WO2023217143A1 - 抗原短肽用于筛选治疗与hpv相关的疾病的药物中的用途及其筛选的tcr - Google Patents

抗原短肽用于筛选治疗与hpv相关的疾病的药物中的用途及其筛选的tcr Download PDF

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WO2023217143A1
WO2023217143A1 PCT/CN2023/093015 CN2023093015W WO2023217143A1 WO 2023217143 A1 WO2023217143 A1 WO 2023217143A1 CN 2023093015 W CN2023093015 W CN 2023093015W WO 2023217143 A1 WO2023217143 A1 WO 2023217143A1
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seq
tcr
cells
amino acid
acid sequence
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PCT/CN2023/093015
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French (fr)
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陈琳
王晓玲
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广州医科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present application relates to the field of medical technology, and in particular to the use of a short antigen peptide for screening drugs for treating HPV-related diseases and its screened TCR.
  • HPV Human Papillomavirus
  • HPV16 An important factor in HPV causing cancer is persistent infection with high-risk HPV types, such as HPV16, HPV18, etc.
  • the E6 and E7 proteins encoded by high-risk HPV can inhibit the activity of the tumor suppressor genes p53 and Rb proteins respectively, leading to cell cycle abnormalities and canceration.
  • High-risk HPV is associated with 90% of cervical and anal cancers, 40% to 60% of vaginal and penile cancers, and, depending on geography, 60% of oropharyngeal cancers.
  • Cervical cancer is the most common malignant tumor of the female reproductive tract, with the global incidence ranking second among female malignant tumors.
  • the incidence of cervical cancer in developing countries is significantly higher than that in developed countries.
  • the incidence rate of cervical cancer in China is 15.30/100,000, and the mortality rate is 4.57/100,000, which is higher than the global average incidence rate (10.61/100,000) and mortality rate (2.98/100,000).
  • the incidence rate of cervical cancer has been on the rise, with the peak age of incidence being 40-60 years old.
  • surgery and Chemotherapy and radiotherapy have good therapeutic effects, but more than half of the patients are diagnosed with locally advanced cervical cancer at the first visit.
  • Specific T cell immunotherapy refers to the method of using specific T cells targeting tumor antigens to kill tumor cells. It is a highly personalized tumor immunotherapy method. Due to the existence of the local immunosuppressive microenvironment of the tumor, the patient's own T cells have limited tumor-killing function. Therefore, people try to improve their ability to kill tumors by genetically modifying T cells.
  • Both TCR-T and CAR-T are genetically modified cell therapy drugs that bind to the corresponding target through the transferred T cell receptor (TCR) or chimeric antigen receptor (CAR) gene, that is, It can activate T cells and use granzyme, perforin, cytokines, etc.
  • TCR-T TCR-T
  • CAR-T the target of CAR-T is the membrane protein on the cell surface.
  • target of TCR-T is the antigen short peptide-MHC complex (peptide-major histocompatibility complex, pMHC).
  • HPV-related proteins are the most ideal T cell immunotherapy targets for cervical cancer.
  • the target recognized by TCR is "antigen short peptide-MHC molecule complex".
  • TCR is also MHC-restricted.
  • a TCR molecule can only specifically recognize short peptides presented by a specific MHC.
  • MHC is polymorphic.
  • the most common HLA type in 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 antigenic short peptides is a prerequisite for TCR-T therapy. Although whether a short peptide binds to HLA can be obtained through affinity prediction, HLA binding assay, etc., whether the short peptide can be naturally presented by HPV-expressing tumor cells determines whether the short peptide-specific TCR can be used in tumors. The key to treatment.
  • the purpose of this application is to provide a short antigen peptide for use in screening drugs for treating HPV-related diseases and the screened T cell receptor (TCR).
  • the short antigen peptide screens specific T cell antibodies, transcription T cells containing the TCR can be specifically activated and have a strong killing effect on tumor cells expressing A1101 and HPV.
  • short antigen peptides for screening drugs for treating diseases related to HPV.
  • the amino acid sequence of the short antigen peptides is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • HPV-related diseases are HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer.
  • short antigen peptides in screening drugs for the treatment of chronic HPV infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer.
  • the amino acid of the short antigen peptide is The sequence is shown as SEQ ID NO:1 or SEQ ID NO:2.
  • TCR T cell receptor
  • the TCR includes an ⁇ chain containing a variable region and/or a ⁇ chain containing a variable region, and the variable region of the ⁇ chain includes an amino acid sequence such as SEQ ID NO :3 or the complementarity determining region 1 (CDR1) shown in SEQ ID NO:9; and/or
  • the amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO:4 or SEQ ID NO:10.
  • T cell receptor according to item 5, wherein the variable region of the ⁇ chain includes the complementarity determining region 1 (CDR1) whose amino acid sequence is as shown in SEQ ID NO: 6; and/or
  • the amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO:7.
  • T cell receptor according to item 5 or 6, wherein the variable region of the ⁇ chain contains an amino acid sequence such as SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 13 or the complementarity determining region 3 (CDR3) shown in SEQ ID NO:15; and/or
  • variable region of the ⁇ chain includes a complementarity determining region 3 (CDR3) containing an amino acid sequence as shown in SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.
  • CDR3 complementarity determining region 3
  • variable region of the alpha chain further includes a first leader sequence
  • variable region of the beta chain also includes a second leader sequence.
  • T cell receptor according to any one of items 5-8, wherein the amino acid sequence of the ⁇ chain variable region is such as SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID
  • amino acid sequence of the variable region of the ⁇ chain is as shown in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27 or is the same as SEQ ID NO:21, SEQ ID NO:23 , SEQ ID NO:25 or SEQ ID NO:27 has an amino acid sequence of at least 90% sequence identity.
  • T cell receptor according to any one of items 5 to 9, wherein the ⁇ chain further comprises an ⁇ constant region and/or the ⁇ chain further comprises a ⁇ constant region, preferably, the The constant region is a mouse constant region or a human constant region.
  • TCR T cell receptor
  • T cell receptor according to any one of items 5-11, wherein the TCR is human.
  • T cell receptor according to any one of items 5-12, wherein the TCR is monoclonal.
  • T cell receptor according to any one of items 5-13, wherein the TCR is single chain.
  • T cell receptor according to any one of items 5-14, wherein the TCR comprises two chains.
  • TCR T cell receptor
  • TCR T cell receptor
  • the amino acid sequence of the short antigen peptide is such as SEQ ID NO:1 or SEQ ID NO:2.
  • nucleic acid molecule wherein the nucleic acid molecule comprises the TCR described in any one of coding items 5-17 or the ⁇ chain or ⁇ chain of the TCR.
  • nucleic acid molecule according to item 18, wherein the nucleotide sequence encoding the ⁇ chain includes a nucleoside shown in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36 acid sequence; and/or
  • the nucleotide sequence encoding the ⁇ chain includes SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID The nucleotide sequence shown in NO:35 or SEQ ID NO:37.
  • a vector wherein the vector contains the nucleic acid molecule described in item 18 or 19.
  • An engineered cell comprising the TCR described in any one of Items 5-17, the nucleic acid molecule described in any one of Items 18-19, or the vector described in any one of Items 20-23.
  • engineered cell according to any one of items 24-26, wherein the engineered cell is a primary cell obtained from a subject, preferably the subject is a mammalian subject , preferably human.
  • the engineered cell according to any one of items 24-27, wherein the engineered cell is a T cell or an NK cell.
  • the T cell is a T cell isolated from peripheral blood.
  • a method for producing the engineered cells described in any one of items 24-29 which includes in vitro or ex vivo the nucleic acid molecule described in any one of items 18-19 or any of items 20-23.
  • the vector of one item is introduced into a cell.
  • a pharmaceutical composition comprising the T cell receptor (TCR) described in any one of items 5-17, the nucleic acid molecule described in any one of items 18-19, and any one of items 20-23 The vector described in Item 2 or the engineered cell described in any one of Items 24-29.
  • TCR T cell receptor
  • composition according to item 32 further comprising a pharmaceutically acceptable carrier or adjuvant.
  • TCR T cell receptor
  • HPV-related diseases are HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer.
  • a method for treating HPV-related diseases comprising combining the T cell receptor (TCR) described in any one of Items 5-17, the nucleic acid molecule described in any one of Items 18-19, and Item 20
  • TCR T cell receptor
  • the vector according to any one of -23 or the engineered cell according to any one of items 24-29 or the pharmaceutical composition according to any one of items 32-33 is administered to a subject in need.
  • 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.
  • the short antigen peptide described in this application can screen specific T cell receptors (TCR), and at the same time, T cells transduced with the TCR can be specifically activated and have strong killing effect on tumor cells expressing A1101 and HPV. It can be used for immunotherapy of HPV-positive tumors such as cervical cancer.
  • TCR T cell receptors
  • the T cells transduced with the TCR described in this application have a strong activation response to cell lines that express E6, but have no activation response to cell lines that do not express E6, and have a strong killing function against cell lines that express E6. And can effectively inhibit the growth of E6-positive tumors.
  • Figure 1 is a schematic diagram of cloning TTLEQQYNK and GTTLEQQYNK antigen short peptide-specific T cells in Example 1, wherein Figure 1A is a schematic diagram of the T cell flow cytometric detection results after two rounds of stimulation, and Figure 1B is a schematic diagram of T cells after three rounds of stimulation. Schematic diagram of cell flow cytometry results.
  • Figure 2 is a schematic diagram of using flow cytometry to identify the positive rate of transfection in Example 3.
  • Figure 3 is a schematic diagram of the expression of NFAT in response to target cells loaded with TTLEQQYNK antigen short peptide in T cells transduced with the TCR described in the present application in Example 3.
  • Figure 4 is a schematic diagram of using flow cytometry to identify the positive rate of transfection in Example 4.
  • FIG. 5 is a schematic diagram of the cell proliferation index of T cells transduced with the TCR described in the present application in Example 4.
  • Figure 6 is a schematic diagram of tumor volume after inoculation of T cells transduced with TCRs described in the present application in Example 5, wherein Figure 6A is a schematic diagram of tumor volume changes over time after inoculation of T cells, and Figure 6B is a schematic diagram of inoculation of T cells containing different TCRs Histogram of tumor weight after T cells.
  • FIGS. 7A to 7B are statistical schematic diagrams of the observed growth status of organoids and the integrity and incompleteness of organoids.
  • Figure 7A is a schematic diagram of the growth status of organoids observed through a microscope
  • Figure 7B is a schematic diagram of the complete morphology of organoids. Schematic diagram of counting the number of organoids with incomplete morphology.
  • This application provides the use of short antigen peptides for screening drugs for treating diseases related to HPV.
  • the amino acid sequence of the short antigen peptides is shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • sequence of SEQ ID NO:1 is as follows: TTLEQQYNK
  • SEQ ID NO:2 The sequence of SEQ ID NO:2 is as follows: GTTLEQQYNK.
  • 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.
  • This application provides the use of short antigen peptides for screening drugs for the treatment of chronic HPV infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer.
  • the short antigen peptide The amino acid sequence is shown in SEQ ID NO:1 or SEQ ID NO:2.
  • the drug is a T cell receptor (TCR) for binding to an antigen peptide-HLA-A1101 complex comprising the antigen peptide.
  • TCR T cell receptor
  • the T cell receptor is obtained through screening of the antigen short peptide, and the T cell receptor (TCR) binds to the TTLEQQYNK-HLA-A1101 complex or to the GTTLEQQYNK-HLA-A1101 complex.
  • T cell receptor or TCR is the only receptor for specific antigen peptides presented on the major histocompatibility complex (MHC). In the immune system, it is triggered by the binding of the antigen-specific TCR to the pMHC complex. T cells have direct physical contact with antigen-presenting cells (APCs), and then other cell surface molecules of T cells and APCs interact, which causes a series of subsequent cell signaling and other physiological reactions, resulting in different Antigen-specific T cells exert immune effects on target cells
  • APCs antigen-presenting cells
  • the TCR is a molecule that contains variable alpha and beta chains or variable gamma and delta chains and is capable of peptide specific binding to an MHC molecule.
  • the TCR is in the alpha beta form.
  • TCRs that exist in the ⁇ and ⁇ forms are generally similar in structure, but the T cells expressing them can have different anatomical locations or functions, and TCRs can be found on the cell surface or in soluble form.
  • the TCR is found on the surface of T cells (T lymphocytes), where it is typically responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.
  • T lymphocytes T cells
  • MHC major histocompatibility complex
  • variable domain of a TCR contains complementarity-determining regions (CDRs), which are often major contributors to the antigen recognition and binding capacity and specificity of peptides, MHC and/or MHC-peptide complexes.
  • CDRs complementarity-determining regions
  • FR framework region
  • CDR3 is the primary CDR responsible for antigen binding or specificity, or the most important of the three CDRs on a given TCR variable region for antigen recognition and/or for interaction with the processed peptide portion of the peptide-MHC complex.
  • the CDR1 of the alpha chain can interact with the N-terminal part of some antigenic peptides; in some cases, the CDR1 of the beta chain can interact with the C-terminal part of some antigenic peptides; in some cases , CDR2 has the strongest effect or is primarily responsible for the interaction or recognition of the MHC part of the MHC-peptide complex; in some cases, the variable region antigen of the ⁇ chain contains other hypervariable regions (CDR4 or HVR4) , which are typically involved in superantigen binding rather than antigen recognition.
  • CDR4 or HVR4 hypervariable regions
  • the TTLEQQYNK-HLA-A1101 complex or GTTLEQQYNK-HLA-A1101 complex refers to a complex combining HLA-A1101 and the antigenic short peptide TTLEQQYNK or GTTLEQQYNK.
  • the protein is degraded by the proteasome in cells into polypeptides of different lengths, and some polypeptides Combined with HLA to form a complex and presented to the cell surface.
  • the TTLEQQYNK-HLA-A1101 complex or GTTLEQQYNK-HLA-A1101 complex recognized by the TCR can be expressed on the cell membrane, or it can also be expressed as a soluble The form of protein exists in solution.
  • amino acid sequence of HLA-A1101 is shown in SEQ ID NO: 38, and its amino acid sequence is:
  • the short antigen peptide is used for screening drugs for treating diseases related to HPV, and the amino acid sequence of the short antigen peptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • 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.
  • the drug is a T cell receptor (TCR) for binding to an antigen peptide-HLA-A1101 complex comprising the antigen peptide.
  • TCR T cell receptor
  • the antigenic short peptide is used for screening drugs for the treatment of chronic HPV infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer, and the antigen
  • the amino acid sequence of the short peptide is shown in SEQ ID NO:1 or SEQ ID NO:2.
  • the drug is a T cell receptor (TCR) for binding to an antigen peptide-HLA-A1101 complex comprising the antigen peptide.
  • TCR T cell receptor
  • the TCR includes an ⁇ chain containing a variable region and/or a ⁇ chain containing a variable region, and the variable region of the ⁇ chain includes an amino acid sequence such as SEQ Complementarity determining region 1 (CDR1) shown in ID NO:3 or SEQ ID NO:9; and/or
  • the amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO:4 or SEQ ID NO:10.
  • amino acid sequence shown in SEQ ID NO:3 is: SSYSPS
  • amino acid sequence shown in SEQ ID NO:9 is: TTLSN
  • amino acid sequence shown in SEQ ID NO:4 is: YTSAATLV
  • amino acid sequence shown in SEQ ID NO:10 is: LVKSGEV
  • variable region of the ⁇ chain includes the complementarity determining region 1 (CDR1) whose amino acid sequence is as shown in SEQ ID NO: 6; and/or
  • the amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO:7.
  • amino acid sequence shown in SEQ ID NO:6 is: SGDLS
  • amino acid sequence shown in SEQ ID NO:7 is: YYNGEE
  • variable region of the alpha chain comprises a complementarity determining region 3 containing an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15 (CDR3); and/or
  • variable region of the ⁇ chain includes a complementarity determining region 3 (CDR3) containing an amino acid sequence as shown in SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.
  • CDR3 complementarity determining region 3
  • amino acid sequence shown in SEQ ID NO:5 is: VVSLSGGYNKLI
  • amino acid sequence shown in SEQ ID NO:11 is: AGPKITGGGNKLT
  • amino acid sequence shown in SEQ ID NO:13 is: AGPILTGGGNKLT
  • amino acid sequence shown in SEQ ID NO:15 is: AGPVLTGGGNKLT
  • amino acid sequence shown in SEQ ID NO:8 is: ASSVTGSGYT
  • amino acid sequence shown in SEQ ID NO:12 is: ASSVGGGPNYGYT
  • amino acid sequence shown in SEQ ID NO:14 is: ASGLSGPNTGELF
  • amino acid sequence shown in SEQ ID NO:16 is: ASSVGGPNTGELF.
  • variable region of the alpha chain further includes a first leader sequence
  • variable region of the beta chain also includes a second leader sequence.
  • the first leader sequence of the variable region of the alpha chain and the second leader sequence of the variable region of the beta chain are well known to those skilled in the art.
  • the first leader sequence of the variable region of the alpha chain can be Using a leader sequence with an amino acid sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 19, the second leader sequence of the variable region of the ⁇ chain can use a leader sequence with an amino acid sequence as shown in SEQ ID NO: 18.
  • amino acid sequence shown in SEQ ID NO:17 is: MLLLLVPVLEVIFTLGGTR
  • amino acid sequence shown in SEQ ID NO:19 is: MLLITSMLVLWMQLSQVN
  • amino acid sequence shown in SEQ ID NO:18 is: MGFRLLCCVAFCLLGAGPV
  • the amino acid sequence of the alpha chain variable region is as shown in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26 or is the same as SEQ ID NO:20 , SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26 have an amino acid sequence of at least 90% sequence identity; and/or
  • amino acid sequence of the variable region of the ⁇ chain is as shown in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27 or is the same as SEQ ID NO:21, SEQ ID NO:23 , SEQ ID NO:25 or SEQ ID NO:27 has an amino acid sequence of at least 90% sequence identity.
  • amino acid sequence shown in SEQ ID NO:20 is:
  • amino acid sequence shown in SEQ ID NO:21 is:
  • amino acid sequence shown in SEQ ID NO:22 is:
  • amino acid sequence shown in SEQ ID NO:23 is:
  • amino acid sequence shown in SEQ ID NO:24 is:
  • amino acid sequence shown in SEQ ID NO:25 is:
  • amino acid sequence shown in SEQ ID NO:26 is:
  • amino acid sequence shown in SEQ ID NO:27 is:
  • the amino acid sequence of the ⁇ chain variable region has at least 90% sequence identity with SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26, such as an amino acid sequence with SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26 have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity Sexual amino acid sequence;
  • amino acid sequence of the variable region of the ⁇ chain having at least 90% sequence identity with SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27 may be an amino acid sequence with SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27 have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity sexual amino acid sequence.
  • the alpha chain further comprises an alpha constant region and/or the beta chain further comprises a beta constant region.
  • the constant region is a mouse constant region or a human constant region.
  • amino acid sequence of the mouse ⁇ constant region is shown in SEQ ID NO: 28;
  • the amino acid sequence of the mouse ⁇ constant region is as SEQ ID NO: 29, that is, the constant regions of the ⁇ chain of the TCR mentioned above can all have the same constant region. Similarly, the constant regions of the ⁇ chains of all TCRs can also be All have the same constant region.
  • amino acid shown in SEQ ID NO:28 is:
  • amino acid shown in SEQ ID NO:29 is:
  • the constant region of the TCR may contain a short linker sequence in which cysteine residues form a disulfide bond, thereby connecting the two chains of the TCR.
  • TCRs can have additional of cysteine residues such that the TCR contains two disulfide bonds in the constant region.
  • the amino acid sequence of the alpha chain variable region of the TCR is as shown in SEQ ID NO: 20 and the amino acid sequence of the beta chain variable region is as shown in SEQ ID NO: 21, or the amino acid sequence of the alpha chain variable region
  • the sequence is as SEQ ID NO:22 and the amino acid sequence of the ⁇ chain variable region is as SEQ ID NO:23, or the amino acid sequence of the ⁇ chain variable region is as SEQ ID NO:24 and the amino acid sequence of the ⁇ chain variable region is as SEQ ID NO:25, or the amino acid sequence of the ⁇ chain variable region is SEQ ID NO:26 and the amino acid sequence of the ⁇ chain variable region is SEQ ID NO:27.
  • artificial disulfide bonds are introduced between the residues of the ⁇ chain and ⁇ chain constant regions of the TCR, and the positions of the disulfide bonds that can be introduced are well known to those skilled in the art.
  • the TCR is isolated or purified or recombinant.
  • the TCR is human.
  • the TCR is monoclonal.
  • the TCR is single chain.
  • the TCR contains two chains.
  • TCRs can be obtained from biological sources, such as from cells (e.g., from T cells (e.g., cytotoxic T cells)), T cell hybridomas, or other publicly available resources.
  • TCRs can be derived from one of multiple animal species, e.g. Human, mouse, rat or other mammal, such as usually from humans.
  • the TCR may be in a cell-bound form or in a soluble form, with the soluble form being preferred.
  • the soluble form of the TCR refers to a TCR that undergoes mutations in its hydrophobic core region.
  • the mutations in these hydrophobic core regions are preferably mutations that can improve the stability of the soluble TCR of the present application.
  • the amino acid sequence of the alpha chain variable region of the TCR is as shown in SEQ ID NO: 20 and the amino acid sequence of the beta chain variable region is as shown in SEQ ID NO: 21, or the amino acid sequence of the alpha chain variable region
  • the sequence is as SEQ ID NO:22 and the amino acid sequence of the ⁇ chain variable region is as SEQ ID NO:23, or the amino acid sequence of the ⁇ chain variable region is as SEQ ID NO:24 and the amino acid sequence of the ⁇ chain variable region is as SEQ ID NO:25, or the amino acid sequence of the ⁇ chain variable region is SEQ ID NO:26 and the amino acid sequence of the ⁇ chain variable region is SEQ ID NO:27.
  • the T cell receptor (TCR) described in the present application includes an alpha chain containing a variable region and/or a beta chain containing a variable region.
  • the variable region of the alpha chain includes an amino acid sequence such as SEQ ID The complementarity determining region 1 (CDR1) shown in NO:3 or SEQ ID NO:9; and/or the amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO:4 or SEQ ID NO:10.
  • variable region of the ⁇ chain includes the complementarity determining region 1 (CDR1) whose amino acid sequence is as shown in SEQ ID NO:6; and/or the amino acid sequence is the complementarity determining region as shown in SEQ ID NO:7 2(CDR2).
  • CDR1 complementarity determining region 1
  • variable region of the alpha chain comprises a complementarity determining region 3 containing an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15 (CDR3); and/or the variable region of the ⁇ chain includes a complementarity determining region containing an amino acid sequence as shown in SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16 3 (CDR3);
  • the variable region of the ⁇ chain also includes a first leader sequence; and/or the variable region of the ⁇ chain also includes a second leader sequence;
  • the variable region of the ⁇ chain The amino acid sequence of the region is as shown in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26 or is the same as SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 Or SEQ ID NO:26 has an amino acid sequence with at least 90% sequence identity
  • the constant region is a mouse constant region or a human constant region.
  • the TCR is isolated or purified or recombinant; preferably, the TCR is human; preferably, the TCR is monoclonal; preferably, the TCR is single chain ; Preferably, the TCR contains two chains; Preferably, the TCR is in a cell-bound form or in a soluble form, preferably in a soluble form; Preferably, the TCR is complexed with the antigen short peptide-HLAA1101 Preferably, the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the present application also provides a nucleic acid molecule, said nucleic acid molecule comprising encoding the TCR or the ⁇ chain or ⁇ chain of the TCR.
  • the nucleotide sequence encoding the alpha chain comprises a nucleotide sequence set forth 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 ⁇ chain includes the amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37;
  • nucleotide sequence shown in SEQ ID NO:30 is:
  • the nucleotide sequence shown in SEQ ID NO:31 is:
  • the nucleotide sequence shown in SEQ ID NO:32 is:
  • the nucleotide sequence shown in SEQ ID NO:33 is:
  • the nucleotide sequence shown in SEQ ID NO:34 is:
  • the nucleotide sequence shown in SEQ ID NO:35 is:
  • the nucleotide sequence shown in SEQ ID NO:36 is:
  • the nucleotide sequence shown in SEQ ID NO:37 is:
  • the nucleotide sequence encoding the alpha chain and/or the nucleotide sequence encoding the beta chain is codon optimized.
  • codon optimization involves balancing the percentage of selected codons with the abundance of published human transfer RNAs so that none is overloaded or limiting. In some cases, this may be necessary because most amino acids are encoded by more than one codon 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.
  • codons are selected to select those that are balanced with human frequency of use. Generally, the redundancy of amino acid codons is such that different codons code for one amino acid.
  • the resulting mutations when selecting codons for substitutions, it may be desirable that the resulting mutations be silent mutations so that the codon changes do not affect the amino acid sequence.
  • the last nucleotide of a codon can be left unchanged without affecting the amino acid sequence.
  • the present application provides a vector, which contains the nucleic acid molecule described above.
  • one or more nucleic acids encoding one or two chains of the above-mentioned TCR are cloned into one or more suitable expression vectors.
  • the expression vector can be any suitable recombinant expression vector and can be used for transformation or transduction. Infect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses.
  • the vector may contain regulatory sequences (such as transcriptional and translational initiation and termination codons) that are specific for the type of host (e.g., bacterial, fungal, plant or animal) into which the vector is to be introduced, as appropriate and taking into account that the vector is DNA-based Still based on RNA.
  • the vector may also contain a non-natural 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 repeats of murine stem cell virus, also contemplated. Other promoters are known to the skilled artisan.
  • CMV cytomegalovirus
  • the vector is an expression vector, preferably a viral vector, preferably a retroviral vector, and further preferably a lentiviral vector.
  • the present application also provides a host cell containing the nucleic acid molecule described above.
  • the nucleic acid encoding TCR can be isolated and inserted into one or more vectors to generate Further cloning/or expression in host cells.
  • Such nucleic acids can be readily isolated and sequenced using conventional techniques (eg, by using oligonucleotide probes capable of binding specifically to genes encoding the alpha and beta chains of the TCR).
  • methods of preparing a TCR include culturing a host cell comprising a nucleic acid encoding a TCR as provided above under conditions suitable for expression of a TCR molecule, and optionally culturing a host cell (or Host cell culture medium) to recover TCR.
  • the host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include transformants and transformed cells, including primary transformed cells and progeny derived therefrom, regardless of passage number.
  • the offspring may not be identical in nucleic acid content to the parent cells, but may contain mutations.
  • the present application also provides an engineered cell, which contains the T cell receptor (TCR), the above-mentioned nucleic acid molecule or the above-mentioned vector.
  • TCR T cell receptor
  • the TCR is heterologous to the cell.
  • the engineered cells are cell lines.
  • the engineered cells are primary cells obtained from a subject, preferably a mammalian subject, preferably a human.
  • the engineered cells are T cells, preferably T cells isolated from peripheral blood.
  • the T cells are CD8+ or CD4+.
  • the engineered cells may be, for example, a cell population or genetically engineered cells expressing TCR, which cells are usually eukaryotic cells, such as mammalian cells, and are usually human cells.
  • the cells are derived from blood, bone marrow, lymph or lymphoid organs and are cells of the immune system, such as cells of innate immunity 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 and multipotent stem cells, including induced pluripotent stem cells (iPSCs).
  • the cells are typically primary cells, such as those isolated directly from the subject and/or isolated from the subject and frozen.
  • cells include one or more subsets of T cells or other cell types, such as the entire population of T cells, CD+ cells, CD8+ cells, and subpopulations thereof.
  • T cells and/or CD+ and/or CD8+ T cells include naive T (TN) cells, T effector cells (TEFF), memory T cells and their subtypes (such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (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, naturally occurring and adaptive regulatory T (Treg) cells etc.
  • TN naive T
  • TEFF T effector cells
  • TSCM stem cell memory T
  • TCM central memory T
  • TEM effector memory T
  • TIL tumor-infiltrating lymphocytes
  • immature T cells immature T cells
  • mature T cells helper T cells
  • cytotoxic T cells cytotoxic T cells
  • Mucosal-associated invariant T (MAIT) cells naturally occurring and adaptive regulatory T (
  • the engineered cells are natural killer (NK) cells, preferably the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells cells, eosinophils and/or basophils.
  • NK natural killer
  • the present application provides a method for producing the above-mentioned engineered cells, which includes introducing the above-mentioned nucleic acid molecules or the above-mentioned vectors into cells in vitro or ex vivo.
  • the vector is a viral vector and the introduction is by transduction.
  • the present application provides a pharmaceutical composition, which contains the above-mentioned T cell receptor (TCR), the above-mentioned nucleic acid molecule, the above-mentioned vector or the above-mentioned engineered cell. In one embodiment, it further comprises a pharmaceutically acceptable carrier or adjuvant.
  • TCR T cell receptor
  • nucleic acid molecule the above-mentioned nucleic acid molecule
  • vector the above-mentioned vector or the above-mentioned engineered cell.
  • it further comprises a pharmaceutically acceptable carrier or adjuvant.
  • the pharmaceutically acceptable carrier or adjuvant refers to the ingredients in the pharmaceutical composition that are non-toxic to the subject except the active ingredients.
  • Pharmaceutically acceptable carriers or adjuvants include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • the pharmaceutical compositions may utilize timed release, delayed release and sustained release delivery systems such that delivery of the composition occurs prior to sensitization of the site to be treated and allows sufficient time to cause sensitization.
  • Many types of release delivery systems are available and known. Such systems may be used to avoid repeated administration of the composition, thereby increasing subject and physician convenience.
  • TCR T cell receptor
  • nucleic acid molecule the above-mentioned nucleic acid molecule
  • vector the above-mentioned vector
  • engineered cell the above-mentioned pharmaceutical composition
  • the disease associated with HPV is HPV chronic infection, cervical intraepithelial neoplasia, cervical cancer, head and neck cancer, anal cancer, penile cancer, vaginal cancer or vulvar cancer, etc.
  • the present application provides a method for treating HPV-related diseases, including using 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 pharmaceutical composition described above is administered to a subject in need.
  • 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.
  • the TCR described in this application can specifically bind to the antigen short peptide of HPV-positive tumor cells, and the T cells transduced by the TCR can be specifically activated and have a strong killing effect on target cells.
  • the TCR can For immunotherapy of HPV-positive tumors such as cervical cancer.
  • this application can screen out specific T cell receptors (TCR), and the T cells after transcribing the TCR can be specifically activated and have strong killing effect on tumor cells expressing A1101 and HPV. It can be used for immunotherapy of HPV-positive tumors such as cervical cancer.
  • TCR T cell receptors
  • the T cells transduced with the TCR in this application have a strong activation response to cell lines that express E6, but have no activation response to cell lines that do not express E6, and have a strong killing function against cell lines that express E6, and Can effectively inhibit the growth of E6-positive tumors.
  • the T cells transduced with the TCR in this application can significantly kill primary cervical cancer organoid cells and can effectively inhibit the growth of E6-positive tumors.
  • % means wt%, that is, weight percentage. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional reagent products that can be purchased commercially.
  • TTLEQQYNK SEQ ID NO: 1, Jiangsu GenScript Biotechnology Co., Ltd.
  • GTTLEQQYNK SEQ ID NO: 2, Jiangsu GenScript Biotechnology Co., Ltd.
  • TTLEQQYNK short peptide and GTTLEQQYNK short peptide were renatured with biotin-labeled HLA-A*11:01 respectively to prepare pHLA monomer.
  • pHLA monomers and tetramers were combined with PE-labeled streptavidin (BD Company, catalog number 554061) to form PE-labeled tetramers, and the tetramers and CD8 double-positive cells were detected by flow cytometry.
  • pHLA monomers and tetramers were The polymer production method refers to the protocol published by the NIH Tetramer Core Facility. The specific steps can be found on the website https://tetramer.yerkes.emory.edu/support/protocols#1. The specific operations are as follows:
  • Test media 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)
  • PBMC peripheral blood of healthy volunteers by density gradient centrifugation, and CD14-positive cells were separated from the PBMC for inducing dendritic cells (DC).
  • CD14-positive cells were cultured in culture medium (1640+10% AuFBS+1% PS+800IU/ml GM-CSF+500IU/ml IL-4) until the third day when 1 ml of culture medium (containing 1600IU/ml GM-CSF+ 1000IU/ml IL-4), add DC maturation inducing factors 10ng/ml IL-1 ⁇ , 10ng/ml IL-6, 10ng/ml TNF- ⁇ , 1ug/ml PGE2 on the fifth day, and continue to culture for two days to obtain Mature DC.
  • TTLEQQYNK and GTTLEQQYNK antigen short peptide-specific T cells were cloned by the method described above, and the cloned antigen short peptide-specific T cells were analyzed by flow cytometry.
  • the T cell flow cytometry results after two and three rounds of stimulation with TTLEQQYNK and GTTLEQQYNK antigen short peptides are shown in Figure 1.
  • Figure 1A is a schematic diagram of the T cell flow cytometry results after two rounds of stimulation.
  • Figure 1B is a schematic diagram of the T cell flow cytometry results after two rounds of stimulation. Schematic diagram of T cell flow cytometry results after three rounds of stimulation.
  • T cells specific for TTLEQQYNK and GTTLEQQYNK antigen short peptides have been successfully cloned.
  • the partial TCR sequence obtained by TTLEQQYNK is detailed in Example 2 below.
  • Example 2 TTLEQQYNK antigen short peptide-specific TCR lentiviral vector construction and lentiviral packaging
  • the CD8 and tetramer double-positive cells in Example 1 were flow sorted to obtain single cells, and the single cells obtained by sorting were amplified using a one-step RT-PCR kit (QIAGEN, catalog number 210212). TCR ⁇ chain and ⁇ chain, and sequence the PCR products. Compare the sequencing results with IMGT (International By comparing the sequences in the public database of the Immunogenetic Information System, you can obtain the TCR alpha chain variable region sequence and the nucleotide sequence of the beta chain variable region and the information of CDR1, CDR2, and CDR3, and get 4 TCRs.
  • IMGT International By comparing the sequences in the public database of the Immunogenetic Information System, you can obtain the TCR alpha chain variable region sequence and the nucleotide sequence of the beta chain variable region and the information of CDR1, CDR2, and CDR3, and get 4 TCRs.
  • nucleotide sequence of the ⁇ chain variable region sequence of TCR010 is shown in SEQ ID NO: 39, and its nucleotide sequence is as follows:
  • nucleotide sequence of the ⁇ chain variable region is shown in SEQ ID NO:40, and its nucleotide sequence is as follows:
  • CDR1 complementarity determining region 1
  • CDR2 complementarity determining region 2
  • CDR3 complementarity determining region 3
  • CDR1 complementarity determining region 1
  • CDR2 complementarity determining region 2
  • CDR3 complementarity determining region 3
  • the nucleotide sequence of the alpha chain variable region sequence of TCR013 is shown in SEQ ID NO: 41,
  • nucleotide sequence of the ⁇ chain variable region is shown in SEQ ID NO:42:
  • CDR1 complementarity determining region 1
  • CDR2 complementarity determining region 2
  • CDR3 complementarity determining region 3
  • CDR1 complementarity determining region 1
  • CDR2 complementarity determining region 2
  • CDR3 complementarity determining region 3
  • the nucleotide sequence of the alpha chain variable region sequence of TCR028 is shown in SEQ ID NO: 43,
  • nucleotide sequence of the ⁇ chain variable region is shown in SEQ ID NO:44:
  • CDR1 complementarity determining region 1
  • CDR2 complementarity determining region 2
  • CDR3 complementarity determining region 3
  • CDR1 complementarity determining region 1
  • CDR2 complementarity determining region 2
  • CDR3 complementarity determining region 3
  • the nucleotide sequence of the alpha chain variable region sequence of TCR090 is shown in SEQ ID NO: 45,
  • nucleotide sequence of the ⁇ chain variable region is shown in SEQ ID NO:46:
  • CDR1 complementarity determining region 1
  • CDR2 complementarity determining region 2
  • CDR3 complementarity determining region 3
  • CDR1 complementarity determining region 1
  • CDR2 complementarity determining region 2
  • CDR3 complementarity determining region 3
  • the TTLEQQYNK TCR ⁇ and ⁇ chain variable region sequences were cloned into a pLKO-based expression plasmid (Addgene), and the ⁇ or ⁇ variable domain was cloned using the standard method of a multi-fragment recombinant cloning kit (Novezan Biotechnology, Cat. No. C113). Cloned into PLKO-based expression plasmids containing murine ⁇ or ⁇ constant regions, the ligated plasmids were transformed into competent E. coli strain Stbl3 cells (Shanghai Vidy Biotechnology Co., Ltd.) and inoculated into cells containing 100 ⁇ g/ml ampicillin. on LB/agar plates.
  • TTLEQQYNK TCR i.e., TCR010, TCR013, TCR028, TCR090.
  • Test medium 10% FBS (Lonsera, catalog number S711-001), DMEM (cytiva, catalog number SH30243.01)
  • NFAT Nuclear factor of activated T cells
  • Test media 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (ThermoFisher, catalog number C11875500BT)
  • the target cells used in this experiment were T2-A11 cells (T2 cells are deposited at ATCC, The registration number is CRL-1992, and T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032).
  • Prepare target cells in the experimental culture medium adjust the target cell concentration to 1.6 ⁇ 10 6 cells/ml, and take 50 ⁇ l from each well to obtain 80,000 cells/well.
  • JK8NF Jurkat-CD8-NFAT
  • JK8NF cells Jurkat cells
  • JK8NF cells were deposited at ATCC under the catalog 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.
  • MOI multipleplicity of infection
  • NFAT The expression of NFAT in T cells transduced by the TCR of the present application in response to target cells loaded with TTLEQQYNK antigen short peptide was examined by the method described above.
  • Graphpad prism8 was used to draw the expression level curve of NFAT, and the results are shown in Figure 3.
  • Test media 10% FBS (ThermoFisher, catalog number 10099-044), RPMI1640 (ThermoFisher, catalog number C11875500BT)
  • the effector cells (T cells) in this experiment are T cells transduced with the TCR of the application, and the T cells of the same volunteer that have not been transfected with the TCR of the application are used as a control group.
  • peripheral blood mononuclear cells The volunteers' peripheral blood was subjected to density gradient centrifugation to obtain peripheral blood mononuclear cells.
  • the peripheral blood mononuclear cells were placed in the wells of a 24-well plate at 5.0 ⁇ 10 5 /500 ⁇ l per well.
  • a total of 1x10 6 cells were collected and treated with anti-CD3 /CD28 magnetic beads stimulated T cells and then cultured in a 37°C, 5% CO 2 incubator. Observe the cell clumps after 24 hours.
  • Add the TCR lentivirus obtained in Example 2 at MOI (multiplicity of infection) 2 for transduction.
  • the target cell used in this experiment is SK-MEL-28-E6.
  • the genotype of SK-MEL-28 (deposited at ATCC) is A*11:01, but it does not express E6 itself, so E6 is overexpressed through a lentiviral vector.
  • Genetically constructed SK-MEL-28-E6 cells were used as target cells.
  • Target cells will be prepared in the experimental culture medium, the target cell concentration is adjusted to 5.0 ⁇ 10 4 cells/ml, and 100 ⁇ l is taken from each well to obtain 5000 cells/well.
  • RTCA x Celligence system monitors target cell proliferation
  • T cells transduced by the TCR of the present application were tested by real-time monitoring of target cell proliferation experiments (as described above).
  • Use Graphpad prism8 to plot the cell index of cell proliferation in each well (a parameter generated by the RTCA x Celligence system to characterize the number of cells).
  • the experimental results are shown in Figure 5.
  • T cells expressing the TCR specific for the TTLEQQYNK-A*11:01 antigen short peptide have a strong killing function against the cell line expressing E6.
  • Example 5 Primary T cells expressing TCR specific for TTLEQQYNK antigen short peptide in vivo Identification of tumor growth inhibition function
  • TCR-transduced T cells The tumor volume changes were observed to evaluate the tumor-killing function of T cells. In vitro experiments showed that the four TCRs had similar tumor-killing effects. Therefore, only TCR013 and TCR028 were selected as representatives to identify the tumor growth-inhibiting function in vivo.
  • Test media 10% FBS (ThermoFisher, catalog number 10099-044), RPMI1640 (ThermoFisher, catalog number C11875500BT)
  • NCG mice purchased from Jicui Yaokang Company, female, 4-6 weeks old, genotypes are (prkdc)ko/ko, (Il2rg female)ko/ko.
  • the tumor cells used in this experiment were SK-MEL-28-E6. Tumor cells were prepared in physiological saline, and the tumor cell concentration was adjusted to 5.0 ⁇ 10 7 cells/ml. Each mouse was subcutaneously inoculated with 200 ⁇ l to obtain 1.0 ⁇ 10 7 cells/mouse. The tumor volume grew to approximately 100 cubic meters. mm, seeded with effector T cells.
  • the effector cells (T cells) in this experiment are T cells transduced with the TCR of the application, and the T cells of the same volunteer that have not been transfected with the TCR of the application are used as a control group. 5 mice per group.
  • peripheral blood mononuclear cells The volunteers' peripheral blood was subjected to density gradient centrifugation to obtain peripheral blood mononuclear cells.
  • the peripheral blood mononuclear cells were placed in the wells of a 24-well plate at 5.0 ⁇ 10 5 /500 ⁇ l per well.
  • a total of 4x10 6 cells were collected and treated with anti-CD3 /CD28 magnetic beads stimulated T cells and then cultured in a 37°C, 5% CO 2 incubator. After 24 hours, observe the cell clumps.
  • TCR genes TCR013 and TCR028 genes
  • MOI multiplicity of infection
  • 1640 medium with % FBS was expanded until 3-4 days after transduction, and TCR transfection efficiency was determined by flow cytometry.
  • the concentration of effector cells after expanded culture was adjusted to 5.0 ⁇ 10 7 positive cells/ml, and each mouse was injected with a dose of 200 ⁇ l through the eye vein to obtain 1.0 ⁇ 10 7 positive cells/mouse.
  • Figure 6A is a schematic diagram of the change in tumor volume over time after inoculation of T cells.
  • Figure 6B is a schematic diagram of the tumor volume after inoculation of T cells containing different TCRs. Histogram of tumor weight.
  • T cells expressing TCR specific for TTLEQQYNK-A*11:01 antigen short peptide can effectively inhibit the growth of E6-positive tumors.
  • Test medium OrganoPro cervical cancer organoid culture medium kit (Ketu Medical, catalog number K2O-M-CC)
  • Figures 7A to 7B Observe the growth status of organoids through a microscope, take pictures of typical visual fields, and count the number of complete and incomplete organoids in the visual field. The results are shown in Figures 7A to 7B, where Figure 7A is the organoids observed through a microscope. Schematic diagram of the growth status of organs. Figure 7B is a schematic diagram of statistics on the number of organoids with complete and incomplete forms.
  • T cells expressing TCR specific for TTLEQQYNK-A*11:01 antigen short peptide can significantly kill primary cervical cancer organoid cells.
  • the T cells conveying the TCR in this application can be specifically activated and have a strong killing effect on tumor cells expressing A1101 and HPV.
  • the T cells have a strong killing function against E6-expressing cell lines and can effectively inhibit the growth of E6-positive tumors.

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Abstract

本申请公开了抗原短肽用于筛选治疗与HPV相关的疾病的药物中的用途及其筛选的TCR，所述抗原短肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示，本申请使用抗原短肽，能够筛选出特异性T细胞受体（TCR），并且经过转录所述TCR之后的T细胞能够被特异性激活并且对表达A1101和HPV的肿瘤细胞具有很强的杀伤作用，其可以用于宫颈癌等HPV阳性的肿瘤的免疫治疗，并且转导了本申请所述TCR的T细胞对表达E6的细胞系有很强的激活反应，对不表达E6的细胞系没有激活反应，并且对表达E6的细胞系有很强的杀伤功能，并能够有效抑制E6阳性肿瘤的生长。

Description

抗原短肽用于筛选治疗与HPV相关的疾病的药物中的用途及其筛选的TCR
相关申请的交叉引用
本申请要求对2022年5月10日提交的中国专利申请号CN202210503002.7的优先权，其公开内容通过引用整体并入本文。
电子序列表的引用
电子序列表的内容(TFG00782PCT-XLB.xml；大小：52,426字节；创建日期：2023年5月6日)通过引用整体并入本文。
技术领域
本申请涉及医药技术领域，尤其涉及一种抗原短肽用于筛选治疗与HPV相关的疾病的药物中的用途及其筛选的TCR。
背景技术
人类乳头瘤病毒(Human Papillomavirus，HPV)是一种DNA病毒，属于乳头瘤病毒科乳头瘤病毒属。该类病毒感染人体的表皮与黏膜组织，目前约有170种类型的HPV被判别出来，有些时候HPV入侵人体后会引起疣甚至癌症，但大多数时候则没有任何临床症状。
HPV导致癌症的一个重要因素是高危型HPV的持续感染，如HPV16、HPV18等。由高风险HPV编码的E6和E7蛋白可以分别抑制抑癌基因p53和Rb蛋白的活性，从而导致细胞周期异常，发生癌变。高危型HPV与90％的宫颈癌和肛门癌，40％至60％的阴道癌和阴茎癌有关，同时根据地理特性，还可能与60％的口咽癌相关。
宫颈癌是最常见的女性生殖道恶性肿瘤，全球发病率在女性恶性肿瘤中居第二位，且发展中国家的宫颈癌发病率显著高于高达国家。根据2018年最新统计，中国宫颈癌发病率为15.30/10万，死亡率为4.57/10万，高于全球平均发病率(10.61/10万)和死亡率(2.98/10万)。近10年来，宫颈癌发病率呈上升趋势，40-60岁为发病高峰年龄。尽管对于早期宫颈癌，手术和 放化疗治疗效果较好，但是一半以上的患者初次就诊即被诊断为局部晚期宫颈癌。30％-70％的局部晚期宫颈癌患者会出现复发及(或)远处转移，而对于局部进展期和转移性宫颈癌，传统治疗的疗效十分有限，其五年生存率仅为57.1％和17.3％。
特异性T细胞免疫治疗是指利用针对肿瘤抗原的特异性T细胞来杀伤肿瘤细胞的方法，是一种高度个性化的肿瘤免疫治疗方法。由于肿瘤局部免疫抑制微环境的存在，病人体内的自身T细胞杀伤肿瘤的功能有限。因此，人们试图通过对T细胞进行基因改造的方法提高其杀伤肿瘤的能力。TCR-T和CAR-T均为基因修饰过的细胞治疗药物，通过转入的T细胞受体(T cell receptor,TCR)或嵌合抗原受体(CAR)基因与相应靶点结合后，即可激活T细胞，利用T细胞释放的颗粒酶、穿孔素、细胞因子等清除肿瘤细胞；但TCR-T和CAR-T的显著的不同点在于：CAR-T的靶点是细胞表面的膜蛋白，而TCR-T的靶点则是抗原短肽-MHC复合物(peptide-major histocompatibility complex,pMHC)。
HPV相关蛋白是宫颈癌最理想的T细胞免疫治疗靶点。TCR识别的靶点是“抗原短肽-MHC分子复合物”。TCR同时具有MHC限制性，理论上一个TCR分子仅能够特异性识别某一特定MHC递呈的短肽。MHC具有多态性，目前已发现的人类MHC(又称为人类白细胞抗原，human leukocyte antigen,HLA)等位基因数量超过15000个，且特定HLA在不同人群中的出现频率有很大差异，中国人群中最常见的HLA类型为HLA-A1101。HLA I类分子呈递的短肽长度为8-11个氨基酸，HLA II类分子呈递的短肽长度为12-24个氨基酸，这些抗原短肽的发现及鉴定是TCR-T治疗的前提。尽管短肽是否与HLA结合可以通过亲和力预测、HLA结合力测定等获得，但是该短肽能否被表达HPV的肿瘤细胞天然递呈，才是决定该短肽特异性的TCR能否用于肿瘤治疗的关键。
因此，本领域技术人员致力于寻找A1101限制性HPV抗原短肽，以及将发现的抗原短肽用于筛选可以特异性识别HPV阳性肿瘤细胞的TCR，从而使他们在T细胞免疫治疗中发挥作用。
发明内容
本申请的目的在于提供一种抗原短肽用于筛选治疗与HPV相关的疾病的药物中的用途以及所筛选的T细胞受体(TCR)，所述抗原短肽筛选特异性T细胞抗体，转录了所述TCR的T细胞能够被特异性激活并且对表达A1101和HPV的肿瘤细胞具有很强的杀伤作用。
本申请具体技术方案如下：
1.抗原短肽用于筛选治疗与HPV相关的疾病的药物中的用途，所述抗原短肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
2.根据项1所述的用途，其中，所述与HPV相关的疾病为HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌。
3.抗原短肽用于筛选治疗HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌的药物中的用途，所述抗原短肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
4.根据项1-3中任一项所述的用途，其中，所述药物为用于与包含所述抗原短肽的抗原短肽-HLA-A1101复合物结合的T细胞受体(TCR)。
5.一种T细胞受体(TCR)，其中，所述TCR包含含有可变区的α链和/或含有可变区的β链，α链的可变区包含氨基酸序列为如SEQ ID NO:3或SEQ ID NO:9所示的互补决定区1(CDR1)；和/或
氨基酸序列为如SEQ ID NO:4或SEQ ID NO:10所示的互补决定区2(CDR2)。
6.根据项5所述的T细胞受体(TCR)，其中，所述β链的可变区包含氨基酸序列为如SEQ ID NO:6所示的互补决定区1(CDR1)；和/或
氨基酸序列为如SEQ ID NO:7所示的互补决定区2(CDR2)。
7.根据项5或6所述的T细胞受体(TCR)，其中，所述α链的可变区包含含有氨基酸序列为如SEQ ID NO:5、SEQ ID NO:11、SEQ ID NO:13或SEQ ID NO:15所示的互补决定区3(CDR3)；和/或
所述β链的可变区包含含有氨基酸序列为如SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:14或SEQ ID NO:16所示的互补决定区3(CDR3)。
8.根据项5-7中任一项所述的T细胞受体(TCR)，所述α链的可变区还包括第一先导序列；和/或
所述β链的可变区还包括第二先导序列。
9.根据项5-8中任一项所述的T细胞受体(TCR)，其中，所述α链可变区的氨基酸序列为如SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24或SEQ ID NO:26所示或者与SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24或SEQ ID NO:26具有至少90％序列相同性的氨基酸序列；和/或
所述β链的可变区的氨基酸序列如SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25或SEQ ID NO:27所示或与SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25或SEQ ID NO:27具有至少90％序列相同性的氨基酸序列。
10.根据项5-9中任一项所述的T细胞受体(TCR)，其中，所述α链还包含α恒定区和/或所述β链还包含β恒定区，优选地，所述恒定区为小鼠恒定区或人恒定区。
11.根据项5-10中任一项所述的T细胞受体(TCR)，其中，所述TCR是分离的或纯化的或者是重组的。
12.根据项5-11中任一项所述的T细胞受体(TCR)，其中，所述TCR是人的。
13.根据项5-12中任一项所述的T细胞受体(TCR)，其中，所述TCR是单克隆的。
14.根据项5-13中任一项所述的T细胞受体(TCR)，其中，所述TCR是单链。
15.根据项5-14中任一项所述的T细胞受体(TCR)，其中，所述TCR包含两条链。
16.根据项5-15中任一项所述的T细胞受体(TCR)，其中，所述TCR为细胞结合的形式或为可溶的形式，优选为可溶的形式。
17.根据项5-16中任一项所述的T细胞受体(TCR)，其中，所述TCR与抗原短肽-HLAA1101复合物结合，优选地，所述抗原短肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
18.一种核酸分子，其中，所述核酸分子包含编码项5-17中任一项所述的TCR或者所述TCR的α链或β链。
19.根据项18所述的核酸分子，其中编码α链的核苷酸序列包含如SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34或SEQ ID NO:36所示的核苷酸序列；和/或
编码β链的核苷酸序列包含如SEQ ID NO:31、SEQ ID NO:33、SEQ ID  NO:35或SEQ ID NO:37所示的核苷酸序列。
20.一种载体，其中，所述载体包含项18或19所述的核酸分子。
21.根据项20所述的载体，其中，所述载体为表达载体。
22.根据项20或21所述的载体，其中，所述载体为病毒载体，优选为逆转录病毒载体。
23.根据项22所述的载体，其中，所述病毒载体为慢病毒载体。
24.一种工程化细胞，其包含项5-17中任一项所述的TCR、项18-19中任一项所述的核酸分子或者项20-23中任一项所述的载体。
25.根据项24所述的工程化细胞，其中，所述TCR对所述细胞是异源的。
26.根据项24或25所述的工程化细胞，其中，所述工程化细胞是细胞系。
27.根据项24-26中任一项所述的工程化细胞，其中，所述工程化细胞是获自受试者的原代细胞，优选地，所述受试者为哺乳动物受试者，优选为人。
28.根据项24-27中任一项所述的工程化细胞，其中，所述工程化细胞是T细胞或NK细胞，优选地，所述T细胞是从外周血分离的T细胞。
29.根据项28所述的工程化细胞，其中，所述T细胞为CD8+或CD4+。
30.一种生产项24-29中任一项所述的工程化细胞的方法，其包括在体外或离体地将项18-19中任一项所述的核酸分子或者项20-23任一项所述的载体引入细胞中。
31.根据项30所述的方法，其中，所述载体为病毒载体，并且所述引入是通过转导进行的。
32.一种药物组合物，其包含项5-17中任一项所述的T细胞受体(TCR)、项18-19中任一项所述的核酸分子、项20-23中任一项所述的载体或者项24-29中任一项所述的工程化细胞。
33.根据项32所述的药物组合物，其还包含药学上可接受的载体或佐剂。
34.项5-17中任一项所述的T细胞受体(TCR)、项18-19中任一项所述的核酸分子、项20-23中任一项所述的载体或者项24-29中任一项所述的工程化细胞或者项32-33中任一项所述的药物组合物在制备治疗与HPV相关的疾病的药物中的用途。
35.根据项34所述的用途，其中，与HPV相关的疾病为HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌。
36.一种治疗与HPV相关的疾病的方法，包括将项5-17中任一项所述的T细胞受体(TCR)、项18-19中任一项所述的核酸分子、项20-23中任一项所述的载体或者项24-29中任一项所述的工程化细胞或者项32-33中任一项所述的药物组合物施用于有需要的受试者。
37.根据项36所述的方法，其中，所述与HPV相关的疾病为HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌。
发明的效果
本申请所述的抗原短肽能够筛选特异性的T细胞受体(TCR)，同时转导了所述TCR的T细胞能够被特异性激活并且对表达A1101和HPV的肿瘤细胞具有很强的杀伤作用，可以用于宫颈癌等HPV阳性的肿瘤的免疫治疗。
并且转导了本申请所述TCR的T细胞对表达E6的细胞系有很强的激活反应，对不表达E6的细胞系没有激活反应，并且对表达E6的细胞系有很强的杀伤功能，并能够有效抑制E6阳性肿瘤的生长。
附图说明
图1是实施例1中克隆TTLEQQYNK和GTTLEQQYNK抗原短肽特异性T细胞的示意图，其中，图1A是经过二轮刺激后的T细胞流式检测结果示意图，图1B是经过三轮刺激后的T细胞流式检测结果示意图。
图2是实施例3中使用流式细胞仪鉴定转染阳性率的示意图。
图3是实施例3中转导本申请所述的TCR的T细胞对负载TTLEQQYNK抗原短肽的靶细胞起反应的NFAT的表达的示意图。
图4是实施例4中使用流式细胞仪鉴定转染阳性率的示意图。
图5是实施例4中转导本申请所述的TCR的T细胞的细胞增殖的细胞指数的示意图。
图6是实施例5中接种转导本申请所述的TCR的T细胞后肿瘤体积的示意图，其中，图6A是接种T细胞后肿瘤体积随时间变化的示意图，图6B是接种含有不同TCR的T细胞后肿瘤重量的柱状图。
图7A至图7B是观察的类器官的生长状态以及类器官的完整性和不完整性的统计示意图，其中，图7A是通过显微镜观察到的类器官的生长状态示意图，图7B是对形态完整和形态不完整的类器官的数量进行统计的示意图。
具体实施方式
下面结合附图所描述的实施方式对本申请做以详细说明，其中所有附图中相同的数字表示相同的特征。虽然附图中显示了本申请的具体实施例，然而应当理解，可以以各种形式实现本申请而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本申请，并且能够将本申请的范围完整的传达给本领域的技术人员。
需要说明的是，在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解，技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异作为区分组件的方式，而是以组件在功能上的差异作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本申请的较佳实施方式，然而所述描述乃以说明书的一般原则为目的，并非用以限定本申请的范围。本申请的保护范围当视所附权利要求所界定者为准。
本申请提供了抗原短肽用于筛选治疗与HPV相关的疾病的药物中的用途，所述抗原短肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
SEQ ID NO:1的序列如下：TTLEQQYNK
SEQ ID NO:2的序列如下：GTTLEQQYNK。
在一个实施方案中，所述与HPV相关的疾病为HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌等。
本申请提供了抗原短肽用于筛选治疗HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌的药物中的用途，所述抗原短肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
在一个实施方案中，所述药物为用于与包含所述抗原短肽的抗原短肽-HLA-A1101复合物结合的T细胞受体(TCR)。
即所述T细胞受体是通过所述抗原短肽的筛选得到，所述T细胞受体 (TCR)与TTLEQQYNK-HLA-A1101复合物结合或者与GTTLEQQYNK-HLA-A1101复合物结合。
所述T细胞受体或TCR是呈递在主组织相容性复合体(MHC)上的特异性抗原肽的唯一受体，在免疫系统中，通过抗原特异性的TCR与pMHC复合物的结合引发T细胞与抗原呈递细胞(APC)直接的物理接触，然后T细胞及APC两者的其他细胞表面分子就发生相互作用，这就引起了一系列后续的细胞信号传递和其他生理反应，从而使得不同抗原特异性的T细胞对靶细胞发挥免疫效应
所述TCR是含有可变α和β链或可变γ和δ链的分子，并且所述分子能够结合至MHC分子的肽特异性结合，在一些实施方案中，TCR呈αβ形式。通常，以αβ和γδ形式存在的TCR在结构上总体上相似，但是表达它们的T细胞可以具有不同的解剖学位置或功能，TCR可以在细胞表面上发现或以可溶形式发现。通常，在T细胞(T淋巴细胞)的表面上发现TCR，在此处它通常负责识别结合至主要组织相容性复合物(MHC)分子的抗原。
TCR的可变结构域含有互补决定区(CDR)，其通常是肽、MHC和/或MHC-肽复合物的抗原识别以及结合能力和特异性的主要贡献者，TCR的CDR或其组合形成给定TCR分子的全部或基本上全部的抗原结合位点，TCR的可变区内的各个CDR通常由框架区(FR)隔开。其中，CDR3是负责抗原结合或特异性的主要CDR，或者在给定TCR可变区上在三个CDR中对于抗原识别和/或对于与肽-MHC复合物的经加工肽部分的相互作用最重要，在一些情况下，α链的CDR1可以与某些抗原肽的N末端部分相互作用；在一些情况下，β链的CDR1可以与某些抗原肽的C末端部分相互作用；在一些情况下，CDR2对于MHC-肽复合物的MHC部分的相互作用或识别具有最强的作用或者是主要的负责CDR；在一些情况下，β链的可变区抗原含有其他高变区(CDR4或HVR4)，其通常参与超抗原结合而非抗原识别。
所述TTLEQQYNK-HLA-A1101复合物或者GTTLEQQYNK-HLA-A1101复合物是指HLA-A1101和抗原短肽TTLEQQYNK或GTTLEQQYNK结合的复合物，蛋白质在细胞中被蛋白酶体降解为不同长度的多肽，一部分多肽与HLA结合形成复合物被递呈至细胞表面。所述TCR识别的TTLEQQYNK-HLA-A1101复合物或者GTTLEQQYNK-HLA-A1101复合物可以表达于细胞膜上，也可以以可溶性 蛋白的形式存在于溶液中。
所述HLA-A1101的氨基酸序列如SEQ ID NO:38所示，其氨基酸序列为：
在一个实施方案中，抗原短肽用于筛选治疗与HPV相关的疾病的药物中的用途，所述抗原短肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。在一个实施方案中，所述与HPV相关的疾病为HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌。在一个实施方案中，所述药物为用于与包含所述抗原短肽的抗原短肽-HLA-A1101复合物结合的T细胞受体(TCR)。
在一个实施方案中，抗原短肽用于筛选治疗HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌的药物中的用途，所述抗原短肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。在一个实施方案中，所述药物为用于与包含所述抗原短肽的抗原短肽-HLA-A1101复合物结合的T细胞受体(TCR)。
本申请提供了一种T细胞受体(TCR)，其中，所述TCR包含含有可变区的α链和/或含有可变区的β链，α链的可变区包含氨基酸序列为如SEQ ID NO:3或SEQ ID NO:9所示的互补决定区1(CDR1)；和/或
氨基酸序列为如SEQ ID NO:4或SEQ ID NO:10所示的互补决定区2(CDR2)。
SEQ ID NO:3所示的氨基酸序列为：SSYSPS
SEQ ID NO:9所示的氨基酸序列为：TTLSN
SEQ ID NO:4所示的氨基酸序列为：YTSAATLV
SEQ ID NO:10所示的氨基酸序列为：LVKSGEV
在一个实施方案中，所述β链的可变区包含氨基酸序列为如SEQ ID NO:6所示的互补决定区1(CDR1)；和/或
氨基酸序列为如SEQ ID NO:7所示的互补决定区2(CDR2)。
SEQ ID NO:6所示的氨基酸序列为：SGDLS
SEQ ID NO:7所示的氨基酸序列为：YYNGEE
在一个实施方案中，所述α链的可变区包含含有氨基酸序列为如SEQ ID NO:5、SEQ ID NO:11、SEQ ID NO:13或SEQ ID NO:15所示的互补决定区3 (CDR3)；和/或
所述β链的可变区包含含有氨基酸序列为如SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:14或SEQ ID NO:16所示的互补决定区3(CDR3)。
SEQ ID NO:5所示的氨基酸序列为：VVSLSGGYNKLI
SEQ ID NO:11所示的氨基酸序列为：AGPKITGGGNKLT
SEQ ID NO:13所示的氨基酸序列为：AGPILTGGGNKLT
SEQ ID NO:15所示的氨基酸序列为：AGPVLTGGGNKLT
SEQ ID NO:8所示的氨基酸序列为：ASSVTGSGYT
SEQ ID NO:12所示的氨基酸序列为：ASSVGGGPNYGYT
SEQ ID NO:14所示的氨基酸序列为：ASGLSGPNTGELF
SEQ ID NO:16示的氨基酸序列为：ASSVGGPNTGELF。
在一个实施方案中，所述α链的可变区还包括第一先导序列；和/或
所述β链的可变区还包括第二先导序列。
所述α链的可变区的第一先导序列和所述β链的可变区的第二先导序列是本领域技术人员公知的，例如所述α链的可变区的第一先导序列可以使用氨基酸序列如SEQ ID NO:17或SEQ ID NO:19所示的先导序列，所述β链的可变区的第二先导序列可以使用氨基酸序列如SEQ ID NO:18所示的先导序列。
SEQ ID NO:17所示的氨基酸序列为：MLLLLVPVLEVIFTLGGTR
SEQ ID NO:19所示的氨基酸序列为：MLLITSMLVLWMQLSQVN
SEQ ID NO:18所示的氨基酸序列为：MGFRLLCCVAFCLLGAGPV
在一个实施方案中，所述α链可变区的氨基酸序列为如SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24或SEQ ID NO:26所示或者与SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24或SEQ ID NO:26具有至少90％序列相同性的氨基酸序列；和/或
所述β链的可变区的氨基酸序列如SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25或SEQ ID NO:27所示或与SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25或SEQ ID NO:27具有至少90％序列相同性的氨基酸序列。
SEQ ID NO:20所示的氨基酸序列为：
SEQ ID NO:21所示的氨基酸序列为：

SEQ ID NO:22所示的氨基酸序列为：
SEQ ID NO:23所示的氨基酸序列为：
SEQ ID NO:24所示的氨基酸序列为：
SEQ ID NO:25所示的氨基酸序列为：
SEQ ID NO:26所示的氨基酸序列为：
SEQ ID NO:27所示的氨基酸序列为：
所述α链可变区的氨基酸序列与SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24或SEQ ID NO:26具有至少90％序列相同性的氨基酸序列如可以是与SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24或SEQ ID NO:26具有90％、91％、92％、93％、94％、95％、96％、97％、98％序列相同性的氨基酸序列；
所述β链的可变区的氨基酸序列与SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25或SEQ ID NO:27具有至少90％序列相同性的氨基酸序列可以是与SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25或SEQ ID NO:27具有90％、91％、92％、93％、94％、95％、96％、97％、98％序列相同性的氨基酸序列。
在一个实施方案中，所述α链还包含α恒定区和/或所述β链还包含β恒定区，优选地，所述恒定区为小鼠恒定区或人恒定区。
例如，小鼠α恒定区的氨基酸序列如SEQ ID NO:28所示；和/或
小鼠β恒定区的氨基酸序列如SEQ ID NO:29，即对于上述所述的TCR的α链的恒定区，可以均具有相同的恒定区，同理，所有TCR的β链的恒定区也可以均具有相同的恒定区。
SEQ ID NO:28所示的氨基酸为：
SEQ ID NO:29所示的氨基酸为：
所述TCR的恒定区可以含有短连接序列，其中半胱氨酸残基形成二硫键，从而连接TCR的两条链。TCR可以在α和β链中的每一个中具有另外 的半胱氨酸残基，使得TCR在恒定区中含有两个二硫键。
在一个实施方案中，所述TCR的α链可变区的氨基酸序列如SEQ ID NO:20且β链可变区的氨基酸序列如SEQ ID NO:21所示，或者α链可变区的氨基酸序列如SEQ ID NO:22且β链可变区的氨基酸序列如SEQ ID NO:23所示，或者α链可变区的氨基酸序列如SEQ ID NO:24且β链可变区的氨基酸序列如SEQ ID NO:25所示，或者α链可变区的氨基酸序列如SEQ ID NO:26且β链可变区的氨基酸序列如SEQ ID NO:27所示。
在一个实施方案中，所述TCR的α链和β链恒定区的残基之间引入人工二硫键，可以引入的二硫键的位置是本领域技术人员公知的。
在一个实施方案中，所述TCR是分离的或纯化的或者是重组的。
在一个实施方案中，所述TCR是人的。
在一个实施方案中，所述TCR是单克隆的。
在一个实施方案中，所述TCR是单链。
在一个实施方案中，所述TCR包含两条链。
TCR可以从生物来源获得，如来自细胞(如来自T细胞(例如细胞毒性T细胞))、T细胞杂交瘤或其他公众可获得的资源，例如，TCR可以源自多个动物物种之一，如人、小鼠、大鼠或其他哺乳动物，如通常来自人。
在一些实施方案中，所述TCR可以是细胞结合的形式的或为可溶形式，优选为可溶的形式。
所述TCR为可溶的形式指的是在其疏水芯区域发生突变的TCR，这些疏水芯区域的突变优选为能够使本申请可溶性TCR的稳定性提高的突变。
在一个实施方案中，所述TCR的α链可变区的氨基酸序列如SEQ ID NO:20且β链可变区的氨基酸序列如SEQ ID NO:21所示，或者α链可变区的氨基酸序列如SEQ ID NO:22且β链可变区的氨基酸序列如SEQ ID NO:23所示，或者α链可变区的氨基酸序列如SEQ ID NO:24且β链可变区的氨基酸序列如SEQ ID NO:25所示，或者α链可变区的氨基酸序列如SEQ ID NO:26且β链可变区的氨基酸序列如SEQ ID NO:27所示。
在一个实施方案中，本申请所述的T细胞受体(TCR)包含含有可变区的α链和/或含有可变区的β链，α链的可变区包含氨基酸序列为如SEQ ID NO:3或SEQ ID NO:9所示的互补决定区1(CDR1)；和/或氨基酸序列为如SEQ ID NO:4或SEQ ID NO:10所示的互补决定区2(CDR2)。在一个实施 方案中，所述β链的可变区包含氨基酸序列为如SEQ ID NO:6所示的互补决定区1(CDR1)；和/或氨基酸序列为如SEQ ID NO:7所示的互补决定区2(CDR2)。在一个实施方案中，所述α链的可变区包含含有氨基酸序列为如SEQ ID NO:5、SEQ ID NO:11、SEQ ID NO:13或SEQ ID NO:15所示的互补决定区3(CDR3)；和/或所述β链的可变区包含含有氨基酸序列为如SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:14或SEQ ID NO:16所示的互补决定区3(CDR3)；优选地，所述α链的可变区还包括第一先导序列；和/或所述β链的可变区还包括第二先导序列；优选地，所述α链可变区的氨基酸序列为如SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24或SEQ ID NO:26所示或者与SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24或SEQ ID NO:26具有至少90％序列相同性的氨基酸序列；和/或所述β链的可变区的氨基酸序列如SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25或SEQ ID NO:27所示或与SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25或SEQ ID NO:27具有至少90％序列相同性的氨基酸序列；优选地，所述α链还包含α恒定区和/或所述β链还包含β恒定区，优选地，所述恒定区为小鼠恒定区或人恒定区。在一个实施方案中，所述TCR是分离的或纯化的或者是重组的；优选地，所述TCR是人的；优选地，所述TCR是单克隆的；优选地，所述TCR是单链；优选地，所述TCR包含两条链；优选地，所述TCR为细胞结合的形式或为可溶的形式，优选为可溶的形式；优选地，所述TCR与抗原短肽-HLAA1101复合物结合，优选地，所述抗原短肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
本申请还提供了一种核酸分子，所述核酸分子包含编码所述的TCR或是TCR的α链或β链。
在一个实施方案中，编码所述α链的核苷酸序列包含如SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34或SEQ ID NO:36所示的核苷酸序列；和/或
编码所述β链的核苷酸序列包含如SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35或SEQ ID NO:37所示的氨基酸序列；
其中，SEQ ID NO:30所示的核苷酸序列为：

SEQ ID NO:31所示的核苷酸序列为：
SEQ ID NO:32所示的核苷酸序列为：
SEQ ID NO:33所示的核苷酸序列为：
SEQ ID NO:34所示的核苷酸序列为：
SEQ ID NO:35所示的核苷酸序列为：
SEQ ID NO:36所示的核苷酸序列为：
SEQ ID NO:37所示的核苷酸序列为：
在一个实施方案中，编码α链的核苷酸序列和/或编码β链的核苷酸序列是经密码子优化的。通常，密码子优化涉及使所选择的密码子的百分比与已公开的人类转移RNA的丰度平衡，使得没有一者过载或受限。在一些情况下，这可能是必要的，因为大多数氨基酸由超过一种密码子编码，并且密码子使用因生物而异。经转染的基因与宿主细胞之间的密码子使用差异可能会影响核酸构建体的蛋白质表达和免疫原性。通常，对于密码子优化，选择密码子以选择与人类使用频率平衡的那些密码子。通常，氨基酸密码子的冗余度使得不同的密码子编码一种氨基酸。在一些实施方案中，在选择用于置换的密码子时，可能需要所得突变是沉默突变，使得密码子改变不影响氨基酸序列。通常，密码子的最后一个核苷酸可以保持不变而不会影响氨基酸序列。
本申请提供了一种载体，所述载体包含上述所述的核酸分子。
例如，将编码上述TCR的一条或两条链的一种或多种核酸克隆到合适的一种或多种表达载体中，表达载体可以是任何合适的重组表达载体，并且可以用于转化或转染任何合适的宿主。合适的载体包括设计用于繁殖和扩增或用于表达或用于两者的那些，如质粒和病毒。
所述载体可以含有调节序列(如转录和翻译起始和终止密码子)，其对待引入载体的宿主的类型(例如，细菌、真菌、植物或动物)具有特异性，酌情并考虑载体是基于DNA还是基于RNA。载体也可以含有与编码TCR的核苷酸序列可操作连接的非天然启动子。所述启动子可以是非病毒启动子或病毒启动子，如巨细胞病毒(CMV)启动子、SV40启动子、RSV启动子和在鼠干细胞病毒的长末端重复序列中发现的启动子，也考虑了熟练技术人员已知的其他启动子。
所述载体为表达载体，优选为病毒载体，优选为逆转录病毒载体，进一步优选为慢病毒载体。
本申请还提供了一种包含上述所述核酸分子的宿主细胞，为了重组产生TCR，可以将编码TCR的核酸分离，并且将其插入一种或多种载体中，以 在宿主细胞中进一步克隆/或表达。可以使用常规技术(例如，通过使用能够与编码TCR的α链和β链的基因特异性结合的寡核苷酸探针)容易地分离和测序这种核酸。在一些实施方案中，提供了制备TCR的方法，其中，所述方法包括在适合于表达TCR分子的条件下培养如上提供的包含编码TCR的核酸的宿主细胞，以及任选地从宿主细胞(或宿主细胞培养基)回收TCR。
所述宿主细胞是指已引入外源核酸的细胞，包括此类细胞的后代。宿主细胞包括转化体和转化细胞，其包括原代转化细胞和源自其的后代，不考虑传代次数。后代在核酸含量上可能与亲代细胞不完全相同，但可能含有突变。
本申请还提供了一种工程化细胞，其包含所述的T细胞受体(TCR)、上述所述的核酸分子或者上述所述的载体。
在一个实施方案中，所述TCR对所述细胞是异源的。
在一个实施方案中，所述工程化细胞是细胞系。
在一个实施方案中，所述工程化细胞是获自受试者的原代细胞，优选地，所述受试者为哺乳动物受试者，优选为人。
在一个实施方案中，所述工程化细胞是T细胞，优选是从外周血分离的T细胞。
在一个实施方案中，所述T细胞为CD8+或CD4+。
所述工程化细胞例如可以是细胞群体或者表达TCR的基因工程化细胞，该细胞通常是真核细胞，如哺乳动物细胞，并且通常是人细胞。在一些实施方案中，细胞源自血液、骨髓、淋巴或淋巴器官，是免疫系统的细胞，如先天免疫或适应性免疫的细胞，例如骨髓或淋巴样细胞(包括淋巴细胞，通常是T细胞和/或NK细胞)。其他示例性细胞包括干细胞，如多潜能干细胞和多能干细胞，包括诱导多能干细胞(iPSC)。细胞通常是原代细胞，如直接从受试者分离和/或从受试者分离并冷冻的那些。在一些实施方案中，细胞包括T细胞或其他细胞类型的一个或多个子集，如整个T细胞群、CD+细胞、CD8+细胞及其亚群。
T细胞和/或CD+和/或CD8+T细胞的亚型和亚群包括幼稚T(TN)细胞、效应T细胞(TEFF)、记忆T细胞及其亚型(如干细胞记忆T(TSCM)、中枢记忆T(TCM)、效应记忆T(TEM)或终末分化的效应记忆T细胞)、肿瘤浸润淋巴细胞(TIL)、未成熟T细胞、成熟T细胞、辅助T细胞、细胞毒性T细胞、粘膜相关恒定T(MAIT)细胞、天然存在和适应性调节T(Treg) 细胞等。
在一些实施方式中，所述工程化细胞是自然杀伤(NK)细胞，优选地，细胞是单核细胞或粒细胞，例如骨髓细胞、巨噬细胞、嗜中性粒细胞、树突细胞、肥大细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
本申请提供了一种生产上述所述的工程化细胞的方法，其包括在体外或离体地将上述所述的核酸分子或者上述所述的载体引入细胞中。
所述载体为病毒载体，并且所述引入是通过转导进行的。
本申请提供了一种药物组合物，其包含上述所述的T细胞受体(TCR)、上述所述的核酸分子、上述所述的载体或者上述所述的工程化细胞。在一个实施方案中，其还包含药学上可接受的载体或佐剂。
所述药学上可接受的载体或佐剂是指药物组合物中除了活性成分之外对受试者无毒的成分。药学上可接受的载体或佐剂包括但不限于缓冲液、赋形剂、稳定剂或防腐剂。
所述药物组合物可以利用定时释放、延迟释放和持续释放递送系统，使得所述组合物的递送发生在待治疗部位的致敏之前并且有足够的时间引起致敏。许多类型的释放递送系统是可用的并且是已知的。此类系统可用避免重复给予所述组合物，从而增加受试者和医生的便利性。
本申请提供了上述所述的T细胞受体(TCR)、上述所述的核酸分子、上述所述的载体、上述所述的工程化细胞或者上述所述的药物组合物在制备治疗与HPV相关的疾病的药物中的用途。
在一个实施方案中，与HPV相关的疾病为HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌等。
本申请提供了一种治疗与HPV相关的疾病的方法，包括将上述所述的T细胞受体(TCR)、上述所述的核酸分子、上述所述的载体或者上述所述的工程化细胞或者上述所述的药物组合物施用于有需要的受试者。在一个实施方案中，所述与HPV相关的疾病为HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌。
本申请所述的TCR能够与HPV阳性肿瘤细胞的抗原短肽特异性结合，转导了所述TCR的T细胞能够被特异性激活并且对靶细胞具有很强的杀伤作用，所述的TCR可以用于宫颈癌等HPV阳性的肿瘤的免疫治疗。
本申请通过使用抗原短肽，能够筛选出特异性T细胞受体(TCR)，并且经过转录所述TCR之后的T细胞能够被特异性激活并且对表达A1101和HPV的肿瘤细胞具有很强的杀伤作用，其可以用于宫颈癌等HPV阳性的肿瘤的免疫治疗。
本申请转导了所述TCR的T细胞对表达E6的细胞系有很强的激活反应，对不表达E6的细胞系没有激活反应，并且对表达E6的细胞系有很强的杀伤功能，并能够有效抑制E6阳性肿瘤的生长。
本申请转导了所述TCR的T细胞能够显著杀伤原代宫颈癌类器官细胞，可以有效地抑制E6阳性肿瘤的生长。
实施例
本申请对试验中所用到的材料以及试验方法进行一般性和/或具体的描述，在下面的实施例中，如果无其他特别的说明，％表示wt％，即重量百分数。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
实施例1克隆TTLEQQYNK和GTTLEQQYNK抗原短肽特异性T细胞
利用合成短肽TTLEQQYNK(SEQ ID NO:1，江苏金斯瑞生物科技有限公司)和GTTLEQQYNK(SEQ ID NO:2，江苏金斯瑞生物科技有限公司)分别刺激来自于基因型为HLA-A*11:01的健康志愿者的外周血淋巴细胞。将TTLEQQYNK短肽和GTTLEQQYNK短肽分别与带有生物素标记的HLA-A*11:01复性，制备pHLA单体。这些单体与用PE标记的链霉亲和素(BD公司，目录号554061)组合成PE标记的四聚体，流式检测该四聚体及CD8双阳性细胞，其中，pHLA单体和四聚体的制作方法参照NIH Tetramer Core Facility公开的protocol，具体步骤见网页https://tetramer.yerkes.emory.edu/support/protocols#1，具体操作如下：
(1)试剂
试验培养基：10％FBS(赛默飞公司(ThermoFisher)，目录号10099-044)，RPMI1640(赛默飞公司(ThermoFisher)，目录号C11875500BT)，10％HS(Gemni，目录号100512)，TexMACS(美天旎(Mitenyi)，目录号170-076-309)
(2)方法
通过密度梯度离心法从健康志愿者的外周血中分出PBMC，从PBMC中分出CD14阳性细胞，用于诱导树突状细胞(dendritic cell,DC)。CD14阳性细胞在培养基(1640+10％AuFBS+1％PS+800IU/ml GM-CSF+500IU/ml IL-4)中培养至第三天补1ml培养基(含1600IU/ml GM-CSF+1000IU/ml IL-4)，第五天加入DC成熟诱导因子10ng/ml IL-1β，10ng/ml IL-6，10ng/ml TNF-α，1ug/ml PGE2，继续培养两天，即可获得成熟的DC。用成熟的DC分别负载TTLEQQYNK短肽和GTTLEQQYNK，终浓度为20μg/ml，然后去刺激从PBMC中分出的CD8阳性T细胞(培养基TexMACS+10％HS+1％PS+60ng/ml IL21+10IU/ml IL2/7/15)。经过三轮刺激后，用流式细胞分选仪分选出四聚体及CD8双阳性的T细胞，此即抗原特异性T细胞，将分选获得的CD8和四聚体双阳性单细胞用一步法RT-PCR试剂盒(QIAGEN凯杰，目录号210212)分别扩增TCRα链及β链，PCR产物送测序。将测序结果与IMGT(国际免疫遗传学信息系统)的公开数据库中的序列进行比对，即可以获得TCRα链可变区序列及β链可变区的核苷酸序列及其CDR1、CDR2、CDR3的信息。
(3)结果
通过如上所述方法克隆TTLEQQYNK和GTTLEQQYNK抗原短肽特异性T细胞，利用流式细胞术分析克隆出的抗原短肽特异性T细胞。经过TTLEQQYNK和GTTLEQQYNK抗原短肽二轮刺激和三轮刺激后的T细胞流式检测结果如图1所示，其中图1A是经过二轮刺激后的T细胞流式检测结果示意图，图1B是经过三轮刺激后的T细胞流式检测结果示意图。
从图1可以看出，已成功克隆TTLEQQYNK和GTTLEQQYNK抗原短肽特异性T细胞，其中TTLEQQYNK获得的部分TCR序列见如下实施例2详述。
实施例2：TTLEQQYNK抗原短肽特异性TCR慢病毒载体构建及慢病毒包装
(1)TCR慢病毒载体构建
将实施例1中CD8和四聚体双阳性的细胞进行流式分选获得单细胞，将分选获得的单细胞用一步法RT-PCR试剂盒(QIAGEN凯杰，目录号210212)分别扩增TCRα链及β链，将PCR产物测序。将测序结果与IMGT(国际 免疫遗传学信息系统)的公开数据库中的序列进行比对，即可以获得TCRα链可变区序列及β链可变区的核苷酸序列及其CDR1、CDR2、CDR3的信息，得到4个TCR(TCR010、TCR013、TCR028和TCR090)的α链可变区序列及β链可变区的核苷酸序列及其CDR1、CDR2、CDR3的信息，其分别如下所示：
TCR010的α链可变区序列的核苷酸序列如SEQ ID NO:39所示，其核苷酸序列如下：
β链可变区的核苷酸序列如SEQ ID NO:40所示，其核苷酸序列如下：
α链的互补决定区1(CDR1)的氨基酸序列如SEQ ID NO:3所示：SSYSPS
互补决定区2(CDR2)的氨基酸序列如SEQ ID NO:4所示：YTSAATLV
互补决定区3(CDR3)的氨基酸序列如SEQ ID NO:5所示：VVSLSGGYNKLI
β链的互补决定区1(CDR1)的氨基酸序列如SEQ ID NO:6所示：SGDLS
互补决定区2(CDR2)的氨基酸序列如SEQ ID NO:7所示：YYNGEE
互补决定区3(CDR3)的氨基酸序列如SEQ ID NO:8所示：ASSVTGSGYT
TCR013的α链可变区序列的核苷酸序列如SEQ ID NO:41所示，
β链可变区的核苷酸序列如SEQ ID NO:42所示：
α链的互补决定区1(CDR1)的氨基酸序列如SEQ ID NO:9所示：TTLSN
互补决定区2(CDR2)的氨基酸序列如SEQ ID NO:10所示：LVKSGEV
互补决定区3(CDR3)的氨基酸序列如SEQ ID NO:11所示：AGPKITGGGNKLT
β链的互补决定区1(CDR1)的氨基酸序列如SEQ ID NO:6所示：SGDLS
互补决定区2(CDR2)的氨基酸序列如SEQ ID NO:7所示：YYNGEE
互补决定区3(CDR3)的氨基酸序列如SEQ ID NO:12所示：ASSVGGGPNYGYT
TCR028的α链可变区序列的核苷酸序列如SEQ ID NO:43所示，
β链可变区的核苷酸序列如SEQ ID NO:44所示：
α链的互补决定区1(CDR1)的氨基酸序列如SEQ ID NO:9所示：TTLSN
互补决定区2(CDR2)的氨基酸序列如SEQ ID NO:10所示：LVKSGEV
互补决定区3(CDR3)的氨基酸序列如SEQ ID NO:13所示：AGPILTGGGNKLT
β链的互补决定区1(CDR1)的氨基酸序列如SEQ ID NO:6所示：SGDLS
互补决定区2(CDR2)的氨基酸序列如SEQ ID NO:7所示：YYNGEE
互补决定区3(CDR3)的氨基酸序列如SEQ ID NO:14所示：ASGLSGPNTGELF
TCR090的α链可变区序列的核苷酸序列如SEQ ID NO:45所示，
β链可变区的核苷酸序列如SEQ ID NO:46所示：
α链的互补决定区1(CDR1)的氨基酸序列如SEQ ID NO:9所示：TTLSN
互补决定区2(CDR2)的氨基酸序列如SEQ ID NO:10所示：LVKSGEV
互补决定区3(CDR3)的氨基酸序列如SEQ ID NO:15所示：AGPVLTGGGNKLT
β链的互补决定区1(CDR1)的氨基酸序列如SEQ ID NO:6所示：SGDLS
互补决定区2(CDR2)的氨基酸序列如SEQ ID NO:7所示：YYNGEE
互补决定区3(CDR3)的氨基酸序列如SEQ ID NO:16所示： ASSVGGPNTGELF
将TTLEQQYNK TCRα和β链可变区序列克隆至基于pLKO的表达质粒(Addgene)，通过多片段重组克隆试剂盒(诺唯赞生物科技公司，目录号C113)标准方法将α或β可变结构域克隆到含有鼠α或β恒定区的基于PLKO的表达质粒中，将连接的质粒转化到感受态大肠杆菌菌株Stbl3细胞(上海唯地生物技术有限公司)中并接种于含有100μg/ml氨苄青霉素的LB/琼脂平板上。在37℃下过夜孵育之后，挑取单个菌落并在37℃下在10ml的含有100μg/ml氨苄青霉素的LB中振荡过夜生长。使用小提中量试剂盒(天根生化科技公司(TIANGEN)目录号#DP118-02)纯化克隆的质粒并且对质粒进行测序得到TTLEQQYNK TCR(即TCR010，TCR013，TCR028，TCR090)。
(2)慢病毒包装
试验培养基：10％FBS(Lonsera，目录号S711-001)，DMEM(思拓凡公司(cytiva)，目录号SH30243.01)
准备293T细胞(保藏机构为美国典型培养物保藏中心即ATCC，保藏目录号为CRL-1573)在10cm皿中培养，在不超过80％满度的时候开始质粒转染，病毒包装质粒和TTLEQQYNK TCR质粒的比例为1:1，共10μg。将上述质粒加在无血清的DMEM培养基中与PEI(polyethylenimine，聚乙烯亚胺)混合，然后将加质粒的混合液加到293T细胞中，37度培养。72h后，将细胞的上清用100kd超滤管浓缩收集病毒载体。
实施例3表达TTLEQQYNK抗原短肽特异性TCR的Jurkat细胞系构建及功能鉴定
活化T细胞核因子(NFAT)报告基因表达方法
进行以下试验以证明TCR转导的T细胞对靶细胞特异性的激活反应。利用流式细胞分析技术检测NFAT表达量作为T细胞激活的读出值。
(1)试剂
试验培养基：10％FBS(Lonsera，目录号S711-001)，RPMI1640(赛默飞公司(ThermoFisher)，目录号C11875500BT)
(2)方法
靶细胞制备
本实验中所用的靶细胞为T2-A11细胞(T2细胞保藏于ATCC，保藏目 录号为CRL-1992，T2-A11细胞是参考Cancer Biology & Therapy,8:21,2025-2032在T2细胞基础上构建的)。在实验培养基中制备靶细胞，靶细胞浓度调至1.6×106个/毫升，每孔取50微升从而得80000个细胞/孔。
效应细胞制备
本实验的效应细胞为转导了本申请TCR的Jurkat-CD8-NFAT(JK8NF)细胞，并以未转染本申请TCR的JK8NF细胞作为对照组。
将JK8NF细胞(Jurkat细胞保藏于ATCC，保藏目录号为TIB-152，JK8NF细胞是参考Cancer Res 2006；66(23):11455-61，Front.Immunol.11:633.在Jurkat细胞基础上构建的)按MOI(感染复数)＝10加入实施例2中获得的携带本申请TCR基因的慢病毒，72小时后用流式细胞仪鉴定转染阳性率100％左右(其结果如图2所示)，将扩大培养后的效应细胞浓度调至1.6×106个/毫升，每孔取50微升从而得80000个细胞/孔。
短肽溶液制备
将原浓度5mg/ml短肽(TTLEQQYNK)浓度稀释为400μg/ml，然后按10倍比例依次往下稀释成40μg/ml、4μg/ml、0.4μg/ml、0.04μg/ml、0.004μg/ml、0.0004μg/ml。
每孔取50微升使短肽在96孔板中的终浓度分别为100μg/ml、10μg/ml、1μg/ml、0.1μg/ml、0.01μg/ml、0.001μg/ml、0.0001μg/ml。
最终每孔加50μl靶细胞，50μl效应细胞，50μl相应浓度短肽稀释液和50ul培养基于96孔平底板中，与37度细胞培养箱中孵育12h。
(3)结果
通过如上所述方法检验本申请TCR转导的T细胞对负载TTLEQQYNK抗原短肽的靶细胞起反应的NFAT的表达。利用Graphpad prism8绘制NFAT的表达水平曲线，其结果如图3所示。
从图3可以看出，转导本申请TCR的T细胞对负载其特异的短肽的靶细胞有很好的激活反应。
实施例4表达TTLEQQYNK抗原短肽特异性TCR的原代T细胞构建及功能鉴定
实时监测靶细胞增殖方法
(1)试剂
试验培养基：10％FBS(赛默飞公司(ThermoFisher)，目录号10099-044)，RPMI1640(赛默飞公司公司(ThermoFisher)，目录号C11875500BT)
(2)方法
效应T细胞制备
本实验的效应细胞(T细胞)为转导了本申请TCR的T细胞，并以同一志愿者未转染本申请TCR的T细胞作为对照组。
将志愿者的外周血经过密度梯度离心，获得外周血单个核细胞，将外周血单个核细胞按照24孔板每孔5.0×105/500μl置于孔中，共收集1x106细胞，用抗CD3/CD28磁珠刺激T细胞后置于37℃，5％CO2培养箱中培养。24h后观察细胞成团情况，按MOI(感染复数)＝2加入实施例2中获得的TCR慢病毒转导后，在含有200IU/ml IL-2的含10％FBS的1640培养基扩增直至转导后3-4天后，用流式细胞仪鉴定TCR转染效率(其结果如图4所示)。将扩大培养后的效应细胞浓度调至5.0×104个阳性细胞/毫升，每孔取100微升从而得5000个阳性细胞/孔。
靶细胞制备
本实验中所用的靶细胞为SK-MEL-28-E6，SK-MEL-28(保藏于ATCC)的基因型为A*11:01，但是本身不表达E6，因此通过慢病毒载体过表达E6基因构建SK-MEL-28-E6细胞作为靶细胞。将在实验培养基中制备靶细胞，靶细胞浓度调至5.0×104个/毫升，每孔取100微升从而得5000个细胞/孔。
RTCA x Celligence系统监测靶细胞增殖
于E-plate中加入上述准备好的100微升T细胞与100微升靶细胞，然后将E-plate安装到RTCA分析仪，与37℃，5％CO2培养箱中静置培养90h，观察靶细胞实时增殖曲线。
(3)结果
通过实时监测靶细胞增殖实验(如上所述)检验本申请TCR转导的T细胞的功能。利用Graphpad prism8绘制各孔中细胞增殖的细胞指数(RTCA x Celligence系统生成的表征细胞数的参数)。实验结果如图5所示。
从图5可以看出，表达TTLEQQYNK-A*11:01抗原短肽特异性TCR的T细胞对表达E6的细胞系有很强的杀伤功能。
实施例5：表达TTLEQQYNK抗原短肽特异性TCR的原代T细胞体内 抑制肿瘤生长功能鉴定
进行以下试验以证明TCR转导的T细胞对肿瘤生长的抑制作用。观察肿瘤的体积变化评估T细胞杀伤肿瘤的功能，体外实验显示四个TCR杀伤肿瘤的效果类似，因此仅选取TCR013和TCR028作为代表，进行体内抑制肿瘤生长功能鉴定。
(1)试剂与小鼠
试验培养基：10％FBS(赛默飞公司(ThermoFisher)，目录号10099-044)，RPMI1640(赛默飞公司公司(ThermoFisher)，目录号C11875500BT)
NCG小鼠：购买于集萃药康公司，雌性，4-6周龄，基因型是(prkdc)ko/ko，(Il2rg雌)ko/ko。
(2)方法
肿瘤细胞皮下接种
本实验用到的肿瘤细胞是SK-MEL-28-E6。将在生理盐水中制备肿瘤细胞，肿瘤细胞浓度调至5.0×107个/毫升，每只小鼠皮下接种200微升从而得1.0×107个细胞/只，待肿瘤体积长到约100立方毫米，接种效应T细胞。
效应T细胞制备
本实验的效应细胞(T细胞)为转导了本申请TCR的T细胞，并以同一志愿者未转染本申请TCR的T细胞作为对照组。每组5只小鼠。
将志愿者的外周血经过密度梯度离心，获得外周血单个核细胞，将外周血单个核细胞按照24孔板每孔5.0×105/500μl置于孔中，共收集4x106细胞，用抗CD3/CD28磁珠刺激T细胞后置于37℃，5％CO2培养箱中培养。24h后观察细胞成团情况，按MOI(感染复数)＝2加入实施例2中获得的TCR基因(TCR013和TCR028的基因)的慢病毒转导后，在含有200IU/ml IL-2的含10％FBS的1640培养基扩增直至转导后3-4天后，用流式细胞仪鉴定TCR转染效率。将扩大培养后的效应细胞浓度调至5.0×107个阳性细胞/毫升，每只小鼠通过眼静脉注射200微升剂量从而得1.0×107个阳性细胞/只。
(3)结果
观察记录接种T细胞后肿瘤的体积，根据肿瘤体积的大小变化评估表达本申请的TCR的原代T细胞治疗肿瘤的效果。利用Graphpad prism8绘制肿瘤的生长曲线，实验结果如图6A和图6B所示，其中，图6A是接种T细胞后肿瘤体积随时间变化的示意图，图6B是接种含有不同TCR的T细胞后肿 瘤重量的柱状图。
从图6A和图6B可以看出，表达TTLEQQYNK-A*11:01抗原短肽特异性TCR的T细胞能够有效抑制E6阳性肿瘤的生长。
实施例6TTLEQQYNK-A*11:01抗原短肽特异性TCR对原代HPV肿瘤的杀伤功能
(1)试剂
试验培养基：OrganoPro宫颈癌类器官培养基试剂盒(科途医学，目录号K2O-M-CC)
(2)方法
将8000个HPV16+HLA-A*11:01+的宫颈癌类器官KOCC-002S4(科途医学)，与8000个转导了TCR013的原代T细胞(TCR013)，或者未转导TCR的对照T细胞(对照组)共培养，72小时后，显微镜下观察类器官的生长状态并拍照。
(3)结果
通过显微镜观察类器官的生长状态，对典型视野拍照，统计视野中形态完整和不完整的类器官的数量，其结果如图7A至图7B所示，其中，图7A是通过显微镜观察到的类器官的生长状态示意图，图7B是对形态完整和形态不完整的类器官的数量进行统计的示意图。
从图7A至图7B可以看出，表达TTLEQQYNK-A*11:01抗原短肽特异性TCR的T细胞能够显著杀伤原代宫颈癌类器官细胞。
综上所述，本申请转达所述TCR的T细胞能够被特异性激活并且对表达A1101和HPV的肿瘤细胞具有很强的杀伤作用。
所述T细胞对表达E6的细胞系有很强的杀伤功能，并能够有效抑制E6阳性肿瘤的生长。
以上所述，仅是本申请的较佳实施例而已，并非是对本申请作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本申请技术方案内容，依 据本申请的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本申请技术方案的保护范围。

Claims (20)

  1. 抗原短肽用于筛选治疗与HPV相关的疾病的药物中的用途，所述抗原短肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
  2. 根据权利要求1所述的用途，其中，所述与HPV相关的疾病为HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌。
  3. 抗原短肽用于筛选治疗HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌的药物中的用途，所述抗原短肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
  4. 根据权利要求1-3中任一项所述的用途，其中，所述药物为用于与包含所述抗原短肽的抗原短肽-HLA-A1101复合物结合的T细胞受体(TCR)。
  5. 一种T细胞受体(TCR)，其中，所述TCR包含含有可变区的α链和/或含有可变区的β链，α链的可变区包含氨基酸序列为如SEQ ID NO:3或SEQ ID NO:9所示的互补决定区1(CDR1)；和/或
    氨基酸序列为如SEQ ID NO:4或SEQ ID NO:10所示的互补决定区2(CDR2)。
  6. 根据权利要求5所述的T细胞受体(TCR)，其中，所述β链的可变区包含氨基酸序列为如SEQ ID NO:6所示的互补决定区1(CDR1)；和/或
    氨基酸序列为如SEQ ID NO:7所示的互补决定区2(CDR2)。
  7. 根据权利要求5或6所述的T细胞受体(TCR)，其中，所述α链的可变区包含含有氨基酸序列为如SEQ ID NO:5、SEQ ID NO:11、SEQ ID NO:13或SEQ ID NO:15所示的互补决定区3(CDR3)；和/或
    所述β链的可变区包含含有氨基酸序列为如SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:14或SEQ ID NO:16所示的互补决定区3(CDR3)；
    优选地，所述α链的可变区还包括第一先导序列；和/或
    所述β链的可变区还包括第二先导序列；
    优选地，所述α链可变区的氨基酸序列为如SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24或SEQ ID NO:26所示或者与SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24或SEQ ID NO:26具有至少90％序列相同性的氨基酸 序列；和/或
    所述β链的可变区的氨基酸序列如SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25或SEQ ID NO:27所示或与SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25或SEQ ID NO:27具有至少90％序列相同性的氨基酸序列；
    优选地，所述α链还包含α恒定区和/或所述β链还包含β恒定区，优选地，所述恒定区为小鼠恒定区或人恒定区。
  8. 根据权利要求5-7中任一项所述的T细胞受体(TCR)，其中，所述TCR是分离的或纯化的或者是重组的；
    优选地，所述TCR是人的；
    优选地，所述TCR是单克隆的；
    优选地，所述TCR是单链；
    优选地，所述TCR包含两条链；
    优选地，所述TCR为细胞结合的形式或为可溶的形式，优选为可溶的形式；
    优选地，所述TCR与抗原短肽-HLAA1101复合物结合，优选地，所述抗原短肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
  9. 一种核酸分子，其中，所述核酸分子包含编码权利要求5-17中任一项所述的TCR或者所述TCR的α链或β链。
  10. 根据权利要求9所述的核酸分子，其中编码α链的核苷酸序列包含如SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34或SEQ ID NO:36所示的核苷酸序列；和/或
    编码β链的核苷酸序列包含如SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35或SEQ ID NO:37所示的核苷酸序列。
  11. 一种载体，其中，所述载体包含权利要求9或10所述的核酸分子。
  12. 根据权利要求11所述的载体，其中，所述载体为表达载体；
    优选地，所述载体为病毒载体，优选为逆转录病毒载体；
    优选地，所述病毒载体为慢病毒载体。
  13. 一种工程化细胞，其包含权利要求5-8中任一项所述的TCR、权利要求9-10中任一项所述的核酸分子或者权利要求11-12中任一项所述的载体。
  14. 根据权利要求13所述的工程化细胞，其中，所述TCR对所述细胞 是异源的；
    优选地，所述工程化细胞是细胞系；
    优选地，所述工程化细胞是获自受试者的原代细胞，优选地，所述受试者为哺乳动物受试者，优选为人；
    优选地，所述工程化细胞是T细胞或NK细胞，优选地，所述T细胞是从外周血分离的T细胞；
    优选地，所述T细胞为CD8+或CD4+。
  15. 一种生产权利要求13-14中任一项所述的工程化细胞的方法，其包括在体外或离体地将权利要求9-10中任一项所述的核酸分子或者权利要求11-12任一项所述的载体引入细胞中。
  16. 根据权利要求15所述的方法，其中，所述载体为病毒载体，并且所述引入是通过转导进行的。
  17. 一种药物组合物，其包含权利要求5-8中任一项所述的T细胞受体(TCR)、权利要求9-10中任一项所述的核酸分子、权利要求11-12中任一项所述的载体或者权利要求13-14中任一项所述的工程化细胞；
    优选地，其还包含药学上可接受的载体或佐剂。
  18. 权利要求5-8中任一项所述的T细胞受体(TCR)、权利要求9-10中任一项所述的核酸分子、权利要求11-12中任一项所述的载体或者权利要求13-14中任一项所述的工程化细胞或者权利要求17所述的药物组合物在制备治疗与HPV相关的疾病的药物中的用途；
    优选地，与HPV相关的疾病为HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌。
  19. 一种治疗与HPV相关的疾病的方法，包括将权利要求5-8中任一项所述的T细胞受体(TCR)、权利要求9-10中任一项所述的核酸分子、权利要求11-12中任一项所述的载体或者权利要求13-14中任一项所述的工程化细胞或者权利要求17所述的药物组合物施用于有需要的受试者。
  20. 根据权利要求19所述的方法，其中，所述与HPV相关的疾病为HPV慢性感染、宫颈上皮内瘤样病变、宫颈癌、头颈癌、肛门癌、阴茎癌、阴道癌或外阴癌。
PCT/CN2023/093015 2022-05-10 2023-05-09 抗原短肽用于筛选治疗与hpv相关的疾病的药物中的用途及其筛选的tcr WO2023217143A1 (zh)

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