Use of short antigen peptides in screening of drugs to treat HPV-related diseases and TCRs screened by them

Abstract

The present application discloses the use of antigen short peptides in screening drugs for treating HPV-related diseases and the TCRs screened thereby, and the amino acid sequence of the antigen short peptides is as shown in SEQ ID NO: 1 or SEQ ID NO: 2. In the present application, the antigen short peptides can be used to screen specific T cell receptors (TCRs), and the TCR-introduced T cells are specifically activated and have a very strong killing effect on tumor cells expressing A1101 and HPV, and can be used for immunotherapy of HPV-positive tumors such as cervical cancer. Furthermore, the TCR-introduced T cells of the present application show a very strong activation response to cell lines expressing E6, but do not show an activation response to cell lines not expressing E6, have a very strong killing function against cell lines expressing E6, and can effectively inhibit the growth of E6-positive tumors.

Description

[Technical field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to Chinese Patent Application No. CN202210503002.7, filed on May 10, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] (Reference to Electronic Sequence Listing) The contents of the electronic sequence listing (TFG00782PCT-XLB.xml, size: 52,426 bytes, created on May 6, 2023) are incorporated herein by reference in their entirety.

[0003] The present application relates to the field of pharmaceutical technology, and in particular to the use of antigenic short peptides for screening drugs for treating HPV-associated diseases and the TCRs screened thereby. [Background technology]

[0004] Human papillomavirus (HPV) is a DNA virus belonging to the genus Papillomavirus in the family Papillomaviridae. This type of virus infects the epidermis and mucosal tissues of the human body, and currently about 170 types of HPV have been identified. When HPV invades the human body, it can cause warts and cancer, but in most cases there are no clinical symptoms.

[0005] The key factor in causing HPV cancer is persistent infection with high-risk HPVs such as HPV16 and HPV18. The E6 and E7 proteins encoded by high-risk HPVs inhibit the activity of the tumor suppressor genes p53 and Rb, respectively, which may lead to cell cycle abnormalities and carcinogenesis. High-risk HPVs are associated with 90% of cervical and anal cancers, 40%-60% of vaginal and penile cancers, and may be associated with 60% of oropharyngeal cancers depending on geographic characteristics.

[0006] Cervical cancer is the most common malignant tumor occurring in the female reproductive organs, with the global incidence ranking second among female malignant tumors, and the incidence of cervical cancer in developing countries is significantly higher than that in developed countries. According to the latest statistics in 2018, the incidence rate of cervical cancer in China is 15.30 per 100,000, and the mortality rate is 4.57 per 100,000, both of which are higher than the global average incidence rate (10.61 per 100,000) and mortality rate (2.98 per 100,000). In the past decade, the incidence rate of cervical cancer has been on the rise, with the peak age of onset being in the 40s to 60s. Although surgery, radiation therapy, and chemotherapy are effective in treating early cervical cancer, more than half of patients are diagnosed with locally advanced cervical cancer at the first visit. Although 30%–70% of patients with locally advanced cervical cancer will experience recurrence and / or distant metastasis, the efficacy of conventional treatments for locally advanced and metastatic cervical cancer is very limited, with 5-year survival rates of only 57.1% and 17.3%, respectively.

[0007] Specific T cell immunotherapy refers to the method of killing tumor cells using specific T cells that target tumor antigens, which is a highly personalized tumor immunotherapy. Because tumors have a local immunosuppressive microenvironment, the tumor-killing function of the patient's own T cells is limited. Therefore, people are trying to improve the tumor-killing ability by genetically modifying T cells. Both TCR-T and CAR-T are genetically modified cell therapy drugs that, when they bind to the corresponding target via the introduced T cell receptor (T cell receptor, TCR) or chimeric antigen receptor (CAR) gene, activate T cells and eliminate tumor cells by utilizing granzymes, perforin, cytokines, etc. released by T cells. However, the major difference between TCR-T and CAR-T is that the target of CAR-T is the membrane protein on the cell surface, while the target of TCR-T is the antigen short peptide-MHC complex (peptide-major histocompatibility complex, pMHC).

[0008] HPV-associated proteins are the most ideal targets for T cell immunotherapy against cervical cancer. The target recognized by TCR is the "antigen short peptide-MHC molecule complex". TCR is also restricted to MHC. In theory, one TCR molecule specifically recognizes only short peptides presented by a certain MHC. MHC is polymorphic, and the number of human MHC (also called human leukocyte antigen, HLA) alleles discovered so far is more than 15,000, and the frequency of specific HLA in different human populations varies greatly. In China, the most common HLA type in the population is HLA-A1101. Short peptides presented by HLA class I molecules are 8-11 amino acids in length, and short peptides presented by HLA class II molecules are 12-24 amino acids in length. The discovery and identification of these antigen short peptides is a prerequisite for TCR-T therapy. Whether a short peptide binds to HLA, which can be determined by affinity prediction or HLA binding assays, and whether the short peptide is naturally presented by HPV-expressing tumor cells is key to determining whether a TCR specific for that short peptide can be used for tumor therapy.

[0009] Therefore, those skilled in the art have been striving to discover A1101-restricted HPV antigen short peptides and use the discovered antigen short peptides to screen TCRs that can specifically recognize HPV-positive tumor cells, so that they can play a role in T cell immunotherapy. Summary of the Invention

[0010] The purpose of this application is to provide the use of antigen short peptides in screening drugs for treating HPV-related diseases and the T cell receptors (TCRs) screened thereby, which are used to screen specific T cell antibodies, and the T cells transcribed with the TCR are specifically activated and have a very strong killing effect on tumor cells expressing A1101 and HPV.

[0011] The specific technical solutions of this application are as follows:

[0012] 1. Use of an antigenic short peptide in screening for drugs to treat HPV-related diseases, wherein the amino acid sequence of said antigenic short peptide is shown in SEQ ID NO:1 or SEQ ID NO:2. 2. The use according to item 1, wherein the HPV-related disease is HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer. 3. Use of an antigenic short peptide in screening for drugs to treat HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer, wherein the amino acid sequence of the antigenic short peptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2. 4. The use according to any one of items 1 to 3, wherein the agent is a T cell receptor (TCR) for binding to an antigenic short peptide-HLA-A1101 complex containing the antigenic short peptide. 5. A T cell receptor (TCR), comprising an alpha chain comprising a variable region and / or a beta chain comprising a variable region, the variable region of the alpha chain comprising a complementarity determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 9, and / or A T cell receptor (TCR) comprising a complementarity determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10. 6. The variable region of the β chain comprises a complementarity determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO: 6, and / or The T cell receptor (TCR) of item 5, comprising a complementarity determining region 2 (CDR2) having the amino acid sequence set forth in SEQ ID NO:7. 7. The variable region of the alpha chain comprises a complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15; and / or The T cell receptor (TCR) of claim 5 or 6, wherein the variable region of the beta chain comprises a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. 8. The alpha chain variable region further comprises a first leader sequence; and / or 8. The T cell receptor (TCR) according to any one of items 5 to 7, wherein the variable region of the β chain further comprises a second leader sequence. 9. The amino acid sequence of the variable region of the alpha chain is the amino acid sequence shown in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26, or is an amino acid sequence having at least 90% sequence identity to SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26; and / or A T cell receptor (TCR) according to any one of items 5 to 8, wherein the amino acid sequence of the variable region of the β chain is the amino acid sequence shown in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27, or is an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27. 10. The T cell receptor (TCR) according to any one of items 5 to 9, wherein the alpha chain further comprises an alpha constant region and / or the beta chain further comprises a beta constant region, preferably, the constant region being a mouse constant region or a human constant region. 11. The T cell receptor (TCR) of any one of paragraphs 5 to 10, wherein the TCR is isolated, purified or recombinant. 12. The T cell receptor (TCR) according to any one of items 5 to 11, wherein the TCR is human. 13. The T cell receptor (TCR) according to any one of items 5 to 12, wherein the TCR is monoclonal. 14. The T cell receptor (TCR) according to any one of items 5 to 13, wherein the TCR is a single chain. 15. The T cell receptor (TCR) of any one of clauses 5 to 14, wherein the TCR comprises two chains. 16. The T cell receptor (TCR) according to any one of items 5 to 15, wherein the TCR is in a cell-associated or soluble form, preferably in a soluble form. 17. The T cell receptor (TCR) according to any one of items 5 to 16, wherein the TCR binds to an antigenic short peptide-HLAA1101 complex, and preferably, the amino acid sequence of the antigenic short peptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 2. 18. A nucleic acid molecule comprising a nucleotide sequence encoding the TCR or the α chain or β chain of said TCR according to any one of items 5 to 17. 19. The nucleotide sequence encoding the alpha chain comprises the nucleotide sequence shown in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36; and / or 19. The nucleic acid molecule of claim 18, wherein the nucleotide sequence encoding the beta chain comprises the nucleotide sequence set forth in SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. 20. A vector comprising the nucleic acid molecule of item 18 or 19. 21. The vector according to paragraph 20, wherein the vector is an expression vector. 22. The vector of clause 20 or 21, wherein the vector is a viral vector, preferably a retroviral vector. 23. The vector of clause 22, wherein the viral vector is a lentiviral vector. 24. An engineered cell comprising the TCR of any one of paragraphs 5 to 17, the nucleic acid molecule of any one of paragraphs 18 to 19, or the vector of any one of paragraphs 20 to 23. 25. The engineered cell of clause 24, wherein the TCR is heterologous to the cell. 26. The engineered cell of paragraph 24 or 25, wherein the engineered cell is a cell line. 27. The engineered cell according to any one of clauses 24 to 26, wherein the engineered cell is a primary cell obtained from a subject, preferably, the subject is a mammalian subject, preferably a human. 28. The engineered cell according to any one of paragraphs 24 to 27, wherein the engineered cell is a T cell or a NK cell, preferably, the T cell is a T cell isolated from peripheral blood. 29. The engineered cell of clause 28, wherein the T cells are CD8+ or CD4+. 30. A method for producing an engineered cell according to any one of paragraphs 24 to 29, comprising introducing into a cell in vitro or ex vivo a nucleic acid molecule according to any one of paragraphs 18 to 19 or a vector according to any one of paragraphs 20 to 23. 31. The method of claim 30, wherein the vector is a viral vector and the introduction is by transduction. 32. A pharmaceutical composition comprising the T cell receptor (TCR) according to any one of items 5 to 17, the nucleic acid molecule according to any one of items 18 to 19, the vector according to any one of items 20 to 23, or the engineered cell according to any one of items 24 to 29. 33. The pharmaceutical composition according to item 32, further comprising a pharma- ceutically acceptable carrier or adjuvant. 34. Use of a T cell receptor (TCR) according to any one of clauses 5 to 17, a nucleic acid molecule according to any one of clauses 18 to 19, a vector according to any one of clauses 20 to 23, or an engineered cell according to any one of clauses 24 to 29, or a pharmaceutical composition according to any one of clauses 32 to 33 in the preparation of a medicament for treating an HPV-associated disease. 35. The use according to clause 34, wherein the HPV-related disease is HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer. 36. A method for treating an HPV-associated disease, comprising administering to a subject in need thereof the T cell receptor (TCR) according to any one of paragraphs 5 to 17, the nucleic acid molecule according to any one of paragraphs 18 to 19, the vector according to any one of paragraphs 20 to 23, or the engineered cell according to any one of paragraphs 24 to 29, or the pharmaceutical composition according to any one of paragraphs 32 to 33. 37. The method of clause 36, wherein the HPV-associated disease is HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer. Effect of the Invention

[0013] The antigen short peptides described in the present application can screen for specific T cell receptors (TCRs), and at the same time, the TCR-transduced T cells can be specifically activated and have a very strong killing effect on tumor cells expressing A1101 and HPV, and can be used for the immunotherapy of HPV-positive tumors such as cervical cancer.

[0014] And the T cells transduced with the TCR described in the present application have a very strong activation response to cell lines expressing E6, no activation response to cell lines not expressing E6, and have a very strong killing function to cell lines expressing E6, and can effectively inhibit the growth of E6-positive tumors. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram of the cloning of TTLEQQYNK and GTTLEQQYNK antigen short peptide-specific T cells in Example 1. Here, FIG. 1A is a schematic diagram of the flow cytometry results of T cells after two rounds of stimulation, and FIG. 1B is a schematic diagram of the flow cytometry results of T cells after three rounds of stimulation. [Diagram 2] FIG. 1 is a schematic diagram of using flow cytometry to determine the positive rate of transfection in Example 3. [Diagram 3] FIG. 1 is a schematic diagram of NFAT expression in T cells transduced with the TCR described in this application in Example 3 in response to target cells loaded with TTLEQQYNK antigenic short peptides. [Figure 4] FIG. 1 is a schematic diagram showing the determination of the positive rate of transfection by flow cytometry in Example 4. [Diagram 5] FIG. 1 is a schematic diagram of the cell proliferation index of T cells transduced with the TCRs described in this application in Example 4. [Figure 6]6A-6B are schematic diagrams of tumor volume following inoculation of T cells transduced with the TCRs described in this application in Example 5, where Fig. 6A is a schematic diagram of the change in tumor volume over time following T cell inoculation, and Fig. 6B is a histogram of tumor weight following inoculation of T cells containing different TCRs. [Figure 7] Figure 7A-Figure 7B are statistical schematic diagrams of the observed organoid proliferation state and the completeness and incompleteness of the organoid. Here, Figure 7A is a schematic diagram showing the proliferation state of the organoid observed under a microscope, and Figure 7B is a schematic diagram showing the statistical number of complete and incomplete organoids. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present application will be described in detail below with reference to embodiments illustrated in the accompanying drawings, in which like numerals represent like features in all drawings. Although specific embodiments of the present application are illustrated in the drawings, it should be understood that the present application can be embodied in various forms and should not be limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0017] It should be noted that the specification and claims use specific terms to refer to specific components. Those skilled in the art will understand that different nouns may be used to refer to the same components. The specification and claims do not use differences in nouns as a way to distinguish components, but rather use differences in the functions of components as a basis for distinction. The terms "containing" or "including" referred to throughout the specification and claims are open terms, so they should be interpreted as "including but not limited to". The following descriptions are preferred embodiments for carrying out the present application, but these descriptions are intended for the general principles of the specification and do not limit the scope of the present application. The scope of protection of the present application shall be specified by the appended claims.

[0018] The present application provides the use of an antigenic short peptide in screening for drugs to treat HPV-related diseases, the amino acid sequence of said antigenic short peptide being shown in SEQ ID NO:1 or SEQ ID NO:2. The sequence of SEQ ID NO:1 is as follows: TTLEQQYNK The sequence of SEQ ID NO:2 is as follows: GTTLEQQYNK.

[0019] In one embodiment, the HPV-associated disease is HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer.

[0020] The present application provides the use of an antigenic short peptide in screening for drugs to treat HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer, the amino acid sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0021] In one embodiment, said agent is a T cell receptor (TCR) for binding to an antigenic short peptide-HLA-A1101 complex comprising said antigenic short peptide.

[0022] That is, the T cell receptor is obtained by screening the antigen short peptide, and the T cell receptor (TCR) binds to the TTLEQQYNK-HLA-A1101 complex or the GTTLEQQYNK-HLA-A1101 complex.

[0023] The T cell receptor or TCR is the only receptor that presents a specific antigen peptide present on the major histocompatibility complex (MHC), and in the immune system, the binding of an antigen-specific TCR to the pMHC complex leads to direct physical contact between the T cell and the antigen-presenting cell (APC), which then interacts with other cell membrane surface molecules of both the T cell and the APC, which then triggers a series of cell signaling and other physiological responses that allow T cells with different antigen specificities to exert immune effects against target cells.

[0024] The TCR is a molecule that includes variable α and β chains, or variable γ and δ chains, and is capable of peptide-specific binding to MHC molecules. In some embodiments, the TCR is of the αβ type. In general, TCRs present in the αβ and γδ types are similar in overall structure, but the T cells expressing them may have different anatomical locations or functions, and the TCRs may be found on the cell surface or in a soluble form. Typically, TCRs are present on the surface of T cells (T lymphocytes) and are responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0025] The variable domain of a TCR contains complementarity determining regions (CDRs), which often contribute significantly to antigen recognition and binding capacity and specificity of peptides, MHC and / or MHC-peptide complexes. The CDRs of a TCR, or a combination thereof, form all or substantially all of the antigen binding site of a given TCR molecule, and the individual CDRs in the variable region of a TCR are typically spaced apart by framework regions (FRs). Of these, CDR3 is the primary CDR involved in antigen binding or specificity, or the most important of the three CDRs on a given TCR variable region for antigen recognition and / or interaction with the processed peptide portion of a peptide-MHC complex. In some cases, CDR1 of the α chain can interact with the N-terminal portion of some antigen peptides. In some cases, CDR1 of the β chain can interact with the C-terminal portion of some antigen peptides. In some cases, CDR2 is the CDR that has the strongest effect or is primarily involved in interaction or recognition of the MHC portion of an MHC-peptide complex. In some cases, the variable region antigen of the β chain contains other hypervariable regions (CDR4 or HVR4), which are usually involved in superantigen binding rather than antigen recognition.

[0026] Said TTLEQQYNK-HLA-A1101 complex or GTTLEQQYNK-HLA-A1101 complex refers to the complex of HLA-A1101 and antigen short peptide TTLEQQYNK or GTTLEQQYNK, protein is degraded by intracellular proteasome into polypeptides of different lengths, some polypeptides bind to HLA to form complexes, and are presented on cell surface.The TTLEQQYNK-HLA-A1101 complex or GTTLEQQYNK-HLA-A1101 complex recognized by said TCR can be expressed on cell membrane or exist in solution in the form of soluble protein.

[0027] The amino acid sequence of HLA-A1101 is shown in SEQ ID NO: 38, and the amino acid sequence is as follows: MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAY LEGRCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSYTQAASSDSAQGSDVSLTACKVSR

[0028] In one embodiment, the antigenic short peptide is used to screen for a drug for treating HPV-related disease, and the amino acid sequence of the antigenic short peptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the HPV-related disease is HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer. In one embodiment, the drug is a T cell receptor (TCR) for binding to an antigenic short peptide-HLA-A1101 complex comprising the antigenic short peptide.

[0029] In one embodiment, the antigenic short peptide is used for screening of a drug for treating HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer, and the amino acid sequence of said antigenic short peptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, said drug is a T cell receptor (TCR) for binding to an antigenic short peptide-HLA-A1101 complex comprising said antigenic short peptide.

[0030] The present application provides a T cell receptor (TCR), the TCR comprising an α chain comprising a variable region and / or a β chain comprising a variable region, the variable region of the α chain comprising a complementarity determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 9, and / or It comprises a complementarity determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10.

[0031] The amino acid sequence shown in SEQ ID NO:3 is SSYSPS. The amino acid sequence shown in SEQ ID NO:9 is TTLSN. The amino acid sequence shown in SEQ ID NO:4 is YTSAATLV. The amino acid sequence shown in SEQ ID NO:10 is LVKSGEV.

[0032] In one embodiment, the variable region of the beta chain comprises a complementarity determining region 1 (CDR1) having the amino acid sequence set forth in SEQ ID NO:6, and / or It contains a complementarity determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO:7.

[0033] The amino acid sequence shown in SEQ ID NO:6 is SGDLS. The amino acid sequence shown in SEQ ID NO:7 is YYNGEE.

[0034] In one embodiment, the variable region of the alpha chain comprises a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15; and / or The variable region of the β chain comprises a complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

[0035] The amino acid sequence shown in SEQ ID NO:5 is VVSLSGGYNKLI. The amino acid sequence shown in SEQ ID NO:11 is AGPKITGGGNKLT. The amino acid sequence shown in SEQ ID NO:13 is AGPILTGGGNKLT. The amino acid sequence shown in SEQ ID NO:15 is AGPVLTGGGNKLT. The amino acid sequence shown in SEQ ID NO:8 is ASSVTGSGYT. The amino acid sequence shown in SEQ ID NO:12 is ASSVGGGPNYGYT. The amino acid sequence shown in SEQ ID NO:14 is ASGLSGPNTGELF. The amino acid sequence shown in SEQ ID NO:16 is ASSVGGPNTGELF.

[0036] In one embodiment, the alpha chain variable region further comprises a first leader sequence, and / or The variable region of the β chain further comprises a second leader sequence.

[0037] The first leader sequence of the α chain variable region and the second leader sequence of the β chain variable region are well known to those skilled in the art. For example, the first leader sequence of the α chain variable region may be a leader sequence having the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 19, and the second leader sequence of the β chain variable region may be a leader sequence having the amino acid sequence shown in SEQ ID NO: 18.

[0038] The amino acid sequence shown in SEQ ID NO:17 is MLLLLVPVLEVIFTLGGTR. The amino acid sequence shown in SEQ ID NO:19 is MLLITSMLVLWMQLSQVN. The amino acid sequence shown in SEQ ID NO:18 is MGFRLLCCVAFCLLGAGPV.

[0039] In one embodiment, the amino acid sequence of the alpha chain variable region is the amino acid sequence set forth in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26; and / or The amino acid sequence of the variable region of the β chain is the amino acid sequence shown in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27.

[0040] The amino acid sequence shown in SEQ ID NO:20 is as follows: MLLLLVPVLEVIFTLGGTRAQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLFWYVQHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCVVSLSGGYNKLIFGAGTRLAVHPY The amino acid sequence shown in SEQ ID NO:21 is as follows: MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVTGSGYTFGSGTRLTVV The amino acid sequence shown in SEQ ID NO:22 is as follows: MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGPKITGGGNKLTFGTGTQLKVELN The amino acid sequence shown in SEQ ID NO:23 is as follows: MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVGGGPNYGYTFGSGTRLTVV The amino acid sequence shown in SEQ ID NO:24 is as follows: MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGPILTGGGNKLTFGTGTQLKVELN The amino acid sequence shown in SEQ ID NO:25 is as follows: MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASGLSGPNTGELFFGEGSRLTVL The amino acid sequence shown in SEQ ID NO:26 is as follows: MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQLTDVGTYFCAGPVLTGGGNKLTFGTGTQLKVELN The amino acid sequence shown in SEQ ID NO:27 is as follows: MGFRLLCCVAFCLLGAGPVDSGVTQTPRYLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVGGPNTGELFFGEGSRLTVL

[0041] The amino acid sequence of the alpha chain variable region having at least 90% sequence identity to SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26 can be, for example, an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26. The amino acid sequence of the variable region of the β chain has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27, and can be, for example, an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27.

[0042] In one embodiment, the alpha chain further comprises an alpha constant region and / or the beta chain further comprises a beta constant region, preferably wherein the constant region is a murine constant region or a human constant region.

[0043] For example, the amino acid sequence of the mouse alpha constant region is set forth in SEQ ID NO:28; and / or The amino acid sequence of the mouse β constant region is as shown in SEQ ID NO: 29, that is, the constant regions of the α chains of the above-mentioned TCRs can all have the same constant region. Similarly, the constant regions of the β chains of all the TCRs can also have the same constant region.

[0044] The amino acids shown in SEQ ID NO:28 are as follows: IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS The amino acids shown in SEQ ID NO:29 are as follows: EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS

[0045] The constant region of the TCR may contain a short linker sequence in which cysteine ​​residues form disulfide bonds to link the two chains of the TCR. The TCR may have additional cysteine ​​residues in each of the α and β chains such that the constant region contains two disulfide bonds.

[0046] In one embodiment, the amino acid sequence of the alpha chain variable region of the TCR is as set forth in SEQ ID NO: 20, and the amino acid sequence of the beta chain variable region is as set forth in SEQ ID NO: 21, or the amino acid sequence of the alpha chain variable region is as set forth in SEQ ID NO: 22, and the amino acid sequence of the beta chain variable region is as set forth in SEQ ID NO: 23, or the amino acid sequence of the alpha chain variable region is as set forth in SEQ ID NO: 24, and the amino acid sequence of the beta chain variable region is as set forth in SEQ ID NO: 25, or the amino acid sequence of the alpha chain variable region is as set forth in SEQ ID NO: 26, and the amino acid sequence of the beta chain variable region is as set forth in SEQ ID NO: 27.

[0047] In one embodiment, an artificial disulfide bond is introduced between a residue of the α chain constant region and a residue of the β chain constant region of the TCR, and the locations of disulfide bonds that can be introduced are well known to those skilled in the art.

[0048] In one embodiment, the TCR is isolated, purified or recombinant.

[0049] In one embodiment, the TCR is human.

[0050] In one embodiment, the TCR is monoclonal.

[0051] In one embodiment, the TCR is a single chain.

[0052] In one embodiment, the TCR comprises two chains.

[0053] The TCR can be obtained from a biological source, e.g., a cell (e.g., a T cell (e.g., a cytotoxic T cell)), a T cell hybridoma, or other publicly available source, e.g., the TCR can be derived from one of several animal species, such as human, mouse, rat, or other mammal, typically human.

[0054] In one embodiment, said TCR is in cell-associated or soluble form, preferably in soluble form.

[0055] The TCR being soluble refers to a TCR having a mutation in its hydrophobic core region, and it is preferable that these mutations in the hydrophobic core region are mutations that can improve the stability of the soluble TCR of the present application.

[0056] In one embodiment, the amino acid sequence of the alpha chain variable region of the TCR is as set forth in SEQ ID NO: 20, and the amino acid sequence of the beta chain variable region is as set forth in SEQ ID NO: 21, or the amino acid sequence of the alpha chain variable region is as set forth in SEQ ID NO: 22, and the amino acid sequence of the beta chain variable region is as set forth in SEQ ID NO: 23, or the amino acid sequence of the alpha chain variable region is as set forth in SEQ ID NO: 24, and the amino acid sequence of the beta chain variable region is as set forth in SEQ ID NO: 25, or the amino acid sequence of the alpha chain variable region is as set forth in SEQ ID NO: 26, and the amino acid sequence of the beta chain variable region is as set forth in SEQ ID NO: 27.

[0057] In one embodiment, a T cell receptor (TCR) described in the present application comprises an alpha chain comprising a variable region and / or a beta chain comprising a variable region, wherein the variable region of the alpha chain comprises a complementarity determining region 1 (CDR1) having the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:9, and / or a complementarity determining region 2 (CDR2) having the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:10. In one embodiment, the variable region of the beta chain comprises a complementarity determining region 1 (CDR1) having the amino acid sequence set forth in SEQ ID NO:6, and / or a complementarity determining region 2 (CDR2) having the amino acid sequence set forth in SEQ ID NO:7. In one embodiment, the variable region of the α chain comprises a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15, and / or the variable region of the β chain comprises a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, preferably the variable region of the α chain further comprises a first leader sequence, and / or the variable region of the β chain further comprises a second leader sequence, preferably the amino acid sequence of the variable region of the α chain is set forth in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26. and / or the amino acid sequence of the variable region of said β chain is the amino acid sequence shown in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27, preferably said α chain further comprises an α constant region, and / or said β chain further comprises a β constant region, preferably said constant region is a mouse constant region or a human constant region.In one embodiment, the TCR is isolated, purified or recombinant, preferably the TCR is human, preferably the TCR is monoclonal, preferably the TCR is single chain, preferably the TCR comprises two chains, preferably the TCR is cell-associated or soluble, preferably soluble, preferably the TCR binds to an antigenic short peptide-HLAA1101 complex, preferably the amino acid sequence of the antigenic short peptide is as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0058] The present application also provides a nucleic acid molecule comprising a nucleotide sequence encoding said TCR or the α or β chain of said TCR.

[0059] In one embodiment, the nucleotide sequence encoding the alpha chain comprises the nucleotide sequence shown in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36; and / or the nucleotide sequence encoding the beta chain comprises the nucleotide sequence set forth in SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37; wherein the nucleotide sequence shown in SEQ ID NO: 30 is ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACTCTGGGAGGAACCAGAGCCCAGTCGGTGACCCAGCTTGACAGCCACGTCTCTGTCTCTGAAGGAACCCCGGTGCTGCTGAGGTGCAACTCATCTTCTTATTCACCATCTCTCTTCTGGTATTGTGCAACACCCCAACAAAGGACTCCAGCTTCTCCTGA AGTACACATCAGCGGCCACCCTGGTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAAAGAAGGTGAAACCTCCTTCCACCTGACGAAACCCTCAGCCCATATGAGCGACGCGGCTGAGTACTTCTGTGTTGTGAGTCTTTCTGGTGGCTACAATAAGCTGATTTTTGGAGCAGGGACCAGGCTGGCTGTACACCCATATATC CAGAATCCAGAGCCCGCCGTGTATCAGCTGAAGGACCCAAGGAGCCAGGATTCCACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAACGTGCCCAAGACCATGGAGTCCGGCACCTTCATCACAGACAAGTGCGTCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACCT CCTTCACATGCCAGGATATCTTTAAGGAGACCAATGCCACATATCCTTCCTCTGACGTGCCATGTGATGCCACCCTGACAGAGAGTCCTTCGAGACCGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGCGCATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCTGAGGCTGTGGAGCTCC and The nucleotide sequence shown in sequence number 31 is, ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAGAGAGAGCAAA AGGAAACATTCTTGAACGATTCTCCGCCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTGACAGGGAGTGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGATCTGAGGAACGTGACACCCCCTAAGGTGTCTCTGTTCGAGCCCAGCAAGGCCGAGATCG CCAATAAGCAGAAGGCCACCCTGGTGTGCCTTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCTGTGCACCGACCCACAGGCCTACAAGGAGTCCAATTACTCTTATTGTCTGAGCTCCCGGCTGAGAGTGTCCGCCCACATTTTGGCACAACCTAGAAATCACTTCAGGTGCCAGGTGCAGTTTCAC GGCCTGAGCGAGGAGGATAAGTGGCCAGAGGGATCCCCAAAGCCTGTGACCCAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGTGGAATCACATCCGCCTTTATCACCAGGGCGTGCTGAGCGCCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCC and The nucleotide sequence shown in sequence number 32 is, ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAGGTGAATGGACAACAGGTAATGCAAATTCCTCAGTACCAGCATGTACAAGAAGGAGAGGACTTCACCACGTACTGCAATTCCTCAACTACTTAAGCAATATACAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTGATACAGTTA GTGAAGAGTGGAGAAGTGAAGAAGCAGAAAAGACTGACATTTCAGTTTGGAGAAGCAAAAAAGAACAGCTCCCTGCCACATCACAGCCACCCAGACTACAGATGTAGGAACCTACTTCTGTGCAGGGCGAAATCACGGGAGGAGGAAACAAACTCACCTTTGGGACAGGCACTCAGCTAAAAGTGGAACTCAATATCCAG AATCCAGAGCCCGCCGTGTATCAGCTGAAGGACCAAGGAGCCAGGATTCCACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAACGTGCCCAAGACCATGGAGTCCGGCACCTTCATCACAGACAAGTGCGTCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACCTCC TTCACATGCCAGGATATCTTTAAGGAGACCAATGCCACATATCCTTCCTCTGACGTGCCATGTGATGCCACCCTGACAGAGAGTCCTTCGAGACCGACATGAACCTGAATTTTCAGAACCTGTCTGATGGGCCTGCGCATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCTGAGGCTGTGGAGCTCC and The nucleotide sequence shown in sequence number 33 is, ATGGGCTTCAGGCTCCTCTGCTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAGAGAGAGCAAAAG GAAACATTCTTGAACGATTCTCCGCCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTCCAGCAGCGTAGGCGGGGGCCCTAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGTACTGAGGAACGTGACACCCCCTAAGGTGTCTCTGTTCGAGCCCAGCAAGGCCGAG ATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAGGAGGTGCACAGCGCGTGTGCACCGACCCACAGGCCTACAAGGAGTCCAATTACTCTTATTGTCTGAGCTCCCGGCTGAGAGTGTCCCGCCACATTTTGGCACAACCTAGAAATCACTTCAGGTCCAGGTGCAGTTTC ACGGCCTGAGCGAGGAGGATAAGTGGCCAGAGGGATCCCCAAAGCCTGTGACCCAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGTGGAATCACATCCGCTCTTATCACCAGGGCGTGCTGAGCGCCACATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCC and The nucleotide sequence shown in sequence number 34 is, ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAGGTGAATGGACAACAGGTAATGCAAATTCCTCAGTACCAGCATGTACAAGAAGGAGAGGACTTCACCACGTACTGCAATTCCTCAACTACTTAAGCAATATACAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTGATACAGTTA GTGAAGAGTGGAGAAGTGAAGAAGCAGAAAAGACTGACATTTCAGTTTGGAGAAGCAAAAAGAACAGCTCCCTGCACATCACAGCCACCAGACTACAGATGTAGGAACCTACTTCTGTGCAGGCCCCATACTCACGGGAGGAGAAACAAACTCACCTTTGGGACAGGCACTCAGCTAAAAGTGGAACTCAATATCCAG AATCCAGAGCCCGCCGTGTATCAGCTGAAGGACCAAGGAGCCAGGATTCCACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAACGTGCCCAAGACCATGGAGTCCGGCACCTTCATCACAGACAAGTGCGTCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACCTCC TTCACATGCCAGGATATCTTTAAGGAGACCAATGCCACATATCCTTCCTCTGACGTGCCATGTGATGCCACCCTGACAGAGAGTCCTTCGAGACCGACATGAACCTGAATTTTCAGAACCTGTCTGATGGGCCTGCGCATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCTGAGGCTGTGGAGCTCC and The nucleotide sequence shown in sequence number 35 is ATGGGCTTCAGGCTCCTCTGCTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAGAGAGAGCAAAAG GAAACATTCTTGAACGATTCTCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTGTATTCTGTGCCAGCGGGCTTTCAGGACCGAACACCGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAGGATCTGAGGAACGTGACACCCCCTAAGGTGTCTCTGTTCGAGCCCAGGCAAGGCCGAG ATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAGGAGGTGCACAGCGCGTGTGCACCGACCCACAGGCCTACAAGGAGTCCAATTACTCTTATTGTCTGAGCTCCCGGCTGAGAGTGTCCCGCCACATTTTGGCACAACCTAGAAATCACTTCAGGTCCAGGTGCAGTTTC ACGGCCTGAGCGAGGAGGATAAGTGGCCAGAGGGATCCCCAAAGCCTGTGACCCAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGTGGAATCACATCCGCTCTTATCACCAGGGCGTGCTGAGCGCCACATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCC and The nucleotide sequence shown in sequence number 36 is ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAGGTGAATGGACAACAGGTAATGCAAATTCCTCAGTACCAGCATGTACAAGAAGGAGAGGACTTCACCACGTACTGCAATTCCTCAACTACTTAAGCAATATACAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTGATACAGTTA GTGAAGAGTGGAGAAGTGAAGAAGCAGAAAAGACTGACATTTCAGTTTGGAGAAGCAAAAAAGAACAGCTCCCTGCCACATCACAGCCACCCAGCACTACAGATGTAGGAACCTACTTCTGTGCAGGCCGGTACTCACGGGAGGAGAAACAAACTCACCTTTGGGACAGGCACTCAGCTAAAAGTGGAACTCAATATCCAG AATCCAGAGCCCGCCGTGTATCAGCTGAAGGACCAAGGAGCCAGGATTCCACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAACGTGCCCAAGACCATGGAGTCCGGCACCTTCATCACAGACAAGTGCGTCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACCTCC TTCACATGCCAGGATATCTTTAAGGAGACCAATGCCACATATCCTTCCTCTGACGTGCCATGTGATGCCACCCTGACAGAGAGTCCTTCGAGACCGACATGAACCTGAATTTTCAGAACCTGTCTGATGGGCCTGCGCATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCTGAGGCTGTGGAGCTCC and The nucleotide sequence shown in sequence number 37 is, ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTGGGGGGACCGAACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAGGATCTGAGGAACGTGACACCCCCTAAGGTGTCTCTGTTCGAGCCCAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTGCACCGACCCACAGGCCTACAAGGAGTCCAATTACTCTTATTGTCTGAGCTCCCGGCTGAGAGTGTCCGCCACATTTTGGCACAACCCTAGAAATCACTTCAGGTGCCAGGTGCAGTTTCACGGCCTGAGCGAGGAGGATAAGTGGCCAGAGGGATCCCCAAAGCCTGTGACCCAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGTGGAATCACATCCGCCTCTTATCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCC is.

[0060] In one embodiment, the nucleotide sequence encoding the α chain and / or the nucleotide sequence encoding the β chain are codon-optimized. Typically, codon optimization involves balancing the proportion of selected codons with the abundance of published human transfer RNAs to avoid overloading or limitation. This may be necessary in some cases, since most amino acids are encoded by multiple codons, and codon usage varies between organisms. Differences in codon usage between the transfected gene and the host cell may affect protein expression and immunogenicity of the nucleic acid construct. Typically, codon optimization involves selecting codons that are balanced with human usage. Generally, redundancy in amino acid codons results in different codons encoding one amino acid. In some embodiments, when selecting codons for substitution, it may be desirable for the resulting mutation to be a silent mutation, so that the change in the codon does not affect the amino acid sequence. Typically, the last nucleotide of the codon can be left unchanged without affecting the amino acid sequence.

[0061] The present application provides a vector comprising the above-described nucleic acid molecule.

[0062] For example, one or more nucleic acids encoding one or two chains of the TCRs described above may be cloned into one or more suitable expression vectors, which may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include vectors designed for propagation and amplification and / or expression, such as plasmids and viruses.

[0063] Considering whether the vector is DNA or RNA based, the vector may include regulatory sequences (such as transcription and translation initiation and termination codons) specific to the type of host into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals). The vector may also include a non-native promoter operably linked to the nucleotide sequence encoding the TCR. The promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat of the murine stem cell virus, and other promoters known to those skilled in the art are also contemplated.

[0064] Said vector is an expression vector, preferably a viral vector, preferably a retroviral vector, more preferably a lentiviral vector.

[0065] The present application also provides host cells comprising the above-mentioned nucleic acid molecules, and for recombinantly producing a TCR, the nucleic acid encoding the TCR can be isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the α and β chains of the TCR). In some embodiments, a method of preparing a TCR is provided, which comprises culturing a host cell comprising a nucleic acid encoding a TCR as provided above under conditions suitable for expression of the TCR molecule, and optionally recovering the TCR from the host cell (or host cell culture medium).

[0066] The host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include transformants and transformed cells, including the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell and may contain mutations.

[0067] The present application also provides an engineered cell comprising said T cell receptor (TCR), said nucleic acid molecule or said vector.

[0068] In one embodiment, the TCR is xenogeneic to the cell.

[0069] In one embodiment, the engineered cell is a cell line.

[0070] In one embodiment, the engineered cells are primary cells obtained from a subject, preferably, the subject is a mammalian subject, preferably a human subject.

[0071] In one embodiment, the engineered cells are T cells, preferably T cells isolated from peripheral blood.

[0072] In one embodiment, the T cells are CD8+ or CD4+.

[0073] The engineered cells can be, for example, cell populations or genetically engineered cells expressing a TCR, which are typically eukaryotic cells, such as mammalian cells, and typically human cells. In some embodiments, the cells are cells of the immune system, such as cells of innate or adaptive immunity, such as myeloid or lymphoid cells (including lymphocytes, typically T cells and / or NK cells), derived from blood, bone marrow, lymph or lymphoid organs. Other exemplary cells include stem cells, such as pluripotent stem cells, including multipotent stem cells and induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as those isolated directly from a subject and / or those isolated and frozen from a subject. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the total population of T cells, CD4+ cells, CD8+ cells, and subpopulations thereof.

[0074] Subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes (such as stem cell memory T cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), or terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, natural and adaptive regulatory T (Treg) cells, and the like.

[0075] In some embodiments, the engineered cells are natural killer (NK) cells, preferably the cells are myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, monocytes or granulocytes such as eosinophils and / or basophils.

[0076] The present application provides a method for producing the above-mentioned engineered cell, comprising introducing the above-mentioned nucleic acid molecule or the above-mentioned vector into a cell in vitro or ex vivo.

[0077] The vector is a viral vector and the introduction is by transduction.

[0078] The present application provides a pharmaceutical composition comprising the above T cell receptor (TCR), the above nucleic acid molecule, the above vector, or the above engineered cell. In one embodiment, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or adjuvant.

[0079] The pharma- ceutically acceptable carrier or adjuvant refers to an ingredient in a pharmaceutical composition that is non-toxic to a subject, except for the active ingredient. Pharmaceutically acceptable carriers or adjuvants include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0080] The pharmaceutical composition may utilize time-release, delayed release, and sustained release delivery systems so that delivery of the composition occurs prior to sensitization of the treatment site, allowing sufficient time for sensitization to occur.Many types of release delivery systems are available and known.Such systems can be used to avoid repeated administration of the composition, thereby improving the convenience of the subject and the physician.

[0081] The present application provides the use of the above T cell receptor (TCR), the above nucleic acid molecule, the above vector, the above engineered cell, or the above pharmaceutical composition in the preparation of a medicament for treating an HPV-associated disease.

[0082] In one embodiment, the HPV-associated disease is HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer, etc.

[0083] The present application provides a method for treating an HPV-associated disease, comprising administering to a subject in need thereof the T cell receptor (TCR), the nucleic acid molecule, the vector, or the engineered cell, or the pharmaceutical composition. In one embodiment, the HPV-associated disease is HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer, or vulvar cancer.

[0084] The TCR described in the present application can specifically bind to antigenic short peptides on HPV-positive tumor cells, and T cells transduced with the TCR can be specifically activated and have a very strong killing effect on target cells, and the TCR can be used for immunotherapy of HPV-positive tumors such as cervical cancer.

[0085] In the present application, by using antigen short peptides, specific T cell receptors (TCRs) can be screened, and the TCR-transduced T cells can be specifically activated and have a very strong killing effect on tumor cells expressing A1101 and HPV, and can be used for immunotherapy of HPV-positive tumors such as cervical cancer.

[0086] The T cells transduced with the TCR described in the present application have a very strong activation response to cell lines expressing E6, no activation response to cell lines not expressing E6, and a very strong killing function to cell lines expressing E6, and can effectively inhibit the growth of E6-positive tumors.

[0087] The TCR-transduced T cells of the present application can significantly kill primary cervical cancer organoid cells and effectively inhibit the growth of E6-positive tumors. EXAMPLES

[0088] This application provides general and / or specific descriptions of the materials and test methods used in the tests. In the following examples, unless otherwise specified, % means wt%, i.e. weight percent. If the manufacturers of the reagents and instruments used are not indicated, they are all conventional reagent products available on the market.

[0089] Example 1 Cloning of TTLEQQYNK and GTTLEQQYNK antigen short peptide-specific T cells The synthesized short peptides TTLEQQYNK (SEQ ID NO: 1, Jiangsu GenScript Biotechnology Co., Ltd.) and GTTLEQQYNK (SEQ ID NO: 2, Jiangsu GenScript Biotechnology Co., Ltd.) were used to stimulate peripheral blood lymphocytes of healthy volunteers with the genotype of HLA-A*11:01, respectively. The TTLEQQYNK short peptide and the GTTLEQQYNK short peptide were regenerated with biotin-labeled HLA-A*11:01, respectively, to prepare pHLA monomers. These monomers were combined with PE-labeled streptavidin (BD, catalog number 554061) to form PE-labeled tetramers, and the tetramers and CD8 double-positive cells were detected by flow cytometry. Among them, the preparation methods of pHLA monomers and tetramers can be referred to the protocols published by the NIH Tetramer Core Facility. The specific procedures can be referred to on the webpage https: / / tetramer.yerkes.emory.edu / support / protocols#1. The specific operations are as follows:

[0090] (1) Reagents Test media: 10% FBS (ThermoFisher, Catalog No. 10099-044), RPMI1640 (ThermoFisher, Catalog No. C11875500BT), 10% HS (Gemni, Catalog No. 100512), TexMACS (Mitenyi, Catalog No. 170-076-309)

[0091] (2) Method PBMCs were isolated from peripheral blood of healthy volunteers by density gradient centrifugation, and CD14 positive cells were isolated from the PBMCs to induce dendritic cells (DCs). CD14 positive cells were cultured in medium (1640+10%AuFBS+1%PS+800IU / ml GM-CSF+500IU / ml IL-4), supplemented with 1ml of medium (containing 1600IU / ml GM-CSF+1000IU / ml IL-4) on the third day, and DC maturation inducers 10ng / ml IL-1β, 10ng / ml IL-6, 10ng / ml TNF-α, and 1ug / ml PGE2 were added on the fifth day. Mature DCs were obtained by continuing the culture for two days. Mature DCs were used to stimulate naive CD8-positive T cells isolated from PBMCs with TTLEQQYNK short peptide and GTTLEQQYNK loaded at a final concentration of 20 μg / ml (medium TexMACS+10% HS+1% PS+60 ng / ml IL21+10 IU / ml IL2 / 7 / 15). After three rounds of stimulation, antigen-specific T cells, tetramer and CD8 double positive T cells, were selected using a flow cytometry sorter. For the selected CD8 and tetramer double positive single cells, TCR α and β chains were amplified using a one-step RT-PCR kit (QIAGEN, catalog number 210212), respectively, and the PCR products were sequenced. The sequence results were aligned with the sequences in the public database of IMGT (International Immunogenetic Information System), and the sequences of the TCR α chain variable region, the nucleotide sequences of the β chain variable region, and their CDR1, CDR2, and CDR3 information could be obtained.

[0092] (3) Results TTLEQQYNK and GTTLEQQYNK antigen short peptide-specific T cells were cloned by the above method, and the cloned antigen short peptide-specific T cells were analyzed by flow cytometry. The flow cytometry results of T cells after 2 rounds and 3 rounds of stimulation with TTLEQQYNK and GTTLEQQYNK antigen short peptides are shown in Figure 1. In this figure, Figure 1A is a schematic diagram of the flow cytometry results of T cells after 2 rounds of stimulation, and Figure 1B is a schematic diagram of the flow cytometry results of T cells after 3 rounds of stimulation.

[0093] As can be seen from Figure 1, TTLEQQYNK and GTTLEQQYNK antigen short peptide-specific T cells were successfully cloned. The partial TCR sequence obtained by TTLEQQYNK is detailed in Example 2 below.

[0094] Example 2: Construction of TTLEQQYNK antigen short peptide-specific TCR lentiviral vector and lentiviral packaging (1) Construction of TCR lentiviral vector The CD8 and tetramer double positive cells of Example 1 were flow sorted to obtain single cells, and the α and β chains of the TCR were amplified from the obtained single cells using a one-step RT-PCR kit (QIAGEN, catalog number 210212), and the PCR products were sequenced. The sequence of the TCR α chain variable region, the nucleotide sequence of the β chain variable region, and information on their CDR1, CDR2, and CDR3 were obtained by aligning the sequencing results with the sequences in the public database of IMGT (International Immunogenetic Information System). The α chain variable region sequence and the nucleotide sequence of the β chain variable region, and information on their CDR1, CDR2, and CDR3 of four TCRs (TCR010, TCR013, TCR028, and TCR090) were obtained, and are shown below. The nucleotide sequence of the α chain variable region sequence of TCR010 is shown in SEQ ID NO:39, and the nucleotide sequence is as follows: ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACTCTGGGAGGAACCAGAGCCCAGTCGGTGACCCAGCTTGACAGCCACGTCTCTGTCTCTGAAGGAACCCCGGTGCTGCTGAGGTGCAACTACTCATCTTCTTATTCACCATCTCTCTTCTGGTATTGCAACACCCCAACAAAGGACTCCAGCTTCTCCTG AAGTACACATCAGCGGCCACCCTGGTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCCACCTGACGAAACCCTCAGCCCATATGAGCGACGCGGCTGAGTACTTCTGTGTTGTGAGTCTTTCTGGTGGCTACAATAAGCTGATTTTTGGAGCAGGGACCAGGCTGGCTGTACACCCATAT The nucleotide sequence of the β chain variable region is shown in SEQ ID NO: 40 and is as follows: ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTC ATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTGACAGGGAGTGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTA The amino acid sequence of the α-chain complementarity determining region 1 (CDR1) is shown in SEQ ID NO:3: SSYSPS The amino acid sequence of the complementarity determining region 2 (CDR2) is shown in SEQ ID NO: 4: YTSAATLV The amino acid sequence of the complementarity determining region 3 (CDR3) is shown in SEQ ID NO:5: VVSLSGGYNKLI The amino acid sequence of the complementarity determining region 1 (CDR1) of the β chain is shown in SEQ ID NO:6: SGDLS The amino acid sequence of the complementarity determining region 2 (CDR2) is shown in SEQ ID NO: 7: YYNGEE The amino acid sequence of the complementarity determining region 3 (CDR3) is shown in SEQ ID NO: 8: ASSVTGSGYT The nucleotide sequence of the alpha chain variable region sequence of TCR013 is shown in SEQ ID NO:41: ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAGGTGAATGGACAACAGGTAATGCAAATTCCTCAGTACCAGCATGTACAAGAAGGAGAGGACTTCACCACGTACTGCAATTCCTCAACTACTTTAAGCAATATACAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTGATACAG TTAGTGAAGAGTGGAGAAGTGAAGAAGCAGAAAAGACTGACATTTCAGTTTGGAGAAGCAAAAAAGAACAGCTCCCTGCACATCACAGCCACCCAGACTACAGATGTAGGAACCTACTTCTGTGCAGGGCCGAAAATCACGGGAGGAGGAAACAAACTCACCTTTGGGACAGGCACTCAGCTAAAAGTGGAACTCAAT The nucleotide sequence of the β chain variable region is shown in SEQ ID NO:42: ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTC AGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTAGGCGGGGGCCCTAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTA The amino acid sequence of the α-chain complementarity determining region 1 (CDR1) is shown in SEQ ID NO: 9: TTLSN The amino acid sequence of the complementarity determining region 2 (CDR2) is shown in SEQ ID NO: 10: LVKSGEV The amino acid sequence of the complementarity determining region 3 (CDR3) is shown in SEQ ID NO: 11: AGPKITGGGNKLT The amino acid sequence of the complementarity determining region 1 (CDR1) of the β chain is shown in SEQ ID NO:6: SGDLS The amino acid sequence of the complementarity determining region 2 (CDR2) is shown in SEQ ID NO: 7: YYNGEE The amino acid sequence of the complementarity determining region 3 (CDR3) is shown in SEQ ID NO: 12: ASSVGGGPNYGYT The nucleotide sequence of the alpha chain variable region sequence of TCR028 is shown in SEQ ID NO:43: ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAGGTGAATGGACAACAGGTAATGCAAATTCCTCAGTACCAGCATGTACAAGAAGGAGAGGACTTCACCACGTACTGCAATTCCTCAACTACTTTAAGCAATATACAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTGATACAG TTAGTGAAGAGTGGAGAAGTGAAGAAGCAGAAAAGACTGACATTTCAGTTTGGAGAAGCAAAAAAGAACAGCTCCCTGCACATCACAGCCACCCAGACTACAGATGTAGGAACCTACTTCTGTGCAGGCCCCATACTCACGGGAGGAGGAAACAAACTCACCTTTGGGACAGGCACTCAGCTAAAAGTGGAACTCAAT The nucleotide sequence of the β chain variable region is shown in SEQ ID NO:44: ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTC AGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCGGGCTTTCAGGACCGAACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTG The amino acid sequence of the α-chain complementarity determining region 1 (CDR1) is shown in SEQ ID NO: 9: TTLSN The amino acid sequence of the complementarity determining region 2 (CDR2) is shown in SEQ ID NO: 10: LVKSGEV The amino acid sequence of the complementarity determining region 3 (CDR3) is shown in SEQ ID NO: 13: AGPILTGGGNKLT. The amino acid sequence of the complementarity determining region 1 (CDR1) of the β chain is shown in SEQ ID NO:6: SGDLS The amino acid sequence of the complementarity determining region 2 (CDR2) is shown in SEQ ID NO: 7: YYNGEE The amino acid sequence of the complementarity determining region 3 (CDR3) is shown in SEQ ID NO: 14: ASGLSGPNTGELF The nucleotide sequence of the alpha chain variable region sequence of TCR090 is shown in SEQ ID NO:45: ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAGGTGAATGGACAACAGGTAATGCAAATTCCTCAGTACCAGCATGTACAAGAAGGAGAGGACTTCACCACGTACTGCAATTCCTCAACTACTTTAAGCAATATACAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTGATACAG TTAGTGAAGAGTGGAGAAGTGAAGAAGCAGAAAAGACTGACATTTCAGTTTGGAGAAGCAAAAAAGAACAGCTCCCTGCACATCACAGCCACCCAGACTACAGATGTAGGAACCTACTTCTGTGCAGGGCCGGTACTCACGGGAGGAGGAAACAAACTCACCTTTGGGACAGGCACTCAGCTAAAAGTGGAACTCAAT The nucleotide sequence of the β chain variable region is shown in SEQ ID NO:46: ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTC AGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTGGGGGGACCGAACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTG The amino acid sequence of the α-chain complementarity determining region 1 (CDR1) is shown in SEQ ID NO: 9: TTLSN The amino acid sequence of the complementarity determining region 2 (CDR2) is shown in SEQ ID NO: 10: LVKSGEV The amino acid sequence of the complementarity determining region 3 (CDR3) is shown in SEQ ID NO: 15: AGPVLTGGGNKLT The amino acid sequence of the complementarity determining region 1 (CDR1) of the β chain is shown in SEQ ID NO:6: SGDLS The amino acid sequence of the complementarity determining region 2 (CDR2) is shown in SEQ ID NO: 7: YYNGEE The amino acid sequence of the complementarity determining region 3 (CDR3) is shown in SEQ ID NO: 16: ASSVGGPNTGELF

[0095] The α and β chain variable region sequences of the TTLEQQYNK TCR were cloned into a pLKO-based expression plasmid (Addgene), and the α or β variable domain was cloned into a PLKO-based expression plasmid containing the mouse α or β constant region by standard methods using a Multi-Fragment Recombinant Cloning Kit (Novezan Biotech, Cat. No. C113), and the ligated plasmids were transformed into competent E. coli strain Stbl3 cells (Shanghai Weidi Biotechnology Co., Ltd.) and inoculated onto LB / agar plates containing 100 μg / ml ampicillin. After overnight incubation at 37°C, single colonies were picked and grown overnight with shaking at 37°C in 10 ml of LB containing 100 μg / ml ampicillin. The cloned plasmids were purified using a miniprep midi kit (TIANGEN Biotech Co., Ltd. (TIANGEN), catalog no. DP118-02), and the plasmids were sequenced to obtain TTLEQQYNK TCRs (i.e., TCR010, TCR013, TCR028, TCR090).

[0096] (2) Packaging of lentivirus Test medium: 10% FBS (Lonsera, Catalog No. S711-001), DMEM (cytiva, Catalog No. SH30243.01) 293T cells (depository institution is American Type Culture Collection, i.e. ATCC, accession number is CRL-1573) were prepared and cultured in 10 cm dishes, and transfection of plasmids was started when the confluence was less than 80%, and the ratio of viral packaging plasmid to TTLEQQYNK TCR plasmid was 1:1, totaling 10 μg. The above plasmids were added to serum-free DMEM medium and mixed with PEI (polyethylenimine), and then the mixture with the added plasmid was added to 293T cells and cultured at 37 ° C. After 72 hours, the cell supernatant was concentrated using a 100 kd ultrafiltration tube to collect the viral vector.

[0097] Example 3 Construction and functional identification of Jurkat cell line expressing TCR specific to TTLEQQYNK antigen short peptide A method for expression of the nuclear factor of activated T cells (NFAT) reporter gene The following experiments were performed to demonstrate the specific activation response of TCR-transduced T cells to target cells. Flow cytometry analysis was used to detect NFAT expression levels as a readout of T cell activation.

[0098] (1) Reagents Test medium: 10% FBS (Lonsera, Catalog No. S711-001), RPMI1640 (ThermoFisher, Catalog No. C11875500BT)

[0099] (2) Method Preparation of target cells The target cells used in this experiment were T2-A11 cells (T2 cells have been deposited with the ATCC and the accession number is CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). The target cells were prepared in the experimental medium, and the target cell concentration was 1.6 × 10 6 Adjusted to cells / ml and removed 50 μl from each well to give 80,000 cells / well.

[0100] Preparation of effector cells The effector cells in this experiment were Jurkat-CD8-NFAT (JK8NF) cells transduced with the TCR of the present application, and JK8NF cells not transfected with the TCR of the present application were used as a control.

[0101] The lentivirus having the TCR gene of the present application obtained in Example 2 was added to JK8NF cells (Jurkat cells have been deposited with ATCC and are given the accession number TIB-152. JK8NF cells were constructed based on Jurkat cells with reference to Cancer Res 2006;66(23):11455-61, Front.Immunol.11:633) at an MOI (multiplicity of infection) of 10. After 72 hours, it was confirmed by flow cytometry that the transfection positivity rate was about 100% (the results are shown in FIG. 2). The concentration of effector cells after expansion culture was adjusted to 1.6×10 6 Adjusted to cells / ml and removed 50 μl from each well to give 80,000 cells / well.

[0102] Preparation of short peptide solutions The short peptide (TTLEQQYNK) was diluted to 400 μg / ml from its original concentration of 5 mg / ml, and then further diluted in 10-fold increments to 40 μg / ml, 4 μg / ml, 0.4 μg / ml, 0.04 μg / ml, 0.004 μg / ml, and 0.0004 μg / ml.

[0103] 50 μl of the short peptides were dispensed into each well of the 96-well plate so that the final concentrations in the plate were 100 μg / ml, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, 0.001 μg / ml, and 0.0001 μg / ml, respectively.

[0104] Finally, 50 μl of target cells, 50 μl of effector cells, 50 μl of short peptide dilutions with corresponding concentrations, and 50 μl of medium were added to each well of a 96-well flat-bottom plate and incubated in a cell culture incubator at 37° C. for 12 hours.

[0105] (3) Results The expression of NFAT in the TCR transduced T cells of the present application in response to target cells loaded with TTLEQQYNK antigen short peptide was tested by the above method.Graphpad prism8 was used to plot the curve of NFAT expression level.The result is shown in Figure 3.

[0106] From FIG. 3, it can be seen that T cells transduced with the TCR of the present application have a good activation response against target cells loaded with their specific short peptide.

[0107] Example 4 Construction and functional identification of primary T cells expressing TCR specific to TTLEQQYNK antigen short peptide Method for monitoring target cell proliferation in real time (1) Reagents Test medium: 10% FBS (ThermoFisher, Catalog No. 10099-044), RPMI1640 (ThermoFisher, Catalog No. C11875500BT)

[0108] (2) Method Preparation of effector T cells The effector cells (T cells) in this experiment were T cells transduced with the TCR of the present application, and T cells from the same volunteer that were not transfected with the TCR of the present application were used as a control group.

[0109] Peripheral blood from volunteers was centrifuged on a density gradient to obtain peripheral blood mononuclear cells, and peripheral blood mononuclear cells were cultured at 5.0 × 10 per well of a 24-well plate. 5 Place a total of 1 x 10 cells / 500 μl into well. 6 The cells were collected. The T cells were stimulated with anti-CD3 / CD28 magnetic beads and then cultured in a 37°C, 5% CO2 incubator. After 24 hours, cell mass formation was observed. The TCR lentivirus obtained in Example 2 was added at an MOI (multiplicity of infection) of 2 for transduction, and the cells were amplified in 1640 medium containing 10% FBS and 200 IU / ml of IL-2 for 3 to 4 days after transduction, and the TCR transfection efficiency was measured using flow cytometry (the results are shown in Figure 4). The concentration of effector cells after expansion culture was adjusted to 5.0 x 10 4 Adjusted to positive cells / ml and took 100 μl per well, giving 5000 positive cells / well.

[0110] Preparation of target cells The target cells used in this experiment were SK-MEL-28-E6. The genotype of SK-MEL-28 (deposited with ATCC) is A*11:01, but it does not express E6 by itself. Therefore, SK-MEL-28-E6 cells were constructed as target cells by overexpressing the E6 gene via a lentiviral vector. The target cells were prepared in the experimental medium, and the target cell concentration was 5.0 × 10 4 Adjusted to cells / ml and dispensed 100 μl into each well to give 5,000 cells / well.

[0111] Monitoring target cell proliferation with the RTCA x Celligence system 100 μl of the T cells and 100 μl of the target cells prepared above were added to an E-plate, the E-plate was attached to an RTCA analyzer, and incubated at 37° C. in a 5% CO 2 incubator for 90 hours to observe the real-time proliferation curve of the target cells.

[0112] (3) Results The functionality of the TCR-transduced T cells of the present application was tested through real-time monitoring of target cell proliferation experiments (as described above). Graphpad prism8 was used to plot the cell index (a parameter generated by the RTCA x Celligence system to characterize cell number) of cell proliferation in each well. The experimental results are shown in Figure 5.

[0113] As can be seen from FIG. 5, T cells expressing TCR specific to the TTLEQQYNK-A*11:01 antigen short peptide have a strong killing function against cell lines expressing E6.

[0114] Example 5: Identification of in vivo tumor growth inhibitory function of primary T cells expressing TCR specific for TTLEQQYNK antigen short peptide The following experiment was carried out to demonstrate the inhibitory effect of TCR-transduced T cells on tumor growth. The change in tumor volume was observed to evaluate the tumor-killing function of T cells. In vitro experiments showed that the four TCRs had similar tumor-killing effects, so only TCR013 and TCR028 were selected as representatives to identify their in vivo tumor growth inhibitory function.

[0115] (1) Reagents and mice Test medium: 10% FBS (ThermoFisher, Catalog No. 10099-044), RPMI1640 (ThermoFisher, Catalog No. C11875500BT) NCG mice: purchased from GemPharmatech Co., Ltd., female, 4 to 6 weeks old, genotypes were (prkdc) ko / ko and (Il2rg female) ko / ko.

[0116] (2) Method Subcutaneous inoculation of tumor cells The tumor cells used in this experiment were SK-MEL-28-E6. Tumor cells were prepared in saline and the tumor cell concentration was 5.0 × 10 7 The cells were adjusted to 1.0 × 10 cells / ml and each mouse was inoculated subcutaneously with 200 μl. 7 Cells / mouse were obtained. Tumor volumes were approximately 100 mm 3 When the mice were grown to 100 μg / mL, effector T cells were inoculated.

[0117] Preparation of effector T cells The effector cells (T cells) in this experiment were T cells transduced with the TCR of the present application, and T cells from the same volunteers not transfected with the TCR of the present application were used as a control group, with 5 mice per group.

[0118] Peripheral blood from volunteers was centrifuged on a density gradient to obtain peripheral blood mononuclear cells, and peripheral blood mononuclear cells were cultured at 5.0 × 10 per well of a 24-well plate. 5 Place a total of 4 x 10 cells / 500 μl into the well. 6The cells were collected. The T cells were stimulated with anti-CD3 / CD28 magnetic beads and then cultured in a 37°C, 5% CO2 incubator. After 24 hours, cell mass formation was observed. The lentivirus of the TCR genes (TCR013 and TCR028 genes) obtained in Example 2 was added at an MOI (multiplicity of infection) of 2 for transduction, and the cells were amplified in 1640 medium containing 10% FBS and 200 IU / ml of IL-2 for 3 to 4 days after transduction, and the TCR transfection efficiency was measured using flow cytometry. The concentration of effector cells after expansion culture was adjusted to 5.0 x 10 7 Adjust the volume to 1.0 x 10 positive cells / ml and inject 200 μl into each mouse via the eye vein. 7 Positive cells / mouse were obtained.

[0119] (3) Results The tumor volume after inoculation of T cells was observed and recorded, and the effect of the primary T cells expressing the TCR of the present application in treating tumors was evaluated based on the change in tumor volume. Tumor growth curves were plotted using Graphpad prism8. The experimental results are shown in Figures 6A and 6B, where Figure 6A is a schematic diagram of the change in tumor volume over time after inoculation of T cells, and Figure 6B is a histogram of tumor weight after inoculation of T cells containing different TCRs.

[0120] Figures 6A and 6B show that T cells expressing TCR specific to the TTLEQQYNK-A*11:01 antigen short peptide can effectively inhibit the growth of E6-positive tumors.

[0121] Example 6 Killing function of TCR specific to TTLEQQYNK-A*11:01 antigen short peptide against primary HPV tumor (1) Reagents Test medium: OrganoPro Cervical Cancer Organoid Media Kit (Ketu Medical, Catalog No. K2O-M-CC)

[0122] (2) Method 8,000 HPV16 + HLA-A*11:01 +Cervical cancer organoid KOCC-002S4 (Science and Technology) was co-cultured with 8,000 primary T cells transduced with TCR013 (TCR013) or control T cells not transduced with TCR (control group), and after 72 hours, the growth status of the organoids was observed under a microscope and photographed.

[0123] (3) Results The growth state of the organoids was observed under a microscope, a typical field of view was photographed, and the number of complete and incomplete organoids in the field of view was statistically calculated. The results are shown in Figures 7A to 7B. Here, Figure 7A is a schematic diagram of the growth state of the organoids observed under a microscope, and Figure 7B is a schematic diagram of the statistical number of complete and incomplete organoids.

[0124] Figures 7A and 7B show that T cells expressing TCR specific to the TTLEQQYNK-A*11:01 antigen short peptide can significantly kill primary cervical cancer organoid cells.

[0125] In summary, the TCR-transduced T cells in this application can be specifically activated and have highly potent killing effects against tumor cells expressing A1101 and HPV.

[0126] The T cells have very strong killing function against E6-expressing cell lines and can effectively inhibit the growth of E6-positive tumors.

[0127] The above is merely a preferred embodiment of the present application, and is not intended to limit the present application to other forms. Those skilled in the art can use the technical content disclosed above to modify the above into equivalent embodiments with modifications or equivalent changes. However, without departing from the content of the technical solution of the present application, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present application still fall within the protection scope of the technical solution of the present application.

Claims

1. 13. Use of an antigenic short peptide in screening for drugs to treat HPV-related diseases, wherein the amino acid sequence of said antigenic short peptide is shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. 2. The use according to claim 1, wherein the HPV-related disease is HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer.

3. 1. Use of an antigenic short peptide in screening for drugs to treat HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer, wherein the amino acid sequence of the antigenic short peptide is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

4. The use according to any one of claims 1 to 3, wherein the agent is a T cell receptor (TCR) for binding to an antigenic short peptide-HLA-A1101 complex comprising said antigenic short peptide.

5. A T cell receptor (TCR), said TCR comprising an α chain comprising a variable region and / or a β chain comprising a variable region, said α chain variable region comprising a complementarity determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:9, and / or A T cell receptor (TCR) comprising a complementarity determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:

10.

6. the variable region of the β chain comprises a complementarity determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO:6, and / or 6. The T cell receptor (TCR) of claim 5, comprising a complementarity determining region 2 (CDR2) having the amino acid sequence set forth in SEQ ID NO:

7.

7. the variable region of the alpha chain comprises a complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15; and / or the variable region of the β chain comprises a complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; Preferably, the alpha chain variable region further comprises a first leader sequence; and / or the variable region of the beta chain further comprises a second leader sequence; Preferably, the amino acid sequence of the variable region of the alpha chain is the amino acid sequence shown in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26, or is an amino acid sequence having at least 90% sequence identity with SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26, and / or the amino acid sequence of the variable region of the β chain is the amino acid sequence shown in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:27, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:27; A T cell receptor (TCR) according to claim 5 or 6, wherein preferably the alpha chain further comprises an alpha constant region and / or the beta chain further comprises a beta constant region, preferably the constant region being a mouse constant region or a human constant region.

8. the TCR is isolated, purified or recombinant; Preferably, the TCR is human, Preferably, the TCR is monoclonal, Preferably, the TCR is single chain, Preferably, the TCR comprises two chains, Preferably, said TCR is in cell-associated or soluble form, preferably in soluble form; A T cell receptor (TCR) according to any one of claims 5 to 7, wherein the TCR binds to an antigenic short peptide-HLAA1101 complex, and preferably the amino acid sequence of the antigenic short peptide is as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

9. A nucleic acid molecule comprising a nucleotide sequence encoding the TCR according to any one of claims 5 to 17, or the α or β chain of said TCR.

10. and / or the nucleotide sequence encoding the alpha chain comprises the nucleotide sequence shown in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36; and / or 10. The nucleic acid molecule of claim 9, wherein the nucleotide sequence encoding the beta chain comprises the nucleotide sequence shown in SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:

37.

11. A vector comprising the nucleic acid molecule of claim 9 or 10.

12. the vector is an expression vector, Preferably, said vector is a viral vector, preferably a retroviral vector; The vector according to claim 11, preferably wherein the viral vector is a lentiviral vector.

13. An engineered cell comprising a TCR according to any one of claims 5 to 8, a nucleic acid molecule according to any one of claims 9 to 10, or a vector according to any one of claims 11 to 12.

14. the TCR is heterologous to the cell; Preferably, the engineered cell is a cell line, Preferably, said engineered cells are primary cells obtained from a subject, preferably said subject is a mammalian subject, preferably a human subject; Preferably, said engineered cells are T cells or NK cells, preferably said T cells are T cells isolated from peripheral blood; 14. The engineered cell of claim 13, wherein the T cells are preferably CD8+ or CD4+.

15. A method for producing an engineered cell according to any one of claims 13 to 14, comprising introducing a nucleic acid molecule according to any one of claims 9 to 10 or a vector according to any one of claims 11 to 12 into a cell in vitro or ex vivo.

16. The method of claim 15, wherein the vector is a viral vector and the introduction is by transduction.

17. A pharmaceutical composition comprising a T cell receptor (TCR) according to any one of claims 5 to 8, a nucleic acid molecule according to any one of claims 9 to 10, a vector according to any one of claims 11 to 12, or an engineered cell according to any one of claims 13 to 14, Preferably, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or adjuvant.

18. Use of a T cell receptor (TCR) according to any one of claims 5 to 8, a nucleic acid molecule according to any one of claims 9 to 10, a vector according to any one of claims 11 to 12, or an engineered cell according to any one of claims 13 to 14, or a pharmaceutical composition according to claim 17 in the preparation of a medicament for treating an HPV-associated disease, comprising: Preferably, the HPV-associated disease is HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer.

19. A method for treating an HPV-associated disease comprising administering to a subject in need thereof a T cell receptor (TCR) according to any one of claims 5 to 8, a nucleic acid molecule according to any one of claims 9 to 10, a vector according to any one of claims 11 to 12, or an engineered cell according to any one of claims 13 to 14, or a pharmaceutical composition according to claim 17.

20. 20. The method of claim 19, wherein the HPV-associated disease is HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer.

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