A T cell receptor, a preparation method thereof and uses thereof

Abstract

The present invention relates to the field of biomedicine, and particularly to a T cell receptor and its preparation method and use. The T cell receptor EBV-TCR of the present invention comprises a variable region of the TCRα chain and a variable region of the TCRβ chain; the variable region of the TCRα chain contains peptide segments CDRα1, CDRα2, and CDRα3; the variable region of the TCRβ chain contains peptide segments CDRβ1, CDRβ2, and CDRβ3; the amino acid sequence of CDRα3 is as shown in SEQ ID NO.7, and the amino acid sequence of CDRβ3 is as shown in SEQ ID NO.3. The TCR of the present invention has high specificity, and binding to T cells can significantly activate cytokine release, providing a solid theoretical and experimental basis for the development of TCR-T therapy targeting Epstein-Barr virus antigens.

Description

Technical Field

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

[0002] Epstein-Barr virus (EBV) is a common double-stranded DNA herpesvirus. Approximately 90% of the population worldwide is infected with EBV (Tzellos, S., Farrell, P. J. Epstein-barr virus sequence variation - biology and disease. Pathogens, 2012, 1(2): 156 - 174). Research has confirmed that the occurrence of a variety of diseases is closely related to EBV infection, including infections after hematopoietic stem cell transplantation (HSCT), lymphoma, nasopharyngeal carcinoma, etc. (Taylor, G. S., Long, H. M., Brooks, J. M., Rickinson, A. B., Hislop, A. D. The immunology of Epstein-Barr virus-induced disease. Annu Rev Immunol, 2015, 33: 787 - 821). In addition, 1 - 2% of all human tumors are caused by EBV, 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, treatment strategies targeting EBV are of great significance.

[0003] The main stages of Epstein - Barr virus (EBV) infection are divided into the proliferative phase and the latent phase. EBV expresses different proteins at different times. The proteins expressed at the beginning of the proliferative phase and the proteins expressed during the latent phase (mainly including 6 nuclear proteins EBNA1, 2, 3A, 3B, and 3C, EBNA - LP, and 3 latent membrane proteins LMP1, LMP2A, LMP2B, where LMP2B is a truncated isoform of LMP2A) are considered to play a role in tumor formation. And EBV - related tumor cells also express EBV antigen proteins, and the main way for the human body to fight against the virus is T - cell - mediated killing. Therefore, targeting EBV antigen proteins is an ideal target for T - cell - mediated immunotherapy.

[0004] TCR - T therapy (T cell receptor engineered T cell therapy) is an adoptive T - cell therapy that has developed rapidly in recent years. Different from CAR - T therapy (Chimeric antigen receptor T cell therapy) which can only recognize membrane - surface targets, TCR - T can recognize targets from both inside and outside the cell with the help of the HLA (Human leukocyte antigen) antigen - presenting system. At the same time, TCR - T has higher sensitivity. In theory, a single antigen on the target cell can activate TCR - T cells and exert a killing function. TCR - T therapy shows great potential in the treatment of blood and solid tumors.

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

[0006] In view of the above - mentioned disadvantages of the prior art, the purpose of the present invention is to provide a T - cell receptor and its preparation method and use to solve the problems in the prior art.

[0007] To achieve the above - mentioned purpose and other related purposes, the present invention provides a T - cell receptor, which is selected from 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:

[0008] I) The T cell receptor BMLF1-TCR comprises a variable region of the TCRα chain and a variable region of the TCRβ chain; the variable region of the TCRα chain contains peptide segments CDRα1, CDRα2, and CDRα3; the variable region of the TCRβ chain contains peptide segments CDRβ1, CDRβ2, and CDRβ3; the amino acid sequence of CDRα3 is as shown in SEQ ID NO.7, and the amino acid sequence of CDRβ3 is as shown in SEQ ID NO.3;

[0009] II) The variable region of the TCRα chain of the T cell receptor LMP1-TCR1 contains peptide segments CDRα1, CDRα2, and CDRα3; the variable region of the TCRβ chain of the T cell receptor LMP1-TCR1 contains peptide segments CDRβ1, CDRβ2, and CDRβ3; the amino acid sequence of CDRα3 is as shown in SEQ ID NO.15, and the amino acid sequence of CDRβ3 is as shown in SEQ ID NO.11;

[0010] III) The variable region of the TCRα chain of the T cell receptor LMP1-TCR2 contains peptide segments CDRα1, CDRα2, and CDRα3; the variable region of the TCRβ chain of the T cell receptor LMP1-TCR2 contains peptide segments CDRβ1, CDRβ2, and CDRβ3; the amino acid sequence of CDRα3 is as shown in SEQ ID NO.23, and the amino acid sequence of CDRβ3 is as shown in SEQ ID NO.19;

[0011] IV) The variable region of the TCRα chain of the T cell receptor EBNA-3B-TCR contains peptide segments CDRα1, CDRα2, and CDRα3; the variable region of the TCRβ chain of the T cell receptor EBNA-3B-TCR contains peptide segments CDRβ1, CDRβ2, and CDRβ3; the amino acid sequence of CDRα3 is as shown in SEQ ID NO.33, and the amino acid sequence of CDRβ3 is as shown in SEQ ID NO.29;

[0012] 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 CDRα3 is as shown in SEQ ID NO.42, and the amino acid sequence of CDRβ3 is as shown in SEQ ID NO.38.

[0013] The present invention also provides an EBV antigen peptide, which comprises antigen peptide BMLF1, antigen peptide LMP1, antigen peptide EBNA3, or antigen peptide LMP2;

[0014] The amino acid sequence of the antigenic peptide BMLF1 is as shown in SEQ ID No. 25; or,

[0015] The amino acid sequence of the antigenic peptide LMP1 is as shown in SEQ ID No. 26; or,

[0016] The amino acid sequence of the antigenic peptide EBNA3 is as shown in SEQ ID No. 35; or,

[0017] The amino acid sequence of the antigenic peptide LMP2 is as shown in SEQ ID No. 44.

[0018] The present invention also provides a preparation method for 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 comprises the following steps:

[0019] 1) Loading the aforementioned EBV antigenic peptide onto in vitro differentiated dendritic cells;

[0020] 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.

[0021] The present invention also provides a biological material, which comprises polynucleotide, nucleic acid construct, virus or cell:

[0022] 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 antigenic peptide;

[0023] The nucleic acid construct comprises the aforementioned polynucleotide and a plasmid backbone.

[0024] The virus contains the aforementioned polynucleotide.

[0025] 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.

[0026] 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 EBV antigen peptide or the aforementioned biomaterial in the preparation of tumor treatment products.

[0027] 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 cells and pharmaceutically acceptable excipients.

[0028] The present invention also provides the use of the aforementioned EBV antigen peptide or the biomaterial as described above in the preparation of the following products:

[0029] 1) Anti-tumor peptide vaccine products;

[0030] 2) In vitro generated T cell receptor products.

[0031] The present invention also provides a method for treating tumors, which is to use 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 cells for tumor patients.

[0032] As described above, a T cell receptor of the present invention, its preparation method and use have the following beneficial effects:

[0033] Combined with the distribution characteristics of HLA subtypes in the Chinese population, the present invention selects four EBV antigen peptides (BMLF1, LMP1, LMP2, EBNA3B) corresponding to the two most frequent HLA subtypes (HLA-A*11:01, HLA-A*02:01) in the Chinese population and the European and American populations, systematically screens for EBV antigen-specific TCRs, and comprehensively verifies the TCR functions respectively. The T cell receptor of the present invention will lay a direct foundation for the development of TCR-T therapy targeting EBV antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shown is the sorting flow chart of the TCR-T cells of the present invention.

[0035] Figure 2 Shown is a schematic diagram of the sorting and single-cell sequencing results of the TCR-T cells of the present invention.

[0036] Figure 3 Shown is a schematic diagram of the in vitro binding experiment results of the TCR-T cells of the present invention.

[0037] Figure 4 Shown is a schematic diagram of the in vitro activation experiment results of the TCR-T cells of the present invention.

[0038] Figure 5 Shown is a schematic diagram of the in vitro killing experiment results of the TCR-T cells of the present invention.

[0039] Figure 6 Shown is a schematic diagram of the in vitro killing experiment results of some TCR-T cells of the present invention. Detailed implementation manners

[0040] The present invention provides a T cell receptor BMLF1-TCR or a fragment thereof. The T cell receptor BMLF1-TCR comprises a variable region of TCR α chain and a variable region of TCR β chain; the variable region of TCR α chain contains peptide segments CDRα1, CDRα2 and CDRα3; the variable region of TCR β chain contains peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of CDRα3 is as shown in SEQ ID NO.7, and the amino acid sequence of CDRβ3 is as shown in SEQ ID NO.3.

[0041] In some specific implementation manners, the amino acid sequence of CDRα1 in the variable region of TCR α chain is as shown in SEQ ID NO.5 or the amino acid sequence of CDRα2 is as shown in SEQ ID NO.6.

[0042] In some specific implementation manners, the amino acid sequence of CDRβ1 in the variable region of TCR β chain is as shown in SEQ ID NO.1 or the amino acid sequence of CDRβ2 is as shown in SEQ ID NO.2.

[0043] The present invention also provides a T cell receptor LMP1-TCR1 or a fragment thereof. The variable region of TCR α chain of the T cell receptor LMP1-TCR1 contains peptide segments CDRα1, CDRα2 and CDRα3; the variable region of TCR β chain of the T cell receptor LMP1-TCR1 contains peptide segments CDRβ1, CDRβ2 and CDRβ3; the amino acid sequence of CDRα3 is as shown in SEQ ID NO.15, and the amino acid sequence of CDRβ3 is as shown in SEQ ID NO.11.

[0044] In some specific implementation manners, the amino acid sequence of CDRα1 in the variable region of TCR α chain is as shown in SEQ IDNO.13 or the amino acid sequence of CDRα2 is as shown in SEQ ID NO.14.

[0045] In some specific embodiments, the amino acid sequence of CDRβ1 in the variable region of the TCRβ chain is as shown in SEQ ID NO.9 or the amino acid sequence of CDRβ2 is as shown in SEQ ID NO.10.

[0046] The present invention also provides a T cell receptor LMP1-TCR2 or a fragment thereof. The variable region of the TCRα chain of the T cell receptor LMP1-TCR2 contains peptide segments CDRα1, CDRα2, and CDRα3; the variable region of the TCRβ chain of the T cell receptor LMP1-TCR2 contains peptide segments CDRβ1, CDRβ2, and CDRβ3; the amino acid sequence of CDRα3 is as shown in SEQ ID NO.23, and the amino acid sequence of CDRβ3 is as shown in SEQ ID NO.19.

[0047] In some specific embodiments, the amino acid sequence of CDRα1 in the variable region of the TCRα chain is as shown in SEQ ID NO.21 or the amino acid sequence of CDRα2 is as shown in SEQ ID NO.22.

[0048] In some specific embodiments, the amino acid sequence of CDRβ1 in the variable region of the TCRβ chain is as shown in SEQ ID NO.17 or the amino acid sequence of CDRβ2 is as shown in SEQ ID NO.18.

[0049] The present invention also provides a T cell receptor EBNA-3B-TCR or a fragment thereof. The variable region of the TCRα chain of the T cell receptor EBNA-3B-TCR contains peptide segments CDRα1, CDRα2, and CDRα3; the variable region of the TCRβ chain of the T cell receptor EBNA-3B-TCR contains peptide segments CDRβ1, CDRβ2, and CDRβ3; the amino acid sequence of CDRα3 is as shown in SEQ ID NO.33, and the amino acid sequence of CDRβ3 is as shown in SEQ ID NO.29.

[0050] In some specific embodiments, the amino acid sequence of CDRα1 in the variable region of the TCRα chain is as shown in SEQ ID NO.31 or the amino acid sequence of CDRα2 is as shown in SEQ ID NO.32.

[0051] In some specific embodiments, the amino acid sequence of CDRβ1 in the variable region of the TCRβ chain is as shown in SEQ ID NO.27 or the amino acid sequence of CDRβ2 is as shown in SEQ ID NO.28.

[0052] The present invention also provides a T cell receptor LMP2-TCR or a fragment thereof. 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 CDRα3 is as shown in SEQ ID NO.42, and the amino acid sequence of CDRβ3 is as shown in SEQ ID NO.38.

[0053] In some specific embodiments, the amino acid sequence of CDRα1 in the variable region of the TCRα chain is as shown in SEQ ID NO.40 or the amino acid sequence of CDRα2 is as shown in SEQ ID NO.41.

[0054] In some specific embodiments, 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.

[0055] In some specific embodiments, the HLA subtypes targeted by the foregoing T cell receptor BMLF1-TCR, the foregoing T cell receptor LMP1-TCR1, the foregoing T cell receptor LMP1-TCR2, the foregoing T cell receptor EBNA-3B-TCR, or the foregoing T cell receptor LMP2-TCR include HLA-A*02:01 type and HLA-A*11:01 type. More specifically, the HLA subtype targeted by the foregoing T cell receptor BMLF1-TCR, the foregoing T cell receptor LMP1-TCR1, or the foregoing T cell receptor LMP1-TCR2 is HLA-A*02:01 type; or, the HLA subtype targeted by the foregoing T cell receptor EBNA-3B-TCR is HLA-A*11:01 type; or, the HLA subtype targeted by the foregoing T cell receptor LMP2-TCR is HLA-A*02:01 type.

[0056] CDR (Complementarity Determining Region) generally refers to the region in an antibody or receptor that can form complementarity with an antigenic determinant in terms of spatial structure. The variability in an antibody or receptor is usually not evenly distributed throughout the variable region of the entire antibody or receptor. The variable regions of the α-chain and β-chain of a monoclonal antibody or receptor usually each have three hypervariable regions (HVRs). These regions can usually form complementarity with an antigenic determinant in terms of spatial structure, so the hypervariable regions are also called complementarity determining regions (CDRs). That is, the variable region of the α-chain usually includes three complementarity determining regions, namely CDRα1, CDRα2, and CDRα3, and the variable region of the β-chain usually also includes three complementarity determining regions, namely CDRβ1, CDRβ2, and CDRβ3.

[0057] In some specific embodiments, the variable regions of the α-chain and β-chain may further include framework regions. The framework regions 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 substituting, deleting, or adding one or more (specifically, it may be 1 - 50, 1 - 30, 1 - 20, 1 - 10, 1 - 5, or 1 - 3) amino acids. This framework region sequence may have a homology of 80%, 85%, 90%, 93%, 95%, 97%, or more than 99% with the framework region sequence of the human monoclonal antibody variable region sequence.

[0058] In some specific embodiments, the amino acid sequence of the TCR α-chain variable region of the T cell receptor BMLF1-TCR is as shown in SEQ ID NO.45; and / or, the amino acid sequence of the TCR β-chain variable region of the T cell receptor BMLF1-TCR is as shown in SEQ ID NO.46.

[0059] In some specific embodiments, the amino acid sequence of the TCR α-chain variable region of the T cell receptor LMP1-TCR1 is as shown in SEQ ID NO.47; and / or, the amino acid sequence of the TCR β-chain variable region of the T cell receptor LMP1-TCR1 is as shown in SEQ ID NO.48.

[0060] In some specific embodiments, the amino acid sequence of the TCR α-chain variable region of the T cell receptor LMP1-TCR2 is as shown in SEQ ID NO.49; and / or, the amino acid sequence of the TCR β-chain variable region of the T cell receptor LMP1-TCR2 is as shown in SEQ ID NO.50.

[0061] In some specific embodiments, the amino acid sequence of the variable region of the TCRα chain of the T cell receptor EBNA-3B-TCR is as shown in SEQ ID NO.51; and / or, the amino acid sequence of the variable region of the TCRβ chain of the T cell receptor EBNA-3B-TCR is as shown in SEQ ID NO.52.

[0062] In some specific embodiments, the amino acid sequence of the variable region of the TCRα chain of the T cell receptor LMP2-TCR is as shown in SEQ ID NO.53; and / or, the amino acid sequence of the variable region of the TCRβ chain of the T cell receptor LMP2-TCR is as shown in SEQ ID NO.54.

[0063] In some specific embodiments, the TCRα chain variable 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%, 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 TCRβ chain variable 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%, 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 NO.54.

[0064] 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 TCRα chain constant region or a TCRβ chain constant region.

[0065] In some specific embodiments, the amino acid sequence of the TCRα chain constant region is as shown in SEQ ID NO.55; and / or, the amino acid sequence of the TCRβ chain constant region is as shown in SEQ ID NO.56.

[0066] In some specific embodiments, the constant region of the TCR α chain 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%, at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO. 55; the constant region of the TCR β chain 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%, at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO. 56.

[0067] In some specific embodiments, the amino acid sequence of the TCR α chain in the T cell receptor BMLF1-TCR is as shown in SEQ ID NO. 8; the amino acid sequence of the TCR β chain in the T cell receptor BMLF1-TCR is as shown in SEQ ID NO. 4.

[0068] In some specific embodiments, the amino acid sequence of the TCR α chain in the T cell receptor LMP1-TCR1 is as shown in SEQ ID NO. 16; the amino acid sequence of the TCR β chain in the T cell receptor LMP1-TCR1 is as shown in SEQ ID NO. 12.

[0069] In some specific embodiments, the amino acid sequence of the TCR α chain in the T cell receptor LMP1-TCR2 is as shown in SEQ ID NO. 24; the amino acid sequence of the TCR β chain in the T cell receptor LMP1-TCR2 is as shown in SEQ ID NO. 20.

[0070] In some specific embodiments, the amino acid sequence of the TCR α chain in the T cell receptor EBNA-3B-TCR is as shown in SEQ ID NO. 34; the amino acid sequence of the TCR β chain in the T cell receptor EBNA-3B-TCR is as shown in SEQ ID NO. 30.

[0071] In some specific embodiments, the amino acid sequence of the TCR α chain in the T cell receptor LMP2-TCR is as shown in SEQ ID NO. 43; the amino acid sequence of the TCR β chain in the T cell receptor LMP2-TCR is as shown in SEQ ID NO. 39.

[0072] In some specific embodiments, in the case of having the aforementioned TCRα and TCRβ chain variable region CDR sequences, the TCRα chain may further 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%, 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 may further 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%, 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 NO.39.

[0073] In some specific embodiments, the TCRα chain is selected from a human TCRα chain, a humanized TCRα chain, a chimeric TCRα chain or a murine TCRα chain; the TCRβ chain is selected from a human TCRβ chain, a humanized TCRβ chain, a chimeric TCRβ chain or a murine TCRβ chain. Wherein, "chimeric TCRα chain" or "chimeric TCRβ" means that the TCRα chain or TCRβ chain contains sequences derived from more than one species, such as sequences derived from humans and mice.

[0074] In some specific embodiments, the T cell receptor BMLF1-TCR binds to the BMLF1 antigen; or, the T cell receptors LMP1-TCR1 and 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.

[0075] 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.

[0076] The present invention also provides an EBV antigen peptide, which comprises antigen peptide BMLF1, antigen peptide LMP1, antigen peptide EBNA3 or antigen peptide LMP2.

[0077] In some specific embodiments, the amino acid sequence of the antigen peptide BMLF1 is as shown in SEQ ID No. 25.

[0078] In some specific embodiments, the amino acid sequence of the antigen peptide LMP1 is as shown in SEQ ID No. 26.

[0079] In some specific embodiments, the amino acid sequence of the antigen peptide EBNA3 is as shown in SEQ ID No. 35.

[0080] In some specific embodiments, the amino acid sequence of the antigen peptide LMP2 is as shown in SEQ ID No. 44.

[0081] In some specific embodiments, the aforementioned antigen peptide BMLF1 can be recognized by the aforementioned T cell receptor BMLF1-TCR; or, the aforementioned antigen peptide LMP1 can be recognized by the aforementioned T cell receptor LMP1-TCR1 or the aforementioned T cell receptor LMP1-TCR2; or, the aforementioned antigen peptide EBNA3 can be recognized by the aforementioned T cell receptor EBNA-3B–TCR; or, the aforementioned antigen peptide LMP2 can be recognized by the aforementioned T cell receptor LMP2-TCR.

[0082] The present invention also provides a preparation method 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 or the aforementioned T cell receptor LMP2-TCR, and the preparation method comprises the following steps:

[0083] 1) Loading the aforementioned antigen peptide BMLF1, the aforementioned antigen peptide LMP1, the aforementioned antigen peptide EBNA3 or the aforementioned antigen peptide LMP2 onto in vitro differentiated dendritic cells (DC);

[0084] 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.

[0085] In some specific embodiments, the loading in step 1) is a process of mixing and incubating the antigen peptide BMLF1, antigen peptide LMP1, antigen peptide EBNA3 or antigen peptide LMP2 with mature dendritic cells and then irradiating.

[0086] The present invention also provides a biological material, which comprises a polynucleotide, a nucleic acid construct, a virus or a cell.

[0087] 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 antigen peptide BMLF1, the aforementioned antigen peptide LMP1, the aforementioned antigen peptide EBNA3 or the aforementioned antigen peptide LMP2.

[0088] In some specific embodiments, the nucleic acid construct comprises the aforementioned polynucleotide and a plasmid backbone.

[0089] In some specific embodiments, the plasmid backbone is a plasmid backbone of a virus, an adenovirus or an adeno-associated virus. More specifically, the plasmid backbone is selected from any one or more of 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-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.

[0090] In some specific embodiments, the virus contains the aforementioned polynucleotide.

[0091] In some specific embodiments, the virus is selected from a lentivirus, an adenovirus or an adeno-associated virus.

[0092] 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.

[0093] In some specific embodiments, the cell is a cell with the aforementioned polynucleotide integrated into its genome.

[0094] In some specific embodiments, the cell is a T cell. Preferably, the T cell is a TCR-T cell.

[0095] In some specific embodiments, the T cell is also selected from helper T cells, suppressor T cells, effector T cells, cytotoxic T cells, delayed hypersensitivity T cells, naive T cells, or memory T cells. Among them, helper T cells are T cells with the function of assisting humoral immunity and cellular immunity; suppressor T cells are T cells with the function of suppressing cellular immunity and humoral immunity; effector T cells are T cells with the function of releasing lymphokines; cytotoxic T cells are T cells with the function of killing target cells; delayed hypersensitivity T cells are T cells that participate in type IV hypersensitivity reactions and can act on helper T cells and suppressor T cells to expand the immune effect; naive T cells are undifferentiated T cells; memory T cells are T cells with the function of remembering specific antigen stimulation.

[0096] In some specific embodiments, the T cell is a Jurkat T cell or a CD8+ T cell.

[0097] 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 the preparation of tumor treatment products.

[0098] The present invention also provides the use of the aforementioned antigen peptide BMLF1, the aforementioned antigen peptide LMP1, the aforementioned antigen peptide EBNA3, the aforementioned antigen peptide LMP2, the aforementioned polynucleotide, or the aforementioned nucleic acid construct in the preparation of anti-tumor peptide vaccine products.

[0099] The present invention also provides the use of the aforementioned antigen peptide BMLF1, the aforementioned antigen peptide LMP1, the aforementioned antigen peptide EBNA3, the aforementioned antigen peptide LMP2, the aforementioned polynucleotide, or the aforementioned nucleic acid construct in the preparation of in vitro generated T cell receptor products.

[0100] 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, cholangiocarcinoma, colorectal adenocarcinoma, lymphoid neoplasm, 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, retinoblastoma.

[0101] In some specific embodiments, the lymphoid neoplasm includes NKT cell lymphoma, Hodgkin lymphoma or diffuse large B cell lymphoma.

[0102] 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 a pharmaceutically acceptable excipient.

[0103] In some specific embodiments, the excipients include various excipients and diluents, which are not essential active ingredients and have no excessive toxicity after administration. The excipients include sterile water or physiological saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (such as phosphoric acid, citric acid, and other organic acids), preservatives, surfactants (such as PEG, Tween, etc.), chelating agents (such as EDTA, etc.), or binders. The excipients also include other low-molecular-weight polypeptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol, or sorbitol. The excipients for aqueous solutions for injection are selected from physiological saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannitol or sorbitol isotonic solution. The aqueous solution for injection contains solubilizers. The solubilizers are selected from alcohols (ethanol), polyols (propylene glycol or PEG), and / or non-ionic surfactants (Tween 80 or HCO-50). In the tumor treatment product provided by the present invention, the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, or the aforementioned T cell is the single active ingredient, or can be combined with one or more other active components useful for tumor treatment to form a combined preparation. The active components are various other drugs for tumor treatment. The content of the active component in the pharmaceutical composition is a safe and effective amount, and the safe and effective amount should be adjustable for those skilled in the art. For example, the dosage of the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, or the aforementioned T cell and the active ingredient of the tumor treatment product depends on the patient's body 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 to 1000 mg / kg / day, 1 to 3 mg / kg / day, 3 to 5 mg / kg / day, 5 to 10 mg / kg / day, 10 to 20 mg / kg / day, 20 to 30 mg / kg / day, 30 to 40 mg / kg / day, 40 to 60 mg / kg / day, 60 to 80 mg / kg / day, 80 to 100 mg / kg / day, 100 to 200 mg / kg / day, 200 to 500 mg / kg / day, or greater than 500 mg / kg / day.

[0104] The present invention also provides a method for treating tumors, which is to use 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 for tumor patients.

[0105] In some specific embodiments, the administered amount is 1 - 1000 mg / kg / day. Specifically, the administered amount 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 - 1000 mg / kg / day.

[0106] In some specific embodiments, the subject of the method can be a mammal; preferably, the subject of the method is a human.

[0107] In the present invention, the T cell receptor (TCR) is a molecule present on the surface of T cells, which is responsible for recognizing peptide - MHC complexes. In some specific embodiments, the TCR is a truncated or full - length TCR. In some specific embodiments, the TCR is a heterodimer composed of α and β chains. In some specific embodiments, the TCR can also be a single - chain TCR (scTCR).

[0108] In the present invention, the term "constant region of TCRα chain" or "constant region of TCRβ chain" includes an extracellular constant region, a transmembrane region, and an intracellular constant region connected in sequence, where the extracellular constant region may include the hinge regions of TCRα chain and TCRβ chain, which participate in the formation of disulfide bonds between TCRα chain and TCRβ chain; the transmembrane region also belongs to the constant region, and its functions include participating in the membrane anchoring of TCRα chain and TCRβ chain and the interaction with CD3 subunits to form the TCR - CD3 complex; the possible functions of the intracellular constant region include participating in the conformational change and signal transduction of the TCR - CD3 complex after TCR signal transduction.

[0109] In the present invention, the term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., the α-chain or β-chain of a TCR (or γδ-chain and δ-chain of TCRs)) involved in the binding of an immunoglobulin superfamily binding protein (e.g., TCR) to an antigen. The variable domains of the α-chain and β-chain of a native TCR (Va and Vβ, respectively) generally have similar structures, each domain containing four conserved framework regions (FRs) and three CDRs. The Va domain is encoded by two separate DNA segments, a variable gene segment and a joining gene segment (VJ); the Vβ domain is encoded by three separate DNA segments, a variable gene segment, a diversity gene segment, and a joining gene segment (VDJ). A single Va or Vβ domain may be sufficient to confer antigen-binding specificity. In addition, a TCR that binds a specific antigen can be used to isolate the Va or Vβ domain from the antigen-binding TCR to screen libraries of complementary Va or Vβ domains, respectively.

[0110] In the present invention, the term "antigen" refers to a molecule presented by a cell surface molecule or intracellularly by an MHC molecule or MHC-like molecule and capable of being 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 may be a tumor antigen, such as a tumor-associated antigen (TAA), or a tumor specific antigen (TSA).

[0111] In the present invention, the term "isolated" refers to a material that has been removed from its natural state or otherwise manipulated, such as the α-chain, β-chain, T cell receptor, and nucleic acid described herein. An isolated material may be substantially or essentially free of the components that normally accompany it in its natural state, or it may be manipulated to be in an artificial state, together with the components that normally accompany it in its natural state. An isolated material may be in a natural, chemically synthesized, or recombinant form. An isolated material may also or alternatively be in an enriched, partially purified, or purified form.

[0112] In the present invention, the term "polynucleotide" is also referred to as "nucleotide" or "nucleic acid" and is a nucleic acid chain composed of deoxyribonucleic acid, ribonucleic acid, modified nucleic acid or bases, and / or their analogs, or any substrate capable of being incorporated into a chain by a DNA or RNA polymerase.

[0113] In the present invention, the term "nucleic acid construct", also referred to as "vector", means a molecule capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the gene or sequence in the host cell. Examples of vectors include, but are not limited to, viral vectors, plasmids, cosmids or phage vectors.

[0114] In the present invention, the term "host cell" means a cell into which exogenous nucleic acid has been introduced, including progeny of these cells.

[0115] In the present invention, to determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence to which the sequences are 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 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.

[0116] Sequence comparison and calculation of the percent identity between two sequences can be achieved using mathematical algorithms. In a preferred embodiment, the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, with the Blossum 62 matrix or PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6, to determine the percent identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, with the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6, to determine the percent identity between two nucleic acid sequences. A particularly preferred set of parameters (and the set of parameters to 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.

[0117] The PAM120 weighted residue table can also be used, with a gap length penalty of 12 and a gap penalty of 4. The percent identity between two amino acid sequences or nucleic acid sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) that has been incorporated into the ALIGN program (version 2.0).

[0118] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0119] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments and not for limiting the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an", and "the" include the plural forms.

[0120] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, any value between the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials similar to or equivalent to the methods, devices, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0121] The sequence information in the present invention is as follows:

[0122] SEQ ID No.1

[0123] SNHLY

[0124] SEQ ID No.2

[0125] FYNNEI

[0126] SEQ ID No.3

[0127] CASSEGQVSPGELFF

[0128] SEQ ID No.4

[0129] MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYN

[0130] NEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEGQVSPGELFFGEGSRLTVLEDLRNVTP

[0131] PKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLR

[0132] VSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEI

[0133] LLGKATLYAVLVSTLVVMAMVKRKNS

[0134] SEQ ID No.5

[0135] NSASQS

[0136] SEQ ID No.6

[0137] VYSSG

[0138] SEQ ID No.7

[0139] CVVNGMDSSYKLIF

[0140] SEQ ID No.8

[0141] MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLM

[0142] SVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNGMDSSYKLIFGSGTRLLVRPDIQNPEPA

[0143] VYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFK

[0144] ETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSSSEQ IDNo.9

[0145] MGHRA

[0146] SEQ ID No.10

[0147] YSYEKL

[0148] SEQ ID No.11

[0149] CASSQEGTGELFF

[0150] SEQ ID No.12

[0151] MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFV

[0152] YSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSQEGTGELFFGEGSRLTVLEDLRNVTP

[0153] PKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLR

[0154] VSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEI

[0155] LLGKATLYAVLVSTLVVMAMVKRKNS

[0156] SEQ ID No.13

[0157] YGGTVN

[0158] SEQ ID No.14

[0159] YFSGDPLV

[0160] SEQ ID No.15

[0161] CALPRWDGGYNKLIF

[0162] SEQ ID No.16

[0163] MLLLLIPVLGMIFALRDARAQSVSQHNHHVILSEAASLELGCNYSYGGTVNLFWYVQYPGQHLQLLLKY

[0164] FSGDPLVKGIKGFEAEFIKSKFSFNLRKPSVQWSDTAEYFCALPRWDGGYNKLIFGAGTRLAVHPYYIQN

[0165] PEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQ

[0166] DIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSSSEQ IDNo.17

[0167] DFQATT

[0168] SEQ ID No.18

[0169] SNEGSKA

[0170] SEQ ID No.19

[0171] CSASGAQPDDPQHF

[0172] SEQ ID No.20

[0173] MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSK

[0174] ATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSASGAQPDDPQHFGDGTRLSILEDLRNVTPPK

[0175] VSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVS

[0176] ATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILL

[0177] GKATLYAVLVSTLVVMAMVKRKNS

[0178] SEQ ID No.21

[0179] TSDPSYG

[0180] SEQ ID No.22

[0181] QGSYDQQN

[0182] SEQ ID No.23

[0183] CAMREVALNAGGTSYGKLTF

[0184] SEQ ID No.24

[0185] MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIY

[0186] QGSYDQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMREVALNAGGTSYGKLTFGQGTILT

[0187] VHPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSN

[0188] QTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSSSEQ ID No.25

[0189] GLCTLVAML

[0190] SEQ ID No.26

[0191] YLLEMLWRL

[0192] SEQ ID No.27

[0193] SGHTA

[0194] SEQ ID No.28

[0195] FQGNSA

[0196] SEQ ID No.29

[0197] CASSLNGGHYEQYF

[0198] SEQ ID No.30

[0199] MGTRLLFWVAFCLLGADHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQSLGQGLEFLIYFQ

[0200] GNSAPDKSGLPSDRFSAERTGGSVSTLTIQRTQQEDSAVYLCASSLNGGHYEQYFGPGTRLTVTEDLRNV

[0201] TPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSR

[0202] LRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATIL

[0203] YEILLGKATLYAVLVSTLVVMAMVKRKNS

[0204] SEQ ID No.31

[0205] SIFNT

[0206] SEQ ID No.32

[0207] LYKAGEL

[0208] SEQ ID No.33

[0209] CAGHRTDKLIF

[0210] SEQ ID No.34

[0211] MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYK

[0212] AGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGHRTDKLIFGTGTRLQVFPNIQNPEPAVYQLKD

[0213] PRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATY

[0214] PSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSSSEQ ID No.35

[0215] IVTDFSVIK

[0216] SEQ ID No.36

[0217] MNHEY

[0218] SEQ ID No.37

[0219] SVGAGI

[0220] SEQ ID No.38

[0221] CASSTQGGGDGYTF

[0222] SEQ ID No.39

[0223] MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHY

[0224] SVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSTQGGGDGYTFGSGTRLTVVEDLRN

[0225] VTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSS

[0226] RLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATI

[0227] LYEILLGKATLYAVLVSTLVVMAMVKRKNS

[0228] SEQ ID No.40

[0229] TSINN

[0230] SEQ ID No.41

[0231] IRSNERE

[0232] SEQ ID No.42

[0233] CATEGDGGYSTLTF

[0234] SEQ ID No.43

[0235] METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIR

[0236] SNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATEGDGGYSTLTFGKGTMLLVSPDIQNPEPAV

[0237] YQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKE

[0238] TNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSSSEQ IDNo.44

[0239] FLYALALLL

[0240] SEQ ID No.45

[0241] MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLM

[0242] SVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNGMDSSYKLIFGSGTRLLVRPDSEQID No.46

[0243] MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFY

[0244] NNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEGQVSPGELFFGEGSRLTVLSEQID No.47

[0245] MLLLLIPVLGMIFALRDARAQSVSQHNHHVILSEAASLELGCNYSYGGTVNLFWYVQYPGQHLQLLLKY

[0246] FSGDPLVKGIKGFEAEFIKSKFSFNLRKPSVQWSDTAEYFCALPRWDGGYNKLIFGAGTRLAVHPYYSEQ ID No.48

[0247] MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMF

[0248] VYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSQEGTGELFFGEGSRLTVLSEQID No.49

[0249] MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIY

[0250] QGSYDQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMREVALNAGGTSYGKLTFGQGTILT

[0251] VHPN

[0252] SEQ ID No.50

[0253] MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSK

[0254] ATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSASGAQPDDPQHFGDGTRLSILSEQ IDNo.51

[0255] MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYK

[0256] AGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGHRTDKLIFGTGTRLQVFPNSEQ IDNo.52

[0257] MGTRLLFWVAFCLLGADHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQSLGQGLEFLIYFQ

[0258] GNSAPDKSGLPSDRFSAERTGGSVSTLTIQRTQQEDSAVYLCASSLNGGHYEQYFGPGTRLTVTSEQID No.53

[0259] METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIR

[0260] SNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATEGDGGYSTLTFGKGTMLLVSPSEQ IDNo.54

[0261] MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHY

[0262] SVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSTQGGGDGYTFGSGTRLTVVSEQID No.55

[0263] IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSF

[0264] TCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSSSEQID No.56

[0265] EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYS

[0266] YCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGV

[0267] LSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS

[0268] SEQ ID No.57

[0269] RAKRGSGATNFSLLKQAGDVEENPGP

[0270] SEQ ID No.58

[0271] ATGGATACATGGCTCGTATGCTGGGCAATTTTTTCTCTCCTGAAAGCCGGGCTGACTGAGCCTGAGG

[0272] TTACACAGACACCCAGCCACCAAGTAACTCAGATGGGACAAGAGGTCATCCTTCGGTGCGTGCCCAT

[0273] TTCAAACCACCTGTACTTCTACTGGTACCGCCAGATTCTGGGTCAGAAAGTGGAGTTTCTGGTGAGC

[0274] TTCTATAACAATGAGATCTCCGAGAAATCAGAGATTTTTGACGACCAGTTCTCTGTGGAACGGCCGG

[0275] ATGGGAGCAATTTCACTCTGAAGATTAGGAGCACTAAGCTGGAAGATTCCGCCATGTATTTCTGCGC

[0276] TTCAAGTGAGGGTCAGGTCAGTCCAGGAGAGCTGTTTTTTGGCGAGGGTAGTAGATTGACCGTCCTG

[0277] GAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAA

[0278] ACAAACAAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTG

[0279] GTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCTGCACGGACCCTCAGGCCTACAAGGAGAGCAA

[0280] TTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGCAACCACT

[0281] TCCGCTGCCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAAC

[0282] CTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCT

[0283] ATCAACAAGGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGC

[0284] TGTGCTTGTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAATTCAAGAGCCAAAAGAGG

[0285] TTCTGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCAT

[0286] GATAAGCCTCAGAGTGTTGTTGGTAATCTTGTGGCTGCAGCTTTCCTGGGTTTGGTCTCAGCGGAAA

[0287] GAGGTTGAGCAGGACCCAGGACCTTTTAATGTCCCAGAGGGTGCAACAGTTGCTTTCAACTGCACCT

[0288] ACTCCAACAGTGCATCCCAGTCCTTCTTTTGGTATCGCCAGGATTGCAGAAAGGAACCAAAACTGCT

[0289] CATGAGCGTTTACTCAAGTGGAAACGAGGACGGTCGATTCACAGCACAGCTGAATCGCGCATCACA

[0290] GTACATAAGTCTTCTCATCAGGGACAGTAAGTTGTCTGATTCTGCCACATACCTGTGTGTGGTAAAC

[0291] GGAATGGATAGCAGTTACAAACTGATCTTCGGCAGTGGAACAAGACTGCTGGTGAGGCCTGACATC

[0292] CAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGT

[0293] TCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAA

[0294] ATGCGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGAC

[0295] AAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGT

[0296] GATGCCACGTTGACCGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTA

[0297] TGGGACTCCGAATCCTCCTGCTGAAAGTAGCGGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTC

[0298] CAGTTGA

[0299] SEQ ID No.59

[0300] ATGGGATGTAGACTGCTGTGCTGCGCTGTCCTTTGCCTGCTCGGAGCAGTACCAATCGACACCGAAG

[0301] TGACCCAAACACCTAAACACCTCGTGATGGGCATGACAAATAAGAAAAGCCTCAAGTGCGAACAAC

[0302] ACATGGGCCACAGAGCAATGTACTGGTATAAGCAGAAAGCTAAGAAGCCTCCAGAGCTGATGTTTG

[0303] TATACTCATACGAGAAGCTGAGCATTAACGAGAGCGTGCCTAGTAGGTTTTCACCAGAGTGCCCTAA

[0304] TTCTTCCCTTTTGAACCTGCACCTGCATGCGTTGCAACCTGAAGACTCCGCCTTGTACCTGTGTGCAT

[0305] CTTCCCAGGAGGGGACTGGAGAGTTGTTTTTCGGAGAAGGAAGTAGACTGACCGTTCTGGAGGATCT

[0306] GAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACA

[0307] AAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTG

[0308] AATGGCAAGGAGGTCCACAGTGGGGTCTGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGC

[0309] TACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGCAACCACTTCCGCTG

[0310] CCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCAC

[0311] ACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACA

[0312] AGGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTT

[0313] GTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAATTCAAGAGCCAAAAGAGGTTCTGGC

[0314] GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGTTGCTC

[0315] CTCCTTATCCCAGTGCTCGGAATGATCTTCGCGCTCCGCGACGCCCGAGCCCAGTCTGTGTCTCAACA

[0316] TAATCATCATGTGATCTTGTCTGAGGCTGCTAGCTTGGAGCTGGGATGTAACTACTCTTATGGTGGCA

[0317] CCGTGAATCTCTTTTGGTATGTGCAGTACCCTGGCCAACATCTCCAACTCTTGCTTAAATACTTTAGC

[0318] GGTGATCCACTGGTAAAAGGGATTAAGGGTTTCGAGGCCGAGTTCATCAAAAGTAAGTTCAGCTTCA

[0319] ATCTGCGGAAGCCTAGCGTTCAGTGGAGTGATACTGCCGAATATTTCTGTGCATTGCCACGCTGGGA

[0320] TGGAGGATATAATAAGCTGATTTTCGGAGCCGGTACACGCCTGGCCGTTCATCCATACTACATCCAG

[0321] AACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCA

[0322] CCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAATG

[0323] CGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAG

[0324] CTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGAT

[0325] GCCACGTTGACCGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGG

[0326] GACTCCGAATCCTCCTGCTGAAAGTAGCGGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAG

[0327] TTGA

[0328] SEQ ID No.60

[0329] ATGCTGTTGCTGCTCCTGCTGCTCGGACCAGGAAGTGGATTGGGAGCCGTGGTCAGTCAGCATCCTAG

[0330] CTGGGTAATATGCAAGTCAGGCACCTCCGTGAAGATCGAGTGCAGGAGTCTTGATTTCCAGGCGACG

[0331] ACCATGTTTTGGTACCGCCAGTTCCCGAAACAGAGCCTCATGCTGATGGCAACGTCAAACGAGGGTT

[0332] CAAAGGCAACCTATGAACAGGGCGTGGAGAAGGACAAGTTCCTGATAAACCATGCATCCCTGACCCT

[0333] CAGCACTTTGACAGTTACTAGCGCTCACCCAGAGGATTCTAGTTTTTACATTTGCTCCGCTAGTGGAGC

[0334] ACAGCCCGATGATCCACAGCACTTTGGAGATGGAACCAGACTCAGCATCCTCGAGGATCTGAGAAAT

[0335] GTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACAAAAGGCTA

[0336] CCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGCAA

[0337] GGAGGTCCACAGTGGGGTCTGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTG

[0338] AGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGCAACCACTTCCGCTGCCAAGTGC

[0339] AGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACA

[0340] TCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAAGGGGTCTT

[0341] GTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGTACACT

[0342] GGTGGTGATGGCTATGGTCAAAAGAAAGAATTCAAGAGCCAAAAGAGGTTCTGGCGCCACCAACTTC

[0343] AGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGTCACTTAGCTCTCTTCTTA

[0344] AAGTAGTAACTGCTTCACTTTGGCTTGGCCCCGGTATTGCACAGAAGATCACCCAGACACAACCCGG

[0345] CATGTTCGTGCAGGAAAAGGAGGCTGTGACGCTCGACTGCACCTATGATACCTCCGACCCAAGTTAC

[0346] GGCCTGTTCTGGTACAAGCAGCCTAGCTCAGGAGAGATGATTTTCCTGATCTACCAGGGGTCATATGA

[0347] CCAACAGAACGCAACAGAAGGGAGGTATAGCCTGAATTTCCAGAAGGCCCGAAAGAGTGCCAACCT

[0348] GGTGATCTCCGCTAGTCAGCTTGGAGACAGCGCAATGTACTTTTGCGCAATGCGCGAAGTGGCACTC

[0349] AATGCCGGGGGAACTTCTTATGGCAAGCTGACTTTCGGACAGGGTACGATACTCACTGTCCATCCAAA

[0350] CATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGC

[0351] CTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGA

[0352] CAAATGCGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAG

[0353] ACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCT

[0354] GTGATGCCACGTTGACCGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTT

[0355] ATGGGACTCCGAATCCTCCTGCTGAAAGTAGCGGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTC

[0356] CAGTTGA

[0357] SEQ ID No.61

[0358] ATGGGAACACGCCTTCTGTTTTGGGTTGCCTTCTGCCTGTTGGGCGCTGACCACACCGGTGCAGGCGT

[0359] CTCTCAGTCACCATCAAACAAGGTGACTGAGAAAGGGAAAGATGTGGAGCTGAGATGTGACCCTATC

[0360] TCTGGACACACCGCCCTGTACTGGTACAGACAGTCTCTTGGTCAGGGGCTGGAGTTCCTGATCTACTT

[0361] TCAGGGAAACAGCGCCCCAGACAAGTCTGGTCTCCCCTCTGACAGATTTTCCGCAGAAAGGACCGGC

[0362] GGTAGCGTGTCAACTCTGACCATACAGCGCACCCAGCAAGAAGATTCCGCCGTGTACCTGTGCGCCT

[0363] CTTCACTCAACGGCGGCCATTATGAGCAGTACTTCGGACCTGGCACCCGCCTCACTGTGACCGAGGAT

[0364] CTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAAC

[0365] AAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGT

[0366] GAATGGCAAGGAGGTCCACAGTGGGGTCTGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGC

[0367] TACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGCAACCACTTCCGCTG

[0368] CCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCAC

[0369] ACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAA

[0370] GGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTC

[0371] AGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAATTCAAGAGCCAAAAGAGGTTCTGGCGCCA

[0372] CCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGCTGCTTGAAC

[0373] ACCTCCTGATTATTCTTTGGATGCAGCTGACTTGGGTCAGTGGGCAGCAGCTGAATCAGAGCCCCCAG

[0374] AGCATGTTCATTCAAGAAGGGGAAGATGTGTCCATGAATTGCACCTCCTCCAGCATCTTCAACACCTG

[0375] GCTTTGGTACAAGCAAGACCCCGGAGAAGGACCAGTTCTCCTTATAGCCCTGTACAAGGCCGGAGAG

[0376] CTGACCAGTAATGGTCGACTGACCGCGCAATTCGGAATAACGCGGAAGGACAGCTTCCTGAATATCA

[0377] GCGCAAGCATTCCAAGTGATGTCGGAATCTATTTTTGCGCTGGACATCGAACCGATAAACTGATTTTCG

[0378] GAACAGGGACCAGACTGCAAGTGTTTCCCAACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAA

[0379] AGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGA

[0380] AAACCATGGAATCTGGAACGTTCATCACTGACAAATGCGTGCTGGACATGAAAGCTATGGATTCCAAG

[0381] AGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCA

[0382] ACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACCGAGAAAAGCTTTGAAACAGA

[0383] TATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCGGGAT

[0384] TTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA

[0385] SEQ ID No.62

[0386] ATGAGCATAGGTCTCCTTTGTTGTGCCGCACTTAGCCTGCTTTGGGCCGGCCCCGTTAACGCTGGTGTT

[0387] ACTCAAACGCCGAAGTTCCAGGTGCTCAAAACTGGCCAGTCAATGACACTGCAGTGCGCTCAGGACA

[0388] TGAACCACGAATATATGAGTTGGTACAGACAGGACCCTGGGATGGGATTGAGGCTGATTCACTACAGT

[0389] GTCGGAGCTGGGATCACAGACCAGGGCGAAGTCCCCAACGGCTACAACGTGTCAAGGTCTACCACC

[0390] GAGGACTTTCCCCTCAGACTTCTCTCTGCTGCACCATCTCAGACGAGTGTGTACTTCTGTGCTAGCAG

[0391] CACTCAAGGGGGCGGTGATGGCTACACGTTTGGAAGTGGCACCCGGCTCACAGTGGTTGAGGATCTG

[0392] AGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACAAA

[0393] AGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAA

[0394] TGGCAAGGAGGTCCACAGTGGGGTCTGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTAC

[0395] TGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGCAACCACTTCCGCTGCCA

[0396] AGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACA

[0397] GAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAAGGG

[0398] GTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGT

[0399] ACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAATTCAAGAGCCAAAAGAGGTTCTGGCGCCACC

[0400] AACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGGAAACACTGCTC

[0401] GGCGTGTCACTCGTTATTCTTTGGCTCCAGCTGGCCCGGGTTAATTCACAACAGGGCGAGGAAGATCC

[0402] TCAAGCGTTGTCTATTCAGGAGGGAGAGAACGCAACCATGAACTGCAGCTATAAAACCTCTATCAAC

[0403] AACCTCCAGTGGTATCGGCAGAACTCAGGCCGAGGTCTGGTCCATCTGATACTGATTCGCAGCAATGA

[0404] GCGCGAGAAGCATTCTGGGAGACTGCGCGTCACACTCGACACATCAAAGAAAAGCTCCTCCCTGCTG

[0405] ATCACGGCTTCAAGAGCCGCAGACACTGCGAGCTACTTCTGTGCAACTGAGGGCGATGGTGGGTATT

[0406] CCACTCTCACATTTGGTAAAGGGACCATGCTGCTGGTGTCCCCTGACATCCAGAACCCAGAACCTGCT

[0407] GTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCA

[0408] AATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAATGCGTGCTGGACATGAAA

[0409] GCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATAT

[0410] CTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACCGAGAAA

[0411] AGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCT

[0412] GAAAGTAGCGGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA

[0413] Example 1 Sorting of TCR-T cells and TCR sequencing

[0414] 1. In vitro differentiation of human peripheral monocytes into dendritic cells (DCs)

[0415] (1) CD14+ monocytes were sorted 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 cells were cultured for another 2 days to induce the differentiation of monocytes into MoDCs cells.

[0416] (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 the maturation of DC cells.

[0417] (3) After 24 h, the mature DC cells were harvested, and the expression of mature DCs markers was detected by flow cytometry. The phenotype of mature DCs: CD14-, CD11c+, CD40+, CD80+, CD83+, CD86+, HLA-ABC+, HLA-DR+. The collected mature DCs cells were used for subsequent loading of EBV antigen polypeptides.

[0418] 2. Co-stimulatory culture of antigen-loaded DC cells with autologous CD8+ T cells

[0419] (1) Add 5 μg / ml of EBV antigen peptides (i.e., antigen peptide BMLF1, antigen peptide LMP1, antigen peptide EBNA3, or antigen peptide LMP2, which are synthesized based on the amino acid sequences respectively) to the above-mentioned Mature DCs. Seal with Parafilm and place in a MACSmix rotator, and rotate and bind in a cell incubator for 4 h. After the incubation of mDCs with EBV antigen peptides is completed, irradiate the DCs loaded with EBV antigen peptides with a dose of 40 Gy using an irradiator. After the irradiation is completed, centrifuge to collect the DCs loaded with EBV antigen peptides into a 15 ml centrifuge tube.

[0420] (2) Obtain PBMCs from the blood of healthy donors by Lymphoprep density gradient centrifugation method, and use the EasySepTM Human CD8 Positive Selection Kit to isolate CD8+ T cells from PBMCs. Add the DCs loaded with EBV antigen peptides and CD8+ T cells to a 12-well plate for culture at a ratio of DC / T of 1:2.5. Add IL-21 with a final concentration of 30 ng / ml and 5 μg / ml of EBV antigen peptides to the culture system.

[0421] (3) Place the 12-well plate in a cell incubator at 37 °C and 5% CO2 and incubate overnight. On Day 1, add 10 ng / ml of IL-2, IL-7, and IL-15 to each well, and then place it in a cell incubator and culture at 37 °C and 5% CO2 for 10 days. During the culture process, perform half-volume medium replacement every 3 days.

[0422] (4) On Day 14, prepare Irradiated EBV-peptide-loaded DCs and perform a second round of in vitro stimulation with the cells in each culture system, and continue to stimulate for 10 days.

[0423] (5) On Day 25, aspirate the cells in each well of the plate, wash twice with FACS buffer, add CD8a-APC and PE-HLA-A0201-restricted-EBV-peptide-Tetramer (MBL company), mix well and incubate at 4 °C in the dark for 30 min. After the incubation is completed, wash 3 times with FACS buffer, resuspend the cells with 2 ml of FACS buffer containing 1× DAPI working solution, and then detect the proportion of CD8+ / tetramer+ (%).

[0424] 3. Sorting of EBV-antigen-specific-T cells and single-cell TCR sequencing

[0425] (1) The process of sorting EBV-antigen-specific-T cells is asFigure 1 As shown, 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 as follows: Figure 2 As shown in A, the proportions of CD8a+ / Tetramer+ T cells from healthy donors were 0.34% for BMLF1, 2.52% for LMP1, 0.4% for EBNA3B, and 0.15% for LMP2, respectively, and the cell populations were clearly separated. This indicates that EBV-antigen-specific-TCR-T cells were specifically amplified during the two rounds of in vitro stimulation. Subsequently, CD8+ / tetramer+ T cells in the co-culture system were specifically sorted using a flow cytometry cell sorter and then sent to Genomics for single-cell TCR+ transcriptome sequencing of 10× Genomics to obtain the TCR sequences and transcriptomic characteristics of CD8+ / tetramer+ T cells.

[0426] (2) After single-cell sequencing was completed, transcriptomic characteristics analysis was performed on the top ten TCRs. The TCR sequencing results and frequency ranking results of this cell population obtained from the analysis are as follows: Figure 2 As shown in B. 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 (where 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 mismatching. Finally, the amino acid sequence of the TCRα chain in the T cell receptor BMLF1-TCR 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.

[0427] 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.

[0428] 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.

[0429] 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.

[0430] 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.

[0431] 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.

[0432] 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.

[0433] 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.

[0434] 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.

[0435] 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.

[0436] Using P2A and Furin-cleavage (where the amino acid sequence of the P2A-Furin-cleavage fusion peptide is shown in SEQ ID No.57) enables the α and β chains of the TCR to be co-expressed in a lentiviral expression vector, and the nucleotide sequence encoding the T cell receptor that co-expresses the TCRα and β chains in the above lentiviral expression vector is shown in any of SEQ ID No.58-62. Among them, the nucleotide sequence encoding the TCRα and β chains that co-express the T cell receptor BMLF1-TCR is shown in SEQ ID No.58; the nucleotide sequence encoding the TCRα and β chains that co-express the T cell receptor LMP1-TCR1 is shown in SEQ ID No.59; the nucleotide sequence encoding the TCRα and β chains that co-express the T cell receptor LMP1-TCR2 is shown in SEQ ID No.60; the nucleotide sequence encoding the TCRα and β chains that co-express the T cell receptor EBNA-3B-TCR is shown in SEQ ID No.61; the nucleotide sequence encoding the TCRα and β chains that co-express the T cell receptor LMP2-TCR is shown in SEQ ID No.62. The above-mentioned peptide segments and polynucleotides can be obtained by chemical synthesis and homologous recombination when the sequences are known.

[0437] Example 2 In vitro binding experiment of EBV-antigen-specific-TCR-T cells

[0438] 1. Preparation of EBV-antigen-specific-TCR-T cells

[0439] (1) The optimized EBV-antigen-TCR lentiviral expression vector obtained in Example 1 was constructed, and then the lentiviral packaging of EBV-antigen-TCR was carried out. The specific operation of virus packaging is as follows: Digest and passage 293T cells, observe the cell density on the day of transfection, and transfection can be carried out when it is 80-90% confluent. There is no need to change the culture medium before transfection. Opti-MEM needs to be preheated in a 37-degree water bath, and the Fugene-HD transfection reagent needs to be restored to room temperature before use and shaken well before use. The composition of the complex for transfecting each T75 flask is as follows: pHR-SFFV-TCR-Puro plasmid containing the target TCR: 10 μg; pMD2.G (envelope plasmid): 5 μg; pSPAX2 (core enzyme): 7.5 μg. After making up to 1 mL with opti-MEM, mix well; add 56 μL of Fugene-HD transfection reagent, pipette gently 5-6 times, place at room temperature for 15 min, and then add it to the T75 flask. Subsequently, collect the virus supernatant at 48 h and 72 h. After collection, centrifuge at 4000 rpm for 5 min, take the supernatant and filter it through a 0.45-μm filter membrane (PES material) before use. Collect the lentivirus solution of EBV-antigen-specific-TCR-TCR at 48 h and 72 h after virus packaging respectively.

[0440] (2) Infect Jurkat T cells with EBV-antigen-TCR lentiviral particles, add 10 μg / ml polybrene, centrifuge at 800×g for 90 min at 30 °C for centrifugal infection. After centrifugation, place it in a cell culture incubator and culture at 37 °C and 5% CO2 for 24 h, and then replace it with fresh 1640 culture medium for culture.

[0441] (3) Add 20 μl of CD3 / 28 beads (Genscript) and 20 ng / ml of IL-2 to the CD8+ T cells of healthy donors. After activating the T cells for 48 h, add 10 μg / ml polybrene for the first round of infection with EBV-antigen-TCR lentivirus. After centrifugation, culture in a cell culture incubator at 37 °C and 5% CO2 for 24 h. Subsequently, carry out the second round of infection with EBV-antigen-TCR lentivirus.

[0442] 2. Detection of the specificity of EBV-antigen-TCR-T cell binding by FACS

[0443] Stain the EBV-antigen cells prepared above with mTCRβC+ / Tetramer. The flow cytometry results show that the Tetramer+ / mTCRβC+ Jurkat T of EBV-BMLF1 / LMP1 / EBNA3B Figure 3A) and CD8+ / Tetramer+ primary CD8+ T( Figure 3 B) The cell population is distinct. This result shows that the EBV-BMLF1 / LMP1 / EBNA3B-TCR screened and identified in vitro can be normally expressed in the Jurkat T cell line and primary CD8+ T, and can specifically bind to HLA-restricted-EBV-Tetramer.

[0444] Example 3 In vitro activation experiment of EBV-antigen-TCR-T cells

[0445] The in vitro activation experiment of EBV-antigen-T cells, namely the IFNγ-Elispot experiment, includes the following steps:

[0446] (1) In vitro, direct the donor's autologous monocytes to differentiate into DC cells. Similar to Example 1, load DC with EBV antigen peptides respectively, and co-culture the EBV-antigen-specific-TCR-T cells prepared in Example 2 with DC loaded with EBV antigen peptides in vitro;

[0447] (2) After 24 hours of in vitro co-culture, discard the cells, then wash 5 times with 200 μl of DPBS to completely remove the cells. Add 100 μl of 1 μg / ml detection antibody 7-B6-1-biotin to the culture system and incubate at 37°C for 2 hours. Subsequently, add 100 μl of streptavidin-HRP (3420-2H, MABTECH) and incubate at room temperature for 1 hour.

[0448] (3) After washing 5 times with 200 μl of DPBS, add 100 μl of substrate TMB to each well until complete plaques appear in the wells, and then add sterile water to interrupt the color development process.

[0449] The results are as Figure 4 shown. DC 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 increases significantly ( Figure 4 A-C). Therefore, this result indicates that the EBV-BMLF1 / LMP1 / EBNA3B-TCR obtained by this screening and identification can specifically recognize EBV-BMLF1 / LMP1 / EBNA3B antigen peptides respectively.

[0450] Example 4 In vitro killing experiment of EBV-antigen-TCR-T cells

[0451] The in vitro killing experiment of EBV-TCR-T cells, namely the Incucyte killing experiment, includes the following steps:

[0452] (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 respectively. Subsequently, EBV-BMLF1 / LMP1 / EBNA3B-TCR-T cells or CD8 + T cells and K562-A1101 control cells or K562-A1101-BMLF1 / LMP1 / EBNA3B target cells were added to 96-well plates at an effector-to-target ratio of 5:1 for co-culture respectively;

[0453] (2) Dissolve Annexin V Dyes (Sartorius) by adding 100 μL of DPBS. Then the Annexin V Dyes reagent was diluted in complete medium to a final dilution of 1:200.

[0454] (3) The 96-well plates containing cells were placed into an Incucyte (Sartorius) live cell analysis instrument, and cell apoptosis was monitored using appropriate fluorescence channels. The specific parameters are as follows: A. Observation magnification: 20× objective lens; B. Channel selection: Phase contrast Green|Red|Orange|NIRc; C. Scanning type: 2 - 4 images per well; D. Scanning interval: Scan once every 30 min until the killing efficiency detection is completed after 48 h of co-culture.

[0455] (4) After the detection, the in vitro killing efficiency of TCR-T was analyzed using the Incucyte live cell analysis system software (Sartorius).

[0456] The results are as Figure 5 shown. In this example, the Incucyte high-throughput long-term dynamic imaging and analysis system was used to detect the killing of target cells by EBV-BMLF1 / LMP1 / EBNA3B-TCR-T cells in real time. The results showed that within 48 h of co-culture, compared with the negative control CD8+ T cells not transfected with EBV-BMLF1 / LMP1 / EBNA3B-TCR, TCR-T cells at an effector-to-target ratio of 5:1 were able to kill target cells that were HLA-A*11:01 positive and expressed the TEBV-BMLF1 / LMP1 / EBNA3B antigen more efficiently ( Figure 5 A - D).

[0457] In Vivo Killing Experiment of EBV-antigen-TCR-T Cells

[0458] The in vivo killing experiment of EBV-EBAN3B-TCR-T cells, namely the CDX animal experiment, includes the following steps:

[0459] (1) Six-week-old Jicuiyaokang NCG female mice were selected and injected with 2E6 K562 cell line expressing Luciferase reporter gene carrying HLA-A*11:01 and EBNA3B antigen into the mice via the lateral tail vein.

[0460] (2) Three days after transplantation of the K562-HLA-EBNA3B-luci cell line, 1E7 TCR-T cells were injected via the lateral tail vein, and untransfected TCR gene control T lymphocytes were used as negative controls.

[0461] (3) On the 7th, 15th, and 27th days after inoculation of tumor cells, small animal in vivo imaging was performed, and at the same time, the body weight changes and survival of the mice were detected to evaluate the in vivo killing effect of EBV-EBAN3B-TCR-T cells on target cells.

[0462] The results are as Figure 6 shown. In this example, the in vivo CDX model was used to evaluate the effectiveness of EBV-EBAN3B-TCR-T cells in killing target cells ( Figure 6 A). The in vivo imaging results showed that obvious tumor cell growth occurred in the mice in the normal saline group and the Mock T cell reinfusion group within 27 days after tumor cell reinfusion, while no obvious tumor growth was observed in some mice (3 / 4) in the EBV-EBAN3B-TCR-T reinfusion group ( Figure 6 B), and the total photon number or average photon number intensity of luciferase in the EBV-EBAN3B-TCR-T group mice was significantly lower than that in the normal saline group and the Mock T control group ( Figure 6 C-D). The CDX experiment results showed that HLA-A*11:01-restricted EBV-EBAN3B-TCR-T cells could efficiently and specifically kill K562-HLA-A*11:01-EBAN3B-luciferase target cells in vivo.

[0463] The above embodiments are intended to illustrate the embodiments disclosed in the present invention and should not be construed as limiting the present invention. In addition, various modifications listed herein and changes in the methods of 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 connection 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, all such modifications that are obvious 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 targeting Epstein-Barr virus antigen, 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) The T cell receptor BMLF1-TCR comprises a TCR α chain variable region and a TCR β chain variable region; the TCR α chain variable region comprises peptide segments CDR α1, CDR α2 and CDR α3; the TCR β chain variable region comprises peptide segments CDR β1, CDR β2 and CDR β3; the amino acid sequence of the CDR α3 is shown in SEQ ID NO.7, and the amino acid sequence of the CDR β3 is shown in SEQ ID NO.3; the amino acid sequence of CDR α1 in the TCR α chain variable region of the T cell receptor BMLF1-TCR is shown in SEQ ID NO.5, and the amino acid sequence of CDR α2 is shown in SEQ ID NO.6; the amino acid sequence of CDR β1 in the TCR β chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of CDR β2 is shown in SEQ ID NO.2; II) 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; 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, and the amino acid sequence of CDRα2 is shown in SEQ ID NO.14; the amino acid sequence of CDRβ1 in the TCRβ chain variable region is shown in SEQ ID NO.9, and the amino acid sequence of CDRβ2 is shown in SEQ ID NO.10; III) 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; 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, and the amino acid sequence of CDRα2 is shown in SEQ ID NO.22; the amino acid sequence of CDRβ1 in the TCRβ chain variable region is shown in SEQ ID NO.17, and the amino acid sequence of CDRβ2 is shown in SEQ ID NO.18; IV) 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; the amino acid sequence of CDRα1 in the TCRα chain variable region of the T cell receptor EBNA-3B-TCR is shown in SEQ ID NO.31, and the amino acid sequence of CDRα2 is shown in SEQ ID NO.32; the amino acid sequence of CDRβ1 in the TCRβ chain variable region is shown in SEQ ID NO.27, and the amino acid sequence of CDRβ2 is shown in SEQ ID NO.28; V) The variable region of the TCR α chain of the T cell receptor LMP2-TCR comprises peptide segments CDR α1, CDR α2 and CDR α3; the variable region of the TCR β chain of the T cell receptor LMP2-TCR comprises peptide segments CDR β1, CDR β2 and CDR β3; the amino acid sequence of the CDR α3 is shown in SEQ ID NO.42, and the amino acid sequence of the CDR β3 is shown in SEQ ID NO.38; the amino acid sequence of CDR α1 in the variable region of the TCR α chain of the T cell receptor LMP2-TCR is shown in SEQ ID NO.40, and the amino acid sequence of CDR α2 is shown in SEQ ID NO.41; the amino acid sequence of CDR β1 in the variable region of the TCR β chain is shown in SEQ ID NO.36, and the amino acid sequence of CDR β2 is shown in SEQ ID NO.

37.

2. 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 of the T cell receptor BMLF1-TCR 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 of the T cell receptor LMP1-TCR1 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 of the T cell receptor LMP1-TCR2 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 of the T cell receptor EBNA-3B-TCR 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 of the T cell receptor LMP2-TCR is shown in SEQ ID NO.

54.

3. 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 of the T cell receptor BMLF1-TCR is shown in SEQ ID NO. 8; and / or the amino acid sequence of the TCRβ chain of the T cell receptor BMLF1-TCR is shown in SEQ ID NO. 4; B2) the amino acid sequence of the TCRα chain of the T cell receptor LMP1-TCR1 is shown in SEQ ID NO. 16; and / or the amino acid sequence of the TCRβ chain of the T cell receptor LMP1-TCR1 is shown in SEQ ID NO. 12; C2) the amino acid sequence of the TCRα chain of the T cell receptor LMP1-TCR2 is shown in SEQ ID NO. 24; and / or the amino acid sequence of the TCRβ chain of the T cell receptor LMP1-TCR2 is shown in SEQ ID NO. 20; D2) the amino acid sequence of the TCRα chain of the T cell receptor EBNA-3B-TCR is shown in SEQ ID NO. 34; and / or the amino acid sequence of the TCRβ chain of the T cell receptor EBNA-3B-TCR is shown in SEQ ID NO. 30; E2) The amino acid sequence of the TCRα chain of the T cell receptor LMP2-TCR is shown in SEQ ID NO.43; and / or, the amino acid sequence of the TCRβ chain of the T cell receptor LMP2-TCR is shown in SEQ ID NO.

39.

4. 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 3; 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 3, the polynucleotide according to a), the nucleic acid construct according to b) or the virus according to c).

5. The biomaterial according to claim 4, characterized in that The plasmid backbone of the nucleic acid construct is a plasmid backbone of a lentivirus, adenovirus or adeno-associated virus; The cells are T cells.

6. The biomaterial according to claim 5, characterized in that 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-Tur Any one or more of boGFP, 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 T cells are TCR-T cells.

7. The biomaterial according to claim 6, characterized in that The T cells are Jurkat T cells or CD8+T cells.

8. Use of the T cell receptor according to any one of claims 1 to 3 or the biomaterial according to any one of claims 4 to 7 in the preparation of a tumor treatment product or in vitro generation of a T cell receptor product, wherein the tumor is selected from lymphoma, gastric cancer, nasopharyngeal carcinoma or chronic myeloid leukemia.

9. A tumor treatment product, characterized in that: The tumor treatment product comprises the T cell receptor according to any one of claims 1 to 3 or the biological material according to any one of claims 4 to 7 and a pharmaceutically acceptable excipient.

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