WO2020038492A1 - 包含核酸及tcr修饰的免疫细胞的治疗剂及其应用 - Google Patents

包含核酸及tcr修饰的免疫细胞的治疗剂及其应用 Download PDF

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WO2020038492A1
WO2020038492A1 PCT/CN2019/102584 CN2019102584W WO2020038492A1 WO 2020038492 A1 WO2020038492 A1 WO 2020038492A1 CN 2019102584 W CN2019102584 W CN 2019102584W WO 2020038492 A1 WO2020038492 A1 WO 2020038492A1
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cancer
cells
virus
her2
tumor
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PCT/CN2019/102584
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French (fr)
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胡放
侯亚非
绳纪坡
谭贤魁
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合成免疫股份有限公司
杭州康万达医药科技有限公司
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Priority to EP19851924.1A priority Critical patent/EP3842523A4/en
Priority to US17/270,943 priority patent/US11819519B2/en
Priority to US16/798,465 priority patent/US11786552B2/en
Publication of WO2020038492A1 publication Critical patent/WO2020038492A1/zh

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Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a therapeutic agent for immune cells containing nucleic acids and TCR modification, a labeled polypeptide, a coding nucleic acid, an expression vector, an oncolytic virus, a kit, and applications thereof.
  • Tumor immunotherapy is mainly to stimulate the function of endogenous anti-tumor T cells or adoptively transfer tumor-specific T cells cultured in vitro to achieve the efficacy of killing and removing cancer cells in vivo.
  • Early tumor vaccines targeted tumor-associated antigens (TAA) derived from their own proteins, and attempted to induce specific anti-tumor immune responses in vivo, but most related clinical trials were unsuccessful (see the article “Nat Rev Clin Oncol .2014 Nov; 11 (11): 630-2. ").
  • TAA tumor-associated antigens
  • One of the main reasons is that high-affinity T cells targeting these tumor-associated antigens are mostly cleared by the central tolerance mechanism of the thymus during development and differentiation.
  • Tumor vaccines are assisted even by strong adjuvants, such as with mature trees Sudden cells present antigens, and the specific T cells induced in vivo are mostly low-affinity T cells that do not recognize tumor cells (see the document “J Immunol. 2008 Feb 01; 180 (3): 1526-34" ).
  • the development of personalized vaccines targeting neoantigen derived from genetic mutations is a development trend, and initial efficacy has been seen in clinical trials, but more clinical trials are needed to further verify this type of tumor The efficacy of the vaccine (see the literature "Nat Rev Drug Disv. 2018 May 30; 17 (6): 393"; "Front Immunol. 2017; 8: 1848”).
  • TAE tumor microenvironment
  • an effective treatment strategy is adoptive transfer of T cells obtained in vitro that can effectively recognize tumor antigens.
  • T cells are derived from infiltrating T lymphocytes (TILs) of tumor tissue, or they can be peripheral blood T cells (TCR-T) that can effectively recognize tumor antigens after being modified with antigen-specific T cell receptor (TCR) genes.
  • TILs infiltrating T lymphocytes
  • TCR-T peripheral blood T cells
  • TCR-T peripheral blood T cells
  • adoptive transfer therapy includes chimeric antigen receptor-based CAR-T therapy and T cell receptor-based TCR-T therapy.
  • CAR-T mainly targets tumor antigens expressed on the cell surface. Despite the limited number of tumor antigens recognized, CAR-T targeting CD19 has shown significant clinical efficacy against hematological tumors including B-cell leukemia and lymphoma, and has The product was approved by the FDA for marketing (see document "N Engl J Med. 2017 Oct 05; 377 (14): 1313-1315"). For the treatment of solid tumors, CAR-T has not seen a clear clinical effect.
  • TCR-T mainly targets polypeptide antigens presented by human major histocompatibility antigen HLA molecules. Epitope polypeptides can be derived from intracellular proteins and cell membrane surface proteins. The types and quantities of target antigens are much greater than those recognized by CAR-T.
  • TCR-T therapy is considered to be the most promising immune cell gene therapy for solid tumors (see document “Adv Immunol. 2016; 130: 279-94”).
  • TCR-T targeting the NY-ESO-1 antigen has also seen clear results in the treatment of solid tumors in phase II clinical trials (see the document “Front Immunol. 2018; 9: 947”).
  • Her2 / neu protein is over-expressed in a variety of epithelial-derived cancer cells, such as breast, gastric, colorectal, ovarian, pancreatic, lung, esophageal, bladder, and kidney cancers (see the document “Trends in Molecular Med” , 2013; 19: 677 "), making Her2 / neu a suitable target for immunotherapy.
  • T cells that specifically recognize Her2 / neu epitope peptides 369-377 can be successfully isolated from the ascites of Her2 / neu high-expressing ovarian cancer (see the document "J. Exp. Med. 1995; 181: 2109- 2117 ").
  • a tumor vaccine targeting Her2 / neu 369-377 peptide antigen has entered clinical trials, but Phase III clinical trials have failed to meet the intended goal of prolonging patient survival (http://www.onclive.com/web-exclusives/phase-iii- nelipepimuts-study-in-breast-cancer-halted-after-futility-review).
  • Adoptive transfer of cultured CAR-T cells targeting Her2 / neu antigens as the first CAR-T therapy for solid tumors has entered clinical trials, but due to the generation of a strong cytokine release syndrome (CRS) Terminated due to patient death (see document "Nature Med, 2016; 22:26").
  • CRS cytokine release syndrome
  • TCR-T therapy in clinical trials has not shown severe cytokine storm toxicity as shown in CAR-T therapy.
  • TCR-T targeting Her2 / neu antigens may avoid severe cytokine storms in solid tumors.
  • Her2 / neu is a tumor-associated antigen derived from its own protein. Most of the T cells that recognize such antigens are cleared by the central tolerance selection mechanism. Therefore, it is difficult to obtain natural T cells that specifically identify tumor cells from peripheral blood.
  • Induced polypeptide-specific T cells often fail to effectively recognize tumor-associated antigens expressed at low levels on the surface of tumor cells (see the literature "Cancer Res. 1998; 58 (21): 4902-8").
  • gene point mutations in complementarity determining regions (CDRs) on TCR are generally used, or those that have never been screened by central tolerance mechanisms are used.
  • Induction was performed in a humanized mouse T cell bank to obtain a high affinity antigen-specific TCR (see document “Front Immunol. 2013; 4: 363").
  • the TCR-T prepared by the high-affinity TCR obtained based on this strategy has produced severe off-target toxicity in normal clinical cells in clinical trials (see the document “Sci Rep. 2016 Jan 13; 6: 18851 ").
  • the targets recognized by TCR-T are HLA molecules on the cell surface, ⁇ 2 -microglobulin (beta 2 -Microglobulin), and antigenic complexes formed by antigen polypeptides (where HLA protein and ⁇ 2 -microglobulin are paired to form MHC Class I molecules).
  • TCR-T's antigen recognition ability is not only restricted by HLA molecules, but also closely related to the expression of HLA molecules and tumor antigens in tumor cells, and the integrity of the HLA antigen peptide presentation pathway.
  • HLA molecules in the process of tumor development and development, the HLA molecule expression of tumor cells is often reduced, or the function of related molecules in the HLA antigen presentation pathway in the cytoplasm is lost.
  • the antigen polypeptide cannot be effectively presented to the surface of tumor cells by HLA molecules. Recognized by T cells (see document "Cancer Gene Ther. 2002 Dec; 9 (12): 1043-55").
  • HLA class I antigen presentation pathway ie, the MHC class I antigen presentation pathway
  • tumor cells include HLA, ⁇ 2 -microglobulin, TAP, tapasin, LMP, and EARP.
  • Gene mutation or abnormal expression of other antigen-presenting molecules may also be related to the excessive functional activation of tumor-transforming molecules such as RAS, MYC, MOS, and Her2 / Neu (see the document “Trends in Molecular Medicine, 2013, 19 (11): 677-681 ").
  • the diversity of tumor tissue is also expressed by the heterogeneity of tumor antigen expression. Tumor cells with low or no expression of tumor antigen may escape the recognition and killing of TCR-T, thereby affecting the efficacy of TCR-T (see literature "Trends Immunol. 2016 06; 37 (6): 349-351").
  • TCR-T therapy a challenge faced by TCR-T therapy is how to promote TCR-T targeting tumor-associated antigens to effectively recognize and kill tumor cells.
  • the present invention provides a therapeutic agent, a labeled polypeptide, a coding nucleic acid, an expression vector, an oncolytic virus, a kit, and an application thereof.
  • the present invention provides:
  • a therapeutic agent for treating tumors and / or cancers comprising:
  • a first composition wherein the first composition comprises a first active ingredient in a first pharmaceutically acceptable carrier, the first active ingredient including or containing Nucleic acid encoding a marker polypeptide; the marker polypeptide has an amino acid sequence of one or more epitope polypeptides, and the epitope polypeptides can be presented by the MHC class I molecule to the tumor cells and / or cancer cells Surface; and
  • a second composition wherein the second composition comprises a second active ingredient in a second pharmaceutically acceptable carrier, the second active ingredient comprises a T cell receptor-modified immune cell; the T cell is affected by Somatically modified immune cells are capable of specifically recognizing and binding the epitope polypeptide presented by the MHC class I molecule.
  • amino acid sequence of the epitope polypeptide is derived from the amino acid sequence of a protein existing in nature, or is an artificially synthesized amino acid sequence that does not exist in nature; preferably, the Proteins found in nature include proteins of human origin and proteins of other species than humans.
  • the labeled polypeptide comprises the following amino acid sequences operatively linked and connected in series: the amino acid sequence of an N-terminal signal peptide, and one or more of the epitopes An amino acid sequence of a polypeptide, and optionally an amino acid sequence of a C-terminal endoplasmic reticulum retention signal, wherein when the labeled polypeptide includes multiple amino acid sequences of the epitope polypeptide, every two adjacent antigens
  • the amino acid sequences of epitope polypeptides are linked by the amino acid sequence of a cleavable linking polypeptide.
  • the therapeutic agent according to (4), wherein the amino acid sequence of the epitope polypeptide includes Her2 / neu 369-377, NY-ESO-1 157-165, NY as shown in SEQ ID NO: 3 -ESO-11-11-1, NY-ESO-153-62, NY-ESO-118-27, N-ras55-64, K-ras224-232, K-ras10-18, K-ras 10-19, H3.3K27M 26-35, SSX-2 41-49, MAGE-C2 336-344, MAGE-C2 191-200, MAGE-C2 307-315, MAGE-C2 42-50, MAGE-A1 120 -129, MAGE-A1 230-238, MAGE-A1 161-169, KK-LC-1 76-84, p53 99-107, HPV16-E6 29-38, HPV16-E7 11-19, HPV16-E7 11- 19. EBV-LMP1 51-59, EBV-LMP
  • the nucleic acid further has an HLA protein coding sequence, wherein the HLA protein coding sequence and the marker polypeptide coding sequence are respectively under the control of a respective promoter, or the The HLA protein coding sequence is under the control of the same promoter as the marker polypeptide coding sequence and the HLA protein coding sequence is operably linked to the marker polypeptide coding sequence via a cleavable linking polypeptide coding sequence.
  • HLA protein is an HLA-A2 protein
  • amino acid sequence of the HLA-A2 is shown in SEQ ID NO: 29.
  • nucleic acid includes DNA or RNA; and the RNA includes mRNA transcribed from the DNA.
  • the therapeutic agent according to (10), wherein the replication-deficient recombinant virus is derived from adenovirus, adenovirus-associated virus (AAV), herpes simplex virus, pox virus, influenza virus, alphavirus, Or Sendai virus.
  • AAV adenovirus-associated virus
  • the selective replication-type recombinant oncolytic virus is derived from a genetically mutated virus having oncolytic effect and a wild-type virus having oncolytic effect; preferably, the Selective replication-type recombinant oncolytic viruses are derived from adenoviruses, poxviruses, herpes simplex virus, measles virus, Semliki forest virus, vesicular stomatitis virus, polio virus, retrovirus, and respiratory virus that have oncolytic effects. Enterovirus, Seneca Valley virus, Echo enterovirus, Coxsackie virus, Newcastle disease virus and Maraba virus.
  • the selective replication-type recombinant oncolytic virus is a recombinant oncolytic adenovirus obtained by genetically modifying type 5 adenovirus
  • the recombinant oncolytic adenovirus has a genome
  • the E1B-55K gene and / or E1B-19K gene are deleted, and the genome of the recombinant oncolytic adenovirus contains the E1A gene coding sequence; preferably, the E1A gene coding sequence is under the control of an exogenous promoter.
  • the gene is changed so that the expressed E1A protein cannot bind to the pRb protein; preferably, the E1A gene coding sequence is under the control of an exogenous promoter.
  • the therapeutic agent according to (1), wherein the immune cells include primitive T cells or precursor cells thereof, NKT cells, or T cell lines.
  • the tumor and / or cancer comprises: breast cancer, head and neck tumor, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon Cancer, rectal cancer, brain cancer, liver cancer, bone cancer, chorionic cancer, gastrinoma, pheochromocytoma, prolactinoma, vonHippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder Cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown site, carcinoid, fibrosarcoma, Paget's disease, cervical Cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, cutaneous squamous cell carcinoma, meso
  • a labeled polypeptide comprising the following amino acid sequences operably linked in tandem: the amino acid sequence of an N-terminal signal peptide, the amino acid sequence of one or more epitope polypeptides, and optionally an C-terminal endoplasm An amino acid sequence of a net retention signal, wherein when the labeled polypeptide includes a plurality of amino acid sequences of the epitope polypeptide, the amino acid sequences of each two adjacent epitope polypeptides are cleavably linked The amino acid sequence of the polypeptide is linked; preferably, the cleavable linked polypeptide is a furin digestion recognition polypeptide.
  • An isolated recombinant virus wherein the genome of the recombinant virus has the nucleic acid according to any one of (30) to (33); and the recombinant virus comprises a selective replication-type recombinant oncolytic virus or Replication-deficient recombinant virus.
  • AAV adenovirus-associated virus
  • the selective replication-type recombinant oncolytic virus is derived from adenovirus, pox virus, herpes simplex virus, measles virus, Semliki forest virus, vesicular Stomatitis virus, poliovirus, retrovirus, reovirus, Seneca virus, Echo enterovirus, Coxsackie virus, Newcastle disease virus and Maraba virus.
  • the E1A gene of the virus is changed so that the expressed E1A protein cannot bind to the pRb protein; preferably, the E1A gene coding sequence is under the control of an exogenous promoter.
  • a kit of synergistic combination drugs for treating tumors and / or cancers comprising:
  • a first container containing the first composition in the therapeutic agent according to any one of (1) to (24);
  • a second container containing the second composition in the therapeutic agent according to any one of (1) to (24), wherein the first container and the second container are independent; as well as
  • the tumor and / or cancer comprises: breast cancer, head and neck tumor, synovial cancer, kidney cancer, connective tissue cancer, melanin Tumor, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, chorionic cancer, gastrinoma, pheochromocytoma, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome , Anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningiomas, spinal tumors, osteochondroma, chondrosarcoma, Ewing's sarcoma, unknown primary cancer, carcinoid, fiber Sarcoma, Paget's disease, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, skin squamous cell carcinoma, me
  • a method for treating tumors and / or cancers comprising:
  • the tumor and / or cancer patient is administered a second composition of the therapeutic agents according to any one of (1) to (24).
  • a second composition of the therapeutic agents is administered to the tumor and / or cancer patient.
  • the tumor and / or cancer comprises: breast cancer, head and neck tumor, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon Cancer, rectal cancer, brain cancer, liver cancer, bone cancer, chorionic cancer, gastrinoma, pheochromocytoma, prolactinoma, vonHippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder Cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown site, carcinoid, fibrosarcoma, Paget's disease, cervical Cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, cutaneous squamous cell carcinoma, mes
  • the present invention has the following advantages and positive effects:
  • the invention proposes to express foreign antigen epitope peptides and / or foreign MHC class I molecules in tumor cells,
  • the concept of combined treatment was to significantly enhance the number of epitope peptide / MHC class I molecular complexes on the surface of tumor cells, and to use TCR-modified immune cells specific to the epitope peptide.
  • oncolytic virus When oncolytic virus is used as a vector to mediate the expression of exogenous epitope peptides and / or exogenous MHC class I molecules in tumor cells, tumor killing by oncolytic viruses and tumor killing by TCR-modified immune cells Can produce synergistic therapeutic effects.
  • TCR-modified immune cells especially TCR-modified T cells
  • MHC class I antigen presenting pathway only Conventional expression of foreign genes in tumor cells does not significantly enhance the presentation of epitope peptides on the surface of tumor cells, especially when tumor cells often suffer from defects in the MHC class I antigen presentation pathway.
  • the present invention first designs a labeled polypeptide amino acid sequence containing one or more epitope peptide amino acid sequences, and a nucleic acid having the coding sequence of the labeled polypeptide, so that the nucleic acid is transfected into tumor cells and / or After the cancer cells, or after inserting the nucleic acid into the viral genome and infecting the tumor cells and / or cancer cells with the resulting recombinant virus, the labeled epitope polypeptide chain can be expressed in the cells and introduced through the amino-terminal signal peptide
  • the endoplasmic reticulum (ER) is then cut / processed into the required epitope polypeptide fragments, which can be finally presented on the surface of tumor cells and / or cancer cells by MHC class I molecules, which can achieve
  • the surface of tumor cells significantly enhances the effect of presenting epitope peptides, which effectively solves the problem of low antigen expression caused by defects in the antigen presentation pathway caused by tumor immune evasion mechanisms.
  • the present invention further inserts an HLA protein coding sequence into the nucleic acid to solve the problem of low or missing endogenous HLA expression.
  • the present invention also proposes to combine the active ingredient including or containing the nucleic acid encoding the labeled polypeptide with a T cell receptor modified immune cell that recognizes an epitope peptide, thereby improving the effect of T cell receptor modified immune cells on tumor cells. Recognition sensitivity further enhances the ability of T cell receptor-modified immune cells to kill tumor cells.
  • the labeled polypeptide and / or HLA protein-encoding nucleic acid is introduced into tumor cells and / or cancer cells by an oncolytic virus, while exerting the role of the oncolytic virus in killing tumor cells and / or cancer cells, further,
  • the synergistic therapeutic effect achieved by significantly enhancing the presentation of the exogenous epitope peptide on the surface of tumor cells and the immune cells modified by T cell receptors is enhanced.
  • oncolytic virus can relieve the tumor's microenvironment's immunosuppressive state while killing the tumor, and improve the homing of T cell receptor-modified immune cells.
  • T cell receptor-modified immune cells can also effectively eliminate the lytic After oncovirus infection, tumor cells that cannot lyse after completing the replication cycle and produce a sufficient number of daughter viruses have lysed; thus achieving a further synergistic effect.
  • the antigen released by the tumor cells lysed by the oncolytic virus can further activate the body's own anti-tumor immunity, which can achieve a better tumor killing effect than using the oncolytic virus or T cell receptor-modified immune cells alone, and achieve The effect of synergistic treatment.
  • the present invention provides a new approach for tumor treatment through the above-mentioned inventive concept.
  • oncolytic virus refers to a virus capable of selectively replicating and lysing tumor cells in tumor cells.
  • terapéuticaally effective amount refers to an amount of a functional agent or pharmaceutical composition capable of exhibiting a detectable therapeutic or inhibitory effect, or an amount that exerts an antitumor effect. The effect can be detected by any test method known in the art.
  • administering refers to providing a compound, complex or composition (including viruses and cells) to a subject.
  • patient refers to a human or non-human organism. Therefore, the methods and compositions described herein are suitable for use in human and veterinary diseases.
  • the patient has a tumor.
  • the patient has one or more types of cancer at the same time.
  • the term "synergistic effect” refers to an effect jointly exerted by two or more agents, which effect is greater than the sum of the individual effects of each agent therein.
  • plaque forming unit refers to: The amount of virus that produces an plaque is called a plaque forming unit (pfu).
  • VP refers to the number of virus particles.
  • VP / kg refers to the number of viral particles per kilogram of patient weight.
  • TCID50 refers to half of the tissue culture infective dose, meaning the dose of virus that infects half of the tissue culture and causes cytopathic disease.
  • MOI Multiplicity of infection
  • FIG. 1 shows a table of specific killer T cells of Her2 / neu 369-377 polypeptide (Her2-E75) induced from HLA-A2 + normal donor PBMC (specifically # 1 PBMC) in Example 1 of the present invention.
  • 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 (# 1 PBMC) provided by one donor, and the right picture relates to PBMC (# 2 PBMC) 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 was 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 indicates the percentage of killing rate of T cells to 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 Of peripheral blood mononuclear cells.
  • the other group was added with paclitaxel 10 ⁇ M as a positive control (shown as separate lower triangle points in Figures 3H-3K).
  • Figure 4 shows that target cells transfected with a vector carrying the HLA-A2 gene and the Her2-E75 minigene can express HLA-A2, and the expressed Her2-E75 polypeptide can form HLA-A2 with Her2 TCR-6A5-mC. Antigen complex recognized by TCR.
  • Figure 4A shows that HLA-A2-negative Her2 / neu-negative 293T cells are transfected with a lentiviral plasmid vector capable of expressing the HLA-A2 gene and Her2-E75 minigene (without KDEL at the C-terminus), which can activate transfection of Her2 TCR -6A5-mC TCR for PBMC.
  • Each test group and control group were duplicated, and the results were shown as the mean ⁇ SME.
  • the abscissa shows that 293T cells as target cells were transfected with different gene vectors.
  • the ordinate indicates the concentration of IFN- ⁇ secreted by T cells.
  • the effective target ratio E: T was 10: 1.
  • the upper diagram of FIG. 4B shows the main functional fragments of the constructed lentiviral vector carrying the HLA-A2 and Her2-E75 minigenes (without KDEL at the C-terminus) (ie, “pCDH-EF1p-A2-PKGp-E75 vector”).
  • the fragments shown express the HLA-A2 gene driven by the EF-1 ⁇ promoter and the Her2-E75 minigene driven by the PKG promoter (without KDEL at the C-terminus).
  • FIG. 4B shows the composition of the Her2-E75 minigene (the C-terminus does not have KDEL), which consists of an INSL5 signal peptide, eight Her2-E75 epitope peptide coding sequences, and a furin digestion fragment therebetween.
  • Figure 4D shows the main functional fragments of the constructed recombinant adenoviral vector (ie, "Adeasy-A2E75 vector") carrying the HLA-A2 and Her2-E75 minigenes (with KDEL at the C-terminus).
  • Adenovirus is a type 5 adenovirus genome that is deficient in E1 and E3 genes.
  • FIG. 4C shows that HLA-A2 negative cells SKOV3 can express HLA-A2 after infection with the vector carrying the HLA-A2 and Her2-E75 minigenes (with KDEL at the C-terminus) shown in FIG. 4D.
  • the abscissa shows that SKOV3 cells were infected with different recombinant adenoviruses.
  • Adeasy-A2E75 is an adenovirus that expresses HLA-A2 and Her2-E75 polypeptides;
  • Ad control is a control adenovirus that does not carry the HLA-A2 and Her2-E75 minigenes.
  • HLA-A2 + (MFI) represents the mean fluorescence intensity of cells after staining with anti-HLA-A2 fluorescent antibodies.
  • MOI is the number of viral infections.
  • FIG. 5 shows that replication-defective adenoviruses carrying the HLA-A2 gene and the Her2E75 minigene can increase the sensitivity of Her2TCR-6A5-mC TCR to different target cells after infecting tumor cells.
  • FIG. 5A shows that T cells expressing Her2, TCR-6A5-mC, and TCR can be activated by different tumor cell lines infected with adenovirus carrying the HLA-A2 gene and Her2 E75 minigene, and secrete IFN- ⁇ . Effector cells are peripheral blood mononuclear cells infected with a replication-deficient lentivirus carrying the Her2TCR-6A5-mCTCR gene.
  • the target cells were HLA-A2 negative Her2 / neu positive ovarian cancer cells SKOV3, HLA-A2 positive Her2 / neu positive breast cancer cells MCF-7, and HLA-A2 negative Her2 / neu negative small cell lung cancer cells NCI-H446.
  • the abscissa shows that different tumor cell line cells as target cells have undergone different treatments.
  • Adeasy-A2E75 is a target cell infected with replication-defective adenovirus carrying the HLA-A2 gene and the Her2E75 minigene alone;
  • PBMC control is a control between the target cell and the TCR gene without transfection of Her2 TCR-6A5-mC Mixed culture of peripheral blood mononuclear cells;
  • PBMC control + Adeasy-A2E75 is the target cell infected with replication-deficient adenovirus carrying the HLA-A2 gene and Her2 E75 minigene 24 hours later without Her2 TCR-6A5-mC and TCR gene Transfection of control peripheral blood mononuclear cells mixed culture;
  • PBMC with Her2 TCR-6A5" as target cells and mixed culture of peripheral blood mononuclear cells transfected with Her2 TCR-6A5-mC TCR gene;
  • Her2 TCR- 6A5 PBMC + Adeasy-A2E75 is a target cell infected with replication-deficient aden
  • the ordinate shows the concentration of IFN- ⁇ secreted by T cells.
  • FIG. 5B shows that T cells expressing Her2, TCR-6A5-mC, and TCR can specifically kill tumor cell lines infected with adenoviruses carrying the HLA-A2 gene and Her2 E75 minigene.
  • the abscissa shows that cells of different tumor cell lines are treated differently as target cells, which is the same as the group shown on the abscissa of FIG. 5A.
  • Cytotoxicity% ((the number of live cells of the initial target cells-the number of live cells of the target cells at the end of the culture) / the number of live cells of the initial target cells) ⁇ 100.
  • FIG. 6 shows that the oncolytic adenovirus carrying the Her2-E75 minigene can increase the recognition sensitivity of Her2 TCR-6A5-mC TCR to target cells expressing HLA-A2 after infecting tumor cells.
  • Figure 6A shows that T cells expressing Her2, TCR-6A5-mC, and TCR can be activated by infection with oncolytic adenoviruses carrying the Her2-E75 minigene and expressing HLA-A2 tumor cell lines, and secrete IFN- ⁇ . Effector cells are peripheral blood mononuclear cells infected with lentivirus carrying the Her2TCR-6A5-mCTCR gene.
  • the target cells were HLA-A2 negative Her2 / neu positive ovarian cancer cells SKOV3, HLA-A2 positive Her2 / neu positive breast cancer cells MCF-7, and HLA-A2 negative Her2 / neu negative small cell lung cancer cells NCI-H446.
  • the abscissa shows that different tumor cell line cells as target cells have undergone different treatments.
  • Ad-E75 is a target cell infected with an oncolytic adenovirus carrying the Her2-E75 minigene alone;
  • PBMC control is a target cell and a control peripheral blood mononuclear cell that is not transfected with the Her2 TCR-6A5-mC TCR gene Mixed culture;
  • PBMC control + Ad-E75 is the target cell infected with oncolytic adenovirus carrying the Her2-E75 microgene 24 hours later and mixed with control peripheral blood mononuclear cells not transfected with Her2 TCR-6A5-mC TCR gene Culture;
  • PBMC with Her2 TCR-6A5" as the target cell and mixed culture of peripheral blood mononuclear cells transfected with Her2 TCR-6A5-mC TCR gene;
  • PBMC + Ad-E75 with Her2 TCR-6A5" as the target The cells were infected with the oncolytic adenovirus carrying the Her2-E75 minigene 24 hours later and mixed with peripheral blood monon
  • the ordinate shows the concentration of IFN- ⁇ secreted by T cells.
  • FIG. 6B shows that T cells expressing Her2, TCR-6A5-mC, and TCR can specifically kill tumor cell lines infected with an oncolytic adenovirus carrying the Her2-E75 minigene.
  • the abscissa shows that the cells of different tumor cell lines are treated differently as target cells, which is the same group as the abscissa shown in FIG. 6A.
  • "**” and "***” show comparison with the oncolytic adenovirus group expressing Her2-E75 polypeptide alone, or with the peripheral blood mononuclear cell group expressing Her2 TCR-6A5-mC TCR alone The combination of the two showed that peripheral blood mononuclear cells expressing Her2, TCR-6A5-mC, and TCR had significant specific lethality against target cells infected with oncolytic adenovirus expressing Her2-E75 polypeptide.
  • Each test group and control group are three wells, and the results are shown as the mean ⁇ SME.
  • Figure 7 shows that replication-defective adenoviruses carrying the HLA-A2 and Her2-E75 minigenes can increase the sensitivity of Her2 TCR-6A5-mC TCR to different target cells after infection with tumor cells.
  • Figures 7A and 7B show the same experimental response.
  • Figure 7A shows the detection of factor levels in the supernatant, and
  • Figure 7B shows the detection of cell-related killer counts.
  • Figure 7A shows that T cells expressing Her2 TCR-6A5-mC TCR can be activated by infection with replication-deficient adenoviruses carrying the HLA-A2 and Her2-E75 minigenes and expressing HLA-A2 tumor cell lines, and secrete IFN - ⁇ .
  • the effector cells are peripheral blood mononuclear cells infected with a lentivirus carrying the Her2 TCR-6A5-mC TCR gene.
  • the target cells are HLA-A2 negative Her2 / neu positive large cell lung cancer NCI-H460, HLA-A2 negative Her2 / neu positive colon cancer cells HT-29, HLA-A2 positive Her2 / neu negative glioblastoma cells U87MG .
  • the abscissa shows that different tumor cell line cells as target cells have undergone different treatments.
  • Adeasy-A2E75 is a target cell infected with an oncolytic adenovirus carrying the HLA-A2 and Her2-E75 minigenes alone;
  • PBMC control is the periphery of the target cell and a control that is not transfected with the Her2 TCR-6A5-mC TCR gene Mixed culture of blood mononuclear cells;
  • PBMC control + Adeasy-A2E75 is a target cell infected with adenovirus carrying HLA-A2 and Her2-E75 microgenes 24 hours later than a control without transfection of Her2 TCR-6A5-mC TCR gene Mixed culture of peripheral blood mononuclear cells;
  • PBMC with Her2 TCR-6A5" as the target cell and mixed culture of peripheral blood mononuclear cells transfected with Her2 TCR-6A5-mC TCR gene;
  • PBMC + with Her2 TCR-6A5" Adeasy-A2E75 is a target cell infected with replication-
  • the ordinate shows the concentration of IFN- ⁇ secreted by T cells.
  • FIG. 7B shows that T cells expressing Her2 TCR-6A5-mC TCR can specifically kill tumor cell lines infected with replication-deficient adenoviruses carrying the HLA-A2 and Her2-E75 minigenes.
  • the abscissa shows that cells of different tumor cell lines are treated differently as target cells.
  • "***", "**” and "*” show comparison with the replication-deficient adenovirus group expressing HLA-A2 and Her2-E75 polypeptides alone, or with Her2 TCR-6A5-mC TCR expressing alone Compared with the peripheral blood mononuclear cell group, the combination of the two showed that peripheral blood mononuclear cells expressing Her2 TCR were significantly specific for target cells infected with replication-deficient adenovirus expressing HLA-A2 and Her2-E75 polypeptides. Sexually lethal.
  • FIG. 7C shows the results of the flow detection of the cells.
  • the abscissa shows the target cell group.
  • the ordinate “HLA-A2 + (MFI)” represents the mean fluorescence intensity of cells after staining with anti-HLA-A2 fluorescent antibodies.
  • the oncolytic adenovirus Ad-E75A2 carrying the Her2-E75 minigene and HLA-A2 and the replication-deficient adenovirus Adeasy-A2E75 carrying the HLA-A2 and Her2-E75 minigenes can infect tumor cell lines to increase HLA-A2 expression High, promoting its sensitivity to TCR-T.
  • FIG. 8 shows that the infection sensitivity of Her2TCR-6A5-mC TCR to different target cells can be increased after infection of tumor cells by oncolytic adenoviruses carrying the Her2-E75 minigene and HLA-A2.
  • Figures 8A and 8B show the same experimental response.
  • Figure 8A shows the detection of factor levels in the supernatant, and
  • Figure 8B shows the detection of cell-related killer counts.
  • FIG. 8A shows that T cells expressing Her2, TCR-6A5-mC, and TCR can be activated by different tumor cell line cells infected with oncolytic adenoviruses carrying the Her2-E75 minigene and HLA-A2 and secrete IFN- ⁇ .
  • Effector cells are peripheral blood mononuclear cells infected with lentivirus carrying the Her2TCR-6A5-mCTCR gene.
  • the target cells are HLA-A2 negative Her2 / neu positive large cell lung cancer NCI-H460, HLA-A2 negative Her2 / neu positive colon cancer cells HT-29, HLA-A2 positive Her2 / neu negative glioblastoma cells U87MG .
  • the abscissa shows that different tumor cell line cells as target cells have undergone different treatments.
  • Ad-E75A2 is a target cell infected with an oncolytic adenovirus carrying the Her2-E75 minigene and HLA-A2 alone;
  • PBMC control is the periphery of the target cell and a control that is not transfected with the Her2 TCR-6A5-mC TCR gene Mixed culture of blood mononuclear cells;
  • PBMC control + Ad-E75A2 as target cells infected with oncolytic adenovirus carrying Her2-E75 minigene and HLA-A2 24 hours later without transfection with Her2 TCR-6A5-mC TCR gene Control peripheral blood mononuclear cells mixed culture;
  • PBMC with Her2 TCR-6A5" as target cells and mixed peripheral blood mononuclear cells transfected with Her2 TCR-6A5-mC TCR gene;
  • Her2 TCR-6A5 PBMC + Ad-E75A2 is a target cell infected with an oncolytic adenovirus carrying the Her2-E75
  • the ordinate shows the concentration of IFN- ⁇ secreted by T cells.
  • "***” and "****” show comparison with the oncolytic adenovirus group expressing Her2-E75 polypeptide and HLA-A2 alone, or with peripheral blood expressing Her2 TCR-6A5-mC TCR alone Compared with the mononuclear cell group, the combined group showed that oncolytic adenovirus expressing Her2-E75 polypeptide and HLA-A2 can significantly increase the peripheral blood mononuclear cells expressing Her2 TCR-6A5-mC to different target cells.
  • FIG. 8B shows that T cells expressing Her2, TCR-6A5-mC, and TCR can specifically kill tumor cell lines infected with oncolytic adenoviruses carrying the Her2-E75 minigene and HLA-A2.
  • the abscissa shows that cells of different tumor cell lines are treated differently as target cells.
  • the ordinate shows the killing rate of T cells to target cells
  • Cytotoxicity% ((the number of live cells of the initial target cells-the number of live cells of the target cells at the end of the culture) / the number of live cells of the initial target cells) ⁇ 100.
  • the effective target ratio E: T was 10: 1
  • FIG. 9 is a schematic diagram showing the main structure of a type 5 oncolytic adenovirus backbone vector pShuttle-MCS-CMV-E1A-SV40pA constructed according to an embodiment of the present invention.
  • FIG. 10 shows an oncolytic adenovirus type 5 genomic DNA (ie, “OAd-E75”, upper panel) that expresses a marker polypeptide alone, and 5 that co-expresses the marker polypeptide and HLA-A2 according to an embodiment of the present invention.
  • Schematic diagram of the main structure of type II oncolytic adenovirus genomic DNA ie, "OAd-E75-A2", below.
  • Figure 11 shows the animal experimental protocol in Example 9, specifically showing the dosing protocol of this example, where the first group is a blank control group; the second group is the OAd-E75A2 group (that is, the oncolytic virus is administered alone) );
  • the third group is Her2 TCR-6A5-mC T (IV) group (that is, TCR T intravenously administered alone);
  • the fourth group is OAd-E75A2 + Her2 TCR-6A5-mC T (IV) group (that is, combined administration Drugs, of which TCR is administered intravenously);
  • the fifth group is Her2 TCR-6A5-mC T (IT) group (that is, TCR alone administered intratumorally);
  • the sixth group is OAd-E75A2 + Her2 TCR-6A5-mC T (IT) group (i.e., combined administration, in which TCR was administered intratumorally).
  • the solid dots in FIG. 11 indicate the start time of subcutaneous inoculation of tumor cells in animals,
  • FIG. 13 shows a tumor volume change curve of a single animal in each group of animals in Example 9.
  • FIG. 14 shows the change curve of the relative tumor proliferation rate (T / C)% of each group of animals with the number of days after administration in Example 9.
  • the relative tumor proliferation rate (T / C)% refers to the The percentage value of the average tumor volume compared to the average subcutaneous tumor volume of the control animals measured on the same day is used to indicate the inhibition of tumor growth in the treatment group relative to the control group.
  • T / C% lower than 40% during drug development is considered the basic standard for drug effectiveness.
  • FIG. 15 shows the average body weight change curve of each group of animals in Example 9.
  • FIG. 16 shows the results of flow cytometric analysis of the number of human T cells in tumor tissues of each group of animals at the termination of the experiment in Example 9.
  • FIG. FIG. 16A is the number of human CD3 + T cells per 20,000 tumor cells in the tumor tissue of the six groups of animals
  • FIG. 16B is the number of human CD8 + T cells per 20,000 tumor cells in the tumor tissue of the six groups of animals
  • FIG. 16C is the number of human CD4 + T cells per 200 million tumor cells in the tumor tissue of the six groups of animals.
  • the abscissa indicates the different groups set in the experiment
  • the ordinate indicates the number of T cells after normalization.
  • the standardized treatment means that the immune cells infiltrated in the tumors of each group of animals are represented by the number of immune cells detected per 20,000 tumor-derived cells.
  • the inventors of the present invention have proposed theoretically and experimentally verified the significant enhancement of the presentation of foreign epitope peptides on the surface of tumor cells and the use of specificity
  • the concept of a combined therapy targeting TCR-modified immune cells of the epitope peptide According to the inventive concept, the present invention effectively solves the problems of low antigen expression amount and / or low or missing HLA expression caused by a defect in the antigen presentation pathway caused by tumor immune evasion mechanism, and improves tumor cell response to T cell receptors.
  • the recognition sensitivity of modified immune cells further improves the ability of T cell receptor-modified immune cells to homing and kill tumor cells.
  • the present invention also expands the applicable range of adoptive immune cells based on TCR gene modification to treat tumors, and avoids the limitation of smaller use range brought by HLA restriction.
  • the present invention provides a therapeutic agent for treating tumors and / or cancers, comprising:
  • a first composition wherein the first composition comprises a first active ingredient in a first pharmaceutically acceptable carrier, the first active ingredient including or containing Nucleic acid encoding a marker polypeptide; the marker polypeptide has an amino acid sequence of one or more epitope polypeptides, and the epitope polypeptides can be presented by the MHC class I molecule to the tumor cells and / or cancer cells Surface; and
  • a second composition wherein the second composition comprises a second active ingredient in a second pharmaceutically acceptable carrier, the second active ingredient comprises a T cell receptor-modified immune cell; the T cell is affected by Somatically modified immune cells are capable of specifically recognizing and binding the epitope polypeptide presented by the MHC class I molecule.
  • antigenic proteins expressed in the cytoplasm can enter the MHC class I antigen presentation pathway.
  • the short peptides containing epitope polypeptides
  • TAP molecules TAP molecules.
  • the plasmoplasmic reticulum and formed a trimer with the HLA protein and ⁇ 2 -microglobulin, it is presented on the cell surface (where the HLA protein and ⁇ 2 -microglobulin are paired to form MHC class I molecules), thereby being immune Cell recognition.
  • tumor antigens expressed in the cytoplasm cannot effectively form epitope polypeptides or enter the endoplasmic reticulum and combine with HLA and ⁇ 2 microglobulin to form complexes.
  • the exogenous marker polypeptide expressed in the tumor cells and / or cancer cells can enter the MHC class I antigen presentation. Pathway, thereby increasing the expression amount of the HLA / antigen epitope polypeptide complex on the surface of tumor cells, thereby enhancing the recognition sensitivity of the T cell receptor-modified immune cells to tumor cells and / or cancer cells.
  • the amino acid sequence of the epitope polypeptide may be derived from the amino acid sequence of a protein existing in nature, or an artificially synthesized amino acid sequence not existing in nature.
  • the proteins existing in nature include human-derived proteins and proteins of other species other than humans.
  • the amino acid sequence of the epitope polypeptide is derived from the amino acid sequence of a tumor-associated antigen or a tumor-specific antigen.
  • Tumor-associated antigen generally refers to a normal protein derived from itself, but is overexpressed or abnormally expressed in tumor cells, and includes carcinoembryonic antigen, tumor-testis antigen (CT antigen), and the like.
  • Tumor-specific antigen generally refers to a mutant protein derived from itself, or a heterologous viral protein associated with tumorigenesis and development.
  • tumor-associated antigen and “tumor-specific antigen” are sometimes collectively referred to as “tumor antigen”.
  • the tumor antigen may be a tumor antigen as described in the Cancer Antigenic Peptide Database (website https://caped.icp.ucl.ac.be).
  • the tumor antigen may be a tumor antigen as described in Table 1 below. It is also preferred that the tumor antigen may be human Her2 / neu, NY-ESO-1, N-ras, K-ras, H3.3K27M, SSX-2, MAGE-C2, MAGE-A1, KK-LC- 1. p53.
  • the amino acid sequence of Her2 / neu is shown in SEQ ID NO: 21.
  • the epitope polypeptide may be a peptide having 8-11 amino acids capable of being presented by MHC class I molecules.
  • the epitope polypeptide may be an epitope polypeptide as described in Cancer Antigenic Peptide Database (website https://caped.icp.ucl.ac.be).
  • the epitope polypeptide may be an epitope polypeptide as described in Table 1 below.
  • the epitope polypeptide is the same as the epitope polypeptide described in Table 1 below with 4-9 consecutive amino acids (e.g., 4, 5, 6, 7, 8, or 9 consecutive Of the same amino acid), and these polypeptides are 8-11 amino acids in length.
  • the epitope polypeptide includes, but is not limited to, Her2 / neu 369-377 as shown in SEQ ID NO: 3, Her2 / neu 373-382 as shown in SEQ ID NO: 22, NY-ESO- 1157-165, NY-ESO-1 1-1-1, NY-ESO-1 53-62, NY-ESO-1 18-27, N-ras 55-64, K-ras 224-232, K-ras 10- 18, K-ras 10-19, H3.3K27M 26-35, SSX-2 41-49, MAGE-C2 336-344, MAGE-C2 191-200, MAGE-C2 307-315, MAGE-C2 42-50 , MAGE-A1 120-129, MAGE-A1 230-238, MAGE-A1 161-169, KK-LC-1 76-84, p53 99-107, HPV16-E6 29-38, HPV16-E7 11-19, HPV16-E7 11-19,
  • Tumor antigen name HLA typing Amino acid sequence position N-ras A1 55-64 MART2 A1 446-455 MATN A11 226-234 CDKN2A A11 125-133 CDK12 A11 924-932 k-ras A2 224-232 hsp70-2 A2 286-295 HAUS3 A2 154-162 GAS7 A2 141-150 CSNK1A1 A2 26-34 CLPP A2 240-248 CDK4 A2 23-32 ⁇ -actinin-4 A2 118-127 beta-catenin A24 29-37 SIRT2 A3 192-200 GPNMB A3 179-188 EFTUD2 A3 668-677 MUM-3 A68 322-330 Elongation factor 2 A68 581-589 CASP-8 B35 476-484 SNRPD1 B38 19-Oct OS-9 B44 438-446 MUM-2 B44 123-133 MUM-1 B44 30-38 KIAAO205 B44 262-270 NFY
  • each of the epitope polypeptides has flexible linking fragments at both ends, which serve as the cleavage site of the proteolytic enzyme in the cytoplasm to release the epitope polypeptide.
  • the flexible connecting fragments include GSGSR, AGSGSR, and AGSGS.
  • the labeled polypeptide has a signal peptide at the amino terminus of the amino acid sequence of the one or more epitope polypeptides that can introduce the labeled polypeptide into the endoplasmic reticulum.
  • the core of the signal peptide contains long stretches of hydrophobic amino acids, forming a single ⁇ -helix.
  • the amino terminus of a signal peptide usually begins with a short positively charged amino acid sequence. There is usually an amino acid cleavage site at the end of the signal peptide that is recognized and cleaved by a signal peptidase.
  • the signal peptide may be a signal peptide (SEQ ID NO: 28) composed of amino acid 1-22 of the amino terminal of insulin-like protein (INSL5).
  • every two of the epitope polypeptides may be linked by a cleavable linking polypeptide.
  • the cleavable linking polypeptide includes a furin digestion recognition polypeptide, which has a standard four amino acid motif that can be cleaved by the Furin enzyme, namely the RX- [KR] -R amino acid sequence (see the document "Molecular Therapy 2007; vol. 15 no. 6,1153-1159 ").
  • the amino acid sequence of the cleavable linking polypeptide is RRKR.
  • the epitope polypeptide linked by the RX- [KR] -R amino acid sequence is cleaved and hydrolyzed by the furin enzyme in the endoplasmic reticulum, releasing the epitope polypeptide, and HLA and ⁇ 2 -microglobulin in the endoplasmic reticulum form an antigen complex.
  • Aminopeptidases and carboxypeptidases in the endoplasmic reticulum may also be involved in the enzymatic hydrolysis and release of epitope polypeptides (see the document "J Immunol. 2009 November 1; 183 (9): 5526-5536"), so they can be cleaved Sexually linked polypeptides may also include aminopeptidase and carboxypeptidase digestion recognition polypeptides.
  • the labeled polypeptide has an endoplasmic reticulum retention signal peptide at the carboxyl terminus of the amino acid sequence of the one or more epitope polypeptides.
  • the amino acid sequence of the endoplasmic reticulum retention signal of the soluble polypeptide is KDEL, and the endoplasmic reticulum retention signal of the ER membrane protein is KKXX (see the document "Molecular Molecular Biology of the Cell. 2003; 14 (3): 889-902 ").
  • the labeled polypeptide is a soluble polypeptide. It is therefore preferred that the endoplasmic reticulum retention signal peptide is a K-D-E-L fragment consisting of lysine-aspartic acid-glutamic acid-leucine residues.
  • the labeled polypeptide includes the following amino acid sequences operably linked and connected in series: the amino acid sequence of an N-terminal signal peptide, the amino acid sequence of one or more of the epitope polypeptides, and optionally An amino acid sequence of a selected C-terminal endoplasmic reticulum retention signal, wherein when the labeled polypeptide includes a plurality of amino acid sequences of the epitope polypeptide, every two adjacent amino acid sequences of the epitope polypeptide
  • the amino acid sequence of the epitope polypeptide and the amino acid sequence of the optional C-terminal endoplasmic reticulum retention signal may be linked by the amino acid sequence of the cleavable linking polypeptide.
  • the labeled polypeptide includes an amino acid sequence of the C-terminal endoplasmic reticulum retention signal.
  • the nucleic acid further has an HLA protein coding sequence, wherein the HLA protein coding sequence and the marker polypeptide coding sequence are respectively in respective Under the control of a promoter, or the HLA protein coding sequence and the marker polypeptide coding sequence are under the same promoter control, and the HLA protein coding sequence can be cleaved to connect the polypeptide coding sequence with the marker polypeptide coding sequence. Operationally connected.
  • the phenotype of the HLA protein is consistent with the phenotype of the HLA protein to which the labeled polypeptide can bind.
  • 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.
  • the present invention further increases the expression amount of the MHC / antigen epitope polypeptide complex on the surface of tumor cells, thereby enhancing the recognition sensitivity of the T cell receptor-modified immune cells to tumor cells.
  • the MHC protein is a HLA class I protein.
  • the HLA includes HLA-A, B, and C.
  • the HLA protein is HLA-A2 protein, and the amino acid sequence of HLA-A2 is shown in SEQ ID NO: 29.
  • cleavable linking polypeptide linking the HLA protein and the labeled polypeptide are known in the art, such as a 2A polypeptide, and the 2A polypeptide includes, but is not limited to, an F2A polypeptide from a picornavirus, and Similar Class 2A polypeptides from other viruses; it may also be a Furin-F2A linker.
  • the first composition and the second composition are each independently present in the therapeutic agent without being mixed with each other.
  • the nucleic acid includes DNA or RNA; and the RNA includes mRNA transcribed from the DNA.
  • the first active ingredient is a recombinant virus
  • the genome of the recombinant virus has a marker polypeptide coding sequence and an optional HLA protein coding sequence; wherein the recombinant virus includes a selective replication-type recombinant Oncoviruses or replication defective recombinant viruses.
  • the replication-deficient recombinant virus is a viral vector that lacks one or several essential functional genes related to virus replication, proliferation, and virus particle assembly.
  • the viral vector cannot replicate in normal cells to form progeny viruses, but can express the virus itself Or an exogenous gene product.
  • the replication-deficient recombinant virus is preferably derived from adenovirus, adenovirus-associated virus (AAV), herpes simplex virus, pox virus, influenza virus, alphavirus, and Sendai virus.
  • the replication-deficient recombinant virus is a recombinant adenovirus obtained by genetically modifying type 5 adenovirus, the E1 gene is deleted in the genome of the recombinant adenovirus, and an insertion is made at the position of the deleted E1 gene.
  • the recombinant oncolytic virus may be derived from a genetically mutated virus having oncolytic effect and a wild-type virus having oncolytic effect.
  • the recombinant oncolytic virus is derived from adenovirus, pox virus, herpes simplex virus, measles virus, Semliki forest virus, vesicular stomatitis virus, polio virus, and retrovirus with oncolytic effect. , Reovirus, Seneca Valley virus, Echo enterovirus, Coxsackie virus, Newcastle disease virus and Maraba virus.
  • oncolytic virus After oncolytic virus infects tumor cells, it selectively replicates in tumor cells, and lyses tumor cells through a large number of daughter virus proliferation to achieve specific killing of tumor cells.
  • the released daughter virus can selectively infect and lyse other tumor cells to clear the tumor tissue to the greatest extent (see the document "Nat Biotechnol. 2012 Jul 10; 30 (7): 658-70").
  • the abnormal signaling pathways of RAS, TP53, RB1, PTEN, and WNT in tumor cells affect the cell's own antiviral mechanism, making it easier for the virus to replicate in tumor cells, which is the main reason for the formation of tumor selectivity.
  • oncolytic viruses Due to the complete molecular mechanism that inhibits virus replication in normal cells, oncolytic viruses cannot effectively replicate and spread after infection with normal cells, thus greatly limiting the damage to normal tissue cells (see the document “Nat Rev. Cancer. 2017 11; 17 (11) : 633 ").
  • the genetic engineering of the virus genome has further enhanced the tumor selectivity of oncolytic viruses, and can carry functional foreign genes to enhance the antitumor activity of oncolytic viruses.
  • oncolytic viruses can also change the microenvironment of tumor tissues, mainly by inducing secretion of cytokines, attracting natural immune cells, releasing tumor antigens, providing immune danger signals, etc., thereby enhancing tumor local anti-tumor Immune response (see document "J. Clin. Invest.
  • Adenovirus is an oncolytic virus that was developed earlier.
  • Ad5 adenovirus H101 based on E1B-55K and E3 gene defects is the first oncolytic virus product to be approved for marketing (see the document “Hum GeneGener. 2018” Feb; 29 (2): 151-159 ").
  • the molecular structure and biological characteristics of adenoviruses have been studied in depth, making it easier for adenoviruses to become oncolytic viruses through genetic engineering.
  • adenovirus Some features of adenovirus, including foreign genes that allow insertion of larger fragments after genome modification; adenovirus genomic DNA will not integrate into the host genome; nor will it cause malignant transformation of human cells; it can infect most human tumor cells; and The preparation of stable high-titer virus particles and the like make the adenovirus more suitable as an oncolytic virus vector (see the document "Curr Opin Virol. 2016; 12: 9-15").
  • the different designs of oncolytic adenoviruses are related to their different mechanisms of tumor selectivity. For example, Ad5 adenoviruses with defective E1B-55K genes infect normal cells with p53 function and induce apoptosis before completing the virus replication cycle.
  • the tumor selectivity of oncolytic adenoviruses carrying VA-RNA gene defects depends on the incomplete activation of RAS signaling pathways and interferon pathways in tumor cells (see the literature “Cancer Res. 2003; 63 (17): 5544-50 ").
  • Another tumor-selective mechanism of oncolytic adenovirus is the use of tumor-specific gene promoters to drive genes necessary for adenovirus replication.
  • the alpha-fetoprotein promoter in liver cancer cells see the document "Hum Gene Gene Ther. 1999; 10 (10): 1721-33”
  • PSA prostate-specific antigen
  • a promoter whose activity depends on free E2F1 transcription factors such as an E2F1 promoter inserted into the palindromic sequence of E2F1
  • it can only be effective in tumor cells rich in free E2F1 Expression of virus-replicated downstream molecules to selectively lyse tumor cells (see literature "Mol Ther. 2007; 15 (9): 1607-15”; “Cancer Cell. 2002; 1 (4): 325-37”).
  • oncolytic adenovirus Although genetically engineered oncolytic adenovirus enhances the selective killing of tumor cells, the virus' oncolytic capacity must be further strengthened to improve its clinical efficacy. Because the oncolytic virus completes the replication cycle, it depends on the cellular components and unique molecular mechanisms of the host tumor cells. The diversity of tumors determines that tumor cells with different growth states and properties are infected with oncolytic viruses. Some tumor cells cannot complete replication. Cycle and produce a sufficient number of daughter viruses to lyse the cells. In addition, the deletion of oncolytic adenovirus E1B-55K may cause the process of exporting mRNA encoding late viral proteins from the nucleus to the cytoplasm for protein translation, thereby affecting virus replication in tumor cells (see the literature " Viruses. 2015 Nov; 7 (11): 5767-5779 "). These factors will limit the clinical efficacy of oncolytic virus when used alone.
  • a nucleic acid containing the marker polypeptide coding sequence and an optional HLA protein coding sequence is introduced into a tumor cell and / or cancer cell by an oncolytic virus, and the oncolytic virus kills the tumor cell and / or
  • immune cells modified by T cell receptors can also effectively eliminate tumor cells that have been lysed after being infected with oncolytic viruses, failing to complete the replication cycle and generate a sufficient number of daughter viruses, thereby achieving synergy. effect.
  • the selective replication-type recombinant oncolytic virus is a recombinant oncolytic adenovirus obtained by genetically modifying type 5 adenovirus.
  • the genome of the recombinant oncolytic adenovirus is deleted from the E1B-55K gene and / or the E1B-19K gene (for example, the E1B-55K gene is deleted, or the E1B-55K gene and the E1B-19K gene are deleted)
  • the genome of the recombinant oncolytic adenovirus contains an E1A gene coding sequence; preferably, the E1A gene coding sequence is under the control of an exogenous promoter.
  • the coding sequence of the marker polypeptide inserted into the oncolytic adenovirus genome can be under the control of the E1B gene promoter, the E1B TATA box sequence, and the E1B polyadenylation signal sequence of the adenovirus itself. Expressed in tumor cells.
  • the coding sequence of the marker polypeptide inserted into the oncolytic virus genome can be under the control of a eukaryotic cell promoter, which includes but is not limited to the CMV promoter, the EF1a promoter, SV40 promoter, PGK1 promoter, Ubc promoter, human ⁇ -actin promoter.
  • a eukaryotic cell promoter which includes but is not limited to the CMV promoter, the EF1a promoter, SV40 promoter, PGK1 promoter, Ubc promoter, human ⁇ -actin promoter.
  • the E1A gene of the recombinant oncolytic adenovirus can be changed so that the expressed E1A protein cannot bind to the pRb protein.
  • the nucleic acid sequence encoding the CR2 region of the E1A protein in the recombinant oncolytic adenovirus genomic DNA is deleted from nucleotides 923-946 of type 5 adenovirus genomic DNA (oncolytic adenovirus E1A - ⁇ 24), the amino acid sequence of the encoded E1A protein is deleted from LTCHEAGF.
  • the deleted amino acid sequence is the binding region between the E1A protein and the Rb protein.
  • the E1A protein lacking this amino acid sequence cannot bind to the Rb protein, resulting in oncolytic adenovirus E1A- ⁇ 24 selectively in tumor cells with defective Rb / E2F1 pathway. Replicate and lyse tumor cells.
  • the E1A gene of the recombinant oncolytic adenovirus is under the control of a tissue-specific promoter or a tumor-specific promoter.
  • the tissue-specific or tumor-specific promoter includes E2F-1 promoter, telomerase hTERT promoter, tyrosinase promoter, prostate-specific antigen promoter, alpha-fetoprotein promoter, and COX-2 Promoter.
  • the tumor-specific promoter is an E2F-1 promoter (its nucleotide sequence is SEQ ID NO: 30). In normal cells, because E2F-1 binds to pRb, the expression of E1A regulated by the E2F-1 promoter is suppressed.
  • E2F-1 In tumor cells, due to the lack of pRb or excessive phosphorylation, the level of "free" E2F-1 increases, and the activation of the E2F-1 promoter drives the expression of E1A, and causes the adenovirus to selectively replicate in tumor cells and lyse the cells .
  • E3 gene of the recombinant oncolytic adenovirus is deleted.
  • the inhibitory effect of E3-19K protein on the HLA class I antigen presentation pathway can be avoided, so that the epitope polypeptide and the endogenous tumor antigen introduced from outside can be more effectively presented to the surface of tumor cells.
  • the structural and functional proteins of the adenovirus are deleted from at least one immunodominant epitope recognized by a T cell.
  • Structural and functional proteins include E1A, E1B, hexon, penton base, fibrin, capsid protein IX, DNA polymerase, and single-stranded DNA binding protein.
  • immunodominant epitopes recognized by T cells on viral proteins are removed by point mutations in genes (see patent document WO2016178167A1).
  • the immune cell modified by a T cell receptor includes a primitive T cell or a precursor cell thereof, an NKT cell, or a T cell strain.
  • the T cell receptor includes at least one of an ⁇ chain and a ⁇ chain, and both the ⁇ chain and the ⁇ chain include a variable region and a constant region, and the T cell receptor can specifically recognize tumor cells and / or cancers The epitope polypeptide on the cell surface.
  • the amino acid sequence of the variable region of the ⁇ chain has at least 98%, preferably at least 98.5%, more preferably at least 99% identity with the amino acid sequence shown in SEQ ID NO: 1, the ⁇ chain
  • the amino acid sequence of the variable region 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: 2 as long as it does not significantly affect the effect of the present invention.
  • the amino acid sequence of the variable region of the ⁇ chain is shown in SEQ ID NO: 1
  • 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.
  • 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 in 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, 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: 8, the amino acid sequence is modified by one two in the constant region. Sulfur bond; for a ⁇ chain with the amino acid sequence shown in SEQ ID NO: 9, its constant region is replaced with a 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 alpha chain of the TCR is shown in SEQ ID NO: 5
  • the amino acid sequence of the beta 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 alpha 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: 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%, and 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 present invention also relates to 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, both of the alpha chain coding sequence and the beta chain coding sequence comprise A variable region coding sequence and a constant region coding sequence, the T cell receptor is capable of specifically recognizing the epitope polypeptide on the surface of tumor cells and / or cancer cells.
  • the amino acid sequence encoded by the alpha chain variable region coding sequence has at least 98%, preferably at least 98.5%, more preferably at least 99% identity with the amino acid sequence shown in SEQ ID NO: 1, the beta chain variable region
  • the amino acid sequence encoded by the coding 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: 2 as long as it does not significantly affect the effect of the present invention.
  • the ⁇ -chain variable region coding sequence encodes an amino acid sequence shown in SEQ ID NO: 1
  • the ⁇ -chain variable region coding sequence encodes an amino acid sequence shown in SEQ ID NO: 2.
  • the nucleic acid may be DNA or RNA.
  • the coding sequence of the ⁇ -chain variable region is shown in SEQ ID NO: 10, and the coding sequence of the ⁇ -chain variable region is shown in SEQ ID NO: 11.
  • 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, the coding sequence is modified by 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, it is modified by one two in the constant region. 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 may be mutated, including removing, inserting, and / or replacing one or more amino acid codons, so that the function of the expressed TCR recognition epitope polypeptide is 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 epitope polypeptide presented by the target cell.
  • 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-binding epitope polypeptide.
  • CDR2 complementarity determining region 2
  • the present invention also relates to a recombinant expression vector for expressing the TCR, which contains a nucleic acid (eg, DNA) encoding a T cell receptor according to the present invention operatively linked to a promoter, and / or a complementary sequence thereof.
  • a nucleic acid eg, DNA
  • the DNA encoding the T cell receptor 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 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.
  • One embodiment includes expressing an autofluorescent protein (such as GFP or other fluorescent protein) for in vivo tracking imaging.
  • Another embodiment includes expression of an inducible suicide gene system, such as induction of herpes simplex virus-thymidine kinase (HSV-TK) protein, or induction of expression of 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: 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 ( IRES (internal ribosome entry site) sequence is linked to the nucleic acid according to the present invention.
  • the coding sequence of the cleavable linking polypeptide may be the cleavable / ribosomal jumping 2A linking sequence described above, which may be derived from different viral genomes, including F2A, T2A, P2A, and E2A.
  • Another embodiment includes the expression of a human chemokine receptor gene, such as CCR2. These chemokine receptors can bind to corresponding chemokine ligands that are highly expressed in tumor tissue, thereby increasing the number of cells modified by the TCR gene according to the present invention. Homing in tumor tissue.
  • the first composition comprises a therapeutically effective amount of the DNA, or a therapeutically effective amount of the mRNA.
  • the first composition comprises a therapeutically effective amount of the recombinant virus.
  • the recombinant virus is a recombinant oncolytic adenovirus
  • the recombinant oncolytic adenovirus is administered at a dose of 5 ⁇ 10 7 to 5 ⁇ 10 12 vp / day, 1-2 times a day, and continuously administered for 1-7 days.
  • the second composition comprises a therapeutically effective amount of the T cell receptor modified immune cells.
  • the T-cell receptor-modified immune cells are included in a total dose range of 1 ⁇ 10 3 to 1 ⁇ 10 9 cells / Kg body weight per course of treatment.
  • the DNA can be formulated for intratumoral injection, for example, direct intratumoral injection in the form of a plasmid, or intratumoral injection after liposome packaging, or it can be linked to nanoparticles (such as poly-L-lysine) Acids, polyamino acids, polymers such as polyethyleneimine and chitosan) can be injected intratumorally.
  • nanoparticles such as poly-L-lysine
  • Acids, polyamino acids, polymers such as polyethyleneimine and chitosan can be injected intratumorally.
  • the transfection rate can also be enhanced by electrotransfection after intratumoral injection.
  • the mRNA can also be formulated in a similar manner for administration by intratumoral injection.
  • the recombinant virus can be formulated for intratumoral injection, intraperitoneal administration, subarachnoid administration, or intravenous administration.
  • the immune cells can be formulated for administration via arterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, intramuscular, or intraperitoneal administration.
  • the therapeutic agent consists of the first composition and the second composition.
  • the therapeutic agent of the present invention may further include suitable pharmaceutically acceptable excipients, including pharmaceutical or physiological carriers, excipients, diluents (including physiological saline, PBS solution), and various additives. , Including sugars, lipids, peptides, amino acids, antioxidants, adjuvants, preservatives, etc.
  • the invention also provides the use of the therapeutic agent in the preparation of a medicament for treating tumors and / or cancers.
  • the tumor and / or cancer includes: head and neck tumor, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, chorionic cancer , Gastrinoma, pheochromocytoma, prolactinoma, hematoma, neuroma, vonHippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureter cancer, glioma, Neuroblastoma, meningiomas, spinal tumors, osteochondroma, chondrosarcoma, Ewing's sarcoma, unknown primary cancer, carcinoid, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer Eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, skin cancer, mesotheli
  • the tumor and / or cancer may include HLA-A2 positive and Her2 / neu negative, HLA-A2 negative and Her2 / neu positive, HLA-A2 and Her2 / neu both positive, or HLA-A2 and Her2 / neu were negative.
  • the therapeutic agent within the scope of the present invention may be administered to a patient according to the actual situation of the tumor and / or cancer patient.
  • the invention also provides the labeled polypeptides of the invention described above.
  • the amino acid sequence of the labeled polypeptide has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 56, or SEQ ID NO: 60, More preferably at least 98.5%, and still more preferably at least 99% consistency. Also preferably, the amino acid sequence of the labeled polypeptide is as shown in SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 56, or SEQ ID NO: 60.
  • the invention also provides an isolated nucleic acid having a coding sequence for a labeled polypeptide according to the invention.
  • the amino acid sequence of the labeled polypeptide has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 56, or SEQ ID NO: 60, and more preferably A consistency of at least 98.5%, and preferably at least 99%.
  • Embodiments of the nucleic acid are as described above.
  • the nucleic acid is DNA with a nucleotide sequence such as SEQ ID NO: 25 (corresponding to a sequence excluding HLA-A2), or SEQ ID NO: 26 (corresponding to a sequence including HLA-A2, where HLA- A2 is before the labeled polypeptide), or SEQ ID NO: 27 (corresponding to the sequence including HLA-A2, where HLA-A2 is after the labeled polypeptide), or SEQ ID NO: 57, or SEQ ID NO: 58, or SEQ ID NO: 61.
  • SEQ ID NO: 25 corresponding to a sequence excluding HLA-A2
  • SEQ ID NO: 26 corresponding to a sequence including HLA-A2, where HLA- A2 is before the labeled polypeptide
  • SEQ ID NO: 27 corresponding to the sequence including HLA-A2, where HLA-A2 is after the labeled polypeptide
  • SEQ ID NO: 57, or SEQ ID NO: 58, or SEQ ID NO: 61 SEQ
  • SEQ ID NO: 24 (corresponding to the sequence excluding HLA-A2), or SEQ ID ID NO: 31 (corresponding to the sequence including HLA-A2, where HLA-A2 precedes the labeled polypeptide)
  • SEQ ID NO: 32 (corresponding to the sequence including HLA-A2, where HLA-A2 is after the labeled polypeptide)
  • SEQ ID NO: 56 SEQ ID NO: 36, or SEQ ID NO: 60 .
  • the invention also provides the use of the nucleic acid in the preparation of a medicament for treating or preventing tumors and / or cancers.
  • the present invention also provides a recombinant expression vector containing the nucleic acid according to the present invention, and / or its complementary sequence.
  • the nucleic acid 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)).
  • the recombinant vector expressing the labeled polypeptide of the present invention can be obtained by conventional recombinant DNA technology in the art.
  • the basic backbone of the recombinant expression vector is an oncolytic adenovirus.
  • the promoter is an endogenous viral gene promoter, such as an E1A promoter, an E1B promoter, or an E3 promoter.
  • the promoter is a tissue-specific or tumor-specific promoter.
  • the promoter is the E2F-1 promoter shown in SEQ ID NO: 30.
  • the recombinant expression vector may also include coding sequences of other functional molecules, such as a reporter gene, which can be used to identify whether a cell is transfected with a recombinant expression vector, or For determination of protein levels and activities, for example by flow cytometry, amplification / expression methods, immunohistochemical methods, FISH and shedding antigen assays, Southern blotting, Western blotting or PCR techniques. Therefore, methods for measuring protein levels in cells are generally known in the art.
  • the invention also provides the use of the recombinant expression vector in the preparation of a medicament for treating or preventing tumors and / or cancers.
  • the present invention also provides an isolated recombinant virus, wherein the genome of the recombinant virus has the nucleic acid according to the present invention; and the recombinant virus includes a selective replication-recombinant oncolytic virus or a replication-deficient recombinant virus.
  • the isolated recombinant virus are as described above.
  • the invention also provides the use of the recombinant virus in the preparation of a medicament for treating or preventing tumors and / or cancers.
  • the invention also provides a kit of synergistic combined drugs for treating tumors and / or cancers, comprising:
  • a second container containing the second composition in the therapeutic agent according to the present invention wherein the first container and the second container are independent;
  • the invention also provides the use of the kit in the preparation of a medicament for treating or preventing tumors and / or cancers.
  • the tumor and / or cancer include: head and neck tumor (including nasopharyngeal cancer), synovial cancer, renal cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, Bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, blood cancer, neuroma, vonHippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer Glioma, neuroblastoma, meningiomas, spinal tumors, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown site, carcinoid, fibrosarcoma, breast cancer, Paget's disease, cervical cancer , Esophageal cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate
  • the tumor and / or cancer may include HLA-A2 positive and Her2 / neu negative, HLA-A2 negative and Her2 / neu positive, HLA-A2 and Her2 / neu both positive, or HLA-A2 and Her2 / neu were negative.
  • the patient can be provided with a kit within the scope of the present invention.
  • the invention also provides a method for treating tumors and / or cancers, including:
  • a second composition of the therapeutic agents according to the invention is administered to said tumor and / or cancer patient.
  • the first composition and the second composition in the therapeutic agent can be administered simultaneously (e.g., intratumorally as a mixture), separately but simultaneously (e.g., administered by intratumoral and intravenous injection, respectively), or sequentially (e.g., The first composition is applied first, and then the second composition is applied; or the second composition is applied first, and then the first composition is applied).
  • the method includes the following steps performed sequentially:
  • a second composition of the therapeutic agents is administered to the tumor and / or cancer patient.
  • the second composition of the therapeutic agents is administered to the tumor and / or cancer patient 1 to 30 days after the first composition is first administered.
  • administering the second composition of said therapeutic agent to said tumor and / or cancer patient from 1-30 days after first administering said first composition refers to the first administration of the second composition with
  • the first first composition is administered at intervals of 1-30 days (e.g., 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days), or the first application of the second composition to the first one that is closest to the previous one
  • the composition is administered at intervals of 1-30 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days).
  • the time interval between the first administration of the second composition and the administration of the first composition immediately before it is 3-14 days (e.g., 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14 days).
  • the first composition comprises the recombinant oncolytic adenovirus, and the recombinant oncolytic adenovirus is administered at a dose of 5 ⁇ 10 7 to 5 ⁇ 10 12 vp / day, 1- 2 times, continuous application for 1-7 days, or any value between the above ranges.
  • the T-cell receptor-modified immune cells are administered at a dose of 1 ⁇ 10 3 to 1 ⁇ 10 9 cells / Kg body weight per course of treatment. Preferably, it is administered 1-3 times a day and continuously for 1-7 days.
  • 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 immune system of the patient to enhance the The number and function of T cell receptor-modified immune cells in the body.
  • the other drugs for treating tumors and / or cancers include, but are not limited to: chemotherapeutic drugs, such as cyclophosphamide, fludarabine; radiotherapy drugs; 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 DNA can be formulated for intratumoral injection, for example, direct intratumoral injection in the form of a plasmid, or intratumoral injection after liposome packaging, or it can be linked to nanoparticles (such as poly-L-lysine) Acid, polyamino acid, polyethyleneimine, chitosan and other polymers) intratumoral injection, and the transfection rate can also be enhanced by electrotransfection after intratumoral injection.
  • the mRNA can also be formulated in a similar manner for administration by intratumoral injection.
  • the recombinant virus can be formulated for intratumoral injection, intraperitoneal administration, subarachnoid administration, or intravenous administration.
  • the T cell receptor-modified immune cells can be formulated for administration via arteries, veins, subcutaneously, intradermally, intratumorally, intralymphatics, intralymph nodes, subarachnoid space, intramedullary, intramuscular, or intraperitoneal administration.
  • the tumor and / or cancer include: head and neck tumor (including nasopharyngeal cancer), synovial cancer, renal cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, Bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, blood cancer, neuroma, vonHippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer Glioma, neuroblastoma, meningiomas, spinal tumors, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown site, carcinoid, fibrosarcoma, breast cancer, Paget's disease, cervical cancer , Esophageal cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate
  • the tumor and / or cancer may include HLA-A2 positive and Her2 / neu negative, HLA-A2 negative and Her2 / neu positive, HLA-A2 and Her2 / neu both positive, or HLA-A2 and Her2 / neu were negative.
  • the method within the scope of the present invention can be provided to the patient according to the actual situation of the tumor and / or cancer patient.
  • 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), small cell lung cancer NCI-H446
  • the cell line was maintained in RPMI-1640 complete medium (Lonza, cat # 12-115F). 10% calf serum FBS (ATCC 30-2020) was added to RPMI-1640 complete medium, 2mmol / L L-glutamic acid , 100 ⁇ g / ml penicillin and 100 ⁇ g / ml streptomycin.
  • Peripheral blood products Unless otherwise specified, human peripheral blood products (including peripheral blood mononuclear cells) from healthy donors used in the test were from the Pacific Blood Center in San Francisco (# 1PBMC and # 2PBMC were collected 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.
  • cytokines After incubating in an incubator at 5% CO 2 and 37 ° C for 16-24 hours, the following final concentrations of cytokines were added: human IL-2 (Peprotech, cat # 200-02) 100IU / ml, human IL-7 (Peprotech, cat # 200-07) 5ng / ml, human IL-15 (Peprotech, cat # 200-15) 5ng / ml.
  • 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.
  • the phenotype of T cells expressing Her2 / neu 369-377-specific TCR was analyzed by flow cytometry.
  • the tested cells were collected and placed in 1.5 ml tubes (the number of cells was about 10e5), and 1 ml of DPBS solution (2.7 mM KCl, 1.5 mM KH 2 PO 4 , 136.9 mM NaCl, 8.9 mM 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-E
  • 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) .
  • a single CD8 + Her2-E75 / HLA-A2 pentamer + cells were sorted into a single culture well of a 96-well culture plate, and HLA-A2-positive PBMC treated with 25 ⁇ g / ml mitomycin C for 2 hours was added.
  • Cells, 10e5 cells per well add 1 ⁇ g / ml Her2 / neu 369-377 peptide and culture overnight, then add RPMI-1640 containing IL-2 100IU / ml, IL-7 5ng / ml, IL-15 5ng / ml completely Medium.
  • the culture medium containing the cytokine is freshly changed every 3-4 days, and the presence of T cell clones is observed under a microscope. Collect proliferating T cells, perform antigen restimulation as described above to obtain a sufficient number of cells, perform phenotypic or functional tests, and extract RNA to clone the TCR gene.
  • T cell function test In order to test the ability of T cells transfected with TCR gene to recognize the epitope polypeptide, 10e5 TCR transfected T cells and 10e5 T2 cells were added to each well of a 96-well plate at 100 ⁇ l / Each well was mixed cultured in RPMI-1640 complete medium, and each test group was duplicated.
  • PBMC cells and tumor cells transfected with TCR gene were added to each well of a 96-well plate according to different effect target ratios, and cultured for 24 hours. Hours later, the supernatant was collected to detect the gamma interferon secreted in the supernatant.
  • Each test group was a multi-well or a three-well.
  • an anti-human CD8 antibody Biolegend, cat # 300912
  • an anti-human CD8 antibody Biolegend, cat # 300912
  • 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.
  • TCR ⁇ and ⁇ full-sequence gene fragments were added and cloned into pRACE vectors (Takara Bio, cat # 634858, USA), respectively.
  • the competent bacteria Stellar (Takara Bio Company, cat # 636763, USA) was transformed and the plasmid was obtained for sequencing.
  • the viral vectors used to express TCR are replication-defective lentiviral vectors, including: the lentiviral vector pCDH-EF1 ⁇ -MCS- (PGK-GFP) expressing GFP, which can be purchased from System Biosciences (Cat # CD811A-1); and pCDH-EF1 ⁇ -MCS, a vector that does not express GFP, was obtained by removing the PGK promoter and GFP gene on the pCDH-EF1 ⁇ -MCS- (PGK-GFP) vector using conventional techniques in the art.
  • PGK-GFP lentiviral vector pCDH-EF1 ⁇ -MCS-
  • 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 insertion of 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 particles were obtained by transfecting 293T / 293FT cells with Lipofectaine 2000 transfection reagent (invitrogen, # 11668019). Prepare 293T / 293FT cells and the transfection procedure according to the manufacturer's instructions. Transfection was performed in a 6-well culture plate. First, a liposome mixed solution of the transfected plasmid was prepared with Opti-MEM 1 medium (Thermo Fisher, cat # 51985091). According to the manufacturer's instructions, 6 ⁇ l of lipofectaine2000 reagent was added to 250 ⁇ l of the culture solution.
  • Opti-MEM 1 medium Thermo Fisher, cat # 51985091
  • 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 and 37 ° C. Replace with DBS-free DMEM medium (Thermo Fisher, cat # 11965092). Continue to incubate for 24 hours and 48 hours. Collect the cell supernatants and centrifuge at 2000g for 10 minutes.
  • the obtained virus supernatant 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
  • Recombinant TCR lentivirus transfected human T cells frozen thawed primary PBMC cells were cultured in RPMI-1640 complete culture for 24 hours, and dead cells were removed by Ficoll-Paque Premium Density Gradient Centrifugation ( ⁇ 400g) for 30 minutes. It was treated with 2 ⁇ g / ml anti-human CD3 antibody (Biolegend, OKT3 clone cat # 317303) and 2 ⁇ g / ml anti-human CD28 antibody (Biolegend, cat # 302914) (where 100 ⁇ l of the above-mentioned CD3 antibody and CD28 antibody were added to each well.
  • DPBS solution in a 24-well 24-well plate culture well with a cell concentration of 2 ⁇ 10e6 / ml.
  • Dynabead human T-CD3 / CD8 magnetic beads (Thermo Fisher Company, cat # 11131D) can also be used. Perform stimulus activation.
  • Phenotypic and functional tests can be performed after 72 hours. Transfection of T cell lines is also performed according to the above steps. If the viral vector is labeled with GFP, generally GFP-positive cells can be observed under a fluorescence microscope 48 hours after transfection.
  • Preparation Example 1 Preparation of a recombinant lentiviral vector expressing a labeled polypeptide.
  • the exogenous expressed gene was obtained first, and the HLA-A201 gene fragment was obtained by RT-PCR.
  • RNA was extracted from HLA-A201 + PBMC cells, using Superscript RT-PCR kit (Thermo Fisher, cat # 12574018), and the HLA-A201 gene was obtained according to the manufacturer's instructions, and the sequence was determined to be the complete sequence (the amino acid sequence is as shown in SEQ ID NO: 29, and the nucleotide sequence is shown in SEQ ID NO: 35).
  • the gene fragment of the marker polypeptide is obtained by DNA synthesis (Integrated DNA Technologies, gblocks Gene Fragments), and includes: a marker polypeptide “E75 ⁇ 1” (C-terminal does not have KDEL) (its amino acid sequence is as shown in SEQ ID NO : 56, the nucleotide sequence is shown in SEQ ID NO: 57); the labeled polypeptide "E75 ⁇ 4" (the C-terminus does not have KDEL) (the amino acid sequence is shown in SEQ ID NO: 36, the core The nucleotide sequence is shown in SEQ ID NO: 58); the labeled polypeptide “E75 ⁇ 8” (the C terminal does not have KDEL) (the amino acid sequence is shown in SEQ ID NO: 60, and the nucleotide sequence is shown in SEQ ID NO : Shown in 61).
  • Lentiviral vector pCDH-EF1 ⁇ -MCS- (PGK-GFP) was purchased from System Biosciences (Cat # CD811A-1).
  • the HLA-A2 gene fragment was inserted into a multiple cloning site downstream of the EF1a promoter by using conventional gene cloning techniques in the art.
  • the plasmid was digested with Xcm-1 to remove the GFP gene fragment, and the gene fragment of the marker polypeptide was inserted into the downstream of the PGK promoter and replaced the GFP gene by using conventional gene cloning techniques in the field, and the "pCDH” -EF1p-A2-PKGp-E75 ⁇ 1 ”,“ pCDH-EF1p-A2-PKGp-E75 ⁇ 4 ”, and“ pCDH-EF1p-A2-PKGp-E75 ⁇ 8 ”.
  • the sequencing results of each plasmid were correct.
  • the HLA-A2 gene is ligated with the furin digestion sequence and the F2A sequence and the coding sequence of the marker polypeptide "E75 ⁇ 8" (the K terminal has KDEL) to form an A2-Her2E75 sequence fragment (the nucleotide sequence of which is as SEQ ID ID NO : Shown in 26).
  • In-fusion cloning kit (Takara Bio, cat # 638909) was used to perform PCR according to the manufacturer's instructions.
  • the primers used were 5'-AGAGCTAGCGAATTCAACATGGCCGTCATG-3 '(SEQ ID ID: 37) and 5'-TGATTGTCGACGCCCTTAAAGCTCTCTATAAGGAAG-3' (SEQ ID ID NO : 38).
  • Preparation Example 2 Preparation of a replication-deficient recombinant adenovirus expressing a marker polypeptide / HLA-A2.
  • Recombinant adenoviruses are mainly prepared with reference to the preparation method of the AdEasy system (see the document "Nature protocols 2007; 2: 1236-1247”).
  • the exogenous expressed gene was obtained first, and the HLA-A201 gene fragment was obtained by RT-PCR.
  • RNA was extracted from HLA-A201 + PBMC cells, and the Superscript RT-PCR kit (Thermo Fisher, cat # 12574018) was used.
  • the HLA-A201 gene was obtained according to the manufacturer's instructions, and sequenced to determine the complete sequence.
  • exogenous genes such as Her2-E75 mini genes, or tandem mini genes and expression control elements, are obtained through DNA synthesis (Integrated DNA Technologie s, gblocks Gene Fragments), including: the labeled polypeptide "E75 ⁇ 8" (C-terminal has KDEL) (its amino acid sequence is shown in SEQ ID NO: 24, and its nucleotide sequence is shown in SEQ ID NO: 59).
  • the HLA-A2 gene was digested with furin and F2A sequence and the coding sequence of the marker polypeptide "E75 ⁇ 8" to form A2-Her2E75 sequence fragment (its nucleotide sequence is as SEQ ID NO: 26 As shown).
  • In-fusion cloning kit (Takara Bio, cat # 638909) was used to perform PCR according to the manufacturer's instructions.
  • the primers used were 5'-TAGAGATCTGGTACCAACATGGCCGTCATGG-3 '(SEQ ID NO: 39) and 5'-GGCTCGAGCGGCCGCTTAAAGCTCGTCTTTAAGGAAG-3' (SEQ ID NO : 40).
  • the A2-Her2E75 fragment is inserted into the multi-gene cloning site of pShttle-CMV vector (Agilent technologies, cat # 24007) by using conventional gene cloning techniques in the field to obtain pShuttle- CMV-A2E75-SV40pA.
  • Virus particles were obtained by transfection of ADENO-X 293 cells (Takara, cat # 632271) with Lipofectaine 3000 transfection reagent (Thermo Fisher Company, cat # L3000001).
  • ADENO-X 293 cells were cultured in 6-well plates to 50-75% confluence and transfected.
  • the purified recombinant adenovirus plasmid was digested with Pac I (NEB Biolabs, cat # R0547s) and purified. Cells were transfected with Lipofectaine 3000 according to the manufacturer's instructions. After 24 hours, fresh DMEN culture medium was replaced. Cytopathies began to appear from 10 to 14 days, showing that the cells were suspended like patches.
  • the cells were collected and suspended in a PBS solution, and freeze-thawed 4 times in dry ice and a water bath at 37 ° C. The supernatant after centrifugation was the initial preparation of the virus and stored at -80 ° C.
  • ADENO-X 293 cells were cultured in a T75 culture dish (Corning, cat # 430661). After 75% confluence, 30% -50% of the original virus cryopreservation solution was added. After 3-5 days of cytopathic changes, the cells were collected and frozen and thawed as described above to obtain a virus suspension.
  • the virus titer was measured using an adenovirus titer kit (Takara, cat # 632270) according to the manufacturer's instructions. When infecting target cells, resuspend the target cells with fresh culture solution, add adenovirus with a quantitative titer according to the number of cells, and detect the expression of foreign genes after 3-4 days of culture. The results showed that foreign gene expression was positive.
  • Preparation Example 3 Preparation of a recombinant oncolytic adenovirus expressing a labeled polypeptide and preparation of a recombinant oncolytic adenovirus expressing a labeled polypeptide / HLA-A2.
  • the obtained PCR fragment was purified and digested with BglII to recover and ligated to the pShuttle-CMV vector (purchased from Agilent Technologies, cat # 24007) between the BglII and EcoRV sites in the multiple cloning site to obtain pShuttle-E1A , Sequencing the E1A sequence in it, confirming that it is correct.
  • the obtained PCR fragment was purified and then digested with SalI and ligated to the SalI site in a multicloning site on a pShuttle vector (purchased from Agilent, cat # 240006) to obtain pShuttle-MCS-CMV-E1A- SV40polyA ( Figure 9).
  • the sequence of CMV-E1A-SV40polyA was sequenced and confirmed to be correct.
  • Oncolytic adenovirus type 5 genomic DNA (“OAd-E75”) and two type 5 oncolytic adenovirus genomic DNA (“OAd-E75-A2”) co-expressing the Her2-E75 minigene with HLA-A2 (see Figure 10).
  • the target gene expression frame includes an EF-1 ⁇ promoter, a Her2-E75 minigene (or Her2-E75 minigene and HLA-A2) coding region sequence and a BGHpolyA sequence. After obtaining the above two fragments, they were inserted into the KpnI and XhoI sites on type 5 oncolytic adenovirus backbone plasmid pShuttle-MCS-CMV-E1A-SV40pA, respectively.
  • the BGHpolyA sequence was amplified from the pcDNA3.1 plasmid (purchased from Invitrogen) by a high-fidelity PCR method.
  • E75 Her2-E75 minigene, referred to as "E75” in this section
  • the obtained PCR fragment was purified and digested with XhoI and ligated to the XhoI site on the pShuttle-EF1a-MCS-BGHpA-CMV-E1A-SV40pA vector to obtain pShuttle-EF1a-E75-BGHpA-CMV-E1A -SV40pA, the E75 sequence was sequenced and confirmed to be correct.
  • the HLA-A2, Furin-F2A, and E75 sequences used in this work were amplified by high-fidelity PCR using the CD811-EF1a-A2-F2A-HerE75 plasmid as a template.
  • the HLAA2 fragment, the Furin-F2A ligation fragment and the E75 fragment are amplified separately first, and the HLAA2 fragment needs to be at its end Add a stop codon, and the E75 fragment needs to remove the stop codon at its end.
  • E75-Furin-F2A-HLAA2 was purified and XhoI digested and recovered and ligated to the XhoI site on the pShuttle-EF1a-MCS-BGHpA-CMV-E1A-SV40pA vector to obtain pShuttle- EF1a-E75-HLA-A2-BGHpA-CMV-E1A-SV40pA, the E75-HLAA2 fragment was sequenced and confirmed to be correct.
  • BJ5183 containing pAdEasy-1 plasmid
  • BJ5183 (containing pAdEasy-1 plasmid) (purchased from Agilent) super competent bacteria, mix them gently and then place them on ice for 30 minutes, and incubate them at 42 ° C for 90 seconds. Put them back on ice to continue incubation 2
  • For 500 minutes add 500 ⁇ l of LB medium to each tube, and shake culture at 37 ° C and 150 RPM for 45 minutes. Spread it on a Kaner-resistant LB plate and incubate at 37 ° C overnight. The next day, the smaller clones appearing on the LB plate were picked and inoculated in 4 ml of Kana resistant LB medium, and cultured at 37 ° C with shaking at 200 RPM overnight.
  • the plasmid DNA of each tube bacterial solution was extracted by using the plasmid small amount extraction kit.
  • the obtained plasmid DNA was analyzed by agarose gel electrophoresis. The significantly smaller plasmid was discarded, and the larger plasmid was analyzed by PacI digestion. Recombinant pAdEasy plasmid digested with PacI will produce a smaller fragment of 4.5kb or 3kb.
  • OAd-E75 and OAd-E75A2 two plasmids that have undergone correct recombination (a small band with a size of 4.5 kb after digestion by PacI) are obtained: OAd-E75 and OAd-E75A2.
  • the above two plasmids can be used to package and express Her2-E75. Or oncolytic adenovirus type 5 co-expressing HLA-A2 and Her2-E75.
  • Linearization of adenovirus genomic DNA 2-3 ⁇ g of plasmid DNA (OAd-E75 and OAd-E75A2) obtained in the previous step were completely digested with PacI, and reacted at 37 ° C for 2-3 hours for phenol / chloroform extraction. And ethanol ammonium acetate precipitation, rinse the linearized DNA twice with 70% ethanol and dry at room temperature for 3 minutes, dissolve in 10 ⁇ l of clean deionized water, use this DNA to directly transfect AD293 cells or store the linearized DNA at -20 ° C spare.
  • Adenovirus packaging One day before the adenovirus packaging, the well-growing AD293 cells were seeded in 6-well plates, and the cell density was preferably ⁇ 70% when the transfection experiment was performed the next day. Lipo2000 or similar transfection reagents were used to transfect PacI linearized DNA into AD293 cells (transfection reagents need not be removed). The DNA transfected AD293 cells were returned to the CO 2 incubator and cultured for about 14 days, until AD293 cells appeared large areas of lesions and floated, indicating that the adenovirus packaging was successful. The diseased AD293 cells and virus mixture in the 6-well plate was pipetted to make it fully float and collected in a clean centrifuge tube. The cell suspension can be shaken briefly and vigorously to release as much adenovirus from the cells as possible, and the mixture can be stored briefly Store at 4 ° C and long-term at -80 ° C.
  • Adenovirus amplification AD293 was inoculated into a 60mm petri dish one day before amplification, and the cell coverage was about 80% when infected with adenovirus the next day. Pipette approximately 1ml of adenovirus mixture collected from a 6-well plate and directly add it to a 60mm petri dish inoculated with AD293. After mixing with light shaking, return to the CO 2 incubator to continue the culture. Most cells can be seen after about 2-3 days The lesions floated up, and the entire cell suspension was collected in a clean centrifuge tube by pipetting the cells as described above.
  • the collected 2 ml of the virus suspension was added to a 10 cm culture dish with an AD293 cell coverage of 80%, and continued in a CO 2 incubator. After 2-3 days of culture, most of the cytopathic lesions can be seen; collect the virus suspension in the 10cm culture dish; also draw 2ml of the virus suspension and add it to a 10cm culture dish with 80% coverage of AD293 cells in a CO 2 incubator After culturing for about 2-3 days, most of the cytopathic lesions were seen floating up; the virus suspension in the 15 cm culture dish was collected, and at this time, the adenovirus titer in the cell supernatant reached about 10 8 PFU / ml.
  • a large amount of adenovirus can be amplified by using the virus suspension in a 15cm petri dish.
  • virus suspension in a 15cm petri dish.
  • Adenovirus purification The virus suspension collected in the previous step was centrifuged and separated into a supernatant and a cell pellet. Adenovirus in the supernatant was precipitated with PEG8000 / NaCl and resuspended in 10 mM Tris ⁇ Cl (pH 8.0); the cell pellet was resuspended in 10 mM Tris ⁇ Cl (pH 8.0) repeatedly freeze-thaw 3-5 times, The adenovirus in the cells was completely released, and the cell suspension was centrifuged again, and the supernatant was retained for purification by the following CsCl density gradient centrifugation.
  • the adenovirus suspension obtained was centrifuged twice with light CsCl (1.2g / ml) and heavy CsCl (1.45g / ml), and the white Ad band after the second centrifugation was aspirated with a syringe, and finally PD was used.
  • the -10 desalting column replaced the adenovirus-dissolved CsCl with an adenovirus storage solution (10 mM Tris (pH 7.4), 1 mM MgCl2, 10% Glycerol, filtered and stored at 4 ° C after sterilization), and stored at -80 ° C after dispensing.
  • Adenovirus titration is performed using an adenovirus titration kit. The basic principle is to infect AD293 cells after diluting the adenovirus to a suitable titer, and to detect the expression of Hexon protein on the cell surface 48 hours later.
  • the titer (PFU / ml) of active adenovirus was determined by counting the number of cells expressing Hexon protein within a specified area.
  • Example 1 Inducing Her2 / neu 369-377 polypeptide (Her2-E75 epitope polypeptide) to specifically kill T cells from HLA-A2-positive normal peripheral blood
  • peptide-specific killer T cells were induced from HLA-A2-positive normal PBMC (# 2) by two rounds of in vitro stimulation with a low concentration of Her2 / neu 369-377 polypeptide at 1 ⁇ g / ml, and flow cytometry was performed. And single cell isolation.
  • the specific method is as described above. The results are as follows:
  • the right panel of Figure 1A shows that 0.024% of the lymphocytes are CD8 positive killer T cells that can bind to the Her2 / neu 369-377 / HLA-A2 pentamer (ie, Her2-E75 pentamer).
  • the peptide-stimulated control cells did not show CD8-positive pentamer-positive cells.
  • the results show that in the natural T cell bank, the number of specific T cells that recognize Her2 / neu 369-377 antigen polypeptide is small. Despite its small number, this population of T cells that recognize Her2 / neu 369-377 polypeptides can still be clearly distinguished.
  • the positive cells include high affinity T cells and low affinity T cells.
  • 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: 2
  • the coding sequence is shown in SEQ ID NO: 1
  • 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: 3 shown.
  • 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.
  • the expression of 6A5 TCR ⁇ 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 ⁇ chain of the constant region linked by a cleavable linking polypeptide from a human-derived sequence to a murine-derived sequence (SEQ ID NO: 20) (the corresponding TCR is Her2TCR-6A5-mC, The amino acid sequence is shown in SEQ ID NO: 23) and ligated to the above vector to obtain Her2TCR-6A5-mC recombinant lentiviral vector.
  • 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.
  • TCR-6A5-mC The fragment was amplified by 5 'primer 5'-AGAGCTAGCGAATTCAACATGGGCTGCAGGCTGCTC-3' (SEQ ID ID NO: 52) and 3 'primer 5'-GGATCGCTTGGCACGTGAATTCTTTCTTTTGACCATAGCCAT-3' (SEQ ID NO: 53); carrying a mouse-derived constant region sequence
  • the ⁇ gene of Her2TCR-6A5-mC was amplified by 5 'primer 5'-TCCAACCCTGGGCCCATGCTCCTGTTGCTCATACCAGTG-3' (SEQ ID ID: 54) and 3 'primer 5'-GTTGATTGTCGACGCCCTCAACTGGACCACAGCCT-3' (SEQ ID ID ID: 55).
  • 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 were performed: 98 ° C for 10 seconds, 65 ° C for 10 seconds, and 72 ° C for 3 minutes.
  • the obtained TCR fragment was cloned into the MCS region downstream of the EF1 ⁇ promoter of the pCDH-EF1 ⁇ -MCS vector.
  • the recombinant TCR lentiviral expression vectors constructed were prepared according to the aforementioned method to obtain respective recombinant TCR lentiviral particles.
  • Example 3 Normal peripheral blood T cells transfected with Her2TCR-6A5-mC recombinant lentivirus expressing specific TCR that recognizes Her2 / neu 369-377 polypeptide
  • 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:
  • FIG. 2A shows that lymphocytes can bind to the Her2-E75 tetramer in the two peripheral blood mononuclear cells (# 1 PBMC and # 2 PBMC respectively), indicating that Her2 TCR-6A5-mC expressed by these cells can Specific recognition of Her2 / neu antigen polypeptide presented by HLA-A2.
  • the results also showed that in Her2-E75 tetramer-positive cells (ie, expressing Her2 TCR-6A5-mC), the positive rate of CD8 + T killer cells was similar to that of CD8 - lymphocytes.
  • 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.
  • Example 4 Her2 / neu 369-377 polypeptide-specific TCR expressed by normal peripheral blood T cells transfected with Her2 TCR-6A5-mC recombinant lentivirus can recognize HLA-A2 + Her2 / neu + tumor cells
  • 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.
  • 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 that 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.
  • FIG. 3C-K shows that T cells expressing Her2 TCR-6A5-mC TCR can specifically recognize and kill HLA-A2 + Her2 / neu + tumor cell line MCF-7 compared to control T cells not transfected with TCR , HCT116, PANC-1 and HEPG-2.
  • the lethality was dose-dependent with the number of Her2 TCR-6A5-mCT cells.
  • -6A5-mC T cell specific killing The results also showed that when Her2 TCR-6A5-mC T cells were increased to a certain number, HLA-A2 + Her2 / neu + tumor cells showed significant specific recognition and killing functions, and the effective target ratio was less than 10: At 1, the specific killing function is not obvious, and may be related to the number of Her2 / neu epitope polypeptides presented by HLA-A2 on the surface of tumor cells.
  • one strategy is to increase the number of tumor target cells expressing HLA-A2 and Her2 / neu.
  • Example 5 Transfection of gene vectors expressing HLA-A2 and Her2 / neu epitope polypeptides allows target cells to express exogenous HLA-A2 and Her2 / neu epitope polypeptides
  • tumor cells can be expressed by the exogenous Her2 / neu antigen to increase the Her2 / neu epitope presented by HLA-A2. achieve. Because tumor cells often have low or absent expression of endogenous HLA class I molecules, transfection vectors can simultaneously express exogenous HLA-A2 genes to increase the expression of HLA class I molecules. In addition, tumor cells often suffer from functional defects in the HLA class I antigen presentation pathway. As a result, tumor antigen proteins cannot be effectively degraded into epitope polypeptides and presented to the cell surface by HLA class I molecules.
  • the epitope polypeptide is directly introduced into the endoplasmic reticulum via a signal peptide, the degradation of the protease in the cytoplasm and the transport function of the TAP molecule can be eliminated, and the epitope polypeptide can directly form the HLA molecule and ⁇ 2 microglobulin in the endoplasmic reticulum. Complex and presented to the surface of tumor cells. Minigenes expressing epitope polypeptides can be linked through the enzyme-cut fragments of Furin enzyme to form a series of minigenes of multiple epitope polypeptides. After entering the endoplasmic reticulum, more genes are released by the furin enzyme. Epitope polypeptide.
  • Adding an endoplasmic reticulum retention signal KDEL fragment to the end of the tandem epitope polypeptide chain can prevent the epitope polypeptide chain from being transferred to downstream secretory organelles, thereby increasing the chance of forming an HLA / polypeptide complex.
  • Three lentiviral vector plasmids were prepared according to the method of Preparation Example 1: "pCDH-EF1p-A2-PKGp-E75 ⁇ 1" (carrying the HLA-A2 coding sequence and a Her2-E75 epitope polypeptide coding sequence), " pCDH-EF1p-A2-PKGp-E75 ⁇ 4 ”(carrying the HLA-A2 coding sequence and 4 repeats of the Her2-E75 epitope polypeptide coding sequence) and“ pCDH-EF1p-A2-PKGp-E75 ⁇ 8 ”(carrying HLA-A2 coding sequence and 8 repeated Her2-E75 epitope polypeptide coding sequences), collectively referred to as "pCDH-EF1p-A2-PKGp-E75 vector”.
  • FIG. 4B is a schematic diagram showing the overall structure of a lentiviral vector containing the HLA-A2 and Her2 / neu epitope polypeptide (Her2-E75) minigene (pCDH-EF1p-A2-PKGp-E75 vector).
  • HLA-A2 is driven by the EF-1 ⁇ promoter
  • the epitope polypeptide (shown as Her2-E75 in the figure) minigene is driven by the PKG promoter.
  • FIG. 4B is a schematic diagram showing the composition of the Her2 / neu epitope polypeptide minigene in “pCDH-EF1p-A2-PKGp-E75 ⁇ 8”, which shows eight Her2-E75s linked by the fragment cut by Furin enzyme Epitope polypeptide (Her2-E75 ⁇ 8 minigene).
  • 293T cells were transfected with the three lentiviral plasmid vectors constructed above, and used as target cells to detect the recognition function of Her2TCR-6A5T cells.
  • 293T cells are human kidney epithelial cell lines and are HLA-A2 negative Her2 / neu negative.
  • the number of epitope polypeptide minigenes on plasmid pCDH-EF1p-A2-PKGp-E75 is 1, 4, and 8 (shown as 293-A2-PKG-E75 ⁇ 1, 293-A2- PKG-E75 ⁇ 4, 293-A2-PKG-E75 ⁇ 8).
  • 293T cells without transfection plasmid were used as the negative control group (shown as the 293 control), and T2 cells presented Her2 / neu 369-377 antigen polypeptide 0.1 ⁇ g / ml as the positive control group (shown as T2 + Her2 in the figure) -E75).
  • FIG. 4A shows that Her2, TCR-6A5-mC, T cells can specifically recognize 293T cells transfected with plasmid vectors expressing HLA-A2 and Her2 / neu epitope polypeptides and secrete IFN- ⁇ , indicating that HLA-A2 and Her2 / neu The 369-377 epitope polypeptides were all expressed by 293T, and presented to the cell surface in the manner of HLA / polypeptide complex and recognized by Her2TCR-6A5-mCTCR.
  • the T cell's recognition activity will also increase, indicating that the number of HLA-A2 / antigen epitope polypeptide complexes presented on the cell surface can follow the Her2 / neu epitope Polypeptide expression increases.
  • the HLA-A2 gene and Her2 / neu epitope polypeptide minigenes were loaded into an adenovirus type 5 vector E1 area.
  • the replication-deficient adenovirus Adeasy-A2-Her2 E75 (abbreviated as "Adeasy-A2E75") was prepared according to the method described in Preparation Example 2, which is a replication that expresses HLA-A2 and eight Her2 / neu epitope polypeptides
  • the vector diagram of the defective adenovirus is shown in Figure 4D (Adeasy-A2E75 vector).
  • This adenoviral vector is derived from an adenovirus type 5 with deletion of the sequences of the E1 and E3 regions (see the document "Nature Protocols 2007; 2: 1236-1247”).
  • the expression of HLA-A2 gene and Her2 / neu epitope polypeptide minigene is driven by exogenous CMV promoter.
  • the HLA-A2 gene and Her2 / neu epitope polypeptide minigene are divided by the cleavable fragment Furin enzyme recognition fragment and F2A fragment, so that on the one hand, it can ensure that the expression levels of HLA-A2 and Her2 / neu epitope polypeptides are similar.
  • the length of the nucleic acid sequence of the source gene can also be kept within the size range of the exogenous gene allowed by the adenovirus genome.
  • the end of the tandem epitope polypeptide chain is added with an endoplasmic reticulum retention signal KDEL fragment to increase the chance of forming an HLA / polypeptide complex. Since E3-19K protein inhibits the antigen presentation function of HLA, adenoviruses lacking the sequence of E3 region will not interfere with the presentation of the expressed foreign epitope polypeptide.
  • the ovarian cancer cell SKOV3 was infected with Easy-A2-Her2 E75 adenovirus particles.
  • SKOV3 cells are HLA-A2 negative.
  • the expression of HLA-A2 on the surface of SKOV3 cells can be used to determine whether the Adeasy-A2-Her2 E75 adenovirus vector can express foreign genes.
  • the SKOV3 cell line was cultured in a 10 cm petri dish, digested with trypsin when the confluence was around 80%, and washed with the culture medium, centrifuged again, resuspended with the medium McCony5A (Gibco # 16600-082), and plated to In a 24-well plate, 1 ⁇ 10e5 per well and 500 ⁇ l of culture medium per well; After 24 hours, add the Easy-A2-Her2 E75 adenovirus, add 0MOI, 5MOI, 10MOI, 20MOI to the same volume of virus preparation, and continue to culture for 24h After discarding the supernatant and digesting with trypsin, the cells were harvested, resuspended in 100 ⁇ l PBS into 1.5 ml EP tubes, and 2 ⁇ l APC-anti-human HLA-A2 antibody (BD # 561341) was added to each tube.
  • BD # 561341 APC-anti-human HLA-A2 antibody
  • FIG. 4C shows that SKOV3 cells can express HLA-A2 within 24 hours after being infected with the Easy-A2-Her2 E75 adenovirus. The amount of HLA-A2 expression is dose-dependent with the titer of the infected virus, while the infection does not express foreign genes.
  • the SKOV3 cells of the adenovirus in the control group do not express HLA-A2, indicating that adenoviruses carrying the HLA-A2 gene and Her2 / neu epitope polypeptide minigene can effectively express exogenous HLA-A2 molecules.
  • Example 6 Infecting tumor cells with replication-deficient adenovirus expressing HLA-A2 and Her2 / neu epitope polypeptide minigenes can significantly enhance the recognition and killing sensitivity of Her2 TCR-6A5-mC T cells to tumor cells.
  • Figure 5A shows that infection of tumor cells with the Easy-A2-Her2 E75 adenovirus can significantly stimulate Her2 TCR-6A5-mC T cells to specifically secrete IFN- ⁇ .
  • the adenovirus infection multiple MOI of tumor cells was 10, and the effective target ratio of co-culture with PBMC cells transfected with Her2 TCR-6A5-mC TCR was 5: 1.
  • the tumor cell lines were HLA-A2 + Her2 / neu + MCF-7 cells, HLA-A2 - Her2 / neu + SKOV3 cells, and HLA-A2 - Her2 / neu - NCI-H446 cells.
  • tumor cells alone did not secrete interferon gamma after being infected with the Adeasy-A2-Her2 E75 adenovirus alone (the experimental group "Adeasy-A2E75”).
  • PBMC cells transfected with Her2 TCR-6A5-mC TCR could not effectively recognize HLA-A2 + Her2 / neu + MCF-7 cells and secrete interferon ⁇ under the condition of low target ratio (the experimental group "has Her2TCR-6A5 PBMC ").
  • PBMC cells For SKOV3 cells, PBMC cells ("PBMC with Her2TCR-6A5") transfected with Her2 TCR-6A5-mC TCR were also unable to specifically secrete interferon ⁇ compared to the control PBMC ("PBMC control" in the experimental group).
  • PBMC control the control PBMC
  • Her2 TCR-6A5-mC T cells could significantly enhance their recognition function, regardless of whether the tumor cells expressed endogenous HLA-A2 and Her2 / neu (experimental group) "PBMC + Adeasy-A2E75 with Her2TCR-6A5").
  • the MOI of the virus infection of the tumor cells was 10, and the effective target ratio of the mixed culture was 8: 1.
  • FIG. 5B shows that, after the Adeasy-A2-Her2 E75 adenovirus alone infected tumor cells, it can cause most NCI-H446 cells to die (the target cell is the experimental group NCI-H446 Adeasy-A2E75). This may be because E1 / E3 deleted replication-deficient adenovirus can sometimes cause apoptosis in certain cells, especially when the infection titer is high (see the document "Gene Ther. 1999 Jun; 6 (6): 1054-63 ").
  • the cell killing rate of the replication-deficient adenovirus expressing HLA-A2 and Her2 and E75 polypeptide alone is about 32.8%, and Her2 expressing TCR alone
  • the cell killing rate of peripheral blood mononuclear cells of -6A5-mC TCR is about 30.9%. Compared with the cell killing rate of a single drug, the cell killing rate of both is increased, and the cell killing rate is about 95.8%. , Showing a significant synergy effect.
  • the cell killing rate of replication-defective adenovirus expressing HLA-A2 and Her2 and E75 polypeptides alone was about 29.7%, and peripheral blood mononuclear cells expressing Her2 and TCR-6A5-mC and TCR alone.
  • the cell killing rate is about 34.3%.
  • the cell killing rate of the two drugs applied at the same time is increased, and the cell killing rate is about 66%, which also shows a significant synergistic effect.
  • T cells can specifically kill tumor cells infected with the Easy-A2-Her2 E75 adenovirus, regardless of whether the tumor cells express endogenous HLA-A2 and Her2 / neu antigens. If adenoviruses expressing HLA class I molecules and tumor antigen epitope polypeptide minigenes are oncolytic adenoviruses, tumor cells infected with oncolytic viruses can not only be selectively killed by oncolytic viruses, but also for tumors that cannot be effectively lysed by oncolytic viruses. Cells can also be recognized and killed by T cells transfected with TCR that recognizes specific tumor antigen epitopes by expressing specific foreign tumor antigen epitope polypeptides. This is the Easy-A2-Her2 E75 gland according to the present invention. Theoretical and experimental basis for the combined application of virus and Her2 TCR-6A5-mC T cells to enhance tumor killing function.
  • Example 7 Infecting tumor cells with an oncolytic adenovirus expressing a Her2 / neu epitope polypeptide mini-gene can significantly enhance the recognition and killing sensitivity of Her2 TCR-6A5-mC T cells to tumor cells.
  • Ad-E75 In order to further verify whether the oncolytic adenovirus Ad-E75 expressing the Her2 / neu epitope polypeptide minigene alone infected tumor cells, increased the cell surface HLA-A2 / Her2 antigen that can be recognized by Her2 TCR-6A5-mC T cells The number of epitope peptide complexes, thereby enhancing the T cell's recognition and killing sensitivity to tumor cells.
  • KDEL Her2 / neu epitope polypeptide minigene
  • Oncoviruses OAd-E75, also known as "Ad-E75").
  • a Herv-neu epitope polypeptide minigene driven by a CMV promoter and an adenovirus E1A gene driven by the EF-1 ⁇ promoter were simultaneously inserted into the E1 region of the replication-deficient adenovirus.
  • Continuously expressed E1A protein can cause E1B-deficient adenovirus to selectively replicate and proliferate in certain tumor cells, resulting in oncolytic effect.
  • the tumor cell lines MCF-7, SKOV3 and NCI-H446 were infected with Ad-E75 oncolytic adenovirus respectively, and then co-cultured with PBMC cells transfected with Her2 TCR-6A5-mC TCR for 24 hours to detect Her2 TCR-6A5-mC T Cells specifically secrete IFN- ⁇ and kill target cells.
  • Figure 6A shows that HLA-A2-positive tumor cells MCF-7 infected with Ad-E75 oncolytic adenovirus can significantly stimulate Her2 TCR-6A5-mC T cells to specifically secrete IFN- ⁇ , while HLA-A2-negative cells SKOV3 and NCI- H446 infection could not increase the sensitivity of Her2 TCR-6A5-mC T cell specific recognition after being infected with Ad-E75 virus.
  • the adenovirus infection multiple MOI of tumor cells was 10, and the effective target ratio of co-culture with PBMC cells transfected with Her2 TCR-6A5-mC TCR was 5: 1.
  • the tumor cell lines were HLA-A2 + Her2 / neu + MCF-7 cells, HLA-A2 - Her2 / neu + SKOV3 cells, and HLA-A2 - Her2 / neu - NCI-H446 cells.
  • the results showed that tumor cells alone did not secrete interferon gamma after being infected with Ad-E75 adenovirus alone ("Ad-E75" in the experimental group).
  • Ad-E75 PBMC cells transfected with Her2 TCR-6A5-mC TCR cannot effectively recognize MCF-7 and secrete interferon gamma ("PBMC with Her2TCR-6A5") even with a low efficiency target ratio.
  • Her2 TCR-6A5-mC T cells could significantly enhance their recognition function (experimental group "PBMC + Ad-E75 with Her2TCR-6A5")
  • Ad-E75 oncolytic adenovirus-infected tumor cells cannot increase the tumor cell recognition activity of control PBMC cells without transfection of Her2 TCR-6A5-mC TCR ("PBMC control" and "PBMC control + Ad-E75" in the experimental group) It shows that the increase in secretion of interferon ⁇ after adenovirus infection is produced by enhancing the recognition activity of Her2 TCR-6A5-mC TCR on tumor cells.
  • the results show that after Ad-E75 oncolytic adenovirus infects tumor cells, HLA-A2 positive tumor cell surface can be increased by HLA-A2 by expressing Her2 / neu epitope polypeptide minigene.
  • Her2 / neu 369-377 peptide complex number thereby enhancing the recognition sensitivity of Her2 TCR-6A5-mC T cells.
  • HLA-A2 negative cells SKOV3 and NCI-H446 infected with Ad-E75 oncolytic adenovirus did not enhance the cell's recognition sensitivity to Her2 TCR-6A5-mC T cells, indicating that Her2 / neu epitope polypeptide requires endogenous HLA-
  • the presentation of A2 molecules further showed that Her2 TCR-6A5-mC T cells recognized Her2 / neu epitopes as HLA-A2 restricted specific recognition.
  • tumor cell lines SKOV3, MCF-7 and NCI-H446 were infected with Ad-E75 Tumor adenovirus was mixed with PBMC cells transfected with Her2, TCR-6A5-mC and TCR 24 hours later and cultured for another 24 hours. Trypan blue was used to detect the number of surviving adherent cells to determine the killing function of T cells on target cells.
  • the MOI of the virus infection of the tumor cells was 10, and the effective target ratio of the mixed culture was 5: 1.
  • FIG. 6B shows that Ad-E75 oncolytic adenovirus alone can cause the death of most SKOV3 and NCI-H446 cells after infecting tumor cells (SKOV3, U87MG and NCI-H446's experimental group "Ad-E75"). This shows that Ad-E75 has obvious oncolytic effect on these tumor cells.
  • the target cell was MCF-7 in this experiment, the cell killing rate of Ad-E75 oncolytic adenovirus alone was approximately 20.9%, and the cell killing rate of peripheral blood mononuclear cells expressing Her2 TCR-6A5-mC TCR alone.
  • Her2, TCR-6A5-mC, and T cells can specifically recognize the Her2 / neu epitope expressed by oncolytic adenovirus presented by endogenous HLA-A2 and kill the infection of Ad-E75 oncolytic glands. Viral tumor cells. And these results show that after the oncolytic adenovirus carrying the Her2 / neu epitope polypeptide minigene infects tumor cells, it can express the Her2 / neu epitope polypeptide and be presented by endogenous HLA-A2 molecules, thereby enhancing T cell pairing. Recognition and killing sensitivity of tumor cells. It also shows that Her2 TCR-6A5-mC T cells have a synergistic effect on the killing function of target cells and the oncolytic effect of oncolytic virus.
  • Example 8 Infecting tumor cells with an oncolytic adenovirus that simultaneously expresses the Her2 / neu epitope polypeptide minigene and exogenous HLA-A2, can not only significantly enhance the recognition and killing of tumor cells by Her2 TCR-6A5-mC T cells Sensitivity can also identify HLA-A2-negative tumor cell lines.
  • Ad-E75A2 which expresses both Her2 / neu epitope polypeptide minigene and HLA-A2 infected tumor cells, whether the cell surface can be increased by HLA recognized by Her2 TCR-6A5-mC T cells -A2 / Her2 epitope peptide complex number, thereby enhancing the T cell's recognition and killing sensitivity to tumor cells.
  • Example 6 It has been shown in Example 6 that replication-defective adenovirus (Adeasy-A2E75) that inserts the HLA-A2 gene linked by a Furin-F2A linking fragment upstream of the Her2 / neu epitope polypeptide minigene can express HLA- A2 and Her2 / neu epitopes, and significantly enhance the recognition activity of Her2 TCR-6A5-mC T cells to target cells.
  • the oncolytic adenovirus constructed in this example is derived from the oncolytic adenovirus described in Example 7, except that the HLA-A2 gene linked by the F2A linking fragment is inserted downstream of the Her2 / neu epitope polypeptide minigene.
  • oncolytic adenovirus Ad-E75A2 Construction of oncolytic adenovirus Ad-E75A2.
  • the tumor cell line HLA-A2-positive Her2-negative U87MG, HLA-A2-negative Her2-positive NCI-H460 and HT-29 cells were infected with Adeasy-A2E75 replication-deficient adenovirus and Ad-E75A2 oncolytic adenovirus, respectively, 24 hours later It was mixed with PBMC cells transfected with Her2 TCR-6A5-mC and TCR and co-cultured for another 24 hours.
  • Her2 TCR-6A5-mC T cells were specifically tested for their ability to secrete IFN- ⁇ and kill target cells.
  • Figure 7A shows that tumor cell U87MG, NCI-H460, and HT-29 cells infected with replication-defective adenovirus Adeasy-A2E75 can significantly stimulate Her2 TCR-6A5-mC T cells to specifically secrete IFN- ⁇ , regardless of whether the target cells express Source HLA-A2 or Her2 antigen. This is consistent with the recognition activity against other target cells shown in FIG. 5A in Example 6.
  • Adenovirus infection with tumor cells infected with a MOI of 10 and co-culture with Her2 TCR-6A5-mC TCR PBMC cells was 10: 1.
  • the tumor cell lines were HLA-A2 + Her2 / neu - U87MG cells, HLA-A2 - Her2 / neu + NCI-H460 and HT-29 cells.
  • the results showed that tumor cells alone did not secrete interferon gamma after being infected with the Adeasy-A2E75 replication-deficient adenovirus alone (the experimental group "Adeasy-A2E75").
  • PBMC cells transfected with Her2 TCR-6A5-mC TCR were also unable to effectively recognize target cells and secrete interferon gamma (experimental group "PBMC with Her2TCR-6A5").
  • Her2 TCR-6A5-mC T cells could significantly enhance their recognition function (experimental group "PBMC + Adeasy-A2E75 with Her2TCR-6A5").
  • Figure 8A shows that after tumor cells U87MG, NCI-H460, and HT-29 cells were infected with oncolytic adenovirus Ad-E75A2, although Her2 TCR-6A5-mC T cells recognized NCI-H460, the activity of secreting IFN- ⁇ was not significantly enhanced.
  • the oncolytic adenovirus carrying HLA-A2 gene and Her2 / neu epitope polypeptide minigene can express exogenous HLA-A2 and Her2 / neu epitopes after infecting tumor cells, thus making Her2 TCR-6A5-mC T cells Can recognize HLA-A2 negative or Her2 negative target cells.
  • tumor cell lines U87MG, NCI-H460, and HT-29 cells were infected with replication defects, respectively.
  • Adenovirus type Adeasy-A2E75 and oncolytic adenovirus Ad-E75A2 were mixed with PBMC cells transfected with Her2 TCR-6A5-mC TCR 24 hours later and cultured for another 24 hours. Trypan blue was used to detect the killing function of T cells on target cells.
  • the MOI of virus infection of tumor cells was 10, and the effective target ratio of mixed culture was 10: 1.
  • FIGs 7B and 8B show that under the MOI conditions used, infection with replication-defective adenovirus Adeasy-A2E75 and oncolytic adenovirus Ad-E75A2 alone showed a certain oncolytic effect ("Adeasy- A2E75 "and" Ad-E75A2 "in each experimental group in Figure 8B).
  • Adeasy- A2E75 and oncolytic adenovirus Ad-E75A2 alone showed a certain oncolytic effect
  • Adeasy- A2E75 and oncolytic adenovirus
  • T cells can significantly enhance the killing function of target cells.
  • the killing function of target cells infected with replication-defective adenovirus Adeasy-A2E75 was significantly enhanced.
  • Infection with oncolytic adenovirus Ad-E75A2 can also enhance the killing function of Her2 TCR-6A5-mC T cells to target cells.
  • the target cell is U87MG in the experiment shown in FIG. 7B
  • the cell killing rate of replication-defective adenovirus alone is about 9.8%
  • the cells of peripheral blood mononuclear cells expressing Her2TCR-6A5-mC TCR alone The killing rate is about 8.8%.
  • the killing rate of the cells applied simultaneously is increased, and the killing rate is about 83.6%, showing a significant synergistic effect.
  • FIG. 7B the cell killing rate of replication-defective adenovirus alone is about 9.8%, and the cells of peripheral blood mononuclear cells expressing Her2TCR-6A5-mC TCR alone The killing rate is about 8.8%.
  • the killing rate of the cells applied simultaneously is increased, and the killing rate is about 83.6%, showing a significant synergistic effect.
  • the cell killing rate of replication-defective adenovirus alone is about 5.4%
  • the cell killing rate of peripheral blood mononuclear cells expressing Her2 TCR-6A5-mC TCR alone is about 7%.
  • the cell killing rate of the two drugs applied at the same time is increased, and the cell killing rate is about 33.4%, which also shows a significant synergistic effect.
  • the cell killing rate of replication-defective adenovirus alone is about 8.4%
  • the cell killing rate of peripheral blood mononuclear cells expressing Her2TCR-6A5-mC TCR alone is about 12.9%.
  • the cell killing rate of the two drugs applied at the same time is increased, and the cell killing rate is about 74.2%, which also shows a significant synergistic effect.
  • Her2 TCR-6A5-mC T cells showed significant killing activity on U87MG and HT-29 cells infected with oncolytic adenovirus.
  • the target cell is U87MG in the experiment shown in FIG. 8B
  • the cell killing rate of oncolytic adenovirus alone is about 10.6%
  • the cell killing of peripheral blood mononuclear cells expressing Her2 TCR-6A5-mC TCR alone The rate is about 8.7%.
  • the cell killing rate of the two drugs applied at the same time is increased, and the cell killing rate is about 38.4%, showing a significant synergistic effect.
  • the cell killing rate of oncolytic adenovirus alone is about 6.5%
  • the cell killing rate of peripheral blood mononuclear cells expressing Her2 TCR-6A5-mC TCR alone is about 9.8%
  • the cell killing rate of the two drugs applied at the same time is increased, and the cell killing rate is about 44.2%, which also shows a significant synergistic effect.
  • Example 9 The combined inhibitory effect of oncolytic adenovirus OAd-E75A2 and human Her2 TCR-6A5-mC T cells on the growth of human ovarian cancer cell SKOV3 subcutaneously inoculated with NCG severely immunodeficiency mice
  • human ovarian cancer cell SKOV3 was inoculated into the NCG severe immunodeficiency mice (provided by Jiangsu Jicui) under the dorsal skin of the right forelimb to prepare a tumor-bearing animal model that simulates the human environment and used to detect the oncolysis prepared according to Preparation Example 3.
  • NCG severe immunodeficiency mice provided by Jiangsu Jicui
  • Adenovirus OAd-E75A2 and human Her2 TCR-6A5-mC T cells obtained by the method described in the section above "Recombinant TCR Lentivirus Transfecting Human T Cells" PB005F, specification 100 million, frozen)) combined inhibitory effect on the growth of human ovarian cancer cell SKOV3 subcutaneously inoculated with NCG in severely immunodeficiency mice.
  • the cell inoculation amount of each animal was 3 ⁇ 10 6 cells. About 9 days after the cell inoculation, 30 tumor-bearing mice with a subcutaneous tumor volume that satisfies the requirements (tumor volume of 90-120 mm 3 ) were selected and divided into random groups. There are 6 groups of 5 animals in each group. The first group was a blank control group. Each mouse in the group was injected intratumorally (IT for short) with 100 ⁇ l adenovirus preservation solution (containing 10 mM Tris (pH 7.4), 1 mM) on days 0, 4, and 8.
  • IT intratumorally
  • the second group was OAd-E75A2 Group (i.e., oncolytic virus was administered alone), each mouse in the group was injected intratumorally with 100 ⁇ l oncolytic adenovirus OAd-E75A2 on day 0, 4 and 8 respectively, and the amount of virus injected per animal was 5 ⁇ 10 8 PFU, intravenous injection of 100 ⁇ l physiological saline on the second, sixth and tenth day;
  • the third group is Her2 TCR-6A5-mC T (IV) group (that is, TCR T intravenously administered alone)
  • Each mouse in the group was injected intratumorally with 100 ⁇ l adenovirus preservation solution on day 0, 4 and 8 and 100 ⁇ l Her2 TCR-6A5 was injected intravenously on tail on day 2, 6 and 10 -mC
  • mice were injected intratumorally at day 0, 4 and 8 l oncolytic adenovirus OAd-E75A2, the amount of virus injected per animal was 5 ⁇ 10 8 PFU, on day 2, 6 and day 10 tail intravenous injection of 100 ⁇ l Her2 TCR-6A5-mC T cells, The number is 2 ⁇ 10 7 ; the fifth group is Her2 TCR-6A5-mC T (IT) group (that is, TCR T is administered alone in the tumor), and each mouse in the group is on day 0, day 4 and On the 8th day, 100 ⁇ l of adenovirus preservation solution was injected intratumorally, and on the 2nd, 6th, and 10th days, 100 ⁇ l of Her2 TCR-6A5-mC T cells were injected intratumorally, and the number of cells was 2 ⁇ 10 7 cells; the sixth group was OAd-E75A2 + Her2 TCR-6A5-mC T (IT) group (that is, combined administration, of which TCR T was administered
  • the cells were as described in the above section "Recombinant TCR lentivirus transfected human T cells"
  • the preparation method is freshly prepared, wherein the culture time is from Her2 TCR-6A5-mC recombinant lentivirus transfection to PBMC, and the cells are first cultured for about 8 days according to the expected different administration time.
  • the cells are Her2 TCR-6A5-mC TCR.
  • the positive rate was about 35-38%.
  • each group of animals was injected with 100,000 IU IL2 subcutaneously in the neck (SC for short) on the 2nd, 3rd, 6th, 7th, 10th and 11th days, respectively.
  • Huan Pharmaceutical Factory product batch number 817660010002383594693
  • the animal experimental protocol is shown in Figure 11. From the day of grouping (day 0), the subcutaneous tumor volume of each mouse and the weight of the mice were measured, and measured and recorded twice a week.
  • the average tumor volume change of each group of animals in the experimental results is shown in Fig. 12, the tumor volume change of each animal in each group is shown in Fig.
  • the average weight of the fourth group of animals increased significantly.
  • This key indicator shows that the intratumoral injection of oncolytic adenovirus and the infusion of human TCR T cells by the tail vein are more favorable for tumor treatment than other modes of administration.
  • the effect on the overall condition of the animal's body is small; it can be seen from FIG. 16 that at the end of the experiment, in the two groups of animals injected with Her2 TCR-6A5-mC T cells in the tail vein, the tumors of the fourth group Compared with the third group, more human T cells (CD3 + , CD8 +, and CD4 + ) still survived. This may be due to the intratumoral injection of oncolytic adenovirus OAd-E75A2, which caused more human Her2 TCR-6A5.
  • the fourth group also showed a significant inhibitory effect on tumor growth.
  • a large number of human T cells CD3 + , CD8 + and CD4 +
  • CD8 + T cells was significantly less than the fifth group of CD8 + T cells in the injection of Her2 TCR-6A5-mC T tumors in tumor-bearing animals in the individual animal tumor coadministration of six groups, the fourth group of animals with human tumors
  • the number of T cells is similar, and more importantly, the growth inhibition effect of subcutaneous tumors in the sixth group of animals is more obvious than that in the fourth group.
  • TCR-T therapy adoptive transfer of tumor-specific TCR gene-modified T cells is the most promising immune cell therapy (TCR-T therapy) for the treatment of malignant solid tumors. If the tumor antigen targeted by TCR-T is a tumor-associated antigen derived from its own protein, the specific TCR affinity of TCR-T may not be sufficient to recognize the trace HLA / epitope polypeptide complex presented by tumor cells And effective killing of tumor cells.
  • the tumor microenvironment can not only cause the immunosuppressive state in tumor tissues, but also cause the expression of HLA class I molecules in tumor cells to be reduced or absent, and the class I antigen presentation mechanism in tumor cells may also be defective, thereby making the tumor antigen table This site cannot be effectively presented by MHC class I molecules, which also limits TCR-T's ability to recognize and kill tumor cells.
  • Oncolytic virus can not only selectively replicate and lyse tumor cells in tumor cells, but also can alleviate the local immunosuppressive state of tumors through its autoimmunity. Oncolytic viruses can also be used as gene vectors to selectively express foreign genes in tumor cells.
  • HLA class I molecules and tumor epitope polypeptides are selectively expressed in tumor cells by oncolytic adenoviruses, the number of HLA class I molecules and epitope polypeptide complexes on the surface of tumor cells can be increased, thereby enhancing TCR-T Sensitivity to identify and kill tumor cells.
  • the combined use of TCR-T and oncolytic viruses expressing HLA class I molecules and epitope polypeptides not only shows the synergistic effect of the two in the process of specifically killing tumor cells, but also can expand the scope of application of TCR-T.
  • TCR-T expressing Her2 and TCR according to the present invention when used alone, it is restricted to HLA-A2 positive Her2 / neu positive tumor patients because of HLA-A2 restriction.
  • the oncolytic adenovirus according to the present invention by selectively infecting tumor cells and expressing exogenous HLA-A2 molecules and Her2 / neu epitope polypeptides, HLA-A2-negative tumors are low-expressed or not expressed.
  • the tumor cells of Her2 / neu antigen can be the target cells of TCR-T based on Her2TCR, which can avoid the limitation of HLA limitation faced by adoptive TCR-T cell therapy, thereby greatly increasing the application range of TCR-T.
  • the technology and method provided by the present invention provide a new approach for treating tumors by adoptively adopting an oncolytic virus that adoptively transfers T cells modified with specific TCR and expresses HLA class I molecules and tumor epitope polypeptides.

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Abstract

一种包含核酸及TCR修饰的免疫细胞的治疗剂及其应用。所述治疗剂包含含有第一活性成分的第一组合物和含有第二活性成分的第二组合物；该第一活性成分包括或含有用于导入肿瘤细胞和/或癌细胞的、具有标记性多肽编码序列的核酸；所述标记性多肽具有一个或多个抗原表位多肽的氨基酸序列，所述抗原表位多肽能够被MHC I类分子提呈在所述肿瘤细胞和/或癌细胞表面；该第二组合物包含位于第二可药用载体中的第二活性成分，该第二活性成分包含T细胞受体修饰的免疫细胞；所述的T细胞受体修饰的免疫细胞能够特异性识别并结合被所述MHC I类分子提呈的所述抗原表位多肽。具有协同治疗效果，为肿瘤治疗提供了一个新的途径。

Description

包含核酸及TCR修饰的免疫细胞的治疗剂及其应用 技术领域
本发明属于生物技术领域，具体而言，涉及包含核酸及TCR修饰的免疫细胞的治疗剂、标记性多肽、编码核酸、表达载体、溶瘤病毒、药盒及其应用。
背景技术
随着针对CTLA-4和PD-1/PD-L1等免疫检查点抑制剂在临床试验中表现出显著的抗肿瘤疗效(参见文献“Cancer Cell 27，450-461(2015)”)，以及以CAR-T(嵌合抗原受体-T细胞)为代表的过继T细胞疗法对B细胞型血液肿瘤所表现出的长期有效的治疗效果(参见文献“N Engl J Med 2017；377：2545-2554”)，肿瘤免疫治疗已成为最具发展前景的领域之一，可望把恶性肿瘤转化成为可控制的慢性疾病，甚至可以治愈某些晚期癌症。肿瘤免疫治疗主要是通过激发内源的抗肿瘤T细胞功能，或过继转输体外培养的肿瘤特异性T细胞，以达到杀伤和清除体内癌细胞的疗效。早期的肿瘤疫苗是靶向来源于自身蛋白的肿瘤相关抗原(TAA)，试图在体内诱导出特异性的抗肿瘤免疫反应，但大部分相关的临床试验均未成功(参见文献“Nat Rev Clin Oncol.2014 Nov；11(11)：630-2.”)。一个主要原因是针对这些肿瘤相关抗原的高亲和性T细胞在发育和分化过程中多数被胸腺的中枢耐受机制所清除，肿瘤疫苗即使在很强的佐剂帮助下，例如用成熟的树突状细胞提呈抗原，在体内所诱导出的特异性T细胞大多为不能识别肿瘤细胞的低亲和性T细胞(参见文献“J Immunol.2008 Feb 01；180(3)：1526-34”)。为了克服这个障碍，靶向来源于基因突变的肿瘤新生抗原(neoantigen)个体化疫苗的开发是一个发展趋势，并在临床试验中看到初步疗效，但需要更多的临床试验进一步验证这类肿瘤疫苗的疗效(参见文献“Nat Rev Drug Discov.2018 May  30；17(6)：393”；“Front Immunol.2017；8：1848”)。肿瘤免疫治疗的另外一个障碍是肿瘤在发生和发展过程中产生的免疫抑制微环境，肿瘤微环境(TME)对特异性T细胞的分化和增殖以及抗肿瘤功能产生较强的抑制作用(参见文献“Curr Opin Immunol.2016 Apr；39：1-6”)。
针对这些问题，一个有效的治疗策略是过继转输在体外获得的、可以有效识别肿瘤抗原的T细胞。这些T细胞来源于肿瘤组织的浸润T淋巴细胞(TILs)，也可以是经抗原特异性T细胞受体(TCR)基因修饰后，能有效识别肿瘤抗原的外周血T细胞(TCR-T)。体外培养避免了肿瘤组织的抑制微环境并提供优化的培养条件，可以获得足够数量的抗肿瘤T细胞用于过继转输治疗(参见文献“Adv Immunol.2016；130：279-94”)。过继T细胞治疗包括基于嵌合抗原受体(chimeric antigen receptor)的CAR-T疗法以及基于T细胞受体的TCR-T疗法。CAR-T主要靶向细胞表面表达的肿瘤抗原，尽管所识别的肿瘤抗原数量有限，靶向CD19的CAR-T针对血液肿瘤包括B细胞型白血病和淋巴瘤表现出了显著的临床疗效，并有产品获得FDA批准上市(参见文献“N Engl J Med.2017 Oct 05；377(14)：1313-1315”)。对于实体瘤的治疗，CAR-T尚未看到明确的临床疗效。TCR-T主要靶向被人类主要组织相容性抗原HLA分子所提呈的多肽抗原，表位多肽可以来源于细胞内蛋白以及细胞膜表面蛋白，靶抗原种类及数量大大多于CAR-T所识别的肿瘤抗原(参见文献“Int Immunol.2016 07；28(7)：349-53”)。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”)。靶向NY-ESO-1抗原的TCR-T治疗实体肿瘤也在临床二期试验中看到明确的疗效(参见文献“Front Immunol.2018；9：947”)。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”)，使得Her2/neu成为免疫治疗的一个适当靶点。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，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、靶向Her2/neu抗原的CAR-T细胞作为第一个治疗实体瘤的CAR-T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”)。目前临床试验中的TCR-T疗法尚未出现如CAR-T疗法中所表现出的严重的细胞因子风暴毒性，靶向Her2/neu抗原的TCR-T治疗实体瘤可能会避免严重的细胞因子风暴。Her2/neu属于来自自身蛋白的肿瘤相关抗原，识别这类抗原的T细胞大多数被中枢耐受选择机制所清除，因此，从外周血中获得特异性识别肿瘤细胞的天然T细胞十分困难，所诱导的多肽特异性T细胞常常不能有效识别肿瘤细胞表面低水平表达的肿瘤相关抗原(参见文献“Cancer Res.1998；58(21)：4902-8”)。为了获得用于制备TCR-T并能有效识别肿瘤细胞的TCR，一般通过对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细胞库中进行诱导以获得高亲和性的抗原特异性TCR(参见文献“Front Immunol.2013；4：363”)。然而，基于这种策略所获得的高亲和性TCR所制备的TCR-T，在临床试验中产生了针对正常组织细胞的严重脱靶毒性反应(参见文献“Sci Rep.2016 Jan 13；6：18851”)。另外，TCR-T所识别的靶点是细胞表面的HLA分子，β 2-微球蛋白(beta2-Microglobulin)以及抗原多肽所形成的抗原复合物(其中HLA蛋白和β 2-微球蛋白配对形成MHC I类分子)。TCR-T的抗原识别能力不仅受HLA分子的限制，还与肿瘤细胞中HLA分子和肿瘤抗原的表达量，以及HLA抗原多肽提呈途径的完整性密切相关。然而肿瘤在发生发展过程常常发生肿瘤细胞的HLA分子表达量降低，或者胞 浆中的HLA抗原提呈途径中相关分子的功能缺失，从而导致抗原多肽不能被HLA分子有效提呈到肿瘤细胞表面而被T细胞所识别(参见文献“Cancer Gene Ther.2002 Dec；9(12)：1043-55”)。这种HLA I类抗原提呈途径(即MHC I类抗原提呈途径)的缺失是肿瘤免疫逃避的一个机制，与肿瘤细胞中包括HLA、β 2-微球蛋白、TAP、tapasin、LMP、ERAP等抗原提呈分子的基因突变或表达异常有关，也可能与RAS、MYC、MOS和Her2/Neu等肿瘤转化分子的过度功能活化有关(参见文献“Trends in Molecular Medicine，2013，19(11)：677-681”)。另外，肿瘤组织的多样性也表现为肿瘤抗原表达的不均一性，低表达或不表达肿瘤抗原的肿瘤细胞就可能会逃避TCR-T的识别和杀伤，从而影响TCR-T的疗效(参见文献“Trends Immunol.2016 06；37(6)：349-351”)。
因此，TCR-T疗法所面临的一个挑战是如何促使靶向肿瘤相关抗原的TCR-T有效识别和杀伤肿瘤细胞。
发明内容
为解决上述现有技术中所存在的问题，本发明提供了治疗剂、标记性多肽、编码核酸、表达载体、溶瘤病毒、药盒及其应用。
具体而言，本发明提供了：
(1)一种用于治疗肿瘤和/或癌症的治疗剂，包含：
(a)第一组合物，其中该第一组合物包含位于第一可药用载体中的第一活性成分，该第一活性成分包括或含有用于导入肿瘤细胞和/或癌细胞的、具有标记性多肽编码序列的核酸；所述标记性多肽具有一个或多个抗原表位多肽的氨基酸序列，所述抗原表位多肽能够被MHC I类分子提呈在所述肿瘤细胞和/或癌细胞表面；和
(b)第二组合物，其中该第二组合物包含位于第二可药用载体中的第二活性成分，该第二活性成分包含T细胞受体修饰的免疫细胞；所述的T细胞受体修饰的免疫细胞能够特异性识别并结合被所述MHC I类分子提呈的所述抗原表位多肽。
(2)根据(1)所述的治疗剂，其中所述抗原表位多肽的氨基 酸序列来源于自然界存在的蛋白的氨基酸序列，或者为人工合成的自然界不存在的氨基酸序列；优选地，所述自然界存在的蛋白包括人源蛋白和除人以外的其它物种的蛋白。
(3)根据(1)所述的治疗剂，其中所述抗原表位多肽的氨基酸序列来源于肿瘤相关抗原或肿瘤特异性抗原的氨基酸序列。
(4)根据(1)所述的治疗剂，其中所述标记性多肽包括可操作地连接的、依次串联的以下氨基酸序列：N端信号肽的氨基酸序列、一个或多个所述抗原表位多肽的氨基酸序列、可任选的C端内质网滞留信号的氨基酸序列，其中当所述标记性多肽包括多个所述抗原表位多肽的氨基酸序列时，每两个相邻的所述抗原表位多肽的氨基酸序列之间由可切割性连接多肽的氨基酸序列连接。
(5)根据(4)所述的治疗剂，其中所述抗原表位多肽的氨基酸序列包括如SEQ ID NO：3所示的Her2/neu 369-377、NY-ESO-1 157-165、NY-ESO-1 1-11、NY-ESO-1 53-62、NY-ESO-1 18-27、N-ras 55-64、K-ras 224-232、K-ras 10-18、K-ras 10-19、H3.3K27M 26-35、SSX-2 41-49、MAGE-C2 336-344、MAGE-C2 191-200、MAGE-C2 307-315、MAGE-C2 42-50、MAGE-A1 120-129、MAGE-A1 230-238、MAGE-A1 161-169、KK-LC-1 76-84、p53 99-107、HPV16-E6 29-38、HPV16-E7 11-19、HPV16-E7 11-19、EBV-LMP1 51-59、EBV-LMP1 125-133。
(6)根据(1)所述的治疗剂，其中所述核酸还具有HLA蛋白编码序列，其中该HLA蛋白编码序列与所述标记性多肽编码序列分别在各自的启动子控制之下，或者该HLA蛋白编码序列与所述标记性多肽编码序列在同一启动子控制之下并且该HLA蛋白编码序列通过可切割性连接多肽编码序列与所述标记性多肽编码序列可操作地连接。
(7)根据(6)所述的治疗剂，其中所述HLA蛋白为HLA-A2蛋白，所述HLA-A2的氨基酸序列如SEQ ID NO：29所示。
(8)根据(1)所述的治疗剂，其中所述第一组合物和所述第二组合物各自独立地存在于所述治疗剂中而互不混合。
(9)根据(1)所述的治疗剂，其中所述核酸包括DNA或RNA；所述RNA包括由所述DNA转录的mRNA。
(10)根据(1)所述的治疗剂，其中所述第一活性成分为重组病毒，所述重组病毒的基因组具有所述标记性多肽编码序列和可任选的HLA蛋白编码序列；其中，所述重组病毒包括选择复制型重组溶瘤病毒或复制缺陷型重组病毒。
(11)根据(10)所述的治疗剂，其中所述复制缺陷型重组病毒来源于腺病毒、腺病毒相关病毒(AAV)、单纯疱疹病毒、痘病毒、流感病毒、甲病毒(Alphavirus)、或仙台病毒。
(12)根据(10)所述的治疗剂，其中所述复制缺陷型重组病毒为对5型腺病毒进行基因改造而得到的重组腺病毒，该重组腺病毒的基因组中缺失了E1基因，并且在所缺失的E1基因的位置包含所述标记性多肽编码序列和可任选的所述HLA蛋白编码序列。
(13)根据(10)所述的治疗剂，其中所述选择复制型重组溶瘤病毒来源于具有溶瘤作用的经基因突变的病毒和具有溶瘤作用的野生型病毒；优选地，所述选择复制型重组溶瘤病毒来源于具有溶瘤作用的腺病毒、痘病毒、单纯疱疹病毒、麻疹病毒、塞姆利基森林病毒、水疱性口炎病毒、脊髓灰质炎病毒、逆转录病毒、呼肠孤病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒和马拉巴病毒。
(14)根据(10)所述的治疗剂，其中所述选择复制型重组溶瘤病毒为对5型腺病毒进行基因改造而得到的重组溶瘤腺病毒，该重组溶瘤腺病毒的基因组中缺失了E1B-55K基因和/或E1B-19K基因，并且所述重组溶瘤腺病毒的基因组中包含E1A基因编码序列；优选地，所述E1A基因编码序列是在外源启动子控制下的。
(15)根据(10)或(14)所述的治疗剂，其中所述重组溶瘤病毒为对5型腺病毒进行基因改造而得到的重组溶瘤腺病毒，该重组溶瘤腺病毒的E1A基因被改变为使得所表达的E1A蛋白无法与pRb蛋白结合；优选地，所述E1A基因编码序列是在外源启动子控制下的。
(16)根据(14)或(15)所述的治疗剂，其中所述重组溶瘤腺病毒的E3基因全部或部分缺失。
(17)根据(1)所述的治疗剂，其中所述免疫细胞包括原始T细胞或其前体细胞，NKT细胞，或T细胞株。
(18)根据(9)所述的治疗剂，其中所述第一组合物包含治疗有效量的所述DNA、或治疗有效量的所述mRNA。
(19)根据(10)所述的治疗剂，其中所述第一组合物包含治疗有效量的所述重组病毒。
(20)根据(1)所述的治疗剂，其中所述第二组合物包含治疗有效量的所述的T细胞受体修饰的免疫细胞。
(21)根据(9)所述的治疗剂，其中所述DNA配制成通过瘤内注射给药；所述mRNA配制成通过瘤内注射给药。
(22)根据(10)所述的治疗剂，其中所述重组病毒配制成通过瘤内注射给药、腹膜内给药、蛛网膜下腔内给药、或静脉给药。
(23)根据(1)所述的治疗剂，其中所述免疫细胞配制成通过动脉、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。
(24)根据(1)所述的治疗剂，其中所述治疗剂由所述第一组合物和所述第二组合物组成。
(25)根据(1)-(24)中任一项所述的治疗剂在制备用于治疗肿瘤和/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/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰 岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
(27)一种标记性多肽，包括可操作地连接的依次串联的以下氨基酸序列：N端信号肽的氨基酸序列、一个或多个抗原表位多肽的氨基酸序列、可任选的C端内质网滞留信号的氨基酸序列，其中当所述标记性多肽包括多个所述抗原表位多肽的氨基酸序列时，每两个相邻的所述抗原表位多肽的氨基酸序列之间由可切割性连接多肽的氨基酸序列连接；优选地，所述可切割性连接多肽是furin酶切识别多肽。
(28)根据(27)所述的标记性多肽，其中所述抗原表位多肽的氨基酸序列包括如SEQ ID NO：3所示的Her2/neu 369-377、NY-ESO-1 157-165、NY-ESO-1 1-11、NY-ESO-1 53-62、NY-ESO-1 18-27、N-ras 55-64、K-ras 224-232、K-ras 10-18、K-ras 10-19、H3.3K27M 26-35、SSX-2 41-49、MAGE-C2 336-344、MAGE-C2 191-200、MAGE-C2 307-315、MAGE-C2 42-50、MAGE-A1 120-129、MAGE-A1 230-238、MAGE-A1 161-169、KK-LC-1 76-84、p53 99-107、HPV16-E6 29-38、HPV16-E7 11-19、HPV16-E7 11-19、EBV-LMP1 51-59、EBV-LMP1 125-133。
(29)根据(27)所述的标记性多肽，其中所述标记性多肽的氨基酸序列具有与如SEQ ID NO：24、SEQ ID NO：36、SEQ ID NO：56、或SEQ ID NO：60所示的氨基酸序列至少98％的一致性；优选的是，其中所述标记性多肽的氨基酸序列如SEQ ID NO：24、SEQ ID NO：36、SEQ ID NO：56、或SEQ ID NO：60所示。
(30)一种分离的、具有根据(27)-(29)中任一项所述的标记性多肽的编码序列的核酸。
(31)根据(30)所述的核酸，其中所述核酸还具有HLA蛋白编码序列，其中该HLA蛋白编码序列与所述标记性多肽编码序列分别在各自的启动子控制之下，或者该HLA蛋白编码序列与所述标记性多肽编码序列在同一启动子控制之下并且该HLA蛋白编码序列通过可切割性连接多肽编码序列与所述标记性多肽编码序列可操作地连接。
(32)根据(30)所述的核酸，其中所述核酸包括DNA和mRNA。
(33)根据(32)所述的核酸，其中所述核酸为DNA，其核苷酸序列如SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：57、SEQ ID NO：58、或SEQ ID NO：61所示。
(34)一种重组表达载体，含有根据(30)-(33)中任一项所述的核酸，和/或其互补序列。
(35)一种分离的重组病毒，其中所述重组病毒的基因组具有根据(30)-(33)中任一项所述的核酸；并且，所述重组病毒包括选择复制型重组溶瘤病毒或复制缺陷型重组病毒。
(36)根据(35)所述的重组病毒，其中所述复制缺陷型重组病毒来源于腺病毒、腺病毒相关病毒(AAV)、单纯疱疹病毒、痘病毒、流感病毒、甲病毒(Alphavirus)、或仙台病毒。
(37)根据(35)所述的重组病毒，其中所述复制缺陷型重组病毒为对5型腺病毒进行基因改造而得到的重组腺病毒，该重组腺病毒的基因组中缺失了E1基因，并且在所缺失的E1基因的位置包含所述标记性多肽编码序列和可任选的所述HLA蛋白编码序列。
(38)根据(35)所述的重组病毒，其中所述重组病毒为选择复制型重组溶瘤病毒，并且所述选择复制型重组溶瘤病毒来源于具有溶瘤作用的经基因突变的病毒和具有溶瘤作用的野生型病毒；优选地，所述选择复制型重组溶瘤病毒来源于具有溶瘤作用的腺病毒、痘病毒、单纯疱疹病毒、麻疹病毒、塞姆利基森林病毒、水疱性口炎病毒、脊髓灰质炎病毒、逆转录病毒、呼肠孤病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒和马拉巴病毒。
(39)根据(35)所述的重组病毒，其中所述选择复制型重组溶瘤病毒为对5型腺病毒进行基因改造而得到的重组溶瘤腺病毒，该重组溶瘤腺病毒的基因组中缺失了E1B-55K和/或E1B-19K基因，并且所述重组溶瘤腺病毒的基因组中包含E1A基因编码序列；优选地，所述E1A基因编码序列是在外源启动子控制下的。
(40)根据(35)或(39)所述的重组病毒，其中所述选择复制型重组溶瘤病毒为对5型腺病毒进行基因改造而得到的重组溶瘤腺病 毒，该重组溶瘤腺病毒的E1A基因被改变为使得所表达的E1A蛋白无法与pRb蛋白结合；优选地，所述E1A基因编码序列是在外源启动子控制下的。
(41)根据(39)或(40)所述的重组病毒，其中所述选择复制型重组溶瘤病毒的E3基因全部或部分缺失。
(42)一种用于治疗肿瘤和/或癌症的具有协同作用的联合药物的药盒，包括：
第一容器，该第一容器装有根据(1)-(24)中任一项所述的治疗剂中的第一组合物；
第二容器，该第二容器装有根据(1)-(24)中任一项所述的治疗剂中的第二组合物，其中所述第一容器和所述第二容器是独立的；以及
载明给药时机和给药方式的说明书。
(43)根据(30)-(33)中任一项所述的核酸在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
(44)根据(34)所述的重组表达载体在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
(45)根据(35)-(41)中任一项所述的重组病毒在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
(46)根据(42)所述的药盒在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
(47)根据(43)-(46)中任一项所述的用途，其中所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰 腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
(48)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(1)-(24)中任一项所述的治疗剂中的第一组合物；和
对所述肿瘤和/或癌症患者施用根据(1)-(24)中任一项所述的治疗剂中的第二组合物。
(49)根据(48)所述的方法，包括以下依次进行的步骤：
1)对所述肿瘤和/或癌症患者施用所述第一组合物；和
2)在施用所述第一组合物之后，对所述肿瘤和/或癌症患者施用所述的治疗剂中的第二组合物。
(50)根据(48)所述的方法，其中所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
本发明与现有技术相比具有以下优点和积极效果：
本发明为了提高TCR修饰的免疫细胞治疗肿瘤的效果，以及增加TCR修饰的免疫细胞治疗肿瘤的适用范围，提出了在肿瘤细胞内表达外源抗原表位肽和/或外源MHC I类分子，以显著加强肿瘤细胞表面的抗原表位肽/MHC I类分子复合物的数量，并采用特异性针对 该抗原表位肽的TCR修饰的免疫细胞进行联合治疗的构思。当通过溶瘤病毒作为载体介导外源抗原表位肽和/或外源MHC I类分子在肿瘤细胞内的表达时，溶瘤病毒对肿瘤的杀伤与TCR修饰的免疫细胞对肿瘤的杀伤还能够产生协同治疗效果。
具体而言，由于TCR修饰的免疫细胞(特别是TCR修饰的T细胞)所识别的是通过MHC I类抗原提呈途径所提呈的抗原表位肽/MHC I类分子复合物，因此仅通过常规的外源基因表达方式在肿瘤细胞内表达外源抗原，并不能显著加强抗原表位肽在肿瘤细胞表面的提呈，尤其当肿瘤细胞常常发生MHC I类抗原提呈途径的缺陷。为此，本发明首先设计了含有一个或多个抗原表位肽氨基酸序列的标记性多肽氨基酸序列，以及具有该标记性多肽的编码序列的核酸，使得所述核酸在转染肿瘤细胞和/或癌细胞后、或在将所述核酸插入病毒基因组并使所得到的重组病毒感染肿瘤细胞和/或癌细胞后，能够在细胞内表达标记性抗原表位多肽链，并通过氨基端的信号肽导入内质网(ER)，进而被切割/加工成需要的抗原表位多肽片段，该抗原表位多肽片段能够被MHC I类分子最终提呈在肿瘤细胞和/或癌细胞表面，从而能够达到在肿瘤细胞表面显著加强抗原表位肽的提呈的效果，有效解决了肿瘤免疫逃避机制所带来的抗原提呈途径缺陷所导致的抗原表达量低的问题。本发明还进一步地在所述核酸中插入HLA蛋白编码序列，以解决内源性HLA表达低下或缺失的问题。本发明还提出将包括或含有该标记性多肽的编码核酸的活性成分与识别抗原表位肽的T细胞受体修饰的免疫细胞联用，提高了T细胞受体修饰的免疫细胞对肿瘤细胞的识别敏感性，从而进一步提高了T细胞受体修饰的免疫细胞杀伤肿瘤细胞的能力。
进一步地，本发明通过溶瘤病毒将所述标记性多肽和/或HLA蛋白编码核酸导入肿瘤细胞和/或癌细胞，在发挥溶瘤病毒杀伤肿瘤细胞和/或癌细胞的作用的同时，进一步增强了上述显著加强外源抗原表位肽在肿瘤细胞表面的提呈与T细胞受体修饰的免疫细胞联合所达到的协同治疗效果。另外，溶瘤病毒在杀伤肿瘤的同时还可以缓解肿瘤微环境的免疫抑制状态，提高T细胞受体修饰的免疫细胞的归 巢；此外，T细胞受体修饰的免疫细胞还可以有效清除被溶瘤病毒感染后，不能完成复制周期并产生足够数量的子病毒而发生裂解的肿瘤细胞；由此实现了进一步的协同作用。此外，被溶瘤病毒裂解的肿瘤细胞所释放的抗原可进一步激活机体自身的抗肿瘤免疫，从而可实现比单独使用溶瘤病毒或T细胞受体修饰的免疫细胞更好的肿瘤杀伤效果，实现了协同治疗效果。
本发明通过上述发明构思为肿瘤治疗提供了一个新的途径。
定义
在本发明中，词语“肿瘤”、“癌症”、“肿瘤细胞”、“癌细胞”涵盖本领域通常认为的含义。
本文所用的词语“溶瘤病毒”是指能够选择性地在肿瘤细胞中复制并裂解肿瘤细胞的病毒。
本文所用的词语“治疗有效量”是指功能药剂或药物组合物能够表现出可检测的治疗效果或抑制效果的量，或者起到抗肿瘤效果的量。所述效果可以通过本领域任何已知的检验方法检测。
本文所用的词语“给药”或“施用”是指向受试者提供化合物、复合物或组合物(包括病毒和细胞)。
本文所用的词语“患者”是指人或非人类生物。因此，本文所述的方法和组合物适用于人类疾病和兽类疾病。在一些实施方案中，患者患有肿瘤。在一些例子中，患者同时患有一种或多种类型的癌症。
本文所用的词语“协同效果”是指两种或多种药剂共同起到的效果，该效果大于其中各药剂的单独效果的总和。
本文所用的术语“pfu”或“蚀斑形成单位”(plague forming unit)是指：产生一个蚀斑的病毒量称为一个蚀斑形成单位(pfu)。
本文所用的术语“VP”是指病毒颗粒的个数。
本文所用的术语“VP/kg”是指病毒颗粒数/千克患者体重。
本文所用的术语“TCID50”是指半数组织培养感染剂量(median tissue culture infective dose)，表示使半数组织培养物遭受感染，而发生细胞病变的病毒剂量。
本文所用的术语“MOI”或“感染复数”(Multiplicity of infection)也即，病毒与细胞个数比，是指用以起始病毒感染的每个细胞感染病毒颗粒的粒数。MOI＝pfu/细胞，即细胞个数×MOI＝总PFU。
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#1 PBMC)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(#1 PBMC)，右图涉及另一个不同供体提供的PBMC(#2 PBMC)。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示出经携带HLA-A2基因和Her2-E75微小基因的载体转染后的靶细胞可表达HLA-A2，所表达的Her2-E75多肽可与HLA-A2形成被Her2 TCR-6A5-mC TCR所识别的抗原复合物。图4A示出HLA-A2阴性Her2/neu阴性的293T细胞被能够表达HLA-A2基因和Her2-E75微小基因(C端不具有KDEL)的慢病毒质粒载体转染后， 可激活转染Her2 TCR-6A5-mC TCR的PBMC。各试验组和对照组均为复孔，结果显示为平均值±SME。横坐标示出作为靶细胞的293T细胞转染不同的基因载体。纵坐标表示T细胞分泌的IFN-γ的浓度。效靶比E∶T为10∶1。图4B的上图示出所构建的携带HLA-A2和Her2-E75微小基因(C端不具有KDEL)的慢病毒载体(即“pCDH-EF1p-A2-PKGp-E75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HLA-A2基因和PKG启动子所驱动的Her2-E75微小基因(C端不具有KDEL)。图4B的下图示出Her2-E75微小基因(C端不具有KDEL)的构成，由INSL5信号肽、8个Her2-E75抗原表位肽编码序列及其之间的furin酶切片段所组成。图4D示出所构建的携带HLA-A2和Her2-E75微小基因(C端具有KDEL)的重组腺病毒载体(即“Adeasy-A2E75载体”)的主要功能片段。腺病毒为E1基因和E3基因缺陷的5型腺病毒基因组。在E1区插入由CMV启动子所驱动的HLA-A2和Her2-E75微小基因及其之间的Furin-F2A连接片段所组成的表达元件。其中Her2-E75微小基因由INSL5信号肽、8个Her2-E75抗原表位肽编码序列及其之间的furin酶切片段、以及羧基端的内质网滞留信号肽KDEL所组成。图4C示出HLA-A2阴性细胞SKOV3感染图4D所示出的携带HLA-A2和Her2-E75微小基因(C端具有KDEL)的载体后可表达HLA-A2。横坐标示出SKOV3细胞感染不同的重组腺病毒。“Adeasy-A2E75”为表达HLA-A2和Her2-E75多肽的腺病毒；“Ad对照”为不携带HLA-A2和Her2-E75微小基因的对照腺病毒。纵坐标“HLA-A2 +(MFI)”表示细胞经抗HLA-A2荧光抗体染色后的荧光强度均值。“MOI”为病毒感染复数。
图5示出携带HLA-A2基因和Her2 E75微小基因的复制缺陷型腺病毒感染肿瘤细胞后可增加Her2 TCR-6A5-mC TCR对不同靶细胞的识别敏感性。图5A示出表达Her2 TCR-6A5-mC TCR的T细胞可被感染携带HLA-A2基因和Her2 E75微小基因的腺病毒的不同肿瘤细胞株细胞所激活，并分泌IFN-γ。效应细胞为经携带Her2 TCR-6A5-mC TCR基因的复制缺陷型慢病毒感染的外周血单个核细 胞。靶细胞为HLA-A2阴性Her2/neu阳性的卵巢癌细胞SKOV3，HLA-A2阳性Her2/neu阳性的乳腺癌细胞MCF-7以及HLA-A2阴性Her2/neu阴性的小细胞肺癌细胞NCI-H446。横坐标示出作为靶细胞的不同肿瘤细胞株细胞经过不同处理。“Adeasy-A2E75”为单独感染携带HLA-A2基因和Her2 E75微小基因的复制缺陷型腺病毒的靶细胞；“PBMC对照”为靶细胞与未经Her2 TCR-6A5-mC TCR基因转染的对照外周血单个核细胞混合培养；“PBMC对照+Adeasy-A2E75”为靶细胞感染携带HLA-A2基因和Her2 E75微小基因的复制缺陷型腺病毒24小时后与未经Her2 TCR-6A5-mC TCR基因转染的对照外周血单个核细胞混合培养；“具有Her2 TCR-6A5的PBMC”为靶细胞与经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞混合培养；“具有Her2 TCR-6A5的PBMC+Adeasy-A2E75”为靶细胞感染携带HLA-A2基因和Her2 E75微小基因的复制缺陷型腺病毒24小时后与经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞混合培养。纵坐标示出T细胞分泌的IFN-γ的浓度。效靶比E∶T为5∶1，腺病毒感染复数为MOI＝10。“****”示出与单独使用表达HLA-A2和Her2 E75多肽的复制缺陷型腺病毒组比较，或者与单独使用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞组相比较，二者联用组示出表达HLA-A2和Her2 E75多肽的复制缺陷型腺病毒可显著增加表达Her2 TCR-6A5-mC TCR的外周血单个核细胞对不同靶细胞的识别活性(p＜＝0.0001)，无论靶细胞自身是否表达HLA-A2和Her2/neu抗原。各试验组和对照组均为三孔，结果显示为平均值±SME。图5B示出表达Her2 TCR-6A5-mC TCR的T细胞可特异性杀伤感染携带HLA-A2基因和Her2 E75微小基因的腺病毒的肿瘤细胞株细胞。横坐标示出不同肿瘤细胞株细胞经不同处理作为靶细胞，与图5A的横坐标所示组别相同。效靶比E∶T为8∶1，腺病毒感染复数为MOI＝10。纵坐标示出T细胞对靶细胞的杀伤率，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。“***”和“**”示出与单独使用表达HLA-A2和Her2 E75多肽的复制缺陷型腺病毒组比 较，或者与单独使用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞组相比较，二者联用组示出表达Her2 TCR-6A5-mC TCR的外周血单个核细胞对感染表达HLA-A2和Her2 E75多肽的腺病毒的靶细胞具有显著的特异性杀伤性。“***”表示p＜＝0.001，“**”表示p＜＝0.01。各试验组和对照组均为三孔，结果显示为平均值±SME。
图6示出携带Her2-E75微小基因的溶瘤腺病毒感染肿瘤细胞后都可增加Her2 TCR-6A5-mC TCR对表达HLA-A2的靶细胞的识别敏感性。图6A示出表达Her2 TCR-6A5-mC TCR的T细胞可被感染携带Her2-E75微小基因的溶瘤腺病毒且表达HLA-A2的肿瘤细胞株细胞所激活，并分泌IFN-γ。效应细胞为经携带Her2 TCR-6A5-mC TCR基因的慢病毒感染的外周血单个核细胞。靶细胞为HLA-A2阴性Her2/neu阳性的卵巢癌细胞SKOV3，HLA-A2阳性Her2/neu阳性的乳腺癌细胞MCF-7以及HLA-A2阴性Her2/neu阴性的小细胞肺癌细胞NCI-H446。横坐标示出作为靶细胞的不同肿瘤细胞株细胞经过不同处理。“Ad-E75”为单独感染携带Her2-E75微小基因的溶瘤腺病毒的靶细胞；“PBMC对照”为靶细胞与未经Her2 TCR-6A5-mC TCR基因转染的对照外周血单个核细胞混合培养；“PBMC对照+Ad-E75”为靶细胞感染携带Her2-E75微小基因的溶瘤腺病毒24小时后与未经Her2 TCR-6A5-mC TCR基因转染的对照外周血单个核细胞混合培养；“具有Her2 TCR-6A5的PBMC”为靶细胞与经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞混合培养；“具有Her2 TCR-6A5的PBMC+Ad-E75”为靶细胞感染携带Her2-E75微小基因的溶瘤腺病毒24小时后与经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞混合培养。纵坐标示出T细胞分泌的IFN-γ的浓度。效靶比E∶T为5∶1，腺病毒感染复数为MOI＝10。“**”示出与单独使用表达Her2-E75多肽的溶瘤腺病毒组相比较，或者与单独使用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞组相比较，二者联用组示出表达Her2-E75多肽的溶瘤腺病毒可显著增加表达Her2 TCR-6A5-mC TCR的外周血单个核细胞对表达HLA-A2靶细胞的识别活性(p＜＝0.01)，其中靶细胞自身主要表达HLA-A2抗原。各试验组和 对照组均为三孔，结果显示为平均值±SME。图6B示出表达Her2 TCR-6A5-mC TCR的T细胞可特异性杀伤感染携带Her2-E75微小基因的溶瘤腺病毒的肿瘤细胞株细胞。横坐标示出不同肿瘤细胞株细胞经不同处理作为靶细胞，与图6A的横坐标所示组别相同。纵坐标示出T细胞对靶细胞的杀伤率，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。效靶比E∶T为5∶1，腺病毒感染复数为MOI＝10。“**”和“***”示出与单独使用表达Her2-E75多肽的溶瘤腺病毒组相比较，或者与单独使用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞组相比较，二者联用组示出表达Her2 TCR-6A5-mC TCR的外周血单个核细胞对感染表达Her2-E75多肽的溶瘤腺病毒的靶细胞具有显著的特异性杀伤性。“**”表示p＜＝0.01，“***”表示p＜＝0.001。各试验组和对照组均为三孔，结果显示为平均值±SME。
图7示出携带HLA-A2和Her2-E75微小基因的复制缺陷型腺病毒感染肿瘤细胞后都可增加Her2 TCR-6A5-mC TCR对不同靶细胞的识别敏感性。图7A和图7B是同一实验反应，图7A为上清液的因子水平检测，图7B为细胞相关杀伤计数检测。图7A示出表达Her2 TCR-6A5-mC TCR的T细胞可被感染携带HLA-A2和Her2-E75微小基因的复制缺陷型腺病毒且表达HLA-A2的肿瘤细胞株细胞所激活，并分泌IFN-γ。效应细胞为经携带Her2 TCR-6A5-mC TCR基因的慢病毒感染的外周血单个核细胞。靶细胞为HLA-A2阴性Her2/neu阳性的大细胞肺癌NCI-H460，HLA-A2阴性Her2/neu阳性的结肠癌细胞HT-29，HLA-A2阳性Her2/neu阴性的胶质细胞瘤细胞U87MG。横坐标示出作为靶细胞的不同肿瘤细胞株细胞经过不同处理。“Adeasy-A2E75”为单独感染携带HLA-A2和Her2-E75微小基因的溶瘤腺病毒的靶细胞；“PBMC对照”为靶细胞与未经Her2 TCR-6A5-mC TCR基因转染的对照外周血单个核细胞混合培养；“PBMC对照+Adeasy-A2E75”为靶细胞感染携带HLA-A2和Her2-E75微小基因的腺病毒24小时后与未经Her2 TCR-6A5-mC TCR基因转染的对照外周血单个核细胞混合培养；“具有Her2  TCR-6A5的PBMC”为靶细胞与经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞混合培养；“具有Her2 TCR-6A5的PBMC+Adeasy-A2E75”为靶细胞感染携带HLA-A2和Her2-E75微小基因的复制缺陷型腺病毒24小时后与经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞混合培养。纵坐标示出T细胞分泌的IFN-γ的浓度。效靶比E∶T为10∶1，腺病毒感染复数为MOI＝10。“**”示出与单独使用表达HLA-A2和Her2-E75多肽的复制缺陷型腺病毒组相比较，或者与单独使用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞组相比较，二者联用组示出表达HLA-A2和Her2-E75多肽的复制缺陷型腺病毒可显著增加表达Her2 TCR-6A5-mC TCR的外周血单个核细胞对不同靶细胞的识别活性(p＜＝0.01)，不论靶细胞自身是否表达HLA-A2和Her2/Neu抗原。各试验组和对照组均为三孔，结果显示为平均值±SME。图7B示出表达Her2 TCR-6A5-mC TCR的T细胞可特异性杀伤感染携带HLA-A2和Her2-E75微小基因的复制缺陷型腺病毒的肿瘤细胞株细胞。横坐标示出不同肿瘤细胞株细胞经不同处理作为靶细胞。纵坐标示出T细胞对靶细胞的杀伤率，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。效靶比E∶T为10∶1，腺病毒感染复数为MOI＝10。“***”、“**”和“*”示出与单独使用表达HLA-A2和Her2-E75多肽的复制缺陷型腺病毒组相比较，或者与单独使用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞组相比较，二者联用组示出表达Her2 TCR的外周血单个核细胞对感染表达HLA-A2和Her2-E75多肽的复制缺陷型腺病毒的靶细胞具有显著的特异性杀伤性。“***”表示p＜＝0.001，“**”表示p＜＝0.01，“*”表示p＜＝0.05。各试验组和对照组均为三孔，结果显示为平均值±SME。图7C示出细胞的流式检测结果。横坐标示出靶细胞组别。纵坐标“HLA-A2 +(MFI)”表示细胞经抗HLA-A2荧光抗体染色后的荧光强度均值。携带Her2-E75微小基因和HLA-A2的溶瘤腺病毒Ad-E75A2和携带HLA-A2和Her2-E75微小基因的复制缺陷型腺病毒Adeasy-A2E75感染肿瘤细胞株可以使其HLA-A2表达升高，促进其 对TCR-T的敏感性。
图8示出携带Her2-E75微小基因和HLA-A2的溶瘤腺病毒感染肿瘤细胞后都可增加Her2 TCR-6A5-mC TCR对不同靶细胞的识别敏感性。图8A和图8B是同一实验反应，图8A为上清液的因子水平检测，图8B为细胞相关杀伤计数检测。图8A示出表达Her2 TCR-6A5-mC TCR的T细胞可被感染携带Her2-E75微小基因和HLA-A2的溶瘤腺病毒的不同肿瘤细胞株细胞所激活，并分泌IFN-γ。效应细胞为经携带Her2 TCR-6A5-mC TCR基因的慢病毒感染的外周血单个核细胞。靶细胞为HLA-A2阴性Her2/neu阳性的大细胞肺癌NCI-H460，HLA-A2阴性Her2/neu阳性的结肠癌细胞HT-29，HLA-A2阳性Her2/neu阴性的胶质细胞瘤细胞U87MG。横坐标示出作为靶细胞的不同肿瘤细胞株细胞经过不同处理。“Ad-E75A2”为单独感染携带Her2-E75微小基因和HLA-A2的溶瘤腺病毒的靶细胞；“PBMC对照”为靶细胞与未经Her2 TCR-6A5-mC TCR基因转染的对照外周血单个核细胞混合培养；“PBMC对照+Ad-E75A2”为靶细胞感染携带Her2-E75微小基因和HLA-A2的溶瘤腺病毒24小时后与未经Her2 TCR-6A5-mC TCR基因转染的对照外周血单个核细胞混合培养；“具有Her2 TCR-6A5的PBMC”为靶细胞与经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞混合培养；“具有Her2 TCR-6A5的PBMC+Ad-E75A2”为靶细胞感染携带Her2-E75微小基因和HLA-A2的溶瘤腺病毒24小时后与经Her2 TCR-6A5-mC TCR基因转染的外周血单个核细胞混合培养。纵坐标示出T细胞分泌的IFN-γ的浓度。效靶比E∶T为10∶1，腺病毒感染复数为MOI＝10。“***”和“****”示出与单独使用表达Her2-E75多肽和HLA-A2的溶瘤腺病毒组相比较，或者与单独使用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞组相比较，二者联用组示出表达Her2-E75多肽和HLA-A2的溶瘤腺病毒可显著增加表达Her2 TCR-6A5-mC TCR的外周血单个核细胞对不同靶细胞的识别活性，不论靶细胞自身是否表达HLA-A2和Her2/Neu抗原。“***”表示p＜＝0.001，“****”表示p＜＝0.0001。各试验组和对照组均为三孔，结果显示为平均值±SME。图8B示出 表达Her2 TCR-6A5-mC TCR的T细胞可特异性杀伤感染携带Her2-E75微小基因和HLA-A2的溶瘤腺病毒的肿瘤细胞株细胞。横坐标示出不同肿瘤细胞株细胞经不同处理作为靶细胞。纵坐标示出T细胞对靶细胞的杀伤率，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。效靶比E∶T为10∶1，腺病毒感染复数为MOI＝10。“**”和“*”示出与单独使用表达Her2-E75多肽和HLA-A2的溶瘤腺病毒组相比较，或者与单独使用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞组相比较，二者联用组示出表达Her2 TCR-6A5-mC TCR的外周血单个核细胞对感染表达Her2-E75多肽和HLA-A2的溶瘤腺病毒的靶细胞具有显著的特异性杀伤性。“**”表示p＜＝0.01，“*”表示p＜＝0.05。各试验组和对照组均为三孔，结果显示为平均值±SME。
图9示出本发明一个实施方案所构建的5型溶瘤腺病毒骨架载体pShuttle-MCS-CMV-E1A-SV40pA的主要结构的示意图。
图10示出本发明一个实施方案所构建的单独表达标记性多肽的5型溶瘤腺病毒基因组DNA(即“OAd-E75”，上图)、以及共表达标记性多肽与HLA-A2的5型溶瘤腺病毒基因组DNA(即“OAd-E75-A2”，下图)的主要结构的示意图。
图11示出实施例9中的动物实验方案，具体示出了该实施例的给药方案，其中第一组为空白对照组；第二组为OAd-E75A2组(即溶瘤病毒单独给药)；第三组为Her2 TCR-6A5-mC T(IV)组(即TCR T静脉单独给药)；第四组为OAd-E75A2+Her2 TCR-6A5-mC T(IV)组(即联合给药，其中TCR T静脉给药)；第五组为Her2 TCR-6A5-mC T(IT)组(即TCR T瘤内单独给药)；第六组为OAd-E75A2+Her2 TCR-6A5-mC T(IT)组(即联合给药，其中TCR T瘤内给药)。图11中实心圆点表示动物皮下接种肿瘤细胞的开始时间，空心圆点表示动物试验的分组时间。
图12示出实施例9中各组动物的肿瘤平均体积变化曲线，其中“*”表示P＜＝0.05，“***”表示P＜＝0.001。
图13示出实施例9中各组动物中单只动物的肿瘤体积变化曲线。
图14示出实施例9中各组动物的相对肿瘤增殖率(T/C)％随给药后天数的变化曲线，相对肿瘤增殖率(T/C)％是指试验中所有处理组动物皮下的平均肿瘤体积与同一天测量的对照组动物皮下的平均肿瘤体积相比的百分比数值，用来表示相对于对照组，处理组中肿瘤生长的抑制情况。通常药物研发过程中T/C％低于40％被认为是药物有效的基本标准。
图15示出实施例9中各组动物的平均体重变化曲线。
图16示出实施例9中各组动物在实验终止时肿瘤组织中人T细胞数量的流式分析结果。其中图16A为六组动物的肿瘤组织中每20000个肿瘤细胞中的人CD3 +T细胞的数量，图16B为六组动物的肿瘤组织中每20000个肿瘤细胞中的人CD8 +T细胞的数量，图16C为六组动物的肿瘤组织中每20000万个肿瘤细胞中的人CD4 +T细胞的数量。各图中横坐标表示实验中设置的不同组别，纵坐标表示标准化处理后的T细胞的数目。其中标准化处理是指每组动物肿瘤内浸润的免疫细胞分别按照每20000个肿瘤来源的细胞中检出的免疫细胞数目来表示。
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。
本发明的发明人针对过继免疫细胞疗法以及溶瘤病毒治疗肿瘤所面临的问题和困难，通过理论研究和实验验证，提出了显著加强外源抗原表位肽在肿瘤细胞表面的提呈以及采用特异性针对该抗原表位肽的TCR修饰的免疫细胞进行联合治疗的构思。通过该发明构思，本发明有效解决了肿瘤免疫逃避机制所带来的抗原提呈途径缺陷所导致的抗原表达量低和/或HLA表达低下或缺失的问题，提高了肿瘤细胞对T细胞受体修饰的免疫细胞的识别敏感性，从而进一步提高了T细胞受体修饰的免疫细胞的归巢和杀伤肿瘤细胞的能力。另外， 本发明也扩大了基于TCR基因修饰的过继免疫细胞治疗肿瘤的适用范围，避免HLA限制性所带来的使用范围较小的局限性。
具体而言，本发明提供了一种用于治疗肿瘤和/或癌症的治疗剂，包含：
(a)第一组合物，其中该第一组合物包含位于第一可药用载体中的第一活性成分，该第一活性成分包括或含有用于导入肿瘤细胞和/或癌细胞的、具有标记性多肽编码序列的核酸；所述标记性多肽具有一个或多个抗原表位多肽的氨基酸序列，所述抗原表位多肽能够被MHC I类分子提呈在所述肿瘤细胞和/或癌细胞表面；和
(b)第二组合物，其中该第二组合物包含位于第二可药用载体中的第二活性成分，该第二活性成分包含T细胞受体修饰的免疫细胞；所述的T细胞受体修饰的免疫细胞能够特异性识别并结合被所述MHC I类分子提呈的所述抗原表位多肽。
通常，表达在细胞浆内的抗原蛋白可进入MHC I类抗原提呈途径，经系列蛋白酶水解后，形成的短肽(含有抗原表位多肽)通过内质网膜上的TAP分子转导进入内质网内，并与其中的HLA蛋白和β 2-微球蛋白形成三聚体后被提呈在细胞表面(其中HLA蛋白和β 2-微球蛋白配对形成MHC I类分子)，从而被免疫细胞识别。由于肿瘤细胞中常发生MHC I类抗原提呈途径的功能缺失，导致细胞浆内表达的肿瘤抗原不能有效形成表位多肽或进入内质网并与HLA和β2微球蛋白结合形成复合物。
在本发明中，通过将所述具有标记性多肽编码序列的核酸导入肿瘤细胞和/或癌细胞，表达在肿瘤细胞和/或癌细胞内的外源标记性多肽可进入MHC I类抗原提呈途径，从而增加了肿瘤细胞表面HLA/抗原表位多肽复合物的表达量，由此增强了所述T细胞受体修饰的免疫细胞对肿瘤细胞和/或癌细胞的识别敏感性。
所述抗原表位多肽的氨基酸序列可以来源于自然界存在的蛋白的氨基酸序列，或者为人工合成的自然界不存在的氨基酸序列。优选地，所述自然界存在的蛋白包括人源蛋白和除人以外的其它物种的蛋白。更优选地，所述抗原表位多肽的氨基酸序列来源于肿瘤相关抗原 或肿瘤特异性抗原的氨基酸序列。
“肿瘤相关抗原”通常指来源于自身的正常蛋白，但在肿瘤细胞中过度表达或异常表达，其包括癌胚抗原、肿瘤-睾丸抗原(CT抗原)等。
“肿瘤特异性抗原”通常指来源于自身的突变蛋白，或异体的与肿瘤发生和发展相关的病毒蛋白。
在本发明中，有时将“肿瘤相关抗原”和“肿瘤特异性抗原”笼统地称为“肿瘤抗原”。
所述肿瘤抗原可以是如癌症抗原性多肽数据库(Cancer Antigenic Peptide Database)(网址https：//caped.icp.ucl.ac.be)中所述的肿瘤抗原。优选的是，所述肿瘤抗原可以是如下表1中所述的肿瘤抗原。还优选的是，所述肿瘤抗原可以是人Her2/neu、NY-ESO-1、N-ras、K-ras、H3.3K27M、SSX-2、MAGE-C2、MAGE-A1、KK-LC-1、p53。Her2/neu的氨基酸序列如SEQ ID NO：21所示。
所述抗原表位多肽可以是能够被MHC I类分子提呈的具有8-11个氨基酸的肽段。所述抗原表位多肽可以是如癌症抗原性多肽数据库(Cancer Antigenic Peptide Database)(网址https：//caped.icp.ucl.ac.be)中所述的抗原表位多肽。优选的是，所述抗原表位多肽可以是如下表1中所述的抗原表位多肽。在其它实施方案中，所述抗原表位多肽为与如下表1中所述的抗原表位多肽具有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。还优选的是，所述抗原表位多肽包括但不限于如SEQ ID NO：3所示的Her2/neu 369-377、SEQ ID NO：22所示的Her2/neu 373-382、NY-ESO-1157-165、NY-ESO-1 1-11、NY-ESO-1 53-62、NY-ESO-1 18-27、N-ras 55-64、K-ras 224-232、K-ras 10-18、K-ras 10-19、H3.3K27M 26-35、SSX-2 41-49、MAGE-C2 336-344、MAGE-C2 191-200、MAGE-C2 307-315、MAGE-C2 42-50、MAGE-A1 120-129、MAGE-A1 230-238、MAGE-A1 161-169、KK-LC-1 76-84、p53 99-107、HPV16-E6 29-38、HPV16-E7 11-19、HPV16-E7 11-19、EBV-LMP1 51-59、EBV-LMP1 125-133。
表1优选的肿瘤抗原及抗原表位多肽表
肿瘤抗原名称 HLA分型 氨基酸序列位置
N-ras A1 55-64
MART2 A1 446-455
MATN A11 226-234
CDKN2A A11 125-133
CDK12 A11 924-932
k-ras A2 224-232
hsp70-2 A2 286-295
HAUS3 A2 154-162
GAS7 A2 141-150
CSNK1A1 A2 26-34
CLPP A2 240-248
CDK4 A2 23-32
α-辅肌动蛋白-4 A2 118-127
β-连环蛋白 A24 29-37
SIRT2 A3 192-200
GPNMB A3 179-188
EFTUD2 A3 668-677
MUM-3 A68 322-330
延伸因子2 A68 581-589
CASP-8 B35 476-484
SNRPD1 B38 19-Oct
OS-9 B44 438-446
MUM-2 B44 123-133
MUM-1 B44 30-38
KIAAO205 B44 262-270
NFYC B52 275-282
RBAF600 B7 329-337
HSDL1 Cw14 20-27
MUM-2 Cw6 126-134
K-ras Cw8 (10-18)
K-ras Cw8 (10-19)
MAGE-A3 A1 168-176
MAGE-A1 A1 161-169
SSX-2 A2 41-49
NY-ESO-1/LAGE-2 A2 157-165
NY-ESO-1/LAGE-2 A2 (1-11)
MAGE-C2 A2 336-344
MAGE-C2 A2 191-200
MAGE-A10 A2 254-262
LAGE-1 A2 (1-11)
HERV-K-MEL A2 (1-9)
GAGE-3，4，5，6，7 A29 (10-18)
NY-ESO-1/LAGE-2 A31 53-62
NY-ESO-1/LAGE-2 A31 (18-27)
LAGE-1 A31 (18-27)
MAGE-A6 A34 290-298
KK-LC-1 B15 76-84
MAGE-A6 B35 168-176
MAGE-A6 B37 127-136
MAGE-A3 B37 127-136
MAGE-A2 B37 127-136
MAGE-A1 B37 120-129
MAGE-C2 B44 307-315
MAGE-C2 B57 42-50
MAGE-A6 Cw16 293-301
MAGE-A1 Cw16 230-238
BAGE-1 Cw16 (2-10)
GAGE-1，2，8 Cw6 (9-16)
MAGE-A12m Cw7 170-178
酪氨酸酶 A1 243-251
酪氨酸酶 A1 146-156
酪氨酸酶 A2 (1-9)
酪氨酸酶 A2 369-377
Melan-A/MART-1 A2 32-40
Melan-A/MART-1 A2 26(27)-35
酪氨酸酶 A24 368-373和336-340e
酪氨酸酶 A24 206-214
酪氨酸酶 A26 90-98
TRP-2 A31 197-205
TRP-2 A33 197-205
酪氨酸酶 B35 312-320
酪氨酸酶 B35 309-320
Melan-A/MART-1 B35 26-35
酪氨酸酶 B38 388-397
酪氨酸酶 B44 192-200
Melan-A/MART-1 B45 24-33(34)
TRP-2 Cw8 387-395
MMP-2 A2 560-568
HER-2/neu A2 369-377
CPSF A2 1360-1369
CPSF A2 250-258
CALCA A2 16-25
PRAME A24 301-309
FGF5 A3 172-176和217-220
p53 B46 99-107
PBF B55 499-510
H3.3K27M A2 26-35
HPV16-E6   29-38
HPV16-E7   11-19
HPV16-E7   11-19
EBV-LMP1   51-59
EBV-LMP1   125-133
在某些实施方案中，每个所述抗原表位多肽的两端具有柔性连接片段，作为细胞浆内蛋白水解酶的酶切位点，以释放出该抗原表位多肽。所述柔性连接片段包括GSGSR、AGSGSR和AGSGS。
在某些实施方案中，所述标记性多肽在所述一个或多个抗原表位多肽氨基酸序列的氨基端具有可把该标记性多肽导入内质网的信号肽。信号肽的核心含有长段疏水性氨基酸，形成单个α-螺旋。信号肽氨基端常以短的带正电荷的氨基酸序列开始，信号肽末端通常存在一段被信号肽酶识别和切割的氨基酸切割位点。所连接的外源多肽进入内质网后，信号肽被信号肽酶识别和切割，在内质网内释放出外源多肽。因此，携带信号肽的标记性多肽可以不通过MHC I类抗原提呈途径中的蛋白酶水解以及TAP分子的转运，即可直接进入内质网。所述信号肽可以为由胰岛素样蛋白(INSL5)氨基端第1-22氨基酸所组成的信号肽(SEQ ID NO：28)。
在所述标记性多肽具有多个所述抗原表位多肽的情况中，每两个所述抗原表位多肽可由可切割性连接多肽所连接。可切割性连接多肽包括furin酶切识别多肽，其具有可被Furin酶切割的标准的四氨基酸基序，即R-X-[KR]-R氨基酸序列(参见文献“Molecular Therapy 2007；vol.15 no.6，1153-1159”)。优选的是，所述可切割性连接多肽的氨基酸序列为R-R-K-R。所述标记性多肽被上述信号肽导入内质网后，由R-X-[KR]-R氨基酸序列连接的抗原表位多肽被内质网中的furin酶切割水解，释放出抗原表位多肽，与内质网中的HLA和β 2-微球蛋白形成抗原复合物。内质网内的氨肽酶和羧肽酶也可能参与抗 原表位多肽的酶解和释放(参见文献“J Immunol.2009 November 1；183(9)：5526-5536”)，因此，可切割性连接多肽还可以包括氨肽酶和羧肽酶酶切识别多肽。
为了使被信号肽导入内质网的标记性多肽能够滞留在内质网腔内，以利于释放出抗原表位多肽并与HLA和β2微球蛋白结合形成抗原复合物，在某些实施方案中，所述标记性多肽在所述一个或多个抗原表位多肽氨基酸序列的羧基端具有内质网滞留信号肽。可溶性多肽(即非跨膜蛋白)的内质网滞留信号(ER retention signal)的氨基酸序列是KDEL，ER膜蛋白的内质网滞留信号是KKXX(参见文献“Molecular Biology of the Cell.2003；14(3)：889-902”)。在本发明中，所述标记性多肽是可溶性多肽。因此优选的是，所述内质网滞留信号肽是由赖氨酸-天冬氨酸-谷氨酸-亮氨酸残基组成的K-D-E-L片段。
在一个具体实施方案中，所述标记性多肽包括可操作地连接的、依次串联的以下氨基酸序列：N端信号肽的氨基酸序列、一个或多个所述抗原表位多肽的氨基酸序列、可任选的C端内质网滞留信号的氨基酸序列，其中当所述标记性多肽包括多个所述抗原表位多肽的氨基酸序列时，每两个相邻的所述抗原表位多肽的氨基酸序列之间由可切割性连接多肽的氨基酸序列连接；所述抗原表位多肽的氨基酸序列和可任选的C端内质网滞留信号的氨基酸序列可以由可切割性连接多肽的氨基酸序列连接。优选的是，所述标记性多肽包括所述C端内质网滞留信号的氨基酸序列。
优选地，所述标记性多肽包括由可切割性连接多肽R-R-K-R所连接的n个所述抗原表位多肽(优选为Her2/neu 369-377表位多肽)，其中n为大于等于1的整数，例如n＝1、2、3、4、5、6、7、8、9、10、11、12...。优选的是，n为1-20之间的整数(例如，2-20之间的整数)；还优选的是，n为1-10之间的整数(例如，2-10之间的整数)；还优选的是，n＝8。
考虑到肿瘤细胞和/或癌细胞中的HLA蛋白也常出现表达缺陷，包括完全缺失，单倍型缺失或等位基因缺失，不同肿瘤类型缺失的范 围从65％到90％不等(参见文献“Immunol Today.1997；18：89-95”)，因此，在一个实施方案中，所述核酸还具有HLA蛋白编码序列，其中该HLA蛋白编码序列与所述标记性多肽编码序列分别在各自的启动子控制之下，或者该HLA蛋白编码序列与所述标记性多肽编码序列在同一启动子控制之下并且该HLA蛋白编码序列通过可切割性连接多肽编码序列与所述标记性多肽编码序列可操作地连接。优选的是，该HLA蛋白的表型与所述标记性多肽能够结合的HLA蛋白的表型一致。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。
这样，本发明进一步增加了肿瘤细胞表面MHC/抗原表位多肽复合物的表达量，从而增强了所述T细胞受体修饰的免疫细胞对肿瘤细胞的识别敏感性。
进一步具体地，所述MHC蛋白是HLA I类蛋白。进一步具体地，所述HLA包括HLA-A、B、C。优选的是，所述HLA蛋白为HLA-A2蛋白，HLA-A2的氨基酸序列如SEQ ID NO：29所示。
连接所述HLA蛋白与所述标记性多肽之间的所述可切割性连接多肽的例子是本领域已知的，例如2A多肽，2A多肽包括但不限于来自微小核糖核酸病毒的F2A多肽、以及来自其它病毒的相似的2A类多肽；还可以是Furin-F2A连接片段。
优选地，在本发明的治疗剂中，所述第一组合物和所述第二组合物各自独立地存在于所述治疗剂中而互不混合。
优选地，在本发明的治疗剂中，所述核酸包括DNA或RNA；所述RNA包括由所述DNA转录的mRNA。
在一个实施方案中，所述第一活性成分为重组病毒，所述重组病毒的基因组具有标记性多肽编码序列和可任选的HLA蛋白编码序列；其中，所述重组病毒包括选择复制型重组溶瘤病毒或复制缺陷型重组病毒。
所述复制缺陷型重组病毒是缺失一个或数个与病毒复制、增殖 和病毒颗粒组装有关的必需功能基因的病毒载体，该病毒载体不能在正常细胞内复制形成子代病毒，但可以表达病毒自身或外源的基因产物。所述复制缺陷型重组病毒优选来源于腺病毒、腺病毒相关病毒(AAV)、单纯疱疹病毒、痘病毒、流感病毒、甲病毒(Alphavirus)、仙台病毒。
优选地，所述复制缺陷型重组病毒为对5型腺病毒进行基因改造而得到的重组腺病毒，该重组腺病毒的基因组中缺失了E1基因，并且在所缺失的E1基因的位置插入有所述标记性多肽的编码序列和可任选的HLA蛋白编码序列。
所述重组溶瘤病毒可以来源于具有溶瘤作用的经基因突变的病毒和具有溶瘤作用的野生型病毒。优选地，所述重组溶瘤病毒来源于具有溶瘤作用的腺病毒、痘病毒、单纯疱疹病毒、麻疹病毒、塞姆利基森林病毒、水疱性口炎病毒、脊髓灰质炎病毒、逆转录病毒、呼肠孤病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒和马拉巴病毒。
溶瘤病毒感染肿瘤细胞后选择性在肿瘤细胞中进行复制，通过子病毒的大量增殖从而裂解肿瘤细胞达到特异性杀伤肿瘤细胞的作用。释放的子病毒又可以选择性感染并裂解其它肿瘤细胞从而最大程度地清除肿瘤组织(参见文献“Nat Biotechnol.2012 Jul 10；30(7)：658-70”)。肿瘤细胞中异常的RAS、TP53、RB1、PTEN、WNT等信号传导途径影响了细胞自身的抗病毒机制，使得病毒在肿瘤细胞中更容易复制，是形成肿瘤选择性的主要原因。由于正常细胞中抑制病毒复制的分子机制完整，感染正常细胞后溶瘤病毒不能有效复制和扩散，因而大大限制了对正常组织细胞的损害(参见文献“Nat Rev Cancer.2017 11；17(11)：633”)。经过对病毒基因组的基因工程改造进一步增强了溶瘤病毒的肿瘤选择性，并且可以携带功能性外源基因以增强溶瘤病毒的抗肿瘤活性。除了本身的溶瘤作用外，溶瘤病毒还可以改变肿瘤组织的微环境，主要是通过诱导分泌细胞因子，吸引天然免疫细胞，释放肿瘤抗原，提供免疫危险信号等，从而增强肿瘤局部的抗肿瘤免疫反应(参见文献“J.Clin.Invest.2018；128， 1258-1260”)。腺病毒作为载体是较早被研发的溶瘤病毒，基于E1B-55K和E3基因缺陷的Ad5型腺病毒H101是第一个批准上市的溶瘤病毒产品(参见文献“Hum Gene Ther.2018 Feb；29(2)：151-159”)。腺病毒的分子结构和生物特性的研究比较深入，使得腺病毒更容易通过基因工程改造成为溶瘤病毒。腺病毒的一些特点，包括其基因组改造后允许插入较大片段的外源基因；腺病毒基因组DNA不会整合到宿主基因组中；也不会导致人体细胞的恶变；可感染多数人类肿瘤细胞；以及稳定高滴度病毒颗粒的制备等，使得腺病毒更适合成为溶瘤病毒载体(参见文献“Curr Opin Virol.2016 12；21：9-15”)。溶瘤腺病毒的不同设计与其肿瘤选择性的不同机制有关，比如E1B-55K基因缺陷的Ad5型腺病毒感染具有p53功能的正常细胞后，在完成病毒复制周期前会诱导细胞凋亡，而在大约50％的由于基因突变而丧失p53功能的肿瘤细胞中则会完成复制并裂解细胞(参见文献“Nat Med 1998；4(9)：1098-72”)。部分去除E1A基因的溶瘤腺病毒也会限制其在正常细胞中的复制，E1A的CR2区去除24个碱基后产生的缺陷蛋白(E1AΔ24)会丧失其从E2F1/pRB复合体中释放出E2F1的功能，而游离的E2F1对启动下游复制至关重要。而肿瘤细胞中的pRB途径常有异常，因而产生大量游离的E2F1以保证病毒在肿瘤细胞中的选择复制(参见文献“Cell 2000；100(1)：57-70”)。携带VA-RNA基因缺陷的溶瘤腺病毒的肿瘤选择性则依赖于肿瘤细胞中活化的RAS信号通路以及干扰素通路的不完整(参见文献“Cancer Res.2003；63(17)：5544-50”)。溶瘤腺病毒的另外一种肿瘤选择性机制则是利用肿瘤特异性基因启动子来驱动腺病毒复制所必需的基因。例如用肝癌细胞中的甲胎蛋白启动子(参见文献“Hum Gene Ther.1999；10(10)：1721-33”)；前列腺癌细胞中的前列腺特异性抗原(PSA)启动子(参见文献“Cancer Res.1997；57(13)：2559-63”)，或者骨钙素(hOC)启动子(参见文献“Cancer Res.2002；62(11)：3084-92”)；或者MUC-1阳性乳腺癌中的DF3/MUC1启动子(参见文献“J Clin Invest.2000；106(6)：763-71”)等主要在特定肿瘤细胞中活化的启动子来驱动E1A缺陷腺病毒E1A的基因表达，以达到在这些肿瘤细胞 中选择性复制的目的。如果利用其活性依赖于游离E2F1转录因子的启动子，比如插入能结合E2F1的回文序列的E2F1启动子，来驱动病毒复制所必需基因的表达，只有在富含游离E2F1的肿瘤细胞中才能有效表达病毒复制的下游分子而达到选择性裂解肿瘤细胞(参见文献“Mol Ther.2007；15(9)：1607-15”；“Cancer Cell.2002；1(4)：325-37”)。
基因工程改造后的溶瘤腺病毒虽然增强了肿瘤细胞的选择性杀伤，但病毒的溶瘤能力要进一步加强才能提高其临床疗效。由于溶瘤病毒完成复制周期需要依赖宿主肿瘤细胞的细胞组分和特有的分子机制，而肿瘤的多样性决定了不同生长状态和性质的肿瘤细胞被溶瘤病毒感染后，有些肿瘤细胞不能完成复制周期并产生足够数量的子病毒以裂解细胞。另外，溶瘤腺病毒缺失E1B-55K后，可能会导致编码后期病毒蛋白的mRNA从细胞核中导出到细胞浆中进行蛋白翻译的过程发生障碍，从而影响病毒在肿瘤细胞中的复制(参见文献“Viruses.2015 Nov；7(11)：5767-5779”)。这些因素都会限制溶瘤病毒在单独使用时的临床疗效。
本发明的一个实施方案中通过溶瘤病毒将含有所述标记性多肽编码序列和可任选的HLA蛋白编码序列的核酸导入肿瘤细胞和/或癌细胞，在发挥溶瘤病毒杀伤肿瘤细胞和/或癌细胞的作用的同时，T细胞受体修饰的免疫细胞还可以有效清除被溶瘤病毒感染后，不能完成复制周期并产生足够数量的子病毒而发生裂解的肿瘤细胞，由此实现了协同作用。
优选地，所述选择复制型重组溶瘤病毒为对5型腺病毒进行基因改造而得到的重组溶瘤腺病毒。在一个实施方案中，该重组溶瘤腺病毒的基因组中缺失了E1B-55K基因和/或E1B-19K基因(例如缺失了E1B-55K基因，或者缺失了E1B-55K基因和E1B-19K基因)，并且所述重组溶瘤腺病毒的基因组中包含E1A基因编码序列；优选地，所述E1A基因编码序列是在外源启动子控制下的。
在一个实施方案中，插入溶瘤腺病毒基因组中的所述标记性多肽的编码序列可以在腺病毒自身的E1B基因启动子、E1B TATA盒序列和E1B多聚腺苷酸添加信号序列的调控下在肿瘤细胞中表达。
在另一个实施方案中，插入溶瘤病毒基因组中的所述标记性多肽的编码序列可以在真核细胞启动子的控制下，真核细胞启动子包括但不限于CMV启动子、EF1a启动子、SV40启动子、PGK1启动子、Ubc启动子、人β-肌动蛋白启动子。
所述重组溶瘤腺病毒的E1A基因可以被改变为使得所表达的E1A蛋白无法与pRb蛋白结合。在具体的实施方案中，所述重组溶瘤腺病毒基因组DNA中编码E1A蛋白CR2区域的核酸序列缺失了5型腺病毒基因组DNA第923-946位的24个核苷酸(溶瘤腺病毒E1A-δ24)，所编码的E1A蛋白的氨基酸序列缺失了L-T-C-H-E-A-G-F。该所缺失的氨基酸序列是E1A蛋白和Rb蛋白的结合区域，缺失这段氨基酸序列的E1A蛋白不能结合Rb蛋白，导致溶瘤腺病毒E1A-δ24选择性地在Rb/E2F1通路缺陷的肿瘤细胞中复制并裂解肿瘤细胞。
优选地，所述重组溶瘤腺病毒的E1A基因是在组织特异性启动子或肿瘤特异性启动子控制下的。所述组织特异性启动子或肿瘤特异性启动子包括E2F-1启动子、端粒酶hTERT启动子、酪氨酸酶启动子、前列腺特异性抗原启动子、甲胎蛋白启动子和COX-2启动子。优选的，所述肿瘤特异性启动子是E2F-1启动子(其核苷酸序列为SEQ ID NO：30)。在正常细胞中，由于E2F-1与pRb结合，E2F-1启动子所调控的E1A的表达被抑制。在肿瘤细胞中，由于pRb的缺失或过度磷酸化，“游离”E2F-1的水平增加，E2F-1启动子活化后驱动E1A的表达，并导致腺病毒在肿瘤细胞中选择性复制并裂解细胞。
优选地，所述重组溶瘤腺病毒的E3基因全部或部分缺失。这样能够避免E3-19K蛋白对HLA I类抗原提呈途径的抑制作用，从而使外源导入的抗原表位多肽和内源肿瘤抗原可更有效地被提呈到肿瘤细胞表面。
在某些实施方案中，所述腺病毒的结构蛋白和功能蛋白上缺失至少一个T细胞所识别的免疫显性的抗原表位。结构蛋白和功能蛋白包括E1A、E1B、六邻体、五邻体基底、纤维蛋白、衣壳蛋白IX、DNA聚合酶和单链DNA结合蛋白。在一个实施方案中，通过基因点 突变去除病毒蛋白上T细胞所识别的免疫显性抗原表位(参见专利文献WO2016178167A1)。
在本发明所述的治疗剂中，被T细胞受体修饰的所述免疫细胞包括原始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，所述T细胞受体能够特异性识别肿瘤细胞和/或癌细胞表面上的所述抗原表位多肽。
优选地，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 thedition，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。
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。
本发明还涉及分离的、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，所述T细胞受体能够特异性识别肿瘤细胞和/或癌细胞表面上的所述抗原表位多肽。
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。
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识别抗原表位多肽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抗原表位多肽的生物活性。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结合抗原表位多肽的亲和性。
本发明还涉及用于表达所述TCR的重组表达载体，其含有与启动子有效连接的根据本发明所述的编码T细胞受体的核酸(例如DNA)，和/或其互补序列。
优选地，在所述重组表达载体中，本发明所述的编码T细胞受体的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。
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。
优选地，所述第一组合物包含治疗有效量的所述DNA、或治疗有效量的所述mRNA。
还优选地，所述第一组合物包含治疗有效量的所述重组病毒。优选地，所述重组病毒为重组溶瘤腺病毒时，重组溶瘤腺病毒的施用剂量为5×10 7-5×10 12vp/天，每天1-2次，连续施用1-7天。
还优选地，所述第二组合物包含治疗有效量的所述的T细胞受体修饰的免疫细胞。优选地，包含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细胞受体修饰的免疫细胞。
所述DNA可以配制成通过瘤内注射给药，例如，以质粒的形式直接肿瘤内注射，也可用脂质体包装后瘤内注射，也可连接到纳米颗粒上(如多聚L-赖氨酸、聚氨基酸、聚亚乙基亚胺(polyethyleneimine) 和壳聚糖等聚合物)瘤内注射，也可瘤内注射后用电转方法加强转染率。所述mRNA也可以相似的方式配制成通过瘤内注射给药。
所述重组病毒可以配制成通过瘤内注射给药、腹膜内给药、蛛网膜下腔内给药、或静脉给药。
所述免疫细胞可以配制成通过动脉、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。
优选地，所述治疗剂由所述第一组合物和所述第二组合物组成。
本领域的技术人员可以理解，本发明的治疗剂还可包含合适的可药用的辅料，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。
本发明还提供了所述的治疗剂在制备用于治疗肿瘤和/或癌症的药物中的用途。
所述肿瘤和/或癌症包括：头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，血液癌，神经瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，乳腺癌，佩吉特病，宫颈癌，食道癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。所述肿瘤和/或癌症可以包括本身是HLA-A2阳性而Her2/neu阴性的、HLA-A2阴性而Her2/neu阳性的、HLA-A2和Her2/neu均为阳性的、或HLA-A2和Her2/neu均为阴性的。可以根据肿瘤和/或癌症患者的自身实际情况，为患者施用本发明范围内的治疗剂。
本发明还提供了本发明上文所述的标记性多肽。
优选地，所述标记性多肽的氨基酸序列具有与如SEQ ID NO：24、SEQ ID NO：36、SEQ ID NO：56、或SEQ ID NO：60所示的氨基酸序列至少98％的一致性、更优选至少98.5％、还优选至少99％的一致性。还优选地，所述标记性多肽的氨基酸序列如SEQ ID NO：24、SEQ ID NO：36、SEQ ID NO：56、或SEQ ID NO：60所示。
本发明还提供了一种分离的、具有根据本发明所述的标记性多肽的编码序列的核酸。其中所述标记性多肽的氨基酸序列具有与如SEQ ID NO：24、SEQ ID NO：36、SEQ ID NO：56、或SEQ ID NO：60所示的氨基酸序列至少98％的一致性、更优选至少98.5％、还优选至少99％的一致性。所述核酸的各实施方案如上文所述。
优选地，所述核酸为DNA，其核苷酸序列如SEQ ID NO：25(对应于不包括HLA-A2序列)、或SEQ ID NO：26(对应于包括HLA-A2的序列，其中HLA-A2在标记性多肽之前)、或SEQ ID NO：27(对应于包括HLA-A2的序列，其中HLA-A2在标记性多肽之后)、或SEQ ID NO：57、或SEQ ID NO：58、或SEQ ID NO：61所示。其对应的氨基酸序列如SEQ ID NO：24(对应于不包括HLA-A2序列)、或SEQ ID NO：31所示(对应于包括HLA-A2的序列，其中HLA-A2在标记性多肽之前)、或SEQ ID NO：32所示(对应于包括HLA-A2的序列，其中HLA-A2在标记性多肽之后)、SEQ ID NO：56、SEQ ID NO：36、或SEQ ID NO：60所示。
本发明还提供了所述的核酸在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
本发明还提供了一种重组表达载体，含有根据本发明所述的核酸，和/或其互补序列。
优选地，在所述重组表达载体中，本发明所述的核酸合适地与启动子、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))。表达本发明所述标记性多肽的重组载体可以通过本领域常规的重组DNA技术来获得。
在一些实施方案中，所述重组表达载体的基本骨架是溶瘤腺病毒。所述启动子是内源的病毒基因启动子，例如E1A启动子、E1B启动子或E3启动子。在某些实施方案中，启动子是组织特异性或肿瘤特异性启动子。优选地，在一些实施方案中，启动子是SEQ ID NO：30所示的E2F-1启动子。
在另一些实施方案中，重组表达载体除了包含本发明所述的核酸外，还可以包含其它功能分子的编码序列，例如报告基因，其可以用于鉴定细胞是否转染有重组表达载体，也可以用于测定蛋白质水平和活性，例如通过流式细胞分析、扩增/表达方法、免疫组织化学方法、FISH和脱落抗原测定、Southern印迹、蛋白质印迹或PCR技术。因此，测量细胞中蛋白质水平的方法通常是本领域已知的。
本发明还提供了所述的重组表达载体在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
本发明还提供了一种分离的重组病毒，其中所述重组病毒的基因组具有根据本发明所述的核酸；并且，所述重组病毒包括选择复制型重组溶瘤病毒或复制缺陷型重组病毒。所述分离的重组病毒的各实施方案如上文所述。
本发明还提供了所述的重组病毒在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
本发明还提供了一种用于治疗肿瘤和/或癌症的具有协同作用的联合药物的药盒，包括：
第一容器，该第一容器装有根据本发明所述的治疗剂中的第一组合物；
第二容器，该第二容器装有根据本发明所述的治疗剂中的第二组合物，其中所述第一容器和所述第二容器是独立的；以及
载明给药时机和给药方式的说明书。
本发明还提供了所述的药盒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/或癌症包括：头颈部肿瘤(包括鼻咽癌)，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，血液癌，神经瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，乳腺癌，佩吉特病，宫颈癌，食道癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。所述肿瘤和/或癌症可以包括本身是HLA-A2阳性而Her2/neu阴性的、HLA-A2阴性而Her2/neu阳性的、HLA-A2和Her2/neu均为阳性的、或HLA-A2和Her2/neu均为阴性的。可以根据肿瘤和/或癌症患者的自身实际情况，为患者提供本发明范围内的药盒。
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治疗剂中的第一组合物；和
对所述肿瘤和/或癌症患者施用根据本发明所述的治疗剂中的第二组合物。
所述治疗剂中的第一组合物和第二组合物可以同时(例如，作为混合物同时瘤内注射)、分开但同时(例如，分别通过瘤内和静脉注射给药)或依次施用(例如，首先施用第一组合物，然后施用第二组合物；或者首先施用第二组合物，然后施用第一组合物)。
优选地，所述方法包括以下依次进行的步骤：
1)对所述肿瘤和/或癌症患者首先施用所述第一组合物；和
2)在施用所述第一组合物之后，对所述肿瘤和/或癌症患者施用所述的治疗剂中的第二组合物。
优选地，在首先施用所述第一组合物之后的第1-30天，对所述肿瘤和/或癌症患者施用所述的治疗剂中的第二组合物。
“在首先施用所述第一组合物之后的第1-30天，对所述肿瘤和/或癌症患者施用所述的治疗剂中的第二组合物”是指首次第二组合物的施用与首次第一组合物施用的时间间隔为1-30天(例如，1，2，3、4、5、6、7、8、9、10、11、12、13、14，15，16，17，18，19，20，21，22，23，24，25，26，27，28，29，30天)，或首次第二组合物的施用与在其之前最相邻一次的所述第一组合物施用的时间间隔为1-30天(例如，1，2，3、4、5、6、7、8、9、10、11、12、13、14，15，16，17，18，19，20，21，22，23，24，25，26，27，28，29，30天)。优选地，首次第二组合物的施用与在其之前最相邻一次的所述第一组合物施用的时间间隔为3-14天(例如，3、4、5、6、7、8、9、10、11、12、13、14天)。
在本发明的一个优选实施方案中，第一组合物包含所述重组溶瘤腺病毒，所述重组溶瘤腺病毒的施用剂量为5×10 7-5×10 12vp/天，每天1-2次，连续施用1-7天，或上述范围间的任何值。
在本发明的一个优选实施方案中，所述T细胞受体修饰的免疫细胞的施用剂量为，每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重。优选地，1天施用1-3次，连续施用1-7天。
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，以增强所述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：化疗药物，例如环磷酰胺、氟达拉滨(fludarabine)；放疗药物；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。
所述DNA可以配制成通过瘤内注射给药，例如，以质粒的形式直接肿瘤内注射，也可用脂质体包装后瘤内注射，也可连接到纳米颗粒上(如多聚L-赖氨酸、聚氨基酸、聚亚乙基亚胺和壳聚糖等聚合物)瘤内注射，也可瘤内注射后用电转方法加强转染率。所述mRNA也可以相似的方式配制成通过瘤内注射给药。
所述重组病毒可以配制成通过瘤内注射给药、腹膜内给药、蛛网膜下腔内给药、或静脉给药。
所述T细胞受体修饰的免疫细胞可以配制成通过动脉、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。
所述肿瘤和/或癌症包括：头颈部肿瘤(包括鼻咽癌)，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，血液癌，神经瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，乳腺癌，佩吉特病，宫颈癌，食道癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。所述肿瘤和/或癌症可以包括本身是HLA-A2阳性而Her2/neu阴性的、HLA-A2阴性而Her2/neu阳性的、HLA-A2和Her2/neu均为阳性的、或HLA-A2和Her2/neu均为阴性的。可以根据肿瘤和/或癌症患者的自身实际情况，为患者提供本发明范围内的方法。
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)、小细胞肺癌NCI-H446细胞(ATCC HTB-171)。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(#1 PBMC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 Analyzer 10(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。
MTT计数法：
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，10分钟后吸取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：33)以及α链3’引物5’-TCAGCTGGACCACAGCCGCAG-3’(SEQ ID NO：34)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.2008 Nov；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10分钟后，用0.4μm过滤膜过滤，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-2 100IU/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。
制备例1：表达标记性多肽的重组慢病毒载体的制备。
首先获得外源表达基因，HLA-A201基因片段是通过RT-PCR获得。从HLA-A201 +PBMC细胞中提取RNA，使用Superscript RT-PCR试剂盒(Thermo Fisher公司，cat#12574018)，并按照厂家说明书获得HLA-A201基因，测序确定为完整序列(其氨基酸序列如SEQ ID NO：29所示，核苷酸序列如SEQ ID NO：35所示)。所述标记性多肽的基因片段是通过DNA合成获得基因片段(Integrated DNA Technologies，gblocks Gene Fragments)，包括：标记性多肽“E75×1” (C端不具有KDEL)(其氨基酸序列如SEQ ID NO：56所示，其核苷酸序列如SEQ ID NO：57所示)；标记性多肽“E75×4”(C端不具有KDEL)(其氨基酸序列如SEQ ID NO：36所示，其核苷酸序列如SEQ ID NO：58所示)；标记性多肽“E75×8”(C端不具有KDEL)(其氨基酸序列如SEQ ID NO：60所示，其核苷酸序列如SEQ ID NO：61所示)。慢病毒载体pCDH-EF1α-MCS-(PGK-GFP)购自System Biosciences公司(Cat#CD811A-1)。通过采用本领域常规的基因克隆技术把HLA-A2基因片段插入到EF1a启动子下游的多克隆位点。质粒用Xcm-1酶切后去除GFP基因片段，通过采用本领域常规的基因克隆技术把合成的所述标记性多肽的基因片段分别插入到PGK启动子下游并置换GFP基因，并分别得到“pCDH-EF1p-A2-PKGp-E75×1”、“pCDH-EF1p-A2-PKGp-E75×4”和“pCDH-EF1p-A2-PKGp-E75×8”。各质粒测序结果均无误。
将HLA-A2基因通过furin酶切序列和F2A序列和标记性多肽“E75×8”(C端具有KDEL)的编码序列相连接，形成A2-Her2E75序列片段(其核苷酸序列如SEQ ID NO：26所示)。采用In-fusion克隆试剂盒(Takara Bio，cat#638909)按照厂家说明书进行PCR，所用引物为5‘-AGAGCTAGCGAATTCAACATGGCCGTCATG-3’(SEQ ID NO：37)和5‘-TGATTGTCGACGCCCTTAAAGCTCGTCTTTAAGGAAG-3’(SEQ ID NO：38)，高保真PCR扩增获得基因片段后，通过采用本领域常规的基因克隆技术把A2-Her2E75片段插入到慢病毒载体pCDH-EF1α-MCS-(PGK-GFP)(购自System Biosciences公司，Cat#CD811A-1)的EF1α启动子下游的多基因克隆位点，得到CD811-EF1a-A2-F2A-HerE75质粒。
制备例2：表达标记性多肽/HLA-A2的复制缺陷型重组腺病毒的制备。
1)表达标记性多肽/HLA-A2的复制缺陷型重组腺病毒载体的制备
重组腺病毒主要参照AdEasy系统的制备方法进行制备(参见文献“Nature protocols 2007；2：1236-1247”)。首先获得外源表达基因，HLA-A201基因片段是通过RT-PCR获得。从HLA-A201 +PBMC细胞中提取RNA，使用Superscript RT-PCR试剂盒(Thermo Fisher公司，cat#12574018)，并按照厂家说明书获得HLA-A201基因，测序确定为完整序列。其它外源基因，例如Her2-E75微小基因，或串联的微小基因以及表达调控元件，是通过DNA合成获得基因片段(Integrated DNA Technologie s，gblocks Gene Fragments)，包括：标记性多肽“E75×8”(C端具有KDEL)(其氨基酸序列如SEQ ID NO：24所示，其核苷酸序列如SEQ ID NO：59所示)。利用In-Fusion克隆技术将HLA-A2基因通过furin酶切序列和F2A序列和标记性多肽“E75×8”的编码序列，形成A2-Her2E75序列片段(其核苷酸序列如SEQ ID NO：26所示)。采用In-fusion克隆试剂盒(Takara Bio，cat#638909)按照厂家说明书进行PCR，所用引物为5‘-TAGAGATCTGGTACCAACATGGCCGTCATGG-3’(SEQ ID NO：39)和5‘-GGCTCGAGCGGCCGCTTAAAGCTCGTCTTTAAGGAAG-3’(SEQ ID NO：40)，PCR扩增获得基因片段后，通过采用本领域常规的基因克隆技术把A2-Her2E75片段插入到pShttle-CMV载体(Agilent technologies，cat#24007)的多基因克隆位点，得到pShuttle-CMV-A2E75-SV40pA。携带外源基因的pShuttle载体质粒纯化后，经Pme I酶(NEB Biolabs，cat#R0560s)酶切，纯化后按照厂家说明书用电转仪进行电转(Bio-Rad Gene Pulse)。在2mm比色皿中加入BJ5183-AD-1细菌菌株(Agilent technologies，cat#200157)和经Pme I酶切的携带外源基因的pShuttle载体质粒。在50μl菌株中加入质粒0.5μg，在2500v，200Ohms以及25micro-FD条件下进行电转。经含有50μg/ml卡那霉素的LB培养板培养过夜后，挑出菌落小量培养后获得质粒进行测序，确定是否有外源基因片段通过重组插入到腺病毒载体质粒中。测序结果无误。
2)表达标记性多肽/HLA-A2的复制缺陷型重组腺病毒颗粒的制备与感染
病毒颗粒是通过Lipofectaine 3000转染试剂(Thermo Fisher公司，cat#L3000001)转染ADENO-X 293细胞(Takara，cat#632271)而获得的。在6-孔板中培养ADENO-X 293细胞至50-75％融合后进行转染。纯化的重组腺病毒质粒经Pac I(NEB Biolabs，cat#R0547s)酶切并纯化后，按照厂家说明书用Lipofectaine 3000转染细胞。24小时后更换新鲜DMEN培养液。10-14天开始出现细胞病变，表现为细胞呈斑落样悬浮。收集细胞悬浮于PBS溶液中，于干冰和37℃水浴中冻融4次，离心后的上清为病毒初始制备，置于-80℃保存。为了获得高滴度病毒，在T75培养皿(Corning，cat#430661)中培养ADENO-X 293细胞，至75％融合后加入30％-50％初始制备的病毒冻存液。3-5天出现细胞病变后，收集细胞按上述方法进行冻融以获得病毒悬液。病毒滴度是利用腺病毒滴度试剂盒(Takara，cat#632270)按照厂家说明书来进行测定。感染靶细胞时，用新鲜培养液重悬靶细胞，根据细胞数加入定量滴度的腺病毒，培养3-4天后检测外源基因的表达。结果显示外源基因表达为阳性。
制备例3：表达标记性多肽的重组溶瘤腺病毒的制备及表达标记性多肽/HLA-A2的重组溶瘤腺病毒的制备。
1)5型溶瘤腺病毒载体的构建
首先提取H101商品化溶瘤腺病毒(溶瘤腺病毒H101购自上海三维生物技术有限公司)的基因组DNA为模板，设计两条引物(P26：5′GGAAGATCTGGACTGAAAATGAG3′(SEQ ID NO：41)和P27：5′TGAGGTCAGATGTA ACCAAGATTA 3′(SEQ ID NO：42))，通过高保真PCR扩增5型腺病毒的E1A编码区，PCR产物大小为1173bp。将获得的PCR片段纯化后进行BglII酶切回收并将其连接至pShuttle-CMV载体(购自Agilent technologies，cat#24007)上多克隆位点中的BglII和EcoRV位点之间，获得pShuttle-E1A，对其中的E1A序列进行测序，确认无误。
再次设计2条引物(P36：5′CGCGTCGACTACTGTAATAGTAATCAATTACG G3′(SEQ ID NO： 43)和P37：5′GACGTCGACTAAGATACATTGATGAGTTTGGAC3′)(SEQ ID NO：44)，以载体pShuttle-E1A为模板通过高保真PCR扩增该载体上包括CMV启动子、E1A编码区和SV40polyA序列在内的2017bp的DNA片段。将获得的PCR片段纯化后进行SalI酶切回收并将其连接至pShuttle载体(购自Agilent公司，cat#240006)上多克隆位点中的SalI位点中，获得pShuttle-MCS-CMV-E1A-SV40polyA(图9)，对其中的CMV-E1A-SV40polyA序列进行测序，确认无误。
2)表达Her2-E75微小基因(C端具有KDEL)和共表达Her2-E75微小基因(C端具有KDEL)与HLA-A2的溶瘤腺病毒的基因组DNA的制备
在上述获得的5型溶瘤腺病毒骨架的基础上分别加入可以表达Her2-E75微小基因和共表达Her2-E75微小基因与HLA-A2的基因表达框，最后获得可以单独表达Her2-E75微小基因的5型溶瘤腺病毒基因组DNA(“OAd-E75”)和共表达Her2-E75微小基因与HLA-A2的两种5型溶瘤腺病毒基因组DNA(“OAd-E75-A2”)(见图10)。
其中目的基因表达框包括EF-1α启动子、Her2-E75微小基因(或Her2-E75微小基因与HLA-A2)编码区序列和BGHpolyA序列三部分。获得上述两种片段后分别将其插入到5型溶瘤腺病毒骨架质粒pShuttle-MCS-CMV-E1A-SV40pA上的KpnI和XhoI位点中。BGHpolyA序列通过高保真PCR方法从pcDNA3.1质粒(购自Invitrogen公司)上扩增获得。获得BGHpolyA片段后将其插入到5型溶瘤腺病毒骨架质粒pShuttle-MCS-CMV-E1A-SV40polyA上的HindIII位点中，得到pShuttle-EF1a-MCS-BGHpA-CMV-E1A-SV40pA质粒。
E75(即Her2-E75微小基因，本部分简称“E75”)和E75-HLAA2片段的克隆
E75片段的克隆
设计2条引物(P105：5′CCGCTCGAGATGAAAGGTTCCATCTTCACATTG 3′(SEQ ID NO：45)和P106：5′CCGCTCGAGTTAAAGCTCGTCTTTAAGGAAGGC 3′(SEQ  ID NO：46))，以制备例1得到的CD811-EF1a-A2-F2A-HerE75质粒为模板，利用上述引物进行高保真PCR，最终获得两侧包含XhoI酶切位点的E75片段，大小为399bp。将获得的PCR片段纯化后进行XhoI酶切回收并将其连接至pShuttle-EF1a-MCS-BGHpA-CMV-E1A-SV40pA载体上的XhoI位点中，获得pShuttle-EF1a-E75-BGHpA-CMV-E1A-SV40pA，对其中的E75序列进行测序，确认无误。
E75-HLAA2片段的克隆
在本部分工作中的HLA-A2、Furin-F2A和E75序列均通过高保真PCR以CD811-EF1a-A2-F2A-HerE75质粒为模板扩增获得。为了将HLAA2调换到Furin-F2A连接片段后面，将E75调换到Furin-F2A连接片段前面，首先单独扩增HLAA2片段、Furin-F2A连接片段和E75片段，并且在扩增HLAA2片段时需要在其末端添加终止密码子，而E75片段需要去掉其末端的终止密码子，最后通过重叠延伸PCR的方法将三条片段按计划依次串联合并起来获得E75-Furin-F2A-HLAA2片段。首先设计5条引物(P107：5′TTCCGGATCGCTTGGCACGAAGCTCGTCTTTAAGGAAGG 3′(SEQ ID NO：47)、P108：5′CCTTCCTTAAAGACGAGCTTCGTGCCA AGCGATCCGGAA 3′(SEQ ID NO：48)、P109：5′CGGGGCGCCATGACGGCCATGGGCCCAGGGTTGGACTC 3′(SEQ ID NO：49)、P110：5′GAGTCCAACCCTGGGCCCATGGCCGTCATGGCGCCCC G 3′(SEQ ID NO：50)和P111：5′CTTCTCGAGTCACACTTTACAAGCTGTGAGAG 3′(SEQ ID NO：51))，以CD811-EF1a-A2-F2A-HerE75质粒为模板，利用P105和P107两条引物进行高保真PCR扩增不包含终止密码子但包含部分Furin-F2A连接片段5’端重复序列的E75片段，片段大小为406bp；利用P108和P109两条引物进行高保真PCR扩增包含部分E753’端重复片段和部分HLA-A25’端重复片段的Furin-F2A连接片段，片段大小为136bp；利用P110和P111两条引物进行高保真PCR扩增包含部分Furin-F2A 连接片段3’端重复片段的HLAA2片段，片段大小为1125bp；然后以E75和Furin-F2A连接片段的PCR产物为模板，利用P105和P109两条引物进行高保真PCR扩增E75-Furin-F2A片段，片段大小为503bp；再次以E75-Furin-F2A和HLAA2的PCR产物为模板，利用P105和P111两条引物进行高保真PCR扩增E75-Furin-F2A-HLAA2片段，片段大小为1587bp。将最终获得的E75-Furin-F2A-HLAA2的PCR片段纯化后进行XhoI酶切回收并将其连接至pShuttle-EF1a-MCS-BGHpA-CMV-E1A-SV40pA载体上的XhoI位点中，获得pShuttle-EF1a-E75-HLA-A2-BGHpA-CMV-E1A-SV40pA，对其中的E75-HLAA2片段进行测序，确认无误。
3)单表达Her2-E75微小基因(C端具有KDEL)和共表达Her2-E75微小基因(C端具有KDEL)与HLA-A2的溶瘤腺病毒基因组DNA的获得
将上一步中获得的经过测序确认的pShuttle-EF1a-E75-BGHpA-CMV-E1A-SV40pA和pShuttle-EF1a-E75-HLA-A2-BGHpA-CMV-E1A-SV40pA质粒(各1μg)进行PmeI酶切，37℃反应2-3hr后进行酚/氯仿抽提和乙醇/乙酸铵沉淀，70％乙醇漂洗三次后弃去上清，室温干燥3分钟后加入10μl洁净去离子水完全溶解线性化的DNA片段；随后各加入100μl BJ5183(含pAdEasy-1质粒)(购自Agilent公司)的超级感受态细菌进行常轻柔混匀后置于冰上30分钟，42℃孵育90秒后放回冰上继续孵育2分钟，每管中加入500μl LB培养基，37℃、150RPM振荡培养45分钟后将其涂布于卡那抗性的LB平板上，37℃过夜培养。第二天挑取LB平板上出现的较小克隆接种于4ml卡那抗性的LB培养基中，37℃、200RPM振荡培养过夜。第三天利用质粒小量提取试剂盒提取各管菌液的质粒DNA，首先对获得质粒DNA进行琼脂糖凝胶电泳分析舍弃明显偏小的质粒，将较大质粒进行PacI酶切分析，发生正确重组的pAdEasy质粒经PacI酶切后将产生4.5kb或3kb的一条较小片段。
本部分共获得两个发生正确重组的质粒(PacI酶切后产生一条大小为4.5kb的较小条带)：OAd-E75和OAd-E75A2，上述两个质粒包 含可以用来包装表达Her2-E75或共表达HLA-A2和Her2-E75的5型溶瘤腺病毒。
4)单表达Her2-E75微小基因(C端具有KDEL)和共表达Her2-E75微小基因(C端具有KDEL)与HLA-A2的溶瘤腺病毒的包装、扩增与纯化
腺病毒基因组DNA的线性化：分别将上一步中获得的2-3μg质粒DNA(OAd-E75和OAd-E75A2)进行完全的PacI酶切，37℃反应2-3小时后进行酚/氯仿抽提和乙醇乙酸铵沉淀，使用70％乙醇漂洗线性化DNA两次后室温干燥3分钟，溶解于10μl洁净去离子水中，使用该DNA直接进行AD293细胞的转染或将线性化DNA保存于-20℃备用。
腺病毒的包装：腺病毒包装前一天将长势良好的AD293细胞接种于6孔板中，细胞密度以第二天进行转染实验时细胞覆盖率达到～70％为佳。使用Lipo2000或相似转染试剂将PacI线性化的DNA转染至AD293细胞中(转染试剂不必去除)。将转染DNA后的AD293细胞中放回CO 2培养箱中继续培养约14天左右，直至AD293细胞出现大面积的病变后浮起，代表腺病毒包装成功。吹打6孔板中病变的AD293细胞和病毒混合物使其完全浮起后收集至洁净离心管中，可短暂剧烈震荡细胞悬液使腺病毒尽可能多地从细胞中释放出来，该混合物可短暂保存于4℃和长期保存于-80℃。
腺病毒的扩增：扩增前一天将长势良好的AD293接种于60mm培养皿中，第二天感染腺病毒时细胞覆盖率为80％左右。吸取约1ml从6孔板中收集的腺病毒混合物直接加入到接种有AD293的60mm的培养皿中，轻微震荡混匀后放回CO 2培养箱中继续培养，约2-3天后可见大部分细胞病变后浮起，同上吹打细胞收集全部的细胞悬液于洁净离心管中；接下来将收集的2ml病毒悬液加入到AD293细胞覆盖率为80％的10cm培养皿中，CO 2培养箱中继续培养约2-3天后可见大部分细胞病变浮起；收集10cm培养皿中的病毒悬液；同样吸取2ml病毒悬液加入到AD293细胞覆盖率为80％的10cm培养皿中，CO 2培养箱中继续培养约2-3天后可见大部分细胞病变浮起；收集15cm培养皿中的病毒悬液，此时细胞上清中的腺病毒滴度约达到10 8PFU/ml。到此可使 用15cm培养皿中的病毒悬液进行大量的腺病毒扩增，通常根据实验需要和病毒产量，需要扩增和收集40-80个15cm培养皿的细胞悬液后再进行腺病毒的CsCl密度梯度离心纯化。
腺病毒的纯化：将上一步收集的病毒悬液离心后分为上清和细胞沉淀两部分。使用PEG8000/NaCl沉淀上清中的腺病毒，并重悬于10mM Tris·Cl(pH8.0)中；将细胞沉淀重悬于10mM Tris·Cl(pH8.0)中反复冻融3-5次，完全释放细胞中的腺病毒，再次离心细胞悬液，保留上清进行下面的CsCl密度梯度离心纯化。接下来分别用轻CsCl(1.2g/ml)和重CsCl(1.45g/ml)对获得的腺病毒悬液进行两次离心，用注射器吸取第二次离心后的Ad白色条带，最后用PD-10脱盐柱将溶解腺病毒的CsCl置换为腺病毒保存液(10mM Tris(pH7.4)，1mM MgCl2，10％Glycerol过滤除菌后保存于4℃)，分装后保存于-80℃。
腺病毒滴度的滴定：腺病毒滴度的滴定是利用腺病毒滴度滴定试剂盒完成的。其基本原理为将腺病毒稀释到合适的滴度后感染AD293细胞，48小时后检测细胞表面的Hexon蛋白的表达。通过计数规定面积内表达Hexon蛋白的细胞的数量来确定有活性的腺病毒的滴度(PFU/ml)。
5)腺病毒中HLAA2蛋白表达的检测
选取HLAA2阴性的细胞株(SKOV3)，实验前一天接种细胞于24孔细胞培养板中。第二天按照不同的感染复数(MOI＝5、10和20)将腺病毒OAd-E75和OAd-E75A2加入到接种细胞的孔中，本实验中H101被用来作为阴性对照病毒。混匀后在CO 2培养箱中继续培养48小时后回收细胞，按照常规FACS流程使用抗-HLAA2流式抗体孵育细胞后上机分析。最终确定HLA-A2在SKOV3细胞表面的表达情况，结果呈阳性。
实施例1：从HLA-A2阳性的正常供体外周血诱导Her2/neu 369-377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/neu 369-377/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：2所示，编码序列如SEQ ID NO：1所示，并且该TCR的β链的氨基酸序列如SEQ ID NO：4所示，编码序列如SEQ ID NO：3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。6A5 TCRα 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：52)和3’引物5’-GGATCGCTTGGCACGTGAATTCTTTCTTTTGACCATAGCCAT-3’(SEQ ID NO：53)扩增而得；携带鼠源恒定区序列的Her2 TCR-6A5-mC的α基因是由5’引物5’-TCCAACCCTGGGCCCATGCTCCTGTTGCTCATACCAGTG-3’(SEQ ID NO：54)和3’引物5’-GTTGATTGTCGACGCCCTCAACTGGACCACAGCCT-3’(SEQ ID NO：55)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(IC50Tool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/neu 369-377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TCR。
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-mCT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：转染表达HLA-A2和Her2/neu抗原表位多肽的基因载体可使靶细胞表达外源HLA-A2和Her2/neu表位多肽
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，可以通过使肿瘤细胞表达外源Her2/neu抗原从而增加被HLA-A2所提呈的Her2/neu抗原表位来实现。由于肿瘤细胞常发生内源性HLA I类分子的表达低下或缺失，转染载体可以同时表达外源HLA-A2基因以增加HLA I类分子的表达量。另外，肿瘤细胞常出现HLA I类抗原提呈途径的功能缺陷。导致肿瘤抗原蛋白不能被有效降解成表位多肽并被HLA I类分子提呈到细胞表面。如果通过信号肽把表位多肽直接导入内质网内，可以不经过细胞浆内蛋白酶的降解以及TAP分子的转运功能，表位多肽可以直接和内质网内的HLA分子和β2微球蛋白形成复合物，并被提呈到肿瘤细胞表面。表达抗原表位多肽的微小基因(minigenes)可以通过Furin酶的酶切片段相连接，形成串联的多个抗原表位多肽的微小基因，进入内质网后被其中的furin酶释放出更多的表位多肽。在串联的表位多肽链末端加上内质网滞留信号KDEL片段，可避免表位多肽链被转输到下游分泌细胞器，从而增加形成HLA/多肽复合物的机会。
按制备例1的方法制备得到3个慢病毒载体质粒：“pCDH-EF1p-A2-PKGp-E75×1”(携带HLA-A2编码序列和1个Her2-E75抗原表位多肽编码序列)、“pCDH-EF1p-A2-PKGp-E75×4”(携带HLA-A2编码序列和4个重复的Her2-E75抗原表位多肽编码序列)和“pCDH-EF1p-A2-PKGp-E75×8”(携带HLA-A2编码序列 和8个重复的Her2-E75抗原表位多肽编码序列)，统称为“pCDH-EF1p-A2-PKGp-E75载体”。图4B上图为含有HLA-A2和Her2/neu抗原表位多肽(Her2-E75)微小基因的慢病毒载体的总体结构示意图(pCDH-EF1p-A2-PKGp-E75载体)。HLA-A2被EF-1α启动子所驱动，抗原表位多肽(图中显示为Her2-E75)微小基因被PKG启动子所驱动。图4B下图为“pCDH-EF1p-A2-PKGp-E75×8”中Her2/neu抗原表位多肽微小基因的组成的示意图，显示为被Furin酶的酶切片段相连接的8个Her2-E75抗原表位多肽(Her2-E75×8微小基因)。
用上述所构建的3个慢病毒质粒载体分别转染293T细胞，作为靶细胞检测Her2 TCR-6A5T细胞的识别功能。293T细胞是人肾脏上皮细胞株，为HLA-A2阴性Her2/neu阴性。混合培养转染Her2 TCR-6A5-mC TCR的PBMC细胞和分别转染pCDH-EF1p-A2-PKGp-E75的293T细胞，效靶比为10∶1。24小时后收集上清检测IFN-γ的分泌。质粒pCDH-EF1p-A2-PKGp-E75上的抗原表位多肽微小基因的数目分别为1个，4个和8个(图中分别显示为293-A2-PKG-E75×1、293-A2-PKG-E75×4、293-A2-PKG-E75×8)。用没有转染质粒的293T细胞作为阴性对照组(图中显示为293对照)，用T2细胞提呈Her2/neu 369-377抗原多肽0.1μg/ml作为阳性对照组(图中显示为T2+Her2-E75)。图4A显示，Her2 TCR-6A5-mC T细胞可特异性识别转染表达HLA-A2和Her2/neu抗原表位多肽的质粒载体的293T细胞而分泌IFN-γ，说明HLA-A2和Her2/neu 369-377表位多肽均被293T所表达，并以HLA/多肽复合物的方式提呈到细胞表面而被Her2 TCR-6A5-mC TCR所识别。另外，微小基因中表位多肽的数目越多，T细胞的识别活性也随之增强，说明细胞表面被提呈的HLA-A2/抗原表位多肽复合物的数量可以随着Her2/neu抗原表位多肽的表达增加而增加。
为了使复制缺陷型腺病毒载体能够表达HLA-A2和Her2/neu抗原表位多肽，HLA-A2基因和Her2/neu抗原表位多肽微小基因(C端具有KDEL)被装载到5型腺病毒载体的E1区域。按照制备例2所 述的方法制备得到复制缺陷型腺病毒Adeasy-A2-Her2 E75(简称为“Adeasy-A2E75”)，其为一个表达HLA-A2和8个Her2/neu抗原表位多肽的复制缺陷型腺病毒，其载体示意图见图4D(Adeasy-A2E75载体)。此腺病毒载体来源于缺失E1区和E3区序列的5型腺病毒(参见文献“Nature Protocols 2007；2：1236-1247”)。HLA-A2基因和Her2/neu抗原表位多肽微小基因的表达由外源CMV启动子所驱动。HLA-A2基因和Her2/neu抗原表位多肽微小基因由可切割片段Furin酶识别片段和F2A片段所分割，这样一方面可保证HLA-A2和Her2/neu抗原表位多肽的表达量相近，外源基因核酸序列的长度也可保持在腺病毒基因组所允许装载的外源基因的大小范围之内。另外在串联的表位多肽链末端加上内质网滞留信号KDEL片段，以增加形成HLA/多肽复合物的机会。由于E3-19K蛋白抑制HLA的抗原提呈功能，缺失E3区序列的腺病毒感染细胞后不会干扰所表达的外源抗原表位多肽的提呈。
为了验证复制缺陷型腺病毒载体Adeasy-A2-Her2 E75是否表达外源基因，用Adeasy-A2-Her2 E75腺病毒颗粒感染卵巢癌细胞SKOV3。SKOV3细胞是HLA-A2阴性的，可通过检测SKOV3细胞表面HLA-A2的表达来确定Adeasy-A2-Her2 E75腺病毒载体是否可以表达外源基因。在10cm培养皿中培养SKOV3细胞株，在其汇合度在80％左右时用胰酶消化液消化后再用培养基洗涤一次离心后用培养基McCony5A(Gibco#16600-082)重悬，铺板到24孔板中，每孔1×10e5，每孔500μl培养基；经过24h后加入Adeasy-A2-Her2 E75腺病毒，以0MOI、5MOI、10MOI、20MOI加入同体积的病毒配制液，再继续培养24h后，弃掉上清用胰酶消化后收获细胞，用100μl PBS重悬至1.5ml EP管中，每管加入2μl APC-抗人HLA-A2抗体(BD#561341)，孵育30分钟后，用含1％BSA的PBS洗涤后进行流式上机检测，检测后用Flowjo软件进行分析。图4C显示，SKOV3细胞感染Adeasy-A2-Her2 E75腺病毒后24小时就可表达HLA-A2，表达HLA-A2的量与感染病毒的滴度呈量效关系，而感染不表达外源基因的对照组腺病毒(图中显示为Ad对照)的SKOV3细胞不表 达HLA-A2，说明携带HLA-A2基因和Her2/neu抗原表位多肽微小基因的腺病毒可有效表达外源HLA-A2分子。
实施例6：用表达HLA-A2和Her2/neu抗原表位多肽微小基因的复制缺陷型腺病毒感染肿瘤细胞，可显著增强Her2 TCR-6A5-mC T细胞对肿瘤细胞的识别和杀伤敏感性。
为了进一步验证复制缺陷型腺病毒载体Adeasy-A2-Her2 E75感染肿瘤细胞后，是否增加细胞表面可被Her2 TCR-6A5-mC T细胞所识别的HLA-A2/Her2抗原表位多肽复合物的数量，从而增强T细胞对肿瘤细胞的识别和杀伤敏感性，使肿瘤细胞株SKOV3、MCF-7和NCI-H446分别感染Adeasy-A2-Her2 E75腺病毒，24小时之后与转染Her2 TCR-6A5-mC TCR的PBMC细胞混合并再共培养24小时，检测Her2 TCR-6A5-mC T细胞特异性分泌IFN-γ和杀伤靶细胞的功能。
图5A显示，肿瘤细胞感染Adeasy-A2-Her2 E75腺病毒后可显著刺激Her2 TCR-6A5-mC T细胞特异性分泌IFN-γ。感染肿瘤细胞的腺病毒感染复数MOI为10，与转染Her2 TCR-6A5-mC TCR的PBMC细胞共培养的效靶比为5∶1。肿瘤细胞株为HLA-A2 +Her2/neu +的MCF-7细胞，HLA-A2 -Her2/neu +的SKOV3细胞和HLA-A2 -Her2/neu -的NCI-H446细胞。结果显示，肿瘤细胞单独感染Adeasy-A2-Her2 E75腺病毒后自身几乎不分泌γ干扰素(实验组“Adeasy-A2E75”)。转染Her2 TCR-6A5-mC TCR的PBMC细胞在低效靶比的情况下也不能有效识别HLA-A2 +Her2/neu +的MCF-7细胞而分泌γ干扰素(实验组“具有Her2TCR-6A5的PBMC”)。针对SKOV3细胞，与对照PBMC相比(实验组“PBMC对照”)，转染Her2 TCR-6A5-mC TCR的PBMC细胞(“具有Her2TCR-6A5的PBMC”)也不能特异性分泌γ干扰素。然而，当肿瘤细胞感染Adeasy-A2-Her2 E75腺病毒，Her2 TCR-6A5-mC T细胞对其的识别功能可显著增强，无论肿瘤细胞是否表达内源性HLA-A2和Her2/neu(实验组“具有Her2TCR-6A5的PBMC+Adeasy-A2E75”)。在各靶细胞组中，Her2 TCR-6A5-mC T细胞识别被Adeasy-A2-Her2 E75腺病毒感染的肿瘤细胞的活性均显 著高于未感染病毒的靶细胞的识别活性(p＜＝0.001)。而Adeasy-A2-Her2 E75腺病毒感染肿瘤细胞不能增加没有转染Her2 TCR-6A5-mC TCR的对照PBMC细胞对肿瘤细胞的识别活性(实验组“PBMC对照”和“PBMC对照+Adeasy-A2E75”)，说明腺病毒感染后分泌γ干扰素的增加是通过增强Her2 TCR-6A5-mC TCR对肿瘤细胞的识别活性而产生的。结果说明，Adeasy-A2-Her2 E75腺病毒感染肿瘤细胞后，不仅使HLA-A2阴性细胞SKOV3和NCI-H446表达外源HLA-A2，还可以通过表达Her2/neu抗原表位多肽微小基因，增加肿瘤细胞表面被Her2 TCR-6A5-mC T细胞识别的HLA-A2/Her2/neu 369-377多肽复合物的数量，从而增强Her2 TCR-6A5-mC T细胞的识别敏感性。
为了进一步验证复制缺陷型腺病毒载体Adeasy-A2-Her2 E75感染肿瘤细胞后，是否增加Her2 TCR-6A5-mC T细胞对靶细胞的杀伤功能，肿瘤细胞株SKOV3、MCF-7和NCI-H446分别感染Adeasy-A2-Her2 E75腺病毒，24小时之后与转染Her2 TCR-6A5-mC TCR的PBMC细胞混合并再培养24小时，台盼蓝检测尚存活的贴壁活细胞的数量来确定T细胞对靶细胞的杀伤功能。感染肿瘤细胞的病毒感染复数MOI为10，混合培养的效靶比为8∶1。
图5B显示，Adeasy-A2-Her2 E75腺病毒单独感染肿瘤细胞后，可导致大部分NCI-H446细胞死亡(靶细胞为NCI-H446的实验组Adeasy-A2E75)。这可能是由于E1/E3缺失的复制缺陷型腺病毒有时可导致某些细胞的凋亡，尤其当感染滴度较高时(参见文献“Gene Ther.1999 Jun；6(6)：1054-63”)。图5A所示的感染腺病毒的NCI-H446细胞可以刺激与其混合培养的Her2 TCR-6A5-mC T细胞分泌γ干扰素，但由于这种靶细胞对Adeasy-A2-Her2 E75过于敏感，所以从图5B所示的试验结果中暂时无法确定靶细胞死亡是Her2 TCR-6A5-mC T细胞对感染腺病毒的靶细胞的杀伤还是腺病毒诱导的细胞凋亡(靶细胞为NCI-H446的实验组“具有Her2 TCR-6A5的PBMC+Adeasy-A2E75”)。然而，从图5B可以看出，本实验中靶细胞为MCF-7时，单用表达HLA-A2和Her2 E75多肽的复制缺陷型腺 病毒的细胞杀伤率为约32.8％，单用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞的细胞杀伤率为约30.9％，与单独施用一种药的细胞杀伤率相比，二者同时施用的细胞杀伤率提高，细胞杀伤率约为95.8％，显示出显著的协同效果。本实验中靶细胞为SKOV3时，单用表达HLA-A2和Her2 E75多肽的复制缺陷型腺病毒的细胞杀伤率为约29.7％，单用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞的细胞杀伤率为约34.3％，与单独施用一种药的细胞杀伤率相比，二者同时施用的细胞杀伤率提高，细胞杀伤率约为66％，也显示出显著的协同效果。
这些结果显示，Her2 TCR-6A5-mC T细胞可特异性杀伤感染Adeasy-A2-Her2 E75腺病毒的肿瘤细胞，无论肿瘤细胞是否表达内源性HLA-A2和Her2/neu抗原。如果表达HLA I类分子和肿瘤抗原表位多肽微小基因的腺病毒是溶瘤腺病毒，感染溶瘤病毒的肿瘤细胞不仅可被溶瘤病毒选择性杀伤，对于不能被溶瘤病毒有效裂解的肿瘤细胞，也可通过表达特定的外源肿瘤抗原表位多肽，被转染可识别特定肿瘤抗原表位的TCR的T细胞所识别和杀伤，这就是本发明所述的Adeasy-A2-Her2 E75腺病毒和Her2 TCR-6A5-mC T细胞联合应用以增强杀瘤功能的理论和试验基础。
实施例7：用表达Her2/neu抗原表位多肽微小基因的溶瘤腺病毒感染肿瘤细胞，可显著增强Her2 TCR-6A5-mC T细胞对肿瘤细胞的识别和杀伤敏感性。
为了进一步验证单独表达Her2/neu抗原表位多肽微小基因的溶瘤腺病毒Ad-E75感染肿瘤细胞后，是否增加细胞表面可被Her2 TCR-6A5-mC T细胞所识别的HLA-A2/Her2抗原表位多肽复合物的数量，从而增强T细胞对肿瘤细胞的识别和杀伤敏感性，首先按照制备例3所述的方法制备表达Her2/neu抗原表位多肽微小基因(C端具有KDEL)的溶瘤腺病毒(OAd-E75，也称为“Ad-E75”)。在所述复制缺陷型腺病毒的E1区同时插入CMV启动子所驱动的Her2/neu抗原表位多肽微小基因，以及EF-1α启动子所驱动的腺病毒 E1A基因。持续表达的E1A蛋白可使E1B缺失腺病毒在某些肿瘤细胞中选择性复制和增殖而产生溶瘤作用。使肿瘤细胞株MCF-7、SKOV3和NCI-H446分别感染Ad-E75溶瘤腺病毒，之后与转染Her2 TCR-6A5-mC TCR的PBMC细胞共培养24小时，检测Her2 TCR-6A5-mC T细胞特异性分泌IFN-γ和杀伤靶细胞的功能。
图6A显示，HLA-A2阳性肿瘤细胞MCF-7感染Ad-E75溶瘤腺病毒后可显著刺激Her2 TCR-6A5-mC T细胞特异性分泌IFN-γ，而HLA-A2阴性细胞SKOV3和NCI-H446感染Ad-E75病毒后不能增加Her2 TCR-6A5-mC T细胞特异性识别的敏感性。感染肿瘤细胞的腺病毒感染复数MOI为10，与转染Her2 TCR-6A5-mC TCR的PBMC细胞共培养的效靶比为5∶1。肿瘤细胞株为HLA-A2 +Her2/neu +的MCF-7细胞，HLA-A2 -Her2/neu +的SKOV3细胞和HLA-A2 -Her2/neu -的NCI-H446细胞。结果显示，肿瘤细胞单独感染Ad-E75腺病毒后自身几乎不分泌γ干扰素(实验组“Ad-E75”)。转染Her2 TCR-6A5-mC TCR的PBMC细胞在低效靶比的情况下也不能有效识别MCF-7而分泌γ干扰素(“具有Her2TCR-6A5的PBMC”)。然而，当HLA-A2阳性肿瘤细胞感染Ad-E75溶瘤腺病毒，Her2 TCR-6A5-mC T细胞对其的识别功能可显著增强(实验组“具有Her2TCR-6A5的PBMC+Ad-E75”)，Her2 TCR-6A5-mC T细胞识别被Ad-E75溶瘤腺病毒感染的肿瘤细胞的活性均显著高于未感染病毒的靶细胞的识别活性(p＜＝0.01)。而Ad-E75溶瘤腺病毒感染肿瘤细胞不能增加没有转染Her2 TCR-6A5-mC TCR的对照PBMC细胞对肿瘤细胞的识别活性(实验组“PBMC对照”和“PBMC对照+Ad-E75”)，说明腺病毒感染后分泌γ干扰素的增加是通过增强Her2 TCR-6A5-mC TCR对肿瘤细胞的识别活性而产生的。结果说明，Ad-E75溶瘤腺病毒感染肿瘤细胞后，可以通过表达Her2/neu抗原表位多肽微小基因，增加HLA-A2阳性肿瘤细胞表面被Her2 TCR-6A5-mC T细胞识别的HLA-A2/Her2/neu 369-377多肽复合物的数量，从而增强Her2 TCR-6A5-mC T细胞的识别敏感性。HLA-A2阴性细胞SKOV3和NCI-H446感染Ad-E75溶瘤腺病毒后并不能增强细胞对Her2  TCR-6A5-mC T细胞的识别敏感性，说明Her2/neu抗原表位多肽需要内源HLA-A2分子的提呈，进一步显示Her2 TCR-6A5-mC T细胞对Her2/neu抗原表位的识别是HLA-A2限制的特异性识别。
为了进一步验证溶瘤腺病毒Ad-E75感染肿瘤细胞后，是否增加Her2 TCR-6A5-mC T细胞对靶细胞的杀伤功能，肿瘤细胞株SKOV3、MCF-7和NCI-H446分别感染Ad-E75溶瘤腺病毒，24小时后与转染Her2 TCR-6A5-mC TCR的PBMC细胞混合并再培养24小时，台盼蓝检测尚存活的贴壁活细胞的数量来确定T细胞对靶细胞杀伤功能。感染肿瘤细胞的病毒感染复数MOI为10，混合培养的效靶比为5∶1。
图6B显示，Ad-E75溶瘤腺病毒单独感染肿瘤细胞后，可导致大部分SKOV3和NCI-H446细胞死亡(SKOV3、U87MG和NCI-H446的实验组“Ad-E75”)。说明Ad-E75对这些肿瘤细胞的溶瘤作用明显。本实验中靶细胞为MCF-7时，单用Ad-E75溶瘤腺病毒的细胞杀伤率为约20.9％，单用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞的细胞杀伤率为约30.9％，与单独施用一种药的细胞杀伤率相比，二者同时施用的细胞杀伤率提高，细胞杀伤率约为85.4％，显示出显著的协同效果。由于其它靶细胞SKOV3、和NCI-H446对Ad-E75溶瘤腺病毒过于敏感，所以在该MOI条件下，当Her2 TCR-6A5-mC T细胞与这些感染Ad-E75的靶细胞混合培养时，所显示的杀伤功能与单独感染溶瘤腺病毒暂时无明显差别，说明杀伤活性主要是溶瘤病毒的溶瘤作用所导致。
这些结果说明，Her2 TCR-6A5-mC T细胞可特异性识别被内源性HLA-A2所提呈的由溶瘤腺病毒所表达的Her2/neu抗原表位从而杀伤感染Ad-E75溶瘤腺病毒的肿瘤细胞。并且这些结果显示携带Her2/neu抗原表位多肽微小基因的溶瘤腺病毒感染肿瘤细胞后，可表达Her2/neu抗原表位多肽并被内源性HLA-A2分子提呈，从而增强T细胞对肿瘤细胞的识别和杀伤敏感性。也表明，Her2 TCR-6A5-mC T细胞对靶细胞的杀伤功能和溶瘤病毒的溶瘤作用有一定的协同作用。
实施例8：用同时表达Her2/neu抗原表位多肽微小基因和外源HLA-A2的溶瘤腺病毒感染肿瘤细胞，不仅可显著增强Her2 TCR-6A5-mC T细胞对肿瘤细胞的识别和杀伤敏感性，还可以识别HLA-A2阴性肿瘤细胞株。
为了进一步验证同时表达Her2/neu抗原表位多肽微小基因和HLA-A2的溶瘤腺病毒载体Ad-E75A2感染肿瘤细胞后，是否增加细胞表面可被Her2 TCR-6A5-mC T细胞所识别的HLA-A2/Her2抗原表位多肽复合物的数量，从而增强T细胞对肿瘤细胞的识别和杀伤敏感性，首先按照制备例3所述的方法制备表达Her2/neu抗原表位多肽微小基因(C端具有KDEL)和HLA-A2的溶瘤腺病毒(OAd-E75A2，也称为Ad-E75A2)。在实施例6中已显示，在Her2/neu抗原表位多肽微小基因上游插入由Furin-F2A链接片段连接的HLA-A2基因的复制缺陷型腺病毒(Adeasy-A2E75)感染靶细胞可表达HLA-A2和Her2/neu抗原表位，并显著增强Her2 TCR-6A5-mC T细胞对靶细胞的识别活性。本实施例所构建的溶瘤腺病毒来源于实施例7中所述溶瘤腺病毒，不同之处在于在Her2/neu抗原表位多肽微小基因下游插入由F2A链接片段连接的HLA-A2基因后构建成溶瘤腺病毒Ad-E75A2。使肿瘤细胞株HLA-A2阳性Her2阴性的U87MG、HLA-A2阴性Her2阳性的NCI-H460和HT-29细胞分别感染Adeasy-A2E75复制缺陷型腺病毒和Ad-E75A2溶瘤腺病毒，24小时之后与转染Her2 TCR-6A5-mC TCR的PBMC细胞混合并再共培养24小时，检测Her2 TCR-6A5-mC T细胞特异性分泌IFN-γ和杀伤靶细胞的功能。
首先检测携带HLA-A2基因的腺病毒感染肿瘤细胞株后是否表达外源HLA-A2。用Adeasy-A2E75和Ad-E75A2腺病毒分别感染U87MG、NCI-H460和HT-29细胞，没有感染腺病毒的细胞为对照。病毒感染24小时后收集细胞并用HLA-A2抗体进行染色和流式细胞分析。图7C显示，U87MG表达内源HLA-A2，NCI-H460和HT-29不表达HLA-A2。复制缺陷型腺病毒Adeasy-A2E75和溶瘤腺病毒Ad-E75A2感染后均可增加细胞表面HLA-A2的表达。
图7A显示，肿瘤细胞U87MG、NCI-H460和HT-29细胞感染 复制缺陷型腺病毒Adeasy-A2E75后可显著刺激Her2 TCR-6A5-mC T细胞特异性分泌IFN-γ，无论靶细胞是否表达内源HLA-A2或Her2抗原。这和实施例6中图5A所显示的针对其他靶细胞的识别活性相一致。感染肿瘤细胞的腺病毒感染复数MOI为10，与转染Her2 TCR-6A5-mC TCR的PBMC细胞共培养的效靶比为10∶1。肿瘤细胞株为HLA-A2 +Her2/neu -的U87MG细胞，HLA-A2 -Her2/neu +的NCI-H460和HT-29细胞。结果显示，肿瘤细胞单独感染Adeasy-A2E75复制缺陷型腺病毒后自身几乎不分泌γ干扰素(实验组“Adeasy-A2E75”)。转染Her2 TCR-6A5-mC TCR的PBMC细胞也不能有效识别靶细胞而分泌γ干扰素(实验组“具有Her2TCR-6A5的PBMC”)。然而，当肿瘤细胞感染Adeasy-A2E75腺病毒，Her2 TCR-6A5-mC T细胞对其的识别功能可显著增强(实验组“具有Her2TCR-6A5的PBMC+Adeasy-A2E75”)。Her2 TCR-6A5-mC T细胞识别被Adeasy-A2E75腺病毒感染的肿瘤细胞的活性均显著高于未感染病毒的靶细胞的识别活性(p＜＝0.01)，也显著高于单独感染复制缺陷型病毒的实验组。
图8A显示，肿瘤细胞U87MG、NCI-H460和HT-29细胞感染溶瘤腺病毒Ad-E75A2后，虽然Her2 TCR-6A5-mC T细胞识别NCI-H460后分泌IFN-γ的活性没有显著增强，与感染复制缺陷型Adeasy-A2E75病毒相比，对U87MG和HT-29的识别活性却更显著地增加(U87MG实验组“具有Her2 TCR-6A5的PBMC+Ad-E75A2”，p＜＝0.001；HT-29实验组“具有Her2 TCR-6A5的PBMC+Ad-E75A2”，p＜＝0.0001)。说明携带HLA-A2基因和Her2/neu抗原表位多肽微小基因的溶瘤腺病毒感染肿瘤细胞后可表达外源HLA-A2和Her2/neu抗原表位，从而使Her2 TCR-6A5-mC T细胞可识别HLA-A2阴性或Her2阴性的靶细胞。
为了进一步验证溶瘤腺病毒载体Ad-E75A2感染肿瘤细胞后，是否增加Her2 TCR-6A5-mC T细胞对靶细胞的杀伤功能，肿瘤细胞株U87MG、NCI-H460和HT-29细胞分别感染复制缺陷型腺病毒Adeasy-A2E75和溶瘤腺病毒Ad-E75A2，24小时之后与转染Her2  TCR-6A5-mC TCR的PBMC细胞混合并再培养24小时，台盼蓝检测T细胞对靶细胞杀伤功能。感染肿瘤细胞的病毒感染复数MOI为10，混合培养的效靶比为10∶1。
图7B和图8B显示，在所用的MOI条件下，单独感染复制缺陷型腺病毒Adeasy-A2E75和溶瘤腺病毒Ad-E75A2均显示出一定的溶瘤作用(图7B各实验组中“Adeasy-A2E75”和图8B中各实验组中“Ad-E75A2”)。而感染腺病毒后，可显著增强Her2 TCR-6A5-mC T细胞对靶细胞的杀伤功能。
感染复制缺陷型腺病毒Adeasy-A2E75可增强Her2 TCR-6A5-mC T细胞对所有靶细胞的杀伤功能，无论与单独感染腺病毒实验组(图7B各实验组“Adeasy-A2E75”；U87MG实验组p＜＝0.01，NCI-H460实验组p＜＝0.05，HT-29实验组p＜＝0.001)相比，还是与单独使用Her2 TCR-6A5-mC T细胞的实验组(图7B中各实验组“具有Her2 TCR-6A5的PBMC”；U87MG实验组p＜＝0.001，NCI-H460实验组p＜＝0.05，HT-29实验组p＜＝0.001)相比，Her2 TCR-6A5-mC T细胞对感染复制缺陷型腺病毒Adeasy-A2E75的靶细胞的杀伤功能显著增强。感染溶瘤腺病毒Ad-E75A2也可增强Her2 TCR-6A5-mC T细胞对靶细胞的杀伤功能。进一步而言，图7B所示实验中靶细胞为U87MG时，单用复制缺陷型腺病毒的细胞杀伤率为约9.8％，单用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞的细胞杀伤率为约8.8％，与单独施用一种药的细胞杀伤率相比，二者同时施用的细胞杀伤率提高，细胞杀伤率约为83.6％，显示出显著的协同效果。图7B所示实验中靶细胞为NCI-H460时，单用复制缺陷型腺病毒的细胞杀伤率为约5.4％，单用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞的细胞杀伤率为约7％，与单独施用一种药的细胞杀伤率相比，二者同时施用的细胞杀伤率提高，细胞杀伤率约为33.4％，也显示出显著的协同效果。图7B所示实验中靶细胞为HT-29时，单用复制缺陷型腺病毒的细胞杀伤率为约8.4％，单用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞的细胞杀伤率为约12.9％，与单独施用一种药的细胞杀伤率相比，二者同时施用的细胞杀伤率提高，细胞杀伤率约为74.2％，也 显示出显著的协同效果。
与单独感染溶瘤腺病毒实验组(图8B各实验组“Ad-E75A2”；U87MG实验组p＜＝0.01，NCI-H460实验组p＜＝0.05，HT-29实验组p＜＝0.01)相比，Her2 TCR-6A5-mC T细胞对所有感染溶瘤腺病毒的靶细胞显示出显著的杀伤活性。与单独使用Her2 TCR-6A5-mC T细胞的实验组(图8B中各实验组“具有Her2 TCR-6A5的BPMC”；U87MG实验组p＜＝0.05，HT-29实验组p＜＝0.01)相比，Her2 TCR-6A5-mC T细胞对感染溶瘤腺病毒的U87MG和HT-29细胞显示出显著的杀伤活性。进一步而言，图8B所示实验中靶细胞为U87MG时，单用溶瘤腺病毒的细胞杀伤率为约10.6％，单用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞的细胞杀伤率为约8.7％，与单独施用一种药的细胞杀伤率相比，二者同时施用的细胞杀伤率提高，细胞杀伤率约为38.4％，显示出显著的协同效果。图8B所示实验中靶细胞为HT-29时，单用溶瘤腺病毒的细胞杀伤率为约6.5％，单用表达Her2 TCR-6A5-mC TCR的外周血单个核细胞的细胞杀伤率为约9.8％，与单独施用一种药的细胞杀伤率相比，二者同时施用的细胞杀伤率提高，细胞杀伤率约为44.2％，也显示出显著的协同效果。
可见，用同时表达Her2/neu抗原表位多肽微小基因和HLA-A2的溶瘤腺病毒载体感染肿瘤靶细胞后可显著增强Her2 TCR-6A5-mC T细胞的杀伤敏感性，无论靶细胞是否表达内源性HLA-A2和Her2抗原。联合使用所述溶瘤腺病毒和所述TCR-T细胞表现出了杀伤肿瘤细胞的协同效应。
实施例9：溶瘤腺病毒OAd-E75A2与人Her2 TCR-6A5-mC T细胞对NCG重度免疫缺陷鼠皮下接种的人卵巢癌细胞SKOV3生长的联合抑制作用
本实验中将人卵巢癌细胞SKOV3接种于NCG重度免疫缺陷鼠(江苏集萃公司提供)右前肢背侧皮下，制备模拟人体环境的荷瘤动物模型，用于检测按制备例3制备得到的溶瘤腺病毒OAd-E75A2与按前述“重组TCR慢病毒转染人T细胞”部分所述的方法得到的人Her2  TCR-6A5-mC T细胞(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))对重度免疫缺陷鼠NCG皮下接种的人卵巢癌细胞SKOV3生长的联合抑制作用。每只动物的细胞接种量为3×10 6个细胞，细胞接种约9天后选取皮下肿瘤体积满足要求(肿瘤体积为90～120mm 3)的30只荷瘤鼠，按照区组随机分组的方式分成6组，每组5只，分组当天时间设定为第0天。第一组为空白对照组，组内每只小鼠分别于第0天、第4天和第8天瘤内注射(简称I.T.)100μl腺病毒保存液(含10mM Tris(pH7.4)、1mM MgCl 2、10％丙三醇，过滤除菌后保存于4℃)，于第2天、第6天和第10天尾静脉内注射(简称I.V.)100μl生理盐水；第二组为OAd-E75A2组(即溶瘤病毒单独给药)，组内每只小鼠分别于第0天、第4天和第8天瘤内注射100μl溶瘤腺病毒OAd-E75A2，每只动物的病毒注射量为5×10 8PFU，于第2天、第6天和第10天尾静脉内注射100μl生理盐水；第三组为Her2 TCR-6A5-mC T(IV)组(即TCR T静脉单独给药)，组内每只小鼠分别于第0天、第4天和第8天瘤内注射100μl腺病毒保存液，于第2天、第6天和第10天尾静脉内注射100μl Her2 TCR-6A5-mC T细胞，细胞数量为2×10 7个；第四组为OAd-E75A2+Her2 TCR-6A5-mC T(IV)组(即联合给药，其中TCR T静脉给药)，组内每只小鼠分别于第0天、第4天和第8天瘤内注射100μl溶瘤腺病毒OAd-E75A2，每只动物的病毒注射量为5×10 8PFU，于第2天、第6天和第10天尾静脉内注射100μl Her2 TCR-6A5-mC T细胞，细胞数量为2×10 7个；第五组为Her2 TCR-6A5-mC T(IT)组(即TCR T瘤内单独给药)，组内每只小鼠分别于第0天、第4天和第8天瘤内注射100μl腺病毒保存液，于第2天、第6天和第10天瘤内注射100μl Her2 TCR-6A5-mC T细胞，细胞数量为2×10 7个；第六组为OAd-E75A2+Her2 TCR-6A5-mC T(IT)组(即联合给药，其中TCR T瘤内给药)，组内每只小鼠分别于第0天、第4天和第8天瘤内注射100μl溶瘤腺病毒OAd-E75A2，每只动物的病毒注射量为5×10 8PFU，于第2天、第6天和第10天瘤内注射100μl Her2 TCR-6A5-mC T细胞。在第三组-第六组中，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“重 组TCR慢病毒转染人T细胞”部分所述的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8天左右的时间，细胞的Her2 TCR-6A5-mC TCR阳性率均为约35-38％。除此之外每组动物分别在第2天、第3天、第6天、第7天、第10天和第11天于颈部皮下注射(简称S.C.)10万IU IL2(购自北京四环制药厂，产品批号为81766010002383594693)，用于刺激T细胞增殖和活化，使人的T细胞在NCG小鼠体内存活更长的时间。该动物实验方案见图11。从分组当天(第0天)开始测量每只小鼠皮下肿瘤体积和称量小鼠体重，每周测量和记录两次。实验结果中各组动物肿瘤平均体积变化情况见图12，各组动物中单只动物肿瘤体积变化情况见图13，动物组间的相对肿瘤增殖率(T/C％)见图14，每组动物平均体重变化情况见图15，实验终止时各组动物的肿瘤中人T细胞数量的流式分析结果见图16。
上述结果显示，人Her2 TCR-6A5-mC T细胞单独给药时(第三组和第五组)对NCG重度免疫缺陷鼠皮下接种的人卵巢癌细胞SKOV3生长没有明显的抑制作用(见图12-14)，这是因为本实验采用的人卵巢癌细胞SKOV3是HLA-A2阴性Her2/neu阳性的。然而从上述结果中可以看出，符合本发明联合给药方案的第四组(OAd-E75A2+Her2 TCR-6A5-mC T(IV)组)和第六组(OAd-E75A2+Her2 TCR-6A5-mC T(IT)组)中动物皮下肿瘤的生长得到了很好的抑制(见图12)，与空白对照组中动物皮下肿瘤平均体积相比存在着极为显著的差异(P＜＝0.001)，与第二组(OAd-E75A2组)中动物皮下肿瘤平均体积相比存在着显著的差异(P＜＝0.05)；从图13中可以看出每组动物皮下肿瘤生长的体积变化曲线相对比较集中，无显著异常值出现；从图14中可以看出第四组(OAd-E75A2+Her2 TCR-6A5-mC T(IV)组)和第六组(OAd-E75A2+Her2 TCR-6A5-mC T(IT)组)中动物皮下肿瘤的相对增殖率(T/C％)在第10天即达到40％(或以下)的抑制率，达到临床用药物有效标准；从图15中可以看出，除第四组(OAd-E75A2+Her2 TCR-6A5-mC T(IV)组)以外，其余动物的平均体重变化不大，未表现出明显毒性。而第四组动物的平均体重显著上升，该关键指标显示瘤 内注射溶瘤腺病毒和尾静脉内输注人TCR T细胞的给药模式相较于其它给药模式更加有利于肿瘤的治疗，对动物身体整体状况的影响较小；从图16中可以看出，到实验终止时，尾静脉注射过Her2 TCR-6A5-mC T细胞的两组动物中，第四组动物的肿瘤内中与第三组相比仍旧存活着较多的人T细胞(CD3 +、CD8 +和CD4 +)，这可能是因为瘤内注射了溶瘤腺病毒OAd-E75A2引起了更多的人Her2 TCR-6A5-mC T在肿瘤内的归巢；因此第四组也体现出了对肿瘤生长的明显抑制作用。瘤内注射了Her2 TCR-6A5-mC T的第五组和第六组动物的皮下肿瘤中，在实验终止时仍然存活着大量的人T细胞(CD3 +、CD8 +和CD4 +)，而且第六组联合给药的动物肿瘤内的CD8 +T细胞明显少于第五组单独瘤内注射Her2 TCR-6A5-mC T的动物肿瘤内的CD8 +T细胞，与第四组动物肿瘤内的人T细胞数量相仿，更为重要的是第六组动物皮下肿瘤的生长抑制作用较第四组更为明显。这可能是因为在瘤内注射OAd-E75A2后再次瘤内输注人Her2 TCR-6A5-mC T，能够使Her2 TCR-6A5-mC T更加方便地作用于肿瘤细胞，从而表现出了比第四组更好的抑瘤效果。
综上所述，两组联合用药组(第四组和第六组)在瘤内注射溶瘤腺病毒OAd-E75A2后再次给予人Her2 TCR-6A5-mC T细胞(静脉内注射和瘤内注射)治疗后都表现出了非常显著的抑瘤效果，这就充分说明了溶瘤病毒OAd-E75A2感染肿瘤细胞并表达HLAA2和E75表位肽后，使得随后给予的Her2 TCR-6A5-mC T可以识别感染了OAd-E75A2后的肿瘤细胞表面由HLAA2和E75组成的复合物继而活化Her2 TCR-6A5-mC T，最终发挥溶瘤腺病毒和TCR T细胞的双重抑瘤作用；而另外两个Her2 TCR-6A5-mC T单药组(静脉内注射和瘤内注射)中动物皮下的肿瘤因为不表达HLAA2而不能有效呈递HER2的E75表位肽，不能被Her2 TCR-6A5-mC T所识别也就不能活化Her2 TCR-6A5-mC T，因此未能表现出明显的抑瘤作用。溶瘤腺病毒单药组(第二组)中的动物只是表现出了较弱的抑瘤效果。上述结果显示本发明的联合用药可以较大地提高肿瘤的治疗效果。
讨论：
过继转输经肿瘤特异性TCR基因修饰的T细胞是治疗恶性实体瘤最有前景的免疫细胞疗法(TCR-T疗法)。如果TCR-T所靶向的肿瘤抗原是来源于自身蛋白的肿瘤相关抗原，TCR-T的特异性TCR的亲和性可能不足以识别被肿瘤细胞所提呈的微量HLA/抗原表位多肽复合物而对肿瘤细胞有效杀伤。另外，肿瘤微环境不仅可以造成肿瘤组织内的免疫抑制状态，还可以导致肿瘤细胞HLA I类分子的表达降低或缺失，肿瘤细胞内I类抗原提呈机制也可能出现缺陷，从而使肿瘤抗原表位不能被MHC I类分子有效提呈，这也限制了TCR-T识别和杀伤肿瘤细胞的功能。溶瘤病毒不仅可以选择性在肿瘤细胞内复制和裂解肿瘤细胞，还可以通过其自身免疫原性缓解肿瘤局部的免疫抑制状态。溶瘤病毒还可以作为基因载体，选择性地在肿瘤细胞内表达外源基因。如果通过溶瘤腺病毒选择性地在肿瘤细胞内表达HLA I类分子和肿瘤抗原表位多肽，则可以增加肿瘤细胞表面HLA I类分子和抗原表位多肽复合物的数量，从而增强TCR-T识别和杀伤肿瘤细胞的敏感性。另外，联合使用TCR-T以及表达HLA I类分子以及抗原表位多肽的溶瘤病毒，不仅表现出两者在特异性杀伤肿瘤细胞过程中的协同效应，还可以扩大TCR-T的应用范围。例如，表达本发明所述的Her2 TCR的TCR-T单独使用时，因受HLA-A2限制，只能针对HLA-A2阳性Her2/neu阳性肿瘤患者。如果联合本发明所述的溶瘤腺病毒，通过选择性感染肿瘤细胞并表达外源性HLA-A2分子和Her2/neu抗原表位多肽，使得HLA-A2阴性的肿瘤，以及低表达甚至不表达Her2/neu抗原的肿瘤细胞均可成为基于Her2 TCR的TCR-T的靶细胞，可以避免过继TCR-T细胞治疗所面临的HLA限制的局限性，从而大大增加了TCR-T的应用范围。
总之，本发明所述技术和方法，为联合应用过继转输经特异性TCR修饰的T细胞以及表达HLA I类分子和肿瘤抗原表位多肽的溶瘤病毒来治疗肿瘤提供了新的途径。

Claims (50)

  1. 一种用于治疗肿瘤和/或癌症的治疗剂，包含：
    (a)第一组合物，其中该第一组合物包含位于第一可药用载体中的第一活性成分，该第一活性成分包括或含有用于导入肿瘤细胞和/或癌细胞的、具有标记性多肽编码序列的核酸；所述标记性多肽具有一个或多个抗原表位多肽的氨基酸序列，所述抗原表位多肽能够被MHC I类分子提呈在所述肿瘤细胞和/或癌细胞表面；和
    (b)第二组合物，其中该第二组合物包含位于第二可药用载体中的第二活性成分，该第二活性成分包含T细胞受体修饰的免疫细胞；所述的T细胞受体修饰的免疫细胞能够特异性识别并结合被所述MHC I类分子提呈的所述抗原表位多肽。
  2. 根据权利要求1所述的治疗剂，其中所述抗原表位多肽的氨基酸序列来源于自然界存在的蛋白的氨基酸序列，或者为人工合成的自然界不存在的氨基酸序列；优选地，所述自然界存在的蛋白包括人源蛋白和除人以外的其它物种的蛋白。
  3. 根据权利要求1所述的治疗剂，其中所述抗原表位多肽的氨基酸序列来源于肿瘤相关抗原或肿瘤特异性抗原的氨基酸序列。
  4. 根据权利要求1所述的治疗剂，其中所述标记性多肽包括可操作地连接的、依次串联的以下氨基酸序列：N端信号肽的氨基酸序列、一个或多个所述抗原表位多肽的氨基酸序列、可任选的C端内质网滞留信号的氨基酸序列，其中当所述标记性多肽包括多个所述抗原表位多肽的氨基酸序列时，每两个相邻的所述抗原表位多肽的氨基酸序列之间由可切割性连接多肽的氨基酸序列连接。
  5. 根据权利要求4所述的治疗剂，其中所述抗原表位多肽的氨基酸序列包括如SEQ ID NO：3所示的Her2/neu 369-377、NY-ESO-1  157-165、NY-ESO-1 1-11、NY-ESO-1 53-62、NY-ESO-1 18-27、N-ras 55-64、K-ras 224-232、K-ras 10-18、K-ras 10-19、H3.3K27M 26-35、SSX-2 41-49、MAGE-C2 336-344、MAGE-C2 191-200、MAGE-C2 307-315、MAGE-C2 42-50、MAGE-A1 120-129、MAGE-A1 230-238、MAGE-A1 161-169、KK-LC-1 76-84、p53 99-107、HPV16-E6 29-38、HPV16-E7 11-19、HPV16-E7 11-19、EBV-LMP1 51-59、EBV-LMP1 125-133。
  6. 根据权利要求1所述的治疗剂，其中所述核酸还具有HLA蛋白编码序列，其中该HLA蛋白编码序列与所述标记性多肽编码序列分别在各自的启动子控制之下，或者该HLA蛋白编码序列与所述标记性多肽编码序列在同一启动子控制之下并且该HLA蛋白编码序列通过可切割性连接多肽编码序列与所述标记性多肽编码序列可操作地连接。
  7. 根据权利要求6所述的治疗剂，其中所述HLA蛋白为HLA-A2蛋白，所述HLA-A2的氨基酸序列如SEQ ID NO：29所示。
  8. 根据权利要求1所述的治疗剂，其中所述第一组合物和所述第二组合物各自独立地存在于所述治疗剂中而互不混合。
  9. 根据权利要求1所述的治疗剂，其中所述核酸包括DNA或RNA；所述RNA包括由所述DNA转录的mRNA。
  10. 根据权利要求1所述的治疗剂，其中所述第一活性成分为重组病毒，所述重组病毒的基因组具有所述标记性多肽编码序列和可任选的HLA蛋白编码序列；其中，所述重组病毒包括选择复制型重组溶瘤病毒或复制缺陷型重组病毒。
  11. 根据权利要求10所述的治疗剂，其中所述复制缺陷型重组 病毒来源于腺病毒、腺病毒相关病毒(AAV)、单纯疱疹病毒、痘病毒、流感病毒、甲病毒(Alphavirus)、或仙台病毒。
  12. 根据权利要求10所述的治疗剂，其中所述复制缺陷型重组病毒为对5型腺病毒进行基因改造而得到的重组腺病毒，该重组腺病毒的基因组中缺失了E1基因，并且在所缺失的E1基因的位置包含所述标记性多肽编码序列和可任选的所述HLA蛋白编码序列。
  13. 根据权利要求10所述的治疗剂，其中所述选择复制型重组溶瘤病毒来源于具有溶瘤作用的经基因突变的病毒和具有溶瘤作用的野生型病毒；优选地，所述选择复制型重组溶瘤病毒来源于具有溶瘤作用的腺病毒、痘病毒、单纯疱疹病毒、麻疹病毒、塞姆利基森林病毒、水疱性口炎病毒、脊髓灰质炎病毒、逆转录病毒、呼肠孤病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒和马拉巴病毒。
  14. 根据权利要求10所述的治疗剂，其中所述选择复制型重组溶瘤病毒为对5型腺病毒进行基因改造而得到的重组溶瘤腺病毒，该重组溶瘤腺病毒的基因组中缺失了E1B-55K基因和/或E1B-19K基因，并且所述重组溶瘤腺病毒的基因组中包含E1A基因编码序列；优选地，所述E1A基因编码序列是在外源启动子控制下的。
  15. 根据权利要求10所述的治疗剂，其中所述重组溶瘤病毒为对5型腺病毒进行基因改造而得到的重组溶瘤腺病毒，该重组溶瘤腺病毒的E1A基因被改变为使得所表达的E1A蛋白无法与pRb蛋白结合；优选地，所述E1A基因编码序列是在外源启动子控制下的。
  16. 根据权利要求14或15所述的治疗剂，其中所述重组溶瘤腺病毒的E3基因全部或部分缺失。
  17. 根据权利要求1所述的治疗剂，其中所述免疫细胞包括原始T细胞或其前体细胞，NKT细胞，或T细胞株。
  18. 根据权利要求9所述的治疗剂，其中所述第一组合物包含治疗有效量的所述DNA、或治疗有效量的所述mRNA。
  19. 根据权利要求10所述的治疗剂，其中所述第一组合物包含治疗有效量的所述重组病毒。
  20. 根据权利要求1所述的治疗剂，其中所述第二组合物包含治疗有效量的所述的T细胞受体修饰的免疫细胞。
  21. 根据权利要求9所述的治疗剂，其中所述DNA配制成通过瘤内注射给药；所述mRNA配制成通过瘤内注射给药。
  22. 根据权利要求10所述的治疗剂，其中所述重组病毒配制成通过瘤内注射给药、腹膜内给药、蛛网膜下腔内给药、或静脉给药。
  23. 根据权利要求1所述的治疗剂，其中所述免疫细胞配制成通过动脉、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。
  24. 根据权利要求1所述的治疗剂，其中所述治疗剂由所述第一组合物和所述第二组合物组成。
  25. 根据权利要求1-24中任一项所述的治疗剂在制备用于治疗肿瘤和/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/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌， 食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
  27. 一种标记性多肽，包括可操作地连接的依次串联的以下氨基酸序列：N端信号肽的氨基酸序列、一个或多个抗原表位多肽的氨基酸序列、可任选的C端内质网滞留信号的氨基酸序列，其中当所述标记性多肽包括多个所述抗原表位多肽的氨基酸序列时，每两个相邻的所述抗原表位多肽的氨基酸序列之间由可切割性连接多肽的氨基酸序列连接；优选地，所述可切割性连接多肽是furin酶切识别多肽。
  28. 根据权利要求27所述的标记性多肽，其中所述抗原表位多肽的氨基酸序列包括如SEQ ID NO：3所示的Her2/neu 369-377、NY-ESO-1 157-165、NY-ESO-1 1-11、NY-ESO-1 53-62、NY-ESO-1 18-27、N-ras 55-64、K-ras 224-232、K-ras 10-18、K-ras 10-19、H3.3K27M 26-35、SSX-241-49、MAGE-C2336-344、MAGE-C2191-200、MAGE-C2 307-315、MAGE-C2 42-50、MAGE-A1 120-129、MAGE-A1 230-238、MAGE-A1 161-169、KK-LC-1 76-84、p53 99-107、HPV16-E6 29-38、HPV16-E7 11-19、HPV16-E7 11-19、EBV-LMP1 51-59、EBV-LMP1 125-133。
  29. 根据权利要求27所述的标记性多肽，其中所述标记性多肽的氨基酸序列具有与如SEQ ID NO：24、SEQ ID NO：36、SEQ ID  NO：56、或SEQ ID NO：60所示的氨基酸序列至少98％的一致性；优选的是，其中所述标记性多肽的氨基酸序列如SEQ ID NO：24、SEQ ID NO：36、SEQ ID NO：56、或SEQ ID NO：60所示。
  30. 一种分离的、具有根据权利要求27-29中任一项所述的标记性多肽的编码序列的核酸。
  31. 根据权利要求30所述的核酸，其中所述核酸还具有HLA蛋白编码序列，其中该HLA蛋白编码序列与所述标记性多肽编码序列分别在各自的启动子控制之下，或者该HLA蛋白编码序列与所述标记性多肽编码序列在同一启动子控制之下并且该HLA蛋白编码序列通过可切割性连接多肽编码序列与所述标记性多肽编码序列可操作地连接。
  32. 根据权利要求30所述的核酸，其中所述核酸包括DNA和mRNA。
  33. 根据权利要求32所述的核酸，其中所述核酸为DNA，其核苷酸序列如SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：57、SEQ ID NO：58、或SEQ ID NO：61所示。
  34. 一种重组表达载体，含有根据权利要求30-33中任一项所述的核酸，和/或其互补序列。
  35. 一种分离的重组病毒，其中所述重组病毒的基因组具有根据权利要求30-33中任一项所述的核酸；并且，所述重组病毒包括选择复制型重组溶瘤病毒或复制缺陷型重组病毒。
  36. 根据权利要求35所述的重组病毒，其中所述复制缺陷型重组病毒来源于腺病毒、腺病毒相关病毒(AAV)、单纯疱疹病毒、痘 病毒、流感病毒、甲病毒(Alphavirus)、或仙台病毒。
  37. 根据权利要求35所述的重组病毒，其中所述复制缺陷型重组病毒为对5型腺病毒进行基因改造而得到的重组腺病毒，该重组腺病毒的基因组中缺失了E1基因，并且在所缺失的E1基因的位置包含所述标记性多肽编码序列和可任选的所述HLA蛋白编码序列。
  38. 根据权利要求35所述的重组病毒，其中所述重组病毒为选择复制型重组溶瘤病毒，并且所述选择复制型重组溶瘤病毒来源于具有溶瘤作用的经基因突变的病毒和具有溶瘤作用的野生型病毒；优选地，所述选择复制型重组溶瘤病毒来源于具有溶瘤作用的腺病毒、痘病毒、单纯疱疹病毒、麻疹病毒、塞姆利基森林病毒、水疱性口炎病毒、脊髓灰质炎病毒、逆转录病毒、呼肠孤病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒和马拉巴病毒。
  39. 根据权利要求35所述的重组病毒，其中所述选择复制型重组溶瘤病毒为对5型腺病毒进行基因改造而得到的重组溶瘤腺病毒，该重组溶瘤腺病毒的基因组中缺失了E1B-55K和/或E1B-19K基因，并且所述重组溶瘤腺病毒的基因组中包含E1A基因编码序列；优选地，所述E1A基因编码序列是在外源启动子控制下的。
  40. 根据权利要求35所述的重组病毒，其中所述选择复制型重组溶瘤病毒为对5型腺病毒进行基因改造而得到的重组溶瘤腺病毒，该重组溶瘤腺病毒的E1A基因被改变为使得所表达的E1A蛋白无法与pRb蛋白结合；优选地，所述E1A基因编码序列是在外源启动子控制下的。
  41. 根据权利要求39或40所述的重组病毒，其中所述选择复制型重组溶瘤病毒的E3基因全部或部分缺失。
  42. 一种用于治疗肿瘤和/或癌症的具有协同作用的联合药物的药盒，包括：
    第一容器，该第一容器装有根据权利要求1-24中任一项所述的治疗剂中的第一组合物；
    第二容器，该第二容器装有根据权利要求1-24中任一项所述的治疗剂中的第二组合物，其中所述第一容器和所述第二容器是独立的；以及
    载明给药时机和给药方式的说明书。
  43. 根据权利要求30-33中任一项所述的核酸在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
  44. 根据权利要求34所述的重组表达载体在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
  45. 根据权利要求35-41中任一项所述的重组病毒在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
  46. 根据权利要求42所述的药盒在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。
  47. 根据权利要求43-46中任一项所述的用途，其中所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰 腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
  48. 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1-24中任一项所述的治疗剂中的第一组合物；和
    对所述肿瘤和/或癌症患者施用根据权利要求1-24中任一项所述的治疗剂中的第二组合物。
  49. 根据权利要求48所述的方法，包括以下依次进行的步骤：
    1)对所述肿瘤和/或癌症患者施用所述第一组合物；和
    2)在施用所述第一组合物之后，对所述肿瘤和/或癌症患者施用所述的治疗剂中的第二组合物。
  50. 根据权利要求48所述的方法，其中所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
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