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