TCR constructs specific for ebv antigens

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of immunotherapy, particularly to diseases associated with Epstein-Barr virus (EBV, also known as human gamma herpesvirus 4), such as cancer or post-transplant lymphoproliferative disorders, especially adoptive T-cell therapy or T-cell receptor (TCR) gene therapy. The invention provides combinations of nucleic acids encoding at least two TCR constructs or corresponding proteins or host cells, wherein each TCR construct is capable of specifically binding its corresponding epitope in the context of a corresponding MHC I, and wherein said epitope is a peptide derived from a different antigen expressed by the same infectious agent or cancer, such as an EBV antigen. The invention also provides specific nucleic acids encoding TCR α-chain constructs (TRA) and / or TCR β-chain constructs (TRB) that are specific to epitopes that complex with human MHC I, wherein said epitopes are epitopes of Epstein-Barr virus proteins, and wherein said TCR constructs are specific to epitopes derived from LMP2A, LMP1, or EBNA3C. Proteins encoded by said nucleic acids, corresponding host cells, and pharmaceutical compositions and kits are also objects of this invention. Background Technology

[0002] TCRs are heterodimeric cell surface proteins belonging to the immunoglobulin superfamily, associated with invariant proteins of the CD3 complex involved in mediating signal transduction. TCRs exist in αβ and γδ forms, which are structurally similar but have distinctly different anatomical locations and potential functions. The α and β chains of the native heterodimeric αβ TCR are transmembrane proteins, each containing two extracellular domains: a constant region proximal to the membrane and a variable region distal to the membrane. Each constant and variable region contains intrachain disulfide bonds.

[0003] The variable region of each TCR strand includes variable segments and connecting segments, and in the case of the β-chain, also includes a diversity segment. Each variable region includes three CDRs (complementarity-determining regions) embedded in the frame sequence, a highly polymorphic loop, one of which is a hypervariable region named CDR3. Multiple types of α-chain variable regions (Vα) and multiple types of β-chain variable regions (Vβ) exist, distinguished by their frame, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. The IMGT nomenclature provides a unique TRAV or TRBV number for Vα or Vβ. The TCR specificity of the identified epitope is primarily determined by the CDR3 region (Danska et al., 1990; Garcia et al., 2005).

[0004] The application of adoptive TCR gene therapy can endow a patient's own T cells with the required specificity and generate a sufficient number of activated, non-exhausted T cells in a short period of time. 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, ensuring better persistence and function after metastasis. TCR-engineered T cells can be infused into patients, for example, cancer patients who have experienced lymphopenia due to chemotherapy or radiotherapy, inducing steady-state expansion, thereby greatly enhancing the engraftment and long-term persistence of metastatic T cells and being associated with higher cure rates.

[0005] TCR-based adoptive T-cell therapy relies on classical TCR recognition of antigen-processing epitopes presented in the MHC molecular context. Therefore, T cells expressing epitope-specific TCRs can only be used to treat patients expressing the corresponding MHC in a specific MHC context.

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

[0007] Diseases associated with EBV include infectious mononucleosis and various non-malignant, precancerous, and malignant EBV-related lymphoproliferative disorders, such as post-transplant lymphoproliferative disorder, Burkitt lymphoma, hemophagocytic lymphohistiocytosis, Hodgkin's lymphoma, and non-Hodgkin's lymphoma; non-lymphocytic malignancies such as gastric cancer, lung cancer, and nasopharyngeal carcinoma; and conditions associated with human immunodeficiency virus (HIV), such as hairy leukoplakia and central nervous system lymphoma. The virus is also associated with childhood illnesses such as Alice in Wonderland syndrome and acute cerebellar ataxia, and, according to some evidence, a higher risk of developing certain autoimmune diseases. Approximately 200,000 cancer cases are believed to be caused by EBV each year (Wikipedia).

[0008] Most EBV-related cancers express only a limited number of EBV-specific antigens, such as latent membrane proteins (LMP1, LMP2A) and nucleoproteins (EBNA1, EBNA3C). These antigens have been shown to be targets of TCR-based immunotherapies, such as TCR gene therapy or adoptive T-cell therapy for EBV-related diseases, such as post-transplant lymphoproliferative disorders or cancers (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 for EBV-related malignancies have used naturally occurring EBV-specific T cells derived from third-party donors or patients, where the T cells have been expanded using EBV lymphoblastoid cells (LCLs) or EBV peptide pools. Adoptive T-cell therapy using EBV-specific TCR-engineered T cells has not yet been tested in clinical trials. Compared to naturally occurring EBV-specific T cells, TCR-engineered T cells have several advantages: 1) Efficacy: The introduced TCR is a predefined receptor with high affinity for EBV-positive tumor cells. Generating naturally occurring T cells from a patient's blood depends on the presence of EBV-specific T cells. However, patients may lack expandable, effective T cells. 2) Feasibility: The success rate of engineered T cell production is over 95%, while the success rate of culturing naturally occurring T cells is below 70%. 3) Cost: In the process of engineered T cells, the time from vein to vein is reduced to less than 21 days, while the time to expand naturally occurring T cells exceeds 40 days.

[0010] To enable TCR gene therapy in patient populations with different MHC I (HLA) alleles, it is necessary to identify TCRs limited to different HLA alleles. Furthermore, identifying TCRs limited to different HLA alleles is a prerequisite for targeting EBV epitopes by two different HLA alleles expressed in the same cell.

[0011] The present invention addresses some of the aforementioned problems and provides novel and preferably advantageous immunotherapeutic agents for the immunotherapy of cancer or infectious agents. The problem is solved by the subject matter of the claims. Summary of the Invention

[0012] Nucleic acid encoding TCR construct

[0013] In one embodiment, the present invention provides specific TCR constructs for treating EBV-related diseases, and nucleic acids encoding them.

[0014] This invention provides nucleic acids encoding TCR α-chain constructs (TRA) and / or TCR β-chain constructs (TRB) that are epitope-specific TCR constructs that complex with human MHC I, wherein the epitope is an epitope of the EBV protein.

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

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

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

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

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

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

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

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

[0023] In the context of this invention, unless otherwise expressly stated, "a" should be understood to mean "one or more". Thus, for example, if the TCR construct of this invention contains both an α-chain construct and a β-chain construct, as is preferred throughout the invention, it can be encoded by one or both nucleic acids. Together, the α and β-chain constructs are capable of specifically binding to epitopes of EBV proteins complexed with human MHC I. The α and β-chain constructs themselves are also the subject of this invention as intermediates. This invention also provides single-stranded nucleic acid constructs, wherein, for example, the TCR α and β-chain constructs are separated by a P2A element.

[0024] The present invention discloses a TRA and / or TRB for a nucleic acid-encoding epitope-specific TCR construct that complexes with human MHC I, wherein the epitope has the sequence of SEQ ID NO:1, the MHC I is HLA-A*02:01, and the TRA includes CDR3 having at least 90% sequence identity with SEQ ID NO:13, and the TRB includes CDR3 having at least 90% sequence identity with SEQ ID NO:18. SEQ ID NO:1 is an epitope from the EBV protein LMP2A, which is known in the art to be presented by cancer cells of EBV-related cancer types in the context of HLA-A2 (e.g., HLA-A*02:01, i.e., a very common HLA-type) (e.g., Orentas et al., 2001, see above). As shown herein, TCR constructs having the CDR3 sequence disclosed herein have particularly high affinity or peptide sensitivity compared to TCRs in the art, and are therefore preferred TCR constructs of the present invention.

[0025] Advantageously, the TCR has high peptide sensitivity and a peptide concentration of 10 -8 mol / L or lower, preferably 10 - 9 Half-maximum IFN-γ release of mol / L or lower. Analysis can be performed using TCR-engineered T cells and target cells cultured at a 1:1 effector-to-target cell ratio (e.g., K562-HLA-A*02:01 cells loaded with peptides, preferably the peptide of SEQ ID NO:1), for example, as shown in Figure 15 or... Figure 16 As shown, preferably, as shown in Figure 15.

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

[0027] TRA may contain the linked amino acid shown in SEQ ID NO:14. TRB may contain the linked amino acid shown in SEQ ID NO:19.

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

[0029] This invention discloses a TCR construct with advantageous features, the construct comprising a TRA having a variable region of SEQ ID NO:15 and a TRB having a variable region of SEQ ID NO:20. It is also referred to as TCR06.

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

[0031] This invention discloses a TRA and / or TRB for a TCR construct that encodes a human MHC I complex specifically for an epitope, wherein the epitope has the sequence of SEQ ID NO:2, the MHC I is HLA-B*57:01, and the TRA includes CDR3 having at least 90% sequence identity with SEQ ID NO:23, and the TRB includes 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 EBV-related cancer types in the HLA-B*57:01 background. As shown herein, TCR constructs having the disclosed CDR3 sequence exhibit high affinity or peptide sensitivity.

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

[0033] TRA may contain the linked amino acid shown in SEQ ID NO:24. TRB may contain the linked amino acid shown in SEQ ID NO:29.

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

[0035] This invention discloses a TCR construct with advantageous features, the construct comprising a TRA having a variable region of SEQ ID NO:25 and a TRB having a variable region of SEQ ID NO:30. It is also referred to as TCR50.

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

[0037] This invention discloses a TRA and / or TRB for a TCR construct specifically targeting an epitope that is complexed with human MHC I, wherein the epitope has the sequence of SEQ ID NO:3, the MHC I is HLA-C*15:02, and the TRA includes CDR3 having at least 90% sequence identity with SEQ ID NO:33, and the TRB includes CDR3 having at least 90% sequence identity with SEQ ID NO:38. SEQ ID NO:3 is an epitope of the EBV protein LMP1, and this is the first time it has been shown that the epitope is presented by cancer cells of an EBV-related cancer type. It is presented in the context of HLA-C*15:02. As shown herein, TCR constructs having the CDR3 sequence disclosed herein exhibit high affinity or peptide sensitivity.

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

[0039] TRA may contain the linked amino acid shown in SEQ ID NO:34. TRB may contain the linked amino acid shown in SEQ ID NO:39.

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

[0041] This invention discloses a TCR construct with advantageous features, the construct comprising a variable region TRA having SEQ ID NO:35 and a variable region TRB having SEQ ID NO:40. It is also referred to as TCR83.

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

[0043] This invention discloses a TRA and / or TRB for a TCR construct specifically targeting an epitope and complexing with human MHC I, wherein the epitope has the sequence of SEQ ID NO:4, the MHC I is HLA-C*06:02, and the TRA includes CDR3 having at least 90% sequence identity with SEQ ID NO:43, and the TRB includes CDR3 having at least 90% sequence identity with SEQ ID NO:48. SEQ ID NO:4 is an epitope from the EBV protein EBNA3C, and this is the first time it has been shown that this epitope is presented by cancer cells of an EBV-related cancer type. It is presented in the context of HLA-C*06:02.

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

[0045] TRA may contain the linked amino acid shown in SEQ ID NO:44. TRB may contain the linked amino acid shown in SEQ ID NO:49.

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

[0047] This invention discloses a TCR construct with advantageous features, the construct comprising a variable region TRA having SEQ ID NO:45 and a variable region TRB having SEQ ID NO:50. It is also referred to as TCR64.

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

[0049] This invention discloses a TRA and / or TRB for a TCR construct specifically targeting an epitope and complexing with human MHC I, wherein the epitope has the sequence of SEQ ID NO:5, the MHC I is HLA-B*44:02, and the TRA includes CDR3 having at least 90% sequence identity with SEQ ID NO:53, and the TRB includes CDR3 having at least 90% sequence identity with SEQ ID NO:58. SEQ ID NO:5 is an epitope from the EBV protein EBNA3C, and this is the first time it has been shown that this epitope is presented by cancer cells of an EBV-related cancer type. It is presented in the context of HLA-B*44:02.

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

[0051] TRA may contain the linked amino acid shown in SEQ ID NO:54. TRB may contain the linked amino acid shown in SEQ ID NO:59.

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

[0053] This invention discloses a TCR construct with advantageous features, the construct comprising a TRA having a variable region of SEQ ID NO:55 and a TRB having a variable region of SEQ ID NO:60. It is also referred to as TCR25.

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

[0055] This invention discloses a TRA and / or TRB for a TCR construct specifically targeting an epitope and complexing with human MHC I, wherein the epitope has the sequence of SEQ ID NO:5, the MHC I is HLA-B*44:02, and the TRA includes CDR3 having at least 90% sequence identity with SEQ ID NO:63, and the TRB includes 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, and this is the first time it has been shown that this epitope is presented by cancer cells of an EBV-related cancer type. It is presented in the context of HLA-B*44:02.

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

[0057] TRA may contain the linked amino acid shown in SEQ ID NO:64. TRB may contain the linked amino acid shown in SEQ ID NO:69.

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

[0059] This invention discloses a TCR construct with advantageous features, the construct comprising a variable region TRA having SEQ ID NO:65 and a variable region TRB having SEQ ID NO:70. It is also referred to as TCR58.

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

[0061] The present invention discloses a TRA and / or TRB of a TCR construct specifically targeting an epitope and complexing with human MHC I, wherein the epitope has the sequence of SEQ ID NO:6, the MHC I is HLA-B*07:02, and the TRA includes CDR3 having at least 90% sequence identity with SEQ ID NO:73, and the TRB includes 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 EBV-related cancer types in the HLA-B*07:02 background.

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

[0063] TRA may contain the linked amino acid shown in SEQ ID NO:74. TRB may contain the linked amino acid shown in SEQ ID NO:79.

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

[0065] This invention discloses a TCR construct with advantageous features, the construct comprising a variable region TRA having SEQ ID NO:75 and a variable region TRB having SEQ ID NO:80. It is also referred to as TCR27.

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

[0067] The present invention discloses a TRA and / or TRB of a TCR construct specifically targeting an epitope and complexing with human MHC I, wherein the epitope has the sequence of SEQ ID NO:7, the MHC I is HLA-B*07:02, and the TRA includes CDR3 having at least 90% sequence identity with SEQ ID NO:83, and the TRB includes CDR3 having at least 90% sequence identity with SEQ ID NO:88. SEQ ID NO:7 is an epitope from the EBV protein EBNA3C, which comprises SEQ ID NO:6 and a C-terminal T.

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

[0069] TRA may contain the linked amino acid shown in SEQ ID NO:84. TRB may contain the linked amino acid shown in SEQ ID NO:89.

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

[0071] This invention discloses a TCR construct with advantageous features, the construct comprising a variable region TRA having SEQ ID NO:85 and a variable region TRB having SEQ ID NO:90. It is also referred to as TCR01.

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

[0073] In any TRA and / or TRB construct of the present invention, CDR1 and CDR3 may independently have at least 80%, at least 90%, or 100% sequence identity with the defined sequence. Preferably, CDR3 has 100% sequence identity with the defined CDR3. Typically, if the sequences are different, at most one amino acid is substituted, deleted, or inserted, usually an amino acid substitution. The substitution can be a conservative substitution, i.e., a specific type of amino acid (e.g., polar, nonpolar, acidic, basic, aromatic) is replaced by another amino acid of the same type. Methods for affinity maturation are known in the art and are described further below.

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

[0075] The TCR α and / or β chain constructs of the present invention may contain all the features or domains corresponding to their natural counterparts, i.e., TCR α or β chains, but this is not required. Preferably, the TCR α and / or β chain constructs contain at least a variable region, or a variable region and a constant region, for example, the variable region and / or constant region having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with human variable or constant TCR regions.

[0076] The TCR α-chain constructs and / or TCR β-chain constructs of the present invention preferably include constant regions. For adoptive TCR therapy, the TCR constructs preferably include full-length TCR α and β chains, which include variable and constant regions, including transmembrane regions. The constant regions can be human constant regions, mouse constant regions, or chimeric constant regions, such as a minimal mouse constant region. The TCR constructs can be substantially or entirely derived from humans to have minimized immunogenicity. However, to prevent pairing with endogenous TCR chains, the constructs of the present invention preferably contain one or more, for example 1-5, 1-10, or 1-20, preferably 9 amino acid substitutions, compared to human sequences (Sommermeyer and Uckert, 2010). For this purpose, the constant regions of the TCR α and β-chain constructs can also be mouse constant regions (Cohen et al., 2006) and / or provide additional cysteine ​​residues to enable the formation of additional disulfide bonds between TCR chains (Cohen et al., 2007, Kuball et al., 2007). In addition, the functional expression of transgenic TCRs can be enhanced by codon modification of the TCR sequence (Scholten et al., 2006) and / or by applying peptides (e.g., P2A) to link two TCR chains to achieve stoichiometric expression of both chains (Leisegang et al., 2008), which also leads to enhanced functional expression of transgenic TCRs.

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

[0078] This includes single-chain constructs (scTCRs) and heterodimeric TCR constructs. scTCRs may contain a first TCR chain construct (e.g., an α chain) and a variable region of the entire (full-length) second TCR chain (e.g., a β chain), or vice versa. Furthermore, scTCRs may optionally contain one or more linkers that connect two or more peptides together. Linkers may be, for example, peptides that link two single chains together. The invention also provides such scTCRs fused with cytokines such as human cytokines (e.g., IL-2, IL-7, IL-12, or IL-15).

[0079] In order to specifically recognize epitopes in the MHC context, partly for therapeutic purposes, the nucleic acids of the present invention encode a TCR α construct and a β-chain construct of the TCR construct of the present invention, such as the epitope-specific TCR construct of SEQ ID NO:1 described herein.

[0080] Typically, the nucleic acid sequences provided in this invention are codon-optimized for expression in human cells.

[0081] In the context of this invention, nucleic acids can be DNA or RNA. DNA is preferred. Nucleic acids can be, for example, viral or non-viral vectors, such as transposons, vectors suitable for CRISPR / CAS-based recombination, or plasmids suitable for in vitro RNA transcription. The nucleic acids of this invention are preferably vectors. Suitable vectors include those designed for proliferation and amplification, or for expression, or both, such as plasmids and viruses. The vector can be an expression vector suitable for expression in host cells selected from human T cells or human T cell precursors, preferably human T cells such as CD8+ T cells. The CD8+ cells can be central memory T cells, effector memory T cells, stem cell-like T cells, or effector T cells, or mixtures thereof. The vector can be a viral vector, such as a retroviral vector, particularly a γ-retroviral vector or a lentiviral vector. Examples of suitable expression vectors include the retroviral vector MP71 (Engels et al., 2003). The expression vector contains regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) to which the vector is to be introduced, and expresses the nucleic acid of the present invention in the host cell. In the context of the present invention, the host cell is typically a human CD8+ T cell. Furthermore, the vector of the present invention may include one or more marker genes that allow selection of the host for transduction or transfection. The recombinant expression vector may contain a natural or preferably heterologous promoter operatively linked to a nucleotide sequence encoding the TCR construct of the present invention, or linked to a nucleotide sequence complementary to or hybridizing with a nucleotide sequence encoding the construct of the present invention. The choice of promoter includes, for example, strong promoters, weak promoters, inducible promoters, tissue-specific promoters, and development-specific promoters. The promoter may be a nonviral promoter or a viral promoter. Preferably, it is a heterologous promoter, i.e., a promoter that is not naturally linked to a 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. In addition, recombinant expression vectors can be prepared for constitutive or inducible expression.

[0082] protein

[0083] The present invention also provides a protein, namely an α or β chain construct, or preferably a TCR construct comprising α and β chain constructs, said construct being capable of specifically binding to epitopes of EBV-proteins described herein in their respective MHC I contexts, for example, the TCR construct herein specific to the epitope of SEQ ID NO:1. The protein is encoded by a nucleic acid of the present invention.

[0084] As used herein, the terms “capable of specifically binding,” “recognizing,” or “specific to” a given antigen have equivalent meanings and imply that the TCR construct can specifically bind to and immunely recognize the epitope, preferably derived from an EBV protein, and more preferably having high affinity. For example, in the presence of a low concentration of the corresponding epitope (e.g., about 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 -5 When T cells are co-cultured with target cells pulsed with a mol / L pulse, rather than with target cells pulsed with no epitope or with an unrelated control peptide epitope, if TCR-expressing T cells secrete 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, 2000 pg / mL or more, 2500 pg / mL or higher, 5000 pg / mL or more), TCR can be considered "capable of specifically binding" to peptides derived from EBV proteins. Preferably, the assay is performed using 10,000 TCR+CD8+ T cells and 20,000-50,000, preferably 50,000 target cells expressing appropriate HLA, for example, using the assay described in the examples below. Alternatively, when co-cultured with target cells pulsed with a low concentration of an appropriate peptide, TCRs can be considered to have "antigen specificity" for epitopes if the IFN-γ secreted by T cells expressing TCRs is at least twice the uninduced background level. This "specificity" can be analyzed using, for example, an ELISA assay.

[0085] As described above regarding the TCR of the present invention, high affinity is associated with high peptide sensitivity, for example, at a peptide concentration of 10. -6 mol / L or lower, preferably 10 -7 mol / L or lower, 10 -8 mol / L or lower or 10-9 At mol / L or lower, half-maximal IFN-γ release is observed. Alternatively, affinity can be analyzed using methods well known to those skilled in the art, such as by BiaCore. 100 μM or higher, more preferably 10 μM or higher, TCR affinity or T cell affinity is considered high affinity.

[0086] Based on the defined CDR3 and variable region sequences provided in this invention, affinity maturation of the TCR sequence can be performed (Chervin et al., 2008; Robbins et al., 2008). Non-synonymous nucleotide substitutions resulting in amino acid exchanges in the CDR3 sequence may lead to enhanced affinity of the TCR for the target antigen. Furthermore, TCR sequence variations in other parts of the variable TRA and TRB regions may alter the affinity of the TCR for the peptide-MHC I complex. This may increase the overall affinity of the TCR for peptide-MHC, but carries the risk of non-specific recognition and increased cross-reactivity (Linette et al., 2013). Preferably, TCRs differing from the provided specific sequences retain exclusive specificity to the provided target antigen, i.e., they are not cross-reactive, and most importantly, they are not cross-reactive to human autopeptides. Potential cross-reactivity of TCRs can be detected by comparing them to known autopeptides loaded on cells with the correct MHC alleles (Morgan et al., 2013). Therefore, preferably, adoptive transfer of T cells expressing the TCR construct of this invention has no or significant negative effects on healthy tissues.

[0087] The TCR constructs of the present invention can 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 wherein the scTCRs are interconnected via biotin-streptavidin interactions to allow the formation of the multimeric complex. The present invention also provides higher-order multimeric complexes comprising more than two, for example, four scTCRs of the present invention.

[0088] The TCR constructs of the present invention can be modified to include detectable markers, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), tags (e.g., HIS-tags), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), or particles (e.g., gold particles or magnetic particles).

[0089] host cells

[0090] The present invention also provides host cells comprising the nucleic acids and / or proteins of the present invention. The host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protozoan. The host cell may be a cultured cell or a primary cell, i.e., a cell directly isolated from an organism such as a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell growing in suspension. For the production of recombinant TCRs, peptides, or proteins, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. Although the host cell may be any cell type, may be derived from any type of tissue, and may be at any developmental stage, the host cell is preferably a peripheral blood leukocyte (PBL) or peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell or a T cell precursor, particularly a human T cell, which may be isolated from PBMCs. The T cell may be any T cell, such as a cultured T cell, such as a primary T cell, or a T cell derived from a cultured T cell line, or a T cell derived from a mammal; preferably, the T cell is a T cell or a T cell precursor derived from a human patient. T cells can be obtained from a variety of sources, such as blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. T cells can be, for example, tumor-infiltrating cells (TILs), effector cells, central effector cells, memory T cells, naive T cells, etc., with central memory T cells being preferred. Alternatively, the host cell can be another type of immune effector cell, such as NK cells or macrophages.

[0091] Preferably, and particularly in the case of treatment of a human, the T cells are human T cells. The T cells are preferably CD8+ cells (e.g., cytotoxic T cells). Preferably, the T cells are isolated from a human being, such as a human patient, particularly a patient to be treated. Alternatively, the T cells may be derived from a third-party donor, whether related or unrelated to the patient. Such T cells can be genetically engineered, for example, in a variety of ways, such as by knocking out endogenous TCR and / or MHC I. T cells can 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 containing the nucleic acid of the present invention as an expression vector, wherein the nucleic acid encoding TRA and / or TRB is operatively linked to a heterologous promoter, wherein the host cell expresses the TCR construct of the present invention.

[0093] This invention also provides host cells for expressing two or more different TCR constructs, such as the two TCR constructs of this invention. Preferably, as described herein, the TCR constructs are single-stranded TCR constructs, wherein the TCR α and β strands of each TCR are bound by a linker to avoid mismatches between transgenic TCR strands. The two single-stranded TCR constructs can be encoded on a single expression vector.

[0094] Pharmaceutical Composition

[0095] The present invention also provides a pharmaceutical composition comprising:

[0096] a) The nucleic acid of the present invention encodes a TCR construct capable of specifically binding to its corresponding epitope in the corresponding MHC I background; or

[0097] b) The protein of the present invention comprises a TCR construct capable of specifically binding its corresponding epitope in the corresponding MHC I background (i.e., the TCR construct of the present invention); or

[0098] c) The host cell of the present invention expresses a TCR construct that can specifically bind to its corresponding epitope in the context of corresponding MHC I.

[0099] Preferably, the TCR construct is an epitope-specific TCR construct of SEQ ID NO:1.

[0100] In another embodiment, the present invention provides a kit or pharmaceutical composition comprising at least two pharmaceutical compositions, including

[0101] a) At least two nucleic acids, each encoding a TCR construct capable of specifically binding to its corresponding epitope in the corresponding MHC I background; or

[0102] b) At least two proteins, each containing a TCR construct capable of specifically binding its corresponding epitope in the corresponding MHC I background; or

[0103] c) At least two host cell types, each expressing a TCR construct capable of specifically binding to its corresponding epitope in the corresponding MHC I background.

[0104] The epitopes mentioned here are peptides derived from different antigens expressed by the same cancer or infectious agent. Typically, immunotherapy is used to treat cancer, so the different antigens are preferably antigens expressed by the cancer itself, i.e., antigens expressed by cells of the same cancer. In this document, an antigen is a protein containing an epitope that can be presented on MHC I. It may be useful, for example, if different cancer cells of the same cancer type each express at least one antigen, and therefore the TCR construct targets different cancer cells of the same cancer if the stage of the cancer is unknown and / or if cells at different stages expressing different antigens are to be treated. However, in general, it is advantageous if the different antigens are expressed by the same cancer cells.

[0105] The inventors have surprisingly discovered that it is advantageous to use a combination of at least two TCR constructs expressed by T cells for adoptive T cell therapy, specifically, two or more T cells each expressing a TCR construct that specifically binds to its corresponding epitope in the context of the corresponding MHC I.

[0106] Unbound by this theory, it is believed that resistance to cancer (or infectious agents) based on the expression of two or more antigens can help maintain resistance and prevent immune evasion, for example, through mutation, through downregulation of a target antigen, or through antigen-deficient variants.

[0107] Three or more TCR constructs can also be used, particularly three or more host cells targeting cancer or infectious agents, each of which contains nucleic acids encoding a TCR construct capable of specifically binding its corresponding epitope in its respective MHC I background, wherein the epitope is a peptide derived from different antigens expressed by the same cancer or infectious agent.

[0108] The kit or pharmaceutical composition is used to treat cancer or infectious agents derived from epitopes targeted by the TCR construct.

[0109] The infection or cancer may be related to, for example, EBV. In this case, different EBV antigens may be LMP2A, LMP1, EBNA1, or EBNA3C. Preferably, one of the EBV antigens targeted by a TCR construct of the present invention is LMP2A. A second EBV antigen targeted by a TCR construct of the present invention may be LMP1 or EBN3C, preferably LMP1. If three EBV antigens are to be targeted, the antigens may be LMP2A, LMP1, and EBNA3C. An alternative or additional EBV antigen is EBNA1.

[0110] The present invention also provides pharmaceutical compositions and kits as described above, comprising:

[0111] a) at least two nucleic acids of the present invention, each nucleic acid encoding a TCR construct capable of specifically binding to its corresponding epitope in the corresponding MHC I background; or

[0112] b) At least two proteins of the present invention, each protein comprising a TCR construct capable of specifically binding its corresponding epitope in the corresponding MHC I background; or

[0113] c) At least two host cells of the present invention, each host cell expressing a TCR construct capable of specifically binding its corresponding epitope in the corresponding MHC I background;

[0114] Optionally, the epitopes may originate from different EBV proteins.

[0115] As disclosed herein, one, two, or three TCR constructs used in the context of the pharmaceutical compositions or kits of the present invention may be TCR constructs of the present invention. Preferably, one of the TCR constructs used is the TCR construct disclosed herein that recognizes the epitope of SEQ ID NO:1 in the HLA-A2 background.

[0116] Therefore, the kit or pharmaceutical composition may comprise human CD8+ T cells containing nucleic acids encoding the TCR construct of the present invention, for example, an epitope that recognizes SEQ ID NO:1 in an HLA-A2 background as defined herein. The TCR construct can be expressed from the nucleic acid under the control of a heterologous promoter.

[0117] In the kits or pharmaceutical compositions of the present invention comprising at least two nucleic acids encoding TCR constructs, at least two TCR constructs, or at least two host cells each comprising nucleic acids encoding TCR constructs, the one, two, or three TCR constructs may also be other TCR constructs, such as those known in the art. For example, if the cancer is EBV-associated, one, two, or three TCR constructs disclosed herein, optionally in combination with one of the TCR constructs provided herein, may be used.

[0118] In this application, adoptive T-cell therapy (Protocol c) is preferred, wherein the host cell is a T cell, preferably a human CD8+ T cell. The host cell typically contains nucleic acids encoding a TCR construct under the control of a heterologous promoter.

[0119] However, gene therapy using the nucleic acids of the present invention (scheme a) is also feasible, wherein, for example, a lentiviral vector may be used.

[0120] The TCR construct of the present invention in protein form (Scheme b) can also be used for treatment, for example, to target toxins to associated cancers, or to target bacterial microcells to cancer, and may contain therapeutic agents such as toxins. The TCR construct of the present invention in protein form can also be used to target diagnostic reagents to cancer. Therefore, the composition can also be a diagnostic composition.

[0121] Each type of T cell expressing its respective TCR construct can be included in the kit, wherein each type of T cell is stored separately in, for example, a pharmaceutically acceptable buffer. The components of the kit of the present invention can be formulated for simultaneous or sequential administration. These components can also be used for repeated administration. A possible administration regimen is described in the literature of Tran et al. (2014). Alternatively, they can be mixed and administered simultaneously before administration. Alternatively, T cells expressing their respective TCR constructs can be included in a single pharmaceutical composition. This also applies to the nucleic acids or proteins of the present invention.

[0122] The pharmaceutical compositions or kits of the present invention are typically intended for intravenous administration. They may also contain pharmaceutically acceptable carriers, such as buffers, such as physiological saline or PBS. They may further contain excipients, such as stabilizers like SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, or protein-containing reagents such as bovine serum or skim milk.

[0123] T cells typically occur at a rate of 1 × 10 5 -1×10 9 Administer to patients at a concentration of approximately 1 × 10⁻⁶ cells / kg. 6 -1×10 11 The cells can be administered to the patient as a single dose. These parameters can be adjusted by healthcare professionals based on factors such as the patient's age, sex, weight, and medical condition. For example, the protocol disclosed in the literature of Doran et al. (2019) can be adapted to use the host cells of this invention.

[0124] The pharmaceutical compositions of the present invention, kits comprising the pharmaceutical compositions of the present invention, or kits of the present invention can be used to treat patients expressing MHC, in which the corresponding TCR recognizes the corresponding epitopes, such as those disclosed herein. Average HLA (MHC I) distributions in different populations can be found, for example, at http: / / allelefrequencies.net / . If a patient expresses the corresponding MHC I, it is advantageous to perform testing prior to treatment.

[0125] Patients may have cancer or infectious diseases, particularly in the case of the specific TCR construct disclosed herein, EBV-related diseases such as those selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, Burkitt's lymphoma, hemophagocytic lymphohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, gastric cancer, lung cancer, hairy leukoplakia, post-transplant lymphoproliferative disorder, and central nervous system lymphoma. Preferably, the EBV-related cancer is a type II malignancy, such as confirmed Hodgkin's lymphoma and nasopharyngeal carcinoma, or a type III malignancy, such as confirmed hairy leukoplakia, post-transplant lymphoproliferative disorder, and central nervous system lymphoma (Orentas et al., 2001), because these cancers typically express high levels of LMP2A and LMP1. In type III malignancies, EBNA2C is additionally expressed.

[0126] The targeted cancer cells express a protein derived from the recognized epitope or, optionally, a variety of proteins, preferably most cancer cells.

[0127] Patients are typically mammals. Patients can be mice, but preferably, they are human patients.

[0128] The present invention also discloses a method for treating cancer or infectious diseases by administering an effective amount of the pharmaceutical composition or kit of the present invention to a patient in need, such as EBV-related diseases, preferably EBV-related cancers, wherein the patient is, for example, a patient suffering from said cancer or disease.

[0129] The pharmaceutical compositions and kits of the present invention can be used in combination with other agents, particularly with other anticancer agents. For example, other anticancer agents may be TCR-engineered T cells expressing TCRs specific to other antigens expressed in EBV-positive tumors (e.g., MAGE, NY-ESO, oa), checkpoint inhibitors or other immunotherapies, as well as antibodies, small molecule inhibitors or other types of agents.

[0130] A preferred pharmaceutical use of the present invention is immunotherapy, preferably adoptive T-cell therapy. The products and methods of the present invention are particularly suitable for adoptive T-cell therapy. Administration of the compounds of the present invention may, for example, include administering the T cells of the present invention, such as by infusion into the patient. Preferably, such T cells are the patient's own T cells transduced in vitro with the nucleic acids of the present invention.

[0131] Alternatively, the nucleic acid of the present invention, particularly the expression vector, can be administered to the patient for in vivo transduction of T cells.

[0132] The protein TCR constructs of the present invention can also be used, for example, for diagnostic purposes to determine whether a subject expresses the corresponding protein, particularly whether an epitope recognized by the TCR construct is presented in the MHC I background. For this purpose, these constructs are preferably labeled to facilitate detection. Preferably, patients presenting such epitopes on the corresponding MHC I are treated with the adoptive T-cell therapy of the present invention.

[0133] The present invention also relates to a method for preparing the host cells of the present invention, comprising introducing an expression vector encoding the TCR construct of the present invention into a suitable host cell, preferably human CD8+ T cells isolated from a patient.

[0134] The invention will be further described below in conjunction with the accompanying drawings and sequences; however, the invention is not limited thereto. For the purposes of this invention, all references cited herein are incorporated herein by reference in their entirety.

[0135] vaccine

[0136] In one embodiment, the present invention provides pharmaceutical compositions, particularly vaccine compositions, comprising a peptide or a nucleic acid (such as RNA) encoding such a peptide, said peptide containing an epitope capable of being presented by human MHC I, said epitope being a peptide identified for the first time herein. The epitope can...

[0137] a) Having at least 88% sequence identity with SEQ ID NO:3, wherein the epitope is present on HLA-C*15:02, and wherein the peptide comprises up to 25, preferably up to 11, consecutive amino acids identical to the amino acid sequence appearing in LMP1 of SEQ ID NO:120.

[0138] b) Having at least 88% sequence identity with SEQ ID NO:4, wherein the epitope is present on HLA-C*06:02, and wherein the peptide comprises up to 25, preferably up to 11, consecutive amino acids identical to the amino acid sequence appearing in EBNA3C of SEQ ID NO:121; and

[0139] c) Having at least 90% sequence identity with SEQ ID NO:5, wherein the epitope is present on HLA-B*44:02, and wherein the peptide comprises up to 25, preferably up to 11, consecutive amino acids identical to the amino acid sequence appearing in EBNA3C of SEQ ID NO:121.

[0140] Preferably, the peptide in a) can be specifically recognized by TCR83 as defined herein.

[0141] Preferably, the peptide in b) can be specifically recognized by TCR64 as defined herein.

[0142] Preferably, the peptide in c) can be specifically recognized by TCR25 or TCR58 as defined herein.

[0143] Peptide vaccines are well known in the art. For example, peptide vaccines can be administered to subjects in combination with adjuvants such as aluminum salts (e.g., aluminum phosphate or aluminum hydroxide), squalene (e.g., MF59), liposomes (e.g., QS21), or monophosphate A.

[0144] Such pharmaceutical compositions of the present invention may comprise peptides containing the epitope of SEQ ID NO:3, wherein the epitope is present on HLA-C*15:02, and wherein the peptide comprises up to 25, up to 15, or preferably up to 11 amino acids identical to the amino acid sequence appearing in LMP1 of SEQ ID NO:120.

[0145] Such pharmaceutical compositions of the present invention may comprise peptides containing the epitope of SEQ ID NO:2, wherein the epitope is present on HLA-B*57:01, and wherein the peptide comprises up to 25, up to 15, or preferably up to 11 consecutive amino acids identical to the amino acid sequence appearing in LMP1 of SEQ ID NO:120.

[0146] Such pharmaceutical compositions of the present invention may comprise peptides containing the epitope of SEQ ID NO:5, wherein the epitope is present on HLA-B*44:02, and wherein the peptide comprises up to 25, up to 15, or preferably up to 11 consecutive amino acids identical to the amino acid sequence appearing in EBNA3C of SEQ ID NO:121.

[0147] Nucleic acids encoding peptides containing epitopes as defined herein may also be used, for example, in combination with suitable adjuvants such as liposomes or CpG nucleotides.

[0148] Such pharmaceutical compositions of the present invention may comprise nucleic acids encoding peptides containing epitopes of SEQ ID NO:3, wherein the epitopes are present on HLA-C*15:02, and wherein the peptides contain up to 25, up to 15, or preferably up to 11 amino acids identical to the amino acid sequence appearing in LMP1 of SEQ ID NO:120.

[0149] Such pharmaceutical compositions of the present invention may comprise nucleic acids encoding peptides containing epitopes of SEQ ID NO:4, wherein the epitopes are present on HLA-C*06:02, and wherein the peptides contain up to 25, up to 15, or preferably up to 11 consecutive amino acids identical to the amino acid sequence appearing in EBNA3C of SEQ ID NO:121.

[0150] Such pharmaceutical compositions of the present invention may comprise nucleic acids encoding peptides containing epitopes of SEQ ID NO:5, wherein the epitopes are present on HLA-B*44:02, and wherein the peptides contain up to 25, up to 15, or preferably up to 11 consecutive amino acids identical to the amino acid sequence appearing in EBNA3C of SEQ ID NO:121.

[0151] Any of the vaccine pharmaceutical compositions described herein may be used for vaccination against EBV-related diseases selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, Burkitt's lymphoma, hemophagocytic lymphohistiocytosis, nasopharyngeal carcinoma, head and neck cancer, hairy leukoplakia of gastric cancer, post-transplant lymphoproliferative disorder, and central nervous system lymphoma. Vaccination may be a prophylactic vaccination, i.e., providing vaccination to subjects who have not yet developed the disease or have not been infected with EBV, with the aim of reducing the subject's risk of developing the disease in the event of EBV exposure. The subject may belong to a risk group for the disease; for example, the subject may be identified as having been exposed to EBV or currently having an EBV infection, wherein the subject (has not yet) developed the disease. Alternatively, vaccination may be a therapeutic vaccination. Patients with EBV-related diseases may receive therapeutic vaccination.

[0152] Advantageously, the subject's or patient's HLA is known, and vaccine administration is able to present epitopes on MHC I molecules, for example, patients with HLA-C*15, particularly HLA-C*15:02, for epitopes defined in a), patients with HLA-B*57, particularly HLA-B*57:01, for epitopes defined in b), or patients with HLA-B*44, for epitopes defined in c). Attached Figure Description

[0153] Figure 1 T cell responses were detected using an MHC class I K562 cell bank. T cells expanded on dendritic cells expressing EBV antigen were co-cultured with K562 cells from the MHC cell bank. Immunogenic EBV antigen-HLA combinations were screened by (A) analysis of CD137 expression and (B) measurement of secreted IFN-γ by ELISA. (C) FACS sorting of CD137-positive T cells (11%) that responded to K562-HLA-B*57:01-positive cells was then used to identify the dominant TCR α and TCR β chains. (MIN – no antigen stimulation, MAX – nonspecific antigen stimulation, us – unstained T cells). This method was used to identify and isolate all other TCRs described herein.

[0154] Figure 2. TCR gene analysis. (A) Next-generation sequencing-based TCR library analysis of FACS-sorted T cells that responded to EBV antigen-positive (LMP 1 / LMP2A / EBNA1) K562-HLA-b*57:01 cells. The y-axis indicates the portion of the total reads allocated to each sequence cluster. TCR α and β chains with frequencies >10% were used to construct single-stranded TCR-retroviruses to identify functional TCRs. (B) For a TCR (designated TCR50), the different v segments of the most dominant TCR α (TRAV) and TCR β (TRBV) chains, their frequencies, and the sequences of the CDR-3 regions (IMGT nomenclature) are shown (AMSDLYAGNNRKLI:SEQ ID NO:122, ALTFLRDDDKII:SEQ ID NO:123, VVMATGFQKLV:SEQ ID NO:24, ASSQDARVSGANVLT:SEQ ID NO:124, ASSVTSGSDEQF:SEQ ID NO:125, ASSFSLGHSYEQY:SEQ ID NO:126). This method is applicable to all other TCRs described herein.

[0155] Figure 3 Identification of functional TCR αβ chain combinations. TCR α and TCR β chains with a frequency >10% were used to construct single-stranded TCR-retroviruses to identify functional TCRs. For TCR50, TRAV8-2*01 and TRBV9*01 formed functional TCRs because the TCR-engineered T cells specifically recognized and bound an HLA antigen (LMP 1) of B*57:01. Other TCR α and TCR β chain combinations resulted in non-specific antigen recognition. The combination method for identifying functional TCR α and TCR β chain combinations, as exemplified in this document for TCR50, was applied to all other TCRs described herein. (MIN – no antigen stimulation, MAX – non-specific stimulation).

[0156] Figure 4 Epitope mapping was used to identify antigenic peptides recognized by TCR50. (A) Truncated forms of the full-length LMP1 antigen (LMP1 / 2, LMP1 / 1) were produced and expressed in K562-HLA-B*57:01 cells. (B) The antigenic region carrying the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant using ELISA and was located between nucleotides (nt) 316 and 624. (MIN – no antigen stimulation, MAX – nonspecific antigen stimulation, UT – untransduced T cells).

[0157] Figure 5. Identification of LMP1 immunogenic epitopes. Candidate peptides were screened using the NetMHCpan 4.0 epitope prediction algorithm for HLA-B*57:01, based on the protein region (LMP1 / 2 nt316-624) identified as an epitope-positive sequence. (A) Epitopes were identified using 13 peptides, categorized as strong binders (SB) and weak binders (WB) based on peptide-MHC I binding affinity (binding level). (B) The selected peptides were loaded into K562-HLA-B*57:01 cells and co-cultured with TCR50 engineered T cells from two donors. IFN-γ secretion was measured by ELISA. The TCR50 engineered T cells recognized four epitopes with the following amino acid sequences (bolded in A): Table 1 below shows the SEQ ID NO of the analyzed peptides. (MIN - no antigen stimulation, MAX - nonspecific stimulation, SB - strong binder, WB - weak binder, UT - untransduced T cells).

[0158] Figure 6 Peptide titration of TCR50-engineered T cells. Untransduced (UT) and TCR50-transduced T cells (TCR50) from two donors were co-cultured with K562-HLA-B*57:01 cells loaded with titration-quantitative indicator peptides (SEQ ID NO:2, 108, 110, 116), and the amount of IFN-γ secreted in the supernatant was measured by ELISA. The peptide IALYLQQNW (9mer, SEQ ID NO:2) was recognized at the lowest concentration and can therefore be considered a homologous EBV LMP1 epitope of TCR50. Interestingly and notably, this epitope recognized by TCR50 did not rank highest in the NetMHCpan 4.0 prediction tool, indicating that the prediction tool is not accurate in predicting related immunodominant epitopes.

[0159] Figure 7 TCR50-engineered T cells recognized LMP1-positive cells. Functional analysis was performed on LMP1-specific TCR50-engineered T cells from two donors using K562-HLA-B*57:01 antigen-loaded cells, EBV-associated cancer cell line (L591-B*57:01), and lymphoblastic cell line (WIN, DEM), respectively. T cell reactivity was determined by ELISA measuring the amount of IFN-γ secreted at an effector cell to target (E:T) cell ratio of 1:1. (MIN – no antigen stimulation, MAX – nonspecific stimulation, UT – untransduced T cells).

[0160] Figure 8. Functional analysis of EBNA3C-reactive TCR01. (A) EBNA3C-specific TCR01-engineered T cells were co-cultured with K562-HLA-B*07:02 antigen pulsed cells and EBV-positive cell lines. T cell function was determined by measuring the amount of secreted IFN-γ using ELISA at an effector cell to target (E:T) cell ratio of 1:1. (B) Truncated forms of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were produced and expressed in K562-HLA-B*07:02 cells. (C) The antigenic region carrying the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant using ELISA, and it was located between nucleotides (nt) 2071 and 2979. (D) Candidate peptides were screened using the NetMHCpan4.0 epitope prediction algorithm for HLA-B*07:02, based on the protein region (EBNA3C nt 2071-2979) identified as an epitope positive sequence.(E) Epitopes were identified using 27 peptides (classified as strong binders (SB) and weak binders (WB) based on peptide-MHC I binding affinity (binding level). The epitopes included QPRAPIRPI (SEQ ID NO: 127), RPIPTRFPPPPM (SEQ ID NO: 128), RPRVEESSHGPA (SEQ ID NO: 129), SPQPRAPI (SEQ ID NO: 130), SPQPRAPIRPI (SEQ ID NO: 131), SPQPRAPIRPIP (SEQ ID NO: 132), PQPRAPIRPI (SEQ ID NO: 133), QPRAPIRPIP (SEQ ID NO: 134), PRAPIRPI (SEQ ID NO: 135), APIRPIPTRF (SEQ ID NO: 136), FPPPPMPL (SEQ ID NO: 137), HGPARCSQAT (SEQ ID NO: 138), RPIPTRFPP (SEQ ID NO: 139), and RPIPTRFFP (SEQ ID NO: 139). The 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. (SEQ ID NO:140), IPTRFPPPMP (SEQ ID NO:141), IPTRFPPPPM (SEQ ID NO:142), IPTRFPPPPMPL (SEQ ID NO:143), GPARCSQATA (SEQ ID NO:144), FPPPPMPLQDSM (SEQ ID NO:145), PPMPLQDSM (SEQ ID NO:146), RPIPTRFPPP (SEQ ID NO:147), MPLQDSMAVG (SEQ ID NO:148), PIPTRFPPPPMP (SEQ ID NO:149), PMPLQDSMAV (SEQ ID NO:150), PMPLQDSM (SEQ ID NO:151), QPRAPIRPIPT (SEQ ID NO:152), QPRAPIRP (SEQ ID NO:153)). Epitopes (boldly shown in D) are recognized by TCR01-engineered T cells. (F) K562-HLA-B*07:02 cells were loaded with a titration of the indicator peptide or without the peptide as a control, and the peptide sensitivity of TCR01-engineered T cells was determined by measuring the amount of secreted IFN-γ by ELISA at an E:T cell ratio of 1:1. (MIN - no antigen stimulation, MAX - nonspecific stimulation, SB - strong binder, WB - weak binder, UT - untransduced T cells).

[0161] Figure 9. Functional analysis of EBNA3C-reactive TCR25. (A) EBNA3C-specific TCR25-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 secreted IFN-γ using ELISA at an effector cell to target (E:T) cell ratio of 1:1. (B) Truncated forms of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were produced and expressed in K562-HLA-B*44:02 cells. (C) The antigenic region carrying the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant using ELISA, and it was located between nucleotides (nt)1 and 567. (D) Candidate peptides were screened using the NetMHCpan4.0 epitope prediction algorithm for HLA-B*44:02, based on the protein region (EBNA3C nt 1-567) identified as an epitope-positive sequence. (E) Epitopes were identified using four peptides classified as strong binders (SB) and weak binders (WB) based on peptide-MHC binding affinity (binding level): AEGGVGWRHW (SEQ ID NO:5), SERLVPEESY (SEQ ID NO:155), WLLTSPSQSW (SEQ ID NO:156), and LLTPSQSW (SEQ ID 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 measured by ELISA. TCR25-engineered T cells recognized an epitope with the amino acid sequence AEGGVGWRHW (bolded in D), which can therefore be considered a TCR25 homologous EBV EBNA3C epitope. (F) K562-HLA-B*44:02 cells were loaded with a titration of the indicator peptide or not loaded with the peptide as a control. The peptide sensitivity of TCR 25-engineered T cells was determined by measuring the amount of secreted IFN-γ using ELISA at a 1:1 E:T cell ratio. (MIN - no antigen stimulation, MAX - nonspecific stimulation, SB - strong binder, WB - weak binder, UT - untransduced T cells).

[0162] Figure 10. Functional analysis of EBNA3C-reactive TCR27. (A) EBNA3C-specific TCR27-engineered T cells were co-cultured with K562-HLA-B*07:02 antigen pulsed cells and EBV-positive cell lines. T cell function was determined by measuring the amount of secreted IFN-γ using ELISA at an effector cell to target (E:T) cell ratio of 1:1. (B) Truncated forms of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were produced and expressed in K562-HLA-B*07:02 cells. (C) The antigenic region carrying the immunogenic epitope, located between nucleotides (nt) 2071 and 2979, was identified by measuring the amount of secreted IFN-γ in the supernatant using ELISA. (D) Candidate peptides were screened using the NetMHCpan4.0 epitope prediction algorithm for HLA-B*07:02, based on the protein region identified as an epitope-positive sequence (EBNA3C nt 2071-2979). (E) Epitopes were identified using 27 peptides categorized as strong binders (SB) and weak binders (WB) based on peptide-MHC binding affinity (binding level). 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 (shown in bold in D) were recognized by TCR27-engineered T cells. (F) K562-HLA-B*07:02 cells were loaded with a titration of the indicator peptide or not loaded with the peptide as a control, and the amount of secreted IFN-γ was measured by ELISA at an E:T cell ratio of 1:1 to determine the peptide sensitivity of TCR27-engineered T cells. (MIN - no antigen stimulation, MAX - nonspecific stimulation, SB - strong binder, WB - weak binder, UT - untransduced T cells, SEQ ID NO refer to Figure 8 in the accompanying description).

[0163] Figure 11. Functional analysis of EBNA3C-reactive TCR58. (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 secreted IFN-γ using ELISA at an effector cell to target (E:T) cell ratio of 1:1. (B) Truncated forms of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were produced and expressed in K562-HLA-B*44:02 cells. (C) The antigenic region carrying the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant using ELISA, and it was located between nucleotides (nt)1 and 567. (D) Candidate peptides were screened using the NetMHCpan4.0 epitope prediction algorithm for HLA-B*44:02, based on the protein region (EBNA3C nt 1-567) identified as an epitope-positive sequence. (E) Epitopes were identified using four peptides categorized as strong binders (SB) and weak binders (WB) based on peptide-MHC binding affinity (binding level). 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. An epitope with the amino acid sequence AEGGVGWRHW (boldly shown in D) was recognized by TCR58 engineered T cells and can therefore be considered a TCR58 homologous EBV EBNA3C epitope. (F) K562-HLA-B*44:02 cells were loaded with a titration of the indicator peptide or not as a control. The peptide sensitivity of TCR 58-engineered T cells was determined by measuring the amount of secreted IFN-γ using an ELISA assay at a 1:1 E:T cell ratio. (MIN – no antigen stimulation, MAX – nonspecific stimulation, SB – strong binder, WB – weak binder, UT – untransduced T cells, SEQ ID NO see Figure 9 in the attached figure description).

[0164] Figure 12. Functional analysis of EBNA3C-reactive TCR64. (A) EBNA3C-specific TCR64-engineered T cells were co-cultured with K562-HLA-C*06:02 antigen pulsed cells and EBV-positive cell lines. T cell function was determined by ELISA of secreted IFN-γ at an effector cell to target (E:T) cell ratio of 1:1. (B) Truncated forms of the full-length EBNA3C antigen (EBNA3C / 3, EBNA3C / 2, EBNA3C / 1) were produced and expressed in K562-HLA-C*06:02 cells. (C) The antigenic region carrying the immunogenic epitope was identified by ELISA of secreted IFN-γ in the supernatant and was located between nucleotides (nt) 568 and 1569. (D) Candidate peptides were screened using the NetMHCpan4.0 epitope prediction algorithm for HLA-C*06:02, based on the protein region (EBNA3C nt 568-1569) identified as an epitope positive sequence.(E) Epitopes were identified using 27 peptides classified as strong binders (SB) and weak binders (WB) based on peptide-MHC binding affinity (binding level). These peptides included RRYRRIYDL (SEQ ID NO:158), FRKAQIQGL (SEQ ID NO:4), AREAEVRFL (SEQ ID NO:159), LRGKWQRRY (SEQ ID NO:160), ERYAREAEV (SEQ ID NO:161), SRRRRGACV (SEQ ID NO:162), NLLDFVRFM (SEQ ID NO:163), RRIYDLIEL (SEQ ID NO:164), RRRRGACVV (SEQ ID NO:165), VRFLRGKWQ (SEQ ID NO:166), RRRGACVVY (SEQ ID NO:167), QRRYRRIYD (SEQ ID NO:168), VRFMGVMSS (SEQ ID NO:169), and YAREAEVRFL (SEQ ID NO:168). The 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. (SEQ ID NO:170), NRVGADSIM (SEQ ID NO:171), LHHIWQNLL (SEQ ID NO:172), RRGIKEHVI (SEQ ID NO:173), YRRIYDLIE (SEQ ID NO:174), RRYRRIYDLI (SEQ ID NO:175), ARRGIKEHV (SEQ ID NO:176), QRRYRRIYDL (SEQ ID NO:177), WQRRYRRIY (SEQ ID NO:178), FLRGKWQRRY (SEQ ID NO:179), RRGACVVYD (SEQ ID NO:180), VYDDDVIEV (SEQ ID NO:181), YAREAEVRF (SEQ ID NO:182), GCQNAARTL (SEQ ID NO:183)). An epitope with the amino acid sequence FRKAQIQGL (boldly shown in D) was recognized by TCR64-engineered T cells and can therefore be considered a TCR64 homologous EBV EBNA3C epitope. (F) K562-HLA-C*06:02 cells were loaded with a titration of the indicator peptide or not loaded with the peptide as a control. The peptide sensitivity of TCR 64-engineered T cells was determined by measuring the amount of secreted IFN-γ using ELISA at a 1:1 E:T cell ratio.(MIN - no antigen stimulation, MAX - nonspecific stimulation, SB - strong binder, WB - weak binder, UT - untransduced T cells).

[0165] Figure 13. Functional analysis of LMP1-reactive TCR83. (A) LMP1-specific TCR83-engineered T cells were co-cultured with K562-HLA-C*15:02 antigen pulsed cells or EBV-related cancer cell lines. T cell function was determined by measuring the amount of secreted IFN-γ using ELISA at an effector cell to target (E:T) cell ratio of 1:1. (B) Truncated forms of the full-length LMP1 antigen (LMP1 / 2, LMP1 / 1) were produced and expressed in K562-HLA-C*15:02 cells. (C) The antigenic region carrying the immunogenic epitope was identified by measuring the amount of secreted IFN-γ in the supernatant using ELISA, and it was located between nucleotides (nt) 316 and 624. (D) Candidate peptides were screened using the NetMHCpan4.0 epitope prediction algorithm for HLA-C*15:02 using the protein region (LMP1 nt 316-624) identified as an epitope-positive sequence. (E) Four peptides, classified as weak binders (WB) based on their peptide-MHC binding affinity (binding level), were used to identify epitopes (NSNEGRHHL (SEQ ID NO:184), QQNWWTLLV (SEQ ID NO:3), DSLPHPQQA (SEQ ID NO:185), and YLQQNWWTL (SEQ ID NO:186)). The selected peptides were loaded into K562-HLA-C*15:02 cells and co-cultured with TCR83 engineered T cells. IFN-γ secretion was measured by ELISA. An epitope with the amino acid sequence QQNWWTLLV (boldly shown in D) was recognized by TCR83 engineered T cells and can therefore be considered a homologous EBV LMP1 epitope of TCR83. (F) K562-HLA-C*15:02 cells were loaded with titrated amounts of the indicator peptide or without the peptide as a control. The peptide sensitivity of TCR83 engineered T cells was determined by measuring the amount of secreted IFN-γ using ELISA at an E:T cell ratio of 1:1 (B). (MIN - no antigen stimulation, MAX - nonspecific stimulation, WB - weak binder, UT - untransduced T cells).

[0166] Figure 14. Functional analysis of LMP2A-reactive TCR06. (A) LMP2A-specific TCR06-engineered T cells were co-cultured with K562-HLA-A*02:01 antigen pulsed cells or EBV-related cancer cell lines. T cell function was determined by measuring the amount of secreted IFN-γ using ELISA at an effector cell to target (E:T) cell ratio of 1:1. (B) Truncated forms of the full-length LMP2A antigen (LMP2A / 2, LMP2A / 1) were produced and expressed in K562-HLA-A*02:01 cells. (C) The antigenic region carrying the immunogenic epitope, located between nucleotides (nt) 1006 and 1494, was identified by measuring the amount of secreted IFN-γ in the supernatant using ELISA. (D) Candidate peptides were screened using the NetMHCpan4.0 epitope prediction algorithm for HLA-A*02:01, based on the protein region (LMP1 nt 1006-1494) identified as an epitope-positive sequence. (E) Epitopes (FMCLGGLLTM (SEQ ID NO:187), MLLLIVAGI (SEQ ID NO:188), NLFCMLLLI (SEQ ID NO:189), LLIVAGILFI (SEQ ID NO:190), NLFCMLLLIV (SEQ ID NO:191)) were identified using 14 peptides classified as strong binders (SB) and weak binders (WB) based on peptide-MHC binding affinity (binding level). (SEQ ID NO:192) (SEQ ID NO:1), LIVAGILFI (SEQ ID NO:193), FIPNLFCML (SEQ ID NO:194), IVAGILFIL (SEQ ID NO:195), MLLLIVAGIL (SEQ ID NO:196), CMLLLIVAGI (SEQ ID NO:197), PNLFCMLLLI (SEQ ID NO:198), FIPNLFCMLL (SEQ ID NO:199)). The selected peptides were loaded into K562-HLA-A*02:01 cells and co-cultured with TCR06-engineered T cells. IFN-γ secretion was measured by ELISA. The epitope (shown in bold in D) was recognized by TCR06-engineered T cells and can therefore be considered as a homologous EBV LMP2A epitope of TCR06. Interestingly and notably, the two epitopes recognized by TCR06 did not rank highest in the NetMHCpan 4.0 prediction tool, indicating that the prediction tool is not accurate in predicting relevant immunodominant epitopes. (F) K562-HLA-A*02:01 cells were loaded with a titration of indicator peptide or not loaded with peptide as a control. The peptide sensitivity of TCR 06-engineered T cells was determined by measuring the amount of secreted IFN-γ by ELISA at an E:T cell ratio of 1:1 (B). (MIN - no antigen stimulation, MAX - nonspecific stimulation, S - strong binder, WB - weak binder, UT - untransduced T cells).

[0167] Figure 15. Comparison of peptide sensitivity of TCR and LMP2A-specific TCR06 provided in patents WO 2015 / 022520 A1 (PUBTCR1) and WO 2011 / 039508 A2 (PUBTCR1). Previously published wt EBV LMP2A TCRs with the TRAV12-3*01 / TRAJ41*01 / TRAC α-chain amino acid sequence (SEQ ID NO:2 of WO 2015 / 022520 A1) and the TRBV11-2*01 / TRBD1 / TRBJ2-7 / TRBC β-chain amino acid sequence (SEQ ID NO:3 of WO 2015 / 022520 A1) are also described in the aforementioned literature. Figure 1This is hereby named PUBTCR1. The previously published LMP2A TCR described in WO 2011 / 039508 A2, specifically the LMP2A TCR based on its SEQ ID NO:8, is named PUBTCR2. K562-HLA-A*02:01 cells were loaded with titrated amounts of the indicator peptides CLGGLLTMV and MCLGGLLTMV, respectively, and co-cultured with TCR06-, PUBTCR 1-, or PUBTCR 2- engineered T cells. The peptide sensitivity of all TCRs was determined by measuring the amount of secreted IFN-γ using ELISA at an effector cell to target (E:T) cell ratio of 1:1. TCR06 exhibited higher peptide sensitivity compared to PUBTCR1 and PUBTCR2. UT – Untransduced T cells.

[0168] Figure 16 Extended comparison of peptide sensitivity of LMP2A-specific TCR06 with TCRs provided in patents WO 2015 / 022520 A1 (PUBTCR 1) and WO 2011 / 039508 A2 (PUBTCR 2) and TCR-JC isolated by the applicant.

[0169] The previously published wt EBV LMP2A TCR with the TRAV12-3*01 / TRAJ41*01 / TRAC α-chain amino acid sequence (SEQ ID NO:2 of WO 2015 / 022520A1) and the TRBV11-2*01 / TRBD1 / TRBJ2-7 / TRBC β-chain amino acid sequence (SEQ ID NO:3 of WO 2015 / 022520A1) is also from the aforementioned literature. Figure 1 This is hereby named PUBTCR1. The previously published LMP2A TCR described in WO2011 / 039508 A2, particularly the LMP2A TCR based on its SEQ ID NO:8, is named PUBTCR2. The applicant isolated another LMP2A-specific TCR, which is hereby named TCR-JC. (A) K562-HLA-A*02:01 cells were loaded with a titration of the 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 cell to target (E:T) cell ratio of 1:10. (B) Sigmoidal 4PL curves (constrained model) based on data points A. For TCR06, PUBTCR1, and PUBTCR2, Sigmoidal 4PL regression showed very good R 2(Approximately 0.95). Data from TCR-JC shows a model fit of 0.81 (lacking an upper limit plateau). (C) Based on the data shown in B, the peptide sensitivity of the four TCRs was calculated and used as a 10-1. -9 EC50 in M ​​(EC50 – the mol / L of peptide required to achieve 50% maximum IFN-γ release, SEM – standard error mean).

[0170] Figure 17 EBV-specific TCR-engineered T cells kill cancer cells. LMP1-specific TCR50-, LMP2A-specific TCR06-, and EBNA3C-specific TCR64-engineered T cells interact with L591 EBV. + Tumor cells were co-cultured, these cells naturally expressing LMP1, LMP2A, and EBNA3C, but transfected with their respective MHC I alleles. Data from triplicate wells were averaged at a specified effector cell:target (E:T) cell ratio, and the percentage of surviving cells was calculated based on values ​​obtained from samples co-cultured with untransduced T cells: % survivability = 100 × (experimental value) / (mean background). (UT – untransduced T cells).

[0171] Figure 18 An in vivo mouse model of tumor rejection (A) was established by administering 5 × 10⁻⁶ mg / L to NOG mice. 6 K562-HLA-A*02:01 tumor cells were injected subcutaneously, followed by intravenous injection of 1×10⁻⁶ cells 24 hours later. 7 TCR06 engineered T cells. Data were obtained from single mice given TCR06 engineered T cells (n=10) from two donors and compared with untransduced T cells (n=6). (B) NSG mice subcutaneously injected with 5 × 10 6 K562-HLA-B*57:01 tumor cells, 5×10⁻⁶ mcg intravenously 9 days later. 6 TCR50 engineered T cells. In both models, tumor size was measured and calculated using a diameter gauge. Example

[0172] Production of EBV-specific TCR constructs and transgenic T cells, and identification of epitopes.

[0173] The inventors used an innovative method (Lorenz et al., 2018; WO2016 / 146618 A1) to generate EBV-specific TCRs that recognize endogenously processed, immunogenic EBV epitopes presented by different MHC class I molecules.

[0174] In short, after selecting the target EBV antigen (e.g., LMP1, LMP2A, or EBNA3C), the following experimental steps are performed:

[0175] (i) Specialized antigen-presenting cells (preferably dendritic cells (DCs)) are pulsed with in vitro transcribed (ivt) RNA encoding the full-length sequence of a selected EBV antigen to stimulate autologous T cells. This process is completely objective, allowing dendritic cells to select the optimal epitopes of the antigen for expression, processing, and presentation on the cell surface along with the most suitable MHC class I (MHC type I) molecules.

[0176] (ii) Identification of EBV antigen-responsive 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 is a key feature of the TCR isolation method, as it is based on the extensive flexibility of MHC. To identify each TCR, the inventors selected up to six MHC I alleles corresponding to the T cell donor from the K562 cell library and transfected the cells with the relevant antigens used for priming in step (i). After co-culturing antigen-presenting K562 cells and antigen-stimulated T cells, responsive T cells were identified using ELISA by the release of interferon-(IFN)γ and the upregulation of the T cell activation marker CD137 as measured by flow cytometry. Subsequently, reactive CD8+ T cells were enriched by FACS sorting.

[0177] (iii) Total RNA was isolated from FACS-sorted CD8+ T cells and PCR amplified TCR α and TCR β chain-specific sequences. The dominant TCR α and TCR β sequences were identified by next-generation sequencing.

[0178] (iv) Reexpression of dominant (at least 10%) TCR α and TCR β chain combinations in primary human T cells using the γ-retroviral vector MP71 (Engels et al., 2003; Leisegang et al., 2008; Sommermeyer and Uckert, 2010) NYW identification of functional TCRαβ chain combinations. This was accomplished by co-culturing TCR-engineered T cells with K562 cells carrying appropriate MHC I molecules and expressing full-length EBV antigens. The antigens recognizing the TCRαβ chain combinations were linked to P2A elements and recloned into the MP71 vector in a TCRβ gene-P2A-TCRα gene conformation. The constant TCRαβ chain regions were replaced with their mouse counterparts to enhance the pairing of transgenic TCR chains. Subsequently, codon optimization was performed on the complete TCR transgenic cassette.

[0179] (v) Identification of EBV antigenic peptides (epitopes) recognized by TCR-engineered T cells. For this purpose, the full-length antigen was truncated at either the C- or N-terminus, cloned into the plasmid vector pcDNA3.1(-), and expressed in K562 cells carrying appropriate MHC I molecules. The remaining protein fragments were then examined to determine their ability to further present TCR-recognized epitopes. Finally, candidate peptides for the corresponding protein regions were identified using the epitope prediction algorithm NetMHCpan4.0 (http: / / www.cbs.dtu.dk / services / NetMHCpan / ). Predicted peptides were generated and loaded onto K562 cells carrying appropriate MHC I molecules, and their ability to be recognized by TCR-engineered T cells was investigated in co-culture experiments. Peptides capable of stimulating the production of most IFN-γ molecules were considered homotopes.

[0180] Table 1: Peptides tested for a single TCR (TCR50). Protein regions identified as epitope-positive sequences (LMP1 / 2 nt 316-624) were used to screen candidate peptides using the NetMHCpan4.0 epitope prediction algorithm for HLA-B*57:01. Epitope identification was performed using 13 peptides (categorized as strong binders (SB) and weak binders (WK) based on their peptide-MHC binding affinity). The results are shown in Figure 5.

[0181]

[0182]

[0183] Tables 2, 3, and 4 below describe the characteristics of the generated TCR constructs.

[0184] Table 2: TCR Summary

[0185]

[0186] TCR06, TCR50, and TCR83 exhibit high peptide sensitivity (half-maximal IFN-γ release), especially TCR06.

[0187] Table 3: CDR sequences of preferred TCR constructs of the present invention. CDR1 IMGT amino acid (aa) positions: 27-38. CDR2 IMGT amino acid positions: 56-65. CDR3 IMGT amino acid positions: 105-117. Numbers in parentheses: SEQ ID NO:

[0188]

[0189]

[0190] Table 4: TRAV and TRBV fragments and linking amino acids (IMGT positions 104-118) of the preferred TCR constructs of this invention. Numbers in parentheses: SEQ ID NO:

[0191]

[0192]

[0193] Functional characterization

[0194] The generated TCR-engineered T cells were functionally characterized using in vitro assays.

[0195] First, the peptide sensitivity (half-maximal IFN-γ release) of the isolated TCR was analyzed in a peptide titration experiment (see [reference]). Figure 6 (and Lorenz et al., 2018). In short, peptides were titrated onto K562 target cells in a range of 10. -5 mol / L to 10 - 13 mol / L. Co-culture assays of TCR-engineered T cells at a 1:1 effector cell to target (E:T) cell ratio (unless otherwise specified) imply 2.5 × 10⁻⁶ mol / L. 4 TCR-engineered T cells and 2.5 × 10 4 K562 cells loaded with peptides were co-cultured. After 24 hours, the ability of TCR-transduced T cells to recognize their target epitopes was assessed by IFN-γ ELISA.

[0196] Secondly, in co-culture experiments, target cell lines expressing EBV antigens (LCL, EBV-associated cancer cell lines that endogenously process and present EBV epitopes, and peptide-loaded K562 cells) were used to determine the amount of IFN-γ released by TCR-engineered T cells (see [link to study]). Figure 7 (and Lorenz et al., 2018). In short, TCR-engineered T cells and target cells (2 × 10⁻⁶ each) 4 T cells were co-cultured. After 24 hours, the amount of secreted IFN-γ was determined by ELISA to assess T cell responsiveness. T cells stimulated with PMA and iodine were used as positive controls, and untransduced T cells were used as negative controls.

[0197] Based on the described isolation and characterization process and the identification of epitopes recognized by TCR-engineered T cells, we isolated a total of eight EBV-specific TCRs and the associated immunogenic peptides (epitopes) recognized by these TCRs. Some epitopes are known in the art, while others, particularly the peptides of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, have been identified for the first time in this paper as being presented by the corresponding MHC I molecules.

[0198] Third, L591 tumor cells that express EBV antigen and are endogenously processed and present LMP1, LMP2A, and EBNA3C epitopes but transfected with the relevant MHC I allele were used in co-culture experiments with TCR-engineered T cells to determine the killing ability of TCR-engineered T cells.

[0199] In addition, in vivo assays were used to functionally characterize the generated TCR-engineered T cells. Therefore, two immunocompromised mouse models (NOD and NSG) were used. Tumor cells simultaneously expressing relevant EBV antigens and MHC I molecules were subcutaneously injected into the animals, and TCR-engineered T cells were transferred into mice via tail vein injection.

[0200] TCR Combinations for Immunotherapy

[0201] Combinations of TCR-engineered T cells that identify different epitopes presented by different MHC I molecules represent an interesting option for improving TCR gene therapy. This approach aims to overcome and prevent two problems associated with immunotherapy: (i) tumor growth due to the loss of variants of specific tumor antigens, and (ii) tumor immune escape due to the downregulation of specific MHC I molecules. Using this combined approach will improve the efficiency of TCR gene therapy.

[0202] To demonstrate this, two or three TCRs capable of recognizing endogenously processed EBV antigens naturally presented by different MHC I molecules were selected, such as TCR06 (LMP2A, MHC A*02:01), TCR50 (LMP1, MHC B*57:01), and optionally TCR64 (EBNA3C, MHC C*06:02). TCR-engineered T cells were generated using these TCRs via retroviral transduction as described above. Tumor cells naturally expressing EBV antigens recognized by the TCRs and carrying the corresponding MHC I molecules (A*02:01, B*57:01, C*06:02) were used in co-culture experiments with TCR-engineered T cells.

[0203] Alternatively, such cells can be generated by transfecting the corresponding EBV antigen gene and MHC I gene (e.g., K562 cells).

[0204] In this experiment, TCR-engineered T cells were used alone or in combination and co-cultured with tumor cells at a 2:1 E:T ratio. IFN-γ secretion was determined by ELISA to evaluate the effectiveness of TCR-engineered T cells alone and in combination.

[0205] In addition, cytotoxicity assays were used to analyze the ability of TCR-engineered T cells to kill tumor cells when applied alone and in combination.

[0206] In further experiments, an in vivo mouse model was established. Tumor cells expressing the corresponding EBV antigen and MHC I molecules, such as K562 cells, were subcutaneously injected into the ventral side of NSG mice (NOD.Cg-Prkdc). scid Il2rg tm1 Wjl / Sz). After a palpable tumor is reached, TCR-engineered T cells are injected intravenously, alone or in combination, to determine the efficacy of TCR gene therapy in tumor rejection.

[0207] References

[0208] Chervin et al., 2008. Journal of Immunological Methods 339:175-184.

[0209] Cho et al.,2018. British Journal of Cancer 118:534-545.

[0210] Cohen et al., 2006. Cancer Research 66:8878–8886.

[0211] Cohen et al., 2007. Cancer Research 67:3898–3903.

[0212] Danska et al.,1990.The Journal of Experimental Medicine 172:27-33.

[0213] Doran et al., 2019. Journal of Clinical Oncology doi:10.1200 / JCO.18.02424. [Epub ahead of print].

[0214] Engels et al., 2003. Human Gene Therapy 14:1155-1168.

[0215] Garcia and Adams,2005. Cell 122:333-336.

[0216] Hart et al.,2008.Gene Therapy 15:625-631.

[0217] Jurgens et al.,2006.Journal of Clinical Immunology 26:22-32.

[0218] Kuball et al.,2007.Blood 109:2331–2338.

[0219] Leisegang et al.,2008.Journal of Molecular Medicine.86:573-583.

[0220] Linette et al.,2013.Blood 122:863-871.

[0221] Lorenz et al.,2018.Human Gene Therapy 28:1158-1168.

[0222] Morgan et al.,2013.Journal of Immunotherapy 36:133-151.

[0223] Orentas et al.,2001.Clinical Immunology 98:220-228.

[0224] Robbins et al.,2008.Journal of Immunology 180:6116-6131.

[0225] Scholten et al.,2006.Clinical Immunology 119:135–145.

[0226] Simpson et al.,2011.Proceedings of the National Academy of Sciencesof the USA 108:21176-21181.

[0227] Sommermeyer and Uckert,2010,Journal of Immunology 184:6223-6231.

[0228] Tran et al.,2014.Science 344:641-645.

[0229] WO 2011 / 039508 A2

[0230] WO 2015 / 022520 A1

[0231] Yang et al.,2011.Clinical and Developmental Immunology,Article ID716926

[0232] Zheng et al.,2015. Cancer Immunology Research 3:1138-1147. sequence list <110> Max de Brook-Helmholtz Association of Molecular Medicine <120> TCR constructs specifically targeting EBV antigens <130> 12011 P 6581WO <160> 199 <170> BiSSAP 1.3.6 <210> 1 <211> 9 <212> PRT <213> people <220> <223> Tablet from LMP2A presented at A*02:01 <400> 1 Cys Leu Gly Gly Leu Leu Thr Met Val 1 5 <210> 2 <211> 9 <212> PRT <213> people <220> <223> Tabletop from LMP1 presented at B*57:01 <400> 2 Ile Ala Leu Tyr Leu Gln Gln Asn Trp 1 5 <210> 3 <211> 9 <212> PRT <213> people <220> <223> Tabletop from LMP1 presented at C*15:02 <400> 3 Gln Gln Asn Trp Trp Thr Leu Leu Val 1 5 <210> 4 <211> 9 <212> PRT <213> people <220> <223> Tablet from EBNA3C presented at C*06:02 <400> 4 Phe Arg Lys Ala Gln Ile Gln Gly Leu 1 5 <210> 5 <211> 10 <212> PRT <213> people <220> <223> Tablet from EBNA3C presented at B*44:02 <400> 5 Ala Glu Gly Gly Val Gly Trp Arg His Trp 1 5 10 <210> 6 <211> 10 <212> PRT <213> people <220> <223> Tablet from EBNA3C submitted at B*07:02 <400> 6 Gln Pro Arg Ala Pro Ile Arg Pro Ile Pro 1 5 10 <210> 7 <211> 11 <212> PRT <213> people <220> <223> Tablet from EBNA3C submitted at B*07:02 <400> 7 Gln Pro Arg Ala Pro Ile Arg Pro Ile Pro Thr 1 5 10 <210> 8 <400> 8 000 <210> 9 <400> 9 000 <210> 10 <400> 10 000 <210> 11 <211> 6 <212> PRT <213> people <220> <223> TCR06 - TRA - CDR1 <400> 11 Asp Ser Ala Ile Tyr Asn 1 5 <210> 12 <211> 7 <212> PRT <213> people <220> <223> TCR06 - TRA - CDR2 <400> 12 Ile Gln Ser Ser Gln Arg Glu 1 5 <210> 13 <211> 11 <212> PRT <213> people <220> <223> TCR06 - TRA - CDR3 <400> 13 Ala Val Leu Met Asp Ser Asn Tyr Gln Leu Ile 1 5 10 <210> 14 <211> 13 <212> PRT <213> people <220> <223> TCR06-TRA-linked amino acids <400> 14 Cys Ala Val Leu Met Asp Ser Asn Tyr Gln Leu Ile Trp 1 5 10 <210> 15 <211> 113 <212> PRT <213> Human <220> <223> TCR06 - TRA - Variable Region <400> 15 Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu Gly 1 5 10 15 Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn 20 25 30 Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu 35 40 45 ​​​​​​​​​​​​​​​​​​​​​​​ <213> people <220> <223> TCR06 - TRB - CDR1 <400> 16 Trp Ser His Ser Tyr 1 5 <210> 17 <211> 6 <212> PRT <213> people <220> <223> TCR06 - TRB - CDR2 <400> 17 Ser Ala Ala Ala Asp Ile 1 5 <210> 18 <211> 12 <212> PRT <213> people <220> <223> TCR06 - TRB - CDR3 <400> 18 Ala Ser Ser Glu Asp Gly Met Asn Thr Glu Ala Phe 1 5 10 <210> 19 <211> 14 <212> PRT <213> people <220> <223> TCR06 - TRB - Linked amino acids <400> 19 Cys Ala Ser Ser Glu Asp Gly Met Asn Thr Glu Ala Phe Phe 1 5 10 <210> 20 <211> 113 <212> PRT <213> people <220> <223> TCR06 - TRB - Variable Region <400> 20 Asp Ala Gly Ile Thr Gln Ser Pro Arg Tyr Lys Ile Thr Glu Thr Gly 1 5 10 15 Arg Gln Val Thr Leu Met Cys His Gln Thr Trp Ser His Ser Tyr Met 20 25 30 Phe Trp Tyr Arg Gln Asp Leu Gly His Gly Leu Arg Leu Ile Tyr Tyr 35 40 45 Ser Ala Ala Ala Asp Ile Thr Asp Lys Gly Glu Val Pro Asp Gly Tyr 50 55 60 Val Val Ser Arg Ser Lys Thr Glu Asn Phe Pro Leu Thr Leu Glu Ser 65 70 75 80 Ala Thr Arg Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Glu Asp 85 90 95 Gly Met Asn Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val 100 105 110 Val <210> 21 <211> 6 <212> PRT <213> Human <220> <223> TCR50 - TRA - CDR1 <400> 21 Ser Ser Tyr Ser Pro Ser 1 5 <210> 22 <211> 8 <212> PRT <213> Human <220> <223> TCR50 - TRA - CDR2 <400> twenty two Tyr Thr Ser Ala Ala Thr Leu Val 1 5 <210> twenty three <211> 11 <212> PRT <213> people <220> <223> TCR50 - TRA - CDR3 <400> twenty three Val Val Met Ala Thr Gly Phe Gln Lys Leu Val 1 5 10 <210> twenty four <211> 13 <212> PRT <213> people <220> <223> TCR50 - TRA - Linked amino acids <400> twenty four Cys Val Val Met Ala Thr Gly Phe Gln Lys Leu Val Phe 1 5 10 <210> 25 <211> 114 <212> PRT <213> people <220> <223> TCR50 - TRA - Variable Zone <400> 25 Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly 1 5 10 15 Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser 20 25 30 Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu 35 40 45 Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile Asn Gly Phe Glu 50 55 60 Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser 65 70 75 80 Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val Met Ala Thr 85 90 95 Gly Phe Gln Lys Leu Val Phe Gly Thr Gly Thr Arg Leu Leu Val Ser 100 105 110<° Pro Asn <210> 26 <211> 5 <212> PRT <213> Human <220> [[ID=°2]]<223> TCR50 - TRB - CDR1 <400> 26 Ser Gly Asp Leu Ser 1 5 <210> 27 <211> 6 <212> PRT <213> Human <220> <223> TCR50 - TRB - CDR2 <400> 27 Tyr Tyr Asn Gly Glu Glu 1 5 <210> 28 <211> 12 <212> PRT <213> Human It should be noted that there is a possible error in the original text where <° and <°2> are likely incorrect tags. They should probably be and <22> respectively. This has been reflected in the translation as best as possible while maintaining the integrity of the provided text. <220> <223> TCR50 - TRB - CDR3 <400> 28 Ala Ser Ser Val Thr Ser Gly Ser Asp Glu Gln Phe 1 5 10 <210> 29 <211> 14 <212> PRT <213> people <220> <223> TCR50 - TRB - Linked amino acids <400> 29 Cys Ala Ser Ser Val Thr Ser Gly Ser Asp Glu Gln Phe Phe 1 5 10 <210> 30 <211> 113 <212> PRT <213> people <220> <223> TCR50 - TRB - Variable Region <400> 30 Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr Ala Thr Gly 1 5 10 15 Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp Leu Ser Val 20 25 30 Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe Leu Ile Gln 35 40 45 Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu Glu Arg Phe 50 55 60 Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn Leu Ser Ser 65 70 75 80 Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Val Thr 85 90 95 Ser Gly Ser Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val 100 105 110 Leu <210> 31 <211> 6 <212> PRT <213> Human <220><00008​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <212> PRT <213> Person <220> <223> TCR83 - TRA - Linking Amino Acids <400> 34 Cys Ala Ala Val Asn Asn Ala Gly Asn Met Leu Thr Phe ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro His 100 105 110 <210> 36 <211> 5 <212> PRT <213> Human ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 1 5 10 15 Phe <210> 40 <211> 116 <212> PRT <213> Human <220> <223> TCR83 - TRB - Variable Region <400> 40 Asp Thr Ala Val Ser Gln Thr Pro Lys Tyr Leu Val Thr Gln Met Gly 1 5 10 15 Asn Asp Lys Ser Ile Lys Cys Glu Gln Asn Leu Gly His Asp Thr Met 20 25 30 Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Phe Leu Lys Ile Met Phe Ser 35 40 45 Tyr Asn Asn Lys Glu Leu Ile Ile Asn Glu Thr Val Pro Asn Arg Phe 50 55 60 Ser Pro Lys Ser Pro Asp Lys Ala His Leu Asn Leu His Ile Asn Ser 65 70 75 80 Leu Glu Leu Gly Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Gln Gly 85 90 95 Tyr Gly Gly Pro Ser Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg 100 105 110<W Leu Thr Val Leu 115 <210> 41 <211> 5 <212> PRT <213> people <220> <223> TCR64 - TRA - CDR1 <400> 41 Ser Val Phe Ser Ser 1 5 <210> 42 <211> 7 <212> PRT <213> people <220> <223> TCR64 - TRA - CDR2 <400> 42 Val Val Thr Gly Gly Glu Val 1 5 <210> 43 <211> 13 <212> PRT <213> people <220> <223> TCR64 - TRA - CDR3 <400> 43 Ala Gly Asp Val Asp Thr Gly Thr Ala Ser Lys Leu Thr 1 5 10 <210> 44 <211> 15 <212> PRT <213> people <220> <223> TCR64-TRA-linked amino acid <400> 44 Cys Ala Gly Asp Val Asp Thr Gly Thr Ala Ser Lys Leu Thr Phe 1 5 10 15 <210> 45 <211> 114 <212> PRT <213> people <220> <223> TCR64 - TRA - Variable Region <400> 45 Thr Gln Leu Leu Glu Gln Ser Pro Gln Phe Leu Ser Ile Gln Glu Gly 1 5 10 15 Glu Asn Leu Thr Val Tyr Cys Asn Ser Ser Ser Val Phe Ser Ser Leu 20 25 30 Gln Trp Tyr Arg Gln Glu Pro Gly Glu Gly Pro Val Leu Leu Val Thr 35 40 45 Val Val Thr Gly Gly Glu Val Lys Lys Leu Lys Arg Leu Thr Phe Gln 50 55 60 Phe Gly Asp Ala Arg Lys Asp Ser Ser Leu His Ile Thr Ala Ala Gln 65 70 75 80 Pro Gly Asp Thr Gly Leu Tyr Leu Cys Ala Gly Asp Val Asp Thr Gly 85 90 95 Thr Ala Ser Lys Leu Thr Phe Gly Thr Gly Thr Arg Leu Gln Val Thr 100 105 110 Leu Asp <210> 46 <211> 5 <212> PRT <213> Human <220> <223> TCR64 - TRB - CDR1 <400> 46 Met Asp His Glu Asn 1 5 <210> 47 <211> 6 <212> PRT <213> people <220> <223> TCR64 - TRB - CDR2 <400> 47 Ser Tyr Asp Val Lys Met 1 5 <210> 48 <211> 14 <212> PRT <213> people <220> <223> TCR64 - TRB - CDR3 <400> 48 Ala Ser Ser Leu Leu Gly Ser Gly Ala Leu Tyr Glu Gln Tyr 1 5 10 <210> 49 <211> 16 <212> PRT <213> people <220> <223> TCR64-TRB-linked amino acid <400> 49 Cys Ala Ser Ser Leu Leu Gly Ser Gly Ala Leu Tyr Glu Gln Tyr Phe 1 5 10 15 <210> 50 <211> 115 <212> PRT <213> people <220> <223> TCR64 - TRB - Variable Region <400> 50 Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly 1 5 10 15 Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met 20 25 30 Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe 35 40 45 Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr 50 55 60 Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser 65 70 75 80 Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Leu Leu 85 90 95 Gly Ser Gly Ala Leu Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <212> PRT <213> people <220> <223> CDR25 - TRA - CDR3 <400> 53 Val Ala Trp Asp Thr Gly Phe Gln Lys Leu Val 1 5 10 <210> 54 <211> 13 <212> PRT <213> people <220> <223> CDR25 - TRA - Linked amino acids <400> 54 Cys Val Ala Trp Asp Thr Gly Phe Gln Lys Leu Val Phe 1 5 10 <210> 55 <211> 114 <212> PRT <213> people <220> <223> CD25 - TRA - Variable Region <400> 55 Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly 1 5 10 15 Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser 20 25 30 Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu 35 40 45 Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile Asn Gly Phe Glu 50 55 60 Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser 65 70 75 80 Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Ala Trp Asp Thr 85 90 95 Gly Phe Gln Lys Leu Val Phe Gly Thr Gly Thr Arg Leu Leu Val Ser 100 105 110 Pro Asn <210> 56 <211> 5 <212> PRT <213> Human <220> <223> CD25 - TRB - CDR1 <400> 56 Ser Asn His Leu Tyr 1 5 <210> 57 <211> 6 <212> PRT <213> Human <220> <223> CD25 - TRB - CDR2 <400> 57 Phe Tyr Asn Asn Glu Ile 1 5 <210> 58 <211> 10 <212> PRT <213> Human <220> <223> CD25 - TRB - CDR3 <400> 58 Ala Ser Lys Ala Leu Ala Asp Thr Gln Tyr 1 5 10 <210> 59 <211> 12 <212> PRT <213> people <220> <223> CD25-TRB-linked amino acids <400> 59 Cys Ala Ser Lys Ala Leu Ala Asp Thr Gln Tyr Phe 1 5 10 <210> 60 <211> 112 <212> PRT <213> people <220> <223> TCR25 - TRB - Variable Region <400> 60 Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr Gln Met Gly 1 5 10 15 Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His Leu Tyr Phe 20 25 30 Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe Leu Val Ser 35 40 45 Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe Asp Asp Gln 50 55 60 Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu Lys Ile Arg 65 70 75 80 Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala Ser Lys Ala 85 90 95 Leu Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 100 105 110 <210> 61 <211> 6 <212> PRT <213> people <220> <223> TCR58 - TRA - CDR1 <400> 61 Asn Ser Ala Ser Gln Ser 1 5 <210> 62 <211> 6 <212> PRT <213> people <220> <223> TCR58 - TRA - CDR2 <400> 62 Val Tyr Ser Ser Gly Asn 1 5 <210> 63 <211> 11 <212> PRT <213> people <220> <223> TCR58 - TRA - CDR3 <400> 63 Val Ala Ser Gly Asp Ser Ser Tyr Lys Leu Ile 1 5 10 <210> 64 <211> 13 <212> PRT <213> people <220> <223> TCR58-TRA-linked amino acid <400> 64 Cys Val Ala Ser Gly Asp Ser Ser Tyr Lys Leu Ile Phe 1 5 10 <210> 65 <211> 111 <212> PRT <213> Human <220> <223> TCR58 - TRA - Variable Region <400> 65 Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe Asn Val Pro Glu Gly 1 5 10 15 Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn Ser Ala Ser Gln Ser 20 25 30 Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu Pro Lys Leu Leu Met 35 40 45 Ser Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg Phe Thr Ala Gln Leu 50 55 60 Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile Arg Asp Ser Lys Leu 65 70 75 80 Ser Asp Ser Ala Thr Tyr Leu Cys Val Ala Ser Gly Asp Ser Ser Tyr 85 90 95 Lys Leu Ile Phe Gly Ser Gly Thr Arg Leu Leu Val Arg Pro Asp 100 105 110 <210> 66 <211> 5 <212> PRT <213> Human <220> <223> TCR58 - TRB - CDR1 <400> 66 Ser Asn His Leu Tyr 1 5 <210> 67 <211> 6 <212> PRT <213> people <220> <223> TCR58 - TRB - CDR2 <400> 67 Phe Tyr Asn Asn Glu Ile 1 5 <210> 68 <211> 13 <212> PRT <213> people <220> <223> TCR58 - TRB - CDR3 <400> 68 Ala Ser Ser Asp Pro Leu Ser Thr Tyr Asn Glu Gln Phe 1 5 10 <210> 69 <211> 15 <212> PRT <213> people <220> <223> TCR58-TRB-linked amino acids <400> 69 Cys Ala Ser Ser Asp Pro Leu Ser Thr Tyr Asn Glu Gln Phe Phe 1 5 10 15 <210> 70 <211> 115 <212> PRT <213> people <220> <223> TCR58 - TRB - Variable Region <400> 70 Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr Gln Met Gly 1 5 10 15 Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His Leu Tyr Phe 20 25 30 Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe Leu Val Ser 35 40 45 Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe Asp Asp Gln 50 55 60 Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu Lys Ile Arg 65 70 75 80 Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Asp 85 90 95 Pro Leu Ser Thr Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Leu 115 <210> 71 <211> 7 <212> PRT <213> Human <220> <223> TCR27 - TRA - CDR1 <400> 71 ​​​​​​​​​​​​​​​​<400> 72 Gly Leu Thr Ser Asn 1 5 <210> 73 <211> 14 <212> PRT <213> people <220> <223> TCR27 - TRA - CDR3 <400> 73 Ile Leu Cys Gly Ala Gly Gly Thr Ser Tyr Gly Lys Leu Thr 1 5 10 <210> 74 <211> 16 <212> PRT <213> people <220> <223> TCR27-TRA-linked amino acid <400> 74 Cys Ile Leu Cys Gly Ala Gly Gly Thr Ser Tyr Gly Lys Leu Thr Phe 1 5 10 15 <210> 75 <211> 114 <212> PRT <213> people <220> <223> TCR27 - TRA - Variable Region <400> 75 Asp Ala Lys Thr Thr Gln Pro Asn Ser Met Glu Ser Asn Glu Glu Glu 1 5 10 15 Pro Val His Leu Pro Cys Asn His Ser Thr Ile Ser Gly Thr Asp Tyr 20 25 30 Ile His Trp Tyr Arg Gln Leu Pro Ser Gln Gly Pro Glu Tyr Val Ile 35 40 45 His Gly Leu Thr Ser Asn Val Asn Asn Arg Met Ala Ser Leu Ala Ile 50 55 60 Ala Glu Asp Arg Lys Ser Ser Thr Leu Ile Leu His Arg Ala Thr Leu 65 70 75 80 Arg Asp Ala Ala Val Tyr Tyr Cys Ile Leu Cys Gly Ala Gly Gly Thr 85 90 95 Ser Tyr Gly Lys Leu Thr Phe Gly Gln Gly Thr Ile Leu Thr Val His 100 105 110 Pro Asn <210> 76 <211> 5 <212> PRT <213> Human <220> <223> TCR27 - TRB - CDR1 <400> 76 Met Asn His Glu Tyr 1 5 <210> 77 <211> 6 <212> PRT <213> Human <220> <223> TCR27 - TRB - CDR2 <400> 77 Ser Met Asn Val Glu Val 1 5 <210> 78 <211> 12 <212> PRT <213> Human <220> <223> TCR27 - TRB - CDR3 <400> 78 Ala Ser Asn Val Gln Gly Ala Asn Asn Glu Gln Phe 1 5 10 <210> 79 <211> 14 <212> PRT <213> Human <220> <223> TCR27 - TRB - Linked Amino Acids <400> 79 Cys Ala Ser Asn Val Gln Gly Ala Asn Asn Glu Gln Phe Phe 1 5 10 <210> 80 <211> 113 <212> PRT <213> Human <220> <223> TCR27 - TRB - Variable Region <400> 80 Glu Ala Gln Val Thr Gln Asn Pro Arg Tyr Leu Ile Thr Val Thr Gly 1 5 10 15 Lys Lys Leu Thr Val Thr Cys Ser Gln Asn Met Asn His Glu Tyr Met 20 25 30 Ser Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Gln Ile Tyr Tyr 35 40 45 Ser Met Asn Val Glu Val Thr Asp Lys Gly Asp Val Pro Glu Gly Tyr 50 55 60 Lys Val Ser Arg Lys Glu Lys Arg Asn Phe Pro Leu Ile Leu Glu Ser 65 70 75 80 Pro Ser Pro Asn Gln Thr Ser Leu Tyr Phe Cys Ala Ser Asn Val Gln 85 90 95 Gly Ala Asn Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val 100 105 110 Leu <210> 81 <211> 7 <212> PRT <213> Human <220> <223> TCR01 - TRA - CDR1 <400> 81 Thr Ile Ser Gly Thr Asp Tyr 1 5 <210> 82 <211> 5 <212> PRT <213> Human <220> <223> TCR01 - TRA - CDR2 <400> 82 Gly Leu Thr Ser Asn 1 5 <210> 83 <211> 14 <212> PRT <213> Human <220> <223> TCR01 - TRA - CDR3 <400> 83 Ile Leu Cys Gly Ala Gly Gly Thr Ser Tyr Gly Lys Leu Thr 1 5 10 <210> 84 <211> 16 <212> PRT <213> Human <220> <223> TCR01 - TRA - Linking Amino Acids <400> 84 Cys Ile Leu Cys Gly Ala Gly Gly Thr Ser Tyr Gly Lys Leu Thr Phe 1 5 10 15 <210> 85 <211> 114 <212> PRT <213> Human <220> <223> TCR01 - TRA - Variable Region <400> 85 Asp Ala Lys Thr Thr Gln Pro Asn Ser Met Glu Ser Asn Glu Glu Glu 1 5 10 15 Pro Val His Leu Pro Cys Asn His Ser Thr Ile Ser Gly Thr Asp Tyr 20 25 30 Ile His Trp Tyr Arg Gln Leu Pro Ser Gln Gly Pro Glu Tyr Val Ile 35 40 45 His Gly Leu Thr Ser Asn Val Asn Asn Arg Met Ala Ser Leu Ala Ile 50 55 60 Ala Glu Asp Arg Lys Ser Ser Thr Leu Ile Leu His Arg Ala Thr Leu 65 70 75 80 Arg Asp Ala Ala Val Tyr Tyr Cys Ile Leu Cys Gly Ala Gly Gly Thr 85 90 95 Ser Tyr Gly Lys Leu Thr Phe Gly Gln Gly Thr Ile Leu Thr Val His 100 105 110 Pro Asn <210> 86 <211> 5 <212> PRT <213> people <220> <223> TCR01 - TRB - CDR1 <400> 86 Met Asn His Glu Tyr 1 5 <210> 87 <211> 6 <212> PRT <213> people <220> <223> TCR01 - TRB - CDR2 <400> 87 Ser Met Asn Val Glu Val 1 5 <210> 88 <211> 12 <212> PRT <213> people <220> <223> TCR01 - TRB - CDR3 <400> 88 Ala Ser Ala Ile Gln Gly Ala Asn Asn Glu Gln Phe 1 5 10 <210> 89 <211> 14 <212> PRT <213> people <220> <223> TCR01 - TRB - Linked amino acids <400> 89 Cys Ala Ser Ala Ile Gln Gly Ala Asn Asn Glu Gln Phe Phe 1 5 10 <210> 90 <211> 113 <212> PRT <213> people <220> <223> TCR01 - TRB - Variable Region <400> 90 Glu Ala Gln Val Thr Gln Asn Pro Arg Tyr Leu Ile Thr Val Thr Gly 1 5 10 15 Lys Lys Leu Thr Val Thr Cys Ser Gln Asn Met Asn His Glu Tyr Met 20 25 30 Ser Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Gln Ile Tyr Tyr 35 40 45 Ser Met Asn Val Glu Val Thr Asp Lys Gly Asp Val Pro Glu Gly Tyr 50 55 60 Lys Val Ser Arg Lys Glu Lys Arg Asn Phe Pro Leu Ile Leu Glu Ser 65 70 75 80 Pro Ser Pro Asn Gln Thr Ser Leu Tyr Phe Cys Ala Ser Ala Ile Gln 85 90 95 Gly Ala Asn Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val 100 105 110 Leu <210> 91 <211> 339 <212> DNA <213> Artificial sequence <220> <223> DNA encoding the TCR06 variable region - TRA <400> 91 aagcaagaag tgacccagat tcctgccgct ctgtccgtgc ctgaaggcga gaatctggtc 60 ctgaactgca gcttcaccga cagcgccatc tacaacctgc agtggttcag gcaggatccc 120 ggcaagggac tgacaagcct gctgctgatt cagagcagcc agagagagca gacctccggc 180 agactgaatg ccagcctgga taagtccagc ggcagaagca ccctgtatat cgccgcttct 240 cagcctggcg atagcgccac atatctgtgt gccgtcctga tggactccaa ctaccagctg 300 atttggggag ccggcaccaa gctgatcatc aagcccgac 339 <210> 92 <211> 339 <212> DNA <213> Artificial sequence <220> <223> DNA encoding the variable region of TCR06 - TRB <400> 92 gatgccggaa tcacacagag ccccagatac aagatcaccg agacaggccg gcaagtgacc 60 ctgatgtgtc accagacatg gtcccacagc tacatgttct ggtacagaca ggacctcggc 120 cacggcctga gactgatcta ctattctgcc gccgctgaca tcaccgacaa gggcgaagtg 180 cctgatggct acgtggtgtc cagaagcaag accgagaact tcccactgac actggaaagc 240 gccacacggt cccagaccag cgtgtacttt tgtgccagca gcgaggacgg aatgaacacc 300 gaggcctttt tcggccaagg caccagactg accgtggtg 339 <210> 93 <211> 342 <212> DNA <213> Artificial sequence <220> <223> DNA encoding the variable region of TCR50 - TRA <400> 93 gcccagagcg tgacacaact ggatagccac gtgtccgtgt ctgagggcac acctgtgctg 60 ctgagatgca actactccag cagctacagc cccagcctgt tttggtacgt gcagcaccct 120 aacaagggac tgcagctgct gctgaagtac acctctgccg ccacactggt caagggcatc 180 aatggcttcg aggccgagtt caagaagtcc gagacaagct tccacctgac caagcctagc 240 gctcacatgt ccgatgccgc cgaatacttc tgcgtggtca tggccaccgg cttccagaaa 300 ctggtgtttg gcacaggcac ccggctgctc gtgtccccaa at 342 <210> 94 <211> 339 <212> DNA <213> Artificial sequence <220> <223> DNA encoding the variable region of TCR50 - TRB <400> 94 gatagcggcg ttacccagac acctaagcac ctgatcacag ccacaggcca gcgcgtgacc 60 ctgagatgtt ctcctagaag cggcgacctg agcgtgtact ggtatcagca gtctctggac 120 cagggcctgc agttcctgat ccagtactac aacggcgagg aaagagccaa gggcaacatc 180 ctggaacggt tcagcgccca gcagttccca gatctgcaca gcgagctgaa cctgagcagc 240 ctggaactgg gagatagcgc cctgtacttc tgtgccagct ctgtgaccag cggcagcgac 300 gagcagtttt ttggccctgg caccagactg accgtgctg 339 <210> 95 <211> 333 <212> DNA <213> Artificial sequence <220> <223> DNA encoding the variable region of TCR83 - TRA <400> 95 ggccagaaca tcgatcagcc tacagagatg accgccaccg agggcgccat cgtgcagatc 60 aattgcacct accagaccag cggcttcaac ggcctgttct ggtatcagca gcatgccggc 120 gaggccccta ccttcctgag ctacaatgtg ctggacggcc tggaagaaaa gggcagattc 180 agcagcttcc tgtccagaag caagggctac tcctacctgc tgctgaaaga actgcagatg 240 aaggacagcg cctcttacct gtgtgccgcc gtgaacaacg ccggcaacat gctgacattt 300 ggcggcggaa cacggctgat ggtcaagccc cac 333 <210> 96 <211> 348 <212> DNA <213> Synthetic sequence <220> <223> DNA encoding the variable region of TCR83 - TRB <400> 96 gatacagccg tgtctcagac ccctaagtac ctggtcaccc agatgggcaa cgacaagagc 60 atcaagtgcg agcagaacct gggccacgac accatgtact ggtacaagca ggacagcaag 120 aaattcctga agatcatgtt cagctacaac aacaaagagc tgatcatcaa cgagacagtg 180 cccaaccggt tcagccctaa gagccctgat aaggcccacc tgaacctgca catcaacagc 240 ctggaactgg gcgacagcgc cgtgtacttt tgtgccagct ctcaaggcta tggcggccct 300 agcaccgaca cacagtattt cggccctggc accagactga ccgtgctg 348 <210> 97 <211> 342 <212> DNA <213> Synthetic sequence <220> <223> DNA encoding the variable region of TCR64 - TRA <400> 97 acacagctgc tggaacagag cccacagttc ctgagcatcc aagagggcga gaacctgacc 60 gtgtactgca acagcagcag cgtgttcagc tccctgcagt ggtacaggca agagcctggc 120 gaaggacctg tgctgctggt cacagttgtg acaggcggcg aagtgaagaa gctgaagcgg 180 ctgaccttcc agttcggcga cgccagaaag gattccagcc tgcacattac cgctgctcag 240 ccaggcgata ccggcctgta tctttgtgct ggcgacgtcg acacaggcac cgccagcaaa 300 ctgacatttg gcaccggcac caggctgcaa gtgaccctgg ac 342 <210> 98 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding the variable region of TCR64 - TRB <400> 98 gacgtgaaag tgacacagag cagcagatac ctggtcaagc ggaccggcga gaaggtgttc 60 ctggaatgcg tgcaggacat ggaccacgag aatatgttct ggtacagaca ggaccccggc 120 ctgggcctga gactgatcta cttcagctac gacgtgaaga tgaaggaaaa gggcgacatc 180 cccgagggct acagcgtgtc cagagagaag aaagagcggt tcagcctgat cctggaaagc 240 gccagcacca accagaccag catgtacctg tgtgccagca gcctgcttgg atctggcgcc 300 ctgtacgagc agtatttcgg ccctggcacc agactgaccg tgacc 345 <210> 99 <211> 342 <212> DNA <213> Artificial sequence <220> <223> DNA encoding the variable region of TCR25 - TRA <400> 99 gcccagagcg tgacacaact ggatagccac gtgtccgtgt ctgagggcac acctgtgctg 60 ctgagatgca actactccag cagctacagc cccagcctgt tttggtacgt gcagcaccct 120 aacaagggcc tgcagctgct gctgaagtac acatctgccg ccacactggt caagggcatc 180 aatggcttcg aggccgagtt caagaagtct gagacaagct tccacctgac caagcctagc 240 gctcacatgt ccgatgccgc cgaatacttc tgcgtggcct gggataccgg cttccagaaa 300 ctggtgtttg gcaccggcac acggctgctc gtgtccccaa at 342 <210> 100 <211> 336 <212> DNA <213> Artificial sequence <220> <223> DNA encoding the variable region of TCR25 - TRB <400> 100 gagcccgaag tgacacagac acccagccac caagtgaccc agatgggcca agaagtgatc 60 ctgcgctgcg tgcccatcag caaccacctg tacttctact ggtacagaca gatcctgggc 120 cagaaagtcg agttcctggt gtccttctac aacaacgaga tcagcgagaa gtccgagatc 180 ttcgacgacc agttcagcgt ggaaagaccc gacggcagca acttcaccct gaagatcaga 240 agcaccaagc tcgaggacag cgccatgtac ttttgcgcct ctaaggccct ggccgacaca 300 cagtattttg gccctggcac cagactgacc gtgctg 336 <210> 101 <211> 333 <212> DNA <213> Artificial sequence <220> <223> DNA encoding the variable region of TCR58 - TRA <400> 101 cggaaagagg tggaacagga ccctggacct ttcaacgttc cagagggcgc caccgtggcc 6 ttcaattgca cctacagcaa tagcgccagc cagagctttt tctggtatcg gcaggactgc 120 cggaaagaac ccaagctgct gatgagcgtg tacagcagcg gcaacgagga cggcagattc 180 acagcccagc tgaacagagc cagccagtac atctccctgc tgatccggga tagcaagctg 240 agcgatagcg ccacctatct gtgtgtggcc agcggcgata gcagctacaa gctgatcttt 300 ggcagcggca ccaggctgct tgtgcggccc gat 333 <210> 102 <211> 345 <212> DNA <213> Artificial sequence <220> <223> DNA encoding the TCR58 variable region - TRB <400> 102 gagcccgaag tgacacagac acccagccac caagtgaccc agatgggcca agaagtgatc 60 ctgcgctgcg tgcccatcag caaccacctg tacttctact ggtacagaca gatcctgggc 120 cagaaagtcg agttcctggt gtccttctac aacaacgaga tcagcgagaa gtccgagatc 180 ttcgacgacc agttcagcgt ggaaagaccc gacggcagca acttcaccct gaagatcaga 240 agcaccaagc tcgaggacag cgccatgtac ttttgcgcca gcagcgatcc cctgagcacc 300 tacaacgagc agttcttcgg ccctggcacc agactgaccg tgctg 345 <210> 103 <211> 342 <212> DNA <213> Artificial sequence <220> <223> DNA encoding the TCR27 variable region - TRA <400> 103 gacgccaaga ccacacagcc caacagcatg gaaagcaacg aagaggaacc cgtgcatctg 60 ccctgcaacc acagcacaat cagcggcacc gactacatcc actggtatag acagctgccc 120 tctcagggcc ccgagtatgt gattcacgga ctgaccagca acgtgaacaa ccggatggcc 180 tctctggcca ttgccgagga cagaaagagc agcaccctga tcctgcacag agccacactg 240 agagatgccg ccgtgtacta ctgcatcctt tgtggcgctg gcggcaccag ctatggcaag 300 ctgacatttg gccagggcac catcctgacc gtgcatccca ac 342 <210> 104 <211> 339 <212> DNA <213> Artificial sequence <220> <223> DNA - TRB encoding the variable region of TCR27 <400> 104 gaagctcaag tgacacagaa ccccagatac ctgatcaccg tgaccggcaa gaaactgacc 60 gtgacctgca gccagaacat gaaccacgag tacatgagct ggtacagaca ggaccctggc 120 ctgggcctga gacagatcta ctacagcatg aacgtggaag tgaccgacaa gggcgacgtg 180 cccgagggct acaaggtgtc cagaaaagag aagcggaact tcccactgat cctggaaagc 240 ccatctccta accagaccag cctgtacttc tgcgccagca atgtgcaggg cgccaacaac 300 gagcagttct tcggccctgg caccagactg acagtgctg 339 <210> 105 <211> 342 <212> DNA <213> Artificial sequence <220> <223> DNA encoding the TCR01 variable region - TRA <400> 105 gacgccaaga ccacacagcc caacagcatg gaaagcaacg aagaggaacc cgtgcatctg 60 ccctgcaacc acagcacaat cagcggcacc gactacatcc actggtatag acagctgccc 120 tctcagggcc ccgagtatgt gattcacgga ctgaccagca acgtgaacaa ccggatggcc 180 tctctggcca ttgccgagga cagaaagagc agcaccctga tcctgcacag agccacactg 240 agagatgccg ccgtgtacta ctgcatcctt tgtggcgctg gcggcaccag ctatggcaag 300 ctgacatttg gccagggcac catcctgacc gtgcatccca ac 342 <210> 106 <211> 339 <212> DNA <213> Artificial sequence <220> <223> DNA encoding the TCR01 variable region - TRB <400> 106 gaagctcaag tgacacagaa ccccagatac ctgatcaccg tgaccggcaa gaaactgacc 60gaagctcaag tgacacagaa ccccagatac ctgatcaccg tgaccggcaa gaaactgacc 60 gtgacctgca gccagaacat gaaccacgag tacatgagct ggtacagaca ggaccctggc 120gtgacctgca gccagaacat gaaccacgag tacatgagct ggtacagaca ggaccctggc 120 ctgggcctga gacagatcta ctacagcatg aacgtggaag tgaccgacaa gggcgacgtg 180ctgggcctga gacagatcta ctacagcatg aacgtggaag tgaccgacaa gggcgacgtg 180 cccgagggct acaaggtgtc cagaaaagag aagcggaact tcccactgat cctggaaagc 240cccgagggct acaaggtgtc cagaaaagag aagcggaact tcccactgat cctggaaagc 240 ccatctccta accagaccag cctgtacttc tgcgccagcg ctattcaggg cgccaacaac 300ccatctccta accagaccag cctgtacttc tgcgccagcg ctattcaggg cgccaacaac 300 gagcagttct tcggccctgg caccagactg acagtgctg 339gagcagttct tcggccctgg caccagactg acagtgctg 339 <210> 107<210> 107 <211> 9<211> 9 <212> PRT<212> PRT <213> 人<213> Human <400> 107<400> 107 Trp Thr Leu Leu Val Asp Leu Leu TrpTrp Thr Leu Leu Val Asp Leu Leu Trp 1 5 1 5 <210> 108<210> 108<000175<210> 110 <211> 10 <212> PRT <213> Human <400> 110 Ile Ile Ala Leu Tyr Leu Gln Gln Asn Trp 1 5 10 <210> 111 <211> 10 <212> PRT <213> Human <400> 111 Trp Thr Leu Leu Val Asp Leu Leu Trp Leu 1 5 10 <210> 112 <211> 9 <212> PRT <213> Human <400> 112 Leu Ala Ile Leu Ile Trp Met Tyr Tyr 1 5 <210> 113 <211> 9 <212> PRT <213> Human <400> 113 Leu Leu Phe Leu Ala Ile Leu Ile Trp 1 5 <210> 114 <211> 10 <212> PRT <213> Human <400> 114 Leu Leu Leu Phe Leu Ala Ile Leu Ile Trp 1 5 10 <210> 115 <211> 10 <212> PRT <213> Human <400> 115 Leu Ala Ile Leu Ile Trp Met Tyr Tyr His 1 5 10 <210> 116 <211> 9 <212> PRT <213> Human <400> 116 Ala Leu Tyr Leu Gln Gln Asn Trp Trp 1 5 <210> 117 <211> 10 <212> PRT <213> Human <400> 117<e Phe Leu Ala Ile Leu Ile Trp Met Tyr Tyr 1 5 10 <210> 118 <211> 9[[ID=#35]] <212> PRT <213> Human <ee001830> Asn Ser Asn Glu Gly Arg His His Leu 1 5 <210> 119 <211> 497 <212> PRT <213> Human <220> <223> LMP#2A <400> 119 Met Gly Ser Leu Glu Met Val Pro Met Gly Ala Gly Pro Pro Ser Pro 1 5 10 15 Gly Gly Asp Pro Asp Gly Tyr Asp Gly Gly Asn Asn Ser Gln Tyr Pro 20 25 30<ecc001843> Ser Ala Ser Gly Ser Ser Gly Asn Thr Pro Thr Pro Pro Asn Asp Glu There seems to be an issue with the original text where some tags like and have been modified in the provided translation task. The modified tags are marked with "ee001830" and "ecc001843" respectively. It's not clear if this is intentional or an error in the description. If it's an error, the translation of those tags should be the same as the original ones. If it's intentional, the translation should follow the modified tags as shown above. 35 40 45 Glu Arg Glu Ser Asn Glu Glu Pro Pro Pro Pro Tyr Glu Asp Pro Tyr 50 55 60 Trp Gly Asn Gly Asp Arg His Ser Asp Tyr Gln Pro Leu Gly Thr Gln 65 70 75 80 Asp Gln Ser Leu Tyr Leu Gly Leu Gln His Asp Gly Asn Asp Gly Leu 85 90 95 Pro Pro Pro Pro Tyr Ser Pro Arg Asp Asp Ser Ser Gln His Ile Tyr 100 105 110 Glu Glu Ala Gly Arg Gly Ser Met Asn Pro Val Cys Leu Pro Val Ile 115 120 125 Val Ala Pro Tyr Leu Phe Trp Leu Ala Ala Ile Ala Ala Ser Cys Phe 130 135 140 Thr Ala Ser Val Ser Thr Val Val Thr Ala Thr Gly Leu Ala Leu Ser 145 150 155 160 Leu Leu Leu Ala Ala Val Ala Ser Ser Tyr Ala Ala Ala Gln Arg 165 170 175 Lys Leu Leu Thr Pro Val Thr Val Leu Thr Ala Val Val Thr Phe Phe 180 185 190 Ala Ile Cys Leu Thr Trp Arg Ile Glu Asp Pro Pro Phe Asn Ser Leu 195 200 205 Leu Phe Ala Leu Leu Ala Ala Ala Gly Gly Leu Gln Gly Ile Tyr Val 210 215 220 Leu Val Met Leu Val Leu Leu Ile Leu Ala Tyr Arg Arg Arg Trp Arg 225 230 235 240 Arg Leu Thr Val Cys Gly Gly Ile Met Phe Leu Ala Cys Val Leu Val 245 250 255 Leu Ile Val Asp Ala Val Leu Gln Leu Ser Pro Leu Leu Gly Ala Val 260 265 270 Thr Val Val Ser Met Thr Leu Leu Leu Leu Ala Phe Val Leu Trp Leu 275 280 285 Ser Ser Pro Gly Gly Leu Gly Thr Leu Gly Ala Ala Leu Leu Thr Leu 290 295 300 Ala Ala Ala Leu Ala Leu Leu Ala Ser Leu Ile Leu Gly Thr Leu Asn 305 310 315 320 Leu Thr Thr Met Phe Leu Leu Met Leu Leu Trp Thr Leu Val Val Leu 325 330 335 Leu Ile Cys Ser Ser Cys Ser Ser Cys Pro Leu Ser Lys Ile Leu Leu 340 345 350 Ala Arg Leu Phe Leu Tyr Ala Leu Ala Leu Leu Leu Leu Ala Ser Ala 355 360 365 Leu Ile Ala Gly Gly Ser Ile Leu Gln Thr Asn Phe Lys Ser Leu Ser 370 375 380 Ser Thr Glu Phe Ile Pro Asn Leu Phe Cys Met Leu Leu Leu Ile Val 385 390 395 400 Ala Gly Ile Leu Phe Ile Leu Ala Ile Leu Thr Glu Trp Gly Ser Gly 405 410 415 Asn Arg Thr Tyr Gly Pro Val Phe Met Cys Leu Gly Gly Leu Leu Thr 420 425 430 Met Val Ala Gly Ala Val Trp Leu Thr Val Met Ser Asn Thr Leu Leu 435 440 445 Ser Ala Trp Ile Leu Thr Ala Gly Phe Leu Ile Phe Leu Ile Gly Phe 450 455 460 Ala Leu Phe Gly Val Ile Arg Cys Cys Arg Tyr Cys Cys Tyr Tyr Cys 465 470 475 480 Leu Thr Leu Glu Ser Glu Glu Arg Pro Pro Thr Pro Tyr Arg Asn Thr 485 490 495 Val <210> 120 <211> 386 <212> PRT <213> Human <220> <223> LMP1 <400> 120 Met Glu His Asp Leu Glu Arg Gly Pro Pro Gly Pro Arg Arg Pro Pro 1 5 10 15 Arg Gly Pro Pro Leu Ser Ser Ser Leu Gly Leu Ala Leu Leu Leu Leu 20 25 30 Leu Leu Ala Leu Leu Phe Trp Leu Tyr Ile Val Met Ser Asp Trp Thr 35 40 45 Gly Gly Ala Leu Leu Val Leu Tyr Ser Phe Ala Leu Met Leu Ile Ile 50 55 60 Ile Ile Leu Ile Ile Phe Ile Phe Arg Arg Asp Leu Leu Cys Pro Leu 65 70 75 80 Gly Ala Leu Cys Ile Leu Leu Leu Met Ile Thr Leu Leu Leu Ile Ala 85 90 95 Leu Trp Asn Leu His Gly Gln Ala Leu Phe Leu Gly Ile Val Leu Phe 100 105 110 Ile Phe Gly Cys Leu Leu Val Leu Gly Ile Trp Ile Tyr Leu Leu Glu 115 120 125 Met Leu Trp Arg Leu Gly Ala Thr Ile Trp Gln Leu Leu Ala Phe Phe 130 135 140 Leu Ala Phe Phe Leu Asp Leu Ile Leu Leu Ile Ile Ala Leu Tyr Leu 145 150 155 160 Gln Gln Asn Trp Trp Thr Leu Leu Val Asp Leu Leu Trp Leu Leu Leu 165 170 175 Phe Leu Ala Ile Leu Ile Trp Met Tyr Tyr His Gly Gln Arg His Ser 180 185 190 Asp Glu His His His Asp Asp Ser Leu Pro His Pro Gln Gln Ala Thr 195 200 205 Asp Asp Ser Gly His Glu Ser Asp Ser Asn Ser Asn Glu Gly Arg His 210 215 220 His Leu Leu Val Ser Gly Ala Gly Asp Gly Pro Pro Leu Cys Ser Gln 225 230 235 240 Asn Leu Gly Ala Pro Gly Gly Gly Pro Asp Asn Gly Pro Gln Asp Pro 245 250 255 Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp 260 265 270 Asn Gly Pro His Asp Pro Leu Pro Gln Asp Pro Asp Asn Thr Asp Asp 275 280 285 Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro His Asp 290 295 300 Pro Leu Pro His Ser Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro 305 310 315 320 Pro Gln Leu Thr Glu Glu Val Glu Asn Lys Gly Gly Asp Gln Gly Pro 325 330 335 Pro Leu Met Thr Asp Gly Gly Gly Gly His Ser His Asp Ser Gly His 340 345 350 Gly Gly Gly Asp Pro His Leu Pro Thr Leu Leu Leu Gly Ser Ser Gly 355 360 365 Ser Gly Gly Asp Asp Asp Asp Pro His Gly Pro Val Gln Leu Ser Tyr 370 375 380 Tyr Asp 385 <210> 121 <211> 992 <212> PRT <213> Human <220> <223> EBNA3C <400> 121 Met Glu Ser Phe Glu Gly Gln Gly Asp Ser Arg Gln Ser Pro Asp Asn 1 5 10 15 Glu Arg Gly Asp Asn Val Gln Thr Thr Gly Glu His Asp Gln Asp Pro 20 25 30 Gly Pro Gly Pro Pro Ser Ser Gly Ala Ser Glu Arg Leu Val Pro Glu 35 40 45 Glu Ser Tyr Ser Arg Asp Gln Gln Pro Trp Gly Gln Ser Arg Gly Asp 50 55 60 Glu Asn Arg Gly Trp Met Gln Arg Ile Arg Arg Arg Arg Arg Arg Arg 65 70 75 80 Ala Ala Leu Ser Gly His Leu Leu Asp Thr Glu Asp Asn Val Pro Pro 85 90 95 Trp Leu Pro Pro His Asp Ile Thr Pro Tyr Thr Ala Arg Asn Ile Arg 100 105 110 Asp Ala Ala Cys Arg Ala Val Lys Gln Ser His Leu Gln Ala Leu Ser 115 120 125 Asn Leu Ile Leu Asp Ser Gly Leu Asp Thr Gln His Ile Leu Cys Phe 130 135 140 Val Met Ala Ala Arg Gln Arg Leu Gln Asp Ile Arg Arg Gly Pro Leu 145 150 155 160 Val Ala Glu Gly Gly Val Gly Trp Arg His Trp Leu Leu Thr Ser Pro 165 170 175 Ser Gln Ser Trp Pro Met Gly Tyr Arg Thr Ala Thr Leu Arg Thr Leu 180 185 190 Thr Pro Val Pro Asn Arg Val Gly Ala Asp Ser Ile Met Leu Thr Ala 195 200 205 Thr Phe Gly Cys Gln Asn Ala Ala Arg Thr Leu Asn Thr Phe Ser Ala 210 215 220 Thr Val Trp Thr Pro Pro His Ala Gly Pro Arg Glu Gln Glu Arg Tyr 225 230 235 240 Ala Arg Glu Ala Glu Val Arg Phe Leu Arg Gly Lys Trp Gln Arg Arg 245 250 255 Tyr Arg Arg Ile Tyr Asp Leu Ile Glu Leu Cys Gly Ser Leu His His 260 265 270 Ile Trp Gln Asn Leu Leu Gln Thr Glu Glu Asn Leu Leu Asp Phe Val 275 280 285 Arg Phe Met Gly Val Met Ser Ser Cys Asn Asn Pro Ala Val Asn Tyr 290 295 300 Trp Phe His Lys Thr Ile Gly Asn Phe Lys Pro Tyr Tyr Pro Trp Asn 305 310 315 320 Ala Pro Pro Asn Glu Asn Pro Tyr His Ala Arg Arg Gly Ile Lys Glu 325 330 335 His Val Ile Gln Asn Ala Phe Arg Lys Ala Gln Ile Gln Gly Leu Ser 340 345 350 Met Leu Ala Thr Gly Gly Glu Pro Arg Gly Asp Ala Thr Ser Glu Thr 355 360 365 Ser Ser Asp Glu Asp Thr Gly Arg Gln Gly Ser Asp Val Glu Leu Glu 370 375 380 Ser Ser Asp Asp Glu Leu Pro Tyr Ile Asp Pro Asn Met Glu Pro Val 385 390 395 400 Gln Gln Arg Pro Val Met Phe Val Ser Arg Val Pro Ala Lys Lys Pro 405 410 415 Arg Lys Leu Pro Trp Pro Thr Pro Lys Thr His Pro Val Lys Arg Thr 420 425 430 Asn Val Lys Thr Ser Asp Arg Ser Asp Lys Ala Glu Ala Gln Ser Thr 435 440 445 Pro Glu Arg Pro Gly Pro Ser Glu Gln Ser Ser Val Thr Val Glu Pro 450 455 460 Ala His Pro Thr Pro Val Glu Met Pro Met Val Ile Leu His Gln Pro 465 470 475 480 Pro Pro Val Pro Lys Pro Val Pro Val Lys Pro Thr Pro Pro Pro Ser 485 490 495 Arg Arg Arg Arg Gly Ala Cys Val Val Tyr Asp Asp Asp Val Ile Glu 500 505 510 Val Ile Asp Val Glu Thr Thr Glu Asp Ser Ser Ser Val Ser Gln Pro 515 520 525 Asn Lys Pro His Arg Lys His Gln Asp Gly Phe Gln Arg Ser Gly Arg 530 535 540 Arg Gln Lys Arg Ala Ala Pro Pro Thr Val Ser Pro Ser Asp Thr Gly 545 550 555 560 Pro Pro Ala Val Gly Pro Pro Ala Ala Gly Pro Pro Ala Ala Gly Pro 565 570 575 Pro Ala Ala Gly Pro Pro Ala Ala Gly Pro Pro Ala Ala Gly Pro Pro 580 585 590 Ala Ala Gly Pro Arg Ile Leu Ala Pro Leu Ser Ala Gly Pro Pro Ala 595 600 605 Ala Gly Pro His Ile Val Thr Pro Pro Ser Ala Arg Pro Arg Ile Met 610 615 620 Ala Pro Pro Val Val Arg Met Phe Met Arg Glu Arg Gln Leu Pro Gln 625 630 635 640 Ser Thr Gly Arg Lys Pro Gln Cys Phe Trp Glu Met Arg Ala Gly Arg 645 650 655 Glu Ile Thr Gln Met Gln Gln Glu Pro Ser Ser His Leu Gln Ser Ala 660 665 670 Thr Gln Pro Thr Thr Pro Arg Pro Ser Trp Ala Pro Ser Val Cys Ala 675 680 685 Leu Ser Val Met Asp Ala Gly Lys Ala Gln Pro Ile Glu Ser Ser His 690 695 700 Leu Ser Ser Met Ser Pro Thr Gln Pro Ile Ser His Glu Glu Gln Pro 705 710 715 720 Arg Tyr Glu Asp Pro Asp Ala Pro Leu Asp Leu Ser Leu His Pro Asp 725 730 735 Val Ala Ala Gln Pro Ala Pro Gln Ala Pro Tyr Gln Gly Tyr Gln Glu 740 745 750 Pro Pro Ala Pro Gln Ala Pro Tyr Gln Gly Tyr Gln Glu Pro Pro Pro 755 760 765 Pro Gln Ala Pro Tyr Gln Gly Tyr Gln Glu Pro Pro Ala His Gly Leu 770 775 780 Gln Ser Ser Ser Tyr Pro Gly Tyr Ala Gly Pro Trp Thr Pro Arg Ser 785 790 795 800 Gln His Pro Cys Tyr Arg His Pro Trp Ala Pro Trp Ser Gln Asp Pro 805 810 815 Val His Gly His Thr Gln Gly Pro Trp Asp Pro Arg Ala Pro His Leu 820 825 830 Pro Pro Gln Trp Asp Gly Ser Ala Gly His Gly Gln Asp Gln Val Ser 835 840 845 Gln Phe Pro His Leu Gln Ser Glu Thr Gly Pro Pro Arg Leu Gln Leu 850 855 860 Ser Leu Val Pro Leu Val Ser Ser Ser Ala Pro Ser Trp Ser Ser Pro 865 870 875 880 Gln Pro Arg Ala Pro Ile Arg Pro Ile Pro Thr Arg Phe Pro Pro Pro 885 890 895 Pro Met Pro Leu Gln Asp Ser Met Ala Val Gly Cys Asp Ser Ser Gly 900 905 910 Thr Ala Cys Pro Ser Met Pro Phe Ala Ser Asp Tyr Ser Gln Gly Ala 915 920 925 Phe Thr Pro Leu Asp Ile Asn Ala Thr Thr Pro Lys Arg Pro Arg Val 930 935 940 Glu Glu Ser Ser His Gly Pro Ala Arg Cys Ser Gln Ala Thr Ala Glu 945 950 955 960 Ala Gln Glu Ile Leu Ser Asp Asn Ser Glu Ile Ser Val Phe Pro Lys 965 970 975 Asp Ala Lys Gln Thr Asp Tyr Asp Ala Ser Thr Glu Ser Glu Leu Asp 980 985 990 <210> 122 <211> 14 <212> PRT <213> Person <400> 122 Ala Met Ser Asp Leu Tyr Ala Gly Asn Asn Arg Lys Leu Ile 1 5 10 <210> 123 <211> 11 <212> PRT <213> Person <400> 123 Ala Leu Thr Phe Leu Arg Asp Asp Lys Ile Ile 1 5 10 <210> 124 <211> 15 <212> PRT <213> Person ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <212> PRT <213> Person <400> 127 Gln Pro Arg Ala Pro Ile Arg Pro Ile 1 5 <210> 128 <211> 12 <212> PRT <213> Person <400> 128 Arg Pro Ile Pro Thr Arg Phe Pro Pro Pro Pro Met 1 5 10 <210> 129 <211> 12 <212> PRT <213> Person <400> 129 Arg Pro Arg Val Glu Glu Ser Ser His Gly Pro Ala 1 5 10 <210> 130 <211> 8 <212> PRT <213> Person <400> 130 Ser Pro Gln Pro Arg Ala Pro Ile 1 5 <210> 131 <211> 11 <212> PRT <213> Person <400> 131 Ser Pro Gln Pro Arg Ala Pro Ile Arg Pro Ile 1 5 10 <210> 132 <211> 12 <212> PRT <213> Person <400> 132 Ser Pro Gln Pro Arg Ala Pro Ile Arg Pro Ile Pro 1 5 10 <210> 133 <211> 10 <212> PRT <213> Human <400> 133 Pro Gln Pro Arg Ala Pro Ile Arg Pro Ile 1 5 10 <210> 134 <211> 10 <212> PRT <213> Human <400> 134 Gln Pro Arg Ala Pro Ile Arg Pro Ile Pro 1 5 10 <210> 135 <211> 8 <212> PRT <213> Human<9002186><400> 135 Pro Arg Ala Pro Ile Arg Pro Ile 1 5 <21() 136 <211> 10 <212> PRT <213> Human <400> 136 Ala Pro Ile Arg Pro Ile Pro Thr Arg Phe 1 5 10 <210> 137 <211> 8 <212> PRT <213> Human <400> 137 Phe Pro Pro Pro Pro Met Pro Leu 1 5 <210> 138 <211> 10 <212> PRT <213> Person <400> 138 His Gly Pro Ala Arg Cys Ser Gln Ala Thr 1 5 10 <210> 139 <211> 9 <212> PRT <213> Person <400> 139 Arg Pro Ile Pro Thr Arg Phe Pro Pro 1 5 <210> 140 <211> 8 <212> PRT <213> Person <400> 140 Arg Pro Ile Pro Thr Arg Phe Pro 1 5 <210> 141 <211> 11 <212> PRT <213> Person <400> 141 Ile Pro Thr Arg Phe Pro Pro Pro Pro Met Pro 1 5 10 <210> 142 <211> 11 <212> PRT <213> Person <400> 142 Pro Ile Pro Thr Arg Phe Pro Pro Pro Pro Met 1 5 10 <210> 143 <211> 12 <212> PRT <213> Person <400> 143 Ile Pro Thr Arg Phe Pro Pro Pro Pro Met Pro Leu 1 5 <210> 144 <211> 10 <212> PRT <213> Person <400> 144 Gly Pro Ala Arg Cys Ser Gln Ala Thr Ala 1 5 10 <210> 145 <211> 12 <212> PRT <213> Person <400> 145 Phe Pro Pro Pro Pro Met Pro Leu Gln Asp Ser Met 1 5 10 <210> 146 <211> 9 <212> PRT <213> Person <400> 146 Pro Pro Met Pro Leu Gln Asp Ser Met 1 5 <210> 147 <211> 10 <212> PRT <213> Person <400> 147 Arg Pro Ile Pro Thr Arg Phe Pro Pro Pro 1 5 10 <210> 148 <211> 10 <212> PRT <213> Person <400> 148 Met Pro Leu Gln Asp Ser Met Ala Val Gly 1 5 10 <210> 149 <211> 12 <212> PRT <213> Person <400> 149 Pro Ile Pro Thr Arg Phe Pro Pro Pro Pro Met Pro 1 5 10 <210> 150 <211> 10 <212> PRT <213> Human <400> 150 Pro Met Pro Leu Gln Asp Ser Met Ala Val 1 5 10 <210> 151 <211> 8 <212> PRT <213> Human <400> 151 Pro Met Pro Leu Gln Asp Ser Met 1 5 <210> 152 <211> 11 <212> PRT <213> Human <400> 152 Gln Pro Arg Ala Pro Ile Arg Pro Ile Pro Thr 1 5 10 <210> 153 <211> 8 <212> PRT <213> Human <400> 153 Gln Pro Arg Ala Pro Ile Arg Pro 1 5 <210> 154 <211> 9 <212> PRT <213> Human <400> 154 Arg Ala Pro Ile Arg Pro Ile Pro Thr 1 5 <210> 155 <211> 10 <212> PRT <213> Human <400> 155 Ser Glu Arg Leu Val Pro Glu Glu Ser Tyr 1 5 10 <210> 156 <211> 10 <212> PRT <213> Human <400> 156 Trp Leu Leu Thr Ser Pro Ser Gln Ser Trp 1 5 10 <210> 157 <211> 9 <212> PRT <213> Human <400> 157 Leu Leu Thr Ser Pro Ser Gln Ser Trp 1 5 <210> 158 <211> 9 <212> PRT <213> Human <400> 158 Arg Arg Tyr Arg Arg Ile Tyr Asp Leu 1 5 <210> 159 <211> 9 <212> PRT <213> Human <400> 159 Ala Arg Glu Ala Glu Val Arg Phe Leu 1 5 <210> 160 <211> 9 <212> PRT <213> Human <400> 160 Leu Arg Gly Lys Trp Gln Arg Arg Tyr 1 5 <210> 161 <211> 9 <212> PRT <213> human <400> 161 Glu Arg Tyr Ala Arg Glu Ala Glu Val 1 5 <210> 162 <211> 9 <212> PRT <213> human <400> 162 Ser Arg Arg Arg Arg Gly Ala Cys Val 1 5 <210> 163 <211> 9 <212> PRT <213> human <400> 163 Asn Leu Leu Asp Phe Val Arg Phe Met 1 5 <210> 164 <211> 9 <212> PRT <213> human <400> 164 Arg Arg Ile Tyr Asp Leu Ile Glu Leu 1 5 <210> 165 <211> 9 <212> PRT <213> human <400> 165 Arg Arg Arg Arg Gly Ala Cys Val Val 1 5 <210> 166 <211> 9 <212> PRT <213> human <400> 166 Val Arg Phe Leu Arg Gly Lys Trp Gln 1 5 <210> 167 <211> 9 <212> PRT <213> Person <400> 167 Arg Arg Arg Gly Ala Cys Val Val Tyr 1 5 <210> 168 <211> 9 <212> PRT <213> Person <400> 168 Gln Arg Arg Tyr Arg Arg Ile Tyr Asp 1 5 <210> 169 <211> 9 <212> PRT <213> Person <400> 169 Val Arg Phe Met Gly Val Met Ser Ser 1 5 <210> 170 <211> 10 <212> PRT <213> Person <400> 170 Tyr Ala Arg Glu Ala Glu Val Arg Phe Leu 1 5 10 <210> 171 <211> 9 <212> PRT <213> Person <400> 171 Asn Arg Val Gly Ala Asp Ser Ile Met 1 5 <210> 172 <211> 9 <212> PRT <213> Person <400> 172 Leu His His Ile Trp Gln Asn Leu Leu 1 5 <210> 173 <211> 9 <212> PRT <213> Human <400> 173 Arg Arg Gly Ile Lys Glu His Val Ile 1 5 <210> 174 <211> 9 <212> PRT <213> Human <400> 174 Tyr Arg Arg Ile Tyr Asp Leu Ile Glu 1 5 <210> 175 <211> 10 <212> PRT <213> Human <400> 175 Arg Arg Tyr Arg Arg Ile Tyr Asp Leu Ile 1 5 10 <210> 176 <211> 9 <212> PRT <213> Human <400> 176 Ala Arg Arg Gly Ile Lys Glu His Val 1 5 <210> 177 <211> 10 <212> PRT <213> Human <400> 177 Gln Arg Arg Tyr Arg Arg Ile Tyr Asp Leu 1 5 10 <210> 178 <211> 9 <212> PRT <213> Human It should be noted that there seems to be an incorrect tag "0002462" in the original which is likely a typo and I've translated it as " " for consistency. Also, "0002482" is likely a typo and translated as " ". And " " is likely incomplete in the original, but I've translated it as is. Please double-check the original text for accuracy. <400> 178 Trp Gln Arg Arg Tyr Arg Arg Ile Tyr 1 5 <210> 179 <211> 10 <212> PRT <213> Human <400> 179 Phe Leu Arg Gly Lys Trp Gln Arg Arg Tyr 1 5 10 <210> 180 <211> 9 <212> PRT <213> Human <400> 180 Arg Arg Gly Ala Cys Val Val Tyr Asp 1 5 <210> 181 <211> 9 <212> PRT <213> Human <400> 181 Val Tyr Asp Asp Asp Val Ile Glu Val 1 5 <210> 182 <211> 9 <212> PRT <213> Human <400> 182 Tyr Ala Arg Glu Ala Glu Val Arg Phe 1 5 <210> 183 <211> 9 [[ID=**67**]]<212> PRT (原文本中此处为<212> PRT,未发现错误,故保留) <213> Human <400> 183 Gly Cys Gln Asn Ala Ala Arg Thr Leu 1 5 <210> 184 <211> 9 <212> PRT <213> Human <400> 184 Asn Ser Asn Glu Gly Arg His His Leu 1 5 <210> 185 <211> 9 <212> PRT <213> Human <400> 185 Asp Ser Leu Pro His Pro Gln Gln Ala 1 5 <210> 186 <211> 9 <212> PRT <213> Human <400> 186 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <211> 10 <212> PRT <213> Person <400> 190 Leu Leu Ile Val Ala Gly Ile Leu Phe Ile 1 5 10 <210> 191 <211> 10 <212> PRT <213> Person <400> 191 Asn Leu Phe Cys Met Leu Leu Leu Ile Val 1 5 10 <210> 192 <211> 10 <212> PRT <213> Person <400> 192 Met Cys Leu Gly Gly Leu Leu Thr Met Val 1 5 10 <210> 193 <211> 9 <212> PRT <213> Person <400> 193 Leu Ile Val Ala Gly Ile Leu Phe Ile 1 5 <210> 194 <211> 9 <212> PRT <213> Person <400> 194 Phe Ile Pro Asn Leu Phe Cys Met Leu 1 5 <210> 195 <211> 9 <​​​​​Ile Val Ala Gly Ile Leu Phe Ile Leu 1 5 <210> 196 <211> 10 <212> PRT <213> Human <400> 196 Met Leu Leu Leu Ile Val Ala Gly Ile Leu 1 5 10 <210> 197 <211> 10 <212> PRT <213> Human <400> 197 Cys Met Leu Leu Leu Ile Val Ala Gly Ile 1 5 10 <210> 198 <211> 10 <212> PRT <213> Human <400> 198 Pro Asn Leu Phe Cys Met Leu Leu Leu Ile 1 5 10 <210> 199 <211> 10 <212> PRT <213> Human <400> 199 Phe Ile Pro Asn Leu Phe Cys Met Leu Leu 1 5 10

Claims

1. A nucleic acid encoding a TCR α-strand construct (TRA) and a TCR β-strand construct (TRB) specific to epitopes of human MHC I, wherein the epitopes are epitopes of ebogenile virus (EBV) proteins. a) wherein the epitope has a sequence as shown in SEQ ID NO:1, the MHC I is HLA-A*02:01, and the TRA comprises CDR1 consisting of the sequence of SEQ ID NO:11, CDR2 consisting of the sequence of SEQ ID NO:12, and CDR3 consisting of the sequence of SEQ ID NO:13, and the TRB comprises CDR1 consisting of the sequence of SEQ ID NO:16, CDR2 consisting of the sequence of SEQ ID NO:17, and CDR3 consisting of the sequence of SEQ ID NO:

18.

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

20.

3. The nucleic acid according to claim 1 or 2, wherein the TCR α chain construct and the TCR β chain construct further comprise a constant region, the constant region being selected from human constant regions, mouse constant regions, or chimeric constant regions.

4. A nucleic acid comprising any one of claims 1-3, and at least a second nucleic acid encoding a second TCR construct capable of specifically binding its corresponding epitope in a corresponding MHC I background. i. wherein the epitope binding to the second TCR construct has the sequence shown in SEQ ID NO:2, the MHC I is HLA-B*57:01, and the TRA comprises CDR1 consisting of SEQ ID NO:21, CDR2 consisting of SEQ ID NO:22, and CDR3 consisting of SEQ ID NO:23, and the TRB comprises CDR1 consisting of SEQ ID NO:26, CDR2 consisting of SEQ ID NO:27, and CDR3 consisting of SEQ ID NO:28; or ii. wherein the epitopes binding to the second TCR construct have the sequence shown in SEQ ID NO:3, the MHC I is HLA-C*15:02, the TRA comprises CDR1 consisting of SEQ ID NO:31, CDR2 consisting of SEQ ID NO:32, and CDR3 consisting of SEQ ID NO:33, and the TRB comprises CDR1 consisting of SEQ ID NO:36, CDR2 consisting of SEQ ID NO:37, and CDR3 consisting of SEQ ID NO:38; or iii. wherein the epitopes binding to the second TCR construct have the sequence shown in SEQ ID NO:4, the MHC I is HLA-C*06:02, the TRA comprises CDR1 consisting of SEQ ID NO:41, CDR2 consisting of SEQ ID NO:42, and CDR3 consisting of SEQ ID NO:43, and the TRB comprises CDR1 consisting of SEQ ID NO:46, CDR2 consisting of SEQ ID NO:47, and CDR3 consisting of SEQ ID NO:48; or iv. Wherein the epitopes binding to the second TCR construct have the sequence shown in SEQ ID NO:5, MHC I is HLA-B*44:02, TRA comprises CDR1 consisting of SEQ ID NO:51, CDR2 consisting of SEQ ID NO:52, and CDR3 consisting of SEQ ID NO:53, and TRB comprises CDR1 consisting of SEQ ID NO:56, CDR2 consisting of SEQ ID NO:57, and CDR3 consisting of SEQ ID NO:58; or v. wherein the epitopes of the second TCR construct have the sequence shown in SEQ ID NO:5, the MHC I is HLA-B*44:02, the TRA comprises CDR1 consisting of SEQ ID NO:61, CDR2 consisting of SEQ ID NO:62, and CDR3 consisting of SEQ ID NO:63, and the TRB comprises CDR1 consisting of SEQ ID NO:66, CDR2 consisting of SEQ ID NO:67, and CDR3 consisting of SEQ ID NO:68; or vi. Wherein the epitopes binding to the second TCR construct have the sequence shown in SEQ ID NO:6, MHC I is HLA-B*07:02, TRA comprises CDR1 consisting of SEQ ID NO:71, CDR2 consisting of SEQ ID NO:72, and CDR3 consisting of SEQ ID NO:73, and TRB comprises CDR1 consisting of SEQ ID NO:76, CDR2 consisting of SEQ ID NO:77, and CDR3 consisting of SEQ ID NO:78; or vii. wherein the epitope of the second TCR construct has the sequence shown in SEQ ID NO:7, the MHC I is HLA-B*07:02, and the TRA comprises CDR1 consisting of SEQ ID NO:81, CDR2 consisting of SEQ ID NO:82 and CDR3 consisting of SEQ ID NO:83, and the TRB comprises CDR1 consisting of SEQ ID NO:86, CDR2 consisting of SEQ ID NO:87 and CDR3 consisting of SEQ ID NO:

88.

5. A viral vector, transposon, vector suitable for CRISPR / CAS-based recombination, or plasmid suitable for in vitro RNA transcription, comprising the nucleic acid of any one of claims 1-4.

6. A protein encoded by any one of the nucleic acids of claims 1-4.

7. A host cell comprising the nucleic acid of any one of claims 1-4 and / or the protein of claim 6.

8. The host cell according to claim 7, wherein the host cell is a human CD8+ T cell.

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

10. A kit comprising the pharmaceutical composition of claim 9.

Images