T cell receptor that specifically recognizes EBV antigen peptide, immune cell prepared therefrom, and use thereof

Abstract

Disclosed is a T cell receptor (TCR) that specifically recognizes an EBV antigen peptide, or an antigen-binding fragment thereof. The provided T cell receptor or antigen-binding fragment thereof is capable of specifically recognizing EBV antigen peptide-MHC complexes of EBV in different HLA subtypes, and is capable of targeting and eliminating cancer cells. Further provided are a T cell expressing the described T cell receptor and a use thereof in treating EBV-related cancers.

Description

T-cell receptors that specifically recognize EBV antigen peptides, immune cells prepared from them, and their applications Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the field of cell therapy technology, and particularly to a T-cell receptor that targets EBV antigen peptides, immune cells constructed based on this receptor, and their applications. Background Technology

[0002] Epstein-Barr virus (EBV) is a member of the herpesvirus family that infects humans. EBV is most commonly transmitted through bodily fluids, primarily saliva. EBV can cause infectious mononucleosis, also known as IM, as well as other diseases including cancer. EBV has evolved a life cycle that mimics the natural differentiation pathway of antigen-activated B cells, allowing the virus to enter its latent infection sites. In proliferating infected B cells, Epstein-Barr virus (EBV) executes a gene expression program, known as the "growth" or "latency phase III" program. This type of latency has been found in in vitro EBV-induced lymphoblastoid cell lines (LCLs), post-transplant lymphoproliferative disorders (Brink AA, 1997, J Clin Pathol 50: 911–918.), and EBV-infected B cells in lymphoid organs during primary and persistent EBV infection, where this process is thought to lead to an increase in EBV load through the proliferation of infected cells (Young LS, 2004, Nat Rev Cancer 4: 757–768; Hochberg D, 2004, Proc Natl Acad Sci USA 101: 239–244). Several immunogenic EBV antigens, called latency membrane proteins (LMP1, LMP2A, LMP2B) and Epstein-Barr nuclear antigens (EBNA1, -2, -3A, -3B, -3C, -LP), are expressed in EBV-infected B cells during latency III.

[0003] EBV infection is associated with certain types of cancer. EBV DNA has been found in patients with nasopharyngeal carcinoma (Mutirangura et al., Clin Cancer Res. 4:665-9 (1998); Lo et al., Cancer Res. 59:1188-91 (1999)), lymphoma (Lei et al., Br J Haematol. 111:239-46 (2000); Gallagher et al., Int J Cancer. 84:442-8 (1999); Dronet et al., J Med Viral. 57:383-9 (1999)), breast cancer (Bonnet, M. et al., J. Natl. Cancer Inst., 91:1376-1381 (1999)), and hepatocellular carcinoma (Sugawara, Y. et al., Virology, 256:196-202 (1999)).

[0004] T-cell receptor therapy (TCR-T) and chimeric antigen receptor T-cell therapy (CAR-T) are both cutting-edge gene therapies that utilize a patient's own T lymphocytes to fight cancer. The T cells in these two types of therapies express specific receptors, precisely targeting and recognizing tumor cells, thus attracting significant attention from the scientific community and resulting in a continuously rising research interest, achieving a crucial leap from basic immunology research to clinical application. Thanks to advancements in synthetic biology, immunology, and genetic engineering technologies, the artificial synthesis and enhancement of T cells with specific functions has become a realistic possibility.

[0005] CAR-T therapy utilizes antibody fragments that bind tightly to specific antigens to precisely target tumor cell surface antigens. In recent years, CAR-T cells targeting the CD19 antigen have demonstrated remarkable efficacy in clinical trials treating B-cell leukemia and lymphoma, consistently showing promising disease remission effects. Chimeric antigen receptors (CARs) endow T cells with the unique ability to recognize tumor antigens independently of human leukocyte antigens (HLA). Compared to the natural T cell surface receptor (TCR), CAR-modified T cells possess a broader antigen recognition spectrum. Currently, CAR-T technology has achieved remarkable results in the treatment of acute leukemia and non-Hodgkin's lymphoma and is considered one of the most promising cancer treatment methods.

[0006] In contrast, TCRs (T-cell receptors), as key molecules on the surface of T cells that specifically recognize antigens and mediate immune responses, primarily recognize antigenic peptides presented by histocompatibility complex (MHC) molecules. In the treatment of solid tumors, TCR-T therapy shows significant advantages over CAR-T therapy. This is because CAR-T cell therapy targets only cell surface proteins, while TCR-T recognizes major histocompatibility complex (MHC) molecules, which can present peptide chains derived from cell surface and intracellular proteins. This gives TCR-T the potential to recognize a wider variety of antigens.

[0007] The TCR is a heterodimer composed of two different peptide chains, commonly α and β. Each peptide chain can be further subdivided into a variable region (V region) and a constant region (C region). The constant region further comprises an extracellular region, a transmembrane region, and an intracellular terminal region, while the intracellular region is relatively short. The TCR molecule belongs to the immunoglobulin superfamily, with antigen specificity concentrated in the V region. TCRs are divided into two types: TCR1, composed of γ and δ chains, and TCR2, composed of α and β chains. 90%-95% of T cells in peripheral blood express TCR2, and any single T cell expresses only one of TCR2 or TCR1. As a hallmark feature of all T cell surfaces, the TCR is non-covalently linked to CD3 (ε, δ, γ, ζ) to form the TCR-CD3 complex. This CD3 complex is composed of a series of non-polymorphic proteins that act as the signal transduction mechanism of the TCR. At the molecular level, during early T cell development, a large number of germline-encoded gene fragments randomly assemble into the TCRα and TCRβ chains. In a process known as V(D)J recombination, the TCRα chain is assembled by the binding of its polymorphic variable and linker gene segments to singlet constant regions. The recombination portion of the TCRβ chain consists of its polymorphic variable and linker gene segments along with other dispersed and diverse gene segments. During gene segment rearrangement, the germline-encoding sequences adjacent to the linker regions are incidentally modified. The resulting hypervariable sequences covering the linkers of each V(D)J segment are known as the CDR3 region (complementarity-determining region 3), which subsequently determines the TCR's recognition of specific epitopes on the MHC molecules of the loaded peptide.

[0008] The current standard procedure for TCR-T therapy is to first isolate endogenous TCRs, then engineer and carefully modify them, introduce new T cells, and then reinfuse them into the human body. This significantly expands the number of T cells capable of targeting cancer cells, laying a solid foundation for conquering various solid tumors and hematological malignancies, and is expected to achieve precise identification and powerful attack on various types of tumor cells. Summary of the Invention

[0009] Through repeated, in-depth, and meticulous extensive research, the inventors have identified TCRs that specifically bind to EBV antigen peptides LMP2A (CLGGLLTMV, SEQ ID NO:307; TYGPVFMSL, SEQ ID NO:308; or SSCSSCPLTK, SEQ ID NO:309), EBNA3A (RYSIFFDYM, SEQ ID NO:305), EBNA3C (LLDFVRFMGV, SEQ ID NO:306), or BRLF1 (ATIGTAMYK, SEQ ID NO:304). In embodiments of the present invention, the antigen peptides RYSIFFDYM and TYGPVFMSL can form complexes with HLA-A2402 and be presented together on the cell surface; the antigen peptides LLDVRFMGV and CLGGLLTMV can form complexes with HLA-A0201 and be presented together on the cell surface; and the antigen peptides SSCSSCPLSK and ATIGTMYK can form complexes with HLA-A1101 and be presented together on the cell surface. The present invention also provides a nucleic acid molecule encoding the TCR and a vector comprising the nucleic acid molecule, and cells transduced with the TCR of the present invention. The present invention further provides the use of the TCR, nucleic acid molecule, vector, and cells for the preparation of medicaments for treating EBV-related tumors.

[0010] This invention first provides a T-cell receptor (TCR) or its antigen-binding fragment that specifically recognizes EBV antigen peptides, comprising an α chain and a β chain; the α chain includes an α chain variable region (Vα region), and the β chain includes a β chain variable region (Vβ region); the Vα region includes α chain complementarity-determining region 1 (αCDR1), α chain complementarity-determining region 2 (αCDR2), and α chain complementarity-determining region 3 (αCDR3), and the Vβ region includes β chain complementarity-determining region 1 (βCDR1), β chain complementarity-determining region 2 (βCDR2), and β chain complementarity-determining region 3 (βCDR3), wherein,

[0011] The βCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 73, 79, 83, 87, 93, 94, 98, 102, 105, 109, 315, 311, 127, 135, 148, 152, 156, 310, 172, 178, 314, 191, 195, 199, 205, 210, 215, and 219; SEQ ID NO: 314 is CASSPLX5X6GX7YEQYF; SEQ ID NO: 315 is CASSX8TGX9SYEQYF; SEQ ID NO: 311 is CASSIX 10 LX 11 X 12EQYF; SEQ ID NO:310 is CASSLX 13 X 14 GSYEQYF; X5 = N or D; X6 = R or Q; X7 = T or G; X8 = V or L; X9 = T, D, or H; X 10 =S or A; X 11 =A or G; X 12 =S or A; X 13 =L or M; X 14 =A or G; and / or, the αCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:76, 80, 84, 90, 312, 101, 106, 112, 116, 313, 130, 133, 138, 141, 149, 155, 157, 162, 168, 169, 175, 181, 187, 194, 196, 202, 207, 213, 218, and 220; the SEQ ID NO:312 is CAX1KX2VDSSYKLIF; the SEQ ID NO:313 is CAX3X4NNAGNMLTF; X1 = V or K; X2 = K or G; X3 = A or V; X4 = V, L, T, or C;

[0012] Preferably, the βCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 73, 79, 83, 87, 93, 94, 98, 102, 105, 109, 115, 119, 123, 127, 132, 135, 140, 142, 144, 148, 152, 156, 159, 165, 172, 178, 184, 188, 191, 195, 199, 205, 210, 215 and 219.

[0013] Preferably, the αCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:76, 80, 84, 90, 226, 95, 101, 103, 106, 112, 116, 122, 124, 130, 133, 138, 141, 143, 145, 149, 155, 157, 162, 168, 169, 175, 181, 187, 194, 196, 202, 207, 213, 218 and 220.

[0014] In one embodiment of the invention, the TCR or its antigen-binding fragment binds to or recognizes peptide epitopes of LMP2A (CLGGLLTMV, SEQ ID NO:307; TYGPVFMSL, SEQ ID NO:308; or SSCSSCPLTK, SEQ ID NO:309), EBNA3A (RYSIFFDYM, SEQ ID NO:305), EBNA3C (LLDFVRFMGV, SEQ ID NO:306), or BRLF1 (ATIGTAMYK, SEQ ID NO:304) presented by major histocompatibility complex (MHC) molecules.

[0015] In one embodiment of the invention, the MHC molecule is an HLA-A24 molecule, an HLA-A02 molecule, or an HLA-A11 molecule.

[0016] In one embodiment of the invention, the TCR or its antigen-binding fragment is selected from the group consisting of peptides comprising the following amino acid sequences:

[0017] βCDR1 is selected from SEQ ID NO: 71, 77, 81, 85, 91, 96, 104, 107, 113, 117, 125, 131, 139, 146, 150, 163, 170, 176, 182, 189, 197, 203, 208 and 214; and / or,

[0018] βCDR2 is selected from SEQ ID NO: 72, 78, 82, 86, 92, 97, 108, 114, 118, 126, 134, 147, 151, 158, 164, 171, 177, 183, 190, 198, 204, and 209; and / or,

[0019] αCDR1 is selected from SEQ ID NO: 74, 88, 99, 110, 120, 128, 136, 153, 160, 166, 173, 179, 185, 192, 200, 206, 211, and 216; and / or,

[0020] αCDR2 is selected from SEQ ID NO:75,89,100,111,121,129,137,154,161,167,174,180,186,193,201,212 and217.

[0021] In one embodiment of the invention, the T cell receptor (TCR) or its antigen-binding fragment is selected from the group consisting of peptides comprising the following amino acid sequences:

[0022] 1) αCDR1 is SEQ ID NO:74, αCDR2 is SEQ ID NO:75, and αCDR3 is SEQ ID NO:76, 80, 84, 95, 103, 106 or 226;

[0023] βCDR1 is SEQ ID NO:91, βCDR2 is SEQ ID NO:92, and βCDR3 is SEQ ID NO:93, 94, and 102; or

[0024] βCDR2 is SEQ ID NO:97, βCDR1 is SEQ ID NO:96 or 104, and βCDR3 is SEQ ID NO:98 or 105; or,

[0025] 2) βCDR1 is SEQ ID NO: 117 or 146, βCDR2 is SEQ ID NO: 118 or 147, and βCDR3 is SEQ ID NO: 119, 123, 142, 148 or 144;

[0026] αCDR1 is SEQ ID NO:120, αCDR2 is SEQ ID NO:121, and αCDR3 is SEQ ID NO:122, 124, 143, 145, 149, or 169; or,

[0027] 3) αCDR1 is SEQ ID NO:88, αCDR2 is SEQ ID NO:89, βCDR2 is SEQ ID NO:114, αCDR3 is SEQ ID NO:116, 133, or 141, βCDR1 is SEQ ID NO:113, 131, or 139, and βCDR3 is SEQ ID NO:115, 132, or 140; or,

[0028] 4) βCDR1 is SEQ ID NO:146, βCDR2 is SEQ ID NO:147, βCDR3 is SEQ ID NO:148 or 219, αCDR1 is SEQ ID NO:120 or 192, αCDR2 is SEQ ID NO:121 or 193, and αCDR3 is SEQ ID NO:149, 169, or 220; or,

[0029] 5) βCDR1 is SEQ ID NO:150, βCDR2 is SEQ ID NO:151, βCDR3 is SEQ ID NO:152 or 156, αCDR1 is SEQ ID NO:153, αCDR2 is SEQ ID NO:154, αCDR3 is SEQ ID NO:155 or 157; or,

[0030] 6) βCDR1 is SEQ ID NO:182, βCDR2 is SEQ ID NO:183, βCDR3 is SEQ ID NO:184 or 188, αCDR1 is SEQ ID NO:185, αCDR2 is SEQ ID NO:186; αCDR3 is SEQ ID NO:187; or,

[0031] 7) βCDR1 is SEQ ID NO:189; βCDR2 is SEQ ID NO:190; βCDR3 is SEQ ID NO:191 or 195; αCDR1 is SEQ ID NO:192; αCDR2 is SEQ ID NO:193; αCDR3 is SEQ ID NO:194 or 196;

[0032] 8) βCDR1 is SEQ ID NO: 85 or 214, βCDR2 is SEQ ID NO: 86, βCDR3 is SEQ ID NO: 87 or 215, αCDR1 is SEQ ID NO: 88 or 216; αCDR2 is SEQ ID NO: 89 or 217, αCDR3 is SEQ ID NO: 90 or 218; or,

[0033] 9) βCDR1 is SEQ ID NO:176 or 203; βCDR2 is SEQ ID NO:177 or 204; βCDR3 is SEQ ID NO:178 or 205; αCDR1 is SEQ ID NO:179 or 206; αCDR2 is SEQ ID NO:180; αCDR3 is SEQ ID NO:181 or 207.

[0034] In one embodiment of the invention, the T cell receptor (TCR) or its antigen-binding fragment is selected from the group consisting of peptides comprising the following amino acid sequences:

[0035] 1) βCDR1 is SEQ ID NO:71; βCDR2 is SEQ ID NO:72; βCDR3 is SEQ ID N:73; αCDR1 is SEQ ID N:74; αCDR2 is SEQ ID N:75; αCDR3 is SEQ ID N:76

[0036] 2) βCDR1 is SEQ ID NO:77; βCDR2 is SEQ ID NO:78; βCDR3 is SEQ ID NO:79; αCDR1 is SEQ ID NO:74; αCDR2 is SEQ ID NO:75; αCDR3 is SEQ ID NO:80;

[0037] 3) βCDR1 is SEQ ID NO:81; βCDR2 is SEQ ID NO:82; βCDR3 is SEQ ID NO:83; αCDR1 is SEQ ID N:74; αCDR2 is SEQ ID N:75; αCDR3 is SEQ ID NO:84;

[0038] 4) βCDR1 is SEQ ID NO:85; βCDR2 is SEQ ID NO:86; βCDR3 is SEQ ID NO:87; αCDR1 is SEQ ID NO:88; αCDR2 is SEQ ID NO:89; αCDR3 is SEQ ID NO:90;

[0039] 5) βCDR1 is SEQ ID NO:91; βCDR2 is SEQ ID NO:92; βCDR3 is SEQ ID NO:93; αCDR1 is SEQ ID N:74; αCDR2 is SEQ ID N:75; αCDR3 is SEQ ID NO:226;

[0040] 6) βCDR1 is SEQ ID NO:91; βCDR2 is SEQ ID NO:92; βCDR3 is SEQ ID NO:94; αCDR1 is SEQ ID N:74; αCDR2 is SEQ ID N:75; αCDR3 is SEQ ID NO:95;

[0041] 7) βCDR1 is SEQ ID NO:96; βCDR2 is SEQ ID NO:97; βCDR3 is SEQ ID NO:98; αCDR1 is SEQ ID NO:99; αCDR2 is SEQ ID NO:100; αCDR3 is SEQ ID NO:101;

[0042] 8) βCDR1 is SEQ ID NO:91; βCDR2 is SEQ ID NO:92; βCDR3 is SEQ ID NO:102; αCDR1 is SEQ ID N:74; αCDR2 is SEQ ID N:75; αCDR3 is SEQ ID NO:103;

[0043] 9) βCDR1 is SEQ ID NO:104; βCDR2 is SEQ ID NO:97; βCDR3 is SEQ ID NO:105; αCDR1 is SEQ ID N:74; αCDR2 is SEQ ID N:75; αCDR3 is SEQ ID NO:106;

[0044] 10) βCDR1 is SEQ ID NO:107; βCDR2 is SEQ ID NO:108; βCDR3 is SEQ ID NO:109; αCDR1 is SEQ ID NO:110; αCDR2 is SEQ ID NO:111; αCDR3 is SEQ ID NO:112;

[0045] 11) βCDR1 is SEQ ID NO:113; βCDR2 is SEQ ID NO:114; βCDR3 is SEQ ID NO:115; αCDR1 is SEQ ID NO:88; αCDR2 is SEQ ID NO:89; αCDR3 is SEQ ID NO:116;

[0046] 12) βCDR1 is SEQ ID NO:117; βCDR2 is SEQ ID NO:118; βCDR3 is SEQ ID NO:119; αCDR1 is SEQ ID NO:120; αCDR2 is SEQ ID NO:121; αCDR3 is SEQ ID NO:122;

[0047] 13) βCDR1 is SEQ ID NO:117; βCDR2 is SEQ ID NO:118; βCDR3 is SEQ ID NO:123; αCDR1 is SEQ ID NO:120; αCDR2 is SEQ ID NO:121; αCDR3 is SEQ ID NO:124;

[0048] 14) βCDR1 is SEQ ID NO:125; βCDR2 is SEQ ID NO:126; βCDR3 is SEQ ID NO:127; αCDR1 is SEQ ID NO:128; αCDR2 is SEQ ID NO:129; αCDR3 is SEQ ID NO:130;

[0049] 15) βCDR1 is SEQ ID NO:131; βCDR2 is SEQ ID NO:114; βCDR3 is SEQ ID NO:132; αCDR1 is SEQ ID NO:88; αCDR2 is SEQ ID NO:89; αCDR3 is SEQ ID NO:133;

[0050] 16) βCDR1 is SEQ ID NO:71; βCDR2 is SEQ ID NO:134; βCDR3 is SEQ ID NO:135; αCDR1 is SEQ ID NO:136; αCDR2 is SEQ ID NO:137; αCDR3 is SEQ ID NO:138;

[0051] 17) βCDR1 is SEQ ID NO:139; βCDR2 is SEQ ID NO:114; βCDR3 is SEQ ID NO:140; αCDR1 is SEQ ID NO:88; αCDR2 is SEQ ID NO:89; αCDR3 is SEQ ID NO:141;

[0052] 18) βCDR1 is SEQ ID NO:117; βCDR2 is SEQ ID NO:118; βCDR3 is SEQ ID NO:142; αCDR1 is SEQ ID NO:120; αCDR2 is SEQ ID NO:121; αCDR3 is SEQ ID NO:143;

[0053] 19) βCDR1 is SEQ ID NO:117; βCDR2 is SEQ ID NO:118; βCDR3 is SEQ ID NO:144; αCDR1 is SEQ ID NO:120; αCDR2 is SEQ ID NO:121; αCDR3 is SEQ ID NO:145;

[0054] 20) βCDR1 is SEQ ID NO:146; βCDR2 is SEQ ID NO:147; βCDR3 is SEQ ID NO:148; αCDR1 is SEQ ID NO:120; αCDR2 is SEQ ID NO:121; αCDR3 is SEQ ID NO:149;

[0055] 21) βCDR1 is SEQ ID NO:150; βCDR2 is SEQ ID NO:151; βCDR3 is SEQ ID NO:152; αCDR1 is SEQ ID NO:153; αCDR2 is SEQ ID NO:154; αCDR3 is SEQ ID NO:155;

[0056] 22) βCDR1 is SEQ ID NO:150; βCDR2 is SEQ ID NO:151; βCDR3 is SEQ ID NO:156; αCDR1 is SEQ ID NO:153; αCDR2 is SEQ ID NO:154; αCDR3 is SEQ ID NO:157;

[0057] 23) βCDR1 is SEQ ID NO:107; βCDR2 is SEQ ID NO:158; βCDR3 is SEQ ID NO:159; αCDR1 is SEQ ID NO:160; αCDR2 is SEQ ID NO:161; αCDR3 is SEQ ID NO:162;

[0058] 24) βCDR1 is SEQ ID NO:163; βCDR2 is SEQ ID NO:164; βCDR3 is SEQ ID NO:165; αCDR1 is SEQ ID NO:166; αCDR2 is SEQ ID NO:167; αCDR3 is SEQ ID NO:168;

[0059] 25) βCDR1 is SEQ ID NO:146; βCDR2 is SEQ ID NO:147; βCDR3 is SEQ ID NO:148; αCDR1 is SEQ ID NO:120; αCDR2 is SEQ ID NO:121; αCDR3 is SEQ ID NO:169;

[0060] 26) βCDR1 is SEQ ID NO:170; βCDR2 is SEQ ID NO:171; βCDR3 is SEQ ID NO:172; αCDR1 is SEQ ID NO:173; αCDR2 is SEQ ID NO:174; αCDR3 is SEQ ID NO:175;

[0061] 27) βCDR1 is SEQ ID NO:176; βCDR2 is SEQ ID NO:177; βCDR3 is SEQ ID NO:178; αCDR1 is SEQ ID NO:179; αCDR2 is SEQ ID NO:180; αCDR3 is SEQ ID NO:181;

[0062] 28) βCDR1 is SEQ ID NO:182; βCDR2 is SEQ ID NO:183; βCDR3 is SEQ ID NO:184; αCDR1 is SEQ ID NO:185; αCDR2 is SEQ ID NO:186; αCDR3 is SEQ ID NO:187;

[0063] 29) βCDR1 is SEQ ID NO:182; βCDR2 is SEQ ID NO:183; βCDR3 is SEQ ID NO:188; αCDR1 is SEQ ID NO:185; αCDR2 is SEQ ID NO:186; αCDR3 is SEQ ID NO:187;

[0064] 30) βCDR1 is SEQ ID NO:189; βCDR2 is SEQ ID NO:190; βCDR3 is SEQ ID NO:191; αCDR1 is SEQ ID NO:192; αCDR2 is SEQ ID NO:193; αCDR3 is SEQ ID NO:194;

[0065] 31) βCDR1 is SEQ ID NO:189; βCDR2 is SEQ ID NO:190; βCDR3 is SEQ ID NO:195; αCDR1 is SEQ ID NO:192; αCDR2 is SEQ ID NO:193; αCDR3 is SEQ ID NO:196;

[0066] 32) βCDR1 is SEQ ID NO:197; βCDR2 is SEQ ID NO:198; βCDR3 is SEQ ID NO:199; αCDR1 is SEQ ID NO:200; αCDR2 is SEQ ID NO:201; αCDR3 is SEQ ID NO:202;

[0067] 33) βCDR1 is SEQ ID NO:203; βCDR2 is SEQ ID NO:204; βCDR3 is SEQ ID NO:205; αCDR1 is SEQ ID NO:206; αCDR2 is SEQ ID NO:180; αCDR3 is SEQ ID NO:207;

[0068] 34) βCDR1 is SEQ ID NO:208; βCDR2 is SEQ ID NO:209; βCDR3 is SEQ ID NO:210; αCDR1 is SEQ ID NO:211; αCDR2 is SEQ ID NO:212; αCDR3 is SEQ ID NO:213;

[0069] 35) βCDR1 is SEQ ID NO:214; βCDR2 is SEQ ID NO:86; βCDR3 is SEQ ID NO:215; αCDR1 is SEQ ID NO:216; αCDR2 is SEQ ID NO:217; αCDR3 is SEQ ID NO:218;

[0070] 36) βCDR1 is SEQ ID NO:146; βCDR2 is SEQ ID NO:147; βCDR3 is SEQ ID NO:219; αCDR1 is SEQ ID NO:192; αCDR2 is SEQ ID NO:193; αCDR3 is SEQ ID NO:220.

[0071] In an embodiment of the invention, the T cell receptor (TCR) or its antigen-binding fragment, wherein the β-chain variable region is selected from a non-CDR region and a peptide with the amino acid sequence SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67 or 69, having at least about 80%, 85%, 90%, 93% or 95% identity; and / or, the α-chain variable region is selected from a non-CDR region and a peptide with the amino acid sequence SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67 or 69; and / or, the α-chain variable region is selected from a non-CDR region and a peptide with the amino acid sequence SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 69. Peptides of NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 or 70 have at least about 80%, 85%, 90%, 93% or 95% peptide identity.

[0072] In an embodiment of the invention, the T cell receptor (TCR) or its antigen-binding fragment is selected from a combination comprising an α-chain variable region and a β-chain variable region consisting of the following amino acid sequence pairs:

[0073] 1) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:1 and an α-chain variable region with the amino acid sequence SEQ ID NO:2; or

[0074] 2) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:3 and an α-chain variable region with the amino acid sequence SEQ ID NO:4; or

[0075] 3) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:5 and an α-chain variable region with the amino acid sequence SEQ ID NO:6; or

[0076] 4) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:7 and an α-chain variable region with the amino acid sequence SEQ ID NO:8; or

[0077] 5) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:9 and an α-chain variable region with the amino acid sequence SEQ ID NO:10; or

[0078] 6) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:11 and an α-chain variable region with the amino acid sequence SEQ ID NO:12; or

[0079] 7) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:13 and an α-chain variable region with the amino acid sequence SEQ ID NO:14; or

[0080] 8) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:15 and an α-chain variable region with the amino acid sequence SEQ ID NO:16; or

[0081] 9) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:17 and an α-chain variable region with the amino acid sequence SEQ ID NO:18; or

[0082] 10) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:19 and an α-chain variable region with the amino acid sequence SEQ ID NO:20; or

[0083] 11) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:21 and an α-chain variable region with the amino acid sequence SEQ ID NO:22; or

[0084] 12) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:23 and an α-chain variable region with the amino acid sequence SEQ ID NO:24; or

[0085] 13) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:25 and an α-chain variable region with the amino acid sequence SEQ ID NO:26; or

[0086] 14) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:27 and an α-chain variable region with the amino acid sequence SEQ ID NO:28; or

[0087] 15) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:29 and an α-chain variable region with the amino acid sequence SEQ ID NO:30; or

[0088] 16) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:31 and an α-chain variable region with the amino acid sequence SEQ ID NO:32; or

[0089] 17) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:33 and an α-chain variable region with the amino acid sequence SEQ ID NO:34; or

[0090] 18) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:35 and an α-chain variable region with the amino acid sequence SEQ ID NO:36; or

[0091] 19) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:37 and an α-chain variable region with the amino acid sequence SEQ ID NO:38; or

[0092] 20) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:39, and an α-chain variable region with the amino acid sequence SEQ ID NO:40 or SEQ ID NO:50; or

[0093] 21) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:41 and an α-chain variable region with the amino acid sequence SEQ ID NO:42; or

[0094] 22) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:43 and an α-chain variable region with the amino acid sequence SEQ ID NO:44; or

[0095] 23) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:45 and an α-chain variable region with the amino acid sequence SEQ ID NO:46; or

[0096] 24) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:47 and an α-chain variable region with the amino acid sequence SEQ ID NO:48; or

[0097] 25) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:49 and an α-chain variable region with the amino acid sequence SEQ ID NO:52; or

[0098] 26) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:51 and an α-chain variable region with the amino acid sequence SEQ ID NO:54; or

[0099] 27) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:53 or SEQ ID NO:55, and a α-chain variable region with the amino acid sequence SEQ ID NO:56; or

[0100] 28) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:57 and an α-chain variable region with the amino acid sequence SEQ ID NO:58; or

[0101] 29) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:59 and an α-chain variable region with the amino acid sequence SEQ ID NO:60; or

[0102] 30) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:61 and an α-chain variable region with the amino acid sequence SEQ ID NO:62; or

[0103] 31) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:63 and an α-chain variable region with the amino acid sequence SEQ ID NO:64; or

[0104] 32) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:65 and an α-chain variable region with the amino acid sequence SEQ ID NO:66; or

[0105] 33) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:67 and an α-chain variable region with the amino acid sequence SEQ ID NO:68; or

[0106] 34) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:69 and an α-chain variable region with the amino acid sequence SEQ ID NO:70.

[0107] In an embodiment of the invention, the TCR or its antigen-binding fragment has an α-chain containing an α-chain constant region, which may be a mouse α-chain constant region or a human α-chain constant region; and a β-chain containing a β-chain constant region, which may be a mouse β-chain constant region or a human β-chain constant region; preferably, the α-chain constant region is selected from peptides having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence SEQ ID NO: 224 or 225; and the β-chain constant region is selected from peptides having at least 80%, 85%, 90%, or 95% identity with the amino acid sequence SEQ ID NO: 221, 222, or 223.

[0108] In one embodiment of the present invention, the TCR or its antigen-binding fragment has the structure: α chain-linker peptide-β chain, or β chain-linker peptide-α chain; in an embodiment of the present invention, the linker peptide is a self-cleaving linker peptide, preferably selected from 2A linkers peptides, P2A, E2A, T2A and F2A; in an embodiment of the present invention, the amino acid sequence of the self-cleaving linker peptide is SEQ ID NO:263.

[0109] In an embodiment of the present invention, the N-terminus of the α-chain and β-chain of the TCR or its antigen-binding fragment further comprises a signal peptide; in an embodiment of the present invention, the signal peptide at the N-terminus of the α-chain and the signal peptide at the N-terminus of the β-chain are each independently selected from any one of the signal peptides with the amino acid sequence SEQ ID NO:264-303, and the signal peptide at the N-terminus of the α-chain and the signal peptide at the N-terminus of the β-chain are different.

[0110] In an embodiment of the present invention, the TCR or its antigen-binding fragment is selected from the amino acid sequences SEQ ID NO: 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 or 262.

[0111] In another aspect, the present invention provides a nucleic acid molecule that is the gene encoding the aforementioned TCR or its antigen-binding fragment.

[0112] In another aspect, the present invention provides a carrier comprising the above-described nucleic acid molecules.

[0113] In an embodiment of the present invention, the carrier comprises:

[0114] a) A first nucleic acid sequence encoding the TCRα chain, said TCRα chain comprising an α-chain variable region and an α-chain constant region of the human anti-EBV antigen peptide TCR; and

[0115] b) A second nucleic acid sequence encoding the TCRβ chain, wherein the TCRβ chain comprises a β-chain variable region and a β-chain constant region of the human anti-EBV antigen peptide TCR, wherein the TCRα chain and the TCRβ chain form the aforementioned TCR or its antigen-binding fragment; the EBV antigen peptide is selected from any polypeptide with the amino acid sequences SEQ ID NO: 305, 306, 307, 308, 309 and 304.

[0116] In an embodiment of the present invention, the vector is a viral vector; in an embodiment of the present invention, the vector is a retroviral vector; preferably, the vector is a lentiviral vector.

[0117] The present invention also provides an isolated host cell containing exogenous nucleic acid molecules integrated into the aforementioned vector or chromosome.

[0118] The present invention also provides a cell that has been transduced by the aforementioned vector.

[0119] In an embodiment of the invention, the cell is a T cell.

[0120] In an embodiment of the invention, the cells are T cells derived from the patient.

[0121] Another aspect of the present invention provides a pharmaceutical composition comprising the above-described TCR or its antigen-binding fragment, a nucleic acid molecule, or a cell, and a pharmaceutically acceptable carrier.

[0122] The present invention further discloses the use of the above-mentioned TCR or its antigen-binding fragment, nucleic acid molecule, cell, or pharmaceutical composition in the preparation of a treatment for EBV-related cancer, or cancer or autoimmune diseases caused by EBV infection or EBV relapse.

[0123] In embodiments of the present invention, the EBV-related cancer is selected from nasopharyngeal carcinoma (NPC), lymphoma, gastric cancer, lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia, Burkitt's lymphoma, immunosuppressive lymphoma, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma associated with chronic inflammation, lymphomatoid granuloma, plasmablastic lymphoma, primary exudative lymphoma, post-transplant lymphoproliferative disorders, gastric adenocarcinoma, lymphoepithelioma-associated carcinoma, osteosarcoma, esophageal cancer, clear cell renal cell carcinoma, immunodeficiency-associated leiomyosarcoma, or Hodgkin's lymphoma. In embodiments of the present invention, the EBV-related cancer is selected from nasopharyngeal carcinoma, osteosarcoma, clear cell renal cell carcinoma, and gastric cancer.

[0124] In an embodiment of the invention, the cancer cells of the EBV-related cancer contain a nucleic acid sequence encoding EBNA3A, EBNA3C, LMP2A, or BRLF1, or an amino acid sequence expressing EBNA3A, EBNA3C, LMP2A, or BRLF1.

[0125] In an embodiment of the invention, the cancer cells are selected from HLA-A restricted to HLA-A*2402, HLA-A*0201, or HLA-A*1101.

[0126] Compared with the prior art, the present invention has the following beneficial effects:

[0127] The TCR and TCR-T cells prepared from the present invention can specifically recognize EBV epitope antigen peptides expressed on the surface of cancer cells. Experiments have shown that they can specifically kill cancer cells expressing EBV epitope antigen peptides and can be used for the treatment of EBV-related cancers. Attached Figure Description

[0128] Figure 1A shows the flow cytometry results of the transduced TCR-T1, TCR-T2, TCR-T3, TCR-T4, TCR-T5, TCR-T6, TCR-T7, TCR-T8, TCR-T9 and TCR-T10.

[0129] Figure 1B shows the flow cytometry results of the transduced TCR-T 11, TCR-T 12, TCR-T 13, TCR-T 14, TCR-T 15, TCR-T 16, TCR-T 17, TCR-T 18 and TCR-T 19.

[0130] Figure 1C shows the flow cytometry results of the transduced TCR-T 20, TCR-T 21, TCR-T 22, TCR-T 23, TCR-T 24 and TCR-T 25 cells.

[0131] Figure 1D shows the flow cytometry results of the transduced TCR-T 26, TCR-T 27, TCR-T 28 and TCR-T 29 cells.

[0132] Figure 1E shows the flow cytometry results of the transduced TCR-T 30, TCR-T 31 and TCR-T 32 cells;

[0133] Figure 1F shows the flow cytometry results of the transduced TCR-T 33, TCR-T 34, TCR-T 35 and TCR-T 36 cells. Detailed Implementation

[0134] The following detailed explanation and description of the present invention, in conjunction with specific embodiments, does not limit the scope of protection of the present invention.

[0135] General Technology

[0136] Unless otherwise indicated, the practice of this invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. This technique is well explained in the literature, including, for example: *Molecular Cloning: A Laboratory Manual, 2nd Edition* (Sambrook et al., 1989), Cold Spring Harbor Press; *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Methods in Molecular Biology*, Humana Press; *Cell Biology: A Laboratory Notebook* (edited by J.E. Cell, 1998), Academic Press; *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Introduction to Cell and Tissue Culture* (edited by J.M. Pather and P.E. Roberts, 1998), Plenum Press; *Cell and Tissue Culture: Laboratory Procedures* (edited by A. Doyle, J.B. Griffiths, and D.G. Newell, 1993-1998), J. Wiley and Sons; *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for...* Mammalian Cells (JMMiller and MPCalos, 1987); Current Protocols in Molecular Biology (FMAusubel et al., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: a practical approach (edited by D. Catty., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (eds. M. Zanetti and JD Capra, Harwood Academic Publishers, 1995).

[0137] In some cases, for clarity and / or ease of reference, terms with their commonly understood meanings are defined herein. The invention will now be described in detail by way of the following definitions and examples. All patents and publications mentioned herein (including all sequences disclosed in such patents and publications) are expressly incorporated by reference.

[0138] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; the remainder is weight / weight percentage.

[0139] definition

[0140] Generally, unless otherwise defined, all terms, symbols and other scientific terms or terminology used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0141] As used herein, the numerical range includes the numbers that define the range.

[0142] The terms “approximately,” “close to,” etc., when preceding a list of values ​​or ranges, independently refer to each individual value in that list or range as if each individual value immediately followed the term. The term implies that the value referred to by the term is exactly the same as, close to, or similar to. For example, in some embodiments, “approximately” or “close to” a particular value may indicate 99%, 95%, or 90% of that value.

[0143] As used in this article, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an amino to carboxyl orientation.

[0144] The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” are used interchangeably herein to refer to an amino acid chain of any length (e.g., 10-100 amino acids). The chain may be linear or branched, may contain modified amino acids, and / or may be broken by non-amino acid components. The term also includes amino acid chains that have been modified naturally or through intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeled component. The definition also includes, for example, analogs containing one or more amino acids (including, for example, non-natural amino acids), and other modified polypeptides known in the art. It should be understood that polypeptides can exist as single chains or associated chains. Preferably, mammalian polypeptides (polypeptides originally derived from mammalian organisms) are used, more preferably those polypeptides secreted directly into the culture medium.

[0145] The term "stimulus" refers to a primary response induced by the binding of a stimulating domain or stimulating molecule (e.g., the TCR / CD3 complex) to its homologous ligand, thereby mediating signal transduction events, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulus can mediate alterations in the expression of certain molecules and / or reorganization of cytoskeleton structures.

[0146] The term "EBV-specific TCR (EBV-TCR)" refers to a T-cell receptor capable of specifically recognizing EBV antigenic peptides and binding to the corresponding antigenic peptide-HLA complex; "EBV-specific T cell (EBV-TCR-T)" refers to a T cell transduced with a nucleic acid or vector encoding the "EBV-specific TCR" to express the "EBV-specific TCR". In embodiments of the present invention, the EBV antigenic peptide is selected from RYSIFFDYM, LLDPVRFMGV, CLGGLLTMV, TYGPVFMSL, SSCSSCPLTK, and ATIGTAMYK.

[0147] Unless otherwise stated, the term "CDR" or "complementation-determining region" for a specific variable region should be understood to encompass the complementation-determining region (or a specific complementation-determining region) as defined by any known scheme. In some cases, the scheme used to identify a particular CDR is specified, such as the CDR defined by the Kabat, Chothia, Contact, IMGT, or AHo methods. In other cases, the specific amino acid sequence of the CDR is provided. The CDR classification is further described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography", J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev Comp. Immunol, January 2003; 27(1):55-77 (“IMGT” numbering scheme); and Honegger APluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool”, J Mol Biol, June 8, 2001; 309(3):657-70 (AHo numbering scheme). Unless otherwise specified, all specific CDR segments involved in this invention are delineated using IMGT.

[0148] The term "antitumor effect" refers to biological effects that can be manifested in a variety of ways, including but not limited to, reductions in tumor volume, number of tumor cells, metastasis, increased life expectancy, reduced tumor cell proliferation, reduced tumor cell survival, or improvement of various physiological symptoms associated with cancer.

[0149] As used herein, “treatment” or “curing” refers to any successful indication of treating or improving a disease or condition. Treatment may include, for example, reducing, delaying, or alleviating the severity of one or more symptoms of a disease or condition, or may include reducing the frequency of the occurrence of symptoms of a disease, defect, condition, or adverse condition.

[0150] Suitable culture media and methods for mammalian cell lines are well known in the art, as described in U.S. Patent No. 5,633,162. Examples of standard cell media used for laboratory culture flasks or low-density cell culture and adapted to the specific needs of cell types include: Roswell Park Memorial Institute (RPMI) 1640 medium (Morre, G., The Journal of the American Medical Association, 199, p. 519, f. 1967), L-15 medium (Leibovitz, A. et al., Amer. J. of Hygiene, 78, 1, p. 173, f. 1963), Dulbecco's modified Eagle's medium (DMEM), etc. Today, mammalian cell culture is a routine procedure well described in science textbooks and manuals, detailed in, for example, R. Ian Fresney, *Culture of Animal Cells, a Manual*, 4th edition, Wiley-Liss Publishing Company / New York, 2000.

[0151] Example 1: Culture and sequencing of EBV-specific T cells

[0152] Antigen-specific T cells:

[0153] Peripheral blood mononuclear cells (PBMCs) from donors were stimulated in vitro using chemically synthesized antigenic peptide X (as shown in Table 1) to induce and culture CD4-CD8+ positive CTL cells that can recognize the antigenic peptide and secrete IFN-γ. The antigenic peptide X used in these embodiments is shown in Table 1.

[0154] Table 1. Antigenic peptides and their corresponding proteins

[0155] Example 2: TCR Sequence Acquisition and Construction

[0156] 1. Isolation and screening of specific T cells

[0157] Peripheral blood mononuclear cells (PBMCs) derived from healthy human donors with the HLA-A*2402 genotype were stimulated in vitro using chemically synthesized antigen peptide 1 (amino acid sequence: SEQ ID NO:305) to induce and culture CD4-CD8+ positive CTL cells that can recognize antigen peptide 1 and secrete IFN-γ, and then monoclonal cell lines were further isolated and screened.

[0158] 2. Constructing TCR

[0159] From monoclonal cell culture, samples were taken for TCR sequence sequencing, obtaining VDJ region sequences of 10 TCRs, yielding α-chain variable regions and β-chain variable regions. In this embodiment, the α-chain variable regions were linked to the α-chain constant regions, and the β-chain variable regions were linked to the β-chain constant regions to construct the corresponding α-chain and β-chain. Then, the α-chain and β-chain were linked together using a self-splicing linker peptide to obtain the complete TCR sequence. The amino acid sequence of the self-splicing linker peptide used in this embodiment is SEQ ID NO:263.

[0160] When linking the α and β chains in this invention, the α chain can be placed at the N-terminus to construct a TCR sequence with an "α chain-LINKER-β chain" structure, such as TCR8, TCR9, and TCR10 obtained in this embodiment; alternatively, the β chain can be placed at the N-terminus to construct a TCR sequence with a "β chain-LINKER-α chain" structure. In the implementation of this invention, signal peptide sequences are also inserted at the N-terminus of both the α and β chains. The amino acid sequence of the signal peptide used in this invention is SEQ ID NO:264-303. However, those skilled in the art should understand that the above signal peptide sequence is for illustrative purposes only and is not a limitation; other suitable signal peptides known in the art can be used instead. The constant region of the TCR in this invention can be selected from human or mouse sources, or it can be specifically modified to improve its pairing and assembly efficiency after in vivo expression. Typically, the following combinations can be selected:

[0161] The amino acid sequence of the α chain constant region is SEQ ID NO:221 and the amino acid sequence of the β chain constant region is SEQ ID NO:224;

[0162] The amino acid sequence of the α chain constant region is SEQ ID NO:222 and the amino acid sequence of the β chain constant region is SEQ ID NO:225;

[0163] The amino acid sequence of the α chain constant region is SEQ ID NO:223 and the amino acid sequence of the β chain constant region is SEQ ID NO:225;

[0164] The amino acid sequence of the α chain constant region is SEQ ID NO:316 and the amino acid sequence of the β chain constant region is SEQ ID NO:317;

[0165] The amino acid sequence of the α chain constant region is SEQ ID NO:318 and the amino acid sequence of the β chain constant region is SEQ ID NO:319;

[0166] The amino acid sequence of the α-chain constant region is SEQ ID NO:320 and the amino acid sequence of the β-chain constant region is SEQ ID NO:321; and,

[0167] The amino acid sequence of the α chain constant region is SEQ ID NO:322 and the amino acid sequence of the β chain constant region is SEQ ID NO:323.

[0168] The specific sequences of TCR1-TCR10 obtained in this embodiment are as follows:

[0169] 1) TCR1 structure: β-chain-P2A-α-chain, amino acid sequence: SEQ ID NO:227

[0170] β-chain variable region SEQ ID NO:1;

[0171] βCDR1 SEQ ID NO:71; βCDR2 SEQ ID NO:72; βCDR3 SEQ ID N:73

[0172] α-chain variable region SEQ ID NO:2

[0173] αCDR1 SEQ ID N:74; αCDR2 SEQ ID N:75; αCDR3 SEQ ID N:76

[0174] 2) TCR 2 structure β-chain-P2A-α chain, amino acid sequence: SEQ ID NO:228

[0175] β-chain variable region SEQ ID NO:3

[0176] βCDR1 SEQ ID NO:77; βCDR2 SEQ ID NO:78; βCDR3 SEQ ID NO:79

[0177] α-chain variable region SEQ ID NO:4

[0178] αCDR1 SEQ ID N:74; αCDR2 SEQ ID N:75; αCDR3 SEQ ID NO:80

[0179] 3) TCR 3 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:229

[0180] β-chain variable region SEQ ID NO:5

[0181] βCDR1 SEQ ID NO:81; βCDR2 SEQ ID NO:82; βCDR3 SEQ ID NO:83

[0182] α-chain variable region SEQ ID NO:6

[0183] αCDR1 SEQ ID N:74; αCDR2 SEQ ID N:75; αCDR3 SEQ ID NO:84

[0184] 4) TCR 4 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:230

[0185] β-chain variable region SEQ ID NO:7

[0186] βCDR1 SEQ ID NO:85; βCDR2 SEQ ID NO:86; βCDR3 SEQ ID NO:87

[0187] α-chain variable region SEQ ID NO:8

[0188] αCDR1 SEQ ID NO:88; αCDR2 SEQ ID NO:89; αCDR3 SEQ ID NO:90

[0189] 5) TCR 5 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:231

[0190] β-chain variable region SEQ ID NO:9

[0191] βCDR1 SEQ ID NO:91; βCDR2 SEQ ID NO:92; βCDR3 SEQ ID NO:93

[0192] α-chain variable region SEQ ID NO:10

[0193] αCDR1 SEQ ID N:74; αCDR2 SEQ ID N:75; αCDR3 SEQ ID NO:226

[0194] 6) TCR 6 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:232

[0195] β-chain variable region SEQ ID NO:11

[0196] βCDR1 SEQ ID NO:91; βCDR2 SEQ ID NO:92; βCDR3 SEQ ID NO:94

[0197] α-chain variable region SEQ ID NO:12

[0198] αCDR1 SEQ ID N:74; αCDR2 SEQ ID N:75; αCDR3 SEQ ID NO:95

[0199] 7) TCR 7 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:233

[0200] β-chain variable region SEQ ID NO:13

[0201] βCDR1 SEQ ID NO:96; βCDR2 SEQ ID NO:97; βCDR3 SEQ ID NO:98

[0202] α-chain variable region SEQ ID NO:14

[0203] αCDR1 SEQ ID NO:99; αCDR2 SEQ ID NO:100; αCDR3 SEQ ID NO:101

[0204] 8) TCR 8 structure: α chain-P2A-β chain, amino acid sequence: SEQ ID NO:234

[0205] β-chain variable region SEQ ID NO:15

[0206] βCDR1 SEQ ID NO:91; βCDR2 SEQ ID NO:92; βCDR3 SEQ ID NO:102

[0207] α-chain variable region SEQ ID NO:16

[0208] αCDR1 SEQ ID N:74; αCDR2 SEQ ID N:75; αCDR3 SEQ ID NO:103

[0209] 9) TCR 9 structure: α chain-P2A-β chain, amino acid sequence: SEQ ID NO:235

[0210] β-chain variable region SEQ ID NO:17

[0211] βCDR1 SEQ ID NO:104; βCDR2 SEQ ID NO:97; βCDR3 SEQ ID NO:105

[0212] α-chain variable region SEQ ID NO:18

[0213] αCDR1 SEQ ID N:74; αCDR2 SEQ ID N:75; αCDR3 SEQ ID NO:106

[0214] 10) TCR 10 structure: α chain-P2A-β chain, amino acid sequence: SEQ ID NO:236

[0215] β-chain variable region SEQ ID NO:19

[0216] βCDR1 SEQ ID NO:107; βCDR2 SEQ ID NO:108; βCDR3 SEQ ID NO:109

[0217] α-chain variable region SEQ ID NO:20

[0218] αCDR1 SEQ ID NO:110; αCDR2 SEQ ID NO:111; αCDR3 SEQ ID NO:112

[0219] Example 3: TCR Sequence Acquisition and Construction

[0220] 1. Isolation and screening of specific T cells

[0221] Peripheral blood mononuclear cells (PBMCs) derived from healthy human donors with the HLA-A*0201 genotype were stimulated in vitro using chemically synthesized antigen peptide 2 (amino acid sequence: SEQ ID NO:306) to induce and culture CD4-CD8+ positive CTL cells that can recognize antigen peptide 2 and secrete IFN-γ, and then monoclonal cell lines were further isolated and screened.

[0222] 2. Constructing TCR

[0223] From monoclonal cell culture, samples were taken for TCR sequencing, obtaining VDJ region sequences of 9 TCRs. Following the methods and steps of Example 2, full-length TCRs containing constant regions were constructed, resulting in TCR11-TCR19, with the specific sequences as follows:

[0224] 11) TCR11 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:237

[0225] β-chain variable region SEQ ID NO:21

[0226] βCDR1 SEQ ID NO:113; βCDR2 SEQ ID NO:114; βCDR3 SEQ ID NO:115

[0227] α-chain variable region SEQ ID NO:22

[0228] αCDR1 SEQ ID NO:88; αCDR2 SEQ ID NO:89; αCDR3 SEQ ID NO:116

[0229] 12) TCR 12 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:238

[0230] β-chain variable region SEQ ID NO:23

[0231] βCDR1 SEQ ID NO:117; βCDR2 SEQ ID NO:118; βCDR3 SEQ ID NO:119

[0232] α-chain variable region SEQ ID NO:24

[0233] αCDR1 SEQ ID NO:120; αCDR2 SEQ ID NO:121; αCDR3 SEQ ID NO:122

[0234] 13) TCR 13 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:239

[0235] β-chain variable region SEQ ID NO:25

[0236] βCDR1 SEQ ID NO:117; βCDR2 SEQ ID NO:118; βCDR3 SEQ ID NO:123

[0237] α-chain variable region SEQ ID NO:26

[0238] αCDR1 SEQ ID NO:120; αCDR2 SEQ ID NO:121; αCDR3 SEQ ID NO:124

[0239] 14) TCR 14 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:240

[0240] β-chain variable region SEQ ID NO:27

[0241] βCDR1 SEQ ID NO:125; βCDR2 SEQ ID NO:126; βCDR3 SEQ ID NO:127

[0242] α-chain variable region SEQ ID NO:28

[0243] αCDR1 SEQ ID NO:128; αCDR2 SEQ ID NO:129; αCDR3 SEQ ID NO:130

[0244] 15) TCR 15 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:241

[0245] β-chain variable region SEQ ID NO:29

[0246] βCDR1 SEQ ID NO:131; βCDR2 SEQ ID NO:114; βCDR3 SEQ ID NO:132

[0247] α-chain variable region SEQ ID NO:30

[0248] αCDR1 SEQ ID NO:88; αCDR2 SEQ ID NO:89; αCDR3 SEQ ID NO:133

[0249] 16) TCR 16 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:242

[0250] β-chain variable region SEQ ID NO:31

[0251] βCDR1 SEQ ID NO:71; βCDR2 SEQ ID NO:134; βCDR3 SEQ ID NO:135

[0252] α-chain variable region SEQ ID NO:32

[0253] αCDR1 SEQ ID NO:136; αCDR2 SEQ ID NO:137; αCDR3 SEQ ID NO:138

[0254] 17) TCR 17 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:243

[0255] β-chain variable region SEQ ID NO:33

[0256] βCDR1 SEQ ID NO:139; βCDR2 SEQ ID NO:114; βCDR3 SEQ ID NO:140

[0257] α-chain variable region SEQ ID NO:34

[0258] αCDR1 SEQ ID NO:88; αCDR2 SEQ ID NO:89; αCDR3 SEQ ID NO:141

[0259] 18) TCR 18 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:244

[0260] β-chain variable region SEQ ID NO:35

[0261] βCDR1 SEQ ID NO:117; βCDR2 SEQ ID NO:118; βCDR3 SEQ ID NO:142

[0262] α-chain variable region SEQ ID NO:36

[0263] αCDR1 SEQ ID NO:120; αCDR2 SEQ ID NO:121; αCDR3 SEQ ID NO:143

[0264] 19) TCR 19 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:245

[0265] β-chain variable region SEQ ID NO:37

[0266] βCDR1 SEQ ID NO:117; βCDR2 SEQ ID NO:118; βCDR3 SEQ ID NO:144

[0267] α-chain variable region SEQ ID NO:38

[0268] αCDR1 SEQ ID NO:120; αCDR2 SEQ ID NO:121; αCDR3 SEQ ID NO:145

[0269] Example 4: TCR Sequence Acquisition and Construction

[0270] 1. Isolation and screening of specific T cells

[0271] Peripheral blood mononuclear cells (PBMCs) derived from healthy human donors with the HLA-A*0201 genotype were stimulated in vitro using chemically synthesized antigen peptide 3 (amino acid sequence: SEQ ID NO:307) to induce and culture CD4-CD8+ positive CTL cells that can recognize antigen peptide 3 and secrete IFN-γ, and then monoclonal cell lines were further isolated and screened.

[0272] 2. Construct TCR 20-25

[0273] Samples were taken from monoclonal cell cultures for TCR sequencing, yielding VDJ region sequences of 6 TCRs. Following the methods and steps in Example 2, full-length TCRs containing constant regions were constructed, resulting in TCR20-TCR25, with the specific sequences as follows:

[0274] 20) TCR 20 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:246

[0275] β-chain variable region SEQ ID NO:39

[0276] βCDR1 SEQ ID NO:146; βCDR2 SEQ ID NO:147; βCDR3 SEQ ID NO:148

[0277] α-chain variable region SEQ ID NO:40

[0278] αCDR1 SEQ ID NO:120; αCDR2 SEQ ID NO:121; αCDR3 SEQ ID NO:149

[0279] 21) TCR 21 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:247

[0280] β-chain variable region SEQ ID NO:41

[0281] βCDR1 SEQ ID NO:150; βCDR2 SEQ ID NO:151; βCDR3 SEQ ID No:152

[0282] α-chain variable region SEQ ID NO:42

[0283] αCDR1 SEQ ID NO:153; αCDR2 SEQ ID NO:154; αCDR3 SEQ ID NO:155

[0284] 22) TCR 22 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:248

[0285] β-chain variable region SEQ ID NO:43

[0286] βCDR1 SEQ ID NO:150; βCDR2 SEQ ID NO:151; βCDR3 SEQ ID NO:156

[0287] α-chain variable region SEQ ID NO:44

[0288] αCDR1 SEQ ID NO:153; αCDR2 SEQ ID NO:154; αCDR3 SEQ ID NO:157

[0289] 23) TCR 23 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:249

[0290] β-chain variable region SEQ ID NO:45

[0291] βCDR1 SEQ ID NO:107; βCDR2 SEQ ID NO:158; βCDR3 SEQ ID NO:159

[0292] α-chain variable region SEQ ID NO:46

[0293] αCDR1 SEQ ID NO:160; αCDR2 SEQ ID NO:161; αCDR3 SEQ ID NO:162

[0294] 24) TCR 24 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:250

[0295] β-chain variable region SEQ ID NO:47

[0296] βCDR1 SEQ ID NO:163; βCDR2 SEQ ID NO:164; βCDR3 SEQ ID NO:165

[0297] α-chain variable region SEQ ID NO:48

[0298] αCDR1 SEQ ID NO:166; αCDR2 SEQ ID NO:167; αCDR3 SEQ ID NO:168

[0299] 25) TCR 25 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:251

[0300] β-chain variable region SEQ ID NO:39

[0301] βCDR1 SEQ ID NO:146; βCDR2 SEQ ID NO:147; βCDR3 SEQ ID NO:148

[0302] α-chain variable region SEQ ID NO:50

[0303] αCDR1 SEQ ID NO:120; αCDR2 SEQ ID NO:121; αCDR3 SEQ ID NO:169

[0304] Example 5: TCR Sequence Acquisition and Construction

[0305] 1. Isolation and screening of specific T cells

[0306] Peripheral blood mononuclear cells (PBMCs) derived from healthy human donors with the HLA-A*2402 genotype were stimulated in vitro using chemically synthesized antigenic peptide 4 (amino acid sequence: SEQ ID NO:308) to induce and culture CD4-CD8+ positive CTL cells that can recognize antigenic peptide 4 and secrete IFN-γ, and then monoclonal cell lines were further isolated and screened.

[0307] 2. Construct TCR 26-29

[0308] From monoclonal cell culture, samples were taken for TCR sequencing to obtain VDJ region sequences of 4 TCRs. Following the methods and steps in Example 2, full-length TCRs containing constant regions were constructed, resulting in TCR26-TCR29, with the specific sequences as follows:

[0309] 26) TCR 26 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:252

[0310] β-chain variable region SEQ ID NO:49

[0311] βCDR1 SEQ ID NO:170; βCDR2 SEQ ID NO:171; βCDR3 SEQ ID NO:172

[0312] α-chain variable region SEQ ID NO:52

[0313] αCDR1 SEQ ID NO:173; αCDR2 SEQ ID NO:174; αCDR3 SEQ ID NO:175

[0314] 27) TCR 27 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:253

[0315] β-chain variable region SEQ ID NO:51

[0316] βCDR1 SEQ ID NO:176; βCDR2 SEQ ID NO:177; βCDR3 SEQ ID NO:178

[0317] α-chain variable region SEQ ID NO:54

[0318] αCDR1 SEQ ID NO:179; αCDR2 SEQ ID NO:180; αCDR3 SEQ ID NO:181

[0319] 28)

[0320] TCR 28 structure: β-chain-P2A-α-chain, amino acid sequence: SEQ ID NO:254

[0321] β-chain variable region SEQ ID NO:53

[0322] βCDR1 SEQ ID NO:182; βCDR2 SEQ ID NO:183; βCDR3 SEQ ID NO:184

[0323] α-chain variable region SEQ ID NO:56

[0324] αCDR1 SEQ ID NO:185; αCDR2 SEQ ID NO:186; αCDR3 SEQ ID NO:187

[0325] 29) TCR 29 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:255

[0326] β-chain variable region SEQ ID NO:55

[0327] βCDR1 SEQ ID NO:182; βCDR2 SEQ ID NO:183; βCDR3 SEQ ID NO:188

[0328] α-chain variable region SEQ ID NO:56

[0329] αCDR1 SEQ ID NO:185; αCDR2 SEQ ID NO:186; αCDR3 SEQ ID NO:187

[0330] Example 6: TCR Sequence Acquisition and Construction

[0331] 1. Isolation and screening of specific T cells

[0332] Peripheral blood mononuclear cells (PBMCs) derived from healthy human donors with the HLA-A*1101 genotype were stimulated in vitro using chemically synthesized antigenic peptide 5 (amino acid sequence: SEQ ID NO:309) to induce and culture CD4-CD8+ positive CTL cells that can recognize antigenic peptide 5 and secrete IFN-γ, and then monoclonal cell lines were further isolated and screened.

[0333] 2. Construct TCR 30-32

[0334] From monoclonal cell culture, samples were taken for TCR sequencing to obtain VDJ region sequences of three TCRs. Following the methods and steps in Example 2, full-length TCRs containing the constant region were constructed, resulting in TCR30-TCR32. The specific sequences are as follows:

[0335] 30) TCR 30 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:256

[0336] β-chain variable region SEQ ID NO:57

[0337] βCDR1 SEQ ID NO:189; βCDR2 SEQ ID NO:190; βCDR3 SEQ ID NO:191

[0338] α-chain variable region SEQ ID NO:58

[0339] αCDR1 SEQ ID NO:192; αCDR2 SEQ ID NO:193; αCDR3 SEQ ID NO:194

[0340] 31) TCR 31 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:257

[0341] β-chain variable region SEQ ID NO:59

[0342] βCDR1 SEQ ID NO:189; βCDR2 SEQ ID NO:190; βCDR3 SEQ ID NO:195

[0343] α-chain variable region SEQ ID NO:60

[0344] αCDR1 SEQ ID NO:192; αCDR2 SEQ ID NO:193; αCDR3 SEQ ID NO:196

[0345] 32) TCR 32 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:258

[0346] β-chain variable region SEQ ID NO:61

[0347] βCDR1 SEQ ID NO:197; βCDR2 SEQ ID NO:198; βCDR3 SEQ ID NO:199

[0348] α-chain variable region SEQ ID NO:62

[0349] αCDR1 SEQ ID NO:200; αCDR2 SEQ ID NO:201; αCDR3 SEQ ID NO:202

[0350] Example 7: TCR Sequence Acquisition and Construction

[0351] 1. Isolation and screening of specific T cells

[0352] Peripheral blood mononuclear cells (PBMCs) derived from healthy human donors with the HLA-A*1101 genotype were stimulated in vitro using chemically synthesized antigenic peptide 6 (amino acid sequence: SEQ ID NO:304) to induce and culture CD4-CD8+ positive CTL cells that can recognize antigenic peptide B3 and secrete IFN-γ, and then monoclonal cell lines were further isolated and screened.

[0353] 2. Construct TCR 33-36

[0354] From monoclonal cell culture, samples were taken for TCR sequencing, obtaining VDJ region sequences of 4 TCRs. Following the methods and steps of Example 2, full-length TCRs containing constant regions were constructed, resulting in TCRs 33-TCR 36, with the specific sequences as follows:

[0355] 33) TCR 33 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:259

[0356] β-chain variable region SEQ ID NO:63

[0357] βCDR1 SEQ ID NO:203; βCDR2 SEQ ID NO:204; βCDR3 SEQ ID NO:205

[0358] α-chain variable region SEQ ID NO:64

[0359] αCDR1 SEQ ID NO:206; αCDR2 SEQ ID NO:180; αCDR3 SEQ ID NO:207

[0360] 34) TCR 34 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:260

[0361] β-chain variable region SEQ ID NO:65

[0362] βCDR1 SEQ ID NO:208; βCDR2 SEQ ID NO:209; βCDR3 SEQ ID NO:210

[0363] α-chain variable region SEQ ID NO:66

[0364] αCDR1 SEQ ID NO:211; αCDR2 SEQ ID NO:212; αCDR3 SEQ ID NO:213

[0365] 35) TCR 35 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:261

[0366] β-chain variable region SEQ ID NO:67

[0367] βCDR1 SEQ ID NO:214; βCDR2 SEQ ID NO:86; βCDR3 SEQ ID NO:215

[0368] α-chain variable region SEQ ID NO:68

[0369] αCDR1 SEQ ID NO:216; αCDR2 SEQ ID NO:217; αCDR3 SEQ ID NO:218

[0370] 36) TCR 36 structure: β chain-P2A-α chain, amino acid sequence: SEQ ID NO:262

[0371] β-chain variable region SEQ ID NO:69

[0372] βCDR1 SEQ ID NO:146; βCDR2 SEQ ID NO:147; βCDR3 SEQ ID NO:219

[0373] α-chain variable region SEQ ID NO:70

[0374] αCDR1 SEQ ID NO:192; αCDR2 SEQ ID NO:193; αCDR3 SEQ ID NO:220

[0375] Example 8: Synthesis of the TCR encoding gene

[0376] 1. Gene synthesis

[0377] Genscript Biotech Inc. was commissioned to synthesize the corresponding coding genes based on the amino acid sequences of TCR1-36 in Examples 2-6 above.

[0378] 2. Construction of retroviral vectors

[0379] The methods of gene cloning and recombination are well known in the field and are described in detail in standard manuals such as (Sambrook and Russell et al., Molecular Cloning - A Laboratory Manual (3rd Edition) (2001) CSHL).

[0380] The coding genes for the synthesized TCR1-36 were cloned into lentiviral vectors and transfected into the viral packaging line 293T cells to prepare viral fluid. The viral fluid was then transduced into T cells to obtain TCR-T 1-36 cells expressing the corresponding target TCR sequences.

[0381] Example 9: Construction of TCR-T cells

[0382] 1. Operating Steps

[0383] On day 0, donor blood was collected, and PBMCs were isolated. Donor PBMCs were activated using anti-human CD3 / CD28 antibody. On day 2, cell transduction was performed using lentivirus packaged in 293T form. On day 3, the cell suspension was collected, centrifuged, and the medium was changed. Cells were cultured using X-VIVO15 containing 200 U / mL IL-2, with fresh culture medium added every two days. One day before harvest, TCR expression levels, i.e., the transduction positivity rate, were measured by flow cytometry.

[0384] In this embodiment, T cells were constructed and transduced based on the amino acid sequence of TCR 1-36 from Example 1 to obtain the corresponding TCR-T cells TCR-T 1-36. The non-transduced group was the NC group. The results of successful TCR expression after transduction are shown in Figures 1A-1F and Table 3. TCR-T 1-36 showed good transduction efficiency, indicating that the transduction was successful.

[0385] Table 3 Transduction efficiency

[0386] Experimental Example 1: In vitro functional verification of TCR-T1-10 cells

[0387] 1. Target cell construction

[0388] Here, target cells overexpressing antigen peptide 1 were constructed by introducing the target antigen gene into tumor cells for in vitro evaluation of the function of TCR-T1-10 cells.

[0389] Tumor cells were transduced using a lentivirus containing the gene encoding the target antigen peptide 1. In this example, the tumor cells were HLA-A*2402 human esophageal cancer cell line TE-1. After one day, the medium was changed and cultured in complete medium for 1-2 days. The medium was then changed to complete medium containing puromycin and cultured for another 1-2 days. The expression of the target antigen peptide in the transduced tumor cells was then detected. The steps were as follows:

[0390] 1) Lentiviral transduction of target antigen gene

[0391] The tumor cell density was adjusted to 4–6 × 10⁶ using complete culture medium (the complete culture medium used for TE-1 was RPMI 1640 medium containing 10% FBS). 5 Add 1 ml of the above-mentioned tumor cells / ml to a 6-well plate, add 8 μl of Polybrene (1 mg / ml) to each well until the final concentration is 8 μg / ml, add 10-30 μl of lentiviral vector containing the target antigen gene to each well, set up cell control wells (untransduced group), and add 1 ml of the above-mentioned tumor cells and 8 μl of Polybrene to each well, mix well, and incubate in a CO2 incubator (37℃, 5% CO2) for 1 day;

[0392] Discard the supernatant, add 2 ml of complete culture medium to each well, and incubate in a CO2 incubator for 2 days;

[0393] 2) Screening of transduced tumor cells with puromycin

[0394] Discard the supernatant, add 2 ml of complete culture medium containing 1 μg / ml puromycin to each well, and incubate in a CO2 incubator; observe the cells every 2 days and replace the complete culture medium containing 1 μg / ml puromycin once; passage the cells when they reach confluence.

[0395] Use a complete culture medium containing 1 μg / ml puromycin to screen and culture for 7 days. If there are still viable cells in the control group, the concentration of puromycin needs to be increased and cultured until all cells in the control group die.

[0396] 3) Monoclonal culture of cells containing the target antigen

[0397] Tumor cells transduced with transgenic antigen 1 and cultured for more than 7 days were plated using the limiting dilution method at 1 cell / well and 3 cells / well. Cells were cultured in complete medium containing puromycin, and the cell count in the wells was observed. Wells with single clones were marked. Cultured continuously until the cell count reached at least 6 × 10⁻⁶ cells / well. 7 When the number of cells exceeds a certain threshold, they can be frozen to obtain TE-1-EBNA3A target cells.

[0398] 2. Lethality Experiment

[0399] 1) Conditions for lethality experiments

[0400] TCRT 1-10 cells prepared according to Example 9 were used as effector cells, and NC cells were untransduced T cells. Each effector cell type was mixed with TE-1-EBNA3A target cells at the following effector-to-target ratio and co-incubated at 37°C. Killing efficiency was obtained using real-time label-free cell function analysis. Target cells were seeded at 8000 cells per well, and effector cells were added at an effector-to-target ratio of 1:3 or 3:1 (both calculated based on the number of Tetramer-detected positive cells). Data were collected 4-40 hours after effector cell seeding to evaluate killing efficiency. Specific killing results are shown in the table below.

[0401] Table 4. Killing efficiency of TCR-T 1-10 cells

[0402] Experimental Example 2: In vitro functional verification of TCR-T 11-19 cells

[0403] 1. Target cell construction

[0404] Here, target cells overexpressing antigen peptide 2 were constructed by introducing the target antigen gene into tumor cells for in vitro evaluation of the function of TCR-T11-19 cells.

[0405] Tumor cells were transduced using a lentivirus containing the gene encoding the target antigen peptide 1. In this example, the tumor cells were HLA-A*0201 human osteosarcoma U-2OS cell line. After one day, the medium was changed and cultured in complete medium for 1-2 days. The medium was then changed to complete medium containing puromycin and cultured for another 1-2 days. The expression of the target antigen peptide in the transduced tumor cells was then detected. The steps were as follows:

[0406] 1) Lentiviral transduction of target antigen gene

[0407] The tumor cell density was adjusted to 4–6 × 10⁶ using complete culture medium (the complete culture medium used for U-2OS was McCoy's 5A medium containing 10% FBS). 5Add 1 ml of the above-mentioned tumor cells / ml to a 6-well plate, add 8 μl of Polybrene (1 mg / ml) to each well to a final concentration of 8 μg / ml, add 10-30 μl of lentiviral vector containing the target antigen gene to each well, and set up cell control wells (untransduced group). The control wells are composed of 1 ml of tumor cells and 8 μl of Polybrene as described above. Mix well and incubate in a CO2 incubator (37°C, 5% CO2) for 1 day.

[0408] Discard the supernatant, add 2 ml of complete culture medium to each well, and incubate in a CO2 incubator for 2 days;

[0409] 2) Screening of transduced tumor cells with puromycin

[0410] Refer to the steps and methods in Experiment Example 1;

[0411] 3) Monoclonal culture of cells containing the target antigen

[0412] For specific steps and methods, please refer to Experiment Example 1 to obtain U-2OS-EBNA3C target cells.

[0413] 2. Lethality Experiment

[0414] 1) Conditions for lethality experiments

[0415] TCRT 11-19 cells prepared according to Example 9 were used as effector cells, and NC cells were untransduced T cells. Each effector cell type was mixed with U-2OS-EBNA3C target cells at the following effector-to-target ratio and co-incubated at 37°C. Killing efficiency was obtained using real-time label-free cell function analysis. Target cells were seeded at 8000 cells per well, and effector cells were added at an effector-to-target ratio of 15:1 (based on viable cell count) or 3:1. Data were collected 4-40 hours after effector cell seeding to evaluate killing efficiency. Specific killing results are shown in the table below.

[0416] Table 5. Killing efficiency of TCR-T 11-19 cells

[0417] Experimental Example 3: In vitro functional verification of TCR-T 20-25 cells

[0418] 1. Target cell construction

[0419] Here, target cells overexpressing antigen peptide 3 were constructed by introducing the target antigen gene into tumor cells for in vitro evaluation of the function of TCR-T 20-25 cells.

[0420] Tumor cells were transduced using a lentivirus containing the gene encoding the target antigen peptide 1. In this example, the tumor cells were HLA-A*0201 human osteosarcoma U-2OS cell line. After one day, the medium was changed and cultured in complete medium for 1-2 days. The medium was then changed to complete medium containing puromycin and cultured for another 1-2 days. The expression of the target antigen peptide in the transduced tumor cells was then detected. The steps were as follows:

[0421] 1) Lentiviral transduction of target antigen gene

[0422] The tumor cell density was adjusted to 4–6 × 10⁶ using complete culture medium (the complete culture medium used for U-2OS was McCoy's 5A medium containing 10% FBS). 5 Add 1 ml of the above-mentioned tumor cells / ml to a 6-well plate, add 8 μl of Polybrene (1 mg / ml) to each well to a final concentration of 8 μg / ml, add 10-30 μl of lentiviral vector containing the target antigen gene to each well, and set up cell control wells (untransduced group). The control wells are composed of 1 ml of tumor cells and 8 μl of Polybrene as described above. Mix well and incubate in a CO2 incubator (37°C, 5% CO2) for 1 day.

[0423] Discard the supernatant, add 2 ml of complete culture medium to each well, and incubate in a CO2 incubator for 2 days;

[0424] 2) Screening of transduced tumor cells with puromycin

[0425] Refer to the steps and methods in Experiment Example 1;

[0426] 3) Monoclonal culture of cells containing the target antigen

[0427] For specific steps and methods, please refer to Experiment Example 1 to obtain U-2OS-LMP2A target cells.

[0428] 2. Lethality Experiment

[0429] 1) Conditions for lethality experiments

[0430] TCR-T cells of 20-25 cells prepared according to Example 9 were used as effector cells, and NC cells were untransduced T cells. Each effector cell type was mixed with U-2OS-LMP2A target cells at the following effector-to-target ratio and co-incubated at 37°C. Killing efficiency was obtained using real-time label-free cell function analysis. Target cells were seeded at 8000 cells per well, and effector cells were added at an effector-to-target ratio of 1:3. Data were collected 4-40 hours after effector cell seeding to evaluate killing efficiency. Specific killing results are shown in the table below.

[0431] Table 6. Killing efficiency of TCR-T 20-25 cells

[0432] Experiment Example 4: In vitro functional verification of TCR-T 26-29 cells

[0433] 1. Target cell construction

[0434] Here, target cells overexpressing antigen peptide 4 were constructed by introducing the target antigen gene into tumor cells for in vitro evaluation of the function of TCR-T 26–29 cells.

[0435] Tumor cells were transduced using a lentivirus containing the gene encoding the target antigen peptide 1. In this example, the tumor cells were the HLA-A*2402 TE-1 cell line. After one day, the medium was changed to complete culture medium and cultured for 1-2 days. The medium was then changed to complete culture medium containing puromycin and cultured for another 1-2 days. The expression of the target antigen peptide in the transduced tumor cells was then detected. The steps are as follows:

[0436] 1) Lentiviral transduction of target antigen gene

[0437] The tumor cell density was adjusted to 4–6 × 10⁶ using complete culture medium (the complete culture medium used for TE-1 was RPMI 1640 medium containing 10% FBS). 5 Add 1 ml of the above-mentioned tumor cells / ml to a 6-well plate, add 8 μl of Polybrene (1 mg / ml) to each well until the final concentration is 8 μg / ml, add 10-30 μl of lentiviral vector containing the target antigen gene to each well, set up cell control wells (untransduced group), and add 1 ml of the above-mentioned tumor cells and 8 μl of Polybrene to each well, mix well, and incubate in a CO2 incubator (37℃, 5% CO2) for 1 day;

[0438] Discard the supernatant, add 2 ml of complete culture medium to each well, and incubate in a CO2 incubator for 2 days;

[0439] 2) Screening of transduced tumor cells with puromycin

[0440] Refer to the steps and methods in Experiment Example 1;

[0441] 3) Monoclonal culture of cells containing the target antigen

[0442] For specific steps and methods, please refer to Experiment Example 1 to obtain TE-1-LMP2A target cells.

[0443] 2. Lethality Experiment

[0444] 1) Conditions for lethality experiments

[0445] TCR-T 26-29 cells prepared according to Example 9 were used as effector cells, and NC cells were untransduced T cells. Each effector cell type was mixed with TE-1-LMP2A target cells at the following effector-to-target ratio and co-incubated at 37°C. Killing efficiency was obtained using real-time label-free cell function analysis. Target cells were seeded at 8000 cells per well, and effector cells were added at an effector-to-target ratio of 1:9. Data were collected 4-40 hours after effector cell seeding to evaluate killing efficiency. Specific killing results are shown in the table below.

[0446] Table 7. Killing efficiency of TCR-T cells from 26 to 29

[0447] Experimental Example 5: In vitro functional verification of TCR-T 30-32 cells

[0448] 1. Target cell construction

[0449] Here, target cells overexpressing antigen peptide 5 were constructed by introducing the target antigen gene into tumor cells for in vitro evaluation of the function of TCR-T 30-32 cells.

[0450] Tumor cells were transduced using a lentivirus containing the gene encoding the target antigen peptide 1. In this example, the tumor cells were HLA-A*1101 human renal clear cell carcinoma Caki-2 cell line cells. After one day, the medium was changed and cultured in complete medium for 1-2 days. The medium was then changed to complete medium containing puromycin and cultured for another 1-2 days. The expression of the target antigen peptide in the transduced tumor cells was then detected. The steps were as follows:

[0451] 1) Lentiviral transduction of target antigen gene

[0452] The tumor cell density was adjusted to 4–6 × 10⁶ using complete culture medium (Caki-2 used McCoy's 5A medium containing 20% ​​FBS). 5 Add 1 ml of the above-mentioned tumor cells / ml to a 6-well plate, add 8 μl of Polybrene (1 mg / ml) to each well until the final concentration is 8 μg / ml, add 10-30 μl of lentiviral vector containing the target antigen gene to each well, set up cell control wells (untransduced group), and add 1 ml of the above-mentioned tumor cells and 8 μl of Polybrene to each well, mix well, and incubate in a CO2 incubator (37℃, 5% CO2) for 1 day;

[0453] Discard the supernatant, add 2 ml of complete culture medium to each well, and incubate in a CO2 incubator for 2 days;

[0454] 2) Screening of transduced tumor cells with puromycin

[0455] Refer to the steps and methods in Experiment Example 1;

[0456] 3) Monoclonal culture of cells containing the target antigen

[0457] For specific steps and methods, please refer to Experiment Example 1 to obtain Caki-2-LMP2A target cells.

[0458] 2. Lethality Experiment

[0459] 1) Conditions for lethality experiments

[0460] TCR-T cells of 30-32 prepared according to Example 9 were used as effector cells, and NC cells were untransduced T cells. Each effector cell type was mixed with Caki-2-LMP2A target cells at the following effector-to-target ratio and co-incubated at 37°C. Killing efficiency was obtained using real-time label-free cell function analysis. Target cells were seeded at 8000 cells per well, and effector cells were added at an effector-to-target ratio of 1:9. Data were collected 4-40 hours after effector cell seeding to evaluate killing efficiency. Specific killing results are shown in the table below.

[0461] Table 8. Killing efficiency of TCR-T cells from 30 to 32

[0462] Experimental Example 6: In vitro functional verification of TCR-T 33-36 cells

[0463] 1. Target cell construction

[0464] Here, target cells overexpressing antigen peptide 6 were constructed by introducing the target antigen gene into tumor cells for in vitro evaluation of the function of TCR-T 33-36 cells.

[0465] Tumor cells were transduced using a lentivirus containing the gene encoding the target antigen peptide 1. In this example, the tumor cells were HLA-A*1101 Caki-2 cell lines. After one day, the medium was changed to complete culture medium and cultured for 1-2 days. The medium was then changed to complete culture medium containing puromycin and cultured for another 1-2 days. The expression of the target antigen peptide in the transduced tumor cells was then detected. The steps are as follows:

[0466] 1) Lentiviral transduction of target antigen gene

[0467] The tumor cell density was adjusted to 4–6 × 10⁶ using complete culture medium (Caki-2 used McCoy's 5A medium containing 20% ​​FBS). 5Add 1 ml of the above-mentioned tumor cells / ml to a 6-well plate, add 8 μl of Polybrene (1 mg / ml) to each well to a final concentration of 8 μg / ml, add 10-30 μl of lentiviral vector containing the target antigen gene to each well, and set up cell control wells (untransduced group). The control wells are composed of 1 ml of tumor cells and 8 μl of Polybrene as described above. Mix well and incubate in a CO2 incubator (37°C, 5% CO2) for 1 day.

[0468] Discard the supernatant, add 2 ml of complete culture medium to each well, and incubate in a CO2 incubator for 2 days;

[0469] 2) Screening of transduced tumor cells with puromycin

[0470] Refer to the steps and methods in Experiment Example 1;

[0471] 3) Monoclonal culture of cells containing the target antigen

[0472] For specific steps and methods, please refer to Experiment Example 1 to obtain Caki-2-BRLF target cells.

[0473] 2. Lethality Experiment

[0474] 1) Conditions for lethality experiments

[0475] TCR-T 33-36 cells prepared according to Example 9 were used as effector cells, and NC cells were untransduced T cells. Each effector cell type was mixed with Caki-2-BRLF target cells at the following effector-to-target ratio and co-incubated at 37°C. Killing efficiency was obtained using real-time label-free cell function analysis. Target cells were seeded at 8000 cells per well, and effector cells were added at an effector-to-target ratio of 1:9. Data were collected 4-40 hours after effector cell seeding to evaluate killing efficiency. Specific killing results are shown in the table below.

[0476] Table 9. Killing efficiency of TCR-T 33-36 cells

[0477] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A T-cell receptor (TCR) or its antigen-binding fragment that specifically recognizes EBV antigen peptides, comprising an α-chain and a β-chain; wherein the α-chain comprises an α-chain variable region (Vα region), and the β-chain comprises a β-chain variable region (Vβ region); the Vα region comprises α-chain complementarity-determining region 1 (αCDR1), α-chain complementarity-determining region 2 (αCDR2), and α-chain complementarity-determining region 3 (αCDR3), and the Vβ region comprises β-chain complementarity-determining region 1 (βCDR1), β-chain complementarity-determining region 2 (βCDR2), and β-chain complementarity-determining region 3 (βCDR3), wherein, The βCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 73, 79, 83, 87, 93, 94, 98, 102, 105, 109, 315, 311, 127, 135, 148, 152, 156, 310, 172, 178, 314, 191, 195, 199, 205, 210, 215, and 219; SEQ ID NO: 314 is CASSPLX5X6GX7YEQYF; SEQ ID NO: 315 is CASSX8TGX9SYEQYF; SEQ ID NO: 311 is CASSIX 10 LX 11 X 12 EQYF; SEQ ID NO:310 is CASSLX 13 X 14 GSYEQYF; X5 = N or D; X6 = R or Q; X7 = T or G; X8 = V or L; X9 = T, D, or H; X 10 =S or A; X 11 =A or G; X 12 =S or A; X 13 =L or M; X 14 =A or G; And / or, The αCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:76, 80, 84, 90, 312, 101, 106, 112, 116, 313, 130, 133, 138, 141, 149, 155, 157, 162, 168, 169, 175, 181, 187, 194, 196, 202, 207, 213, 218, and 220; the SEQ ID NO:312 is CAX1KX2VDSSYKLIF; the SEQ ID NO:313 is CAX3X4NNAGNMLTF; X1 = V or A; X2 = K or G; X3 = A or V; X4 = V, L, T, or C; Preferably, the βCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 73, 79, 83, 87, 93, 94, 98, 102, 105, 109, 115, 119, 123, 127, 132, 135, 140, 142, 144, 148, 152, 156, 159, 165, 172, 178, 184, 188, 191, 195, 199, 205, 210, 215 and 219; Preferably, the αCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:76, 80, 84, 90, 226, 95, 101, 103, 106, 112, 116, 122, 124, 130, 133, 138, 141, 143, 145, 149, 155, 157, 162, 168, 169, 175, 181, 187, 194, 196, 202, 207, 213, 218 and 220.

2. The T cell receptor (TCR) or its antigen-binding fragment according to claim 1, wherein, The TCR or its antigen-binding fragment binds to or recognizes peptide epitopes of LMP2A (CLGGLLTMV, SEQ ID NO:307; TYGPVFMSL, SEQ ID NO:308; or SSCSSCPLTK, SEQ ID NO:309), EBNA3A (RYSIFFDYM, SEQ ID NO:305), EBNA3C (LLDFVRFMGV, SEQ ID NO:306), or BRLF1 (ATIGTAMYK, SEQ ID NO:304) presented by major histocompatibility complex (MHC) molecules.

3. The TCR or its antigen-binding fragment according to claim 1 or 2, wherein the MHC molecule is an HLA-A24 molecule, an HLA-A02 molecule, or an HLA-A03 molecule; preferably an HLA-A2402 molecule, an HLA-A0201 molecule, or an HLA-A1101 molecule.

4. The T-cell receptor (TCR) or its antigen-binding fragment according to any one of claims 1-3, wherein the TCR is selected from the group consisting of peptides comprising the following amino acid sequences: βCDR1 is selected from SEQ ID NO: 71, 77, 81, 85, 91, 96, 104, 107, 113, 117, 125, 131, 139, 146, 150, 163, 170, 176, 182, 189, 197, 203, 208 and 214; and / or, βCDR2 is selected from SEQ ID NO: 72, 78, 82, 86, 92, 97, 108, 114, 118, 126, 134, 147, 151, 158, 164, 171, 177, 183, 190, 198, 204, and 209; and / or, αCDR1 is selected from SEQ ID NO: 74, 88, 99, 110, 120, 128, 136, 153, 160, 166, 173, 179, 185, 192, 200, 206, 211, and 216; and / or, αCDR2 is selected from SEQ ID NO:75,89,100,111,121,129,137,154,161,167,174,180,186,193,201,212 and217.

5. The T-cell receptor (TCR) or its antigen-binding fragment according to any one of claims 1-4, wherein the TCR is selected from the group consisting of peptides comprising the following amino acid sequences: 1) αCDR1 is SEQ ID NO:74, αCDR2 is SEQ ID NO:75, and αCDR3 is SEQ ID NO:76, 80, 84, 95, 103, 106 or 226; βCDR1 is SEQ ID NO:91, βCDR2 is SEQ ID NO:92, and βCDR3 is SEQ ID NO:93, 94, and 102; or, βCDR2 is SEQ ID NO:97, βCDR1 is SEQ ID NO:96 or 104, and βCDR3 is SEQ ID NO:98 or 105; or, 2) βCDR1 is SEQ ID NO: 117 or 146, βCDR2 is SEQ ID NO: 118 or 147, and βCDR3 is SEQ ID NO: 119, 123, 142, 148 or 144; αCDR1 is SEQ ID NO:120, αCDR2 is SEQ ID NO:121, and αCDR3 is SEQ ID NO:122, 124, 143, 145, 149, or 169; or, 3) αCDR1 is SEQ ID NO:88, αCDR2 is SEQ ID NO:89, βCDR2 is SEQ ID NO:114, αCDR3 is SEQ ID NO:116, 133, or 141, βCDR1 is SEQ ID NO:113, 131, or 139, and βCDR3 is SEQ ID NO:115, 132, or 140; or, 4) βCDR1 is SEQ ID NO:146, βCDR2 is SEQ ID NO:147, βCDR3 is SEQ ID NO:148 or 219, αCDR1 is SEQ ID NO:120 or 192, αCDR2 is SEQ ID NO:121 or 193, and αCDR3 is SEQ ID NO:149, 169, or 220; or, 5) βCDR1 is SEQ ID NO:150, βCDR2 is SEQ ID NO:151, βCDR3 is SEQ ID NO:152 or 156, αCDR1 is SEQ ID NO:153, αCDR2 is SEQ ID NO:154, αCDR3 is SEQ ID NO:155 or 157; or, 6) βCDR1 is SEQ ID NO:182, βCDR2 is SEQ ID NO:183, βCDR3 is SEQ ID NO:184 or 188, αCDR1 is SEQ ID NO:185, αCDR2 is SEQ ID NO:186; αCDR3 is SEQ ID NO:187; or, 7) βCDR1 is SEQ ID NO:189; βCDR2 is SEQ ID NO:190; βCDR3 is SEQ ID NO:191 or 195; αCDR1 is SEQ ID NO:192; αCDR2 is SEQ ID NO:193; αCDR3 is SEQ ID NO:194 or 196; 8) βCDR1 is SEQ ID NO: 85 or 214, βCDR2 is SEQ ID NO: 86, βCDR3 is SEQ ID NO: 87 or 215, αCDR1 is SEQ ID NO: 88 or 216; αCDR2 is SEQ ID NO: 89 or 217, αCDR3 is SEQ ID NO: 90 or 218; or, 9) βCDR1 is SEQ ID NO:176 or 203; βCDR2 is SEQ ID NO:177 or 204; βCDR3 is SEQ ID NO:178 or 205; αCDR1 is SEQ ID NO:179 or 206; αCDR2 is SEQ ID NO:180; αCDR3 is SEQ ID NO:181 or 207.

6. The T-cell receptor (TCR) or its antigen-binding fragment as described in any one of claims 1-4, wherein the TCR is selected from the group consisting of peptides comprising the following amino acid sequences: 1) βCDR1 is SEQ ID NO:71; βCDR2 is SEQ ID NO:72; βCDR3 is SEQ ID N:73; αCDR1 is SEQ ID N:74; αCDR2 is SEQ ID N:75; αCDR3 is SEQ ID N:76 2) βCDR1 is SEQ ID NO:77; βCDR2 is SEQ ID NO:78; βCDR3 is SEQ ID NO:79; αCDR1 is SEQ ID NO:74; αCDR2 is SEQ ID NO:75; αCDR3 is SEQ ID NO:80; 3) βCDR1 is SEQ ID NO:81; βCDR2 is SEQ ID NO:82; βCDR3 is SEQ ID NO:83; αCDR1 is SEQ ID N:74; αCDR2 is SEQ ID N:75; αCDR3 is SEQ ID NO:84; 4) βCDR1 is SEQ ID NO:85; βCDR2 is SEQ ID NO:86; βCDR3 is SEQ ID NO:87; αCDR1 is SEQ ID NO:88; αCDR2 is SEQ ID NO:89; αCDR3 is SEQ ID NO:90; 5) βCDR1 is SEQ ID NO:91; βCDR2 is SEQ ID NO:92; βCDR3 is SEQ ID NO:93; αCDR1 is SEQ ID N:74; αCDR2 is SEQ ID N:75; αCDR3 is SEQ ID NO:226; 6) βCDR1 is SEQ ID NO:91; βCDR2 is SEQ ID NO:92; βCDR3 is SEQ ID NO:94; αCDR1 is SEQ ID N:74; αCDR2 is SEQ ID N:75; αCDR3 is SEQ ID NO:95; 7) βCDR1 is SEQ ID NO:96; βCDR2 is SEQ ID NO:97; βCDR3 is SEQ ID NO:98; αCDR1 is SEQ ID NO:99; αCDR2 is SEQ ID NO:100; αCDR3 is SEQ ID NO:101; 8) βCDR1 is SEQ ID NO:91; βCDR2 is SEQ ID NO:92; βCDR3 is SEQ ID NO:102; αCDR1 is SEQ ID N:74; αCDR2 is SEQ ID N:75; αCDR3 is SEQ ID NO:103; 9) βCDR1 is SEQ ID NO:104; βCDR2 is SEQ ID NO:97; βCDR3 is SEQ ID NO:105; αCDR1 is SEQ ID N:74; αCDR2 is SEQ ID N:75; αCDR3 is SEQ ID NO:106; 10) βCDR1 is SEQ ID NO:107; βCDR2 is SEQ ID NO:108; βCDR3 is SEQ ID NO:109; αCDR1 is SEQ ID NO:110; αCDR2 is SEQ ID NO:111; αCDR3 is SEQ ID NO:112; 11) βCDR1 is SEQ ID NO:113; βCDR2 is SEQ ID NO:114; βCDR3 is SEQ ID NO:115; αCDR1 is SEQ ID NO:88; αCDR2 is SEQ ID NO:89; αCDR3 is SEQ ID NO:116; 12) βCDR1 is SEQ ID NO:117; βCDR2 is SEQ ID NO:118; βCDR3 is SEQ ID NO:119; αCDR1 is SEQ ID NO:120; αCDR2 is SEQ ID NO:121; αCDR3 is SEQ ID NO:122; 13) βCDR1 is SEQ ID NO:117; βCDR2 is SEQ ID NO:118; βCDR3 is SEQ ID NO:123; αCDR1 is SEQ ID NO:120; αCDR2 is SEQ ID NO:121; αCDR3 is SEQ ID NO:124; 14) βCDR1 is SEQ ID NO:125; βCDR2 is SEQ ID NO:126; βCDR3 is SEQ ID NO:127; αCDR1 is SEQ ID NO:128; αCDR2 is SEQ ID NO:129; αCDR3 is SEQ ID NO:130; 15) βCDR1 is SEQ ID NO:131; βCDR2 is SEQ ID NO:114; βCDR3 is SEQ ID NO:132; αCDR1 is SEQ ID NO:88; αCDR2 is SEQ ID NO:89; αCDR3 is SEQ ID NO:133; 16) βCDR1 is SEQ ID NO:71; βCDR2 is SEQ ID NO:134; βCDR3 is SEQ ID NO:135; αCDR1 is SEQ ID NO:136; αCDR2 is SEQ ID NO:137; αCDR3 is SEQ ID NO:138; 17) βCDR1 is SEQ ID NO:139; βCDR2 is SEQ ID NO:114; βCDR3 is SEQ ID NO:140; αCDR1 is SEQ ID NO:88; αCDR2 is SEQ ID NO:89; αCDR3 is SEQ ID NO:141; 18) βCDR1 is SEQ ID NO:117; βCDR2 is SEQ ID NO:118; βCDR3 is SEQ ID NO:142; αCDR1 is SEQ ID NO:120; αCDR2 is SEQ ID NO:121; αCDR3 is SEQ ID NO:143; 19) βCDR1 is SEQ ID NO:117; βCDR2 is SEQ ID NO:118; βCDR3 is SEQ ID NO:144; αCDR1 is SEQ ID NO:120; αCDR2 is SEQ ID NO:121; αCDR3 is SEQ ID NO:145; 20) βCDR1 is SEQ ID NO:146; βCDR2 is SEQ ID NO:147; βCDR3 is SEQ ID NO:148; αCDR1 is SEQ ID NO:120; αCDR2 is SEQ ID NO:121; αCDR3 is SEQ ID NO:149; 21) βCDR1 is SEQ ID NO:150; βCDR2 is SEQ ID NO:151; βCDR3 is SEQ ID NO:152; αCDR1 is SEQ ID NO:153; αCDR2 is SEQ ID NO:154; αCDR3 is SEQ ID NO:155; 22) βCDR1 is SEQ ID NO:150; βCDR2 is SEQ ID NO:151; βCDR3 is SEQ ID NO:156; αCDR1 is SEQ ID NO:153; αCDR2 is SEQ ID NO:154; αCDR3 is SEQ ID NO:157; 23) βCDR1 is SEQ ID NO:107; βCDR2 is SEQ ID NO:158; βCDR3 is SEQ ID NO:159; αCDR1 is SEQ ID NO:160; αCDR2 is SEQ ID NO:161; αCDR3 is SEQ ID NO:162; 24) βCDR1 is SEQ ID NO:163; βCDR2 is SEQ ID NO:164; βCDR3 is SEQ ID NO:165; αCDR1 is SEQ ID NO:166; αCDR2 is SEQ ID NO:167; αCDR3 is SEQ ID NO:168; 25) βCDR1 is SEQ ID NO:146; βCDR2 is SEQ ID NO:147; βCDR3 is SEQ ID NO:148; αCDR1 is SEQ ID NO:120; αCDR2 is SEQ ID NO:121; αCDR3 is SEQ ID NO:169; 26) βCDR1 is SEQ ID NO:170; βCDR2 is SEQ ID NO:171; βCDR3 is SEQ ID NO:172; αCDR1 is SEQ ID NO:173; αCDR2 is SEQ ID NO:174; αCDR3 is SEQ ID NO:175; 27) βCDR1 is SEQ ID NO:176; βCDR2 is SEQ ID NO:177; βCDR3 is SEQ ID NO:178; αCDR1 is SEQ ID NO:179; αCDR2 is SEQ ID NO:180; αCDR3 is SEQ ID NO:181; 28) βCDR1 is SEQ ID NO:182; βCDR2 is SEQ ID NO:183; βCDR3 is SEQ ID NO:184; αCDR1 is SEQ ID NO:185; αCDR2 is SEQ ID NO:186; αCDR3 is SEQ ID NO:187; 29) βCDR1 is SEQ ID NO:182; βCDR2 is SEQ ID NO:183; βCDR3 is SEQ ID NO:188; αCDR1 is SEQ ID NO:185; αCDR2 is SEQ ID NO:186; αCDR3 is SEQ ID NO:187; 30) βCDR1 is SEQ ID NO:189; βCDR2 is SEQ ID NO:190; βCDR3 is SEQ ID NO:191; αCDR1 is SEQ ID NO:192; αCDR2 is SEQ ID NO:193; αCDR3 is SEQ ID NO:194; 31) βCDR1 is SEQ ID NO:189; βCDR2 is SEQ ID NO:190; βCDR3 is SEQ ID NO:195; αCDR1 is SEQ ID NO:192; αCDR2 is SEQ ID NO:193; αCDR3 is SEQ ID NO:196; 32) βCDR1 is SEQ ID NO:197; βCDR2 is SEQ ID NO:198; βCDR3 is SEQ ID NO:199; αCDR1 is SEQ ID NO:200; αCDR2 is SEQ ID NO:201; αCDR3 is SEQ ID NO:202; 33) βCDR1 is SEQ ID NO:203; βCDR2 is SEQ ID NO:204; βCDR3 is SEQ ID NO:205; αCDR1 is SEQ ID NO:206; αCDR2 is SEQ ID NO:180; αCDR3 is SEQ ID NO:207; 34) βCDR1 is SEQ ID NO:208; βCDR2 is SEQ ID NO:209; βCDR3 is SEQ ID NO:210; αCDR1 is SEQ ID NO:211; αCDR2 is SEQ ID NO:212; αCDR3 is SEQ ID NO:213; 35) βCDR1 is SEQ ID NO:214; βCDR2 is SEQ ID NO:86; βCDR3 is SEQ ID NO:215; αCDR1 is SEQ ID NO:216; αCDR2 is SEQ ID NO:217; αCDR3 is SEQ ID NO:218; 36) βCDR1 is SEQ ID NO:146; βCDR2 is SEQ ID NO:147; βCDR3 is SEQ ID NO:219; αCDR1 is SEQ ID NO:192; αCDR2 is SEQ ID NO:193; αCDR3 is SEQ ID NO:

220.

7. The T-cell receptor (TCR) or its antigen-binding fragment according to any one of claims 1-6, wherein, Its β-chain variable region is selected from non-CDR regions and peptides with amino acid sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67 or 69 having at least about 80%, 85%, 90%, 93% or 95% identity; and / or The α-chain variable region is selected from non-CDR regions and has at least about 80%, 85%, 90%, 93%, or 95% identity with peptides whose amino acid sequences are SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70.

8. The T-cell receptor (TCR) or its antigen-binding fragment according to claim 7, wherein the TCR is selected from a combination comprising an α-chain variable region and a β-chain variable region consisting of the following amino acid sequence pairs: 1) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:1 and an α-chain variable region with the amino acid sequence SEQ ID NO:2; or 2) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:3 and an α-chain variable region with the amino acid sequence SEQ ID NO:4; or 3) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:5 and an α-chain variable region with the amino acid sequence SEQ ID NO:6; or 4) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:7 and an α-chain variable region with the amino acid sequence SEQ ID NO:8; or 5) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:9 and an α-chain variable region with the amino acid sequence SEQ ID NO:10; or 6) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:11 and an α-chain variable region with the amino acid sequence SEQ ID NO:12; or 7) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:13 and an α-chain variable region with the amino acid sequence SEQ ID NO:14; or 8) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:15 and an α-chain variable region with the amino acid sequence SEQ ID NO:16; or 9) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:17 and an α-chain variable region with the amino acid sequence SEQ ID NO:18; or 10) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:19 and an α-chain variable region with the amino acid sequence SEQ ID NO:20; or 11) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:21 and an α-chain variable region with the amino acid sequence SEQ ID NO:22; or 12) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:23 and an α-chain variable region with the amino acid sequence SEQ ID NO:24; or 13) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:25 and an α-chain variable region with the amino acid sequence SEQ ID NO:26; or 14) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:27 and an α-chain variable region with the amino acid sequence SEQ ID NO:28; or 15) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:29 and an α-chain variable region with the amino acid sequence SEQ ID NO:30; or 16) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:31 and an α-chain variable region with the amino acid sequence SEQ ID NO:32; or 17) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:33 and an α-chain variable region with the amino acid sequence SEQ ID NO:34; or 18) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:35 and an α-chain variable region with the amino acid sequence SEQ ID NO:36; or 19) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:37 and an α-chain variable region with the amino acid sequence SEQ ID NO:38; or 20) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:39, and an α-chain variable region with the amino acid sequence SEQ ID NO:40 or SEQ ID NO:50; or 21) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:41 and an α-chain variable region with the amino acid sequence SEQ ID NO:42; or 22) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:43 and an α-chain variable region with the amino acid sequence SEQ ID NO:44; or 23) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:45 and an α-chain variable region with the amino acid sequence SEQ ID NO:46; or 24) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:47 and an α-chain variable region with the amino acid sequence SEQ ID NO:48; or 25) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:49 and an α-chain variable region with the amino acid sequence SEQ ID NO:52; or 26) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:51 and an α-chain variable region with the amino acid sequence SEQ ID NO:54; or 27) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:53 or SEQ ID NO:55, and an α-chain variable region with the amino acid sequence SEQ ID NO:56; or 28) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:57 and an α-chain variable region with the amino acid sequence SEQ ID NO:58; or 29) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:59 and an α-chain variable region with the amino acid sequence SEQ ID NO:60; or 30) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:61 and an α-chain variable region with the amino acid sequence SEQ ID NO:62; or 31) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:63 and an α-chain variable region with the amino acid sequence SEQ ID NO:64; or 32) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:65 and an α-chain variable region with the amino acid sequence SEQ ID NO:66; or 33) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:67 and an α-chain variable region with the amino acid sequence SEQ ID NO:68; or 34) Contains a β-chain variable region with the amino acid sequence SEQ ID NO:69 and an α-chain variable region with the amino acid sequence SEQ ID NO:

70.

9. The TCR or its antigen-binding fragment according to any one of claims 1-8, wherein, The α chain includes an α chain constant region, which may be a mouse α chain constant region or a human α chain constant region; and the β chain includes a β chain constant region, which may be a mouse β chain constant region or a human β chain constant region; preferably. The α-chain constant region is selected from peptides that have at least 80%, 85%, 90%, or 95% identity with the amino acid sequence SEQ ID NO: 224, 225, 316, 318, 320, or 322. The β-chain constant region is selected from peptides that have at least 80%, 85%, 90%, or 95% identity with the amino acid sequence SEQ ID NO: 221, 222, 223, 317, 319, 321, or 323. Preferably, the α-chain constant region and the β-chain constant region are selected from one combination of peptides having the following amino acid sequences: The amino acid sequence of the α chain constant region is SEQ ID NO:221 and the amino acid sequence of the β chain constant region is SEQ ID NO:224; The amino acid sequence of the α chain constant region is SEQ ID NO:222 and the amino acid sequence of the β chain constant region is SEQ ID NO:225; The amino acid sequence of the α chain constant region is SEQ ID NO:223 and the amino acid sequence of the β chain constant region is SEQ ID NO:225; The amino acid sequence of the α chain constant region is SEQ ID NO:316 and the amino acid sequence of the β chain constant region is SEQ ID NO:317; The amino acid sequence of the α chain constant region is SEQ ID NO:318 and the amino acid sequence of the β chain constant region is SEQ ID NO:319; The amino acid sequence of the α-chain constant region is SEQ ID NO:320 and the amino acid sequence of the β-chain constant region is SEQ ID NO:321; and, The amino acid sequence of the α chain constant region is SEQ ID NO:322 and the amino acid sequence of the β chain constant region is SEQ ID NO:

323.

10. The TCR or its antigen-binding fragment according to any one of claims 1-8, having the structure: α chain-linking peptide-β chain, or β chain-linking peptide-α chain; Preferably, the linker peptide is a self-cleaving linker peptide; preferably, the linker peptide is selected from any one of 2A linkers, P2A, E2A, T2A, and F2A; preferably, the amino acid sequence of the self-cleaving linker peptide is SEQ ID NO:

263.

11. The TCR or its antigen-binding fragment according to any one of claims 1-10, wherein, The N-terminus of the α chain and the β chain also contains a signal peptide; preferably, the signal peptide at the N-terminus of the α chain and the signal peptide at the N-terminus of the β chain are each independently selected from any one of the signal peptides with the amino acid sequence SEQ ID NO:264-303, and the signal peptide at the N-terminus of the α chain and the signal peptide at the N-terminus of the β chain are different.

12. The TCR or its antigen-binding fragment according to any one of claims 1-11, wherein the amino acid sequence is SEQ ID NO: 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 or 262.

13. A nucleic acid molecule, which is the gene encoding the TCR or its antigen-binding fragment as described in any one of claims 1-12.

14. A vector comprising the nucleic acid molecule as described in claim 13.

15. The carrier of claim 14, comprising: a) A first nucleic acid sequence encoding the TCRα chain, said TCRα chain comprising an α-chain variable region and an α-chain constant region of the human anti-EBV antigen peptide TCR; and b) A second nucleic acid sequence encoding the TCR β chain, wherein the TCR β chain comprises the β chain variable region and the β chain constant region of the human anti-EBV antigen peptide TCR; in, The TCRα chain and the TCRβ chain form a TCR or its antigen-binding fragment according to any one of claims 1-13; The EBV antigen peptide is selected from any polypeptide with the amino acid sequence SEQ ID NO:305, 306, 307, 308, 309 and 304; Preferably, the vector is a viral vector; more preferably, the vector is a retroviral vector; and most preferably, the vector is a lentiviral vector.

16. An isolated host cell, characterized in that, The host cell contains the vector or chromosome of claim 14 or 15, in which an exogenous nucleic acid molecule of claim 13 is integrated.

17. A type of cell, in which, The cells are cells transduced by the nucleic acid molecule of claim 13 or the vector of claim 14 or 15.

18. The cell as claimed in claim 17, characterized in that, The cells in question are T cells.

19. The cell as claimed in claim 17 or 18, characterized in that, The cells in question are T cells derived from the patient.

20. A pharmaceutical composition, characterized in that, The composition comprises any one of the TCRs or their antigen-binding fragments according to claims 1-12, the nucleic acid molecule according to claim 13, or the cell according to any one of claims 17-19, and a pharmaceutically acceptable carrier.

21. The use of the TCR or its antigen-binding fragment as claimed in any one of claims 1-12, the nucleic acid molecule as claimed in claim 13, the cell as claimed in any one of claims 17-19, or the pharmaceutical composition as claimed in claim 20 in the preparation of a treatment for EBV-related cancers, or cancers or autoimmune diseases caused by EBV infection or EBV relapse.

22. The use as described in claim 21, wherein, The EBV-related cancers are selected from nasopharyngeal carcinoma (NPC), lymphoma, gastric cancer, lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma and leukemia, Burkitt's lymphoma, immunosuppressive lymphoma, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma associated with chronic inflammation, lymphomatoid granuloma, plasmablastic lymphoma, primary exudative lymphoma, post-transplant lymphoproliferative disorders, gastric adenocarcinoma, lymphoepithelioma-associated carcinoma, osteosarcoma, esophageal cancer, clear cell renal cell carcinoma, immunodeficiency-associated leiomyosarcoma, or Hodgkin's lymphoma.

23. The use as described in claim 21 or 22, wherein, The cancer cells of the EBV-related cancer contain nucleic acid sequences encoding EBNA3A, EBNA3C, LMP2A, or BRLF1, or amino acid sequences expressing EBNA3A, EBNA3C, LMP2A, or BRLF1.

24. The use according to any one of claims 21-23, wherein, Cancer cells were selected from HLA-A restricted to HLA-A*2402, HLA-A*0201, or HLA-A*1101.

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