TCR constructs specific for EBV-derived antigens

Abstract

The present invention relates to the field of immunotherapy, particularly immunotherapy for Epistein-Barr virus-associated diseases (EBV, also known as human gammaherpesvirus type 4), such as cancer or post-transplant lymphoproliferative disorders, particularly adoptive T cell therapy or T cell receptor (TCR) gene therapy. The present invention provides a combination of nucleic acids encoding at least two TCR constructs, or respective proteins, or host cells, each capable of specifically binding to a respective epitope associated with a respective MHCI, where the epitopes are peptides derived from different antigens expressed by the same infectious agent or cancer, such as EBV antigens. The present invention also provides specific nucleic acids encoding TCR alpha chain constructs (TRAs) and / or TCR beta chain constructs (TBRs) specific for epitopes complexed with human MHCI, where the epitopes are epitopes of Epistein-Barr virus proteins, and where the TCR constructs are specific for epitopes derived from LMP2A, LMP1, or EBNA3C. The proteins encoded by said nucleic acids, the corresponding host cells, and pharmaceutical compositions and kits are also subject of the present invention.

Description

[Technical field]

[0001] The present invention relates to the field of immunotherapy, in particular to Epistein-Barr Virus-associated diseases (EBV, also known as human gamma-herpesvirus type 4), such as cancer or post-transplant lymphoproliferative diseases, in particular to adoptive T cell therapy, or T cell receptor (TCR) gene therapy. The present invention provides a combination of nucleic acids encoding at least two TCR constructs, or respective proteins, or host cells, where each TCR construct is capable of specifically binding to a respective epitope associated with a respective MHCI, and where the epitopes are peptides derived from different antigens expressed by the same infectious agent or cancer, for example EBV antigens. The present invention also provides specific nucleic acids encoding TCR alpha chain constructs (TRA) and / or TCR beta chain constructs (TBR) of TCR constructs specific for an epitope complexed with human MHCI, where the epitope is an epitope of an Epistein-Barr Virus protein, where the TCR construct is specific for an epitope derived from LMP2A, LMP1 or EBNA3C. The proteins encoded by said nucleic acids, the corresponding host cells, and pharmaceutical compositions and kits are also subject of the present invention. [Background technology]

[0002] TCRs are heterodimeric cell surface proteins of the immunoglobulin superfamily that associate with invariant proteins of the CD3 complex, which are involved in mediating signal transduction. There are αβ and γδ types of TCRs, which are structurally similar but have significant differences in anatomical location and possibly function. The alpha and beta chains of the naturally occurring heterodimeric αβTCR are transmembrane proteins, each with two extracellular domains, a membrane-proximal constant domain and a membrane-distal variable domain. Each constant and variable domain contains an intrachain disulfide bond.

[0003] The variable region of each TCR chain contains a variable segment and a binding segment, and in the case of the beta chain, a diversity segment. Each variable region contains three CDRs (complementarity determining regions) embedded in framework sequences, highly polymorphic loops, and one hypervariable region called CDR3. There are different types of α chain variable regions (Vα) and different types of beta chain variable regions (Vβ) that are distinguished by their own framework sequences, CDR1 and CDR2, and partially defined CDR3 sequences. A unique TRAV or TRBV number is given to Vα or Vβ by the IMGT nomenclature. The specificity of the TCR for the recognized epitope is mainly determined by the CDR3 region (Danska et al., 1990; Garcia et al., 2005.).

[0004] Using adoptive TCR gene therapy, it is possible to rapidly generate sufficient numbers of activated, non-exhausted T cells with the desired specificity on the patient's own T cells. TCRs can be introduced into all T cells or into a subset of T cells, such as CD8+ T cells, central memory T cells, or T cells with stem cell characteristics, to better ensure persistence and function upon introduction. TCR-transduced T cells can be infused into patients, such as cancer patients, for example, those with lymphopenia due to chemotherapy or radiation therapy, to induce homeostatic proliferation, which greatly improves the engraftment and long-term persistence of the transduced T cells and is associated with higher cure rates.

[0005] TCR-based adoptive T cell therapy classically relies on TCR recognition of epitopes on processed antigens presented in the context of MHC molecules, so that T cells expressing a particular TCR specific for an epitope associated with a particular MHC can only be used to treat patients expressing the respective MHC.

[0006] Epistein-Barr virus (EBV), a human herpesvirus, infects approximately 90% of the world's population. In healthy individuals, disease caused by EBV is normally eliminated by immune cells, with T cells playing the most important role.

[0007] EBV-associated diseases are infectious mononucleosis and various non-malignant, pre-malignant and malignant EBV-associated lymphoproliferative disorders such as post-transplant lymphoproliferative disease, Burkitt's lymphoma, hemophagocytic lymphohistiocytosis, Hodgkin's and non-Hodgkin's lymphoma; non-lymphoid malignancies such as gastric cancer, lung cancer, and nasopharyngeal carcinoma; and diseases associated with the human immunodeficiency virus such as hairy leukoplakia and central nervous system lymphoma. The virus is also associated with childhood disorders such as Alice in Wonderland syndrome and acute cerebellar ataxia, and, based on some evidence, with a higher risk of developing certain autoimmune diseases. Approximately 200,000 cases of cancer per year are thought to be attributable to EBV (Wikipedia).

[0008] The majority of EBV-associated cancers express only a limited number of EBV-specific antigens, such as latent membrane proteins (LMP1, LMP2), and nuclear proteins (EBNA1, EBNA3C). These antigens have been shown to be interesting targets for TCR-based immunotherapy, e.g., TCR gene therapy for EBV-associated diseases such as post-transplant lymphoproliferative disorders or cancer, or adoptive T cell therapy (Orentas et al., 2001; Jurgens et al., 2006; Hart et al., 2008; Simpson et al., 2011; Yang et al., 2011; Zheng et al., 2015; Cho et al., 2018; WO 2015 / 022520A1; WO 2011 / 039508A2). Summary of the Invention [Problem to be solved by the invention]

[0009] However, the majority of T cell-based immunotherapies targeting EBV-associated malignancies use natural EBV-specific T cells produced from third-party donors or patients, where the T cells have been expanded using EBV lymphoblastoid cell lines (LCLs) or EBV peptide pools. Adoptive T cell therapy using EBV-specific TCR-transduced T cells has not been tested in clinical trials. TCR-transduced T cells have several advantages compared to natural EBV-specific T cells: 1) Efficacy: The transduced TCR is a defined receptor with high affinity for EBV-positive tumor cells. The expansion of natural T cells derived from the patient's blood depends on the presence of EBV-specific T cells to be expanded. However, patients may lack effective T cells that can be expanded. 2) Feasibility: The success rate of producing artificial T cells is over 95%, while the success rate of the method of expanding natural T cells is below 70%. 3) Cost: The expansion of natural T cells takes more than 40 days, whereas the vein-to-vein time for artificial T cell methods has been shortened to less than 21 days.

[0010] To perform TCR gene therapy on patient populations with different MHCI (HLA) alleles, it is necessary to identify TCRs restricted by different HLA alleles, which is a prerequisite for targeting EBV epitopes via two different HLA alleles expressed by the same cells.

[0011] The present invention addresses some of these problems and provides novel, and preferably advantageous, immunotherapeutic agents useful in the immunotherapy of cancer or infectious agents. This problem is solved by the subject matter of the claims. [Means for solving the problem]

[0012] Nucleic Acids Encoding TCR Constructs In one embodiment, the invention provides specific TCR constructs and the nucleic acids encoding them that are useful for the treatment of EBV-associated disease.

[0013] The present invention relates to a nucleic acid encoding a TCR construct, a TCR alpha chain construct (TRA) and / or a TCR beta chain construct (TBR), specific for an epitope complexed with human MHCI, wherein the epitope is an epitope of an EBV protein, wherein the epitope has the sequence of SEQ ID NO:1, MHCI is HLA-A*02:01, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:13, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:18; wherein the epitope has the sequence of SEQ ID NO:2, MHCI is HLA-B*57:01, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:23, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:28; wherein the epitope has the sequence of SEQ ID NO:3, MHCI is HLA-C*15:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:33, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:38; wherein the epitope has the sequence of SEQ ID NO:4, MHCI is HLA-C*06:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:43, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:48; wherein the epitope has the sequence of SEQ ID NO:5, MHCI is HLA-B*44:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:53, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:58; wherein the epitope has the sequence of SEQ ID NO:5, MHCI is HLA-B*44:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:63, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:68; wherein the epitope has the sequence of SEQ ID NO:6, MHCI is HLA-B*07:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:73, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:78; and / or Here, a nucleic acid is provided, wherein the epitope has the sequence of SEQ ID NO:7, MHCI is HLA-B*07:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:83, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:88.

[0014] In the context of the present invention, "a" is understood to mean "one or more", unless expressly stated otherwise. Thus, for example, as preferred throughout the present invention, when the TCR construct of the present invention comprises both alpha and beta chain constructs, it may be encoded by one or two nucleic acids. The alpha and beta chain constructs together are capable of specifically binding to an epitope derived from EBV complexed with human MHCI. As intermediate products, the alpha and beta chain constructs are also objects of the present invention per se. The present invention also provides single stranded nucleic acid constructs, e.g., in which the TCR alpha and beta chain constructs are separated by a P2A element.

[0015] One nucleic acid of the invention encodes a TRA and / or TRB of a TCR construct specific for an epitope complexed with human MHCI, where the epitope has the sequence of SEQ ID NO: 1, where MHCI is HLA-A*02:01, and where TRA comprises a CDR3 with at least 90% sequence identity to SEQ ID NO: 13, and where TRB comprises a CDR3 with at least 90% sequence identity to SEQ ID NO: 18. SEQ ID NO: 1 is an epitope derived from the EBV protein LMP2A, known in the art to be presented by cancer cells of EBV-associated cancer types in association with HLA-A2, e.g., the very common HLA type HLA-A*02:01 (see, e.g., Orentas et al., 2001, supra). TCR constructs with the CDR3 sequences disclosed herein have particularly high affinity or peptide sensitivity, as shown herein, compared to TCRs in the art, and are therefore preferred TCR constructs of the invention.

[0016] Advantageously, said TCR is stimulated with a peptide concentration of 10 -8 mol / L or less, preferably 10 -9 The peptide has high peptide sensitivity, with a 50% effective concentration of maximum IFN-γ release at 0.5 mol / L or less. This analysis is performed by culturing TCR-transduced T cells with target cells (e.g., K562-HLA-A*02:01 cells carrying a peptide, preferably the peptide of SEQ ID NO: 1) at an effector to target cell ratio of 1:1, for example, as shown in Figure 15 or Figure 16, preferably Figure 15.

[0017] 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. Optionally, 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.

[0018] The TRA may comprise the conjugated amino acids shown in SEQ ID NO: 14. The TRB may comprise the conjugated amino acids shown in SEQ ID NO: 19.

[0019] The TRA may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 15. The TRB may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO:20.

[0020] A TCR construct comprising a TRA with a variable region of SEQ ID NO: 15 and a TRB with a variable region of SEQ ID NO: 20 is shown herein to have particularly advantageous characteristics, and is also referred to as TCR06.

[0021] The variable region of TRA in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:91, and the variable region of TRB in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:92.

[0022] One nucleic acid of the invention encodes a TRA and / or TRB of a TCR construct specific for an epitope complexed with human MHCI, where the epitope has the sequence of SEQ ID NO:2, MHCI is HLA-B*57:01, and TRA comprises a CDR3 with at least 90% sequence identity to SEQ ID NO:23, and TRB comprises a CDR3 with at least 90% sequence identity to SEQ ID NO:28. SEQ ID NO:2 is an epitope derived from the EBV protein LMP1 presented by cancer cells of EBV-associated cancer types in the context of HLA-B*57:01. TCR constructs having the CDR3 sequences disclosed herein have high affinity or peptide sensitivity, as shown herein.

[0023] Optionally, said 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. Optionally, said 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.

[0024] TRA may comprise the conjugated amino acids shown in SEQ ID NO: 24. TRB may comprise the conjugated amino acids shown in SEQ ID NO: 29.

[0025] The TRA may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 25. The TRB may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 30.

[0026] A TCR construct comprising a TRA with a variable region of SEQ ID NO: 25 and a TRB with a variable region of SEQ ID NO: 30 is shown herein to have advantageous characteristics, also referred to as TCR50.

[0027] The variable region of TRA in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:93, and the variable region of TRB in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:94.

[0028] One nucleic acid of the invention encodes a TRA and / or TRB of a TCR construct specific for an epitope complexed with human MHCI, where the epitope has the sequence of SEQ ID NO:3, where MHCI is HLA-C*15:02, and where TRA comprises a CDR3 with at least 90% sequence identity to SEQ ID NO:33, and where TRB comprises a CDR3 with at least 90% sequence identity to SEQ ID NO:38. SEQ ID NO:3 is an epitope derived from the EBV protein LMP1, which has now been shown for the first time to be presented by cancer cells of EBV-associated cancer types. It is presented in the context of HLA-C*15:02. TCR constructs with the CDR3 sequences disclosed herein have high affinity or peptide sensitivity, as shown herein.

[0029] Optionally, said 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. Optionally, said 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.

[0030] TRA may comprise the junction amino acids shown in SEQ ID NO: 34. TRB may comprise the junction amino acids shown in SEQ ID NO: 39.

[0031] The TRA may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 35. The TRB may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 40.

[0032] A TCR construct comprising a TRA with a variable region of SEQ ID NO: 35 and a TRB with a variable region of SEQ ID NO: 40 is shown herein to have advantageous characteristics, also referred to as TCR83.

[0033] The variable region of TRA in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:95, and the variable region of TRB in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:96.

[0034] One nucleic acid of the invention encodes a TRA and / or TRB of a TCR construct specific for an epitope complexed with human MHCI, where the epitope has the sequence of SEQ ID NO:4, MHCI is HLA-C*06:02, and TRA comprises a CDR3 with at least 90% sequence identity to SEQ ID NO:43, and TRB comprises a CDR3 with at least 90% sequence identity to SEQ ID NO:48. SEQ ID NO:4 is an epitope derived from the EBV protein EBNA3C, which has now been shown for the first time to be presented by cancer cells of EBV-associated cancer types. It is presented in the context of HLA-C*06:02.

[0035] Optionally, said 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. Optionally, said 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.

[0036] TRA may comprise the junction amino acids shown in SEQ ID NO: 44. TRB may comprise the junction amino acids shown in SEQ ID NO: 49.

[0037] Preferably, the TRA may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 45. Preferably, the TRB may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 50.

[0038] A TCR construct comprising a TRA with a variable region of SEQ ID NO: 45 and a TRB with a variable region of SEQ ID NO: 50 is shown herein to have advantageous characteristics, also referred to as TCR64.

[0039] The variable region of TRA in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:97, and the variable region of TRB in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:98.

[0040] One nucleic acid of the invention encodes a TRA and / or TRB of a TCR construct specific for an epitope complexed with human MHCI, where the epitope has the sequence of SEQ ID NO:5, MHCI is HLA-B*44:02, and TRA comprises a CDR3 with at least 90% sequence identity to SEQ ID NO:53, and TRB comprises a CDR3 with at least 90% sequence identity to SEQ ID NO:58. SEQ ID NO:5 is an epitope derived from the EBV protein EBNA3C, which has now been shown for the first time to be presented by cancer cells of EBV-associated cancer types. It is presented in the context of HLA-B*44:02.

[0041] Optionally, said 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. Optionally, said 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.

[0042] TRA may comprise the junction amino acids shown in SEQ ID NO: 54. TRB may comprise the junction amino acids shown in SEQ ID NO: 59.

[0043] Preferably, the TRA comprises a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 55. Preferably, the TRB comprises a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 60.

[0044] A TCR construct comprising a TRA having a variable region of SEQ ID NO: 55 and a TRB having a variable region of SEQ ID NO: 60 is shown herein to have advantageous characteristics, also referred to as TCR25.

[0045] The variable region of TRA in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:99, and the variable region of TRB in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:100.

[0046] One nucleic acid of the invention encodes a TRA and / or TRB of a TCR construct specific for an epitope complexed with human MHCI, where the epitope has the sequence of SEQ ID NO:5, MHCI is HLA-B*44:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:63, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:68. As noted above, SEQ ID NO:5 is an epitope derived from the EBV protein EBNA3C, now shown for the first time to be presented by cancer cells of EBV-associated cancer types, which is presented in the context of HLA-B*44:02.

[0047] Optionally, said 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. Optionally, said 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.

[0048] The TRA may comprise the junction amino acids shown in SEQ ID NO: 64. The TRB may comprise the junction amino acids shown in SEQ ID NO: 69.

[0049] Preferably, the TRA may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 65. Preferably, the TRB may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 70.

[0050] A TCR construct comprising a TRA having a variable region of SEQ ID NO: 65 and a TRB having a variable region of SEQ ID NO: 70 is shown herein to have advantageous characteristics, also referred to as TCR58.

[0051] The variable region of TRA in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:101, and the variable region of TRB in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:102.

[0052] One nucleic acid of the invention encodes a TRA and / or TRB of a TCR construct specific for an epitope complexed with human MHCI, where the epitope has the sequence of SEQ ID NO:6, MHCI is HLA-B*07:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:73, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:78. SEQ ID NO:6 is an epitope derived from the EBV protein EBNA3C, presented by cancer cells of EBV-associated cancer types in the context of HLA-B*07:02.

[0053] Optionally, said 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. Optionally, said 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.

[0054] TRA may comprise the junction amino acids shown in SEQ ID NO: 74. TRB may comprise the junction amino acids shown in SEQ ID NO: 79.

[0055] Preferably, the TRA may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 75. Preferably, the TRB may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 80.

[0056] A TCR construct comprising a TRA having a variable region of SEQ ID NO: 75 and a TRB having a variable region of SEQ ID NO: 80 is shown herein to have advantageous characteristics, also referred to as TCR27.

[0057] The variable region of TRA in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:103, and the variable region of TRB in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:104.

[0058] One nucleic acid of the invention encodes a TRA and / or TRB of a TCR construct specific for an epitope complexed with human MHCI, where the epitope has the sequence of SEQ ID NO:7, MHCI is HLA-B*07:02, and TRA comprises a CDR3 with at least 90% sequence identity to SEQ ID NO:83, and TRB comprises a CDR3 with at least 90% sequence identity to SEQ ID NO:88. SEQ ID NO:7 is an epitope derived from the EBV protein EBNA3C, which contains SEQ ID NO:6 and a T at the C-terminus.

[0059] Optionally, said 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. Optionally, said 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.

[0060] TRA may comprise the junction amino acids shown in SEQ ID NO: 84. TRB may comprise the junction amino acids shown in SEQ ID NO:89.

[0061] Preferably, the TRA may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 85. Preferably, the TRB may comprise a variable region having at least 90%, optionally at least 95% or 100% sequence identity to SEQ ID NO: 90.

[0062] A TCR construct comprising a TRA having a variable region of SEQ ID NO: 85 and a TRB having a variable region of SEQ ID NO: 90 is shown herein to have advantageous characteristics, and is also referred to as TCR01.

[0063] The variable region of TRA in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:105, and the variable region of TRB in the TCR construct may be encoded by a nucleic acid having the sequence of SEQ ID NO:106.

[0064] In any TRA and / or TRB construct of the present invention, CDR1 and CDR3 can independently have at least 80%, at least 90% or 100% sequence identity to the described sequence.Preferably, CDR3 has 100% sequence identity to the described and defined CDR3.Typically, when the sequence is not identical, there is at most one amino acid substitution, deletion or insertion, typically an amino acid substitution.This substitution is a conservative substitution, i.e., one amino acid of a particular type (e.g., polar, non-polar, acidic, basic, aromatic) is replaced with another amino acid of the same type.Methods of affinity maturation are known in the art and are further described below.

[0065] Optionally, sequence identity for the described CDR1 and CDR2 and CDR3 regions in the TRA or TRB, preferably in both the TRA and TRB, of the TCR construct of the invention is 100%.

[0066] The TCR alpha and / or beta chain constructs of the invention may, but need not, comprise all the features or domains corresponding to their natural counterparts, i.e. the TCR alpha or beta chain. Preferably, the TCR alpha and / or beta chain constructs comprise at least a variable region, or a variable region and a constant region, e.g. a variable region and / or constant region having at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to a human variable or constant TCR region.

[0067] The TCR alpha chain constructs and / or TCR beta chain constructs of the invention preferably comprise a constant region. For adoptive TCR therapy, it is preferred that the TCR construct comprises a full-length TCR alpha and beta chain, including a variable and constant region, including a transmembrane region. The constant region may be a human constant region, a mouse constant region, or a chimeric constant region, e.g. a minimal mouse constant region. The TCR construct may be substantially or exclusively of human origin to minimize immunogenicity. To prevent association with endogenous TCR chains, the constructs of the invention preferably comprise one or more, e.g. 1-5, 1-10, or 1-20, preferably 9 amino acid substitutions, compared to the human sequence (Sommermeyer and Uckert, 2010). For this purpose, the constant regions of the TCR alpha and beta chain constructs can also be murine constant regions (Cohen et al., 2006) or / and additional cysteines can be provided to allow the formation of additional disulfide bonds between the TCR chains (Cohen et al., 2007, Kuball et al., 2007). In addition, codon modifications of the TCR sequence can be used to enhance the functional expression of the recombinant TCR (Scholten et al., 2006) and / or a peptide (e.g. P2A) can be applied to the linkage of both TCR chains to achieve stoichiometric expression of both chains (Leisegang et al., 2008), which also results in an enhanced functional expression of the recombinant TCR.

[0068] The construct may also be a chimeric antigen receptor or a portion thereof, where, for example, a human TCR variable region may be linked to a different immunoglobulin constant region, e.g. an IgG constant region, or may be linked to an antibody domain capable of specifically binding to an antigen, such as LMP2A.

[0069] Not only heterodimeric TCR constructs are encompassed, but also single chain constructs (scTCRs). The scTCRs may comprise the variable region of a first TCR chain construct (e.g., alpha chain) and the entire (full length) of a second TCR chain (e.g., beta chain), or vice versa. Additionally, the scTCRs may optionally comprise one or more linkers linking two or more polypeptides together. The linker may, for example, be a peptide linking two single chains together. Also provided are such scTCRs of the invention fused to a cytokine, e.g., a human cytokine, e.g., IL-2, IL-7, IL-12, or IL-15.

[0070] In order to be able to specifically recognise an epitope associated with MHC, particularly for therapeutic purposes, the nucleic acid of the invention encodes one TCR alpha chain construct and one beta chain construct of the TCR construct of the invention, e.g. a TCR construct specific for the epitope of SEQ ID NO: 1 as described herein.

[0071] Typically, the nucleic acid sequences provided herein are codon optimized for expression in human cells.

[0072] In the context of the present invention, the nucleic acid is either DNA or RNA. Preferably, the nucleic acid is DNA. The nucleic acid may be, for example, a viral vector or a non-viral vector, for example, a vector suitable for transposon, CRISPR / CAS-based recombination, or a plasmid suitable for in vitro RNA transcription. The nucleic acid of the present invention is preferably a vector. Suitable vectors include vectors designed for propagation and propagation, or for expression, or both, such as plasmids and viruses. The vector may be an expression vector suitable for expression in a host cell selected from the group including human T cells or human T cell precursors, preferably human T cells, such as CD8+ T cells. The CD8+ T cells may be central memory T cells, effector memory T cells, stem cell-like T cells, or effector T cells, or a mixture thereof. The vector may be a viral vector, such as a retrovirus, in particular a gamma-retrovirus or lentivirus vector. Examples of suitable expression vectors include the retroviral vector MP71 (Engels et al., 2003). The expression vector comprises regulatory sequences such as transcription and translation initiation and termination codons, which are specific for the type of host cell (e.g., bacterial, fungal, plant or animal), where the host cell is the host cell into which the vector is to be introduced and where expression of the nucleic acid of the invention is to occur, typically human CD8+ T cells in the context of the present invention. Furthermore, the vector of the present invention may comprise one or more marker genes, which allow for the selection of transduced or transfected host cells. The recombinant expression vector may comprise a native or preferably heterologous promoter operably linked to the nucleotide sequence encoding the TCR construct of the present invention, or a nucleotide sequence complementary or hybridizing to the nucleotide sequence encoding the construct of the present invention. The choice of promoter includes, for example, strong, weak, inducible, tissue-specific and development-specific promoters. The promoter may be a non-viral promoter or a viral promoter.Preferably, the promoter is a heterologous promoter, i.e., a promoter that is not naturally associated with the TCR in human T cells, such as a long terminal repeat promoter, and is suitable for expression in human T cells. The recombinant expression vectors of the invention can be designed for either transient expression, stable expression, or both. Also, the recombinant expression vectors can be made for constitutive or inducible expression.

[0073] protein The invention also provides proteins, i.e. TCR constructs comprising either alpha or beta chain constructs, or preferably both alpha and beta chain constructs capable of specifically binding to epitopes from the above EBV proteins associated with each MHCI, as described herein, for example a TCR construct specific for the epitope of SEQ ID NO: 1, as described herein. The proteins are encoded by the nucleic acids of the invention.

[0074] As used herein, the terms "capable of specifically binding to" or "recognizing" or "being specific for" a given antigen are synonymous and mean that the TCR construct is capable of specifically binding to and immunologically recognizing said epitope, preferably the epitope is derived from an EBV protein, and more preferably can bind or recognize with high affinity. For example, T cells expressing the TCR are stimulated with low concentrations of each epitope, e.g., about 10, except when no epitope or an irrelevant control peptide epitope is used. -11 mol / L, 10 -10 mol / L, 10 -9 mol / L, 10 -8 mol / L, 10 -7 mol / L, 10 -6 mol / L, 10 -5A TCR may be considered to be "capable of specifically binding" to a peptide derived from an EBV protein if it secretes at least about 200 pg / mL or more (e.g., 250 pg / mL or more, 300 pg / mL or more, 400 pg / mL or more, 500 pg / mL or more, 600 pg / mL or more, 700 pg / mL or more, 1000 pg / mL or more, 2,000 pg / mL or more, 2,500 pg / mL or more, 5,000 pg / mL or more) of interferon gamma (IFN-γ) when co-cultured with target cells pulsed with 100 mol / L of each epitope. Preferably, this is tested using 10,000 TCR+CD8+ T cells and 20,000-50,000, preferably 50,000, target cells expressing the appropriate HLA, e.g., by the assay described in the Examples below. Alternatively, or in addition, a TCR may be considered to have "antigen specificity" for an epitope if T cells expressing the TCR secrete at least twice as much IFN-γ as untransfected background IFN-γ when co-cultured with target cells pulsed with a low concentration of the appropriate peptide. Such "specificity," as described above, may be analyzed, for example, by ELISA.

[0075] As described above for the TCRs of the present invention, high affinity correlates with high peptide sensitivity, e.g., a 50% effective concentration for maximal IFN-γ release of 10 -6 mol / L or less, preferably 10 -7 mol / L or less, 10 -8 mol / L or less or 10 -9 The affinity can be obtained at a peptide concentration of 100 mol / L or less. Alternatively, the affinity can be analyzed by methods well known to those skilled in the art, such as BiaCore. Affinity of TCR or avidity of T cell is considered to be high affinity if it is 100 μM or more, preferably 10 μM or more.

[0076] Based on the defined CDR3 and variable region sequences provided in the present invention, it is possible to carry out affinity maturation of TCR sequences (Chervin et al., 2008; Robbins et al., 2008). Nonsynonymous nucleotide substitutions resulting in amino acid changes in the CDR3 sequence may improve the affinity of the TCR to the target antigen. Furthermore, changes in the TCR sequence in other parts of the variable TRA and TRB regions may modify the affinity of the TCR to the peptide-MHCI complex. This may improve the overall affinity of the TCR to peptide-MHC, but at the risk of nonspecific recognition and increased cross-reactivity (Linette et al., 2013). It is preferred that TCRs different from a given specific sequence maintain their exclusive specificity for a given target antigen, i.e., they do not cross-react and, most importantly, they do not have cross-reactivity with human self-peptides. The potential cross-reactivity of the TCR can be tested against known self-peptides carried on cells with the correct MHC alleles (Morgan et al., 2013). It is therefore preferred that adoptive transfer of T cells expressing the TCR constructs of the present invention has no, or significantly no, side effects on healthy tissue.

[0077] The TCR constructs of the invention may also be provided in the form of a multimeric complex comprising at least two scTCR molecules, each of which is fused to at least one biotin moiety and where the scTCRs are connected by a biotin-streptavidin interaction to allow the formation of the multimeric complex. Also provided are higher order multimeric complexes comprising two or more, for example four, scTCRs of the invention.

[0078] The TCR constructs of the invention may be modified to contain a detectable label, such as, for example, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), a tag such as a HIS tag, an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), or a particle (e.g., a gold particle, or a magnetic particle).

[0079] host cell The present invention also provides a host cell comprising the nucleic acid and / or protein of the present invention. The host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or a protist. The host cell may be a cultured cell or a primary cell, i.e. a cell directly isolated from an organism, such as a human. The host cell may be an adherent cell or a suspension cell, i.e. a cell that grows in suspension. For the purposes of producing recombinant TCRs, polypeptides, or proteins, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. The host cell may be of any cell type, may be from any type of tissue, and may be at any stage of development, although preferred host cells are peripheral blood leukocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). More preferably, the host cell is a T cell or a T cell precursor, in particular a human T cell isolated from a PBMC. The T cell can be any T cell, such as cultured T cells, such as primary T cells or T cells from cultured T cell lines, or T cells taken from a mammal, but preferably T cells or T cell precursors from a human patient. The T cells can be obtained from a number of sources, such as blood, bone marrow, lymph nodes, thymus or other tissues or fluids. The T cells can also be enriched or purified. It can be, for example, tumor infiltrating cells (TIL), effector cells, central effector cells, memory T cells, naive T cells, etc., and preferably central memory T cells. Alternatively, the host cell can be another immune effector cell, such as NK cells or macrophages.

[0080] Preferably, the T cells are human T cells, especially when related to human treatment. The T cells are preferably CD8+ T cells (e.g. cytotoxic T cells). Preferably, the T cells are T cells isolated from a human, e.g. a human patient, especially the patient to be treated. Alternatively, the T cells may be derived from a third-party donor, related or unrelated to the patient. Such T cells may be genetically engineered, for example, by multiple methods, e.g., by knocking out endogenous TCR and / or MHCI. The T cells may be generated from autologous or third-party donor stem cells, and optionally, the stem cells are not human embryonic stem cells.

[0081] Preferably, the host cell is a human CD8+ T cell comprising a nucleic acid of the invention that is an expression vector, wherein the nucleic acid encoding TRA and / or TRB is operably linked to a heterologous promoter, and wherein the host cell expresses the TCR construct of the invention.

[0082] The present invention also provides a host cell expressing two or more different TCR constructs, for example two TCR constructs of the present invention.Preferably, in this context, the TCR constructs may be single chain TCR constructs in which the TCR alpha and beta chains of each TCR are linked by a linker to avoid incorrect association between the recombinant TCR chains.The two single chain TCR constructs may be encoded by a single expression vector.

[0083] Pharmaceutical Compositions The present invention also provides a) a nucleic acid of the invention encoding a TCR construct capable of specifically binding to each epitope associated with each MHCI; or b) a protein of the invention comprising a TCR construct capable of specifically binding to each epitope associated with each MHCI (i.e., a TCR construct of the invention); or c) a host cell of the invention expressing a TCR construct capable of specifically binding to each epitope associated with each MHCI; Preferably, the TCR construct is specific for the epitope of SEQ ID NO:1.

[0084] In another embodiment, the present invention a) at least two nucleic acids each encoding a TCR construct capable of specifically binding to a respective epitope associated with a respective MHCI; or b) at least two proteins, each comprising a TCR construct capable of specifically binding to a respective epitope associated with a respective MHCI; or c) at least two host cells, each expressing a TCR construct capable of specifically binding to each epitope associated with each MHCI; or a kit comprising at least two pharmaceutical compositions, wherein the epitopes are peptides from different antigens expressed by the same cancer or infectious agent. Typically, immunotherapy is performed for the treatment of cancer, and thus the different antigens are preferably antigens expressed by cancer, i.e. by cells of the same cancer.In this context, antigen is a protein that contains an epitope that can be presented by MHCI.Different cancer cells of the same type of cancer each express at least one antigen, and therefore TCR constructs can target different cancer cells of the same cancer, which is useful, for example, when the stage of cancer is unknown and / or when cells of different stages that express different antigens should be treated.However, it is generally beneficial when different antigens are expressed by the same cancer cell.

[0085] The inventors have surprisingly found that using a combination of at least two TCR constructs expressed by a T cell, in particular two or more T cells each expressing a TCR construct capable of specifically binding to a respective epitope associated with a respective MHCI, is advantageous in adoptive T cell therapy.

[0086] Without intending to be bound by theory, it is believed that attacking a cancer (or infectious agent) based on the expression of two or more antigens may help to sustain the attack and avoid immune evasion, for example, by downregulation of one target antigen by mutation or by becoming an antigen-loss mutant.

[0087] It is also possible to target a cancer or infectious agent using three or more TCR constructs, particularly three or more host cells each containing nucleic acid encoding a TCR construct capable of specifically binding to a respective epitope associated with a respective MHCI, where the epitopes are peptides derived from different antigens expressed by the same cancer or infectious agent.

[0088] The kit or pharmaceutical composition is for use in the treatment of cancer or an infectious agent which bears the epitope targeted by the TCR construct.

[0089] The infectious disease or cancer may be, for example, related to EBV. In this case, the different EBV antigens may be LMP2A, LMP1, EBNA1 or EBNA3C. Preferably, one of the EBV antigens targeted by one TCR construct of the present invention is LMP2A. The second EBV antigen targeted by one TCR construct of the present invention may be LMP1 or EBNA3C, preferably LMP1. When three EBV antigens are targeted, the antigens may be LMP2A, LMP1 and EBNA3C. An alternative or additional EBV antigen is EBNA1.

[0090] The present invention also relates to a method for producing a method for manufacturing a pharmaceutical composition comprising the steps of: a) at least two nucleic acids of the invention each encoding a TCR construct capable of specifically binding to each epitope associated with each MHCI; or b) at least two proteins of the invention, each comprising a TCR construct capable of specifically binding to a respective epitope associated with a respective MHCI; or c) at least two host cells of the invention, each expressing a TCR construct capable of specifically binding to each epitope associated with each MHCI; wherein optionally the epitopes are derived from different EBV proteins.

[0091] The one, two or three TCR constructs used in connection with the pharmaceutical composition or kit of the present invention may be the TCR constructs of the present invention as disclosed herein.Preferably, one of the TCR constructs used is the TCR construct disclosed herein that recognizes the epitope of SEQ ID NO: 1 associated with HLA-A2.

[0092] Thus, the kit or pharmaceutical composition may comprise a human CD8+ T cell comprising a nucleic acid encoding a TCR construct of the invention recognizing the epitope of SEQ ID NO: 1 in association with HLA-A2, for example as defined herein, which may be expressed from the nucleic acid under the control of a heterologous promoter.

[0093] In the kit or pharmaceutical composition of the present invention, which comprises at least two nucleic acids encoding TCR constructs, at least two TCR constructs, or at least two host cells each comprising a nucleic acid encoding a TCR construct, one, two or three of the TCR constructs can also be other TCR constructs, such as TCR constructs known in the art.For example, when the cancer is EBV-related, one, two or three of the TCR constructs disclosed in Cho et al., 2018; Jurgens et al., 2006; Zheng et al., 2015; Simpson et al., 2011; Yang et al., 2011; Orentase et al., 2001; Hart et al., 2008; WO 2015 / 022520A1; or WO 2011 / 039508 can be used, optionally in combination with one of the TCR constructs provided herein.

[0094] Adoptive T cell therapy (option c) is preferred throughout the application, where the host cell is a T cell, preferably a human CD8+ T cell. The host cell typically comprises a nucleic acid encoding a TCR construct under the control of a heterologous promoter.

[0095] However, gene therapy (option a) with the nucleic acids of the invention is also possible, for example lentiviral vectors can be used.

[0096] The TCR constructs of the invention in protein form (option b) may also be used in therapy, for example to target a bacterial minicell, which may contain a therapeutic agent such as a toxin, to a cancer, to target a toxin to which it is bound. The TCR constructs of the invention in protein form may also be used to target a diagnostic agent to a cancer. Thus the composition may also be a diagnostic composition.

[0097] The T cells, each expressing its respective TCR construct, may be included in a kit, where each T cell is stored separately, for example in a pharma- ceutically acceptable buffer. The components of the kit of the present invention may be prepared for simultaneous administration, or for administration in any order. The components may also be for repeated administration. Tran et al., 2014 describes possible regimes of administration. Alternatively, they may be mixed before administration and administered together. Alternatively, the T cells, each expressing its respective TCR construct, may be included in a single pharmaceutical composition. This is also true for the nucleic acid or protein of the present invention.

[0098] The pharmaceutical composition or kit of the present invention is typically for intravenous injection.They may further comprise a pharma- ceutically acceptable carrier, such as a buffer, for example, physiological saline or PBS.They may further comprise excipients, such as stabilizers, such as SPGA, carbohydrates (sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins, such as albumin or casein, or protein-containing agents, such as bovine serum or skim milk.

[0099] T cells are typically 1x10 per kg 5 -1x10 9 The cells are administered to patients at a concentration of approximately 1x10 cells per dose. 6 -1x10 11 The cells are administered to the patient. These parameters can be adapted by the physician, for example, depending on the age, sex, weight and medical condition of the patient. For example, the protocol disclosed in Doran et al., 2019 can be adapted for use with the host cells of the present invention.

[0100] The pharmaceutical composition of the invention or the kit comprising the pharmaceutical composition or the kit of the invention is for use in treating patients expressing MHC in the context of each TCR recognizing each epitope, e.g. as disclosed herein. The average HLA (MHC I) distribution in different populations can be found, e.g., at http: / / allelefrequencies.net / . It is advantageous to test whether the patient expresses each MHC I before treatment.

[0101] The patient may have cancer or infectious disease, particularly in the context of the specific TCR constructs disclosed herein, EBV-associated disease, such as Hodgkin's and non-Hodgkin's lymphoma, Burkitt's lymphoma, hemophagocytic lymphohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, gastric cancer, lung cancer, hairy leukoplakia, post-transplant lymphoproliferative disease and central nervous system lymphoma.Preferably, the EBV-associated cancer is a type II malignant tumor, exemplified by Hodgkin's lymphoma and nasopharyngeal carcinoma, or a type III malignant tumor, exemplified by hairy leukoplakia, post-transplant lymphoproliferative disease and central nervous system lymphoma (Orentas et al., 2001), which typically express high levels of LMP2A and LMP1. In type III malignant tumors, EBNA2C is additionally expressed.

[0102] The target cancer cells express a protein, or optionally a protein which gives rise to the epitope to be recognised, preferably expressed by a majority of the cancer cells.

[0103] The patient is typically a mammalian patient. The patient may be a mouse, but preferably the patient is a human patient.

[0104] The present invention also discloses a method for treating cancer or an infectious disease, such as an EBV-associated disease, preferably an EBV-associated cancer, by administering an effective amount of the pharmaceutical composition or kit of the present invention to a patient in need thereof, such as a patient having said cancer or disease.

[0105] The pharmaceutical composition and kit of the present invention can be used in combination with other agents, particularly other anti-cancer agents.For example, other anti-cancer agents can be TCR-transduced T cells expressing TCR specific to other antigens (e.g., MAGE, NY-ESO) expressed in EBV-positive tumors, checkpoint inhibitors, or other immunotherapies such as antibodies, small molecule inhibitors, or other types of reagents.

[0106] One preferred medical use of the present invention is immunotherapy, preferably adoptive T cell therapy.The product and method of the present invention are particularly useful in the context of adoptive T cell therapy.The administration of the compound of the present invention can involve, for example, administration, including infusion, of the T cells of the present invention to said patient.Preferably, such T cells are autologous T cells of the patient that are introduced with the nucleic acid of the present invention in vitro.

[0107] Alternatively, nucleic acids, particularly expression vectors, of the invention may be administered to a patient for in vivo transduction of T cells.

[0108] The protein TCR constructs of the present invention can also be used for diagnostic purposes, such as to determine whether a subject expresses each protein, and in particular whether the epitope recognized by the TCR construct is presented in the context of MHCI.For this purpose, such constructs are preferably labeled to facilitate detection.Preferably, the patient who presents such epitopes on each MHCI is treated by adoptive T cell therapy of the present invention.

[0109] The present invention also relates to a method for preparing a host cell of the invention, which comprises introducing an expression vector encoding a TCR construct of the invention into a suitable host cell, preferably a human CD8+ T cell isolated from a patient.

[0110] The present invention is further described in the following examples with reference to the accompanying figures and sequences, but is not limited thereto. For the purposes of the present invention, all documents cited herein are hereby incorporated by reference in their entirety as part of this specification.

[0111] vaccine In one embodiment, the present invention provides a pharmaceutical composition, particularly a vaccine composition, comprising a peptide or a nucleic acid (such as RNA) encoding such a peptide, which comprises the newly identified epitope herein capable of being presented by human MHCI. a) has at least 88% sequence identity to SEQ ID NO:3, the epitope being capable of being presented on HLA-C*15:02, and the peptide comprises up to 25, preferably up to 11, consecutive amino acids identical to a sequence of amino acids occurring in LMP1 of SEQ ID NO:120; b) having at least 88% sequence identity to SEQ ID NO: 4, the epitope being capable of being presented on HLA-C*06:02, and the peptide comprising up to 25, preferably up to 11, consecutive amino acids identical to a sequence of amino acids occurring in EBNA3C of SEQ ID NO: 121; and c) has 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 contains up to 25, preferably up to 11, consecutive amino acids identical to the sequence of amino acids occurring in EBNA3C of SEQ ID NO:121. Preferably, the peptide in a) can be specifically recognized by TCR83 as defined herein. Preferably, the peptide in b) can be specifically recognized by TCR64 as defined herein. Preferably, the peptide in c) can be specifically recognized by TCR25 or 58 as defined herein.

[0112] Peptide vaccination is well known in the art.For example, peptide vaccine can be used for administering to subjects in combination with adjuvants such as aluminum salt, for example aluminum phosphate or aluminum hydroxide, squalene, for example MG59, liposome, for example QS21 or monophosphoryl lipid A.

[0113] Such a pharmaceutical composition of the invention may comprise a peptide comprising an epitope of SEQ ID NO:3, wherein the epitope may be presented on HLA-C*15:02, and wherein the peptide comprises up to 25, up to 15, preferably up to 11 amino acids identical to a sequence of amino acids occurring in the amino acid sequence occurring in LMP1 of SEQ ID NO:120.

[0114] Such a pharmaceutical composition of the invention may comprise a peptide comprising an epitope of SEQ ID NO:2, wherein the epitope may be presented on HLA-B*57:01, and wherein the peptide comprises up to 25, up to 15, preferably up to 11 consecutive amino acids identical to a sequence of amino acids occurring in the amino acid sequence occurring in LMP1 of SEQ ID NO:120.

[0115] Such a pharmaceutical composition of the present invention may comprise a peptide comprising an epitope of SEQ ID NO:5, wherein the epitope may be presented on HLA-B*44:02, and wherein the peptide comprises up to 25, up to 15, preferably up to 11 consecutive amino acids identical to an amino acid sequence occurring in the amino acid sequence occurring in EBNA3C of SEQ ID NO:121.

[0116] Nucleic acids encoding epitope-containing peptides, as defined herein, may also be used in combination with a suitable adjuvant, such as, for example, liposomes or CpG nucleotides.

[0117] Such a pharmaceutical composition of the invention may comprise a nucleic acid encoding a peptide comprising an epitope of SEQ ID NO:3, wherein the epitope may be presented on HLA-C*15:02, and wherein the peptide comprises up to 25, up to 15, preferably up to 11 amino acids identical to a sequence of amino acids occurring in LMP1 of SEQ ID NO:120.

[0118] Such a pharmaceutical composition of the present invention may comprise a nucleic acid encoding a peptide comprising an epitope of SEQ ID NO:4, wherein the epitope may be presented on HLA-C*06:02, and wherein the peptide comprises up to 25, up to 15, or preferably up to 11 consecutive amino acids identical to a sequence of amino acids occurring in EBNA3C of SEQ ID NO:121.

[0119] Such a pharmaceutical composition of the present invention may comprise a nucleic acid encoding a peptide comprising an epitope of SEQ ID NO:5, wherein the epitope may be presented on HLA-B*44:02, and wherein the peptide comprises up to 25, up to 15, preferably up to 11 consecutive amino acids identical to a sequence of amino acids occurring in EBNA3C of SEQ ID NO:121.

[0120] Any of the above vaccine pharmaceutical compositions may be for use in vaccination against EBV-associated disease selected from the group including Hodgkin's and non-Hodgkin's lymphoma, Burkitt's lymphoma, hemophagocytic lymphohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, gastric cancer, hairy leukoplakia, post-transplant lymphoproliferative disease and central nervous system lymphoma. This vaccination may be a vaccination, i.e. a vaccination provided to a subject that does not yet have the disease or is not infected with EBV, with the intention of reducing the risk of the subject developing the disease if contacted with EBV. The subject may belong to a risk group for the disease, for example, a subject that has been contacted with EBV or identified as having a current EBV infection, but does not (yet) have the disease. Alternatively, this vaccination may be a therapeutic vaccination. Patients with EBV-associated disease may be treated with a therapeutic vaccination.

[0121] Advantageously, the HLA of the subject or patient is known and the vaccine is administered to the patient capable of presenting an epitope on an MHCI molecule, for example a) for an epitope as defined in patients with HLA-C*15, in particular HLA-C*15:02, b) for an epitope as defined in patients with HLA-B*57, in particular HLA-B*57:01, or c) for an epitope as defined in patients with HLA-B*44, in particular HLA-B*44:02. [Brief description of the drawings]

[0122] Legend [Figure 1]Figure 1. Detection of T cell responses using the MHC class I K562 cell library. T cells grown on dendritic cells expressing EBV antigens were co-cultured with K562 cells from the MHC cell library. Screening of immunogenic EBV antigen-HLA combinations was performed by (A) analysis of CD137 expression and (B) measuring the amount of secreted IFN-γ by ELISA. (C) FACS sorting of CD137 positive T cells (11%) reacting to K562-HLA-B*57:01 positive cells was used to identify the dominant TCRα and TCRβ chains (min: no antigen stimulation, max: non-specific antigen stimulation, us: unstained T cells). This approach was applied to the identification and isolation of all further TCRs described herein.

[0123] [Figure 2-1] Figure 2. TCR genetic analysis. (A) Next generation sequencing-based TCR repertoire analysis of FACS-sorted T cells responding to EVB antigen-positive (LMP1 / LMP2A / EBNA1) K562-HLA-B*57:01 cells. Percentage of total reads assigned to each sequence cluster with representative sequences shown on the y-axis. TCRα and TCRβ chains with frequencies >10% were used to construct single-chain TCR-retroviruses and functional TCRs were identified. [Figure 2-2] (B) The different V-segments of the most predominant TCRα (TRAV) and TCRβ (TRBV) in one TCR (designated TCR50) are shown with their frequency and the sequences of the CDR3 regions (IMGT nomenclature) (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 for all further TCRs described herein.

[0124] [Diagram 3]Figure 3. Identification of functional TCRαβ chain combinations. TCRα and TCRβ chains with frequencies above 10% were used to construct single-chain TCR-retroviruses to identify functional TCRs. For TCR50, TCR50TRAV8-2*01 and TRBV9*01, which form functional TCRs as TCR-transduced T cells, specifically recognize one antigen (LMP1) in combination with one HLA (B*57:01). Other TCRα and TCRβ chain combinations were non-specific antigen recognition. This combinatorial approach to identify functional TCRα and TCRβ chain combinations, shown here as an example for TCR50, was applied to all further TCRs described herein (minimum: no antigen stimulation, maximum: non-specific antigen stimulation).

[0125] [Figure 4] Figure 4. Epitope mapping to identify antigenic peptides recognized by TCR50. (A) Truncated forms of the full-length LMP1 antigen (LMP1 / 2, LMP1 / 1) were generated and expressed in K562-HLA-B*57:01 cells. (B) The antigenic region with the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant by ELISA, and the region was located between nucleotides (nt) 316 and 624 (min: no antigen stimulation, max: nonspecific antigen stimulation, UT: untransduced T cells).

[0126] [Figure 5-1] Figure 5. Identification of immunogenic epitopes of LMP1. The protein region (LMP1 / 2 nt 316-624) identified as an epitope positive sequence was used to select candidate peptides by applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*57:01. (A) According to peptide-MHCI binding affinity (binding level), 13 peptides classified as strong binding (SB) and weak binding (WB) were used for epitope identification, respectively. [Figure 5-2](B) K562-HLA-B*57:01 cells were loaded with selected peptides and co-cultured with TCR50-transduced T cells from two donors, and IFN-γ secretion was measured by ELISA. Four epitopes (highlighted in A) with the following amino acid sequences were recognized by TCR50-transduced T cells: IALYLQQNWW (SEQ ID NO: 108), IALYLQQNW (SEQ ID NO: 2), IIALYLQQNW (SEQ ID NO: 110), ALYLQQNWW (SEQ ID NO: 116). The sequence numbers of the analyzed peptides are shown in Table 1 below (minimum: no antigenic stimulation, maximum: non-specific stimulation, SB: strong binding, WB: weak binding, UT: non-transduced T cells).

[0127] [Figure 6] Figure 6. Peptide titration of TCR50-transduced T cells. Untransduced (UT) and TCR50-transduced (TCR50) T cells from two donors were co-cultured with K562-HLA-B*57:01 cells carrying titrated amounts of the indicated peptides (SEQ ID NO:2, 108, 110, 116) and the amount of secreted IFN-γ in the supernatant was measured by ELISA. Peptide IALYLQQNW (9mer, SEQ ID NO:2) was recognized at the lowest concentration and is therefore considered to be the cognate EBV LMP1 epitope of TCR50. Interestingly and worth mentioning, this epitope recognized by TCR50 is not ranked at the top by the NetMHCpan4.0 prediction tool, suggesting that the prediction tool is not accurate in predicting relevant immunodominant epitopes.

[0128] [Figure 7]Figure 7. TCR50-transduced T cells recognize LMP1-positive cells. Functional analysis of LMP1-specific TCR50-transduced T cells from two donors using K562-HLA-B*57:01 antigen-bearing cells, 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 IFN-γ secreted by ELISA at an effector-to-target (E:T) cell ratio of 1:1 (minimum: no antigen stimulation, maximum: nonspecific stimulation, UT: nontransduced T cells).

[0129] [Figure 8-1] Figure 8. Functional analysis of EBNA3C-reactive TCR01. (A) EBNA3C-specific TCR01-transduced T cells were co-cultured with antigen-pulsed K562-HLA-B*07:02 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 forms of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were generated and expressed in K562-HLA-B*07:02 cells. [Figure 8-2] (C) The antigen region with the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant by ELISA, and the region is located between nucleotides (nt) 2071 and 2979. (D) The protein region identified as the epitope positive sequence (EBNA3C nt 2071-2979) was used to select candidate peptides by applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*07:02. [Figure 8-3](E) According to the peptide-MHCI binding affinity (binding level), 27 peptides were classified into strong binding (SB) and weak binding (WB) epitopes (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), and ), 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 selected peptides, co-cultured with TCR01-transduced T cells, and IFN-γ secretion was measured by ELISA. Epitopes (highlighted in D) recognized by TCR01-transduced T cells. (F) K562-HLA-B*07:02 cells were loaded with titrated amounts of the indicated peptides or no peptide control, and peptide sensitivity of TCR01-transduced T cells was determined by measuring the amount of IFN-γ secreted by ELISA at a 1:1 effector-to-target (E:T) cell ratio (minimum: no antigen stimulation; maximum: nonspecific stimulation; SB: strong binding; WB: weak binding; UT: untransduced T cells).

[0130] [Figure 9-1]Figure 9. Functional analysis of EBNA3C-reactive TCR25. (A) EBNA3C-specific TCR25-transduced T cells were cocultured with antigen-pulsed K562-HLA-B*44:02 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 forms of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were generated and expressed in K562-HLA-B*44:02 cells. (C) The antigenic region with the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant by ELISA, and the region is located between nucleotides (nt) 1 and 567. (D) Protein regions identified as epitope positive sequences (EBNA3C nt 1-567) were used to select candidate peptides by applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*44:02. [Figure 9-2] (E) Four peptides, classified as strong (SB) and weak (WB) binding according to peptide-MHCI binding affinity (binding level), were used to identify epitopes (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 selected peptides, co-cultured with TCR25-transduced T cells, and IFN-γ secretion was measured by ELISA. One epitope with amino acid sequence AEGGVGWRHW (highlighted in D) was recognized by TCR25-transduced T cells and therefore is considered to be the cognate EBV EBNA3C epitope of TCR25. (F) K562-HLA-B*44:02 cells were loaded with titrated amounts of the indicated peptides or no peptide control. Peptide sensitivity of TCR25-transduced T cells was determined by measuring the amount of IFN-γ secreted by ELISA at a 1:1 effector-to-target (E:T) cell ratio (minimum: no antigen stimulation; maximum: nonspecific stimulation; SB: strong binding; WB: weak binding; UT: untransduced T cells).

[0131] [Figure 10-1] Figure 10. Functional analysis of EBNA3C-reactive TCR27. (A) EBNA3C-specific TCR27-transduced T cells were co-cultured with antigen-pulsed K562-HLA-B*07:02 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 forms of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were generated and expressed in K562-HLA-B*07:02 cells. [Figure 10-2] (C) The antigenic region with the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant (ELISA) and is located between nucleotides (nt) 2071 and 2979. (D) The protein region identified as the epitope positive sequence (EBNA3C nt 2071-2979) was used to select candidate peptides by applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*07:02. [Figure 10-3] (E) Twenty-seven peptides, classified as strong (SB) and weak (WB) binding according to peptide-MHCI binding affinity (binding level), were used for epitope identification. K562-HLA-B*07:02 cells were loaded with selected peptides and co-cultured with TCR27-transduced T cells, and the amount of IFN-γ secreted was measured by ELISA. The epitopes (highlighted in D) were recognized by TCR27-transduced T cells. (F) K562-HLA-B*07:02 cells were loaded with titrated amounts of the indicated peptides or no peptide as a control, and the peptide sensitivity of TCR27-transduced T cells was determined by measuring the amount of IFN-γ secreted by ELISA at an effector-to-target (E:T) cell ratio of 1:1 (minimum: no antigen stimulation, maximum: nonspecific stimulation, SB: strong binding, WB: weak binding, UT: non-transduced T cells, see legend of FIG. 8 for sequence numbers).

[0132] [Figure 11-1]Figure 11. Functional analysis of EBNA3C-reactive TCR58. (A) EBNA3C-specific TCR58-transduced T cells were cocultured with antigen-pulsed K562-HLA-B*44:02 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 forms of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were generated and expressed in K562-HLA-B*44:02 cells. (C) The antigenic region with the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant by ELISA, and the region is located between nucleotides (nt) 1 and 567. (D) Protein regions identified as epitope positive sequences (EBNA3C nt 1-567) were used to select candidate peptides by applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*44:02. [Figure 11-2] (E) Four peptides, classified as strong binding (SB) and weak binding (WB) according to peptide-MHCI binding affinity (binding level), were used for epitope identification, respectively. K562-HLA-B*44:02 cells were loaded with selected peptides and co-cultured with TCR58-transduced T cells, and IFN-γ secretion was measured by ELISA. One epitope with the amino acid sequence AEGGVGWRHW (highlighted in D) was recognized by TCR58-transduced T cells and thus is considered to be the cognate EBV EBNA3C epitope of TCR58. (F) K562-HLA-B*44:02 cells were loaded with titrated amounts of the indicated peptides or no peptide control. Peptide sensitivity of TCR58-transduced T cells was determined by measuring the amount of IFN-γ secreted by ELISA at an effector to target (E:T) cell ratio of 1:1 (minimum: no antigen stimulation; maximum: nonspecific stimulation; SB: strong binding; WB: weak binding; UT: non-transduced T cells; see legend of Figure 9 for sequence numbers).

[0133] [Figure 12-1]Figure 12. Functional analysis of EBNA3C-reactive TCR64. (A) EBNA3C-specific TCR64-transduced T cells were co-cultured with antigen-pulsed K562-HLA-C*06:02 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 forms of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were generated and expressed in K562-HLA-C*06:02 cells. [Figure 12-2] (C) The antigen region with the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant by ELISA, and the region is located between nucleotides (nt) 568 and 1569. (D) The protein region identified as the epitope-positive sequence (EBNA3C nt 568-1569) was used to select candidate peptides by applying the epitope prediction algorithm NetMHCpan4.0 for HLA-C*06:02. [Figure 12-3](E) According to the peptide-MHCI binding affinity (binding level), 27 peptides were classified into strong binding (SB) and weak binding (WB) epitopes (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), RRRRGACVV (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), RRGACVVYD (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 selected peptides, co-cultured with TCR64-transduced T cells, and IFN-γ secretion was measured by ELISA. One epitope (highlighted in D) with amino acid sequence FRKAQIQGL was recognized by TCR64-transduced T cells and is therefore considered to be the cognate EBV EBNA3C epitope of TCR64. (F) K562-HLA-C*06:02 cells were loaded with titrated amounts of the indicated peptides or no peptide control, and peptide sensitivity of TCR64-transduced T cells was determined by ELISA measuring the amount of IFN-γ secreted at a 1:1 effector-to-target (E:T) cell ratio (min: no antigen stimulation, max: nonspecific stimulation, SB: strong binding, WB: weak binding, UT: nontransduced T cells).

[0134] [Figure 13-1] Figure 13. Functional analysis of LMP1-reactive TCR83. (A) LMP1-specific TCR83-transduced T cells were cocultured with antigen-pulsed K562-HLA-C*15:02 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 forms of the full-length LMP1 antigen (LMP1 / 2, LMP1 / 1) were generated and expressed in K562-HLA-C*15:02 cells. (C) The antigen region with the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant by ELISA, and the region is located between nucleotides (nt) 316 and 624. (D) The protein region (LMP1nt 316-624) identified as an epitope positive sequence was used to select candidate peptides by applying the epitope prediction algorithm NetMHCpan4.0 for HLA-C*15:02. [Figure 13-2] (E) Four peptides classified as weak binders (WB) according to peptide-MHCI binding affinity (binding level) were used to identify epitopes (NSNEGRHHL (SEQ ID NO: 184), QQNWWTLLV (SEQ ID NO: 3), DSLPHPQQA (SEQ ID NO: 185), and YLQQNWWTL (SEQ ID NO: 186)). K562-HLA-C*15:02 cells were loaded with selected peptides, co-cultured with TCR83-transduced T cells, and IFN-γ secretion was measured by ELISA. One epitope with amino acid sequence QQNWWTLLV (highlighted in D) was recognized by TCR83-transduced T cells and is therefore considered to be the cognate EBV LMP1 epitope of TCR83. (F) K562-HLA-C*15:02 cells were loaded with titrated amounts of the indicated peptides or no peptide control. Peptide sensitivity of TCR83-transduced T cells was determined by measuring the amount of IFN-γ secreted by ELISA at an effector-to-target (E:T) cell ratio of 1:1 (B) (minimum: no antigen stimulation; maximum: nonspecific stimulation; WB: weak binding; UT: untransduced T cells).

[0135] [Figure 14-1] Figure 14. Functional analysis of LMP2A-reactive TCR06. (A) LMP2A-specific TCR06-transduced T cells were co-cultured with antigen-pulsed K562-HLA-A*02:01 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 forms of the full-length LMP2A antigen (LMP2A / 2, LMP2A / 1) were generated and expressed in K562-HLA-A*02:01 cells. [Figure 14-2](C) The antigenic region with the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant (ELISA) and is located between nucleotides (nt) 1006 and 1494. (D) The protein region identified as the epitope positive sequence (LMP1 nt 1006-1494) was used to select candidate peptides by applying the epitope prediction algorithm NetMHCpan4.0 for HLA-A*02:01. (E) Fourteen peptides classified according to peptide-MHCI binding affinity (binding level) into strong binding (SB) and weak binding (WB) were used to identify the epitopes (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 selected peptides, co-cultured with TCR06-transduced T cells, and IFN-γ secretion was measured by ELISA. The epitopes (highlighted in D) were recognized by TCR06-transduced T cells and therefore are considered to be the cognate EBV LMP2A epitopes of TCR06. Interestingly and worth mentioning, the two epitopes recognized by TCR06 were not ranked at the top by the NetMHCpan4.0 prediction tool, suggesting that the prediction tool is not accurate in predicting relevant immunodominant epitopes. [Figure 14-3](F) K562-HLA-A*02:01 cells were loaded with titrated amounts of the indicated peptides or no peptide control. Peptide sensitivity of TCR06-transduced T cells was determined by measuring the amount of IFN-γ secreted by ELISA at an effector-to-target (E:T) cell ratio of 1:1 (B) (min: no antigen stimulation; max: nonspecific stimulation; SB: strong binding; WB: weak binding; UT: untransduced T cells).

[0136] [Figure 15] Figure 15. Comparison of peptide sensitivity of LMP2A-specific TCR06 with the TCRs provided in patents WO2015 / 022520A1 (PUBTCR1) and WO2011 / 039508A2 (PUBTCR2). The previously published wt EBV LMP2A TCR with the TRAV12-3*01 / TRAJ41*01 / TRAC alpha chain amino acid sequence (SEQ ID NO:2 in WO 2015 / 022520A1) and the TRBV11-2*01 / TRBD1 / TRBJ2-7 / TRBC beta chain amino acid sequence (SEQ ID NO:3 in WO 2015 / 022520A1), also shown in Figure 1 of said document, is herein named PUBTCR1. The previously published LMP2A TCR described in WO 2011 / 039508A2, especially based on its SEQ ID NO:8, is named PUBTCR2. K562-HLA-A*02:01 cells were loaded with titrated amounts of the indicated peptides CLGGLLTMV and MCLGGLLTMV, respectively, and co-cultured with TCR06-, PUBTCR1-, or PUBTCR2-transduced T cells, and the 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. Compared with PUBTCR1 and PUBTCR2, TCR06 has the highest peptide sensitivity. UT: non-transduced T cells.

[0137] [Figure 16]Figure 16. Further comparison of peptide sensitivity of LMP2A-specific TCR06 with the TCRs provided in patents WO 2015 / 022520A1 (PUBTCR1) and WO 2011 / 039508A2 (PUBTCR2) and the TCR-JC isolated in the present application. The previously published wt EBV LMP2A TCR with the TRAV12-3*01 / TRAJ41*01 / TRAC alpha chain amino acid sequence (SEQ ID NO:2 in WO 2015 / 022520A1) and the TRBV11-2*01 / TRBD1 / TRBJ2-7 / TRBC beta chain amino acid sequence (SEQ ID NO:3 in WO 2015 / 022520A1), also Figure 1 of the above documents, is herein designated PUBTCR1. The LMP2A TCR described previously in WO 2011 / 039508A2, specifically based on its SEQ ID NO:8, was named PUBTCR2. Another LMP2A-specific TCR, named herein TCR-JC, was isolated in this application. (A) K562-HLA-A*02:01 cells were loaded with titrated amounts of peptide CLGGLLTMV and co-cultured with TCR06- or PUBTCR1- or PUBTCR2- or TCR-JC-transduced 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 curve (constrained model) based on the data points in A. Sigmoidal regression shows very good R2 (approximately 0.95) for TCR06, PUBTCR1, and PUBTCR2. Data from TCR-JC showed a model fit of 0.81 (lacking an upper plateau). (C) Based on the data shown in B, peptide sensitivity of the four TCRs was calculated and an EC50 of 10-9M was shown (EC50: mol / l of peptide required to reach 50% of maximum IFN-γ release, SEM: standard error).

[0138] [Figure 17]Figure 17. EBV-specific TCR-transduced T cells kill cancer cells. LMP1-specific TCR50, LMP2A-specific TCR06, and EBNA3C-specific TCR64-transduced T cells were co-cultured with L591 EBV+ tumor cells that naturally express LMP1, LMP2A, and EBNA3C and were transduced with the respective MHCI alleles. At the indicated effector-to-target (E:T) cell ratios, data were averaged from triplicate wells and the percentage of viable cells was calculated relative to values ​​obtained from samples co-cultured with untransduced T cells: % specific viability = 100 x (test value) / (background mean). UT: untransduced T cells.

[0139] [Figure 18] Figure 18. In vivo mouse model of tumor rejection. (A) NOG mice were injected subcutaneously with 5x106 K562-HLA-A*02:01 tumor cells and iv with 1x107 TCR06-transduced T cells 24 hours later. Data from individual mice (n=10) receiving TCR06-transduced T cells from two donors are shown compared to non-transduced T cells (n=6). (B) NSG mice were injected subcutaneously with 5x106 K562-HLA-B*57:01 tumor cells and iv with 5x106 TCR50-transduced T cells 9 days later. Tumor size was measured and calculated in both models using calipers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0140] Generation of EBV-specific TCR constructs and recombinant T cells and identification of epitopes. Using an innovative approach (Lorenz et al., 2018; WO2016 / 146618A1), the inventors generated EBV-specific TCRs that recognize immunogenic EBV epitopes endogenously processed and presented by distinct MHC class I molecules.

[0141] Briefly, after selecting an EBV antigen of interest, e.g., LMP1, LMP2A or EBNA3C, the following experimental steps are performed: (i) pulsing professional antigen presenting cells, preferably dendritic cells (DCs), with in vitro transcribed (ivt) RNA encoding the full-length sequence of a selected EBV antigen to stimulate autologous T cells: this process is completely unbiased, allowing the DCs to select the best epitopes of the antigen for expression, processing and presentation on the cell surface in association with the most suitable MHC class I (MHCI) molecules. (ii) Identifying EBV antigen-reactive T cells. This step is carried out by a method using a newly established MHC class I cell library consisting of a cell line expressing a single MHCI derived from the K562 cell line. This part is the basis for utilizing a wide range of MHC diversity, and is therefore an important feature of the TCR isolation approach. To identify each TCR, we selected six MHCIs from the K562 cell library that correspond to the MHCI alleles of the T cell donor, and transfected these cells with the antigen used for priming in step (i). After co-culture of antigen-presenting K562 cells with antigen-stimulated T cells, reactive T cells were identified by measuring interferon (IFN) gamma release using ELISA and upregulation of T cell activation marker CD137 by flow cytometry. Subsequently, reactive CD8+ T cells were enriched by FACS sorting. (iii) isolating total RNA from FACS-sorted CD8+ T cells and amplifying TCRα and TCRβ chain-specific sequences by PCR. Identifying the predominant TCRα and TCRβ sequences by next generation sequencing. (iv) Re-expressing the predominant (at least 10%) TCRα and TCRβ chain combinations in primary human T cells using the γ-retroviral vector MP71 (Engels et al., 2003; Leisegang et al., 2008; Sommermeyer and Uckert, 2010) and identifying functional TCRαβ chain combinations. This is done by co-culturing TCR-transduced T cells with K562 cells carrying the appropriate MHCI molecules and expressing full-length EBV antigens. Antigen-recognizing TCRαβ chain combinations were linked with P2A elements and recloned into the MP71 vector in the TCRβ gene-P2A-TCRα gene configuration. To enhance the association of the recombinant TCR chains, the constant TCRαβ chain regions were replaced with their corresponding mouse regions. This complete TCR gene transfer cassette was subsequently codon-optimized. (v) Identifying the antigenic peptides (epitopes) of EBV recognized by TCR-transduced T cells. For this purpose, the C- or N-terminus of the full-length antigen was truncated, cloned into the plasmid vector pcDNA3.1(-) and expressed in K562 cells carrying the appropriate MHCI molecule. The remaining protein fragments were then tested for their ability to still present epitopes recognized by TCR. Finally, candidate peptides of the corresponding protein regions were identified by the epitope prediction algorithm NetMHCpan4.0 (http: / / www.cbs.dtu.dk / services / NetMHCpan / ). The predicted peptides were generated and loaded onto K562 cells carrying the appropriate MHCI molecule, and examined by co-culture tests for their recognition by TCR-transduced T cells. The peptides most capable of stimulating IFNγ production are considered to be cognate epitopes.

[0142] [Table 1] Evaluation of the generated TCR constructs is shown in Tables 2, 3 and 4 below.

[0143] [Table 2] TCR06, TCR50 and TCR83, especially TCR06, showed high peptide sensitivity (50% effective concentration for maximum IFN-γ release).

[0144] [Table 3]

[0145] [Table 4]

[0146] Functional evaluation The generated TCR-transduced T cells were functionally assessed by in vitro assays.

[0147] First, we analyzed the peptide sensitivity (50% effective concentration for maximal IFNγ release) of isolated TCRs by peptide titration experiments (see Figure 6 and Lorenz et al., 2018). -5 mol / l to 10 -13 Titrated amounts of peptides in the mol / l range were loaded onto K562 target cells. Co-culture assays were performed with TCR-transduced T cells at an effector to target (E:T) cell ratio of 1:1 (unless otherwise indicated), i.e., 2.5x10 4 2.5x10 TCR-transduced T cells 4 The TCR-transduced T cells were co-cultured with peptide-bearing K562 cells. The ability of the TCR-transduced T cells to recognize their target epitopes was assessed 24 hours later by IFN-γ ELISA.

[0148] Second, target cell lines expressing EBV antigens (LCL and peptide-loaded K562 cells, which are EBV-associated cancer cell lines that endogenously process and present EBV epitopes) were applied in co-culture experiments to measure the amount of IFN-γ released by TCR-transduced T cells (see Figure 7 and Lorenz et al., 2018). Briefly, TCR-transduced T cells and target cells (2 × 10 4(1000 cells) 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 a positive control, and non-transduced T cells were used as a negative control.

[0149] Following the isolation and evaluation steps described and the identification of epitopes recognized by the TCR-transduced T cells, the inventors isolated a total of eight EBV-specific TCRs and the associated immunogenic peptides (epitopes) recognized by those TCRs. Some epitopes were already known in the art, whereas others were newly identified herein, in particular the peptides of SEQ ID NOs: 3, 4 and 5, which are presented by the respective MHCI molecules.

[0150] Third, EBV antigen-expressing L591 tumor cells, which endogenously process and present LMP1, LMP2A, and EBNA3C epitopes and are transfected with the relevant MHCI alleles, were used in coculture experiments with TCR-transduced T cells to measure their cytotoxicity by the TCR-transduced T cells.

[0151] Furthermore, the generated TCR-transduced T cells were functionally assessed by in vivo assays, using two immunodeficient mouse models (NOD, NSG): tumor cells expressing both relevant EBV and MHCI molecules were injected subcutaneously into the animals, and TCR-transduced T cells were transferred into the mice by tail vein injection.

[0152] TCR Combinations for Immunotherapy Combining TCR-transduced T cells that recognize different epitopes presented by separate MHCI molecules is an interesting option to improve TCR gene therapy. This approach aims to overcome and prevent two challenges of immunotherapy: (i) tumor overgrowth due to mutants that lose specific tumor antigens, and (ii) tumor immune evasion by downregulating specific MHCI molecules. Using such a combinatorial approach, the efficiency of TCR gene therapy can be improved.

[0153] To demonstrate this concept, two or three TCRs were selected that can recognize EBV antigens endogenously processed and naturally presented by different MHCI molecules, e.g., TCR06 (LMP2A, MHC A*02:01), TCR50 (LMP1, MHC B*57:01), and optionally, TCR64 (EBNA3C, MHC C*06:02). TCR-transduced T cells were generated by retroviral transduction with these TCRs as described. Tumor cells that naturally express the EBV antigens recognized by the TCR and carry each MHCI molecule (A*02:01, B*57:01, C*06:02) are used for co-culture experiments with TCR-transduced T cells.

[0154] Alternatively, such cells (eg, K562 cells) are generated by transfection of the corresponding EBV antigen genes and the MHCI gene.

[0155] In the experiments, TCR-transduced T cells were used independently or in combination and co-cultured with tumor cells at an E:T ratio of 2:1. To evaluate the effect of applying TCR-transduced T cells alone and in combination, IFN-γ secretion was measured by ELISA.

[0156] In addition, cytotoxicity assays were used to analyze the tumor cytotoxicity of TCR-transduced T cells applied alone and in combination.

[0157] In further experiments, an in vivo mouse model was established. Tumor cells expressing the respective EBV antigens and MHCI molecules, such as K562 cells, were transfected into NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl After tumors are palpable, TCR-transduced T cells are injected intravenously (iv) either alone or in combination, and the efficacy of TCR gene therapy in terms of tumor rejection is determined. The present invention may provide the following aspects. [Section 1] A nucleic acid encoding a TCR construct, a TCR alpha chain construct (TRA) and / or a TCR beta chain construct (TRB), specific for an epitope complexed with human MHCI, wherein the epitope is an epitope of an Epistein-Barr Virus (EBV) protein, a) the epitope has the sequence of SEQ ID NO:1, MHCI is HLA-A*02:01, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:13, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:18; b) the epitope has the sequence of SEQ ID NO:2, MHCI is HLA-B*57:01, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:23, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:28; c) the epitope has the sequence of SEQ ID NO:3, MHCI is HLA-C*15:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:33, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:38; d) the epitope has the sequence of SEQ ID NO:4, MHCI is HLA-C*06:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:43, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:48; e) the epitope has the sequence of SEQ ID NO:5, MHCI is HLA-B*44:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:53, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:58; f) the epitope has the sequence of SEQ ID NO:5, MHCI is HLA-B*44:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:63, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:68; g) the epitope has the sequence of SEQ ID NO:6, MHCI is HLA-B*07:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:73, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:78; and / or h) A nucleic acid wherein the epitope has the sequence of SEQ ID NO:6, MHCI is HLA-B*07:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:83, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:88. [Section 2] the epitope has the sequence of SEQ ID NO:1, MHCI is HLA-A*02:01, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:13, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:18; Optionally, wherein 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 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; Most preferably, the nucleic acid of item 1, wherein TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 15, and TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 20. [Section 3] The epitope has the sequence of SEQ ID NO:2, MHCI is HLA-B*57:01, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:23, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:28; Optionally, wherein 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 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, Most preferably, the nucleic acid of item 1, wherein TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO:25, and TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO:30. [Section 4] The epitope has the sequence of SEQ ID NO:3, MHCI is HLA-C*15:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:33, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:38; Optionally, wherein 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 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, Most preferably, the nucleic acid of item 1, wherein TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO:35, and TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO:40. [Section 5] The epitope has the sequence of SEQ ID NO:4, MHCI is HLA-C*06:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:43, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:48; Optionally, wherein 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 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, Most preferably, the nucleic acid of item 1, wherein TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO:45, and TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO:50. [Section 6] The epitope has the sequence of SEQ ID NO:5, MHCI is HLA-B*44:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:53, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:58; Optionally, wherein 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 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, Most preferably, the nucleic acid of item 1, wherein TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO:55, and TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO:60. [Section 7] The epitope has the sequence of SEQ ID NO:5, MHCI is HLA-B*44:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:63, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:68; Optionally, wherein 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 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, Most preferably, the nucleic acid of item 1, wherein TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO:65, and TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO:70. [Section 8] The epitope has the sequence of SEQ ID NO:6, MHCI is HLA-B*07:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:73, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:78; Optionally, wherein 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 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, Most preferably, the nucleic acid of item 1, wherein TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO: 75, and TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO: 80. [Section 9] The epitope has the sequence of SEQ ID NO:6, MHCI is HLA-B*07:02, and TRA comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:83, and TRB comprises a CDR3 having at least 90% sequence identity to SEQ ID NO:88; Optionally, wherein 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 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, Most preferably, the nucleic acid of item 1, wherein TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO:85, and TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO:90. [Section 10] 10. The nucleic acid according to any one of items 1 to 9, wherein the sequence identity in the CDR1, CDR2 and CDR3 regions is 100%. [Section 11] 10. The nucleic acid according to any one of items 1 to 9, encoding at least one alpha and beta chain construct of a TCR construct, wherein said nucleic acid is selected from the group comprising viral vectors, transposons, vectors suitable for CRISPR / CAS based recombination, or plasmids suitable for in vitro RNA transcription, preferably said TCR alpha chain construct and / or TCR beta chain construct further comprises a constant region selected from the group comprising human constant region, murine constant region or chimeric constant region. [Section 12] 12. A protein encoded by the nucleic acid according to any one of items 1 to 11. [Section 13] A host cell, which comprises the nucleic acid and / or protein according to any one of items 1 to 12, and is preferably a human CD8+ T cell. [Section 14] a) a nucleic acid according to any one of claims 1 to 11, capable of encoding a TCR construct capable of specifically binding to said epitope associated with said MHCI; or b) a protein according to claim 12, comprising a TCR construct capable of specifically binding to said epitope associated with said MHCI; or c) a host cell according to claim 13, expressing a TCR construct capable of specifically binding to said epitope associated with said MHCI; wherein the TCR construct is preferably a TCR construct according to item 2, [Section 15] a) at least two nucleic acids, each nucleic acid encoding a TCR construct capable of specifically binding to a respective epitope associated with a respective MHCI; or b) at least two proteins, each of which comprises a TCR construct capable of specifically binding to a respective epitope associated with a respective MHCI; or c) at least two host cells, each host cell expressing a TCR construct capable of specifically binding to a respective epitope associated with each MHCI; A pharmaceutical composition or kit comprising at least two pharmaceutical compositions comprising A pharmaceutical composition or kit, wherein the epitopes are peptides derived from different antigens expressed by the same cancer or infectious agent, optionally wherein the cancer is associated with EBV and the different EBV antigens are selected from the group including LMP2A, LMP1, EBNA1 and EBNA3C, preferably wherein the kit of pharmaceutical compositions is for use in the treatment of said cancer or infectious agent. [Section 16] a) at least two nucleic acids according to any one of paragraphs 1-11, each nucleic acid encoding a TCR construct capable of specifically binding to a respective epitope associated with a respective MHCI; or b) at least two proteins according to clause 12, each of which comprises a TCR construct capable of specifically binding to a respective epitope associated with a respective MHCI; or c) at least two host cells according to clause 13, each host cell expressing a TCR construct capable of specifically binding to a respective epitope associated with each MHCI; Item 14 or 15, a pharmaceutical composition or a kit comprising the pharmaceutical composition, or the kit according to item 14 or 15, Preferably, in the pharmaceutical composition or kit, one of the TCR constructs is a TCR construct according to item 2. [Section 17] 17. The pharmaceutical composition or kit comprising the pharmaceutical composition according to any one of items 14 to 16, for use in treating a patient having an EBV-associated disease selected from the group including Hodgkin's and non-Hodgkin's lymphoma, Burkitt's lymphoma, hemophagocytic lymphohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, lung cancer, gastric cancer, hairy leukoplakia, post-transplant lymphoproliferative disease and central nervous system lymphoma, and expressing said MHCI, or the kit according to any one of items 14 to 16, preferably wherein the treatment is an immunotherapy selected from the group of adoptive T cell therapy and TCR gene therapy. [Section 18] A pharmaceutical composition comprising a peptide comprising an epitope capable of being presented by human MHCI or comprising a nucleic acid encoding said peptide, wherein said epitope is one of the following: a) an epitope capable of being presented on HLA-C*15:02 and having at least 88% sequence identity to SEQ ID NO:3, wherein the peptide comprises up to 11 consecutive amino acids identical to an amino acid sequence occurring in LMP1 of SEQ ID NO:120; b) an epitope capable of being presented on HLA-C*06:02, having at least 88% sequence identity to SEQ ID NO:4, wherein the peptide comprises up to 11 consecutive amino acids identical to an amino acid sequence occurring in EBNA3C of SEQ ID NO:121; and c) an epitope capable of being presented on HLA-B*44:02, having at least 90% sequence identity to SEQ ID NO:5, wherein the peptide comprises up to 11 consecutive amino acids identical to an amino acid sequence occurring in EBNA3C of SEQ ID NO:121; is selected from the group comprising: wherein the pharmaceutical composition is preferably for use in vaccination against EBV-associated diseases selected from the group including Hodgkin's and non-Hodgkin's lymphoma, Burkitt's lymphoma, hemophagocytic lymphohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, lung cancer, gastric cancer, hairy leukoplakia, post-transplant lymphoproliferative disease and central nervous system lymphoma.

[0158] References Chervin et al., 2008. Journal of Immunological Methods 339: 175-184. Cho et al., 2018. British Journal of Cancer 118: 534-545. Cohen et al., 2006. Cancer Research 66: 8878-8886. Cohen et al., 2007. Cancer Research 67: 3898-3903. Danska et al.,1990. The Journal of Experimental Medicine 172: 27-33. Doran et al., 2019. Journal of Clinical Oncology doi: 10.1200 / JCO.18.02424. [Epub ahead of print]. Engels et al., 2003. Human Gene Therapy 14: 1155-1168. Garcia and Adams, 2005. Cell 122: 333-336. Hart et al., 2008. Gene Therapy 15: 625-631. Jurgens et al., 2006. Journal of Clinical Immunology 26: 22-32. Kuball et al., 2007. Blood 109: 2331-2338. Leisegang et al., 2008. Journal of Molecular Medicine. 86: 573-583. Linette et al., 2013. Blood 122: 863-871. Lorenz et al., 2018. Human Gene Therapy 28: 1158-1168. Morgan et al., 2013. Journal of Immunotherapy 36: 133-151. Orentas et al., 2001. Clinical Immunology 98: 220-228. Robbins et al., 2008. Journal of Immunology 180: 6116-6131. Scholten et al., 2006. Clinical Immunology 119: 135-145. Simpson et al., 2011. Proceedings of the National Academy of Sciences of the USA 108: 21176-21181. Sommer-meyer and Uckert, 2010, Journal of Immunology 184: 6223-6231. Tran et al., 2014. Science 344: 641-645. WO 2011 / 039508 A2 WO 2015 / 022520 A1 Yang et al., 2011. Clinical and Developmental Immunology, Article ID 716926 Zheng et al., 2015. Cancer Immunology Research 3: 1138-1147.

Claims

1. A nucleic acid encoding a TCR construct TCR alpha chain construct (TRA) and / or a TCR beta chain construct (TRB) specific for an epitope complexed with human MHCI, wherein the epitope is an epitope of the Epistein-Barr Virus (EBV) protein having the sequence of SEQ ID NO:1, the MHCI is HLA-A*02:01, TRA comprises a CDR1 having SEQ ID NO:11, a CDR2 having SEQ ID NO:12 and a CDR3 having SEQ ID NO:13, and TRB comprises a CDR1 having SEQ ID NO:16, a CDR2 having SEQ ID NO:17 and a CDR3 having SEQ ID NO:

18.

2. The nucleic acid described in claim 1, wherein TRA comprises a variable region having at least 90% sequence identity to SEQ ID NO:15, and TRB comprises a variable region having at least 90% sequence identity to SEQ ID NO:

20.

3. The nucleic acid described in claim 1 or 2, selected from the group including a viral vector, a transposon, a vector suitable for CRISPR / CAS-based recombination, or a plasmid suitable for in vitro RNA transcription.

4. 4. The nucleic acid of claim 3, wherein the TCR alpha chain construct and / or the TCR beta chain construct further comprises a constant region selected from the group comprising a human constant region, a mouse constant region or a chimeric constant region.

5. A protein encoded by the nucleic acid according to any one of claims 1 to 4.

6. A host cell comprising a nucleic acid and / or a protein according to any one of claims 1 to 5.

7. The host cell of claim 6, which is a human CD8+ T cell.

8. a) a nucleic acid according to any one of claims 1 to 4, encoding a TCR construct capable of specifically binding to said epitope in association with said MHCI; or b) a protein according to claim 5 comprising a TCR construct capable of specifically binding said epitope in the context of said MHC I; or c) a host cell according to claim 6 or 7, expressing a TCR construct capable of specifically binding said epitope in association with said MHCI; 13. A pharmaceutical composition comprising:

9. The pharmaceutical composition of claim 8, wherein the TCR construct is the TCR construct of claim 2.

10. 10. The pharmaceutical composition of claim 8 or 9, for use in treating a patient with an EBV-associated disease selected from the group including Hodgkin's and non-Hodgkin's lymphoma, Burkitt's lymphoma, hemophagocytic lymphohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, lung cancer, gastric cancer, hairy leukoplakia, post-transplant lymphoproliferative disease and central nervous system lymphoma, and expressing said MHCI.

11. The pharmaceutical composition of claim 10, wherein the treatment is an immunotherapy selected from the group of adoptive T cell therapy and TCR gene therapy.

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