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TCR CONSTRUCTS SPECIFIC FOR EBV-DERIVED ANTIGENS

20220267407 · 2022-08-25

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to the filed of immunotherapy, in particular, of Epstein-Barr virus-associated diseases (EBV, also designated Human gammaherpesvirus 4), e.g., cancer or post-transplant lymphoproliferative disease, in particular, to adoptive T cell therapy or T cell receptor (TCR) gene therapy. The invention provides a combination of nucleic acids encoding at least two TCR constructs, or the respective proteins or host cells, wherein each TCR construct is capable of specifically binding to its respective epitope in the context of the respective MHC I, and wherein the epitopes are peptides from different antigens expressed by the same infective agent or cancer, e.g., EBV antigens. The invention also provides specific nucleic acids encoding a TCR alpha chain construct (TRA) and/or a TCR beta chain construct (TRB) of a TCR construct specific for an epitope in complex with a human MHC I, wherein the epitope is an epitope of an Epstein-Barr-virus protein, wherein the TCR constructs are specific for epitopes from LMP2A, LMP1 or EBNA3C. Proteins encoded by said nucleic acids, corresponding host cells and pharmaceutical compositions and kits are also objects of the invention.

    Claims

    1. A nucleic acid encoding a TCR alpha chain construct (TRA) and/or a TCR beta chain construct (TRB) of a TCR construct specific for an epitope in complex with a human MHC I, wherein the epitope is an epitope of an Epstein-Barr-virus (EBV) protein, a) wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; b) wherein the epitope has the sequence of SEQ ID NO: 2, the MHC I is HLA-B*57:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 23 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 28; c) wherein the epitope has the sequence of SEQ ID NO: 3, the MHC I is HLA-C*15:02, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 33 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 38; d) wherein the epitope has the sequence of SEQ ID NO: 4, the MHC I is HLA-C*06:02, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 43 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 48; e) wherein the epitope has the sequence of SEQ ID NO: 5, the MHC I is HLA-B*44:02, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 53 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 58; f) wherein the epitope has the sequence of SEQ ID NO: 5, the MHC I is HLA-B*44:02, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 63 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 68; g) wherein the epitope has the sequence of SEQ ID NO: 6, the MHC I is HLA-B*07:02, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 73 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 78; and/or h) wherein the epitope has the sequence of SEQ ID NO: 6, the MHC I is HLA-B*07:02, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 83 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 88.

    2. The nucleic acid of claim 1, wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20.

    3. The nucleic acid of claim 1, wherein the epitope has the sequence of SEQ ID NO: 2, the MHC I is HLA-B*57:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 23 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 28; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 21, a CDR2 having at least 80% sequence identity to SEQ ID NO: 22 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 23, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 26, a CDR2 having at least 80% sequence identity to SEQ ID NO: 27 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 28; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 25 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 30.

    4. The nucleic acid of claim 1, wherein the epitope has the sequence of SEQ ID NO: 3, the MHC I is HLA-C*15:02, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 33 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 38; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 31, a CDR2 having at least 80% sequence identity to SEQ ID NO: 32 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 33, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 36, a CDR2 having at least 80% sequence identity to SEQ ID NO: 37 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 38; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 35 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 40.

    5. The nucleic acid of claim 1, wherein the epitope has the sequence of SEQ ID NO: 4, the MHC I is HLA-C*06:02, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 43 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 48; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 41, a CDR2 having at least 80% sequence identity to SEQ ID NO: 42 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 43, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 46, a CDR2 having at least 80% sequence identity to SEQ ID NO: 47 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 48; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 45 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 50.

    6. The nucleic acid of claim 1, wherein the epitope has the sequence of SEQ ID NO: 5, the MHC I is HLA-B*44:02, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 53 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 58; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 51, a CDR2 having at least 80% sequence identity to SEQ ID NO: 52 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 53, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 56, a CDR2 having at least 80% sequence identity to SEQ ID NO: 57 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 58; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 55 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 60.

    7. The nucleic acid of claim 1, wherein the epitope has the sequence of SEQ ID NO: 5, the MHC I is HLA-B*44:02, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 63 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 68; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 61, a CDR2 having at least 80% sequence identity to SEQ ID NO: 62 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 63, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 66, a CDR2 having at least 80% sequence identity to SEQ ID NO: 67 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 68; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 65 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 70.

    8. The nucleic acid of claim 1, wherein the epitope has the sequence of SEQ ID NO: 6, the MHC I is HLA-B*07:02, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 73 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 78; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 71, a CDR2 having at least 80% sequence identity to SEQ ID NO: 72 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 73, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 76, a CDR2 having at least 80% sequence identity to SEQ ID NO: 77 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 78; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 75 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 80.

    9. The nucleic acid of claim 1, wherein the epitope has the sequence of SEQ ID NO: 6, the MHC I is HLA-B*07:02, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 83 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 88; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 81, a CDR2 having at least 80% sequence identity to SEQ ID NO: 82 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 83, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 86, a CDR2 having at least 80% sequence identity to SEQ ID NO: 87 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 88; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 85 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 90.

    10. The nucleic acid of claim 1, wherein the sequence identity to the recited CDR1 and CDR2 and CDR3 regions is 100%.

    11. The nucleic acid of claim 1, encoding at least one TCR alpha and beta chain construct of the TCR construct, wherein the nucleic acid is selected from the group comprising a viral vector, a transposon, a vector suitable for CRISPR/CAS based recombination or a plasmid suitable for in vitro RNA transcription wherein, preferably, the TCR alpha chain construct and/or the TCR beta chain construct further comprise a constant region selected from the group comprising a human constant region, a murine constant region or a chimeric constant region.

    12. A protein encoded by the nucleic acid of claim 1.

    13. A host cell comprising the nucleic acid claim 1, wherein the host cell preferably is a human CD8+ T cell.

    14. A pharmaceutical composition comprising a) a nucleic acid of claim 1 encoding a TCR construct capable of specifically binding to said epitope in the context of said MHC I wherein the TCR construct preferably is specific for an epitope in complex with a human MHC I wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEO ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20.

    15. A pharmaceutical composition or a kit comprising at least two pharmaceutical compositions comprising a) at least two nucleic acids, each encoding a TCR construct capable of specifically binding to its respective epitope in the context of the respective MHC I; or b) at least two proteins, each comprising a TCR construct capable of specifically binding to its respective epitope in the context of the respective MHC I; or c) at least two host cells, each expressing a TCR construct capable of specifically binding to its respective epitope in the context of the respective MHC I, wherein the epitopes are peptides from different antigens expressed by the same cancer or infectious agent, wherein, optionally, the cancer is associated with EBV and the different EBV antigens are selected from the group comprising LMP2A, LMP1, EBNA1 and EBNA3C, wherein, preferably, the kit of the pharmaceutical composition is for use in treatment of said cancer or infectious agent.

    16. A pharmaceutical composition of claim 14, comprising at least two nucleic acids of, each encoding a TCR construct capable of specifically binding to its respective epitope in the context of the respective MHC I; wherein one of said TCR constructs preferably is specific for an epitope in complex with a human MHC I wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEO ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20.

    17. The pharmaceutical composition of claim 14 for use in the treatment of a patient expressing said MHC I and having an EBV-associated disease selected from the group comprising Hodgkin's and non-Hodgkin lymphoma, Burkitt lymphoma, hemophagocytic lympohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, lung cancer, gastric cancer hairy leukoplakia, post transplant lymphoproliferative disorder and central nervous system lymphoma. wherein the treatment preferably is an immunotherapy selected from the group of adoptive T cell therapy and TCR gene therapy.

    18. A pharmaceutical composition comprising a peptide comprising an epitope capable of being presented by a human MHC I, wherein the epitope is selected from the group comprising epitopes a) having at least 88% sequence identity to SEQ ID NO: 3, wherein the epitope is capable of being presented on HLA-C*15:02, and wherein the peptide comprises at most 11 consecutive amino acids identical to the sequence of amino acids occurring in LMP1 of SEQ ID NO: 120; b) having at least 88% sequence identity to SEQ ID NO: 4, wherein the epitope is capable of being presented on HLA-C*06:02, and wherein the peptide comprises at most 11 consecutive amino acids identical to the sequence of amino acids occurring in EBNA3C of SEQ ID NO: 121; or c) having at least 90% sequence identity to SEQ ID NO: 5, wherein the epitope is capable of being presented on HLA-B*44:02, and wherein the peptide comprises at most 11 consecutive amino acids identical to the sequence of amino acids occurring in EBNA3C of SEQ ID NO: 121, or comprising a nucleic acid encoding said peptide, wherein said pharmaceutical composition preferably is for use in vaccination against an EBV-associated disease selected from the group comprising Hodgkin's and non-Hodgkin lymphoma, Burkitt lymphoma, hemophagocytic lympohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, lung cancer, gastric cancer hairy leukoplakia, post transplant lymphoproliferative disorder and central nervous system lymphoma.

    19. A host cell comprising the protein of claim 12, wherein the host cell preferably is a human CD8+ T cell.

    20. A pharmaceutical composition comprising a protein of claim 12 comprising a TCR construct capable of specifically binding to said epitope in the context of said MHC I; wherein the TCR construct preferably is-specific for an epitope in complex with a human MHC I wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20.

    21. A pharmaceutical composition comprising a host cell of claim 13 expressing a TCR construct capable of specifically binding to said epitope in the context of said MHC I wherein the TCR construct preferably is-specific for an epitope in complex with a human MHC I wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20.

    22. A pharmaceutical composition comprising a host cell of claim 19 expressing a TCR construct capable of specifically binding to said epitope in the context of said MHC I wherein the TCR construct preferably is-specific for an epitope in complex with a human MHC I wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20.

    23. A pharmaceutical composition of claim 20 comprising at least two proteins, each comprising a TCR construct capable of specifically binding to its respective epitope in the context of the respective MHC I; wherein one of said TCR constructs preferably is-specific for an epitope in complex with a human MHC I wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20.

    24. A pharmaceutical composition of claim 21 comprising at least two host cells, each expressing a TCR construct capable of specifically binding to its respective epitope in the context of the respective MHC I; wherein one of said TCR constructs preferably is-specific for an epitope in complex with a human MHC I wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20.

    25. A pharmaceutical composition of claim 22 comprising at least two host cells, each expressing a TCR construct capable of specifically binding to its respective epitope in the context of the respective MHC I; wherein one of said TCR constructs preferably is-specific for an epitope in complex with a human MHC I wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20.

    26. A pharmaceutical composition or kit comprising at least two nucleic acids of claim 1, each encoding a TCR construct capable of specifically binding to its respective epitope in the context of the respective MHC I; wherein one of said TCR constructs preferably is-specific for an epitope in complex with a human MHC I wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20

    27. A pharmaceutical composition or kit comprising at least two proteins of claim 12, each comprising a TCR construct capable of specifically binding to its respective epitope in the context of the respective MHC I; wherein one of said TCR constructs preferably is-specific for an epitope in complex with a human MHC I wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20

    28. A pharmaceutical composition or kit comprising at least two host cells of claim 13, each expressing a TCR construct capable of specifically binding to its respective epitope in the context of the respective MHC I, wherein one of said TCR constructs preferably is-specific for an epitope in complex with a human MHC I wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20.

    29. A pharmaceutical composition or kit comprising at least two host cells of claim 19, each expressing a TCR construct capable of specifically binding to its respective epitope in the context of the respective MHC I, wherein one of said TCR constructs preferably is-specific for an epitope in complex with a human MHC I wherein the epitope has the sequence of SEQ ID NO: 1, the MHC I is HLA-A*02:01, and the TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 13 and the TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO: 18; wherein, optionally, the TRA comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 11, a CDR2 having at least 80% sequence identity to SEQ ID NO: 12 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 13, and the TRB comprises a CDR1 having at least 80% sequence identity to SEQ ID NO: 16, a CDR2 having at least 80% sequence identity to SEQ ID NO: 17 and a CDR3 having at least 90%, preferably, 100% sequence identity to SEQ ID NO: 18; wherein, most preferably, the TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15 and the TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20.

    30. The pharmaceutical composition or kit of claim 15 for use in the treatment of a patient expressing said MHC I and having an EBV-associated disease selected from the group comprising Hodgkin's and non-Hodgkin lymphoma, Burkitt lymphoma, hemophagocytic lympohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, lung cancer, gastric cancer hairy leukoplakia, post transplant lymphoproliferative disorder and central nervous system lymphoma. wherein the treatment preferably is an immunotherapy selected from the group of adoptive T cell therapy and TCR gene therapy.

    Description

    LEGENDS

    [0149] FIG. 1. Detection of T cell responses using a MHC class I K562 cell library. T cells expanded on EBV antigen-expressing dendritic cells were co-cultured with K562 cells of the MHC cell library. Screening for immunogenic EBV antigen-HLA combinations was performed by (A) analysing the expression of CD137 and (B) determining the amount of secreted IFN-γ by ELISA. (C) FACS sorting of CD137-positive T cells (11%) responding to K562-HLA-B*57:01-positive cells, which then were used to identify dominant TCRα- and TCRβ chains. (MIN—no antigen stimulation, MAX—unspecific antigen stimulation, us—unstained T cells). This approach was applied for the identification and isolation of all further TCRs described herein.

    [0150] FIG. 2. TCR gene analysis. (A) Next generation sequencing-based TCR repertoire analysis of FACS-sorted T cells responding to EBV antigen-positive (LMP1/LMP2A/EBNA1) K562-HLA-B*57:01 cells. Indicated are parts of total reads assigned to each sequence cluster, which has a sequence representative indicated on the y-axis. TCRα- and TCRβ chains with a frequency of >10% were utilized to construct single chain TCR-retroviruses to identify the functional TCR. (B) For one TCR (designated as TCR50) the different V segments of the most dominant TCRα-(TRAV) and TCRβ (TRBV) chains, their frequency, and the sequences of the CDR-3 region (IMGT nomenclature) are shown (AMSDLYAGNNRKLI: SEQ ID NO: 122, ALTFLRDDKII: SEQ ID NO: 123, VVMATGFQKLV: SEQ ID NO: 24, ASSQDARVSGANVLT: SEQ ID NO: 124, ASSVTSGSDEQF: SEQ ID NO: 125, ASSFSLGHSYEQY: SEQ ID NO: 126). This approach was applied to all further TCRs described herein.

    [0151] FIG. 3. Identification of the functional TCRαβ chain combination. TCRα- and TCRβ chains with a frequency of >10% were used to construct single chain TCR-retroviruses for the identification of the functional TCR. For TCR50 TRAV8-2*01 and TRBV9*01 formed a functional TCR as TCR-engineered T cells specifically recognize one antigen (LMP1) in combination with one HLA (B*57:01). Other TCRα- and TCRβ chain combinations resulted in unspecific antigen recognition. The combinatorial approach to identify functional TCRα- and TCRβ chain combinations, as exemplarily shown here for TCR50, was applied to all further TCRs described herein. (MIN—no antigen stimulation, MAX—unspecific stimulation).

    [0152] FIG. 4. Epitope mapping to identify the antigenic peptide recognized by TCR50. (A) Truncated versions (LMP1/2, LMP1/1) of the full-length LMP1 antigen were generated and expressed in K562-HLA-B*57:01 cells. (B) The antigen region harboring the immunogenic epitope was identified by measuring the amount of secreted IFN-γ by ELISA in the supernatant and is located between nucleotide (nt) 316 and 624. (MIN—no antigen stimulation, MAX—unspecific antigen stimulation, UT—untransduced T cells).

    [0153] FIG. 5. Identification of the immunogenic epitope of LMP1. The protein region identified as epitope-positive sequence (LMP1/2 nt 316-624) was used to select for candidate peptides applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*57:01. (A) 13 peptides, according to their peptide-MHC I binding affinity (bind level) classified as strong binder (SB) and weak binder (WB), respectively, were utilized for the identification of the epitope. (B) K562-HLA-B*57:01 cells were loaded with the selected peptides, co-cultured with TCR50-engineered T cells of two donors, and IFN-γ secretion was determined by ELISA. Four epitopes (highlighted in A) with the following amino acid sequences were recognized by TCR50-engineered T cells: IALYLQQNWW (SEQ ID NO: 108), IALYLQQNW (SEQ ID NO: 2), IIALYLQQNW (SEQ ID NO: 110), ALYLQQNWW (SEQ ID NO: 116). SEQ ID NOs of the analyzed peptides are shown in Table 1 below. (MIN—no antigen stimulation, MAX—unspecific stimulation, SB—strong binder, WB—weak binder, UT—untransduced T cells).

    [0154] FIG. 6. Peptide titration of TCR50-engineered T cells. Untransduced (UT) and TCR50-transduced T cells (TCR50) of two donors were co-cultured with K562-HLA-B*57:01 cells loaded with titrated amounts of the indicated peptides (SEQ ID NO: 2, 108, 110, 116) and the amount of secreted IFN-γ in the supernatant was determined by ELISA. Peptide IALYLQQNW (9mer, SEQ ID NO: 2), was recognized at lowest concentrations and can therefore be considered as cognate EBV LMP1 epitope of TCR50. Interestingly and worth to mentioning, this epitope recognized by TCR50 was not ranked highest in the NetMHCpan4.0 prediction tool indicating that the prediction tool is not precise in forecasting relevant immunodominant epitopes.

    [0155] FIG. 7. TCR50-engineered T cells recognize LMP1-positive cells. Functional analysis of LMP1-specific TCR50-engineered T cells of two donors using K562-HLA-B*57:01 antigen-loaded cells, an EBV-associated cancer cell line (L591-B*57:01), and lymphoblastoid cell lines (WIN, DEM), respectively. T cell reactivity was determined by measuring the amount of secreted IFN-γ by ELISA at an effector to target (E:T) cell ratio of 1:1. (MIN—no antigen stimulation, MAX—unspecific stimulation, UT—untransduced T cells).

    [0156] FIG. 8. Functional analysis of the EBNA3C-reactive TCR01. (A) EBNA3C-specific TCR01-engineered T cells were co-cultured with K562-HLA-B*07:02 antigen-pulsed cells and EBV-positive cell lines. T cell functionality was determined by measuring the amount of secreted IFN-γ by ELISA at at an effector to target (E:T) cell ratio of 1:1. (B) Truncated versions (EBNA3C/3, EBNA3C/2, EBNA3C/1) of the full-length EBNA3C antigen were generated and expressed in K562-HLA-B*07:02 cells. (C) The antigen region harboring the immunogenic epitope was identified by measuring the amount of secreted IFN-γ by ELISA in the supernatant and is located between nucleotide (nt) 2071 and 2979. (D) The protein region identified as epitope-positive sequence (EBNA3C nt 2071-2979) was used to select for candidate peptides applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*07:02. (E) 27 peptides according to their peptide-MHC I binding affinity (bind level) classified as strong binder (SB) and weak binder (WB), respectively, were utilized for the identification of the epitope (QPRAPIRPI (SEQ ID NO: 127), RPIPTRFPPPPM (SEQ ID NO: 128), RPRVEESSHGPA (SEQ ID NO: 129), SPQPRAPI (SEQ ID NO: 130), SPQPRAPIRPI (SEQ ID NO: 131), SPQPRAPIRPIP (SEQ ID NO: 132), PQPRAPIRPI (SEQ ID NO: 133), QPRAPIRPIP (SEQ ID NO: 134), PRAPIRPI (SEQ ID NO: 135), APIRPIPTRF (SEQ ID NO: 136), FPPPPMPL (SEQ ID NO: 137), HGPARCSQAT (SEQ ID NO: 138), RPIPTRFPP (SEQ ID NO: 139), RPIPTRFP (SEQ ID NO: 140), IPTRFPPPPMP (SEQ ID NO: 141), PIPTRFPPPPM (SEQ ID NO: 142), IPTRFPPPPMPL (SEQ ID NO: 143), GPARCSQATA (SEQ ID NO: 144), FPPPPMPLQDSM (SEQ ID NO: 145), PPMPLQDSM (SEQ ID NO: 146), RPIPTRFPPP (SEQ ID NO: 147), MPLQDSMAVG (SEQ ID NO: 148), PIPTRFPPPPMP (SEQ ID NO: 149), PMPLQDSMAV (SEQ ID NO: 150), PMPLQDSM (SEQ ID NO: 151), QPRAPIRPIPT (SEQ ID NO: 152), QPRAPIRP (SEQ ID NO: 153)). K562-HLA-B*07:02 cells were loaded with the selected peptides, co-cultured with TCR01-engineered T cells, and IFN-γ secretion was determined by ELISA. Epitopes (highlighted in D) were recognized by TCR01-engineered T cells. (F) K562-HLA-B*07:02 cells were loaded with titrated amounts of the indicated peptides or no peptide as control and peptide sensitivity of TCR01-engineered T cells was determined by measuring the amount of secreted IFN-γ by ELISA at an E:T cell ratio of 1:1. (MIN—no antigen stimulation, MAX—unspecific stimulation, SB—strong binder, WB—weak binder, UT—untransduced T cells).

    [0157] FIG. 9. Functional analysis of the EBNA3C-reactive TCR25. (A) EBNA3C-specific TCR25-engineered T cells were co-cultured with K562-HLA-B*44:02 antigen-pulsed cells or an EBV-associated cancer cell line. T cell functionality was determined by measuring the amount of secreted IFN-γ by ELISA at an effector to target (E:T) cell ratio of 1:1. (B) Truncated versions (EBNA3C/3, EBNA3C/2, EBNA3C/1) of the full-length EBNA3C antigen were generated and expressed in K562-HLA-B*44:02 cells. (C) The antigen region harboring the immunogenic epitope was identified by measuring the amount of secreted IFN-γ by ELISA in the supernatant and is located between nucleotide (nt) 1 and 567. (D) The protein region identified as epitope-positive sequence (EBNA3C nt 1-567) was used to select for candidate peptides applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*44:02. (E) Four peptides according to their peptide-MHC binding affinity (bind level) classified as strong binder (SB) and weak binder (WB), respectively, were utilized for the identification of the epitope (AEGGVGWRHW (SEQ ID NO: 5), SERLVPEESY (SEQ ID NO: 155), WLLTSPSQSW (SEQ ID NO: 156), LLTSPSQSW (SEQ ID NO: 157)). K562-HLA-B*44:02 cells were loaded with the selected peptides, co-cultured with TCR25-engineered T cells, and IFN-γ secretion was determined by ELISA. One epitope (highlighted in D) with the amino acid sequence AEGGVGWRHW was recognized by TCR25-engineered T cells and can therefore be considered as cognate EBV EBNA3C epitope of TCR25. (F) K562-HLA-B*44:02 cells were loaded with titrated amounts of the indicated peptide or no peptide as control. Peptide sensitivity of TCR25-engineered T cells was determined by measuring the amount of secreted IFN-γ by ELISA at an E:T cell ratio of 1:1. (MIN—no antigen stimulation, MAX—unspecific stimulation, SB—strong binder, WB—weak binder, UT—untransduced T cells).

    [0158] FIG. 10. Functional analysis of the EBNA3C-reactive TCR27. (A) EBNA3C-specific TCR27-engineered T cells were co-cultured with K562-HLA-B*07:02 antigen-pulsed cells and EBV-positive cell lines. T cell functionality was determined by measuring the amount of secreted IFN-γ by ELISA at an effector to target (E:T) cell ratio of 1:1. (B) Truncated versions (EBNA3C/3, EBNA3C/2, EBNA3C/1) of the full-length EBNA3C antigen were generated and expressed in K562-HLA-B*07:02 cells. (C) The antigen region harboring the immunogenic epitope was identified by measuring the amount of secreted IFN-γ (ELISA) in the supernatant and is located between nucleotide (nt) 2071 and 2979. (D) The protein region identified as epitope-positive sequence (EBNA3C nt 2071-2979) was used to select for candidate peptides applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*07:02. (E) 27 peptides according to their peptide-MHC binding affinity (bind level) classified as strong binder (SB) and weak binder (WB), respectively, were utilized for the identification of the epitope. K562-HLA-B*07:02 cells were loaded with the selected peptides, co-cultured with TCR27-engineered T cells, and IFN-γ secretion was determined by ELISA. Epitopes (highlighted in D) were recognized by TCR27-engineered T cells. (F) K562-HLA-B*07:02 cells were loaded with titrated amounts of the indicated peptides or no peptide as control and peptide sensitivity of TCR27-engineered T cells was determined by measuring the amount of secreted IFN-γ by ELISA at an E:T cell ratio of 1:1. (MIN—no antigen stimulation, MAX—unspecific stimulation, SB—strong binder, WB—weak binder, UT—untransduced T cells, SEQ ID NOs cf. legend to FIG. 8).

    [0159] FIG. 11. Functional analysis of the EBNA3C-reactive TCR58. (A) EBNA3C-specific TCR58-engineered T cells were co-cultured with K562-HLA-B*44:02 antigen-pulsed cells or an EBV-associated cancer cell line. T cell functionality was determined by measuring the amount of secreted IFN-γ by ELISA at an effector to target (E:T) cell ratio of 1:1. (B) Truncated versions (EBNA3C/3, EBNA3C/2, EBNA3C/1) of the full-length EBNA3C antigen were generated and expressed in K562-HLA-B*44:02 cells. (C) The antigen region harboring the immunogenic epitope was identified by measuring the amount of secreted IFN-γ by ELISA in the supernatant and is located between nucleotide (nt) 1 and 567. (D) The protein region identified as epitope-positive sequence (EBNA3C nt 1-567) was used to select for candidate peptides applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*44:02. (E) Four peptides according to their peptide-MHC binding affinity (bind level) classified as strong binder (SB) and weak binder (WB), respectively, were utilized for the identification of the epitope. K562-HLA-B*44:02 cells were loaded with the selected peptides, co-cultured with TCR58-engineered T cells, and IFN-γ secretion was determined by ELISA. One epitope (highlighted in D) with the amino acid sequence AEGGVGWRHW was recognized by TCR58-engineered T cells and can therefore be considered as cognate EBV EBNA3C epitope of TCR58. (F) K562-HLA-B*44:02 cells were loaded with titrated amounts of the indicated peptide or no peptide as control. Peptide sensitivity of TCR58-engineered T cells was determined by measuring the amount of secreted IFN-γ by ELISA at an E:T cell ratio of 1:1. (MIN—no antigen stimulation, MAX—unspecific stimulation, SB—strong binder, WB—weak binder, UT—untransduced T cells, SEQ ID NOs cf. legend to FIG. 9).

    [0160] FIG. 12. Functional analysis of the EBNA3C-reactive TCR64. (A) EBNA3C-specific TCR64-engineered T cells were co-cultured with K562-HLA-C*06:02 antigen-pulsed cells and EBV-positive cell lines. T cell functionality was determined by measuring the amount of secreted IFN-γ by ELISA at an effector to target (E:T) cell ratio of 1:1. (B) Truncated versions (EBNA3C/3, EBNA3C/2, EBNA3C/1) of the full-length EBNA3C antigen were generated and expressed in K562-HLA-C*06:02 cells. (C) The antigen region harboring the immunogenic epitope was identified by measuring the amount of secreted IFN-γ by ELISA in the supernatant and is located between nucleotide (nt) 568 and 1569. (D) The protein region identified as epitope-positive sequence (EBNA3C nt 568-1569) was used to select for candidate peptides applying the epitope prediction algorithm NetMHCpan4.0 for HLA-C*06:02. (E) 27 peptides according to their peptide-MHC binding affinity (bind level) classified as strong binder (SB) and weak binder (WB), respectively, were utilized for the identification of the epitope (RRYRRIYDL (SEQ ID NO: 158), FRKAQIQGL (SEQ ID NO: 4), AREAEVRFL (SEQ ID NO: 159), LRGKWQRRY (SEQ ID NO: 160), ERYAREAEV (SEQ ID NO: 161), SRRRRGACV (SEQ ID NO: 162), NLLDFVRFM (SEQ ID NO: 163), RRIYDLIEL (SEQ ID NO: 164), RRRRGACW (SEQ ID NO: 165), VRFLRGKWQ (SEQ ID NO: 166), RRRGACVVY (SEQ ID NO: 167), QRRYRRIYD (SEQ ID NO: 168), VRFMGVMSS (SEQ ID NO: 169), YAREAEVRFL (SEQ ID NO: 170), NRVGADSIM (SEQ ID NO: 171), LHHIWQNLL (SEQ ID NO: 172), RRGIKEHVI (SEQ ID NO: 173), YRRIYDLIE (SEQ ID NO: 174), RRYRRIYDLI (SEQ ID NO: 175), ARRGIKEHV (SEQ ID NO: 176), QRRYRRIYDL (SEQ ID NO: 177), WQRRYRRIY (SEQ ID NO: 178), FLRGKWQRRY (SEQ ID NO: 179), RRGACWYD (SEQ ID NO: 180), VYDDDVIEV (SEQ ID NO: 181), YAREAEVRF (SEQ ID NO: 182), GCQNAARTL (SEQ ID NO: 183)). K562-HLA-C*06:02 cells were loaded with the selected peptides, co-cultured with TCR64-engineered T cells, and IFN-γ secretion was determined by ELISA. One epitope (highlighted in D) with the amino acid sequence FRKAQIQGLwas recognized by TCR64-engineered T cells and can therefore be considered as cognate EBV EBNA3C epitope of TCR64. (F) K562-HLA-C*06:02 cells were loaded with titrated amounts of the indicated peptide or no peptide as control and peptide sensitivity of TCR64-engineered T cells was determined by measuring the amount of secreted IFN-γ by ELISA at an E:T cell ratio of 1:1. (MIN—no antigen stimulation, MAX—unspecific stimulation, SB—strong binder, WB—weak binder, UT—untransduced T cells).

    [0161] FIG. 13. Functional analysis of the LMP1-reactive TCR83. (A) LMP1-specific TCR83-engineered T cells were co-cultured with K562-HLA-C*15:02 antigen-pulsed cells or an EBV-associated cancer cell line. T cell functionality was determined by measuring the amount of secreted IFN-γ by ELISA at an effector to target (E:T) cell ratio of 1:1. (B) Truncated versions (LMP1/2, LMP1/1) of the full-length LMP1 antigen were generated and expressed in K562-HLA-C*15:02 cells. (C) The antigen region harboring the immunogenic epitope was identified by measuring the amount of secreted IFN-γ by ELISA in the supernatant and is located between nucleotide (nt) 316 and 624. (D) The protein region identified as epitope-positive sequence (LMP1 nt 316-624) was used to select for candidate peptides applying the epitope prediction algorithm NetMHCpan4.0 for HLA-C*15:02. (E) Four peptides according to their peptide-MHC binding affinity (bind level) classified as weak binder (WB) were utilized for the identification of the epitope (NSNEGRHHL (SEQ ID NO: 184), QQNWWTLLV (SEQ ID NO: 3), DSLPHPQQA (SEQ ID NO: 185), YLQQNWWTL (SEQ ID NO: 186)). K562-HLA-C*15:02 cells were loaded with the selected peptides, co-cultured with TCR83-engineered T cells, and IFN-γ secretion was determined by ELISA. One epitope (highlighted in D) with the amino acid sequence QQNWWTLLV was recognized by TCR83-engineered T cells and can therefore be considered as cognate EBV LMP1 epitope of TCR83. (F) K562-HLA-C*15:02 cells were loaded with titrated amounts of the indicated peptide or no peptide as control. Peptide sensitivity of TCR83-engineered T cells was determined by measuring the amount of secreted IFN-γ by ELISA at an E:T cell ratio of 1:1 (B). (MIN—no antigen stimulation, MAX—unspecific stimulation, WB—weak binder, UT—untransduced T cells).

    [0162] FIG. 14. Functional analysis of the LMP2A-reactive TCR06. (A) LMP2A-specific TCR06-engineered T cells were co-cultured with K562-HLA-A*02:01 antigen-pulsed cells or EBV-associated cancer cell lines. T cell functionality was determined by measuring the amount of secreted IFN-γ by ELISA at an effector to target (E:T) cell ratio of 1:1. (B) Truncated versions (LMP2A/2, LMP2A/1) of the full-length LMP2A antigen were generated and expressed in K562-HLA-A*02:01 cells. (C) The antigen region harboring the immunogenic epitope was identified by measuring the amount of secreted IFN-γ (ELISA) in the supernatant and is located between nucleotide (nt) 1006 and 1494. (D) The protein region identified as epitope-positive sequence (LMP1 nt 1006-1494) was used to select for candidate peptides applying the epitope prediction algorithm NetMHCpan4.0 for HLA-A*02:01. (E) 14 peptides according to their peptide-MHC binding affinity (bind level) classified as strong binder (SB) and weak binder (WB), respectively, were utilized for the identification of the epitope (FMCLGGLLTM (SEQ ID NO: 187), MLLLIVAGI (SEQ ID NO: 188), NLFCMLLLI (SEQ ID NO: 189), LLIVAGILFI (SEQ ID NO: 190), NLFCMLLLIV (SEQ ID NO: 191), MCLGGLLTMV (SEQ ID NO: 192), CLGGLLTMV (SEQ ID NO: 1), LIVAGILFI (SEQ ID NO: 193), FIPNLFCML (SEQ ID NO: 194), IVAGILFIL (SEQ ID NO: 195), MLLLIVAGIL (SEQ ID NO: 196), CMLLLIVAGI (SEQ ID NO: 197), PNLFCMLLLI (SEQ ID NO: 198), FIPNLFCMLL (SEQ ID NO: 199)). K562-HLA-A*02:01 cells were loaded with the selected peptides, co-cultured with TCR06-engineered T cells, and IFN-γ secretion was determined by ELISA. Epitopes (highlighted in D) were recognized by TCR06-engineered T cells and can therefore be considered as cognate EBV LMP2A epitopes of TCR06. Interestingly and worth to mentioning, that the two epitopes recognized by TCR06 were not ranked highest in the NetMHCpan4.0 prediction tool indicating that the prediction tool is not precise in forecasting relevant immunodominant epitopes. (F) K562-HLA-A*02:01 cells were loaded with titrated amounts of the indicated peptides or no peptide as control. Peptide sensitivity of TCR06-engineered T cells was determined by measuring the amount of secreted IFN-γ by ELISA at an E:T cell ratio of 1:1(B). (MIN—no antigen stimulation, MAX—unspecific stimulation, S—strong binder, WB—weak binder, UT—untransduced T cells).

    [0163] FIG. 15. Comparison of peptide sensitivity of LMP2A-specific TCR06 and TCRs provided in patents WO 2015/022520 A1 (PUBTCR1) and WO 2011/039508 A2 (PUBTCR2). The previously published wt EBV LMP2A TCR with a TRAV12-3*01/TRAJ41*01/TRAC alpha chain amino acid sequence (SEQ ID No: 2 of WO 2015/022520 A1) and a TRBV11-2*01/TRBD1/TRBJ2-7/TRBC beta chain amino acid sequence (SEQ ID No: 3 of WO 2015/022520 A1), also FIG. 1 of said document, is herein designated PUBTCR1. The previously published LMP2A TCR described in WO 2011/039508 A2, in particular, based on SEQ ID NO: 8 thereof, is designated PUBTCR2. K562-HLA-A*02:01 cells were loaded with titrated amounts of the indicated peptides CLGGLLTMV and MCLGGLLTMV, respectively, co-cultured with TCR06- or PUBTCR1- or PUBTCR2-engineered T cells, and peptide sensitivity of all TCRs was determined by measuring the amount of secreted IFN-γ by ELISA at an effector to target (E:T) cell ratio of 1:1. TCR06 has a higher peptide sensitivity in comparison to PUBTCR1 and PUBTCR2. UT—untransduced T cells.

    [0164] FIG. 16. Extended comparison of peptide sensitivity of LMP2A-specific TCR06 with TCRs provided in patents WO 2015/022520 A1 (PUBTCR1) and WO 2011/039508 A2 (PUBTCR2) and TCR-JC isolated by the applicants.

    [0165] The previously published wt EBV LMP2A TCR with a TRAV12-3*01/TRAJ41*01/TRAC alpha chain amino acid sequence (SEQ ID NO: 2 of WO 2015/022520 A1) and a TRBV11-2*01/TRBD1/TRBJ2-7/TRBC beta chain amino acid sequence (SEQ ID NO: 3 of WO 2015/022520 A1), also FIG. 1 of said document, is herein designated PUBTCR1. The previously published LMP2A TCR described in WO 2011/039508 A2, in particular, based on SEQ ID NO: 8 thereof, is designated PUBTCR2. Another LMP2A-specific TCR, herein designated as TCR-JC, was isolated by the applicants. (A) K562-HLA-A*02:01 cells were loaded with titrated amounts of the peptide CLGGLLTMV, co-cultured with TCR06-, PUBTCR1-, PUBTCR2- or TCR-JC-engineered T cells, and peptide sensitivity of all TCRs was determined by measuring the amount of secreted IFN-γ by ELISA at an effector to target (E:T) cell ratio of 1:10. (B) Sigmoidal 4PL curves (constrained model) based on data points from A. The sigmoidal regression shows a very good R.sup.2 (approx. 0.95) for TCR06, PUBTCR1, and PUBTCR2. Data from TCR-JC shows a model fit of 0.81 (lack of upper plateau). (C) Based on data shown in B, the peptide sensitivity of the four TCRs was calculated and indicated as EC50 in 10.sup.−9 M (EC50—mol/l of peptide needed to achieve 50% of the maximal IFN-γ release, SEM—standard error mean).

    [0166] FIG. 17. EBV-specific TCR-engineered T cells kill cancer cells. LMP1-specific TCR50-, LMP2A-specific TCR06-, and EBNA3C-specific TCR64-engineered T cells were co-cultured with L591 EBV.sup.+ tumor cells, which naturally express LMP1, LMP2A, and EBNA3C but were transfected with the respective MHC I allele. At indicated effector:target (E:T) cell ratios, data of triplicate wells were averaged and the percentage of surviving cells was calculated in relation to the values obtained from the samples co-cultured with untransduced T cells: % specific survival=100×(test value)/(average background). (UT—untransduced T cells).

    [0167] FIG. 18. In vivo mouse models of tumor rejection (A) NOG mice received subcutaneously 5×10.sup.6 K562-HLA-A*02:01 tumor cells and 24 h later intravenously 1×10.sup.7 TCR06-engineered T cells. Data obtained from individual mice receiving TCR06-engineered T cells (n=10) from two donors are shown in comparison to untransduced T cells (n=6). (B) NSG mice received subcutaneously 5×10.sup.6 K562-HLA-B*57:01 tumor cells and nine days later intravenously 5×10.sup.6 TCR50-engineered T cells. In both models, tumor size was measured using a caliper and calculated.

    EXAMPLES

    [0168] Generation of EBV-Specific TCR Constructs and Transgenic T Cells and Identification of Epitopes

    [0169] The inventors used an innovative method (Lorenz et al., 2018; WO2016/146618 A1) to generate EBV-specific TCRs, which recognize endogenously processed, immunogenic EBV epitopes presented by different MHC class I molecules.

    [0170] Briefly, after selecting the EBV antigen of interest, e.g., LMP1, LMP2A or EBNA3C, the following experimental steps were performed: [0171] (i) Pulsing of professional antigen-presenting cells (preferably dendritic cells (DCs)) with in vitro-transcribed (ivt)RNA encoding the full-length sequence of the selected EBV antigen to stimulate autologous T cells. This procedure is completely unbiased and allows the DCs to select the best epitope of the antigen for expression, processing, and presentation on the cell surface in conjunction with the most suitable MHC class I (MHC I) molecule. [0172] (ii) Identification of EBV antigen-reactive T cells. This step was performed by means of a newly established MHC class I cell library, which is composed of single MHC I-expressing cell lines originated from K562 cells. This part is an important feature of the TCR isolation approach, because it is basic to take advantage of the broad MHC flexibility. For the identification of each TCR, the inventors selected up to six MHC I from the K562 cell library that correspond to MHC I alleles of the T cell donor, and transfected the cells with the relevant antigens used for priming in step (i). After co-culture of antigen-presenting K562 cells and antigen-stimulated T cells, responding T cells were identified by interferon-(IFN)γ release using an ELISA, and up-regulation of the T cell activation marker CD137 measured by flow cytometry. Subsequently, reactive CD8+ T cells were enriched by FACS sorting. [0173] (iii) Isolation of total RNA from FACS-sorted CD8+ T cells and PCR amplification of TCRα- and TCRβ chain-specific sequences. Identification of dominant TCRα- and TCRβ sequences by next generation sequencing. [0174] (iv) Re-expression of dominant (at least 10%) TCRα- and TCRβ chain combinations using the γ-retrovirus vector MP71 (Engels et al., 2003; Leisegang et al., 2008; Sommermeyer and Uckert, 2010) in primary human T cells to identify the functional TCRαβ chain combination. This was done by co-cultivation of TCR-engineered T cells with K562 cells carrying the proper MHC I molecule and expressing the full-length EBV antigen. Antigen recognizing TCRαβ chain combinations were linked with a P2A element and recloned in the configuration of TCRβ gene-P2A-TCRα gene into the MP71 vector. Constant TCRαβ chain regions were replaced by their murine counterparts to enhance pairing of transgenic TCR chains. Subsequently, the complete TCR transgene cassette was codon optimized. [0175] (v) Identification of the antigenic peptide (epitope) of EBV, which is recognized by the TCR-engineered T cells. For this, the full-length antigen was C- or N-terminally truncated, cloned into the plasmid vector pcDNA3.1(−) and expressed in K562 cells carrying the proper MHC I molecule. Then, the remaining protein fragments were tested for their ability to furthermore present the epitope recognized by the TCR. Finally, candidate peptides of the corresponding protein region were identified by the epitope prediction algorithm NetMHCpan4.0 (http://www.cbs.dtu.dk/services/NetMHCpan/). Predicted peptides were generated and loaded onto K562 cells carrying the proper MHC I molecule and investigated in co-culture experiments for their ability to be recognized by TCR-engineered T cells. The peptides capable of stimulating most IFN-γ production are considered as cognate epitope.

    TABLE-US-00001 TABLE 1 Peptides tested for one of the TCR (TCR50). The protein region identified as epitope-positive sequence (LMP1/2 nt 316-624) was used to select for candidate peptides applying the epitope  prediction algorithm NetMHCpan4.0 for HLA- B*57:01. 13 peptides, according to their peptide-MHC binding affinity classified as strong binder (SB) and weak binder (WK), respectively, were utilized for epitope identification. Results are shown in FIG. 5. Epitope Length Bind (SEQ ID NO:) (aa) level Affinity WTLLVDLLW  9 SB 12.25 (107) IALYLQQNWW 10 SB 14.84 (108) IALYLQQNW  9 SB 16.32 (2) WWTLLVDLLW 10 SB 32.51 (109) IIALYLQQNW 10 SB 57.84 (110) WTLLVDLLWL 10 SB 111.95 (111) LAILIWMYY  9 SB 231.02 (112) LLFLAILIW  9 SB 234.00 (113) LLLFLAILIW 10 WB 749.75 (114) LAILIWMYYH 10 WB 789.96 (115) ALYLQQNWW  9 WB 853.78 (116) FLAILIWMYY 10 WB 1515.23 (117) NSNEGRHHL  9 WB 12828.84 (118)

    [0176] TCR constructs generated are characterized in Tables 2, 3 and 4 below.

    TABLE-US-00002 TABLE 2 TCR summary Recog- half- SEQ nition maximum EBV- ID of tumor TCR IFN-γ Antigen Epitope NO: MHC I  cells 01 6 × 10.sup.−8 EBNA3C QPRAPIRPIPT 7 B*07:02 + 25 6 × 10.sup.−7 EBNA3C AEGGVGWRHW 5 B*44:02 + 27 6 × 10.sup.−8 EBNA3C QPRAPIRPIP 6 B*07:02 + 58 3 × 10.sup.−7 EBNA3C AEGGVGWRHW 5 B*44:02 + 64 7 × 10.sup.−6 EBNA3C FRKAQIQGL 4 C*06:02 + 50 3 × 10.sup.−8 LMP1 IALYLQQNW 2 B*57:01 + 83 2 × 10.sup.−9 LMP1 QQNWWTLLV 3 C*15:02 + 06 6 × 10.sup.−9 LMP2A CLGGLLTMV 1 A*02:01 +

    [0177] TCR06, TCR50 and TCR83 have a high peptide sensitivity (half maximum IFN-γ release), in particular, TCR06.

    TABLE-US-00003 TABLE 3 CDR sequences of preferred TCR constructs of the invention. CDR1 IMGT aa location: 27-38. CDR2 IMGT aa location: 56-65. CDR3 IMGT aa location: 105-117. Numbers in parentheses: SEQ ID NO: TCR CDR-1  CDR-2  CDR-3  aa seq aa seq aa seq TCR  TRA DSAIYN  IQSSQRE  AVLMDSNYQLI  06 (11) (12) (13) TRB WSHSY  SAAADI  ASSEDGMNTEAF  (16) (17) (18) TCR  TRA SSYSPS  YTSAATLV  VVMATGFQKLV  50 (21) (22) (23) TRB SGDLS  YYNGEE  ASSVTSGSDEQF  (26) (27) (28) TCR  TRA TSGFNG  NVLDGL  AAVNNAGNMLT  83 (31) (32) (33) TRB LGHDT  YNNKEL  ASSQGYGGPSTDTQY  (36) (37) (38) TCR  TRA SVFSS  VVTGGEV  AGDVDTGTASKLT  64 (41) (42) (43) TRB MDHEN  SYDVKM  ASSLLGSGALYEQY  (46) (47) (48) TCR  TRA SSYSPS  YTSAATLV  VAWDTGFQKLV  25 (51) (52) (53) TRB SNHLY  FYNNEI  ASKALADTQY  (56) (57) (58) TCR  TRA NSASQS  VYSSGN  VASGDSSYKLI  58 (61) (62) (63) TRB SNHLY  FYNNEI  ASSDPLSTYNEQF  (66) (67) (68) TCR  TRA TISGTDY  GLTSN  ILCGAGGTSYGKLT  27 (71) (72) (73) TRB MNHEY  SMNVEV  ASNVQGANNEQF  (76) (77) (78) TCR  TRA TISGTDY  GLTSN  ILCGAGGTSYGKLT  01 (81) (82) (83) TRB MNHEY  SMNVEV  ASAIQGANNEQF  (86) (87) (88)

    TABLE-US-00004 TABLE 4 TRAV and TRBV segments and junction aa (IMGT location 104-118) of preferred TCR constructs of the invention. Numbers in parentheses: SEQ ID NO: V TCR segment aa junction aa seq TCR 06 TRA TRAV21*01 CAVLMDSNYQLIW (14) TRB TRBV10-2*02 CASSEDGMNTEAFF (19) TCR 50 TRA TRAV8-2*01 CVVMATGFQKLVF (24) TRB TRBV9*01 CASSVTSGSDEQFF (29) TCR 83 TRA TRAV1-2*01 CAAVNNAGNMLTF (34) TRB TRBV3-1*01 CASSQGYGGPSTDTQYF  (39) TCR 64 TRA TRAV27*01 CAGDVDTGTASKLTF  (44) TRB TRBV28*01 CASSLLGSGALYEQYF  (49) TCR 25 TRA TRAV8-2*01 CVAWDTGFQKLVF (54) TRB TRBV2*01 CASKALADTQYF (59) TCR 58 TRA TRAV12-1*01 CVASGDSSYKLIF (64) TRB TRBV2*01 CASSDPLSTYNEQFF  (69) TCR 27 TRA TRAV26-2*01 CILCGAGGTSYGKLTF  (74) TRB TRBV27*01 CASNVQGANNEQFF (79) TCR 01 TRA TRAV26-2*01 CILCGAGGTSYGKLTF  (84) TRB TRBV27*01 CASAIQGANNEQFF (89)

    [0178] Functional Characterization

    [0179] The generated TCR-engineered T cells were functionally characterized using in vitro assays.

    [0180] Firstly, the peptide sensitivity (half maximum IFN-γ release) of the isolated TCRs was analyzed in peptide titration experiments (cf. FIG. 6 and Lorenz et al., 2018). Briefly, peptides were loaded in titrating amounts onto K562 target cells, ranging from 10.sup.−5 mol/l to 10.sup.−13 mol/l. A co-culture assay was performed with TCR-engineered T cells at an effector to target (E:T) cell ratio of 1:1 (if not otherwise indicated), meaning 2.5×10.sup.4 TCR-engineered T cells were co-cultured with 2.5×10.sup.4 peptide-loaded K562 cells. The ability of TCR-transduced T cells to recognize their target epitope was assessed after 24 h by an IFN-γ ELISA.

    [0181] Secondly, EBV antigen-expressing target cell lines (LCLs, EBV-associated cancer cell lines which endogenously process and present EBV epitopes and peptide-loaded K562 cells) were applied in co-culture experiments to determine the amount of IFN-γ released by TCR-engineered T cells (cf. FIG. 7 and Lorenz et al., 2018). In brief, TCR-engineered T cells and target cells (each 2×10.sup.4) were co-cultured. After 24 h, T cell reactivity was determined by measuring the amount of secreted IFN-γ by ELISA. T cells stimulated with PMA and ionomycin were used as positive control, untransduced T cells as negative control.

    [0182] According to the described isolation and characterization procedure and the identification of the epitopes recognized by TCR-engineered T cells, we have isolated in total eight EBV-specific TCRs, and the relevant, immunogenic peptides (epitopes) recognized by these TCRs. Some of the epitopes were already known in the art, others, in particular, peptides of SEQ ID NO: 3, 4 and 5, have been newly identified herein to be presented by the respective MHC I molecules.

    [0183] Thirdly, EBV antigen-expressing L591 tumor cells which endogenously process and present epitopes of LMP1, LMP2A, and EBNA3C but where transfected with the relevant MHC I alleles were applied in co-culture experiments with TCR-engineered T cells to determine the killing capacity of TCR-engineered T cells.

    [0184] Furthermore, the generated TCR-engineered T cells were functionally characterized using in vivo assays. Therefore, two models of immunocompromised mice (NOD, NSG) were applied. Tumor cells expressing both the relevant EBV antigen and MHC I molecule were injected subcutaneously into the animals and TCR-engineered T cells were transferred by tail vein injection into the mice.

    [0185] Combination of TCRs for Immunotherapy

    [0186] The combination of TCR-engineered T cells which recognize different epitopes presented by distinct MHC I molecules is an interesting option to improve TCR gene therapy. This approach aims to overcome and prevent two problems of immunotherapy: (i) tumor outgrowth due to loss variants of a specific tumor antigen and (ii) tumor immune evasion due to downregulation of a specific MHC I molecule. Using such a combinatorial approach, the efficiency of TCR gene therapy will be enhanced.

    [0187] To prove this concept, two or three TCRs, which are able to recognize endogenously processed EBV antigens naturally presented by different MHC I molecules, are selected, e.g. TCR06 (LMP2A, MHC A*02:01), TCR50 (LMP1, MHC B*57:01), and, optionally, TCR64 (EBNA3C, MHC C*06:02). TCR-engineered T cells are generated with these TCRs by retroviral transduction as described. Tumor cells which naturally express the EBV antigens recognized by the TCR and which harbor the respective MHC I molecules (A*02:01, B*57:01, C*06:02) are used in co-culture experiments with TCR-engineered T cells.

    [0188] Alternatively, such cells (e.g. K562 cells) are generated by transfection of the corresponding EBV antigen genes and the MHC I genes.

    [0189] In the experiment, TCR-engineered T cells are used individually or in combinations, and will be co-cultured with tumor cells in an E:T ration of 2:1. To evaluate the effectiveness of the single and combinatorial application of TCR-engineered T cells, IFN-γ secretion is determined by ELISA.

    [0190] In addition, a cytotoxicity assay is used to analyze the capacity of the single and combinatorial application of TCR-engineered T cells to kill tumor cells.

    [0191] In a further experiment, an in vivo mouse model is established. Tumor cells, e.g. K562 cells, expressing the respective EBV antigens and MHC I molecules, are s.c. injected into the flank of NSG mice (NOD.Cg-Prkdc.sup.scid Il2rg.sup.tm1 Wj1/Sz). After tumors are palpable, TCR-engineered T cells are i.v. injected, individually or in combination, to determine the efficacy of TCR gene therapy in terms of tumor rejection.

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