRRVVAGEGSGSVSLALH LOpLRSAAGAAALALTVDLPPAS SEARNSAFGFQGRLLHLSAGQRLGVHLHETEARARHAWQLT QGATVLGLFRVTipt IpAGLPSPRSE Amino acid sequence of Pritumumab light chain fusion protein: DIOMTOSPSSLSASVGDRVTITCRASODISNYLAWFOOKPGKAPKSLIYAASSLH SKVPTOFSGSGSGTDFTLTISSLOPEDFATYYCLOYSTYPITFGGGTKVEIKRTVAA PSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEO DSKDSTYSLSSTLTLSKADYEKHKWYACEVTHOGLSSPVTKSFNRGECGGGGSGG GGSGGGGSTIPPHVOKSVNNDMIVTDNNGAVKFPQLCKFCDVRF STCDNQKSCMSNCSITSICEKPOEVCVAVWRKNDENITLETVC HDPKLPYHDFILEDAASPKCMKEKKKPGETFFMCSCSSDECN DNIFSEEYNTSNPD Figure 53 U.S. Patent Aug. 26, 2014 Sheet 54 of 65 US 8,815,247 B2

Cantuzumab - HC-PD1 and LC-TGFBRI fusion protein Amino acid sequence of heavy chain-PD1 fusion protein: OVOLVOSGAEVKKPGETVKISCKASDYTFTYYGMNWWKOAPGOGLKWMGW DTTTGEPTYAOKFOGRIAFSLETSASTAYLOKSLKSEDTATYFCARRGPYNWYFD VWGOGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYCNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKC KVSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDI AVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSpGWFLDSPORpWNPPT FSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPE DRSQpGQDCRFRVTQLPNGRDFHMSWRARRNDSGTYLCGAISLA pKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV Amino acid sequence of Cantuzumab light chain fusion protein: DIVMTOSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLORPGOSPOLLIYR MSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLOHLEYPFTFGPGTKLE LKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNS OESVTEODSKDSTYSLSSTLTLSKADYEKHKWYACEVTHOGLSSPVTKSFNRGEC GGGGSGGGGSGGGGSTPPHVOKSVNNDMVTDNNGAVKFPQLCK FCDVRFSTCDNQKSCMSNCSITSICEKPOEVCVAVWRKNDEN TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSC SSDECNDNIFSEEYNTSNPD

Figure 54 U.S. Patent Aug. 26, 2014 Sheet 55 Of 65 US 8,815,247 B2

Cixutumumab HC-PD1 and LC-TGFBRI fusion protein Amino acid sequence of heavy chain-PD1 fusion protein:

EVOLVOSGAEVKKPGSSVKVSCKASGGTFSSYAISWVROAPGOGLEWMGGIP FGTANYAOKFOGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLEWSTO DHYYYYYMDWWGKGTTWTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODW LNGKEYKCKVSNKALPAPEKTISKAKGOPREPOVYTLPPSREEMTKNOWSLTCL WKGFYPSDAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGN VFSCSVMHEALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSPGWFLDSPD rpWNPPTFSPALLVVTEGDNATFTCSFSNTSESTVLNWYRMSPSNQT pKLAAFPEDRSQpGQOCRFRVTQL:PNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAitpSpSpRPAGQFQTL V Amino acid sequence of Cixutumumab light chain fusion protein:

SSELTODPAVSVALGOTVRITCOGDSLRSYYATWYOOKPGOAPILVIYGENKRPS GPDRFSGSSSGNTASLTITGACAEDEADYYCKSRDGSGOHLVFGGGTKLTVLG OPKAAPSVTLFPPSSEELOANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKOSNNKYAASSYLSLTPEQWKSHRSYSCOVTHEGSTVEKTVAPAECSGGGG SGGGGSGGGGSTIPPHVORKSVNNDMIVTDNNGAVKFPQLCKFCD WRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIFSEEYNTSNPD

Figure 55 U.S. Patent Aug. 26, 2014 Sheet 56 of 65 US 8,815,247 B2

Clivatuzumab HC-PD1 and LC-TGFBRIl-fusion protein Amino acid Amino acid sequence of heavy chain-PD1 fusion protein:

OVOLOOSGAEVKKFGASVKVSCEASGYTFPSYWLHWVKOAPGOGLEWIGYINP YNDGTOTNKKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYGFA YNGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVNTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKV SNKALPAPIEKTISKAKGOPREPOVYTLPPSREEMTKNOWSLTCLVKGFYPSDAV EWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVNHEAL HNHYTOKSLSLSPGKGGGGSGGGGSGGGGSpGWFLDSPORPWNPPTFSp ALLVVTEGONATFTCSTSNTSESFVLNWYRMSPSNQTDKLAAFPEDRS QPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKA QIKESLRAELRVTERRAEVPTAitpSPSPRPAGQFQTLV Amino acid sequence of Clivatuzumab light chain fusion protein:

DOLTOSPSSLSASVGDRVTMTCSASSSVSSSYLYWYOOKPGKAPKLWIYSTSNL ASGVPARFSGSGSGTDFTLTISSLOPEDSASYFCHOWNRYPYTFGGGTRLEKRT VAAPSVFIFPPSDEOLKSGTASVVCLLNNFYEAKVOWKVDNALOSGNSOESVTE ODSKDSTYSLSSTLTLSPRKADYEKHKWYACEVTHOGLSSPVTKSFNRGECGGGG SGGGGSGGGGSTIPPHVOKSVNNDMIVTDNNGAVKFPOLCKFCD VRFSTCDNQKSCMSNCSTSCEKPOEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIFSEEYNTSNPD

Figure 56 U.S. Patent Aug. 26, 2014 Sheet 57 Of 65 US 8,815,247 B2

Pritumumab HC-PD1 and LC-TGFBRI fusion protein Amino acid Amino acid sequence of heavy chain-PD1 fusion protein: EVOLLESGGDLVOPGGSLRLSCAASGFTFSNYAMSWVROAPGKGLEWVSAITP SGGSTNYADSVKGRFTISRDNSONTLYLOMNSLRVEDTAVYICGRVPYRSTWYP LYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYCNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYK CKVSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDI AVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSpGWFLDSPDRpWNppT FSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPE DRSQpGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLA pKAc2IKESLRAELRVTERRAEVPTAitpSpSpRpAGQFQTLV Amino acid sequence of Pritumumab light chain fusion protein:.

DIOMTOSPSSLSASVG DRVTITCRASODISNYLAWFOOKPGKAPKSLIYAASSLH SKVPTOFSGSGSGTDFTLTISSLOPEDFATYYCLOYSTYPITFGGGTKVEIKRTVAA PSVFIFPPSDEOLKSGTASVVCLLNN FYPREAKVOWKVDNALOSGNSOESVTEO DSKDSTYSLSSTLTLSKADYEKHKWYACEVTHOGLSSPVTKSFNRGECGGGGSGG GGSGGGGSTIPPHVOKSVNNDMVTDNNGAVKFPQLCKFCDVRF STCDNQKSCMSNCSTSCEKPQEWCVAVWRKNDENITLETVC HDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN DNIFSEEYNTSNPD

Figure 57 U.S. Patent Aug. 26, 2014 Sheet 58 of 65 US 8,815,247 B2

Cantuzumab HC-TGF3RIl-4-1BB fusion protein Amino acid sequence of heavy chain-TGFBRIl-4-1BB fusion protein: OVOLVOSGAEVKKPGETVKISCKASDYTFTYYGMNWVKOAPGOGLKWMGW DTTTGEPTYAOKFOGRIAFSLETSASTAYLOKSLKSEDTATYFCARRGPYNWYFD VWGOGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKC KVSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDI AVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSTPPHVORKSVNNDMIV TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPO EVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepkscdkAC pWAVSGARASPGSAASPRLREGPELSPDDpAGLLDLROGMF AQLVAQNVLLIDGPLSWYSDpGLAGVSLTGGLSYKEDTKELV VAKAGVYYVFFQLELRRVVAGEGSGSVSLALHELQpLRSAAGA AALALTVDLPPASSEARNSAFGFQGRLLHELSAGQRLGVHLH TEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE Amino acid Sequence of light chain DIVMTOSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLORPGOSPOLLIYR MSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLOHLEYPFTFGPGTKLE LKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNS OESVTEODSKDSTYSLSSTLTLSKADYEKHKWYACEVTHOGLSSPVTKSFNRGEC Figure 58 U.S. Patent Aug. 26, 2014 Sheet 59 of 65 US 8,815,247 B2

Cixutumumab HC-TGFBRIl-4-1BB fusion protein Amino acid sequence of heavy chain-TGFBRIl-4-1BB fusion protein: EVOLVOSGAEVKKPGSSVKVSCKASGGTFSSYAISWVROAPGOGLEWMGGIIP FGTANYAOKFOGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLEWSTO DHYYYYYMDWWGKGTTWTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODW LNGKEYKCKVSNKALPAPIEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCL VKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGN VFSCSVMHEALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVORKS WNNDMVTDNNGAVKFPOLCKFCDVRFSTCDNQKSCMSNCS TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAA SPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDep kscdka CpWAVSGARASPGSAASPRLREGPELSPODPAGLLD LRQGMFAQLVAQNVLLIDGPLSWYSDpGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHELQp LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHELSAGQ RLGVHLHTEARARHAWQLTQGATVLGLFRVTipt IPAGLPSP 1RSE Amino acid sequence of light chain:

Clivatuzumab HC-TGFBRIl-4-1BB fusion protein Amino acid sequence of heavy chain-TGFBRIl-4-1BB fusion protein: OVOLOOSGAEVKKFGASVKVSCEASGYTFPSYWLHWVKOAPGOGLEWIGYINP YNDGTOTNKKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYGFA YNGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVNTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKV SNKALPAPIEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCLVKGFYPSDIAV EWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVNHEAL HNHYTOKSLSLSPGKGGGGSGGGGSGGGGSTPPHVORKSVNNDMIVTD NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV CVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK KKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepkscdkACPW) AVSGARASPGSAASPRLREGPELSPDD-PAGLLDLRQGMFAQ LVAQNVLLIDGPLSWYSDpGLAGVSLTGGLSYKEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALht LopLRSAAGAAA LALTVDLPPASSEARNSAFGFQGRLLHELSAGQRLGVHLHCTE ARARHAWQLTQGATVLGLFRVTptIPAGLPSPRSE Amino acid sequence of light chain: DIOLTOSPSSLSASVGDRVTMTCSASSSVSSSYLYWYOOKPGKAPKLWIYSTSNL ASGVPARFSGSGSGTDFTLTISSLOPEDSASYFCHOWNRYPYTFGGGTRLEKRT VAAPSVFIFPPSDEOLKSGTASVVCLLNN FYEAKVOWKVDNALOSGNSOESVTE ODSKDSTYSLSSTLTLSPRKADYEKHKWYACEVTHOGLSSPVTKSFNRGEC Figure 60 U.S. Patent Aug. 26, 2014 Sheet 61 of 65 US 8,815,247 B2

Pritumumab HC-TGFBRIl-4-1BB fusion protein Amino acid sequence of heavy chain-TGFBRIl-4-1BB fusion protein:

EVOLLESGGDLVOPGGSLRLSCAASGFTFSNYAMSWVROAPGKGLEWVSAITP SGGSTNYADSVKGRFTISRDNSONTLYLOMNSLRVEDTAVYICGRVPYRSTWYP LYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYK CKVSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDI AVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSTPPHVOKSVNNDMIV TDNNGAVKFPOLCKFCDVRFSTCDNQKSCMSNCSITSICEKPO EVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepkscdkAC pWAVSGARASPGSAASPRLREGPELSPDDpAGLLDLRQGMF AQLVAQNVLLIDGPLSWYSDpGLAGVSLTGGLSYKEDTKELV VAKAGVYYVFFQLELRRVVAGEGSGSVSLA Lit LQPLRSAAGA AALALTVDLPPASSEARNSAFGFQGRLLHELSAGQRLGVHLHE TEARARHAWQLTQGATVLGLFRVTipt IPAGLPSPRSE Amino acid sequence of light chain:

DIOMTOSPSSLSASVGDRVTITCRASODSNYLAWFOOKPGKAPKSLIYAASSLH SKVPTOFSGSGSGTDFTLTISSLOPEDFATYYCLOYSTYPITFGGGTKVEIKRTVAA PSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEO DSKDSTYSLSSTLTLSKADYEKHKWYACEVTHOGLSSPVTKSFNRGEC

Figure 61 U.S. Patent Aug. 26, 2014 Sheet 62 of 65 US 8,815,247 B2

Cantuzumab HC-TGFBRIl-PD1 fusion protein Amino acid sequence of heavy chain-TGFBRIl-PD1 fusion protein: OVOLVOSGAEWKKPGETVKISCKASDYTFTYYGMNWWKOAPGOGLKWMGW DTTTGEPTYAOKFOGRIAFSLETSASTAYLOKSLKSEDTATYFCARRGPYNWYFD VWGOGTTWTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKC KVSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSD AVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVOKSVNNDMIV TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQ EVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepkscdkpg WFLDSPORpWNPPTFSPALLVVTEGDNATFTCSFSNTSESTVLNWYR MSPSNQTOKLAAFPEDRSQpGQDCRFRVTQLpNGRDFEMSVVRAR RNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAH pSPSPRP AGQFQTLV Amino acid sequence of light chain: DIVMTOSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLORPGOSPOLLIYR MSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLOHLEYPFTFGPGTKLE LKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNS OESVTEODSKDSTYSLSSTLTLSKADYEKHKWYACEVTHOGLSSPVTKSFNRGEC Figure 62 U.S. Patent Aug. 26, 2014 Sheet 63 of 65 US 8,815,247 B2

Cixutumumab HC-TGFBRIl-PD1 fusion protein Amino acid sequence of heavy chain-TGFBRI-PD1 fusion protein: EVOLVOSGAEVKKPGSSVKVSCKASGGTFSSYAISWVROAPGOGLEWMGGIP FGTANYAOKFOGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLEWSTO DHYYYYYMDWWGKGTTWTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYCNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODW LNGKEYKCKVSNKALPAPIEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCL WKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGN VFSCSVMHEALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVOKS VNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCS TSICEKPOEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAA SPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDep kscdkpGWFLDSPDRPWN PipTFSPALLVVTEGONATFTCSFSNTSESF VLNWYRMSPSNQTOKLAAFPEDRSQpGQDCRFRVTQLPNGRDFHM SVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAH pSpSpRipAGQFQTLV Amino acid sequence of light chain: SSELTODPAVSVALGOTVRITCOGDSLRSYYATWYOOKPGOAPILVIYGENKRPS GIPDRFSGSSSGNTASLTITGAOAEDEADYYCKSRDGSGOHLVFGGGTKLTVLG OPKAAPSVTLFPPSSEELOANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKOSNNKYAASSYLSLTPEOWKSHRSYSCOVTHEGSTVEKTVAPAECS

Figure 63 U.S. Patent Aug. 26, 2014 Sheet 64 of 65 US 8,815,247 B2

Clivatuzumab HC-TGFBRI-PD1 fusion protein Amino acid sequence of heavy chain-TGFBRI-PD1 fusion protein: OVOLOOSGAEVKKFGASVKVSCEASGYTFPSYWLHWVKOAPGOGLEWIGYINP YNDGTOTNKKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYGFA YNGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVNTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKV SNKALPAPIEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCLVKGFYPSDIAV EWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVNHEAL HNHYTOKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVOKSVNNDMIVTD NNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPOEV CVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK KKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepkscalkipGWFL OSporpwNPPTFSPALLVVTEGONATFTCSFSNTSESTVLNWYRMSPS NQTDKLAAFPEDRSQPGQDCRFRVTQL?PNGRDFHMSVVRARRND SGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSpRpAGQ FQTLV Amino acid sequence of light chain: DIOLTOSPSSLSASVGDRVTMTCSASSSVSSSYLYWYOOKPGKAPKLWIYSTSNL ASGVPARFSGSGSGTDFTLTISSLOPEDSASYFCHOWNRYPYTFGGGTRLEKRT VAAPSVFIFPPSDEOLKSGTASVVCLLNN FYEAKVOWKVDNALOSGNSOESVTE ODSKDSTYSLSSTLTLSPRKADYEKHKWYACEVTHOGLSSPVTKSFNRGEC Figure 64 U.S. Patent Aug. 26, 2014 Sheet 65 of 65 US 8,815,247 B2

Pritumumab HC-TGFBRI-PD1 fusion protein Amino acid sequence of heavy chain-TGFBRIl-PD1 fusion protein: EVOLLESGGDLVOPGGSLRLSCAASGFTFSNYAMSWVROAPGKGLEWVSAITP SGGSTNYADSVKGRFTISRDNSONTLYLOMNSLRVEDTAVYICGRVPYRSTWYP LYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMSRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYK CKVSNKALPAPIEKTISKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSD AVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMH EALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSTPPHVORKSVNNDMIV TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQ EVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDepkscdkiPG WFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYR MSPSNQTDKLAAFPEDRSQpGQDCRFRVTQL?PNGRDFHMSWRAR RNOSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAH-pSpSpRP AGQFQTLV Amino acid sequence of light chain: DOMTOSPSSLSASVG DRVTITCRASODISNYLAWFOOKPGKAPKSLIYAASSLH SKVPTOFSGSGSGTDFTLTISSLOPEDFATYYCLOYSTYPITFGGGTKVEKRTVAA PSVFIFPPSDEOLKSGTASVVCLLNN FYPREAKVOWKVDNALOSGNSOESVTEO DSKDSTYSLSSTLTLSKADYEKHKWYACEVTHOGLSSPVTKSFNRGEC Figure 65 US 8,815,247 B2 1. 2 TARGETED/IMMUNOMODULATORY The chimeric/fusion polypeptides of the invention are useful FUSION PROTEINS for binding to a cancer cell receptor and reducing the ability of cancer cells to avoid an immune response. CROSS REFERENCE TO RELATED The present invention is based on preparing chimeric/fu APPLICATIONS sion proteins by expression of polynucleotides encoding the fusion proteins that counteract or reverse immune tolerance The application claims priority to Indian Patent Applica of cancer cells. Cancer cells are able to escape elimination by tion No. 1689/CHF/2012 filed on Apr. 30, 2012 and Indian chemotherapeutic agents or tumor-targeted antibodies via Patent Application No. 1690/CHF/2012 filed on Apr. 30, specific immunosuppressive mechanisms in the tumor 2012, the contents of both is hereby incorporated by reference 10 microenvironment and Such ability of cancer cells is recog herein for all purposes. nized as immune tolerance. Such immunosuppressive mecha nisms include immunosuppressive cytokines (for example, BACKGROUND OF THE INVENTION Transforming growth factor beta (TGF-B)) and regulatory T cells and/or immunosuppressive myeloid dendritic cells 1. Technical Field 15 (DCs). By counteracting tumor-induced immune tolerance, The present invention relates generally to the field of gen the present invention provides effective compositions and erating fusion proteins to be used in cancer therapy, and more methods for cancer treatment, optional in combination with specifically, to nucleotide sequences encoding the fusion pro another existing cancer treatment. The present invention pro teins, wherein the fusion or chimeric polypeptides comprises vides strategies to counteract tumor-induced immune toler at least one targeting moiety and at least one immunomodu ance and enhance the antitumor efficacy of chemotherapy by latory moiety that counteracts the immune tolerance of cancer activating and leveraging T cell-mediated adaptive antitumor cells. against resistant or disseminated cancer cells. 2. Related Art In another aspect, the present invention provides a mol The immune system provides the human body with a ecule including at least one targeting moiety fused with at means to recognize and defend itself against microorganisms 25 least one immunomodulatory moiety. The targeting moiety and Substances recognized as foreign or potentially harmful. specifically binds a target molecule, and the immunomodu While passive immunotherapy of cancer with monoclonal latory moiety specifically binds one of the following mol antibodies and passive transfer of T cells to attack tumor cells ecules: (i) Transforming growth factor-beta (TGF-B); (ii) Pro have demonstrated clinical efficacy, the goal of active thera grammed death-1 ligand 1 (PD-L1) or Programmed death-1 peutic vaccination to induce these immune effectors and 30 ligand 2 (PD-L2); (iii) Receptor activator of nuclear factor establish immunological memory against tumor cells has KB (RANK) ligand (RANKL); (iv) Transforming growth remained challenging. Several tumor-specific and tumor-as factor-beta receptor (TGF-pR); (v) Programmed death-1 Sociated antigens have been identified, yet these antigens are (PD-1); (vi) 4-1BB receptor or (vii) Receptor activator of generally weakly immunogenic and tumors employ diverse nuclear factor-KB (RANK). mechanisms to create a tolerogenic environment that allows 35 In a further aspect, the targeting moiety includes an anti them to evade immunologic attack. Strategies to overcome body, antibody fragment including the light or heavy chains Such immune tolerance and activating robust levels of anti of the antibody, ScPV, or Fc-containing polypeptide that spe body and/or T cell responses hold the key to effective cancer cifically binds a component of a tumor cell, tumor antigen, immunotherapy. More important, the individual proteins and tumor vasculature, tumor microenvironment, or tumor-infil how to create an active chimeric polypeptide with an active 40 trating immune cell. Preferably, the targeting moiety is an tertiary structure needs to be explored. antibody or a fragment thereof having binding affinity for a component on a tumor cell. Notably each of the heavy chain SUMMARY OF THE INVENTION and light chain may individually be linked to a separate and distinct immunomodulatory moiety. Further, a heavy or light The present invention provides polynucleotides, as well as 45 chain of an antibody targeting moiety may be linked to an polypeptides encoded thereby, that are expressed in cancer immunomodulatory moiety which in turn can be further cells. These polynucleotides and expressed polypeptides are linked to a second immunomodulatory moiety wherein there useful in a variety of therapeutic methods for the treatment of is a linker between the two immunomodulatory moieties. cancer. The present invention further provides methods of In a still further aspect, there is provided a chimeric reducing growth of cancer cells by counteracting immune 50 polypeptide that comprised a tumor targeting moiety and an tolerance of cancer cells, wherein T cell remain active and immunomodulatory moiety comprising a molecule that binds inhibit the recruitment of T-regulatory that are known to sup transforming growth factor beta (TGF-B), wherein the tumor press the immune system's response to the tumor. Thus the targeting moiety is an antibody that binds to EGFR1, where in chimeric polypeptides generated by the polynucleotides the antibody can be the full antibody, heavy chain or light sequences of the present invention are useful for treating 55 chain. The tumor targeting moiety may include monoclonal cancer because of the expressed fusion or chimeric polypep antibodies that target a cancer cell, including but not limited tides. to cetuximab, trastuzumab, ritubximab, ipilimumab, tremeli In one aspect, the present invention provides for chimeric mumab, muromonab-CD3, abciximab, daclizumab, basilix polypeptides containing at least one targeting moiety to target imab, palivizumab, infliximab. gemtuzumab ozogamicin, a cancer cell and at least one immunomodulating moiety that 60 alemtuzumab, ibritumomab tiuXetan, adalimumab, omali counteracts immune tolerance of cancer cell, wherein the Zumab, to situmomab, I-131 to situmomab, efalizumab, beva targeting moiety and the immunomodulating moiety are cizumab, panitumumab, pertuZumab, natalizumab, etaner linked by a amino acid spacer of Sufficient length of amino cept, IGN101 (Aphton), Volociximab (Biogen Idec and PDL acid residues so that both moieties can Successfully bond to BioPharm), Anti-CD80 mAb (Biogen Idec), Anti-CD23 mAb their individual target. In the alternative, the targeting moiety 65 (Biogen Idel), CAT-3888 (Cambridge Antibody Technol and the immunomodulating moiety that counteract immune ogy), CDP-791 (Imclone), eraptuzumab (Immunomedics), tolerance of cancer cell may be bound directly to each other. MDX-010 (Medarex and BMS), MDX-060 (Medarex), US 8,815,247 B2 3 4 MDX-070 (Medarex), matuzumab (Merck), CP-675,206 and wherein, the immunomodulatory moiety includes an (Pfizer), CAL (Roche), SGN-30 (Seattle Genetics), Zanoli extracellular ligand-binding domain or ectodomain of Pro mumab (Serono and Genmab), adecatumumab (Sereno), grammed Death-1 (PD-1). oregovomab (United Therapeutics), nimotuzumab (YM Bio In a still further aspect, the targeting moiety includes an science), ABT-874 (Abbott Laboratories), denosumab (Am antibody or antibody fragment that specifically binds to gen), AM 108 (Amgen), AMG 714 (Amgen), fontolizumab CD20, and the immunomodulatory moiety includes a (Biogen Idec and PDL BioPharm), daclizumab (Biogent Idec sequence from transforming growth factor-B (TGF-3). and PDL BioPharm), golimumab (Centocor and Schering In one aspect, the present invention provides for optimized Plough), CNTO 1275 (Centocor), ocrelizumab (Genetech genes encoding for a fusion polypeptide comprising at least and Roche), HuMax-CD20 (Genmab), belimumab (HGS and 10 one targeting moiety and at least one immunomodulatory GSK), epratuzumab (Immunomedics), MLN1202 (Millen moiety for treating cancer in a human Subject wherein the nium Pharmaceuticals), visilizumab (PDL BioPharm), tocili optimized genes have been modified to increase expression in Zumab (Roche), ocrerlizumab (Roche), certolizumab pegol a human Subject. preferably the optimized genes comprise (UCB, formerly Celltech), eculizumab (Alexion Pharmaceu sequences for encoding a targeting moiety or an immuno ticals), pexelizumab (Alexion Pharmaceuticals and Procter & 15 modulatory moiety selected from SEQID NOs: 12 to 28. Gamble), abciximab (Centocor), ranibizimumab (Genetech), In another aspect, the present invention provides for a mepolizumab (GSK), TNX-355 (Tanox), or MYO-029 (Wy vector comprising optimized genes for treating cancer in a eth). human Subject wherein the optimized genes have been modi In an another aspect, the tumor targeting moiety is a mono fied to increase CG sequences. Preferably, the vector includes clonal antibody that binds to HER2/Neu, CD20, CTLA4, sequences for encoding at least one targeting moiety and at EGFR1 and wherein the antibody can be the full antibody, least one immunomodulatory moiety selected from SEQID heavy chain or light chain. NOS: 12 to 28. In yet another aspect, the targeting moiety is a molecule In yet another aspect, the present invention provides for a that specifically binds epidermal growth factor receptor method of treating cancer in a Subject, the method compris (EGFR1, Erb-B 1), HER2/neu (Erb-B2), CD20, cytotoxic 25 ing: T-lymphocyte antigen-4 (CTLA-4) which is essential for a. providing at least one recombinant vector comprising Treg function (CD 152); H-1 and Interleukin-6 (IL-6). nucleotide sequences that encode at least one targeting In a still further aspect, the targeting moiety specifically moiety and at least one immunomodulatory moiety binds a component of a regulatory T cell (treg), myeloid selected from SEQID NOs: 12 to 28; and Suppressor cell, or dendritic cell. In another aspect, the tar 30 b. administering the recombinant vector to the Subject geting moiety specifically binds one of the following mol underconditions such that said nucleotide sequences are ecules: (i) CD4; (ii) CD25 (IL-2ct receptor; IL-2aR); (iii) expressed at a level which produces a therapeutically Transforming growth factor-beta receptor (TGF-pR); (vi) effective amount of the encoded fusion proteins in the Transforming growth factor-beta (TGF-B): (vii) Programmed Subject. Death-1 (PD-1); (viii) Programmed death-1 ligand (PD-L1 or 35 In an alternative aspect, the present invention provides an PD-L2. expression vector comprising polynucleotides of optimized In another aspect, the immunomodulatory moiety specifi genes that encode at least one targeting moiety and at least cally binds one of the following molecules: (i) Transforming one immunomodulatory moiety selected from SEQID NOs: growth factor-beta (TGF-3): (ii) Programmed death-1 ligand 12 to 28. (PD-L1 or PD-L2); or 4-1BB receptor. 40 In yet another aspect, the present invention provides a In yet another aspect, the immunomodulatory moiety recombinant host cell transfected with a polynucleotide that includes a molecule that binds TGF-B and inhibits the func encodes a fusion protein peptide of the present invention. tion thereof. Specifically the immunomodulatory moiety In a still further aspect, the present invention contemplates includes an extracellular ligand-binding domain of Trans a process of preparing a fusion protein of the present inven forming growth factor-beta receptor TGF-BRII. TGF-BRIIb, 45 tion comprising: or TGF-BRIII. In another aspect the immunomodulatory moi a. transfecting a host cell with polynucleotide sequences ety includes an extracellular ligand-binding domain (ECD) of that encode chimeric fusion proteins to produce a trans TGF-BRII. Still further the immunomodulatory moiety may formed host cell, wherein the polynucleotide sequences include H-4-1BB ligand which binds to the 4-1BB receptor to encode at least one targeting moiety and at least one stimulate T-cells to help eradiate tumor. 50 immunomodulatory moiety selected from SEQID NOs: In a still further aspect, the targeting moiety includes an 12 to 28; and antibody, antibody fragment, or polypeptide that specifically b. maintaining the transformed host cell under biological binds to HER2/neu, EGFR1, CD20, or cytotoxic T-lympho conditions sufficient for expression of the peptide. cyte antigen-4 (CTLA-4) and wherein the immunomodula In another aspect, the present invention relates to the use of tory moiety includes an extracellular ligand-binding domain 55 a chimeric fusion protein, as shown in FIGS. 1 to 15, in the use of TGF-BRII. of a medicament for the treatment of cancer. Preferably, the In yet another aspect, the immunomodulatory moiety fusion protein is expressed in a host cell and Such expressed includes a molecule that specifically binds to and inhibit the proteins are administered in a therapeutic amount to reduce activity of Programmed death-1 ligand 1 (PD-L 1) or Pro the effects of cancer in a subject in need thereof. grammed death-1 ligand 2 (PD-L2). In another aspect, the 60 In a still further aspect, the present invention provides a immunomodulatory moiety includes an extracellular ligand method of preventing or treating a neoplastic disease. The binding domain or ectodomain of Programmed Death-1 (PD method includes administration to a subject in need thereof 1). one or more fusion proteins of the invention, in various In a further aspect, the targeting moiety includes an anti aspects, the Subject is administered one or more molecule of body, antibody fragment, or polypeptide that specifically 65 the invention in combination with another anticancer therapy, binds to HER2/neu, EGFR1, CD20, cytotoxic T-lymphocyte in one aspect, the anticancer therapy includes a chemothera antigen-4 (CTLA-4), CD25 (IL-2a receptor; IL-2aR), or CD4 peutic molecule, antibody, Small molecule kinase inhibitor, US 8,815,247 B2 5 6 hormonal agent or cytotoxic agent. The anticancer therapy LC constant region with the amino acid sequence of anti may also include ionizing radiation, ultraviolet radiation, HER2/neu heavy chain (SEQID NO: 1) and anti-HER2/neu cryoablation, thermal ablation, or radiofrequency ablation. light chain (SEQ ID NO: 2) attached to amino residues for In yet another aspect, the present invention provides for a TGF-BRII (immunomodulatory moiety) (SEQ ID NO: 4) method of preparing therapeutically active antibody-peptide identified in bold letters and wherein a linker (SEQID NO:3) fusion proteins, the method comprising: is positioned between the anti-HER2/neu light chain and a. preparing a codon optimized sequence of the said fusion protein; TGF-BRII and shown in italics. b. cloning the optimized sequence of said fusion protein in FIG. 2 shows the amino acid sequences of Anti-EGFR1 a host cell capable of transient or continued expression; TGFBRII fusion protein at LC constant region with amino c. growing the host cell in a media under Suitable condi 10 acid sequence of Anti-EGFR1 heavy chain (SEQID NO. 5) tions for growing and allowing the host cell to express and the amino acid sequence of Anti-EGFR1 light chain the cloned protein; and (SEQ ID NO: 6) attached to amino acid residues for TGF d. Subjecting the expressed protein to purification and BRII (immunomodulatory moiety) (SEQID NO: 4) identified optionally checking the bi-specific binding capabilities in bold letters and wherein a linker (SEQID NO:3) is posi of the protein to its targets. 15 tioned between the Anti-EGFR1 light chain and TGF-BRII In a preferred embodiment the therapeutically active anti and shown in italics. body-peptide fusion proteins is a targeting antibody fused to FIG. 3 shows the amino acid sequences of Anti-CTLA4 one or more immunomodulating moiety that counteracts TGFBRII fusion protein at LC constant region with amino immune tolerance of a cancer cell. In one aspect, the immu acid sequence of anti-CTLA4 heavy chain (SEQ ID NO: 7) nomodulating moiety may be linked by an amino acid spacer and amino acid sequence of anti-CTLA4 light chain (SEQID of sufficient length to allow bi-specific binding of the mol NO: 8) attached to amino acid residues for TGF-BRII (immu ecule. The immunomodulating moiety may be bound to either nomodulatory moiety) (SEQ ID NO: 4) identified in bold the C-terminus of the heavy or light chain of the antibody letters and wherein a linker (SEQ ID NO: 3) is positioned In a preferred method as described above, the immuno between the anti-CTLA4 light chain and TGF-BRII and modulating moiety is (i) Transforming growth factor-beta 25 shown in italics. (TGF-3), (ii) Programmed death-1 (PD-1), (iii) CTLA-4 or FIG. 4 shows the amino acid sequences of Anti-HER2/neu (iv) 4-1BB or parts thereof and the targeting antibody binds HC-4-1BB and LC-TGFBRII fusion protein with amino acid epidermal growth factor receptor (EGFR1, Erb-B 1), HER2/ sequence of Anti-HER2/neu/HC-4-1BB fusion protein neu (Erb-B2), CD20, CD6, CTLA-4, Mucin 1 (MUC-1), wherein the amino acid sequence for Anti-HER2/neu heavy Interleukin-2 (IL-2) or Interleukin-6 (IL-6). 30 chain (SEQID NO: 1) is attached to a linker (SEQID NO:3) The method of the present invention provides nucleotide shown in italics and the sequence for 4-1BB (immunomodu sequences that encode the therapeutically active antibody latory moiety) (SEQ ID NO: 9) is in written text font and peptide fusion proteins and Such expression may be con amino acid sequence of anti-HER2/neu light chain (SEQ ID ducted in a transient cell line or a stable cell line. The transient NO: 2) attached to amino residues for TGF-BRII (immuno expression is accomplished by transfecting or transforming 35 modulatory moiety) (SEQID NO: 4) identified in bold letters the host with vectors carrying the fusion proteins into mam and wherein a linker (SEQID NO: 3) is positioned between malian host cells the anti-HER2/neu light chain and TGF-BRII and shown in Once the fusion peptides are expressed, they are preferably italics. subjected to purification and in-vitro tests to check its bi FIG. 5 shows the amino acid sequence of Anti-EGFR1 specificity, that being, having the ability to bind to both the 40 HC-4-1BB and LC-TGFBRII fusion protein with amino acid target moiety and immunomodulating moiety. Such tests may sequence of Anti-EGFR1 heavy chain-4-1BB fusion protein include in-vitro test such as ELISA or NK/T-cell binding wherein the amino acid sequence for Anti-EGFR1 heavy assays to validate bi-functional target binding or immune cell chain (SEQID NO: 5) is attached to a linker (SEQID NO:3) stimulation. is shown in italics and the sequence for 4-1BB (immuno Notably once the specific fusion peptides demonstrate the 45 modulatory moiety) (SEQ ID NO: 9) is in written text font desired bi-specificity, such fusion peptides are selected for and amino acid sequence of light chain Anti-EGFR1 (SEQID Sub-cloning into a stable cell line for larger scale expression NO: 6) attached to amino residues for TGF-BRII (immuno and purification. Such stable cell lines are previously dis modulatory moiety) (SEQID NO: 4) identified in bold letters closed, such as a mammalian cell line, including but not with a linker (SEQID NO: 3) therebetween. limited to HEK293, CHO or NSO. 50 FIG. 6 shows the amino acid sequence of Anti-CTLA4 In a further aspect, the culture medium can be improved by HC-4-1BB and LC-TGFBRII fusion protein with amino acid additions to such medium. For example, the culture medium sequence of Anti-CTLA4 heavy chain-4-1BB fusion protein may include a divalent transitional metallic salt which is wherein the amino acid sequence for Anti-CTLA4 heavy added to the cell culture either initially or in fed-batch mode chain (SEQID NO: 7) is attached to a linker (SEQID NO:3) to reduce accumulation of lactate during culturing and/or 55 is shown in italics and the sequence for 4-1BB (immuno reduce heterogeneity of the fusion proteins. A desirable tran modulatory moiety) (SEQ ID NO: 9) is in written text font sitional metallic salt includes a Zinc ion and the addition of the and amino acid sequence of Anti-CTLA4 light chain (SEQID metal ion may be carried out during different phases of the NO: 8) is attached to amino residues for TGF-BRII (immu production. nomodulatory moiety) (SEQ ID NO: 4) identified in bold Other features and advantages of the invention will be 60 letters with a linker (SEQ ID NO: 3) therebetween. apparent from the following detailed description, drawings FIG. 7 shows the amino acid sequence of Anti-HER2/neu and claims. HC-PD1 and LC-TGFBRII fusion protein with amino acid sequence of Anti-HER2/neu heavy chain-PD1 fusion protein BRIEF DESCRIPTION OF THE DRAWINGS wherein the amino acid sequence for the Anti-HER2/neu 65 heavy chain (SEQID NO: 1) is attached to a linker (SEQ ID FIG. 1 shows the amino acid sequences of with the amino NO: 3) is shown in italics and the sequence for PD1 (immu acid sequence of Anti-HER2/neu-TGFBRII fusion protein at nomodulatory moiety) (SEQID NO: 10) is in written text font US 8,815,247 B2 7 8 and amino acid sequence of Anti-HER2/neu light chain (SEQ shown in italics and the sequence for TGFBRII (immuno ID NO: 2) is attached to amino residues for TGF-BRII (immu modulatory moiety) (SEQ ID NO: 4) is identified in bold nomodulatory moiety) (SEQ ID NO: 4) identified in bold letters and the amino acid sequence for PD-1 (immunomodu letters with a linker (SEQ ID NO: 3) therebetween. latory moiety) (SEQID NO: 10) is in written text font with FIG. 8 shows the amino acid sequence of Anti-EGFR1 linker between (SEQID No. 11) and including the amino acid HC-PD1 and LC-TGFBRII fusion protein with amino acid sequence of Anti-HER2/neu light chain (SEQID NO: 2). sequence of Anti-EGFR1 heavy chain-PD1 fusion protein FIG. 14 shows the amino acid sequence of Anti-EGFR1 wherein the amino acid sequence Anti-EGFR1 heavy chain HC-TGFBRII-PD1 fusion protein with amino acid sequence (SEQIDNO:5) is attached to a linker (SEQIDNO:3) shown of Anti-EGFR1 heavy chain-TGFBRII-PD1 fusion protein in italics and the sequence for PD1 (immunomodulatory moi 10 ety) (SEQID NO: 10) is in written text font and amino acid wherein the amino acid sequence Anti-EGFR1 heavy chain sequence of Anti-EGFR1 light chain (SEQ ID NO: 6) (SEQIDNO:5) is attached to a linker (SEQIDNO:3) shown attached to amino residues for TGF-BRII (immunomodula in italics and the sequence for TGFBRII (immunomodulatory tory moiety) (SEQID NO: 4) identified in bold letters with a moiety) (SEQ ID NO: 4) is identified in bold letters and the linker (SEQID NO:3) therebetween. 15 amino acid sequence for PD-1 (immunomodulatory moiety) FIG. 9 shows the amino acid sequence of Anti-CTLA4 (SEQ ID NO: 10) is in written text font with linker between HC-PD1 and LC-TGFBRII fusion protein with amino acid (SEQ ID No: 11) and including the amino acid sequence of sequence of Anti-CTLA4 heavy chain-PD1 fusion protein Anti-EGFR1 light chain (SEQID NO: 6). wherein the amino acid sequence Anti-CTLA4 heavy chain FIG. 15 shows the of Anti-CTLA4 HC-TGFBRII-PD1 (SEQIDNO: 7) is attached to a linker (SEQIDNO:3) shown fusion protein with amino acid sequence of Anti-CTLA4 in italics and the sequence for PD1 (immunomodulatory moi heavy chain-TGFBRII-PD1 fusion protein wherein the amino ety) (SEQID NO: 10) is in written text font and amino acid acid sequence Anti-CTLA4 heavy chain (SEQID NO: 7) is sequence of Anti-CTLA4 light chain (SEQ ID NO: 8) attached to a linker (SEQID NO: 3) shown in italics and the attached to amino residues for TGF-BRII (immunomodula sequence for TGFBRII (immunomodulatory moiety) (SEQ tory moiety) (SEQID NO: 4) identified in bold letters with a 25 ID NO: 4) is identified in bold letters and the amino acid linker (SEQID NO:3) therebetween. sequence for PD-1 (immunomodulatory moiety) (SEQ ID FIG. 10 shows the amino acid sequence of Anti-HER2/neu NO: 8) is in written text font with linker between (SEQ ID HC-TGFBRII-4-1BB fusion protein with amino acid NO: 11) and including the amino acid sequence of Anti sequence of Anti-HER2/neu heavy chain-TGFBRII-4-1BB CTLA4 light chain (SEQID NO: 8). fusion protein wherein the amino acid sequence for Anti 30 FIG.16 shows the nucleotide sequence of Anti-HER2/neu HER2/neu heavy chain (SEQIDNO: 1) is attached to a linker heavy chain constant region with linker (SEQID NO: 12) and (SEQID NO:3) shown in italics and the sequence for TGF TGFBRII ECD (SEQID NO: 13) that have been codon opti BRII (immunomodulatory moiety) (SEQ ID NO: 4) is iden mized for expression in CHO cell. tified in bold letters and the amino acid sequence for 4-1BB FIG. 17 shows the nucleotide sequence of Anti-HER2/neu (immunomodulatory moiety) (SEQ ID NO: 9) is in written 35 heavy chain variable region (SEQ ID NO: 14), Anti-HER2/ text font with linker between (SEQID No: 11) and including neu light chain variable region (SEQ ID NO: 15) and Anti the amino acid sequence of Anti-HER2/neu light chain (SEQ EGFR1 heavy chain constant region with linker (SEQID NO: ID NO: 2). 16) that have been codon optimized for expression in CHO FIG. 11 shows the amino acid sequence of Anti-EGFR1 cell. HC-TGFBRII-4-1BB fusion protein with amino acid 40 FIG. 18 shows the nucleotide sequence of Anti-EGFR1 sequence of Anti-EGFR1 heavy chain-TGFBRII-4-1BB heavy chain variable region (SEQID NO: 17), Anti-EGFR1 fusion protein wherein the amino acid sequence for Anti light chain variable region (SEQ ID NO: 18), Anti-CTLA4 EGFR1 heavy chain (SEQID NO: 5) sequence is attached to heavy chain variable region (SEQ ID NO: 19) and Anti a linker (SEQID NO:3) shown in italics and the sequence for CTLA4 light chain variable region (SEQ ID NO: 20) that TGFBRII (immunomodulatory moiety) (SEQ ID NO: 4) is 45 have been codon optimized for expression in CHO cell. identified in bold letters and the amino acid sequence for FIG. 19 shows the nucleotide sequence of Anti CD20 IgG1 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in molecule (SEQID NO: 21), Anti-CD20 heavy chain variable written text font with linker between (SEQ ID NO: 11) and region (SEQID NO: 22) and Anti-CD20 light chain variable including the amino acid sequence of Anti-EGFR1 light chain region (SEQID NO. 23) that have been codon optimized for (SEQ ID NO: 6). 50 expression in CHO cell. FIG. 12 shows the amino acid sequence of Anti-CTLA4 FIG. 20 shows the nucleotide sequence of 4-1BB (SEQID HC-TGFBRII-4-1BB fusion protein with amino acid NO: 24) and Anti-IL6R heavy chain (SEQ ID NO: 25) that sequence of Anti-CTLA4 heavy chain-TGFBRII-4-1BB have been codon optimized for expression in CHO cell. fusion protein wherein the amino acid sequence Anti-CTLA4 FIG. 21 shows the nucleotide sequence of Anti-IL6R light heavy chain (SEQID NO: 7) is attached to a linker (SEQ ID 55 chain variable region (SEQ ID NO: 26), Anti-4-1BB heavy NO: 3) shown in italics and the sequence for TGFBRII (im chain (SEQID NO: 27) and Anti-4-1BB light chain variable munomodulatory moiety) (SEQ ID NO: 4) is identified in region (SEQID NO: 28) that have been codon optimized for bold letters and the amino acid sequence for 4-1BB (immu expression in CHO cell. nomodulatory moiety) (SEQID NO: 9) is in written text font FIG. 22 shows the analysis of Protein A purified Anti with linker between (SEQ ID NO: 11) and including the 60 HER2/neu-TGFBRII and Anti-EGFR1-TGFBRII at 12% amino acid sequence of Anti-CTLA4 light chain (SEQ ID PAGE NO:8). FIG. 23 A shows Anti-HER2/neu-TGFBRII samples ana FIG. 13 shows the amino acid sequence of Anti-HER2/neu lyzed by Protein A/SEC Chromtography and B Anti-EGFR1 HC-TGFBRII-PD1 fusion protein with amino acid sequence TGFBRII samples analyzed by Protein A/SEC Chromtogra of Anti-HER2/neu heavy chain-TGFBRII-PD1 fusion protein 65 phy. wherein the amino acid sequence Anti-HER2/neu heavy FIG. 24. A shows that Anti-HER2/neu-TGFBRII and Anti chain (SEQID NO: 1) is attached to a linker (SEQID NO:3) EGFR1-TGFBRII molecules bind to the TGFB indicating that US 8,815,247 B2 10 the fusion protein is functional and B shows that Anti-HER2 letters and wherein a linker (SEQ ID NO: 3) is positioned TGFBRII inhibits the proliferation of BT474 cell line similar between the Cantuzumab light chain and TGF-BRII and to the Bmab200 (Herceptin). shown in italics. FIG. 25 shows that Anti-EGFR1-TGFBRII-inhibits the FIG. 47 shows the amino acid sequences of Cixutumumab proliferation of A431 cell line similar to the Cetuximab. TGFBRII fusion protein at LC constant region with amino FIG. 26 shows the ADCC activity of Anti-HER2-TGFBRII acid sequence of Cixutumumab heavy chain (SEQ ID NO: on BT474 cells is similar to that of Bmab200 (Herceptin). 31) and amino acid sequence of Cixutumumab light chain FIG. 27 shows the ADCC activity of Anti-EGFR1-TGF (SEQ ID NO:32) attached to amino acid residues for TGF BRII on A431 cells wherein the ADCC activities are similar to BRII (immunomodulatory moiety) (SEQID NO: 4) identified that of Cetuximab. 10 in bold letters and wherein a linker (SEQID NO:3) is posi FIG. 28 shows the ADCC activity of ADCC activity of tioned between the Cixutumumab light chain and TGF-BRII Anti-EGFR1-4-1BB in comparison with Anti-EGFR1-TGF and shown in italics. BRII and cetuximab. FIG. 48 shows the amino acid sequences of Clivatuzumab FIG. 29 A shows that the binding activity of Anti-CTLA4 TGFBRII fusion protein at LC constant region with amino TGFBRII to TGFB1 is comparable to Anti-EGFR1-TGFBRII 15 acid sequence of Clivatuzumab heavy chain (SEQID NO:33) and B shows that the binding activity of Anti-CTLA4-TGF and amino acid sequence of ClivatuZumab light chain (SEQ |BRII to CTLA4. ID NO. 34) attached to amino acid residues for TGF-BRII FIG. 30 A shows the binding activity of Anti-CTLA4 (immunomodulatory moiety) (SEQ ID NO: 4) identified in TGFBRII to determine the level of PD1-Fc binding and B bold letters and wherein a linker (SEQIDNO:3) is positioned shows the binding activity of Anti-EGRF1-4-1BB to deter between the Clivatuzumab light chain and TGF-BRII and mine the binding of 4-1BBL. shown in italics. FIG. 31A shows the binding activity of Anti-EGFR1-4- FIG. 49 shows the amino acid sequences of Pritumumab 1BB to EGFR and B shows the binding activity of PD1-Fc TGFBRII fusion protein at LC constant region with amino 4-1BB to find out PDLL-Fc. acid sequence of Pritumumab heavy chain (SEQID NO: 35) FIG.32 shows the binding activity of Anti-EGFR1-PD1 to 25 and amino acid sequence of Pritumumab light chain (SEQID EGFR and PD1. NO:36) attached to amino acid residues for TGF-BRII (im FIG. 33 shows photographs of expressed proteins and munomodulatory moiety) (SEQID NO: 4) identified in bold reduction alkylation thereof. letters and wherein a linker (SEQ ID NO: 3) is positioned FIG.34A shows the mass spectrum Mass Spectrum of light between the Pritumumab light chain and TGF-BRII and chain (LC) (Reduced) of Anti-HER2/neu-TGFBRII ECD 30 shown in italics. fusion and B shows Deconvoluted Mass Spectrum of LC FIG. 50 shows the amino acid sequence of Cantuzumab (Reduced) of Anti-HER2/neu-TGFBRII ECD fusion. 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 35 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 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. 40 letters with a linker (SEQ ID NO: 3) therebetween. FIG. 38 A shows the UV Chromatogram of Tryptic Pep FIG. 51 shows the amino acid sequence of Cixutumumab tides of Anti-HER2/neu-TGFBRII ECD fusion protein and B HC-4-1BB and LC-TGFBRII fusion protein wherein the shows the Total Ion Chromatogram (TIC) of Tryptic Peptides amino acid sequence for the Cixutumumab heavy chain (SEQ of Anti-HER2/neu-TGFBRII ECD fusion protein. ID NO:31) is attached to a linker (SEQ ID NO: 3) shown in FIGS. 39, 40 and 41 provide lists of expected/observed 45 italics and the sequence for 4-1BB (immunomodulatory moi tryptic peptide of the light chain, heavy chain and linked motif ety) (SEQ ID NO: 9) is in written text font and amino acid of the Anti-HER2/neu-TGFBRII ECD fusion protein, respec sequence of Cixutumumab light chain (SEQ ID NO:32) is tively. attached to amino residues for TGF-BRII (immunomodula FIG. 42 A shows the UV Chromatogram of Tryptic Pep tory moiety) (SEQID NO: 4) identified in bold letters with a tides of Anti-EGFR1-TGFBRII ECD fusion protein and B 50 linker (SEQID NO:3) therebetween. shows the Total Ion Chromatogram (TIC) of Tryptic Peptides FIG. 52 shows the amino acid sequence of ClivatuZumab of Anti-EGFR1-TGFBRII ECD fusion protein. HC-4-1BB and LC-TGFBRII fusion protein wherein the FIG. 43 provides a list of expected/observed tryptic peptide amino acid sequence for the Clivatuzumab heavy chain (SEQ of the light chain of the Anti-EGFR1-TGFBRII ECD fusion ID NO: 33) is attached to a linker (SEQ ID NO: 3) shown in protein. 55 italics and the sequence for 4-1BB (immunomodulatory moi FIG. 44 shows the list of expected/observed tryptic peptide ety) (SEQ ID NO: 9) is in written text font and amino acid of the heavy chain of the Anti-EGFR1-TGFBRII ECD fusion sequence of Clivatuzumab light chain (SEQ ID NO. 34) is protein. attached to amino residues for TGF-BRII (immunomodula FIG. 45 shows the list of expected/observed tryptic peptide tory moiety) (SEQID NO: 4) identified in bold letters with a of the heavy chain of the Anti-EGFR1-TGFBRII ECD fusion 60 linker (SEQID NO:3) therebetween. protein. FIG. 53 shows the amino acid sequence of Pritumumab FIG. 46 shows the amino acid sequences of Cantuzumab HC-4-1BB and LC-TGFBRII fusion protein wherein the TGFBRII fusion protein at LC constant region with amino amino acid sequence for the Pritumumab heavy chain (SEQ acid sequence of Cantuzumab heavy chain (SEQID NO: 29) ID NO:35) is attached to a linker (SEQ ID NO: 3) shown in and amino acid sequence of Cantuzumab light chain (SEQID 65 italics and the sequence for 4-1BB (immunomodulatory moi NO: 30) attached to amino acid residues for TGF-BRII (im ety) (SEQ ID NO: 9) is in written text font and amino acid munomodulatory moiety) (SEQID NO: 4) identified in bold sequence of Pritumumab light chain (SEQ ID NO: 36) is US 8,815,247 B2 11 12 attached to amino residues for TGF-BRII (immunomodula sequence for TGFBRII (immunomodulatory moiety) (SEQ tory moiety) (SEQID NO: 4) identified in bold letters with a ID NO: 4) is identified in bold letters and the amino acid linker (SEQID 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 (SEQID No: HC-PD1 and LC-TGFBRII fusion protein wherein the amino 11) and including the amino acid sequence of ClivatuZumab acid sequence for the Cantuzumab heavy chain (SEQID NO: light chain (SEQID NO:34). 29) is attached to a linker (SEQID NO:3) shown in italics and FIG. 61 shows the amino acid sequence of Pritumumab the sequence for PD1 (immunomodulatory moiety) (SEQID HC-TGFBRII-4-1BB fusion protein wherein the amino acid NO: 10) is in written text font and amino acid sequence of sequence for Pritumumab heavy chain (SEQ ID NO: 35) is Cantuzumab light chain (SEQ ID NO: 30) is attached to 10 attached to a linker (SEQID NO: 3) shown in italics and the amino residues for TGF-BRII (immunomodulatory moiety) sequence for TGFBRII (immunomodulatory moiety) (SEQ (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 4) is identified in bold letters and the amino acid ID NO: 3) therebetween. sequence for 4-1BB (immunomodulatory moiety) (SEQ ID FIG.55 shows the amino acid sequence of Cixutumumab NO:9) is in written text font with linker between (SEQID No: HC-PD1 and LC-TGFBRII fusion protein wherein the amino 15 11) and including the amino acid sequence of Pritumumab acid sequence for the Cixutumumab heavy chain (SEQ ID light chain (SEQID NO:36). NO: 31) is attached to a linker (SEQ ID NO: 3) shown in FIG. 62 shows the amino acid sequence of Cantuzumab italics and the sequence for PD1 (immunomodulatory moi HC-TGFBRII-PD1 fusion protein wherein the amino acid ety) (SEQID NO: 10) is in written text font and amino acid sequence for Cantuzumab heavy chain (SEQ ID NO: 29) is sequence of Cixutumumab light chain (SEQ ID NO:32) is attached to a linker (SEQID NO: 3) shown in italics and the attached to amino residues for TGF-BRII (immunomodula sequence for TGFBRII (immunomodulatory moiety) (SEQ tory moiety) (SEQID NO: 4) identified in bold letters with a ID NO: 4) is identified in bold letters and the amino acid linker (SEQID NO:3) therebetween. sequence for PD1 (immunomodulatory moiety) (SEQ ID FIG. 56 shows the amino acid sequence of Clivatuzumab NO: 10) is in written text font with linker between (SEQ ID HC-PD1 and LC-TGFBRII fusion protein wherein the amino 25 No: 11) and including the amino acid sequence of Cantu acid sequence for the Clivatuzumab heavy chain (SEQ ID Zumab light chain (SEQID NO:30). NO: 33) is attached to a linker (SEQ ID NO: 3) shown in FIG. 63 shows the amino acid sequence of Cixutumumab italics and the sequence for PD1 (immunomodulatory moi HC-TGFBRII-PD1 fusion protein wherein the amino acid ety) (SEQID NO: 10) is in written text font and amino acid sequence for Cixutumumab heavy chain (SEQID NO:31) is sequence of Clivatuzumab light chain (SEQ ID NO. 34) is 30 attached to a linker (SEQID NO: 3) shown in italics and the attached to amino residues for TGF-BRII (immunomodula sequence for TGFBRII (immunomodulatory moiety) (SEQ tory moiety) (SEQID NO: 4) identified in bold letters with a ID NO: 4) is identified in bold letters and the amino acid linker (SEQID NO:3) therebetween. sequence for PD1 (immunomodulatory moiety) (SEQ ID FIG. 57 shows the amino acid sequence of Pritumumab NO: 10) is in written text font with linker between (SEQ ID HC-PD1 and LC-TGFBRII fusion protein wherein the amino 35 No: 11) and including the amino acid sequence of Cixutu acid sequence for the Pritumumab heavy chain (SEQID NO: mumab light chain (SEQID NO:32). 35) is attached to a linker (SEQID NO:3) shown in italics and FIG. 64 shows the amino acid sequence of ClivatuZumab the sequence for PD1 (immunomodulatory moiety) (SEQID HC-TGFBRII-PD1 fusion protein wherein the amino acid NO: 10) is in written text font and amino acid sequence of sequence for ClivatuZumab heavy chain (SEQID NO: 33) is Pritumumab light chain (SEQ ID NO: 36) is attached to 40 attached to a linker (SEQID NO: 3) shown in italics and the amino residues for TGF-BRII (immunomodulatory moiety) sequence for TGFBRII (immunomodulatory moiety) (SEQ (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 4) is identified in bold letters and the amino acid ID NO: 3) therebetween. sequence for PD1 (immunomodulatory moiety) (SEQ ID FIG. 58 shows the amino acid sequence of Cantuzumab NO: 10) is in written text font with linker between (SEQ ID HC-TGFBRII-4-1BB fusion protein wherein the amino acid 45 No: 11) and including the amino acid sequence of Clivatu sequence for Cantuzumab heavy chain (SEQ ID NO: 29) is Zumab light chain (SEQID NO:34). attached to a linker (SEQID NO: 3) shown in italics and the FIG. 65 shows the amino acid sequence of Pritumumab sequence for TGFBRII (immunomodulatory moiety) (SEQ HC-TGFBRII-PD1 fusion protein wherein the amino acid ID NO: 4) is identified in bold letters and the amino acid sequence for Pritumumab heavy chain (SEQ ID NO: 35) is sequence for 4-1BB (immunomodulatory moiety) (SEQ ID 50 attached to a linker (SEQID NO: 3) shown in italics and the NO:9) is in written text font with linker between (SEQID No: sequence for TGFBRII (immunomodulatory moiety) (SEQ 11) and including the amino acid sequence of Cantuzumab ID NO: 4) is identified in bold letters and the amino acid light chain (SEQID NO:30). sequence for PD1 (immunomodulatory moiety) (SEQ ID FIG. 59 shows the amino acid sequence of Cixutumumab NO: 10) is in written text font with linker between (SEQ ID HC-TGFBRII-4-1BB fusion protein wherein the amino acid 55 No: 11) and including the amino acid sequence of Pritu sequence for Cixutumumab heavy chain (SEQID NO:31) is mumab light chain (SEQID NO:36). attached to a linker (SEQID NO: 3) shown in italics and the sequence for TGFBRII (immunomodulatory moiety) (SEQ DETAILED DESCRIPTION OF THE INVENTION ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory moiety) (SEQ ID 60 The practice of the present invention will employ, unless NO:9) is in written text font with linker between (SEQID No: otherwise indicated, conventional techniques of immunol 11) and including the amino acid sequence of Cixutumumab ogy, molecular biology, microbiology, cell biology and light chain (SEQID NO:32). recombinant DNA, which are within the skill of the art. See, FIG. 60 shows the amino acid sequence of Clivatuzumab e.g., Sambrook, et al. MOLECULAR CLONING: A LABO HC-TGFBRII-4-1BB fusion protein wherein the amino acid 65 RATORY MANUAL, 2nd edition (1989); CURRENT PRO sequence for ClivatuZumab heavy chain (SEQID NO: 33) is TOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et attached to a linker (SEQID NO: 3) shown in italics and the al, eds., (1987)); the series METHODS IN ENZYMOLOGY US 8,815,247 B2 13 14 (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH gene in the target cell. An example of a transgene is a nucleic (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. acid encoding a chimeric fusion protein of the present inven (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A tion. LABORATORY MANUAL, and ANIMAL CELL CUL The term “expression vector” as used herein means a vec TURE (R. I. Freshney, ed. (1987)). tor containing a nucleic acid sequence coding for at least part Definitions of a gene product capable of being transcribed. Expression Unless otherwise defined, all technical and scientific terms vectors can contain a variety of control sequences, which used herein have the meaning commonly understood by one refer to nucleic acid sequences necessary for the transcription ofordinary skill in the art to which this invention belongs. The and possibly translation of an operatively linked coding 10 sequence in a particular host organism. In addition to control terminology used herein is for the purpose of describing sequences that govern transcription and translation, Vectors particular embodiments only and is not intended to be limit and expression vectors may contain nucleic acid sequences ing of the invention. As used in the description of the inven that serve other functions as well. The term also includes a tion and the appended claims, the singular forms 'a', 'an recombinant plasmidor virus that comprises a polynucleotide and “the are intended to include the plural forms as well, 15 to be delivered into a host cell, either in vitro or in vivo. unless the context clearly indicates otherwise. The following Preferably the host cell is a transient cell line or a stable cell terms have the meanings given: line and more preferably a mammalian host cell and selected The term “polynucleotide' as used herein means a from the group consisting of HEK293, CHO and NSO. sequence of nucleotides connected by phosphodiester link The term “subject,” as used herein means a human or ages. Polynucleotides are presented herein in the direction Vertebrate animal including a dog, cat, horse, cow, pig, sheep, from the 5' to the 3' direction. A polynucleotide of the present goat, chicken, monkey, rat, and mouse. invention can be a deoxyribonucleic acid (DNA) molecule or The term “therapeutically effective amount” as used herein ribonucleic acid (RNA) molecule. Where a polynucleotide is means the amount of the subject compound that will elicit the a DNA molecule, that molecule can be a gene or a cDNA biological or medical response of a tissue, system, animal or molecule. Nucleotide bases are indicated herein by a single 25 human that is being sought by the researcher, Veterinarian, letter code: adenine (A), guanine (G), thymine (T), cytosine medical doctor or other clinician. (C), inosine (I) and uracil (U). A polynucleotide of the present The term “pharmaceutically acceptable' as used herein invention can be prepared using standard techniques well means the carrier, diluent or excipient must be compatible known to one of skill in the art. with the other ingredients of the formulation and not delete The term, “optimized as used herein means that a nucle 30 rious to the recipient thereof. otide sequence has been altered to encode an amino acid The term “recombinant’ as used herein means a genetic sequence using codons that are preferred in the production entity distinct from that generally found in nature. As applied cell or organism, generally a eukaryotic cell, for example, a to a polynucleotide or gene, this means that the polynucle cell of Pichia, a cell of Trichoderma, a Chinese Hamster otide is the product of various combinations of cloning, Ovary cell (CHO) or a human cell. The optimized nucleotide 35 restriction and/or ligation steps, and other procedures that sequence is engineered to retain completely or as much as result in the production of a construct that is distinct from a possible the amino acid sequence originally encoded by the polynucleotide found in nature. starting nucleotide sequence, which is also known as the The term “substantial identity” or “substantial similarity.” "parental” sequence. The optimized sequences herein have as used herein when referring to a nucleic acid or fragment been engineered to have codons that are preferred in CHO 40 thereof, indicates that when optimally aligned with appropri mammalian cells; however optimized expression of these ate nucleotide insertions or deletions with another nucleic sequences in other eukaryotic cells is also envisioned herein. acid (or its complementary strand), there is nucleotide The amino acid sequences encoded by optimized nucleotide sequence identity in at least about 95 to 99% of the sequence. sequences are also referred to as optimized. The term "expres The term "peptide.” “polypeptide' and “protein’ are used sion” as used herein is defined as the transcription and/or 45 interchangeably to denote a sequence polymer of at least two translation of a particular nucleotide sequence driven by its amino acids covalently linked by an amide bond. promoter. The term "homologous' as used herein and relating to The term “transfection' of a cell as used herein means that peptides refers to amino acid sequence similarity between genetic material is introduced into a cell for the purpose of two peptides. When an amino acid position in both of the genetically modifying the cell. Transfection can be accom 50 peptides is occupied by identical amino acids, they are plished by a variety of means known in the art, Such as homologous at that position. Thus by 'substantially homolo transduction or electroporation. gous' means an amino acid sequence that is largely, but not The term "cancer as used herein is defined as disease entirely, homologous, and which retains most or all of the characterized by the rapid and uncontrolled growth of aber activity as the sequence to which it is homologous. As used rant cells. Cancer cells can spread locally or through the 55 herein, “substantially homologous' as used herein means that bloodstream and lymphatic system to other parts of the body. a sequence is at least 50% identical, and preferably at least Examples of various cancers include but are not limited to, 75% and more preferably 95% homology to the reference breast cancer, prostate cancer, ovarian cancer, cervical cancer, peptide. Additional peptide sequence modification are skin cancer, ocular cancer, pancreatic cancer, colorectal can included, such as minor variations, deletions, Substitutions or cer, renal cancer, liver cancer, brain cancer, lymphoma, leu 60 derivitizations of the amino acid sequence of the sequences kemia, lung cancer and the like. disclosed herein, so long as the peptide has Substantially the The term “transgene is used in a broad sense to mean any same activity or function as the unmodified peptides. Notably, heterologous nucleotide sequence incorporated in a vector for a modified peptide will retain activity or function associated expression in a target cell and associated expression control with the unmodified peptide, the modified peptide will gen sequences, such as promoters. It is appreciated by those of 65 erally have an amino acid sequence “substantially homolo skill in the art that expression control sequences will be gous' with the amino acid sequence of the unmodified selected based on ability to promote expression of the trans Sequence. US 8,815,247 B2 15 16 The term “administering as used herein is defined as the seminated cancers (for prevention or treatment of diverse actual physical introduction of the composition into or onto cancers); (ii) To produce immune cell compositions for adop (as appropriate) the host Subject. Any and all methods of tive cellular therapy of diverse cancers; and (iii) To serve as introducing the composition into the Subject are contem immune adjuvants or vaccines for prophylaxis of diverse plated according to the present invention; the method is not cancers or infectious diseases. dependent on any particular means of introduction and is not The targeted immunostimulatory antibodies and/or fusion to be so construed. Means of introduction are well-known to proteins of the invention provide the ability to disrupt immu those skilled in the art, and preferably, the composition is nosuppressive networks in the tumor microenvironment. administered subcutaneously or intratumorally. One skilled Tumors employ a wide array of regulatory mechanisms to in the art will recognize that, although more than one route 10 avoid or Suppress the immune response. Cancer cells actively can be used for administration, a particular route can provide promote immune tolerance in the tumor microenvironment a more immediate and more effective reaction than another via the expression of cytokines and molecules that inhibit the route. Local or systemic delivery can be accomplished by differentiation and maturation of antigen-presenting den administration comprising application or instillation of the dritic cells (DC). The immunosuppressive cytokines and immunovaccines into body cavities, inhalation or insufflation 15 ligands produced by tumor cells include the following: (i) of anaerosol, or by parenteral introduction, comprising intra Transforming growth factor-beta (TGF-B); (ii) Programmed muscular, intravenous, intraportal, intrahepatic, peritoneal, death-1 ligand 1 (PD-L1; B7-H1); (iii) Vascular endothelial Subcutaneous, or intradermal administration. In the event that growth factor (VEGF); and (iv) Interleukin-10 (IL-10). the tumor is in the central nervous system, the composition In addition to blocking dendritic cell (DC) maturation, must be administered intratumorally because there is no these molecules promote the development of specialized sub priming of the immune system in the central nervous system. sets of immunosuppressive CD4 T cells (regulatory T cells; Although chemotherapeutic agents can induce “immuno Treg cells) and myeloid-derived suppressor cells (MDSC). genic tumor cell death and facilitate cross-presentation of Tregs are a minority sub-population of CD4 T cells that antigens by dendritic cells, tumors create a tolerogenic envi constitutively express CD25 the interleukin-2 (IL-2) recep ronment that allows them to Suppress the activation of innate 25 tor cc-chain and the forkhead box P3 (FOXP3) transcription and adaptive immune responses and evade immunologic factor. Tregs (CD4+CD25+FoxP3+cells) maintain immune attack by immune effector cells. The present invention pro tolerance by restraining the activation, proliferation, and vides strategies to counteract tumor-induced immune toler effector functions of a wide range of immune cells, including ance in the tumor microenvironment and can enhance the CD4 and CDS T cells, natural killer (NK) and NKT cells, B antitumor efficacy of chemotherapy by activating and lever 30 cells and antigen presenting cells (APCs) in vitro and in vivo. aging T cell-mediated adaptive antitumor immunity against The accumulation of Treg cells in the tumor microenviron disseminated cancer cells. ment reinforces tumor immune tolerance and facilitates The present invention is based on the discovery that tar tumor progression and metastases. The increased expression geted immunomodulatory antibodies or fusion proteins of the of immunosuppressive cytokines (TGF-B; PD-L1) and present invention can counteract or reverse immune tolerance 35 tumor-infiltrating Tregs is correlated with a reduction of Sur of cancer cells. Cancer cells are able to escape elimination by vival of patients with diverse types of cancers. The fusion chemotherapeutic agents or tumor-targeted antibodies via proteins of the present invention inhibit key immunosuppres specific immunosuppressive mechanisms in the tumor sive molecules expressed by the targeted tumor cell or tumor microenvironment and Such ability of cancer cells is recog infiltrating Treg cells and myeloid suppressor cells (DCs or nized as immune tolerance. By counteracting tumor-induced 40 MDSC). As such, they provide the targeted ability to inhibit immune tolerance, the present invention provides effective the development or function of Tregs within the tumor compositions and methods for cancer treatment, optional in microenvironment. combination with another existing cancer treatment. The present invention provides a method of preventing or The present invention provides compositions and methods treating a neoplastic disease. The method includes adminis for producing fusion proteins that counteract immune toler 45 tration to a Subject in need thereof one or more fusion proteins ance in the tumor microenvironment and promote T cell of the present invention in combination with another antican mediated adaptive antitumor immunity for maintenance of cer therapy, wherein the anticancer therapy is a chemothera durable long-term protection against recurrent or dissemi peutic molecule, antibody, Small molecule kinase inhibitor, nated cancers. These fusion proteins are designed to facilitate hormonal agent, cytotoxic agent, targeted therapeutic agent, effective long term T cell-mediated immune responses 50 anti-angiogenic agent, ionizing radiation, ultraviolet radia against tumor cells by at least one of the following: tion, cryoablation, thermal ablation, or radiofrequency abla a. promoting death of tumor cells via enhancement of anti tion. body-dependent cellular cytotoxicity (ADCC); and As used herein, the term “antibody' includes natural or b. increasing activation and proliferation of antitumor CD8+ artificial mono- or polyvalent antibodies including, but not T cells by negating immune Suppression mediated by regu 55 limited to, polyclonal, monoclonal, multispecific, human, latory T cells and myeloid suppressor cells. These antitu humanized or chimeric antibodies, single chain antibodies, mor immune responses may be activated in tandem with Fab fragments. F(ab') fragments, fragments produced by a the sensitization of tumor cells to immune effector-medi Fab expression library, anti-idiotypic (anti-Id) antibodies (in ated cytotoxicity, thereby establishing a positive feedback cluding, e.g., anti-Id antibodies to antibodies of the inven loop that augments tumor cytoreduction and reinforces 60 tion), and epitope-binding fragments of any of the above. The adaptive antitumor immunity. antibody may be from any animal origin including birds and In addition, the fusion proteins of the present invention are mammals. In one aspect, the antibody is, or derived from, a distinguished from and Superior to existing therapeutic, mol human, murine (e.g., mouse and rat), donkey, sheep, rabbit, ecules in at least one of the following aspects: (i) To counter goat, guinea pig, camel, horse, or chicken. Further, Such anti act immune tolerance in the tumor microenvironment and 65 body may be a humanized version of an antibody. The anti promote T cell-mediated adaptive antitumor immunity for body may be monospecific, bispecific, trispecific, or of maintenance of long-term protection against recurrent or dis greater multispecificity. The antibody herein specifically US 8,815,247 B2 17 18 include a “chimericantibody in which a portion of the heavy tions of a humanized antibody often come from CDRs in and/or light chain is identical with or homologous to corre murine antibodies. In any event, these regions are crucial to sponding sequences in antibodies derived from a particular allow the antibody to recognize and bind to a specific antigen. species or belonging to aparticular antibody class or Subclass, In one embodiment, a hybridoma can produce a targeted while the remainder of the chain(s) is identical with or 5 fusion protein comprising a targeting moiety and an immu homologous to corresponding sequences in antibodies nomodulatory moiety. In one embodiment, a targeting moiety derived from another species or belonging to another anti comprising an antibody, antibody fragment, or polypeptide is body class or Subclass, as well as fragments of Such antibod linked or fused to an immunomodulatory moiety consisting ies, so long as they exhibit the desired biological activity. of a polypeptide, with a linker or without a linker. The linker Examples of antibodies which can be incorporated into 10 compositions and methods disclosed herein include, but are can be an amino acid linker. In one embodiment, a linker is not limited, to antibodies such as trastuzumab (anti-HER2/ (GGGGS)n wherein n is 1, 2, 3, 4, 5, 6, 7, or 8. For example, neu antibody); Pertuzumab (anti-HER2 mAb); cetuximab GGGGSGGGGSGGGGS (SEQ ID NO: 4). In another (chimeric monoclonal antibody to epidermal growth factor embodiment, a linker is EPKSCDK (SEQ ID NO: 11). In receptor EGFR): panitumumab (anti-EGFR antibody); nimo 15 various aspects, the length of the linker may be modified to tuzumab (anti-EGFR antibody); Zalutumumab (anti-EGFR optimize binding of the target moiety or the function of the mAb); Necitumumab (anti-EGFR mab); MDX-210 (human immunomodulatory moiety. In various aspects, the immuno ized anti-HER-2 bispecific antibody); MDX-210 (humanized modulatory moiety is a polypeptide that is fused to the C-ter anti-HER-2 bispecific antibody); MDX-447 (humanized minus of the Fc region of the heavy chain of a targeting anti-EGF receptor bispecific antibody); Rituximab (chimeric antibody or Fc-containing fusion protein. In another aspect, murine/human anti-CD20 mAb): Obinutuzumab (anti-CD20 the immunomodulatory moiety is a polypeptide that is fused mAb): Ofatumumab (anti-CD20 mAb); Tositumumab-1 131 to the C-terminus of the light chain of a targeting antibody. (anti-CD20 mAb); ibritumomab tiuxetan (anti-CD20 mAb); An antibody fragment can include a portion of an intact, Bevacizumab (anti-VEGF mab); Ramucirumab (anti antibody, e.g. including the antigen-binding or variable VEGFR2 mAb); Ranibizumab (anti-VEGF mAb): Afiliber 25 region thereof. Examples of antibody fragments include Fab, cept (extracellular domains of YEGFR1 and VEGFR2 fused Fab'. F(ab')2, and Fv fragments; Fc fragments or Fc-fusion to IgG1 Fc): AMG386 (angiopoietin-1 and -2 binding peptide products; diabodies; linear antibodies; single-chain antibody fused to IgG1 Fc); Dalotuzumab (anti-1GF-1R mAb): Gem molecules; and multispecific antibodies formed from anti tuzumab ozogamicin (anti-CD33 mAb); Alemtuzumab (anti body fragment(s). An intactantibody is one which includes an Campath-1/CD52 mAb); Brentuximab vedotin (anti-CD30 30 mAb); Catumaxomab (bispecific mAb that targets epithelial antigen-binding variable region as well as a light chain con cell adhesion molecule and CD3): Naptumomab (anti-5T4 stant domain (CL) and heavy chain constant domains, CHI, mAb); Girentuximab (anti-Carbonic anhydrase ix): or Farl CH2 and CH3. The constant domains may be native sequence etuzumab (anti-folate receptor). Other examples include anti constant domains (e.g., human native sequence constant bodies such as PanorexTM (17-1A) (murine monoclonal anti 35 domains) or amino acid sequence variant thereof for any other body); Panorex ((a) (17-1 A) (chimeric murine monoclonal modified Fc (e.g. glycosylation or other engineered Fc). antibody); BEC2 (ami-idiotypic mAb, mimics the GD The fusion proteins of the present invention may be syn epitope) (with BCG): Oncolym (Lym-1 monoclonal anti thesized by conventional techniques known in the art, for body); SMART M 195 Ab, humanized 13'1 LYM-1 (Onco example, by chemical synthesis Such as Solid phase peptide lym), Ovarex (B43.13, anti-idiotypic mousemab); 3622W94 40 synthesis. Such methods are known to those skilled in the art. mAb that binds to EGP40 (17-1A) pancarcinoma antigen on In general, these methods employ either Solid or Solution adenocarcinomas; Zenapax (SMART Anti-Tac (IL-2 recep phase synthesis methods, well known in the art. Specifically, tor); SMART M1 95 Ab, humanized Ab, humanized): the methods comprise the sequential addition of one or more NovoMAb-G2 (pancarcinoma specific Ab): TNT (chimeric amino acids or Suitably protected amino acids to a growing mAb to histone antigens); TNT (chimeric mAb to histone 45 peptide chain. Normally, either the amino or carboxyl group antigens); GJiomab-H (Monoclonals—Humanized Abs); of the first amino acid is protected by a Suitable protecting GN1-250 Mab; EMD-72000 (chimeric-EGF antagonist); group. The protected or derivatized amino acid can then be LymphoCide (humanized IL.L.2 antibody); and MDX-260 either attached to an inert solid support or utilized in solution bispecific, targets GD-2, ANA Ab, SMART lDiO Ab, by adding the next amino acid in the sequence having the SMART ABL 364 Ab or ImmuRAIT-CEA. 50 complementary (amino or carboxyl) group Suitably pro Various methods have been employed to produce antibod ies. Hybridoma technology, which refers to a cloned cell line tected, under conditions suitable for forming the amide link that produces a single type of antibody, uses the cells of age. The protecting group is then removed from this newly various species, including mice (murine), hamsters, rats, and added amino acid residue and the next amino acid (Suitably humans. Another method to prepare an antibody uses genetic 55 protected) is then added, and so forth. After all the desired engineering including recombinant DNA techniques. For amino acids have been linked in the proper sequence, any example, antibodies made from these techniques include, remaining protecting groups and any solid Support are among others, chimeric antibodies and humanized antibod removed either sequentially or concurrently to afford the final ies. A chimeric antibody combines DNA encoding regions polypeptide. By simple modification of this general proce from more than one type of species. For example, a chimeric 60 dure, it is possible to add more than one amino acid at a time antibody may derive the variable region from a mouse and the to a growing chain, for example, by coupling (under condition constant region from a human. A humanized antibody comes that do not racemize chiral centers) a protected tripeptide with predominantly from a human, even though it contains non a properly protected dipeptide to form, after deprotection, a human portions. Like a chimeric antibody, a humanized anti pentapeptide. body may contain a completely human constant region. But 65 Typical protecting groups include t-butyloxycarbonyl unlike a chimeric antibody, the variable region may be par (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benxyloxycar tially derived from a human. The nonhuman, synthetic por bonyl (Cbz), p-toluenesulfonyl (Tos); 2,4-dinitrophenyl, ben US 8,815,247 B2 19 20 Zyl (BZl), biphenylisopropyloxy-carboxycarbonyl, cyclo coli, Bacillus subtilis, and Streptococcus spp., will find use hexyl, isopropyl, acetyl, o-nitrophenylsulfonyl, and the like. with the present expression constructs. Yeast hosts useful in Of these, Boc and Fmoc are preferred. the present invention include interalia, Saccharomyces cer Typical Solid Supports are generally cross-linked poly evisiae, Candida albicans, Candida maltosa, Hansenula meric materials. These include divinylbenzene cross-linked 5 polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, styrene-based polymers, for example, divinylbenzene-hy Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces droxymethylstyrene copolymers, divinylbenzene-chlorom pombe and Yarrowia lipolytica. Insect cells for use with bacu ethylstyrene copolymers, and divinylbenzene-benzhydry lovirus expression vectors include, interalia, Aedes aegypti, laminopolystyrene copolymers. The divinylbenzene Autographa Californica, Bombyx mori, Drosophila melano benzhydrylaminopolystyrene copolymers, as illustrated 10 gaster; Spodoptera frugiperda, and Trichoplusia ni. The pro herein using p-methyl-benzhydrylamine resin, offers the teins may also be expressed in Trypanosomes. advantage of directly introducing a terminal amidefunctional Depending on the expression system and host selected, the group into the peptide chain, which function is retained by the proteins of the present invention are produced by growing chain when the chain is cleaved from the Support. host cells transformed by an expression vector described In one method, the polypeptides are prepared by conven- 15 above under conditions whereby the protein of interest is tional Solid phase chemical synthesis on, for example, an expressed. The protein is then isolated from the host cells and Applied Biosystems, Inc. (ABI) 430A peptide synthesizer purified. If the expression system secretes the protein into using a resin that permits the synthesis of the amide peptide growth media, the protein can be purified directly from the form and using t-Boc amino acid derivatives (Peninsula media. If the protein is not secreted, it is isolated from cell Laboratories, Inc.) with standard solvents and reagents. 20 lysates. The selection of the appropriate growth conditions Polypeptides containing either L- or D-amino acids may be and recovery methods are within the skill of the art. Once synthesized in this manner. Polypeptide composition is con purified, the amino acid sequences of the proteins can be firmed by quantitative amino acid analysis and the specific determined, i.e., by repetitive cycles of Edman degradation, sequence of each peptide may be determined by sequence followed by amino acid analysis by HPLC. Other methods of analysis. 25 amino acid sequencing are also known in the art. Preferably, the polypeptides can be produced by recombi Once synthesized or otherwise produced, the inhibitory nant DNA techniques by synthesizing DNA encoding the activity of a candidate polypeptide can be tested by assessing desired polypeptide. Once coding sequences for the desired the ability of the candidate to inhibit the lipopolysaccharide polypeptides have been synthesized or isolated, they can be induced nuclear translocation of NF-kappa.B by, for cloned into any suitable vector for expression. Numerous 30 example, using murine endothelial cells. cloning vectors are known to those of skill in the art, and the The fusion proteins of the present invention can be formu selection of an appropriate cloning vector is a matter of lated into therapeutic compositions in a variety of dosage choice. The gene can be placed under the control of a pro forms such as, but not limited to, liquid Solutions or Suspen moter, ribosome binding site (for bacterial expression) and, sions, tablets, pills, powders, Suppositories, polymeric micro optionally, an operator (collectively referred to herein as 35 capsules or microvesicles, liposomes, and injectable or infus “control elements), so that the DNA sequence encoding the ible solutions. The preferred form depends upon the mode of desired polypeptide is transcribed into RNA in the host cell administration and the particular cancer type targeted. The transformed by a vector containing this expression construc compositions also preferably include pharmaceutically tion. The coding sequence may or may not contain a signal acceptable vehicles, carriers or adjuvants, well known in the peptide or leader sequence. Heterologous leader sequences 40 art, Such as human serum albumin, ion exchangers, alumina, can be added to the coding sequence that causes the Secretion lecithin, buffer Substances such as phosphates, glycine, Sorbic of the expressed polypeptide from the host organism. Other acid, potassium Sorbate, and salts or electrolytes Such as regulatory sequences may also be desirable which allow for protamine sulfate. Suitable vehicles are, for example, water, regulation of expression of the protein sequences relative to saline, dextrose, glycerol, ethanol, or the like, and combina the growth of the host cell. Such regulatory sequences are 45 tions thereof. Actual methods of preparing Such compositions known to those of skill in the art, and examples include those are known, or will be apparent, to those skilled in the art. See, which cause the expression of a gene to be turned on or offin e.g., Remington's Pharmaceutical Sciences, Mack Publish response to a chemical or physical stimulus, including the ing Company, Easton, Pa., 18th edition, 1990. presence of a regulatory compound. Other types of regulatory The above compositions can be administered using con elements may also be present in the vector, for example, 50 ventional modes of delivery including, but not limited to, enhancer sequences. intravenous, intraperitoneal, oral, intralymphatic, or Subcuta The control sequences and other regulatory sequences may neous administration. Local administration to a tumor in be ligated to the coding sequence prior to insertion into a question, or to a site of inflammation, e.g., direct injection vector, Such as the cloning vectors described above. Alterna into an arthritic joint, will also find use with the present tively, the coding sequence can be cloned directly into an 55 invention. expression vector which already contains the control Therapeutically effective doses will be easily determined sequences and an appropriate restriction site. by one of skill in the art and will depend on the severity and The expression vector may then used to transform an course of the disease, the patient's health and response to appropriate host cell. A number of mammalian cell lines are treatment, and the judgment of the treating physician. known in the art and include immortalized cell lines available 60 from the AmericanType Culture Collection (ATCC), such as, EXPERIMENTAL but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293, baby hamster kidney (BHK) cells, monkey Below are examples of specific embodiments for carrying kidney cells (COS), human hepatocellular carcinoma cells out the present invention. The examples are offered for illus (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK') 65 trative purposes only, and are not intended to limit the scope cells, NOS cells derived from carcinoma cells, such as sar of the present invention in any way. Efforts have been made to coma, as well as others. Similarly, bacterial hosts such as E. ensure accuracy with respect to numbers used (e.g., amounts, US 8,815,247 B2 21 22 temperatures, etc.), but some experimental error and devia glycosylation sites as there are three additional N-glycosyla tion should, of course, be allowed for. tion sites are present in the TGFBRII region. Again, storage at -80C did not causing aggregation. The shift in the position or Example 1 appearance of the peak early in SEC column indicates that the increase in the molecular weight is because of the fusion The Fusion proteins comprising of IgG heavy chain linked partner. This once again confirms that the full length molecule to immunomodulator (either Suppressor or activator) ligands is being expressed. were expressed by codon optimized genes for the expression of CHO cells. The codon optimized nucleotide sequences Example 3 defined by SEQID NOs: 12 to 28 were expressed in (CHO) 10 cells and the expressed chimeric/fusion proteins are shown in Functional assays for the Fusion proteins. ELISA experi ment was carried out to check the binding ability of Anti TABLE 1. HER2/neu-TGFBRII and Anti-EGFR1-TGFBRII to TGFB. FIG. 24. A shows that Anti-HER2/neu-TGFBRII and Anti Fusion protein Details 15 EGFR1-TGFBRII molecules bind to the TGFB indicating that i-H R2/neu heavy chain + TGFB-RII ECD and Anti-HER2/neu. the fusion protein is functional. FIG. 24 B shows that Anti An HER2-TGFBRII inhibits the proliferation of BT474 cell line Anti-EGFR1 heavy chain + TGFB-RII ECD and Anti- EGFR1 light similar to the Bmab200 (Herceptin). FIG. 25 shows that Anti TLA4 heavy chain + TGFB-RII ECD and Anti-CTLA4 light 2O EGFR1-TGFBRII-inhibits the proliferation of A431 cell line similar to the Cetuximab. TLA4 heavy chain + PD1 ectodomain and Anti-CTLA4 Example 4 Anti-H R2/neu heavy chain + 4-1BBL and Anti-HER2/neu light i-EGFR1 heavy chain + 4-1BBL and Anti- EGFR1 light chain Antibody dependent cellular cytotoxicity ADCC activity TLA4 heavy chain + 4-1BBL and Anti-CTLA4 light chain 25 for Anti-HER2/neu-TGFBRII fusion protein was conducted odomain-Fc-4-1BBL to determine that the protein binds to the target receptors on ECD-Fc-4-1BBL i-EGFR1 heavy chain + PD ectodomain and Anti- EGFR1 light the cells. The results are shown in FIG. 26 wherein the activity C h 8. l is determined in BT474 cells and it is evident that ADCC i-CD20 heavy chain + 4-1BBL and Anti-CD20 light chain activity (% lysis of cells) of Anti-HER2-TGFBRII on BT474 i-HER2/neu heavy chain + PD1 ectodomain and Anti-HER2/neu. 30 cells is similar to that ofBmab200(Herceptin). FIG. 27 shows ht chain i-IL6Rheavy chain + PD1 ectodomain and Anti-IL6R light ADCC activity of Anti-EGFR1-TGFBRII on A431 cells in wherein the ADCC activities are similar to that of Cetuximab. i-IL6Rheavy chain + TGFB-RII ECD and Anti-IL6R light FIG. 28 shows the ADCC activity of ADCC activity of Anti C h 8.in EGFR1-4-1BB in comparison with Anti-EGFR1-TGFBRII An i-4-1BB heavy chain + PD1 ectodomain and Anti-4-1BB light 35 and cetuximab. ain Example 5 The expressed protein were characterized by using SDS PAGE and the expressed fusion proteins Anti-HER2/neu Binding Activity of the expressed proteins. The aim of this TGFBRII and Anti-EGFR1-TGFBRII were purified from cul 40 assay is to test the functionality of the fusion proteins to bind ture Supernatants using Protein A column and the results are to the target receptors on the cells in a dose dependent manner. shown in FIG. 22. Notably, Anti-EGFR1-TGFBRII light FIG. 29 A shows that the binding activity of Anti-CTLA4 chain mass is higher and it may be because of the presence of TGFBRII to TGFB1 is comparable to Anti-EGFR1-TGFBRII two glycosylation sites on the variable regions light and heavy and B shows that the binding activity of Anti-CTLA4-TGF chain. Both the Anti-HER2/neu-TGFBRII & Anti-EGFR1- 45 |BRII to CTLA4. FIG. 30 A shows the binding activity of TGFBRII heavy chains mass are higher because of the TGF Anti-CTLA4-TGFBRII to determine the level of PD1-Fc |BRII. Also Anti-HER2/neu-TGFBRII heavy chain has four binding and B shows the binding activity of Anti-EGRF1-4- N-glycosylation sites while Anti-EGFR1-TGFBRII has five 1BB to determine the binding of 4-1BBL. FIG. 31 A shows N-glycosylation sites. the binding activity of Anti-EGFR1-4-1BB to EGFR and B 50 shows the binding activity of PD1-Fc-4-1BB to find out Example 2 PDLL-Fc. FIG. 32 shows the binding activity of Anti EGFR1-PD1 to EGFR and PD1. Protein A/SEC chromatography. The Anti-HER2/neu-TG FBRII and Anti-EGFR1-TGFBRII samples were analyzed by Example 6 Protein A/SEC chromatography and the results are shown in 55 FIG.23. FIG.23A shows a sharp peak of elution of Bmab200 Confirmation of primary structure of molecule. As shown (Herceptin) vs a broader elution peak is believed to be a in FIG.33, the expressed proteins are evaluated to determine measure of heterogeneity due to presence of glycosylation as the molecular weight and the presence of glycosylation. The there are three additional N-glycosylation sites that are samples were analyzed by reducing and non-reducing SDS present in the TGFBRII region. Notably storage at -80C did 60 PAGE. The heavy and light chains of the antibody are sepa not causing aggregation. The shift in the position or appear rated by reduction alkylation so that the reduced structures ance of the peak early in SEC column indicates that the can be evaluated. Tryptic digestion of the fusion proteins increase in the molecular weight is because of the fusion provides for the identification of the primary sequence. partner. This once again confirms that the full length molecule MS/MS analysis of the proteins is performed. is being expressed. FIG. 23 B shows a sharp peak of elution of 65 Mass Spectrometry Analysis of Anti-HER2/neu-TGFBRII Bmab200(Herceptin) vs a broader elution peak which is and Anti-EGFR1-TGFBRII. The fusion protein shown in believed to be a measure of heterogeneity due to presence of FIG. 1 was expressed and tested. FIG. 34. A shows the mass US 8,815,247 B2 23 24 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 shows Decon cells/CHOK1 cells. The high producer single, clonal cell voluted Mass Spectrum of LC (Reduced) of Anti-HER2/neu strain is selected for producing the recombinant fusion pro TGFBRII ECD fusion. FIG. 35 shows the Mass Spectrum of tein. Prepare the MCB and characterize for cell viability, heavy chain (HC) (Reduced) of Anti-HER2/neu-TGFBRII 5 productivity, stability and other parameters. The cells are 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 luted Mass Spectrum of LC (Reduced) of Anti-EGFR1-TG 10 The cell culture is performed in feed-batch mode. In the FBRII ECD. FIG. 37 shows the Mass Spectrum of HC (Re cell culture, the mammalian host cells used is Chinese Ham ster Ovary (CHO) cells and culture medium are supplied duced) of Anti-EGFR1-TGFBRII ECD. initially. The CHO cells are genetically engineered to produce Example 7 the Antibody-peptide fusion protein. The zinc sulphate hepta 15 hydrate salt is added in the medium at a concentration of 0.4 The fusion proteins having amino acid sequences as mM. In contrast, there is no addition of any Zinc salt in the described in FIGS. 1 and 2 were inspected using UV chro control medium. The production fermentation run starts with matography and providing chromatograms resulting from the an initial cell count of 0.3-0.45x10° cells/ml at 37+1°C., the first 3-4 days are dedicated to grow the cells in batch phase. chromatographic separation of the tryptic digest of the fusion Next step involves lowering the temperature to 31+1° C. and proteins and tested with UV 218-222 nm wavelength. Total continuing the run till 7th day. Lactate reduces by almost Ion Current (TIC) corresponding to UV trace was also evalu 10-40% throughout the run. The produced fusion protein is ated. FIG. 38 A shows the UV Chromatogram of Tryptic then collected from the media using the technique of affinity Peptides of Anti-HER2/neu-TGFBRII ECD fusion protein chromatography. and B shows the Total Ion Chromatogram (TIC) of Tryptic 25 Peptides of Anti-HER2/neu-TGFBRII ECD fusion protein. Example 10 FIGS. 39, 40 and 41 provide lists of expected/observed tryptic peptide of the light chain, heavy chain and linked motif of the The cell culture is performed in a feed-batch mode is Anti-HER2/neu-TGFBRII ECD fusion protein, respectively. employed. In the cell cultures the mammalian host cells and Notably, all the expected peptides of the molecules were 30 culture medium which is Hyclone CDM4Mab are supplied identified including the light and heavy chain peptides and the initially. The salts (Zinc) is also added in the medium (0.3 peptides of the linked motif (TGF BRII). mM). The production fermentation run starts with an initial FIG. 42 A shows the UV Chromatogram of Tryptic Pep cell count of 0.3-0.45x10° cells/ml at 37+1° C., the first 3-4 tides of Anti-EGFR1-TGFBRII ECD fusion protein and B days are dedicated to growing the cells in batch phase. Next shows the Total Ion Chromatogram (TIC) of Tryptic Peptides 35 step involves lowering the temperature to 31+1-1° C. and of Anti-EGFR1-TGFBRII ECD fusion protein. FIGS. 43,44, continuing the run till 7th day. and 45 provide lists of expected/observed tryptic peptide of the light chain, heavy chain and linked motif of the Anti Example 11 EGFR1-TGFBRIIECD fusion protein, respectively. Again all the expected peptides of the molecules were identified includ 40 Purification of antibody-peptide fusion immunostimula ing the light and heavy chain peptides and the peptides of the tory molecules using protein A column. Supernatant culture linked motif (TGF BRII). secreted from recombinant CHO cell line containing the fusion monoclonal antibodies is tested for titer and endotox Example 8 ins under sterile conditions. The Supernatant is Subjected to 45 affinity chromatography using Mab Select Xtra Protein A The host cell line used for the expression of recombinant affinity resin, washed and equilibrated with binding buffer. fusion protein expression is CHO cells or the derivative of the The pH of the supernatant is adjusted using 0.5M phosphate CHO cells. The CHO cells referred here is either freedom to the same pH as the column; the Supernatant is allowed to CHO-S cells: CHO-S Cells are CHO-derived cells adapted to bind to the column/pass through the column at the flow rate of high density, serum-free Suspension culture in chemically 50 0.5 ml/minute to achieve the maximum binding. All the Anti defined medium that are capable of producing high levels of body-proteins fusion molecules bind through the Fc region secreted, recombinant protein or CHO K1 cells; having the while impurities are eliminated as flow through. The column same as ATCC No. CCL-61. It is basically an adherent cell is washed with equilibration buffer and the bound fusion line. The vectors used for stable cell line: molecules are eluted using 0.1 M glycine at pH 3.0. The pH of The Freedom pCHO 1.0 vector, designed by ProBioGen 55 the eluted proteins is adjusted to neutral pH or the stable AG, to express one or two genes of interest downstream of the formulation pH and the purified protein are stored at -20°C. vector's two different hybrid CMV promoters. This vector or at 2-8° C. contains the dihydrofolate reductase (DHFR) selection marker and a puromycin resistance gene, allowing selection Example 12 using MTX and Puromycin simultaneously. 60 The light chain or the light chain fusion protein coding Differentiating Trastuzumab from Trastuzumab-TGF BRII nucleic acid sequences are cloned into the restriction enzyme Receptor Fusion Molecule sites Avril and Bst/17 under the control of EF2/CMV pro A breast cancer tumor overexpressing the ErbB2 receptor moter. The heavy chain or the heavy chain fusion protein will either by constitutive activation or heterodimerization coding nucleic acid sequences are cloned, in restriction 65 with other members of the ErbB family of receptors lead to enzyme sites EcoRV and Pad under the control of CMV/EF1 tumor progression. This will involve the binding of growth promoter. factors associated with the ErbB signaling pathway. In addi US 8,815,247 B2 25 26 tion to this, the tumor creates a milieu wherein the immune icity observed in Trastuzumab-TGFBRII receptor fusion system is suppressed by activating TGF B and specific cytok treated cells over cells treated with Trastuzumab alone. The ines involved in the Subdued immune response. A novel mol readout for the experiment will use Alamar Blue a resaZurin ecule is generated wherein Trastuzumab (anti ErbB2) is fused dye which will get activated directly proportional to live cells 5 present. Another method could be to measure cytotoxicity by with the TGF BRII receptor as a fusion protein. While it is using cytotox glo which measures protease release which hypothesized that Trastuzumab will act as a targeted mol directly corresponds to proportional dead cells. Yet another ecule homing into the ErbB2 overexpressing breast cancer method could be the use of the flow cytometer directly mea cells, the TGFBRII receptor will sequester TGFB leading to Suring apoptotic and necrotic cell population by using immune activation. The experiment will utilize the growth of Annexin V and propidium iodide. Results from these multiple Herceptin resistant ErbB2 expressing cell lines (selected by 10 experiments will elucidate understanding of the activity of the growing BT474 cells in the presence of Herceptin) in the conjugate molecule as compared to Trastuzumab alone. presence of TGFB, cytotoxic CD8 positive cells and NK cells. Although the invention has been described with reference While Trastuzumab will be ineffective in inducing cytotox to the above example, it will be understood that modifications icity Trastuzumab TGFBRII receptor fusion molecule will 15 and variations are encompassed within the spirit and scope of sequester the TGFB thereby preventing the inhibition of cyto the invention. Accordingly, the invention is limited only by toxic CD8 and NK cells. This will lead to enhanced cytotox the following claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 36

<21 Oc SEO ID NO 1 <211 LENGTH: 449 <212> TYPE PRT <213> ORGANISM: Artificial Sequence <22 Os FEATURE; OTHER INFORMATION: Synthetic construct <4 OOs SEQUENCE: 1

Glu Wall Glin Lieu. Wall Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 15

Lieu. Arg Luell Ser Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 25 3 O

Ile His Trp Wall Arg Glin Ala Pro Gly Gly Luell Glu Trp Val 35 45

Ala Arg Ile Pro Thir Asn Gly Thir Arg Tyr Ala Asp Ser Wall SO 55 60

Lys Gly Arg Phe Thir Ile Ser Ala Asp Thir Ser Asn Thir Ala Tyr 65 70

Luell Gln Met Asn Ser Luell Glu Asp Thir Ala Wall Tyr 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Trp Gly Glin 105 110

Gly Thr Lell Wall. Thir Wall Ser Ser Ala Ser Thr Gly Pro Ser Wall 115 12O 125

Phe Pro Leu Ala Pro Ser Ser Ser Thir Ser Gly Gly Thir Ala Ala 13 O 135 14 O

Luell Gly Luell Wall Lys Asp Phe Pro Glu Pro Wall Thir Wall Ser 145 15 O 155 16 O

Trp Asn Ser Gly Ala Luell Thir Ser Gly Wall His Thr Phe Pro Ala Wall 1.65 17 O 17s

Luell Glin Ser Ser Gly Luell Tyr Ser Leu Ser Ser Wall Wall Thir Wall Pro 18O 185 190

Ser Ser Ser Luell Gly Thr Gin. Thir Tyr Ile Asn Wall ASn His 195 2 OO 2O5

Pro Ser Asn Thir Wall Asp Wall Glu Pro Ser Asp 210 215 22 O

Lys Thir His Thir Pro Pro Pro Ala Pro Glu Luell Tell Gly Gly 225 23 O 235 24 O US 8,815,247 B2 27 28 - Continued

Pro Ser Val Phe Lieu. Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 26 O 265 27 O Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 27s 28O 285 Asn Ala Lys Thr Llys Pro Arg Glu Glu Glin Tyr Asn. Ser Thr Tyr Arg 29 O 295 3 OO Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys 3. OS 310 315 32O Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu 3.25 330 335 Llys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr 34 O 345 35. O Thr Lieu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser Lieu. 355 360 365 Thr Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 37 O 375 38O Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 4 OO Lieu. Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Llys Lieu. Thr Val Asp 4 OS 41O 415 Lys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His 42O 425 43 O Glu Ala Lieu. His Asn His Tyr Thr Gln Llys Ser Lieu Ser Lieu Ser Pro 435 44 O 445 Gly

<210s, SEQ ID NO 2 &211s LENGTH: 214 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 2 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1. 5 1O 15 Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Asp Wall Asn. Thir Ala 2O 25 3O Val Ala Trp Tyr Glin Gln Llys Pro Gly Lys Ala Pro Llys Lieu. Lieu. Ile 35 4 O 45 Tyr Ser Ala Ser Phe Lieu. Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly SO 55 6 O Ser Arg Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Ser Leu Gln Pro 65 70 7s 8O Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin His Tyr Thr Thr Pro Pro 85 90 95

Thr Phe Gly Glin Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 1OO 105 11 O

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 12 O 125

Thir Ala Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe Tyr Pro Arg Glu Ala 13 O 135 14 O

Llys Val Glin Trp Llys Val Asp Asn Ala Lieu. Glin Ser Gly Asn. Ser Glin US 8,815,247 B2 29 30 - Continued

145 150 155 160 Glu Ser Val Thr Glu Glin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 1.65 17O 17s Ser Thr Lieu. Thir Lieu. Ser Lys Ala Asp Tyr Glu Lys His Llys Val Tyr 18O 185 19 O Ala Cys Glu Val Thr His Glin Gly Lieu Ser Ser Pro Val Thr Lys Ser 195 2OO 2O5 Phe Asn Arg Gly Glu. Cys 21 O

<210s, SEQ ID NO 3 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 3 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1. 5 1O 15

<210s, SEQ ID NO 4 &211s LENGTH: 137 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 4 Thir Ile Pro Pro His Val Glin Lys Ser Val Asn Asn Asp Met Ile Val 1. 5 1O 15 Thir Asp Asn. Asn Gly Ala Val Llys Phe Pro Gln Lieu. Cys Llys Phe Cys 2O 25 3O Asp Val Arg Phe Ser Thr Cys Asp Asn Glin Llys Ser Cys Met Ser Asn 35 4 O 45 Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Glin Glu Val Cys Val Ala SO 55 6 O Val Trp Arg Lys Asn Asp Glu Asn. Ile Thr Lieu. Glu Thr Val Cys His 65 70 7s 8O Asp Pro Llys Lieu Pro Tyr His Asp Phe Ile Lieu. Glu Asp Ala Ala Ser 85 90 95 Pro Llys Cys Ile Met Lys Glu Lys Llys Llys Pro Gly Glu Thr Phe Phe 1OO 105 11 O Met Cys Ser Cys Ser Ser Asp Glu. Cys Asn Asp Asn Ile Ile Phe Ser 115 12 O 125 Glu Glu Tyr Asn Thr Ser Asn Pro Asp 13 O 135

<210s, SEQ ID NO 5 &211s LENGTH: 448 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Construct

<4 OOs, SEQUENCE: 5 Glin Val Glin Leu Lys Glin Ser Gly Pro Gly Lieu Val Glin Pro Ser Glin 1. 5 1O 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Lieu. Thir Asn Tyr 2O 25 3O US 8,815,247 B2 31 32 - Continued

Gly Wall His Trp Wall Arg Glin Ser Pro Gly Lys Gly Lieu. Glu Trp Luell 35 4 O 45

Gly Wall Ile Trp Ser Gly Gly Asn Thir Asp Asn Thir Pro Phe Thir SO 55 6 O

Ser Arg Luell Ser Ile Asn Asp Asn Ser Lys Ser Glin Wall Phe Phe 65 70 7s

Met Asn Ser Lell Glin Ser Asn Asp Thir Ala Ile Cys Ala 85 90 95

Arg Ala Luell Thir Tyr Asp Glu Phe Ala Trp Gly Glin Gly 105 11 O

Thir Luell Wall Thir Wall Ser Ala Ala Ser Thir Gly Pro Ser Wall Phe 115 12 O 125

Pro Luell Ala Pro Ser Ser Lys Ser Thir Ser Gly Gly Thir Ala Ala Luell 13 O 135 14 O

Gly Cys Luell Wall Lys Asp Phe Pro Glu Pro Wall Thir Wall Ser Trp 145 150 155 160

Asn Ser Gly Ala Lell Thir Ser Gly Wall His Thir Phe Pro Ala Wall Luell 1.65 17s

Glin Ser Ser Gly Lell Tyr Ser Luell Ser Ser Wall Wall Thir Wall Pro Ser 18O 185 19 O

Ser Ser Luell Gly Thir Glin Thir Tyr Ile ASn Wall Asn His Pro 195

Ser Asn Thir Wall Asp Lys Arg Wall Glu Pro Lys Ser Asp 21 O 215 22O

Thir His Thir Pro Pro Pro Ala Pro Glu Lell Lell Gly Gly Pro 225 23 O 235 24 O

Ser Wall Phe Luell Phe Pro Pro Pro Lys Asp Thir Lell Met Ile Ser 245 250 255

Arg Thir Pro Glu Wall Thir Wall Wall Wall Asp Wall Ser His Glu Asp 26 O 265 27 O

Pro Glu Wall Phe Asn Trp Tyr Wall Asp Gly Wall Glu Wall His Asn 28O 285

Ala Lys Thir Pro Arg Glu Glu Glin Tyr ASn Ser Thir Arg Wall 29 O 295 3 OO

Wall Ser Wall Luell Thir Wall Lell His Glin Asp Trp Lell Asn Gly Glu 3. OS 310 315

Wall Ser Asn Ala Luell Pro Ala Pro Ile Glu 3.25 330 335

Thir Ile Ser Lys Ala Gly Glin Pro Arg Glu Pro Glin Wall Thir 34 O 345 35. O

Lell Pro Pro Ser Arg Asp Glu Luell Thir ASn Glin Wall Ser Luell Thir 355 360 365

Luell Wall Gly Phe Tyr Pro Ser Ile Ala Wall Glu Trp Glu 37 O 375

Ser Asn Gly Glin Pro Glu Asn Asn Tyr Thir Thir Pro Pro Wall Luell 385 390 395 4 OO

Asp Ser Asp Gly Ser Phe Phe Luell Tyr Ser Lys Lell Thir Wall Asp Lys 4 OS 41O 415

Ser Arg Trp Glin Glin Gly Asn Wall Phe Ser Cys Ser Wall Met His Glu 425 43 O

Ala Luell His Asn His Thir Glin Ser Luell Ser Lell Ser Pro Gly 435 44 O 445 US 8,815,247 B2 33 34 - Continued

<210s, SEQ ID NO 6 &211s LENGTH: 214 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 6 Asp Ile Leu Lieu. Thr Glin Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1. 5 1O 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Glin Ser Ile Gly Thr Asn 2O 25 3O Ile His Trp Tyr Glin Glin Arg Thr Asn Gly Ser Pro Arg Lieu. Lieu. Ile 35 4 O 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly SO 55 6 O Ser Gly Ser Gly Thr Asp Phe Thr Lieu Ser Ile Asn Ser Val Glu Ser 65 70 7s 8O Glu Asp Ile Ala Asp Tyr Tyr Cys Glin Glin Asn. Asn. Asn Trp Pro Thr 85 90 95 Thir Phe Gly Ala Gly Thr Llys Lieu. Glu Lieu Lys Arg Thr Val Ala Ala 1OO 105 11 O Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 12 O 125 Thir Ala Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe Tyr Pro Arg Glu Ala 13 O 135 14 O Llys Val Glin Trp Llys Val Asp Asn Ala Lieu. Glin Ser Gly Asn. Ser Glin 145 150 155 160 Glu Ser Val Thr Glu Glin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 1.65 17O 17s Ser Thr Lieu. Thir Lieu. Ser Lys Ala Asp Tyr Glu Lys His Llys Val Tyr 18O 185 19 O Ala Cys Glu Val Thr His Glin Gly Lieu Ser Ser Pro Val Thr Lys Ser 195 2OO 2O5 Phe Asn Arg Gly Glu. Cys 21 O

<210s, SEQ ID NO 7 &211s LENGTH: 447 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OO > SEQUENCE: 7 Glin Val Glin Lieu Val Glu Ser Gly Gly Gly Val Val Glin Pro Gly Arg 1. 5 1O 15

Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 2O 25 3O Thr Met His Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val 35 4 O 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Llys Tyr Tyr Ala Asp Ser Val SO 55 6 O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn. Ser Lys Asn. Thir Lieu. Tyr 65 70 7s 8O

Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 US 8,815,247 B2 35 36 - Continued

Ala Arg Thr Gly Trp Lieu. Gly Pro Phe Asp Tyr Trp Gly Glin Gly Thr 1OO 105 11 O Lieu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 12 O 125 Lieu Ala Pro Ser Ser Lys Ser Thir Ser Gly Gly Thr Ala Ala Lieu. Gly 13 O 135 14 O Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala Val Lieu. Glin 1.65 17O 17s Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 18O 185 19 O Ser Leu Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His Llys Pro Ser 195 2OO 2O5 Asn. Thir Lys Val Asp Lys Arg Val Glu Pro Llys Ser Cys Asp Llys Thr 21 O 215 22O His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly Gly Pro Ser 225 23 O 235 24 O Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 26 O 265 27 O Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 27s 28O 285 Lys Thr Llys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 29 O 295 3 OO Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr 3. OS 310 315 32O Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu Lys Thr 3.25 330 335 Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr Lieu. 34 O 345 35. O Pro Pro Ser Arg Asp Glu Lieu. Thir Lys Asn Glin Val Ser Lieu. Thir Cys 355 360 365 Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 37 O 375 38O Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. Asp 385 390 395 4 OO Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Lys Lieu. Thr Val Asp Llys Ser 4 OS 41O 415 Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 42O 425 43 O Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 44 O 445

<210s, SEQ ID NO 8 &211s LENGTH: 215 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct

<4 OOs, SEQUENCE: 8 Glu Ile Val Lieu. Thr Glin Ser Pro Gly Thr Lieu Ser Leu Ser Pro Gly 1. 5 1O 15 US 8,815,247 B2 37 38 - Continued

Glu Arg Ala Thr Lieu. Ser Cys Arg Ala Ser Glin Ser Val Gly Ser Ser 2O 25 3O Tyr Lieu Ala Trp Tyr Glin Gln Llys Pro Gly Glin Ala Pro Arg Lieu. Lieu. 35 4 O 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser SO 55 6 O Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Arg Lieu. Glu 65 70 7s 8O Pro Glu Asp Phe Ala Val Tyr Tyr Cys Glin Glin Tyr Gly Ser Ser Pro 85 90 95 Trp. Thir Phe Gly Glin Gly Thr Llys Val Glu Ile Lys Arg Thr Val Ala 1OO 105 11 O Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 12 O 125 Gly. Thir Ala Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe Tyr Pro Arg Glu 13 O 135 14 O Ala Lys Val Glin Trp Llys Val Asp Asn Ala Lieu. Glin Ser Gly Asn. Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Lieu. 1.65 17O 17s Ser Ser Thr Lieu. Thir Lieu. Ser Lys Ala Asp Tyr Glu Lys His Llys Val 18O 185 19 O Tyr Ala Cys Glu Val Thr His Glin Gly Leu Ser Ser Pro Val Thir Lys 195 2OO 2O5 Ser Phe Asn Arg Gly Glu. Cys 21 O 215

<210s, SEQ ID NO 9 &211s LENGTH: 2O5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 9 Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala 1. 5 1O 15 Ala Ser Pro Arg Lieu. Arg Glu Gly Pro Glu Lieu. Ser Pro Asp Asp Pro 2O 25 3O Ala Gly Lieu. Lieu. Asp Lieu. Arg Glin Gly Met Phe Ala Glin Lieu Val Ala 35 4 O 45 Glin Asn Val Lieu. Lieu. Ile Asp Gly Pro Lieu. Ser Trp Tyr Ser Asp Pro SO 55 6 O Gly Lieu Ala Gly Val Ser Lieu. Thr Gly Gly Lieu. Ser Tyr Lys Glu Asp 65 70 7s 8O

Thr Lys Glu Lieu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe 85 90 95

Glin Lieu. Glu Lieu. Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val 1OO 105 11 O

Ser Lieu Ala Lieu. His Lieu. Glin Pro Lieu. Arg Ser Ala Ala Gly Ala Ala 115 12 O 125

Ala Lieu Ala Lieu. Thr Val Asp Lieu Pro Pro Ala Ser Ser Glu Ala Arg 13 O 135 14 O

Asn Ser Ala Phe Gly Phe Glin Gly Arg Lieu. Lieu. His Lieu. Ser Ala Gly 145 150 155 160 US 8,815,247 B2 39 40 - Continued

Glin Arg Lieu. Gly Val His Lieu. His Thr Glu Ala Arg Ala Arg His Ala 1.65 17O 17s Trp Gln Lieu. Thr Glin Gly Ala Thr Val Lieu. Gly Lieu Phe Arg Val Thr 18O 185 19 O Pro Glu Ile Pro Ala Gly Lieu Pro Ser Pro Arg Ser Glu 195 2OO 2O5

<210s, SEQ ID NO 10 &211s LENGTH: 150 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 10 Pro Gly Trp Phe Lieu. Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr 1. 5 1O 15 Phe Ser Pro Ala Lieu. Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe 2O 25 3O Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Lieu. Asn Trp Tyr 35 4 O 45 Arg Met Ser Pro Ser Asn Glin Thr Asp Llys Lieu Ala Ala Phe Pro Glu SO 55 6 O Asp Arg Ser Glin Pro Gly Glin Asp Cys Arg Phe Arg Val Thr Glin Lieu 65 70 7s 8O Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn 85 90 95 Asp Ser Gly Thr Tyr Lieu. Cys Gly Ala Ile Ser Lieu Ala Pro Lys Ala 1OO 105 11 O Glin Ile Lys Glu Ser Lieu. Arg Ala Glu Lieu. Arg Val Thr Glu Arg Arg 115 12 O 125 Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly 13 O 135 14 O

Glin Phe Glin. Thir Lieu Wall 145 150

<210s, SEQ ID NO 11 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 11 Glu Pro Llys Ser Cys Asp Llys 1. 5

<210s, SEQ ID NO 12 &211s LENGTH: 1032 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct

<4 OOs, SEQUENCE: 12 gctago acca agggcc cct c cqtgttcc ct ctdgcc.ccct coagcaagtic cacct ctdgc 6 O ggcaccgc.cg Ctctgggctg. cctggtcaag gaCtact tcc ccgagc.ccgt gaccgtgtcC 12 O tggaactctg gcgctctgac Ctc.cggcgtg cacacct tcc ctg.ccgtgct gcagt cct cc 18O

US 8,815,247 B2 55 - Continued

<210s, SEQ ID NO 29 &211s LENGTH: 449 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 29 Glin Val Glin Lieu Val Glin Ser Gly Ala Glu Val Llys Llys Pro Gly Glu 1. 5 1O 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Asp Tyr Thr Phe Thr Tyr Tyr 2O 25 3O Gly Met Asn Trp Val Lys Glin Ala Pro Gly Glin Gly Lieu Lys Trp Met 35 4 O 45 Gly Trp Ile Asp Thr Thr Thr Gly Glu Pro Thr Tyr Ala Glin Llys Phe SO 55 6 O Gln Gly Arg Ile Ala Phe Ser Lieu. Glu Thir Ser Ala Ser Thr Ala Tyr 65 70 7s 8O Lieu. Glin Ile Llys Ser Lieu Lys Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Arg Gly Pro Tyr Asn Trp Tyr Phe Asp Val Trp Gly Glin Gly 1OO 105 11 O Thir Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 12 O 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 13 O 135 14 O Gly Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala Val Lieu. 1.65 17O 17s Gln Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 18O 185 19 O Ser Ser Leu Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His Llys Pro 195 2OO 2O5 Ser Asn. Thir Lys Val Asp Llys Llys Val Glu Pro Llys Ser Cys Asp Llys 21 O 215 22O Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly Gly Pro 225 23 O 235 24 O Ser Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 26 O 265 27 O Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 27s 28O 285

Ala Lys Thir Lys Pro Arg Glu Glu Glin Tyr Asn. Ser Thr Tyr Arg Val 29 O 295 3 OO

Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu 3. OS 310 315 32O

Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu Lys 3.25 330 335

Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr 34 O 345 35. O

Lieu Pro Pro Ser Arg Asp Glu Lieu. Thir Lys Asn. Glin Val Ser Lieu. Thir 355 360 365 US 8,815,247 B2 57 - Continued

Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 37 O 375 38O Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. 385 390 395 4 OO Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Lys Lieu. Thr Val Asp Llys 4 OS 41O 415 Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 42O 425 43 O Ala Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 44 O 445 Lys

<210s, SEQ ID NO 3 O &211s LENGTH: 219 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 30 Asp Ile Val Met Thr Glin Ser Pro Leu Ser Val Pro Val Thr Pro Gly 1. 5 1O 15 Glu Pro Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Lieu. Lieu. His Ser 2O 25 3O Asn Gly Asn Thr Tyr Lieu. Tyr Trp Phe Leu Glin Arg Pro Gly Glin Ser 35 4 O 45 Pro Gln Leu Lieu. Ile Tyr Arg Met Ser Asn Lieu Val Ser Gly Val Pro SO 55 6 O Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Lieu. Arg Ile 65 70 7s 8O Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Lieu. Glin His 85 90 95 Lieu. Glu Tyr Pro Phe Thr Phe Gly Pro Gly Thr Lys Lieu. Glu Lieu Lys 1OO 105 11 O Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 12 O 125 Glin Lieu Lys Ser Gly Thr Ala Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe 13 O 135 14 O Tyr Pro Arg Glu Ala Lys Val Glin Trp Llys Val Asp Asn Ala Lieu. Glin 145 150 155 160 Ser Gly Asn. Ser Glin Glu Ser Val Thr Glu Glin Asp Ser Lys Asp Ser 1.65 17O 17s Thr Tyr Ser Leu Ser Ser Thr Lieu. Thir Leu Ser Lys Ala Asp Tyr Glu 18O 185 19 O Lys His Llys Val Tyr Ala Cys Glu Val Thr His Glin Gly Lieu. Ser Ser 195 2OO 2O5

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu. Cys 21 O 215

<210s, SEQ ID NO 31 &211s LENGTH: 460 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct

<4 OOs, SEQUENCE: 31 US 8,815,247 B2 59 60 - Continued

Glu Wall Glin Luell Wall Glin Ser Gly Ala Glu Wall Lys Lys Pro Gly Ser 1O 15

Ser Wall Wall Ser Ala Ser Gly Gly Thir Phe Ser Ser 25

Ala Ile Ser Trp Wall Arg Glin Ala Pro Gly Glin Gly Lell Glu Trp Met 35 4 O 45

Gly Gly Ile Ile Pro Ile Phe Gly Thir Ala ASn Tyr Ala Glin Phe SO 55 6 O

Glin Gly Arg Wall Thir Ile Thir Ala Asp Ser Thir Ser Thir Ala 65 70

Met Glu Luell Ser Ser Lell Arg Ser Glu Asp Thir Ala Wall Tyr 85 90 95

Ala Arg Ala Pro Lell Arg Phe Luell Glu Trp Ser Thir Glin Asp His 105 11 O

Tyr Met Asp Wall Trp Gly Gly Thir Thir Wall Thir Wall 115 12 O 125

Ser Ser Ala Ser Thir Gly Pro Ser Wall Phe Pro Lell Ala Pro Ser 13 O 135 14 O

Ser Ser Thir Ser Gly Gly Thir Ala Ala Luell Gly Luell Wall Lys 145 150 155 160

Asp Phe Pro Glu Pro Wall Thir Wall Ser Trp Asn Ser Gly Ala Luell 1.65 17O

Thir Ser Gly Wall His Thir Phe Pro Ala Wall Luell Glin Ser Ser Gly Luell 18O 185 19 O

Ser Luell Ser Ser Wall Wall Thir Wall Pro Ser Ser Ser Luell Gly Thir 195

Glin Thir Ile Cys Asn Wall Asn His Pro Ser Asn Thir Wall 21 O 215 22O

Asp Wall Glu Pro Ser Asp Lys Thir His Thir Pro 225 23 O 235 24 O

Pro Pro Ala Pro Glu Lell Luell Gly Gly Pro Ser Wall Phe Luell Phe 245 250 255

Pro Pro Pro Lys Asp Thir Luell Met Ile Ser Arg Thir Pro Glu Wall 26 O 265 27 O

Thir Wall Wall Wall Asp Wall Ser His Glu Asp Pro Glu Wall Phe 27s 28O 285

Asn Trp Wall Asp Gly Wall Glu Wall His ASn Ala Thir Pro 29 O 295 3 OO

Arg Glu Glu Glin Tyr Asn Ser Thir Arg Wall Wall Ser Wall Luell Thir 3. OS 310 315

Wall Luell His Glin Asp Trp Lell Asn Gly Lys Glu Lys Wall 3.25 330 335

Ser Asn Ala Lell Pro Ala Pro Ile Glu Thir Ile Ser Ala 34 O 345 35. O

Gly Glin Pro Arg Glu Pro Glin Wall Thir Lell Pro Pro Ser Arg 355 360 365

Glu Glu Met Thir Lys Asn Glin Wall Ser Luell Thir Cys Lell Wall Gly 37 O 375

Phe Pro Ser Asp Ile Ala Wall Glu Trp Glu Ser Asn Gly Glin Pro 385 390 395 4 OO

Glu Asn Asn Lys Thir Thir Pro Pro Wall Luell Asp Ser Asp Gly Ser 4 OS 41O 415 US 8,815,247 B2 61 - Continued Phe Phe Leu Tyr Ser Lys Lieu. Thr Val Asp Llys Ser Arg Trp Glin Glin 42O 425 43 O Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Lieu. His Asn His 435 44 O 445 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 45.5 460

<210s, SEQ ID NO 32 &211s LENGTH: 213 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 32 Ser Ser Glu Lieu. Thr Glin Asp Pro Ala Val Ser Val Ala Lieu. Gly Glin 1. 5 1O 15 Thr Val Arg Ile Thr Cys Glin Gly Asp Ser Lieu. Arg Ser Tyr Tyr Ala 2O 25 3O Thir Trp Tyr Glin Gln Lys Pro Gly Glin Ala Pro Ile Leu Val Ile Tyr 35 4 O 45 Gly Glu Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser SO 55 6 O Ser Ser Gly Asn Thr Ala Ser Lieu. Thir Ile Thr Gly Ala Glin Ala Glu 65 70 7s 8O Asp Glu Ala Asp Tyr Tyr Cys Llys Ser Arg Asp Gly Ser Gly Gln His 85 90 95 Lieu Val Phe Gly Gly Gly. Thir Lys Lieu. Thr Val Lieu. Gly Glin Pro Llys 1OO 105 11 O

Ala Ala Pro Ser Wall. Thir Lieu. Phe Pro Pro Ser Ser Glu Glu Lieu. Glin 115 12 O 125 Ala Asn Lys Ala Thr Lieu Val Cys Lieu. Ile Ser Asp Phe Tyr Pro Gly 13 O 135 14 O Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Wall Lys Ala Gly 145 150 155 160 Val Glu Thir Thr Thr Pro Ser Lys Glin Ser Asn Asn Lys Tyr Ala Ala 1.65 17O 17s Ser Ser Tyr Lieu Ser Lieu. Thr Pro Glu Glin Trp Llys Ser His Arg Ser 18O 185 19 O Tyr Ser Cys Glin Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 2OO 2O5 Ala Pro Ala Glu. Cys 21 O

<210s, SEQ ID NO 33 &211s LENGTH: 449 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct

<4 OOs, SEQUENCE: 33 Glin Val Glin Lieu. Glin Glin Ser Gly Ala Glu Val Llys Llys Phe Gly Ala 1. 5 1O 15

Ser Val Llys Val Ser Cys Glu Ala Ser Gly Tyr Thr Phe Pro Ser Tyr 2O 25 3O Val Lieu. His Trp Val Lys Glin Ala Pro Gly Glin Gly Lieu. Glu Trp Ile 35 4 O 45 US 8,815,247 B2 63 64 - Continued

Gly Tyr Ile Asn Pro Asn Asp Gly Thir Glin Thir Asn Lys Lys Phe SO 55 6 O

Lys Gly Ala Thir Lell Thir Arg Asp Thir Ser Ile Asn Thir Ala Tyr 65 70 7s

Met Glu Luell Ser Arg Lell Arg Ser Asp Asp Thir Ala Wall Tyr Cys 85 90 95

Ala Arg Gly Phe Gly Gly Ser Gly Phe Ala Asn Gly Glin Gly 105 11 O

Thir Luell Wall Thir Wall Ser Ser Ala Ser Thir Gly Pro Ser Wall Phe 115 12 O 125

Pro Luell Ala Pro Ser Ser Lys Ser Thir Ser Gly Gly Thir Ala Ala Luell 13 O 135 14 O

Gly Luell Wall Lys Asp Phe Pro Glu Pro Wall Thir Wall Ser Trp 145 150 155 160

Asn Ser Gly Ala Lell Thir Ser Gly Wall Asn Thir Phe Pro Ala Wall Luell 1.65 17O 17s

Glin Ser Ser Gly Lell Ser Luell Ser Ser Wall Wall Thir Wall Pro Ser 18O 185 19 O

Ser Ser Luell Gly Thir Glin Thir Tyr Ile ASn Wall Asn His Pro 195

Ser Asn Thir Wall Asp Lys Arg Wall Glu Pro Lys Ser Asp Lys 21 O 215 22O

Thir His Thir Pro Pro Pro Ala Pro Glu Lell Lell Gly Gly Pro 225 23 O 235 24 O

Ser Wall Phe Luell Phe Pro Pro Pro Lys Asp Thir Lell Asn Ile Ser 245 250 255

Arg Thir Pro Glu Wall Thir Wall Wall Wall Asp Wall Ser His Glu Asp 26 O 265 27 O

Pro Glu Wall Phe Asn Trp Tyr Wall Asp Gly Wall Glu Wall His Asn 28O 285

Ala Lys Thir Pro Arg Glu Glu Glin ASn Ser Thir Arg Wall 29 O 295 3 OO

Wall Ser Wall Luell Thir Wall Lell His Glin Asp Trp Lell Asn Gly Glu 3. OS 310 315

Wall Ser Asn Ala Luell Pro Ala Pro Ile Glu Lys 3.25 330 335

Thir Ile Ser Lys Ala Gly Glin Pro Arg Glu Pro Glin Wall Thir 34 O 345 35. O

Lell Pro Pro Ser Arg Glu Glu Met Thir ASn Glin Wall Ser Luell Thir 355 360 365

Luell Wall Gly Phe Tyr Pro Ser Asp Ile Ala Wall Glu Trp Glu 37 O 375

Ser Asn Gly Glin Pro Glu Asn Asn Lys Thir Thir Pro Pro Wall Luell 385 390 395 4 OO

Asp Ser Asp Gly Ser Phe Phe Luell Ser Lys Lell Thir Wall Asp Lys 4 OS 41O 415

Ser Arg Trp Glin Glin Gly Asn Wall Phe Ser Cys Ser Wall Asn His Glu 425 43 O

Ala Luell His Asn His Thir Glin Ser Luell Ser Lell Ser Pro Gly 435 44 O 445 US 8,815,247 B2 65 - Continued

<210s, SEQ ID NO 34 &211s LENGTH: 215 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 34 Asp Ile Glin Lieu. Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1. 5 1O 15 Asp Arg Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser 2O 25 3O Tyr Lieu. Tyr Trp Tyr Glin Gln Llys Pro Gly Lys Ala Pro Llys Lieu. Trp 35 4 O 45 Ile Tyr Ser Thr Ser Asn Lieu Ala Ser Gly Val Pro Ala Arg Phe Ser SO 55 6 O Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Ser Leu Gln 65 70 7s 8O Pro Glu Asp Ser Ala Ser Tyr Phe Cys His Gln Trp Asn Arg Tyr Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Arg Lieu. Glu Ile Lys Arg Thr Val Ala 1OO 105 11 O Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 12 O 125 Gly. Thir Ala Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe Tyr Glu Ala Lys 13 O 135 14 O Val Glin Trp Llys Val Asp Asn Ala Lieu Gln Ser Gly ASn Ser Glin Glu 145 150 155 160 Ser Val Thr Glu Glin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 1.65 17O 17s Thir Lieu. Thir Lieu. Ser Pro Arg Lys Ala Asp Tyr Glu Lys His Llys Val 18O 185 19 O Tyr Ala Cys Glu Val Thr His Glin Gly Leu Ser Ser Pro Val Thir Lys 195 2OO 2O5 Ser Phe Asn Arg Gly Glu. Cys 21 O 215

<210s, SEQ ID NO 35 &211s LENGTH: 450 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 35 Glu Val Glin Lieu. Lieu. Glu Ser Gly Gly Asp Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 2O 25 3O

Ala Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val 35 4 O 45

Ser Ala Ile Thr Pro Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val SO 55 6 O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn. Ser Glin Asn. Thir Lieu. Tyr 65 70 7s 8O

Lieu. Glin Met Asn. Ser Lieu. Arg Val Glu Asp Thr Ala Val Tyr Ile Cys 85 90 95 US 8,815,247 B2 67 - Continued Gly Arg Val Pro Tyr Arg Ser Thr Trp Tyr Pro Leu Tyr Trp Gly Glin 1OO 105 11 O Gly Thr Lieu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 12 O 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 13 O 135 14 O Lieu. Gly Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala Val 1.65 17O 17s Lieu. Glin Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val Pro 18O 185 19 O Ser Ser Ser Leu Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His Lys 195 2OO 2O5 Pro Ser Asn. Thir Lys Val Asp Llys Llys Val Glu Pro Llys Ser Cys Asp 21 O 215 22O Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly Gly 225 23 O 235 24 O Pro Ser Val Phe Lieu. Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 26 O 265 27 O Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 27s 28O 285 Asn Ala Lys Thr Llys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 29 O 295 3 OO Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys 3. OS 310 315 32O Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu 3.25 330 335 Llys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr 34 O 345 35. O Thir Lieu Pro Pro Ser Arg Asp Glu Lieu. Thir Lys Asn Glin Val Ser Lieu. 355 360 365 Thr Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 37 O 375 38O Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 4 OO Lieu. Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Llys Lieu. Thr Val Asp 4 OS 41O 415 Lys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His 42O 425 43 O Glu Ala Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 44 O 445 Gly Lys 450

<210s, SEQ ID NO 36 &211s LENGTH: 214 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct

<4 OOs, SEQUENCE: 36