WO2020038491A1 - 一种分离的t细胞受体、其修饰的细胞、编码核酸及其应用 - Google Patents

一种分离的t细胞受体、其修饰的细胞、编码核酸及其应用 Download PDF

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WO2020038491A1
WO2020038491A1 PCT/CN2019/102562 CN2019102562W WO2020038491A1 WO 2020038491 A1 WO2020038491 A1 WO 2020038491A1 CN 2019102562 W CN2019102562 W CN 2019102562W WO 2020038491 A1 WO2020038491 A1 WO 2020038491A1
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cells
cell
her2
chain
tcr
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侯亚非
侯大炜
谭贤魁
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杭州康万达医药科技有限公司
合成免疫股份有限公司
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    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464406—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/50—Colon
    • 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

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to an isolated T cell receptor, a modified cell thereof, a coding nucleic acid, an expression vector, a preparation method, a pharmaceutical composition and an application.
  • Her2 / neu is a transmembrane protein belonging to the EGFR family. It forms a dimer with other proteins in the family and regulates cell proliferation, differentiation, and canceration through a series of intracellular signaling pathways (see the literature “Growth Factors, 2008; 26: 263 “,” Oncol Biol. Phys, 2004; 58: 903 "). Her2 / neu protein is over-expressed in a variety of epithelial-derived cancer cells, such as breast, gastric, colorectal, ovarian, pancreatic, lung, esophageal, bladder, kidney, etc. , 2013; 19: 677 "), and the expression is relatively uniform in primary and metastatic cancer cells (see the document” J Clin Oncol, 1998; 8: 103 "), therefore, Her2 / neu has become a targeted therapy Proper target.
  • epithelial-derived cancer cells such as breast, gastric, colorectal, ovarian, pancreatic, lung, esophageal, bladder, kidney,
  • Herceptin a humanized monoclonal antibody drug targeting Her2 / neu, can significantly prolong the survival of Her2 / neu-positive breast cancer patients (see the literature "N Engl J Med, 2001, 344: 783”), but Herceptin alone is used to treat Her2 positive
  • the clinical response rate of metastatic breast cancer is only 11% to 26% (see the document “J Clin Oncol, 2002; 20: 7169”), indicating that Herceptin alone is not ideal for most Her2 overexpressing metastatic breast cancer.
  • Herceptin combined with chemotherapy can increase the clinical response rate, most breast cancer patients with Her2 / neu overexpression will develop resistance to Herceptin after one year (see the document "J Clin Oncol, 2001; 19: 2587”).
  • a tumor vaccine targeting Her2 / neu 369-377 peptide antigen has entered clinical trials, although Phase 1/2 clinical trials have shown that this vaccine can induce specific T killer cells directed against Her2 / neu 369-377 peptide antigen (see the document "Breast, Care , 2016; 11: 116 "), but the clinical phase III failed to meet the intended goal of extending patient survival (http://www.onclive.com/web-exclusives/phase-iii-nelipepimuts-study-in-breast- cancer-halted-after-futility-review).
  • CRS cytokine release syndrome
  • TCR-T therapy for adoptive transfer of T cells modified by specific T cell receptor (ie TCR) genes is considered to be the most promising immune cell gene therapy for solid tumors (see the literature “Adv Immunol. 2016; 130: 279 -94 ").
  • clinical studies of TCR-T therapy targeting the NY-ESO-1 antigen have shown encouraging clinical treatment effects (see the document “Nat Med. 2015 Aug; 21 (8): 914-921”).
  • the number of specific TCRs currently known to target tumor antigens and effectively recognize tumor cells is very limited, thus limiting the widespread application of TCR-T therapy.
  • TCR-T therapy does not show the severe cytokine storm toxicity shown in CAR-T therapy
  • the target antigen is derived from its own protein
  • targeting the target antigen with low expression in normal tissue cells may cause serious Autoimmune response, that is, on-target off-tumor (on-target off-tumor) toxicity (see the document “Blood 2009; 114: 535-546").
  • the general strategy is to perform gene point mutations on complementarity determining regions (CDRs) on the TCR in vitro, or by never passing through the central tolerance mechanism Induction was performed in a screened humanized mouse T cell bank (see document "Front Immunol.
  • the present invention provides an isolated T cell receptor, a modified cell thereof, a coding nucleic acid, an expression vector, a preparation method, a pharmaceutical composition, and an application.
  • the present invention provides:
  • An isolated T cell receptor including at least one of an ⁇ chain and a ⁇ chain, each of which includes a variable region and a constant region, characterized in that the T cell receptor It can specifically recognize the antigen Her2 / neu expressed by tumor cells, and the amino acid sequence of the variable region of the ⁇ chain has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 1, the ⁇ The amino acid sequence of the variable region of the chain has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 2.
  • T cell receptor (2) The T cell receptor according to (1), wherein the T cell receptor is capable of specifically recognizing the antigen epitope polypeptide of the antigen Her2 / neu presented by the HLA-A2 molecule; preferably Yes, the epitope polypeptide includes Her2 / neu 369-377 as shown in SEQ ID NO: 3.
  • An isolated nucleic acid encoding a T cell receptor comprising a coding sequence of at least one of an ⁇ chain and a ⁇ chain of the T cell receptor, wherein the ⁇ chain coding sequence and the ⁇ chain coding sequence are both It comprises a variable region coding sequence and a constant region coding sequence, wherein the T cell receptor can specifically recognize the antigen Her2 / neu expressed by a tumor cell, and the amino acid sequence encoded by the ⁇ chain variable region coding sequence has It has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence encoded by the ⁇ -chain variable region coding sequence has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 2.
  • nucleic acid according to (6) wherein the nucleic acid is DNA or RNA.
  • the T cell receptor encoded by the nucleic acid can specifically recognize an epitope polypeptide of the antigen Her2 / neu presented by an HLA-A2 molecule;
  • the epitope polypeptide includes Her2 / neu 369-377 as shown in SEQ ID NO: 3.
  • the promoter may be a eukaryotic cell promoter, including a continuous expression promoter and an inducible expression promoter, including, for example, a PGK1 promoter, an EF-1 ⁇ promoter, a CMV promoter, an SV40 promoter, and a Ubc promoter. , CAG promoter, TRE promoter, CaMKIIa promoter, human beta actin promoter.
  • a T cell receptor-modified cell whose surface is modified by the T cell receptor according to any one of (1) to (5), wherein the cell includes a primitive T cell or a precursor thereof Cells, NKT cells, or T cell lines.
  • the method of transfection includes: a method of transfection with a viral vector, preferably, the viral vector includes a gamma retrovirus vector or a lentiviral vector; a chemical method
  • the chemical method includes a method using liposome transfection
  • the physical method preferably, the physical method includes an electric transfection method.
  • composition wherein the pharmaceutical composition comprises, as an active ingredient, a T cell receptor-modified cell according to (19) or (20), and a pharmaceutically acceptable excipient.
  • composition (28), wherein the pharmaceutical composition is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, Intramuscular or intraperitoneal administration.
  • a method for treating a tumor and / or cancer comprising administering a T cell receptor-modified cell according to (19) or (20) to a tumor and / or cancer patient.
  • T cell receptor-modified cells pass through an artery, vein, subcutaneous, intradermal, intratumor, intralymphatic, intralymphatic, subarachnoid, intramedullary, Intramuscular or intraperitoneal administration.
  • the present invention has the following advantages and positive effects:
  • the present invention successfully induces T cell clones specific to Her2 / neu epitope polypeptides (such as Her2 / neu 369-377 polypeptides) presented by HLA-A2 from peripheral blood of HLA-A2-positive healthy donors.
  • T-cell clones carrying natural TCRs that specifically recognize Her2 / neu epitope polypeptides were screened out, and the full TCR sequence was obtained.
  • This TCR is non-CD8 molecule-dependent and has moderate to high affinity for Her2 / neu epitope polypeptides (such as Her2 / neu 369-377 polypeptide), and can specifically recognize HLA-A2 positive and express Her2 / neu antigens.
  • Tumor cells are non-CD8 molecule-dependent and has moderate to high affinity for Her2 / neu epitope polypeptides (such as Her2 / neu 369-377 polypeptide), and can specifically recognize HLA-A2 positive and express Her2 / neu antigens.
  • the T cell clones carrying this TCR were screened by the central immune tolerance mechanism and entered the peripheral T cell bank. Killer T cells carrying this TCR were once present in normal human peripheral blood and did not cross-react with normal tissue cells that express a small amount of Her2 / neu protein.
  • the normal protein database searches all normal proteins of all humans that contain the key amino acid sites recognized by the TCR, and further screens for epitope polypeptides that may bind the HLA-A2 molecule. Experiments have shown that the TCR does not recognize these epitope polypeptides from normal proteins with potential cross-reactivity. Therefore, the present invention obtains a new type of TCR that can specifically recognize tumor antigens while avoiding possible off-target side effects.
  • the constant region of the TCR is also modified (such as disulfide modification or murine engineering), so that the TCR can further reduce or avoid mismatches with endogenous TCR when expressed on immune cells. occur.
  • Immune cells (such as primitive T cells, precursor cells, NKT cells, and T cell strains) modified with this TCR can specifically recognize a variety of HLA-A2 + and Her2 / neu + tumor cell lines, and have significant anti-tumor properties. effect. Therefore, TCR-T therapy based on this TCR is expected to treat a variety of solid tumors.
  • TCR modified immune cells of the present invention When used to treat tumors, the cytokine storm and immune rejection caused by CAR-T treatment can be effectively avoided.
  • the TCR-modified immune cells of the present invention provide a new option for treating patients with HLA-A2 + and Her2 / neu + tumors, and have good industrial application prospects.
  • FIG. 1 shows a phenotype of a specific killer T cell of Her2 / neu 369-377 polypeptide (Her2-E75) induced from HLA-A2 + normal donor PBMC (specifically # 1PBMC) in Example 1 of the present invention And function test results.
  • Figure 1A is the result of flow cytometry analysis of PBMC cells stained with CD8-APC antibody and Her2-E75 pentamer-PE after two rounds of Her2-E75 antigen-peptide stimulation in vitro.
  • the right picture shows the peptide-stimulated cells.
  • CD8 + pentamer + killer T cell populations were subjected to FACS sorting to obtain T cell clones.
  • the left picture shows the control cells without peptide stimulation.
  • FIG. 1B is a phenotypic analysis of CD8 + E75-tetramer + killer T cell clones after CD8-APC and Her2-E75 tetramer-PE staining.
  • the right figure shows CD8 + Her2 tetramer + T cells.
  • the clone Her2 CTL 6A5 was a purified Her2-E75 peptide-specific CTL cell clone.
  • the left picture shows the control CTL cells without peptide stimulation.
  • FIG. 1C shows the main functional fragments of the constructed lentiviral vector carrying the Her2 TCR-6A5-mC gene (ie, “pCDH-EF1 ⁇ -Her2 TCR vector”).
  • the fragment shown expresses the TCR gene driven by the EF-1 ⁇ promoter.
  • the ⁇ and ⁇ chains of each TCR are invariant regions of mouse origin.
  • the ⁇ and ⁇ chains of TCR are cleavably linked.
  • the polypeptide coding sequence (furin-F2A) is linked.
  • FIG. 2 shows the results of phenotype and function detection of peripheral blood mononuclear cells (PBMC) transfected with Her2 TCR-6A5-mC TCR gene.
  • FIG. 2A shows the results of flow cytometry analysis after transfection of PBMC from two different donors with a lentiviral vector encoding Her2 TCR-6A5-mC, staining with Her2-E75 tetramer-PE and anti-CD8-APC antibody. Firstly, the lymphocyte population was separated according to the cell morphology and size. The Her2-E75 tetramer + cell population was cells expressing Her2 TCR-6A5-mC TCR.
  • the abscissa indicates the fluorescence intensity expressed by the CD8 molecule, and the ordinate indicates the fluorescence intensity of the bound Her2-E75 tetramer.
  • the percentages shown are the ratio of each positive cell population to the number of lymphocytes divided.
  • the left picture relates to peripheral blood mononuclear cells (# 1PBMC) provided by one donor, and the right picture relates to PBMC (# 2PBMC) provided by another donor.
  • CD8 + Her2-E75 tetramer + cells are killer T cells expressing Her2 TCR-6A5-mC.
  • CD8 - Her2-E75 tetramer + cells may be CD4 + helper T cells expressing Her2 TCR-6A5-mC.
  • FIG. 2B shows that T cells expressing Her2 TCR-6A5-mC can recognize the Her2-E75 polypeptide presented by T2 cells.
  • Two different donor PBMCs transfected with a lentiviral vector encoding Her2 TCR-6A5-mC were cultured for 16 hours with T2 cells presenting Her2-E75 polypeptides with different concentration gradients, and the cell supernatants were taken for IFN- ⁇ ELISA analysis.
  • the target cells in the control group were T2 cells (not shown in the figure) that presented the EBV virus antigen polypeptide LMP2 426-434 that could bind to the HLA-A2 molecule.
  • 0.1 ⁇ g / ml in the figure indicates a T2 cell group presenting a 0.1 ⁇ g / ml Her2-E75 polypeptide
  • 0.01 ⁇ g / ml indicates a T2 cell group presenting a 0.01 ⁇ g / ml Her2-E75 polypeptide
  • 0.001 “ ⁇ g / ml” means a T2 cell group that presents a 0.001 ⁇ g / ml Her2-E75 polypeptide
  • 0.0001 ⁇ g / ml means a T2 cell group that presents a Her2-E75 polypeptide of 0.0001 ⁇ g / ml.
  • the ordinate indicates the concentration of IFN- ⁇ secreted by T cells.
  • FIG. 2C shows the results of a CD8 antibody blocking test for T cell function.
  • # 2 PBMC transfected with a lentiviral vector encoding Her2 TCR-6A5-mC was co-cultured with Her2-E75 antigen polypeptide presented by T2 cells, and an anti-human CD8 antibody was added to test whether T cells secrete IFN- ⁇ function. suppressed.
  • T2 + Her2-E75 represents the T2 cell group presenting Her2-E75 polypeptide without anti-human CD8 antibody
  • T2 + Her2-E75 + anti-CD8 represents the addition of anti-human CD8 antibody.
  • the abscissa indicates different experimental groups, and the ordinate indicates the concentration of IFN- ⁇ secreted by T cells. "Ns" indicates no significant difference between the two experimental groups.
  • the experimental and control groups in Figures 2B and 2C are duplicated. The results are shown as the mean ⁇ SEM.
  • FIG. 3 shows the results of functional detection of peripheral blood mononuclear cells (PBMCs) transfected with Her2, TCR-6A5-mC, and TCR genes to identify tumor cell lines.
  • Figure 3A shows the expression of HLA-A2 and Her2 / neu by cells of different tumor cell lines.
  • the abscissa indicates different human tumor cell lines.
  • Colo205" and “HCT116” are colon cancer cells;
  • MDA-MB-231” and “MCF-7” are breast cancer cells;
  • PANC-1 is a pancreatic cancer cell;
  • U87MG is a glioma cell;
  • NCI-H446 is a lung cancer cell.
  • FIG. 3B shows the result of transfection of # 2 PBMC with lentiviral vector encoding Her2, TCR-6A5-mC, and TCR gene. After mixed culture with cells of different tumor cell lines for 24 hours, the cell supernatant was taken for IFN- ⁇ ELISA analysis. Each test group and control group are three wells, and the results are shown as the mean ⁇ SME.
  • the abscissa shows different target cells, and the ordinate shows the concentration of IFN- ⁇ secreted by T cells.
  • the effective target ratio E: T is 5: 1.
  • White bars show that the effector cells are control peripheral blood mononuclear cells that have not been transfected with the Her2 TCR-6A5-mC TCR gene, and black bars show that the effector cells are peripheral blood that has been transfected with the Her2 TCR-6A5-mC TCR gene Mononuclear cells.
  • Figures 3C, D, E, F, G, H, I, J, and K show that # 2PBMC was transfected with a lentiviral vector encoding the Her2TCR-6A5-mC TCR gene to different tumor cell lines. .
  • FIG. 3C and 3H show the results for the tumor cell line MCF-7
  • FIG. 3D shows the results for the tumor cell line HCT116
  • FIG. 3E shows the results for the tumor cell line U87MG
  • FIG. 3F shows the results for the tumor cell line Results of NCI-H446,
  • FIG. 3G shows the results for the tumor cell line SKOV3
  • FIG. 3I shows the results for the tumor cell line PANC-1
  • FIG. 3J shows the results for the tumor cell line HEPG2
  • FIG. 3K shows the results for the tumor Results for cell line HT-29.
  • Each test group and control group are three wells, and the results are shown as the mean ⁇ SME.
  • the abscissa shows different effective target ratios E: T.
  • the ordinate shows the percentage killing rate of T cells against target cells.
  • the dot-shaped diagram shows that the effector cells are control peripheral blood mononuclear cells that have not been transfected with the Her2 TCR-6A5-mC gene.
  • the upper triangle diagram shows that the effector cells are transfected with the Her2 TCR-6A5-mC gene.
  • peripheral blood mononuclear cells In the MTT killing experiment, the other group was added with paclitaxel 10 ⁇ M as a positive control (shown as separate lower triangle points in Figures 3H-3K).
  • FIG. 4 shows the results of the detection of the recognition function of the amino acid key sites on the Her2-E75 polypeptide recognized by Her2, TCR-6A5-mC and TCR, and the epitope polypeptide derived from human normal proteins with potential cross-reactivity.
  • FIG. 4A shows that nine new epitope polypeptides formed in Example 5 were mixed with T2 cells and # 2 PBMC transfected with a lentiviral vector encoding Her2TCR-6A5-mC TCR gene for 24 hours, and then the cells were removed. The supernatant was analyzed by ELISA for IFN- ⁇ detection. Each test group and control group were duplicated, and the results were shown as the mean ⁇ SME.
  • T2 cells present different epitope polypeptides T2 + polypeptides
  • E75 is Her2 / neu 369-377 polypeptide
  • E75-K1A Her2 / I2A
  • E75-F3A Her2 / neu 369-377 polypeptide
  • E75-G4A Her2 / neu amino acids at the corresponding positions of the 369-377 polypeptide were replaced by alanine
  • E75 "A7G” is the seventh alanine of the Her2 / neu 369-377 polypeptide replaced by glycine.
  • the ordinate shows the concentration of IFN- ⁇ secreted by T cells.
  • the effective target ratio E: T is 5: 1.
  • Fig. 4B shows the recognition function of Her2, TCR-6A5-mC, TCR, which recognizes epitope polypeptides derived from human normal proteins and has potential cross-reactivity.
  • E75 is a Her2 / neu 369-377 polypeptide
  • polypeptide "B” is an NSMA3 93-101 polypeptide
  • C is an O11A1 103-111 polypeptide
  • D is an SV2C 687-695 polypeptide.
  • PBMC transfected with a lentiviral vector encoding Her2, TCR-6A5-mC, and TCR gene was mixed and cultured with T2 cells presenting the polypeptide with different gradients for 24 hours, and the cell supernatant was taken for IFN- ⁇ ELISA analysis.
  • Each test group and control group are three wells, and the results are shown as the mean ⁇ SME.
  • the abscissa shows that T2 cells present epitope polypeptides at different concentrations. The ordinate indicates the concentration of IFN- ⁇ secreted by T cells.
  • FIG. 5 shows the main functional fragments of the Her2TCR-6A5-mC-PGKp-tEGFR lentiviral vector carrying the tEGFR gene and the Her2 TCR-6A5-mC gene constructed in Example 6.
  • FIG. The fragments shown include: 1) the expression of the TCR gene driven by the EF-1 ⁇ promoter, the ⁇ and ⁇ chain invariant regions of each TCR are mouse-derived constant region fragments, and the ⁇ and ⁇ chains of TCR are Linked by a cleavable linker coding sequence (furin-F2A);
  • Figure 6 shows the killing activity of human T cells expressing Her2, TCR-6A5-mC and TCR and human T cells simultaneously expressing Her2, TCR-6A5-mC and tEGFR in experiment of Example 7 against different human tumor cell lines, of which Figure 6A shows The experimental results for the tumor cell line C33A are shown, FIG. 6B shows the experimental results for the tumor cell line CFPAC-1, and FIG. 6C shows the experimental results for the tumor cell line Saos-2. Each test group and control group are three wells, and the results are shown as the mean ⁇ SME. 6A-C abscissas show different effect target ratios E: T. The ordinate shows the percentage value of cell killing rate.
  • the dot-shaped figure shows that the effector cells are control human T cells that have not been transfected with any TCR.
  • the square figure shows that the effector cells are human T cells that express Her2, TCR-6A5-mC, TCR, and the upper triangle shows the effector cells.
  • FIG. 7 shows the change curve of tumor volume of each group of animals in the experiment of Example 8.
  • the abscissa is the time (days) after administration, and the ordinate is the tumor volume (mm 3 ).
  • the words “tumor”, “cancer”, “tumor cell”, “cancer cell”, “T cell”, “T cell receptor”, “T cell receptor modification”, “TCR variable region” , “TCR constant region”, “antigen”, “antigenic epitope polypeptide”, “homologous sequence”, “coding”, “antigen presentation”, “recombinant DNA expression vector”, “promoter”, “complementary sequence” “Transfection”, “autologous”, “allogene”, “specific recognition”, “TCR-T therapy” encompasses the meanings commonly understood in the art.
  • Her2 / neu antigens are tumor-associated antigens. Most of the high-affinity T cells that recognize Her2 / neu antigens are cleared by the central tolerance mechanism to avoid possible autoimmune reactions (see the literature “Immunol Rev. 2016; 271 (1 ): 127-40 "). Therefore, it has become very difficult to induce T cell clones from peripheral blood T cell banks that specifically recognize Her2 / neu antigen expressed by tumor cells.
  • Her2 / neu 369-377 polypeptide naturally presented in the cell may be glycosylated and cause differences in TCR recognition configuration (see the document “Proc.Natl.Acad.Sci.USA 2003; 100: 15029–15034 ").
  • high-affinity T cell clones that can only recognize foreign-loaded antigen peptides often gain dominant expansion, and can specifically recognize cells that have been extracted by cells.
  • the growth of T cell clones of the present endogenous Her2 / neu antigen polypeptide is inhibited (see the document "J Exp Med.
  • TCR-T therapy based on allo-TCR is at risk of generating allo-reactions against other normal autoprotein epitopes (see documents "Int. J. Cancer 2009; 125,649–655", “Nat Immunol 2007; 8: 388–97 ").
  • Another research team induced Her2 / neu 369-377 peptide-specific T cells from the peripheral blood of tumor patients immunized with the Her2 / neu 369-377 peptide vaccine, and paired alpha and beta chains derived from different T cells and matched them.
  • T cells transfected with this high-affinity TCR could recognize a variety of HLA-A2 + Her2 / neu + tumor cells (see the literature "HUMAN GENE THERAPY 2014; 25: 730–739” ).
  • This TCR is not obtained from monoclonal T cells, so it cannot be determined whether this TCR is a natural TCR that exists in a T cell bank that has been screened for central tolerance.
  • point mutations in the functional regions of TCR-recognizing epitope polypeptides can be used to screen high-affinity TCRs.
  • Her2 / neu protein is also expressed in trace amounts in important organs such as myocardium, lung, esophagus, kidney, and bladder (see the document "Oncogene. 1990Jul; 5 (7): 953-62"), non-natural High affinity Her2 / neu antigen-specific TCRs are at risk for off-target toxicity in normal tissues.
  • Tumor cells highly express Her2 / neu protein, so the number of antigenic polypeptides presented by HLA on the cell surface will increase accordingly. The difference in the number of HLA / antigen-polypeptide complexes on tumor cells and normal cells can make specific T cells distinguish normal. And tumor tissue window.
  • the invention proposes to obtain the sequence of natural TCR from an auto-T cell repertoire, and then express TCR on T cells in vitro, so that the TCR-expressing T cells can recognize tumor cells to increase expression.
  • Her2 / neu antigen is the key to the successful development of effective and low-toxicity TCR-T therapy.
  • the present invention induces Her2 / neu presentation of HLA-A2 from peripheral blood of HLA-A2-positive healthy donors.
  • the peptide has specific T-cell clones, and T-cell clones carrying natural TCRs with moderate affinity for Her2 / neu 369-377 polypeptides were selected from the clones.
  • the present invention also does not adopt the method of other research groups to induce polypeptide-specific T cells from HLA-A2-negative peripheral blood (see the document "The Journal of Immunology, 2010, 184: 1617-1629"), although it is easier to obtain it from allogeneic PBMCs. Allo-T cells with high affinity and recognition of Her2 / neu 369-377 polypeptide antigens were obtained, but this also increased the T cell cross recognition of other polypeptides presented by HLA-A2 molecules and caused an allogeneic response.
  • the present invention provides an isolated T cell receptor, including at least one of an alpha chain and a beta chain, both of which include a variable region and a constant region, and are characterized in that,
  • the T cell receptor can specifically recognize the antigen Her2 / neu expressed by tumor cells, and the amino acid sequence of the variable region of the ⁇ chain has at least 98%, preferably the amino acid sequence shown in SEQ ID NO: 1.
  • At least 98.5%, more preferably at least 99% identity the amino acid sequence of the variable region of the ⁇ chain has at least 98%, preferably at least 98.5%, more preferably at least 98.5% of the amino acid sequence shown in SEQ ID NO: 2 99% consistency as long as it does not significantly affect the effect of the invention.
  • the amino acid sequence of the variable region of the ⁇ chain is shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the ⁇ chain is shown in SEQ ID NO: 2.
  • variable regions of the TCR alpha and beta chains are used to bind the antigen-polypeptide / major histocompatibility complex (MHC I) and include three hypervariable regions or complementarity determining regions (CDRs), respectively, that is, CDR1, CDR2, CDR3.
  • CDRs complementarity determining regions
  • the CDR3 region is essential for specifically recognizing antigenic polypeptides presented by MHC molecules.
  • TCR ⁇ chain is recombined from different V and J gene fragments, while ⁇ chain is recombined from different V, D and J gene fragments.
  • the MHC class I molecule includes human HLA.
  • the HLA includes: HLA-A, B, and C.
  • the T cell receptor can specifically recognize the antigen epitope polypeptide of the antigen Her2 / neu presented by the HLA-A2 molecule.
  • the amino acid sequence of the antigen Her2 / neu is shown in SEQ ID NO: 21.
  • the epitope polypeptide includes Her2 / neu 369-377 as shown in SEQ ID NO: 3.
  • HLA-A2 alleles expressed by HLA-A2 positive cells include HLA-A * 0201, 0202, 0203, 0204, 0205, 0206, and 0207.
  • the HLA-A2 molecule is preferably HLA-A * 0201.
  • the epitope polypeptide of the antigen Her2 / neu is a Her2 / neu 369-377 polypeptide (SEQ ID NO: 3).
  • the epitope polypeptide of the antigen Her2 / neu is 4-9 consecutive identical amino acids (e.g., 4, 5, 6, 7, 8 or 9) as the Her2 / neu 369-377 polypeptide. Epitope polypeptides of consecutive identical amino acids), and these polypeptides are 8-11 amino acids in length.
  • the epitope polypeptide of the antigen Her2 / neu is a Her2 / neu373-382 polypeptide (SEQ ID NO: 22).
  • the maximum half-reactive polypeptide concentration of the T cell receptor-recognizing Her2 / neu 369-377 polypeptide is between 1.0-10 ng / ml. In one embodiment of the invention, the maximum semi-reactive polypeptide concentration is about 1.6 ng / ml-2.9 ng / ml.
  • maximum half-reactive polypeptide concentration refers to the concentration of the polypeptide required to induce T cell response to 50% of the maximum.
  • the maximum half-reactive polypeptide concentration of specific T cells against cytomegalovirus (CMV) antigen CMV pp65 (495-503) polypeptide is between 0.1-1 ng / ml, and this TCR is considered to have high CMV antigen polypeptide Affinity (see document "Journal of Immunogical Methds 2007; 320: 119-131").
  • the T cell receptor has a medium to high affinity for the Her2 / neu antigen, thereby avoiding off-target toxicity that may be caused by high affinity (the maximum half-reactive polypeptide concentration is less than 0.1 ng / ml).
  • the T cell receptor recognizes the Her2 / neu 369-377 polypeptide does not rely on the help of CD8 molecules.
  • CD8 negative CD4 positive T cells expressing the T cell receptor can also specifically recognize Her2 presented by HLA-A2 / neu 369-377 polypeptide secretes cytokines, thereby enhancing the function of killer T cells expressing the T cell receptor.
  • exogenous TCR ⁇ and ⁇ chains expressed by T cells may mismatch with the ⁇ and ⁇ chains of the TCR itself, which will not only dilute the expression of correctly paired exogenous TCRs, but also the antigen specificity of the mismatched TCRs. It is clear that there is a potential danger of recognizing self-antigens, so it is preferable to modify the constant regions of the TCR ⁇ chain and ⁇ chain to reduce or avoid mismatches.
  • the constant region of the ⁇ chain and / or the constant region of the ⁇ chain are derived from humans; preferably, the present invention finds that the constant region of the ⁇ chain may be wholly or partially Grounds are replaced by homologous sequences derived from other species, and / or the constant region of the beta strand may be replaced in whole or in part by homologous sequences derived from other species. More preferably, the other species is a mouse.
  • the replacement can increase the expression of TCR in the cell, and can further increase the specificity of the cells modified by the TCR to the Her2 / neu antigen.
  • the constant region of the alpha chain may be modified with one or more disulfide bonds, and / or the constant region of the beta chain may be modified with one or more disulfide bonds, such as one or two.
  • two differently modified TCRs are prepared.
  • One way is to add a disulfide bond to the TCR constant region by point mutation.
  • the method is described in the document "Cancer Res. 2007Apr 15; 67 (8): 3898-903. ", which is incorporated herein by reference in its entirety.
  • Her2TCR-1B5-mC is the replacement of the corresponding human TCR constant region sequence with the mouse TCR constant region sequence. The method is described in the document "Eur. J. Immunol. 2006 36: 3052-3059", which is incorporated by reference in its entirety. This article.
  • amino acid sequence of the ⁇ chain is shown in SEQ ID NOs: 4, 5, or 6, and the amino acid sequence of the ⁇ chain is shown in SEQ ID NOs: 7, 8, or 9.
  • the sequence is the original human sequence; for the ⁇ chain shown in SEQ ID No: 5, the amino acid sequence is modified by one two in the constant region. Sulfur bond; for an ⁇ chain whose amino acid sequence is shown in SEQ ID NO: 6, the constant region is replaced with a mouse-derived constant region.
  • the sequence is the original human sequence; for the ⁇ chain shown in SEQ ID NO: 8, the amino acid sequence is modified by one two in the constant region. Sulfur bond; for ⁇ chain with amino acid sequence as shown in SEQ ID NO: 9, its constant region is replaced with mouse-derived constant region.
  • the amino acid sequence of the alpha chain of the TCR is shown in SEQ ID NO: 4, and the amino acid sequence of the beta chain is shown in SEQ ID NO: 7.
  • the amino acid sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 5
  • the amino acid sequence of the ⁇ chain is shown in SEQ ID NO: 8.
  • the amino acid sequence of the alpha chain of the TCR is shown in SEQ ID NO: 6, and the amino acid sequence of the beta chain is shown in SEQ ID NO: 9.
  • the alpha chain of the TCR has an amino acid sequence obtained by replacing, deleting, and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID NOs: 4, 5, or 6.
  • the ⁇ chain has at least 90%, preferably at least 95%, and more preferably at least 99% identity with the amino acid sequence shown in SEQ ID NOs: 4, 5, or 6.
  • the ⁇ chain of the TCR has an amino acid sequence obtained by replacing, deleting, and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID NOs: 7, 8 or 9 ;
  • the ⁇ chain has at least 90%, preferably at least 95%, more preferably at least 99% identity with the amino acid sequence shown in SEQ ID NOs: 7, 8 or 9.
  • the alpha and / or beta chains of the TCR of the present invention may also bind other functional sequences at the ends (such as the C-terminus), such as the functional sequence of the co-stimulatory signals CD28, 4-1BB, and / or CD3zeta.
  • the invention also provides an isolated nucleic acid encoding a T cell receptor, comprising a coding sequence of at least one of an alpha chain and a beta chain of the T cell receptor, the alpha chain coding sequence and the beta chain encoding
  • the sequences each include a variable region coding sequence and a constant region coding sequence, characterized in that the T cell receptor can specifically recognize the antigen Her2 / neu expressed by a tumor cell, and the amino acid encoded by the ⁇ chain variable region coding sequence
  • the sequence has at least 98%, preferably at least 98.5%, and more preferably at least 99% identity with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence encoded by the ⁇ -chain variable region coding sequence has the same identity as SEQ ID NO:
  • the amino acid sequence shown in 2 has at least 98%, preferably at least 98.5%, and more preferably at least 99% identity, as long as the effect of the present invention is not significantly affected. It is also preferred that the ⁇ -chain variable region
  • the nucleic acid may be DNA or RNA.
  • the ⁇ -chain variable region coding sequence is shown in SEQ ID NO: 10, and the ⁇ -chain variable region coding sequence is shown in SEQ ID NO: 11.
  • the T cell receptor encoded by the nucleic acid can specifically recognize an epitope polypeptide of the antigen Her2 / neu presented by an HLA-A2 molecule.
  • the epitope polypeptide of the antigen Her2 / neu is a Her2 / neu 369-377 polypeptide (SEQ ID NO: 3).
  • the epitope polypeptide of the antigen Her2 / neu is 4-9 consecutive identical amino acids (e.g., 4, 5, 6, 7, 8 or 9) as the Her2 / neu 369-377 polypeptide. Epitope polypeptides of consecutive identical amino acids), and these polypeptides are 8-11 amino acids in length.
  • the epitope polypeptide of the antigen Her2 / neu is a Her2 / neu373-382 polypeptide (SEQ ID NO: 22).
  • the maximum half-response polypeptide concentration of the Her2 / neu 369-377 polypeptide recognized by the T cell receptor encoded by the nucleic acid is between 1.0-10 ng / ml (e.g., 3.0-8.0 ng / ml, 5.0- 7.0ng / ml). In one embodiment of the invention, the maximum semi-reactive polypeptide concentration is about 1.6-2.9 ng / ml. In this case, the T cell receptor has medium to high affinity for Her2 / neu antigen, which can avoid off-target toxicity that may be caused by high affinity (the maximum half-reactive polypeptide concentration is less than 0.1 ng / ml).
  • the constant region of the ⁇ chain and / or the constant region of the ⁇ chain are derived from humans; preferably, the ⁇ chain constant region coding sequence is wholly or partially derived from other The homologous sequence of a species is replaced, and / or the ⁇ -chain constant region coding sequence is replaced in whole or in part by a homologous sequence derived from another species. More preferably, the other species is a mouse.
  • the replacement can increase the expression of TCR in the cell, and can further increase the specificity of the cells modified by the TCR to the Her2 / neu antigen.
  • the alpha chain constant region coding sequence may include one or more disulfide bond coding sequences, and / or the beta chain constant region coding sequence may include one or more disulfide bond coding sequences.
  • the ⁇ -chain coding sequence is shown in SEQ ID NOs: 12, 13, or 14, and the ⁇ -chain coding sequence is shown in SEQ ID NOs: 15, 16, or 17.
  • the sequence is the original human sequence; for the ⁇ chain shown in SEQ ID NO: 13, it is modified with one two in the constant region. Sulfur bond; for an ⁇ chain whose coding sequence is shown in SEQ ID NO: 14, its constant region is replaced with a mouse-derived constant region.
  • the sequence is the original human sequence; for the ⁇ chain shown in SEQ ID NO: 16, the sequence is modified by one two Sulfur bond; for a ⁇ chain whose coding sequence is shown in SEQ ID NO: 17, its constant region is replaced with a mouse-derived constant region.
  • the coding sequence of the alpha chain of the TCR is shown in SEQ ID NO: 12, and the coding sequence of the beta chain is shown in SEQ ID NO: 15.
  • the coding sequence of the alpha chain of the TCR is shown in SEQ ID NO: 13
  • the coding sequence of the beta chain is shown in SEQ ID NO: 16.
  • the coding sequence of the alpha chain of the TCR is shown in SEQ ID NO: 14, and the coding sequence of the beta chain is shown in SEQ ID NO: 17.
  • the ⁇ -chain coding sequence and the ⁇ -chain coding sequence are linked by a coding sequence of a cleavable linking polypeptide, which can increase the expression of TCR in a cell.
  • cleavable linked polypeptide means that the polypeptide plays a connecting role and can be cleaved by a specific enzyme, or the nucleic acid sequence encoding the polypeptide is translated by ribosome skipping, so that it is linked by The polypeptides are separated from each other.
  • cleavable linking polypeptides are known in the art, such as F2A polypeptides, F2A polypeptide sequences include, but are not limited to, F2A polypeptides from picornaviruses, and similar class 2A sequences from other viruses.
  • the cleavable / ribosome jumping 2A linking sequences can come from different viral genomes, including F2A (foot-and-mouth disease virus 2A), T2A (thosea signa virus 2A), P2A (piglet virus 1 Porcine teschovirus-1 2A) and E2A (equine rhinitis A virus 2A).
  • the cleavable linking polypeptide also includes a standard four amino acid motif that can be cleaved by the Furin enzyme, namely the R-X- [KR] -R amino acid sequence.
  • the TCR encoded by this embodiment is a single-chain chimeric T cell receptor. After expression of the single-chain chimeric T cell receptor is completed, the cleavable linking polypeptide connecting the ⁇ chain and the ⁇ chain will be cleaved by a specific enzyme in the cell. , Thereby forming equal amounts of free ⁇ and ⁇ chains.
  • the alpha and beta chains that make up a single-chain chimeric TCR can also be replaced as described above.
  • the constant region (and its corresponding coding sequence) is replaced in whole or in part by homologous sequences derived from other species, and / or modified with (Encodes) one or more disulfide bonds.
  • sequence of the nucleic acid is as shown in SEQ ID NOs: 18, 19, or 20.
  • the nucleotide sequence of the nucleic acid is coded to increase gene expression, protein translation efficiency, and protein expression, thereby enhancing TCR's ability to recognize antigens.
  • Codon optimization includes, but is not limited to, modification of the translation initiation region, alteration of mRNA structural fragments, and use of different codons encoding the same amino acid.
  • the TCR-encoding nucleic acid sequence can be mutated, including removing, inserting, and / or replacing one or more amino acid codons, so that the expressed TCR recognizes Her2 / neu antigen's function unchanged or enhanced.
  • a conservative amino acid substitution is performed, including replacing one amino acid in the variable region of the TCR alpha chain and / or beta chain described above with another amino acid having similar structural and / or chemical properties.
  • similar amino acids refers to amino acid residues having similar properties as polarity, electrical load, solubility, hydrophobicity, hydrophilicity and the like.
  • the mutated TCR still has the biological activity of recognizing the aforementioned Her2 / neu antigen polypeptide presented by the target cells.
  • a TCR maturation modification is performed, that is, an amino acid of the complementarity determining region 2 (CDR2) and / or CDR3 region in the variable region of the TCR ⁇ and / or ⁇ chain described above is performed. Removal, insertion, and / or substitution, thereby altering the affinity of the TCR for binding to the Her2 / neu antigen.
  • the invention also provides an isolated mRNA transcribed from the DNA according to the invention.
  • the present invention also provides a recombinant expression vector containing a nucleic acid (such as DNA) according to the present invention operatively linked to a promoter, and / or a complementary sequence thereof.
  • a nucleic acid such as DNA
  • the DNA according to the present invention is suitably operably linked to a promoter, an enhancer, a terminator and / or a polyA signal sequence.
  • the combination of the above-mentioned action elements of the recombinant expression vector of the present invention can promote the transcription and translation of DNA and enhance the stability of mRNA.
  • the basic backbone of the recombinant expression vector can be any known expression vector, including plasmids or viruses.
  • Viral vectors include, but are not limited to, for example, retroviral vectors (the virus prototype is Moloney Murine Leukemia Virus (MMLV)) and lentivirus.
  • Vector viral prototype is human immunodeficiency virus type 1 (HIV)).
  • Recombinant vectors expressing the TCR of the present invention can be obtained by conventional recombinant DNA technology in the art.
  • the expression of the ⁇ -chain and ⁇ -chain genes on the recombinant expression vector can be driven by two different promoters.
  • the promoters include various known types, such as strongly expressed, weakly expressed, sustained Expressed, inducible, tissue-specific, and differentiation-specific promoters.
  • Promoters can be of viral or non-viral origin (such as eukaryotic cell promoters), such as the CMV promoter, the promoter on the MSCV LTR, the EF1- ⁇ promoter, and the PGK-1 promoter, the SV40 promoter , Ubc promoter, CAG promoter, TRE promoter, CaMKIIa promoter, human ⁇ -actin promoter.
  • the two promoters can be driven in the same direction or in the opposite direction.
  • the expression of the ⁇ -chain and ⁇ -chain genes on the recombinant expression vector can be driven by the same promoter, for example, in the case of encoding a single-chain chimeric T cell receptor, the nucleotide sequence of the ⁇ -chain and The ⁇ -chain nucleotide sequence is linked by the Furin-F2A polypeptide coding sequence.
  • the recombinant expression vector may include coding sequences of other functional molecules in addition to the alpha chain and beta chain genes.
  • an autofluorescent protein such as GFP or other fluorescent protein
  • Another embodiment includes expressing an inducible suicide gene system, such as inducing expression of a herpes simplex virus-thymidine kinase (HSV-TK) protein, or inducing expression of a Caspase 9 (iCasp9) protein.
  • HSV-TK herpes simplex virus-thymidine kinase
  • iCasp9 Caspase 9
  • the recombinant expression vector may contain a suicide gene coding sequence, and the suicide gene may be selected from the group consisting of: iCasp9, HSV-TK, mTMPK, truncated EGFR, truncated CD19, truncated CD20, or a combination thereof.
  • the suicide gene coding sequence is under the control of a promoter, and the promoter for controlling the suicide gene coding sequence and the promoter to which the nucleic acid according to the present invention is linked may be the same or different, and are independent of each other.
  • the suicide gene coding sequence and the nucleic acid according to the present invention are under the control of the same promoter, and the suicide gene coding sequence can be linked to the coding sequence of the polypeptide or the internal ribosome entry site through cleavability.
  • IRS internal ribosome entry site
  • the coding sequence of the cleavable linking polypeptide may be the cleavable / ribosomal hopping described above.
  • 2A linker sequence which can come from different viral genomes, including F2A, T2A, P2A, and E2A.
  • Another embodiment includes the expression of human chemokine receptor genes, such as CCR2, which can bind to tumor tissue.
  • CCR2 human chemokine receptor genes
  • Corresponding chemokine ligands with high and medium expression can increase the homing of cells modified by the TCR gene according to the present invention in tumor tissues.
  • the present invention also provides a T cell receptor modified cell, the surface of which is modified by the T cell receptor according to the present invention, wherein the cell includes a primitive T cell or a precursor cell thereof, a NKT cell, or a T cell. Cell line.
  • the "modification" in the "T cell receptor modification” means that the cell expresses the T cell receptor according to the present invention by gene transfection, that is, the T cell receptor is anchored in a transmembrane region.
  • the modified cell has the function of recognizing the antigen polypeptide / MHC complex on the cell membrane.
  • the T cell receptor modified cell expresses a suicide gene protein on its cell surface or in the cell, and the suicide gene may be selected from the group consisting of: iCasp9, HSV-TK, mTMPK, truncated EGFR, truncated CD19, truncated CD20, or a combination thereof. This can increase the safety of the T cell receptor modified cells for use in vivo.
  • the invention also provides a method for preparing a T cell receptor modified cell according to the invention, comprising the following steps:
  • the cells in step 1) can be derived from mammals, including humans, dogs, mice, rats and their transgenic animals.
  • the cells may be autologous or allogeneic.
  • Allogeneic cells include cells from identical twins, allogeneic stem cells, and genetically modified allogeneic T cells.
  • the cells in step 1) include primitive T cells or their precursor cells, NKT cells, or T cell lines.
  • the term "naive T cell” refers to a mature T cell in peripheral blood that has not been activated by the corresponding antigen.
  • These cells can be isolated by methods known in the art.
  • T cells can be obtained from different tissues and organs, including peripheral blood, bone marrow, lymphoid tissue, spleen, umbilical cord blood, and tumor tissue.
  • the T cells can be derived from hematopoietic stem cells (HSCs), including from bone marrow, peripheral blood, or umbilical cord blood, and isolated by stem cell marker molecules such as CD34.
  • HSCs hematopoietic stem cells
  • the T cells can be derived from induced pluripotent stem cells (iPS cells), including introducing specific genes or specific gene products into somatic cells, and converting the somatic cells into stem cells, and then inducing differentiation into T cells or their precursors in vitro. cell.
  • T cells can be obtained by common methods such as density gradient centrifugation. Examples of density gradient centrifugation include Ficoll or Percoll density centrifugation.
  • density gradient centrifugation include Ficoll or Percoll density centrifugation.
  • One embodiment is to obtain an enriched T cell product from peripheral blood using apheresis or leukapheresis.
  • One embodiment is to label specific cell populations with antibodies and then use magnetic bead separation (such as System (Miltenyi Biotec), or flow cytometry to obtain enriched CD8 + or CD4 + T cells.
  • the T cell precursor cell is a hematopoietic stem cell.
  • the coding gene of the TCR according to the present invention can be directly introduced into hematopoietic stem cells and then transduced into the body to further differentiate into mature T cells; the coding gene can also be introduced into T cells differentiated and matured from hematopoietic stem cells under specific conditions in vitro.
  • the cells can be resuspended in cryopreserved solution and stored in liquid nitrogen.
  • cryopreserved solutions include, but are not limited to, PBS solutions containing 20% DMSO and 80% human serum albumin.
  • the cells were frozen at -80 ° C at a temperature lowered by 1 ° C per minute, and then stored in the gas phase portion of the liquid nitrogen tank.
  • Other cryopreservation methods are to directly place cells in cryopreservation solution at -80 ° C or liquid nitrogen for cryopreservation.
  • the nucleic acid is a nucleic acid according to the present invention, including the DNA and RNA.
  • the transfection includes physical, biological, and chemical means.
  • the physical method is to introduce the TCR gene into the cell in the form of DNA or RNA through calcium phosphate precipitation, liposomes, microinjection, electroporation, and gene gun.
  • electrotransfer instruments such as Amaxa Nucleofector-II (Amaxa Biosystems, Germany), ECM 830 (BTX) (Harvard Instruments, USA), Gene Pulser II (BioRad, USA), Multiporator (Eppendort, Germany) ).
  • the biological method is to introduce TCR genes into cells through DNA or RNA vectors.
  • Retroviral vectors are common tools for transfection and insertion of foreign gene fragments into animal cells (including human cells).
  • the vectors are derived from lentivirus, poxvirus, herpes simplex virus, adenovirus and adenovirus-related viruses.
  • the chemical approach is to introduce polynucleotides into cells, including colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, microbeads, micelles, and liposomes.
  • various detection methods are used to analyze whether the target gene is introduced into the target cell.
  • the detection methods include common molecular biological methods (such as Southern and Northern blots, RT-PCR, PCR, etc.) ), Or common biochemical methods (such as ELISA and Western blotting), and the methods mentioned in the present invention.
  • the transfection is performed by a retroviral vector or a lentiviral vector.
  • T cells can be expanded in vitro by co-activation of the TCR / CD3 complex on the surface and co-stimulatory molecules such as CD28.
  • Stimulators that activate TCR, CD3, and CD28 can be adsorbed on the surface of a culture container, or the surface of a co-culture (such as magnetic beads), or directly added to the cell culture solution for co-culture.
  • Another embodiment is co-culturing T cells with trophoblasts, which express co-stimulatory molecules or corresponding ligands, including but not limited to HLA-A2, ⁇ 2-microglobulin, CD40, CD83, CD86, CD127, 4-1BB.
  • co-stimulatory molecules or corresponding ligands including but not limited to HLA-A2, ⁇ 2-microglobulin, CD40, CD83, CD86, CD127, 4-1BB.
  • T cells are cultured and expanded under appropriate culture conditions.
  • the cells can be passaged when they reach a confluence of more than 70%.
  • fresh culture medium is replaced in 2 to 3 days.
  • In vitro culture can be performed within 24 hours or as long as 14 days or longer. The thawed cells can be used for the next application after thawing.
  • the cells can be cultured in vitro for several hours to 14 days, or any number of hours in between.
  • T cell culture conditions include the use of basal medium, including but not limited to RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo.
  • Conditions required for cell survival and proliferation include, but are not limited to, the use of serum (human or fetal bovine serum), interleukin-2 (IL-2), insulin, IFN- ⁇ , IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGF- ⁇ and TNF-a, other culture additives (including amino acids, sodium pyruvate, vitamin C, 2-mercaptoethanol, growth hormone, growth factors) .
  • IL-2 interleukin-2
  • insulin insulin
  • IFN- ⁇ interleukin-2
  • IL-4 interleukin-7
  • GM-CSF GM-CSF
  • IL-10 interleukin-12
  • IL-15 IL-21
  • TGF- ⁇ and TNF-a other culture additives
  • Can be placed in an appropriate cell culture conditions e.g., temperature may be at 37 °C, 32 °C, 30 °C or room temperature, atmospheric conditions and may be (e.g.) containing 5% CO 2
  • the invention also provides the use of T cell receptor modified cells according to the invention in the manufacture of a medicament for the treatment or prevention of tumors and / or cancers.
  • the tumor and / or cancer is antigen Her2 / neu positive and HLA-A2 positive, including but not limited to breast cancer, ovarian cancer, gastric cancer, esophageal cancer, colon cancer, pancreatic cancer, bladder cancer, kidney cancer, Prostate cancer, cervical cancer, endometrial cancer, salivary gland cancer, skin cancer, lung cancer, bone cancer, and brain cancer.
  • the invention also provides the use of T cell receptor modified cells according to the invention in the preparation of a medicament for detecting tumors and / or cancers in a host.
  • a sample of tumor and / or cancer cells removed from the host can be contacted with the T cell receptor-modified cells of the present invention at a certain concentration, and it can be judged based on the degree of reaction between the two Whether the tumor and / or cancer is HLA-A2 positive or HLA-A2 negative, and whether the antigen Her2 / neu is expressed.
  • the present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition comprises, as an active ingredient, a T cell receptor modified cell according to the present invention, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition preferably comprises the T cell receptor modified cells in a total dose range of 1 ⁇ 10 3 to 1 ⁇ 10 9 cells / Kg body weight per patient per course, including any between the two endpoints. Number of cells.
  • each course of treatment is 1-3 days and is administered 1-3 times a day. Patients can be treated with one or more courses according to actual conditions and needs.
  • the pharmaceutically acceptable excipients include medicinal or physiological carriers, excipients, diluents (including physiological saline, PBS solution), and various additives, including sugars, lipids, polypeptides, amino acids, antioxidants, adjuvants, Preservatives, etc.
  • the pharmaceutical composition can be administered by a suitable route of administration, which is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, intramuscular, or peritoneal Within administration.
  • the present invention also provides a method for treating tumors and / or cancers, comprising administering a T cell receptor modified cell according to the present invention to a tumor and / or cancer patient.
  • the tumor and / or cancer is antigen Her2 / neu positive and HLA-A2 positive, including but not limited to breast cancer, ovarian cancer, gastric cancer, esophageal cancer, colon cancer, pancreatic cancer, bladder cancer, kidney cancer, Prostate cancer, cervical cancer, endometrial cancer, salivary gland cancer, skin cancer, lung cancer, bone cancer, and brain cancer.
  • the administered dose of the T-cell receptor-modified cells is preferably a total dose range of 1 ⁇ 10 3 to 1 ⁇ 10 9 cells / Kg body weight per patient per course of treatment.
  • each course of treatment is 1-3 days and is administered 1-3 times a day.
  • Patients can be treated with one or more courses according to actual conditions and needs.
  • the T cell receptor modified cells can be administered by a suitable administration route, which is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, Intramuscular or intraperitoneal administration.
  • the T cell receptor modified cells can eliminate tumor cells expressing Her2 / neu antigen after entering the body of the treatment subject, and / or change the microenvironment of tumor tissue and induce other anti-tumor immune responses.
  • the method for treating tumors and / or cancers further comprises administering to the tumors and / or cancer patients other drugs for treating tumors, and / or drugs for regulating the immune system of patients to enhance the T cell receptor modified The number and function of cells in the body.
  • the other drugs for treating tumors include, but are not limited to: chemotherapeutic drugs, such as cyclophosphamide, fludarabine; radiotherapy; immunosuppressive agents, such as cyclosporine, azathioprine, methotrexate, Mycophenolate, FK50; antibodies, such as antibodies against CD3, IL-2, IL-6, IL-17, TNF ⁇ .
  • the method for treating tumors and / or cancers further comprises administering to the patient other drugs for treating tumors and / or cancers, and / or drugs for regulating the patient's immune system, and
  • the T cell receptor-modified immune cell has serious toxic and side effects, the number and function of the T cell receptor-modified immune cell carrying the suicide gene are eliminated in the body.
  • the other drugs for treating tumors and / or cancers include, but are not limited to: chemically induced dimerization (CID) drugs, AP1903, phosphorylated ganciclovir, anti-Cd20 antibodies, anti-CMYC antibodies, anti-EGFR antibody.
  • the percentage concentration (%) of each reagent refers to the volume percentage concentration (% (v / v)) of the reagent.
  • the cell line used to prepare the lentiviral particles is 293T cells (ATCC CRL-3216).
  • the presenting cell line for presenting the antigen polypeptide is a T2 cell (174xCEM.T2, ATCC CRL-1992).
  • the tumor cell lines used for detection are human colorectal cancer colo205 cells (ATCC CCL-222), HT-29 cells (HTB-38) and HCT116 cells (ATCC CCL-247), human breast cancer MDA-MB-231 cells ( ATCC (HTB-26) and MCF7 cells (ATCCHTB-22), human ovarian cancer SKOV3 cells (ATCC, HTB-77), human pancreatic cancer PANC-1 cells (ATCCCRL-1469), human glioblastoma U87MG cells (ATCC, ATHT) -14), human hepatocellular carcinoma HepG2 cells (ATCC HB-8065), human non-small cell lung cancer NCI-H460 cells (ATCC HTB177) and small cell lung cancer NCI-H4
  • Peripheral blood products Unless otherwise specified, human peripheral blood products (including peripheral blood mononuclear cells) from healthy donors used in the following tests were obtained from the Pacific Blood Center in San Francisco (# 1PBMC and # 2. PBMC are from the Apheresis method collection kit, respectively Trima residual cell components # R32334 and # R33941).
  • Counting by trypan blue staining After washing the cells with PBS, trypsinize them, suspend the cells in PBS, add trypan blue staining solution with a final concentration of 0.04% (w / v), and count under the microscope. Dead cells will Stained light blue, living cells resist staining. Take the number of live cells as the final data.
  • RNA of positive cells was extracted, reverse transcribed into cDNA and cloned into the vector, and then HLA gene sequencing analysis was performed to determine that the cell type was HLA-A * 0201.
  • HLA-A2-positive PBMC cells were cultured in the culture wells of a 24-well culture plate, and the culture medium was the above-mentioned RPMI-1640 complete medium.
  • antigen restimulation of the cultured T cells Add 10e6 cultured cells obtained in each well to a 24-well plate, and add 2 ⁇ 10e6 25 ⁇ g / ml mitomycin C (Santa Cruz Biotechnology, cat # SC-3514) treated with HLA-A2-positive PBMC cells for 2 hours as trophoblasts, adding Her2 / neu 369-377 peptide at a final concentration of 1 ⁇ g / ml to each well, and adding IL-2 after overnight culture 100IU / ml, IL-7 5ng / ml, IL-15 5ng / ml (final concentration). After two rounds of the above antigen stimulation and restimulation, the expanded T cells were collected for phenotypic analysis and T cell cloning.
  • T cells expressing Her2 / neu 369-377-specific TCR were analyzed by flow cytometry. Collect the cells to be tested and place them in 1.5ml tubes (the number of cells is about 10e5), and use 1ml DPBS solution (2.7mM KCl, 1.5mM KH 2 PO 4 , 136.9mM NaCl, 8.9mM Na 2 HPO 4 ⁇ 7H 2 O, (pH 7.4) Wash once and reset to 100 ⁇ l DPBS containing 1% calf serum, add 5 ⁇ l fluorescein APC-labeled anti-human CD8 antibody (Biolegend, cat # 300912), and 10 ⁇ l fluorescein PE-labeled Her2- E75 / HLA-A2 tetramer (Her2-E75 tetramer, MBL International Co., cat # T01014) or Her2-E75 / HLA-A2 pentamer (Her2-E75 pent
  • the flow cytometer was a MACSQuant Analyzer 10 (Miltenyi Biotec), and the results were analyzed using Flowjo software (Flowjo). T cell clones are obtained by single cell isolation and culture using a flow cytometer (FACS sorter). Her2 / neu369-377 peptide antigen-stimulated PBMCs were stained with APC-labeled anti-human CD8 antibody and PE-labeled Her2-E75 / HLA-A2 pentamer, and then subjected to flow cytometry (model: Sony cell sorter SH800) .
  • 10e5 TCR transfected T cells and 10e5 T2 cells were added to each well of a 96-well plate, and a final concentration of 0.1 ⁇ g / ml was added.
  • the epitope polypeptide to be tested is then cultured overnight in an incubator at 5% CO 2 and 37 ° C. After 24 hours, the supernatant was collected, and the human IFN- ⁇ ELISA Read-set-Go kit (eBioscience, cat # 88-7316) or human IFN- ⁇ DuoSet ELISA kit (R & D Systems, cat # DY285B) was used. IFN- ⁇ was detected in the supernatant.
  • Collect cell supernatant 18-24 hours and use human IFN- ⁇ ELISA Read-set-Go kit (eBioscience, cat # 88-7316) or human IFN- ⁇ DuoSet ELISA kit (R & D Systems, cat # DY285B), according to the manufacturer Instructions for detection of IFN- ⁇ in the supernatant.
  • human IFN- ⁇ ELISA Read-set-Go kit eBioscience, cat # 88-7316
  • human IFN- ⁇ DuoSet ELISA kit R & D Systems, cat # DY285B
  • Trypsinize the cells in the logarithmic growth phase collect them after centrifugation, and blow them evenly to prepare a single cell suspension; adjust the cell concentration to 0.1 ⁇ 10 ⁇ 10 4 / ml with cell culture solution (adjust the inoculated cells according to different cell growth conditions Number), inoculated in a 96-well cell culture plate, the culture system was 100 ⁇ l / well, placed at 37 ° C, and incubated in a 5% CO 2 incubator overnight, so that the cells were completely adherent and reached 70 to 80% the next day; The counting board counts and the countstar is used to verify the correctness of the count.
  • the full gene sequence of the TCR ⁇ chain and ⁇ chain and the cleavable F2A sequence and the Furin digestion fragment are synthesized, and linked to the multicloning site downstream of the EF-1 ⁇ promoter of the vector
  • the sequence of the inserted TCR is TCR ⁇ chain (without stop codon), Furin digestion fragment, F2A fragment, and TCR ⁇ chain (for the method, see the document "Gene Ther. 2008 Nov; 15 (21): 1411–1423").
  • the GFP-expressing vector is driven by the inverted PGK promoter.
  • the vector that does not express GFP has the PGK promoter and the GFP fragment removed.
  • TCR lentiviral vector plasmid and 1.8 ⁇ g of virus packaging plasmid of pCDH system (SBI, cat # LV500A-1), mixed and incubated for 25 minutes, and then added to 293T / 293FT cell culture wells. Incubate for 16 hours at 5% CO 2 at 37 ° C. Replace with DBS-free DMEM culture medium (Thermo Fisher, cat # 11965092). Continue to incubate the cells for 24 hours and 48 hours. Collect the cell supernatants and centrifuge at 2000g for 10 minutes. The virus supernatant obtained after filtration with a 0.4 ⁇ m filter membrane was concentrated using a lentivirus concentrate (GeneCopoeia TM # LPR-LCS-01) according to the manufacturer's instructions and used to infect cells.
  • a lentivirus concentrate GeneCopoeia TM # LPR-LCS-01
  • Her2 CTL 6A5 The cell clone Her2 CTL clone 6A5 (referred to as Her2 CTL 6A5).
  • the right panel of FIG. 1B shows that 97.9% of the CD8 + CTL cells can bind to the Her2 / neu 369-377 / HLA-A2 tetramer (ie, Her2-E75 tetramer), showing that this purified T cell clone was not intermixed with other unrelated cells.
  • Left panel is a control T cell that cannot bind Her2-E75 tetramer.
  • Example 2 Obtaining the full sequence of Her2 / neu 369-377 polypeptide-specific TCR
  • total RNA is directly purified from a certain number of Her2, CTL, and 6A5 cells obtained in Example 1, and the paired TCR ⁇ chain and ⁇ chain gene sequences are obtained by 5'-RACE RT-PCR method (that is, the two chains can be common It constitutes a functional TCR that recognizes an antigen polypeptide, and the TCR encoded by it is called "Her2TCR-6A5".
  • the amino acid sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 4, the coding sequence is shown in SEQ ID NO: 12, and the amino acid sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 7, and the coding sequence is shown in SEQ ID: NO: 15.
  • FIG. 1C shows a schematic diagram of the structural fragment of the constructed TCR lentiviral vector. The constant regions of the TCR alpha chain and beta chain were replaced by human-derived sequences by murine-derived sequences and linked by a cleavable linking polypeptide.
  • 6A5TCR ⁇ chain and ⁇ chain is driven by the EF-1 ⁇ promoter.
  • This promoter is a highly expressed promoter in eukaryotic cells, and will not be affected by factors such as methylation to cause loss of function, which is suitable for long-term expression of foreign genes in vivo.
  • the TCR ⁇ chain and ⁇ chain are connected by F2A polypeptide sequences.
  • the TCR ⁇ chain and ⁇ chain genes can be transcribed simultaneously and translated by ribosome skipping, thereby separating the TCR ⁇ chain and ⁇ chain polypeptides from each other. This ensures the consistency of the expression of the TCR ⁇ chain and ⁇ chain, thereby more efficiently constituting the TCR dimer.
  • There is also a furin cleavage site between the TCR ⁇ chain and the ⁇ chain which is used to remove extra peptides at the carboxyl terminus of the ⁇ chain.
  • the nucleotide sequence of the TCR ⁇ chain and the ⁇ chain of the constant region linked by a cleavable linking polypeptide from a human-derived sequence to a mouse-derived sequence (SEQ ID NO: 20) (the corresponding TCR is Her2TCR-6A5-mC, The amino acid sequence is shown in SEQ ID NO: 23) to the above vector to obtain Her2TCR-6A5-mC recombinant lentiviral vector.
  • the Her2 TCR-6A5-mC gene fragment was amplified by PCR, it was cloned downstream of the EF1-promoter of the above lentiviral vector (ie, pCDH-EF1 ⁇ -MCS): Her2 carrying a mouse-derived constant region sequence.
  • the recombinant TCR lentiviral expression vectors constructed were prepared according to the aforementioned method to obtain respective recombinant TCR lentiviral particles.
  • TCR obtained by the present invention can be expressed in primary T cells and has the function of recognizing Her2 / neu antigen polypeptide
  • a recombinant lentiviral particle carrying the Her2 TCR-6A5-mC gene was transfected with CD3 / CD28 antibody-activated peripheral blood T cells from two different normal donors, and the cells were collected for Her2-E75 tetramer staining 14 days later.
  • the specific method is as described above. The results are as follows:
  • CD8 - lymphocytes probably CD4 + T-helper cells, if infected with lentivirus CD8 + and CD4 + T cells, like transfection efficiency, indicating that CD4 + cells on exogenous Her2 / neu 369-377 specific TCR can Effectively binds Her2-E75 tetramer.
  • the 369-377 epitope polypeptide is CD8 independent.
  • CD4 cells expressing Her2 TCR-6A5-mC TCR secrete cytokines after recognizing Her2 antigen, which can not only help kill the function of T cells and the survival time in vivo, but also can induce the targeting of endogenous tumor antigens by regulating the tumor microenvironment. Specific T cells, thereby enhancing anti-tumor immunity.
  • IFN- ⁇ secreted by T cells was used to determine the function of this TCR-expressing PBMC cell to specifically recognize the Her2 / neu 369-377 polypeptide.
  • Figure 2B shows that PBMCs expressing Her2 and TCR-6A5-mC can be activated by Her2 / neu and 369-377 antigen polypeptides presented by T2 cells to secrete IFN- ⁇ , indicating that primary T expressing exogenous Her2 and TCR-6A5-mC Cells can specifically recognize Her2 / neu 369-377 polypeptides presented by HLA-A2 molecules.
  • the ability to recognize antigenic polypeptides is related to the expression of exogenous TCR on T cells.
  • EC50 half-maximum response
  • FIG. 2C shows that the T cells secreted the IFN- ⁇ function after adding anti-human CD8 antibodies when co-culturing the T cells with the antigen polypeptide (T2 + Her2-E75, Her2 / neu 369-377 polypeptide) presented by T2 cells. inhibition.
  • T2 + Her2-E75, Her2 / neu 369-377 polypeptide presented by T2 cells. inhibition.
  • Tumor cell lines include colorectal cancer Colo205 and HCT116, breast cancer MDA-MB-231 and MCF-7, pancreatic cancer PANC-1, glioma U87MG, and small cell lung cancer NCI-H446.
  • Tumor cells were stained with anti-HLA-A2 antibody (BD Bioscences, cat # 561341) and anti-human CD340 (erbB2) antibody (Biolegend, cat # 324406) and then subjected to flow cytometry. The results are shown in FIG.
  • NCI-H446 of HLA -A2 and Her2 / neu are both negative.
  • These tumor cell lines not only originate from different tissues, but also express different HLA-A2 and Her2 / neu.
  • U87MG and NCI-H446 cells can be used as negative control for Her2 TCR-6A5-mC T cell function test.
  • FIG. 3B shows that T cells expressing Her2 TCR-6A5-mC can be activated by HLA-A2 + Her2 / neu + tumor cell lines and secrete IFN- ⁇ .
  • the tumor cell lines include colon cancer Colo205 and HCT116, and breast cancer MDA -MB-231 and MCF-7, pancreatic cancer PANC-1.
  • the control group HLA-A2 + Her2 / neu - glioma U87MG and HLA-A2 - Her2 / neu - lung cancer NCI-H446 failed to activate T cells transfected with Her2 TCR-6A5-mC, indicating Her2 TCR -6A5-mC TCR can specifically recognize the Her2 / neu antigen presented by HLA-A2 on the surface of tumor cells.
  • Control T cells derived from the same donor PBMC and cultured in parallel but not transfected with Her2 TCR-6A5-mC could not be activated by the listed tumor cell lines, indicating that the response to tumor cells was not non-specific.
  • the results also showed that the ability of Her2 TCR-6A5-mC T cells to recognize the Her2 / neu antigen presented by HLA-A2 was not related to the expression of HLA-A2 and Her2 / neu molecules on the surface of tumor cells. Different tumor cells may have different inhibitory effects on T cells. On the other hand, the expression on the cell surface does not necessarily reflect the total Her2 / neu expression. Some tumor cells express Her2 / neu mainly in the cell cytoplasm. These antigens are more easily presented by HLA-A2 (see document "J Immunol 2006; 177: 5088-5097").
  • Example 5 Her2 / neu 369-377 polypeptide-specific TCR expressed after transfection of normal peripheral blood T cells with Her2 TCR-6A5-mC recombinant lentivirus does not recognize potential potential human normal proteins that can bind HLA-A2 molecules. Cross-reacted epitope polypeptide.
  • the nine new epitope polypeptides (final concentration: 0.1 ⁇ g / ml) were cultured with T2 cells and # 2PBMC transfected with the lentiviral vector encoding Her2 TCR-6A5-mC TCR gene for 24 hours, and then the cells were removed. The supernatant was analyzed by ELISA for IFN- ⁇ detection.
  • the effective target ratio E: T is 5: 1.
  • the amino acid side chains of these sites may form an epitope polypeptide that binds to the HLA-A2 molecule. Locating points or binding sites that are specifically recognized by TCR. Changing the amino acid residues at these sites will cause the polypeptide to lose the antigen specificity recognized by Her2. TCR-6A5-mC. TCR, while the amino acid residues at other sites are specific to Her2. TCR-6A5-mC The contribution of TCR recognition function is relatively small. Therefore, normal human proteins containing isoleucine at position 2, phenylalanine at position 3, glycine at position 4, serine at position 5, and leucine residue at position 6 are all possible. Recognized by Her2TCR-6A5-mCTCR and cross-reacted.
  • the human normal protein database https://prosite.expasy.org/cgi-bin/prosite/PSScan.cgi) was searched using the "XIFGSLXXX" sequence, where " "X” can be any of the 21 common amino acids.
  • a total of 13 different human normal protein sequences contain the -2I-3F-4G-5S-6L- sequence.
  • Table 1 shows the protein name, the position of the epitope containing the -2I-3F-4G-5S-6L- sequence, and Epitope sequence.
  • HLA / polypeptide binding prediction software http://www.cbs.dtu.dk/services / NetMHC /
  • Table 1 also shows the predicted affinity of the polypeptide to HLA-A2, and the ordering of the polypeptide's binding HLA-A2 affinity among the known affinity of the natural epitope polypeptide that binds HLA-A2.
  • Affinity (nM) refers to the prediction of the affinity of the epitope polypeptide to HLA-A2.
  • the results showed that, like the Her2 / neu 369-377 polypeptide, the NSMA3 93-101 polypeptide, the O11A1 103-111 polypeptide, and the SV2C 687-695 polypeptide all contained the -2I-3F-4G-5S-6L-sequence, and may be highly parental.
  • FIG. 4B shows the results of examining the secretion of IFN- ⁇ in the supernatant after mixed culture of peripheral blood mononuclear cells (PBMC) transfected with Her2, TCR-6A5-mC, TCR gene and epitope polypeptides presented by T2 cells at different concentrations.
  • PBMC peripheral blood mononuclear cells
  • Example 6 Preparation of Her2 TCR-6A5-mC-PGKp-tEGFR lentiviral vector plasmid carrying tEGFR gene and Her2 TCR-6A5-mC gene
  • tEGFR truncated human EGFR
  • tEGFR consists of a nucleotide sequence encoding a human GM-CSF receptor signal peptide fragment (a nucleic acid fragment encoding a human granulocyte-macrophage colony stimulating factor (GM-CSF) receptor leader peptide) and a human EGFR functional fragment (EGFR domains) III, IV and transmembrane spanning components (SEQ ID NO: 30) (the 2233-3306bp fragment in the nucleotide sequence of GenBank number KX055828, see the document "Cancer Immunol Res.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • Extracellular domain III has Cetuximab (Cetuximab, trade name Erbitux It was the earliest binding site of the FDA-approved IgG1 monoclonal antibody against the EGF receptor (see the document "Blood. 2011 Aug 4; 118 (5): 1255–1263.”).
  • 5 'primer 5'-GACGAGAGCGGCCTGACCATGCTTC-3' SEQ ID NO: 31
  • 3 'primer 5'-GCACAGTCGCTCGAGTCACATGAAGAG-3' SEQ ID NO: 32
  • the Q5 high-fidelity PCR kit (NEB, cat # M0543S) was used for PCR.
  • the reaction conditions were: after 30 seconds at 98 ° C, 25 cycles of 98 ° C for 10 seconds, 60 ° C for 15 seconds, and 72 ° C for 1 minute.
  • the tEGFR fragment obtained by PCR amplification was cloned downstream of the PGK promoter on the Her2 TCR-6A5-mC recombinant lentiviral vector. Downstream of tEGFR is a BGH poly A signal (bovine growth hormone polyadenylation signal).
  • anti-EGFR antibody can be anti-EGFR antibody [EGFR1] (PE / Cy7 from Abcam) (ab239309)
  • EGFR1 anti-EGFR antibody [EGFR1] (PE / Cy7 from Abcam)
  • ab239309 anti-EGFR antibody
  • Example 7 Killing effect of human T cells expressing Her2 TCR-6A5-mC and human T cells expressing Her2 TCR-6A5-mC and tEGFR on different human tumor cells
  • Human tumor cell lines include cervical cancer cell C33A, osteosarcoma cell Saos-2, and pancreatic cancer cell CFPAC-1. Tumor cells were stained with anti-HLA-A2 antibody (BD Bioscences, cat # 561341) and anti-human CD340 (erbB2) antibody (Biolegend, cat # 324406) and then subjected to flow cytometry. The results showed that cervical cancer cell C33A, osteosarcoma cell Saos-2, and pancreatic cancer cell CFPAC-1 were HLA-A2 + Her / neu + .
  • Human T cells expressing Her2 TCR-6A5-mC and human T cells expressing both Her2 TCR-6A5-mC and tEGFR are prepared by referring to the aforementioned method (that is, the "recombinant TCR lentivirus transfected human T cell" method), in which the expression Her2 TCR-6A5-mC human T cells are normal peripheral blood T cells transfected with Her2 TCR-6A5-mC recombinant lentivirus, and human T cells expressing both Her2 TCR-6A5-mC and tEGFR are Her2 TCR-6A5 -mC-PGKp-tEGFR recombinant lentivirus-transfected normal peripheral blood T cells, and the peripheral blood mononuclear cells used in the preparation thereof were obtained from Allcells Corporation (Cat. No. PB005F, specification 100 million, frozen).
  • the peripheral blood mononuclear cells used in the preparation thereof were obtained from Allcells Corporation (Cat. No. PB005F,
  • Figures 6A-C show that both human T cells expressing Her2 TCR-6A5-mC TCR and human T cells expressing both Her2 TCR-6A5-mC and tEGFR can be specific compared to control human T cells that have not been transfected with any TCR.
  • the lethality was dose-dependent with human T cells expressing Her2 TCR-6A5-mC TCR or human T cells expressing both Her2 TCR-6A5-mC and tEGFR.
  • a mouse model of Colo205 colon cancer with NOD-SCID immunodeficiency after cyclophosphamide (Cy) injection was used to simulate the human immune internal environment, and human T cells expressing Her2 TCR-6A5-mC (refer to the aforementioned " Recombinant TCR lentivirus transfected human T cells was prepared by the method, in which the peripheral blood mononuclear cells used were obtained from the American allcells company (Cat. No. PB005F, specification 100 million, frozen) to inhibit tumor growth, and proved the efficacy of its basic drugs.
  • the NOD-SCID immunodeficient mice used in this experiment were 6-week-old female mice (Vitalivar). Each mouse was inoculated with Colo205 colon cancer cells on the ventral subcutaneous tumor, and the inoculation amount was 3 ⁇ 10 6 Eight cells were selected 7 days after inoculation, and 18 tumor-bearing mice with an average tumor size of about 100 mm 3 were randomly divided into 3 groups of 6 mice in each group. The grouping day is set to day 0. The first group was a blank control group (Cy + IL-2 + PBS (it)). Cyclophosphamide (Baxter, 8D231A) 200 mg / Kg per mouse was intraperitoneally injected on day 0 with a dose of 100 ⁇ l.
  • each mouse was injected intratumorally (“it”) with 100 ⁇ l of PBS solution (available from Cellmax, article number CBS101.05); at the same time, the neck was injected subcutaneously with IL-2 (from Jiangsu Kingsley Company, trade name) For Intercan-180350101), 100,000 IU each.
  • the neck was injected subcutaneously with IL-2, 100,000 IU each.
  • each mouse was injected intratumorally with 100 ⁇ l of PBS solution; at the same time, IL-2 was injected subcutaneously in the neck, each 100,000 IU.
  • the neck was injected subcutaneously with IL-2, 100,000 IU each.
  • each mouse was injected with 100 ⁇ l of PBS solution intratumorally; at the same time, IL-2 was injected subcutaneously in the neck, each with 100,000 IU. On day 6, IL-2 was injected subcutaneously in the neck, each 100,000 IU. On day 7, IL-2 was injected subcutaneously in the neck, each 100,000 IU.
  • the second group is the control T cell group (Cy + IL-2 + T (it)).
  • the dosing schedule is basically the same as the first group, except that it is not intratumoral on day 1, day 3, and day 5.
  • the PBS solution was injected, but 100 ⁇ l of a control T-cell suspension (PBS in suspension medium) without transfection of any TCR was injected intratumorally, and the number of cells was 2 ⁇ 10 7 cells / each.
  • the third group is the Her2 TCR-6A5-mC T cell group (Cy + IL-2 + TCR-6A5T (it)) of the present invention.
  • the dosing schedule is basically the same as the first group, except that on the first day, the first On day 3 and day 5, instead of intratumoral injection of PBS solution, 100 ⁇ l of human T cell suspension expressing Her2 TCR-6A5-mC was intratumorally injected, and the number of cells was 2 ⁇ 10 7 cells / each.
  • the cells were freshly prepared according to the aforementioned preparation method, wherein the culture time was reconstituted from Her2 TCR-6A5-mC After lentivirus transfection of PBMC, the cells were first cultured for about 10 days according to the expected different administration time, and the Her2 TCR-6A5-mC TCR positive rate of the cells was about 40%. From day 0, tumor volume was measured every 2-4 days. The change of the average tumor volume of each group of animals is shown in Fig. 7.
  • Her2 / neu 369-377 polypeptide binding to the HLA-A2 molecule is different from that of the polypeptide / HLA complex presented in the cell (see the "Journal of Immunology, 2008, 180" : 8135–8145 ").
  • Her2 / neu 369-377 polypeptide is used as mimotope antigen, and the specific TCR induced can recognize both Her2 / neu369-377 polypeptide and similar polypeptides presented by tumor cells.
  • Her2 / neu373-382 polypeptide see the document "J Immunol.
  • TCR has a Her2 / neu 369-377 polypeptide presented by HLA-A2 High affinity, but can not effectively recognize the corresponding epitope peptides presented by tumor cells and kill tumor cells.
  • the TCR that specifically recognizes the Her2 / neu 369-377 polypeptide according to the present invention can target the Her2 / neu 369-377 polypeptide presented by tumor cells to specifically recognize and kill tumor cells.
  • TCRs that recognize self-antigens are mostly cleared by the central tolerance mechanism, most of the TCRs that naturally exist in peripheral T-cell pools that can recognize Her2 / neu antigens have low-affinity.
  • Another CD8 function-independent, high-affinity TCR that can recognize tumor cells is derived from the pairing of multiple alpha chains and beta chains of Her2 / neu 373-382 peptide-specific T cell population, and then screening through functional testing. (See document "HUMAN GENE THERAPY 2024, 25: 730-739"; WO / 2016/133779). Since it is not directly obtained from specific monoclonal T cells, it is not possible to determine whether this TCR exists in the peripheral natural T cell bank.
  • TCR recognition Her2 / neu 369-377 polypeptide of the present invention belongs to medium to high affinity, and the function of TCR does not depend on the auxiliary function of CD8, so it is suitable for the modification of T cells in adoptive transfer therapy.
  • the TCR according to the present invention cannot recognize all potential epitope polypeptides derived from normal proteins obtained by comparison and screening methods and computer-aided prediction software, thereby further avoiding potential cross-reaction risks against normal proteins.

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Abstract

提供了一种分离的T细胞受体、其修饰的细胞、编码核酸及其应用。所述分离的T细胞受体(TCR)包括α链和β链中的至少一者，所述α链和β链均包含可变区和恒定区，其特征在于，所述T细胞受体能够特异性识别肿瘤细胞所表达的抗原Her2/neu，并且所述α链的所述可变区的氨基酸序列具有与SEQ ID NO:1所示的氨基酸序列至少98％的一致性，所述β链的所述可变区的氨基酸序列具有与SEQ ID NO:2所示的氨基酸序列至少98％的一致性。所述TCR可特异性识别肿瘤抗原，同时可避免可能的脱靶毒副反应。用此TCR修饰的免疫细胞具有显著的抗肿瘤效果。

Description

一种分离的T细胞受体、其修饰的细胞、编码核酸及其应用 技术领域
本发明属于生物技术领域，具体而言，涉及分离的T细胞受体、其修饰的细胞、编码核酸、表达载体、制备方法、药物组合物和应用。
背景技术
Her2/neu(ERBB2)是属于EGFR家族的一个跨膜蛋白，和家族其它蛋白形成二聚体并通过一系列细胞内信号传导途径来调控细胞增值、分化以及癌变等过程(参见文献“Growth Factors,2008；26:263”、“Oncol Biol.Phys,2004；58:903”)。Her2/neu蛋白在多种上皮来源的癌细胞中过度表达，如乳腺癌，胃癌，大肠癌，卵巢癌，胰腺癌，肺癌，食管癌，膀胱癌，肾癌等(参见文献“Trends in Molecular Med,2013；19:677”)，并且在原发灶和转移灶癌细胞中的表达相对均一(参见文献“J Clin Oncol,1998；8:103”)，因此，Her2/neu成为靶向治疗的适当靶点。
靶向Her2/neu的人源化单抗药物Herceptin可以显著延长Her2/neu阳性的乳腺癌患者生存期(参见文献“N Engl J Med,2001,344:783”)，然而单独使用Herceptin治疗Her2阳性的转移乳腺癌的临床反应率只有11％到26％(参见文献“J Clin Oncol,2002；20:7169”)，表明单用Herceptin对大部分Her2高表达的转移乳腺癌的疗效并不理想。尽管Herceptin联合化疗可提高临床应答率，但大多数Her2/neu过度表达的乳腺癌病人在一年后会对Herceptin产生抗性(参见文献“J Clin Oncol,2001；19:2587”)。
Her2/neu阳性肿瘤患者体内会产生针对Her2/neu抗原的内源性抗体和T细胞反应(参见文献“Cancer Res,2005；65:650”)，因而，靶向Her2/neu抗原的特异性免疫治疗成为一种颇有前景的治疗手段。特异性识别Her2/neu抗原表位多肽(epitope peptide)369-377的T细胞 可以从Her2/neu高表达的卵巢癌腹水中成功分离(参见文献“J.Exp.Med.1995；181:2109–2117”)。靶向Her2/neu 369-377多肽抗原的肿瘤疫苗进入临床试验，尽管临床1/2期显示此疫苗可以诱导出针对Her2/neu 369-377多肽抗原的特异性T杀伤细胞(参见文献“Breast Care,2016；11:116”)，但临床三期却没达到延长病人生存期的预定目标(http://www.onclive.com/web-exclusives/phase-iii-nelipepimuts-study-in-breast-cancer-halted-after-futility-review)。过继转输经体外培养的、基于嵌合抗原受体(chimeric antigen receptors，CARs)的肿瘤特异性T细胞疗法被开发后，作为第一个针对实体瘤的、靶向Her2/neu抗原的CAR-T细胞疗法进入临床试验，但由于产生强烈的细胞因子风暴(cytokine release syndrome，CRS)导致病人死亡而被终止(参见文献“Nature Med,2016；22:26”)。严重的细胞因子风暴以及神经毒性是CAR-T治疗中常见的毒性反应(参见文献“Blood,2016；127:3321”)，部分原因可能和CAR这种非天然的T细胞受体不受限制的细胞活化有关(参见文献“Nat Ned,2015；21:581”)，或者与不需抗原刺激的细胞因子的自分泌有关(参见文献“Cancer Immunol Res,2015；3:356”)。
过继转输经过特异性T细胞受体(即TCR)基因修饰的T细胞的TCR-T疗法被认为是针对实体瘤最具前景的免疫细胞基因疗法(参见文献“Adv Immunol.2016；130:279-94”)。其中，靶向NY-ESO-1抗原的TCR-T疗法的临床研究显示出令人鼓舞的临床治疗效果(参见文献“Nat Med.2015Aug；21(8):914-921”)。然而，目前已知的靶向肿瘤抗原、并有效识别肿瘤细胞的特异性TCR的数量十分有限，因此限制了TCR-T疗法的广泛应用。另外，尽管TCR-T疗法没有出现CAR-T疗法中所表现出的严重的细胞因子风暴毒性，但如果靶抗原来源于自身蛋白，针对正常组织细胞中低表达的靶抗原有可能会导致严重的自身免疫反应，即转接脱靶(或称中靶脱瘤)(on target off tumor)毒性反应(参见文献“Blood 2009；114:535-546”)。另外，为了获得有效识别肿瘤细胞的高亲和性TCR，一般的策略是体外通过对TCR上的互补性决定区(complementarity determining regions,CDRs)进行基因点突变，或者通过从未经过中枢耐受机制筛选的人源化小鼠T细胞库中 进行诱导(参见文献“Front Immunol.2013；4:363”)。然而，这种高亲和性TCR可能会对正常自身蛋白产生交叉反应而导致对正常组织细胞的杀伤作用，即严重甚至致命的脱靶(off-target)毒性(参见文献“Curr Opin Immunol 2015；33:16–22”、参见文献“Sci Transl Med.2013；5(197):197ra103”)。因此，获得特异性靶向肿瘤抗原并有效识别肿瘤细胞，同时避免可能出现的脱靶毒副反应的新型TCR基因，是成功开发TCR-T免疫细胞基因疗法面临的主要挑战。
发明内容
为解决上述现有技术中所存在的问题，本发明提供了分离的T细胞受体、其修饰的细胞、编码核酸、表达载体、制备方法、药物组合物和应用。
具体而言，本发明提供了：
(1)一种分离的T细胞受体，包括α链和β链中的至少一者，所述α链和β链均包含可变区和恒定区，其特征在于，所述T细胞受体能够特异性识别肿瘤细胞所表达的抗原Her2/neu，并且所述α链的所述可变区的氨基酸序列具有与SEQ ID NO:1所示的氨基酸序列至少98％的一致性，所述β链的所述可变区的氨基酸序列具有与SEQ ID NO:2所示的氨基酸序列至少98％的一致性。
(2)根据(1)所述的T细胞受体，其中所述的T细胞受体能够特异性识别被HLA-A2分子所提呈的所述抗原Her2/neu的抗原表位多肽；优选的是，所述抗原表位多肽包括如SEQ ID NO:3所示的Her2/neu 369-377。
(3)根据(1)所述的T细胞受体，其中所述α链的所述恒定区和/或所述β链的所述恒定区来源于人；优选地，所述α链的所述恒定区全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链的所述恒定区全部或部分地被来源于其它物种的同源序列所替换；更优选地，所述其它物种为小鼠。
(4)根据(1)所述的T细胞受体，其中所述α链的所述恒定区修饰有一个或多个二硫键，并且/或者所述β链的所述恒定区修饰有 一个或多个二硫键。
(5)根据(1)所述的T细胞受体，其中所述α链的氨基酸序列如SEQ ID NOs:4、5或6所示，所述β链的氨基酸序列如SEQ ID NOs:7、8或9所示。
(6)一种分离的、编码T细胞受体的核酸，包含所述T细胞受体的α链和β链中的至少一者的编码序列，所述α链编码序列和β链编码序列均包含可变区编码序列和恒定区编码序列，其特征在于，所述T细胞受体能够特异性识别肿瘤细胞表达的抗原Her2/neu，并且所述α链可变区编码序列编码的氨基酸序列具有与SEQ ID NO:1所示的氨基酸序列至少98％的一致性，所述β链可变区编码序列编码的氨基酸序列具有与SEQ ID NO:2所示的氨基酸序列至少98％的一致性。
(7)根据(6)所述的核酸，其中所述核酸为DNA或RNA。
(8)根据(6)所述的核酸，其中所述α链可变区编码序列如SEQ ID NO:10所示，所述β链可变区编码序列如SEQ ID NO:11所示。
(9)根据(6)所述的核酸，其中被所述核酸编码的所述T细胞受体能够特异性识别被HLA-A2分子所提呈的所述抗原Her2/neu的抗原表位多肽；优选的是，所述抗原表位多肽包括如SEQ ID NO:3所示的Her2/neu 369-377。
(10)根据(6)所述的核酸，其中所述α链恒定区编码序列和/或所述β链恒定区编码序列来源于人；优选地，所述α链恒定区编码序列全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链恒定区编码序列全部或部分地被来源于其它物种的同源序列所替换；更优选地，所述其它物种为小鼠。
(11)根据(6)所述的核酸，其中所述α链恒定区编码序列包含一个或多个二硫键编码序列，并且/或者所述β链恒定区编码序列包含一个或多个二硫键编码序列。
(12)根据(6)所述的核酸，其中所述α链编码序列如SEQ ID NOs:12、13或14所示，所述β链编码序列如SEQ ID NOs:15、16或17所示。
(13)根据(6)-(11)中任一项所述的核酸，其中所述α链编码序列和所述β链编码序列之间由可切割性连接多肽的编码序列连接。
(14)根据(13)所述的核酸，其序列如SEQ ID NOs:18、19、或20所示。
(15)一种重组表达载体，其含有与启动子有效连接的、根据(6)-(14)中任一项所述的核酸，和/或其互补序列。所述启动子可以是真核细胞启动子，包括持续表达启动子和可诱导表达启动子，包括(例如)：PGK1启动子、EF-1α启动子、CMV启动子、SV40启动子、Ubc启动子、CAG启动子、TRE启动子、CaMKIIa启动子、人β肌动蛋白(human beta actin)启动子。
(16)根据(15)所述的重组表达载体，其中所述重组表达载体含有自杀基因编码序列；其中所述自杀基因选自：iCasp9、HSV-TK、mTMPK、截短的EGFR、截短的CD19、截短的CD20或其组合。
(17)根据(16)所述的重组表达载体，其中所述自杀基因编码序列是在启动子控制下的，并且该用于控制所述自杀基因编码序列的启动子与(6)-(14)中任一项所述的核酸所连接的启动子相同或不同，并且是彼此独立的。
(18)根据(16)所述的重组表达载体，其中所述自杀基因编码序列和(6)-(14)中任一项所述的核酸是在同一个启动子控制下的，并且所述自杀基因编码序列通过可切割性连接多肽的编码序列或者内部核糖体进入位点(internal ribosome entry site，IRES)序列，与(6)-(14)中任一项所述的核酸相连接。
(19)一种T细胞受体修饰的细胞，该细胞的表面被(1)-(5)中任一项所述的T细胞受体修饰，其中所述细胞包括原始T细胞或其前体细胞，NKT细胞，或T细胞株。
(20)根据(19)所述的T细胞受体修饰的细胞，其中该细胞在其细胞表面或细胞内表达自杀基因蛋白；其中所述自杀基因选自：iCasp9、HSV-TK、mTMPK、截短的EGFR、截短的CD19、截短的CD20或其组合。
(21)一种制备根据(19)或(20)所述的T细胞受体修饰的 细胞的方法，包括以下步骤：
1)提供细胞；
2)提供编码根据(1)-(5)中任一项所述的T细胞受体的核酸；
3)将所述核酸转染入所述细胞中。
(22)根据(21)所述的方法，其中步骤1)所述的细胞来自自体或异体。
(23)根据(21)所述的方法，其中所述转染的方式包括：采用病毒载体转染的方式，优选的是，所述病毒载体包括γ逆转录病毒载体或慢病毒载体；化学方式，优选的是，所述化学方式包括采用脂质体转染的方式；物理方式，优选的是，所述物理方式包括电转染方式。
(24)根据(21)所述的方法，其中步骤2)所述的核酸为根据(6)-(14)中任一项所述的核酸。
(25)根据(19)或(20)所述的T细胞受体修饰的细胞在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
(26)根据(25)所述的用途，其中所述肿瘤和/或癌症是抗原Her2/neu阳性的，并且是HLA-A2阳性的。
(27)根据(19)或(20)所述的T细胞受体修饰的细胞在制备用于检测宿主的肿瘤和/或癌症的药物中的用途。
(28)一种药物组合物，其中该药物组合物包括作为活性成分的根据(19)或(20)所述的T细胞受体修饰的细胞，及可药用辅料。
(29)根据(28)所述的药物组合物，其中所述药物组合物包含每个患者每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重的所述T细胞受体修饰的细胞。
(30)根据(28)所述的药物组合物，其中所述药物组合物适于经动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内或腹膜内给药。
(31)一种治疗肿瘤和/或癌症的方法，包括对肿瘤和/或癌症患 者施用根据(19)或(20)所述的T细胞受体修饰的细胞。
(32)根据(31)所述的方法，其中所述T细胞受体修饰的细胞的施用剂量为每个患者每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重。
(33)根据(31)所述的方法，其中所述T细胞受体修饰的细胞通过动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内或腹膜内给药。
(34)根据(31)所述的方法，其中所述肿瘤和/或癌症是抗原Her2/neu阳性的，并且是HLA-A2阳性的。
(35)根据(31)所述的方法，还包括对所述肿瘤和/或癌症患者施用其它用于治疗肿瘤的药物，和/或用于调节患者免疫系统的药物。
本发明与现有技术相比具有以下优点和积极效果：
本发明从HLA-A2阳性的健康供体外周血中成功诱导出对HLA-A2提呈的Her2/neu抗原表位多肽(如Her2/neu 369-377多肽)有特异性的T细胞克隆，并从中筛选出携带有特异性识别Her2/neu抗原表位多肽(如Her2/neu 369-377多肽)的天然TCR的T细胞克隆，进而获得了该TCR全序列。此TCR属于非CD8分子依赖性，对Her2/neu抗原表位多肽(如Her2/neu 369-377多肽)具有中等到高亲和性，可特异性识别HLA-A2阳性并表达Her2/neu抗原的肿瘤细胞。另外，携带此TCR的T细胞克隆经过中枢免疫耐受机制筛选后进入外周T细胞库。携带此TCR的杀伤T细胞曾存在于正常人外周血，并未对微量表达Her2/neu蛋白的正常组织细胞产生交叉反应。另外，为了避免所述TCR对正常蛋白产生脱靶交叉反应而导致自身免疫毒性，首先获得Her2/neu 369-377多肽上与所述TCR识别功能相关的关键氨基酸位点的信息，据此，从人正常蛋白数据库中搜索出包含所述TCR识别的关键氨基酸位点的所有人正常蛋白，并进一步筛选出可能结合HLA-A2分子的抗原表位多肽。实验显示，所述TCR不识别这些来自正常蛋白，具有潜在交叉反应的表位多肽。因此，本 发明获得了能够特异性识别肿瘤抗原，同时能够避免可能出现的脱靶毒副反应的新型TCR。
在进一步的发明中还对TCR的恒定区进行了改造(例如进行二硫键修饰或鼠源化改造)，以使得此TCR在免疫细胞上表达时能够进一步减少或避免与内源TCR错配的发生。
用此TCR修饰的免疫细胞(例如原始T细胞、其前体细胞、NKT细胞、T细胞株)可特异性识别多种HLA-A2 +且Her2/neu +的肿瘤细胞株，具有显著的抗肿瘤效果。因此，基于此TCR的TCR-T疗法可望治疗多种实体瘤。
用本发明的TCR修饰的免疫细胞治疗肿瘤时，可有效避免采用CAR-T治疗时所引起的细胞因子风暴和免疫排斥反应。
本发明的TCR修饰的免疫细胞为治疗HLA-A2 +且Her2/neu +肿瘤患者提供了一种新的选择，具有良好的产业应用前景。
附图说明
图1示出本发明实施例1中从HLA-A2 +正常供体PBMC(具体为#1PBMC)中诱导出的Her2/neu 369-377多肽(Her2-E75)特异性杀伤性T细胞的表型和功能检测结果。图1A为经过两轮Her2-E75抗原多肽体外刺激后，PBMC细胞经CD8-APC抗体和Her2-E75五聚体-PE染色后进行流式细胞分析的结果，右图是经多肽刺激的细胞，对CD8 +五聚体 +杀伤T细胞群进行FACS分选，以获得T细胞克隆。左图为没有多肽刺激的对照组细胞。横坐标表示CD8分子表达的荧光强度，纵坐标表示结合的Her2-E75五聚体的荧光强度。图1B为CD8 +E75-四聚体 +杀伤T细胞克隆经CD8-APC和Her2-E75四聚体-PE染色后流式细胞的表型分析，右图显示CD8 +Her2四聚体 +T细胞克隆Her2 CTL 6A5为纯化的Her2-E75多肽特异性CTL细胞克隆。左图为没有多肽刺激的对照组CTL细胞。横坐标表示CD8分子表达的荧光强度，纵坐标表示结合的Her2-E75四聚体的荧光强度。图1C示出所构建的携带Her2 TCR-6A5-mC基因的慢病毒载体(即“pCDH-EF1α-Her2 TCR载体”)的主要功能片段。示出的片段表达 EF-1α启动子所驱动的TCR基因，各TCR的β链和α链的不变区片段均为鼠源不变区片段，TCR的β链和α链被可切割性连接多肽的编码序列(furin-F2A)所链接。
图2示出经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞(PBMC)的表型和功能检测结果。图2A为编码Her2 TCR-6A5-mC的慢病毒载体转染来自两个不同供体的PBMC，经Her2-E75四聚体-PE和抗CD8-APC抗体染色后进行流式细胞分析的结果。首先根据细胞形态和大小分出淋巴细胞群，Her2-E75四聚体 +细胞群为表达Her2 TCR-6A5-mC TCR的细胞。横坐标表示CD8分子表达的荧光强度，纵坐标表示结合的Her2-E75四聚体的荧光强度。所示百分率为各阳性细胞群占分出的淋巴细胞数的比率。左图涉及一个供体所提供的外周血单个核细胞(#1PBMC)，右图涉及另一个不同供体提供的PBMC(#2PBMC)。CD8 +Her2-E75四聚体 +细胞为表达Her2 TCR-6A5-mC的杀伤性T细胞。CD8 -Her2-E75四聚体 +细胞可能为表达Her2 TCR-6A5-mC的CD4 +辅助T细胞。图2B示出表达Her2 TCR-6A5-mC的T细胞可以识别被T2细胞所提呈的Her2-E75多肽。经编码Her2 TCR-6A5-mC的慢病毒载体转染的两个不同供体PBMC分别与提呈不同浓度梯度Her2-E75多肽的T2细胞混合培养16小时，取细胞上清进行IFN-γ的ELISA分析。对照组中靶细胞为提呈可以结合HLA-A2分子的EBV病毒抗原多肽LMP2 426-434的T2细胞(图中未显示)。图中“0.1μg/ml”表示提呈0.1μg/ml的Her2-E75多肽的T2细胞组，“0.01μg/ml”表示提呈0.01μg/ml的Her2-E75多肽的T2细胞组，“0.001μg/ml”表示提呈0.001μg/ml的Her2-E75多肽的T2细胞组，“0.0001μg/ml”表示提呈0.0001μg/ml的Her2-E75多肽的T2细胞组。纵坐标表示T细胞分泌的IFN-γ的浓度。图2C示出T细胞功能的CD8抗体阻断试验结果。其中，经编码Her2 TCR-6A5-mC的慢病毒载体转染的#2 PBMC与T2细胞提呈的Her2-E75抗原多肽共培养时加入抗人CD8抗体，检测T细胞分泌IFN-γ的功能是否被抑制。图中“T2+Her2-E75”表示未加入抗人CD8抗体的、提呈0.1μg/ml的Her2-E75多肽的T2细胞组，“T2+Her2-E75+抗-CD8”表示加入 抗人CD8抗体的、提呈0.1μg/ml的Her2-E75多肽的T2细胞组。横坐标表示不同实验组别，纵坐标表示T细胞分泌的IFN-γ的浓度。“ns”表示两实验组无显著性差异。图2B和2C中各试验组和对照组均为复孔，结果显示为平均值±SEM。
图3示出经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞(PBMC)识别肿瘤细胞株的功能检测结果。图3A示出不同肿瘤细胞株细胞表达HLA-A2和Her2/neu的情况。横坐标表示不同的人肿瘤细胞株。“Colo205”和“HCT116”为结肠癌细胞；“MDA-MB-231”和“MCF-7”为乳腺癌细胞；“PANC-1”为胰腺癌细胞；“U87MG”为神经胶质瘤细胞；“NCI-H446”为肺癌细胞。纵坐标“MFI”表示细胞经抗HLA-A2荧光抗体或抗Her2/neu荧光抗体染色后的荧光强度均值。白色条柱为Her2/neu在细胞表面的表达量，黑色条柱为HLA-A2在细胞表面的表达量。图3B示出编码Her2 TCR-6A5-mC TCR基因的慢病毒载体转染#2 PBMC，与不同肿瘤细胞株细胞混合培养24小时后，取细胞上清进行IFN-γ的ELISA分析结果。各试验组和对照组均为三孔，结果显示为平均值±SME。横坐标示出不同靶细胞，纵坐标示出T细胞分泌的IFN-γ的浓度。效靶比E:T为5:1。白色条柱示出效应细胞为未经Her2 TCR-6A5-mC TCR基因转染的对照外周血单个核细胞，黑色条柱示出效应细胞为经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞。图3C、D、E、F、G、H、I、J、和K示出#2 PBMC经编码Her2 TCR-6A5-mC TCR基因的慢病毒载体转染后，对不同肿瘤细胞株的杀伤活性。图3C-3G的杀伤活性是通过对活细胞计数获得的，图3H-3K的杀伤活性是MTT方法测定的，反应时间为24小时。其中，图3C和3H示出针对肿瘤细胞株MCF-7的结果、图3D示出针对肿瘤细胞株HCT116的结果、图3E示出针对肿瘤细胞株U87MG的结果、图3F示出针对肿瘤细胞株NCI-H446的结果、图3G示出针对肿瘤细胞株SKOV3的结果、图3I示出针对肿瘤细胞株PANC-1的结果、图3J示出针对肿瘤细胞株HEPG2的结果、图3K示出针对肿瘤细胞株HT-29的结果。各试验组和对照组均为三孔，结果显示为平均值±SME。横坐标示出不同的效靶比E:T。纵坐标示 出T细胞对靶细胞的杀伤率百分比数值。圆点形图注示出效应细胞为未经Her2 TCR-6A5-mC TCR基因转染的对照外周血单个核细胞，上三角图注示出效应细胞为经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞。MTT杀伤实验中，另外一组为加紫杉醇10μM作为阳性对照(图3H-3K中示出为单独的下三角点)。
图4示出Her2 TCR-6A5-mC TCR所识别的Her2-E75多肽上的氨基酸关键位点，以及对来自人正常蛋白并具有潜在交叉反应的表位多肽的识别功能检测的结果。图4A示出实施例5中所形成的9个新表位多肽分别与T2细胞以及转染有编码Her2 TCR-6A5-mC TCR基因的慢病毒载体的#2 PBMC混合培养24小时后，取细胞上清进行IFN-γ检测的ELISA分析结果。各试验组和对照组均为复孔，结果显示为平均值±SME。横坐标示出T2细胞提呈不同的表位多肽(T2+多肽)，“E75”为Her2/neu 369-377多肽，“E75-K1A”、“E75-I2A”、“E75-F3A”、“E75-G4A”、“E75-S5A”、“E75-L6A”、“E75-F8A”、“E75-L9A”分别为Her2/neu 369-377多肽相应位点的氨基酸被丙氨酸所替换，“E75-A7G”为Her2/neu 369-377多肽的第七个丙氨酸被甘氨酸替代。纵坐标示出T细胞分泌的IFN-γ的浓度。效靶比E:T为5:1。图4B示出Her2 TCR-6A5-mC TCR识别来自人正常蛋白且具有潜在交叉反应的表位多肽的识别功能。“E75”为Her2/neu 369-377多肽，多肽“B”是NSMA3 93-101多肽，“C”是O11A1 103-111多肽，“D”是SV2C 687-695多肽。转染有编码Her2 TCR-6A5-mC TCR基因的慢病毒载体的#2 PBMC与提呈不同浓度梯度所述多肽的T2细胞混合培养24小时，取细胞上清进行IFN-γ的ELISA分析。各试验组和对照组均为三孔，结果显示为平均值±SME。横坐标示出T2细胞提呈不同浓度的表位多肽。纵坐标表示T细胞分泌的IFN-γ的浓度。
图5示出实施例6所构建的携带tEGFR基因和Her2 TCR-6A5-mC基因的Her2 TCR-6A5-mC-PGKp-tEGFR慢病毒载体的主要功能片段。示出的片段包括：1)表达EF-1α启动子所驱动的TCR基因，各TCR的β链和α链的不变区片段均为鼠源不变区片段，TCR的β链和α链被可切割性连接多肽的编码序列(furin-F2A)所链接；
2)表达PGK启动子所驱动的截短的人EGFR基因片段。
图6示出实施例7实验中表达Her2 TCR-6A5-mC TCR的人T细胞和同时表达Her2 TCR-6A5-mC和tEGFR的人T细胞对不同人肿瘤细胞株的杀伤活性，其中图6A示出针对肿瘤细胞株C33A的实验结果，图6B示出针对肿瘤细胞株CFPAC-1的实验结果，图6C示出针对肿瘤细胞株Saos-2的实验结果。各试验组和对照组均为三孔，结果显示为平均值±SME。图6A-C横坐标示出不同的效靶比E:T。纵坐标示出细胞杀伤率百分比数值。圆点形图注示出效应细胞为没有转染任何TCR的对照人T细胞，方形图注示出效应细胞为表达Her2 TCR-6A5-mC TCR的人T细胞，上三角形图注示出效应细胞为同时表达Her2 TCR-6A5-mC和tEGFR的人T细胞。
图7示出实施例8实验中各组动物给药处理后肿瘤体积的变化曲线，横坐标为给药后时间(天)，纵坐标为肿瘤体积(mm 3)。
具体实施方式
以下通过具体实施方式的描述并参照附图对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
在本发明中，词语“肿瘤”、“癌症”、“肿瘤细胞”、“癌细胞”、“T细胞”、“T细胞受体”、“T细胞受体修饰”、“TCR可变区”、“TCR恒定区”、“抗原”、“抗原表位多肽”、“同源序列”、“编码”、“抗原提呈”、“重组DNA表达载体”、“启动子”、“互补序列”、“转染”、“自体”、“异体”、“特异性识别”、“TCR-T疗法”涵盖本领域通常认为的含义。
Her2/neu抗原属于肿瘤相关抗原，识别Her2/neu抗原的高亲和性T细胞多数被中枢耐受机制所清除，以避免导致可能的自身免疫反应(参见文献“Immunol Rev.2016；271(1):127-40”)。因此，从外周血T细胞库中诱导出具有特异性识别肿瘤细胞所表达的Her2/neu抗原的T细胞克隆变得十分困难。利用树突状细胞(Dendritic cell)提呈 Her2/neu 369-377多肽抗原，进而从Her2/neu 369-377多肽疫苗免疫过的患者外周血中诱导出的高亲和性TCR尽管可以识别极低量外源所负载的(exogenously loaded)Her2/neu 369-377多肽，但不能识别肿瘤细胞内源提呈的(endogenously presented)抗原多肽(参见文献“Cancer Res.1998；58:4902–4908”)。这可能是由于外源负载的多肽/HLA复合物在构型(conformation)上与细胞内部自然提呈的HLA/多肽复合物有所不同而导致，或者由于Her2/neu 369-377多肽位于Her2蛋白高度糖化区，细胞内部自然提呈的Her2/neu 369-377多肽可能被糖基化而导致TCR识别构型的差异(参见文献“Proc.Natl.Acad.Sci.USA 2003；100:15029–15034”)。体外通过Her2/neu 369-377抗原多肽诱导T细胞的过程中，仅能识别外源负载抗原多肽的高亲和性T细胞克隆往往获得优势生长(dominant expansion)，而能特异性识别被细胞提呈的内源性Her2/neu抗原多肽的T细胞克隆生长受到抑制(参见文献“J Exp Med.2016 Nov 14；213(12):2811-2829”)，因而增加了获得可识别肿瘤细胞的功能性TCR的难度。有研究小组从HLA-A2阴性的外周血中诱导出异体T细胞(Allo–T cells)，可以特异性识别HLA-A2限制的Her2/neu 369-377抗原多肽，用获得的TCR基因转染T细胞后，不仅可以识别肿瘤细胞提呈的Her2/neu 369-377抗原多肽，也可交叉识别同家族的Her3以及Her4抗原表位(参见文献“Journal of Immunology,2008,180:8135–8145”)。然而，基于异体allo-TCR的TCR-T疗法存在产生针对其它正常自身蛋白抗原表位的异体反应(allo-reaction)的风险(参见文献“Int.J.Cancer 2009；125,649–655”、“Nat Immunol2007；8:388–97”)。另外一个研究小组从Her2/neu 369-377多肽疫苗免疫过的肿瘤患者外周血中诱导出Her2/neu 369-377多肽特异性T细胞，并把来源于不同T细胞的alpha和beta链进行配对并筛选出一个高亲和性TCR，转染此高亲和性TCR的T细胞可识别HLA-A2 +Her2/neu +的多种肿瘤细胞(参见文献“HUMAN GENE THERAPY 2014；25:730–739”)。这个TCR不是从单克隆T细胞获得，因此不能确定此TCR是否为存在于经过中枢耐受筛选过的T细胞库的天然TCR。为了提高TCR对HLA I类分子提呈的表位多肽的亲和性，可以通过对 TCR识别表位多肽的功能性区域进行点突变，并筛选出高亲和性TCR。由于Her2/neu蛋白也在心肌、肺、食道、肾、膀胱这些重要脏器有微量表达(参见文献“Oncogene.1990Jul；5(7):953-62”)，因此经上述方法获得非天然的高亲和性Her2/neu抗原特异性TCR存在对正常组织产生脱靶毒性反应的风险。
肿瘤细胞高表达Her2/neu蛋白，因此细胞表面被HLA提呈的抗原多肽的数量也会相应增加，在肿瘤细胞和正常细胞上HLA/抗原多肽复合物数量的差异可成为特异性T细胞区分正常和肿瘤组织的窗口。本发明提出从自体T细胞库(auto-T cell repertoire)中获得天然TCR的序列，进而在体外使TCR表达在T细胞上，以使所得到的表达TCR的T细胞可识别肿瘤细胞增加表达的Her2/neu抗原，是成功开发有效低毒的TCR-T疗法的关键。
为了获得能够特异性识别肿瘤抗原，同时能够避免可能出现的脱靶毒副反应的TCR，本发明从HLA-A2阳性的健康供体外周血中诱导对HLA-A2提呈的Her2/neu 369-377多肽有特异性的T细胞克隆，并从中筛选出携带有对Her2/neu 369-377多肽具有中等亲和性的天然TCR的T细胞克隆。这不同于其他研究小组从经过Her2/neu 369-377多肽疫苗免疫过的肿瘤患者外周血诱导Her2/neu 369-377多肽特异性T细胞的策略(参见文献“HUMAN GENE THERAPY 2014,25:730–739”)，本发明认为经过Her2/neu 369-377抗原多肽免疫后，针对Her2/neu 369-377多肽的特定T细胞克隆会优势增殖，因而不能代表体内T细胞库(repertoire)中自然存在的可识别靶细胞所提呈的Her2/neu 369-377多肽抗原的特异性T细胞群。本发明也没有采取其他研究小组从HLA-A2阴性外周血中诱导多肽特异性T细胞的方式(参见文献“The Journal of Immunology,2010,184:1617–1629”)，尽管从异体PBMC中更容易获得高亲和性的识别Her2/neu 369-377多肽抗原的allo-T细胞，但这也同时增加了T细胞交叉识别被HLA-A2分子提呈的其它多肽而导致的异体反应。
基于上述构思，本发明提供了一种分离的T细胞受体，包括α链和β链中的至少一者，所述α链和β链均包含可变区和恒定区，其 特征在于，所述T细胞受体能够特异性识别肿瘤细胞所表达的抗原Her2/neu，并且所述α链的所述可变区的氨基酸序列具有与SEQ ID NO:1所示的氨基酸序列至少98％、优选至少98.5％、更优选至少99％的一致性，所述β链的所述可变区的氨基酸序列具有与SEQ ID NO:2所示的氨基酸序列至少98％、优选至少98.5％、更优选至少99％的一致性，只要不显著影响本发明的效果即可。还优选的是，所述α链的所述可变区的氨基酸序列如SEQ ID NO:1所示，所述β链的所述可变区的氨基酸序列如SEQ ID NO:2所示。
TCRα链和β链的可变区用于结合抗原多肽/主要组织相容性复合体(MHC I)，分别包括三个超变区或称为互补决定区(complementarity determining regions，CDRs)，即，CDR1、CDR2、CDR3。其中CDR3区域对特异性识别被MHC分子提呈的抗原多肽至关重要。TCRα链是不同的V和J基因片段重组而成，β链则是不同的V、D和J基因片段重组而成。特定基因片段重组结合所形成的相应CDR3区域，以及结合区域回文以及随机插入的核苷酸(palindromic and random nucleotide additions)形成了TCR对抗原多肽识别的特异性(参见文献“Immunobiology:The immune system in health and disease.5 th edition,Chapter 4,The generation of Lymphocyte antigen receptors”)。所述MHC I类分子包括人HLA。所述HLA包括：HLA-A、B、C。
进一步具体地，所述的T细胞受体能够特异性识别被HLA-A2分子所提呈的所述抗原Her2/neu的抗原表位多肽。抗原Her2/neu的氨基酸序列如SEQ ID NO:21所示。优选的是，所述抗原表位多肽包括如SEQ ID NO:3所示的Her2/neu 369-377。HLA-A2阳性细胞表达的HLA-A2等位基因包括HLA-A*0201、0202、0203、0204、0205、0206和0207。优选的是，所述HLA-A2分子优选为HLA-A*0201。
在一个实施方案中，所述抗原Her2/neu的抗原表位多肽为Her2/neu 369-377多肽(SEQ ID NO:3)。在其它实施方案中，所述抗原Her2/neu的抗原表位多肽为与Her2/neu 369-377多肽具有4-9个连续的相同氨基酸(例如，4、5、6、7、8或9个连续的相同氨基 酸)的抗原表位多肽，并且这些多肽的长度为8-11个氨基酸。例如，在一个实施方案中，所述抗原Her2/neu的抗原表位多肽为Her2/neu373-382多肽(SEQ ID NO:22)。
优选地，所述T细胞受体识别Her2/neu 369-377多肽的最大半反应多肽浓度在1.0-10ng/ml之间。在本发明的一个实施方案中，所述最大半反应多肽浓度约为1.6ng/ml-2.9ng/ml。术语“最大半反应多肽浓度”是指诱导T细胞反应达到最大值的50％所需多肽的浓度。据报道，针对巨细胞病毒(CMV)抗原CMV pp65(495-503)多肽的特异性T细胞的最大半反应多肽浓度在0.1-1ng/ml之间，而此TCR对CMV抗原多肽被认为具有高亲和性(参见文献“Journal of Immunogical Methds 2007；320:119-131”)。在本发明中，所述T细胞受体对Her2/neu抗原具有中等到高亲和性，从而可避免高亲和性(最大半反应多肽浓度小于0.1ng/ml)可能带来的脱靶毒性。另外，所述T细胞受体识别Her2/neu 369-377多肽不依赖CD8分子的辅助作用，CD8阴性CD4阳性T细胞表达所述T细胞受体也可特异性识别被HLA-A2提呈的Her2/neu 369-377多肽而分泌细胞因子，从而增强表达所述T细胞受体的杀伤性T细胞的功能。
T细胞表达的外源TCRα链和β链有可能和T细胞本身TCR的α链和β链发生错配，不仅会稀释正确配对的外源TCR的表达量，错配TCR的抗原特异性也不明确，因而有识别自身抗原的潜在危险，因此优选将TCRα链和β链的恒定区修饰以减少或避免错配。
在一个实施方案中，所述α链的所述恒定区和/或所述β链的所述恒定区来源于人；优选地，本发明发现所述α链的所述恒定区可以全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链的所述恒定区可以全部或部分地被来源于其它物种的同源序列所替换。更优选地，所述其它物种为小鼠。所述替换可以增加细胞中TCR的表达量，并且可以进一步提高被该TCR修饰的细胞对Her2/neu抗原的特异性。
所述α链的所述恒定区可以修饰有一个或多个二硫键，并且/或者所述β链的所述恒定区可以修饰有一个或多个二硫键，例如1个或 2个。
在具体的实施方式中，制备了两种不同方式修饰的TCR，一种方式是通过点突变在TCR恒定区增加一个二硫键，方法在文献“Cancer Res.2007 Apr 15；67(8):3898-903.”中描述，其全文通过引用方式并入本文。Her2 TCR-1B5-mC是用小鼠TCR恒定区序列置换相应的人TCR恒定区序列，方法在文献“Eur.J.Immunol.2006 36:3052–3059”中描述，其全文通过引用方式并入本文。
在具体的实施方案中，所述α链的氨基酸序列如SEQ ID NOs:4、5或6所示，所述β链的氨基酸序列如SEQ ID NOs:7、8或9所示。
其中，对于氨基酸序列如SEQ ID NO:4所示的α链，其序列为原始的人源序列；对于氨基酸序列如SEQ ID NO:5所示的α链，其在恒定区修饰有1个二硫键；对于氨基酸序列如SEQ ID NO:6所示的α链，其恒定区替换为鼠源恒定区。
其中，对于氨基酸序列如SEQ ID NO:7所示的β链，其序列为原始的人源序列；对于氨基酸序列如SEQ ID NO:8所示的β链，其在恒定区修饰有1个二硫键；对于氨基酸序列如SEQ ID NO:9所示的β链，其恒定区替换为鼠源恒定区。
在一个具体实施方案中，所述TCR的α链的氨基酸序列如SEQ ID NO:4所示，β链的氨基酸序列如SEQ ID NO:7所示。在另一个具体实施方案中，所述TCR的α链的氨基酸序列如SEQ ID NO:5所示，β链的氨基酸序列如SEQ ID NO:8所示。在又一个具体实施方案中，所述TCR的α链的氨基酸序列如SEQ ID NO:6所示，β链的氨基酸序列如SEQ ID NO:9所示。
在本发明其它具体的实施方案中，所述TCR的α链具有在SEQ ID NOs:4、5或6所示氨基酸序列中替换、删除、和/或添加一个或多个氨基酸而得到的氨基酸序列；例如，所述α链具有与SEQ ID NOs:4、5或6所示氨基酸序列至少90％、优选至少95％、更优选至少99％的一致性。
在本发明其它具体的实施方案中，所述TCR的β链具有在SEQ ID NOs:7、8或9所示氨基酸序列中替换、删除、和/或添加一个或 多个氨基酸而得到的氨基酸序列；例如，所述β链具有与SEQ ID NOs:7、8或9所示氨基酸序列至少90％、优选至少95％、更优选至少99％的一致性。
本发明的TCR的α链和/或β链还可以在末端(例如C末端)结合其它功能性序列，例如共刺激信号CD28、4-1BB和/或CD3zeta的功能区序列。
本发明还提供了一种分离的、编码T细胞受体的核酸，包含所述T细胞受体的α链和β链中的至少一者的编码序列，所述α链编码序列和β链编码序列均包含可变区编码序列和恒定区编码序列，其特征在于，所述T细胞受体能够特异性识别肿瘤细胞表达的抗原Her2/neu，并且所述α链可变区编码序列编码的氨基酸序列具有与SEQ ID NO:1所示的氨基酸序列至少98％、优选至少98.5％、更优选至少99％的一致性，所述β链可变区编码序列编码的氨基酸序列具有与SEQ ID NO:2所示的氨基酸序列至少98％、优选至少98.5％、更优选至少99％的一致性，只要不显著影响本发明的效果即可。还优选的是，所述α链可变区编码序列编码如SEQ ID NO:1所示的氨基酸序列，所述β链可变区编码序列编码如SEQ ID NO:2所示的氨基酸序列。
所述核酸可以为DNA或RNA。
优选地，所述α链可变区编码序列如SEQ ID NO:10所示，所述β链可变区编码序列如SEQ ID NO:11所示。
进一步具体地，被所述核酸编码的所述T细胞受体能够特异性识别被HLA-A2分子所提呈的所述抗原Her2/neu的抗原表位多肽。
在一个实施方案中，所述抗原Her2/neu的抗原表位多肽为Her2/neu 369-377多肽(SEQ ID NO:3)。在其它实施方案中，所述抗原Her2/neu的抗原表位多肽为与Her2/neu 369-377多肽具有4-9个连续的相同氨基酸(例如，4、5、6、7、8或9个连续的相同氨基酸)的抗原表位多肽，并且这些多肽的长度为8-11个氨基酸。例如，在一个实施方案中，所述抗原Her2/neu的抗原表位多肽为Her2/neu373-382多肽(SEQ ID NO:22)。
优选地，被所述核酸编码的所述T细胞受体识别Her2/neu 369-377多肽的最大半反应多肽浓度在1.0-10ng/ml之间(例如，在3.0-8.0ng/ml、5.0-7.0ng/ml之间)。在本发明的一个实施方案中，所述最大半反应多肽浓度约为1.6-2.9ng/ml。在此情况下，所述T细胞受体对Her2/neu抗原具有中高等亲和性，可避免高亲和性(最大半反应多肽浓度小于0.1ng/ml)可能带来的脱靶毒性。
在一个实施方案中，所述α链的所述恒定区和/或所述β链的所述恒定区来源于人；优选地，所述α链恒定区编码序列全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链恒定区编码序列全部或部分地被来源于其它物种的同源序列所替换。更优选地，所述其它物种为小鼠。所述替换可以增加细胞中TCR的表达量，并且可以进一步提高被该TCR修饰的细胞对Her2/neu抗原的特异性。
所述α链恒定区编码序列可以包含一个或多个二硫键的编码序列，并且/或者所述β链恒定区编码序列可以包含一个或多个二硫键的编码序列。
在具体的实施方案中，所述α链编码序列如SEQ ID NOs:12、13或14所示，所述β链编码序列如SEQ ID NOs:15、16或17所示。
其中，对于编码序列如SEQ ID NO:12所示的α链，其序列为原始的人源序列；对于编码序列如SEQ ID NO:13所示的α链，其在恒定区修饰有1个二硫键；对于编码序列如SEQ ID NO:14所示的α链，其恒定区替换为鼠源恒定区。
其中，对于编码序列如SEQ ID NO:15所示的β链，其序列为原始的人源序列；对于编码序列如SEQ ID NO:16所示的β链，其在恒定区修饰有1个二硫键；对于编码序列如SEQ ID NO:17所示的β链，其恒定区替换为鼠源恒定区。
在一个具体实施方案中，所述TCR的α链的编码序列如SEQ ID NO:12所示，β链的编码序列如SEQ ID NO:15所示。在另一个具体实施方案中，所述TCR的α链的编码序列如SEQ ID NO:13所示，β链的编码序列如SEQ ID NO:16所示。在又一个具体实施方案中，所述TCR的α链的编码序列如SEQ ID NO:14所示，β链的编码序列如SEQ ID NO:17所示。
在另外的实施方案中，所述α链编码序列和所述β链编码序列之间由可切割性连接多肽的编码序列连接，这样可以增加TCR在细胞内的表达。术语“可切割性连接多肽”是指该多肽起到连接作用，并且可以被特定的酶切割，或者编码此多肽的核酸序列通过核糖体跳跃方式(ribosome skipping)进行翻译，从而使被其连接的多肽彼此分离。可切割性连接多肽的例子是本领域已知的，例如F2A多肽，F2A多肽序列包括但不限于来自微小核糖核酸病毒的F2A多肽、以及来自其它病毒相似的2A类序列。例如，可切割/核糖体跳跃2A链接序列可来自不同的病毒基因组，包括F2A(口蹄疫病毒(foot-and-mouth disease virus)2A)、T2A(thosea asigna virus 2A)、P2A(猪捷申病毒1型(porcine teschovirus-1)2A)和E2A(马鼻炎A病毒(equine rhinitis A virus)2A)。另外，可切割性连接多肽也包括可被Furin酶切割的标准的四氨基酸基序(canonical four amino acid motif)，即R-X-[KR]-R氨基酸序列。该实施方案所编码的TCR为单链嵌合T细胞受体，该单链嵌合T细胞受体表达完成后，连接α链和β链的可切割性连接多肽会被细胞中的特定酶切割，从而形成等量游离的α链和β链。
组成单链嵌合TCR的α链和β链也可如上文所述，恒定区(及其相应的编码序列)全部或部分地被来源于其它物种的同源序列所替换，并且/或者修饰有(编码)一个或多个二硫键。
在具体的实施方案中，所述核酸的序列如SEQ ID NOs:18、19、或20所示。
优选地，对所述核酸的核苷酸序列进行编码子优化以增加基因表达、蛋白翻译效率以及蛋白表达，从而增强TCR识别抗原的能力。编码子优化包括但不限于翻译启动区域的修饰、改变mRNA结构片段、以及使用编码同一氨基酸的不同密码子。
在其它的实施方案中，可以对上述TCR编码核酸的序列进行突变，包括去除、插入和/或置换一个或多个氨基酸密码子，使得所表达的TCR识别Her2/neu抗原的功能不变或者增强。例如，在一个实施方案中，进行保守氨基酸置换，包括对上述TCRα链和/或β链的可变区中的一 个氨基酸用结构和/或化学属性相似的另一个氨基酸进行置换。术语“相似的氨基酸”是指具有相似的极性、电负荷、可溶性、疏水性、亲水性等属性的氨基酸残基。突变后的TCR仍具有识别上述被靶细胞提呈的Her2/neu抗原多肽的生物活性。在另一个实施方案中，进行TCR成熟性(TCR maturation)修饰，即，包括对上述TCRα链和/或β链的可变区中的互补决定区2(CDR2)和/或CDR3区域的氨基酸进行去除、插入和/或置换，从而改变TCR结合Her2/neu抗原的亲和性。
本发明还提供了一种分离的、由根据本发明所述的DNA转录的mRNA。
本发明还提供了一种重组表达载体，其含有与启动子有效连接的根据本发明所述的核酸(例如DNA)，和/或其互补序列。
优选地，在所述重组表达载体中，本发明所述的DNA合适地与启动子、增强子、终止子和/或polyA信号序列有效连接。
本发明的重组表达载体的上述作用元件的组合能够促进DNA的转录和翻译，并增强mRNA的稳定性。
重组表达载体的基本骨架可以是任何已知的表达载体，包括质粒或病毒，病毒载体包括但不限于(例如)逆转录病毒载体(病毒原型为莫洛尼鼠白血病病毒(MMLV))和慢病毒载体(病毒原型为人类免疫缺陷I型病毒(HIV))。表达本发明所述TCR的重组载体可以通过本领域常规的重组DNA技术来获得。
在一个实施方案中，重组表达载体上的α链和β链基因的表达可以由两个不同的启动子所驱动，启动子包括各种已知的类型，例如强表达的、弱表达的、持续表达的、可诱导的、组织特异性的、和分化特异性的启动子。启动子可以是病毒来源的或者非病毒来源的(例如真核细胞启动子)，例如CMV启动子、MSCV的LTR上的启动子、EF1-α启动子、和PGK-1启动子、SV40启动子、Ubc启动子、CAG启动子、TRE启动子、CaMKIIa启动子、人β肌动蛋白启动子。两个启动子的驱动方向可以是同向也可以是反向的。
在另一个实施方案中，重组表达载体上的α链和β链基因的表达可以由同一个启动子所驱动，例如编码单链嵌合T细胞受体的情 况，α链的核苷酸序列和β链的核苷酸序列由Furin-F2A多肽编码序列相连接。
在另一些实施方案中，重组表达载体除了包含α链和β链基因外，还可以包含其它功能分子的编码序列。一个实施方案包括表达自发荧光蛋白(如GFP或其它荧光蛋白)以用于体内追踪成像。另一个实施方案包括表达可诱导的自杀基因系统，例如诱导表达单纯疱疹病毒-胸腺嘧啶核苷激酶(HSV-TK)蛋白，或者诱导表达Caspase 9(iCasp9)蛋白。表达这些“安全转换分子”(safety-switch)可以增加经本发明所述TCR基因修饰的细胞在体内使用的安全性(参见文献“Front.Pharmacol.,2014；5:1-8)。因此，所述重组表达载体可含有自杀基因编码序列，所述自杀基因可选自：iCasp9、HSV-TK、mTMPK、截短的EGFR、截短的CD19、截短的CD20或其组合。可选地，所述自杀基因编码序列是在启动子控制下的，并且该用于控制所述自杀基因编码序列的启动子与本发明所述的核酸所连接的启动子可以相同或不同，并且是彼此独立的。或者，所述自杀基因编码序列和本发明所述的核酸是在同一个启动子控制下的，并且所述自杀基因编码序列可通过可切割性连接多肽的编码序列或者内部核糖体进入位点(IRES(internal ribosome entry site))序列，与本发明所述的核酸相连接。所述可切割性连接多肽的编码序列可以是前文所述的可切割/核糖体跳跃2A链接序列，其可来自于不同的病毒基因组，包括F2A、T2A、P2A和E2A。另一个实施方案包括表达人趋化因子受体基因，例如CCR2，这些趋化因子受体可结合肿瘤组织中高表达的相应趋化因子配体，从而可以增加经本发明所述TCR基因修饰的细胞在肿瘤组织中的归巢。
本发明还提供了一种T细胞受体修饰的细胞，该细胞的表面被本发明所述的T细胞受体修饰，其中所述细胞包括原始T细胞或其前体细胞，NKT细胞，或T细胞株。
所述“T细胞受体修饰”中的“修饰”是指，通过基因转染使细胞表达有本发明所述的T细胞受体，即，所述T细胞受体通过跨膜区锚定在所修饰的细胞的细胞膜上，并具有识别抗原多肽/MHC复 合物的功能。
在一些实施方案中，所述T细胞受体修饰的细胞在其细胞表面或细胞内表达自杀基因蛋白，所述自杀基因可选自：iCasp9、HSV-TK、mTMPK、截短的EGFR、截短的CD19、截短的CD20或其组合。这样可增加所述T细胞受体修饰的细胞在体内使用的安全性。
本发明还提供了一种制备根据本发明所述的T细胞受体修饰的细胞的方法，包括以下步骤：
1)提供细胞；
2)提供编码本发明T细胞受体的核酸；
3)将所述核酸转染入所述细胞中。
步骤1)所述的细胞可以来源于哺乳动物，包括人、犬、小鼠、大鼠及其转基因动物。所述细胞可以来自自体或异体。异体细胞包括来自同卵双胞胎的细胞、异体干细胞、经基因改造的异体T细胞。
步骤1)所述的细胞包括原始T细胞或其前体细胞、NKT细胞、或T细胞株。术语“原始T细胞(naive T cell)”是指外周血中尚未被相应抗原活化的成熟T细胞。这些细胞可以通过本领域已知的方法分离得到。例如，T细胞可以从不同组织器官获得，包括外周血、骨髓、淋巴组织、脾脏、脐带血、肿瘤组织。一个实施方案中，T细胞可以来自造血干细胞(HSCs)，包括来自骨髓、外周血或者脐带血，通过干细胞标记分子例如CD34而分离获得。一个实施方案中，T细胞可以来自诱导性多功能干细胞(iPS cells)，包括把特定基因或特定基因产物导入体细胞，使该体细胞转化为干细胞后，体外诱导分化成T细胞或其前体细胞。T细胞可以通过常用方法如密度梯度离心法而获得，密度梯度离心法的例子包括Ficoll或者Percoll密度离心。一个实施方案是利用血浆分离置换法(apheresis)或白细胞去除法(leukapheresis)从外周血获得富集的T细胞的产物。一个实施方案是用抗体标记特定细胞群后，通过磁珠分离的方式(如
Figure PCTCN2019102562-appb-000001
系统(Miltenyi Biotec))、或流式细胞分离的方式获得富集的CD8 +或CD4 +T细胞。
优选地，所述T细胞前体细胞为造血干细胞。可以将本发明所 述TCR的编码基因直接引入造血干细胞，然后转输到体内，进一步分化成为成熟T细胞；也可以将编码基因引入在体外特定条件下由造血干细胞分化成熟的T细胞中。
所述细胞可以被重悬于冻存溶液里置于液氮中保存。常用冻存溶液包括但不限于包含20％DMSO和80％人血清白蛋白的PBS溶液。细胞以每分钟降低温度1℃的条件冻存于-80℃，然后保存于液氮罐的气相部分。其它冻存方法是把置于冻存液的细胞直接放入-80℃或液氮中进行冻存。
步骤2)所述的核酸为根据本发明所述的核酸，包括所述DNA和RNA。
所述转染包括物理方式、生物方式和化学方式。物理方式是通过磷酸钙沉淀、脂质体、微注射、电穿孔、基因枪等途径把TCR基因以DNA或RNA的形式导入细胞内。目前已有商业化的仪器，包括电转移仪，例如Amaxa Nucleofector-II(德国Amaxa Biosystems公司)、ECM 830(BTX)(美国Harvard Instruments)、Gene Pulser II(美国BioRad公司)、Multiporator(德国Eppendort公司)。生物方式是通过DNA或RNA载体把TCR基因引入细胞内，逆转录病毒载体(例如γ逆转录病毒载体)是转染并插入外源基因片段到动物细胞(包括人细胞)的常用工具，其它病毒载体来源于慢病毒、痘病毒、单纯疱疹病毒、腺病毒以及腺病毒相关病毒等。化学方式是把多核苷酸引入细胞内，包括胶态分散系统，比如大分子复合物、纳米胶囊、微球体、微珠、微团和脂质体。无论以什么方式把TCR基因引入细胞，要用各种检测方法分析目的基因是否引入靶细胞内，所述检测方法包括常见的分子生物学方法(例如Southern印迹和Northern印迹、RT-PCR和PCR等)，或者常见的生物化学方法(例如ELISA和Western印迹)，以及本发明所提及的方法。
优选地，所述转染通过逆转录病毒载体或慢病毒载体进行。
转染后所述细胞的培养可以根据实际应用通过其各自的常规方法和条件进行。例如，T细胞通过表面的TCR/CD3复合体，以及辅助刺激分子(如CD28)共同激活后，可获得体外扩增。激活TCR、CD3 和CD28的刺激物(如抗TCR、CD3或CD28的抗体)可以吸附在培养容器表面，或者共培养物(比如磁珠)表面，也可以直接加入细胞培养液中共同培养。另一个实施方案是将T细胞与滋养细胞共同培养，所述滋养细胞表达辅助刺激分子或者相应的配体，包括但不限于HLA-A2、β2-微球蛋白、CD40、CD83、CD86、CD127、4-1BB。
依照通常的哺乳动物细胞体外培养的方法，将T细胞在适当培养条件下进行培养和扩增。例如，细胞达到70％以上融合状态(confluence)时可进行传代，一般2到3天换新鲜培养液。当细胞达到一定数目时直接使用，或按上述描述进行冻存。体外培养的时间可以是24小时之内，也可以长达14天或更长。冻存细胞解冻后可进行下一步应用。
在一个实施方案中，细胞可以在体外培养数小时到14天，或者之间任何小时数。T细胞培养条件包括使用基础培养液，包括但不限于RPMI 1640、AIM-V、DMEM、MEM、a-MEM、F-12、X-Vivo 15和X-Vivo。其它细胞生存和增殖所需要的条件包括但不限于使用血清(人或胎牛血清)、白介素-2(IL-2)、胰岛素、IFN-γ、IL-4、IL-7、GM-CSF、IL-10、IL-12、IL-15、IL-21、TGF-β和TNF-a，其它培养添加物(包括氨基酸、丙酮酸钠、维生素C、2-巯基乙醇、生长激素、生长因子)。细胞可置于适当的培养条件，例如，温度可处于37℃、32℃、30℃或者室温，并且空气条件可为(例如)含5％CO 2的空气。
本发明还提供了根据本发明所述的T细胞受体修饰的细胞在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
所述肿瘤和/或癌症是抗原Her2/neu阳性的，并且是HLA-A2阳性的，包括但不限于乳腺癌、卵巢癌、胃癌、食管癌、肠癌、胰腺癌、膀胱癌、肾癌、前列腺癌、子宫颈癌、子宫内膜癌、唾液腺癌、皮肤癌、肺癌、骨癌以及脑癌。
本发明还提供了根据本发明所述的T细胞受体修饰的细胞在制备用于检测宿主的肿瘤和/或癌症的药物中的用途。
在本发明的一个实施方案中，可将从宿主取出的肿瘤和/或癌症细胞的样本与本发明所述的T细胞受体修饰的细胞以一定浓度进行接触，根据二者的反应程度可以判断所述肿瘤和/或癌症是HLA-A2 阳性的还是HLA-A2阴性的，以及是否表达抗原Her2/neu。
本发明还提供了一种药物组合物，其中该药物组合物包括作为活性成分的根据本发明所述的T细胞受体修饰的细胞，及可药用辅料。
所述药物组合物优选包含每个患者每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重的所述T细胞受体修饰的细胞，包括两个端点之间的任何数量的细胞。优选的是，每个疗程1-3天，每天施用1-3次。可以根据实际情况和需要对患者进行一个或多个疗程的治疗。
所述可药用辅料包括药用或生理载体、赋形剂、稀释剂(包括生理盐水、PBS溶液)、以及各种添加剂，包括糖类、脂类、多肽、氨基酸、抗氧化剂、佐剂、保鲜剂等。
所述药物组合物可通过合适的给药途径给药，其适于经动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内或腹膜内给药。
本发明还提供了一种治疗肿瘤和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据本发明所述的T细胞受体修饰的细胞。
所述肿瘤和/或癌症是抗原Her2/neu阳性的，并且是HLA-A2阳性的，包括但不限于乳腺癌、卵巢癌、胃癌、食管癌、肠癌、胰腺癌、膀胱癌、肾癌、前列腺癌、子宫颈癌、子宫内膜癌、唾液腺癌、皮肤癌、肺癌、骨癌以及脑癌。
所述T细胞受体修饰的细胞的施用剂量优选为每个患者每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重。优选的是，每个疗程1-3天，每天施用1-3次。可以根据实际情况和需要对患者进行一个或多个疗程的治疗。
所述T细胞受体修饰的细胞可通过合适的给药途径给药，其适于经动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内或腹膜内给药。
所述T细胞受体修饰的细胞进入治疗对象体内后可以消除表达Her2/neu抗原的肿瘤细胞，和/或改变肿瘤组织的微环境而诱发其它抗 肿瘤免疫反应。
所述治疗肿瘤和/或癌症的方法还包括对肿瘤和/或癌症患者施用其它用于治疗肿瘤的药物，和/或用于调节患者免疫系统的药物，以增强所述T细胞受体修饰的细胞在体内的数量和功能。所述其它用于治疗肿瘤的药物包括但不限于：化疗药物，例如环磷酰胺、氟达拉滨(fludarabine)；放疗；免疫抑制剂，例如环孢素、硫唑嘌呤、甲氨蝶呤、麦考酚酯(mycophenolate)、FK50；抗体，例如抗CD3、IL-2、IL-6、IL-17、TNFα的抗体。
在某些实施方案中，所述治疗肿瘤和/或癌症的方法还包括对患者施用其它用于治疗肿瘤和/或癌症的药物，和/或用于调节患者免疫系统的药物，用于当所述T细胞受体修饰的免疫细胞产生严重毒副作用时，清除携带自杀基因的所述T细胞受体修饰的免疫细胞在体内的数量和功能。所述其它用于治疗肿瘤和/或癌症的药物包括但不限于：化学诱导二聚化(CID)药物、AP1903、磷酸化更昔洛韦(ganciclovir)、抗Cd20抗体、抗CMYC抗体、抗EGFR抗体。
本发明还提供了所述分离的T细胞受体用于检测接受该TCR修饰的T细胞(即TCR-T细胞)治疗的患者体内的该TCR-T细胞的增殖或生存情况的应用，从而进行药物代谢研究，和了解该TCR-T细胞的疗效和毒性。具体而言，TCR序列可作为引物，通过PCR方法检测体内携带此TCR的TCR-T细胞的数量。与荧光标记的HLA/多肽复合物多聚体染色后用流式细胞法进行分析的方法相比，所述应用所需要的细胞量少，也更敏感。
以下通过例子的方式进一步解释或说明本发明的内容，但这些例子不应被理解为对本发明的保护范围的限制。
例子
除非特别说明，否则以下例子中所用实验方法均使用生物工程领域的常规实验流程、操作、材料和条件进行。
以下除非特别说明，否则各试剂的百分浓度(％)均指该试剂的体积百分浓度(％(v/v))。
材料和方法
细胞株：用于制备慢病毒颗粒的细胞株为293T细胞(ATCC CRL-3216)。用于提呈抗原多肽的提呈细胞株为T2细胞(174xCEM.T2，ATCC CRL-1992)。用于检测功能的肿瘤细胞株为人结直肠癌colo205细胞(ATCC CCL-222)、HT-29细胞(HTB-38)和HCT116细胞(ATCC CCL-247)、人乳腺癌MDA-MB-231细胞(ATCC HTB-26)和MCF7细胞(ATCCHTB-22)、人卵巢癌SKOV3细胞(ATCC HTB-77)、人胰腺癌PANC-1细胞(ATCCCRL-1469)、人神经胶质细胞瘤U87MG细胞(ATCC HTB-14)、人肝细胞癌HepG2细胞(ATCC HB-8065)、人非小细胞肺癌NCI-H460细胞(ATCC HTB177)和小细胞肺癌NCI-H446细胞(ATCC HTB-171)、人宫颈癌细胞C33A(ATCC HTB-31)、人骨肉瘤细胞Saos-2(ATCC HTB-85)、人胰腺癌细胞CFPAC-1(ATCC CRL-1918)。细胞株用RPMI-1640完全培养基(Lonza，cat#12-115F)维持培养，RPMI-1640完全培养基中加入10％小牛血清FBS(ATCC 30-2020)，2mmol/L L-谷氨酸，100μg/ml青霉素和100μg/ml链霉素。
外周血制品：除非特别说明，否则以下试验所用健康供者的人外周血制品(包括外周血单个核细胞)来自位于旧金山的Pacific血液中心(#1PBMC和#2 PBMC分别为来自Apheresis法收集试剂盒的Trima残留细胞组分#R32334和#R33941)。
台盼蓝染色法计数：将细胞用PBS洗后，用胰蛋白酶消化，细胞悬浮在PBS中，加入终浓度为0.04％(w/v)的台盼蓝染液，显微镜下计数，死细胞会染成浅蓝色，活细胞拒染。取活细胞数为最终数据。
体外诱导Her2/neu 369-377特异性杀伤T细胞(CTL)：外周血经Ficoll-Paque Premium(Sigma-Aldrich公司，cat#GE-17-5442-02)密度梯度离心(×400g)30分钟后获得单个核细胞(PBMC)。首先用荧光素FITC标记的抗HLA-A2抗体(Biolegend公司，cat#343303)染色检测细胞的HLA-A2表型，流式细胞分析(流式细胞仪为MACSQuant Analyzer 10(Miltenyi Biotec公司)，用Flowjo软件 (Flowjo公司)进行结果分析)后提取阳性细胞的RNA，逆转录为cDNA并克隆到载体上，之后进行HLA基因测序分析，确定细胞配型为HLA-A*0201。HLA-A2阳性的PBMC细胞培养在24-孔培养板的培养孔，培养液为上述RPMI-1640完全培养基。每孔2×10e6/ml PBMC，加入Her2/neu 369-377多肽(Her2-E75，用Peptide2.0合成，10μg/ml溶于DMSO)，终浓度为1μg/ml。置于5％CO 2、37℃条件下的培养箱培养16-24小时后加入以下终浓度的细胞因子：人IL-2(Peprotech公司，cat#200-02)100IU/ml，人IL-7(Peprotech公司，cat#200-07)5ng/ml，人IL-15(Peprotech公司，cat#200-15)5ng/ml。培养10到14天，对培养的T细胞进行抗原再刺激：在24-孔板中每孔加入10e6个上述所得的培养细胞，同时加入2×10e6个经25μg/ml丝裂霉素C(Santa Cruz Biotechnology公司，cat#SC-3514)处理2小时的HLA-A2阳性的PBMC细胞作为滋养细胞，每孔加入终浓度为1μg/ml的Her2/neu 369-377多肽，培养过夜后加入IL-2 100IU/ml，IL-7 5ng/ml，IL-15 5ng/ml(终浓度)。经两轮上述抗原刺激和再刺激后，收集扩增的T细胞进行表型分析以及T细胞克隆。
流式细胞分析及单细胞分离：表达Her2/neu 369-377特异性TCR的T细胞表型是通过流式细胞来分析的。收集被检测的细胞置于1.5ml管(细胞数目约为10e5个)，用1ml DPBS溶液(2.7mM KCl，1.5mM KH 2PO 4，136.9mM NaCl,8.9mM Na 2HPO 4·7H 2O，pH 7.4)洗一遍，并重置于100μl含有1％小牛血清的DPBS中，加入5μl荧光素APC标记的抗人CD8抗体(Biolegend公司，cat#300912)，以及10μl荧光素PE标记的Her2-E75/HLA-A2四聚体(Her2-E75四聚体，MBL International Co.公司，cat#T01014)或者Her2-E75/HLA-A2五聚体(Her2-E75五聚体，Proimmune公司，cat#F214-2A-D)，冰上孵育30分钟后用DPBS溶液洗两遍，重悬于100μl PBS溶液(8mM Na 2HPO 4、136mM NaCl、2mM KH 2PO 4、2.6mM KCl，pH7.2-7.4)进行流式细胞分析。流式细胞仪为MACSQuant Analyzer10(Miltenyi Biotec公司)，用Flowjo软件(Flowjo公司)进行结果分析。T细胞克隆是利用流式细胞分离仪(FACS sorter)进行单细胞 分离后培养获得。对Her2/neu369-377多肽抗原刺激过的PBMC用APC标记的抗人CD8抗体和PE标记的Her2-E75/HLA-A2五聚体染色，然后进行流式细胞分离(型号：Sony cell sorter SH800)。单个CD8 +Her2-E75/HLA-A2五聚体 +细胞被分选到96-孔培养板的单个培养孔后，加入经25μg/ml丝裂霉素C处理2小时的HLA-A2阳性的PBMC细胞，每孔10e5个细胞，加入1μg/ml Her2/neu 369-377多肽培养过夜后，加入含有IL-2 100IU/ml、IL-7 5ng/ml、IL-15 5ng/ml的RPMI-1640完全培养液。每3-4天换新鲜含有所述细胞因子的培养液，显微镜下观察是否有T细胞克隆生长。收集增殖的T细胞，按上述方法进行抗原再刺激以获得足够数量的细胞，进行表型或功能检测，以及提取RNA进行TCR基因的克隆。
T细胞功能检测：为了检测转染TCR基因的T细胞识别抗原表位多肽的能力，在96-孔板的每孔中加入10e5个转染TCR基因的T细胞以及10e5个T2细胞，在100μl/每孔RPMI-1640完全培养基中进行混合培养，各试验组为复孔。再加入不同终浓度(分别为1μg/ml、0.5μg/ml、0.1μg/ml、0.05μg/ml、0.01μg/ml、0.005μg/ml、0.001μg/ml和0.0001μg/ml)的Her2/neu 369-377多肽后置于5％CO 2、37℃条件下的孵育箱过夜培养。为了确定所述TCR识别表位抗原的关键氨基酸位点，在96-孔板的每孔中加入10e5个转染TCR基因的T细胞以及10e5个T2细胞，再加入终浓度为0.1μg/ml的待测表位多肽后置于5％CO 2、37℃条件下的孵育箱过夜培养。24小时后收集上清，用人IFN-γELISA Read-set-Go试剂盒(eBioscience公司，cat#88-7316)或人IFN-γDuoSet ELISA试剂盒(R&D Systems，cat#DY285B)，按照厂家说明书，对上清中的IFN-γ进行检测。
为了检测转染TCR基因的T细胞识别肿瘤细胞株的能力，根据不同效靶比在96-孔板的每孔中加入一定数量的转染TCR基因的PBMC细胞和肿瘤细胞作为靶细胞，培养24小时后，收集上清检测上清中分泌的γ干扰素。各试验组为复孔或三孔。抗体功能阻断试验中，细胞培养孔中同时加入10μg/ml终浓度的抗人CD8抗体(Biolegend公司，cat#300912)，细胞置于5％CO 2、37℃条件下的 孵育箱过夜培养。18-24小时收集细胞上清，并用人IFN-γELISA Read-set-Go试剂盒(eBioscience公司，cat#88-7316)或人IFN-γDuoSet ELISA试剂盒(R&D Systems，cat#DY285B)，按照厂家说明书，对上清中的IFN-γ进行检测。
为了检测转染TCR基因的T细胞杀伤肿瘤细胞的能力，在24-孔培养板中每孔加入靶细胞1×10e4培养24小时使靶细胞完全贴壁,去除悬浮细胞，根据设定的效靶比加入一定数量的转染TCR基因的T细胞。培养24小时后，去除悬浮细胞，并用胰酶消化收集贴壁细胞进行台盼蓝染色计数活细胞。杀伤率(Cytotoxicity)％＝(初始靶细胞的活细胞数-培养终止时的靶细胞的活细胞数)/初始靶细胞的活细胞数×100。各实验组为复孔或三孔，差异显著性用学生t-检验分析。或者用MTT方法检测杀伤活性。
MTT方法说明：
胰酶消化对数生长期细胞，终止后离心收集，吹散均匀，制备单细胞悬液；用细胞培养液将细胞浓度调整至0.1～10×10 4/ml(根据不同细胞生长状况调整接种细胞数)，接种于96孔细胞培养板，培养体系为100μl/孔，置于37℃，5％CO 2培养箱培养过夜，使细胞完全贴壁，第二天达到70～80％；计数方式用计数板计数，同时用countstar计数仪来验证计数的正确性。取出96孔板，加入100μl预先配制的T细胞和TCR-T细胞悬液，加样前轻微涡旋，空白对照孔加100μl的相应细胞培养的无血清培养基；置于37℃，5％CO 2培养箱分别培养24小时；于24h后取细胞，离心400g，10min后吸取180μl培养基放入新的96孔板中，留样用于后面ELISA检测上清IFN-γ水平，检测步骤可参照检测说明书。注：上清可-80℃冻存以用于后续检测。每孔加入新的100μl完全培养基，每孔加入10μl MTT溶液(5mg/ml，即0.5％MTT)，继续培养4～6h；设立效应细胞对照组，在加入MTT 4小时后，300g离心5分钟，将染上MTT的效应细胞离心至板底以后，再弃去上清，再加入DMSO检测。每孔加入150μl DMSO，置摇床上低速震荡10分钟，使结晶物充分溶解，在酶标仪上检测其在490nm处的吸光值。
获得单克隆TCR基因：利用Zymo Quick-RNA Microprep试剂盒(Zymo Research公司，cat#R1050)从T细胞克隆提纯总RNA，以此为模板利用Smarter RACE 5’/3’试剂盒获得cDNA(美国Takara Bio公司，cat#634858)。用5’-CDS引物和TCRβ链3’引物5’-GCCTCTGGAATCCTTTCTCTTG-3’(SEQ ID NO:24)以及α链3’引物5’-TCAGCTGGACCACAGCCGCAG-3’(SEQ ID NO:25)进行PCR，扩增出TCRα和β全序列基因片段，并分别克隆到pRACE载体(美国Takara Bio，cat#634858)上。转化感受态细菌Stellar(美国Takara Bio公司，cat#636763)并获得质粒后进行测序。
重组TCR慢病毒表达载体的制备：用于表达TCR的病毒载体为复制缺陷型慢病毒载体，包括：表达GFP的慢病毒载体pCDH-EF1α-MCS-(PGK-GFP)，可购自System Biosciences公司(Cat#CD811A-1)；以及不表达GFP的载体pCDH-EF1α-MCS，通过采用本领域常规技术去除pCDH-EF1α-MCS-(PGK-GFP)载体上的PGK启动子及GFP基因而得到。根据所获得的TCR基因序列，合成TCRβ链和α链以及之间可切割的F2A序列和Furin酶切片段的全基因序列，并链接到所述载体的EF-1α启动子下游的多克隆位点，插入TCR的转录顺序依次为TCRβ链(无终止密码子)，Furin酶切片段，F2A片段，TCRα链(方法参见文献“Gene Ther.2008Nov；15(21):1411–1423”)。表达GFP的载体是被反向的PGK启动子驱动的。不表达GFP的载体则是去除了PGK启动子以及GFP片段。
重组TCR慢病毒颗粒的制备：TCR慢病毒颗粒是通过Lipofectaine 2000转染试剂(invitrogen，#11668019)转染293T/293FT细胞而获得的。依照厂家说明书准备293T/293FT细胞以及转染流程。转染在6孔培养板进行，首先用Opti-MEM 1培养液(Thermo Fisher公司，cat#51985091)制备转染质粒的脂质体混合溶液，依照厂家说明在250μl培养液中加入lipofectaine2000试剂6μl、以及TCR慢病毒载体质粒0.8μg和pCDH系统的病毒包装质粒1.8μg(SBI公司，cat#LV500A-1)，混合孵育25分钟后加入293T/293FT细胞培养孔。5％CO 2、37℃条件下培养16小时，换不含FBS的DMEM培养液 (Thermo Fisher公司，cat#11965092)，继续培养24小时和48小时后分别收集细胞上清，2000g离心10min后，用0.4μm过滤膜过滤后得到的病毒上清使用慢病毒浓缩液(GeneCopoeiaTM#LPR-LCS-01)按厂家说明书浓缩后用于感染细胞。
重组TCR慢病毒转染人T细胞：冻存的原代PBMC细胞解冻后在RPMI-1640完全培养液中培养24小时，经Ficoll-Paque Premium密度梯度离心(×400g)30分钟去除死细胞，置于用2μg/ml抗人CD3抗体(Biolegend公司，OKT3克隆cat#317303)和2μg/ml抗人CD28抗体(Biolegend公司，cat#302914)处理(其中每孔加入100μl含有上述CD3抗体和CD28抗体的DPBS溶液)24小时的24孔板培养孔中，细胞浓度为2×10e6/ml，也可以用Dynabead人T-CD3/CD8磁珠(Thermo Fisher公司，cat#11131D)，按照厂家说明书对PBMC细胞进行刺激活化。培养24小时后收集细胞，加入100μl浓缩后TCR慢病毒颗粒(3×10e8Tu/ml)中置于24孔板的孔中，用含有IL-2100IU/ml、IL-7 5ng/ml、IL-15 5ng/ml的RPMI-1640完全培养液或X-VIVO15(Lonza#04-418Q)继续培养，每3天换新鲜含有上述细胞因子的培养液。也可以使用RestroNectin预处理的培养板(Takara公司，cat#T110A)，按照厂家说明书用病毒感染活化的PBMC细胞。一般72小时后可进行表型和功能检测。转染T细胞株也依照上述步骤进行，如果病毒载体上带有GFP标记，一般转染后48小时即可在荧光显微镜下观察到GFP阳性细胞。
实施例1：从HLA-A2阳性的正常供体外周血诱导Her2/neu369-377多肽(Her2-E75表位多肽)特异性杀伤T细胞
本实施例用1μg/ml的低浓度Her2/neu 369-377多肽经过两轮体外刺激从HLA-A2阳性的正常PBMC(#2)中诱导出多肽特异性杀伤T细胞，并进行流式细胞分析及单细胞分离。具体方法如上文所述。结果如下：
图1A右图显示，0.024％的淋巴细胞为可结合Her2/neu369-377/HLA-A2五聚体(即Her2-E75五聚体)的CD8阳性杀伤性T 细胞，左图中没有经Her2多肽刺激的对照细胞没有出现CD8阳性五聚体阳性细胞。结果说明在自然T细胞库中，识别Her2/neu 369-377抗原多肽的特异性T细胞数量很少。尽管数量少，这群可识别Her2/neu 369-377多肽的T细胞仍可被清晰地区分出来。另外根据结合Her2-E75五聚体的荧光强度，阳性细胞中又包含高亲和性T细胞和低亲和性T细胞。通过流式细胞分离出300个CD8阳性五聚体阳性细胞后进行单克隆培养，经过两轮抗原多肽再刺激以及细胞因子扩增，从这300个分离出的单个T细胞中获得一个增殖的T细胞克隆Her2 CTL克隆6A5(称为Her2 CTL 6A5)。图1B右图显示97.9％的CD8 +CTL细胞可结合Her2/neu 369-377/HLA-A2四聚体(即Her2-E75四聚体)，显示此纯化的T细胞克隆没有混杂其他无关细胞。左图为不能结合Her2-E75四聚体的对照T细胞。
实施例2：Her2/neu 369-377多肽特异性TCR全序列的获得
本实施例直接从由实施例1得到的一定数量的Her2 CTL 6A5细胞提纯总RNA，通过5’-RACE RT-PCR的方法获得配对的TCRα链和β链基因序列(即，两条链可共同组成识别抗原多肽的功能性TCR)，其编码的TCR称为“Her2 TCR-6A5”。该TCR的α链的氨基酸序列如SEQ ID NO:4所示，编码序列如SEQ ID NO:12所示，并且该TCR的β链的氨基酸序列如SEQ ID NO:7所示，编码序列如SEQ ID NO:15所示。此TCR存在于HLA-A2阳性正常人的外周T细胞库中，不会对微量表达Her2/neu蛋白的正常细胞产生交叉反应而导致自身免疫反应。为了检测所获TCR的抗原特异性及其功能，TCRα链和β链序列被克隆到复制缺陷型慢病毒表达载体中。图1C显示所构建的TCR慢病毒载体结构片段示意图。TCRα链和β链的恒定区由人源序列替换为鼠源序列，并由可切割性连接多肽连接。6A5TCRα链和β链的表达由EF-1α启动子所驱动。此启动子属于真核细胞中高表达启动子，而且不会受到甲基化等因素的影响而导致功能丧失，适于外源基因在体内的长期表达。TCRα链和β链之间由F2A多肽序列所连接，TCRα链和β链基因可同时被转录，通过核糖体跳跃方式 (ribosome skipping)进行翻译，从而使TCRα链和β链多肽彼此分离。这样保证了TCRα链和β链表达量的一致性，从而更有效率的组成TCR二聚体。TCRα链和β链之间还链有furin酶切位点，用于去除β链羧基端的多余肽段。
将由可切割性连接多肽链接的、恒定区由人源序列替换为鼠源序列的TCRβ链和α链的核苷酸序列(SEQ ID NO:20)(对应的TCR为Her2 TCR-6A5-mC，氨基酸序列如SEQ ID NO:23所示)连接至上述载体，以得到Her2 TCR-6A5-mC重组慢病毒载体。Her2 TCR-6A5-mC基因片段通过PCR扩增后，克隆到上述慢病毒载体(即pCDH-EF1α-MCS)的EF1-启动子下游：携带鼠源恒定区序列的Her2 TCR-6A5-mC的β片段是由5’引物5’-AGAGCTAGCGAATTCAACATGGGCTGCAGGCTGCTC-3’(SEQ ID NO:26)和3’引物5’-GGATCGCTTGGCACGTGAATTCTTTCTTTTGACCATAGCCAT-3’(SEQ ID NO:27)扩增而得；携带鼠源恒定区序列的Her2TCR-6A5-mC的α基因是由5’引物5’-TCCAACCCTGGGCCCATGCTCCTGTTGCTCATACCAGTG-3’
(SEQ ID NO:28)和3’引物5’-GTTGATTGTCGACGCCCTCAACTGGACCACAGCCT-3’(SEQ ID NO:29)扩增而得。PCR使用Q5高保真PCR试剂盒(NEB,cat#M0543S)，反应条件为：98℃ 30秒后，进行25个循环：98℃ 10秒，65℃ 10秒，以及72℃ 3分钟。获得的TCR片段克隆到pCDH-EF1α-MCS载体的EF1α启动子下游的MCS区域。
将构建得到的重组TCR慢病毒表达载体按前述方法制备得到各自的重组TCR慢病毒颗粒。
实施例3：正常外周血T细胞经Her2 TCR-6A5-mC重组慢病毒转染后表达可识别Her2/neu 369-377多肽的特异性TCR。
为了进一步验证本发明所获得的TCR能否在原代T细胞表达并具有识别Her2/neu抗原多肽的功能，用携带Her2 TCR-6A5-mC基因 的重组慢病毒颗粒(Her2 TCR-6A5-mC重组慢病毒载体)转染经CD3/CD28抗体活化的、来自两个不同正常供体的外周血T细胞，14天后收集细胞进行Her2-E75四聚体染色。具体方法如上文所述。结果如下：
图2A显示，两个供体外周血单个核细胞(分别为#1 PBMC和#2 PBMC)中均有淋巴细胞可以结合Her2-E75四聚体，说明这些细胞表达的Her2 TCR-6A5-mC可以特异性识别被HLA-A2提呈的Her2/neu抗原多肽。结果还显示，Her2-E75四聚体阳性细胞(即表达Her2 TCR-6A5-mC)中，CD8 +T杀伤细胞的阳性率和CD8 -淋巴细胞的阳性率相近。CD8 -的淋巴细胞很可能是CD4 +的T辅助细胞，如果慢病毒感染CD8 +和CD4 +T细胞的转染效率一样，说明CD4 +细胞上的外源Her2/neu 369-377特异性TCR能有效结合Her2-E75四聚体。这也进一步说明转染的Her2 TCR-6A5-mC不需要CD8分子的辅助功能也能有效结合Her2/HLA-A2复合物，即Her2 TCR-6A5-mC识别被HLA-A2提呈的Her2/neu 369-377表位多肽是CD8非依赖型。表达Her2 TCR-6A5-mC TCR的CD4细胞识别Her2抗原后分泌细胞因子，不仅可以辅助杀伤T细胞的功能及在体内的存活时间，也可以通过调节肿瘤微环境来诱导针对内源性肿瘤抗原的特异性T细胞，从而增强抗肿瘤免疫。
在96-孔板的每孔中加入10e5个转染TCR的PBMC细胞，与不同浓度被T2细胞(每孔1×10e5个)提呈的Her2/neu 369-377抗原多肽(Her2/neu 369-377抗原多肽从0.1μg/ml开始进行10倍稀释，从而得到终浓度为0.1μg/ml、0.01μg/ml、0.001μg/ml和0.0001μg/ml的不同组)混合培养后，检测上清中T细胞分泌的IFN-γ，用以确定此表达TCR的PBMC细胞特异性识别Her2/neu 369-377多肽的功能。图2B显示，表达Her2 TCR-6A5-mC的PBMC可以被T2细胞提呈的Her2/neu 369-377抗原多肽所激活而分泌IFN-γ，说明表达外源Her2 TCR-6A5-mC的原代T细胞可以特异性识别被HLA-A2分子提呈的Her2/neu 369-377多肽。识别抗原多肽的能力与外源TCR在T细胞上的表达量相关。两个不同供体PBMC转染Her2 TCR-6A5-mC后识 别抗原多肽的最大半反应(half-maximum reaction，EC50)多肽浓度经曲线拟合推算分别为约1.6ng/ml和2.9ng/ml(IC50 Tool程序，http://www.ic50.tk/)。尽管此反应敏感度低于识别病毒抗原等外源抗原的高亲和性TCR的EC50(EC50约10e-10M)(参见文献“CANCER RESEARCH 1998,58.4902-4908”和“HUMAN GENE THERAPY 2014,25:730–739”)，但仍处于可识别常见肿瘤相关抗原的中高TCR亲和力范围之内(如文献“Eur J Immunol(2012)42:3174–9”所述)。
图2C显示T细胞与T2细胞提呈的抗原多肽(T2+Her2-E75，即Her2/neu 369-377多肽)共培养时加入抗人CD8抗体后，T细胞分泌IFN-γ的功能没有被显著抑制。这说明外源TCR识别Her2/neu369-377抗原多肽的功能不需要CD8分子的辅助作用，也显示本发明所述的Her2 TCR-6A5-mC TCR的识别功能是非CD8功能依赖型。
实施例4：正常外周血T细胞经Her2 TCR-6A5-mC重组慢病毒转染后表达的Her2/neu 369-377多肽特异性TCR可识别HLA-A2 +Her2/neu +肿瘤细胞
首先检测所选肿瘤细胞株表达HLA-A2和Her2/neu的情况。肿瘤细胞株包括结直肠癌Colo205和HCT116、乳腺癌MDA-MB-231和MCF-7、胰腺癌PANC-1、神经胶质瘤U87MG以及小细胞肺癌NCI-H446。肿瘤细胞经抗HLA-A2抗体(BD Bioscences,cat#561341)以及抗人CD340(erbB2)抗体(Biolegend,cat#324406)染色后进行流式细胞分析。图3A结果显示，Colo205、MDA-MB-231、MCF-7、HCT116、PANC-1均为HLA-A2 +Her/neu +；U87MG为HLA-A2 +，Her2/neu -；NCI-H446的HLA-A2和Her2/neu均为阴性。这些肿瘤细胞株不仅来源于不同组织，所表达的HLA-A2和Her2/neu也各异，其中U87MG和NCI-H446细胞可作为Her2 TCR-6A5-mC T细胞功能检测的阴性对照。
在96-孔板的每孔中加入1×10e4个肿瘤细胞后，根据效靶比(5:1)在96-孔板的每孔中加入一定数量的转染Her2 TCR-6A5-mC TCR的PBMC细胞或没有转染Her2 TCR-6A5-mC TCR的PBMC细胞作为对 照组。效靶比为5:1。T细胞与不同肿瘤细胞株混合培养，之后检测上清液中分泌的IFN-γ。具体方法如上文所述。结果如下：
图3B显示，表达Her2 TCR-6A5-mC的T细胞均可被HLA-A2 + Her2/neu +的肿瘤细胞株所激活并分泌IFN-γ，肿瘤细胞株包括结肠癌Colo205和HCT116、乳腺癌MDA-MB-231和MCF-7、胰腺癌PANC-1。而对照组HLA-A2 + Her2/neu -的神经胶质瘤U87MG、以及HLA-A2 - Her2/neu -的肺癌NCI-H446却不能激活转染Her2 TCR-6A5-mC的T细胞，说明Her2 TCR-6A5-mC TCR可以特异性识别肿瘤细胞表面被HLA-A2提呈的Her2/neu抗原。来源于同一供体PBMC、平行培养但没有转染Her2 TCR-6A5-mC的对照组T细胞不能被所列肿瘤细胞株所激活，说明对肿瘤细胞的反应不是非特异性的。结果也显示，Her2 TCR-6A5-mC T细胞识别HLA-A2提呈的Her2/neu抗原的能力与肿瘤细胞表面HLA-A2和Her2/neu分子的表达量不太相关。不同肿瘤细胞可能存在对T细胞不同的抑制作用，另一方面，细胞表面的表达量不一定反映出Her2/neu总的表达量，某些肿瘤细胞表达的Her2/neu主要存在于细胞胞浆内，这些抗原更容易被HLA-A2所提呈(参见文献“J Immunol 2006；177:5088-5097”)。
在培养板中每孔加入靶细胞1×10e4，根据设定的效靶比(1:1、5:1、10:1、20:1、40:1)加入一定数量的转染TCR基因的PBMC细胞，24小时后测定T细胞对肿瘤细胞的杀伤活性。图3C-K显示，与没有转染TCR的对照T细胞相比，表达Her2 TCR-6A5-mC TCR的T细胞可以特异性识别和杀伤HLA-A2 + Her2/neu +的肿瘤细胞株MCF-7，HCT116，PANC-1和HEPG-2。杀伤能力与Her2 TCR-6A5-mC T细胞的数量呈量效关系。而对照组HLA-A2 + Her2/neu -的神经胶质瘤U87MG、HLA-A2-Her2/neu+的SKOV3和HT-29以及HLA-A2 - Her2/neu -的肺癌NCI-H446却不能被Her2 TCR-6A5-mC T细胞特异性杀伤。结果也显示，当Her2 TCR-6A5-mC T细胞增加到一定数量时，对HLA-A2 + Her2/neu +的肿瘤细胞表现出显著的特异性识别和杀伤功能，当效靶比低于10:1时，特异性杀伤功能并不明显，可能与肿瘤细胞表面被HLA-A2所提呈的Her2/neu表位多肽的数量有关。 为了进一步增强Her2 TCR-6A5-mC T细胞对肿瘤细胞的识别和杀伤敏感性，一个策略是增加肿瘤靶细胞表达HLA-A2和Her2/neu的数量。
实施例5：正常外周血T细胞经Her2 TCR-6A5-mC重组慢病毒转染后表达的Her2/neu 369-377多肽特异性TCR不识别可结合HLA-A2分子的来自人正常蛋白的具有潜在交叉反应的表位多肽。
所述Her2 TCR-6A5-mC TCR来源于健康供体外周血的T细胞，由于所述TCR存在于外周血的正常T细胞(T cell repertoire)，通常情况下不会识别正常组织的自身蛋白而产生脱靶毒性反应。为了进一步提高临床使用表达所述TCR的T细胞的安全性，本实施例首先通过抗原表位多肽的比对筛选(alanine scanning)来确定与Her2 TCR-6A5-mC TCR识别功能相关的氨基酸关键位点(motif)。把Her2-E75多肽KIFGSLAFL上每一个氨基酸各自分别用丙氨酸替代，以进行单突变。由于Her2-E75多肽的第七个氨基酸本身是丙氨酸，因此单突变时用甘氨酸替代。合成所形成的新表位多肽，并检测这些多肽是否能激活表达Her2 TCR-6A5-mC TCR的T细胞。由于丙氨酸保持多肽链二级结构的基本骨架，又拥有较小的残基侧链，因此可以确定被其所置换的特定残基对多肽生物活性所起的作用，对于抗原表位多肽，可以确定与Her2 TCR-6A5-mC TCR识别功能相关的氨基酸关键位点。所形成的9个新表位多肽(终浓度为0.1μg/ml)分别与T2细胞以及转染有编码Her2 TCR-6A5-mC TCR基因的慢病毒载体的#2PBMC混合培养24小时后，取细胞上清进行IFN-γ检测的ELISA分析。效靶比E:T为5:1。图4A结果显示，Her2-E75多肽第1、2、3、4、5、6、8、9位的氨基酸残基各自被丙氨酸替换后或第7位的丙氨酸被甘氨酸替换后，分别形成的新表位多肽激活Her2 TCR-6A5-mC TCR分泌干扰素的能力各不相同。与Her2-E75相比，第1位赖氨酸残基被替换后，抗原表位多肽激活Her2 TCR-6A5-mC TCR的能力有所增强，当第7位的丙氨酸被甘氨酸所替换，第8位的苯丙氨酸和第9位的亮氨酸被丙氨酸所替换后，表位多肽激活Her2 TCR-6A5-mC  TCR的能力有所降低，然而当第2位的异亮氨酸，第3位的苯丙氨酸，第4位的甘氨酸，第5位的丝氨酸和第6位的亮氨酸被丙氨酸替换后，抗原表位多肽激活Her2 TCR-6A5-mC TCR的能力明显降低。结果说明第2、3、4、5、6位氨基酸残基对于Her2 TCR-6A5-mC TCR的识别功能至关重要，这些位点的氨基酸侧链可能形成表位多肽结合HLA-A2分子的锚定位点或者是TCR特异性识别的结合位点，改变这些位点的氨基酸残基将导致多肽失去被Her2 TCR-6A5-mC TCR所识别的抗原特异性，而其他位点的氨基酸残基对Her2 TCR-6A5-mC TCR识别功能的贡献相对较小。因此，包含第2位的异亮氨酸、第3位的苯丙氨酸、第4位的甘氨酸、第5位的丝氨酸和第6位的亮氨酸残基的正常人蛋白质，都有可能被Her2 TCR-6A5-mC TCR所识别而产生交叉反应。为了获得所有包含上述关键氨基酸残基位点的人正常蛋白，用“X-I-F-G-S-L-X-X-X”序列搜索人正常蛋白数据库(https://prosite.expasy.org/cgi-bin/prosite/PSScan.cgi)，其中“X”可以是21个常见氨基酸中的任何一个。共13个不同的人正常蛋白序列中包含-2I-3F-4G-5S-6L-序列，表1示出蛋白名称、包含-2I-3F-4G-5S-6L-序列的抗原表位位置和抗原表位序列。这些多肽若要成为被Her2 TCR-6A5-mC TCR所识别的抗原表位多肽，首先要能够结合HLA-A2，通过HLA/多肽结合预测软件(http://www.cbs.dtu.dk/services/NetMHC/)可以预测多肽与HLA-A2的结合能力。表1还示出预测的多肽与HLA-A2的亲和性，以及多肽结合HLA-A2的亲和性在已知的与HLA-A2结合的天然表位多肽的亲和性中的排序。“亲和性(nM)”是指该表位多肽与HLA-A2的亲和性预测。“％排序”是指该表位多肽结合HLA-A2的亲和性在已知的和HLA-A2结合的天然表位多肽的亲和性排序，数目越小亲和性越高。“结合水平”是预测该表位多肽结合HLA-A2的能力。“SB”(强结合)是指所述多肽与HLA-A2具有高度亲和性，通常％排序<0.5设定为强亲和，0.5<％排序<2设定为弱亲和，％排序>2设定为不结合。通常亲和性<50nM，％排序<0.5被认为多肽与HLA-A2结合是高亲和性的。结果显示，和Her2/neu 369-377多肽一样，NSMA3 93-101多肽、 O11A1 103-111多肽和SV2C 687-695多肽均包含-2I-3F-4G-5S-6L-序列，并且可能为高亲和性结合HLA-A2的所预测的表位多肽。为了检测上述来源于人正常蛋白、包含-2I-3F-4G-5S-6L-序列并高亲和性结合HLA-A2分子的潜在表位多肽是否能被Her2 TCR-1B5-mC TCR所识别，检测表达Her2 TCR-1B5-mC的T细胞是否能被T2细胞所提呈的表位多肽激活并分泌γ干扰素。转染有编码Her2 TCR-6A5-mC TCR基因的慢病毒载体的#2 PBMC与提呈不同浓度梯度所述多肽的T2细胞混合培养24小时，取细胞上清进行IFN-γ的ELISA分析。图4B示出经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞(PBMC)与T2细胞提呈的不同浓度的表位多肽混合培养后检查上清中分泌IFN-γ的结果。结果示出，除了Her2/neu 369-377多肽外，其他3个所预测的抗原表位多肽均不能激活Her2 TCR-1B5-mC T细胞，说明所预测的来源于人正常蛋白的表位多肽均不能被Her2TCR-1B5-mC TCR所识别，从而降低了Her2 TCR-6A5-mC TCR识别正常蛋白而产生脱靶副反应的风险。
表1
Figure PCTCN2019102562-appb-000002
实施例6：携带tEGFR基因和Her2 TCR-6A5-mC基因的Her2 TCR-6A5-mC-PGKp-tEGFR慢病毒载体质粒的制备
首先合成截短的人EGFR(truncated human EGFR，tEGFR)基因片段(通过美国Integrated DNA Technologies公司合成)。tEGFR由编码人GM-CSF受体信号肽片段的核苷酸序列(编码人粒细胞-巨噬细胞集落刺激因子(GM-CSF)受体前导肽的核酸片段)与编码人EGFR功能片段(EGFR domains)III、IV和跨膜片段(transmembrane spanning components)的核苷酸序列融合而成(SEQ ID NO:30)(即GenBank编号KX055828的核苷酸序列中2233-3306bp的片段，参见文献“Cancer Immunol Res.2016Jun；4(6):509-19.”)。细胞外功能区III上具有西妥昔单抗(Cetuximab，商品名可为爱必妥
Figure PCTCN2019102562-appb-000003
其最早为美国FDA批准上市的针对EGF受体的IgG1单克隆抗体)的结合位点(参见文献“Blood.2011Aug 4；118(5):1255–1263.”)。以合成的tEGFR基因片段为模板，由5’引物5’-GACGAGAGCGGCCTGACCATGCTTC-3’(SEQ ID NO:31)和3’引物5’-GCACAGTCGCTCGAGTCACATGAAGAG-3’(SEQ ID NO:32)通过PCR扩增获得tEGFR片段。PCR使用Q5高保真PCR试剂盒(NEB,cat#M0543S)，反应条件为：98℃30秒后，进行25个循环98℃10秒，60℃15秒，72℃1分钟。PCR扩增获得的tEGFR片段克隆到Her2 TCR-6A5-mC重组慢病毒载体上的PGK启动子的下游。tEGFR下游是加BGH poly A信号(牛生长激素多聚腺苷酸化(bovine growth hormone polyadenylation)信号)。对携带tEGFR基因的Her2 TCR-6A5-mC-PGKp-tEGFR重组慢病毒载体进行全序列测序，确定插入的各基因序列无误。图5显示所构建的TCR慢病毒载体结构片段示意图。
通过抗EGFR抗体(可为得自Abcam公司的Anti-EGFR抗体[EGFR1](PE/Cy7
Figure PCTCN2019102562-appb-000004
)(ab239309))对转染Her2 TCR-6A5-mC-PGKp-tEGFR慢病毒的PBMC细胞进行染色和流式细胞分析，确认tEGFR的表达。
转染Her2 TCR-6A5-mC-PGKp-tEGFR慢病毒的人T细胞可同时表达Her2 TCR-6A5-mC TCR和“安全转换分子”tEGFR，tEGFR表达后可增加经本发明所述TCR基因修饰的细胞在体内使用的安全性，在需要的时候可以使用抗EGFR抗体(例如西妥昔单抗)来清除患者体内的本发明所述TCR基因修饰的细胞。
实施例7：表达Her2 TCR-6A5-mC的人T细胞和同时表达Her2 TCR-6A5-mC和tEGFR的人T细胞对不同人肿瘤细胞的杀伤作用
首先检测所选肿瘤细胞株表达HLA-A2和Her2/neu的情况。人肿瘤细胞株包括宫颈癌细胞C33A、骨肉瘤细胞Saos-2、胰腺癌细胞CFPAC-1。肿瘤细胞经抗HLA-A2抗体(BD Bioscences,cat#561341)以及抗人CD340(erbB2)抗体(Biolegend,cat#324406)染色后进行流式细胞分析。检测结果显示宫颈癌细胞C33A、骨肉瘤细胞Saos-2、胰腺癌细胞CFPAC-1均为HLA-A2 +Her/neu +。
表达Her2 TCR-6A5-mC的人T细胞和同时表达Her2 TCR-6A5-mC和tEGFR的人T细胞参照前述方法(即“重组TCR慢病毒转染人T细胞”的方法)制备得到，其中表达Her2 TCR-6A5-mC的人T细胞为经Her2 TCR-6A5-mC重组慢病毒转染的正常外周血T细胞，同时表达Her2 TCR-6A5-mC和tEGFR的人T细胞为经Her2 TCR-6A5-mC-PGKp-tEGFR重组慢病毒转染的正常外周血T细胞，并且其中制备所用的外周血单个核细胞得自美国allcells公司(货号PB005F，规格100million，冷冻)。
在96孔培养板中每孔加入作为靶细胞的各肿瘤细胞2×10e4个，再同时以效靶比(E:T)为1:1、5:1、10:1、20:1分别加入对照T细胞(“MOCK-T”)、表达Her2 TCR-6A5-mC的人T细胞(“6A5-TCR-T”)、同时表达Her2 TCR-6A5-mC和tEGFR的人T细胞(“6A5-EGFR-TCR-T”)到对应的孔中，每组均设置3个复孔。37℃，5％二氧化碳培养箱中孵育24h后进行MTT检测，得出相对应的杀伤结果。
图6A-C显示，与没有转染任何TCR的对照人T细胞相比，表 达Her2 TCR-6A5-mC TCR的人T细胞和同时表达Her2 TCR-6A5-mC和tEGFR的人T细胞均可以特异性识别和杀伤HLA-A2 + Her2/neu +的肿瘤细胞株C33A、CFPAC-1、Saos-2。杀伤能力与表达Her2 TCR-6A5-mC TCR的人T细胞或同时表达Her2 TCR-6A5-mC和tEGFR的人T细胞呈量效关系。
由此可以看出，正常外周血T细胞经Her2 TCR-6A5-mC重组慢病毒和Her2 TCR-6A5-EGFR-mC转染后表达的Her2/neu 369-377多肽特异性TCR以及携带EGFR自杀基因的TCR可识别进而杀伤宫颈癌细胞C33A、骨肉瘤细胞Saos-2、胰腺癌细胞CFPAC-1。
实施例8：表达Her2 TCR-6A5-mC的人T细胞对小鼠皮下肿瘤生长的影响
本实验将在环磷酰胺(Cy)注射后的NOD-SCID免疫缺陷的Colo205结肠癌小鼠模型用于模拟人免疫内环境，并检测表达Her2 TCR-6A5-mC的人T细胞(参照前述“重组TCR慢病毒转染人T细胞”方法制备得到，其中所用外周血单个核细胞得自美国allcells公司(货号PB005F，规格100million，冷冻))对肿瘤生长的抑制作用，证明其基本药物疗效。
本实验所用的NOD-SCID免疫缺陷小鼠为6周龄雌性小鼠(维通利华)，每只小鼠在腹侧皮下荷瘤接种Colo205结肠癌细胞，每只接种量为3×10 6个细胞，接种7日后选取平均瘤体在100mm 3左右的18只荷瘤小鼠随机分成3组，每组6只小鼠。分组当天设为第0天。第一组为空白对照组(Cy+IL-2+PBS(i.t))，其中第0天腹腔注射环磷酰胺(Baxter，8D231A)200mg/Kg每只小鼠，给药剂量100μl。第1天每只小鼠瘤内注射(简称“i.t”)100μl PBS溶液(得自Cellmax公司，货号CBS101.05)；同时颈部皮下注射IL-2(得自江苏金丝利公司，商品名为因特康-180350101)，每只10万IU。第2天颈部皮下注射IL-2，每只10万IU。第3天每只小鼠瘤内注射100μl PBS溶液；同时颈部皮下注射IL-2，每只10万IU。第4天颈部皮下注射IL-2，每只10万IU。第5天每只小鼠瘤内注射100μl PBS溶液；同时颈部 皮下注射IL-2，每只10万IU。第6天颈部皮下注射IL-2，每只10万IU。第7天颈部皮下注射IL-2，每只10万IU。第二组为对照T细胞组(Cy+IL-2+T(i.t))，给药方案基本与第一组相同，不同之处在于在第1天、第3天和第5天不是瘤内注射PBS溶液，而是瘤内注射100μl没有转染任何TCR的对照T细胞悬液(悬浮介质为PBS)，细胞数量为2×10 7个/每只。第三组为本发明Her2 TCR-6A5-mC T细胞组(Cy+IL-2+TCR-6A5T(i.t))，给药方案基本与第一组相同，不同之处在于在第1天、第3天和第5天不是瘤内注射PBS溶液，而是瘤内注射100μl表达Her2 TCR-6A5-mC的人T细胞悬液，细胞数量为2×10 7个/每只。在第三组中，当对每只小鼠给予100μl Her2 TCR-6A5-mC T细胞时，所述细胞均是按照前述制备方法新鲜制备的，其中培养时间均为自Her2 TCR-6A5-mC重组慢病毒转染PBMC起，根据预计的不同给药时间先行培养10天左右的时间，细胞的Her2 TCR-6A5-mC TCR阳性率均为约40％。从第0天开始，每2-4天测量一次瘤体体积。各组动物肿瘤平均体积变化情况见图7。
如图7所示，在环磷酰胺注射后的NOD-SCID免疫缺陷的Colo205结肠癌小鼠模型中，瘤内注射表达Her2 TCR-6A5-mC的人T细胞悬液可以抑制肿瘤生长，第二组和第三组之间对比有p值小于0.001的显著差异(图中表示为“***”)，由此可以看出表达Her2 TCR-6A5-mC的人T细胞相对没有转染任何TCR的对照T细胞是有明显疗效的。
讨论
不同肿瘤细胞株对特异性T细胞的反应敏感性差异可能与肿瘤细胞表达不同水平Her2/neu抗原多肽/HLA-A2复合体有关，也可能与肿瘤细胞本身对T细胞功能的不同抑制作用有关。尽管特异性识别Her2/neu 369-377多肽的高亲和性TCR可以通过Her2/neu 369-377多肽体外诱导而获得，但这些高亲和性TCR往往不能识别肿瘤细胞所提呈的Her2/neu抗原(参见文献“Cancer Res.1998；58:4902–4908”、“Cancer Immunol.Immunother.2008；57:271–280”)。一个原因可能是外源 Her2/neu 369-377多肽结合HLA-A2分子的构型与细胞内所提呈的多肽/HLA复合物的构型有所不同(参见文献“Journal of Immunology,2008,180:8135–8145”)。另一个可能原因为，Her2/neu 369-377多肽作为模拟表位(mimotope)抗原，所诱导的特异性TCR既可识别Her2/neu369-377多肽，也可识别被肿瘤细胞提呈的相似多肽，例如Her2/neu373-382多肽(参见文献“J Immunol.2013Jan 1；190(1):479–488”)，然而高亲和性TCR虽然对HLA-A2提呈的Her2/neu 369-377多肽具有高亲和力，却不能有效识别相应的被肿瘤细胞提呈的模拟表位多肽而杀伤肿瘤细胞。本发明所述的特异性识别Her2/neu 369-377多肽的TCR能够靶向肿瘤细胞所提呈的Her2/neu 369-377多肽而特异性识别和杀伤肿瘤细胞。
由于识别自身抗原的高亲和性T细胞大多被中枢耐受机制所清除，外周T细胞库中自然存在的可以识别Her2/neu抗原的TCR大多为中低亲和性。另外一个可以识别肿瘤细胞的CD8功能非依赖型的高亲和性TCR是来自经Her2/neu 373-382多肽特异性T细胞群的多个α链和β链进行配对后，通过功能检测筛选而出(参见文献“HUMAN GENE THERAPY 2024,25:730–739”；WO/2016/133779)。由于不是从特异性的单克隆T细胞直接获得，不能确定此TCR是否存在于外周自然T细胞库。一般认为，高亲和性T细胞的过继转输治疗的疗效要优于靶向同一抗原的低亲和性T细胞(参见文献“Clin Exp Immunol(2015)180:255–70”)。然而，高亲和性TCR本身容易产生识别自身抗原的自身免疫性反应(参见文献“Blood(2009)114:535–46”)，没有经过中枢耐受机制筛选的TCR也会识别抗原低表达的正常组织，或者针对其它类似的自身抗原表位产生交叉反应的脱靶毒性(参见文献“Sci Transl Med(2013)5:197ra103”、“Blood(2013)122:863–71”)。选择高亲和性TCR的另一个原因是这些TCR的功能不依赖CD8的辅助功能，因而可以通过转染CD4 +T细胞而获得对CD8 +杀伤T细胞功能的辅助作用。本发明所述的TCR识别Her2/neu 369-377多肽属于中到高亲和性，而且TCR的功能不依赖CD8的辅助功能，因而适合用于过继转输治疗中T细胞的修饰。本发明所述TCR不能识别通过比对筛选方 法和计算机辅助预测软件所获得的来源于正常蛋白的所有潜在的表位多肽，从而进一步避免了针对正常蛋白的潜在的交叉反应风险。
总之，本发明提供了一种从HLA-A2 +的自体外周T细胞库中诱导而来的Her2/neu 369-377多肽特异性TCRα链和β链全序列，经转染后表达此TCR及恒定区被修饰的TCR的原代杀伤性T细胞可以识别多种HLA-A2 +Her2/neu +的肿瘤细胞。为开发和临床应用过继转输经特异性TCR修饰的T细胞来治疗肿瘤提供了新的方法和途径。

Claims (35)

  1. 一种分离的T细胞受体，包括α链和β链中的至少一者，所述α链和β链均包含可变区和恒定区，其特征在于，所述T细胞受体能够特异性识别肿瘤细胞所表达的抗原Her2/neu，并且所述α链的所述可变区的氨基酸序列具有与SEQ ID NO:1所示的氨基酸序列至少98％的一致性，所述β链的所述可变区的氨基酸序列具有与SEQ ID NO:2所示的氨基酸序列至少98％的一致性。
  2. 根据权利要求1所述的T细胞受体，其中所述的T细胞受体能够特异性识别被HLA-A2分子所提呈的所述抗原Her2/neu的抗原表位多肽；优选的是，所述抗原表位多肽包括如SEQ ID NO:3所示的Her2/neu 369-377。
  3. 根据权利要求1所述的T细胞受体，其中所述α链的所述恒定区和/或所述β链的所述恒定区来源于人；优选地，所述α链的所述恒定区全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链的所述恒定区全部或部分地被来源于其它物种的同源序列所替换；更优选地，所述其它物种为小鼠。
  4. 根据权利要求1所述的T细胞受体，其中所述α链的所述恒定区修饰有一个或多个二硫键，并且/或者所述β链的所述恒定区修饰有一个或多个二硫键。
  5. 根据权利要求1所述的T细胞受体，其中所述α链的氨基酸序列如SEQ ID NOs:4、5或6所示，所述β链的氨基酸序列如SEQ ID NOs:7、8或9所示。
  6. 一种分离的、编码T细胞受体的核酸，包含所述T细胞受体的α链和β链中的至少一者的编码序列，所述α链编码序列和β链编 码序列均包含可变区编码序列和恒定区编码序列，其特征在于，所述T细胞受体能够特异性识别肿瘤细胞表达的抗原Her2/neu，并且所述α链可变区编码序列编码的氨基酸序列具有与SEQ ID NO:1所示的氨基酸序列至少98％的一致性，所述β链可变区编码序列编码的氨基酸序列具有与SEQ ID NO:2所示的氨基酸序列至少98％的一致性。
  7. 根据权利要求6所述的核酸，其中所述核酸为DNA或RNA。
  8. 根据权利要求6所述的核酸，其中所述α链可变区编码序列如SEQ ID NO:10所示，所述β链可变区编码序列如SEQ ID NO:11所示。
  9. 根据权利要求6所述的核酸，其中被所述核酸编码的所述T细胞受体能够特异性识别被HLA-A2分子所提呈的所述抗原Her2/neu的抗原表位多肽；优选的是，所述抗原表位多肽包括如SEQ ID NO:3所示的Her2/neu 369-377。
  10. 根据权利要求6所述的核酸，其中所述α链恒定区编码序列和/或所述β链恒定区编码序列来源于人；优选地，所述α链恒定区编码序列全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链恒定区编码序列全部或部分地被来源于其它物种的同源序列所替换；更优选地，所述其它物种为小鼠。
  11. 根据权利要求6所述的核酸，其中所述α链恒定区编码序列包含一个或多个二硫键编码序列，并且/或者所述β链恒定区编码序列包含一个或多个二硫键编码序列。
  12. 根据权利要求6所述的核酸，其中所述α链编码序列如SEQ ID NOs:12、13或14所示，所述β链编码序列如SEQ ID NOs:15、16或17所示。
  13. 根据权利要求6-11中任一项所述的核酸，其中所述α链编码序列和所述β链编码序列之间由可切割性连接多肽的编码序列连接。
  14. 根据权利要求13所述的核酸，其序列如SEQ ID NOs:18、19、或20所示。
  15. 一种重组表达载体，其含有与启动子有效连接的、根据权利要求6-14中任一项所述的核酸，和/或其互补序列。
  16. 根据权利要求15所述的重组表达载体，其中所述重组表达载体含有自杀基因编码序列；其中所述自杀基因选自：iCasp9、HSV-TK、mTMPK、截短的EGFR、截短的CD19、截短的CD20或其组合。
  17. 根据权利要求16所述的重组表达载体，其中所述自杀基因编码序列是在启动子控制下的，并且该用于控制所述自杀基因编码序列的启动子与所述的核酸所连接的启动子相同或不同，并且是彼此独立的。
  18. 根据权利要求16所述的重组表达载体，其中所述自杀基因编码序列和所述的核酸是在同一个启动子控制下的，并且所述自杀基因编码序列通过可切割性连接多肽的编码序列或者内部核糖体进入位点序列，与所述的核酸相连接。
  19. 一种T细胞受体修饰的细胞，该细胞的表面被权利要求1-5中任一项所述的T细胞受体修饰，其中所述细胞包括原始T细胞或其前体细胞，NKT细胞，或T细胞株。
  20. 根据权利要求19所述的T细胞受体修饰的细胞，其中该细 胞在其细胞表面或细胞内表达自杀基因蛋白；其中所述自杀基因选自：iCasp9、HSV-TK、mTMPK、截短的EGFR、截短的CD19、截短的CD20或其组合。
  21. 一种制备根据权利要求19或20所述的T细胞受体修饰的细胞的方法，包括以下步骤：
    1)提供细胞；
    2)提供编码根据权利要求1-5中任一项所述的T细胞受体的核酸；
    3)将所述核酸转染入所述细胞中。
  22. 根据权利要求21所述的方法，其中步骤1)所述的细胞来自自体或异体。
  23. 根据权利要求21所述的方法，其中所述转染的方式包括：采用病毒载体转染的方式，优选的是，所述病毒载体包括γ逆转录病毒载体或慢病毒载体；化学方式，优选的是，所述化学方式包括采用脂质体转染的方式；物理方式，优选的是，所述物理方式包括电转染方式。
  24. 根据权利要求21所述的方法，其中步骤2)所述的核酸为根据权利要求6-14中任一项所述的核酸。
  25. 根据权利要求19或20所述的T细胞受体修饰的细胞在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
  26. 根据权利要求25所述的用途，其中所述肿瘤和/或癌症是抗原Her2/neu阳性的，并且是HLA-A2阳性的。
  27. 根据权利要求19或20所述的T细胞受体修饰的细胞在制 备用于检测宿主的肿瘤和/或癌症的药物中的用途。
  28. 一种药物组合物，其中该药物组合物包括作为活性成分的根据权利要求19或20所述的T细胞受体修饰的细胞，及可药用辅料。
  29. 根据权利要求28所述的药物组合物，其中所述药物组合物包含每个患者每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重的所述T细胞受体修饰的细胞。
  30. 根据权利要求28所述的药物组合物，其中所述药物组合物适于经动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内或腹膜内给药。
  31. 一种治疗肿瘤和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据权利要求19或20所述的T细胞受体修饰的细胞。
  32. 根据权利要求31所述的方法，其中所述T细胞受体修饰的细胞的施用剂量为每个患者每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重。
  33. 根据权利要求31所述的方法，其中所述T细胞受体修饰的细胞通过动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内或腹膜内给药。
  34. 根据权利要求31所述的方法，其中所述肿瘤和/或癌症是抗原Her2/neu阳性的，并且是HLA-A2阳性的。
  35. 根据权利要求31所述的方法，还包括对所述肿瘤和/或癌症患者施用其它用于治疗肿瘤的药物，和/或用于调节患者免疫系统的药物。
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