Modified EBV-targeting enhanced immune cells and their medical uses

Abstract

The present invention relates to modified immune cells expressing T cell receptors (TCR) targeting Epstein-Barr virus (EBV) and their pharmaceutical uses. The present invention provides compositions comprising LMP2A specific TCR-T cell populations for the treatment of EBV-associated cancers and other indications, as well as methods for preparing and using the compositions. The present invention also provides nucleic acid sequences encoding the TCR and vectors thereof. The modified EBV-targeting immune cells of the present invention are activated upon binding tumor cells; they recognize targets and activate T-cell functions depending on presentation by major histocompatibility complex (MHC) molecules, while maintaining proliferation and antitumor activity. The EBV-targeting immune cells have demonstrated favorable antitumor efficacy in preclinical studies, offering a novel therapeutic strategy for patients with EBV-associated cancers.

Description

[0001] Modified EBV-Targeting Enhanced Immune Cells and Their Medical Uses

[0002] Technical Field

[0003] The present invention relates to the field of immunotherapy technology, and specifically to a modified immune cell and its medical uses.

[0004] Background

[0005] 1.1 Virology of Epstein-Barr Virus

[0006] Epstein-Barr virus (EBV) is a widely transmitted herpesvirus (human herpesvirus type 4), transmitted through close contact between susceptible individuals and asymptomatic EBV shedders. Most primary EBV infections worldwide are subclinical and inapparent. EBV antibodies are present in all populations and distributed worldwide, with 90%-95% of adults being EBV seropositive.

[0007] Like other herpesviruses, EBV has a latent phase. The host cells of EBV in humans are B lymphocytes, T lymphocytes, epithelial cells, and muscle cells. Unlike herpes simplex virus (HSV) or cytomegalovirus (CMV), EBV can transform B cells and generally does not cause cytopathic effects in cell culture.

[0008] EBV is the main pathogen of infectious mononucleosis. Its infection persists lifelong without symptoms in most adults, but can cause B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, and nasopharyngeal carcinoma (NPC) in some patients. Compared with other major herpesviruses, disease reactivation is not a prominent issue for EBV, but it is associated with aggressive lymphoproliferative disorders in transplant recipients.

[0009] EBV belongs to the Gamma Herpesviridae subfamily and is the prototype virus of the Lymphocryptoviral genus. In vitro, all gamma herpesviruses can replicate in lymphoid cells, and some can undergo lytic replication in epithelial cells and fibroblasts. Infection of primate B lymphocytes usually results in latent infection, characterized by the persistent presence of the viral genome and the expression of a limited set of latent gene products, which promote the transformation process and help drive cell proliferation.

[0010] B lymphocytes infected with EBV are characterized by the establishment of latent infection. Persistent EBV infection may result from the dynamic interaction between viral evasion i strategies and host immune responses. The 172Kbp EBV genome encodes approximately 100 genes, 10 of which are expressed during latency and involved in the establishment and maintenance of the "immortalized" state. These 10 genes include 6 nuclear proteins (EBNA1, 2, 3A, 3B, 3C, and LP), 2 latent membrane proteins (LMP-1 and LMP-2), and 2 EBV-encoded RNAs (EBER1 and EBER2).

[0011] Unlike lytic replication, latency does not result in the production of viral particles. Instead, the circular EBV genomic DNA exists as an episome in the nucleus and is replicated by cellular DNA polymerase. During latency, only a subset of EBV genes is expressed. Latent EBV expresses its genes in one of three patterns, referred to as latent transcription. EBV can latently infect B cells and epithelial cells, but different latent transcription patterns may exist in the two cell types.

[0012] EBV can exhibit one of three latency stages: latency I, latency II, or latency III. Each latency stage results in the production of a limited and unique set of viral proteins and viral RNAs.

[0013] In B cells, all three latency stages are possible. EBV latency in B cells typically progresses from latency III to latency II, and then to latency I. Each stage of latency uniquely affects B cell behavior. After infecting resting naive B cells, EBV enters latency III. The set of proteins and RNAs produced in latency III activates B cells. Later, the virus restricts its gene expression and enters latency II. The more restricted set of proteins and RNAs produced in latency II can induce B cell differentiation into memory B cells. Finally, EBV further restricts gene expression and enters latency I. When memory B cells divide, the expression of EBNA-1 allows the replication of the EBV genome.

[0014] In epithelial cells, only latent transcription II occurs.

[0015] EBNA-1 is required for the replication of episomal genes and the maintenance of the viral genome after cellular immortalization. EBNA-1 tyrosine 518 (Y518) forms DNA-protein crosslinks, promoting the termination of EBV OriP replication and the maintenance of viral episomes. In vivo experiments have shown that dormant (i.e., non-dividing) B cells infected with EBV express only EBNA-1. EBNA-1 is the only EBV protein expressed by all EBV-associated malignancies and can bind to specific palindromic DNA sequences on chromosome 11, leading to breaks and genomic instability. Studies have shown that EBNA-1 acts on deaminases and purine metabolism during B cell immortalization. LMP-2 is an integral membrane protein that, together with LMP-1, is present in the plasma membrane of EBV-infected lymphocytes. Among EBV proteins associated with transformation in NPC and EBV-associated malignancies, EBNA-1, LMP-1, and LMP-2 are most frequently co-expressed. Since both LMP-1 and LMP-2 contain T cell epitopes, their persistent expression in vivo suggests an important role in the persistence of EBV in the human host. The function of LMP-2 is to serve as a substrate for B lymphocyte src family tyrosine kinases and is associated with a 70kDa tyrosine-phosphorylated cellular protein. Considering the important role of tyrosine kinases in transmembrane signaling mediated by growth factor receptors, the association between LMP-2 and tyrosine kinases reflects the significant role of LMP-2 in influencing cell growth.

[0016] It is currently believed that latently EBV-infected B cells have oncogenic potential, as they can proliferate indefinitely in vitro. In patients with congenital or acquired immunodeficiency, these cells can also cause lymphoproliferative disorders, including lymphoma.

[0017] 1.2 Epidemiology and Etiology of Nasopharyngeal Carcinoma

[0018] Nasopharyngeal carcinoma is the main type of tumor occurring in the nasopharynx (the tubular passage connecting the back of the nasal cavity to the oropharynx below). The epidemiology, histology, natural course, and response to treatment of nasopharyngeal carcinoma differ from other head and neck squamous cell carcinomas.

[0019] In 2020, there were more than 133,000 new cases of nasopharyngeal carcinoma and 80,000 deaths from nasopharyngeal carcinoma worldwide. Nasopharyngeal carcinoma has ethnic and geographical distribution differences, reflecting its multifactorial etiology.

[0020] Geographical and ethnic distribution: The incidence of nasopharyngeal carcinoma shows significant geographical variation: it is rare in the United States and Western Europe, with an incidence of 0.5-2 cases per 100,000; it is more common in southern China (including Hong Kong), with an incidence that can reach 25 cases per 100,000 per year; moderate-risk areas include Southeast Asia, North Africa, the Middle East, and the Arctic region. Populations migrating from high-risk areas to low-risk areas still have a higher risk of disease, but it usually decreases after several generations. Gender and age distribution: The incidence of nasopharyngeal carcinoma in men is 2-3 times that in women.

[0021] The geographical distribution differences in the incidence of nasopharyngeal carcinoma indicate that its etiology is multifactorial. In endemic populations, the risk appears to result from the interaction of several factors: EBV (Epstein-Barr virus) infection (the main pathogenic factor), environmental factors (such as heavy intake of pickled foods and smoking), and genetic susceptibility. In addition, the increased incidence in younger adults in high-risk and moderate-risk areas suggests that early-life exposure to common pathogenic substances is a key factor. In the United States and Europe, nasopharyngeal carcinoma is more often associated with alcohol consumption and smoking, which are also typical risk factors for other head and neck tumors.

[0022] 1.3 EBV-Positive Gastric Cancer

[0023] EBV-associated gastric cancer (EBVaGC) has unique clinicopathological and molecular characteristics, such as high DNA methylation, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit a (PIK3CA) gene mutations, and high programmed death ligand 1 (PD-L1) expression. According to research reports, EBVaGC patients account for approximately 8.7% of gastric cancer patients, but the incidence of EBVaGC varies by region. Immunotherapy or targeted therapy for EBVaGC patients based on EBV and its related carcinogenic mechanisms can benefit patients.

[0024] EBV latent membrane protein 2A (LMP2A) is a transmembrane protein expressed by EBV during latent infection and is involved in the development of EBV-associated tumors. According to research reports, LMP2A can be detected in the tumor tissues of 53.8% of EBVaGC patients. After EBV infects gastric mucosal cells in the body, LMP2A can phosphorylate signal transducer and activator of transcription 3 (STAT3), activate the transcription of DNA methyltransferase 1 (DNMT1), induce extensive methylation of host genes, affect the cell cycle and microenvironment of host cells, thereby leading to gastric cancer. In addition, LMP2A can also cooperate with certain miRNAs to inhibit the demethylation of methyl cytosine dioxygenase 2 (TET2), leading to an imbalance in cellular methylation / demethylation, thereby promoting the development of EBVaGC. LMP2A can also inhibit the expression of aquaporin 3 (AQP3) and participate in the proliferation and migration of tumor cells through the mTOR / AQP3 / extracellular regulated protein kinase (ERK) / 4E binding protein 1 (4E-BP1) signaling pathway. PD-L1 high expression is also an important factor promoting the occurrence and development of EBVaGC. Cytokines produced by CD8+ T cells accumulated in the tumor microenvironment of EBVaGC can lead to increased PD-L1 expression, and EBV miRNAs can also mediate PD-L1 transcription in tumor cells, allowing them to escape immune surveillance. Although EBVaGC has no specific clinical manifestations compared with EBV-negative gastric cancer, it has unique pathological features. EBVaGC patients are mostly male, with a median age of 66.5 years. Compared with patients with the other three gastric cancer subtypes, EBVaGC patients have a lower lymph node metastasis rate and a better prognosis. EBVaGC is often located in the proximal stomach or residual stomach, mostly forming ulcerative masses, which can have obvious gastric wall thickening. Pathologically, it is moderately to poorly differentiated adenocarcinoma with a large number of lymphocyte infiltrates, most of which are CD8+ T cells. Under low magnification, infiltrating lymphocytes and irregular glandular structures form a lace-like or reticular structure, which is a characteristic pathological manifestation of EBVaGC. However, some EBVaGCs present as moderately differentiated adenocarcinomas without lymphocyte infiltration, which are easily missed, so EBER in situ hybridization is required for a clear diagnosis.

[0025] 1.4 Role of T Cells in Immunotherapy of EBV-Associated Tumors and Basic Principles of TCR-T Cell Use

[0026] T cells are immune cells derived from bone marrow and lymphoid tissues and matured in the thymus. They express T cell receptors (TCRs) on their surface and play an important role in eliminating infections and cancer cells in cell-mediated immunity. TCRs can recognize and specifically bind to target antigen epitopes presented by major histocompatibility complex (MHC) molecules. Once T cells recognize their targets, they can kill target cells through massive proliferation, release of cytokines, and cytotoxicity.

[0027] Regarding the application scope of TCR-T therapy, people are currently generally attempting to use adoptive T cell immunotherapy to treat human malignant tumors (such as leukemia) and viral diseases (such as hepatitis B virus (HBV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV)). However, isolating and expanding virus- or tumor-specific T cells from patients' blood is very difficult and time-consuming. Therefore, researchers have adopted a new therapeutic strategy: by introducing TCRs or TCR a / p heterodimers targeting specific antigens, these gene-edited T lymphocytes act on specific viral or tumor antigens, endowing T cells with clear antigen specificity. TCR-T has high clinical application value and significance for solid tumors, especially cervical cancer associated with HBV infection.

[0028] TCR-T cell therapy relies on the presentation of MHC molecules to recognize targets and activate T cell functions and can recognize intracellular antigen fragments presented by MHC molecules. This feature determines that TCR-T cell therapy has a wider range of targets, including intracellular antigens, cell surface antigens, and neoantigens produced after tumor cell mutations, which can overcome the differences in antigen expression of malignant tumor cells caused by tumor heterogeneity, reflecting higher therapeutic value.

[0029] Summary of the Invention

[0030] In view of the current technical problems, the present invention provides a modified immune cell, which is a cell therapy product specifically targeting EBV tumor-specific antigens. Structurally, its T cell receptor exhibits MHC restriction (specifically for HLA-A11:O1), which is capable of specifically recognizing EBV LMP2 antigen polypeptide presented by HLA-A11:O1 and then produces cytokines such as interferon-y (IFN-y) and tumor necrosis factor-a (TNF-a) and exert cytolytic effects on tumor cells. By targeting the expression of unique antigens infected by EBV to kill tumor cells and simultaneously clear viral infections, it has potential therapeutic effects on the diseases associated with EBV infection, which comprise post-transplant lymphoproliferative disorders caused by EBV and various malignant tumors, preferably, nasopharyngeal carcinoma, NKT cell lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, and gastric cancer.

[0031] The first aspect of the present invention provides a T cell receptor specifically targets the EBV LMP2A antigen.

[0032] In one embodiment, the EBV TCR comprises a TCR a-chain variable region and a TCR P-chain variable region; wherein the amino acid sequence of aCDR3 of the TCR a-chain variable region is set forth in SEQ ID NO: 17, or a variant thereof in which one or two amino acids are substituted with other amino acids; and the amino acid sequence of CDR3 of the TCR P-chain variable region is set forth in SEQ ID NO: 10, or a variant thereof in which one or two amino acids are substituted with other amino acids. In one embodiment, the EBV TCR comprises a TCR a-chain variable region and a TCR P-chain variable region; wherein the amino acid sequence of aCDR3 of the TCR a-chain variable region is set forth in SEQ ID NO:3, or a variant thereof in which one or two amino acids are substituted with other amino acids; and the amino acid sequence of CDR3 of the TCR P-chain variable region is set forth in SEQ ID NO:6, or a variant thereof in which one or two amino acids are substituted with other amino acids.

[0033] In one embodiment, the EBV TCR comprises a TCR a-chain variable region and a TCR P-chain variable region; wherein the amino acid sequence of aCDR3 of the TCR a-chain variable region is set forth in SEQ ID NO: 13, or a variant thereof in which one or two amino acids are substituted with other amino acids; and the amino acid sequence of PCDR3 of the TCR P-chain variable region is set forth in SEQ ID NO: 14, or a variant thereof in which one or two amino acids are substituted with other amino acids.

[0034] In some embodiments, the TCR a-chain variable regions comprise complementarity determining regions aCDRl, aCDR2, and aCDR3, and the TCR P-chain variable regions comprise complementarity determining regions PCDR1, PCDR2, and PCDR3.

[0035] Preferably, aCDRl, aCDR2, and aCDR3 are set forth in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 respectively; PCDR1, PCDR2, and PCDR3 are set forth in SEQ ID NO: 18, SEQ ID NO:7, and SEQ ID NO: 10 respectively; or comprise the above CDR variants in which one or two amino acids in one or more CDRs are substituted with other amino acids.

[0036] Preferably, aCDRl, aCDR2, and aCDR3 are set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively; PCDR1, PCDR2, and PCDR3 are set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO: 6 respectively; or comprise the above CDR variants in which one or two amino acids in one or more CDRs are substituted with other amino acids.

[0037] Preferably, aCDRl, aCDR2, and aCDR3 are set forth in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO:13 respectively; PCDR1, PCDR2, and PCDR3 are set forth in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 14 respectively; or comprise the above CDR variants in which one or two amino acids in one or more CDRs are substituted with other amino acids.

[0038] In some embodiments, the TCR a-chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:23, SEQ ID NO: 19, or SEQ ID NO:21; in some embodiments, the TCR P-chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:24, SEQ ID NO:20, or SEQ ID NO:22.

[0039] Preferably, the TCR a-chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:23; the TCR P-chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:24.

[0040] Preferably, the TCR a-chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19; the TCR P-chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:20.

[0041] Preferably, the TCR a-chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:21; the TCR P-chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:22.

[0042] In some embodiments, the TCR a-chain comprises the amino acid sequence of a human T cell receptor constant region; in some embodiments, the TCR P-chain comprises the amino acid sequence of a human T cell receptor constant region; or comprises variants of the above constant regions in which one or more amino acids are substituted with other amino acids.

[0043] In some embodiments, the TCR a-chain comprises the amino acid sequence of a murine T cell receptor constant region; in some embodiments, the TCR P-chain comprises the amino acid sequence of a murine T cell receptor constant region; or comprises variants of the above constant regions in which one or more amino acids are substituted with other amino acids.

[0044] Preferably, the amino acid sequence of the murine constant region of the TCR a-chain is set forth in SEQ ID NO:26; the amino acid sequence of the murine constant region of the TCR P-chain is set forth in SEQ ID NO:27.

[0045] In some embodiments, the TCR or fragment thereof is a single-chain TCR fragment, and the single-chain TCR fragment links the TCR a-chain and TCR P-chain via a self-cleaving P2A polypeptide.

[0046] Preferably, the amino acid sequence of the self-cleaving P2A polypeptide is set forth in SEQ ID NO:25.

[0047] In some embodiments, the TCR or fragment thereof has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:32, SEQ ID NO:28, or SEQ ID NO:30. Preferably, the TCR or fragment thereof is the amino acid sequence of TCR101 set forth in SEQ ID NO:32.

[0048] Preferably, the TCR or fragment thereof is the amino acid sequence of TCR073 set forth in SEQ ID NO:28.

[0049] Preferably, the TCR or fragment thereof is the amino acid sequence of TCR092 set forth in SEQ ID NO:30.

[0050] In some embodiments, a conjugate is bound to the C-terminus or N-terminus of the TCR a-chain and / or the TCR P-chain, and the conjugate comprises a detectable label, a therapeutic agent, a PK-modifying moiety, or a combination of any of these substances.

[0051] In some embodiments, the TCR or fragment is capable of binding to an EBV LMP2A antigen polypeptide presented by HLA-A*11.

[0052] Preferably, the EBV LMP2A antigen polypeptide is SSCSSCPLSK set forth in SEQ ID NO:34. Preferably, the EBV LMP2A antigen polypeptide is SSCSSCPLTK set forth in SEQ ID NO:35. The second aspect of the present invention provides a nucleic acid molecule, which comprises a nucleic acid sequence encoding the TCR or fragment thereof according to the first aspect of the present invention.

[0053] In a preferred embodiment, the nucleic acid molecule encodes the amino acid sequence of TCR101 set forth in SEQ ID NO:32.

[0054] Preferably, the nucleic acid molecule comprises the nucleotide sequence of TCR101 set forth in SEQ ID NO:33.

[0055] In a preferred embodiment, the nucleic acid molecule encodes the amino acid sequence of TCR073 set forth in SEQ ID NO:28.

[0056] Preferably, the nucleic acid molecule comprises the nucleotide sequence of TCR073 set forth in SEQ ID NO:29.

[0057] In a more preferred embodiment, the nucleic acid molecule encodes the amino acid sequence of TCR092 set forth in SEQ ID NO: 30.

[0058] Preferably, the nucleic acid molecule comprises the nucleotide sequence of TCR092 set forth in SEQ ID NO:31.

[0059] The third aspect of the present invention provides a vector, which comprises the nucleic acid molecule according to the second aspect of the present invention. The vector is selected from any one of plasmids, binary vectors, DNA vectors, mRNA vectors, retroviral vectors, lentiviral vectors, transposon-based vectors, and artificial chromosomes.

[0060] The fourth aspect of the present invention provides a host cell, which comprises the TCR or fragment thereof according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, or the vector according to the third aspect of the present invention.

[0061] The fifth aspect of the present invention provides an immune cell, which comprises the TCR or fragment thereof according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, or the vector according to the third aspect of the present invention.

[0062] In some embodiments, the immune cell is selected from lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells; in some preferred embodiments, the immune cell is a T cell.

[0063] The sixth aspect of the present invention provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR or fragment thereof according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, the host cell according to the fourth aspect of the present invention, or the immune cell according to the fifth aspect of the present invention.

[0064] The seventh aspect of the present invention provides use of the TCR or fragment thereof according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, the host cell according to the fourth aspect of the present invention, the immune cell according to the fifth aspect of the present invention, or the pharmaceutical composition according to the sixth aspect of the present invention in the preparation of a medicament for preventing or treating diseases caused by EBV infection; the diseases associated with EBV infection comprise post-transplant lymphoproliferative disorders caused by EBV and various malignant tumors, preferably, wherein the diseases associated with EBV infection comprise at least one selected from nasopharyngeal carcinoma, NKT cell lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, and gastric cancer.

[0065] Beneficial Effects of the Application

[0066] The present invention provides a novel TCR or fragment thereof specifically targeting the EBV LMP2A antigen. It is surprisingly found that the TCR of the present invention has extremely strong affinity for the LMP2A-HLA-A* 11 complex and can simultaneously target and recognize LMP2A or LMP2A mutant peptides (the S9T mutation has a high proportion in many nasopharyngeal carcinoma patients). Moreover, the proportion of the HLA-A11 subtype is relatively high in Asian regions such as China. Therefore, the TCR of this application has a wide range of applicable populations.

[0067] In addition, the TCR-T cells of the present invention can specifically recognize EBV LMP2 viral peptides presented by HLA-A* 11:01, and then produce cytokines, and exert cytolytic effects on tumor cells. By targeting the expression of unique antigens infected by EBV to kill tumor cells and simultaneously clear viral infections, it has potential therapeutic effects on the diseases associated with EBV infection comprise post-transplant lymphoproliferative disorders caused by EBV and various malignant tumors, nasopharyngeal carcinoma, NKT cell lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, and gastric cancer.

[0068] Anti-tumor therapy targeting EBV LMP2 can overcome the protective effect of the tumor barrier on solid malignant tumor tissues. EBV LMP2A is a foreign antigen and is not expressed in normal tissues. The CRS and ICANS reactions of TCR-T drugs are mild and well-tolerated, and the main adverse reactions are mostly caused by the drug attacking normal tissue cells expressing the corresponding target outside the tumor. Therefore, EBV-targeting enhanced immune cells have more excellent safety characteristics.

[0069] The unique TCR technology screens out characteristic high-affinity TCRs, and the present invention modifies the TCR constant region to reduce the risk of mismatching with endogenous TCR chains.

[0070] Brief Description of the Drawings

[0071] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings needed in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained according to these drawings without creative work.

[0072] Figure 1: Structure of EBV TCR sequence;

[0073] Figure 2: Production process of EBV TCR-T;

[0074] Figure 3: Detection of EBV TCR-T cell subset expression;

[0075] Figure 4: Functional activity of EBV TCR-T cell subsets against LMP2A or LMP2A-S9T peptides;

[0076] Figure 5: In vitro killing function of EBV TCR-T cell subsets against PANC1-LMP2A target cells;

[0077] Figure 6: In vitro cytokine function of EBV TCR-T cell subsets against PANC1-LMP2A target cells;

[0078] Figure 7: Identification of key amino acids recognized by EBV TCR-T cells using alanine scanning library technology;

[0079] Figure 8: Cross-reactivity of EBV TCR-T cells with epitopes in human peptide libraries;

[0080] Figure 9: Flow cytometry detection of expression and mismatch rate of EBV TCR-T cells and sorted CD4 subsets;

[0081] Figure 10: Function of CD4+ EBV TCR-T cells against PANC1-LMP2A-B95.8 tumor cells;

[0082] Figure 11: Function of CD4+ EBV TCR-T cells against PANC1-LMP2A-GD1(S9T) tumor cells;

[0083] Figure 12: Repeat effector function of EBV TCR-T cells against gastric cancer AGS-A11-LMP2A tumor cells;

[0084] Figure 13: Protocol of EBV TCR-T in immunodeficient mouse PANC1-LMP2A xenograft model;

[0085] Figure 14: Efficacy experiment of EBV TCR-T in immunodeficient mouse PANC1-LMP2A xenografts;

[0086] Figure 15: In vivo expansion and cytokines of EBV TCR-T in immunodeficient mouse PANC1-LMP2A xenografts.

[0087] Detailed Implementation Methods

[0088] To make this invention easier to understand, some technical and scientific terms are explained before describing the examples. Unless clearly defined otherwise elsewhere in the application documents of this invention, all other technical and scientific terms used in this invention have the meanings commonly understood by those of ordinary skill in the art to which this invention belongs.

[0089] The term “T cell receptor (TCR)” is defined herein as a TCR that binds to an EBV surface antigen in the context of a major histocompatibility complex (MHC) molecule, thereby inducing a helper or cytotoxic response in cells expressing the recombinant TCR. The TCR or fragment thereof of the present invention can recognize the corresponding peptide segment of EBV LMP2A that matches HLA-A*ll:01, regardless of whether the complete LMP2A antigen is expressed.

[0090] The term “MHC molecule” refers to a protein of the immunoglobulin superfamily, which can be a class I or class II MHC molecule. Therefore, it has specificity for antigen presentation; different individuals have different MHCs, which can present different short peptides from a protein antigen on the surface of their respective APC cells. The human MHC is usually called the HLA gene or HLA complex.

[0091] TCR is a glycoprotein on the cell membrane surface existing as a heterodimer of a / p chains or y / 8 chains. In 95% of T cells, the TCR heterodimer is composed of a and chains, while 5% of T cells have TCRs composed of y and 5 chains. The natural aP heterodimeric TCR has a and P chains, which constitute the subunits of the aP heterodimeric TCR. Each of the a and P chains contains a variable region and a constant region; each variable region contains 3 CDRs (complementarity determining regions) embedded in framework regions: CDR1, CDR2, and CDR3. The CDR regions of the a and P chains of the TCR in the present invention are defined using the IMGT numbering rule. The CDR regions determine the binding of TCR to the pMHC complex. The sequences of TCR constant domains can be found in the public database of the International ImMunoGeneTics Information System (IMGT).

[0092] In the present invention, the terms “T cell receptor”, “TCR”, and “TCR molecule” are used interchangeably.

[0093] Nucleic Acid Molecules

[0094] The nucleic acid molecule of the present invention comprises a nucleic acid sequence encoding an EBV TCR molecule; and / or comprises a nucleic acid sequence encoding a costimulatory molecule.

[0095] The present invention provides a nucleic acid molecule encoding the aforementioned TCR molecule or fragment thereof, where the fragment can be one or more CDRs, variable region s of a and / or P chains, and a and / or P chains.

[0096] In some embodiments, the nucleic acid encodes one or more structural features for increasing and / or stabilizing the association between the expressed TCR a and P chains. In some embodiments, the feature can be a specific amino acid or amino acid sequence. In some embodiments, the nucleic acid can encode one or more non-natural cysteine residues for forming one or more disulfide bonds between the TCR a and P chains. In some embodiments, the nucleic acid can encode one or more non-natural cysteine residues in the constant region s of the TCR a and P chains.

[0097] The nucleotide sequence of the nucleic acid molecule of the present invention can be single-stranded or double-stranded; the nucleic acid molecule can be RNA or DNA, which may or may not comprise introns. Preferably, the nucleic acid molecule of the present invention does not comprise introns but is capable of encode the TCR of the present invention.

[0098] The nucleotide sequence can be codon-optimized. Different cells use specific codons differently; the codons in the sequence can be changed according to the cell type to increase expression levels. Codon usage tables for mammalian cells and various other organisms are well known to those skilled in the art.

[0099] In some embodiments, the coding sequence of the present invention is a single chain, linking the TCR P-chain coding sequence and the TCR a-chain coding sequence via a P2A coding sequence; and the single-chain coding nucleotide is in the same reading frame.

[0100] It should be understood that in gene cloning operations, it is often necessary to design appropriate restriction enzyme sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed amino acid sequence, but this does not affect the activity of the target sequence. To construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other appropriate regions of the recombinant protein, including but not limited to suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. Therefore, the amino terminus or carboxyl terminus of the fusion protein of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used herein. For example, the tags can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-Tagll, AU1, EE, T7, 4A6, 8, B, gE, and Tyl. These tags can be used for protein purification.

[0101] Vectors

[0102] The present invention also relates to vectors containing the nucleic acid molecule sequences described herein and one or more regulatory sequences operably linked to these sequences. The nucleic acid molecules of the present invention can be manipulated in various ways to ensure the expression of the fusion protein. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0103] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is usually operably linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutated, truncated, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, a non-translated region of mRNA important for translation in the host cell. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator functional in the selected host cell can be used in the present invention.

[0104] The nucleic acid molecules of the present invention can be cloned into many types of vectors. For example, they can be cloned into plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Further, the vector is an expression vector. Expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology handbooks. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0105] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO 01 / 96584 and US 6,326,193).

[0106] For example, in some embodiments, the present invention uses a lentiviral vector, which comprises an origin of replication, 3' LTR, 5' LTR, the polynucleotide sequence described herein, and optionally a selectable marker.

[0107] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation factor-la (EF-la). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Further, inducible promoters can also be considered. The use of inducible promoters provides a molecular switch that can turn on the expression of the polynucleotide sequence operably linked to the inducible promoter when expression is desired and turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0108] To evaluate the expression of the target gene, the expression vector introduced into the cell can also comprise either or both of a selectable marker gene or a reporter gene, which facilitates the identification and selection of expressing cells from a population of cells that are intended to be transfected or infected via the viral vector. In other aspects, the selectable marker can be carried on a separate piece of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0109] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Expression of the reporter gene is measured at an appropriate time after the DNA has been introduced into the recipient cell. Suitable reporter genes comprise genes encoding luciferase, P-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.

[0110] Methods for introducing genes into cells and expressing genes in cells are known in the art. Vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0111] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Biological methods for introducing interesting polynucleotides into host cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0112] Biological methods for introducing polynucleotides into host cells include the use of viral vectors, especially lentiviral vectors, which have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses, etc. Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used. Immune Cells

[0113] The immune cells of the present invention are selected from lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, and mast cells.

[0114] In some embodiments, the immune cell is a lymphocyte; in some embodiments, the immune cell is a NK cell; in some embodiments, the immune cell is a B cell; in some embodiments, the immune cell is a TIL cell. In some embodiments, the immune cell is a T cell, which can be derived from T cells isolated from a subject, or can be part of a mixed cell population isolated from a subject, such as a peripheral blood lymphocyte (PBL) population. For example, the cell can be isolated from peripheral blood mononuclear cells (PBMCs) and can be a CD4+ helper T cell or a CD8+ cytotoxic T cell. The cell can be in a mixed population of CD4+ helper T cells / CD8+ cytotoxic T cells. Generally, the cell can be activated with an antibody (e.g., anti-CD3 antibody) to make them more receptive to transfection.

[0115] The modified immune cell of the present invention is an immune cell comprising the aforementioned TCR receptor molecule and the aforementioned immune costimulatory molecule; in some embodiments, the modified immune cell is constructed by introducing a coding sequence encoding the aforementioned TCR receptor molecule and the aforementioned immune costimulatory molecule or a vector comprising the aforementioned coding sequence into isolated immune cells; in some embodiments, the modified immune cell is constructed by introducing a coding sequence encoding the aforementioned TCR receptor molecule and the aforementioned immune costimulatory molecule or a vector comprising the aforementioned coding sequence into immune cells in vivo; in some embodiments, the coding sequences of the TCR receptor molecule and the immune costimulatory molecule are expressed in tandem and are in the same reading frame.

[0116] Compositions

[0117] The present invention also provides compositions comprising the modified immune cells, nucleic acids, or vectors of the present invention. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is suitable for use in research, treatment, prevention, and / or diagnosis.

[0118] In some embodiments, the modified immune cells, nucleic acids, or vectors of the present invention are preferably formulated into a medicament or drug together with one or more other pharmaceutically acceptable ingredients known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants, masking agents, colorants, flavoring agents, and sweeteners. The term "pharmaceutically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for contact with the tissues of the subject in question (e.g., humans) without excessive toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Each carrier, adjuvant, excipient, etc., must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, adjuvants, excipients, etc., can be found in standard pharmaceutical textbooks, such as “Remington's Pharmaceutical Sciences”; and “Handbook of Pharmaceutical Excipients”.

[0119] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease.

[0120] When referring to an “immunologically effective amount”, “anti-tumor effective amount”, “tumor-inhibiting effective amount”, or “therapeutic amount”, the precise amount of the composition of the present invention to be administered can be determined by a physician, considering individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can generally be stated that pharmaceutical compositions including T cells described herein can be administered at a dose of 104to 109cells / kg body weight, preferably 105to 107cells / kg body weight. T cell compositions can also be administered multiple times at these doses. Cells can be administered to patients by injection techniques known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by those skilled in the medical field by monitoring signs of the patient's disease and adjusting treatment accordingly.

[0121] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection, or intraperitoneally to a patient.

[0122] Medical Uses

[0123] In another aspect, use of the modified immune cells, nucleic acids, vectors, or pharmaceutical compositions of the present invention in the preparation of a medicament for treating or preventing a disease or condition is provided. In some embodiments, the modified immune cells, nucleic acids, vectors, or pharmaceutical compositions of the present invention can be used for preventing or treating the diseases associated with EBV infection comprise post-transplant lymphoproliferative disorders caused by EBV and various malignant tumors, preferably, the diseases associated with EBV infection comprise at least one selected from nasopharyngeal carcinoma, NKT cell lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, and gastric cancer.

[0124] Methods of Treatment and Prevention

[0125] Treatment can be performed by isolating T cells from a patient or volunteer with the diseases associated with EBV infection, introducing the nucleic acid molecule or vector of the present invention into the aforementioned T cells, and then infusing these genetically engineered cells back into the patient. Therefore, the present invention provides a method for treating the diseases associated with EBV infections, comprising infusing isolated T cells expressing the TCR of the present invention, preferably T cells derived from the patient themselves, into the patient. Generally, it includes (1) isolating the patient's T cells, (2) transducing the T cells in vitro with the nucleic acid molecule or vector of the present invention, (3) infusing the genetically engineered T cells into the patient. The number of cells isolated, transfected, and infused can be determined by a physician.

[0126] In some embodiments of the present invention, the modified immune cells, nucleic acids, vectors, or pharmaceutical compositions of the present invention can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiotherapy, immunosuppressants, and viral inhibitors. For example, treatment can be combined with nucleoside analogs or interferons known in the art for treating the diseases associated with EBV infection.

[0127] “Patient”, “subject”, “individual”, etc., are used interchangeably herein to refer to a living organism, such as a mammal, that can mount an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

[0128] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific examples. Obviously, the examples described are only part of the examples of the present invention, not all of them. Based on the examples in the present invention, all other examples obtained by those skilled in the art without creative work belong to the protection scope of the present invention. It should be noted that the description order of the following examples is not intended to limit the preferred order of the examples.

[0129] The experimental methods without specific conditions in the following examples usually follow conventional conditions such as those in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition. 2001), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise specified, the reagents and materials involved in the text are commercially available or can be prepared by those skilled in the art according to common knowledge. Any methods and materials similar or equivalent to those described can be used in the present invention. The preferred implementation methods and materials in the text are only for demonstration but cannot limit the content of the present invention.

[0130] Example 1: Design of EBV TCR Sequences and Construction of Vectors

[0131] The structures of EBV TCR-T were shown in Figure 1. The coding sequence of the EBV TCR fragment was fully gene-synthesized, and the synthesized gene was inserted into a lentiviral vector. The EBV TCR-T is comprised of an EBV LMP2A T cell receptor P-chain variable region (LMP2A TCR VP), a T cell receptor P-chain constant region (TCR CP), a 2A self-cleaving peptide, an EBV LMP2A T cell receptor a-chain variable region (LMP2A TCR Va), and a T cell receptor a-chain constant region (TCR Ca), wherein the TCR constant region was modified to reduce endogenous TCR mismatch. The five TCR clones, TCR073, TCR074, TCR092, TCR101, and TCR150, all had high TCR affinity and did not require further modification (e.g., affinity maturation). The CDR1, CDR2 and CDR3 amino acid sequences from TCR a variable region and

[0132] TCR 0 variable region corresponding to TCR073 / TCR092 / TCR101 were shown in Table 1.

[0133] Table 1. Amino acid sequences of CDRs, TCR a variable regions, and TCR 0 variable regions of TCR073 /

[0134] TCR092 / TCR101

[0135] Example 2: Production Process of EBV TCR-T

[0136] Preparation of T cell complete medium: CTS™ OpTmizer™ Supplement (Gibco, A379040-01), 5% CTS™ Immune Cell SR (Gibco, A25961-01) , 2% Glutamax (Gibco, A12860-01) and 400 lU / mL IL-2 for injection (Shandong Quangang Pharmaceutical Co., Ltd., 08-102) were mixed by inversion and stored in a 4°C refrigerator for later use.

[0137] Preparation of T cell cryopreservation solution: 75%CS10 (ThermoFisher, A2596101) + 25%HSA (FLEXBUMIN, S20181007).

[0138] The production process was shown in Figure 2:

[0139] Day 0: CD3+ T cells were isolated from apheresis blood, the cell concentration was adjusted to lxlO6 / mL with T cell complete medium. T cells were activated using Transact (CD3 / CD28 microspheres, Macs, 6201000014) with the volume ratio of Transact to T cells =1:30, and infected with the lentivirus vectors after 24 hours of stimulation and culture.

[0140] Day 1: After 24 hours of activation, T cells were counted, adjusted the cell density to 5xlO5 / mL, and added the EBV TCR lentivirus.

[0141] Day 2—11: After cell infection, the cell state was observed daily, and T cell complete medium was added to maintain the density of T cells at 5xlO5 / mL for cell expansion.

[0142] Day 12: The cells were harvested by centrifugation at 300g for 5 minutes and then washed with a physiological saline solution containing 5% human serum albumin. Subsequently, the cells were frozen in T cell cryopreservation solution at an appropriate density and finally stored in liquid nitrogen.

[0143] Example 3: Detection of EBV TCR-T Cell Subset Expression

[0144] Preparation of flow cytometry buffer: DPBS (Gibco, 14190250) and 2% FBS (Gibco, 10099141) were mixed and stored in a 4°C refrigerator for later use.

[0145] EBV TCR-T cells and Mock T cells (control group) were washed once with flow cytometry buffer, and the supernatant was discarded. Subsequently, PE HLA-A* 11:01 LMP-2A Tetramer (MBL, TS-M111-1), APC Hamster Anti-mTCRp (BD, 553174), PE-Cy7 anti-human CD4 (BIOLEGEND, 300512), and PerCP / Cy5.5 anti-human CD8a (BIOLEGEND, 301032) were added. The cells were incubated at 4°C for 60 minutes in the dark, then washed again with flow cytometry buffer, resuspended, and finally detected with a flow cytometer (Beckman CytoFLEX). The results showed that the positive rates of EBV TCR-T cells TCR-073, TCR-074, TCR-092, TCR-101, and TCR- 150 were above 60%, and the proportions of CD4+ and CD8+ T cells were basically maintained at around 50%, showing no difference compared with Mock T cells (Figure 3). This indicated that EBV TCR-T can be stably expressed, and the exogenous TCR sequence had no significant impact on the proportions of CD4+ and CD8+ T cells.

[0146] Example 4: Functional Activity of EBV TCR-T Cell Subsets Against LMP2A or LMP2A-S9T Peptides

[0147] In this experiment, the Miltenyi magnetic bead sorting system was used to sort EBV TCR-T cells to obtain different subset cells. When different subset cells were co-cultured with K562-A11 cells loaded with peptides, the secretion of IFN-y was detected to determine the function of EBV TCR-T cell subsets against LMP2A or LMP2A-S9T peptides. Preparation of RIO cell complete medium: RPMI 1640 (Gibco, 22400-089) and 10% FBS (Gibco, 10099141) were mixed by inversion and stored in a 4°C refrigerator for later use.

[0148] Preparation of T cell medium: CTSTM OpTmizer™ , Supplement (Gibco, A379040-01), 5% CTS™ Immune Cell SR (Gibco, A25961-01) and 2% Glutamax (Gibco, A12860-01) were mixed by inversion and stored in a 4°C refrigerator for later use.

[0149] Peptide preparation: 2 mg of LMP2A or LMP2A-S9T peptide was dissolved in DMSO to a final concentration to 10 mM and diluted to 1010M in a 10-fold gradient.

[0150] Preparation of the target cells: K562-A11 cells with good growth status after several generations were centrifuged at 500g for 5 min, discard the supernatant and resuspend the cells in complete R10 cell culture medium. After counting, K562-A11 cells of 2.4 x 106were resuspended in 12ml TCM culture medium. Then, a 96-well U-plate was used, and K562-A11 cells were added at lOOpl / well and 2 x 104 / well. Subsequently, LMP2A or LMP2A-S9T peptide solution was added according to the concentration gradient, and the final concentration was adjusted to 10’5M~ 10"11M; Finally, the effector cells were added to a 96-well U-plate, which was placed in a 37°C incubator for 2 hours of incubation.

[0151] Cell sorting: EBV TCR-T cells were tested for cell viability and density using NC250. According to the counting results, IxlO7cells were centrifuged and the supernatant was discarded. The cells were then resuspended in 80ul of eluent. Add 20ul CD8 sorting magnetic beads to the cells, followed by co-incubation at 4°C for 15 minutes. A Miltenyi-specific LM sorting column was rinsed once with 5 ml of elution buffer and then mounted on the magnetic stand for later use. Then, the co-incubated EBV TCR-T cells were taken out, resuspended in 3ml of eluent, and loaded onto the sorting column. Subsequently, the sorting column was eluted three times, and the eluent was collected and labeled as CD4+cells; After the elution was completed, the magnetic frame was removed, and the sorting column was eluted with 5ml of eluent. The eluent is collected and labeled as CD8+cells; both groups of cells were centrifuged and resuspended in 2ml complete T cell culture medium, and NC250 was used to detect cell viability and density.

[0152] Preparation of Effector Cells: 3.6x10sEBV TCR-T cells were resuspended in 4.5 ml T cell medium, and the cell density was adjusted to 4xl05cells / ml. The effector cells were added at 50pl / well, and then placed in a 37°C incubator for co-incubation for 24 hours. The co-culture supernatant was collected to detect cytokine secretion by flow cytometry.

[0153] The results showed that EBV TCR-T CD8+ cells secreted strong cytokines in response to K562-A11 cells loaded with LMP2A and LMP2A-S9T peptides, and the intensity of cytokine secretion was positively correlated with the loading concentration of peptides (Figures 4A, B); EBV TCR-T CD4+ cells also had a certain degree of cytokine secretion in response to K562-A11 cells loaded with LMP2A and LMP2A-S9T peptides, which was positively correlated with the loading concentration of peptides (Figure 4C).

[0154] Example 5: In vitro Killing and Cytokine Secretion of EBV TCR-T Cell Subsets Against Target Cells

[0155] The real-time cell analysis (RTCA) technology was used to evaluate the killing effect of EBV TCR-T on target cells. We cultured human pancreatic cancer cells PANC1-LMP2A (LMP2+) as target cells in a 96-well RTCA plate. After the target cells were incubated for approximately 16 hours, the sorted EBV TCR-T CD4+, CD8+subsets, and Mock T cells were added respectively for co-culture. Subsequently, continuous observation was conducted for 48 hours, and the survival curves of the target cells were plotted into continuous graphs for comparison. After the killing process was completed, the co-culture supernatants were collected for cytokine detection.

[0156] Killing of tumor cells by EBV TCR-T CD4+ and CD8+ subset cells (RTCA)

[0157] Day 0: Human pancreatic cancer PANCI -LMP2A cells in good growth status were adjusted to a density of 4xl05cells / ml to serve as target cells for later use. Take a 96-well RTCA plate, 50pl / well of the corresponding medium was added for instrument baseline measurement, and 50ul of target cells with a density of 4xl05 / ml were added to each well. After standing for approximately 5 minutes, the 96-well plate was placed on the instrument, and the growth curve was continuously detected for about 16 hours.

[0158] Day 1: Take EBV TCR-T CD4+, CD8+subsets and Mock T cells with pre-determined positive rates and cell viability, calculate the number of effector cells based on the positive rates. The cells were then added at 50pl / well at effector-to-target ratios of 5:1 and 1:1, and the killing curves were continuously detected by the instrument.

[0159] Day 3: The growth and killing curves of RTCA were collected, and the co-culture supernatant was collected for cytokine detection.

[0160] Take Human Thl / Th2 Cytokine Cytometric Bead Array Kit II (BD, 551809) and equilibrate it to room temperature.

[0161] Aspirate 2 ml of Assay Diluent to reconstitute the standard to a concentration of 5000pg / ml and equilibrated at room temperature for 30 minutes.

[0162] Standard preparation: The standard labeled as SI is serially diluted 2-fold to obtain S2 to S9, and the blank control is labeled as S10.

[0163] Preparation of Human Thl / Th2 Cytokine Capture Beads mixture: Microsphere solutions A1-A6 were mixed in equal proportions.

[0164] Take a 96-well U plate, add 50pl Human Thl / Th2 Cytokine Capture Beads mixture to each sample well.

[0165] Add 50pl of test samples, standard curve samples (S1-S10).

[0166] Add 50pl of Human Thl / Th2 PE Detection Reagent to each sample well.

[0167] Then, the 96-well U-bottom plate was incubated at room temperature for 180 minutes in the dark. Subsequently, lOOpl of Wash buffer was added, followed by centrifugation at 300g for 5 minutes, and the supernatant was discarded. The pellets were resuspended with lOOpl of Wash buffer, the samples were loaded for flow cytometry detection, and the flow cytometry results were analyzed using FCAP Array v3 software.

[0168] The results showed that both EBV TCR-T CD4+ and CD8+ subset cells had significant specific killing effects on PANCI -LMP2A cells, and the killing speed was positively correlated with the effector-target ratio. After 48 hours of co-culture, the killing rate of EBV TCR-T subset cells on positive target cells in the E:T=1:1 group was nearly 100% (Figures 5A, B); the secretion levels of IFN-y and TNF in EBV TCR-T subset cells in the effector-target ratio 5:1 group were significantly higher than those in the Mock T group (Figures 6A, B); at the same effector-target ratio, the half -killing time of the EBV TCR-T CD8+ subset was significantly shorter than that of the CD4+ subset, and the concentration of released cytokines was positively correlated with the killing speed. The results indicated that both EBV TCR-T CD4+ and CD8+ subset cells had strong anti-tumor effects on target cells expressing LMP2A+. Example 6: Identification of Key Amino Acids Recognized by EBV TCR-T Cells Using Alanine Scanning Library Technology

[0169] This study aims to analyze the impact of each amino acid residue in the LMP2A 340-349 epitope peptide on TCR affinity and activity by individually mutating each amino acid residue to alanine (A) using alanine scanning library technology, thereby identifying the recognition sites of EBV TCR-T. K562-A11 cells were HLA-A*ll:01-positive human chronic myeloid leukemia cells. K562-A11 cells loaded with mutated peptides were incubated with EBV TCR-T in a 37°C incubator, and the secretion of cytokine IFN-y in the supernatant was detected after 24 hours.

[0170] The functional activity of EBV TCR-T binding to the EBV LMP2A epitope peptide-MHC complex in vitro could be evaluated by studying the ability of EBV TCR-T cells to specifically recognize K562-A11 cells loaded with EBV LMP2A epitope peptides at different concentration gradients and secrete cytokines.

[0171] Preparation of peptide: 2mg of polypeptide was dissolved in 190pl of DMSO to obtain a concentration of lOmM and then diluted to InM with TCM. Specific information was shown on the table below.

[0172] Preparation of the target cells: K562-A11 cells with good growth status after several generations were centrifuged at 500g / 5 min, discard the supernatant and resuspend the cells in complete RIO cell culture medium. After counting, K562-A11 cells of 2.4 x 106were resuspended in 12ml TCM culture medium. Take a 96-well U-plate and add K562-A11 cells at lOOpl / well and 2 x 104 / well. Subsequently, add the corresponding peptide solution according to the concentration gradient, adjust the final concentration to 10’5M~1012M. Finally, the effector cells were added to a 96-well U-plate, which was placed in a 37 °C incubator for 2 hours of incubation.

[0173] Preparation of Effector Cells: EBV TCR-T cells of 3.6x10swas resuspended in 6 ml T cell medium, and the cell density was adjusted to 4xl05cells / ml. The effector cells were added at 50pl / well, and then placed in a 37°C incubator for co-incubation for 24 hours. The co-culture supernatant was collected to detect cytokine secretion by flow cytometry.

[0174] After K562-Al 1 cells were loaded with EBV LMP2A and mutated epitope peptides at concentrations ranging from 106M to 108M and co-incubated with EBV TCR-T, the secretion of IFN-y was positively correlated with the concentration of the loaded EBV LMP2A epitope peptide, while Mock T could not recognize the EBV LMP2A epitope peptide loaded by K562-A11 cells. The results showed that cytokine secretion was significantly reduced when TCR092, TCR101, and TCR150 were incubated with EBV LMP2A peptides with amino acid mutations at positions 3 / 6 / 8 / 10, 3 / 6 / 7 / 8 / 10, and 3 / 6 / 7 / 10, respectively (Figure 7A-C), which proved that the recognition sites at positions 3 / 6 / 8 / 10 for TCR092, 3 / 6 / 7 / 8 / 10 for TCR101, and 3 / 6 / 7 / 10 for TCR150 were key amino acids. These key amino acid residues were believed to play critical roles in the direct binding of TCR to the EBV LMP2A epitope peptide.

[0175] The results indicated that EBV TCR-T can recognize the key binding sites of the EBV LMP2A epitope peptide and had strong binding functional activity.

[0176] Example 7: Cross-Reactivity of EBV TCR-T with Human Peptide Library

[0177] The human TCR repertoire exhibited inherent cross-reactivity, which 108unique TCRs were capable of recognizing more than 1015potential peptides. In clinical trials, TCR cross-reactivity can lead to unexpected targeting of healthy human tissues, causing severe toxicity. The recognition sites at positions 3 / 6 / 8 / 10 for TCR092, 3 / 6 / 7 / 8 / 10 for TCR101, and 3 / 6 / 7 / 10 for TCR150 were key amino acids. These key amino acid residues were believed to play crucial roles in the direct binding of TCR to the EBV LMP2A epitope peptide or in controlling the spatial structure of the EBV LMP2A epitope peptide. Using Expitope 2.0 (http: / / webclu.bio.wzw.tum.de / expitope2 / ) screening for self-antigen peptides that meet the criteria: at least six amino acids were identical to the LMP2A 340-349 peptide (SSCSSCPLSK (SEQ ID NO: 34), and a total of 27 candidate peptides were identified. In the experiment, K562-A11 cells loaded with the above peptides were co-incubated with EBV TCR-T, and the IFN-y level in the supernatant was detected to study whether there was cross-reactivity between EBV TCR-T and these peptides, which provided support for further non-clinical safety studies.

[0178] The preparation of peptide: 2mg of the peptides (No. 1-29) were dissolved with 150ul-200ul DMSO respectively to a final concentration of lOmM. Specific information was shown on the table below.

[0179] Preparation of the target cells: K562-A11 cells with good growth status after several generations were centrifuged at 500g for 5 min, discard the supernatant and resuspend the cells in complete RIO cell culture medium. Take a 96-well U-plate and add K562-A11 cells at lOOpl / well and 2 x 104 / well. Subsequently, 11 pl of the polypeptide solutions (No.l~29) at a concentration of lOOpM was added to each well, and the concentration was adjusted to lOuM; the 96-well U-plate was placed in a 37°C incubator for 2 hours, then add the effector cells.

[0180] Preparation of the effector cells: EBV TCR-T cells were resuspended in T cell medium, counted and the cell density was adjusted to 4xl05cells / ml. The effector cells were added at 50pl / well, and then placed in a 37 °C incubator for co-incubation for 24 hours. The co-culture supernatant was collected to detect cytokine secretion by flow cytometry.

[0181] The results showed that under the condition of lOpM, EBV TCR-T significantly secreted the cytokine IFN-y in response to K562-A11 cells loaded with LMP2A 340-349 peptide, but no cytokine secretion was detected in response to 27 experimental group peptides and negative control groups (HPV E6 , Vehicle) , which indicated that EBV TCR-T had no cross-reactivity with peptides that had the highest consistency (6 identical amino acids) with LMP2A 340-349 (Figure 8 A-D), suggesting that EBV TCR-T had a low potential off target toxicity risk against endogenous antigen peptides in the human body.

[0182] Example 8: TCR Expression and Mismatch Rate of EBV TCR-T Subsets

[0183] EBV TCR-T cells and Mock T cells (control group) were stained with PE HLA-A* 11:01

[0184] LMP-2A Tetramer (MBL, TS-M111-1) to detect specific TCR in the EBV TCR-T cell product; APC Hamster Anti-mTCRP (BD, 553174) was used to detect all TCR chain expressions in the EBV TCR-T cell product; BV421 Anti-human CD8 (BD, 743064) was used for the detection of EBV TCR-T subset cells. The EBV TCR-T subset cells were incubated at 4°C in the dark for 60 minutes, then washed with flow cytometry buffer, resuspended, and finally detected using a flow cytometer (Beckman CytoFLEX).

[0185] EBV TCR-T mismatch rate = [1 - (LMP2A tetramer+ / mTCRP+)] x 100%

[0186] The results showed that the positive rate of TCR in EBV TCR-T was approximately 80%, and the positive rate of EBV TCR-T CD4+cells was also approximately 85% (Figure 9); the ratio of CD8+to CD4+in EBV TCR-T was similar; meanwhile, the mismatch rate of EBV TCR-T was lower than 5%.

[0187] In summary, EBV TCR-T was also expressed normally in the CD4+subset and had reduced the mismatch rate and potential safety risks through structural optimization.

[0188] Example 9: Function of CD4+EBV TCR-T Cells against PANC1-LMP2A-B95.8 Tumor Cells

[0189] The real-time cell analysis (RTCA) technology was used to evaluate the killing effect of EBV TCR-T on target cells. Human pancreatic cancer cell line PANC1-LMP2A-B95.8 was cultured as target cell in a 96-well RTCA plate. After the target cells were incubated for approximately 16 hours, the sorted EBV TCR-T CD4+and Mock T cells were added respectively for co-culture. Continuous observation was conducted for 48 hours, and the survival curves of the target cells were plotted into continuous graphs for comparison. After the killing process was completed, the co-culture supernatants were collected for cytokine detection

[0190] The results showed that EBV TCR-T CD4+cells had a significant specific killing effect on PANC1-LMP2A-B95.8 cells. After 48 hours of co-culture, the killing rate of EBV TCR-T CD4+cells on positive target cells reached 80% (Figure 10 A); the IFN-y secretion levels in the TCR101 and TCR092 groups were significantly higher than those in the TCR073 group, while almost no cytokine secretion was detected in the Mock-T group (Figure 10 B). The results indicated that EBV TCR-T CD4+cells had a strong anti-tumor effect against PANC1-LMP2A-B95.8 target cells. Example 10: Function of CD4+EBV TCR-T Cells against PANC1-LMP2A-GD1(S9T)

[0191] Tumor Cells

[0192] The real-time cell analysis (RTCA) technology was used to evaluate the killing effect of EBV TCR-T on target cells. Human pancreatic cancer cell line expressing the LMP2A mutant peptide (PANC1-LMP2A-GD1(S9T)) were cultured in 96-well RTCA plates as target cells. After incubation for approximately 16 hours, EBV TCR-T CD4+cells and Mock T were added respectively for co-culture. Continuous observation was conducted for 48 hours, and the survival curves of target cells were plotted into continuous graphs for comparison. After the killing was completed, the co-culture supernatant was collected for cytokine detection.

[0193] The results showed that EBV TCR-T CD4+cells had a significant specific killing effect on PANCI -LMP2A-GD1(S9T) cells. After 48 hours of co-culture, the killing rate of TCR101 cells on positive target cells reached 100%, and the killing rates of the TCR092 group and TCR073 group on positive target cells also exceeded 50% (Figure 11 A); significant IFN-y secretion was detected in the TCR101 group (Figure 11 B). The results indicated that EBV TCR-T CD4+cells also had a strong anti-tumor effect on target cells expressing the LMP2A mutant peptide PANC 1 -LMP2A-GD 1 (S9T).

[0194] Example 11: Rechallenge Efficacy of EBV TCR-T Cells on Gastric Cancer AGS-A11-LMP2A Tumor Cells

[0195] The real-time cell analysis (RTCA) technology was used to evaluate the killing effect of EBV TCR-T on target cells. Human gastric cancer cell line AGS-A11-LMP2A was cultured as target cells in a 96-well RTCA plate. After the target cells were incubated for approximately 16 hours, the sorted EBV TCR-T and Mock T cells were added respectively for co-culture. Continuous observation was conducted for approximately 72 hours. After 3 days of killing, T cells were collected and then added to the RTCA plate pre-seeded with target cells for 24 hours to perform multiple rounds of killing. The survival curves of the target cells were plotted into continuous graphs for comparison; meanwhile, the co-culture supernatant after re-stimulation was collected for cytokine detection. The results showed that EBV TCR-T cells had obvious anti-tumor effects on gastric cancer AGS-A11-LMP2A cells in both rounds of coculture. Most tumor cells were killed in the first round, and tumor proliferation was still inhibited in the second round (Figure 12A); in the second round of killing, significant IFN-y secretion was detected (Figure 12B). The results indicated that EBV TCR-T can maintain a long-term anti-tumor effect after multiple rounds of tumor stimulation.

[0196] Example 12: Function of EBV TCR-T Cells in NPG Immunodeficient Mice Bearing PANC1-LMP2A Cells

[0197] To investigate the killing, expansion, and persistence of EBV TCR-T on PANCI -LMP2A tumor cells in vivo, 48 female NPG immunodeficient mice were used in the experiment. On Dayl7, PANCI -LMP2A cells were subcutaneously inoculated into mice. The mice were randomly divided into 8 groups, including 6 groups of TCR073, TCR092, TCR101 with low and high doses, and UT group and Vehicle group were set as controls. On Day 0, EBV TCR-T was injected via tail vein once at doses of 3x10sand IxlO7positive T cells / mouse; the negative control group was injected with IxlO7UT cells / mouse; meanwhile, Vehicle group mice were injected with the same amount of normal saline; the mice were observed for 4 weeks after administration. The body weight of animals and tumor length and short diameter of tumors were measured twice a week to calculate the tumor volume. Peripheral blood of the animals was collected weekly to detect the expansion and persistence of EBV TCR-T cells, and plasma was collected by centrifugation to detect cytokine secretion levels (Figure 13).

[0198] Note: “N / A” means not applicable.

[0199] The results showed that there was no significant difference in body weight between the low and high dose groups of EBV TCR-T (Figure 14 A, C) compared with the Vehicle control group and the UT control group. On Day 28, the tumor volumes in the low and high dose groups of EBV TCR-T were less than 100 mm3, and the tumor volume inhibition rates in the low and high dose groups of EBV TCR-T TCR073, TCR092, and TCR101 all exceeded 90% (Figure 14 B, D). It indicated that the low and high dose groups of EBV TCR-T could significantly inhibit tumor cell proliferation (P < 0.05).

[0200] On Day7~14 after administration, the expansion of EBV TCR-T in the peripheral blood of animals reached a peak and gradually decreased over time (Figure 15 A). The cytokine level reached a peak on Day 2-7 and then decreased rapidly (Figure 15 B).

[0201] Under experimental conditions, intravenous administration of EBV TCR-T (0.3xl07~1.0xl07TCR-T cells / animal) significantly inhibited the growth of PANCI -LMP2A tumor cells. Meanwhile, significant expansion of TCR-T and cytokine secretion were observed in vivo, even at the minimum effective dose of 0.3xl07TCR-T cells / animal.

[0202] The above is a detailed introduction to the present invention. Specific examples are used herein to explain the principles and implementations of the present invention. The descriptions of the above examples are only used to help understand the method and core idea of the present invention; meanwhile, for those skilled in the art, according to the idea of the present invention, there will be changes in the specific implementations and application scopes. In conclusion, the content of this specification should not be understood as a limitation to the present invention.

Claims

Claims1. AT cell receptor (TCR) or fragment thereof, comprising a TCR a-chain variable region and a TCR P-chain variable region; wherein: the amino acid sequence of aCDR3 of the TCR a-chain variable region is set forth in SEQ ID NO: 17, and the amino acid sequence of CDR3 of the TCR P-chain variable region is set forth in SEQ ID NO: 10; or the amino acid sequence of aCDR3 of the TCR a-chain variable region is set forth in SEQ ID NO: 3, and the amino acid sequence of PCDR3 of the TCR P-chain variable region is set forth in SEQ ID NO:6; or the amino acid sequence of aCDR3 of the TCR a-chain variable region is set forth in SEQ ID NO: 13, and the amino acid sequence of PCDR3 of the TCR P-chain variable region is set forth in SEQ ID NO: 14.

2. The TCR or fragment thereof according to claim 1, wherein the TCR a-chain variable regions comprise complementarity determining regions aCDRl, aCDR2, and aCDR3, and the TCR P-chain variable region comprises complementarity determining regions PCDR1, PCDR2, and PCDR3, wherein: aCDRl, aCDR2, and aCDR3 are set forth in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 respectively, PCDR1, PCDR2, and PCDR3 are set forth in SEQ ID NO: 18, SEQ ID NO:7, and SEQ ID NO: 10 respectively; or aCDRl, aCDR2, and aCDR3 are set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively, PCDR1, PCDR2, and PCDR3 are set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO: 6 respectively; or aCDRl, aCDR2, and aCDR3 are set forth in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO: 13 respectively, and PCDR1, PCDR2, and PCDR3 are set forth in SEQ ID NO: 8, SEQ ID NO:9, and SEQ ID NO: 14 respectively.

3. The TCR or fragment thereof according to claim 1, wherein the amino acid sequence of the TCR a-chain variable region is set forth in SEQ ID NO:23, SEQ ID NO: 19, or SEQ ID NO:21, or the TCR a-chain variable region comprises the amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 23, SEQ ID NO: 19 or SEQ ID NO: 21; and / orthe amino acid sequence of the TCR P-chain variable region is set forth in SEQ ID NO:24, SEQ ID NO:20, or SEQ ID NO:22, or the TCR P-chain variable region comprises the amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 24, SEQ ID NO: 20 and SEQ ID NO: 22.

4. The TCR or fragment thereof according to claim 1, wherein the TCR a-chain and TCR P-chain each comprise a constant region, and the constant region is selected from human T cell receptor constant regions or murine T cell receptor constant regions; preferably, the amino acid sequence of the TCR a-chain constant region is set forth in SEQ ID NO:26, and the amino acid sequence of the TCR P-chain constant region is set forth in SEQ ID NO:27.

5. The TCR or fragment thereof according to any one of claims 1-4, wherein the TCR or fragment is a single-chain fragment; the single-chain TCR fragment links the TCR a-chain and TCR P-chain via a self-cleaving P2A polypeptide; preferably, the amino acid sequence of the self-cleaving P2A polypeptide is set forth in SEQ ID NO:25.

6. The TCR or fragment thereof according to any one of claims 1-5, wherein the amino acid sequence of the TCR or fragment thereof is selected from any one of the following: preferably, the amino acid sequence is set forth in SEQ ID NO:32 or the amino acid sequence has at least 90% identical to SEQ ID NO:32; preferably, the amino acid sequence is a set forth in SEQ ID NO:28 or the amino acid sequence has at least 90% identical to SEQ ID NO:28; preferably, the amino acid sequence is set forth in SEQ ID NO:30 or the amino acid sequence has at least 90% identical to SEQ ID NO:30.

7. The TCR or fragment thereof according to claim 1, wherein a conjugate is bound to the C-terminus or N-terminus of the TCR a-chain and / or TCR P-chain , and the conjugate comprises a detectable label, a therapeutic agent, a PK-modifying moiety, or a combination of any of these substances.

8. The TCR or fragment thereof according to any one of claims 1-7, wherein the TCR or fragment is capable of binding to an EBV LMP2A antigen polypeptide presented by HLA-A*11.

9. The TCR or fragment thereof according to claim 8, wherein the EBV LMP2A antigen polypeptide comprises an amino acid sequence SSCSSCPLSK set forth in SEQ ID NO:34 or SSCSSCPLTK set forth in SEQ ID NO:35.

10. A nucleic acid molecule encoding the TCR or fragment thereof according to any one of claims 1-9, or a complementary sequence thereof; preferably, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:33; preferably, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:29; preferably, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:31.

11. A vector comprising the nucleic acid molecule according to claim 10, wherein the vector is selected from plasmids, binary vectors, DNA vectors, mRNA vectors, retroviral vectors, lentiviral vectors, transposon-based vectors, and artificial chromosomes.

12. An isolated polypeptide encoded by the nucleic acid molecule according to claim 10 or the vector according to claim 11.

13. A host cell comprising the isolated polypeptide according to claim 12.

14. An immune cell comprising the TCR or fragment thereof according to any one of claims 1-9, the nucleic acid molecule according to claim 10, the vector according to claim 11, or the isolated polypeptide according to claim 12.

15. The immune cell according to claim 14, wherein the immune cell is selected from lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells; preferably, the immune cell is a T cell.

16. A pharmaceutical composition, comprising a pharmaceutically acceptable carrier and the TCR or fragment thereof according to any one of claims 1-9, the nucleic acid molecule according to claim 10, the vector according to claim 11, the isolated polypeptide according to claim 12, the host cell according to claim 13, or the immune cell according to claims 14 and 15.

17. Use of the TCR or fragment thereof according to any one of claims 1-9, the nucleic acid molecule according to claim 10, the vector according to claim 11, the isolated polypeptideaccording to claim 12, the host cell according to claim 13, the immune cell according to claims 14 and 15, or the pharmaceutical composition according to claim 16 in the preparation of a medicament for preventing or treating diseases associated with EBV infection.

18. The use according to claim 17, wherein the diseases associated with EBV infection comprise post-transplant lymphoproliferative disorders caused by EBV and various malignant tumors, preferably, wherein the diseases associated with EBV infection comprise at least one selected from nasopharyngeal carcinoma, NKT cell lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, and gastric cancer.

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