T cell receptor as well as preparation method and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a T cell receptor as well as a preparation method and application thereof. The T cell receptor EBV-TCR provided by the invention comprises a TCR alpha chain variable region and a TCR beta chain variable region; the TCR alpha chain variable region comprises peptide fragments CDR alpha 1, CDR alpha 2 and CDR alpha 3; the TCR [beta] chain variable region comprises peptide fragments CDR [beta] 1, CDR [beta] 2 and CDR [beta] 3; the amino acid sequence of the CDR alpha 3 is as shown in SEQ ID NO.7, and the amino acid sequence of the CDR beta 3 is as shown in SEQ ID NO.3. The TCR provided by the invention has high specificity, can obviously activate the release of cytokines when being combined with T cells, and provides a solid theoretical and experimental basis for developing TCR-T therapy of targeted EB virus antigen.

Description

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. About 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 many diseases is closely related to EBV infection, including post-transplantation infection after hematopoietic stem cell transplantation (HSCT), lymphoma, nasopharyngeal carcinoma, etc. (Taylor, GS, Long, HM, Brooks, JM, Rickinson, AB, Hislop, AD 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 Epstein-Barr virus, which causes 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 infection phase of EB virus is mainly divided into the proliferation phase and the latent phase. EB virus expresses different proteins in different phases. The proteins expressed at the beginning of the proliferation phase and the proteins expressed in the latent phase (mainly including 6 nuclear proteins EBNA1, 2, 3A, 3B and 3C, EBNA-LP and 3 latent membrane proteins LMP1, LMP2A, LMP2B, among which LMP2B is a truncated isoform of LMP2A) are believed to play a role in the tumor formation process. In addition, EB virus-related tumor cells also express EB virus antigen proteins, and the main way for the human body to fight against 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 an adoptive T cell therapy that has developed rapidly in recent years. Unlike CAR-T therapy (Chimeric antigen receptor T cell therapy), which can only recognize targets on the membrane surface, TCR-T can recognize targets from inside and outside the cell with the help of the HLA (Human leukocyte antigen) antigen presentation 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 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, 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. Summary of the invention

[0006] 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.

[0007] 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 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) 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;

[0009] 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;

[0010] 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;

[0011] 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;

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

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

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

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

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

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

[0018] 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:

[0019] 1) loading the aforementioned EBV antigen peptides onto dendritic cells differentiated in vitro;

[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 biomaterial, which comprises a polynucleotide, a nucleic acid construct, a virus or a 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 antigen 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 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 cell and pharmaceutically acceptable excipients.

[0028] The present invention also provides the use of the aforementioned EBV antigen peptide or the aforementioned biological material in preparing the following products:

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

[0030] 2) Generate T cell receptor products in vitro.

[0031] The present invention also provides a method for treating tumors, which comprises using 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 treat tumor patients.

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

[0033] The present invention combines the distribution characteristics of HLA subtypes in 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 population and the European and American population, systematically performs Epstein-Barr virus antigen-specific TCR screening, and conducts comprehensive TCR function verification respectively. 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

[0034] Figure 1 Shown is a flow chart for sorting 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 results of an in vitro binding experiment of the TCR-T cells of the present invention.

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

[0038] Figure 5 Shown is a schematic diagram of the results of an in vitro killing experiment 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 DESCRIPTION

[0040] The present invention provides a T cell receptor BMLF1-TCR or a fragment thereof, wherein 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 CDRα1, CDRα2 and CDRα3; the TCR β 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] In some specific embodiments, the HLA subtypes 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 include 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.

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

[0057] 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 complementary determining regions or at both ends of the complementary 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 mouse 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] In some specific embodiments, the TCR α chain variable region can also be: a peptide segment having a sequence similarity of 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% 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 a sequence similarity of 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% 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%, and at least 99.5% sequence similarity.

[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 TCR α chain constant region or a TCR β chain constant region.

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

[0066] In some specific embodiments, the TCR α chain constant 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.55; the TCR β chain constant 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.56.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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 a sequence similarity of 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% 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 a sequence similarity of 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% 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.

[0073] In some embodiments, the TCR α chain is selected from human TCR α chain, humanized TCR α chain, chimeric TCR α chain or murine TCR α chain; the TCR β chain is selected from human TCR β chain, humanized TCR β chain, chimeric TCR β chain or murine TCR β chain. Wherein "chimeric TCR α chain" or "chimeric TCR β" means that the TCR α chain or TCR β 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 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.

[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 an antigen peptide BMLF1, an antigen peptide LMP1, an antigen peptide EBNA3 or an antigen peptide LMP2.

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

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

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

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

[0081] 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.

[0082] 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:

[0083] 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;

[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 the antigenic peptide BMLF1, antigenic peptide LMP1, antigenic peptide EBNA3 or antigenic peptide LMP2 with mature dendritic cells, incubating them and then irradiating them.

[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 antigenic peptide BMLF1, the aforementioned antigenic peptide LMP1, the aforementioned antigenic peptide EBNA3 or the aforementioned antigenic 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 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 urboGFP, 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, any one or more of pHR-SFFV-Puro.

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

[0091] In some 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 having the aforementioned polynucleotide integrated into its genome.

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

[0095] In some specific embodiments, the T cells are further selected from helper T cells, suppressor T cells, effector T cells, cytotoxic T cells, delayed hypersensitivity T cells, natural 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 and can act on helper T cells and suppressor T cells to expand the immune effect; natural T cells are T cells that have not yet differentiated; memory T cells are T cells with memory-specific antigen stimulation function.

[0096] In some embodiments, the T cells are Jurkat T cells or CD8+ T cells.

[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 preparing tumor treatment products.

[0098] 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 the preparation of an anti-tumor peptide vaccine product.

[0099] The present invention also provides the 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.

[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, 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.

[0101] In some embodiments, the lymphoid tumor comprises 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 pharmaceutically acceptable excipients.

[0103] 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 saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), 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. The adjuvant is selected from saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannoses or sugar alcohol isotonic solution when used for injection of aqueous solution. The injection aqueous solution 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 product provided by the present invention, the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned T cell is a single effective ingredient, and can also be combined with one or more other active components useful for tumor treatment to form a combined preparation. The active component is 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 a person 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 an 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.

[0104] The present invention also provides a method for treating tumors, which comprises using 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 treat tumor patients.

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

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

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

[0108] In the present invention, the term "TCRα chain constant region" or "TCRβ chain constant region" includes an extracellular constant region, a transmembrane region and an 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 the disulfide bond of 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 transformation of the conformation of the TCR-CD3 complex and signal transduction after TCR signal transduction.

[0109] In the present invention, the term "variable region" or "variable domain" refers to the domain of the immunoglobulin superfamily binding protein (e.g., TCR α-chain or β-chain (or gd chain and d chain) TCRs) involved in the binding of the immunoglobulin superfamily binding protein (e.g., TCR) to the antigen. The variable domains of the a chain and b chain of the natural TCR (Va and nb, respectively) generally have similar structures, each domain containing four conserved framework regions (FR) and three CDRs. The Va domain is encoded by two independent DNA fragments, the variable gene fragment and the connecting gene fragment (VJ); the nb domain is encoded by three independent DNA fragments, namely the variable gene fragment, the diversity gene fragment and the connecting gene fragment (VDJ). A single Va or nb domain may be sufficient to confer antigen binding specificity. In addition, TCRs that bind to a specific antigen can be separated from the TCR that binds to the antigen using the Va or nb domain to screen the library of complementary Va or nb domains, respectively.

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

[0111] In the present invention, the term "isolated" refers to materials that have been removed from their natural state or otherwise artificially manipulated, such as the α chains, β chains, T cell receptors and nucleic acids described herein. The 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, together with components that normally accompany it in its natural state. The isolated material may be in natural, chemically synthesized or recombinant form. The isolated material may also or alternatively be in an enriched, partially purified or purified form.

[0112] 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.

[0113] 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.

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

[0115] 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 may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison or non-homologous sequences may 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, the molecules are identical at this position.

[0116] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) that has been integrated into the GAP program of the GCG software package is used, using a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences. In another preferred embodiment, the GAP program in the GCG software package is used (available at http: / / www.gcg.com) to determine the percent identity between two nucleic acid sequences using the NWSgapdna.CMP matrix and gap weights 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.

[0117] 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.

[0118] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways 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 scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the 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.

[0120] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical 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 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 grasp of the prior art by those skilled in the art and the record 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 can also be used to realize 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] PKVSLFEPSKAEIANKQKATLVCLARGFFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLR

[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] MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSSNSASQSFFWYRQDCRKEPKLLM

[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] MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRIHY

[0262] SVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSTQGGGGDGYTFGSGTRLTVVSEQID No.55

[0263] IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSF

[0264] TCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLKVAGFNLLMTLRLWSSSEQID No.56

[0265] EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYS

[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] CTTCAAAGAGACCAACGCCACCTAACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACCGAGAAA

[0411] AGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCT

[0412] GAAAGTAGCGGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA

[0413] Example 1 TCR-T cell sorting and TCR sequencing

[0414] 1. Differentiation of human peripheral monocytes into dendritic cells (DC) in vitro

[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 culture 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 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 (1 μg / mL, Peprotech) were added to induce DC maturation.

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

[0418] 2. DC cells loaded with antigen peptides and co-stimulated with autologous CD8+T cells

[0419] (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 DC. After sealing with Parafilm, place it in a MACSmix rotator and rotate and bind it in a cell culture incubator for 4 hours. After the incubation of mDCs and EBV antigen peptides, use an irradiator to irradiate the DCs loaded with EBV antigen peptides at a dose of 40Gy. After irradiation, collect the DCs loaded with EBV antigen peptides by centrifugation into a 15 ml centrifuge tube.

[0420] (2) PBMCs were obtained from the blood of healthy donors by Lymphoprep density gradient centrifugation. CD8+T cells in PBMCs were separated using EasySepTMHuman 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 with a final concentration of 30 ng / ml and 5 μg / ml of EBV antigen peptides were added to the culture system.

[0421] (3) The 12-well plate was placed in a 37°C, 5% CO2 cell culture incubator and incubated overnight. On Day 1, 10 ng / ml of IL-2, IL-7, and IL-15 were added to each well, and then placed in a cell culture incubator at 37°C, 5% CO2 for 10 days. During the culture process, half of the medium was replaced every 3 days.

[0422] (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.

[0423] (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 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.

[0424] 3. EBV-antigen-specific-T cell sorting and single-cell TCR sequencing

[0425] (1) The process of sorting EBV-antigen-specific-T cells is as follows Figure 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 shown in Figure 2 As shown in A, the proportions of CD8a+ / Tetramer+T in healthy donors are BMLF1-0.34%, LMP1-2.52%, EBNA3B-0.4% and LMP2-0.15%, respectively, with obvious grouping. This indicates that EBV-antigen-specific-TCR-T cells were specifically amplified in two rounds of in vitro stimulation. Subsequently, a flow cytometer was used to specifically sort the CD8+ / tetramer+T cells in the co-culture system, 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.

[0426] (2) After the single-cell sequencing is completed, the transcriptomic characteristics of the top ten TCRs are analyzed, and the TCR sequencing results and frequency ranking results of the cell population obtained by the analysis are as follows: Figure 2 B. The TCR sequence with the highest frequency was then optimized, and the specific optimization content was to replace its TCR-C region, i.e., 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, and the amino acid sequence of the TCRα chain in the T cell receptor BMLF1-TCR obtained in the end 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 as 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 as 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 as 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 as 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 as 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 as 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 as 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 as SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38, respectively.

[0436] P2A and Furin-cleavage (wherein the amino acid sequence of the P2A-Furin-cleavage fusion peptide is shown as SEQ ID No.57) are used to enable the α and β chains of TCR to be expressed simultaneously in a lentiviral expression vector, wherein the nucleotide sequence encoding the T cell receptor that simultaneously expresses TCR α and β chains in the above-mentioned lentiviral expression vector is shown as any one of SEQ ID No.58-62. Among them, the nucleotide sequence encoding the simultaneous expression of TCRα and β chains in the T cell receptor BMLF1-TCR is shown in SEQ ID No. 58; the nucleotide sequence encoding the simultaneous expression of TCRα and β chains in the T cell receptor LMP1-TCR1 is shown in SEQ ID No. 59; the nucleotide sequence encoding the simultaneous expression of TCRα and β chains in the T cell receptor LMP1-TCR2 is shown in SEQ ID No. 60; the nucleotide sequence encoding the simultaneous expression of TCRα and β chains in the T cell receptor EBNA-3B-TCR is shown in SEQ ID No. 61; the nucleotide sequence encoding the simultaneous expression of TCRα and β chains in 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.

[0437] Example 2 EBV-antigen-specific-TCR-T cell binding experiment in vitro

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

[0439] (1) The optimized EBV-antigen-TCR obtained in Example 1 was used to construct a lentiviral expression vector, and then the lentiviral packaging of EBV-antigen-TCR was performed. The specific operation of virus packaging is as follows: 293T cells were digested and passaged, and the cell density was observed on the day of transfection. Transfection can be performed when it is 80-90% full. 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. It needs to be shaken before use. The complex components of each bottle of T75 for transfection are 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, blow normally 5-6 times, leave at room temperature for 15min, and then add it to the T75 bottle. Then, the virus supernatant was collected at 48h and 72h, and the supernatant was filtered through a 0.45um filter membrane (PES material) at 4000rpm for 5min before use. The lentiviral solution of EBV-antigen-specific-TCR-TCR was collected at 48h and 72h after virus packaging.

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

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

[0442] 2. FACS detection of EBV-antigen-TCR-T cell binding specificity

[0443] The EBV-antigen cells prepared above were stained with mTCRβC+ / Tetramer, and the flow cytometry results showed that the Tetramer+ / mTCRβC+Jurkat T( Figure 3A) and CD8+ / Tetramer+primary CD8+T( Figure 3 B) The cell clustering is obvious. The results show 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, 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) Directly differentiating the donor's autologous monocytes into DC cells 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;

[0447] (2) After 24 h of in vitro co-culture, the cells were discarded and then washed five times with 200 μl 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.

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

[0449] The results are as follows Figure 4 As shown in Figure 2, DCs loaded with EBV antigen peptides can specifically activate EBV-antigen-specific-TCR-T cells in vitro, and the IFNγ expression level of EBV-antigen-specific-TCR-T cells increased significantly after activation ( Figure 4 AC), so the results show that the EBV-BMLF1 / LMP1 / EBNA3B-TCR obtained in this screening and identification can specifically recognize the 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 infecting T cells or K562-A1101 cells with lentivirus. + T cells and K562-A1101 control cells or K562-A1101-BMLF1 / LMP1 / EBNA3B target cells were added to 96-well plates for co-culture;

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

[0454] (3) Place the 96-well plate containing cells into the Incucyte (Sartorius) live cell analyzer and use appropriate fluorescence channels to monitor cell apoptosis. The 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 test is completed after 48 hours of co-culture.

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

[0456] The results are as follows Figure 5 As shown, this example uses the Incucyte high-throughput long-term dynamic imaging and analysis system to detect the killing of target cells by EBV-BMLF1 / LMP1 / EBNA3B-TCR-T cells in real time. The results show that within 48 hours of co-culture, compared with negative control CD8+T cells that are not transfected with EBV-BMLF1 / LMP1 / EBNA3B-TCR, TCR-T cells with an effector-target ratio of 5:1 can more efficiently kill target cells that are HLA-A*11:01 positive and express TEBV-BMLF1 / LMP1 / EBNA3B antigens ( Figure 5 AD).

[0457] Example 5 In vivo killing experiment of EBV-antigen-TCR-T cells

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

[0459] (1) Six-week-old female NCG mice were selected and injected with 2E6 K562 cells carrying HLA-A*11:01 and EBNA3B antigens and expressing the Luciferase reporter gene into the mice through 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 control T lymphocytes without TCR gene transfer were used as negative control.

[0461] (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.

[0462] The results are as follows Figure 6 As shown, this example uses an in vivo CDX model to evaluate the effectiveness of EBV-EBAN3B-TCR-T cells in killing target cells ( Figure 6 A). In vivo imaging results showed that mice in the saline group and the Mock T cell infusion group showed obvious tumor cell growth within 27 days after tumor cell infusion, while some mice (3 / 4) in the EBV-EBAN3B-TCR-T infusion group showed no obvious tumor growth ( Figure 6 B), and the total photon number or average photon number intensity of luciferase in the EBV-EBAN3B-TCR-T group was significantly lower than that in the normal saline group and the Mock T control group ( Figure 6 CD). The results of CDX experiments 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 examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious 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 as described above that are obvious to those skilled in the art 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 contains 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; III) The variable region of the TCRα chain of the T cell receptor LMP1-TCR2 contains 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; IV) the variable region of the TCRα chain of the T cell receptor EBNA-3B-TCR contains 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; V) The variable region of the TCRα chain of the T cell receptor LMP2-TCR contains 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.

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 amino acid sequence of CDRβ1 in the TCRβ chain variable region 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 in 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.

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