Patent ID: 12209137

EXAMPLES

Material and Methods

TCR-Identification

The alpha and beta TCR chain sequences were isolated from T cells of healthy donors. To ensure enrichment of peptide-specific T cells, the cells were either repeatedly stimulated with artificial antigen-presenting cells coated with CT45-IP-MHC and CD28 (Priming) as described in Walter et al., 2003 J Immunol., November 15; 171(10):4974-8 and subsequently, single-cell sorted using CT45-IP-HLA-A*02 tetramers or alternatively stimulated with CT45-IP-loaded T2 cells. After sufficient expansion, the cells were sorted using CT45-IP-HLA-A*02 tetramers.

The TCR nucleotide sequences were obtained via standard methods such as 5′ RACE and Sanger sequencing as described e.g. in Molecular Cloning, Laboratory Manual, Fourth Edition by Green and Sambrook. The genes encoding the V regions and J regions of the TCRs are listed in Table 2. The annotation was performed by GeneData 11.0.1 using the IMGT/GENE-DB (Version: 28 Nov. 2019) as reference database. The TCR amino acid sequences are listed in Table 3.

TABLE 2Identified TCRsIDValphaJalphaVbetaJbetaTCR-1TRAV38-1TRAJ22TRBV7-9TRBJ2-7TCR-2TRAV14/DV4TRAJ52TRBV13TRBJ1-1TCR-3TRAV14/DV4TRAJ33TRBV27TRBJ1-5TCR-4TRAV3TRAJ30TRBV6-2TRBJ2-1TCR-5TRAV35TRAJ26TRBV9TRBJ2-7TCR-6TRAV12-3TRAJ7TRBV4-1TRBJ2-1TCR-7TRAV38-2/DV8TRAJ32TRBV13TRBJ2-1TCR-8TRAV19TRAJ40TRBV13TRBJ2-1TCR-9TRAV5TRAJ17TRBV2TRBJ2-1TCR-10TRAV1-2TRAJ44TRBV6-1TRBJ2-1TCR-11TRAV22TRAJ44TRBV6-1TRBJ2-7TCR-12TRAV27TRAJ50TRBV4-1TRBJ1-2TCR-13TRAV14/DV4TRAJ5TRBV19TRBJ2-1TCR-14TRAV14/DV4TRAJ22TRBV11-2TRBJ1-6TCR-15TRAV21TRAJ37TRBV5-1TRBJ2-7

TABLE 3TCR amino acid sequencesSEQIDRegion/NO:TCRChainDomianSequence1TCR-5alphavariableQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPdomainVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGGYNYGQNFVFGPGTRLSVLP2TCR-5alphaCDR1SIFNT3TCR-5alphaCDR2LYKAGEL4TCR-5alphaCDR3AGGYNYGQNFV5TCR-5alphaconstantYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS6TCR-5alphafull-QQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPlengthVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGGYNYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS7TCR-5betavariableGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIdomainQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSAGLAGGYEQYFGPGTRLTVT8TCR-5betaCDR1SGDLS9TCR-5betaCDR2YYNGEE10TCR-5betaCDR3ASSAGLAGGY EQY11TCR-5betaconstantEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWdomainWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG12TCR-5betafull-GVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIlengthQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSAGLAGGYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG13TCR-1alphavariableQTVTQSQPEM SVQEAETVTL SCTYDTSENN YYLFWYKQPPdomainSRQMILVIRQ EAYKQQNATE NRFSVNFQKA AKSFSLKISDSQLGDTAMYF CAPLGLVAGS ARQLTFGSGT QLTVLP14TCR-1alphaCDR1TSENNYY15TCR-1alphaCDR2QEAYKQQN16TCR-1alphaCDR3APLGLVAGSA RQLT750TCR-1alphaconstantDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS17TCR-1alphafull-QTVTQSQPEM SVQEAETVTL SCTYDTSENN YYLFWYKQPPlengthSRQMILVIRQ EAYKQQNATE NRFSVNFQKA AKSFSLKISDSQLGDTAMYF CAPLGLVAGS ARQLTFGSGT QLTVLPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS18TCR-1betavariableGVSQDPRHKI TKRGQNVTFR CDPISEHNRL YWYRQTLGQGdomainPEFLTYFQNE AQLEKSRLLS DRFSAERPKG SFSTLEIQRTEQGDSAMYLC ASSTDITSYE QYFGPGTRLT VT19TCR-1betaCDR1SEHNR20TCR-1betaCDR2FQNEAQ21TCR-1betaCDR3ASSTDITSYE QY11TCR-1betaconstantEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWdomainWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG22TCR-1betafull-GVSQDPRHKI TKRGQNVTFR CDPISEHNRL YWYRQTLGQGlengthPEFLTYFQNE AQLEKSRLLS DRFSAERPKG SFSTLEIQRTEQGDSAMYLC ASSTDITSYE QYFGPGTRLT VTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG23TCR-14alphavariableQKITQTQPGM FVQEKEAVTL DCTYDTSDQS YGLFWYKQPSdomainSGEMIFLIYQ GSYDEQNATE GRYSLNFQKA RKSANLVISASQLGDSAMYF CAINGLPGSA RQLTFGSGTQ LTVLP24TCR-14alphaCDR1TSDQSYG25TCR-14alphaCDR2QGSYDEQN26TCR-14alphaCDR3AINGLPGSAR QLT750TCR-14alphaconstantDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS27TCR-14alphafull-QKITQTQPGM FVQEKEAVTL DCTYDTSDQS YGLFWYKQPSlengthSGEMIFLIYQ GSYDEQNATE GRYSLNFQKA RKSANLVISASQLGDSAMYF CAINGLPGSA RQLTFGSGTQ LTVLPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS28TCR-14betavariableGVAQSPRYKI IEKRQSVAFW CNPISGHATL YWYQQILGQGdomainPKLLIQFQNN GVVDDSQLPK DRFSAERLKG VDSTLKIQPAKLEDSAVYLC ASSRFVATGT SPLHFGNGTR LTVT29TCR-14betaCDR1SGHAT30TCR-14betaCDR2FQNNGV31TCR-14betaCDR3ASSRFVATGT SPLH32TCR-14betaconstantEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWdomainWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF33TCR-14betafull-GVAQSPRYKI IEKRQSVAFW CNPISGHATL YWYQQILGQGlengthPKLLIQFQNN GVVDDSQLPK DRFSAERLKG VDSTLKIQPAKLEDSAVYLC ASSRFVATGT SPLHFGNGTR LTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF34TCR-13alphavariableQKITQTQPGM FVQEKEAVTL DCTYDTSDQS YGLFWYKQPSdomainSGEMIFLIYQ GSYDEQNATE GRYSLNFQKA RKSANLVISASQLGDSAMYF CAMSDPIMDT GRRALTFGSG TRLQVQP24TCR-13alphaCDR1TSDQSYG25TCR-13alphaCDR2QGSYDEQN35TCR-13alphaCDR3AMSDPIMDTG RRALT751TCR-13alphaconstantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS36TCR-13alphafull-QKITQTQPGM FVQEKEAVTL DCTYDTSDQS YGLFWYKQPSlengthSGEMIFLIYQ GSYDEQNATE GRYSLNFQKA RKSANLVISASQLGDSAMYF CAMSDPIM DT GRRALTFGSG TRLQVQPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS37TCR-13betavariableGITQSPKYLF RKEGQNVTLS CEQNLNHDAM YWYRQDPGQGdomainLRLIYYSQIV NDFQKGDIAE GYSVSREKKE SFPLTVTSAQKNPTAFYLCA SKSRGPNLAD TQYFGPGTRL TVL38TCR-13betaCDR1LNHDA39TCR-13betaCDR2SQIVND40TCR-13betaCDR3ASKSRGPNLA DTQY11TCR-13betaconstantEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWdomainWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG41TCR-13betafull-GITQSPKYLF RKEGQNVTLS CEQNLNHDAM YWYRQDPGQGlengthLRLIYYSQIV NDFQKGDIAE GYSVSREKKE SFPLTVTSAQKNPTAFYLCA SKSRGPNLAD TQYFGPGTRL TVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG42TCR-15alphavariableKQEVTQIPAA LSVPEGENLV LNCSFTDSAI YNLQWFRQDPdomainGKGLTSLLLI QSSQREQTSG RLNASLDKSS GRSTLYIAASQPGDSATYLC AVLLTGKLIF GQGTTLQVKP43TCR-15alphaCDR1DSAIYN44TCR-15alphaCDR2IQSSQRE45TCR-15alphaCDR3AVLLTGKLI750TCR-15alphaconstantDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS46TCR-15alphafull-KQEVTQIPAA LSVPEGENLV LNCSFTDSAI YNLQWFRQDPlengthGKGLTSLLLI QSSQREQTSG RLNASLDKSS GRSTLYIAASQPGDSATYLC AVLLTGKLIF GQGTTLQVKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS47TCR-15betavariableGVTQTPRYLI KTRGQQVTLS CSPISGHRSV SWYQQTPGQGdomainLQFLFEYFSE TQRNKGNFPG RFSGRQFSNS RSEMNVSTLELGDSALYLCA SSGGPGPSGE QYFGPGTRLT VT48TCR-15betaCDR1SGHRS49TCR-15betaCDR2YFSETQ50TCR-15betaCDR3ASSGGPGPSG EQY11TCR-15betaconstantEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWdomainWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG51TCR-15betafull-GVTQTPRYLI KTRGQQVTLS CSPISGHRSV SWYQQTPGQGlengthLQFLFEYFSE TQRNKGNFPG RFSGRQFSNS RSEMNVSTLELGDSALYLCA SSGGPGPSGE QYFGPGTRLT VTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG52TCR-6alphavariableQQKEVEQDPG PLSVPEGAIV SLNCTYSNSA FQYFMWYRQYdomainSRKGPELLMY TYSSGNKEDG RFTAQVDKSS KYISLFIRDSQPSDSATYLC AMSGNSGARD YGNNRLAFGK GNQVVVIP53TCR-6alphaCDR1NSAFQY54TCR-6alphaCDR2TYSSGN55TCR-6alphaCDR3AMSGNSGARD YGNNRLA751TCR-6alphaconstantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS56TCR-6alphafull-QQKEVEQDPG PLSVPEGAIV SLNCTYSNSA FQYFMWYRQYlengthSRKGPELLMY TYSSGNKEDG RFTAQVDKSS KYISLFIRDSQPSDSATYLC AMSGNSGARD YGNNRLAFGK GNQVVVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS57TCR-6betavariableEVTQTPKHLV MGMTNKKSLK CEQHMGHRAM YWYKQKAKKPdomainPELMFVYSYE KLSINESVPS RFSPECPNSS LLNLHLHALQPEDSALYLCA SATWEEAGPY NEQFFGPGTR LTVL58TCR-6betaCDR1MGHRA59TCR-6betaCDR2YSYEKL60TCR-6betaCDR3ASATWEEAGP YNEQF11TCR-6betaconstantEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWdomainWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG61TCR-6betafull-EVTQTPKHLV MGMTNKKSLK CEQHMGHRAM YWYKQKAKKPlengthPELMFVYSYE KLSINESVPS RFSPECPNSS LLNLHLHALQPEDSALYLCA SATWEEAGPY NEQFFGPGTR LTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG62TCR-3alphavariableQKITQTQPGM FVQEKEAVTL DCTYDTSDQS YGLFWYKQPSdomainSGEMIFLIYQ GSYDEQNATE GRYSLNFQKA RKSANLVISASQLGDSAMYF CGYSNYQLIW GAGTKLIIKP24TCR-3alphaCDR1TSDQSYG25TCR-3alphaCDR2QGSYDEQN63TCR-3alphaCDR3GYSNYQLI750TCR-3alphaconstantDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS64TCR-3alphafull-QKITQTQPGM FVQEKEAVTL DCTYDTSDQS YGLFWYKQPSlengthSGEMIFLIYQ GSYDEQNATE GRYSLNFQKA RKSANLVISASQLGDSAMYF CGYSNYQLIW GAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKWLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS65TCR-3betavariableQVTQNPRYLI TVTGKKLTVT CSQNMNHEYM SWYRQDPGLGdomainLRQIYYSMNV EVTDKGDVPE GYKVSRKEKR NFPLILESPSPNQTSLYFCA SREGTGGYQP QHFGDGTRLS IL66TCR-3betaCDR1MNHEY67TCR-3betaCDR2SMNVEV68TCR-3betaCDR3ASREGTGGYQ PQH32TCR-3betaconstantEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWdomainWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF69TCR-3betafull-QVTQNPRYLI TVTGKKLTVT CSQNMNHEYM SWYRQDPGLGlengthLRQIYYSMNV EVTDKGDVPE GYKVSRKEKR NFPLILESPSPNQTSLYFCA SREGTGGYQP QHFGDGTRLS ILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF70TCR-7alphavariableQTVTQSQPEM SVQEAETVTL SCTYDTSESD YYLFWYKQPPdomainSRQMILVIRQ EAYKQQNATE NRFSVNFQKA AKSFSLKISDSQLGDAAMYF CTFRYGGATN KLIFGTGTLL AVQP71TCR-7alphaCDR1TSESDYY15TCR-7alphaCDR2QEAYKQQN72TCR-7alphaCDR3TFRYGGATNK LI751TCR-7alphaconstantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS73TCR-7alphafull-QTVTQSQPEM SVQEAETVTL SCTYDTSESD YYLFWYKQPPlengthSRQMILVIRQ EAYKQQNATE NRFSVNFQKA AKSFSLKISDSQLGDAAMYF CTFRYGGATN KLIFGTGTLL AVQPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS74TCR-7betavariableGVIQSPRHLI KEKRETATLK CYPIPRHDTV YWYQQGPGQDdomainPQFLISFYEK MQSDKGSIPD RFSAQQFSDY HSELNMSSLELGDSALYFCA SRGGWAETPD TQYFGPGTRL TVL75TCR-7betaCDR1PRHDT76TCR-7betaCDR2FYEKMQ77TCR-7betaCDR3ASRGGWAETP DTQY11TCR-7betaconstantEDLKNVFPPE VAVFEPSEAE ISHTQKATLV CLATGFYPDHdomainVELSWWVNGK EVHSGVSTDP QPLKEQPALN DSRYCLSSRLRVSATFWQNP RNHFRCQVQF YGLSENDEWT QDRAKPVTQIVSAEAWGRAD CGFTSESYQQ GVLSATILYE ILLGKATLYAVLVSALVLMA MVKRKDSRG78TCR-7betafull-GVIQSPRHLI KEKRETATLK CYPIPRHDTV YWYQQGPGQDlengthPQFLISFYEK MQSDKGSIPD RFSAQQFSDY HSELNMSSLELGDSALYFCA SRGGWAETPD TQYFGPGTRL TVL EDLKNVFPPEVAVFEPSEAE ISHTQKATLV CLATGFYPDH VELSWWVNGKEVHSGVSTDP QPLKEQPALN DSRYCLSSRL RVSATFWQNPRNHFRCQVQF YGLSENDEWT QDRAKPVTQI VSAEAWGRADCGFTSESYQQ GVLSATILYE ILLGKATLYA VLVSALVLMAMVKRKDSRG79TCR-9alphavariableEDVEQSLFLS VREGDSSVIN CTYTDSSSTY LYWYKQEPGAdomainGLQLLTYIFS NMDMKQDQRL TVLLNKKDKH LSLRIADTQTGDSAIYFCAE KETAGNKLTF GGGTRVLVKP80TCR-9alphaCDR1DSSSTY81TCR-9alphaCDR2IFSNMDM82TCR-9alphaCDR3AEKETAGNKL T751TCR-9alphaconstantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS83TCR-9alphafull-EDVEQSLFLS VREGDSSVIN CTYTDSSSTY LYWYKQEPGAlengthGLQLLTYIFS NMDMKQDQRL TVLLNKKDKH LSLRIADTQTGDSAIYFCAE KETAGNKLTF GGGTRVLVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS84TCR-9betavariableEPEVTQTPSH QVTQMGQEVI LRCVPISNHL YFYWYRQILGdomainQKVEFLVSFY NNEISEKSEI FDDQFSVERP DGSNFTLKIRSTKLEDSAMY FCASTVQSPR TNEQFFGPGT RLTVL85TCR-9betaCDR1SNHLY86TCR-9betaCDR2FYNNEI87TCR-9betaCDR3ASTVQSPRTN EQF11TCR-9betaconstantEDLKNVFPPE VAVFEPSEAE ISHTQKATLV CLATGFYPDHdomainVELSWWVNGK EVHSGVSTDP QPLKEQPALN DSRYCLSSRLRVSATFWQNP RNHFRCQVQF YGLSENDEWT QDRAKPVTQIVSAEAWGRAD CGFTSESYQQ GVLSATILYE ILLGKATLYAVLVSALVLMA MVKRKDSRG88TCR-9betafull-EPEVTQTPSH QVTQMGQEVI LRCVPISNHL YFYWYRQILGlengthQKVEFLVSFY NNEISEKSEI FDDQFSVERP DGSNFTLKIRSTKLEDSAMY FCASTVQSPR TNEQFFGPGT RLTVLEDLKNVFPPE VAVFEPSEAE ISHTQKATLV CLATGFYPDHVELSWWVNGK EVHSGVSTDP QPLKEQPALN DSRYCLSSRLRVSATFWQNP RNHFRCQVQF YGLSENDEWT QDRAKPVTQIVSAEAWGRAD CGFTSESYQQ GVLSATILYE ILLGKATLYAVLVSALVLMA MVKRKDSRG89TCR-4alphavariableQSVAQPEDQV NVAEGNPLIV KCTYSVSGNP YLFWYVQYPNdomainRGLQFLLKYI TGDNLVKGSY GFEAEFNKSQ TSFHLKKPSALVSDSALYFC AAPRDDKIIF GKGTRLHILP90TCR-4alphaCDR1VSGNPY91TCR-4alphaCDR2YITGDNLV92TCR-4alphaCDR3AAPRDDKII751TCR-4alphaconstantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS93TCR-4alphafull-QSVAQPEDQV NVAEGNPLIV KCTYSVSGNP YLFWYVQYPNlengthRGLQFLLKYI TGDNLVKGSY GFEAEFNKSQTSFHLKKPSALVSDSALYFC AAPRDDKIIF GKGTRLHILPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS94TCR-4betavariableGVTQTPKFRV LKTGQSMTLL CAQDMNHEYMdomainYWYRQDPGMG LRLIHYSVGE GTTAKGEVPD GYNVSRLKKQNFLLGLESAA PSQTSVYFCA SSYLRTGGNE QFFGPGTRLT VL66TCR-4betaCDR1MNHEY95TCR-4betaCDR2SVGEGT96TCR-4betaCDR3ASSYLRTGGN EQF11TCR-4betaconstantEDLKNVFPPE VAVFEPSEAE ISHTQKATLV CLATGFYPDHdomainVELSWWVNGK EVHSGVSTDP QPLKEQPALN DSRYCLSSRLRVSATFWQNP RNHFRCQVQF YGLSENDEWT QDRAKPVTQIVSAEAWGRAD CGFTSESYQQ GVLSATILYE ILLGKATLYAVLVSALVLMA MVKRKDSRG97TCR-4betafull-GVTQTPKFRV LKTGQSMTLL CAQDMNHEYMlengthYWYRQDPGMG LRLIHYSVGE GTTAKGEVPD GYNVSRLKKQNFLLGLESAA PSQTSVYFCA SSYLRTGGNE QFFGPGTRLT VLEDLKNVFPPE VAVFEPSEAE ISHTQKATLV CLATGFYPDHVELSWWVNGK EVHSGVSTDP QPLKEQPALN DSRYCLSSRLRVSATFWQNP RNHFRCQVQF YGLSENDEWT QDRAKPVTQIVSAEAWGRAD CGFTSESYQQ GVLSATILYE ILLGKATLYAVLVSALVLMA MVKRKDSRG98TCR-8alphavariableQKVTQAQTEI SVVEKEDVTL DCVYETRDTT YYLFWYKQPPdomainSGELVFLIRR NSFDEQNEIS GRYSWNFQKS TSSFNFTITASQVVDSAVYF CALSGRGSGT YKYIFGTGTR LKVLA99TCR-8alphaCDR1TRDTTYY100TCR-8alphaCDR2RNSFDEQN101TCR-8alphaCDR3ALSGRGSGTY KYI751TCR-8alphaconstantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS102TCR-8alphafull-QKVTQAQTEI SVVEKEDVTL DCVYETRDTT YYLFWYKQPPlengthSGELVFLIRR NSFDEQNEIS GRYSWNFQKS TSSFNFTITASQVVDSAVYF CALSGRGSGT YKYIFGTGTR LKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS103TCR-8betavariableGVIQSPRHLI KEKRETATLK CYPIPRHDTV YWYQQGPGQDdomainPQFLISFYEK MQSDKGSIPD RFSAQQFSDY HSELNMSSLELGDSALYFCA SSAGTSYNEQ FFGPGTRLTV L75TCR-8betaCDR1PRHDT76TCR-8betaCDR2FYEKMQ104TCR-8betaCDR3ASSAGTSYNE QF11TCR-8betaconstantEDLKNVFPPE VAVFEPSEAE ISHTQKATLV CLATGFYPDHdomainVELSWWVNGK EVHSGVSTDP QPLKEQPALN DSRYCLSSRLRVSATFWQNP RNHFRCQVQF YGLSENDEWT QDRAKPVTQIVSAEAWGRAD CGFTSESYQQ GVLSATILYE ILLGKATLYAVLVSALVLMA MVKRKDSRG105TCR-8betafull-GVIQSPRHLI KEKRETATLK CYPIPRHDTV YWYQQGPGQDlengthPQFLISFYEK MQSDKGSIPD RFSAQQFSDY HSELNMSSLELGDSALYFCA SSAGTSYNEQ FFGPGTRLIV L EDLKNVFPPEVAVFEPSEAE ISHTQKATLV CLATGFYPDH VELSWWVNGKEVHSGVSTDP QPLKEQPALN DSRYCLSSRL RVSATFWQNPRNHFRCQVQF YGLSENDEWT QDRAKPVTQI VSAEAWGRADCGFTSESYQQ GVLSATILYE ILLGKATLYA VLVSALVLMAMVKRKDSRG106TCR-10alphavariableQNIDQPTEMT ATEGAIVQIN CTYQTSGFNG LFWYQQHAGEdomainAPTFLSYNVL DGLEEKGRFS SFLSRSKGYS YLLLKELQMKDSASYLCAVP APYTGTASKL TFGTGTRLQV TL107TCR-10alphaCDR1TSGFNG108TCR-10alphaCDR2NVLDGL109TCR-10alphaCDR3AVPAPYTGTA SKLT750TCR-10alphaconstantDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS110TCR-10alphafull-QNIDQPTEMT ATEGAIVQIN CTYQTSGFNG LFWYQQHAGElengthAPTFLSYNVL DGLEEKGRFS SFLSRSKGYS YLLLKELQMKDSASYLCAVP APYTGTASKL TFGTGTRLQV TLDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS111TCR-10betavariableGVTQTPKFQV LKTGQSMTLQ CAQDMNHNSMdomainYWYRQDPGMG LRLIYYSASE GTTDKGEVPN GYNVSRLNKREFSLRLESAA PSQTSVYFCA SSEGYSTYNE QFFGPGTRLT VL112TCR-10betaCDR1MNHNS113TCR-10betaCDR2SASEGT114TCR-10betaCDR3ASSEGYSTYN EQF11TCR-10betaconstantEDLKNVFPPE VAVFEPSEAE ISHTQKATLV CLATGFYPDHdomainVELSWWVNGK EVHSGVSTDP QPLKEQPALN DSRYCLSSRLRVSATFWQNP RNHFRCQVQF YGLSENDEWT QDRAKPVTQIVSAEAWGRAD CGFTSESYQQ GVLSATILYE ILLGKATLYAVLVSALVLMA MVKRKDSRG115TCR-10betafull-GVTQTPKFQV LKTGQSMTLQ CAQDMNHNSMlengthYWYRQDPGMG LRLIYYSASE GTTDKGEVPN GYNVSRLNKREFSLRLESAA PSQTSVYFCA SSEGYSTYNE QFFGPGTRLT VLEDLKNVFPPE VAVFEPSEAE ISHTQKATLV CLATGFYPDHVELSWWVNGK EVHSGVSTDP QPLKEQPALN DSRYCLSSRLRVSATFWQNP RNHFRCQVQF YGLSENDEWT QDRAKPVTQIVSAEAWGRAD CGFTSESYQQ GVLSATILYE ILLGKATLYAVLVSALVLMA MVKRKDSRG116TCR-12alphavariableQLLEQSPQFL SIQEGENLTV YCNSSSVFSS LQWYRQEPGEdomainGPVLLVTVVT GGEVKKLKRL TFQFGDARKD SSLHITAAQPGDTGLYLCAG GLSDSYDKVI FGPGTSLSVI P117TCR-12alphaCDR1SVFSS118TCR-12alphaCDR2VVTGGEV119TCR-12alphaCDR3AGGLSDSYDK VI751TCR-12alphaconstantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS120TCR-12alphafull-QLLEQSPQFL SIQEGENLTV YCNSSSVFSS LQWYRQEPGElengthGPVLLVTVVT GGEVKKLKRL TFQFGDARKD SSLHITAAQPGDTGLYLCAG GLSDSYDKVI FGPGTSLSVI PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS121TCR-12betavariableEVTQTPKHLV MGMTNKKSLK CEQHMGHRAM YWYKQKAKKPdomainPELMFVYSYE KLSINESVPS RFSPECPNSS LLNLHLHALQPEDSALYLCA SSQGVGQEYG YTFGSGTRLT VV58TCR-12betaCDR1MGHRA59TCR-12betaCDR2YSYEKL122TCR-12betaCDR3ASSQGVGQEY GYT32TCR-12betaconstantEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWdomainWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF123TCR-12betafull-EVTQTPKHLV MGMTNKKSLK CEQHMGHRAM YWYKQKAKKPlengthPELMFVYSYE KLSINESVPS RFSPECPNSS LLNLHLHALQPEDSALYLCA SSQGVGQEYG YTFGSGTRLT VVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF124TCR-11alphavariableIQVEQSPPDL ILQEGANSTL RCNFSDSVNN LQWFHQNPWGdomainQLINLFYIPS GTKQNGRLSA TTVATERYSL LYISSSQTTDSGVYFCAVNT GTASKLTFGT GTRLQVTL125TCR-11alphaCDR1DSVNN126TCR-11alphaCDR2IPSGT127TCR-11alphaCDR3AVNTGTASKL T750TCR-11alphaconstantDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS128TCR-11alphafull-IQVEQSPPDL ILQEGANSTL RCNFSDSVNN LQWFHQNPWGlengthQLINLFYIPS GTKQNGRLSA TTVATERYSL LYISSSQTTDSGVYFCAVNT GTASKLTFGT GTRLQVTLDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS129TCR-11betavariableGVTQTPKFQV LKTGQSMTLQ CAQDMNHNSMdomainYWYRQDPGMG LRLIYYSASE GTTDKGEVPN GYNVSRLNKREFSLRLESAA PSQTSVYFCA SSPRGQGRSY EQYFGPGTRL TVT112TCR-11betaCDR1MNHNS113TCR-11betaCDR2SASEGT130TCR-11betaCDR3ASSPRGQGRS YEQY11TCR-11betaconstantEDLKNVFPPE VAVFEPSEAE ISHTQKATLV CLATGFYPDHdomainVELSWWVNGK EVHSGVSTDP QPLKEQPALN DSRYCLSSRLRVSATFWQNP RNHFRCQVQF YGLSENDEWT QDRAKPVTQIVSAEAWGRAD CGFTSESYQQ GVLSATILYE ILLGKATLYAVLVSALVLMA MVKRKDSRG131TCR-11betafull-GVTQTPKFQV LKTGQSMTLQ CAQDMNHNSMlengthYWYRQDPGMG LRLIYYSASE GTTDKGEVPN GYNVSRLNKREFSLRLESAA PSQTSVYFCA SSPRGQGRSY EQYFGPGTRL TVTEDLKNVFPPE VAVFEPSEAE ISHTQKATLV CLATGFYPDHVELSWWVNGK EVHSGVSTDP QPLKEQPALN DSRYCLSSRLRVSATFWQNP RNHFRCQVQF YGLSENDEWT QDRAKPVTQIVSAEAWGRAD CGFTSESYQQ GVLSATILYE ILLGKATLYAVLVSALVLMA MVKRKDSRG132TCR-2alphavariableQKITQTQPGM FVQEKEAVTL DCTYDTSDQS YGLFWYKQPSdomainSGEMIFLIYQ GSYDEQNATE GRYSLNFQKA RKSANLVISASQLGDSAMYF CAMREGSDAG GTSYGKLTFG QGTILTVHP24TCR-2alphaCDR1TSDQSYG25TCR-2alphaCDR2QGSYDEQN133TCR-2alphaCDR3AMREGSDAGG TSYGKLT751TCR-2alphaconstantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITdomainDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS134TCR-2alphafull-QKITQTQPGM FVQEKEAVTL DCTYDTSDQS YGLFWYKQPSlengthSGEMIFLIYQ GSYDEQNATE GRYSLNFQKA RKSANLVISASQLGDSAMYF CAMREGSDAG GTSYGKLTFG QGTILIVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKIVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS135TCR-2betavariableGVIQSPRHLI KEKRETATLK CYPIPRHDTV YWYQQGPGQDdomainPQFLISFYEK MQSDKGSIPD RFSAQQFSDY HSELNMSSLELGDSALYFCA SSPSTGRLNT EAFFGQGTRL TVV75TCR-2betaCDR1PRHDT76TCR-2betaCDR2FYEKMQ136TCR-2betaCDR3ASSPSTGRLN TEAF32TCR-2betaconstantEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWdomainWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF137TCR-2betafull-GVIQSPRHLI KEKRETATLK CYPIPRHDTV YWYQQGPGQDlengthPQFLISFYEK MQSDKGSIPD RFSAQQFSDY HSELNMSSLELGDSALYFCA SSPSTGRLNT EAFFGQGTRL TVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF
Re-Expression of TCRs

The human constant chain domains were exchanged by their murine counterparts with additional mutations in the transmembrane domain to enhance hydrophobicity. Both modifications are described in Jin et al., JCI Insight. 2018; 3(8):e99488.

In order to assess their functional characteristics, the identified TCRs were re-expressed in primary T cells of healthy donors using TCR mRNA electroporation technique. In brief, a T7 transcription was performed on DNA templates of the TCR alpha and beta chain nucleotide sequence. The resulting TCR mRNA was used for electroporation of pre-activated T cells for transient expression of the exogenous TCR which was validated in co-culture experiments.

Co-Culture Assays (Assessment of Functional Avidity, Specificity, and TCR-Motif)

The functional characteristics of the TCRs were assessed in co-culture experiments with T2 cells. CD8+ T cells were pre-stimulated and electroporated with TCR mRNA. At the day of co-culture, luciferase-transduced T2 cells were either loaded with peptide CT45-IP (SEQ ID NO: 138) at different concentrations (assessment of functional avidity, EC50), with sequence similar peptides (SEQ ID NO: 146-155) at a concentration of 10 μM or with alanine-substitution variants of the CT45-IP peptide (SEQ ID NO: 139-145) at a concentration range of 10-30 fold above the average EC50of the respective TCR (TCR-motif determination). As control the irrelevant peptide NYESO1-001 (SEQ ID NO: 188) at a concentration of 10 μM and unloaded T2 cells were used. In brief, T2 cells were incubated for 2 hours with the respective amount of peptide and subsequently washed and harvested.

Cells electroporated with the model TCR 1G4-a95Ly as well as mock-electroporated T cells without exogenous TCR served as control. T cells and peptide-loaded T2 cells were seeded at a ratio of 1:1 and incubated for 24 h until supernatant harvest. Supernatants were subjected to an analysis for the presence of luciferase, released by apoptotic/necrotic T2 cells, killed by peptide-specific T cells. By adding specific substrate, the amount of luciferase present in the supernatant was determined by measuring the chemiluminescent signal in a microplate reader.

Test for Functionality in CD4+ T Cells

CD3+ T cells were stimulated and electroporated with TCR mRNA. After overnight incubation, the TCR-transfected T cells were co-cultivated at a 1:1 ratio with T2 cells loaded either with 10 μM CT45-IP (SEQ ID NO: 138) or 10 μM irrelevant NYESO1-001 (SEQ ID NO: 188) peptide. Directly after start of co-culture a cytokine secretion blocking reagent was added and incubated for 5 h at 37° C. Afterwards, the T cells were stained with fluorescently labeled antibodies for different surface markers such as CD4 and CD8. After fixation and permeabilization, the cells were stained for intracellular cytokines (TNF-α and IFN-γ) and analyzed on a flow cytometer (data not shown).

Lentiviral Co-Transduction of the Co-Stimulatory Molecule CD8

In order to engage CD4 T cells in the immune response after transduction with the MHC class I-restricted TCRs, a co-transduction with the co-stimulatory molecule CD8 was conducted. For that purpose, a lentiviral vector encoding the TCR chains as well as the CD8 molecule was used for transduction of pre-stimulated CD3+ T cells. The T cells were activated using plate-coated CD3 and anti-CD28 together with addition of IL-2 for 24 h. Pre-titrated concentrated lentiviral supernatant was added to the cells together with an adjuvant for transduction enhancement of lentivirus particles, e.g., Lentiboost® reagent (Sirion Biotech). The T cells were expanded over the course of 10 days using increasing volumes of media and decreasing concentration of IL-2 while transferring the cells consecutively into larger cell culture flasks. Transduction efficiency and resting state of T cells was checked prior to freezing of cells via flow cytometry. Subsequently, the effect of CD8 co-transfection on the killing efficiency against CT45-IP presenting tumor cells was analyzed in a live-cell monitoring killing assay (data not shown).

TCR Surface-Staining

A surface marker staining was performed with electroporated and pre-activated CD8+ T cells. To this end, anti-CD8, CD3 and/or mTCRB antibodies were used as well as TCR staining with fluorescently labeled Dextramers (dextramer backbone with conjugated CT45-IP-HLA-A*02 or irrelevant NYESO1-001-HLA-A*02). After 30 minutes cells were washed, fixed and subsequently analyzed by flow cytometry. Gates, to define dextramer-positive cells, were set according to the signal of cells stained with irrelevant peptide-MHC dextramer.

Live-Cell Monitoring Killing Assays

Proliferation of tumor cell lines expressing red fluorescent protein (RFP) was monitored using a live cell imaging system by quantifying red object counts over time. The cell lines NCIH1703 and A375 were co-cultivated with T cells expressing TCRs of the invention at an E:T ratio of 9:1, 3:1 or 1:1 or without T cells and monitored over a period of 48 h. As positive control, target cells were loaded with 10 μM of peptide CT45-IP. Decline of tumor cell line proliferation over time is an indicator for tumor cell killing.

Example 1: Functional Avidity

All fifteen identified CT45-IP-specific TCRs show high functional avidity as measured by peptide titration experiments which is expressed as half maximal killing capacity EC50shown inFIG.1. The measured EC50values range from 0.15 nM to 59.5 nM. In particular the EC50values are 7.78 nM (TCR-1); 4.69 nM (TCR-2); 1.02 nM (TCR-3); 1.31 nM (TCR-4); 1.32 nM (TCR-5); 3.25 nM (TCR-6); 0.48 nM (TCR-7); 6.52 nM (TCR-8); 0.15 nM (TCR-9); 8.75 nM (TCR-10); 59.50 nM (TCR-11); 47.47 nM (TCR-12); 11.38 nM (TCR-13); 17.69 nM (TCR-14); 18.60 nM (TCR-15).

Example 2: Specificity and TCR-Motif

The herein described TCRs were tested for their specificity profiles by testing their ability to recognize 10 CT45-IP sequence-similar peptides (SEQ ID NOs: 146-155). Loading with CT45-IP and CT45-IP sequence-similar peptides occurred at a very high concentration of 10 μM, to detect low signals. TCRs showed no binding to another peptide except CT45-IP. Minor binding signals were detected for TCR-9 and TCR-6 to peptide SP-05-0004 (SEQ ID NO: 149) as depicted inFIG.2. The TCR characterization also involved the determination of the TCR binding motif to CT45-IP peptide as listed in Table 4. For this purpose, CT45-IP alanine exchange peptide variants (SEQ ID NO: 139-145) were tested in co-culture experiments with all fifteen TCRs. Alanine exchange peptides were tested at a concentration range of 10-30 fold above the average EC50of the respective TCR. A relevant position is indicated by a number (referring to the position of the amino acid in the peptide), a non-relevant position is represented by a hyphen, and a position that was not tested is marked by an x.

TABLE 4Consensus motifIDConsensus motifTCR-11x34567-xTCR-2-x3(4)567-xTCR-3-x34567-xTCR-4-x34-678xTCR-5-x3--67(8)xTCR-61x34-6--xTCR-71x-4-67-xTCR-8-x---5-7-xTCR-91x34567(8)xTCR-10-x--567-xTCR-111x3456-8xTCR-12-x34-67-xTCR-131x34-67-xTCR-141x345---xTCR-151x34567-x

Example 3: CD4-Functionality

A test for functionality in CD4+ T cells was performed as described above. Among the fifteen tested TCRs, the inventors identified TCRs, which show functionality in CD8+ T cells as well as CD4+ T cells (data not shown).

Example 4: Surface Expression

The surface expression of the herein described TCRs was measured by CT45-IP-HLA-A2*02 dextramer staining and is shown inFIG.3. The surface expression varied from 0.53% (TCR-11) to 55.5% (TCR-5) positive events after gating on CD3+ T cells after TCR mRNA electroporation. In particular, surface expression was 8.9% (TCR-1); 39.1% (TCR-2); 27.2% (TCR-3); 39.1% (TCR-4); 61.1% (TCR-5); 2.9% (TCR-6); 53.3% (TCR-7); 60.7% (TCR-8); 44.0% (TCR-9); 52.8% (TCR-10); 6.6% (TCR-11); 20.1% (TCR-12); 53.1% (TCR-13); 11.6% (TCR-14); 22.6% (TCR-15).

Example 5: Efficacy Against Tumor Cell Lines

Two tumor cell lines, the A375 with ˜30 copies per cell and the NCIH1703 with ˜150 copies per cell of CT45-IP were co-cultivated with CD8+ T cells expressing TCR-9, TCR-7 or Mock-TCR (FIG.4). The efficacy of those TCRs for killing the two cell lines was assessed in a live cell monitoring experiment. While Mock-TCR does not lead to significant reduction of tumor cell proliferation, TCR-9 and TCR-7 efficiently kill the tumor cell lines loaded with CT45-IP peptide at an E:T ratio of 6:1 (TCR-9) or 1.8:1 (TCR-7) (NCIH1703 & A375), without additional peptide loading at an E:T ratio of 6:1 (TCR-9) or 1.8:1 (TCR-7) (NCIH1703 & A375), and at an E:T ratio of 2:1 (TCR-9) or 0.6:1 (TCR-7) and 0.6:1 (TCR-9) or 0.2:1 (TCR-7) (NICIH1703) as measured by the normalized red object count of <2.

Example 6: Safety Window

Co-culture assays were performed using T2 cells loaded with a titration series of peptide CT45-IP and SP-05-0004, respectively. The safety window was determined as EC50SP 0004 divided by EC50CT45-IP (data not shown).

Example 7: CT45-IP Presentation

The detection frequency of the CT45-IP antigenic peptide was analysed in primary and cultured tumor samples. A summary of the results is shown in Table 5. In the table, an expression of >0% is indicated as +, an expression of 10% is indicated as ++ and an expression of 30% is indicated as +++. The tumor entities in which presentation was detected are bile duct cancer (CCC), liver cancer (HCC), skin cancer (MEL, due to cell line identifications), lymph node cancer (NHL), non-small cell lung cancer (NSCLC), ovarian cancer (OC), esophageal cancer (OSCAR) and uterus cancer (UEC).

TABLE 5Target presentationTarget detectionEntityfrequency (%)CCC+HCC+MEL+++NHL+NSCLC+OC++OSCAR++UEC+

Example 8: Efficacy of CD4+ T Cells Co-Transduced with a CT45-IP-Specific TCR and CD8 Against Tumor Cells

Tumor cells presenting CT45-IP were co-cultivated with CD4+ T cells or CD3+ T cells co-transduced with the herein described CT45-IP-specific TCRs and CD8. The killing efficacy of the T cells was assessed in a live cell monitoring experiment. Upon co-transduction with CT45-IP-specific TCR and CD8, efficient killing of the tumor cell lines as measured by the normalized red object count of <2 was observed (data not shown).

Items

1. An antigen binding protein specifically binding to a CT45 antigenic peptide that is in a complex with a major histocompatibility complex (MHC) protein, wherein the CT45 antigenic peptide comprises or consists of the amino acid sequence of SEQ ID NO: 138 (KIFEMLEGV) and wherein the antigen binding protein comprises a first polypeptide comprising a variable domain VAcomprising complementarity determining regions (CDR) CDRa1, CDRa2 and CDRa3 and a second polypeptide comprising a variable domain VBcomprising CDRb1, CDRb2 and CDRb3, wherein1) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 14, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 16, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 19, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 21,2) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 24, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 133, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 75, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 136,3) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 24, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 63, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 66, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 68,4) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 90, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 92, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 66, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 96,5) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 2, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 4, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 8, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 10,6) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 53, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 55, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 58, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 60,7) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 71, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 72, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 75, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 77,8) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 99, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 101, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 75, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 104,9) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 80, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 82, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 85, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 87,10) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 107, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 109, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 112, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 114,11) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 125, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 127, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 112, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 130, or12) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 117, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 119, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 58, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 122,13) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 24, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 35, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 38, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 40,14) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 24, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 26, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 29, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 31, or15) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 43, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 45, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 48, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 50,wherein the antigen binding protein comprises said CDRa1, CDRa3, CDRb1 and CDRb3 sequence(s) with not more than one, two or three amino acid mutations, wherein each of CDRa1, CDRa3, CDRb1 and/or CDRb3 may comprise one, two or three amino acid mutations.2. The antigen binding protein of item 1, wherein1) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 15, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 20,2) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 25, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 76,3) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 25, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 67,4) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 91, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 95,5) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 3, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 9,6) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 54, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 59,7) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 15, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 76,8) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 100, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 76,9) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 81, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 86,10) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 108, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 113,11) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 126, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 113, or12) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 118, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 59,13) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 25, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 39,14) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 25, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 30, or15) CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 44, and CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 49,wherein the antigen binding protein comprises said CDRa2 and CDRb2 sequence(s) with not more than one, two, three or four amino acid mutations, wherein each of CDRa2 and/or CDRb2 may comprise one, two, three or four amino acid mutations.3. The antigen binding protein of item 1 or 2, wherein the antigen binding protein specifically binds to a complex of the CT45 antigenic peptide and an MHC protein.4. The antigen binding protein of any one of items 1 to 3, wherein the MHC protein is an HLA protein, more particularly HLA-A, even more particularly HLA-A*02.5. The antigen binding protein of any one of items 1 to 4, wherein the EC50of CT45-IP for inducing killing of CT45-IP:MHC complex presenting cells by T cells expressing the antigen binding protein is less than about 60 nM, less than about 50 nM, less than about 30 nM, less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 5 nM, less than about 2.5 nM, less than about 1.5 nM or less than about 1 nM.6. The antigen binding protein of any one of items 1 to 5, wherein the antigen binding protein specifically binds to a functional epitope comprising or consisting of 2, 3 or 4 amino acid positions selected from the group consisting of positions 3, 4, 5, 6 and 7 of SEQ ID NO: 138.7. The antigen binding protein of any one of items 1 to 6, wherein the antigen binding protein does not significantly bind to at least 1, at least 2, at least 3, at least 4, at least 5, or all similar peptides selected from the group consisting of SEQ ID NO: 146 (SP-05-0001), SEQ ID NO: 147 (SP-05-0002), SEQ ID NO: 148 (SP-05-0003), SEQ ID NO: 149 (SP-05-0004), SEQ ID NO: 150 (SP-05-0005), SEQ ID NO: 151 (SP-05-0006), SEQ ID NO: 152 (SP-05-0007), SEQ ID NO: 153 (SP-05-0008), SEQ ID NO: 154 (SP-05-0009) and SEQ ID NO: 155 (SP-05-0010), preferably from the group consisting of SEQ ID NO: 146 (SP-05-0001), SEQ ID NO: 147 (SP-05-0002), SEQ ID NO: 148 (SP-05-0003), SEQ ID NO: 150 (SP-05-0005), SEQ ID NO: 151 (SP-05-0006), SEQ ID NO: 152 (SP-05-0007), SEQ ID NO: 153 (SP-05-0008), SEQ ID NO: 154 (SP-05-0009) and SEQ ID NO: 155 (SP-05-0010), more preferably from the group consisting of SEQ ID NO: 147 (SP-05-0002), SEQ ID NO: 151 (SP-05-0006), SEQ ID NO: 152 (SP-05-0007) and SEQ ID NO: 155 (SP-05-0010).8. The antigen binding protein of any one of items 1 to 7, wherein the first and the second polypeptide are comprised on two polypeptide chains, preferably wherein VAis comprised in a first polypeptide chain and VBis comprised in a second polypeptide chain.9. The antigen binding protein of any one of items 1 to 7, wherein the antigen binding protein is a single chain antigen binding protein, preferably a single chain TCR, or a single chain bispecific antigen binding protein, preferably a single chain bispecific TCR.10. The antigen binding protein of any one of items 1 to 9, wherein the antigen binding protein is monovalent or multivalent, e.g. tetra-, tri- or bivalent.11. The antigen binding protein of any one of items 1 to 10, wherein the antigen binding protein is monospecific or multispecific, e.g. tetra-, tri- or bispecific.12. The antigen binding protein of any one of items 1 to 11, wherein the antigen binding protein is a soluble protein.13. The antigen binding protein of any one of items 1 to 12, wherein the antigen binding protein is a TCR.14. The antigen binding protein of item 13, wherein the TCR is selected from the group consisting of an α/β TCR, a γ/δ TCR, a single chain TCR, a membrane-bound TCR, a soluble TCR, a monovalent, bivalent or multivalent TCR, a monospecific, bispecific or multispecific TCR, a functional fragment of a TCR, and a fusion protein or chimeric protein comprising a functional fragment of a TCR.15. The antigen binding protein of item 13, wherein the TCR is an α/β TCR or a γ/δ TCR, preferably an α/β TCR.16. The antigen binding protein of any one of items 1 to 15, further comprising one or more of the following:(i) one or more further antigen binding sites;(ii) a transmembrane domain, optionally including a cytoplasmic signaling region;(iii) a diagnostic agent;(iv) a therapeutic agent.17. The antigen binding protein of item 16, wherein the one or more further antigen binding sites comprise an antibody-derived antigen binding site, preferably comprising or consisting of VLand VH.18. The antigen binding protein of any one of items 1 to 17, wherein VAcomprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 132, 62, 89, 1, 52, 70, 98, 79, 106, 124, 116, 34, 23, and 42, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 13, 132, 62, 89, 1, 52, 70, 98, 79, 106, 124, 116, 34, 23, or 42 and comprising the CDRa1, CDRa2 and CDRa3 according to item 1 or 2; and wherein VBcomprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 135, 65, 94, 7, 57, 74, 103, 84, 111, 129, 121, 37, 28 and 47 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 18, 135, 65, 94, 7, 57, 74, 103, 84, 111, 129, 121, 37, 28 or 47 and comprising the CDRb1, CDRb2 and CDRb3 according to item 1 or 2, wherein the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may comprise one, two or three amino acid mutations, preferably amino acid substitutions.19. The antigen binding protein of any one of items 1 to 18, wherein VAand VBare TCR variable domains, preferably TCR alpha, beta, gamma or delta variable domains, more preferably wherein VAis a TCR alpha or gamma, preferably alpha, variable domain, and VBis a TCR beta or delta, preferably beta, variable domain.20. The antigen binding protein of any one of items 1 to 19, further comprising a constant domain, wherein the constant domain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 750, 751, 156, 11, 32, and 157, preferably selected from the group consisting of SEQ ID NO: 5, 750, 751, 11 and 32, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 5, 750, 751, 156, 11, 32 or 157.21. The antigen binding protein of any one of items 1 to 20, wherein the first polypeptide is a TCR alpha chain and the second polypeptide is a TCR beta chain or the first polypeptide is a TCR gamma chain and the second polypeptide is a TCR delta chain preferably wherein the first polypeptide is a TCR alpha chain and the second polypeptide is a TCR beta chain.22. The antigen binding protein of any one of items 1 to 21, wherein the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27, 46 and 158-172, preferably selected from the group consisting of SEQ ID NO: 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27, 46 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 17, 134, 64, 93, 6, 56, 73, 102, 83, 110, 128, 120, 36, 27, 46 or 158-172, and the second polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33, 51 and 173-187, preferably selected from the group consisting of SEQ ID NO: 22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33, 51, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 22, 137, 69, 97, 12, 61, 78, 105, 88, 115, 131, 123, 41, 33, 51 or 173-187.23. The antigen binding protein of any one of items 1 to 22, wherein the antigen binding protein specifically binds to a functional epitope comprising or consisting ofi. amino acid positions 1, 3 and 4 of SEQ ID NO: 138, preferably amino acid positions 1, 3, 4 and 5, or 1, 3, 4 and 6 or 1, 3, 4, 5 and 6 or 1, 3, 4, 5, 6 and 7 of SEQ ID NO: 138;ii. amino acid positions 4, 6 and 7 of SEQ ID NO: 138, preferably amino acid positions 1, 4, 6 and 7, or 3, 4, 6 and 7 or 1, 3, 4, 6 and 7 of SEQ ID NO: 138; oriii. amino acid positions 5 and 7 of SEQ ID NO: 138, preferably amino acid positions 5, 6 and 7, or 3, 4, 5, 6 and 7 of SEQ ID NO: 138.24. The antigen binding protein of any one of items 1 to 23, wherein the antigen binding protein does not significantly bind to the similar peptides of the group consisting ofi. SEQ ID NO: 146 (SP-05-0001), SEQ ID NO: 147 (SP-05-0002), SEQ ID NO: 148 (SP-05-0003), SEQ ID NO: 149 (SP-05-0004), SEQ ID NO: 150 (SP-05-0005), SEQ ID NO: 151 (SP-05-0006), SEQ ID NO: 152 (SP-05-0007), SEQ ID NO: 153 (SP-05-0008), SEQ ID NO: 154 (SP-05-0009) and SEQ ID NO: 155 (SP-05-0010); orii. SEQ ID NO: 146 (SP-05-0001), SEQ ID NO: 147 (SP-05-0002), SEQ ID NO: 148 (SP-05-0003), SEQ ID NO: 150 (SP-05-0005), SEQ ID NO: 151 (SP-05-0006), SEQ ID NO: 152 (SP-05-0007), SEQ ID NO: 153 (SP-05-0008), SEQ ID NO: 154 (SP-05-0009) and SEQ ID NO: 155 (SP-05-0010).25. The antigen binding protein of any one of items 1 to 24, wherein the antigen binding protein has a mean expression of at least 5%, at least 10%, at least 20%, at least 30%, or at least 40%.26. The antigen binding protein of any one of items 1 to 25, wherein VAcomprises a V segment encoded by TRAV14, in particular TRAV14/DV4, and a CDRa1 according to SEQ ID NO: 24 and a CDRa2 according to SEQ ID NO: 25.27. The antigen binding protein of any one of items 1 to 26, wherein VBcomprisesi. a V segment encoded by TRBV13, and a CDRb1 according to SEQ ID NO: 75 and a CDRb2 according to SEQ ID NO: 76;ii. a V region encoded by TRBV4-1, and a CDRb1 according to SEQ ID NO: 58 and a CDRb2 according to SEQ ID NO: 59, oriii. a V region encoded by TRBV6-1, and a CDRb1 according to SEQ ID NO: 112 and a CDRb2 according to SEQ ID NO: 113.28. The antigen binding protein of any one of items 1 to 27, wherein the antigen binding protein is capable of activatinga CD4+ T cell, in particular a CD4+CD8− T cell, and/ora CD8+ T cell, in particular a CD8+CD4-T cell, and wherein the antigen binding protein is preferably a TCR, more preferably an α/β TCR or γ/δ TCR.29. A nucleic acid or nucleic acids comprising one or more sequences encoding the antigen binding protein of any one of items 1 to 28.30. A vector or a collection of vectors comprising the nucleic acid(s) of item 29.31. A host cell comprising the antigen binding protein of any one of items 1 to 28, or the nucleic acid(s) of item 29, or the vector or collection of vectors of item 30.32. The host cell of item 31, wherein the host cell isa lymphocyte, preferably a T cell, a T cell progenitor or an NK cell, more preferably a CD4 or CD8 positive T cell; ora cell for recombinant expression, such as a Chinese Hamster Ovary (CHO) cell or a yeast cell.33. A pharmaceutical composition comprising the antigen binding protein of any one of items 1 to 28, the nucleic acid(s) of item 29, the vector or collection of vectors of item 30, or the host cell of item 31 or 32 and optionally a pharmaceutically acceptable carrier.34. The antigen binding protein of any one of items 1 to 28, the nucleic acid(s) of item 29, the vector or collection of vectors of item 30, the host cell of item 31 or 32, or the pharmaceutical composition of item 33 for use in medicine.35. The antigen binding protein of any one of items 1 to 28, the nucleic acid(s) of item 29, the vector or collection of vectors of item 30, the host cell of item 31 or 32, or the pharmaceutical composition of item 33 for use in a method of treatment and/or diagnosis of a proliferative disease, in particular cancer.36. A method of treatment of a proliferative disease, in particular cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the antigen binding protein of any one of items 1 to 28, the nucleic acid(s) of item 29, the vector or collection of vectors of item 30, the host cell of item 31 or 32, or the pharmaceutical composition of item 33.37. The antigen binding protein, nucleic acid(s), vector or collection of vectors, host cell or pharmaceutical composition for use of item 35 or the method of treatment of item 36, wherein the cancer is a CT45 expressing cancer, more particularly selected from the group of cancers consisting of lung cancer, NSCLC, gall bladder cancer, bile duct cancer, lymph node cancer, ovarian cancer, esophageal cancer, liver cancer, uterus cancer and melanoma.38. The antigen binding protein, nucleic acid(s), vector or collection of vectors, host cell or pharmaceutical composition for use of item 35 or 37 or the method of treatment of item 36 or 37, wherein the method of treatment comprises immune therapy, in particular adoptive autologous or heterologous T-cell therapy.39. The antigen binding protein, nucleic acid(s), vector or collection of vectors, host cell or pharmaceutical composition for use of any one of items 35, 37 or 38 or the method of treatment of any one of items 36 to 38, wherein the antigen binding protein is a TCR.40. The antigen binding protein, nucleic acid(s), vector or collection of vectors, host cell or pharmaceutical composition for use of any one of items 35 or 37 to 39 or the method of treatment of any one of items 36 to 39, wherein the antigen binding protein is expressed on the surface of a host cell.41. The antigen binding protein, nucleic acid(s), vector or collection of vectors, host cell or pharmaceutical composition for use of any one of items 35 or 37 to 40 or the method of treatment of any one of items 36 to 40, wherein the method of treatment comprises administration of a host cell expressing the antigen binding protein, wherein the host cell is a T cell, a T cell progenitor or an NK cell, preferably a T cell.42. The antigen binding protein, nucleic acid(s), vector or collection of vectors, host cell or pharmaceutical composition for use of item 40 or the method of treatment of item 40, wherein the host cell, preferably the T cell, T cell progenitor or NK cell, more preferably the T cell, is autologous.43. The antigen binding protein, nucleic acid(s), vector or collection of vectors, host cell or pharmaceutical composition for use of item 40 or the method of treatment of item 40, wherein the host cell, preferably the T cell, T cell progenitor or NK cell, more preferably the T cell, is allogeneic.44. The antigen binding protein, nucleic acid(s), vector or collection of vectors, host cell or pharmaceutical composition for use of any one of items 35 or 37 to 43 or the method of treatment of any one of items 36 to 43, wherein the antigen binding protein is conjugated to a therapeutically active agent.45. The antigen binding protein, nucleic acid(s), vector or collection of vectors, host cell or pharmaceutical composition for use of any one of items 44 or the method of treatment of any one of items 44, wherein the therapeutically active agent is selected from the group consisting of a radionuclide, a chemotherapeutic agent and a toxin.46. The antigen binding protein, nucleic acid(s), vector or collection of vectors, host cell or pharmaceutical composition for use of any one of items 35 or 37 to 45 or the method of treatment of any one of items 36 to 45, wherein the method of treatment further comprises administering at least one chemotherapeutic agent to the subject in need of treatment.47. The antigen binding protein, nucleic acid(s), vector or collection of vectors, host cell or pharmaceutical composition for use of any one of items 35 or 37 to 46 or the method of treatment of any one of items 36 to 46, wherein the method of treatment further comprises administering radiation therapy to the subject in need of treatment.48. A method of treating cancer in a subject in need thereof, comprising:a) isolating a cell from said subject;b) transforming the cell with a vector or collection of vectors encoding the antigen binding protein of any one of items 1 to 28 to produce a transformed cell;c) expanding the transformed cell to produce a plurality of transformed cells; andd) administering the plurality of transformed cells to said subject.49. A method of treating cancer in a subject in need thereof, comprising:a) isolating a cell from a healthy donor;b) transforming the cell with a vector or collection of vectors encoding the antigen binding protein of any one of items 1 to 28 to produce a transformed cell;c) expanding the transformed cell to produce a plurality of transformed cells; andd) administering the plurality of transformed cells to said subject.50. The method of item 48 or 49, wherein the transformed cell is a lymphocyte, preferably an NK cell or T cell or T cell progenitor, more preferably a T cell.51. Use of the antigen binding protein of any one of items 1 to 28 for the manufacture of a medicament for the treatment of a proliferative disease.52. An in-vitro method of detecting cancer, in particular cancer expressing CT45, in a biological sample comprising:a) contacting the biological sample with the antigen binding protein of any one of items 1 to 28, andb) detecting binding of the antigen binding protein to the biological sample.53. A method of producing the antigen binding protein according to any one of items 1 to 28, comprisinga) providing a host cell,b) providing a genetic construct comprising a nucleic acid or nucleic acids encoding the antigen binding protein of any of items 1 to 28,c) introducing the genetic construct into the host cell, andd) expressing the genetic construct by the host cell.54. The method of item 53, further comprising the isolation and purification of the antigen binding protein from the host cell and, optionally, reconstitution of the antigen binding protein in a T cell.55. The method of item 53 or 54, further comprising cell surface presentation of said antigen binding protein.56. The method of any one of items 53 to 55, wherein the genetic construct is an expression construct comprising a promoter sequence operably linked to the nucleic acid encoding the antigen binding protein.57. The method according to any one of items 53 to 56, wherein the genetic construct is introduced into the host cell by retroviral transfection.