Use of antigen short peptides in screening for drugs for treating HPV-related diseases and TCR screened thereby

Abstract

This application discloses the use of an antigen short peptide in the screening of drugs for treating HPV-related diseases and the TCR screened thereby. The amino acid sequence of the antigen short peptide is as shown in SEQ ID NO: 1. The antigen short peptide of this application can screen for specific T cell receptors (TCRs). At the same time, the T cells transduced with TCRs are 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. Furthermore, the T cells transduced with the TCR of this application show a very strong activation reaction to cell lines expressing E7, do not show an activation reaction to cell lines not expressing E7, have a very strong killing function against cell lines expressing E7, and can effectively inhibit the growth of E7-positive tumors.

Description

Cross - reference to related applications

[0001] This application claims priority to Chinese Patent Application No. CN202210503907.4 filed on May 10, 2022, the disclosure of which is hereby incorporated by reference in its entirety. Reference to the electronic sequence listing

[0002] The content of the electronic sequence listing (TFG00783PCT - XLB.xml, size: 49,700 bytes, creation date: May 6, 2023) is hereby incorporated by reference in its entirety.

Technical Field

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

Background Art

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

[0005] An important factor for HPV to cause cancer is the persistent infection of high - risk types of HPV such as HPV16 and HPV18. The E6 protein and E7 protein encoded by high - risk HPV inhibit the activities of the tumor suppressor genes p53 and Rb protein respectively, which may thereby cause cell cycle abnormalities and canceration. High - risk types of HPV are associated with 90% of cervical cancer and anal cancer, 40% - 60% of vaginal cancer and penile cancer, and may also be associated with 60% of oropharyngeal cancer depending on geographical characteristics.

[0006] Cervical cancer is the most common malignant tumor that occurs in women's reproductive organs. The global incidence rate ranks second among women's malignant tumors, and the incidence rate of cervical cancer in developing countries is significantly higher than that in developed countries. According to the latest statistics in 2018, the morbidity rate of cervical cancer in China is 15.30 per 100,000 people, and the mortality rate is 4.57 per 100,000 people, which is higher than the world average morbidity rate (10.61 per 100,000 people) and mortality rate (2.98 per 100,000 people). In the past 10 years, the morbidity rate of cervical cancer has shown an increasing trend, and the peak age of morbidity is in the 40-60 age group. Surgery, radiotherapy, and chemotherapy are effective in the treatment of early-stage cervical cancer, but more than half of the patients are diagnosed with locally advanced cervical cancer at the first diagnosis. 30% - 70% of the patients with locally advanced cervical cancer experience recurrence and / or distant metastasis. In the case of locally advanced cervical cancer and metastatic cervical cancer, the effectiveness of conventional treatments is very limited, and the 5-year survival rates are only 57.1% and 17.3% respectively.

[0007] Specific T cell immunotherapy refers to a method of killing tumor cells using specific T cells that target tumor antigens, and this is a highly individualized tumor immunotherapy. Since there is a local immunosuppressive microenvironment in tumors, the tumor-killing function of the patient's own T cells in the body is limited. Therefore, people are trying to improve the ability to kill tumors by genetically modifying T cells. Both TCR-T and CAR-T are genetically modified cell therapy drugs. When they bind to the corresponding targets through the introduced T cell receptor (TCR) or chimeric antigen receptor (CAR) gene, they activate T cells and utilize granzyme, perforin, cytokines, etc. released by T cells to eliminate tumor cells. However, the big difference between TCR-T and CAR-T is that the target of CAR-T is a membrane protein on the cell surface, while the target of TCR-T is an antigen short peptide-major histocompatibility complex (peptide-major histocompatibility complex, pMHC).

[0008] HPV-related 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 by MHC. In theory, one TCR molecule specifically recognizes only the short peptides presented by a specific MHC. MHC is polymorphic, and the number of human MHC (also called human leukocyte antigen; HLA) alleles discovered so far exceeds 15,000. The frequency of specific HLA varies greatly among human populations. In China, the most common HLA type within the population is HLA-A1101. The short peptides presented by HLA class I molecules are 8-11 amino acids in length, and the short peptides presented by HLA class II molecules are 12-24 amino acids in length. The discovery and identification of these antigen short peptides are preconditions for TCR-T therapy. Whether a short peptide binds to HLA can be known by affinity prediction, HLA binding assay, etc. Whether the short peptide is naturally presented by HPV-expressing tumor cells is the key to determining whether a TCR specific to the short peptide can be used for tumor treatment.

[0009] Therefore, those skilled in the art are working hard 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.

Disclosure of the Invention

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

[0011] The specific technical solution of this application is as follows.

[0012] 1. Use of an antigen short peptide in screening for a drug for treating HPV-related diseases, wherein the amino acid sequence of the antigen short peptide is shown in SEQ ID NO: 1. 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 antigen short peptide in screening for 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, wherein the amino acid sequence of the antigen short peptide is shown in SEQ ID NO: 1. 4. The use according to any one of items 1 to 3, wherein the drug is a T cell receptor (TCR) for binding to an antigen short peptide-HLA-A1101 complex containing the antigen short peptide. 5. A T cell receptor (TCR), wherein the TCR comprises an α chain containing a variable region and / or a β chain containing a variable region, and the variable region of the α chain has a complementarity-determining region 1 (CDR1) with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 14, and / or A T cell receptor (TCR) comprising a complementarity-determining region 2 (CDR2) with the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 15. 6. The T cell receptor (TCR) according to item 5, wherein the variable region of the β chain has a complementarity-determining region 1 (CDR1) with the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 17, and / or A complementarity-determining region 2 (CDR2) with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 12, or SEQ ID NO: 18. 7. The variable region of the α chain contains a complementarity-determining region 3 (CDR3) with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 16, and / or The T cell receptor (TCR) according to item 5 or 6, wherein the variable region of the β chain contains a complementarity-determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 13, or SEQ ID NO: 19. 8. The T cell receptor (TCR) according to any one of items 5 to 7, wherein the variable region of the α chain further contains a first leader sequence and / or The T cell receptor (TCR) according to any one of items 5 to 7, wherein the variable region of the β chain further contains a second leader sequence. 9. The amino acid sequence of the variable region of the α chain is the amino acid sequence shown in SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, and / or the amino acid sequence of the variable region of the β chain is the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31. The T cell receptor (TCR) according to any one of items 5 to 8. 10. The T cell receptor (TCR) according to any one of items 5 to 9, wherein the α chain further contains an α constant region and / or the β chain further contains a β constant region. Preferably, the constant region is a mouse constant region or a human constant region. 11. The T cell receptor (TCR) according to any one of items 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 of human origin. 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 single-chain. 15. The T cell receptor (TCR) according to any one of items 5 to 14, wherein the TCR contains two chains. 16. The T cell receptor (TCR) according to any one of items 5 to 15, wherein the TCR is cell-bound or soluble, preferably soluble. 17. The TCR according to any one of items 5 to 16, wherein the TCR binds to an antigen short peptide - HLA - A1101 complex, and preferably, the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO: 1. 18. A nucleic acid molecule comprising the TCR according to any one of items 5 to 17 or a nucleotide sequence encoding the α - chain or β - chain of the TCR. 19. The nucleotide sequence encoding the α - chain comprises the nucleotide sequence shown in SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38, and / or The nucleotide sequence encoding the β - chain comprises the nucleotide sequence shown in SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39. The nucleic acid molecule according to item 18. 20. A vector comprising the nucleic acid molecule according to item 18 or 19. 21. The vector according to item 20, wherein the vector is an expression vector. 22. The vector according to item 20 or 21, wherein the vector is a viral vector, preferably a retroviral vector. 23. The vector according to item 22, wherein the viral vector is a lentiviral vector. 24. An engineered cell comprising the TCR according to any one of items 5 to 17, the nucleic acid molecule according to any one of items 18 to 19, or the vector according to any one of items 20 to 23. 25. The engineered cell according to item 24, wherein the TCR is heterologous to the cell. 26. The engineered cell according to item 24 or 25, wherein the engineered cell is a cell line. 27. The engineered cell according to any one of items 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 items 24 to 27, wherein the engineered cell is a T cell or an NK cell, preferably, the T cell is a T cell isolated from peripheral blood. 29. The engineered cell according to item 28, wherein the T cell is CD8+ or CD4+. 30. A method for producing an engineered cell according to any one of items 24 - 29, comprising introducing a nucleic acid molecule according to any one of items 18 - 19 or a vector according to any one of items 20 - 23 into a cell in vitro or ex vivo. 31. The method according to item 30, wherein the vector is a viral vector and the introduction is performed by transduction. 32. A pharmaceutical composition comprising a T cell receptor (TCR) according to any one of items 5 - 17, a nucleic acid molecule according to any one of items 18 - 19, a vector according to any one of items 20 - 23, or an engineered cell according to any one of items 24 - 29. 33. The pharmaceutical composition according to item 32, further comprising a pharmaceutically acceptable carrier or adjuvant. 34. Use of a T cell receptor (TCR) according to any one of items 5 - 17, a nucleic acid molecule according to any one of items 18 - 19, a vector according to any one of items 20 - 23, an engineered cell according to any one of items 24 - 29, or a pharmaceutical composition according to any one of items 32 - 33 in the preparation of a medicament for treating HPV - related diseases. 35. The use according to item 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 HPV - related diseases, comprising administering to a subject in need thereof a T cell receptor (TCR) according to any one of items 5 - 17, a nucleic acid molecule according to any one of items 18 - 19, a vector according to any one of items 20 - 23, an engineered cell according to any one of items 24 - 29, or a pharmaceutical composition according to any one of items 32 - 33. 37. The method according to item 36, 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.

[0013] The antigen short peptide described in this application can screen for specific T cell receptors (TCRs). At the same time, the T cells transduced with the TCR 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] Moreover, the T cells transduced with the TCR described in this application have a very strong activation reaction against cell lines expressing E7, have no activation reaction against cell lines not expressing E7, have a very strong killing function against cell lines expressing E7, and can effectively inhibit the growth of E7-positive tumors.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0016] This application will be described in detail below with reference to the embodiments described in the accompanying drawings, where the same numbers in all the drawings represent the same features. Although specific embodiments of this application are shown in the drawings, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments described in this specification. Rather, these embodiments are provided to provide a complete understanding of this application and to fully convey the scope of this application to those skilled in the art.

[0017] It should be noted that in the specification and claims, specific terms are used to refer to specific components. Those skilled in the art will understand that different nouns may be used to refer to the same component. This specification and the claims do not use the difference in nouns as a way to distinguish components, but rather use the difference in the functions of components as the criterion for distinction. Since the terms "comprising" or "including" mentioned throughout the specification and claims are open terms, they should be construed as "including but not limited to". The following description is a preferred embodiment for implementing this application, but these descriptions are for the purpose of the general principles of the specification and do not limit the scope of this application. The protection scope of this application shall be determined by the appended claims.

[0018] This application provides the use of an antigen short peptide in the screening of drugs for treating HPV-related diseases, and the amino acid sequence of the antigen short peptide is shown in SEQ ID NO: 1. The sequence of SEQ ID NO: 1 is as follows: IVCPICSQK.

[0019] In one embodiment, the HPV-related diseases include HPV chronic infections, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer, or vulvar cancer, etc.

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

[0021] In one embodiment, the drug is a T cell receptor (TCR) for binding to an antigen short peptide-HLA-A1101 complex containing the antigen 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 IVCPICSQK-HLA-A1101 complex.

[0023] The T cell receptor or TCR is the only receptor that presents specific antigen peptides present on the major histocompatibility complex (MHC). In the immune system, the binding of the antigen-specific TCR to the pMHC complex causes direct physical contact between the T cell and the antigen-presenting cell (APC). Subsequently, other cell membrane surface molecules of both the T cell and the APC interact, thereby triggering a series of subsequent cell signaling and other physiological reactions, enabling T cells with different antigen specificities to exert an immune effect on target cells.

[0024] The TCR is a molecule containing variable α and β chains, or variable γ and δ chains, and the molecule can specifically bind to a peptide on an MHC molecule. In some embodiments, the TCR is of the αβ type. Generally, TCRs existing in the αβ and γδ types are overall similar in structure, but the T cells expressing them may have different anatomical locations or functions, and TCRs may be found on the cell surface or in a soluble form. Usually, the TCR is present on the surface of a T cell (T lymphocyte) and plays a role in recognizing an antigen bound to a major histocompatibility complex (MHC) molecule.

[0025] The variable domains of the TCR contain complementarity-determining regions (CDRs), which often greatly contribute to the antigen recognition, binding ability, and specificity for peptides, MHCs, and / or MHC-peptide complexes. The CDRs of the TCR, or combinations thereof, form all or substantially all of the antigen-binding site of a given TCR molecule, and the individual CDRs within the variable region of the TCR are typically separated by framework regions (FRs). Among these, CDR3 is the major 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 the 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 has the strongest effect on or is mainly involved in the interaction or recognition of the MHC portion of the 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] The -IVCPICSQK-HLA-A1101 complex refers to a complex formed by the binding of HLA-A1101 and the antigen short peptide IVCPICSQK. Proteins are degraded by proteasomes in cells into polypeptides of different lengths, and some polypeptides bind to HLA to form a complex and are presented on the cell surface. The IVCPICSQK-HLA-A1101 complex recognized by the TCR can be expressed on the cell membrane or exist in solution in the form of a soluble protein.

[0027] The amino acid sequence of the HLA-A1101 is shown in SEQ ID NO: 40, and its amino acid sequence is as follows. MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSYTQAASSDSAQGSDVSLTACKVSR

[0028] In one embodiment, the antigen short peptide is used for screening drugs for treating HPV-related diseases, and the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO: 1. 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 antigen short peptide-HLA-A1101 complex containing the antigen short peptide.

[0029] In one embodiment, the antigen short peptide is used for screening drugs 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 the antigen short peptide is shown in SEQ ID NO: 1. In one embodiment, the drug is a T cell receptor (TCR) for binding to an antigen short peptide-HLA-A1101 complex containing the antigen short peptide.

[0030] This application provides a T cell receptor (TCR), the TCR includes an α chain containing a variable region and / or a β chain containing a variable region, and the variable region of the α chain has a complementarity-determining region 1 (CDR1) with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 14, and / or includes a complementarity-determining region 2 (CDR2) with the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 15.

[0031] The amino acid sequence shown in SEQ ID NO: 2 is TSDPSYG. The amino acid sequence shown in SEQ ID NO: 8 is DSVNN. The amino acid sequence shown in SEQ ID NO: 14 is NYSPAY. The amino acid sequence shown in SEQ ID NO: 3 is QGSYDQQN. The amino acid sequence shown in SEQ ID NO: 9 is IPSGT. The amino acid sequence shown in SEQ ID NO: 15 is IRENEKE.

[0032] In one embodiment, the variable region of the β chain has a complementarity-determining region 1 (CDR1) with the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 17, and / or includes a complementarity-determining region 2 (CDR2) with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 12, or SEQ ID NO: 18.

[0033] The amino acid sequence shown in SEQ ID NO: 5 is LNHNV. The amino acid sequence shown in SEQ ID NO: 11 is MNHEY. The amino acid sequence shown in SEQ ID NO: 17 is GTSNPN. The amino acid sequence shown in SEQ ID NO: 6 is YYDKDF. The amino acid sequence shown in SEQ ID NO: 12 is SMNVEV. The amino acid sequence shown in SEQ ID NO: 18 is SVGIG.

[0034] In one embodiment, the variable region of the α chain includes a complementarity-determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 16, and / or the variable region of the β chain includes a complementarity-determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 13, or SEQ ID NO: 19.

[0035] The amino acid sequence shown in SEQ ID NO: 4 is AMRIDAGGTSYGKLT. The amino acid sequence shown in SEQ ID NO: 10 is AVEGDNAGGTSYGKLT. The amino acid sequence shown in SEQ ID NO: 16 is ALAGYQKVT. The amino acid sequence shown in SEQ ID NO: 7 is ATSRDRVNTGELF. The amino acid sequence shown in SEQ ID NO: 13 is ASSWSTSYGYT. The amino acid sequence shown in SEQ ID NO: 19 is AWSLRTSGSEQF.

[0036] In one embodiment, the TCR can bind to an antigen short peptide-HLA-A1101 complex, and preferably, the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO: 1.

[0037] The antigen short peptide-HLA-A1101 complex refers to a complex formed by the binding of HLA-A1101 and the antigen short peptide. Proteins are decomposed by proteasomes in cells into polypeptides of different lengths, and some polypeptides bind to HLA to form a complex and are presented on the cell surface. The antigen short peptide-HLA-A1101 complex recognized by the TCR can be expressed on the cell membrane or exist in solution in the form of a soluble protein.

[0038] In one embodiment, the variable region of the α chain further includes a first leader sequence, and / or the variable region of the β chain further includes a second leader sequence.

[0039] The first leader sequence of the variable region of the α chain and the second leader sequence of the variable region of the β chain are well known to those skilled in the art. For example, as the first leader sequence of the variable region of the α chain, a leader sequence having the amino acid sequence shown in SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24 can be used, and as the second leader sequence of the variable region of the β chain, a leader sequence having the amino acid sequence shown in SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25 can be used.

[0040] The amino acid sequence shown in SEQ ID NO: 20 is MSLSSLLKVVTASLWLGPGI. The amino acid sequence shown in SEQ ID NO: 22 is MKRILGALLGLLSAQVCCVR. The amino acid sequence shown in SEQ ID NO: 24 is MESFLGGVLLILWLQVDWVK. The amino acid sequence shown in SEQ ID NO: 21 is MGPGLLHWMALCLLGTGHG. The amino acid sequence shown in SEQ ID NO: 23 is MGPQLLGYVVLCLLGAGPL. The amino acid sequence shown in SEQ ID NO: 25 is MLCSLLALLLGTFFGVR.

[0041] In one embodiment, the amino acid sequence of the α-chain variable region is the amino acid sequence shown in SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, and / or the amino acid sequence of the variable region of the β-chain is the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31.

[0042] The amino acid sequence shown in SEQ ID NO: 26 is as follows. MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMRIDAGGTSYGKLTFGQGTILTVHPN The amino acid sequence shown in SEQ ID NO: 27 is as follows. MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSRDRVNTGELFFGEGSRLTVL The amino acid sequence shown in SEQ ID NO: 28 is as follows. MKRILGALLGLLSAQVCCVRGIQVEQSPPDLILQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLYISSSQTTDSGVYFCAVEGDNAGGTSYGKLTFGQGTILTVHPN The amino acid sequence shown in SEQ ID NO: 29 is as follows. MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSWSTSYGYTFGSGTRLTIV The amino acid sequence shown in SEQ ID NO: 30 is as follows. MESFLGGVLLILWLQVDWVKSQKIEQNSEALNIQEGKTATLTCNYTNYSPAYLQWYRQDPGRGPVFLLLIRENEKEKRKERLKVTFDTTLKQSLFHITASQPADSATYLCALAGYQKVTFGTGTKLQVIPN The amino acid sequence shown in SEQ ID NO: 31 is as follows. MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWSLRTSGSEQFFGPGTRLTVL

[0043] The amino acid sequence of the variable region of the α-chain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30 can be, for example, an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity with SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30. The amino acid sequence of the variable region of the β-chain having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31 can be, for example, an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity with SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31.

[0044] In one embodiment, the amino acid sequence of the α-chain variable region of the TCR is as shown in SEQ ID NO: 26, and the amino acid sequence of the β-chain variable region is as shown in SEQ ID NO: 27, or the amino acid sequence of the α-chain variable region is as shown in SEQ ID NO: 28, and the amino acid sequence of the β-chain variable region is as shown in SEQ ID NO: 29, or the amino acid sequence of the α-chain variable region is as shown in SEQ ID NO: 30, and the amino acid sequence of the β-chain variable region is as shown in SEQ ID NO: 31.

[0045] In one embodiment, the α-chain further includes an α constant region, and / or the β-chain further includes a β constant region. Preferably, the constant region is a mouse constant region or a human constant region. For example, the amino acid sequence of the mouse α constant region is shown in SEQ ID NO: 32, and / or the amino acid sequence of the mouse β constant region is as shown in SEQ ID NO: 33.

[0046] The amino acids shown in SEQ ID NO: 32 are as follows. IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS The amino acids shown in SEQ ID NO: 33 are as follows. EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS

[0047] 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 can have additional cysteine residues in each of the α and β chains such that the constant region contains two disulfide bonds.

[0048] In one embodiment, an artificial disulfide bond is introduced between a residue in the constant region of the α chain of the TCR and a residue in the constant region of the β chain, and the positions at which disulfide bonds can be introduced are well known to those skilled in the art.

[0049] In one embodiment, the TCR is isolated, purified, or recombinant.

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

[0051] In one embodiment, the TCR is monoclonal.

[0052] In one embodiment, the TCR is single-chain.

[0053] In one embodiment, the TCR comprises two chains.

[0054] The TCR can be obtained from a biological source, for example, from cells (e.g., T cells (e.g., cytotoxic T cells)), T cell hybridomas, or other publicly available resources, and can be derived from, for example, one of a plurality of animal species such as humans, mice, rats, or other mammals, typically humans.

[0055] In one embodiment, the TCR is cell-bound or soluble, preferably soluble.

[0056] The fact that the soluble TCR is soluble refers to a TCR in which a mutation has occurred in its hydrophobic core region, and the mutations in these hydrophobic core regions are preferably mutations that can improve the stability of the soluble TCR of the present application.

[0057] In one embodiment, the TCR includes an α chain containing a variable region and / or a β chain containing a variable region. The variable region of the α chain includes a complementarity-determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 14, and / or a complementarity-determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 15. In one embodiment, the variable region of the β chain includes a complementarity-determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 17, and / or a complementarity-determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 12, or SEQ ID NO: 18. In one embodiment, the variable region of the α chain includes a complementarity-determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 16, and / or the variable region of the β chain includes a complementarity-determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 13, or SEQ ID NO: 19. Preferably, the variable region of the α chain further includes a first leader sequence, and / or the variable region of the β chain further includes a second leader sequence. Preferably, the amino acid sequence of the variable region of the α chain is the amino acid sequence shown in SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, and / or the amino acid sequence of the variable region of the β chain is the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31. Preferably, the α chain further includes an α constant region, and / or the β chain further includes a β constant region. Preferably, the 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 of human origin, preferably the TCR is monoclonal, preferably the TCR is single-chain, preferably the TCR comprises two chains, preferably the TCR is cell-bound or soluble, preferably soluble, preferably the TCR binds to an antigen short peptide-HLA-A1101 complex, and preferably the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO: 1.

[0058] This application provides a nucleic acid molecule comprising a nucleotide sequence encoding a TCR or an α-chain or β-chain of a TCR.

[0059] In one embodiment, the nucleotide sequence encoding the α-chain comprises the nucleotide sequence shown in SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38, and / or the nucleotide sequence encoding the β-chain comprises the nucleotide sequence shown in SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39, wherein the nucleotide sequence shown in SEQ ID NO: 34 is ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGACCTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATGTTCGTGCAGGAAAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGTGATCCAAGTTATGGTCTATTCTGGTACAAGCAGCCCAGCAGTGGGGAAATGATTTTTCTTATTTATCAGGGGTCTTATGACCAGCAAAATGCAACAGAAGGTCGCTACTCATTGAATTTCCAGAAGGCAAGAAAATCCGCCAACCTTGTCATCTCCGCTTCACAACTGGGGGACTCAGCAATGTACTTCTGTGCAATGAGAATTGATGCTGGTGGTACTAGCTATGGAAAGCTGACATTTGGACAAGGGACCATCTTGACTGTCCATCCAAATATCCAGAATCCAGAGCCCGCCGTGTATCAGCTGAAGGACCCAAGGAGCCAGGATTCCACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAACGTGCCCAAGACCATGGAGTCCGGCACCTTCATCACAGACAAGTGCGTGCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACCTCCTTCACATGCCAGGATATCTTTAAGGAGACCAATGCCACATATCCTTCCTCTGACGTGCCATGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACCGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGCGCATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGGCTGTGGAGCTCC and the nucleotide sequence shown in SEQ ID NO: 35 is ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACAGGTCATGGGGATGCCATGGTCATCCAGAACCCAAGATACCAGGTTACCCAGTTTGGAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTCATGTACTGGTACCAGCAGAAGTCAAGTCAGGCCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTAACAATGAAGCAGACACCCCTGATAACTTCCAATCCAGGAGGCCGAACACTTCTTTCTGCTTTCTTGACATCCGCTCACCAGGCCTGGGGGACGCAGCCATGTACCTGTGTGCCACCAGCAGAGATAGGGTCAACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAGGATCTGAGGAACGTGACACCCCCTAAGGTGTCTCTGTTCGAGCCCAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTGCACCGACCCACAGGCCTACAAGGAGTCCAATTACTCTTATTGTCTGAGCTCCCGGCTGAGAGTGTCCGCCACATTTTGGCACAACCCTAGAAATCACTTCAGGTGCCAGGTGCAGTTTCACGGCCTGAGCGAGGAGGATAAGTGGCCAGAGGGATCCCCAAAGCCTGTGACCCAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGTGGAATCACATCCGCCTCTTATCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCC and the nucleotide sequence shown in SEQ ID NO: 36 is ATGAAGAGGATATTGGGAGCTCTGCTGGGGCTCTTGAGTGCCCAGGTTTGCTGTGTGAGAGGAATACAAGTGGAGCAGAGTCCTCCAGACCTGATTCTCCAGGAGGGAGCCAATTCCACGCTGCGGTGCAATTTTTCTGACTCTGTGAACAATTTGCAGTGGTTTCATCAAAACCCTTGGGGACAGCTCATCAACCTGTTTTACATTCCCTCAGGGACAAAACAGAATGGAAGATTAAGCGCCACGACTGTCGCTACGGAACGCTACAGCTTATTGTACATTTCCTCTTCCCAGACCACAGACTCAGGCGTTTATTTCTGTGCTGTGGAGGGGGATAATGCTGGTGGTACTAGCTATGGAAAGCTGACATTTGGACAAGGGACCATCTTGACTGTCCATCCAAATATCCAGAATCCAGAGCCCGCCGTGTATCAGCTGAAGGACCCAAGGAGCCAGGATTCCACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAACGTGCCCAAGACCATGGAGTCCGGCACCTTCATCACAGACAAGTGCGTGCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACCTCCTTCACATGCCAGGATATCTTTAAGGAGACCAATGCCACATATCCTTCCTCTGACGTGCCATGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACCGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGCGCATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGGCTGTGGAGCTCC and the nucleotide sequence shown in SEQ ID NO: 37 is ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCAGTTGGTCGACCTCCTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCATTGTAGAGGATCTGAGGAACGTGACACCCCCTAAGGTGTCTCTGTTCGAGCCCAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTGCACCGACCCACAGGCCTACAAGGAGTCCAATTACTCTTATTGTCTGAGCTCCCGGCTGAGAGTGTCCGCCACATTTTGGCACAACCCTAGAAATCACTTCAGGTGCCAGGTGCAGTTTCACGGCCTGAGCGAGGAGGATAAGTGGCCAGAGGGATCCCCAAAGCCTGTGACCCAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGTGGAATCACATCCGCCTCTTATCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCC and the nucleotide sequence shown in SEQ ID NO: 38 is ATGGAGTCATTCCTGGGAGGTGTTTTGCTGATTTTGTGGCTTCAAGTGGACTGGGTGAAGAGCCAAAAGATAGAACAGAATTCCGAGGCCCTGAACATTCAGGAGGGTAAAACGGCCACCCTGACCTGCAACTATACAAACTATTCCCCAGCATACTTACAGTGGTACCGACAAGATCCAGGAAGAGGCCCTGTTTTCTTGCTACTCATACGTGAAAATGAGAAAGAAAAAAGGAAAGAAAGACTGAAGGTCACCTTTGATACCACCCTTAAACAGAGTTTGTTTCATATCACAGCCTCCCAGCCTGCAGACTCAGCTACCTACCTCTGTGCTCTCGCGGGTTACCAGAAAGTTACCTTTGGAACTGGAACAAAGCTCCAAGTCATCCCAAATATCCAGAATCCAGAGCCCGCCGTGTATCAGCTGAAGGACCCAAGGAGCCAGGATTCCACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAACGTGCCCAAGACCATGGAGTCCGGCACCTTCATCACAGACAAGTGCGTGCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACCTCCTTCACATGCCAGGATATCTTTAAGGAGACCAATGCCACATATCCTTCCTCTGACGTGCCATGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACCGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGCGCATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGGCTGTGGAGCTCC and the nucleotide sequence shown in SEQ ID NO: 39 is ATGCTCTGCTCTCTCCTTGCCCTTCTCCTGGGCACTTTCTTTGGGGTCAGATCTCAGACTATTCATCAATGGCCAGCGACCCTGGTGCAGCCTGTGGGCAGCCCGCTCTCTCTGGAGTGCACTGTGGAGGGAACATCAAACCCCAACCTATACTGGTACCGACAGGCTGCAGGCAGGGGCCTCCAGCTGCTCTTCTACTCCGTTGGTATTGGCCAGATCAGCTCTGAGGTGCCCCAGAATCTCTCAGCCTCCAGACCCCAGGACCGGCAGTTCATCCTGAGTTCTAAGAAGCTCCTTCTCAGTGACTCTGGCTTCTATCTCTGTGCCTGGAGTCTCCGGACTAGCGGGAGCGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAGGATCTGAGGAACGTGACACCCCCTAAGGTGTCTCTGTTCGAGCCCAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTGCACCGACCCACAGGCCTACAAGGAGTCCAATTACTCTTATTGTCTGAGCTCCCGGCTGAGAGTGTCCGCCACATTTTGGCACAACCCTAGAAATCACTTCAGGTGCCAGGTGCAGTTTCACGGCCTGAGCGAGGAGGATAAGTGGCCAGAGGGATCCCCAAAGCCTGTGACCCAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGTGGAATCACATCCGCCTCTTATCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCC is.

[0060] The nucleic acid molecule may include natural and / or non-natural nucleotides and bases, for example, those having backbone modifications. The nucleic acid molecule refers to a polymer of nucleotides. Such a polymer of nucleotides may include natural and / or non-natural nucleotides and includes, but is not limited to, DNA, RNA, and PNA. The nucleotide sequence refers to the linear sequence constituting the nucleic acid molecule.

[0061] In some cases, the nucleic acid molecule includes cDNA, and in some cases, the nucleic acid molecule can be modified for use in the constructs described herein, for example, for codon optimization. In some cases, the sequence can be designed to include terminal restriction site sequences for the purpose of cloning into a vector.

[0062] In some cases, the nucleic acid molecule encoding the TCR can be obtained from various sources, for example, by polymerase chain reaction (PCR) amplification of the coding nucleic acid isolated from within one or more predetermined cells or from said one or more predetermined cells.

[0063] In one embodiment, the nucleotide sequence encoding the α-chain and / or the nucleotide sequence encoding the β-chain is codon-optimized. Generally, codon optimization involves balancing the proportion of selected codons with the abundance of published human transfer RNAs so that no overload or limitation occurs. Since most amino acids are encoded by multiple codons and codon usage varies among organisms, this may be necessary in some cases. Differences in codon usage between the transfected gene and the host cell can affect protein expression and immunogenicity of the nucleic acid construct. Generally, in codon optimization, codons balanced with human usage frequency are selected. Generally, due to the redundancy of amino acid codons, one amino acid will be encoded by different codons. In some embodiments, when selecting a codon for substitution, it may be desirable for the resulting mutation to be a silent mutation so that the codon change does not affect the amino acid sequence. Generally, the last nucleotide of the codon can be left unchanged without affecting the amino acid sequence.

[0064] This application provides a vector comprising the above nucleic acid molecule.

[0065] For example, one or more nucleic acids encoding one or two chains of the above TCR can be cloned into one or more suitable expression vectors, and the expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include vectors designed for propagation and amplification or expression or both, such as plasmids and viruses.

[0066] Considering whether the vector is DNA-based or RNA-based, the vector may contain regulatory sequences (such as start and stop codons for transcription and translation) specific to the type of host into which the vector is introduced (e.g., bacteria, fungi, plants, or animals). The vector may also contain 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, SV40 promoter, RSV promoter, and the promoter found in the long terminal repeat sequence of murine stem cell virus, and other promoters known to those skilled in the art are also contemplated.

[0067] In one embodiment, the vector is an expression vector.

[0068] In one embodiment, the vector is a viral vector, preferably a retroviral vector.

[0069] In one embodiment, the vector is a lentiviral vector.

[0070] This application also provides a host cell containing such a nucleic acid. To recombinantly produce a TCR, the nucleic acid encoding the TCR can be isolated and inserted into one or more vectors for further cloning / expression in the host cell. Such nucleic acids can be easily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the α and β chains of the TCR). In some embodiments, a method for preparing a TCR is provided, which includes culturing a host cell containing the nucleic acid encoding the TCR provided above under conditions suitable for the expression of the TCR molecule, and optionally, recovering the TCR from the host cell (or the host cell culture medium).

[0071] The host cell refers to a cell into which exogenous nucleic acid has been introduced, and includes the progeny of such a cell. Host cells include transformants and transformed cells (including primary transformed cells and their descendants), regardless of the number of passages. The progeny may not have the same nucleic acid content as the parental cell and may contain mutations.

[0072] This application also provides engineered cells comprising the above TCR, the above nucleic acid molecule, or the above vector.

[0073] In one embodiment, the TCR is heterologous to the cell.

[0074] In one embodiment, the engineered cell is a cell line.

[0075] In one embodiment, the engineered cell is a primary cell obtained from a subject, preferably the subject is a mammalian subject, preferably a human.

[0076] In one embodiment, the engineered cell is a T cell, preferably a T cell isolated from peripheral blood.

[0077] In one embodiment, the T cell is CD8+ or CD4+.

[0078] The manipulated cells can be, for example, a cell population or genetically engineered cells expressing a TCR, and these cells are typically eukaryotic cells such as mammalian cells, usually human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph or lymphoid organs, and are cells of the immune system such as cells of innate or adaptive immunity such as bone marrow or lymphoid cells (including lymphocytes, typically T cells and / or NK cells). 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 directly isolated from a subject and / or those isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the entire T cell population, CD+ cells, CD8+ cells, and their subpopulations.

[0079] Subtypes and subpopulations of T cells and / or CD+ and / or CD8+ T cells include naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes (stem cell memory T cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), or terminally differentiated effector memory T cells, etc.), 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.

[0080] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes such as bone marrow cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and / or basophils.

[0081] This application provides a method for producing the above-described manipulated cells, which includes introducing the above-described nucleic acid molecule or the above-described vector into cells in vitro or ex vivo.

[0082] In one embodiment, the vector is a viral vector and the introduction is performed by transduction.

[0083] The present application provides a pharmaceutical composition comprising the above-described T cell receptor (TCR), the above-described nucleic acid molecule, the above-described vector, or the above-described engineered cell. In one embodiment, it further comprises a pharmaceutically acceptable carrier or adjuvant.

[0084] The pharmaceutically acceptable carrier or adjuvant refers to a component in a pharmaceutical composition that is non-toxic to a subject, excluding the active ingredient. Examples of pharmaceutically acceptable carriers or adjuvants include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0085] The pharmaceutical composition may utilize timed release, delayed release, and sustained release delivery systems such that delivery of the composition occurs prior to sensitization of the treatment site and provides sufficient time to cause sensitization. Many types of release delivery systems are available and known. Such systems can be used to avoid repeated administration of the composition, thereby improving convenience for the subject and the physician.

[0086] The present application provides the use of the above-described T cell receptor (TCR), the above-described nucleic acid molecule, the above-described vector, the above-described engineered cell, or the above-described pharmaceutical composition in the preparation of a medicament for treating HPV-related diseases.

[0087] 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, etc.

[0088] This application provides a method for treating HPV-related diseases, including administering the above-mentioned T cell receptor (TCR), the above-mentioned nucleic acid molecule, the above-mentioned vector, or the above-mentioned engineered cell, or the above-mentioned pharmaceutical composition, to a subject in need thereof. 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.

[0089] The TCR described in this application can specifically bind to the antigen short peptide of HPV-positive tumor cells. The T cells transduced with the TCR can be specifically activated and have a very strong killing effect on target cells. The TCR can be used for the immunotherapy of HPV-positive tumors such as cervical cancer.

[0090] The T cells transduced with the TCR of this application can significantly kill primary cervical cancer organoid cells and effectively inhibit the growth of E7-positive tumors.

Example

[0091] This application provides a general and / or specific description of the materials and test methods used in the tests. In the following examples, unless otherwise specifically indicated, % means wt%, that is, weight percentage. When the manufacturers of the reagents and equipment used are not indicated, they are all commercially available conventional reagent products.

[0092] Example 1 Cloning of IVCPICSQK Antigen Short Peptide-Specific T Cells The synthesized short peptide IVCPICSQK (SEQ ID NO: 1, Jiangsu GenScript Biotechnology Co., Ltd.) was used to stimulate peripheral blood lymphocytes of healthy volunteers with the HLA-A*11:01 genotype. The IVCPICSQK short peptide was reconstituted with biotin-labeled HLA-A*11:01 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. For the preparation methods of pHLA monomers and tetramers, please refer to the protocol published by the NIH Tetramer Core Facility. The preparation method can be found on the web page https: / / tetramer.yerkes.emory.edu / support / protocols#1.

[0093] (1) Reagents Test medium: 10% FBS (ThermoFisher, catalog number 10099-044), RPMI1640 (ThermoFisher, catalog number C11875500BT), 10% HS (Gemni, catalog number 100512), TexMACS (Mitenyi, catalog number 170-076-309)

[0094] (2) Methods PBMCs were isolated from the peripheral blood of healthy volunteers by density gradient centrifugation. CD14-positive cells were isolated from PBMCs to induce dendritic cells (DCs). The CD14-positive cells were cultured in a medium (1640 + 10% AuFBS + 1% PS + 800 IU / ml GM-CSF + 500 IU / ml IL-4), supplemented with 1 ml of medium (containing 1600 IU / ml GM-CSF + 1000 IU / ml IL-4) on the 3rd day, and 10 ng / ml IL-1β, 10 ng / ml IL-6, 10 ng / ml TNF-α, and 1 μg / ml PGE2, which are DC maturation-inducing factors, were added on the 5th day, and the culture was continued for 2 days to obtain mature DCs. Using the mature DCs, the IVCPICSQK short peptide was loaded to a final concentration of 20 μg / ml to stimulate naive CD8-positive T cells isolated from PBMCs (medium TexMACS + 10% HS + 1% PS + 60 ng / ml IL21 + 10 IU / ml IL2 / 7 / 15). After 3 rounds of stimulation, tetramers and CD8 double-positive T cells, which are antigen-specific T cells, were sorted using a flow cytometer sorter.

[0095] (3) Results T cells specific for the IVCPICSQK antigen short peptide were cloned by the above method, and the cloned antigen short peptide-specific T cells were analyzed by flow cytometry. The results of flow cytometry of T cells after 2 rounds and 3 rounds of stimulation with the IVCPICSQK antigen short peptide are shown in Figure 1.

[0096] As can be seen from Figure 1, the cloning of T cells specific for the IVCPICSQK antigen short peptide was successful.

[0097] Example 2: Construction of an IVCPICSQK antigen short peptide-specific TCR lentiviral vector and packaging of lentivirus (1) Construction of the TCR lentiviral vector The CD8 and tetramer double-positive cells of Example 1 were flow sorted to obtain single cells. For the obtained single cells, the α-chain and β-chain of the TCR were amplified using a one-step RT-PCR kit (QIAGEN, catalog number 210212), respectively, and the PCR products were sequenced. By aligning the sequencing results with the sequences in the public database of IMGT (International Immunogenetic Information System), the sequence of the variable region of the α-chain of the TCR, the nucleotide sequence of the variable region of the β-chain, and the information of their CDR1, CDR2, and CDR3 can be obtained. The variable region sequence of the α-chain, the nucleotide sequence of the variable region of the β-chain, and the information of their CDR1, CDR2, and CDR3 of three TCRs (TCR074, TCR095, and TCR123) were obtained and are shown below, respectively. The nucleotide sequence of the variable region of the α-chain of TCR074 is shown in SEQ ID NO: 41: ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGACCTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATGTTCGTGCAGGAAAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGTGATCCAAGTTATGGTCTATTCTGGTACAAGCAGCCCAGCAGTGGGGAAATGATTTTTCTTATTTATCAGGGGTCTTATGACCAGCAAAATGCAACAGAAGGTCGCTACTCATTGAATTTCCAGAAGGCAAGAAAATCCGCCAACCTTGTCATCTCCGCTTCACAACTGGGGGACTCAGCAATGTACTTCTGTGCAATGAGAATTGATGCTGGTGGTACTAGCTATGGAAAGCTGACATTTGGACAAGGGACCATCTTGACTGTCCATCCAAAT The nucleotide sequence of the variable region of the β-chain is shown in SEQ ID NO: 42: ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACAGGTCATGGGGATGCCATGGTCATCCAGAACCCAAGATACCAGGTTACCCAGTTTGGAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTCATGTACTGGTACCAGCAGAAGTCAAGTCAGGCCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTAACAATGAAGCAGACACCCCTGATAACTTCCAATCCAGGAGGCCGAACACTTCTTTCTGCTTTCTTGACATCCGCTCACCAGGCCTGGGGGACGCAGCCATGTACCTGTGTGCCACCAGCAGAGATAGGGTCAACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTG The amino acid sequence of the complementarity-determining region 1 (CDR1) of the α chain is shown in SEQ ID NO: 2: TSDPSYG The amino acid sequence of the complementarity-determining region 2 (CDR2) is shown in SEQ ID NO: 3: QGSYDQQN The amino acid sequence of the complementarity-determining region 3 (CDR3) is shown in SEQ ID NO: 4: AMRIDAGGTSYGKLT The amino acid sequence of the complementarity-determining region 1 (CDR1) of the β chain is shown in SEQ ID NO: 5: LNHNV The amino acid sequence of the complementarity-determining region 2 (CDR2) is shown in SEQ ID NO: 6: YYDKDF The amino acid sequence of the complementarity-determining region 3 (CDR3) is shown in SEQ ID NO: 7: ATSRDRVNTGELF The nucleotide sequence of the α chain variable region sequence of TCR095 is shown in SEQ ID NO: 43: ATGAAGAGGATATTGGGAGCTCTGCTGGGGCTCTTGAGTGCCCAGGTTTGCTGTGTGAGAGGAATACAAGTGGAGCAGAGTCCTCCAGACCTGATTCTCCAGGAGGGAGCCAATTCCACGCTGCGGTGCAATTTTTCTGACTCTGTGAACAATTTGCAGTGGTTTCATCAAAACCCTTGGGGACAGCTCATCAACCTGTTTTACATTCCCTCAGGGACAAAACAGAATGGAAGATTAAGCGCCACGACTGTCGCTACGGAACGCTACAGCTTATTGTACATTTCCTCTTCCCAGACCACAGACTCAGGCGTTTATTTCTGTGCTGTGGAGGGGGATAATGCTGGTGGTACTAGCTATGGAAAGCTGACATTTGGACAAGGGACCATCTTGACTGTCCATCCAAAT The nucleotide sequence of the variable region of the β chain is shown in SEQ ID NO: 44: ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCAGTTGGTCGACCTCCTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCATTGTA The amino acid sequence of the complementarity-determining region 1 (CDR1) of the α chain is shown in SEQ ID NO: 8: DSVNN The amino acid sequence of the complementarity-determining region 2 (CDR2) is shown in SEQ ID NO: 9: IPSGT The amino acid sequence of complementarity-determining region 3 (CDR3) is shown in SEQ ID NO: 10: AVEGDNAGGTSYGKLT The amino acid sequence of complementarity-determining region 1 (CDR1) of the β chain is shown in SEQ ID NO: 11: MNHEY The amino acid sequence of complementarity-determining region 2 (CDR2) is shown in SEQ ID NO: 12: SMNVEV The amino acid sequence of complementarity-determining region 3 (CDR3) is shown in SEQ ID NO: 13: ASSWSTSYGYT The nucleotide sequence of the variable region of the α chain of TCR123 is shown in SEQ ID NO: 45: ATGGAGTCATTCCTGGGAGGTGTTTTGCTGATTTTGTGGCTTCAAGTGGACTGGGTGAAGAGCCAAAAGATAGAACAGAATTCCGAGGCCCTGAACATTCAGGAGGGTAAAACGGCCACCCTGACCTGCAACTATACAAACTATTCCCCAGCATACTTACAGTGGTACCGACAAGATCCAGGAAGAGGCCCTGTTTTCTTGCTACTCATACGTGAAAATGAGAAAGAAAAAAGGAAAGAAAGACTGAAGGTCACCTTTGATACCACCCTTAAACAGAGTTTGTTTCATATCACAGCCTCCCAGCCTGCAGACTCAGCTACCTACCTCTGTGCTCTCGCGGGTTACCAGAAAGTTACCTTTGGAACTGGAACAAAGCTCCAAGTCATCCCAAAT The nucleotide sequence of the variable region of the β chain is shown in SEQ ID NO: 46: ATGCTCTGCTCTCTCCTTGCCCTTCTCCTGGGCACTTTCTTTGGGGTCAGATCTCAGACTATTCATCAATGGCCAGCGACCCTGGTGCAGCCTGTGGGCAGCCCGCTCTCTCTGGAGTGCACTGTGGAGGGAACATCAAACCCCAACCTATACTGGTACCGACAGGCTGCAGGCAGGGGCCTCCAGCTGCTCTTCTACTCCGTTGGTATTGGCCAGATCAGCTCTGAGGTGCCCCAGAATCTCTCAGCCTCCAGACCCCAGGACCGGCAGTTCATCCTGAGTTCTAAGAAGCTCCTTCTCAGTGACTCTGGCTTCTATCTCTGTGCCTGGAGTCTCCGGACTAGCGGGAGCGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTA The amino acid sequence of the complementarity-determining region 1 (CDR1) of the α chain is shown in SEQ ID NO: 14: NYSPAY The amino acid sequence of the complementarity-determining region 2 (CDR2) is shown in SEQ ID NO: 15: IRENEKE The amino acid sequence of the complementarity-determining region 3 (CDR3) is shown in SEQ ID NO: 16: ALAGYQKVT The amino acid sequence of the complementarity-determining region 1 (CDR1) of the β chain is shown in SEQ ID NO: 17: GTSNPN The amino acid sequence of the complementarity-determining region 2 (CDR2) is shown in SEQ ID NO: 18: SVGIG The amino acid sequence of the complementarity-determining region 3 (CDR3) is shown in SEQ ID NO: 19: AWSLRTSGSEQF

[0098] The variable region sequences of the α and β chains of the IVCPICSQK 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 a standard method using a multi-fragment recombination cloning kit (Novezan Biotech, catalog number C113). The ligated plasmid was transformed into competent E. coli strain Stbl3 cells (Shanghai Weidi Biotechnology Co., Ltd.), and inoculated onto an LB / agar plate containing 100 μg / ml ampicillin. After incubating overnight at 37 °C, a single colony was picked up and grown overnight at 37 °C with shaking in 10 ml of LB containing 100 μg / ml ampicillin. The cloned plasmid was purified using a mini-prep midi kit (TIANGEN Biotech Co., Ltd. (TIANGEN), catalog number DP118-02), and the sequence of the plasmid was determined to obtain the IVCPICSQK TCR (i.e., TCR074, TCR095, TCR123).

[0099] (2) Lentivirus packaging Test medium: 10% FBS (Lonsera, catalog number S711-001), DMEM (cytiva, catalog number SH30243.01) 293T cells (the depository institution is American Type Culture Collection, i.e., ATCC, and the deposit number is CRL-1573) were prepared and cultured in a 10 cm dish. Transfection of the plasmid was started when the confluence was less than 80%. The ratio of the virus packaging plasmid to the IVCPICSQK TCR plasmid was 1:1, with a total of 10 μg. The above plasmids were added to serum-free DMEM medium and mixed with PEI (polyethylenimine). Then, the mixed solution with the added plasmids was added to 293T cells and cultured at 37 °C. After 72 hours, the cell supernatant was concentrated using a 100 kd ultrafiltration tube, and the virus vector was collected.

[0100] Example 3: Construction and functional identification of a Jurkat cell line expressing a TCR specific for the IVCPICSQK antigen short peptide Method for expressing 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 the NFAT expression level as a readout of T cell activation.

[0101] (1) Reagents Test medium: 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (ThermoFisher, catalog number C11875500BT)

[0102] (2) Methods Preparation of target cells The target cells used in this experiment were T2-A11 cells (T2 cells are deposited with ATCC, deposit number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). Target cells were prepared in experimental medium and the target cell concentration was adjusted to 1.6×10 6 cells / ml, and 50 μl was collected from each well to obtain 80,000 cells / well.

[0103] 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 group.

[0104] The JK8NF cells (Jurkat cells are deposited with ATCC, and the deposit number is TIB-152. The JK8NF cells were constructed based on Jurkat cells with reference to Cancer Res 2006;66(23):11455-61, Front. Immunol. 11:633) were added with the lentivirus having the TCR gene of the present application obtained in Example 2 at an MOI (multiplicity of infection) = 10. After 72 hours, it was confirmed by flow cytometry that the transfection positive rate was about 100% (the results are shown in Figure 2). After expansion culture, the concentration of effector cells was adjusted to 1.6×10 6 cells / ml, and 50 μl was collected from each well to obtain 80,000 cells / well.

[0105] Preparation of short peptide solution The original short peptide (IVCPICSQK) with a concentration of 5 mg / ml was diluted to 400 μg / ml, and further diluted in order from the higher concentration at a ratio of 10-fold to 40 μg / ml, 4 μg / ml, 0.4 μg / ml, 0.04 μg / ml, 0.004 μg / ml, 0.0004 μg / ml.

[0106] 50 μl was collected from each well so that the final concentrations of the short peptides in the 96-well plate were 100 μg / ml, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, 0.001 μg / ml, 0.0001 μg / ml, respectively.

[0107] Finally, 50 μl of target cells, 50 μl of effector cells, 50 μl of the corresponding concentration of short peptide dilution, 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.

[0108] (3) Results The expression of NFAT in the T cells transduced with the TCR of the present application in response to the target cells loaded with the IVCPICSQK antigen short peptide was tested by the above method. A curve of the NFAT expression level was drawn using Graphpad prism8. The results are shown in Figure 3.

[0109] From Figure 3, it can be seen that the T cells transduced with the TCR of the present application have a good activation response to the target cells loaded with their specific short peptides.

[0110] Example 4 Construction and functional identification of primary T cells expressing a TCR specific for the IVCPICSQK antigen short peptide Schedule for real-time monitoring of target cell proliferation (1) Reagents Test medium: 10% FBS (ThermoFisher, catalog number 10099-044), RPMI1640 (ThermoFisher, catalog number C11875500BT)

[0111] (2) Methods Preparation of effector T cells The effector cells (T cells) in this experiment were the T cells transduced with the TCR of the present application, and the T cells of the same volunteer not transfected with the TCR of the present application were used as the control group.

[0112] The peripheral blood of the volunteer was centrifuged by density gradient to obtain peripheral blood mononuclear cells, and the peripheral blood mononuclear cells were placed in each well of a 24-well plate at 5.0×10 5 cells / 500 μl, and a total of 1×10 6 cells were collected. After stimulating the T cells with anti-CD3 / CD28 magnetic beads, they were cultured in an incubator at 37°C and 5% CO 2 . After 24 hours, cell mass formation was observed. After adding the lentivirus of the TCR (TCR074, TCR095, and TCR123) genes obtained in Example 2 at an MOI (multiplicity of infection) = 2 for transduction, they 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 (shown in Figure 4). The concentration of the effector cells after expansion culture was adjusted to 5.0×10 4 positive cells / ml, 100 μl was taken from each well, and 5000 positive cells / well were obtained.

[0113] Preparation of target cells The target cells used in this experiment were SK-MEL-28-E7. The genotype of SK-MEL-28 (deposited with ATCC) is A*11:01, but it does not express E7 itself. Therefore, SK-MEL-28-E7 cells were constructed as target cells by overexpressing the E7 gene via a lentiviral vector. The target cells were prepared in an experimental medium, and the target cell concentration was adjusted to 5.0×10 4 cells / ml, and 100 μl was collected from each well to obtain 5,000 cells / well.

[0114] Monitoring the growth of target cells by RTCA x Celligence system 100 μl of the T cells prepared above and 100 μl of the target cells were added to an E-plate. The E-plate was attached to an RTCA analyzer and incubated in an incubator at 37°C and 5% CO 2 for 90 hours, and the real-time growth curve of the target cells was observed.

[0115] (3) Results The function of the T cells transduced with the TCR of the present application was tested through real-time monitoring of the target cell growth experiment (as described above). The cell index of cell growth in each well was plotted using Graphpad prism8. The experimental results are shown in Figure 5.

[0116] As can be seen from Figure 5, the T cells expressing the TCR specific to the IVCPICSQK-A*11:01 antigen short peptide have a strong killing function against the cell line expressing E7.

[0117] Example 5: Identification of the in vivo tumor growth inhibitory function of primary T cells expressing the TCR specific to the IVCPICSQK antigen short peptide The following experiments were conducted to demonstrate the inhibitory effect of TCR-transduced T cells on tumor growth. Changes in tumor volume were observed to evaluate the tumor-killing function of T cells. In in vitro experiments, since three TCRs were shown to have similar tumor-killing effects, only TCR095 and TCR123 were selected as representatives to identify the in vivo tumor growth inhibitory function.

[0118] (1) Reagents and Mice Test medium: 10% FBS (ThermoFisher, catalog number 10099-044), RPMI1640 (ThermoFisher, catalog number C11875500BT) NCG mice: Purchased from GemPharmatech Co., Ltd, female, 4 - 6 weeks old, genotype (prkdc)ko / ko, (Il2rg female)ko / ko.

[0119] (2) Methods Subcutaneous inoculation of tumor cells The tumor cells used in this experiment were SK-MEL-28-E7. The tumor cells were prepared with physiological saline, and the tumor cell concentration was adjusted to 5.0×10 7 cells / ml, and 200 μl was subcutaneously inoculated into each mouse to obtain 1.0×10 7 cells / mouse. Effector T cells were inoculated when the tumor volume grew to approximately 100 mm 3 .

[0120] 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 of the same volunteer not transfected with the TCR of the present application were used as the control group. Five mice were used per group.

[0121] The peripheral blood of the volunteer was centrifuged by density gradient to obtain peripheral blood mononuclear cells, and the peripheral blood mononuclear cells were placed in each well of a 24-well plate at 5.0×10 5 cells / 500 μl, for a total of 4×10 6Individual cells were collected. After stimulating T cells with anti-CD3 / CD28 magnetic beads, they were cultured in an incubator at 37 °C and 5% CO 2 After culturing in an incubator, cell mass formation was observed after 24 hours. After transduction by adding the lentiviruses of TCRs (TCR095 and TCR123) obtained in Example 2 at MOI (multiplicity of infection) = 2, they were amplified in RPMI 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 5.0×10 7 Positive cells / ml, and 200 μl was injected into each mouse via the ophthalmic vein to obtain 1.0×10 7 Positive cells / mouse.

[0122] (3) Results The volume of the tumor after inoculation of T cells was observed and recorded, and the effect of primary T cells expressing the TCR of the present application in tumor treatment was evaluated based on the change in tumor volume. The growth curve of the tumor was drawn using GraphPad Prism 8. The experimental results are shown in FIGS. 6A and 6B. Here, FIG. 6A is a schematic diagram of the change in tumor volume over time after T cell inoculation, and FIG. 6B is a histogram of tumor weight after inoculation of T cells containing different TCRs.

[0123] From FIGS. 6A and 6B, it can be seen that T cells expressing TCR specific to the IVCPICSQK-A*11:01 antigen short peptide can effectively inhibit the growth of E7-positive tumors.

[0124] Example 6 T cells expressing TCR specific to the IVCPICSQK antigen short peptide can inhibit the growth of cervical cancer organoids After co-culturing cervical cancer organoids with T cells, the growth state of the organoids was observed to evaluate the killing effect of TCR-transduced T cells on primary cervical cancer cells.

[0125] (1) Reagents Test medium: OrganoPro Cervical Cancer Organoid Medium Kit (Ketu Medical, catalog number K2O-M-CC)

[0126] (2) Method 8,000 HPV16 + HLA-A*11:01 + The cervical cancer organoid KOCC-002S4 (Ketu Medicine) was co-cultured with 8,000 primary T cells (TCR123) transduced with TCR123 or control T cells without TCR transduction (control group). After 72 hours, the growth state of the organoids was observed under a microscope and photographed.

[0127] (3) Results The growth state of the organoids was observed under a microscope, and photos of typical fields of view were taken. The numbers of complete and incomplete organoids within the fields of view were counted. The results are shown in FIGS. 7A to 7B. Here, FIG. 7A is a schematic diagram of the growth status of the organoids observed under a microscope, and FIG. 7B is a schematic diagram of the counted numbers of complete and incomplete organoids.

[0128] It can be seen from FIGS. 7A to 7B that T cells expressing a TCR specific for the IVCPICSQK-A*11:01 antigen short peptide can significantly kill primary cervical cancer organoid cells.

[0129] In summary, the T cells transduced with the TCR in the present application can be specifically activated and have a very strong killing effect on tumor cells expressing A1101 and HPV.

[0130] The T cells have a very strong killing function against cell lines expressing E7 and can effectively inhibit the growth of E7-positive tumors.

[0131] The above are only preferred embodiments of the present application and are not intended to limit the present application to other forms. A person skilled in the art can make modifications or corrections to equivalent embodiments with changes or equivalent changes by using the technical content disclosed above. However, any simple modification, equivalent change, and correction made to the above embodiments based on the technical essence of the present application without departing from the content of the technical solution of the present application are still included within the protection scope of the technical solution of the present application.

Claims

1. Use of an antigen short peptide in screening for a drug for treating an HPV-related disease, wherein the amino acid sequence of the antigen short peptide is shown in SEQ ID NO:

1.

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. Use of an antigen short peptide in screening for 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, wherein the amino acid sequence of the antigen short peptide is shown in SEQ ID NO:

1.

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

5. A T cell receptor (TCR), wherein the TCR comprises an α chain containing a variable region and / or a β chain containing a variable region, and the variable region of the α chain has a complementarity-determining region 1 (CDR1) with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 14, and / or A T cell receptor (TCR) comprising a complementarity-determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO:

15.

6. The variable region of the β chain has a complementarity-determining region 1 (CDR1) with the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 17, and / or The T cell receptor (TCR) according to claim 5, comprising a complementarity-determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 12, or SEQ ID NO:

18.

7. The variable region of the α chain comprises a complementarity-determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 16, 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: 7, SEQ ID NO: 13, or SEQ ID NO: 19, 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 the amino acid sequence shown in SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, and / or the amino acid sequence of the variable region of the β chain is the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31, Preferably, the α chain further comprises an α constant region, and / or the β chain further comprises a β constant region, preferably, the constant region is a mouse constant region or a human constant region, The T cell receptor (TCR) according to claim 5 or 6.

8. The TCR is isolated, purified, or recombinant, Preferably, the TCR is of human origin, Preferably, the TCR is monoclonal, Preferably, the TCR is single-chain, Preferably, the TCR comprises two chains, Preferably, the TCR is cell-bound or soluble, preferably soluble, Preferably, the TCR binds to an antigenic short peptide-HLA-A1101 complex, preferably, the amino acid sequence of the antigenic short peptide is as shown in SEQ ID NO: 1, The T cell receptor (TCR) according to any one of claims 5 to 7.

9. A nucleic acid molecule comprising the TCR according to any one of claims 5 to 8 or a nucleotide sequence encoding the α chain or β chain of the TCR.

10. The nucleotide sequence encoding the α chain comprises the nucleotide sequence shown in SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38, and / or The nucleotide sequence encoding the β chain comprises the nucleotide sequence shown in SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39, The nucleic acid molecule according to claim 9.

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

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

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, the engineered cell is a primary cell obtained from a subject, preferably the subject is a mammalian subject, preferably a human, Preferably, the engineered cell is a T cell or an NK cell, preferably the T cell is a T cell isolated from peripheral blood, Preferably, the T cell is CD8+ or CD4+, the engineered cell according to claim 13.

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 according to claim 15, wherein the vector is a viral vector and the introduction is performed 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, further comprising a pharmaceutically acceptable carrier or adjuvant, the pharmaceutical composition.

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, 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-related disease, Preferably, the HPV-related disease is an HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer, the use.

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

20. The method according to claim 19, wherein the HPV-related disease is an 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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