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NOVEL ENGINEERED T CELL RECEPTORS AND IMMUNE THERAPY USING THE SAME

20190135914 ยท 2019-05-09

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention pertains to antigen recognizing constructs against COL6A3 antigens. The invention in particular provides novel engineered T cell receptor (TCR) based molecules which are selective and specific for the tumor expressing antigen COL6A3. The TCR of the invention, and COL6A3 antigen binding fragments derived therefrom, are of use for the diagnosis, treatment and prevention of COL6A3 expressing cancerous diseases. Further provided are nucleic acids encoding the antigen recognizing constructs of the invention, vectors comprising these nucleic acids, recombinant cells expressing the antigen recognizing constructs and pharmaceutical compositions comprising the compounds of the invention.

    Claims

    1. An antigen recognizing construct comprising a first domain comprising three complementary determining regions (CDRs) comprising the amino acid sequences according to SEQ ID NOs: 5 (CDRa1), 6 (CDRa2), and 7 (CDRa3), and a second domain comprising three complementary determining regions (CDRs) comprising the amino acid sequences according to SEQ ID NOs: 13 (CDRb1), 14 (CDRb2), and 15 (CDRb3), wherein at least one of said complementary determining regions is replaced with at least one amino acid sequence selected from the group of: a) SEQ ID NO: 26 (CDRa1 mutant 1) and SEQ ID NOs: 37 to 49 (CDRb1-mut1 to CDRb1-mut13), and b) a mutated sequence comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 26 and SEQ ID NOs: 37 to 49, wherein the at least one amino acid sequence contains one, two, or three amino acid substitutions.

    2. The antigen recognizing construct according to claim 1, wherein said antigen recognizing construct is stable, and capable of specifically and/or selectively binding to a COL6A3 antigenic peptide comprising the amino acid sequence according to SEQ ID NO: 1, optionally in the context with MHC.

    3. The antigen recognizing construct according to claim 1, wherein the antigen recognizing construct is an antibody, or derivative such as a bispecific molecule or fragment thereof, or a T cell receptor (TCR), or a derivative such as a bispecific molecule or fragment thereof.

    4. The antigen recognizing construct according to claim 1, wherein said first domain is part of a TCR ? or ? chain; and/or wherein said second domain is part of a TCR ? or ? chain.

    5. A nucleic acid encoding for an antigen recognizing construct according to claim 1, or a nucleic acid vector comprising said nucleic acid.

    6. A recombinant host cell comprising an antigen recognizing construct according to claim 1 or a nucleic acid or a vector thereof, wherein said host cell optionally is a) a lymphocyte, optionally a T lymphocyte or T lymphocyte progenitor cell, optionally a CD4 or CD8 positive T cell or b) a non-lymphocyte, optionally a Chinese Hamster Ovary (CHO) cell.

    7. A pharmaceutical composition comprising the antigen recognizing construct according to claim 1, a nucleic acid or vector thereof, or the host cell comprising said construct or nucleic acid or vector, and a pharmaceutically acceptable carrier, diluent stabilizer and/or excipient.

    8. A method of producing a COL6A3-specific antigen recognizing construct according to claim 2, comprising a. providing a suitable host cell, b. providing a genetic construct comprising a coding sequence encoding the antigen recognizing construct, c. introducing said genetic construct into said suitable host cell, and d. expressing said genetic construct by said suitable host cell.

    9. The method according to claim 8, further comprising isolating and purifying the antigen recognizing construct from the suitable host cell and, optionally, reconstituting the antigen recognizing construct in a T-cell.

    10. The antigen recognizing construct according to claim 1, a nucleic acid or vector thereof, or a host cell comprising said construct, nucleic acid or vector, or a pharmaceutical composition thereof, adapted for use in medicine.

    11. The antigen recognizing construct according to claim 1, a nucleic acid or vector thereof, a host cell comprising said construct, nucleic acid or vector, or a pharmaceutical composition thereof, adapted for use in the diagnosis, prevention, and/or treatment of a proliferative disease, optionally cancer.

    12. The antigen recognizing construct according to claim 1, a nucleic acid or vector thereof, a host cell comprising said construct, nucleic acid or vector, or a pharmaceutical composition thereof adapted for use in the diagnosis, prevention, and/or treatment of a proliferative disease, optionally cancer, wherein said cancer is selected from a cancer where COL6A3 is overexpressed, mutated, and/or a COL6A3-derived tumor-associated antigen is presented, such as, for example, colorectal cancer, colon cancer, liver cancer, ovarian cancer, and gastric cancer.

    13. The antigen recognizing construct according to claim 1, wherein the mutated sequence is a combination of a first and a second amino acid sequence, such that the mutated sequence is provided by replacing one, two or three amino acids in the first sequence with the corresponding one, two or three amino acids of the second sequence.

    14. The antigen recognizing construct according to claim 13, wherein the mutated sequence is a combination of a first and a second amino acid sequence, such that the mutated sequence is provided by replacing one, two or three consecutive amino acids in the first sequence with the corresponding one, two or three consecutive amino acids of the second sequence.

    15. The antigen recognizing construct according to claim 1, wherein the mutated sequence of (b) contains at least one conservative amino acid substitution compared to the at least one amino acid sequence of (a).

    16. The antigen recognizing construct according to claim 1, wherein said at least one of said complementary determining regions is replaced with SEQ ID NO: 40 and wherein SEQ ID NO: 40 contains one, two, or three amino acid substitutions.

    17. The antigen recognizing construct according to claim 1, wherein said at least one of said complementary determining regions is replaced with SEQ ID NO: 40.

    18. The antigen recognizing construct according to claim 1, comprising a variant selected from the group consisting of 75-1-75-25 of Table 2 and C-1-C-18 of Table 5.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0101] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

    [0102] The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures and Sequences:

    [0103] FIG. 1 shows the conversion of a TCR into stabilized V?/V? single-chain TCR (scTv) via yeast surface display. ScTv molecules displayed on the surface of transformed Saccharomyces cerevisiae EBY100 were stained with FITC-labeled anti-Vbeta1 antibody and PE-labeled HLA-A*02/COL6A3-002 tetramer. The unmodified scTv R4P3F9 (left panel, SEQ ID NO: 22) is compared to an scTv clone bearing single point mutations to stabilize the scTv scaffold (right panel), which was derived from the selection of a random mutation scTv library.

    [0104] FIG. 2 shows scTv affinity maturation via yeast surface display. Stabilized scTv molecules with and without affinity maturated CDR1 beta were stained with HLA-A*02 tetramers containing COL6A3-002 (SEQ ID NO: 1) and counterstained with a mix of HLA-A*02 tetramers containing 9 peptides (SEQ ID NO: 28 to 36) with high sequence similarity to COL6A3-002. Stabilized scTv (SEQ ID NO 27) with non-maturated beta chain CDR1 sequence RSGDLS (SEQ ID NO: 13) is compared with scTv clones bearing the affinity maturated beta chain CDR1 sequences AMDHPY (SEQ ID NO: 40) and ARWHRN (SEQ ID NO: 39).

    [0105] FIG. 3 shows size exclusion chromatography elution profiles of anti-CD3 Fab-scTv R4P3F9S fusion variants 75-1 to 75-25.

    [0106] FIG. 4 shows the HLA-A*02/COL6A3-002 binding kinetics of anti-CD3 Fab-scTv R4P3F9S fusion variants 75-1 to 75-25 as measured by biolayer interferometry. Analyzed concentrations of HLA-A*02/COL6A3-002 are indicated.

    [0107] FIG. 5 shows binding analysis of anti-CD3 Fab-scTv R4P3F9S fusion variants 75-1 to 75-25 as measured via biolayer interferometry (BLI). 1 ?M of HLA-A*02 in complex with the indicated similar peptides was analyzed.

    [0108] FIG. 6 shows a comparison of HLA-A*02/COL6A3-002 and HLA-A*02/COL6A1-001 (SEQ ID NO: 30) binding kinetics of different anti-CD3 Fab-scTv R4P3F9S fusion variants. Analyzed concentrations of Fab-scTv molecules are indicated.

    [0109] FIG. 7 shows staining of maturated R4P3F9 TCR variant expressing human CD8.sup.+ T cells with PE-labeled HLA-A*02/COL6A3-002 tetramers. For control purpose, no TCR (Mock) or the 1G4 TCR specific for NYESO1-001 was expressed and staining with PE-labeled HLA-A*02/NYESO1-001 tetramers was used.

    [0110] FIG. 8 shows IFN-gamma release of maturated R4P3F9 TCR variant expressing human CD8.sup.+ T cells in response to COL6A3-002. For control purpose, no TCR (mock) or the 1G4 TCR specific for NYESO1-001 was expressed. IFN-gamma release was determined by ELISA after co-culture of electroporated CD8.sup.+ T cells with T2 cells loaded with a serial dilution of COL6A3-002.

    [0111] FIG. 9 shows IFN-gamma release of maturated R4P3F9 TCR variant expressing human CD8.sup.+ T cells in response to COL6A3-002 and different similar peptides. For control purpose, no TCR (mock) or the 1G4 TCR specific for NYESO1-001 was expressed. IFN-gamma release was determined by ELISA after co-culture of electroporated CD8.sup.+ T cells with T2 cells loaded with 10 ?M of COL6A3-002 or similar peptides.

    [0112] FIG. 10 shows staining of maturated R4P3F9 TCR variant expressing human CD8.sup.+ T cells with PE-labeled peptide-HLA-A*02 tetramers. For control purpose, no TCR (Mock) or the 1G4 TCR specific for NYESO1-001 was expressed and staining with PE-labeled HLA-A*02/NYESO1-001 tetramers was used.

    [0113] FIG. 11 shows IFN-gamma release of maturated R4P3F9 TCR variant expressing human CD8.sup.+ T cells in response to COL6A3-002 or COL6A1-001. For control purpose, no TCR (mock) or the 1G4 TCR specific for NYESO1-001 was expressed. IFN-gamma release was determined by ELISA after co-culture of electroporated CD8.sup.+ T cells with T2 cells loaded with a serial dilution of COL6A3-002 or COL6A1-001.

    [0114] FIG. 12 shows IFN-gamma release of primary human CD8.sup.+ T cells expressing R4P3F9 TCR variants upon co-culture with different tumor cell lines. SF539, SW982 and Hs840.T cells present the target peptide at different levels. MCF-7 cells do not present the target peptide. As controls, effector cells without exogenous TCR were analyzed along with cells with TCRs of interest. IFN-gamma release was determined by ELISA. * marks a data point that is out of scale.

    TABLE-US-00001 TABLE1 Peptidesequencesoftheinvention(positionsareaccordingtoIMGTnumbering: (Fran?oisEhrenmann,PatriceDuroux,ChantalGinestoux;Proteindisplays:human(Homosapiens) TRAV;IMGTRepertoire.IMGT?,theinternationalImMunoGeneticsinformationsystem? www.imgt.org.; Created:16Mar.2011.Version:03Jun.2016;Fran?oisEhrenmann,PatriceDuroux,Chantal Ginestoux;Proteindisplays:human(Homosapiens)TRBV;IMGTRepertoire.IMGT ImMunoGeneticsinformationsystem? www.imgt.org.;Created:16Mar.2011.Version:03Jun.2016.) SEQ ID NO: Name Description Sequence 1 COL6A3- FLLDGSANV 002 2 R4P3F9 R4P3F9TCR MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIA alpha alpha SLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDG chain- RFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQ full LTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTD length FDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD TNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 3 R4P3F9 R4P3F9TCR MKSLRVLLVILWLQLSWVWSQ alpha alpha leader chain- leader peptide 4 R4P3F9 R4P3F9TCR QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS alpha alpha GKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ variable chain- PSDSATYLCAAYSGAGSYQLTFGKGTKLSVIP variable domain 5 R4P3F9 R4P3F9TCR DRGSQS CDRa1 alpha chain- CDR1 6 R4P3F9 R4P3F9TCR IYSNGD CDRa2 alpha chain- CDR2 7 R4P3F9 R4P3F9TCR CAAYSGAGSYQLT CDRa3 alpha chain- CDR3 8 R4P3F9- R4P3F9TCR NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS alpha alpha DVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS constant chain- IIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF constant RILLLKVAGFNLLMTLRLWSS domain 9 R4P3F9- R4P3F9TCR NIQN alpha alpha constant chain- start constant domain start 10 R4P3F9 R4P3F9TCR MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTL beta betachain- RCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNIL full ERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGY length TFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPAL NDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW TQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILY EILLGKATLYAVLVSALVLMAMVKRKDF 11 R4P3F9 R4P3F9TCR MGFRLLCCVAFCLLGAGPV beta betachain- leader leader peptide 12 R4P3F9 R4P3F9TCR DSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLD beta betachain- QGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSS variable variable LELGDSALYFCASSVESSYGYTFGSGTRLTVV domain 13 R4P3F9 R4P3F9TCR RSGDLS CDRb1 betachain- CDR1 14 R4P3F9 R4P3F9TCR YYNGEE CDRb2 betachain- CDR2 15 R4P3F9 R4P3F9TCR CASSVESSYGYT CDRb3 betachain- CDR3 16 R4P3F9 R4P3F9TCR EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDH beta betachain- VELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL constant constant RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI domain VSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDF 17 R4P3F9 R4P3F9TCR EDLNK beta betachain- constant constant start1 domain start1 18 R4P3F9 R4P3F9TCR EDLKN beta betachain- constant constant start2 domain start2 19 Aga2p- Aga2p MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSL R4P3F9 fusion STTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSP protein INTQYVFGGGGSDYKDDDDKGGGASQKEVEQNSGPLSVPE withscTv GAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGD R4P3F9and KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGA tags GSYQLTFGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGG SGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQ GLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSL ELGDSALYFCASSVESSYGYTFGSGTRLTVVEDLNKAAAG GSGGEQKLISEEDL 20 Aga2p Leader MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSL sequence STTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSP andAga2p INTQYVF 21 FLAGtag FLAGtag GGGGSDYKDDDDKGGGAS plus linkers 22 scTv Single QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS R4P3F9 chain GKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ variable PSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGOGG domainsof SGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPR R4P3F9 SGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSA with QQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSG linker; TRLTVVEDLNK aF55Sin alpha variable domain 23 Myctag Linkerand AAAGGSGGEQKLISEEDL Myctag 24 scTv scTv QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS R4P3F9- R4P3F9 GKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ bQ43K with PSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGG stabilizing SGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPR mutation SGDLSVYWYKQSLDQGLQFLIQYYNGEERAKGNILERFSA bQ43Kin QQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSG beta TRLTVVEDLNK variable domain 25 scTv scTv QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS R4P3F9- R4P3F9 GKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ bL72S with PSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGG stabilizing SGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPR mutation SGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNISERFSA bL72Sin QQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSG beta TRLTVVEDLNK variable domain 26 CDRa1 aG29R DRRSQS mutant1 mutation 27 scTv Stabilized QKEVEQNSGPLSVPEGAIASLNCTYSDRRSQSFFWYRQYS R4P3F95 versionof GKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ scTv PSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPGGGGSGGG R4P3F9 GSGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSP RSGDLSVYWYKQSLDQGLQFLIQYYNGEERAKGNISERFS AQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGS GTRLTVV 28 AGRN-001 Similar ALLDGRVQL peptides 29 CLASP1- Similar RLLDGAFKL 001 peptides 30 COL6A1- Similar ILLDGSASV 001 peptides 31 COL6A2- Similar FLLDGSERL 001 peptides 32 COL6A3- Similar FLFDGSANLV 006 peptides 33 COL6A3- Similar FLFDGSANL 008 peptides 34 COL6A3- Similar FLLDGSEGV 014 peptides 35 VWA2-001 Similar FLLDGSNSV peptides 36 VWF-001 Similar FLLDGSSRL peptides 37 CDRb1 Betachain- ARWHNN mutant1 CDR1 variant1 38 CDRb1 Betachain- AKDHLN mutant2 CDR1 variant2 39 CDRb1 Betachain- ARWHRN mutant3 CDR1 variant3 40 CDRb1 Betachain- AMDHPY mutant4 CDR1 variant4 41 CDRb1 Betachain- ATDHYN mutant5 CDR1 variant5 42 CDRb1 Betachain- ARYHTN mutant6 CDR1 variant6 43 CDRb1 Betachain- APYHLN mutant7 CDR1 variant7 44 CDRb1 Betachain- AKDHTN mutant8 CDR1 variant8 45 CDRb1 Betachain- ARYHRN mutant9 CDR1 variant9 46 CDRb1 Betachain- ARWHSN mutant10 CDR1 variant10 47 CDRb1 Betachain- ATDHYN mutant11 CDR1 variant11 48 CDRb1 Betachain- RWGDLN mutant12 CDR1 variant12 49 CDRb1 Betachain- ARDHLN mutant13 CDR1 variant13 50 75-1 Fabheavy MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC chainwith AASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ stabilized KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY scTv GDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG R4P3F9S GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTSPPSPAPPVAGQKEVEQNSGPLSVPEGAIASL NCTYSDRGSQSFFWYRQYSGKSPELIMSTYSNGDKEDGRF TAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLT FGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGGSGGGGS GVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYKQSLDQG LQFLIQYYNGEERAKGNISERFSAQQFPDLHSELNLSSLE LGDSALYFCASSVESSYGYTFGSGTRLTVVEDLKN 51 75-Fab Fabheavy MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC heavy chain AASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ chain KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY GDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTSPPSPAPPVAG 52 75-Fab Fablight MKWVTFISLLFLFSSAYSDIQMTQSPSSLSASVGDRVTIT light chain CRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRF chain SGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVIEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 53 1G4alpha 1G4TCR METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVL alpha NCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGR chain- LNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYI full PTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTD length FDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD TNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 54 1G4alpha 1G4TCR METLLGLLILWLQLQWVSSK leader alpha chain- leader peptide 55 1G4alpha 1G4TCR QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPG variable alpha KGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQ chain- PGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHP variable domain 56 1G4alpha 1G4TCR YIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS constant alpha DVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS chain- IIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF constant RILLLKVAGFNLLMTLRLWSS domain 57 1G4beta 1G4TCR MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTL betachain- QCAQDMNHEYMSWYRQDP0MGLRLIHYSVGAGITDQ0EVP full NGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGE length LFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKAT LVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPA LNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATIL YEILLGKATLYAVLVSALVLMAMVKRKDSRG 58 1G4beta 1G4TCR MSIGLLCCAALSLLWAGPVNA leader betachain- leader peptide 59 1G4beta 1G4TCR GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDP0M0 variable betachain- LRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAA variable PSQTSVYFCASSYVGNTGELFFGEGSRLTVL domain 60 1G4beta Betachain- EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDH constant constant VELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL domain RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI VSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDSRG 61 NYESO1- Control SLLMWITQV 001 peptide 62 C-14 C-14;C-5 MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTL beta;C-5 TCRfull RCSPAMDHPYVYWYQQSLDQGLQFLIQYYNGEERAKGNIL beta length ERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGY betachain TFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL withCDRb1 VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPAL mutant4 NDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW TQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILY EILLGKATLYAVLVSALVLMAMVKRKDF 63 C-14 C-14TCR MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIA alpha full SLNCTYSDRRSQSFFWYRQYSGKSPELIMFIYSNGDKEDG length RFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQ alpha LTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTD chainwith FDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN CDRa1 KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD mutant1 TNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 64 75-5 Fabheavy MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC chainwith AASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ stabilized KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY scTv GDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG R4P3F9S GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS andCDRb1 GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP mutant4 KSCDKTHTSPPSPAPPVAGQKEVEQNSGPLSVPEGAIASL NCTYSDRGSQSFFWYRQYSGKSPELIMSTYSNGDKEDGRF TAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLT FGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGGSGGGGS GVTQTPKHLITATGQRVTLRCSPAMDHPYVYWYKQSLDQG LQFLIQYYNGEERAKGNISERFSAQQFPDLHSELNLSSLE LGDSALYFCASSVESSYGYTFGSGTRLTVVEDLKN 65 75-14 Fabheavy MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC chainwith AASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ stabilized KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY scTv GDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG R4P3F9S, GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS CDRa1 GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP mutant1 KSCDKTHTSPPSPAPPVAGQKEVEQNSGPLSVPEGAIASL andCDRb1 NCTYSDRRSQSFFWYRQYSGKSPELIMSTYSNGDKEDGRF mutant4 TAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLT FGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGGSGGGGS GVTQTPKHLITATGQRVTLRCSPAMDHPYVYWYKQSLDQG LQFLIQYYNGEERAKGNISERFSAQQFPDLHSELNLSSLE LGDSALYFCASSVESSYGYTFGSGTRLTVVEDLKN 66 75-25 Fabheavy MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC chainwith AASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ stabilized KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY scTv GDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG R4P3F95in GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS beta/alpha GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP orientation, KSCDKTHTSPPSPAPPVAGGVTQTPKHLITATGQRVTLRC CDRa1 SPRSGDLSVYWYKQSLDQGLQFLIQYYNGEERAKGNISER mutant1 FSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTF GSGTRLTVVEDLKNGGGGSGGGGSGGGGSGGGGSGGGGSQ KEVEQNSGPLSVPEGAIASLNCTYSDRRSQSFFWYRQYSG KSPELIMSTYSNGDKEDGRFTAQLNKASQYVSLLIRDSQP SDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQN

    EXAMPLES

    [0115] Native T cell receptors (TCRs) against cancer antigens are often of lower affinity when compared to TCRs targeting viral antigens, and this may be one possible explanation for tumor immune escape (Aleksic et al. 2012). Therefore, it is desirable to have higher affinity TCR variants designed for the use as antigen recognizing constructs in an adoptive cell therapy, or as recognition module of a soluble approach, i.e., using bispecific molecules (Hickman et al. 2016). This invention thus relates to the modification and optimization of the naturally occurring T cell receptor R4P3F9 (SEQ ID NOs: 2 and 10) targeting the tumor associated peptide COL6A3-002 (SEQ ID NO: 1) with an affinity of about 60 ?M (DE102016115246).

    Example 1: Generation of Stable scTv

    [0116] For the present invention, the previously investigated TCR R4P3F9 (SEQ ID NOs: 2 and 10) was converted into a single chain TCR construct (scTv, SEQ ID NO: 22) for maturation via yeast surface display by combination of the variable alpha (SEQ ID NO: 4) and beta (SEQ ID NO: 12) domain with appendages of the respective constant domains (SEQ ID NOs: 9 and 17) and an appropriate glycine-serine linker sequence. The DNA of the corresponding sequence was synthesized and transformed into Saccharomyces cerevisiae EBY100 (MATa AGA1::GAL1AGA1::URA3 ura352 trp1 leu2delta200 his3delta200 pep4::HIS3 prbd1.6R can1 GAL) (ATCC? MYA 4941?) together with a yeast display vector containing a leader sequence and the Aga2p yeast mating protein (SEQ ID NO: 20), based on pCT302 (Boder et al. 2000). The resulting fusion protein after homologous recombination in the yeast (SEQ ID NO: 19) contains a leader peptide at the N-terminus of the Aga2p protein, responsible for the display of the protein of interest (Boder et al. 1997), short peptide tags including linker sequences (SEQ ID NOs: 21 and 23) for expression controls and the protein of interest, namely the scTv R4P3F9 (SEQ ID NO: 22) or its variants. The transformation was performed as described in DE102016121899 and resulted in up to 10.sup.9 yeast clones per library. The libraries were generated via a random mutation PCR approach spanning the whole gene sequence of the scTv R4P3F9.

    [0117] The selection process for the yeast clones bearing the best expressing scTv that is selectively binding to COL6A3-002 in context of HLA-A*02 was essentially performed as described in Smith et al 2015. In order to ascertain high expression and correct conformation of the scTv R4P3F9 variant, displayed on the yeast surface, an anti-Vbeta1 (Beckman Coulter, clone BL37.2) antibody was used, together with HLA-A*02/COL6A3-002 tetramer (FIG. 1). The scTv conversion by yeast surface display revealed two crucial stabilizing mutations in the framework region together with the original CDR sequences for the proper presentation of the scTv on the cell surface, namely bQ43K (SEQ ID NO: 24) and bL72S (SEQ ID NO: 25), both located on the beta chain. Furthermore, during stability maturation position 29 in the CDR1 of the alpha chain (SEQ ID NO: 5) was converted from glycine to arginine (CDRa1 mutant 1, SEQ ID NO: 26), which resulted in improved tetramer binding.

    Example 2: Affinity Maturation of Stabilized scTv

    [0118] To generate scTv molecules with higher binding affinity towards HLA-A*02/COL6A3-002, the CDRb1 (SEQ ID NO: 13) was degenerated using the previously identified stabilized scTv R4P3F9S scaffold (SEQ ID NO: 27) expressing the stabilizing mutations aG29R, bQ43K and bL72S. The CDRb1 residues were randomized by using degenerate DNA oligo primers essentially as described previously (Smith et al. 2015). The resulting DNA library was transformed as described in example 1. To retain scTv binding selectivity, negative selection was employed against HLA-A*02 tetramers comprising peptides derived from normal tissues (SEQ ID NOs: 28 to 36), which show high sequence similarity to COL6A3-002 peptide.

    [0119] For the selection of affinity enhanced and selective scTv R4P3F9S variants a decreasing concentration of HLA-A*02/COL6A3-002 tetramer was used for each sorting round. After three selection rounds, single scTv clones were isolated and sequenced, resulting in a variety of affinity maturated CDRb1 sequences (SEQ ID NOs: 37 to 49). For scTv with maturated CDRb1 sequences a strong improvement in COL6A3-002 binding could be demonstrated while the selectivity of COL6A3-002 binding was retained as no binding of 9 similar peptides was observed (FIG. 2).

    Example 3: Production of Bispecific Antibody-scTv Fusion Proteins

    [0120] Stabilized and affinity maturated scTv against HLA-A*02/COL6A3-002 can be expressed in fusion with an antibody moiety directed against CD3 allowing tumor-specific retargeting and activation of T cells independent of their natural specificity. The inventors generated bispecific antibody-TCR fusion proteins comprising an anti-CD3 Fab (UCHT1) heavy chain (SEQ ID NO: 51) fused to scTv R4P3F9S variants (SEQ ID NOs: 50, 64, 65 and 66) and an anti-CD3 Fab (UCHT1) light chain (SEQ ID NO: 52). The resulting Fab-scTv fusion proteins have a molecular mass of approximately 75 kDa. Based on different CDR1 sequences of the scTv R4P3F9S alpha (SEQ ID NOs: 5 and 26) and beta chain (SEQ ID NOs: 13 and 37 to 49) different Fab-scTv fusion variants (75-1 to 75-25, Table 2) were expressed in transiently transfected ExpiCHO cells as recommended by the manufacturer. Proteins were purified by protein L and size exclusion chromatography. All fusion variants could be produced with yields ranging from 80 ?g up to 1 mg (Table 2) and homogeneously formed heterodimers at the expected size as analyzed by size exclusion chromatography (FIG. 3).

    TABLE-US-00002 TABLE2 NomenclatureandyieldsofbispecificFab-scTvfusionproteins.Themoleculesarebased onSEQIDNOs50and52andtheindicatedCDRa1andCDRb1variants. Variant CDRa1/SEQ CDRb1/SEQ Yield[?g] 75-1 DRGSQS(SEQIDNO.5) RSGDLS(SEQIDNO.13) 267.9 75-2 DRGSQS(SEQIDNO.5) ARWHNN(SEQIDNO.37) 78.4 75-3 DRGSQS(SEQIDNO.5) AKDHLN(SEQIDNO.38) 646.7 75-4 DRGSQS(SEQIDNO.5) ARWHRN(SEQIDNO.39) 704.3 75-5 DRGSQS(SEQIDNO.5) AMDHPY(SEQIDNO.40) 397.2 75-6 DRGSQS(SEQIDNO.5) ATDHYN(SEQIDNO.41) 268.1 75-7 DRGSQS(SEQIDNO.5) ARYHTN(SEQIDNO.42) 83.2 75-8 DRGSQS(SEQIDNO.5) APYHLN(SEQIDNO.43) 765.7 75-9 DRGSQS(SEQIDNO.5) AKDHTN(SEQIDNO.44) 1067.2 75-10 DRRSQS(SEQIDNO.26) RSGDLS(SEQIDNO.13) 389.6 75-11 DRRSQS(SEQIDNO.26) ARWHNN(SEQIDNO.37) 270.4 75-12 DRRSQS(SEQIDNO.26) AKDHLN(SEQIDNO.38) 943.6 75-13 DRRSQS(SEQIDNO.26) ARWHRN(SEQIDNO.39) 560.3 75-14 DRRSQS(SEQIDNO.26) AMDHPY(SEQIDNO.40) 360.7 75-15 DRRSQS(SEQIDNO.26) ATDHYN(SEQIDNO.41) 541.5 75-16 DRRSQS(SEQIDNO.26) ARYHTN(SEQIDNO.42) 403.6 75-17 DRRSQS(SEQIDNO.26) APYHLN(SEQIDNO.43) 195.5 75-18 DRRSQS(SEQIDNO.26) AKDHTN(SEQIDNO.44) 731.3 75-19 DRRSQS(SEQIDNO.26) ARYHRN(SEQIDNO.45) 794 75-20 DRRSQS(SEQIDNO.26) ARWHSN(SEQIDNO.46) 85.5 75-21 DRRSQS(SEQIDNO.26) ATDHYN(SEQIDNO.47) 276 75-22 DRRSQS(SEQIDNO.26) RWGDLN(SEQIDNO.48) 255 75-23 DRRSQS(SEQIDNO.26) ARDHLN(SEQIDNO.49) 217 75-24.sup.a DRGSQS(SEQIDNO:5) RSGDLS(SEQIDNO.13) 166.6 75-25.sup.a DRRSQS(SEQIDNO.26) RSGDLS(SEQIDNO.13) 267 .sup.abeta-alpha orientation of scTv

    Example 4: Fab-scTv Fusion Protein Binding to COL6A3-002 and Similar Peptides

    [0121] Binding affinity of anti-CD3-scTv R4P3F9S fusion proteins towards HLA-A*02 monomers with COL6A3-002 or different similar peptides was measured by biolayer interferometry. Measurements were done on an Octet RED384 system using settings recommended by the manufacturer. Briefly, purified Fab-scTv molecules were loaded onto biosensors (FAB2G) prior to analyzing serial dilutions of HLA-A*02/COL6A3-002. Compared to variants 75-1 and 75-24 comprising wild-type CDRa1 and wild-type CDRb1 increased binding affinities of up to 40-fold were observed for Fab-scTv variants with maturated CDRa1 and/or CDRb1 sequences (Table 3, FIG. 4). In order to evaluate the selectivity of binding to HLA-A*02/COL6A3-002, purified Fab-scTv molecules loaded onto FAB2G biosensors were screened for binding to 1 ?M similar peptides (SEQ ID NOs: 28 to 36), each in complex with HLA-A*02. Except of HLA-A*02/COL6A1-001 (SEQ ID NO: 30), which was bound by most of the Fab-scTv variants containing maturated CDRa1 (SEQ ID NO: 26), Fab-scTv variants showed no binding to similar peptides (FIG. 5) arguing for high binding selectivity. For some Fab-scTv variants the therapeutic window between HLA-A*02/COL6A3-002 and HLA-A*02/COL6A1-001 binding was investigated by loading biotinylated peptide-HLA complexes onto biosensors (SA) and analyzing dilution series of Fab-scTv variants. While variant 75-10 comprising maturated CDRa1 (SEQ ID NO: 26) and wild-type CDRb1 (SEQ ID NO: 13) sequence showed 8-fold increased binding affinity to HLA-A*02/COL6A3-002 over HLA-A*02/COL6A1-001, an up to 57-fold increased binding affinity was detected for Fab-scTv variant 75-13 comprising a maturated CDRb1 (SEQ ID NO: 39) arguing for an improvement in therapeutic window (Table 4, FIG. 6).

    TABLE-US-00003 TABLE 3 Binding affinity of Fab-scTv fusion proteins to HLA-A*02/COL6A3-002. Variant KD (M) kon (1/Ms) koff (1/s) 75-1 8.06E?06 1.01E+05 8.17E?01 75-2 3.69E?06 1.59E+05 5.86E?01 75-3 4.92E?06 9.71E+04 4.78E?01 75-4 5.76E?06 9.78E+04 5.63E?01 75-5 4.32E?04 2.21E+03 9.55E?01 75-6 1.13E?06 2.06E+05 2.32E?01 75-7 1.79E?06 1.93E+05 3.44E?01 75-8 3.45E?06 1.36E+05 4.69E?01 75-9 1.41E?05 6.02E+04 8.51E?01 75-10 1.78E?06 1.69E+05 3.01E?01 75-11 2.82E?07 4.16E+05 1.18E?01 75-12 3.74E?07 2.67E+05 1.00E?01 75-13 4.05E?07 3.28E+05 1.33E?01 75-14 3.10E?06 8.41E+04 2.61E?01 75-15 7.78E?07 2.33E+05 1.81E?01 75-16 5.87E?07 3.37E+05 1.98E?01 75-17 2.27E?07 3.62E+05 8.20E?02 75-18 1.93E?06 1.51E+05 2.91E?01 75-19 6.00E?07 2.96E+05 1.78E?01 75-20 5.31E?07 6.08E+05 3.23E?01 75-21 5.52E?07 2.72E+05 1.50E?01 75-22 8.22E?07 2.48E+05 2.04E?01 75-23 3.24E?07 3.18E+05 1.03E?01 75-24 5.20E?06 1.08E+05 5.62E?01 75-25 8.33E?06 6.23E+04 5.19E?01

    TABLE-US-00004 TABLE 4 Comparative binding affinity of Fab-scTv fusion proteins to HLA-A*02/COL6A3-002 and HLA-A*02/COL6A1-001. Variant pHLA-A*02 KD (M) KD.sub.COL6A1-001/KD.sub.COL6A3-002 75-10 COL6A3-002 1.37E?05 8 COL6A1-001 1.08E?04 75-11 COL6A3-002 8.50E?07 8 COL6A1-001 6.46E?06 75-12 COL6A3-002 7.24E?07 12 COL6A1-001 8.98E?06 75-13 COL6A3-002 7.39E?07 57 COL6A1-001 4.23E?05 75-17 COL6A3-002 8.25E?07 9 COL6A1-001 7.10E?06 75-23 COL6A3-002 1.15E?06 22 COL6A1-001 2.55E?05

    Example 5: Use of Affinity-Maturated TCRs for Cellular Expression

    [0122] Modification of T cells to express TCRs recognizing a tumor-specific peptide-HLA is a promising alternative of redirecting T cells to cancer cells. As the usage of maturated CDR1 sequences could improve cell-bound TCRs against HLA-A*02/COL6A3-002, the identified CDRa1 and CDRb1 mutant sequences were grafted onto the parental TCR R4P3F9 (SEQ ID NOs: 2 and 10). The resulting mutant TCR variants (C-1 to C-18, Table 5) were expressed in human CD8.sup.+ T cells after electroporation of respective mRNA generated by in vitro transcription of PCR-amplified DNA constructs. For control purpose, the 1G4 TCR (SEQ ID NOs: 53 and 57) against NYESO1-001 peptide (SEQ ID NO: 61) was expressed. After overnight incubation of RNA-electroporated CD8.sup.+ T cells, expression of introduced TCR variants was analyzed by staining with PE-labeled HLA-A*02/COL6A3-002 tetramers or HLA-A*02/NYESO1-001 tetramers. While the parental TCR R4P3F9 variant C-1 showed only minimal staining with HLA-A*02/COL6A3-002 tetramers, the R4P3F9 TCR variants C-2 to C-18 with maturated CDRa1 and/or CDRb1 showed increased tetramer staining (FIG. 7). Functional activation of CD8.sup.+ T cells (20,000 cells/well) expressing different maturated R4P3F9 TCR variants was investigated by determining levels of released IFN-gamma upon co-culture with T2 cells (20,000 cells/well) loaded with either a dilution series of COL6A3-002 (SEQ ID NO: 1) or 10 ?M of COL6A3-002 and similar peptides (SEQ ID NOs: 28 to 36). Compared to the parental R4P3F9 TCR variant C-1, maturated TCR variants C-2 to C-18 showed increased IFN-gamma release with maximum levels reached already at lower peptide concentrations (FIG. 8). As expected no IFN-gamma release was observed with T cells expressing no TCR or the 1G4 control TCR specific for NYESO1-001. To analyze the selectivity of COL6A3-002 recognition of the maturated R4P3F9 TCR variants, the IFN-gamma release in response to T2 cells loaded with different similar peptides (SEQ ID NOs: 28 to 36) was analyzed and revealed different selectivity profiles for the maturated R4P3F9 TCR variants. Most interestingly, the TCR variants C-5 (SEQ ID Nos 62 and 2) and C-14 (SEQ ID NOs: 62 and 63) comprising the same maturated CDRb1 (SEQ ID NO: 40) did not show any cross-reactivity towards COL6A1-001 or other similar peptides (FIG. 9) making affinity maturated R4P3F9 TCR variants C-5 and C14 most promising candidates for cellular TCR-based tumor targeting.

    TABLE-US-00005 TABLE5 NomenclatureofcellularTCRvariants.ThemoleculesarebasedonSEQIDNOs2and 10andtheindicatedCDRa1andCDRb1variants. Variant CDRa1 CDRb1 C-1 DRGSQS(SEQIDNO.5) RSGDLS(SEQIDNO.13) C-2 DRGSQS(SEQIDNO.5) ARWHNN(SEQIDNO.37) C-3 DRGSQS(SEQIDNO.5) AKDHLN(SEQIDNO.38) C-4 DRGSQS(SEQIDNO.5) ARWHRN(SEQIDNO.39) C-5 DRGSQS(SEQIDNO.5) AMDHPY(SEQIDNO.40) C-6 DRGSQS(SEQIDNO.5) ATDHYN(SEQIDNO.41) C-7 DRGSQS(SEQIDNO.5) ARYHTN(SEQIDNO.42) C-8 DRGSQS(SEQIDNO.5) APYHLN(SEQIDNO.43) C-9 DRGSQS(SEQIDNO.5) AKDHTN(SEQIDNO.44) C-10 DRRSQS(SEQIDNO.26) RSGDLS(SEQIDNO.13) C-11 DRRSQS(SEQIDNO.26) ARWHNN(SEQIDNO.37) C-12 DRRSQS(SEQIDNO.26) AKDHLN(SEQIDNO.38) C-13 DRRSQS(SEQIDNO.26) ARWHRN(SEQIDNO.39) C-14 DRRSQS(SEQIDNO.26) AMDHPY(SEQIDNO.40) C-15 DRRSQS(SEQIDNO.26) ATDHYN(SEQIDNO.41) C-16 DRRSQS(SEQIDNO.26) ARYHTN(SEQIDNO.42) C-17 DRRSQS(SEQIDNO.26) APYHLN(SEQIDNO.43) C-18 DRRSQS(SEQIDNO.26) AKDHTN(SEQIDNO.44)

    Example 6: Window of COL6A3-002 and COL6A1-001 Recognition of Cellular TCR Variants

    [0123] Cellular expression and analysis of R4P3F9 variants was performed as described above. In accordance with previous experiments (FIG. 7), staining of T cells expressing R4P3F9 TCR variants C-2 to C-18 with PE-labeled HLA-A*02/COL6A3-002 tetramers were increased compared to the parental TCR C-1. Additionally, TCR variants C-12 and C-17 showed binding to HLA-A*02/COL6A1-001 (FIG. 10). Expression of all maturated R4P3F9 variants improved functional activation of CD8.sup.+ T cells in response to T2 cells loaded with a dilution series of COL6A3-002 (SEQ ID NO: 1) reaching 5- to 90-fold lower EC.sub.50 values compared to the parental TCR C-1 (FIG. 11, Table 6). The lowest EC.sub.50 value was found for variant C-14. Again, TCR variants C-5 (SEQ ID NOs 62 and 2) and C-14 (SEQ ID NOs: 62 and 63) comprising the same maturated CDRb1 (SEQ ID NO: 40) did not show any cross-reactivity towards COL6A1-001, while other variants showed strong recognition with EC.sub.50 windows (COL6A3-002 vs. COL6A1-001) as low as factor 5.

    TABLE-US-00006 TABLE 6 EC.sub.50 values [nM] of IFN-? release of T cells expressing R4P3F9 variants after coculture with T2 cells loaded with COL6A3-002 or COL6A1-001. Variant EC.sub.50 COL6A3-002 [nM] EC.sub.50 COL6A1-001 [nM] C-1 2.51 C-2 0.16 C-3 0.14 871.sup.a C-4 0.13 C-5 0.15 C-6 0.48 C-7 0.29 C-8 0.20 350 C-9 0.55 C-10 0.32 1.5 C-11 0.32 8.2 C-12 0.20 1.9 C-13 0.23 9.7 C-14 0.03 C-15 0.31 69 C-16 0.34 78 C-17 0.33 4.1 C-18 0.14 280089.sup.a .sup.aplateau not reached

    Example 7: Efficacy of Maturated R4P3F9 Variants C-5 and C-14 on Tumor Cell Lines

    [0124] Cellular expression and analysis of R4P3F9 variants was performed as described above. Expression of the maturated R4P3F9 variants C-5 (SEQ ID NOs 62 and 2) and C-14 (SEQ ID NOs: 62 and 63) improved functional activation of CD8.sup.+ T cells in response to COL6A3-002 (SEQ ID NO: 1)-presenting tumor cell lines as compared to the parental TCR C-1 (FIG. 12). The tumor cell lines used during this study present different amounts of target peptide. SF539 cells carry ?4000 copies of HLA-A*02/COL6A3-002 per cell and SW982 cells carry ?460 copies per cell. Whereas the parental TCR C-1 did not mediate strong T-cell activation upon co-culture with target-positive cell lines, TCR variant C-14 showed even stronger improvement of functional activation than TCR variant C-5. These data are in line with EC.sub.50 improvements from TCR C-1 to C-5 and to C-14 (table 6). The target-negative tumor cell line MCF-7 was not recognized by any of these TCRs.

    REFERENCES

    [0125] Aleksic et al. 2012: Different affinity windows for virus and cancer-specific T-cell receptorsimplications for therapeutic strategies, Eur J Immunol. 2012 December; 42 (12):3174-9;

    [0126] Hickman et al. 2016: Antigen Selection for Enhanced Affinity T-Cell Receptor-Based Cancer Therapies, J Biomol Screen. 2016 September; 21 (8):769-85;

    [0127] Boder and Wittrup 2000: Yeast surface display for directed evolution of protein expression, affinity, and stability, Methods Enzymol. 2000; 328:430-44;

    [0128] Boder and Wittrup 1997: Yeast surface display for screening combinatorial polypeptide libraries, Nat Biotechnol. 1997 June; 15 (6):553-7;

    [0129] Smith et al. 2015: T Cell Receptor Engineering and Analysis Using the Yeast Display Platform, Methods Mol Biol. 2015; 1319:95-141;

    [0130] DE102016121899.5

    [0131] DE102016115246