TCR constructs specific to EBV-derived antigens

Abstract

The present invention relates to immunotherapy, in particular to the field of Epstein-Barr virus-associated disease (EBV, also designated as human gamma herpesvirus 4), e.g., cancer or post-transplant lymphoproliferative disease, in particular to adoptive T cell therapy or T cell receptor (TCR) gene therapy. The present invention provides at least two TCR constructs, or a combination of nucleic acids encoding each protein or host cell, wherein each TCR construct can specifically bind to its respective epitope in the context of each MHC I, and the epitope is a peptide from a different antigen expressed by the same infectious pathogen or cancer, e.g., an EBV antigen. The present invention also provides specific nucleic acids encoding a TCR alpha chain construct (TRA) and / or a TCR beta chain construct (TRB) of a TCR construct that is specific to an epitope forming a complex with human MHC I, wherein the epitope is an epitope of the Epstein-Barr virus protein and the TCR construct is specific to an epitope from LMP2A, LMP1, or EBNA3C. Proteins encoded by said nucleic acids, corresponding host cells, pharmaceutical compositions, and kits are also the subject of the present invention.

Description

Technology Field

[0001] The present invention relates to immunotherapy, in particular to the field of Epstein-Barr virus-associated disease (EBV, also designated as human gamma herpesvirus 4), e.g., cancer or post-transplant lymphoproliferative disease, in particular to adoptive T cell therapy or T cell receptor (TCR) gene therapy. The present invention provides at least two TCR constructs, or a combination of nucleic acids encoding each protein or host cell, wherein each TCR construct can specifically bind to its respective epitope in the context of each MHC I, and the epitope is a peptide from different antigens expressed by the same infectious pathogen or cancer, e.g., EBV antigen. The present invention also provides specific nucleic acids encoding a TCR alpha chain construct (TRA) and / or a TCR beta chain construct (TRB) of a TCR construct that is specific to an epitope forming a complex with human MHC I, wherein the epitope is an epitope of the Epstein-Barr virus protein and the TCR construct is specific to an epitope from LMP2A, LMP1, or EBNA3C. Proteins encoded by said nucleic acids, corresponding host cells, pharmaceutical compositions, and kits are also the subject of the present invention. Background Technology

[0002] TCRs are heterodimeric cell surface proteins of the immunoglobulin superfamily associated with the constant protein of the CD3 complex involved in the mediation of signal transduction. TCRs exist in αβ and γδ forms, which are structurally similar but differ significantly in anatomical location and possibly function. The alpha and beta chains of the natural heterodimeric αβTCR are transmembrane proteins and each contain two extracellular domains, a membrane-proximal constant domain, and a membrane-distal variable domain. Each of the constant and variable domains contains an intrachain disulfide bond.

[0003] The variable region of each TCR chain includes a variable segment and a linkage segment, and in the case of the beta chain, also includes a diversity segment. Each variable region contains three CDRs (Complementarity Determining Regions) embedded in the framework sequence, highly polymorphic loops, and one hypervariable region designated as CDR3. There are several types of alpha chain variable (Vα) and several types of beta chain variable (Vβ) regions, distinguished by their framework regions, CDR1 and CDR2 sequences, and a partially defined CDR3 sequence. Unique TRAV or TRBV numbers are assigned as Vα or Vβ by the IMGT nomenclature. TCR specificity for recognized epitopes is primarily determined by the CDR3 region [cf. Danska et al., 1990; Garcia et al., 2005.].

[0004] Adoptive TCR gene therapy allows for the conferring desired specificity to a patient's own T cells and the generation of a sufficient number of activated, non-consumed T cells in a short period. TCRs can be transduced into all T cells or T cell subsets, such as CD8+ T cells, central memory T cells, or T cells with stem cell characteristics, which can enhance persistence and function upon delivery. TCR-engineered T cells can be injected into patients, for example, cancer patients who develop lymphopenia due to chemotherapy or radiation, to induce the expansion of homeostasis. This significantly enhances the engraftment and long-term persistence of transplanted T cells and may be associated with higher cure rates.

[0005] TCR-based adoptive T cell therapy relies on classical TCR recognition of processed epitopes of antigens presented in relation to MHC molecules. Therefore, T cells expressing specific TCRs specific to epitopes in the context of specific MHCs can only be used to treat patients expressing each respective MHC.

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

[0007] EBV-related diseases include various non-malignant, precancerous, and malignant EBV-associated lymphoproliferative diseases such as infectious mononucleosis, post-transplant lymphoproliferative disorders, Burkitt lymphoma, hemophagocytic lymphohistiocytosis, and Hodgkin and non-Hodgkin lymphomas; non-lymphatic malignancies such as gastric cancer, lung cancer, and nasopharyngeal cancer; and pathologies associated with the human immunodeficiency virus, such as leukoplakia and central nervous system lymphomas. This virus is also associated with the childhood disorder of Alice in Wonderland syndrome and acute cerebellar ataxia, and based on some evidence, there is an increased risk of developing certain autoimmune diseases. It is estimated that approximately 200,000 cases of cancer annually are attributed to EBV (Wikipedia).

[0008] Most EBV-associated cancers express only a limited number of EBV-specific antigens, such as latent membrane proteins (LMP1, LMP2A) and nuclear proteins (EBNA1, EBNA3C). These antigens have been identified as interesting targets for TCR-based immunotherapy, e.g., TCR gene therapy, or proton therapy for EBV-associated diseases, e.g., post-transplant lymphoproliferative disorders, or cancers [Refs: 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 / 022520 A1; WO 2011 / 039508 A2].

[0009] However, most T-cell-based immunotherapies targeting EBV-related malignancies utilize natural EBV-specific T cells generated from third-party donors or patients, and these T cells are expanded using EBV lymphoblast cell lines (LCL) or EBV peptide pools. Adoptive T-cell therapy using EBV-specific TCR-engineered T cells has not been tested in clinical trials. TCR-engineered T cells have several advantages compared to natural EBV-specific T cells: 1) Efficacy: The introduced TCR is a predefined receptor with high affinity for EBV-positive tumor cells. Growing natural T cells from a patient's blood depends on the presence of EBV-specific T cells. However, patients may lack effective T cells that can be expanded. 2) Feasibility: The success rate of manufacturing engineered T cells is over 95%, whereas the success rate of the procedure to grow natural T cells is less than 70%. 3) Cost: Compared to the expansion of natural T cells which takes more than 40 days, the time from vein to vein in the engineered T cell process is shortened to less than 21 days.

[0010] Performing TCR gene therapy on a patient population with different MHC I (HLA) alleles requires identifying TCRs restricted to different HLA alleles. Additionally, the identification of TCRs restricted to different HLA alleles is a prerequisite for targeting EBV epitopes through two different HLA alleles expressed by the same cell.

[0011] The present invention solves part of these problems and provides a novel and preferably advantageous immunotherapeutic agent useful for immunotherapy of cancer or infectious pathogens. This problem is solved by the subject matter of the claims. means of solving the problem

[0012] TCR The work nucleic acids that code for

[0013] In one embodiment, the present invention provides a specific TCR construct useful for the treatment of EBV-related diseases, and a nucleic acid encoding the same.

[0014] The present invention relates to a nucleic acid encoding a TCR alpha chain construct (TRA) and / or a TCR beta chain construct (TRB) of a TCR construct specific to an epitope that forms a complex with human MHC I, wherein the epitope is an epitope of an Epstein-Barr virus (EBV) protein, and

[0015] a) The epitope has the sequence of SEQ ID NO. 1, the MHC I is HLA-A*02:01, the TRA comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 13, and the TRB comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 18;

[0016] b) The epitope has the sequence of SEQ ID NO. 2, the MHC I is HLA-B*57:01, the TRA comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 23, and the TRB comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 28;

[0017] c) The epitope has the sequence of SEQ ID NO. 3, the MHC I is HLA-C*15:02, the TRA comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 33, and the TRB comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 38;

[0018] d) The epitope has the sequence of SEQ ID NO. 4, the MHC I is HLA-C*06:02, the TRA comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 43, and the TRB comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 48;

[0019] e) The epitope has the sequence of SEQ ID NO. 5, the MHC I is HLA-B*44:02, the TRA comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 53, and the TRB comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 58;

[0020] f) The epitope has the sequence of SEQ ID NO. 5, the MHC I is HLA-B*44:02, the TRA comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 63, and the TRB comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 68;

[0021] g) The epitope has the sequence of SEQ ID NO. 6, the MHC I is HLA-B*07:02, the TRA comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 73, and the TRB comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 78; and / or

[0022] h) Provides a nucleic acid in which the epitope has the sequence of SEQ ID NO. 7, the MHC I is HLA-B*07:02, the TRA comprises CDR3 having at least 90% sequence identity with SEQ ID NO. 83, and the TRB comprises CDR3 having at least 90% sequence identity with SEQ ID NO. 88.

[0023] In the context of the present invention, "a" is understood to mean "one or more" unless otherwise explicitly stated. Thus, for example, if the TCR construct of the present invention contains both alpha and beta chain constructs as is preferred throughout the present invention, it may be encoded by one or two nucleic acids. The alpha and beta chain constructs can specifically bind to the epitopes of EBV proteins that together form a complex with human MHC I. As intermediate products, the alpha and beta chain constructs are themselves the subject of the present invention. The present invention also provides, for example, a single-chain nucleic acid construct in which the TCR alpha and beta chain constructs are separated by a P2A element.

[0024] One nucleic acid of the present invention codes for a TRA and / or TRB of a TCR construct specific to an epitope that forms a complex with human MHC I, wherein the epitope has the sequence of SEQ ID NO. 1, the MHC I is HLA-A*02:01, the TRA comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 13, and the TRB comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 18. SEQ ID NO. 1 is an epitope from the EBV protein LMP2A known in the art as being presented by EBV-associated cancer types, i.e., cancer cells of very common HLA-types, in the context of HLA-A2, e.g., HLA-A*02:01 [Reference: e.g., Orentas et al., 2001, see above]. The TCR construct having the CDR3 sequence disclosed herein has, compared to TCRs in the art, particularly high affinity or peptide sensitivity as presented in this specification, and is therefore a preferred TCR construct of the present invention.

[0025] Advantageously, the above TCR is 10 - 8 mol / L or less, preferably 10 -9 It has high peptide sensitivity accompanied by half-maximum IFN-γ release at peptide concentrations of mol / L or less. The analysis can be performed, for example, by culturing TCR-engineered T cells with target cells (e.g., K562-HLA-A*02:01 cells loaded with a peptide, preferably the peptide of SEQ ID NO. 1) at a 1:1 effector-to-target cell ratio, as described in FIG. 15 or 16, preferably as described for FIG. 15.

[0026] Optionally, TRA comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO. 11, CDR2 having at least 80% sequence identity with respect to SEQ ID NO. 12, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO. 13. Optionally, TRB comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO. 16, CDR2 having at least 80% sequence identity with respect to SEQ ID NO. 17, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO. 18.

[0027] TRA may include the conjugated amino acid presented in SEQ ID NO. 14. TRB may include the conjugated amino acid presented in SEQ ID NO. 19.

[0028] TRA may include a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 15. TRB may include a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 20.

[0029] A TCR composition comprising a TRA having a variable region of SEQ ID NO. 15 and a TRB having a variable region of SEQ ID NO. 20 has been found to have particularly advantageous features in this specification. This is also designated as TCR06.

[0030] The variable region of the TRA of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 91, and the variable region of the TRB of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 92.

[0031] One nucleic acid of the present invention codes for a TRA and / or TRB of a TCR construct specific to an epitope that forms a complex with human MHC I, wherein the epitope has the sequence of SEQ ID NO. 2, the MHC I is HLA-B*57:01, the TRA comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 23, and the TRB comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 28. SEQ ID NO. 2 is an epitope from the EBV protein LMP1 presented by cancer cells of an EBV-associated cancer type in the context of HLA-B*57:01. A TCR construct having the CDR3 sequence disclosed herein has high affinity or peptide sensitivity as presented herein.

[0032] Optionally, the TRA comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO 21, CDR2 having at least 80% sequence identity with respect to SEQ ID NO 22, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO 23. Optionally, the TRB comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO 26, CDR2 having at least 80% sequence identity with respect to SEQ ID NO 27, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO 28.

[0033] TRA may include the conjugated amino acid presented in SEQ ID NO. 24. TRB may include the conjugated amino acid presented in SEQ ID NO. 29.

[0034] TRA may include a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO 25. TRB may include a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO 30.

[0035] A TCR composition comprising a TRA having a variable region of SEQ ID NO. 25 and a TRB having a variable region of SEQ ID NO. 30 has been found to have advantageous features in this specification. This is also designated as TCR50.

[0036] The variable region of the TRA of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 93, and the variable region of the TRB of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 94.

[0037] One nucleic acid of the present invention codes for a TRA and / or TRB of a TCR construct specific to an epitope that forms a complex with human MHC I, wherein the epitope has the sequence of SEQ ID NO. 3, the MHC I is HLA-C*15:02, the TRA comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 33, and the TRB comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 38. SEQ ID NO. 3 is an epitope from the EBV protein LMP1, which has been revealed for the first time to be presented by cancer cells of an EBV-associated cancer type. It is presented in the context of HLA-C*15:02. A TCR construct having the CDR3 sequence disclosed herein has high affinity or peptide sensitivity as presented herein.

[0038] Optionally, the TRA comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO. 31, CDR2 having at least 80% sequence identity with respect to SEQ ID NO. 32, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO. 33. Optionally, the TRB comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO. 36, CDR2 having at least 80% sequence identity with respect to SEQ ID NO. 37, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO. 38.

[0039] TRA may include the conjugated amino acid presented in SEQ ID NO. 34. TRB may include the conjugated amino acid presented in SEQ ID NO. 39.

[0040] TRA may include a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 35. TRB may include a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 40.

[0041] A TCR composition comprising a TRA having a variable region of SEQ ID NO. 35 and a TRB having a variable region of SEQ ID NO. 40 has been found to have advantageous features in this specification. This is also designated as TCR83.

[0042] The variable region of the TRA of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 95, and the variable region of the TRB of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 96.

[0043] One nucleic acid of the present invention codes for a TRA and / or TRB of a TCR construct specific to an epitope that forms a complex with human MHC I, wherein the epitope has the sequence of SEQ ID NO. 4, the MHC I is HLA-C*06:02, the TRA comprises CDR3 having at least 90% sequence identity with SEQ ID NO. 43, and the TRB comprises CDR3 having at least 90% sequence identity with SEQ ID NO. 48. SEQ ID NO. 4 is an epitope from the EBV protein EBNA3C, which has been revealed to be presented by cancer cells of an EBV-associated cancer type for the first time now. It is presented in the context of HLA-C*06:02.

[0044] Optionally, the TRA comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO 41, CDR2 having at least 80% sequence identity with respect to SEQ ID NO 42, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO 43. Optionally, the TRB comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO 46, CDR2 having at least 80% sequence identity with respect to SEQ ID NO 47, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO 48.

[0045] TRA may include the conjugated amino acid presented in SEQ ID NO. 44. TRB may include the conjugated amino acid presented in SEQ ID NO. 49.

[0046] Preferably, TRA comprises a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 45. Preferably, TRB comprises a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 50.

[0047] A TCR composition comprising a TRA having a variable region of SEQ ID NO. 45 and a TRB having a variable region of SEQ ID NO. 50 has been found to have advantageous features in this specification. This is also designated as TCR64.

[0048] The variable region of the TRA of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 97, and the variable region of the TRB of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 98.

[0049] One nucleic acid of the present invention codes for a TRA and / or TRB of a TCR construct specific to an epitope that forms a complex with human MHC I, wherein the epitope has the sequence of SEQ ID NO. 5, the MHC I is HLA-B*44:02, the TRA comprises CDR3 having at least 90% sequence identity with SEQ ID NO. 53, and the TRB comprises CDR3 having at least 90% sequence identity with SEQ ID NO. 58. SEQ ID NO. 5 is an epitope from the EBV protein EBNA3C, which has been revealed to be presented by cancer cells of an EBV-associated cancer type for the first time now. It is presented in the context of HLA-B*44:02.

[0050] Optionally, the TRA comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO. 51, CDR2 having at least 80% sequence identity with respect to SEQ ID NO. 52, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO. 53. Optionally, the TRB comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO. 56, CDR2 having at least 80% sequence identity with respect to SEQ ID NO. 57, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO. 58.

[0051] TRA may include the conjugated amino acid presented in SEQ ID NO. 54. TRB may include the conjugated amino acid presented in SEQ ID NO. 59.

[0052] Preferably, TRA comprises a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 55. Preferably, TRB comprises a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 60.

[0053] A TCR composition comprising a TRA having a variable region of SEQ ID NO. 55 and a TRB having a variable region of SEQ ID NO. 60 has been found to have advantageous features in this specification. This is also designated as TCR25.

[0054] The variable region of the TRA of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 99, and the variable region of the TRB of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 100.

[0055] One nucleic acid of the present invention codes for a TRA and / or TRB of a TCR construct specific to an epitope that forms a complex with human MHC I, wherein the epitope has the sequence of SEQ ID NO. 5, the MHC I is HLA-B*44:02, the TRA comprises CDR3 having at least 90% sequence identity with SEQ ID NO. 63, and the TRB comprises CDR3 having at least 90% sequence identity with SEQ ID NO. 68. As mentioned above, SEQ ID NO. 5 is an epitope from the EBV protein EBNA3C, which has been revealed to be presented by cancer cells of an EBV-associated cancer type for the first time now. It is presented in the context of HLA-B*44:02.

[0056] Optionally, the TRA comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO. 61, CDR2 having at least 80% sequence identity with respect to SEQ ID NO. 62, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO. 63. Optionally, the TRB comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO. 66, CDR2 having at least 80% sequence identity with respect to SEQ ID NO. 67, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO. 68.

[0057] TRA may include the conjugated amino acid presented in SEQ ID NO. 64. TRB may include the conjugated amino acid presented in SEQ ID NO. 69.

[0058] Preferably, TRA comprises a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 65. Preferably, TRB comprises a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 70.

[0059] A TCR composition comprising a TRA having a variable region of SEQ ID NO. 65 and a TRB having a variable region of SEQ ID NO. 70 has been found to have advantageous features in this specification. This is also designated as TCR58.

[0060] The variable region of the TRA of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 101, and the variable region of the TRB of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 102.

[0061] One nucleic acid of the present invention codes for a TRA and / or TRB of a TCR construct specific to an epitope that forms a complex with human MHC I, wherein the epitope has the sequence of SEQ ID NO. 6, the MHC I is HLA-B*07:02, the TRA comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 73, and the TRB comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 78. SEQ ID NO. 6 is an epitope from the EBV protein EBNA3C presented by cancer cells of an EBV-associated cancer type in the context of HLA-B*07:02.

[0062] Optionally, the TRA comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO. 71, CDR2 having at least 80% sequence identity with respect to SEQ ID NO. 72, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO. 73. Optionally, the TRB comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO. 76, CDR2 having at least 80% sequence identity with respect to SEQ ID NO. 77, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO. 78.

[0063] TRA may include the conjugated amino acid presented in SEQ ID NO. 74. TRB may include the conjugated amino acid presented in SEQ ID NO. 79.

[0064] Preferably, TRA comprises a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 75. Preferably, TRB comprises a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 80.

[0065] A TCR composition comprising a TRA having a variable region of SEQ ID NO. 75 and a TRB having a variable region of SEQ ID NO. 80 has been found to have advantageous features in this specification. This is also designated as TCR27.

[0066] The variable region of the TRA of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 103, and the variable region of the TRB of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 104.

[0067] One nucleic acid of the present invention codes for a TRA and / or TRB of a TCR construct specific to an epitope that forms a complex with human MHC I, wherein the epitope has the sequence of SEQ ID NO. 7, the MHC I is HLA-B*07:02, the TRA comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 83, and the TRB comprises a CDR3 having at least 90% sequence identity with SEQ ID NO. 88. SEQ ID NO. 7 is an epitope of the EBV protein EBNA3C and comprises SEQ ID NO. 6 and a C-terminal T.

[0068] Optionally, the TRA comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO. 81, CDR2 having at least 80% sequence identity with respect to SEQ ID NO. 82, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO. 83. Optionally, the TRB comprises CDR1 having at least 80% sequence identity with respect to SEQ ID NO. 86, CDR2 having at least 80% sequence identity with respect to SEQ ID NO. 87, and CDR3 having at least 90%, preferably 100%, sequence identity with respect to SEQ ID NO. 88.

[0069] TRA may include the conjugated amino acid presented in SEQ ID NO. 84. TRB may include the conjugated amino acid presented in SEQ ID NO. 89.

[0070] Preferably, TRA comprises a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 85. Preferably, TRB comprises a variable region having at least 90%, optionally at least 95% or 100% sequence identity with respect to SEQ ID NO. 90.

[0071] A TCR composition comprising a TRA having a variable region of SEQ ID NO. 85 and a TRB having a variable region of SEQ ID NO. 90 has been found to have advantageous features in this specification. This is also designated as TCR01.

[0072] The variable region of the TRA of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 105, and the variable region of the TRB of the above TCR construct can be coded by a nucleic acid having the sequence of SEQ ID NO. 106.

[0073] In any TRA and / or TRB composition of the present invention, independently, CDR1 and CDR3 may have at least 80%, at least 90%, or 100% sequence identity with respect to the listed sequences. Preferably, CDR3 has 100% sequence identity with respect to the listed defined CDR3. Typically, where the sequences are not identical, at most one amino acid exchange, deletion, or insertion is present, and typically an amino acid exchange is present. The exchange may be a conserved substitution, that is, one amino acid of a specific type (e.g., polar, nonpolar, acidic, basic, aromatic) is exchanged for another amino acid of the same type. Methods of affinity maturation are known in the art and are further described below.

[0074] Optionally, the sequence identity for the listed CDR1, CDR2, and CDR3 regions is 100% in the TRA or TRB of the TCR construct of the present invention, preferably in both the TRA and TRB.

[0075] The TCR alpha and / or beta chain constructs of the present invention may include all features or domains corresponding to their natural counterparts, i.e., the TCR alpha or beta chain, but this is not essential. Preferably, the TCR alpha and / or beta chain constructs include at least a variable region, or a variable and invariant region, for example, a variable and / or invariant region having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with respect to a human variable or invariant TCR region.

[0076] The TCR alpha chain constructs and / or TCR beta chain constructs of the present invention preferably comprise a constant region. For adoptive TCR therapy, it is preferable that the TCR construct comprises full-length TCR alpha and beta chains comprising variable and constant regions including a transmembrane region. The constant region may be a chimeric constant region, such as a human constant region, a murine constant region, or a minimal murine constant region. The TCR construct may be of human origin, either essentially or exclusively, to minimize immunogenicity. However, to prevent pairing with endogenous TCR chains, the constructs of the present invention preferably contain one or more, for example, 1 to 5, 1 to 10, or 1 to 20, preferably 9, amino acid exchanges compared to the human sequence [Ref. Sommermeyer and Uckert, 2010]. For this purpose, the invariant regions of the TCR alpha and beta chain constructs may also be murine invariant regions [cf. Cohen et al., 2006], or additional cysteine ​​is provided to enable the formation of additional disulfide bonds between the TCR chains [cf. Cohen et al., 2007, Kuball et al., 2007]. Additionally, the functional expression of the transformed TCR can be enhanced by using codon modifications of the TCR sequence [cf. Scholten et al., 2006], or a peptide (e.g., P2A) can be applied to link the two TCR chains, achieve stoichiometric expression of both chains [cf. Leisegang et al., 2008], and also enhance the functional expression of the transformed TCR.

[0077] The construct may also be a chimeric antigen receptor or part thereof, for example, a human TCR variable region may be connected to a different immunoglobulin constant domain, for example, an IgG constant domain, or an antibody domain capable of specifically binding to an antigen such as LMP2A.

[0078] The scTCR also includes heterodimeric TCR constructs. The scTCR may include a variable region of a first TCR construct (e.g., alpha chain) and a full (full-length) second TCR chain (e.g., beta chain), or vice versa. Additionally, the scTCR may optionally include one or more linkers that link two or more polypeptides together. The linker may be, for example, a peptide that links two chains together. Furthermore, such scTCR of the present invention is provided for fusion with human cytokines, such as IL-2, IL-7, IL-12, or IL-15.

[0079] In the context of MHC, particularly to enable specific recognition of an epitope for therapeutic purposes, the nucleic acid of the present invention codes for one TCR alpha and one beta chain construct of the TCR construct of the present invention, for example, as described herein, a TCR construct specific to the epitope of SEQ ID NO. 1.

[0080] Typically, the nucleic acid sequence provided by the present invention is codon-optimized for expression in human cells.

[0081] In the context of the present invention, the nucleic acid may be DNA or RNA. Preferably, it is DNA. The nucleic acid may be, for example, a viral vector, or a non-viral vector, for example, a transposon, a vector suitable for 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 plasmids and viruses designed for proliferation and expansion, or for expression, or both. The vector may be an expression vector suitable for expression in host cells selected from the group comprising human T cells or human T cell precursors, preferably CD8+ T cells. The CD8+ 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, for example, a retrovirus, particularly a gamma-retrovirus or lentivirus vector. An example of a suitable expression vector includes the retrovirus vector MP71 [Ref. Engels et al., 2003]. The expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is introduced, and the expression of the nucleic acid of the present invention is typically performed in human CD8+ T cells in the context of the present invention. Additionally, the vector of the present invention may comprise one or more marker genes that enable the selection of the transfected or transfected host. The recombinant expression vector may comprise a natural or preferably heterogeneous promoter operably linked to a nucleotide sequence encoding the TCR construct of the present invention, or to a nucleotide sequence that is complementary to or hybridizes with the nucleotide sequence encoding the construct of the present invention. The selection of the 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, it is a heterogeneous promoter, that is, a promoter not naturally linked to the TCR in human T cells, such as a long terminal repeat promoter suitable for expression in human T cells. The recombinant expression vector of the present invention may be designed for transient expression, stable expression, or both. Additionally, the recombinant expression vector may be prepared for constitutive expression or inducible expression.

[0082] protein

[0083] The present invention also provides a TCR construct comprising a protein, i.e., an alpha or beta chain construct, or preferably, both alpha and beta chain constructs capable of specifically binding to an epitope from the aforementioned EBV-protein in the context of each MHC I as described herein, for example, a TCR construct specific to the epitope of SEQ ID NO. 1 as described herein. The protein is encoded by the nucleic acid of the present invention.

[0084] As used herein, the terms “capable of specifically binding to a given antigen,” “recognizing” a given antigen, or “specific” to a given antigen are synonyms and mean that a TCR construct can specifically bind to and immunologically recognize said epitopes, preferably from EBV proteins, more preferably with high affinity. For example, a TCR is such that T cells expressing the TCR, in the absence of epitopes or in the presence of irrelevant control peptide epitopes, at low concentrations of each epitope, e.g., 10 -11 mol / L, 10 -10 mol / L, 10 -9 mol / L, 10 -8 mol / L, 10 -7 mol / L, 10 -6 mol / L, 10 -5When co-cultured with target cells pulsed at mol / L, 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 γ (IFN-γ) is secreted, it can be considered "capable of specifically binding" to peptides from EBV proteins. Preferably, this is tested with 10,000 TCR+CD8+ T cells and 20,000 to 50,000, preferably 50,000, target cells expressing appropriate HLA, for example, by the assay as described below in the examples. Alternatively or additionally, if T cells expressing a TCR secrete at least twice the amount of IFN-γ at the background level of untransduced IFN-γ when co-cultured with target cells pulsed with a low concentration of an appropriate peptide, the TCR may be considered to have "antigen specificity" for the epitope. This "specificity" described above can be analyzed, for example, by ELISA.

[0085] High affinity is high peptide sensitivity as described above for the TCR of the present invention, for example, 10 -6 mol / L or less, preferably 10 -7 mol / L or less, 10 -8 mol / L or less or 10 -9 It correlates with half-max IFN-γ release at peptide concentrations of mol / L or less. Alternatively, affinity can be analyzed by methods known to those skilled in the art, for example, by ViaCore. TCR affinity or T cell binding strength of 100 μM or more, more preferably 10 μM or more, is considered high affinity.

[0086] Based on the defined CDR3 and variable region sequences provided by the present invention, it is possible to perform affinity maturation of the TCR sequence [cf. Chervin et al., 2008; Robbins et al., 2008]. Non-synonymous nucleotide substitutions that induce amino acid exchange in the CDR3 sequence can induce an enhancement of the TCR's affinity for the target antigen. Additionally, changes in the TCR sequence in other parts of the variable TRA and TRB regions can alter the TCR's affinity for the peptide-MHC I complex. While this can increase the overall affinity of the TCR for the peptide-MHC, there is a risk of non-specific recognition and increased cross-reactivity [cf. Linette et al., 2013]. It is desirable for TCRs different from the provided specific sequence to maintain exclusive specificity for the provided target antigen; that is, they are non-cross-reactive, and most importantly, they are non-cross-reactive to human autopeptides. The potential cross-reactivity of TCRs can be tested against known autopeptides loaded into cells with the correct MHC allele [Ref. Morgan et al., 2013]. Therefore, it is desirable that adoptive delivery of T cells expressing the TCR construct of the present invention have no or no significant adverse effects on healthy tissues.

[0087] The TCR composition according to the present invention may also be provided in the form of a multimeric complex comprising at least two scTCR molecules, wherein each scTCR molecule is fused to at least one biotin moiety and the scTCRs are interconnected by biotin-streptavidin interactions to enable the formation of the multimeric complex. Additionally, a higher-order multimeric complex comprising more than two, for example, four scTCRs, according to the present invention is provided.

[0088] The TCR composition of the present invention may be modified to include detectable labels such as, for example, radioactive isotopes, fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), tags such as HIS-tags, enzymes (e.g., alkaline phosphatase, horseradish peroxidase), or particles (e.g., gold particles or magnetic particles).

[0089] host cell

[0090] 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, e.g., a plant, animal, fungus, or algae, or a prokaryotic cell, e.g., a bacterium or protozoa. The host cell may be a cultured cell or a primary cell, i.e., a cell isolated directly from an organism, e.g., a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell growing in a suspension. For the purpose of producing a recombinant TCR, polypeptide, or protein, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. The host cell may be any cell type, may originate from any type of tissue, and may be at any stage of development, but the host cell is preferably a peripheral blood leukocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a human T cell that can be isolated from a T cell or a T cell precursor, particularly from a PBMC. The T cells may be any T cells, such as cultured T cells, for example, primary T cells, T cells from cultured T cell lines, or T cells obtained from mammals, preferably T cells or T cell precursors from human patients. The T cells may be obtained from various sources, such as blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. The T cells may also be concentrated or purified. These may be, for example, tumor-infiltrating cells (TILs), effector cells, central effector cells, memory T cells, naive T cells, etc., preferably central memory T cells. Alternatively, the host cells may be other immune effector cells, for example, NK cells or macrophages.

[0091] Preferably, particularly in the context of human therapy, the T cells are human T cells. The T cells are preferably CD8+ cells (e.g., cytotoxic T cells). Preferably, the T cells are T cells isolated from a human, for example, a human patient, particularly the patient to be treated. Alternatively, the T cells may be derived from a third-party donor who may or may not be associated with the patient. These T cells may be genetically engineered in various ways, for example, by knockout of the endogenous TCR and / or MHC I. The T cells may also be generated from autologous or third-party donor stem cells, wherein optionally, the stem cells are not human embryonic stem cells.

[0092] Preferably, the host cell is a human CD8+ T cell comprising the nucleic acid of the present invention, which is an expression vector, wherein the nucleic acid encoding TRA and / or TRB is operably linked to a heterologous promoter, and the host cell expresses the TCR construct of the present invention.

[0093] The present invention also provides two or more different TCR constructs, for example, a host cell expressing two TCR constructs of the present invention. Preferably, in this context, the TCR constructs are Japanese-chain TCR constructs, and the TCR alpha and beta chains of each TCR are combined by a linker to avoid mispairing between transgenic TCR chains. The two Japanese-chain TCR constructs may be encoded on a single expression vector.

[0094] Pharmaceutical composition

[0095] The present invention also

[0096] a) a nucleic acid of the present invention encoding a TCR construct capable of specifically binding to each epitope thereof in the context of each MHC I; or

[0097] b) a protein of the present invention comprising a TCR construct capable of specifically binding to each epitope thereof in the context of each MHC I (i.e., the TCR construct of the present invention); or

[0098] c) The host cell of the present invention expressing a TCR construct capable of specifically binding to each epitope thereof in the context of each MHC I

[0099] Provides a pharmaceutical composition comprising

[0100] Preferably, the TCR construct is a TCR construct specific to the epitope of SEQ ID NO. 1.

[0101] In another embodiment, the present invention

[0102] a) at least two nucleic acids each encoding a TCR construct capable of specifically binding to each epitope thereof in the context of each MHC I; or

[0103] b) at least two proteins each comprising a TCR construct capable of specifically binding to each epitope thereof in the context of each MHC I; or

[0104] c) At least two host cells each expressing a TCR construct capable of specifically binding to its respective epitope in the context of each MHC I

[0105] A pharmaceutical composition comprising, or a kit comprising at least two pharmaceutical compositions, is provided.

[0106] Here, an epitope is a peptide from a different antigen expressed by the same cancer or infectious pathogen. Typically, immunotherapy is to be performed for the treatment of cancer, and therefore the different antigens are preferably antigens expressed by the cancer, that is, by cells of the same cancer. In this context, an antigen is a protein containing an epitope that can be presented on MHC I. Different cancer cells of the same cancer type may each express at least one antigen, and thus TCR constructs can be directed to different cancer cells of the same cancer, which may be useful, for example, when the stage of the cancer is unknown and / or when treating cells of different stages that express different antigens. However, generally, it is beneficial when different antigens are expressed by the same cancer cells.

[0107] The inventors have surprisingly discovered that for adoptive T cell therapy, it is advantageous to use a combination of two or more T cells each expressing at least two TCR constructs expressed by T cells, particularly TCR constructs capable of specifically binding to each of their respective epitopes in the context of each MHC I.

[0108] Without being bound by theory, attacking cancer (or infectious pathogens) based on the expression of two or more antigens is thought to play a role in sustaining the attack and evading immune evasion, for example, through mutations, downregulation of a single target antigen, or antigen-losing variants.

[0109] It is also possible to target a cancer or infectious pathogen to three or more host cells each containing a nucleic acid encoding a TCR construct capable of specifically binding to each of its epitopes in the context of each MHC I, wherein the epitopes are peptides from different antigens expressed by the same cancer or infectious pathogen.

[0110] The kit or pharmaceutical composition is intended for use in the treatment of cancer or infectious pathogens from which epitopes targeted by TCR compositions originate.

[0111] Infection or cancer may be associated with, for example, 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. The alternative or additional EBV antigen is EBNA1.

[0112] The present invention also,

[0113] a) at least two nucleic acids of the present invention, each encoding a TCR construct capable of specifically binding to each epitope thereof in the context of each MHC I; or

[0114] b) at least two proteins of the present invention, each comprising a TCR construct capable of specifically binding to each epitope thereof in the context of each MHC I; or

[0115] c) At least two host cells of the present invention each expressing a TCR construct capable of specifically binding to each epitope thereof in the context of each MHC I

[0116] Providing the aforementioned pharmaceutical composition or kit comprising,

[0117] Here, optionally, the epitope is derived from a different EBV protein.

[0118] One or two or three TCR constructs used in the context of the pharmaceutical composition or kit of the present invention may be TCR constructs of the present invention as disclosed herein. Preferably, one of the TCR constructs used is a TCR construct disclosed herein that recognizes the epitope of SEQ ID NO. 1 in the context of HLA-A2.

[0119] Accordingly, the kit or pharmaceutical composition may comprise human CD8+ T cells comprising a nucleic acid encoding a TCR construct of the present invention that recognizes the epitope of SEQ ID NO. 1 in the context of HLA-A2 as defined herein, for example. The TCR construct may be expressed from the nucleic acid under the control of a heterologous promoter.

[0120] In a kit or pharmaceutical composition of the present invention comprising at least two nucleic acids encoding a TCR construct, at least two TCR constructs each comprising a nucleic acid encoding a TCR construct, or at least two host cells, one, two, or three of the TCR constructs may also be other TCR constructs, for example, TCR constructs known in the art. For example, in cases where cancer is associated with EBV, references [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 / 022520 A1; One, two, or three of the TCR compositions disclosed in [or WO 2011 / 039508] may optionally be used in combination with one of the TCR compositions provided in this specification.

[0121] Adoptive T cell therapy (option c) is preferred throughout this application, wherein 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.

[0122] However, gene therapy using the nucleic acid of the present invention (option a) is also possible, and here, for example, a lentivirus vector can be used.

[0123] The TCR construct of the present invention in protein form (option b) may also be used in therapy, for example, to target bacterial micelles to cancer, for targeting toxins associated with cancer, and this may include therapeutic agents such as toxins. The TCR construct of the present invention in protein form may also be used to target diagnostic agents to cancer. Thus, the composition may also be a diagnostic composition.

[0124] T cells expressing each TCR construct individually may be included in the kit, wherein each T cell is individually stored, for example, in a pharmaceutically acceptable buffer. The components of the kit of the present invention may be formulated for simultaneous administration or for administration in any order. The components may also be intended for repeated administration. The literature [Reference: Tran et al., 2014] describes possible administration regimens. Alternatively, they may be mixed and administered together prior to administration. Alternatively, T cells expressing each TCR construct individually may be included in a single pharmaceutical composition. The same applies to the nucleic acids or proteins of the present invention.

[0125] The pharmaceutical compositions or kits of the present invention are typically intended for intravenous administration. They may further comprise a pharmaceutically acceptable carrier, such as a buffer solution, e.g., physiological saline or PBS. They may further comprise excipients, such as stabilizers such as SPGA, carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, or protein-containing preparations such as bovine serum or skim milk.

[0126] T cells are typically 1 × 10 per kg. 5 Up to 1 × 10 9 It is administered to the patient at a concentration equivalent to that of dog cells. Approximately 1 × 10⁶ 6 Up to 1 × 10 11 Dog cells can be administered to a patient in a single dose. These parameters can be adjusted by a healthcare professional according to, for example, the patient's age, sex, weight, and medical condition. For example, a protocol disclosed in the literature [Ref. Doran et al., 2019] can be adapted to use the host cells of the present invention.

[0127] The pharmaceutical composition of the present invention, or a kit comprising the pharmaceutical composition, or the kit of the present invention may be intended for use in the treatment of patients expressing MHC in a context where each TCR recognizes each epitope, for example, as disclosed herein. The average HLA (MHC I) distribution in various populations can be found at http: / / allelefrequencies.net / . If a patient expresses each MHC I, it is advantageous to test before treatment.

[0128] The patient may have a cancer or infectious disease, particularly in the context of certain TCR constructs disclosed herein, an EBV-associated disease selected from the group including, for example, Hodgkin's and non-Hodgkin lymphoma, Burkitt lymphoma, hemophagocytic lymphohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, gastric cancer, lung cancer, hairy leukoplakia, post-transplant lymphoproliferative disorder, and central nervous system lymphoma. Preferably, EBV-related cancers are type II malignancies exemplified by Hodgkin lymphoma and nasopharyngeal carcinoma, or type III malignancies exemplified by leukoplakia, post-transplant lymphoproliferative disorder, and central nervous system lymphoma [cf. Orentas et al., 2001], because these cancers typically express high levels of LMP2A and LMP1. In type III malignancies, EBNA2C is additionally expressed.

[0129] Targeted cancer cells express a protein, or optionally, a protein from which the recognized epitope originates, preferably most cancer cells.

[0130] The patient is typically a mammalian patient. The patient may be a mouse, but preferably, the patient is a human patient.

[0131] The present invention also discloses a method for treating cancer or an infectious disease, e.g., an EBV-related disease, preferably an EBV-related cancer, by administering an effective amount of the pharmaceutical composition or kit of the present invention to a patient in need thereof, e.g., a patient having the cancer or disease.

[0132] The pharmaceutical compositions and kits of the present invention may be used in combination with other agents, particularly other anticancer agents. For example, other anticancer agents may be TCR-engineered T cells expressing a TCR specific to other antigens expressed in EBV-positive tumors (e.g., MAGE, NY-ESO, OA), checkpoint inhibitors or other immunotherapies, in addition, antibodies, small molecule inhibitors or other types of reagents.

[0133] One preferred medical use of the present invention is immunotherapy, preferably adoptive T cell therapy. The products and methods of the present invention are particularly useful in the context of adoptive T cell therapy. Administration of the compounds of the present invention may include, for example, administering, or injecting, the T cells of the present invention to the patient. Preferably, these T cells are the patient's own T cells transduced in vitro with the nucleic acid of the present invention.

[0134] Alternatively, the patient may also be administered the nucleic acid of the present invention, in particular the expression vector, for the in vivo transduction of T cells.

[0135] The protein TCR constructs of the present invention may also be used for diagnostic purposes, for example, to investigate whether a subject expresses each protein, particularly whether an epitope recognized by the TCR construct is presented in the context of MHC I. To this end, such constructs are preferably labeled to facilitate detection. Preferably, patients presenting such epitopes on each MHC I are treated with the adoptive T cell therapy of the present invention.

[0136] The present invention also relates to a method for producing a host cell of the present invention, comprising introducing an expression vector encoding a TCR construct of the present invention into a suitable host cell, preferably a human CD8+ T cell isolated from a patient.

[0137] The present invention is further described in the following examples with reference to the accompanying drawings and sequences, though not limited thereto. For the purposes of the invention, all references cited herein are incorporated by reference in their entirety.

[0138] vaccine

[0139] In one embodiment, the present invention provides a pharmaceutical composition, particularly a vaccine composition, comprising a peptide comprising an epitope that can be presented by human MHC I, wherein the epitope is a nucleic acid (RNA, etc.) that is newly identified herein or that codes for such peptide. The epitope

[0140] a) having at least 88% sequence identity with respect to SEQ ID NO. 3, wherein the epitope may be presented on HLA-C*15:02, and the peptide comprises up to 25, preferably up to 11, consecutive amino acids identical to the sequence of amino acids present in LMP1 of SEQ ID NO. 120;

[0141] b) having at least 88% sequence identity with respect to SEQ ID NO. 4, wherein the epitope may be presented on HLA-C*06:02, and the peptide comprises up to 25, preferably up to 11, consecutive amino acids identical to the sequence of amino acids present in EBNA3C of SEQ ID NO. 121;

[0142] c) having at least 90% sequence identity with respect to SEQ ID NO. 5, wherein the epitope may be presented on HLA-B*44:02, and the peptide comprises up to 25, preferably up to 11, consecutive amino acids identical to the sequence of amino acids present in EBNA3C of SEQ ID NO. 121.

[0143] Preferably, the peptide of a) can be specifically recognized by TCR83 as defined herein. Preferably, the peptide of b) can be specifically recognized by TCR64 as defined herein. Preferably, the peptide of c) can be specifically recognized by TCR25 or 58 as defined herein.

[0144] Peptide vaccines are well known in the art. For example, peptide vaccines can be used to administer to a subject in combination with an adjuvant such as an aluminum salt, e.g., aluminum phosphate or aluminum hydroxide, squalene, e.g., MF59, a liposome, e.g., QS21, or monophosphoryl lipid A.

[0145] The pharmaceutical composition of the present invention may comprise a peptide comprising the epitope of SEQ ID NO. 3, wherein the epitope may be presented on HLA-C*15:02, and the peptide comprises up to 25, up to 15, or preferably up to 11 amino acids identical to the sequence of amino acids present in LMP1 of SEQ ID NO. 120.

[0146] The pharmaceutical composition of the present invention may comprise a peptide comprising the epitope of SEQ ID NO. 2, wherein the epitope may be presented on HLA-B*57:01, and the peptide comprises up to 25, up to 15, or preferably up to 11 consecutive amino acids identical to the sequence of amino acids present in LMP1 of SEQ ID NO. 120.

[0147] The pharmaceutical composition of the present invention may comprise a peptide comprising the epitope of SEQ ID NO. 5, wherein the epitope may be presented on HLA-B*44:02, and the peptide comprises up to 25, up to 15, or preferably up to 11 consecutive amino acids identical to the sequence of amino acids present in EBNA3C of SEQ ID NO. 121.

[0148] As defined herein, nucleic acids encoding peptides containing epitopes may also be used in combination with suitable adjuvants, such as liposomes or CpG nucleotides.

[0149] The pharmaceutical composition of the present invention may comprise a nucleic acid encoding a peptide containing the epitope of SEQ ID NO. 3, wherein the epitope may be presented on HLA-C*15:02, and the peptide comprises up to 25, up to 15, or preferably up to 11 amino acids identical to the sequence of amino acids present in LMP1 of SEQ ID NO. 120.

[0150] The pharmaceutical composition of the present invention may comprise a nucleic acid encoding a peptide containing the epitope of SEQ ID NO. 4, wherein the epitope may be presented on HLA-C*06:02, and the peptide comprises up to 25, up to 15, or preferably up to 11 consecutive amino acids identical to the sequence of amino acids present in EBNA3C of SEQ ID NO. 121.

[0151] The pharmaceutical composition of the present invention may comprise a nucleic acid encoding a peptide containing the epitope of SEQ ID NO. 5, wherein the epitope may be presented on HLA-B*44:02, and the peptide comprises up to 25, up to 15, or preferably up to 11 consecutive amino acids identical to the sequence of amino acids present in EBNA3C of SEQ ID NO. 121.

[0152] Any of the above-mentioned vaccine pharmaceutical compositions may be intended for use in vaccination against EBV-related diseases selected from the group comprising Hodgkin lymphoma and non-Hodgkin lymphoma, Burkitt lymphoma, hemophagocytic lymphohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, gastric cancer, leukoplakia pilaris, post-transplant lymphoproliferative disorder, and central nervous system lymphoma. The vaccination may be a prophylactic vaccination, that is, a vaccination provided to a subject who does not yet have the disease or does not have an EBV infection, with the aim of reducing the risk of the subject developing the disease upon EBV contact. The subject may belong to a risk group for the disease, for example, a subject who has had EBV contact or is identified as having a current EBV infection, who does not (yet) have the disease. Alternatively, the vaccination may be a therapeutic vaccination. Patients with EBV-related diseases may be treated with therapeutic vaccination.

[0153] Advantageously, the HLA of the subject or patient is known, and the vaccine is administered to a patient who can present an epitope on an MHC I molecule, for example, a patient having HLA-C*15 in the case of an epitope as defined in a), particularly HLA-C*15:02, a patient having HLA-B*57 in the case of an epitope as defined in b), particularly HLA-C*57:01, a patient having HLA-B*44 in the case of an epitope as defined in c), and a patient having HLA-B*44 in the case of an epitope as defined in c). Brief explanation of the drawing

[0154] Fig. 1. MHC Detection of T cell responses using a Class I K562 cell libraryT cells expanded from EBV antigen-expressing dendritic cells were co-cultured with K562 cells from an MHC cell library. Screening for immunogenic EBV antigen-HLA combinations was performed by (A) analyzing CD137 expression and (B) determining the amount of secreted IFN-γ by ELISA. (C) FACS screening of CD137-positive T cells (11%) responding to K562-HLA-B*57:01-positive cells, followed by the identification of 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 additional TCRs described herein. Fig. 2. TCR Genetic analysis (A) Next-generation sequencing-based TCR repertoire analysis of FACS-selected T cells reacting with EBV antigen-positive (LMP1 / LMP2A / EBNA1) K562-HLA-B*57:01 cells. Presented are a portion of the total reads assigned to each sequence cluster, and sequence representatives are shown on the y-axis. We constructed Japanese-stranded TCR-retroviruses to identify functional TCRs using TCRα- and TCRβ strands with a frequency of more than 10%. (B) For one TCR (designated as TCR50), different V segments of the most dominant TCRα-(TRAV) and TCRβ(TRBV) chains, their frequencies, and sequences of the CDR-3 region (IMGT nomenclature) are presented (AMSDLYAGNNRKLI: SEQ NO. 122, ALTFLRDDKII: SEQ NO. 123, VVMATGFQKLV: SEQ NO. 24, ASSQDARVSGANVLT: SEQ NO. 124, ASSVTSGSDEQF: SEQ NO. 125, ASSFSLGHSYEQY: SEQ NO. 126). This approach has been applied to all additional TCRs described herein. Fig. 3. Functional TCRαβ Sympathy of the chain combinationJapanese-strand TCR-retroviruses for functional TCR identification were constructed using TCRα- and TCRβ chains with a frequency of 10% or more. In the case of TCR50, TRAV8-2*01 and TRBV9*01 formed functional TCRs because TCR-engineered T cells specifically recognized a single antigen (LMP1) in combination with a single HLA (B*57:01). Other TCRα- and TCRβ chain combinations resulted in non-specific antigen recognition. As presented herein exemplarily for TCR50, the combinatorial approach for identifying functional TCRα- and TCRβ chain combinations was applied to all additional TCRs described herein (MIN - no antigen stimulation, MAX - non-specific stimulation). Fig. 4. TCR50 To identify antigenic peptides recognized by Epitope Map ping (A) Cleaved versions 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 containing the immunogenic epitope was identified by measuring the amount of IFN-γ secreted by ELISA in the supernatant and is located between nucleotides (nt) 316 and 624. (MIN - no antigen stimulation, MAX - non-specific antigen stimulation, UT - non-transduced T cells). Fig. 5. LMP1's Immunogenicity Epitope's sympathyCandidate peptides were selected by applying the NetMHCpan 4.0 epitope prediction algorithm for HLA-B*57:01 using the protein region identified as the epitope-positive sequence (LMP1 / 2 nt 316–624). (A) Thirteen peptides were used for epitope identification based on their peptide-MHC I binding affinity (binding level), classified as strong binders (SB) and weak binders (WB), respectively. (B) The selected peptides were loaded into K562-HLA-B*57:01 cells, co-cultured with TCR50-engineered T cells from two donors, and IFN-γ secretion was measured by ELISA. Four epitopes with the following amino acid sequences (highlighted in A) were recognized by TCR50-engineered T cells: IALYLQQNWW (Sequence No. 108), IALYLQQNW(Sequence No. 2), IIALYLQQNW (Sequence No. 110), ALYLQQNWW (Sequence No. 116) The sequence numbers of the analyzed peptides are as shown in Table 1 below. (MIN - No antigen stimulation, MAX - Non-specific stimulation, SB - Strong binder, WB - Weak binder, UT - Non-transduced T cells). Fig. 6. TCR50 - Peptide titration of engineered T cells . Non-transformed (UT) and TCR50-transformed T cells (TCR50) from two donors were co-cultured with K562-HLA-B*57:01 cells loaded with appropriate amounts of the labeled peptides (SEQ Nos. 2, 108, 110, 116), and the amount of secreted IFN-γ in the supernatant was measured by ELISA. The peptide IALYLQQNW (9mer, SEQ No. 2) was recognized at the lowest concentration and thus can be considered a homologous EBV LMP1 epitope of TCR50. Interestingly and worth noting, this epitope recognized by TCR50 was not ranked highest in the NetMHCpan4.0 prediction tool, which indicates that the prediction tool is inaccurate in predicting the relevant immunodominant epitope. Fig. 7. TCR50 - Engineered T cells are LMP1 Recognizes positive cells Functional analysis of LMP1-specific TCR50-enhanced T cells from two donors using K562-HLA-B*57:01 antigen-loading cells, EBV-associated cancer cell line (L591-B*57:01), and lymphoblastic cell line, respectively (WIN, DEM). T cell responsiveness was determined by measuring the amount of IFN-γ secreted by ELISA at an effector-to-target (E:T) cell ratio of 1:1. (MIN - no antigen stimulation, MAX - non-specific stimulation, UT - non-transgenic T cells). Fig. 8. EBNA3C Reactivity TCR01's Functional analysis(A) EBNA3C-specific TCR01-engineered T cells were co-cultured with K562-HLA-B*07:02 antigen pulsed cells and EBV-positive cell lines. T cell function 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) Cleaved versions of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were generated and expressed in K562-HLA-B*07:02 cells. (C) The antigen region containing the immunogenic epitope was identified by measuring the amount of IFN-γ secreted by ELISA in the supernatant and is located between nucleotides (nt) 2071 and 2979. (D) Candidate peptides were selected by applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*07:02 using the protein region identified as the epitope-positive sequence (EBNA3C nt 2071-2979).(E) Twenty-seven peptides were used for epitope identification based on their peptide-MHC I binding affinity (binding level), classified into strong binders (SB) and weak binders (WB), respectively (QPRAPIRPI (SEQ No. 127), RPIPTRFPPPPM (SEQ No. 128), RPRVEESSHGPA (SEQ No. 129), SPQPRAPI (SEQ No. 130), SPQPRAPIRPI (SEQ No. 131), SPQPRAPIRPIP (SEQ No. 132), PQPRAPIRPI (SEQ No. 133), QPRAPIRPIP (SEQ No. 134), PRAPIRPI (SEQ No. 135), APIRPIPTRF (SEQ No. 136), FPPPPMPL (SEQ No. 137), HGPARCSQAT (SEQ No. 138), RPIPTRFPP (SEQ No. 139), RPIPTRFP(SEQ No. 140), IPTRFPPPPMP(SEQ No. 141), PIPTRFPPPPM(SEQ No. 142), IPTRFPPPPMPL(SEQ No. 143), GPARCSQATA(SEQ No. 144), FPPPPMPLQDSM(SEQ No. 145), PPMPLQDSM(SEQ No. 146), RPIPTRFPPP(SEQ No. 147), MPLQDSMAVG(SEQ No. 148), PIPTRFPPPPMP(SEQ No. 149), PMPLQDSMAV(SEQ No. 150), PMPLQDSM(SEQ No. 151), QPRAPIRPIPT(SEQ No. 152), QPRAPIRP(SEQ No. 153)). Selected peptides were loaded into K562-HLA-B*07:02 cells, co-cultured with TCR01-engineered T cells, and IFN-γ secretion was measured by ELISA. The epitope (highlighted as D) was recognized by TCR01-engineered T cells. (F) K562-HLA-B*07:02 cells were loaded with a titratable amount of the labeled peptide or without the peptide as a control, and the peptide sensitivity of TCR01-engineered T cells was determined by measuring the amount of IFN-γ secreted by ELISA at an E:T cell ratio of 1:1.(MIN - No antigen stimulation, MAX - Non-specific stimulation, SB - Strong binder, WB - Weak binder, UT - Non-transduced T cells). Fig. 9. EBNA3C Reactivity TCR25's Functional analysis(A) EBNA3C-specific TCR25-manipulated T cells were co-cultured with K562-HLA-B*44:02 antigen pulsed cells or EBV-associated cancer cell lines. T cell function 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) Cleaved versions 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 antigen region containing the immunogenic epitope was identified by measuring the amount of IFN-γ secreted by ELISA in the supernatant and is located between nucleotides (nt) 1 and 567. (D) Candidate peptides were selected by applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*44:02 using the protein region identified as the epitope-positive sequence (EBNA3C nt 1-567). (E) Four peptides were used for epitope identification based on their peptide-MHC binding affinity (binding level), classified as strong binders (SB) and weak binders (WB), respectively (AEGGVGWRHW (SEQ No. 5), SERLVPEESY (SEQ No. 155), WLLTSPSQSW (SEQ No. 156), LLTSPSQSW (SEQ No. 157)). The selected peptides were loaded into K562-HLA-B*44:02 cells, co-cultured with TCR25-engineered T cells, and IFN-γ secretion was determined by ELISA. One epitope (highlighted as D) having the amino acid sequence AEGGVGWRHW was recognized by TCR25-engineered T cells and can therefore be considered a cognate of TCR25, the EBV EBNA3C epitope. (F) K562-HLA-B*44:02 cells were loaded with an appropriate amount of the labeled peptide, or no peptide was loaded as a control. The peptide sensitivity of TCR25-engineered T cells was determined by measuring the amount of secreted IFN-γ by ELISA at an E:T cell ratio of 1:1.(MIN - No antigen stimulation, MAX - Non-specific stimulation, SB - Strong binder, WB - Weak binder, UT - Non-transduced T cells). Fig. 10. EBNA3C Reactivity TCR27's Functional analysis(A) EBNA3C-specific TCR27-engineered T cells were co-cultured with K562-HLA-B*07:02 antigen-pulse cells and EBV-positive cell lines. T cell function 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) Cleaved versions of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were generated and expressed in K562-HLA-B*07:02 cells. (C) The antigen region containing the immunogenic epitope was identified by measuring the amount of IFN-γ secreted in the supernatant (ELISA) and is located between nucleotides (nt) 2071 and 2979. (D) Candidate peptides were selected by applying the epitope prediction algorithm NetMHCpan4.0 to HLA-B*07:02 using the protein region identified as the epitope-positive sequence (EBNA3C nt 2071-2979). (E) Twenty-seven peptides were used for epitope identification based on their peptide-MHC binding affinity (binding level), classified into strong binders (SB) and weak binders (WB), respectively. The selected peptides were loaded into K562-HLA-B*07:02 cells, co-cultured with TCR27-engineered T cells, and IFN-γ secretion was measured by ELISA. Epitopes (highlighted in D) were recognized by TCR27-engineered T cells. (F) Peptide sensitivity of TCR27-engineered T cells was determined by loading an appropriate amount of the labeled peptide into K562-HLA-B*07:02 cells or by loading no peptide as a control, and by measuring the amount of secreted IFN-γ by ELISA at a 1:1 E:T cell ratio. (MIN - no antigen stimulation, MAX - non-specific stimulation, SB - strong binder, WB - weak binder, UT - non-transgenic T cells, see legend for SEQ ID NO. 8). Fig. 11. EBNA3C Reactivity TCR58's Functional analysis(A) EBNA3C-specific TCR58-engineered T cells were co-cultured with K562-HLA-B*44:02 antigen pulsed cells or EBV-associated cancer cell lines. T cell function 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) Cleaved versions 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 antigen region containing the immunogenic epitope was identified by measuring the amount of IFN-γ secreted by ELISA in the supernatant and is located between nucleotides (nt) 1 and 567. (D) Candidate peptides were selected by applying the epitope prediction algorithm NetMHCpan4.0 to HLA-B*44:02 using the protein region identified as the epitope-positive sequence (EBNA3C nt 1-567). (E) Four peptides were used for epitope identification based on their peptide-MHC binding affinity (binding level), classified as strong binders (SB) and weak binders (WB), respectively. The selected peptides were loaded into K562-HLA-B*44:02 cells, co-cultured with TCR58-engineered T cells, and IFN-γ secretion was measured by ELISA. One epitope (highlighted in D) having the amino acid sequence AEGGVGWRHW was recognized by TCR58-engineered T cells and can therefore be considered a cognate EBV EBNA3C epitope of TCR58. (F) K562-HLA-B*44:02 cells were loaded with an appropriate amount of the labeled peptide, or no peptide was loaded as a control. Peptide sensitivity of TCR58-engineered T cells was determined by measuring the amount of secreted IFN-γ by ELISA at an E:T cell ratio of 1:1. (MIN - no antigen stimulation, MAX - non-specific stimulation, SB - strong binder, WB - weak binder, UT - non-transgenic T cells, see legend for SEQ ID NO. 9). Fig. 12. EBNA3C Reactivity TCR64's Functional analysis(A) EBNA3C-specific TCR64-engineered T cells were co-cultured with K562-HLA-C*06:02 antigen pulsed cells and EBV-positive cell lines. T cell function 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) Cleaved versions of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were generated and expressed in K562-HLA-C*06:02 cells. (C) The antigen region containing the immunogenic epitope was identified by measuring the amount of IFN-γ secreted by ELISA in the supernatant and is located between nucleotides (nt) 568 and 1569. (D) Candidate peptides were selected by applying the epitope prediction algorithm NetMHCpan4.0 for HLA-C*06:02 using the protein region (EBNA3C nt 568-1569) identified as an epitope-positive sequence.(E) Twenty-seven peptides were used for epitope identification based on their peptide-MHC binding affinity (binding level), classified as strong binders (SB) and weak binders (WB), respectively (RRYRRIYDL (SEQ No. 158), FRKAQIQGL (SEQ No. 4), AREAEVRFL (SEQ No. 159), LRGKWQRRY (SEQ No. 160), ERYAREAEV (SEQ No. 161), SRRRRGACV (SEQ No. 162), NLLDFVRFM (SEQ No. 163), RRIYDLIEL (SEQ No. 164), RRRRGACVV (SEQ No. 165), VRFLRGKWQ (SEQ No. 166), RRRGACVVY (SEQ No. 167), QRRYRRIYD (SEQ No. 168), VRFMGVMSS (SEQ No. 169), YAREAEVRFL(SEQ No. 170), NRVGADSIM(SEQ No. 171), LHHIWQNLL(SEQ No. 172), RRGIKEHVI(SEQ No. 173), YRRIYDLIE(SEQ No. 174), RRYRRIYDLI(SEQ No. 175), ARRGIKEHV(SEQ No. 176), QRRYRRIYDL(SEQ No. 177), WQRRYRRIY(SEQ No. 178), FLRGKWQRRY(SEQ No. 179), RDRGACVY(SEQ No. 180), VYDDDVIEV(SEQ No. 181), YAREAEVRF(SEQ No. 182), GCQNAARTL(SEQ No. 183)). Selected peptides were loaded into K562-HLA-C*06:02 cells, co-cultured with TCR64-engineered T cells, and IFN-γ secretion was measured by ELISA. One epitope (highlighted as D) having the amino acid sequence FRKAQIQGL was recognized by TCR64-engineered T cells and can therefore be considered a TCR64 homologous EBV EBNA3C epitope. (F) Peptide sensitivity of TCR64-engineered T cells was determined by loading an appropriate amount of the labeled peptide into K562-HLA-C*06:02 cells or by not loading the peptide as a control, by measuring the amount of IFN-γ secreted by ELISA at an E:T cell ratio of 1:1.(MIN - No antigen stimulation, MAX - Non-specific stimulation, SB - Strong binder, WB - Weak binder, UT - Non-transduced T cells). Fig. 13. LMP1 Reactivity TCR83's Functional analysis (A) LMP1-specific TCR83-engineered T cells were co-cultured with K562-HLA-C*15:02 antigen pulsed cells or EBV-associated cancer cell lines. T cell functionality 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) Cleaved versions 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 containing the immunogenic epitope was identified by measuring the amount of IFN-γ secreted by ELISA in the supernatant and is located between nucleotides (nt) 316 and 624. (D) Candidate peptides were selected by applying the NetMHCpan4.0 epitope prediction algorithm for HLA-C*15:02 using the protein region identified as the epitope-positive sequence (LMP1 nt 316-624). (E) Four peptides were used for epitope identification based on peptide-MHC binding affinity (binding level) classified as weak binders (WB) (NSNEGRHHL (SEQ No. 184), QQNWWTLLV(SEQ No. 3), DSLPHPQQA (SEQ No. 185), YLQQNWWTL (SEQ No. 186)). K562-HLA-C*15:02 cells were loaded with the selected peptides, co-cultured with TCR83-engineered T cells, and IFN-γ secretion was measured by ELISA. One epitope (highlighted as D) having the amino acid sequence QQNWWTLLV was recognized by TCR83-engineered T cells and can therefore be considered a cognate EBV LMP1 epitope of TCR83. (F) K562-HLA-C*15:02 cells were loaded with an appropriate amount of the labeled peptides, or no peptides were loaded as a control. The peptide sensitivity of TCR83-engineered T cells was determined by measuring the amount of IFN-γ secreted by ELISA at an E:T cell ratio of 1:1 (B). (MIN - No antigen stimulation, MAX - Non-specific stimulation, WB - Weak binder, UT - Non-transduced T cells). Fig. 14. LMP2A Reactivity TCR06's Functional analysis(A) LMP2A-specific TCR06-engineered T cells were co-cultured with K562-HLA-A*02:01 antigen pulsed cells or EBV-associated cancer cell lines. T cell function 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) Cleaved versions of the full-length LMP2A antigen (LMP2A / 2, LMP2A / 1) were generated and expressed in K562-HLA-A*02:01 cells. (C) The antigen region containing the immunogenic epitope was identified by measuring the amount of IFN-γ secreted in the supernatant (ELISA) and is located between nucleotides (nt) 1006 and 1494. (D) Candidate peptides were selected by applying the NetMHCpan4.0 epitope prediction algorithm for HLA-A*02:01 using the protein region identified as the epitope-positive sequence (LMP1 nt 1006-1494). (E) Based on their peptide-MHC binding affinity (binding level), classified as strong binders (SB) and weak binders (WB), 14 peptides were used for epitope identification (FMCLGGLLTM (SEQ No. 187), MLLLIVAGI (SEQ No. 188), NLFCMLLLI (SEQ No. 189), LLIVAGILFI (SEQ No. 190), NLFCMLLLIV (SEQ No. 191), MCLGGLLTMV (Sequence No. 192), CLGGLLTMV(SEQ No. 1), LIVAGILFI (SEQ No. 193), FIPNLFCML (SEQ No. 194), IVAGILFIL (SEQ No. 195), MLLLIVAGIL (SEQ No. 196), CMLLLIVAGI (SEQ No. 197), PNLFCMLLLI (SEQ No. 198), FIPNLFCMLL (SEQ No. 199)). Selected peptides were loaded into K562-HLA-A*02:01 cells, co-cultured with TCR06-engineered T cells, and IFN-γ secretion was measured by ELISA. The epitope (highlighted as D) was recognized by TCR06-engineered T cells and can therefore be considered as a homologous EBV LMP2A epitope of TCR06. Interestingly and worth noting, the two epitopes recognized by TCR06 were not ranked highest in the NetMHCpan4.0 prediction tool, indicating that the prediction tool is inaccurate in predicting relevant immunogenic epitopes. (F) K562-HLA-A*02:01 cells were loaded with an appropriate amount of the labeled peptide, or no peptide was loaded as a control. Peptide sensitivity of TCR06-engineered T cells was determined by measuring the amount of IFN-γ secreted by ELISA at an E:T cell ratio of 1:1 (B). (MIN - no antigen stimulation, MAX - non-specific stimulation, S - strong binder, WB - weak binder, UT - non-transgenic T cells). FIG. 15. Patent WO 2015 / 022520 A1( PUBTCR1 ) and WO 2011 / 039508 A2( PUBTCR2 Provided in ) LMP2A - Specific TCR06 and TCR's Comparison of peptide sensitivityA previously disclosed wt EBV LMP2A TCR having the alpha-chain amino acid sequence TRAV12-3*01 / TRAJ41*01 / TRAC (SEQ No. 2 of WO 2015 / 022520 A1) and the beta-chain amino acid sequence TRBV11-2*01 / TRBD1 / TRBJ2-7 / TRBC (SEQ No. 3 of WO 2015 / 022520 A1) (also FIG. 1 of the said document) is designated herein as PUBTCR1. In particular, based on its SEQ No. 8, a previously disclosed LMP2A TCR described in WO 2011 / 039508 A2 is designated as PUBTCR2. K562-HLA-A*02:01 cells were loaded with appropriate amounts of the labeled peptides CLGGLLTMV and MCLGGLLTMV, respectively, and co-cultured with TCR06-, PUBTCR1-, or PUBTCR2-engineered T cells. 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. TCR06 exhibited higher peptide sensitivity compared to PUBTCR1 and PUBTCR2 UT-non-transduced T cells. Fig. 16. LMP2A specific TCR06's Peptide sensitivity and, patents WO 2015 / 022520 A1(PUBTCR1) and WO 2011 / 039508 A2( PUBTCR2 Provided in ) TCR and isolates by the applicant made Extended comparison with TCR-JC . A previously disclosed wt EBV LMP2A TCR having the alpha-chain amino acid sequence TRAV12-3*01 / TRAJ41*01 / TRAC (SEQ No. 2 of WO 2015 / 022520 A1) and the beta-chain amino acid sequence TRBV11-2*01 / TRBD1 / TRBJ2-7 / TRBC (SEQ No. 3 of WO 2015 / 022520 A1) (also FIG. 1 of the said document) is designated herein as PUBTCR1. In particular, based on its SEQ No. 8, the previously disclosed LMP2A TCR described in WO 2011 / 039508 A2 is designated as PUBTCR2. Another LMP2A-specific TCR designated herein as TCR-JC was isolated by the applicant. (A) K562-HLA-A*02:01 cells were loaded with an appropriate amount of peptide CLGGLLTMV and co-cultured with TCR06-, PUBTCR1-, PUBTCR2-, or TCR-JC-engineered T cells. 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 1:10. (B) S-shaped 4PL curves based on data points from A (pharmaceutical model). S-shaped regression showed very good R-values ​​for TCR06, PUBTCR1, and PUBTCR2. 2 It indicates (approx. 0.95). The data from TCR-JC shows a model fit of 0.81 (lack of upper stable phase). (C) Based on the data presented in B, the peptide sensitivity of the four TCRs was calculated, and 10 -9 Indicated as EC50 in M ​​(EC50 - mol / l of peptide required to achieve 50% of maximum IFN-γ release, SEM - mean of standard error). Fig. 17. EBV - Specific TCR - Engineered T cells are cancer cells killLMP1-specific TCR50-, LMP2A-specific TCR06-, and EBNA3C-specific TCR64-engineered T cells were co-cultured with L591 EBV+ tumor cells, which naturally express LMP1, LMP2A, and EBNA3C but were transfected with their respective MHC I alleles. For the indicated effector:target (E:T) cell ratio, the triple-well data were averaged, and the percentage of viable cells was calculated relative to the values ​​obtained from samples co-cultured with non-transfected T cells: % specific viability = 100 × (test value) / (mean background). (UT - non-transfected T cells). Fig. 18. Tumor rejection reaction In vivo Mouse model (A) NOG mice are 5×10 6 K562-HLA-A*02:01 tumor cells were administered subcutaneously, and 1×10⁶ were administered 24 hours later. 7 TCR06-engineered T cells were administered intravenously. Data obtained from individual mice that received TCR06-engineered T cells (n=10) from two donors are presented in comparison to non-transgenic T cells (n=6). (B) NSG mice were 5×10 6 K562-HLA-B*57:01 tumor cells were administered subcutaneously, and 5×10⁶ were administered after 9 days. 6 TCR50-manipulated T cells were administered intravenously. In both models, tumor size was measured and calculated using calipers. Specific details for implementing the invention

[0155] Examples

[0156] EBV - Specific TCR Works and generation of transformed T cells and Epitope's sympathy

[0157] The inventors used an innovative method [Ref. Lorenz et al., 2018; WO2016 / 146618 A1] to generate an EBV-specific TCR that recognizes endogenously processed immunogenic EBV epitopes presented by different MHC class I molecules.

[0158] Simply put, after selecting the target EBV antigen, e.g., LMP1, LMP2A, or EBNA3C, the following experimental steps were performed:

[0159] (i) Pulse of professional antigen-presenting cells (preferably dendritic cells (DCs)) with in vitro transcribed (ivt) RNA encoding the full-length sequence of an EBV antigen selected to stimulate autologous T cells. This procedure is not completely biased, and the DC can select the best epitope of the antigen for expression, processing, and presentation at the cell surface in combination with the most suitable MHC class I (MHC I) molecule.

[0160] (ii) Identification of EBV antigen-reactive T cells. This step was performed using a newly established MHC class I cell library consisting of single MHC I-expressing cell lines derived from K562 cells. This part is an important function of the TCR isolation approach, as it is fundamental to utilizing extensive MHC flexibility. To identify each TCR, the inventors selected up to six MHC I alleles from the K562 cell library corresponding to the MHC I alleles of the T cell donors and transfected the cells with the relevant antigens used for priming in step (i). After co-culture of antigen-presenting K562 cells and antigen-stimulated T cells, reactive T cells were identified by interferon-(IFN)γ release using ELISA and the upregulation of the T cell activation marker CD137 measured by flow cytometry. Subsequently, reactive CD8+ T cells were enriched by FACS screening.

[0161] (iii) Isolation of total RNA from FACS-selected CD8+ T cells, and PCR amplification of TCRα and TCRβ chain-specific sequences. Identification of dominant TCRα- and TCRβ sequences by next-generation sequencing.

[0162] (iv) To confirm functional TCRαβ chain combinations, re-expression of dominant (at least 10%) TCRα- and TCRβ chain combinations was performed in primary human T cells using the γ-retroviral vector MP71 [Refs. Engels et al., 2003; Leisegang et al., 2008; Sommermeyer and Uckert, 2010]. This was performed by co-culture of TCR-engineered T cells with K562 cells carrying appropriate MHC I molecules and expressing full-length EBV antigens. Antigens recognizing the TCRαβ chain combinations were linked to the P2A element and recloned into the MP71 vector with the configuration of TCRβ gene-P2A-TCRα gene. The invariant TCRαβ chain regions were substituted with their murine counterparts to enhance the pairing of the transformed TCR chains. Subsequently, the complete TCR introduction gene cassette was codon-optimized.

[0163] (v) Identification of antigenic peptides (epitopes) of EBV recognized by TCR-engineered T cells. For this purpose, the full-length antigen was cleaved at the C- or N-terminus, cloned into the plasmid vector pcDNA3.1(-), and expressed in K562 cells carrying appropriate MHC I molecules. Subsequently, the remaining protein fragments were tested for their ability to further present epitopes recognized by the 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, loaded into K562 cells carrying appropriate MHC I molecules, and investigated in co-culture experiments for their ability to be recognized by TCR-engineered T cells. Peptides capable of stimulating most IFN-γ production are considered to be homologous epitopes.

[0164] Peptides tested against one of the TCRs (TCR50). Candidate peptides were selected by applying the epitope prediction algorithm NetMHCpan4.0 for HLA-B*57:01 using a protein region identified as an epitope-positive sequence (LMP1 / 2 nt 316-624). Thirteen peptides were used for epitope identification based on peptide-MHC binding affinities classified as strong binders (SB) and weak binders (WK), respectively. The results are presented in Figure 5. Epitope (sequence number) Length (aa) Combination level Affinity WTLLVDLLW (107) 9 SB 12.25 IALYLQQNWW (108) 10 SB 14.84 IALYLQQNW (2) 9 SB 16.32 WWTLLVDLLW (109) 10 SB 32.51 IIALYLQQNW (110) 10 SB 57.84 WTLLVDLLWL (111) 10 SB 111.95 LAILIWMYY (112) 9 SB 231.02 LLFLAILIW (113) 9 SB 234.00 LLLFLAILIW (114) 10 WB 749.75 LAILIWMYYH (115) 10 WB 789.96 ALYLQQNWW (116) 9 WB 853.78 FLAILIWMYY (117) 10 WB 1515.23 NSNEGRHHL (118) 9 WB 12828.84

[0165] The generated TCR constructs are characterized in Tables 2, 3, and 4 below.

[0166] TCR Summary. TCR 절반-최대 IFN-η EBV-항원 에피토프 서열번호 MHC I 종양 세포의 인식 01 6x10 -8 EBNA3C QPRAPIRPIPT 7 B*07:02 + 25 6x10 -7 EBNA3C AEGGVGWRHW 5 B*44:02 + 27 6x10 -8 EBNA3C QPRAPIRPIP 6 B*07:02 + 58 3x10 -7 EBNA3C AEGGVGWRHW 5 B*44:02 + 64 7x10 -6 EBNA3C FRKAQIQGL 4 C*06:02 + 50 3x10 -8 LMP1 IALYLQQNW 2 B*57:01 + 83 2x10 -9 LMP1 QQNWWTLLV 3 C*15:02 + 06 6x10 -9 LMP2A CLGGLLTMV 1 A*02:01 +

[0167] TCR06, TCR50, and TCR83, particularly TCR06, have high peptide sensitivity (half-max of IFN-γ release).

[0168] CDR sequence of a preferred TCR construct of the present invention. CDR1 IMGT aa positions: 27-38. CDR2 IMGT aa positions: 56-65. CDR3 IMGT aa positions: 105-117. Numbers in parentheses: Sequence numbers TCR CDR -1 aa 서열 CDR -2 aa 서열 CDR -3 aa 서열 TCR 06 TRA DSAIYN (11) IQSSQRE (12) AVLMDSNYQLI (13) TRB WSHSY (16) SAAADI (17) ASSEDGMNTEAF (18) TCR 50 TRA SSYSPS (21) YTSAATLV (22) VVMATGFQKLV (23) TRB SGDLS (26) YYNGEE (27) ASSVTSGSDEQF (28) TCR 83 TRA TSGFNG (31) NVLDGL (32) AAVNNAGNMLT (33) TRB LGHDT (36) YNNKEL (37) ASSQGYGGPSTDTQY (38) TCR 64 TRA SVFSS (41) VVTGGEV (42) AGDVDTGTASKLT (43) TRB MDHEN (46) SYDVKM (47) ASSLLGSGALYEQY (48) TCR 25 TRA SSYSPS (51) YTSAATLV (52) VAWDTGFQKLV (53) TRB SNHLY (56) FYNNEI (57) ASKALADTQY (58) TCR 58 TRA NSASQS (61) VYSSGN (62) VASGDSSYKLI (63) TRB SNHLY (66) FYNNEI (67) ASSDPLSTYNEQF (68) TCR 27 TRA TISGTDY (71) GLTSN (72) ILCGAGGTSYGKLT (73) TRB MNHEY (76) SMNVEV (77) ASNVQGANNEQF (78) TCR 01 TRA TISGTDY (81) GLTSN (82) ILCGAGGTSYGKLT (83) TRB MNHEY (86) SMNVEV (87) ASAIQGANNEQF (88)

[0169] TRAV and TRBV segments and junction aa (IMGT positions 104-118) of a preferred TCR construct of the present invention. Number in parentheses: Sequence number TCR V 세그먼트 aa junction aa sequence TCR 06 TRA TRAV21*01 CAVLMDSNYQLIW (14) TRB TRBV10-2*02 CASSEDGMNTEAFF (19) TCR 50 TRA TRAV8-2*01 CVVMATGFQKLVF (24) TRB TRBV9*01 CASSVTSGSDEQFF (29) TCR 83 TRA TRAV1-2*01 CAAVNNAGNMLTF (34) TRB TRBV3-1*01 CASSQGYGGPSTDTQYF (39) TCR 64 TRA TRAV27*01 CAGDVDTGTASKLTF (44) TRB TRBV28*01 CASSLLGSGALYEQYF (49) TCR 25 TRA TRAV8-2*01 CVAWDTGFQKLVF (54) TRB TRBV2*01 CASKALADTQYF (59) TCR 58 TRA TRAV12-1*01 CVASGDSSYKLIF (64) TRB TRBV2*01 CASSDPLSTYNEQFF (69) TCR 27 TRA TRAV26-2*01 CILCGAGGTSYGKLTF (74) TRB TRBV27*01 CASNVQGANNEQFF (79) TCR 01 TRA TRAV26-2*01 CILCGAGGTSYGKLTF (84) TRB TRBV27*01 CASAIQGANNEQFF (89)

[0170] Functional characterization

[0171] The generated TCR-engineered T cells were functionally characterized using in vitro assays.

[0172] First, the peptide sensitivity of the isolated TCR (half-max of IFN-γ release) was analyzed in peptide titration experiments (see Figure 6 and reference [Reference: Lorenz et al., 2018]). Briefly, the peptide is 10 -5 mol / l to 10 -13 K562 target cells were loaded with titratable amounts in the mol / l range. Co-culture assays were performed using TCR-engineered T cells at an effector-to-target (E:T) cell ratio of 1:1 (unless otherwise indicated), which is 2.5 × 10⁶ 4 TCR-engineered T cells are 2.5 × 10 4 This means that they were co-cultured with peptide-loaded K562 cells. The ability of TCR-transformed T cells to recognize their target epitopes was evaluated by IFN-γ ELISA after 24 hours.

[0173] Second, EBV antigen-expressing target cell lines (EBV-associated cancer cell lines endogenously treated and presenting LCL, EBV epitope, and peptide-loaded K562 cells) were applied to co-culture experiments to determine the amount of IFN-γ released by TCR-engineered T cells (see Fig. 7 and reference [Lorenz et al., 2018]). Briefly, TCR-engineered T cells and target cells (2×10⁶, each) 4 ) were co-cultured. After 24 hours, T cell responsiveness was determined by measuring the amount of secreted IFN-γ by ELISA. T cells stimulated with PMA and ionomycin were used as positive controls, and non-transgenic T cells were used as negative controls.

[0174] Based on the described isolation and characterization procedures and the identification of epitopes recognized by TCR-engineered T cells, a total of eight EBV-specific TCRs and associated immunogenic peptides (epitopes) recognized by these TCRs were isolated. Some epitopes are already known in the art, and other epitopes, in particular the peptides of SEQ ID NOs. 3, 4, and 5, were newly identified in this specification as being presented by their respective MHC I molecules.

[0175] Third, EBV antigen-expressing L591 tumor cells, which are endogenously treated with and presenting epitopes of LMP1, LMP2A, and EBNA3C but transfected with the relevant MHC I allele, were subjected to co-culture experiments with TCR-engineered T cells to determine the apoptotic ability of the TCR-engineered T cells.

[0176] Additionally, the generated TCR-engineered T cells were functionally characterized using in vivo assays. Thus, two models of immunodeficient mice (NOD, NSG) were applied. Tumor cells expressing both the relevant EBV antigen and MHC I molecules were injected subcutaneously into the animals, and TCR-engineered T cells were delivered to the mice by tail vein injection.

[0177] For immunotherapy TCR's mixture

[0178] A combination of TCR-engineered T cells that recognize different epitopes presented by different MHC I molecules is an interesting option for improving TCR gene therapy. This approach aims to overcome and prevent two problems of immunotherapy: (i) tumor growth caused by loss variants of specific tumor antigens and (ii) tumor immune evasion caused by the downregulation of specific MHC I molecules. Using this combination approach enhances the efficiency of TCR gene therapy.

[0179] To demonstrate this concept, two or three TCRs capable of recognizing endogenously processed EBV antigens naturally presented by different MHC I molecules, e.g., TCR06 (LMP2A, MHC A*02:01), TCR50 (LMP1, MHC B*57:01), and optionally TCR64 (EBNA3C, MHC C*06:02), are selected. TCR-engineered T cells are generated with these TCRs by retroviral transduction as described. Tumor cells naturally expressing the EBV antigen recognized by the TCRs and possessing the respective MHC I molecules (A*02:01, B*57:01, C*06:02) are used in co-culture experiments with TCR-engineered T cells.

[0180] Alternatively, these cells (e.g., K562 cells) are produced by transfection of the corresponding EBV antigen gene and MHC I gene.

[0181] In the experiment, TCR-engineered T cells are used individually or in combination and co-cultured with tumor cells at an E:T ratio of 2:1. To evaluate the efficacy of single and combination applications of TCR-engineered T cells, IFN-γ secretion is measured by ELISA.

[0182] In addition, cytotoxicity assays are used to analyze the ability of single and combination applications of TCR-engineered T cells to kill tumor cells.

[0183] In further experiments, an in vivo mouse model is established. Tumor cells, for example, K562 cells expressing respective EBV antigens and MHC I molecules, were subcutaneously injected into the flanks of NSG mice (NOD.Cg-Prkdc scid Il2rg tm1 Wjl / Sz). After the tumor becomes palpable, TCR-engineered T cells are injected intravenously, either individually or in combination, to determine the efficacy of TCR gene therapy in terms of tumor rejection.

[0184] References

[0185] Chervin et al., 2008. Journal of Immunological Methods 339: 175-184.

[0186] Cho et al., 2018. British Journal of Cancer 118: 534-545.

[0187] Cohen et al., 2006. Cancer Research 66: 8878-8886.

[0188] Cohen et al., 2007. Cancer Research 67: 3898-3903.

[0189] Danska et al., 1990. The Journal of Experimental Medicine 172: 27-33.

[0190] Doran et al., 2019. Journal of Clinical Oncology doi: 10.1200 / JCO.18.02424. [Epub ahead of print].

[0191] Engels et al., 2003. Human Gene Therapy 14: 1155-1168.

[0192] Garcia and Adams, 2005. Cell 122: 333-336.

[0193] Hart et al., 2008. Gene Therapy 15: 625-631.

[0194] Jurgens et al., 2006. Journal of Clinical Immunology 26: 22-32.

[0195] Kuball et al., 2007. Blood 109: 2331-2338.

[0196] Leisegang et al., 2008. Journal of Molecular Medicine. 86: 573-583.

[0197] Linette et al., 2013. Blood 122: 863-871.

[0198] Lorenz et al., 2018. Human Gene Therapy 28: 1158-1168.

[0199] Morgan et al., 2013. Journal of Immunotherapy 36: 133-151.

[0200] Orentas et al., 2001. Clinical Immunology 98: 220-228.

[0201] Robbins et al., 2008. Journal of Immunology 180: 6116-6131.

[0202] Scholten et al., 2006. Clinical Immunology 119: 135-145.

[0203] Simpson et al., 2011. Proceedings of the National Academy of Sciences of the USA 108: 21176-21181.

[0204] Sommermeyer and Uckert, 2010, Journal of Immunology 184: 6223-6231.

[0205] Tran et al., 2014. Science 344: 641-645.

[0206] WO 2011 / 039508 A2

[0207] WO 2015 / 022520 A1

[0208] Yang et al., 2011. Clinical and Developmental Immunology, Article ID 716926

[0209] Zheng et al., 2015. Cancer Immunology Research 3: 1138-1147.

Claims

Claim 1 A nucleic acid encoding a TCR alpha chain construct (TRA) and a TCR beta chain construct (TRB) of a TCR construct specific to an epitope that forms a complex with human MHC I, wherein the epitope is an epitope of an Epstein-Barr-virus (EBV) protein, the epitope has the sequence of SEQ ID NO. 1, the MHC I is HLA-A*02:01, the TRA comprises CDR1 having the sequence of SEQ ID NO. 11, CDR2 having the sequence of SEQ ID NO. 12, and CDR3 having the sequence of SEQ ID NO. 13, and the TRB comprises CDR1 having the sequence of SEQ ID NO. 16, CDR2 having the sequence of SEQ ID NO. 17, and CDR3 having the sequence of SEQ ID NO.

18. Claim 2 A nucleic acid according to claim 1, wherein the TRA comprises a variable region having at least 90% sequence identity with respect to SEQ ID NO. 15, and the TRB comprises a variable region having at least 90% sequence identity with respect to SEQ ID NO.

20. Claim 3 The nucleic acid of claim 1, wherein the nucleic acid is selected from the group consisting of viral vectors, transposons, vectors suitable for CRISPR / CAS-based recombination, and plasmids suitable for in vitro RNA transcription. Claim 4 In paragraph 3, the nucleic acid further comprises the TCR alpha chain construct and the TCR beta chain construct comprising an invariant region selected from the group comprising a human invariant region, a murine invariant region, and a chimeric invariant region. Claim 5 A protein encoded by the nucleic acid of any one of paragraphs 1 to 4. Claim 6 An isolated host cell comprising the nucleic acid of any one of claims 1 to 4. Claim 7 In paragraph 6, the isolated host cell, wherein the host cell is a human CD8+ T cell. Claim 8 A pharmaceutical composition for treating an EBV-associated disease in a patient expressing HLA-A*02:01, comprising: a) a nucleic acid of any one of claims 1 to 4 encoding a TCR construct capable of specifically binding to the epitope in the context of MHC I; or b) a protein comprising a TCR construct encoded by the nucleic acid and capable of specifically binding to the epitope in the context of MHC I; or c) an isolated host cell comprising the nucleic acid and expressing a TCR construct capable of specifically binding to the epitope in the context of MHC I, wherein the EBV-associated disease is an EBV-associated cancer, Alice in Wonderland syndrome, acute cerebellar ataxia, or an EBV-associated autoimmune disease. Claim 9 A pharmaceutical composition according to claim 8, wherein the EBV-associated cancer is selected from the group consisting of Hodgkin's and non-Hodgkin lymphoma, Burkitt lymphoma, hemophagocytic lymphohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, lung cancer, gastric cancer, hairy leukoplakia, post-transplant lymphoproliferative disorder, and central nervous system lymphoma. Claim 10 A pharmaceutical composition according to claim 9, wherein the treatment is an immunotherapy selected from the group of adoptive T cell therapy and TCR gene therapy. Claim 11 In claim 8, the pharmaceutical composition is administered in combination with a pharmaceutical composition comprising: a) at least one second nucleic acid encoding a second TCR construct capable of specifically binding to its respective epitope in the context of each MHC I, i. a second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 2, an MHC I being HLA-B*57:01, a TRA comprising CDR1 having SEQ ID NO. 21, a CDR2 having SEQ ID NO. 22, and a CDR3 having SEQ ID NO. 23, and a TRB comprising CDR1 having SEQ ID NO. 26, a CDR2 having SEQ ID NO. 27, and a CDR3 having SEQ ID NO. 28; ii. iii. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 3, MHC I being HLA-C*15:02, TRA including CDR1 having SEQ ID NO. 31, CDR2 having SEQ ID NO. 32, and CDR3 having SEQ ID NO. 33, and TRB including CDR1 having SEQ ID NO. 36, CDR2 having SEQ ID NO. 37, and CDR3 having SEQ ID NO. 38; iii. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 4, MHC I being HLA-C*06:02, TRA including CDR1 having SEQ ID NO. 41, CDR2 having SEQ ID NO. 42, and CDR3 having SEQ ID NO. 43, and TRB including CDR1 having SEQ ID NO. 46, CDR2 having SEQ ID NO. 47, and CDR3 having SEQ ID NO. 48; iv. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 5, MHC I being HLA-B*44:02, TRA including CDR1 having SEQ ID NO. 51, CDR2 having SEQ ID NO. 52, and CDR3 having SEQ ID NO. 53, and TRB including CDR1 having SEQ ID NO. 56, CDR2 having SEQ ID NO. 57, and CDR3 having SEQ ID NO. 58; ⅴ.A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 5, MHC I being HLA-B*44:02, TRA including CDR1 having SEQ ID NO. 61, CDR2 having SEQ ID NO. 62, and CDR3 having SEQ ID NO. 63, and TRB including CDR1 having SEQ ID NO. 66, CDR2 having SEQ ID NO. 67, and CDR3 having SEQ ID NO. 68; vi. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 6, MHC I being HLA-B*07:02, TRA including CDR1 having SEQ ID NO. 71, CDR2 having SEQ ID NO. 72, and CDR3 having SEQ ID NO. 73, and TRB including CDR1 having SEQ ID NO. 76, CDR2 having SEQ ID NO. 77, and CDR3 having SEQ ID NO. 78; and / or vii. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 7, MHC I being HLA-B*07:02, TRA comprising CDR1 having SEQ ID NO. 81, CDR2 having SEQ ID NO. 82, and CDR3 having SEQ ID NO. 83, and TRB comprising CDR1 having SEQ ID NO. 86, CDR2 having SEQ ID NO. 87, and CDR3 having SEQ ID NO. 88; or b) at least one second protein comprising said second TCR construct capable of specifically binding to its respective epitope in the context of each MHC I; or c) at least one isolated host cell expressing said second TCR construct capable of specifically binding to its respective epitope in the context of each MHC I. Claim 12 A pharmaceutical composition according to claim 8, further comprising: a) at least one second nucleic acid encoding a second TCR construct capable of specifically binding to its respective epitope in the context of each MHC I, i. a second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 2, an MHC I being HLA-B*57:01, a TRA comprising CDR1 having SEQ ID NO. 21, a CDR2 having SEQ ID NO. 22, and a CDR3 having SEQ ID NO. 23, and a TRB comprising CDR1 having SEQ ID NO. 26, a CDR2 having SEQ ID NO. 27, and a CDR3 having SEQ ID NO. 28; ii. iii. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 3, MHC I being HLA-C*15:02, TRA including CDR1 having SEQ ID NO. 31, CDR2 having SEQ ID NO. 32, and CDR3 having SEQ ID NO. 33, and TRB including CDR1 having SEQ ID NO. 36, CDR2 having SEQ ID NO. 37, and CDR3 having SEQ ID NO. 38; iii. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 4, MHC I being HLA-C*06:02, TRA including CDR1 having SEQ ID NO. 41, CDR2 having SEQ ID NO. 42, and CDR3 having SEQ ID NO. 43, and TRB including CDR1 having SEQ ID NO. 46, CDR2 having SEQ ID NO. 47, and CDR3 having SEQ ID NO. 48; iv. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 5, MHC I being HLA-B*44:02, TRA including CDR1 having SEQ ID NO. 51, CDR2 having SEQ ID NO. 52, and CDR3 having SEQ ID NO. 53, and TRB including CDR1 having SEQ ID NO. 56, CDR2 having SEQ ID NO. 57, and CDR3 having SEQ ID NO. 58; ⅴ.A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 5, MHC I being HLA-B*44:02, TRA including CDR1 having SEQ ID NO. 61, CDR2 having SEQ ID NO. 62, and CDR3 having SEQ ID NO. 63, and TRB including CDR1 having SEQ ID NO. 66, CDR2 having SEQ ID NO. 67, and CDR3 having SEQ ID NO. 68; vi. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 6, MHC I being HLA-B*07:02, TRA including CDR1 having SEQ ID NO. 71, CDR2 having SEQ ID NO. 72, and CDR3 having SEQ ID NO. 73, and TRB including CDR1 having SEQ ID NO. 76, CDR2 having SEQ ID NO. 77, and CDR3 having SEQ ID NO. 78; and / or vii. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 7, MHC I being HLA-B*07:02, TRA comprising CDR1 having SEQ ID NO. 81, CDR2 having SEQ ID NO. 82, and CDR3 having SEQ ID NO. 83, and TRB comprising CDR1 having SEQ ID NO. 86, CDR2 having SEQ ID NO. 87, and CDR3 having SEQ ID NO. 88; or b) at least one second protein comprising said second TCR construct capable of specifically binding to its respective epitope in the context of each MHC I; or c) at least one isolated host cell expressing said second TCR construct capable of specifically binding to its respective epitope in the context of each MHC I. Claim 13 A kit for treating an EBV-associated disease in a patient expressing HLA-A*02:01, wherein the EBV-associated disease is an EBV-associated cancer, Alice in Wonderland syndrome, acute cerebellar ataxia, or an EBV-associated autoimmune disease, and wherein the kit comprises the pharmaceutical composition of claim 8 and a pharmaceutical composition comprising the following: a) at least one second nucleic acid encoding a second TCR construct capable of specifically binding to its respective epitope in the context of each MHC I, i. a second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 2, an MHC I being HLA-B*57:01, a TRA comprising CDR1 having SEQ ID NO. 21, a CDR2 having SEQ ID NO. 22, and a CDR3 having SEQ ID NO. 23, and a TRB comprising CDR1 having SEQ ID NO. 26, a CDR2 having SEQ ID NO. 27, and a CDR3 having SEQ ID NO. 28; ii. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 3, MHC I being HLA-C*15:02, TRA including CDR1 having SEQ ID NO. 31, CDR2 having SEQ ID NO. 32, and CDR3 having SEQ ID NO. 33, and TRB including CDR1 having SEQ ID NO. 36, CDR2 having SEQ ID NO. 37, and CDR3 having SEQ ID NO. 38; iii. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 4, MHC I being HLA-C*06:02, TRA including CDR1 having SEQ ID NO. 41, CDR2 having SEQ ID NO. 42, and CDR3 having SEQ ID NO. 43, and TRB including CDR1 having SEQ ID NO. 46, CDR2 having SEQ ID NO. 47, and CDR3 having SEQ ID NO. 48; iv.v. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 5, MHC I being HLA-B*44:02, TRA including CDR1 having SEQ ID NO. 51, CDR2 having SEQ ID NO. 52, and CDR3 having SEQ ID NO. 53, and TRB including CDR1 having SEQ ID NO. 56, CDR2 having SEQ ID NO. 57, and CDR3 having SEQ ID NO. 58; vi. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 5, MHC I being HLA-B*44:02, TRA including CDR1 having SEQ ID NO. 61, CDR2 having SEQ ID NO. 62, and CDR3 having SEQ ID NO. 63, and TRB including CDR1 having SEQ ID NO. 66, CDR2 having SEQ ID NO. 67, and CDR3 having SEQ ID NO. 68; vi. A second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 6, an MHC I of HLA-B*07:02, a TRA comprising CDR1 having SEQ ID NO. 71, CDR2 having SEQ ID NO. 72, and CDR3 having SEQ ID NO. 73, and a TRB comprising CDR1 having SEQ ID NO. 76, CDR2 having SEQ ID NO. 77, and CDR3 having SEQ ID NO. 78; and / or vii. a second nucleic acid comprising an epitope having the sequence of SEQ ID NO. 7, an MHC I of HLA-B*07:02, a TRA comprising CDR1 having SEQ ID NO. 81, CDR2 having SEQ ID NO. 82, and CDR3 having SEQ ID NO. 83, and a TRB comprising CDR1 having SEQ ID NO. 86, CDR2 having SEQ ID NO. 87, and CDR3 having SEQ ID NO. 88; or b) at least one second protein comprising said second TCR construct capable of specifically binding to each of its epitopes in each MHC I context; or c) at least one isolated host cell expressing said second TCR construct capable of specifically binding to each of its epitopes in each MHC I context. Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete

Images