T cell receptors and immune therapy using the same

Abstract

The present invention pertains to antigen recognizing constructs against COL6A3 antigens. The invention in particular provides novel T cell receptor (TCR) based molecules which are selective and specific for the tumor expressed 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. A method of treating a patient who has cancer that presents a peptide consisting of the amino acid sequence of FLLDGSANV (SEQ ID NO: 58) in a complex with HLA-A*02, comprising administering to the patient a population of transformed CD8+ T cells expressing at least one vector encoding a T cell receptor (TCR), wherein the TCR comprises SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, wherein the TCR is capable of binding to a peptide consisting of the amino acid sequence of FLLDGSANV (SEQ ID NO: 58) in a complex with HLA-A*02, and wherein the cancer is selected from gastrointestinal cancer, gastric cancer, breast cancer, colon cancer, esophageal cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, rectal cancer, gallbladder cancer, and urinary bladder cancer.

2. The method of claim 1, wherein the population of transformed cells are produced by a method comprising isolating a cell from a subject, transforming the cell with at least one vector encoding the TCR to produce a transformed cell, and expanding the transformed cell to produce the population of transformed cells.

3. The method of claim 2, wherein the subject is the patient.

4. The method of claim 2, wherein the subject is a healthy donor.

5. The method of claim 1, wherein the TCR comprises an chain comprising the amino acid sequence of SEQ ID NO: 18 and a chain comprising the amino acid sequence of SEQ ID NO: 24.

6. The method of claim 1, wherein the population of transformed cells are administered in the form of a pharmaceutical composition.

7. The method of claim 6, wherein the pharmaceutical composition comprises a chemotherapeutic agent selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and vincristine.

8. The method of claim 1, wherein the TCR comprises: a CDR1 chain comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 chain comprising the amino acid sequence of SEQ ID NO: 14, a CDR3 chain comprising the amino acid sequence of SEQ ID NO: 15, a CDR1 chain comprising the amino acid sequence of SEQ ID NO: 19, a CDR2 chain comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 chain comprising the amino acid sequence of SEQ ID NO: 21.

9. The method of claim 1, wherein the TCR comprises a CDR1 chain consisting of the amino acid sequence of SEQ ID NO: 13, a CDR2 chain consisting of the amino acid sequence of SEQ ID NO: 14, a CDR3 chain consisting of the amino acid sequence of SEQ ID NO: 15, a CDR1 chain consisting of the amino acid sequence of SEQ ID NO: 19, a CDR2 chain consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR3 chain consisting of the amino acid sequence of SEQ ID NO: 21.

10. The method of claim 1, wherein the TCR comprises a CDR1 chain consisting of the amino acid sequence of SEQ ID NO: 13, a CDR2 chain comprising the amino acid sequence of SEQ ID NO: 14, a CDR3 chain consisting of the amino acid sequence of SEQ ID NO: 15, a CDR1 chain consisting of the amino acid sequence of SEQ ID NO: 19, a CDR2 chain comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 chain consisting of the amino acid sequence of SEQ ID NO: 21.

11. The method of claim 1, wherein the cancer is gastrointestinal cancer.

12. The method of claim 1, wherein the TCR comprises a CDR1 chain comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 chain consisting of the amino acid sequence of SEQ ID NO: 14, a CDR3 chain consisting of the amino acid sequence of SEQ ID NO: 15, a CDR1 chain comprising the amino acid sequence of SEQ ID NO: 19, a CDR2 chain consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR3 chain consisting of the amino acid sequence of SEQ ID NO: 21.

13. The method of claim 1, wherein the TCR comprises a CDR1 chain consisting of the amino acid sequence of SEQ ID NO: 13, a CDR2 chain consisting of the amino acid sequence of SEQ ID NO: 14, a CDR3 chain comprising the amino acid sequence of SEQ ID NO: 15, a CDR1 chain consisting of the amino acid sequence of SEQ ID NO: 19, a CDR2 chain consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR3 chain comprising the amino acid sequence of SEQ ID NO: 21.

14. The method of claim 1, wherein the TCR comprises a CDR1 chain comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 chain comprising the amino acid sequence of SEQ ID NO: 14, a CDR3 chain consisting of the amino acid sequence of SEQ ID NO: 15, a CDR1 chain comprising the amino acid sequence of SEQ ID NO: 19, a CDR2 chain comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 chain consisting of the amino acid sequence of SEQ ID NO: 21.

15. The method of claim 1, wherein the TCR comprises a CDR1 chain comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 chain consisting of the amino acid sequence of SEQ ID NO: 14, a CDR3 chain comprising the amino acid sequence of SEQ ID NO: 15, a CDR1 chain comprising the amino acid sequence of SEQ ID NO: 19, a CDR2 chain consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR3 chain comprising the amino acid sequence of SEQ ID NO: 21.

16. The method of claim 1, wherein the TCR comprises a CDR1 chain consisting of the amino acid sequence of SEQ ID NO: 13, a CDR2 chain comprising the amino acid sequence of SEQ ID NO: 14, a CDR3 chain comprising the amino acid sequence of SEQ ID NO: 15, a CDR1 chain consisting of the amino acid sequence of SEQ ID NO: 19, a CDR2 chain comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 chain comprising the amino acid sequence of SEQ ID NO: 21.

17. The method of claim 1, wherein the cancer is breast cancer.

18. The method of claim 1, wherein the cancer is colon cancer.

19. The method of claim 1, wherein the cancer is lung cancer.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) 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.

(2) 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:

(3) FIG. 1: IFN release (left axis) and HLA-A*02/COL6A3-002 tetramer staining (right axis) of human primary CD8+ T-cells of one donor electroporated with alpha and beta chain RNA of TCR R4P1D10 (Table 1), respectively, after co-incubation with K562-A2 target cells (see Hirano N. et al; Blood. 2006 Feb. 15; 107(4):1528-36) loaded with COL6A3-002 peptide (SEQ ID NO:58), various COL6A3-002 alanine or glycine substitution variants at positions 1-9 of (SEQ ID NO:59-67), or NYESO1-001 control peptide (SEQ ID NO:68).

(4) FIG. 2: IFN release (left axis) and HLA-A*02/COL6A3-002 tetramer staining (right axis) of human primary CD8+ T-cells of one donor electroporated with alpha and beta chain RNA of TCR R4P1D10 (Table 1), respectively, after co-incubation with K562-A2 target cells loaded with COL6A3-002 peptide (SEQ ID NO:58), homologous but unrelated peptide AGRN-001, CLASP-001, COL6A1-001, COL6A2-001, COL6A3-006, COL6A3-008, COL6A3-014, VWA2-001, VWF-001 (SEQ ID NO:49-57) or NYESO1-001 control peptide (SEQ ID NO:68). Electroporated CD8+ T-cells only (E only) serve as control.

(5) FIG. 3: HLA-A*02/COL6A3-002 tetramer and HLA-A*02/NYESO1-001 tetramer staining, respectively, of J.RT3-T3.5 cells electroporated with alpha and beta chain RNA of TCR R4P1D10 or NYESO1-001-specific control TCR 1G4 (Table 1). Mock electroporated J.RT3-T3.5 cells serve as control.

(6) FIG. 4: HLA-A*02/COL6A3-002 tetramer and HLA-A*02/NYESO1-001 tetramer staining, respectively, of SUP-T1 cells electroporated with alpha and beta chain RNA of TCR R4P1D10 or NYESO1-001-specific control TCR 1G4 (Table 1). Mock electroporated SUP-T1 cells serve as control.

(7) FIG. 5: HLA-A*02/COL6A3-002 tetramer and HLA-A*02/NYESO1-001 tetramer staining, respectively, of human primary CD8+ T-cells of one donor electroporated with alpha and beta chain RNA of TCR R4P1D10 or NYESO1-001-specific control TCR 1G4 (Table 1). Mock electroporated CD8+ T-cells serve as control.

(8) FIG. 6: IFN release of human primary CD8+ T-cells of one donor electroporated with alpha and beta chain RNA of TCR R4P3F9 (Table 1), respectively, after co-incubation with K562-A2 target cells loaded with COL6A3-002 peptide (SEQ ID NO:58), various COL6A3-002 alanine or glycine substitution variants at positions 1-9 of (SEQ ID NO:59-67), or NYESO1-001 control peptide (SEQ ID NO:68).

(9) FIG. 7: IFN release of human primary CD8+ T-cells of one donor electroporated with alpha and beta chain RNA of TCR R4P3F9 (Table 1), respectively, after co-incubation with K562-A2 target cells loaded with COL6A3-002 peptide (SEQ ID NO:58), homologous but unrelated peptide AGRN-001, CLASP-001, COL6A1-001, COL6A2-001, COL6A3-006, COL6A3-008, COL6A3-014, VWA2-001, VWF-001 (SEQ ID NO:49-57) or NYESO1-001 control peptide (SEQ ID NO:68). Mock electroporated CD8+ T-cells (E only) serve as control.

(10) FIG. 8: HLA-A*02/COL6A3-002 tetramer and HLA-A*02/NYESO1-001 tetramer staining, respectively, of J.RT3-T3.5 cells electroporated with alpha and beta chain RNA of TCR R4P3F9 or NYESO1-001-specific control TCR 1G4 (Table 1). Mock electroporated J.RT3-T3.5 cells serve as control.

(11) FIG. 9: HLA-A*02/COL6A3-002 tetramer and HLA-A*02/NYES01-001 tetramer staining, respectively, of SUP-T1 cells electroporated with alpha and beta chain RNA of TCR R4P3F9 or NYESO1-001-specific control TCR 1G4 (Table 1). Mock electroporated SUP-T1 cells serve as control.

(12) FIG. 10: IFN release of human primary CD8+ T-cells of one donor electroporated with alpha and beta chain RNA of TCR R4P3H3 (Table 1), respectively, after co-incubation with K562-A2 target cells loaded with COL6A3-002 peptide (SEQ ID NO:58), various COL6A3-002 alanine or glycine substitution variants at positions 1-9 of (SEQ ID NO:59-67) or NYESO1-001 control peptide (SEQ ID NO:68).

(13) FIG. 11: IFN release of human primary CD8+ T-cells of one donor electroporated with alpha and beta chain RNA of TCR R4P3H3 (Table 1), respectively, after co-incubation with K562-A2 target cells loaded with COL6A3-002 peptide (SEQ ID NO:58), homologous but unrelated peptide AGRN-001, CLASP-001, COL6A1-001, COL6A2-001, COL6A3-006, COL6A3-008, COL6A3-014, VWA2-001, VWF-001 (SEQ ID NO:49-57) or NYESO1-001 control peptide (SEQ ID NO:68). Mock electroporated CD8+ T-cells (E only) serve as control.

(14) FIG. 12: HLA-A*02/COL6A3-002 tetramer and HLA-A*02/NYESO1-001 tetramer staining, respectively, of SUP-T1 cells electroporated with alpha and beta chain RNA of TCR R4P3H3 or NYESO1-001-specific control TCR 1G4 (Table 1). Mock electroporated SUP-T1 cells serve as control.

DETAILED DESCRIPTION OF THE INVENTION

(15) TABLE-US-00001 TABLE1 TCRsequencesoftheinvention SEQIDNO: TCR Chain Region Sequence 1 R4P1D10 alpha CDR1 DRGSQS 2 R4P1D10 alpha CDR2 IY 3 R4P1D10 alpha CDR3 CAVNFHDKIIF 4 R4P1D10 alpha variabledomain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVP EGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMF IYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDS ATYLCAVN 5 R4P1D10 alpha constantdomain NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVS QSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSD FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFE TDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 6 R4P1D10 alpha full-length MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVP EGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMF IYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDS ATYLCAVNFHDKIIFGKGTRLHILPNIQNPDPAVY QLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS IIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL SVIGFRILLLKVAGFNLLMTLRLWSS 7 R4P1D10 beta CDR1 SGDLS 8 R4P1D10 beta CDR2 YYNGEE 9 R4P1D10 beta CDR3 CASSVASAYGYTF 10 R4P1D10 beta variabledomain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATG QRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIHYYN GEERAKGNILERFSAQQFPDLHSELNLSSLELGDS ALYFCASSV 11 R4P1D10 beta constantdomain EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATG FFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALN DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSE NDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQ GVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DF 12 R4P1D10 beta full-length MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATG QRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIHYYN GEERAKGNILERFSAQQFPDLHSELNLSSLELGDS ALYFCASSVASAYGYTFGSGTRLTVVEDLNKVFPP EVAVFEPSEAEISHTQKATLVCLATGFFPDHVELS WWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSR LRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRA KPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILY EILLGKATLYAVLVSALVLMAMVKRKDF 13 R4P3F9 alpha CDR1 DRGSQS 14 R4P3F9 alpha CDR2 IY 15 R4P3F9 alpha CDR3 CAAYSGAGSYQLTF 16 R4P3F9 alpha variabledomain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVP EGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMF IYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDS ATYLCA 17 R4P3F9 alpha constantdomain NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVS QSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSD FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFE TDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 18 R4P3F9 alpha full-length MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVP EGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMF IYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDS ATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNPDP AVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDV YITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAF NNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNF QNLSVIGFRILLLKVAGFNLLMTLRLWSS 19 R4P3F9 beta CDR1 SGDLS 20 R4P3F9 beta CDR2 YYNGEE 21 R4P3F9 beta CDR3 CASSVESSYGYTF 22 R4P3F9 beta variabledomain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATG QRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIHYYN GEERAKGNILERFSAQQFPDLHSELNLSSLELGDS ALYFCASSV 23 R4P3F9 beta constantdomain EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATG FFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALN DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSE NDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQ GVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DF 24 R4P3F9 beta full-length MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATG QRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYN GEERAKGNILERFSAQQFPDLHSELNLSSLELGDS ALYFCASSVESSYGYTFGSGTRLTVVEDLNKVFPP EVAVFEPSEAEISHTQKATLVCLATGFFPDHVELS WWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSR LRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRA KPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILY EILLGKATLYAVLVSALVLMAMVKRKDF 25 R4P3H3 alpha CDR1 DRGSQS 26 R4P3H3 alpha CDR2 IY 27 R4P3H3 alpha CDR3 CAVKAGNQFYF 28 R4P3H3 alpha variabledomain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVP EGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMF IYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDS ATYLCAV 29 R4P3H3 alpha constantdomain NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVS QSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSD FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFE TDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 30 R4P3H3 alpha full-length MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVP EGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMF IYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDS ATYLCAVKAGNQFYFGTGTSLTVIPNIQNPDPAVY QLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS IIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL SVIGFRILLLKVAGFNLLMTLRLWSS 31 R4P3H3 beta CDR1 SGHVS 32 R4P3H3 beta CDR2 FQNEAQ 33 R4P3H3 beta CDR3 CASSLLTSGGDNEQFF 34 R4P3H3 beta variabledomain MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRG QDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQN EAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQED SAVYLCASSL 35 R4P3H3 beta constantdomain EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATG FYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALN DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSE NDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQ GVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 36 R4P3H3 beta full-length MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRG QDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQN EAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQED SAVYLCASSLLTSGGDNEQFFGPGTRLTVLEDLKN VFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDH VELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWT QDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSA TILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 37 1G4 alpha CDR1 DSAIYN 38 1G4 alpha CDR2 IQS 39 1G4 alpha CDR3 CAVRPTSGGSYIPTF 40 1G4 alpha variabledomain METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEG ENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQ SSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSA TYLCAVR 41 1G4 alpha constantdomain YIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVS QSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSD FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFE TDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 42 1G4 alpha full-length METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEG ENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQ SSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSA TYLCAVRPTSGGSYIPTFGRGTSLIVHPYIQNPDP AVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDV YITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAF NNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNF QNLSVIGFRILLLKVAGFNLLMTLRLWSS 43 1G4 beta CDR1 MNHEY 44 1G4 beta CDR2 SVGAGI 45 1G4 beta CDR3 CASSYVGNTGELFF 46 1G4 beta variabledomain MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTG QSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVG AGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQT SVYFCASSY 47 1G4 beta constantdomain EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATG FYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALN DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSE NDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQ GVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 48 1G4 beta full-length MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTG QSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVG AGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQT SVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFP PEVAVFEPSEAEISHTQKATLVCLATGFYPDHVEL SWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSS RLRVSATFWQNPRNHPRCQVQFYGLSENDEWTQDR AKPVTQIVSAEAWGRADCGFTSESYQQGVLSATIL YEILLGKATLYAVLVSALVLMAMVKRKDSRG

(16) TABLE-US-00002 TABLE2 Peptidesequencesoftheinvention PeptideCode Sequence SEQIDNO: AGRN-001 ALLDGRVQL 49 CLASP1-001 RLLDGAFKL 50 COL6A1-001 ILLDGSASV 51 COL6A2-001 FLLDGSERL 52 COL6A3-006 FLFDGSANLV 53 COL6A3-008 FLFDGSANL 54 COL6A3-014 FLLDGSEGV 55 VWA2-001 FLLDGSNSV 56 VWF-001 FLLDGSSRL 57 COL6A3-002 FLLDGSANV 58 A1 ALLDGSANV 59 A2 FALDGSANV 60 A3 FLADGSANV 61 A4 FLLAGSANV 62 AS FLLDASANV 63 A6 FLLDGAANV 64 A7 FLLDGSGNV 65 A8 FLLDGSAAV 66 A9 FLLDGSANA 67 NYESO1-001 SLLMWITQV 68

EXAMPLES

(17) In an aspect, allo-reactive settings are used to circumvent self-tolerance and yield T-cells with a higher avidity when compared to T-cells derived from autologous settings, i.e., patients. Examples of such settings include in vitro generation of allo-HLA reactive, peptide-specific T-cells (Sadovnikova et al. 1998; Savage et al. 2004; Wilde et al. 2012), and immunization of mice transgenic for human-MHC or human TCR (Stanislawski et al. 2001; Li et al. 2010), each of which are incorporated by reference in their entireties.

(18) To isolate high avidity T-cells from allo-reactive setting, PBMCs from HLA-A*02-negative healthy donors are used after obtaining informed consent. Recombinant biotinylated HLA-A*02 class I monomers and A2 fluorescent tetramers containing COL6A3-002 are obtained from MBLI (Woburn, Mass.). PBMCs are incubated with anti-CD2OSA diluted in phosphate buffered saline (PBS) for 1 hour at room temperature, washed, and incubated with the biotinylated HLA-A*02/COL6A3-002 monomers for 30 minutes at room temperature, washed, and plated at 310.sup.6 cells/well in 24-well plates in RPMI with 10% human AB serum. Interleukin 7 (IL-7; R&D Systems, Minneapolis, Minn.) was added on day 1 at 10 ng/mL and IL-2 (Chiron, Harefield, United Kingdom) was added at 10 U/mL on day 4. Over a 5-week period cells were restimulated weekly with fresh PBMCs, mixed with responder cells at a 1:1 ratio, and plated at 310.sup.6/well in 24-well plates.

(19) To obtain high avidity T-cells, approximately 10.sup.6 PBMCs with HLA-A*02/COL6A3-002 tetramer-phycoerythrin (PE) (obtained from MBLI) were incubated for 30 minutes at 37 C., followed by anti-CD8-fluorescein isothiocyanate (FITC)/allophycocyanin (APC) for 20 minutes at 4 C., followed by fluorescence activated cell sorting. Sorted tetramer-positive cells were expanded in 24-well plates using, per well, 210.sup.5 sorted cells, 210.sup.6 irradiated A2-negative PBMCs as feeders, 210.sup.4 CD3/CD28 beads/mL (Dynal, Oslo Norway), and IL-2 (1000 U/mL). The high avidity T-cells, thus obtained, were then used to identify and isolate TCRs using techniques known in the art, such as single cell 5 RACE (Rapid Amplification of cDNA Ends). Non-redundant TCR DNAs were then analyzed for amino acid/DNA sequences determination and cloning into expression vectors.

(20) Three COL6A3-002-specific TCRs (R4P1D10, R4P3F9 and R4P3H3, see Table 2), each encoding tumor specific TCR-alpha and TCR-beta chains, were isolated and amplified from T-cells of healthy donors. Cells from healthy donors were in vitro stimulated according to a method previously described (Walter et al., 2003 J Immunol., November 15; 171(10):4974-8). COL6A3 peptide presentation was performed as described previously (Hirano N. et al; Blood. 2006 Feb. 15; 107(4):1528-36). Target-specific cells were single-cell sorted using HLA-A*02 multimers and then used for subsequent TCR isolation. TCR sequences were isolated via 5 RACE by standard methods as described by e.g. Molecular Cloning a laboratory manual fourth edition by Green and Sambrook. The alpha and beta variable regions of TCRs R4P1D10, R4P3F9 and R4P3H3 were sequenced and cloned for further functional characterization. R4P1D10 and R4P3H3 are derived from HLA-A*02 positive donors and R4P3F9 is derived from a HLA-A*02 negative donor (allo-reactive setting).

(21) TABLE-US-00003 TABLE 3 SPR affinity of COL6A3-002 and NYESO1-001 TCRs Equilibrium dissociation con- Equilibrium dissociation con- stant (K.sub.D) for HLA-A02/ stant (K.sub.D) for HLA-A02/ TCR COL6A3-002 complex in M NYESO1-001 complex in M R4P1D10 16 no binding R4P3F9 62 no binding R4P3H3 102 no binding 1G4 no binding 7

Example 1: T-Cell Receptor R4P1D10

(22) The TCR R4P1D10 alpha and beta chains were cloned as described previously, for example, as described in U.S. Pat. No. 8,519,100, the content of which is hereby incorporated by reference in its entirety for said methods. TCR R4P1D10 is restricted towards HLA-A2-presented COL6A3-002 (see table 3 above).

(23) TABLE-US-00004 TABLE4 FeaturesofR4P1D10alphachain: Start Stop Description Sequence 1 21 Lsegment MKSLRVLLVILWLQLSWVWSQ(SEQIDNO:69) 1 113 Vchain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTY (TRAV12-2) SDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQY VSLLIRDSQPSDSATYLCAVN 48 53 CDR1 DRGSQS 71 72 CDR2 IY 110 120 CDR3 CAVNFHDKIIF 116 130 Jsegment DKIIFGKGTRLHILP (TRAJ30) 131 272 Constantregion NIQNDPAVYQLRDSKSSKDSVCLFTDFDSQTNVSQSKDSDVYITDK (TRAC) TVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPES SCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLW SS

(24) TABLE-US-00005 TABLE5 FeaturesofR4P1D10betachain: Start Stop Description Sequence 1 19 Lsegment MGFRLLCCVAFCLLGAGPV(SEQIDNO:70) (TRBV9) 1 114 Vchain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCS (TRBV9) PRSGDLSVYWYQQSLDQGLQFLIHYYNGEERAKGNILERFSAQ QFPDLHSELNLSSLELGDSALYFCASSV 46 50 CDR1 SGDLS 68 73 CDR2 YYNGEE 110 122 CDR3 CASSVASAYGYTF 118 131 Jchain YGYTFGSGTRLTVV 132 308 constantregion EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVEL (TRBC1) SWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATF WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRA DCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV KRKDF

(25) R4P1D10 specifically recognizes COL6A3-002 as human primary CD8+ T-cells re-expressing this TCR release IFN upon co-incubation with HLA-A*02+ target cells and bind HLA-A*02 tetramers, respectively, loaded either with COL6A3-002 peptide or alanine and glycine substitution variants of COL6A3-002 (FIG. 1) or different peptides showing high degree of sequence similarity to COL6A3-002 (FIG. 2). NYESO1-001 peptide is used as negative control.

(26) Re-expression of R4P1D10 leads to selective binding of HLA-A*02/COL6A3-002 tetramers but not HLA-A*02/NYESO1-001 tetramers in J.RT3-T3.5 Jurkat cells (FIG. 3), SUP-T1 cells (FIG. 4) and human primary CD8+ T-cells (FIG. 5). For each cell type, re-expression of the NYESO1-001-specific TCR 1G4 and mock expression are used as control.

(27) SPR (Surface Plasmon Resonance) binding analysis for R4P1D10, expressed as soluble TCR according to a previously described method (Willcox B E et al., 1999 Protein Sci., November; 8(11):2418-23), and HLA-A*02/COL6A3-002 complex reveals an affinity of K.sub.D=16 M (Table 3). SPR binding data for 1G4 TCR and HLA-A*02/NYESO1-001 are used as control.

Example 2: T-Cell Receptor R4P3F9

(28) The TCR R4P3F9 alpha and beta chains were cloned as described before, for example, as described in U.S. Pat. No. 8,519,100, which is hereby incorporated by reference in its entirety for said methods. TCR R4P3F9 is restricted towards HLA-A2-presented COL6A3-002 (see table 3 above).

(29) TABLE-US-00006 TABLE6 FeaturesofR4P3F9alphachain Start Stop Description Sequence 1 21 Lsegment MKSLRVLLVILWLQLSWVWSQ(SEQIDNO:71) (TRAV12-2) 1 111 Vchain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTY (TRAV12-2) SDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQY VSLLIRDSQPSDSATYLCA 48 53 CDR1 DRGSQS 71 72 CDR2 IY 110 123 CDR3 CAAYSGAGSYQLTF 113 133 Jsegment YSGAGSYQLTFGKGTKLSVIP 134 274 Constantregion NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITD (TRAC) KTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPE SSCDVKLVEKSFETDTNLNFQNLSVIGFRILLKVAGFNLLMTLRLW SS

(30) TABLE-US-00007 TABLE7 FeaturesofR4P3F9betachain Start Stop Description Sequence 1 19 Lsegment MGFRLLCCVAFCLLGAGPV(SEQIDNO:72) (TRBV9) 1 114 Vchain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGD (TRBV9) LSVYWYQQSLDQGLQFLIHYYNGEERAKGNILERFSAQQFPDLHSELN LSSLELGDSALYFCASSV 46 50 CFR2 SGDLS 68 73 CDR2 YYNGEE 110 122 CDR3 CASSVESSYGYTF 118 131 Jchain YGYTFGSGTRLTVV (TRBJ1-2) 132 308 constantregion EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVH (TRBC1) SGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLY AVLVSALVLMAMVKRKDF

(31) R4P3F9 specifically recognizes COL6A3-002 as human primary CD8+ T-cells re-expressing this TCR release IFN upon co-incubation with HLA-A*02+ target cells, respectively, loaded either with COL6A3-002 peptide or alanine and glycine substitution variants of COL6A3-002 (FIG. 6) or different peptides showing high degree of sequence similarity to COL6A3-002 (FIG. 7). NYESO1-001 peptide is used as negative control.

(32) Re-expression of R4P3F9 leads to selective binding of HLA-A*02/COL6A3-002 tetramers but not HLA-A*02/NYESO1-001 tetramers in J.RT3-T3.5 Jurkat cells (FIG. 8) and SUP-T1 cells (FIG. 9). For each cell type, re-expression of the NYESO1-001-specific TCR 1G4 and mock expression are used as control.

(33) SPR binding analysis for R4P3F9, expressed as soluble TCR according to a previously described method (Willcox B E et al., 1999 Protein Sci., November; 8(11):2418-23), and HLA-A*02/COL6A3-002 complex reveals an affinity of K.sub.D=62 M (Table 3). SPR binding data for 1G4 TCR and HLA-A*02/NYESO1-001 are used as control.

Example 3: T-Cell Receptor R4P3H3

(34) The TCR R4P3H3 alpha and beta chains were cloned as described before, for example, as described in U.S. Pat. No. 8,519,100, which is hereby incorporated by reference in its entirety. TCR R4P3H3 is restricted towards HLA-A2-presented COL6A3-002 (see table 3 above).

(35) TABLE-US-00008 TABLE8 FeaturesofR4P3H3alphachain Start Stop Description Sequence 1 21 Lsegment MKSLRVLLVILWLQLSWVWSQ(SEQIDNO:73) (TRAV12-2) 1 112 Vchain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNC (TRAV12-2) TYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNK ASQYVSLLIRDSQPSDSATYLCAV 48 53 CDR1 DRGSQS 71 72 CDR2 IY 110 120 CDR3 CAVKAGNQFYF 115 130 Jsegment GNQFYFGTGTSLTVIP (TRAJ49) 131 271 Constantregion NIQNPDPAVYQLRDSKSSKDSVCLFTDFDSQTNVSQSKDSDVYI (TRAC) TKDTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFF PSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNL LMTLRLWSS

(36) TABLE-US-00009 TABLE9 FeaturesofR4P3H3betachain Start Stop Description Sequence 1 19 Lsegment MGTRLLCWVVLGFLGTDHT(SEQIDNO:74) (TRBV7-8) 1 115 Vchain MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALR (TRBV7-8) CDPISGHVSLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSD RFFAERPEGSVSTLKIQRTQQEDSAVYLCASSL 46 50 CDR1 SGHVS 68 73 CDR2 FQNEAQ 111 126 CDR3 CASSLLTSGGDNEQFF 122 135 Jchain NEQFFGPGTRLTVL (TRBJ2-1) 136 314 constantregion EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHV (TRBC2) ELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRV SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA EAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVS ALVLMAMVKRKDSRG

(37) R4P3H3 specifically recognizes COL6A3-002 as human primary CD8+ T-cells re-expressing this TCR release IFN upon co-incubation with HLA-A*02+ target cells, respectively, loaded either with COL6A3-002 peptide or alanine and glycine substitution variants of COL6A3-002 (FIG. 10) or different peptides showing high degree of sequence similarity to COL6A3-002 (FIG. 11). NYESO1-001 peptide is used as negative control.

(38) Re-expression of R4P3H3 leads to selective binding of HLA-A*02/COL6A3-002 tetramers but not HLA-A*02/NYESO1-001 tetramers in SUP-T1 cells (FIG. 12). Re-expression of the NYESO1-001-specific TCR 1G4 and mock expression are used as control.

(39) SPR binding analysis for R4P3H3, expressed as soluble TCR according to a previously described method (Willcox B E et al., 1999 Protein Sci., November; 8(11):2418-23), and HLA-A*02/COL6A3-002 complex reveals an affinity of K.sub.D=102 M (Table 3). SPR binding data for 1G4 TCR and HLA-A*02/NYESO1-001 are used as control.