T-cell receptor, and preparation method therefor and use thereof

Abstract

The present invention relates to the field of biomedicine. Provided are a T-cell receptor, and a preparation method therefor and the use thereof. The T-cell receptor EBV-TCR comprises a TCRα chain variable region and a TCRβ chain variable region; the TCRα chain variable region comprises peptide fragments CDRα1, CDRα2 and CDRα3; the TCRβ chain variable region comprises peptide fragments CDRβ1, CDRβ2 and CDRβ3; and the amino acid sequence of the CDRα3 is as set forth in SEQ ID NO. 7, and the amino acid sequence of the CDRβ3 is as set forth in SEQ ID NO. 3. The TCR has a high specificity and can significantly activate the release of cytokines when binding to T-cells, thus providing a theoretical and experimental basis for developing a TCR-T therapy targeting an EB virus antigen.

Description

A T cell receptor and its preparation method and use Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a T cell receptor and a preparation method and use thereof. Background Art

[0002] Epstein-Barr virus (EBV) is a common double-stranded DNA herpes virus. Approximately 90% of the world's population is infected with EBV (Tzellos, S., Farrell, PJ. Epstein-Barr virus sequence variation - biology and disease. Pathogens, 2012, 1(2): 156-174). Studies have shown that the occurrence of a variety of diseases is closely related to EBV infection, including post-transplant infection after hematopoietic stem cell transplantation (HSCT), lymphoma, and nasopharyngeal carcinoma (Taylor, GS, Long, HM, Brooks, JM, Rickinson, AB, Hislop, AD. The immunology of Epstein-Barr virus-induced disease. Annu Rev Immunol, 2015, 33787-821). In addition, 1-2% of all human tumors are caused by Epstein-Barr virus, resulting in approximately 200,000 new cancer patients each year (Münz, C. Latency and lytic replication in Epstein-Barr virus-associated oncogenesis. Nat Rev Microbiol, 2019, 17(11):691-700). Therefore, therapeutic strategies targeting Epstein-Barr virus are of great significance.

[0003] The stages of EB virus infection are mainly divided into the proliferation phase and the latent phase. EB virus expresses different proteins at different stages. Proteins expressed at the beginning of the proliferation phase and proteins expressed during the latent phase (mainly including six nuclear proteins EBNA1, 2, 3A, 3B and 3C, EBNA-LP and three latent membrane proteins LMP1, LMP2A, LMP2B, of which LMP2B is a truncated isoform of LMP2A) are believed to play a role in tumor formation. In addition, EB virus-related tumor cells also express EB virus antigen proteins, and the main way for the human body to fight the virus is T cell-mediated killing. Therefore, targeting EB virus antigen proteins is an ideal target for T cell-mediated immunotherapy.

[0004] TCR-T therapy (T cell receptor engineered T cell therapy) is a form of adoptive T cell therapy that has rapidly developed in recent years. Unlike CAR-T therapy (Chimeric antigen receptor T cell therapy), which can only recognize surface targets, TCR-T cells utilize the HLA (Human leukocyte antigen) antigen presentation system to recognize targets both inside and outside the cell. Furthermore, TCR-T cells possess greater sensitivity; in theory, a single antigen on a target cell can activate a TCR-T cell and exert its killing function. TCR-T therapy has shown great potential in the treatment of both hematologic and solid tumors.

[0005] Most of the previously disclosed inventions published TCRs targeting a single EB virus antigen peptide or a single HLA subtype, and did not provide complete TCR function verification data (including CN101182531A, CN106632659A, CN107001444B, and CN111690050A); CN108602875A and CN109306005A provided complete TCR function verification data, but only published TCRs targeting a single EB antigen peptide or a single HLA subtype.

[0006] Summary of the Invention

[0007] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a T cell receptor and a preparation method and use thereof, so as to solve the problems in the prior art.

[0008] To achieve the above objectives and other related objectives, the present invention provides a T cell receptor, wherein the T cell receptor is selected from the group consisting of T cell receptor BMLF1-TCR, T cell receptor LMP1-TCR1, T cell receptor LMP1-TCR2, T cell receptor EBNA-3B-TCR or T cell receptor LMP2-TCR, wherein:

[0009] I) The T cell receptor BMLF1-TCR comprises a TCRα chain variable region and a TCRβ chain variable region; the TCRα chain variable region comprises peptides CDRα1, CDRα2, and CDRα3; the TCRβ chain variable region comprises peptides CDRβ1, CDRβ2, and CDRβ3; the amino acid sequence of CDRα3 is shown in SEQ ID NO. 7, and the amino acid sequence of CDRβ3 is shown in SEQ ID NO. 3;

[0010] II) the TCRα chain variable region of the T cell receptor LMP1-TCR1 comprises peptides CDRα1, CDRα2, and CDRα3; the TCRβ chain variable region of the T cell receptor LMP1-TCR1 comprises peptides CDRβ1, CDRβ2, and CDRβ3; the amino acid sequence of CDRα3 is shown in SEQ ID NO. 15, and the amino acid sequence of CDRβ3 is shown in SEQ ID NO. 11;

[0011] III) the TCRα chain variable region of the T cell receptor LMP1-TCR2 comprises peptides CDRα1, CDRα2, and CDRα3; the TCRβ chain variable region of the T cell receptor LMP1-TCR2 comprises peptides CDRβ1, CDRβ2, and CDRβ3; the amino acid sequence of CDRα3 is shown in SEQ ID NO. 23, and the amino acid sequence of CDRβ3 is shown in SEQ ID NO. 19;

[0012] IV) the TCRα chain variable region of the T cell receptor EBNA-3B-TCR comprises peptides CDRα1, CDRα2, and CDRα3; the TCRβ chain variable region of the T cell receptor EBNA-3B-TCR comprises peptides CDRβ1, CDRβ2, and CDRβ3; the amino acid sequence of CDRα3 is shown in SEQ ID NO. 33, and the amino acid sequence of CDRβ3 is shown in SEQ ID NO. 29;

[0013] V) The variable region of the TCRα chain of the T cell receptor LMP2-TCR contains peptide segments CDRα1, CDRα2 and CDRα3; the variable region of the TCRβ chain of the T cell receptor LMP2-TCR contains peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.42, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.38.

[0014] The present invention also provides an EBV antigen peptide, wherein the EBV antigen peptide comprises an antigen peptide BMLF1, an antigen peptide LMP1,

[0015] Antigenic peptide EBNA3 or antigenic peptide LMP2;

[0016] The amino acid sequence of the antigenic peptide BMLF1 is shown in SEQ ID No. 25; or

[0017] The amino acid sequence of the antigenic peptide LMP1 is shown in SEQ ID No. 26; or

[0018] The amino acid sequence of the antigenic peptide EBNA3 is shown in SEQ ID No. 35; or

[0019] The amino acid sequence of the antigen peptide LMP2 is shown in SEQ ID No.44.

[0020] The present invention also provides a method for preparing the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR, or the aforementioned T cell receptor LMP2-TCR, the preparation method comprising the following steps:

[0021] 1) loading the aforementioned EBV antigen peptides onto dendritic cells differentiated in vitro;

[0022] 2) Co-culturing the dendritic cells in step 1) with CD8+ T cells to obtain the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR, or the aforementioned T cell receptor LMP2-TCR.

[0023] The present invention also provides a biomaterial, comprising a polynucleotide, a nucleic acid construct, a virus or a cell:

[0024] The polynucleotide encodes the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR or the aforementioned T cell receptor LMP2-TCR; or, the polynucleotide encodes the aforementioned EBV antigen peptide;

[0025] The nucleic acid construct comprises the aforementioned polynucleotide and a plasmid backbone.

[0026] The virus contains the aforementioned polynucleotide.

[0027] The cell contains the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR, the aforementioned T cell receptor LMP2-TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned virus.

[0028] The present invention also provides use of the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR, the aforementioned T cell receptor LMP2-TCR, the aforementioned EBV antigen peptide or the aforementioned biomaterial in preparing tumor treatment products.

[0029] The present invention also provides a tumor treatment product, which comprises the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR, the aforementioned T cell receptor LMP2-TCR, the aforementioned EBV antigen peptide or the aforementioned T cell and pharmaceutically acceptable excipients.

[0030] The present invention also provides the use of the aforementioned EBV antigen peptide or the aforementioned biomaterial in preparing the following products:

[0031] 1) Anti-tumor peptide vaccine products;

[0032] 2) Generate T cell receptor products in vitro.

[0033] The present invention also provides a method for treating tumors, which comprises administering the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR, the aforementioned T cell receptor LMP2-TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned virus or the aforementioned T cell to a tumor patient.

[0034] As described above, the T cell receptor of the present invention and its preparation method and use have the following beneficial effects:

[0035] The present invention combines the HLA subtype distribution characteristics of the Chinese population, selects four Epstein-Barr virus antigen peptides (BMLF1, LMP1, LMP2, EBNA3B) corresponding to the two most frequent HLA subtypes (HLA-A*11:01 and HLA-A*02:01) in the Chinese and European and American populations, systematically conducts Epstein-Barr virus antigen-specific TCR screening, and conducts comprehensive TCR function verification. The T cell receptor of the present invention will lay a direct foundation for the development of TCR-T therapy targeting Epstein-Barr virus antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG1 shows a flow chart of the TCR-T cell sorting process of the present invention.

[0037] FIG2 is a schematic diagram showing the results of sorting and single-cell sequencing of TCR-T cells of the present invention.

[0038] FIG3 is a schematic diagram showing the results of an in vitro TCR-T cell binding experiment of the present invention.

[0039] FIG4 is a schematic diagram showing the results of an in vitro activation experiment of TCR-T cells of the present invention.

[0040] FIG5 is a schematic diagram showing the results of an in vitro killing experiment of TCR-T cells of the present invention.

[0041] FIG6 is a schematic diagram showing the results of in vitro killing experiments of some TCR-T cells of the present invention. DETAILED DESCRIPTION

[0042] The present invention provides a T cell receptor BMLF1-TCR or a fragment thereof, wherein the T cell receptor BMLF1-TCR comprises a TCRα chain variable region and a TCRβ chain variable region; the TCRα chain variable region comprises peptide segments CDRα1, CDRα2 and CDRα3; the TCRβ chain variable region comprises peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.7, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.3.

[0043] In some specific embodiments, the amino acid sequence of CDRα1 in the TCRα chain variable region is shown as SEQ ID NO.5 or the amino acid sequence of CDRα2 is shown as SEQ ID NO.6.

[0044] In some specific embodiments, the amino acid sequence of CDRβ1 in the TCRβ chain variable region is shown as SEQ ID NO.1 or the amino acid sequence of CDRβ2 is shown as SEQ ID NO.2.

[0045] The present invention also provides a T cell receptor LMP1-TCR1 or a fragment thereof, wherein the TCRα chain variable region of the T cell receptor LMP1-TCR1 comprises peptide segments CDRα1, CDRα2 and CDRα3; the TCRβ chain variable region of the T cell receptor LMP1-TCR1 comprises peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.15, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.11.

[0046] In some specific embodiments, the amino acid sequence of CDRα1 in the TCRα chain variable region is shown as SEQ ID NO.13 or the amino acid sequence of CDRα2 is shown as SEQ ID NO.14.

[0047] In some specific embodiments, the amino acid sequence of CDRβ1 in the TCRβ chain variable region is shown as SEQ ID NO.9 or the amino acid sequence of CDRβ2 is shown as SEQ ID NO.10.

[0048] The present invention also provides a T cell receptor LMP1-TCR2 or a fragment thereof, wherein the TCRα chain variable region of the T cell receptor LMP1-TCR2 comprises peptide segments CDRα1, CDRα2 and CDRα3; the TCRβ chain variable region of the T cell receptor LMP1-TCR2 comprises peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.23, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.19.

[0049] In some specific embodiments, the amino acid sequence of CDRα1 in the TCRα chain variable region is shown as SEQ ID NO.21 or the amino acid sequence of CDRα2 is shown as SEQ ID NO.22.

[0050] In some specific embodiments, the amino acid sequence of CDRβ1 in the TCRβ chain variable region is shown as SEQ ID NO.17 or the amino acid sequence of CDRβ2 is shown as SEQ ID NO.18.

[0051] The present invention also provides a T cell receptor EBNA-3B-TCR or a fragment thereof, wherein the TCRα chain variable region of the T cell receptor EBNA-3B-TCR comprises peptide segments CDRα1, CDRα2 and CDRα3; the TCRβ chain variable region of the T cell receptor EBNA-3B-TCR comprises peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.33, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.29.

[0052] In some specific embodiments, the amino acid sequence of CDRα1 in the TCRα chain variable region is shown as SEQ ID NO.31 or the amino acid sequence of CDRα2 is shown as SEQ ID NO.32.

[0053] In some specific embodiments, the amino acid sequence of CDRβ1 in the TCRβ chain variable region is shown as SEQ ID NO. 27 or the amino acid sequence of CDRβ2 is shown as SEQ ID NO. 28.

[0054] The present invention also provides a T cell receptor LMP2-TCR or a fragment thereof, wherein the TCRα chain variable region of the T cell receptor LMP2-TCR comprises peptide segments CDRα1, CDRα2 and CDRα3; the TCRβ chain variable region of the T cell receptor LMP2-TCR comprises peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.42, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.38.

[0055] In some specific embodiments, the amino acid sequence of CDRα1 in the TCRα chain variable region is shown as SEQ ID NO.40 or the amino acid sequence of CDRα2 is shown as SEQ ID NO.41.

[0056] In some specific embodiments, the amino acid sequence of CDRβ1 in the TCRβ chain variable region is shown as SEQ ID NO.36 or the amino acid sequence of CDRβ2 is shown as SEQ ID NO.37.

[0057] In some specific embodiments, the HLA subtype targeted by the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR, or the aforementioned T cell receptor LMP2-TCR includes HLA-A*02:01 and HLA-A*11:01. More specifically, the HLA subtype targeted by the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, or the aforementioned T cell receptor LMP1-TCR2 is HLA-A*02:01; or the HLA subtype targeted by the aforementioned T cell receptor EBNA-3B-TCR is HLA-A*11:01; or the HLA subtype targeted by the aforementioned T cell receptor LMP2-TCR is HLA-A*02:01.

[0058] A CDR (complementarity determining region) generally refers to the region of an antibody or receptor that spatially complements an antigenic determinant. The variability in an antibody or receptor is typically not evenly distributed throughout the variable region. Both the α and β chain variable regions of a monoclonal antibody or receptor typically have three hypervariable regions (HVRs). These regions typically complement the antigenic determinant spatially, and are therefore also called complementarity determining regions (CDRs). The α chain variable region typically includes three CDRs: CDRα1, CDRα2, and CDRα3, while the β chain variable region also typically includes three CDRs: CDRβ1, CDRβ2, and CDRβ3.

[0059] In some specific embodiments, the α chain variable region and the β chain variable region may further include a framework region, and the framework region may be located between the complementarity determining regions or at both ends of the complementarity determining regions. In certain specific embodiments of the present invention, the sequence of the framework region is a human monoclonal antibody variable region, or a framework region sequence of a murine monoclonal antibody variable region, obtained by replacing, deleting, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and the framework region sequence may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology with the framework region sequence of the human monoclonal antibody variable region sequence.

[0060] In some specific embodiments, the amino acid sequence of the TCRα chain variable region of the T cell receptor BMLF1-TCR is shown as SEQ ID NO.45; and / or, the amino acid sequence of the TCRβ chain variable region of the T cell receptor BMLF1-TCR is shown as SEQ ID NO.46.

[0061] In some specific embodiments, the amino acid sequence of the TCRα chain variable region of the T cell receptor LMP1-TCR1 is shown as SEQ ID NO.47; and / or the amino acid sequence of the TCRβ chain variable region of the T cell receptor LMP1-TCR1 is shown as SEQ ID NO.48.

[0062] In some specific embodiments, the amino acid sequence of the TCRα chain variable region of the T cell receptor LMP1-TCR2 is shown as SEQ ID NO.49; and / or the amino acid sequence of the TCRβ chain variable region of the T cell receptor LMP1-TCR2 is shown as SEQ ID NO.50.

[0063] In some specific embodiments, the amino acid sequence of the TCRα chain variable region of the T cell receptor EBNA-3B-TCR is shown as SEQ ID NO.51; and / or, the amino acid sequence of the TCRβ chain variable region of the T cell receptor EBNA-3B-TCR is shown as SEQ ID NO.52.

[0064] In some specific embodiments, the amino acid sequence of the TCRα chain variable region of the T cell receptor LMP2-TCR is shown as SEQ ID NO.53; and / or the amino acid sequence of the TCRβ chain variable region of the T cell receptor LMP2-TCR is shown as SEQ ID NO.54.

[0065] In some specific embodiments, the TCR α chain variable region can also be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.49, SEQ ID NO.51, or SEQ ID NO.53; the TCR β chain variable region can also be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO.46, SEQ ID NO.48, SEQ ID NO.50, SEQ ID NO.52, or SEQ ID The amino acid sequence shown in NO.54 has a peptide segment with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity.

[0066] In some specific embodiments, the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR, or the aforementioned T cell receptor LMP2-TCR further comprises a TCR α chain constant region or a TCR β chain constant region.

[0067] In some specific embodiments, the amino acid sequence of the TCRα chain constant region is shown as SEQ ID NO.55; and / or, the amino acid sequence of the TCRβ chain constant region is shown as SEQ ID NO.56.

[0068] In some specific embodiments, the TCR α chain constant region may also be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO. 55; and the TCR β chain constant region may also be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO. 56.

[0069] In some specific embodiments, the amino acid sequence of the TCRα chain in the T cell receptor BMLF1-TCR is shown as SEQ ID NO.8; the amino acid sequence of the TCRβ chain in the T cell receptor BMLF1-TCR is shown as SEQ ID NO.4.

[0070] In some specific embodiments, the amino acid sequence of the TCRα chain in the T cell receptor LMP1-TCR1 is shown as SEQ ID NO.16; the amino acid sequence of the TCRβ chain in the T cell receptor LMP1-TCR1 is shown as SEQ ID NO.12.

[0071] In some specific embodiments, the amino acid sequence of the TCRα chain in the T cell receptor LMP1-TCR2 is shown as SEQ ID NO.24; the amino acid sequence of the TCRβ chain in the T cell receptor LMP1-TCR2 is shown as SEQ ID NO.20.

[0072] In some specific embodiments, the amino acid sequence of the TCRα chain in the T cell receptor EBNA-3B-TCR is shown as SEQ ID NO.34; the amino acid sequence of the TCRβ chain in the T cell receptor EBNA-3B-TCR is shown as SEQ ID NO.30.

[0073] In some specific embodiments, the amino acid sequence of the TCRα chain in the T cell receptor LMP2-TCR is shown as SEQ ID NO.43; the amino acid sequence of the TCRβ chain in the T cell receptor LMP2-TCR is shown as SEQ ID NO.39.

[0074] In some specific embodiments, in the case of having the aforementioned TCRα and TCRβ chain variable region CDR sequences, the TCRα chain can also be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO.8, SEQ ID NO.16, SEQ ID NO.24, SEQ ID NO.34, or SEQ ID NO.43; the TCRβ chain can also be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO.4, SEQ ID NO.12, SEQ ID NO.20, SEQ ID NO.30, or SEQ ID The amino acid sequence shown in NO.39 has a peptide segment with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5% sequence similarity.

[0075] In some embodiments, the TCRα chain is selected from a human TCRα chain, a humanized TCRα chain, a chimeric TCRα chain, or a murine TCRα chain; and the TCRβ chain is selected from a human TCRβ chain, a humanized TCRβ chain, a chimeric TCRβ chain, or a murine TCRβ chain. "Chimeric TCRα chain" or "chimeric TCRβ" refers to a TCRα chain or TCRβ chain comprising sequences derived from more than one species, such as sequences derived from human and mouse.

[0076] In some specific embodiments, the T cell receptor BMLF1-TCR binds to the BMLF1 antigen; or, the T cell receptor LMP1-TCR1 and the T cell receptor LMP1-TCR2 bind to the LMP1 antigen; or, the T cell receptor EBNA-3B-TCR binds to the EBNA3 antigen; or, the T cell receptor LMP2-TCR binds to the LMP2 antigen.

[0077] The present invention also provides an antigen-binding protein, which comprises the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR or the aforementioned T cell receptor LMP2-TCR.

[0078] The present invention also provides an EBV antigen peptide, which comprises an antigen peptide BMLF1, an antigen peptide LMP1, an antigen peptide EBNA3 or an antigen peptide LMP2.

[0079] In some specific embodiments, the amino acid sequence of the antigenic peptide BMLF1 is shown as SEQ ID No. 25.

[0080] In some specific embodiments, the amino acid sequence of the antigenic peptide LMP1 is shown as SEQ ID No.26.

[0081] In some specific embodiments, the amino acid sequence of the antigenic peptide EBNA3 is shown as SEQ ID No.35.

[0082] In some specific embodiments, the amino acid sequence of the antigenic peptide LMP2 is shown as SEQ ID No.44.

[0083] In some specific embodiments, the aforementioned antigenic peptide BMLF1 can be recognized by the aforementioned T cell receptor BMLF1-TCR; or, the aforementioned antigenic peptide LMP1 can be recognized by the aforementioned T cell receptor LMP1-TCR1 or the aforementioned T cell receptor LMP1-TCR2; or, the aforementioned antigenic peptide EBNA3 can be recognized by the aforementioned T cell receptor EBNA-3B–TCR; or, the aforementioned antigenic peptide LMP2 can be recognized by the aforementioned T cell receptor LMP2-TCR.

[0084] The present invention also provides a method for preparing the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR, or the aforementioned T cell receptor LMP2-TCR, the preparation method comprising the following steps:

[0085] 1) loading the aforementioned antigenic peptide BMLF1, the aforementioned antigenic peptide LMP1, the aforementioned antigenic peptide EBNA3, or the aforementioned antigenic peptide LMP2 onto dendritic cells (DCs) differentiated in vitro;

[0086] 2) Co-culturing the dendritic cells in step 1) with CD8+ T cells to obtain the T cell receptor BMLF1-TCR, the T cell receptor LMP1-TCR1, the T cell receptor LMP1-TCR2, the T cell receptor EBNA-3B-TCR or the T cell receptor LMP2-TCR.

[0087] In some specific embodiments, the loading in step 1) is a process of mixing the antigenic peptide BMLF1, antigenic peptide LMP1, antigenic peptide EBNA3 or antigenic peptide LMP2 with mature dendritic cells, incubating the mixture and then irradiating the mixture.

[0088] The present invention also provides a biomaterial comprising a polynucleotide, a nucleic acid construct, a virus or a cell.

[0089] In some specific embodiments, the polynucleotide encodes the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR or the aforementioned T cell receptor LMP2-TCR; or, the polynucleotide encodes the aforementioned antigenic peptide BMLF1, the aforementioned antigenic peptide LMP1, the aforementioned antigenic peptide EBNA3 or the aforementioned antigenic peptide LMP2.

[0090] In some specific embodiments, the nucleic acid construct comprises the aforementioned polynucleotide and a plasmid backbone.

[0091] In some specific embodiments, the plasmid backbone is a plasmid backbone of a virus, adenovirus or adeno-associated virus. More specifically, the plasmid backbone is selected from pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-T Any one or more of puroGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP / VSV-G, pENTR / U6, pLenti6 / BLOCK-iT-DEST, pcDNA1.2 / V5-GW / lacZ, pLenti6.2 / N-Lumio / V5-DEST, pGCSIL-GFP, and pHR-SFFV-Puro.

[0092] In some embodiments, the virus contains the aforementioned polynucleotide.

[0093] In some embodiments, the virus is selected from a lentivirus, an adenovirus, or an adeno-associated virus.

[0094] In some specific embodiments, the cell contains the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR or the aforementioned T cell receptor LMP2-TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned virus.

[0095] In some specific embodiments, the cell is a cell having the aforementioned polynucleotide integrated into its genome.

[0096] In some embodiments, the cell is a T cell. Preferably, the T cell is a TCR-T cell.

[0097] In some embodiments, the T cells are further selected from helper T cells, suppressor T cells, effector T cells, cytotoxic T cells, delayed-type hypersensitivity T cells, naive T cells, or memory T cells. Helper T cells are T cells that assist humoral and cellular immunity; suppressor T cells are T cells that suppress cellular and humoral immunity; effector T cells are T cells that release lymphokines; cytotoxic T cells are T cells that kill target cells; delayed-type hypersensitivity T cells are T cells that participate in type IV hypersensitivity reactions and can act on helper and suppressor T cells to amplify the immune effect; naive T cells are T cells that have not yet differentiated; and memory T cells are T cells that have the ability to stimulate memory-specific antigens.

[0098] In some embodiments, the T cells are Jurkat T cells or CD8+ T cells.

[0099] The present invention also provides the use of the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR, the aforementioned T cell receptor LMP2-TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned virus or the aforementioned cell in preparing tumor treatment products.

[0100] The present invention also provides use of the aforementioned antigenic peptide BMLF1, the aforementioned antigenic peptide LMP1, the aforementioned antigenic peptide EBNA3, the aforementioned antigenic peptide LMP2, the aforementioned polynucleotide or the aforementioned nucleic acid construct in preparing an anti-tumor peptide vaccine product.

[0101] The present invention also provides use of the aforementioned antigenic peptide BMLF1, the aforementioned antigenic peptide LMP1, the aforementioned antigenic peptide EBNA3, the aforementioned antigenic peptide LMP2, the aforementioned polynucleotide or the aforementioned nucleic acid construct in preparing an in vitro generated T cell receptor product.

[0102] In some specific embodiments, the tumor is selected from one or more of nasopharyngeal carcinoma, adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct cancer, colon adenocarcinoma, lymphoid tumors, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial cancer, chronic myeloid leukemia, lung cancer, anal cancer, and retinoblastoma.

[0103] In some embodiments, the lymphoid neoplasm comprises NKT cell lymphoma, Hodgkin lymphoma, or diffuse large B cell lymphoma.

[0104] The present invention also provides a tumor treatment product, which comprises the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR, the aforementioned T cell receptor LMP2-TCR, the aforementioned EBV antigen peptide, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned virus or the aforementioned T cell and pharmaceutically acceptable excipients.

[0105] In some embodiments, the adjuvant includes various excipients and diluents, which are not necessary active ingredients and have no excessive toxicity after administration. The adjuvant includes sterile water or normal saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.) or adhesives. The adjuvant also includes other low molecular weight polypeptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol or sorbitol. When the adjuvant is used for an aqueous solution for injection, it is selected from normal saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannose or sugar alcohol isotonic solution. The aqueous solution for injection includes a solubilizing agent. The solubilizing agent is selected from alcohol (ethanol), polyol (propylene glycol or PEG) and / or nonionic surfactant (Tween 80 or HCO-50). In the tumor treatment products provided by the present invention, the aforementioned TCR, polynucleotide, nucleic acid construct, or T cell may be a single active ingredient, or may be combined with one or more other active ingredients useful for tumor treatment to form a combination preparation. The active ingredients may be other various drugs used for tumor treatment. The content of the active ingredient in the pharmaceutical composition is a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of the active ingredient of the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned T cell and tumor treatment product depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the bifunctional compound as the active ingredient is 1-1000 mg / kg / day, 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or greater than 500 mg / kg / day.

[0106] The present invention also provides a method for treating tumors, which comprises administering the aforementioned T cell receptor BMLF1-TCR, the aforementioned T cell receptor LMP1-TCR1, the aforementioned T cell receptor LMP1-TCR2, the aforementioned T cell receptor EBNA-3B-TCR, the aforementioned T cell receptor LMP2-TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned virus or the aforementioned T cell to a tumor patient.

[0107] In some embodiments, the amount administered is 1-1000 mg / kg / day. Specifically, the amount administered is 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or 500 mg-1000 mg / kg / day.

[0108] In some embodiments, the subject of the method can be a mammal; preferably, the subject of the method is a human.

[0109] In the present invention, a T cell receptor (TCR) is a molecule present on the surface of a T cell that is responsible for recognizing a peptide-MHC complex. In some embodiments, the TCR is a truncated or intact full-length TCR. In some embodiments, the TCR is a heterodimer composed of α and β chains. In some embodiments, the TCR may also be a single-chain TCR (scTCR).

[0110] In the present invention, the term "TCRα chain constant region" or "TCRβ chain constant region" includes the extracellular constant region, transmembrane region and intracellular constant region connected in sequence, wherein the extracellular constant region may include the hinge region of the TCRα chain and the TCRβ chain, and participate in the formation of disulfide bonds between the TCRα chain and the TCRβ chain; the transmembrane region also belongs to the constant region, and its functions include participating in the cell membrane anchoring of the TCRα chain and the TCRβ chain and the interaction with the CD3 subunit to form a TCR-CD3 complex; the possible functions of the intracellular constant region include participating in the conformational transition of the TCR-CD3 complex and signal transduction after TCR signal transduction.

[0111] In the present invention, the term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., TCR α-chain or β-chain (or gd chain and d chain) TCRs) involved in the binding of an immunoglobulin superfamily binding protein (e.g., TCR) to an antigen. The variable domains of the a chain and b chain of a natural TCR (Va and nb, respectively) generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. The Va domain is encoded by two independent DNA segments, the variable gene segment and the joining gene segment (VJ); the nb domain is encoded by three independent DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (VDJ). A single Va or nb domain may be sufficient to confer antigen binding specificity. In addition, a TCR that binds to a specific antigen can be separated from the antigen-binding TCR using the Va or nb domain to screen a library of complementary Va or nb domains, respectively.

[0112] In the present invention, the term "antigen" refers to a cell surface molecule or an intracellular molecule presented by an MHC molecule or an MHC-like molecule that can be bound by an antibody or a T cell receptor (TCR), including but not limited to polypeptide antigens (e.g., NYESO-1, AFP, and MART-1), lipid antigens (e.g., β-GlcCer, eLPA, and LPE), or polysaccharide antigens (e.g., CA199, CA72-4, and CA125). The antigen can be a tumor antigen, such as a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

[0113] As used herein, the term "isolated" refers to a material that has been removed from its natural state or otherwise artificially manipulated, such as the α chains, β chains, T cell receptors, and nucleic acids described herein. An isolated material may be substantially or essentially free of components that normally accompany it in its natural state, or may be manipulated to be in an artificial state with components that normally accompany it in its natural state. An isolated material may be in natural, chemically synthesized, or recombinant form. An isolated material may also or alternatively be in an enriched, partially purified, or purified form.

[0114] In the present invention, the term "polynucleotide" also referred to as "nucleotide" or "nucleic acid" refers to a nucleic acid chain composed of deoxyribonucleic acid, ribonucleic acid, modified nucleic acid or base, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0115] In the present invention, the term "nucleic acid construct" also referred to as "vector" refers to a vector capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, plasmids, cosmids, or phage vectors.

[0116] In the present invention, the term "host cell" refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of these cells.

[0117] In the present invention, to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleic acids at corresponding amino acid positions or nucleic acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleic acid at the corresponding position in the second sequence, then the molecules are identical at that position.

[0118] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, the percent identity between two nucleic acid sequences is determined using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0119] The percent identity between two amino acid or nucleic acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0120] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0121] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0122] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified herein, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0123] The sequence information of the present invention is as follows:

[0124] Example 1 TCR-T cell sorting and TCR sequencing

[0125] 1. Differentiation of human peripheral monocytes into dendritic cells (DCs) in vitro

[0126] (1) CD14+ monocytes were isolated from the peripheral blood of healthy donors using magnetic beads (Miltenyi Biotec, Cat. No. 130-050-201) and cultured in AIM-V medium (Thermo) containing 1% human serum, human GM-CSF (Peprotech, 100 ng / mL), and IL-4 (50 ng / mL) for 3 days. After changing the medium, the culture was continued for 2 days to induce the differentiation of monocytes into MoDCs.

[0127] (2) After 5 days of differentiation, cytokines such as TNF-α (10 ng / mL), IL-1β (10 ng / mL), IL-6 (10 ng / mL), and Prostaglandin E2 (Peprotech, 1 μg / mL) were added to induce DC maturation.

[0128] (3) After 24 hours, mature DCs were harvested and expression of mature DC markers was detected by flow cytometry. Phenotype of mature DCs: CD14-, CD11c+, CD40+, CD80+, CD83+, CD86+, HLA-ABC+, HLA-DR+. The obtained mature DCs were collected and used for subsequent loading with EBV antigen peptides.

[0129] 2. DC cells loaded with antigen peptides are co-stimulated with autologous CD8+ T cells and cultured

[0130] (1) Add 5 μg / ml of EBV antigen peptide (i.e., antigen peptide BMLF1, antigen peptide LMP1, antigen peptide EBNA3, or antigen peptide LMP2, which are synthesized based on the amino acid sequence) to the above-mentioned mature DCs. After sealing with Parafilm, place the cells in a MACSmix rotator and rotate and bind in a cell culture incubator for 4 hours. After the mDCs are incubated with the EBV antigen peptide, use an irradiator to irradiate the DCs loaded with the EBV antigen peptide at a dose of 40 Gy. After irradiation, collect the DCs loaded with the EBV antigen peptide by centrifugation and place them in a 15 ml centrifuge tube.

[0131] (2) PBMCs were obtained from the blood of healthy donors by Lymphoprep density gradient centrifugation. CD8+ T cells in PBMCs were isolated using the EasySepTM Human CD8 Positive Selection Kit. DCs loaded with EBV antigen peptides and CD8+ T cells were added to 12-well plates and cultured at a DC / T ratio of 1:2.5. IL-21 at a final concentration of 30 ng / ml and 5 μg / ml of EBV antigen peptides were added to the culture system.

[0132] (3) Place the 12-well plate in a 37°C, 5% CO2 cell culture incubator and incubate overnight. On Day 1, add 10 ng / ml of IL-2, IL-7, and IL-15 to each well. The plate is then placed in a cell culture incubator at 37°C, 5% CO2 for 10 days. Change the medium halfway every 3 days during the culture process.

[0133] (4) On Day 14, Irradiated EBV-peptide-loaded DCs were prepared and subjected to a second round of in vitro stimulation with cells in each culture system, and the stimulation was continued for 10 days.

[0134] (5) On Day 25, cells were aspirated from each well of the plate, washed twice with FACS buffer, and then CD8a-APC and PE-HLA-A0201-restricted-EBV-peptide-Tetramer (MBL company) were added. After mixing, the cells were incubated at 4°C in the dark for 30 min. After the incubation, the cells were washed three times with FACS buffer, and the cells were resuspended in 2 ml of FACS buffer containing 1× DAPI working solution and the CD8+ / tetramer+ (%) ratio was detected.

[0135] 3. EBV-antigen-specific T cell sorting and single-cell TCR sequencing

[0136] (1) The process of sorting EBV-antigen-specific-T cells is shown in Figure 1. After two rounds of in vitro stimulation of CD8+ T cells from healthy donors with autologous EBV-antigen-specific loaded DCs, the cells in the co-stimulation system were collected for flow cytometry analysis. The specific flow cytometry results are shown in Figure 2A. The proportions of CD8a+ / Tetramer+ T cells from healthy donors were BMLF1-0.34%, LMP1-2.52%, EBNA3B-0.4% and LMP2-0.15%, respectively, with obvious clustering. This indicates that EBV-antigen-specific-TCR-T cells were specifically amplified in two rounds of in vitro stimulation. Subsequently, the CD8+ / tetramer+ T cells in the co-culture system were specifically sorted using a flow cytometer and then sent to Jingneng Bio for 10×Genomics single-cell TCR+ transcriptome sequencing to obtain the TCR sequence and transcriptomic characteristics of CD8+ / tetramer+ T cells.

[0137] (2) After the single-cell sequencing was completed, the transcriptomic characteristics of the top ten TCRs were analyzed, and the TCR sequencing results and frequency ranking results of the cell population obtained by analysis are shown in Figure 2B. Subsequently, the TCR sequence with the highest frequency was optimized. The specific optimization content was to replace its TCR-C region, that is, the TCR constant region, with mTCR-C (wherein mTCR-C includes mTCRα-C and mTCRβ-C, the amino acid sequence of mTCRα-C is shown in SEQ ID No.55, and the amino acid sequence of mTCRβ-C is shown in SEQ ID No.56) to avoid TCR mismatch. The amino acid sequence of the TCRα chain in the T cell receptor BMLF1-TCR obtained is shown in SEQ ID NO.8; the amino acid sequence of the TCRβ chain in the T cell receptor BMLF1-TCR is shown in SEQ ID NO.4.

[0138] The amino acid sequence of the TCRα chain in the T cell receptor LMP1-TCR1 is shown in SEQ ID NO.16; the amino acid sequence of the TCRβ chain in the T cell receptor LMP1-TCR1 is shown in SEQ ID NO.12.

[0139] The amino acid sequence of the TCRα chain in the T cell receptor LMP1-TCR2 is shown in SEQ ID NO.24; the amino acid sequence of the TCRβ chain in the T cell receptor LMP1-TCR2 is shown in SEQ ID NO.20.

[0140] The amino acid sequence of the TCRα chain in the T cell receptor EBNA-3B-TCR is shown in SEQ ID NO.34; the amino acid sequence of the TCRβ chain in the T cell receptor EBNA-3B-TCR is shown in SEQ ID NO.30.

[0141] The amino acid sequence of the TCRα chain in the T cell receptor LMP2-TCR is shown in SEQ ID NO.43; the amino acid sequence of the TCRβ chain in the T cell receptor LMP2-TCR is shown in SEQ ID NO.39.

[0142] The amino acid sequences of CDRα1, CDRα2 and CDRα3 in the TCRα chain of the T cell receptor BMLF1-TCR are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively; the amino acid sequences of CDRβ1, CDRβ2 and CDRβ3 in the TCRβ chain are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0143] The amino acid sequences of CDRα1, CDRα2 and CDRα3 in the TCRα chain of the T cell receptor LMP1-TCR1 are shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively; the amino acid sequences of CDRβ1, CDRβ2 and CDRβ3 in the TCRβ chain are shown in SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11, respectively.

[0144] The amino acid sequences of CDRα1, CDRα2 and CDRα3 in the TCRα chain of the T cell receptor LMP1-TCR2 are shown in SEQ ID NO.21, SEQ ID NO.22 and SEQ ID NO.23, respectively; the amino acid sequences of CDRβ1, CDRβ2 and CDRβ3 in the TCRβ chain are shown in SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO.19, respectively.

[0145] The amino acid sequences of CDRα1, CDRα2 and CDRα3 in the TCRα chain of the T cell receptor EBNA-3B-TCR are shown in SEQ ID NO.31, SEQ ID NO.32 and SEQ ID NO.33, respectively; the amino acid sequences of CDRβ1, CDRβ2 and CDRβ3 in the TCRβ chain are shown in SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.29, respectively.

[0146] The amino acid sequences of CDRα1, CDRα2 and CDRα3 in the TCRα chain of the T cell receptor LMP2-TCR are shown in SEQ ID NO.40, SEQ ID NO.41 and SEQ ID NO.42, respectively; the amino acid sequences of CDRβ1, CDRβ2 and CDRβ3 in the TCRβ chain are shown in SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38, respectively.

[0147] P2A and Furin-cleavage (the amino acid sequence of the P2A-Furin-cleavage fusion peptide is shown in SEQ ID No. 57) are used to enable the simultaneous expression of the α and β chains of the TCR in a lentiviral expression vector, wherein the nucleotide sequence encoding the T cell receptor that simultaneously expresses the TCR α and β chains in the above-mentioned lentiviral expression vector is shown in any one of SEQ ID Nos. 58-62. Among them, the nucleotide sequence encoding the simultaneous expression of the TCRα and β chains of the T cell receptor BMLF1-TCR is shown in SEQ ID No. 58; the nucleotide sequence encoding the simultaneous expression of the TCRα and β chains of the T cell receptor LMP1-TCR1 is shown in SEQ ID No. 59; the nucleotide sequence encoding the simultaneous expression of the TCRα and β chains of the T cell receptor LMP1-TCR2 is shown in SEQ ID No. 60; the nucleotide sequence encoding the simultaneous expression of the TCRα and β chains of the T cell receptor EBNA-3B-TCR is shown in SEQ ID No. 61; and the nucleotide sequence encoding the simultaneous expression of the TCRα and β chains of the T cell receptor LMP2-TCR is shown in SEQ ID No. 62. The above-mentioned peptides and polynucleotides can be obtained by chemical synthesis or homologous recombination when the sequences are known.

[0148] Example 2 EBV-antigen-specific-TCR-T cell binding experiment in vitro

[0149] 1. Preparation of EBV-antigen-specific-TCR-T cells

[0150] (1) The optimized EBV-antigen-TCR obtained in Example 1 was used to construct a lentiviral expression vector, and then the EBV-antigen-TCR was packaged with lentivirus. The specific operation of virus packaging was as follows: 293T cells were digested and passaged, and the cell density was observed on the day of transfection. Transfection was performed when the cell density was 80-90% full. No culture medium change was required before transfection. Opti-MEM needed to be preheated in a 37-degree water bath, and the Fugene-HD transfection reagent needed to be restored to room temperature before use. It needed to be shaken before use. The complex components for transfection of each bottle of T75 were as follows: pHR-SFFV-TCR-Puro plasmid containing the target TCR: 10ug; pMD2.G (envelope plasmid): 5ug; pSPAX2 (core enzyme): 7.5ug. After adding Opti-MEM to 1mL, mix well; add 56ul Fugene-HD transfection reagent, pipette normally 5-6 times, let it stand at room temperature for 15 minutes, and then add it to the T75 bottle. Viral supernatants were then collected 48 and 72 hours after collection. After collection, the supernatants were filtered through a 0.45 μm filter membrane (PES material) for use. EBV-antigen-specific-TCR-TCR lentiviral fluids were collected 48 and 72 hours after viral packaging.

[0151] (2) Jurkat T cells were infected with EBV-antigen-TCR lentiviral particles by adding 10 μg / ml polybrene and centrifuging at 800 × g for 90 min at 30°C. After centrifugation, the cells were placed in a cell culture incubator at 37°C, 5% CO2 for 24 h, and then replaced with fresh 1640 medium for further culture.

[0152] (3) 20 μl of CD3 / 28 beads (Genscript) and 20 ng / ml of IL-2 were added to CD8+ T cells from healthy donors. After activating the T cells for 48 h, 10 μg / ml of polybrene was added for the first round of infection with EBV-antigen-TCR lentivirus. After centrifugation, the cells were cultured in a cell incubator at 37°C, 5% CO2 for 24 h. Subsequently, a second round of infection with EBV-antigen-TCR lentivirus was performed.

[0153] 2. FACS detection of EBV-antigen-TCR-T cell binding specificity

[0154] EBV-antigen cells prepared above were stained for mTCRβC+ / Tetramer. Flow cytometry results showed distinct cell clusters of EBV-BMLF1 / LMP1 / EBNA3B Tetramer+ / mTCRβC+ Jurkat T cells (Figure 3A) and CD8+ / Tetramer+ primary CD8+ T cells (Figure 3B). These results demonstrate that the EBV-BMLF1 / LMP1 / EBNA3B-TCR screened and identified in vitro can be normally expressed in Jurkat T cell lines and primary CD8+ T cells and can specifically bind to the HLA-restricted EBV-Tetramer.

[0155] Example 3 In vitro activation experiment of EBV-antigen-TCR-T cells

[0156] The in vitro activation assay for EBV-antigen-T cells, i.e., the IFNγ-Elispot assay, includes the following steps:

[0157] (1) Directly differentiating donor autologous monocytes into DCs in vitro, loading the DCs with EBV antigen peptides as in Example 1, and co-culturing the EBV-antigen-specific-TCR-T cells prepared in Example 2 with the DCs loaded with EBV antigen peptides in vitro;

[0158] (2) After 24 h of in vitro co-culture, the cells were discarded and washed five times with 200 μl of DPBS to completely remove the cells. 100 μl of 1 μg / ml detection antibody 7-B6-1-biotin was added to the culture system and incubated at 37°C for 2 h. Subsequently, 100 μl of streptavidin-HRP (3420-2H, MABTECH) was added and incubated at room temperature for 1 h.

[0159] (3) After washing five times with 200 μl DPBS, 100 μl TMB substrate was added to each well until complete spots appeared in the wells. Sterile water was then added to interrupt the color development process.

[0160] The results are shown in Figure 4 . DCs loaded with EBV antigen peptides can specifically activate EBV-antigen-specific-TCR-T cells in vitro. After activation, the IFNγ expression level of EBV-antigen-specific-TCR-T cells was significantly increased ( Figure 4A-C ). Therefore, these results indicate that the EBV-BMLF1 / LMP1 / EBNA3B-TCRs obtained in this screening and identification can specifically recognize EBV-BMLF1 / LMP1 / EBNA3B antigen peptides, respectively.

[0161] Example 4 In vitro killing experiment of EBV-antigen-TCR-T cells

[0162] The in vitro killing assay of EBV-TCR-T cells, also known as the Incucyte killing assay, includes the following steps:

[0163] (1) In vitro, EBV-BMLF1 / LMP1 / EBNA3B-TCR-T effector cells and K562-A1101-BMLF1 / LMP1 / EBNA3B target cells were constructed by lentiviral infection of T cells or K562-A1101 cells. + T cells and K562-A1101 control cells or K562-A1101-BMLF1 / LMP1 / EBNA3B target cells were added to 96-well plates for co-culture;

[0164] (2) Dissolve by adding 100 μL of DPBS Annexin V Dyes (Sartorius). Then Annexin V Dyes reagent was diluted in complete culture medium to a final dilution of 1:200.

[0165] (3) Place the 96-well plate containing cells into the Incucyte (Sartorius) live cell analyzer and monitor cell apoptosis using appropriate fluorescence channels. Specific parameters are as follows: A. Observation magnification: 20× objective lens; B. Channel selection: phase contrast Green|Red|Orange|NIRc; C. Scan type: 2-4 images per well; D. Scan interval: scan once every 30 minutes until the killing efficiency is completed after 48 hours of co-culture.

[0166] (4) After the test, the in vitro killing efficiency of TCR-T was analyzed using the Incucyte live cell analysis system software (Sartorius).

[0167] The results are shown in Figure 5. This example utilizes the Incucyte high-throughput, long-term dynamic imaging and analysis system to monitor target cell killing by EBV-BMLF1 / LMP1 / EBNA3B-TCR-T cells in real time. The results show that within 48 hours of co-culture, TCR-T cells with an effector-target ratio of 5:1 were more efficient at killing target cells that were HLA-A*11:01-positive and expressed the EBV-BMLF1 / LMP1 / EBNA3B antigen, compared to negative control CD8+ T cells not transfected with EBV-BMLF1 / LMP1 / EBNA3B-TCR (Figures 5A-D).

[0168] Example 5 In vivo killing experiment of EBV-antigen-TCR-T cells

[0169] The in vivo killing experiment of EBV-EBAN3B-TCR-T cells, also known as the CDX animal experiment, includes the following steps:

[0170] (1) Six-week-old Jicui Yaokang NCG female mice were selected and injected into the lateral tail vein with 2E6 K562 cell line carrying HLA-A*11:01 and EBNA3B antigens and expressing Luciferase reporter gene.

[0171] (2) Three days after transplantation of the K562-HLA-EBNA3B-luci cell line, 1E7 TCR-T cells were injected into the lateral tail vein, and control T lymphocytes without TCR gene transfection were used as negative controls.

[0172] (3) In vivo imaging of small animals was performed on days 7, 15, and 27 after tumor cell inoculation, and the weight changes and survival of mice were detected to evaluate the in vivo killing effect of EBV-EBAN3B-TCR-T cells on target cells.

[0173] The results are shown in Figure 6. This example used an in vivo CDX model to evaluate the effectiveness of EBV-EBAN3B-TCR-T cells in killing target cells (Figure 6A). In vivo imaging results showed that mice in the saline group and the mock T cell-reinfused group showed significant tumor cell growth within 27 days after tumor cell reinfusion, while some mice (3 / 4) in the EBV-EBAN3B-TCR-T cell-reinfused group showed no significant tumor growth (Figure 6B). Furthermore, the total photon count and average photon count intensity of luciferase in the EBV-EBAN3B-TCR-T group were significantly lower than those in the saline group and the mock T control group (Figures 6C-D). The CDX experimental results demonstrate that HLA-A*11:01-restricted EBV-EBAN3B-TCR-T cells can efficiently and specifically kill K562-HLA-A*11:01-EBAN3B-luciferase target cells in vivo.

[0174] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A T cell receptor, characterized in that The T cell receptor is selected from the group consisting of T cell receptor BMLF1-TCR, T cell receptor LMP1-TCR1, T cell receptor LMP1-TCR2, T cell receptor EBNA-3B-TCR or T cell receptor LMP2-TCR, wherein: I) T cell receptor BMLF1-TCR comprises a TCRα chain variable region and a TCRβ chain variable region; the TCRα chain variable region comprises peptide segments CDRα1, CDRα2 and CDRα3; the TCRβ chain variable region comprises peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.7, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.3; II) the variable region of the TCRα chain of the T cell receptor LMP1-TCR1 comprises peptide segments CDRα1, CDRα2 and CDRα3; the variable region of the TCRβ chain of the T cell receptor LMP1-TCR1 comprises peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.15, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.11; III) the variable region of the TCRα chain of the T cell receptor LMP1-TCR2 comprises peptide segments CDRα1, CDRα2 and CDRα3; the variable region of the TCRβ chain of the T cell receptor LMP1-TCR2 comprises peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.23, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.19; IV) the variable region of the TCRα chain of the T cell receptor EBNA-3B-TCR comprises peptide segments CDRα1, CDRα2 and CDRα3; the variable region of the TCRβ chain of the T cell receptor EBNA-3B-TCR comprises peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.33, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.29; V) The variable region of the TCRα chain of the T cell receptor LMP2-TCR contains peptide segments CDRα1, CDRα2 and CDRα3; the variable region of the TCRβ chain of the T cell receptor LMP2-TCR contains peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of the CDRα3 is shown in SEQ ID NO.42, and the amino acid sequence of the CDRβ3 is shown in SEQ ID NO.

38.

2. The T cell receptor according to claim 1, characterized in that Also includes any of the following features: A) the amino acid sequence of CDRα1 in the TCRα chain variable region of the T cell receptor BMLF1-TCR is as shown in SEQ ID NO.5 or the amino acid sequence of CDRα2 is as shown in SEQ ID NO.6; and / or, the amino acid sequence of CDRβ1 in the TCRβ chain variable region is as shown in SEQ ID NO.1 or the amino acid sequence of CDRβ2 is as shown in SEQ ID NO.2; B) the amino acid sequence of CDRα1 in the TCRα chain variable region of the T cell receptor LMP1-TCR1 is shown in SEQ ID NO.13 or the amino acid sequence of CDRα2 is shown in SEQ ID NO.14; and / or, the TCRβ chain variable region The amino acid sequence of CDRβ1 is shown in SEQ ID NO.9 or the amino acid sequence of CDRβ2 is shown in SEQ ID NO.10; C) the amino acid sequence of CDRα1 in the TCRα chain variable region of the T cell receptor LMP1-TCR2 is shown in SEQ ID NO.21 or the amino acid sequence of CDRα2 is shown in SEQ ID NO.22; and / or, the amino acid sequence of CDRβ1 in the TCRβ chain variable region is shown in SEQ ID NO.17 or the amino acid sequence of CDRβ2 is shown in SEQ ID NO.18; D) the amino acid sequence of CDRα1 in the TCRα chain variable region of the T cell receptor EBNA-3B-TCR is as shown in SEQ ID NO.31 or the amino acid sequence of CDRα2 is as shown in SEQ ID NO.32; and / or, the amino acid sequence of CDRβ1 in the TCRβ chain variable region is as shown in SEQ ID NO.27 or the amino acid sequence of CDRβ2 is as shown in SEQ ID NO.28; E) The amino acid sequence of CDRα1 in the variable region of the TCRα chain of the T cell receptor LMP2-TCR is as shown in SEQ ID NO.40 or the amino acid sequence of CDRα2 is as shown in SEQ ID NO.41; and / or, the amino acid sequence of CDRβ1 in the variable region of the TCRβ chain is as shown in SEQ ID NO.36 or the amino acid sequence of CDRβ2 is as shown in SEQ ID NO.

37.

3. The T cell receptor according to claim 1, characterized in that Also includes any of the following features: A1) the amino acid sequence of the TCRα chain variable region of the T cell receptor BMLF1-TCR is shown in SEQ ID NO.45; and / or the amino acid sequence of the TCRβ chain variable region is shown in SEQ ID NO.46; B1) the amino acid sequence of the TCRα chain variable region of the T cell receptor LMP1-TCR1 is shown in SEQ ID NO.47; and / or the amino acid sequence of the TCRβ chain variable region is shown in SEQ ID NO.48; C1) the amino acid sequence of the TCRα chain variable region of the T cell receptor LMP1-TCR2 is shown in SEQ ID NO.49; and / or the amino acid sequence of the TCRβ chain variable region is shown in SEQ ID NO.50; D1) the amino acid sequence of the TCRα chain variable region of the T cell receptor EBNA-3B-TCR is shown in SEQ ID NO.51; and / or the amino acid sequence of the TCRβ chain variable region is shown in SEQ ID NO.52; E1) The amino acid sequence of the TCRα chain variable region of the T cell receptor LMP2-TCR is shown in SEQ ID NO.53; and / or, the amino acid sequence of the TCRβ chain variable region is shown in SEQ ID NO.

54.

4. The T cell receptor according to claim 1, characterized in that Also includes any of the following features: A2) the amino acid sequence of the TCRα chain in the T cell receptor BMLF1-TCR is shown in SEQ ID NO.8; and / or the amino acid sequence of the TCRβ chain in the T cell receptor BMLF1-TCR is shown in SEQ ID NO.4; B2) the amino acid sequence of the TCRα chain in the T cell receptor LMP1-TCR1 is shown in SEQ ID NO.16; and / or the amino acid sequence of the TCRβ chain in the T cell receptor LMP1-TCR1 is shown in SEQ ID NO.12; C2) the amino acid sequence of the TCRα chain in the T cell receptor LMP1-TCR2 is shown in SEQ ID NO.24; and / or the amino acid sequence of the TCRβ chain in the T cell receptor LMP1-TCR2 is shown in SEQ ID NO.20; D2) The amino acid sequence of the TCRα chain in the T cell receptor EBNA-3B-TCR is shown as SEQ ID NO.34; and / or, the amino acid sequence of the TCRβ chain in the T cell receptor EBNA-3B-TCR is shown as SEQ ID NO.30; E2) The amino acid sequence of the TCRα chain in the T cell receptor LMP2-TCR is shown as SEQ ID NO.43; and / or, the amino acid sequence of the TCRβ chain in the T cell receptor LMP2-TCR is shown as SEQ ID NO.

39.

5. An EBV antigen peptide, characterized in that: The EBV antigenic peptide comprises antigenic peptide BMLF1, antigenic peptide LMP1, antigenic peptide EBNA3 or antigenic peptide LMP2; The amino acid sequence of the antigen peptide BMLF1 is shown in SEQ ID No. 25; or, The amino acid sequence of the antigen peptide LMP1 is shown in SEQ ID No. 26; or, The amino acid sequence of the antigen peptide EBNA3 is shown in SEQ ID No.35; or, The amino acid sequence of the antigen peptide LMP2 is shown in SEQ ID No.

44.

6. A biomaterial, characterized in that The biological material is selected from any of the following: a) a polynucleotide encoding the T cell receptor according to any one of claims 1 to 4; or, the polynucleotide encoding the EBV antigen peptide according to claim 5; b) a nucleic acid construct, comprising the polynucleotide described in a) and a plasmid backbone; c) a virus, said virus comprising the polynucleotide described in a); d) A cell comprising the T cell receptor according to any one of claims 1 to 4, the EBV antigen peptide according to claim 5, the polynucleotide according to a), the nucleic acid construct according to b), or the virus according to c).

7. The biomaterial according to claim 6, characterized in that The plasmid backbone of the nucleic acid construct is a plasmid backbone of a lentivirus, an adenovirus or an adeno-associated virus; preferably, the plasmid backbone is selected from pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, p Any one or more of LKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP / VSV-G, pENTR / U6, pLenti6 / BLOCK-iT-DEST, pcDNA1.2 / V5-GW / lacZ, pLenti6.2 / N-Lumio / V5-DEST, pGCSIL-GFP, pHR-SFFV-Puro; The cell is a T cell; preferably, the T cell is a TCR-T cell; and / or, the T cell is selected from helper T cells, suppressor T cells, effector T cells, cytotoxic T cells, delayed allergic reaction T cells, natural T cells or memory T cells; preferably, the T cell is a Jurkat T cell or a CD8+T cell.

8. Use of the T cell receptor according to any one of claims 1 to 4, the EBV antigen peptide according to claim 5, or the biomaterial according to any one of claims 6 to 7 in the preparation of tumor treatment products, anti-tumor peptide vaccine products, or in vitro generated T cell receptor products.

9. The use according to claim 8, characterized in that The tumor is selected from one or more of adrenal cortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, lymphoid tumors, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial carcinoma, chronic myeloid leukemia, lung cancer, anal cancer, and retinoblastoma.

10. A tumor treatment product, characterized in that: The tumor treatment product comprises the T cell receptor according to any one of claims 1 to 4, the EBV antigen peptide according to claim 5 or the biological material according to any one of claims 6 to 7 and a pharmaceutically acceptable excipient.

11. A method for treating tumors, characterized in that: The method comprises administering to the subject an effective dose of the T cell receptor according to any one of claims 1 to 4, the EBV antigen peptide according to claim 5, the biomaterial according to any one of claims 6 to 7, or the tumor treatment product according to claim 10.

12. The method according to claim 11, characterized in that The method comprises one or more of the following features: 1) The amount of administration is 1-1000 mg / kg / day; 2) The subject is a mammal; preferably, the subject is a human.

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