WO2015197016A1 - 时空可调性抑制病理性靶细胞的系统 - Google Patents

时空可调性抑制病理性靶细胞的系统 Download PDF

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WO2015197016A1
WO2015197016A1 PCT/CN2015/082460 CN2015082460W WO2015197016A1 WO 2015197016 A1 WO2015197016 A1 WO 2015197016A1 CN 2015082460 W CN2015082460 W CN 2015082460W WO 2015197016 A1 WO2015197016 A1 WO 2015197016A1
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cells
tumor
wte
antibody
polypeptide
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French (fr)
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王华茂
宋波
王鹏
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科济生物医药（上海）有限公司
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Publication of WO2015197016A1 publication Critical patent/WO2015197016A1/zh

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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/47—Brain; Nervous system
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2319/00—Fusion polypeptide
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    • C07—ORGANIC CHEMISTRY
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Definitions

  • the present invention relates to the field of tumor immunology, and more particularly to a system for suppressing pathological target cells by spatiotemporal temperability.
  • CAR tumor-specific chimeric antigen receptor
  • TAA tumor-associated antigen
  • scFv tumor-associated antigen
  • NK cell activation sequence T cell or NK cell activation sequence in vitro to form a recombinant plasmid, which is purified by transfection in vitro.
  • TAA tumor-associated antigen
  • scFv tumor-associated antigen
  • NK cells T cell or NK cells
  • CAR mainly comprises an antigen binding portion (extracellular domain) of a TAA-specific antibody, as well as a T cell costimulatory structure (CD137 and CD28) and a signaling structure (CD3 ⁇ intracellular domain).
  • CAR T cells The expansion of CAR T cells demonstrated by the Institute, its sustained activity, its transformation into memory cells, and its anti-tumor effects are outstanding. However, its toxic effects cannot be ignored. In some tumors, CAR T cells recognize that their normal tissues express target antigens or activate their own T cells to induce autoimmune responses. Continuously activated T cells and memory T cells may constitute substantial harm, such as organ targets due to cross-reactivity. To toxicity and so on.
  • a system for inhibiting pathological target cells comprising:
  • a fusion protein comprising a polypeptide tag and a binding molecule that specifically recognizes a pathological target cell
  • a chimeric antigen receptor (CAR) immune effector cell that expresses a binding molecule (including an antibody or a ligand that recognizes the tag of the polypeptide, etc.) that specifically recognizes the polypeptide tag.
  • a binding molecule including an antibody or a ligand that recognizes the tag of the polypeptide, etc.
  • the polypeptide tag is an unrelated antigen having low immunogenicity (including non-immunogenicity) that is low or not expressed in non-tumor tissue.
  • polypeptide tag is an endogenous or exogenous polypeptide.
  • polypeptide tag is selected from the group consisting of, but not limited to, WTE, E-tag, Flag, Myc, His6, and the like.
  • polypeptide tag is a WTE tag.
  • polypeptide tag is a polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO:38.
  • polypeptide tag may be fused to the N-terminus or C-terminus of the binding molecule that specifically recognizes a pathological target cell, or may be fused at both the N-terminus and the C-terminus of the antibody.
  • the pathological target cell is a tumor cell
  • the binding molecule that specifically recognizes a pathological target cell binds to a tumor-associated antigen on the tumor cell.
  • the tumor associated antigen is selected from, but not limited to:
  • EGFR EGFRvIII, de4 EGFR, EpCAM, CD19, CD20, CD33, HER2, EphA2, IL13R, GD2, LMP1, Claudin 18.A2, PLAC1, NY-ESO-1, MAGE4, MUC1, MUC16, LeY, CEA, GPC3, Mesothelin , CAIX (carbonic anhydrase IX), CD123, IL13R, EphA2.
  • the binding molecule is a ligand or an antibody, including but not limited to: Fab, F(ab'), F(ab') 2 , Fv, dAb, Fd, complementary Determining region (CDR) fragment, single chain antibody (scFv), bivalent single chain antibody, single chain phage antibody, bispecific diaborating antibody, triple chain antibody, four chain antibody; monoclonal antibody.
  • the tumor includes, but is not limited to, liver cancer, lung cancer, glioma, breast cancer, gastric cancer, prostate cancer, brain tumor, ovarian cancer, bone tumor, colon cancer, thyroid tumor, mediastinum Tumor, intestinal tumor, renal tumor, adrenal tumor, bladder tumor, testicular tumor, malignant lymphoma, multiple myeloma, nervous system tumor, esophageal cancer, thymic mesothelioma, pancreatic cancer, leukemia, head and neck cancer, cervical cancer , skin cancer, melanoma, vaginal epithelial cancer, gallbladder cancer, malignant fibrous histiocytoma.
  • the immune effector cells comprise: T lymphocytes (including CD4 + or CD8 + T lymphocytes), NK cells.
  • the pathological target cell is a tumor cell that expresses (preferably, highly expresses) EGFRvIII;
  • the binding molecule that specifically recognizes a pathological target cell is an antibody (preferably a CH12 antibody) that specifically binds to EGFRvIII.
  • the chimeric antigen receptor immune effector cells recombinantly express CD28 (preferably including CD28a, CD28b), CD137, CD3 ⁇ (preferably CD3 ⁇ intracellular domain), CD27, CD8, CD19 One or more of CD134, CD20, FcR ⁇ .
  • the chimeric antigen receptor immune effector cell comprises a construct comprising the following operably linked elements: a binding molecule coding sequence that specifically recognizes the polypeptide tag, a CD8 hinge Region, CD28a, CD28b, CD137, CD3 ⁇ (preferably also containing eGFP, F2A). More preferably, the elements of the construct are joined in the following order (5' ⁇ 3'): a binding molecule coding sequence that specifically recognizes the polypeptide tag, a CD8 hinge region, CD28a, CD28b, CD137, CD3 ⁇ (preferably The 5' end of the ground also contains (5' ⁇ 3') eGFP, F2A).
  • the use of any of the systems described above for the preparation of a pathological target Cellular kit Preferably, the use is for non-therapeutic use.
  • kits for preparing the kit comprising:
  • an expression construct a comprising an expression cassette of a fusion protein (which can be expressed in an immune cell), the fusion protein comprising a polypeptide tag and a binding molecule that specifically recognizes a pathological target cell;
  • an expression construct b comprising an expression cassette (which can be expressed in an immune cell) that expresses a binding molecule that specifically recognizes the polypeptide tag (including an antibody or ligand that recognizes the polypeptide tag, etc.);
  • the pathological target cell is a tumor cell
  • the binding molecule that specifically recognizes a pathological target cell binds to a tumor-associated antigen on the tumor cell
  • the chimeric antigen receptor immune effector cells recombinantly express CD28 (preferably including CD28a, CD28b), CD137, CD3 ⁇ (preferably CD3 ⁇ intracellular domain), CD27, CD8, CD19, CD134, CD20, FcR ⁇ One or more.
  • the expression construct a or the expression construct b may be a vector or a plurality of vectors.
  • a method of inhibiting a pathological target cell comprising: administering to the subject the system of inhibiting a pathological target cell.
  • a method for inhibiting pathological target cells by spatial-temporal tunability comprising: administering to a subject a chimeric antigen receptor immune effector cell, which expresses a binding of a specific recognition polypeptide tag a molecule; when it is desired to inhibit a pathological target cell, the fusion protein is administered to the subject, the fusion protein comprising a polypeptide tag and a binding molecule that specifically recognizes the pathological target cell, thereby mediating the immune effector cell to kill the pathological target cell effect.
  • an isolated polypeptide (WTE tag) is provided, the amino acid sequence of which is encoded by the nucleotide sequence set forth in SEQ ID NO:38.
  • an isolated polynucleotide is provided, the nucleotide sequence of which is set forth in SEQ ID NO: 38 or a degenerate sequence thereof.
  • a single chain antibody that specifically binds to the polypeptide is provided, the single chain antibody being encoded by the nucleotide sequence set forth in SEQ ID NO:35.
  • a polynucleotide encoding the single chain antibody is provided, the nucleotide sequence of which is set forth in SEQ ID NO: 35 or a degenerate sequence thereof.
  • Figure 1 Schematic diagram of the structure of the pK/WTE-CH12L expression vector.
  • FIG. 1 Schematic diagram of the structure of the pH/WTE-CH12H expression vector.
  • FIG. Schematic diagram of the structure of the antibody WTE-CH12.
  • Figure 4 is a schematic diagram showing the structure of a lentiviral vector pWPT/eGFP-2D8 (anti-WTE)-CD28a-CD28b-CD137-CD3 ⁇ encoding a CAR sequence.
  • FIG. 6A Determination of specific binding of WTE-CH12 antibodies to U87MG tumor cells as indicated by fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • FIG. 6B Determination of specific binding of WTE-CH12 antibodies to U87MG-EGFRvIII tumor cells as indicated by fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • FIG. 6C Determination of specific binding of WTE-CH12 antibodies to Huh-7 tumor cells as indicated by fluorescence activated cell sorter (FACS).
  • FIG. 6D Determination of specific binding of WTE-CH12 antibodies to Huh-7-EGFRvIII tumor cells as indicated by fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • FIG. 7A Proportion of CAR + T cells after lentiviral vector infection by fluorescence activated cell sorter (FACS).
  • FIG. 7B Proportion of CAR + CD4 + T cells after lentiviral vector infection by fluorescence activated cell sorter (FACS).
  • FIG. 7C Proportion of CAR + CD8 + T cells displayed by fluorescence activated cell sorter (FACS).
  • Figure 8A is a comparison of the killing rates of CAR + T cells against U87MG, U87MG-EGFRvIII tumor cells induced by serial dilutions of WTE-CH12 antibody.
  • Figure 8B Comparison of the killing rates of CAR + T cells against Huh-7, Huh-7-EGFRvIII tumor cells induced by serial dilutions of WTE-CH12 antibody.
  • Figure 9 In vitro competitive inhibition assay of WTE-CH12 mediated cytotoxic effects of WTE-CH12-mediated T lymphocytes expressing chimeric antigen receptors.
  • FIG. 10 Anti-tumor activity assay of WTE-CH12 antibody-induced CAR + T cell treated and control groups in a NOD/SCID tumor-bearing (U87MG-EGFRvIII) mouse model.
  • CAR immune effector cells such as CAR T modified by the present inventors
  • Cells can only target pathological target cells in the presence of mediators, achieving sustained expansion of CAR immune effector cells and exerting a killing effect on tumor cells; whereas in the absence of mediators, CAR Immune effector cells do not function (or play a weaker role).
  • the present invention provides a solution for avoiding the toxic effects of CAR immune effector cells in vivo expansion and cross-reaction to their normal tissues.
  • the present invention replaces a binding molecule that recognizes a pathological target cell-associated antigen (such as a tumor-associated antigen) in a chimeric antigen receptor (CAR) immune effector cell with a binding molecule (such as a single-chain antibody) that recognizes a polypeptide tag (unrelated antigen),
  • a binding molecule such as a single-chain antibody
  • the binding molecule recognizing the pathological target cell-associated antigen and the polypeptide tag are fused to obtain a fusion protein.
  • the selective regulation of the recognition signal can be achieved by using a pattern in which the binding molecule recognizing the pathological target cell-associated antigen is released from the conventional CAR immune effector cell, and the fusion protein is stopped after the pathological target cell is cleared.
  • the blocking of this signal prevents the CAR T cells from recognizing the target antigen that is under-expressed in its normal tissues and its continuous amplification, which may solve this problem. Toxic effects.
  • chimeric antigen receptor (CAR) immune effector cells is well known in the art and is an immune effector cell that expresses a tumor-specific chimeric antigen receptor using genetic engineering techniques and exhibits certain in vitro and clinical trials. Targeting, killing activity and persistence are adoptive cell immunotherapy methods.
  • the immune effector cells include, for example, T cells, NK cells.
  • chimeric antigen receptor immune effector cells Conventional methods for preparing "chimeric antigen receptor immune effector cells” are known to those skilled in the art, including allowing them to express intracellular co-stimulatory cellular intracellular domains, such as CD28 (preferably including CD28a, CD28b). , CD137, CD27, CD3 ⁇ (preferably CD3 ⁇ intracellular domain), one or more of CD8, CD19, CD134, CD20, FcR ⁇ .
  • CD28 preferably including CD28a, CD28b
  • CD137, CD27, CD3 ⁇ preferably CD3 ⁇ intracellular domain
  • CD8 preferably CD19, CD134, CD20, FcR ⁇
  • the pathological target cell may be various harmful cells that are not healthy for the body, and cells that are necessary to be removed from the body.
  • the pathological target cells include tumor cells. Any tumor known in the art can be included in the present invention as long as the tumor is capable of expressing a tumor-associated antigen that is lowly expressed in normal tissues.
  • the tumor includes, but is not limited to, liver cancer, lung cancer, glioma, breast cancer, gastric cancer, prostate cancer, brain tumor, ovarian cancer, bone tumor, colon cancer, thyroid tumor, mediastinal tumor, intestinal tumor, Renal tumor, adrenal tumor, bladder tumor, testicular tumor, malignant lymphoma, multiple myeloma, nervous system tumor, esophageal cancer, thymic mesothelioma, pancreatic cancer, leukemia, head and neck cancer, cervical cancer, skin cancer, melanin Tumor, vaginal epithelial cancer, gallbladder cancer, malignant fibrous histiocytoma.
  • the tumor associated antigens include, but are not limited to, EGFR, EGFRvIII, de4 EGFR, EpCAM, CD19, CD20, CD33, HER2, EphA2, IL13R, GD2, LMP1, Claudin 18.A2, PLAC1, NY-ESO- 1, MAGE4, MUC1, MUC16, LeY, CEA, GPC3, Mesothelin, CAIX (carbonated Anhydride IX), CD123, IL13R, EphA2.
  • the polypeptide tag is an antigen which is lowly expressed (negatively expressed) or not expressed in non-pathological tissues and has low immunogenicity, and does not induce significant immunity in vivo.
  • the reaction which may be an endogenous or exogenous polypeptide. Any unrelated antigen that satisfies the above requirements may be included in the present invention, such as, but not limited to, WTE, E-tag, Flag, Myc, His6, and the like.
  • the "binding molecule that recognizes a polypeptide tag” is a binding molecule that specifically recognizes or binds to the tag of the polypeptide, and may be an antibody or a ligand.
  • Such antibodies include, but are not limited to, Fab, F(ab'), F(ab') 2 , Fv, dAb, Fd, complementarity determining region (CDR) fragments, single chain antibodies (scFv), bivalent single strands Antibody, single-chain phage antibody, bispecific di-chain antibody, tri-chain antibody, tetra-chain antibody; monoclonal antibody.
  • the antibody is a single chain antibody.
  • the action of targeting a pathological target cell is exerted by the binding molecule that specifically recognizes a pathological target cell.
  • the binding molecule that specifically recognizes the pathological target cell carries the immune effector cell to the pathological target cell while targeting the pathological target cell. Play a killing effect.
  • the "binding molecule that specifically recognizes a pathological target cell” may be any binding molecule that specifically recognizes a pathological target cell-associated antigen. Clinically, which binding molecule is applied can be determined depending on the type of pathological target cell to be killed. For example, when the pathological target cell is a glioma cell (such as U87MG) or a liver cancer cell (Huh-7) that specifically expresses EGFRvIII, it is suitable to use an antibody that specifically binds to EGFRvIII.
  • the present inventors replaced a single-chain antibody recognizing the tumor antigen EGFRvIII in a CAR T cell with a single-chain antibody recognizing an unrelated antigen (polypeptide WTE), and simultaneously recognizes a tumor-associated antigen (EGFRvIII).
  • the cloned antibody CH12 and the unrelated antigenic polypeptide WTE (derived from the EGFR NM_005228 intracellular domain 1189-1210 amino acid) were respectively ligated via a ligation peptide (see SEQ ID: 44, SEQ ID NO: 47 in US Pat. No. 7,612,181) for expression of the recombinant protein and preparation.
  • the invention also relates to a kit comprising the system for inhibiting pathological target cells, comprising (1) a fusion protein comprising a polypeptide tag and a binding molecule that specifically recognizes a pathological target cell; and (2) a chimeric antigen A receptor immune effector cell that expresses a binding molecule that specifically recognizes the tag of the polypeptide. Instructions for use indicating the usage may also be included in the kit.
  • the antibody pH/CH12 was used as a template (see SEQ ID NO: 36 for the sequence).
  • the upstream primer 5'-gatgtgcagcttcaggagtcggg-3' (SEQ ID NO: 1) and the downstream primer 5'-acaataatatgtggctgtg tcc-3' (SEQ ID NO: 2) were PCR-amplified for CH12VH fragment, and the PCR amplification conditions were pre-denaturation: 94 °C. , 4 min; denaturation: 94 ° C, 40 s; annealing: 58 ° C, 40 s; extension: 68 ° C, 40 s; 27 cycles, then total extension 68 ° C, 10 min.
  • the size of the amplified product was 288 bp, which was in agreement with expectations.
  • the first step uses Overlap PCR with SEQ ID NO: 3 to SEQ ID NO: 8 as primers for synthesizing WTE fragment (SEQ ID NO: 38) and heavy chain signal peptide sequence (SEQ ID NO: 39), PCR amplification
  • the conditions were pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 58 ° C, 40 s; extension: 68 ° C, 40 s; 7 cycles, then total extension 68 ° C, 10 min.
  • the first step of the Overlap PCR bridge product is used as a template, and the heavy chain signal peptide-WTE fragment is amplified by using SEQ ID NO: 3 and SEQ ID NO: 8 as upstream and downstream primers respectively.
  • the PCR amplification conditions are: Denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 58 ° C, 40 s; extension: 68 ° C, 40 s; 27 cycles, then total extension 68 ° C, 10 min.
  • the size of the amplified product was 134 bp, which was in agreement with expectations.
  • SEQ ID NO: 11 to SEQ ID NO: 16 were used as primers for synthesizing WTE fragment and light chain signal peptide sequence (SEQ ID NO: 40); the first step of OverlapPCR amplification was pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 58 ° C, 40 s; extension: 68 ° C, 40 s; 7 cycles, then total extension 68 ° C, 10 min.
  • the first step PCR bridge product is used as a template, and the light chain signal peptide-WTE fragment is amplified by using SEQ ID NO: 11 and SEQ ID NO: 16 as upstream and downstream primers respectively, and the PCR amplification condition is pre-denaturation. : 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 58 ° C, 40 s; extension: 68 ° C, 40 s; 27 cycles, then a total extension of 68 ° C, 10 min.
  • the size of the amplified product was 179 bp, which was in agreement with expectations.
  • Light chain signal peptide-WTE-CH12Vk fragment splicing conditions light chain signal peptide-WTE (50ng) + CH12Vk (50ng) pre-denaturation: 94 ° C, 4min; denaturation: 94 ° C, 30s; annealing: 60 ° C , 30s; extension: 68 ° C, 30 s, 7 cycles, then total extension 68 ° C, 10 min, supplement DNA polymerase and upstream primer 5 '-gatcgatatccaccatggacatgatggtccttgctcagtttcttgcattcttgttg-3' (SEQ ID NO: 11) and downstream primer 5'- Gaagacagatggtgcagccac-3' (SEQ ID NO: 10) was PCR amplified for 25 cycles and spliced to obtain the light chain signal peptide - WTE-CH12Vk.
  • the amplification conditions were pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 30 s; annealing: 60 ° C, 30 s; extension: 68 ° C, 30 s, for 25 cycles, then total extension 68 ° C, 10 min.
  • the theoretical size is 509 bp.
  • the amplified product was confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.
  • the amplified sequence heavy chain signal peptide-WTE-CH12VH and pH/CH12 were simultaneously digested with restriction endonuclease NheI/EcoRI, and double-digested according to the reaction conditions recommended by the enzyme supplier (New England Biolabs, NEB). .
  • the double-digested heavy chain signal peptide-WTE-CH12 VH fragment and pH/CH12 vector fragment were then ligated with T4 DNA ligase according to the reaction conditions recommended by the enzyme supplier (NEB).
  • the nucleotide sequence encoding the WTE-CH12 VH antibody polypeptide is thus cloned into a vector.
  • the resulting new vector containing the coding sequence for the WTE-CH12 VH antibody polypeptide was designated pH/WTE-CH12H and its structure is shown in Figure 2.
  • the amplified sequence light chain signal peptides -WTE-CH12Vk and pK/CH12K were simultaneously digested with restriction endonuclease EcoRV/BsiWI, and double-digested according to the reaction conditions recommended by the enzyme supplier (New England Biolabs, NEB). .
  • the double-digested heavy chain signal peptide-WTE-CH12 VK fragment and pK/CH12 vector fragment were then ligated with T4 DNA ligase according to the reaction conditions recommended by the enzyme supplier (NEB).
  • the nucleotide sequence encoding the WTE-CH12VK antibody polypeptide is thus cloned into a vector.
  • the resulting new vector containing the WTE-CH12VK antibody polypeptide was named pK/WTE-CH12L, and its detailed structure is shown in FIG.
  • FreeStyle 293-F cells Invitrogen Corp.
  • suspension culture and transfection of FreeStyle TM 293Expression System A method according to instructions. Specifically, the cell density was adjusted to 1 ⁇ 10 6 /mL before transfection, the cells were blown off and the cells were not agglomerated, and the cell viability was determined to be >95% by trypan blue staining.
  • Transfection step 52 ⁇ g of pH/WTE-CH12H and 48 ⁇ g of pK/WTE-CH12K (molar ratio 1:1) recombinant plasmid and 200 ⁇ L of Free-Style 293-F cell liposome transfection reagent "293fectin" with Opti-MEM Dilute to 3.33mL, let stand for 5min, slowly mix the plasmid with the transfection reagent, react at room temperature for 20min, form DNA-fectin mixture and add 93.3mL Free-Style 293-F cells (density 1 ⁇ 10 6 /mL) to Final volume 100 mL, 37 ° C, 8% CO 2 and 130 r / min shake flask culture. After 7 days, the culture supernatant was obtained by centrifugation for purification of the next antibody.
  • Antibody purification was performed using a Protein G affinity chromatography column (Protein G Sepharose Fast Flow from GE Healthcare) for protein purification. Specifically, the Protein G affinity column was returned to room temperature and the PBS was equilibrated by 5 column volumes. The cell expression supernatant in step 1 was applied to the column at a flow rate of 3 ml/min. After the end of the loading, 5 column volumes were equilibrated with PBS, eluted with a pH of 2.7, 0.1 M glycine hydrochloride solution, and the eluate was neutralized by adding 1/10 volume of 1 M disodium hydrogen phosphate solution pH 9.0.
  • a Protein G affinity chromatography column Protein G Sepharose Fast Flow from GE Healthcare
  • the purified sample was desalted by a desalting column (Sephadex G-25F from GE), and the desalted sample was filtered through 0.22 um.
  • Membrane filtration preservation that is, purification of the antibody solution, the resulting antibody structure pattern is shown in Figure 3, the purification results are shown in Figure 5, after one step purification method to obtain an antibody with a purity of >95%, referred to as WTE-CH12 antibody.
  • Example 3 Detection of binding activity of anti-human EGFRvIII WTE-CH12 antibody to tumor cells
  • the binding ability of the WTE-CH12 antibody to EGFRVIII was analyzed by a fluorescence activated cell sorter (FACS, also commonly referred to as flow cytometry) (FACScalibu, supplied by BD).
  • FACS fluorescence activated cell sorter
  • the cells were digested with 10 mM EDTA, and the cells were collected by centrifugation at 200 g x 5 min. Resuspended in 1% calf serum-containing phosphate buffer (NBS PBS) at a concentration of 5 ⁇ 10 6 /mL, and added to a flow-type dedicated tube in an amount of 100 ⁇ l/tube. Centrifuge at 200 g x 5 min and discard the supernatant. The blank control PBS and the test antibody WTE-CH12 were added to the two tubes, and the final concentration of each antibody was 5 ⁇ g/ml, and 100 ⁇ l was added to each tube.
  • NBS PBS 1% calf serum-containing phosphate buffer
  • the antibody hardly bound to U87MG cells.
  • the fluorescence peak of the WTE-CH12 antibody shown in black was significantly different from the blank control (PBS), indicating its ability to efficiently bind to U87MG-EGFRvIII cells.
  • the first strand of cDNA was synthesized by reverse transcription of RT-PCR Kit using the mRNA of hybridoma 2D8 cell line (obtained from Shanghai Ruijin Biotechnology Co., Ltd.) against WTE polypeptide as a template.
  • the first strand of cDNA was used as a template, and Heavy Primers and Light Primer Mix were used as primers (primers were purchased from Shanghai Ruijin Biotechnology Co., Ltd.) to amplify VH.
  • VL gene PCR conditions: pre-denaturation at 94 ° C for 4 min, denaturation at 94 ° C for 40 s, annealing at 55 ° C for 40 s, extension at 68 ° C for 40 s, extension at 68 ° C for 7 min after 30 cycles.
  • the PCR products were detected by agarose gel electrophoresis, and the VH and VL fragments were recovered by the gel recovery kit.
  • VH and VL fragments were used as templates, Linker-Primer Mix was used as primers (primer was purchased from Shanghai Ruijin Biotechnology Co., Ltd.), and the VH and VL fragments were spliced into scFv by overlapping PCR. PCR conditions: denaturation at 94 °C for 1 min, 63 °C Annealing was extended for 4 min for a total of 7 cycles. After 7 cycles, Linker-Primer Mix, polymerase buffer and double distilled water were added to 50 ⁇ l of the reaction system to continue PCR.
  • PCR conditions pre-denaturation at 94 ° C for 4 min, denaturation at 94 ° C for 40 s, annealing at 58 ° C for 40 s, extension at 68 ° C for 1 min, extension at 68 ° C for 7 min after 30 cycles.
  • the PCR product was detected by agarose electrophoresis, and the scFv fragment was recovered by a gel recovery kit.
  • SfiI and NotI were double-digested with the scFv fragment and the pCANTAB 5E vector (purchased from Pharmacia) in the above steps, and the gel-removed fragment was ligated overnight, and then transformed into competent E. coli HB2151 at 16 ° C, and picked up from the transformation plate the next day.
  • Twenty monoclonal cells were cultured at 30 ° C, and cultured until the OD600 was 0.4-0.6, and the expression was induced by adding 0.05 mmol/L IPTG to the overnight concentration (18 h). The supernatant was centrifuged, and the expression of soluble scFv in the culture supernatant was analyzed by ELISA.
  • the antigen WTE-BSA (prepared by Shanghai Ruijin Biotechnology Co., Ltd.) was coated with a 96-well plate at 50 ng/well (1 ng/ ⁇ l, 50 ⁇ l/well), and incubated at 37 ° C for 2 h, 5% PBS skim milk powder ( Bright Dairy Co., Ltd. was blocked at 37 ° C for 2 h, washed 3 times with 0.1 M phosphate buffer (PBS), and the above-mentioned medium-inducible culture supernatant was added to a 96-well plate at 50 ⁇ l per well, and incubated at 37 ° C for 1 h.
  • PBS phosphate buffer
  • the HRP-labeled anti-E tag antibody (purchased from Shanghai Ruijin Biotechnology Co., Ltd.) was diluted 1:1000, 50 ⁇ l/well, and incubated at 37 ° C for 1 h.
  • the cells were washed 3 times with PBST, and goat anti-mouse IgG-HRP (purchased from Santa Cruz) diluted 1:1000 was added and incubated at 37 ° C for 1 h.
  • the absorbance value was measured at a wavelength of 405 nm using a Bio-Rad Model 680 microplate reader, and was judged to be positive by more than 2 times the absorbance value of the negative control well.
  • the clone 2D8-3 with the highest OD value was sequenced, and the single-chain antibody (scfv) sequence of 2D8-3 was determined as SEQ ID NO:35.
  • the plasmid pCANTAB 5E 2D8-3scfv was extracted as a construction template for a lentiviral plasmid which subsequently expressed the chimeric antigen receptor of the present invention.
  • the chimeric antigen receptor protein encoded by the nucleic acid of the present invention may be a chimeric antigen receptor protein selected from the group consisting of a sequence-linked extracellular binding region, a transmembrane region and an intracellular signal region, and the joining sequence is:
  • F2A is a ribosomal hopping sequence from food and mouthvires disease (FMDV) Skipping sequence 2A) (referred to as F2A) to achieve co-expression of eGFP and CAR.
  • CD28a represents its transmembrane region and the second CD28b represents its intracellular signal region.
  • the primer pair used for amplification was the upstream primer 5'-gccggccgaggtccagctg-3' (SEQ ID NO: 17) and the downstream primer 5'-cgtggtccgttttatttccaac-3' ( SEQ ID NO: 18), the amplified bands of interest are all 723 bp.
  • the PCR amplification conditions were pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 58 ° C, 40 s; extension: 68 ° C, 40 s; 27 cycles, then total extension 68 ° C, 10 min.
  • the PCR amplified bands were confirmed by agarose gel electrophoresis to match the expected fragment size.
  • PCR amplification of eGFP sequence for downstream primers the target amplification band size is 1297 bp
  • PCR amplification conditions are pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 58 ° C, 40 s; Extension: 68 ° C, 90 s; 27 cycles, then a total extension of 68 ° C, 10 min.
  • the PCR amplified bands were confirmed by agarose gel electrophores
  • the CD8 ⁇ hinge region was obtained by amplification with the upstream primer 5'-ttggaaataaacggaccacgacgccagcg-3' (SEQ ID NO: 21) and the downstream primer 5'-ggtgataaccagtgacaggag-3' (SEQ ID NO: 22), respectively.
  • CD8 transmembrane region, PCR amplification conditions were pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 30 s; annealing: 58 ° C, 30 s; extension: 68 ° C, 30 s; 25 cycles, then total extension 68 ° C, 10min.
  • the theoretical size of the band was 198 bp, and the amplified product was confirmed by agarose gel electrophoresis to be consistent with the theoretical size.
  • the CD28 transmembrane region-CD28 intracellular signal was obtained by amplification with the upstream primer 5'-gacttcgcctgtgattttttgggtgctggtggtggttgg-3' (SEQ ID NO: 23) and the downstream primer 5'-ctttctgccccgtttggagcgataggct-3' (SEQ ID NO: 24).
  • the fragment was PCR-amplified as above, and the theoretical size of the band was 465 bp.
  • the amplified product was confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.
  • the CD137 intracellular region was obtained by amplification with the upstream primer 5'-aaacggggcagaaagaaactc-3' (SEQ ID NO: 25) and the downstream primer 5'-cagttcacatcctccttc-3' (SEQ ID NO: 26), and the PCR amplification conditions were the same as above.
  • the theoretical size is 126 bp, and the amplified product is confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.
  • the CD3zeta signal region was amplified by the upstream primer 5'-gaaggaggatgtgaactgagagtgaagttcagcaggagc 3' (SEQ ID NO: 27) and the downstream primer 5'-cgaggtcgacctagcgagggggcagggcctgcatg-3' (SEQ ID NO: 28), and the PCR amplification conditions were the same as above, and the theoretical size of the band At 339 bp, the amplified product was confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.
  • SEQ ID NO: 29 to SEQ ID NO: 32 were used as primers for synthesizing F2A-CD8 ⁇ signal peptide fragments; the first step of OverlapPCR amplification conditions was pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; Annealing: 58 ° C, 40 s; extension: 68 ° C, 40 s; 7 cycles, then a total extension of 68 ° C, 5 min.
  • the second step PCR uses the first Overlap PCR bridge product as a template to amplify the F2A-CD8 ⁇ signal peptide fragment with SEQ ID NO: 29 and SEQ ID NO: 32 as upstream and downstream primers respectively.
  • the PCR amplification conditions are pre-denaturation. : 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 58 ° C, 40 s; extension: 68 ° C, 30 s; 27 cycles, then total extension 68 ° C, 5 min.
  • the size of the amplified product was 142 bp, which was in agreement with expectations.
  • the CD137 intracellular signal region and the CD3 ⁇ signal region are obtained by splicing the aforementioned amplification with the upstream primer 5'-gccccaccacgcgacttcgcagcctatcgctccaaacggggcagaaag-3' (SEQ ID NO: 33) and the downstream primer 5'-cgaggtcgacctagcgagggggcagggccggcatg-3' (SEQ ID NO: 34), ie For BBZ (abbreviated as CD137-CD3 ⁇ ), the splicing and PCR amplification conditions are the same as above.
  • the theoretical size of the band was 512 bp, and the amplified product was confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.
  • CD28a CD8 hinge region-CD28 transmembrane region
  • CD28b CD28 intracellular signal region fragment
  • the CD8 hinge region obtained in the above (a) and (b) are taken as the upstream primer 5'-ttggaaataaaacggaccacgacgccagcg-3' (SEQ ID NO: 21) and the downstream primer 5'-ctttctgccccgtttggagcgataggct-3' (SEQ ID NO: 24).
  • the obtained CD28 transmembrane region-CD28 intracellular signal region was spliced to obtain the target fragment: CD8 hinge region-CD28a-CD28b, the theoretical size was 369 bp, the splicing and PCR amplification conditions were the same, and the splicing amplification products were confirmed by agarose gel electrophoresis. Consistent with the theoretical size.
  • CD137-CD3 ⁇ and (2) obtained in the above (1) were ligated with the upstream primer 5'-ttggaaataaaacggaccacgacgccagcg-3' (SEQ ID NO: 21) and the downstream primer 5'-cgaggtcgacctagcgagggggcagggccgggg-3' (SEQ ID NO: 34) by OverlapPCR.
  • the eGFP, F2A-CD8 ⁇ signal peptide-2D8scfv, CD8 hinge region-CD28a were spliced with the upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 19) and the downstream primer 5'-tagcgtaaaaggagcaacatag-3' (SEQ ID NO: 34).
  • -CD28b-CD137-CD3 ⁇ obtained eGFP-F2A-CD8 ⁇ -2D8scFv(WTE)-CD8 hinge region-CD28a-CD28b-CD137-CD3 ⁇ .
  • Stitching conditions eGFP 65ng+F2A-CD8 ⁇ -2D8scFv (anti-WTE) 50ng+CD8 hinge region-CD28a-CD28b-CD137-CD3 ⁇ 85ng (molar ratio 1:1:1), pre-denaturation: 94°C, 4min; denaturation: 94 °C, 30s; annealing: 60 ° C, 30 s; extension: 68 ° C, 30 s, 7 cycles, then total extension 68 ° C, 10 min, supplement DNA polymerase and the above upstream and downstream primers, PCR amplification 27 cycles, amplification
  • the conditions were pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 30 s; annealing: 60 ° C, 30 s; extension: 68 ° C, 120 s, for 25 cycles, then total extension 68 ° C, 10 min.
  • the theoretical size is 2910 bp.
  • the amplified product was confirmed
  • the above 2 was spliced to obtain eGFP-F2A-CD8 ⁇ -2D8scFv(anti-WTE)-CD28a-CD28b-CD137-CD3 ⁇ , and MluI and SalI cleavage sites were introduced upstream and downstream of the open reading frame.
  • the above-obtained target gene was digested with MluI and SalI, and ligated into the same double-cut pWPT vector (see Huamao Wang., et al., Epidermal growth factor receptor vIII enhances tumorigenicity and resistance to 5-fluorouracil in human hepatocellular carcinoma).
  • the recombinant plasmid was correctly sequenced and packaged in lentivirus.
  • the plasmid map is shown in Figure 4.
  • the lentivirus packaging in this example was prepared using 293T cells, specifically, 293T cells (ATCC: CRL-11268) cultured in 10th to 20th passages at a density of 5 ⁇ 10 6 in a 10 cm culture dish, 37 Incubate at °C, 5% CO 2 overnight for transfection.
  • the medium was DMEM (PAA) containing 10% fetal calf serum (PAA), and the medium was changed to serum-free DMEM 2 hours before the next day of transfection.
  • the transfection procedure was as follows: 20 ⁇ g of the target gene plasmid pWPT/eGFP-scFv(anti-WTE)-CD28a-CD28b-CD137-CD3 ⁇ , respectively, with 15 ⁇ g of the packaging plasmid PAX2 and 6 ⁇ g of the envelope plasmid pMD2.G (see literature Huamao Wang., Et al., Epidermal growth factor receptor vIII enhances tumorigenicity and resistance to 5-fluorouracil in human hepatocellular carcinoma. Cancer Letters 279 (2009) 30–38.), dissolved in 500 ⁇ l of MillQ water, mixed, and added 62 ⁇ l of 2.5 M CaCl dropwise.
  • the virus supernatant collected in the above procedure was centrifuged at 28000 rpm in a Beckman Optima L-100XP ultracentrifuge for 2 hours at 4 ° C, and the supernatant was discarded.
  • the resulting precipitate was centrifuged with 1/10 to 1/30 of the volume of Quantum 007 (PAA).
  • PAA Quantum 007
  • Example 7 Sorting of CD4 + or CD8 + T lymphocytes and infection with lentivirus
  • Peripheral blood mononuclear cells (provided by Shanghai Blood Center) of healthy people can be sorted by CD4 + or CD8 + T lymphocyte sorting magnetic beads (Stem Cell Technologies) to obtain CD4 + or CD8 + T lymphocytes.
  • CD4 + or CD8 + T lymphocyte sorting magnetic beads Stem Cell Technologies
  • CD4 + and CD8 + T lymphocytes were added at a density of 1 ⁇ 10 6 /mL, Quantum 007 lymphocyte medium (PAA), and the ratio of cell: magnetic beads was 1:1.
  • Magnetic beads coated with anti-CD3 and CD28 antibodies (Invitrogen) and recombinant human IL-2 (Shanghai Huaxin Biotech Co., Ltd.) at a final concentration of 100 U/mL were cultured at 37 ° C, 5% CO 2 and stimulated for 24 h.
  • the cells obtained by sorting were stimulated for 24 h, and CD4 + or CD8 + T lymphocytes were infected with the recombinant lentivirus of MOI ⁇ 5.
  • the infected T lymphocytes were passaged at a density of 5 ⁇ 10 5 /mL, the culture density was not more than 2 ⁇ 10 6 /mL, and the recombinant human IL-2 was added at a final concentration of 100 U/mL during the culture.
  • Infected T lymphocytes were tested for the positive rate of the target gene by flow cytometry one day before the next experiment. Since eGFP was co-expressed with CAR, the positive cells detecting eGFP were positive cells expressing chimeric antigen receptor, as shown in Fig. 7A. The positive rate of transfection was 57.9%.
  • the ratio of CD4 + eGFP + and CD8 + eGFP + cells in mixed infected T lymphocytes was detected by flow cytometry. Specifically, the infected T lymphocytes were centrifuged at 200 g ⁇ 5 min to collect cells at 5 ⁇ 10 6 . The cell density of /mL was resuspended in 1% phosphate buffered saline (NBS PBS) containing calf serum and added to the flow tube in an amount of 100 ⁇ l/tube.
  • NBS PBS phosphate buffered saline
  • Blank control PBS, anti-CD4 murine monoclonal antibody and anti-CD8 murine monoclonal antibody were diluted 1:50 (purchased from Santa Cruz), 100 ⁇ l per tube, ice bath, and 2 ml 1% NBS PBS per tube after 45 minutes. , centrifuged at 200g ⁇ 5min, a total of two times. The supernatant was discarded, and a 1:50 dilution of goat anti-mouse-PE mouse monoclonal antibody (purchased from Santa Cruz) was added, 100 ⁇ l was added to each tube, and after ice bath for 45 minutes, 2 ml of 1% NBS PBS was added to each tube, and centrifuged at 200 g ⁇ 5 min. A total of two times.
  • Example 8 WTE-CH12 mediated expression of chimeric antigen receptor T lymphocytes in vitro on tumor cells Toxicity test
  • In vitro toxicity test target cells were U87MG, U87MG-EGFRvIII, Huh-7, Huh-7-EGFRvIII, and the effector cells were cultured in vitro for 12 days. FACS detection of chimeric antigen receptor-positive cells was recorded as chimeric antigen receptor-positive T. lymphocytes (CAR + CD4 + and mixed CAR + CD8 + cells).
  • the ratio of U87MG, U87MG-EGFRvIII effector cells to target cells was 10:1, the ratio of Huh-7, Huh-7-EGFRvIII effector cells to target cells was 3:1, and the number of target cells was 10000/well.
  • the maximum concentration of WTE-CH12 antibody in the experimental group was 10 4 ng/ml, and the ten-fold concentration was sequentially diluted by four gradients. Five replicate wells were set in each concentration of the experimental group and the control group, and the average of five replicate wells was taken. The detection time is 18h.
  • the experimental group and the control group are as follows:
  • Control group 1 The maximum release of LDH from target cells
  • Control group 2 The target cells spontaneously release LDH
  • Control group 3 effector cells + target cells.
  • the specific detection method was carried out by referring to the CytoTox 96 non-radioactive cytotoxicity test kit (Promega). This method is based on the colorimetric detection method and can replace the 51Cr release method.
  • the assay quantitatively measures lactate dehydrogenase (LDH).
  • LDH lactate dehydrogenase
  • LDH is a stable cytoplasmic enzyme that is released when cells are lysed and released in much the same way as 51 Cr is released in radioactive analysis.
  • the released LDH medium supernatant can be detected by a 30-minute coupled enzyme reaction in which LDH converts a tetrazolium salt (INT) into red formazan (formazan), resulting in red
  • INT tetrazolium salt
  • formazan red formazan
  • the cytotoxicity calculation formula is:
  • the experimental results show that the 2D8scFv(anti-WTE)-CD28a-CD28b-CD137-CD3 ⁇ CAR + lymphocytes (CD4 + and CD8 + mixed lymphocytes) expressed by the present invention are in the presence of WTE-CH12 antibody against tumor cell U87MG-EGFRvIII.
  • Huh-7-EGFRvIII showed very significant cytotoxicity, and the cytotoxic effect produced showed a significant antibody concentration gradient dependence.
  • the cell killing effect was as high as 98.3% and 93.0% at the antibody concentration of 10 4 ng/ml, respectively.
  • Example 9 In vitro competitive inhibition assay of WTE-CH12-mediated TTE lymphocytes expressing chimeric antigen receptors on tumor cells
  • target cells were U87MG-EGFRvIII, and the effector cells were cultured in vitro for 12 days. FACS detection of chimeric antigen receptor-positive cells was recorded as chimeric antigen receptor-positive T lymphocytes (CAR + CD4 + and CAR + CD8 + mixed cells). The ratio of U87MG-EGFRvIII effector cells to target cells was 10:1, and the number of target cells was 10000/well.
  • concentration of WTE-CH12 antibody in the experimental group was 10 4 ng/ml, and the WTE-CH12 antibody was not added in the control group. Five replicate wells were set in each concentration of the experimental group and the control group, and the average of five replicate wells was taken. The detection time is 18h.
  • the experimental group and the control group are as follows:
  • Experimental group 1 target cell + chimeric antigen receptor-positive T lymphocyte + WTE-CH12 antibody
  • Experimental group 2 target cell + chimeric antigen receptor-positive T lymphocyte + WTE-CH12 antibody + WTE polypeptide (2 times WTE-CH12 antibody molar concentration)
  • Experimental group 3 target cell + chimeric antigen receptor-positive T lymphocyte + WTE-CH12 antibody + WTE polypeptide (20-fold WTE-CH12 antibody molar concentration)
  • Control group 1 The maximum release of LDH from target cells
  • Control group 2 The target cells spontaneously release LDH
  • Control group 3 effector cells + target cells.
  • the experimental results show that when the molar concentration of free WTE polypeptide is twice the concentration of WTE-CH12 antibody, it has a certain inhibitory effect on the toxic effect of WTE-CH12 antibody-mediated CAR + T cells on U87MG-EGFRvIII. In group 1, the inhibitory effect was decreased by 31.1%. When the molar concentration of free WTE polypeptide was 20 times that of WTE-CH12 antibody, its inhibitory effect on WTE-CH12 antibody-mediated cytotoxicity of CAR + T cells to U87MG-EGFRvIII was observed. It is very significant, and its inhibitory effect is reduced by 87.82% compared with experimental group 1. The specific results are shown in Fig. 9.
  • Example 10 WTE-CH12 mediated antitumor activity in tumor-bearing mice expressing chimeric antigen receptor T lymphocytes
  • mice aged 6-8 weeks were grouped as shown above (6/group), and 100 mg/Kg of cyclophosphamide was given to the peritoneal cavity on the day before inoculation (working solution was 20 mg/ml, and the working dose was 5 ⁇ l/ g mouse).
  • working solution was 20 mg/ml, and the working dose was 5 ⁇ l/ g mouse.
  • mice in groups 1 and 3 were subcutaneously inoculated with U87MG-EGFRvIII cell suspension (2.5 ⁇ 10 6 /ml, 200 ⁇ l).
  • Groups 2, 4, and 5 were subcutaneously inoculated with U87MG-EGFRvIII and CART cell suspension.
  • the suspension was obtained by mixing 100 ⁇ l of U87MG-EGFRvIII having a cell concentration of 5 ⁇ 10 6 /ml and a concentration of 1:1 by volume of a cell concentration of 5 ⁇ 10 7 /ml CAR + T cells in 100 ⁇ l.
  • mice in groups 1 and 2 were injected with PBS (100 ⁇ l) in the tail vein, respectively, and mice in groups 3 and 4 were injected with 50 ⁇ g of WTE-CH12 antibody (0.5 mg/ml, 100 ⁇ l) in the tail vein, respectively.
  • the size of the tumor was measured on a designated daily vernier caliper, and the tumor volume was calculated according to the following formula:
  • the reduction in tumor volume in the mouse model was set as the basis for WTE-CH12 mediated CAR + T cell tumor suppressive effects.
  • the formula for calculating the tumor inhibition rate is as follows:
  • mice in control group 2 (only U87MG-EGFRvIII tumor cells and CAR T effector cells were injected) on day 25 after cell inoculation.
  • control group 1 only the tumor cell group was injected
  • no significant intervention was observed in the growth of the U87MG-EGFRvIII tumor, and the inhibition rate was 21.2%.
  • the control group 3 (only injected tumor cells and WTE-CH12 antibody group) had a certain anti-tumor effect, and the inhibition rate was 33.5%, but the effect and experimental group 4 (injection of tumor cells, effector cells and WTE) -CH12 antibody group)
  • the tumor inhibition effect was 67.6%, and the difference was significant, indicating that CART cells expressing the anti-WTE polypeptide single chain antibody mediated by WTE-CH12 antibody showed strong ability to inhibit the growth of U87MG-EGFRvIII.
  • the experimental mice were the same as described above.
  • the number of cells inoculated with Huh-7-EGRFRvIII was 3 ⁇ 10 6 /only, and the number of CAR + T cells was 3 ⁇ 10 6 /only (the ratio of effector cells to target cells was 1:1).
  • 9 ⁇ 10 6 / only effector cells, target cells ratio of 3:1
  • WTE-CH12 antibody 50 ⁇ g / only the experimental grouping is as follows:
  • Huh-7-EGFRvIII+CAR T+PBS effector cell, target cell ratio 3:1
  • mice aged 6-8 weeks were grouped as shown above (5/group), and 100 mg/Kg of cyclophosphamide was given to the peritoneal cavity on the day before inoculation (working solution was 20 mg/ml, and the working dose was 5 ⁇ l/ g mouse).
  • group 1 mice were subcutaneously inoculated with Huh-7-EGFRvIII cell suspension (1.5 ⁇ 10 7 /ml, 200 ⁇ l), and groups 2 and 3 were subcutaneously inoculated with Huh-7-EGFRvIII and CART cell suspension.
  • the cell concentration of the suspension was 1.5 ⁇ 10 7 / ml of Huh-7-EGFRvIII and a cell concentration of 1.5 ⁇ 10 7 / ml CAR + T cells in each 100 ⁇ l to 1: 1 volume ratio mixing obtained.
  • the right side of group 5 and 5 were subcutaneously inoculated with a suspension of Huh-7-EGFRvIII and CART cells.
  • the suspension was composed of Huh-7-EGFRvIII with a cell concentration of 1.5 ⁇ 10 7 /ml and a cell concentration of 4.5 ⁇ 10 7 /ml CAR. + 100 ⁇ l of each of T cells were mixed in a 1:1 volume ratio.
  • mice in groups 1, 2, and 4 were injected with PBS (100 ⁇ l) in the tail vein, respectively, and mice in groups 3 and 5 were injected with 50 ⁇ g of WTE-CH12 antibody (0.5 mg/ml, 100 ⁇ l) in the tail vein, respectively.
  • Tumor volume measurement and tumor inhibition rate calculation formulas are as described in Example 10-1.

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Abstract

本发明提供了时空可调性抑制病理性靶细胞的系统，其中包括（1）融合蛋白，包括多肽标签和特异性识别病理性靶细胞的结合分子；和（2）嵌合抗原受体免疫效应细胞，其表达特异性识别所述多肽标签的结合分子。本发明揭示了一种基于肿瘤特异性嵌合抗原受体（CAR）技术的技术方案，免疫效应细胞仅在介导物质存在的条件下才能够靶向病理性靶细胞，实现CAR免疫效应细胞的持续扩增并发挥对肿瘤细胞的杀伤作用；而在介导物质不存在的条件下，CAR免疫效应细胞不发挥作用。

Description

时空可调性抑制病理性靶细胞的系统 技术领域
本发明涉及肿瘤免疫学领域，更具体地，涉及到时空可调性抑制病理性靶细胞的系统。
背景技术
随着肿瘤免疫学理论和技术的发展，免疫治疗在肿瘤治疗中的作用日益受到重视。T淋巴细胞在肿瘤免疫应答中起主要作用，近年来发展的利用基因改造技术表达肿瘤特异性嵌合抗原受体(Chimeric antigen receptor，CAR)的免疫效应细胞显示出的靶向性、杀伤活性和持久性，为过继性细胞免疫治疗注入了新的解决方案。CAR是将识别肿瘤相关抗原(TAA)的单链抗体(scFv)或抗体片段和T细胞或NK细胞的活化序列在体外进行基因重组，形成重组质粒，在体外通过转染技术，转染经纯化与大规模扩增后的T细胞或NK细胞，称之为CAR T细胞或CAR NK细胞。CAR主要包含TAA特异性抗体的抗原结合部(细胞外域)以及T细胞协同刺激结构(CD137和CD28)和信号传导结构(CD3ζ细胞内域)。
研究所展示的CAR T细胞在体内的扩增、持续活性、转化为记忆细胞以及其抗肿瘤效果都非常出众。但是，其毒性作用不可忽视。在一些肿瘤中，CAR T细胞识别自身正常组织表达靶抗原或者激活本身的T细胞诱导自身免疫反应，持续活化的T细胞和记忆T细胞可能会构成实质性危害，例如由于交叉反应所致器官靶向毒性等。
因此，本领域迫切需要研究化解CAR T细胞的毒性作用、但能有效甚至高效杀伤肿瘤细胞的方法。
发明内容
本发明的目的在于提供一种时空可调性抑制病理性靶细胞的系统。
在本发明的第一方面，提供用于抑制病理性靶细胞的系统，其中包括：
(1)融合蛋白，包括多肽标签和特异性识别病理性靶细胞的结合分子；和
(2)嵌合抗原受体(CAR)免疫效应细胞，其表达特异性识别所述多肽标签的结合分子(包括识别所述多肽标签的抗体或配体等)。
在一个优选例中，所述的多肽标签为具有低免疫原性(包括无免疫原性)的无关抗原，其在非肿瘤组织中低表达或不表达。
在另一优选例中，所述的多肽标签是内源或外源的多肽。
在另一优选例中，所述的多肽标签选自但不限于：WTE，E-tag，Flag，Myc，His6等。
在另一优选例中，所述的多肽标签是WTE标签。
在另一优选例中，所述的多肽标签是SEQ ID NO:38所示核苷酸序列编码的多肽。
在另一优选例中，所述的多肽标签可以融合在所述特异性识别病理性靶细胞的结合分子的N端或C端，也可以同时融合在抗体的N端和C端。
在另一优选例中，所述的病理性靶细胞是肿瘤细胞，所述的特异性识别病理性靶细胞的结合分子结合于肿瘤细胞上的肿瘤相关抗原。
在另一优选例中，所述的肿瘤相关抗原选自(但不限于)：
EGFR，EGFRvIII，de4EGFR，EpCAM，CD19，CD20，CD33，HER2，EphA2，IL13R，GD2，LMP1，Claudin 18.A2，PLAC1，NY-ESO-1，MAGE4，MUC1，MUC16，LeY，CEA，GPC3，Mesothelin，CAIX(碳酸酐酶IX)，CD123，IL13R，EphA2。
在另一优选例中，所述的结合分子是配体或抗体，所述抗体包括(但不限于)：Fab、F(ab’)、F(ab’)2、Fv、dAb、Fd、互补决定区(CDR)片段、单链抗体(scFv)、二价单链抗体、单链噬菌体抗体、双特异双链抗体、三链抗体、四链抗体；单克隆抗体。
在另一优选例中，所述的肿瘤包括(但不限于)：肝癌、肺癌、胶质瘤、乳腺癌、胃癌、前列腺癌、脑肿瘤、卵巢癌、骨肿瘤、结肠癌、甲状腺肿瘤、纵隔肿瘤、肠肿瘤、肾肿瘤、肾上腺肿瘤、膀胱肿瘤、睾丸肿瘤、恶性淋巴瘤、多发性骨髓瘤、神经系统肿瘤、食管癌、胸腺间皮瘤、胰腺癌、白血病、头颈部肿瘤、宫颈癌、皮肤癌、黑色素瘤、阴道上皮癌、胆囊癌、恶性纤维组织细胞瘤。
在另一优选例中，所述的免疫效应细胞包括：T淋巴细胞(包括CD4+或CD8+T淋巴细胞)，NK细胞。
在另一优选例中，所述的病理性靶细胞是表达(较佳地高表达)EGFRvIII的肿瘤细胞；且
所述的特异性识别病理性靶细胞的结合分子是特异性结合EGFRvIII的抗体(较佳地为CH12抗体)。
在另一优选例中，所述的嵌合抗原受体免疫效应细胞重组表达CD28(较佳地包括CD28a，CD28b)，CD137，CD3ζ(较佳地为CD3ζ细胞内域)，CD27，CD8，CD19，CD134，CD20，FcRγ中的一种或多种。
在另一优选例中，所述的嵌合抗原受体免疫效应细胞中包含构建物，所述构建物包含以下操作性连接的元件：特异性识别所述多肽标签的结合分子编码序列、CD8铰链区、CD28a、CD28b、CD137、CD3ζ(较佳地还包含eGFP、F2A)。更佳地，所述构建物中各元件按照以下次序(5’→3’)连接：特异性识别所述多肽标签的结合分子编码序列、CD8铰链区、CD28a、CD28b、CD137、CD3ζ(较佳地5’端还包含(5’→3’)eGFP、F2A)。
在本发明的另一方面，提供前面任一所述的系统的用途，用于制备抑制病理性靶细 胞的药盒。较佳地，该用途为非治疗性的用途。
在本发明的另一方面，提供一种用于制备所述的药盒的试剂盒，所述的试剂盒中包括：
(a)表达构建物a，其包括融合蛋白的表达盒(能在免疫细胞中表达)，所述融合蛋白包括多肽标签和特异性识别病理性靶细胞的结合分子；
(b)表达构建物b，其包括表达特异性识别所述多肽标签的结合分子(包括识别所述多肽标签的抗体或配体等)的表达盒(能在免疫细胞中表达)；和
(c)免疫效应细胞。
在一个优选例中，所述的病理性靶细胞是肿瘤细胞，所述的特异性识别病理性靶细胞的结合分子结合于肿瘤细胞上的肿瘤相关抗原；和/或
所述的嵌合抗原受体免疫效应细胞重组表达CD28(较佳地包括CD28a，CD28b)，CD137，CD3ζ(较佳地为CD3ζ细胞内域)，CD27，CD8，CD19，CD134，CD20，FcRγ中的一种或多种。
在另一优选例中，所述的表达构建物a或表达构建物b可以是一个载体或多个载体。
在本发明的另一方面，提供一种抑制病理性靶细胞的方法，所述方法包括：给予受试者所述的抑制病理性靶细胞的系统。
在本发明的另一方面，提供一种时空可调性抑制病理性靶细胞的方法，所述方法包括：给予受试者嵌合抗原受体免疫效应细胞，其表达特异性识别多肽标签的结合分子；当需要抑制病理性靶细胞时，给予受试者融合蛋白，所述融合蛋白包括多肽标签和特异性识别病理性靶细胞的结合分子，从而介导免疫效应细胞发挥杀伤病理性靶细胞的作用。
在本发明的另一方面，提供一种分离的多肽(WTE标签)，所述的多肽的氨基酸序列由SEQ ID NO:38所示的核苷酸序列所编码。
在本发明的另一方面，提供一种分离的多核苷酸，其核苷酸序列如SEQ ID NO:38所示或其简并序列。
在本发明的另一方面，提供一种特异性结合所述多肽(WTE标签)的单链抗体，所述的单链抗体由SEQ ID NO:35所示的核苷酸序列所编码。
在本发明的另一方面，提供一种编码所述单链抗体的多核苷酸，其核苷酸序列如SEQ ID NO:35所示或其简并序列。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、pK/WTE-CH12L表达载体结构示意图。
图2、pH/WTE-CH12H表达载体结构示意图。
图3、抗体WTE-CH12结构模式图。
图4、本发明中包含编码CAR序列的慢病毒载体pWPT/eGFP-2D8(anti-WTE)-CD28a-CD28b-CD137-CD3ζ的结构示意图。
图5、纯化的WTE-CH12抗体的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测，M为分子量标记,泳道1表示纯化的WTE-CH12抗体。
图6A、通过荧光激活细胞分选仪(FACS)显示的WTE-CH12抗体与U87MG肿瘤细胞的特异性结合的测定。
图6B、通过荧光激活细胞分选仪(FACS)显示的WTE-CH12抗体与U87MG-EGFRvIII肿瘤细胞的特异性结合的测定。
图6C、通过荧光激活细胞分选仪(FACS)显示的WTE-CH12抗体与Huh-7肿瘤细胞的特异性结合的测定。
图6D、通过荧光激活细胞分选仪(FACS)显示的WTE-CH12抗体与Huh-7-EGFRvIII肿瘤细胞的特异性结合的测定。
图7A、通过荧光激活细胞分选仪(FACS)显示的慢病毒载体感染后CAR+T细胞的比例。
图7B、通过荧光激活细胞分选仪(FACS)显示的慢病毒载体感染后CAR+CD4+T细胞的比例。
图7C、通过荧光激活细胞分选仪(FACS)显示的CAR+CD8+T细胞的比例。
图8A、系列梯度稀释的WTE-CH12抗体诱导的CAR+T细胞对U87MG、U87MG-EGFRvIII肿瘤细胞的杀伤率比较。
图8B、系列梯度稀释的WTE-CH12抗体诱导的CAR+T细胞对Huh-7、Huh-7-EGFRvIII肿瘤细胞的杀伤率比较。
图9、WTE多肽对WTE-CH12介导的表达嵌合抗原受体的T淋巴细胞对肿瘤细胞毒性效果的体外竞争抑制试验。
图10、NOD/SCID荷瘤(U87MG-EGFRvIII)小鼠模型显示的WTE-CH12抗体诱导的CAR+T细胞治疗组和对照组的抗肿瘤活性测定。
图11、NOD/SCID荷瘤(Huh-7-EGFRvIII)小鼠模型显示的WTE-CH12抗体诱导的CAR+T细胞治疗组和对照组的抗肿瘤活性测定。
具体实施方式
本发明人经过广泛的研究，揭示了一种基于肿瘤特异性嵌合抗原受体(CAR)技术的时空可调性抑制病理性靶细胞的方法。经本发明人改造的CAR免疫效应细胞(如CAR T 细胞)只有在介导物质存在的条件下才能够靶向病理性靶细胞，实现CAR免疫效应细胞的持续扩增并发挥对肿瘤细胞的杀伤作用；而在介导物质不存在的条件下，CAR免疫效应细胞不发挥作用(或发挥较弱的作用)。本发明为避免CAR免疫效应细胞在体内持续扩增和对自身正常组织的交叉反应产生毒性作用提供了解决方案。
本发明将嵌合抗原受体(CAR)免疫效应细胞中识别病理性靶细胞相关抗原(如肿瘤相关抗原)的结合分子替换为识别多肽标签(无关抗原)的结合分子(如单链抗体)，同时将识别病理性靶细胞相关抗原的结合分子和多肽标签进行融合获得融合蛋白。采用这种将识别病理性靶细胞相关抗原的结合分子游离于传统的CAR免疫效应细胞之外的模式可以实现对该识别信号的选择性调控，在病理性靶细胞被清除后，融合蛋白的停用使患者体内的CAR免疫效应细胞不具有靶向性，对此信号的阻断使CAR T细胞不能够识别自身正常组织低表达的靶抗原和其持续的扩增，解决了这一问题可能引发的毒性作用。
术语“嵌合抗原受体(CAR)免疫效应细胞”是本领域公知的，其是利用基因改造技术表达肿瘤特异性嵌合抗原受体的免疫效应细胞，在体外和临床试验中显示出一定的靶向性、杀伤活性和持久性，为过继性细胞免疫治疗方法。所述的免疫效应细胞例如包括T细胞，NK细胞。
常规的制备“嵌合抗原受体免疫效应细胞”的方法是本领域技术人员已知的，包括让其表达胞内共刺激细胞分子胞内结构域，例如CD28(较佳地包括CD28a，CD28b)，CD137，CD27，CD3ζ(较佳地为CD3ζ细胞内域)，CD8，CD19，CD134，CD20，FcRγ中的一种或多种。通过它们与相应配体结合，激活免疫效应细胞的第二信号，增强免疫细胞的增殖能力及细胞因子的分泌功能，延长活化免疫细胞的存活时间。
本发明中，所述的病理性靶细胞可以是机体内的各种不利于健康的有害细胞，有必要从机体内去除的细胞。所述的病理性靶细胞包括肿瘤细胞。任何本领域已知的肿瘤均可包含在本发明中，只要该肿瘤能够表达正常组织中低表达的肿瘤相关抗原。
例如，所述的肿瘤包括(但不限于)：肝癌、肺癌、胶质瘤、乳腺癌、胃癌、前列腺癌、脑肿瘤、卵巢癌、骨肿瘤、结肠癌、甲状腺肿瘤、纵隔肿瘤、肠肿瘤、肾肿瘤、肾上腺肿瘤、膀胱肿瘤、睾丸肿瘤、恶性淋巴瘤、多发性骨髓瘤、神经系统肿瘤、食管癌、胸腺间皮瘤、胰腺癌、白血病、头颈部肿瘤、宫颈癌、皮肤癌、黑色素瘤、阴道上皮癌、胆囊癌、恶性纤维组织细胞瘤。
例如，所述的肿瘤相关抗原包括(但不限于)：EGFR，EGFRvIII，de4EGFR，EpCAM，CD19，CD20，CD33，HER2，EphA2，IL13R，GD2，LMP1，Claudin 18.A2，PLAC1，NY-ESO-1，MAGE4，MUC1，MUC16，LeY，CEA，GPC3，Mesothelin，CAIX(碳酸 酐酶IX)，CD123，IL13R，EphA2。
本发明中，所述的多肽标签为一种在非病理性组织中低表达(表达量可忽略不计)或不表达且具有较低的免疫原性的抗原，其在体内不诱导显著的机体免疫反应，其可以是内源或外源的多肽。任何满足上述要求的无关抗原均可包含在本发明中，例如但不限于：WTE，E-tag，Flag，Myc，His6等。
所述的“识别多肽标签的结合分子”是特异性识别或结合所述多肽标签的结合分子，可以是抗体或配体。所述抗体包括(但不限于)：Fab、F(ab’)、F(ab’)2、Fv、dAb、Fd、互补决定区(CDR)片段、单链抗体(scFv)、二价单链抗体、单链噬菌体抗体、双特异双链抗体、三链抗体、四链抗体；单克隆抗体。较佳地，所述的抗体是单链抗体。
藉由所述的特异性识别病理性靶细胞的结合分子发挥靶向病理性靶细胞的作用。当多肽标签与所述识别多肽标签的结合分子结合后，所述的特异性识别病理性靶细胞的结合分子在靶向病理性靶细胞的同时，将免疫效应细胞携带到病理性靶细胞上，发挥杀伤作用。
所述的“特异性识别病理性靶细胞的结合分子”可以是特异性识别病理性靶细胞相关抗原的任何结合分子。在临床上，可以根据所需杀灭的病理性靶细胞的种类来确定应用哪种结合分子。例如，当所述的病理性靶细胞为特异性表达EGFRvIII的胶质瘤细胞(如U87MG)或肝癌细胞(Huh-7)时，应用特异性结合EGFRvIII的抗体是合适的。
在本发明的具体实施例中，本发明人将CAR T细胞中识别肿瘤抗原EGFRvIII的单链抗体替换为识别无关抗原(多肽WTE)的单链抗体，同时将识别肿瘤相关抗原(EGFRvIII)的单克隆抗体CH12和无关抗原多肽WTE(来源于EGFR NM_005228胞内段第1189-1210氨基酸)分别经连接肽连接(参见专利US 7612181中SEQ ID:44，SEQ ID NO:47)进行重组蛋白的表达和制备。采用这种将识别靶抗原的抗体游离于传统的CART之外的模式可以实现对该识别信号的选择性调控，在肿瘤清除后，抗EGFRvIII抗体WTE-CH12的停用使患者体内的CAR T细胞不具有靶向性，对此信号的阻断使CAR T细胞不能够识别自身正常组织低表达的靶抗原和其持续的扩增。
本发明还涉及包含所述用于抑制病理性靶细胞的系统的药盒，其中包括(1)融合蛋白，包括多肽标签和特异性识别病理性靶细胞的结合分子；和(2)嵌合抗原受体免疫效应细胞，其表达特异性识别所述多肽标签的结合分子。所述的药盒中还可包含说明用法的使用说明书。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的 条件，或按照制造厂商所建议的条件。
实施例1、本发明中抗人EGFRVIII WTE-CH12抗体重组质粒的构建
1、核酸片段的扩增
(1)以抗体pH/CH12为模板(序列参见SEQ ID NO:36)。上游引物5’-gatgtgcagcttcaggagtcggg-3’(SEQ ID NO:1)和下游引物5’-acaataatatgtggctgtg tcc-3’(SEQ ID NO:2)PCR扩增CH12VH片段，PCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，40s；进行27个循环，然后总延伸68℃，10min。扩增产物大小为288bp，与预期相符。
(2)重链信号肽-WTE片段的扩增，引物如下：
5’-cctagctagccaccatgagagtgctgattcttttgtggctgttcacagcctttcct-3’(SEQ ID NO:3)，
5’-agctgtggagccagacaggaaaccaggaaaggctgtgaacagccac-3’(SEQ ID NO:4)，
5’-ggtttcctgtctggctccacagctgaaaatgcagaatacctaagggtcgcg-3’(SEQ ID NO:5)，
5’-tgctccaataaattcactgctttgtggcgcgacccttaggtattctgcattttc-3’(SEQ ID NO:6)，
5’-ccacaaagcagtgaatttattggagcagcatcaaccaaaggtcctgatgtg-3’(SEQ ID NO:7)，
5’-ctcctgaagctgcacatcaggacctttggttgatgc-3’(SEQ ID NO:8)；
第一步采用Overlap PCR，以SEQ ID NO:3至SEQ ID NO:8为引物，用于合成WTE片段(SEQ ID NO:38)及重链信号肽序列(SEQ ID NO:39)，PCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，40s；进行7个循环，然后总延伸68℃，10min。
第二步PCR以第一步Overlap PCR搭桥产物为模板，分别以SEQ ID NO:3和SEQ ID NO:8为上下游引物进行扩增重链信号肽-WTE片段，PCR扩增条件为，预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，40s；进行27个循环，然后总延伸68℃，10min。扩增产物大小为134bp，与预期相符。
(3)以抗体pK/CH12为模板(序列参见SEQ ID NO:37)，上游引物5’-gacatcctgatgacccaatctcc-3’(SEQ ID NO:9)和下游引物5’-gaagacagatggtgcagccac-3’(SEQ ID NO:10)扩增CH12Vk片段，PCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：55℃，40s；延伸：68℃，40s；进行27个循环，然后总延伸68℃，10min。扩增产物大小为348bp，与预期相符。
(4)轻链信号肽-WTE片段的扩增，引物如下：
5’-gatcgatatccaccatggacatgatggtccttgctcagtttcttgcattcttgttg-3’(SEQ ID NO:11)；
5’-aaaccaaagcaacaagaatgcaagaaactgagcaaggaccatcatgtcc-3’(SEQ ID NO:12)；
5’-ctttggtttccaggtgcaagatgtggctccacagctgaaaatgcagaatacc-3’(SEQ ID NO:13)；
5’-tggcgcgacccttaggtattctgcattttcagctgtggagccacatcttgcacctgg-3’(SEQ ID NO:14)；
5’-taagggtcgcgccacaaagcagtgaatttattggagcaacggtggctgcaccagac-3’(SEQ ID NO:15)；
5’-ttgggtcatcaggatgtctggtgcagccaccgttgctccaataaattcactgctttg-3’(SEQ ID NO:16)；
采用OverlapPCR，以SEQ IDNO:11至SEQ ID NO:16为引物，用于合成WTE片段及轻链信号肽序列(SEQ ID NO:40)；第一步OverlapPCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，40s；进行7个循环，然后总延伸68℃，10min。
第二步PCR以第一步PCR搭桥产物为模板，分别以SEQ ID NO:11和SEQ ID NO:16为上下游引物进行扩增轻链信号肽-WTE片段，PCR扩增条件为，预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，40s；进行27个循环，然后总延伸68℃，10min。扩增产物大小为179bp，与预期相符。
2、核酸片段的拼接
(1)重链信号肽-WTE-CH12VH片段的拼接，以上游引物SEQ ID NO:3和下游引物5’-acaataatatgtggctgtgtcc-3’(SEQ ID NO:2)拼接获得重链信号肽-WTE-CH12VH，拼接条件：重链信号肽-WTE(50ng)+CH12VH(50ng)预变性：94℃，4min；变性：94℃，30s；退火：60℃，30s；延伸：68℃，30s，进行7个循环，然后总延伸68℃，10min，补充DNA聚合酶及上下游引物后PCR扩增25个循环，扩增条件为预变性：94℃，4min；变性：94℃，30s；退火：60℃，30s；延伸：68℃，30s，进行25个循环，然后总延伸68℃，10min。理论大小为441bp。扩增产物经琼脂糖电泳确认与理论大小一致。
(2)轻链信号肽-WTE-CH12Vk片段的拼接条件为：轻链信号肽-WTE(50ng)+CH12Vk(50ng)预变性：94℃，4min；变性：94℃，30s；退火：60℃，30s；延伸：68℃，30s，进行7个循环，然后总延伸68℃，10min，补充DNA聚合酶及上游引物5’-gatcgatatccaccatggacatgatggtccttgctcagtttcttgcattcttgttg-3’(SEQ ID NO:11)和下游引物5’-gaagacagatggtgcagccac-3’(SEQ ID NO:10)后PCR扩增25个循环，拼接获得轻链信号肽-WTE-CH12Vk。扩增条件为预变性：94℃，4min；变性：94℃，30s；退火：60℃，30s；延伸：68℃，30s，进行25个循环，然后总延伸68℃，10min。理论大小为509bp。扩增产物经琼脂糖电泳确认与理论大小一致。
3、包含编码WTE-CH12抗体的核苷酸序列的表达载体的构建
(1)pH/WTE-CH12H载体的构建
扩增得到的序列重链信号肽-WTE-CH12VH及pH/CH12分别用限制性内切酶NheI/EcoRI同时酶切，按照酶供应商(New England Biolabs，NEB)建议的反应条件进行双酶切。然后按照酶供应商(NEB)建议的反应条件用T4DNA连接酶连接双酶切后的重链信号肽-WTE-CH12VH片段和pH/CH12载体片段。由此编码WTE-CH12VH抗体多肽的核苷酸序列被克隆到载体中。所得含有WTE-CH12VH抗体多肽的编码序列的新载体命名为pH/WTE-CH12H，其结构如图2所示。
(2)pK/WTE-CH12L载体的构建
扩增得到的序列轻链信号肽-WTE-CH12Vk及pK/CH12K分别用限制性内切酶EcoRV/BsiWI同时酶切，按照酶供应商(New England Biolabs，NEB)建议的反应条件进行双酶切。然后按照酶供应商(NEB)建议的反应条件用T4DNA连接酶连接双酶切后的重链信号肽-WTE-CH12VK片段和pK/CH12载体片段。由此编码WTE-CH12VK抗体多肽的核苷酸序列被克隆到载体中。所得含有WTE-CH12VK抗体多肽的新载体命名为pK/WTE-CH12L，其详细结构如图1所示。
实施例2、抗人EGFRvIII WTE-CH12抗体的表达和纯化
1、抗人EGFRvIII WTE-CH12抗体的表达
抗体表达应用Free-Style 293-F细胞(购自Invitrogen公司)，悬浮培养及转染方法按照FreeStyleTM 293Expression System说明书操作。具体为转染前将细胞密度调整为1×106个/mL，吹散且使细胞无结团，采用台盼蓝染色测定细胞存活率＞95％。转染步骤：分别将52μg pH/WTE-CH12H和48μg pK/WTE-CH12K(摩尔比1:1)重组质粒及200μL Free-Style 293-F细胞脂质体转染试剂“293fectin”用Opti-MEM稀释至3.33mL，静置5min后将质粒与转染试剂缓慢混合，室温反应20min，形成DNA-fectin混合物后加入93.3mL Free-Style 293-F细胞(密度1×106个/mL)中至终体积100mL，37℃、8％CO2和130r/min摇瓶培养。7天后通过离心获得培养上清用于下一步抗体的纯化。
2、抗人EGFRvIII WTE-CH12抗体的纯化
抗体纯化采用Protein G亲和层析柱(Protein G Sepharose Fast Flow购自GE Healthcare)进行蛋白纯化。具体而言，蛋白G亲和柱恢复室温，PBS平衡5个柱体积。将步骤1中细胞表达上清上柱，流速为3ml/min。上样结束后用PBS平衡5个柱体积，以pH2.7，0.1M甘氨酸盐酸溶液洗脱，洗脱液加入1/10体积1M磷酸氢二钠溶液pH9.0中和。纯化样品经脱盐柱(SephadexG-25F购自GE)脱盐，脱盐样品经0.22um滤 膜过滤保存，即为纯化抗体溶液，所得抗体结构模式如图3，纯化结果如图5所示，经过一步纯化法获得了纯度>95％的抗体，简称WTE-CH12抗体。
实施例3、抗人EGFRvIII WTE-CH12抗体与肿瘤细胞的结合活性检测
通过荧光激活细胞分选仪(FACS，通常又称为流式细胞仪)(FACScalibu，由BD公司提供)分析WTE-CH12抗体与EGFRVIII的结合能力。
具体而言，取对数生长期U87MG(购自ATCC)，U87MG-EGFRvIII(即WO/2011/035465中的U87-EGFRvIII细胞)，Huh-7(购自ATCC)，Huh-7-EGFRvIII(将EGFRvIII编码基因转入Huh-7细胞的构建方法根据：Huamao Wang.，et al.，Epidermal growth factor receptor vIII enhances tumorigenicity and resistance to 5-fluorouracil in human hepatocellular carcinoma.Cancer Letters 279(2009)30-38.)，细胞接种到6cm平皿中，细胞汇合度约为90％，37℃孵箱过夜培养。使用10mM的EDTA消化细胞，200g×5min离心收集细胞。以5×106/mL的浓度重悬于1％含小牛血清的磷酸盐缓冲液(NBS PBS)中，按100μl/管的量加入流式专用管中。200g×5min离心，弃上清。两管中分别加入空白对照PBS及待测抗体WTE-CH12，各抗体的终浓度均为5μg/ml，每管加入100μl。冰浴，45分钟后每管加入2ml 1％NBS PBS，以200g×5min离心，共二遍。弃上清，加入1:50稀释的羊抗人-FITC抗体(购自上海业力生物技术有限公司)，每管加入100μl。冰浴45分钟后每管加入2ml1％NBS PBS，以200g×5min离心，共二遍。弃上清，重悬于300μl 1％NBS PBS中，流式细胞仪检测。应用流式细胞仪数据分析软件WinMDI 2.9分析数据。
如图6A所示，该抗体几乎不能与U87MG细胞结合。如图6B所示，黑色所显示的WTE-CH12抗体的荧光峰，与空白对照(PBS)相比有显著的差异，表明其具有和U87MG-EGFRvIII细胞高效结合的能力。这些结果表明WTE-CH12抗体可以特异性地和过表达的人U87MG-EGFRvIII的肿瘤细胞结合。
如图6C所示，该抗体几乎不能与Huh-7细胞结合。如图6D所示，黑色所显示的WTE-CH12抗体的荧光峰，与空白对照(PBS，图中峰下有灰色阴影)相比有显著的差异，表明其具有和Huh-7-EGFRvIII细胞高效结合的能力。这些结果表明，WTE-CH12抗体可以特异性地和过表达的人的Huh-7-EGFRvIII肿瘤细胞结合。
实施例4、抗WTE多肽2D8单链抗体序列的获得及活性检测
1、抗WTE多肽2D8单链抗体核酸片段的获得
以抗WTE多肽的杂交瘤2D8细胞株(获自上海锐劲生物科技有限公司)mRNA为模板，用RT-PCR Kit反转录，合成cDNA第一链。以cDNA第一链为模板，分别以Heavy Primers、Light Primer Mix为引物(引物购自上海锐劲生物科技有限公司)，扩增VH、 VL基因，PCR条件：94℃预变性4min，94℃变性40s，55℃退火40s，68℃延伸40s，30个循环后68℃延伸7min。琼脂糖凝胶电泳检测PCR产物，胶回收试剂盒分别回收VH、VL片段。
再以VH、VL片段互为模板，Linker-Primer Mix为引物(引物购自上海锐劲生物科技有限公司)，重叠PCR法拼接VH和VL片段成scFv，PCR条件：94℃变性1min，63℃退火延伸4min，共进行7个循环。7个循环后在50μl反应体系中补加Linker-Primer Mix、聚合酶缓冲液和双蒸水，继续PCR。PCR条件：94℃预变性4min，94℃变性40s，58℃退火40s，68℃延伸1min，30个循环后68℃延伸7min。琼脂糖电泳检测PCR产物，胶回收试剂盒回收scFv片段。
2、抗WTE多肽2D8单链抗体的表达及活性检测
SfiⅠ、NotⅠ双酶切上述步骤中scFv片段和pCANTAB 5E载体(购自Pharmacia公司)，胶回收酶切片段，16℃连接过夜后转化至感受态E.coli HB2151，次日从转化平板上挑取20个单克隆进行30℃培养，培养至OD600为0.4～0.6时，加入终浓度为0.05mmol/L IPTG诱导表达过夜(18h)。离心取上清，ELISA分析培养上清中可溶性scFv表达情况。具体而言，抗原WTE-BSA(由上海锐劲生物科技有限公司制备)分别以50ng/孔(1ng/μl，50μl/孔)包被96孔板，37℃孵育2h，5％PBS脱脂奶粉(光明乳业股份有限公司)37℃封闭2h，用0.1M的磷酸缓冲液(PBS)洗涤3次，将上述中诱导表达培养上清加入96孔板，每孔50μl，37℃孵育1h。PBST(PBS+0.05％Tween20)洗涤3次后，以HRP标记的anti-E tag抗体(购自上海锐劲生物科技有限公司)1:1000稀释，50μl/孔，37℃孵育1h。PBST洗涤3次，加入以1:1000稀释的羊抗鼠IgG-HRP(购自Santa Cruz公司)，37℃孵育1h。PBST洗涤5次，加ABTS显色液100μL/孔，37℃避光显色10min。使用Bio-Rad Model 680酶标仪，在波长405nm下检测吸光度值，比阴性对照孔吸光度值高2倍以上判断为阳性。
取OD值最高的克隆2D8-3测序，测得2D8-3的单链抗体(scfv)序列如SEQ ID NO:35。抽取质粒pCANTAB 5E 2D8-3scfv，作为后续表达本发明的嵌合抗原受体的慢病毒质粒的构建模板。
实施例5、表达本发明的嵌合抗原受体的慢病毒质粒的构建
本发明的核酸所编码的嵌合抗原受体蛋白可以是选自包含顺序连接的胞外结合区，跨膜区和胞内信号区的如下的嵌合抗原受体蛋白，连接顺序为：
eGFP-F2A-2D8scFv(anti-WTE)-CD8铰链区-CD28a-CD28b-CD137-CD3ζ，其中F2A为来自口蹄疫病毒(food and mouthvires disease，FMDV)的核糖体跳跃序列(ribosomal  skipping sequence 2A)(简称F2A),实现eGFP与CAR的共表达。CD28a代表其跨膜区，第二个CD28b代表其胞内信号区。具体构建步骤如下：
1、核酸片段的获得
(1)2D8单链抗体scfv(2D8scFv(anti-WTE))序列的扩增
以实施例4中重组质粒pCANTAB 5E 2D8-3scfv为模板，扩增所采用的引物对为上游引物5’-gccggccgaggtccagctg-3’(SEQ ID NO:17)和下游引物5’-cgtggtccgttttatttccaac-3’(SEQ ID NO:18)，目的扩增条带大小均为723bp。PCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，40s；进行27个循环，然后总延伸68℃，10min。PCR扩增条带通过琼脂糖凝胶电泳确认符合预计的片段大小。
(2)eGFP序列的扩增
以pWPT-eGFP(获自University of Geneva,Switzerland；Dr.Didier Trono)为模板，以5’-gcaggggaaagaatagtagaca-3’(SEQ ID NO:19)为上游引物，5’-caaagtctgtttcacgctactagctagtcgagatctgagtccggacttgtacagctcgtc-3’(SEQ ID NO:20)为下游引物进行PCR扩增eGFP序列，目的扩增条带大小为1297bp，PCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，90s；进行27个循环，然后总延伸68℃，10min。PCR扩增条带通过琼脂糖凝胶电泳确认符合预计的片段大小。
(3)嵌合抗原受体其他部分的核酸序列的扩增
嵌合抗原受体蛋白的其他部分及连接这些部分的铰链区的扩增如下：加1ml Trizol(Invitrogen公司)于1×107健康人外周血单个核细胞(上海市血液中心提供)中裂解细胞后，采用酚-氯仿法抽提总RNA，采用ImProm-IITM逆转录试剂盒(promega公司)逆转录制备cDNA。
(a)CD8α铰链区-CD8跨膜区的扩增
以上述制备的cDNA为模板，分别以上游引物5’-ttggaaataaaacggaccacgacgccagcg-3’(SEQ ID NO:21)和下游引物5’-ggtgataaccagtgacaggag-3’(SEQ ID NO:22)扩增获得CD8α铰链区-CD8跨膜区，PCR扩增条件为预变性：94℃，4min；变性：94℃，30s；退火：58℃，30s；延伸：68℃，30s；进行25个循环，然后总延伸68℃，10min。条带理论大小为198bp，扩增产物经琼脂糖电泳确认与理论大小一致。
(b)CD28跨膜区-CD28胞内信号区片段
以上游引物5’-gacttcgcctgtgatttttgggtgctggtggtggttgg-3’(SEQ ID NO:23)和下游引物5’-ctttctgccccgtttggagcgataggct-3’(SEQ ID NO:24)扩增获得CD28跨膜区-CD28胞内信号 区片段，PCR扩增条件同上，条带理论大小为465bp，扩增产物经琼脂糖电泳确认与理论大小一致。
(c)CD137胞内信号区
以上游引物5’-aaacggggcagaaagaaactc-3’(SEQ ID NO:25)和下游引物5’-cagttcacatcctccttc-3’(SEQ ID NO:26)扩增获得CD137胞内区，PCR扩增条件同上，条带理论大小为126bp，扩增产物经琼脂糖电泳确认与理论大小一致
(d)CD3ζ信号区
以上游引物5’-gaaggaggatgtgaactgagagtgaagttcagcaggagc 3’(SEQ ID NO:27)和下游引物5’-cgaggtcgacctagcgagggggcagggcctgcatg-3’(SEQ ID NO:28)扩增获得CD3zeta信号区，PCR扩增条件同上，条带理论大小为339bp，扩增产物经琼脂糖电泳确认与理论大小一致。
(e)F2A-CD8α信号肽片段的拼接：引物如下：
5’-actagctagtagcgtgaaacagactttgaattttgaccttctgaagttggc-3’(SEQ ID NO:29)；
5’-tggtaaggccatgggcccagggttggactcaacgtctcctgccaacttcagaa-3’(SEQ ID NO:30)；
5’-ccatggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgc-3’(SEQ NO:ID NO:31)；
5’-gctggacctcggccggcctggcggcgtggagcag-3’(SEQ ID NO:32)。
采用OverlapPCR，以SEQ IDNO:29至SEQ ID NO:32为引物，用于合成F2A-CD8α信号肽片段；第一步OverlapPCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，40s；进行7个循环，然后总延伸68℃，5min。第二步PCR以第一步Overlap PCR搭桥产物为模板，分别以SEQ ID NO:29和SEQ ID NO:32为上下游引物进行扩增F2A-CD8α信号肽片段，PCR扩增条件为，预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，30s；进行27个循环，然后总延伸68℃，5min。扩增产物大小为142bp，与预期相符。
2、核酸片段的拼接
(1)CD137胞内信号区和CD3ζ片段的拼接
以上游引物5’-gccccaccacgcgacttcgcagcctatcgctccaaacggggcagaaag-3’(SEQ ID NO:33)和下游引物5’-cgaggtcgacctagcgagggggcagggcctgcatg-3’(SEQ ID NO:34)拼接前述扩增获得CD137胞内信号区和CD3ζ信号区，即为BBZ(简称为CD137-CD3ζ)，拼接和PCR扩增条件同上。条带理论大小为512bp，扩增产物经琼脂糖电泳确认与理论大小一致。
(2)CD8铰链区-CD28跨膜区(简称为CD28a)-CD28胞内信号区片段(简称为CD28b) 片段的拼接
以上游引物5’-ttggaaataaaacggaccacgacgccagcg-3’(SEQ ID NO:21)和下游引物5’-ctttctgccccgtttggagcgataggct-3’(SEQ ID NO:24)对上述(a)中获得的CD8铰链区与(b)中获得的CD28跨膜区-CD28胞内信号区进行拼接，获得目的片段：CD8铰链区-CD28a-CD28b，其理论大小为369bp，拼接和PCR扩增条件同上，拼接扩增产物经琼脂糖电泳确认与理论大小一致。
(3)CD8铰链区-CD28a-CD28b-CD137-CD3ζ片段的拼接
以上游引物5’-ttggaaataaaacggaccacgacgccagcg-3’(SEQ ID NO:21)和下游引物5’-cgaggtcgacctagcgagggggcagggcctgcatg-3’(SEQ ID NO:34)采用OverlapPCR拼接上述(1)中获得的CD137-CD3ζ与(2)中获得的CD8铰链区-CD28a-CD28b，获得目的片段CD8铰链区-CD28a-CD28b-CD137-CD3ζ，其理论大小为832bp，拼接和PCR扩增条件同上，拼接扩增产物经琼脂糖电泳确认与理论大小一致。
(4)F2A-CD8α信号肽与2D8scfv(anti-WTE)片段的拼接：以上游引物5’-actagctagtagcgtgaaacagactttgaattttgaccttctgaagttggc-3’(SEQ ID NO:29)和下游引物5’-cgtggtccgttttatttccaac-3’(SEQ ID NO:18)采用OverlapPCR拼接上述中获得的F2A-CD8α信号肽与2D8scfv(anti-WTE)片段，获得目的片段F2A-CD8α信号肽-2D8scfv，其理论大小为871bp，拼接和PCR扩增条件同上，拼接扩增产物经琼脂糖电泳确认与理论大小一致。
(5)eGFP-F2A-CD8α-2D8scFv(anti-WTE)-CD8铰链区-CD28a-CD28b-CD137-CD3ζ片段的拼接
以上游引物5’-gcaggggaaagaatagtagaca-3’(SEQ ID NO:19)和下游引物5’-tagcgtaaaaggagcaacatag-3’(SEQ ID NO:34)拼接eGFP、F2A-CD8α信号肽-2D8scfv、CD8铰链区-CD28a-CD28b-CD137-CD3ζ，获得eGFP-F2A-CD8α-2D8scFv(WTE)-CD8铰链区-CD28a-CD28b-CD137-CD3ζ。拼接条件：eGFP 65ng+F2A-CD8α-2D8scFv(anti-WTE)50ng+CD8铰链区-CD28a-CD28b-CD137-CD3ζ85ng(摩尔比1:1:1)，预变性：94℃，4min；变性：94℃，30s；退火：60℃，30s；延伸：68℃，30s，进行7个循环，然后总延伸68℃，10min，补充DNA聚合酶及上述上下游引物后PCR扩增27个循环，扩增条件为预变性：94℃，4min；变性：94℃，30s；退火：60℃，30s；延伸：68℃，120s，进行25个循环，然后总延伸68℃，10min。理论大小为2910bp。扩增产物经琼脂糖电泳确认与理论大小一致。
3、pWPT/eGFP-F2A-CD8α-2D8scFv(anti-WTE)-CD28a-CD28b-CD137-CD3ζ质粒 载体的构建
上述2中拼接获得eGFP-F2A-CD8α-2D8scFv(anti-WTE)-CD28a-CD28b-CD137-CD3ζ，在其开放阅读框的上下游引入MluI和SalI酶切位点。上述获得的目的基因由MluI和SalI双酶切，连入同样双酶切的pWPT载体(参见文献Huamao Wang.，et al.，Epidermal growth factor receptor vIII enhances tumorigenicity and resistance to 5-fluorouracil in human hepatocellular carcinoma.Cancer Letters 279(2009)30–38.)中，重组质粒经测序正确后进行慢病毒包装，质粒图谱见图4。
实施例6、感染T淋巴细胞慢病毒的制备
1、感染T淋巴细胞慢病毒的包装
本实施例中慢病毒的包装采用293T细胞进行制备，具体而言，以5×106的密度接种培养至第10～20代的293T细胞(ATCC：CRL-11268)于10cm培养皿中，37℃，5％CO2培养过夜准备用于转染。培养基为含10％胎牛血清(PAA公司)的DMEM(PAA公司)，次日转染前2小时更换培养液为无血清DMEM。转染步骤如下：将20μg目的基因质粒pWPT/eGFP-scFv(anti-WTE)-CD28a-CD28b-CD137-CD3ζ，分别与15μg包装质粒PAX2和6μg包膜质粒pMD2.G(参见文献Huamao Wang.，et al.，Epidermal growth factor receptor vIII enhances tumorigenicity and resistance to 5-fluorouracil in human hepatocellular carcinoma.Cancer Letters 279(2009)30–38.)，溶入500μl MillQ水中，混匀，逐滴加入62μl2.5M CaCl2(Sigma公司)，以1200rpm/min vortex混匀，最后逐滴加入500μl2×HeBS(280mM NaCl，10mM KCl，1.5mM Na2HPO4·2H2O，12mM葡萄糖，50mM Hepes(Sigma公司)，pH7.05，0.22μM过滤除菌)，立即逐滴加入培养皿中，轻轻摇匀，37℃，5％CO2，培养4～6h后，更换为含10％胎牛血清的DMEM。次日，观察转染效率(即呈绿色荧光的细胞比例)，约80％的阳性转染效率即为转染实验成功。在转染48h及72h，分别使用0.45μm滤器(Millipore公司)过滤收集病毒，-80℃保存。
2、感染T淋巴细胞慢病毒的纯化与滴度测定
(1)感染T淋巴细胞慢病毒的纯化
将上述步骤中所收集的病毒上清采用Beckman Optima L-100XP超速离心机28000rpm，4℃离心2小时，弃上清，所得沉淀用1/10～1/30原液体积的Quantum 007培养液(PAA公司)进行重悬，以100μl/管分装冻存于-80℃备用。
(2)感染T淋巴细胞慢病毒滴度的测定
慢病毒滴度测定具体方法如下：以1×105/mL接种293T细胞于96孔培养板，50μl/ 孔，培养液为含10％胎牛血清的DMEM。每孔中加5μl/孔的病毒浓缩液，并将其终体积补至50μl，每个样品3倍稀释，6个梯度，两个复孔，病毒梯度稀释液与细胞混匀后37℃，5％CO2培养。感染48h后，流式细胞仪检测eGFP，以阳性率为5～20％的细胞数为宜，计算滴度(U/mL)＝阳性率×稀释倍数×100×104。
实施例7、CD4+或CD8+T淋巴细胞的分选及慢病毒的感染
1、CD4+或CD8+T淋巴细胞的分选
健康人外周血单个核细胞(上海市血液中心提供)通过CD4+或CD8+T淋巴细胞分选磁珠(Stem Cell Technologies)分选获得CD4+或CD8+T淋巴细胞，具体操作步骤同说明书所述。
分选后1:1混合CD4+和CD8+T淋巴细胞，以1×106/mL密度，Quantum 007淋巴细胞培养基液(PAA公司)，并以细胞：磁珠比例为1:1加入同时包被有抗CD3和CD28抗体的磁珠(Invitrogen公司)和终浓度100U/mL的重组人IL-2(上海华新生物高技术有限公司)37℃，5％CO2培养，刺激培养24h。
2、CD4+或CD8+T淋巴细胞慢病毒的感染及阳性率测定
分选获得的细胞刺激培养24h，以MOI≈5的重组慢病毒感染CD4+或CD8+T淋巴细胞。感染后的T淋巴细胞采用5×105/mL的密度进行传代，培养密度不超过2×106/mL，培养过程中补加终浓度100U/mL的重组人IL-2。感染的T淋巴细胞在下一步实验前一天通过流式细胞仪检测目的基因的阳性率，由于eGFP与CAR共表达，检测eGFP的阳性细胞即为表达嵌合抗原受体的阳性细胞，如图7A所示，转染阳性率为57.9％。
并通过流式细胞仪检测混合感染的T淋巴细胞中CD4+eGFP+和CD8+eGFP+细胞的比例，具体而言，感染后的T淋巴细胞以200g×5min离心收集细胞，以5×106/mL的细胞密度重悬于1％含小牛血清的磷酸盐缓冲液(NBS PBS)中，按100μl/管的量加入流式管中。三管中分别加入空白对照PBS，抗CD4鼠单抗及抗CD8鼠单抗1:50稀释(购自Santa Cruz)，每管加入100μl，冰浴，45分钟后每管加入2ml 1％NBS PBS，以200g×5min离心，共二遍。弃上清，加入1:50稀释的羊抗鼠-PE鼠单抗(购自Santa Cruz)，每管加入100μl，冰浴45分钟后每管加入2ml 1％NBS PBS，以200g×5min离心，共二遍。弃上清，重悬于300μl 1％NBS PBS中，流式细胞仪检测。应用流式细胞仪数据分析软件WinMDI2.9分析数据。结果如图7B及7C所示，CD4+eGFP+和CD8+eGFP+细胞的比例分别为17.6％、39.9％。
实施例8、WTE-CH12介导的表达嵌合抗原受体的T淋巴细胞对肿瘤细胞的体外 毒性实验
体外毒性实验靶细胞分别为U87MG，U87MG-EGFRvIII，Huh-7，Huh-7-EGFRvIII，效应细胞为体外培养12天的FACS检测嵌合抗原受体表达阳性细胞记为嵌合抗原受体阳性T淋巴细胞(CAR+CD4+与CAR+CD8+混合细胞)。U87MG，U87MG-EGFRvIII效应细胞与靶细胞的作用比例为10:1，Huh-7，Huh-7-EGFRvIII效应细胞与靶细胞的作用比例为3:1，靶细胞数量为10000/孔。实验组中WTE-CH12抗体最大浓度为104ng/ml，十倍浓度依次稀释四个梯度。实验组各浓度及对照组均设5个复孔，取五个复孔的平均值。检测时间为第18h。其中实验组和对照组如下：
实验组：靶细胞+嵌合抗原受体阳性的T淋巴细胞+WTE-CH12抗体
对照组1：靶细胞最大释放LDH，
对照组2：靶细胞自发释放LDH，
对照组3：效应细胞+靶细胞。
具体检测方法参照CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行。该方法是基于比色法的检测方法，可替代51Cr释放法。
Figure PCTCN2015082460-appb-000001
检测定量地测量乳酸脱氢酶(LDH)。LDH是一种稳定的胞质酶，在细胞裂解时会释放出来，其释放方式与51Cr在放射性分析中的释放方式基本相同。释放出的LDH培养基上清中，可通过30分钟偶联的酶反应来检测，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)，生成的红色产物的量与裂解的细胞数成正比。
细胞毒性计算公式为：
Figure PCTCN2015082460-appb-000002
实验结果表明，本发明表达的2D8scFv(anti-WTE)-CD28a-CD28b-CD137-CD3ζCAR+淋巴细胞(CD4+及CD8+混合淋巴细胞)在WTE-CH12抗体存在的条件下对肿瘤细胞U87MG-EGFRvIII及Huh-7-EGFRvIII表现出非常显著的细胞毒性，产生的细胞毒性效果呈现明显的抗体浓度梯度依赖性，在抗体浓度104ng/ml时细胞杀伤效果分别高达98.3％和93.0％。而在同样的条件下对U87MG细胞没有明显的细胞毒性3.28％，在102-104ng/ml对Huh-7有一定的杀伤效果，但同样浓度下Huh-7-EGFRvIII杀伤效果更为显著，具体结果见图8。
实施例9、WTE多肽对WTE-CH12介导的表达嵌合抗原受体的T淋巴细胞对肿瘤细胞毒性效果的体外竞争抑制试验
体外毒性竞争抑制实验靶细胞分别为U87MG-EGFRvIII，效应细胞为体外培养12天的FACS检测嵌合抗原受体表达阳性细胞记为嵌合抗原受体阳性T淋巴细胞 (CAR+CD4+与CAR+CD8+混合细胞)。U87MG-EGFRvIII效应细胞与靶细胞的作用比例为10:1，靶细胞数量为10000/孔。本实验中实验组WTE-CH12抗体浓度为104ng/ml，对照组均不加WTE-CH12抗体，实验组各浓度及对照组均设5个复孔，取五个复孔的平均值。检测时间为第18h。其中实验组和对照组如下：
实验组1：靶细胞+嵌合抗原受体阳性的T淋巴细胞+WTE-CH12抗体
实验组2：靶细胞+嵌合抗原受体阳性的T淋巴细胞+WTE-CH12抗体+WTE多肽(2倍WTE-CH12抗体摩尔浓度)
实验组3：靶细胞+嵌合抗原受体阳性的T淋巴细胞+WTE-CH12抗体+WTE多肽(20倍WTE-CH12抗体摩尔浓度)
对照组1：靶细胞最大释放LDH，
对照组2：靶细胞自发释放LDH，
对照组3：效应细胞+靶细胞。
具体试验方法和计算公式参见实施例8中说明。
实验结果表明，在游离WTE多肽的摩尔浓度为WTE-CH12抗体浓度的2倍时，其对WTE-CH12抗体介导的CAR+T细胞对U87MG-EGFRvIII的毒性效果有一定的抑制作用，相对实验组1，其抑制作用下降31.1％，当游离WTE多肽的摩尔浓度为WTE-CH12抗体浓度的20倍时，其对WTE-CH12抗体介导的CAR+T细胞对U87MG-EGFRvIII的毒性效果抑制作用十分显著，相对与实验组1其抑制作用下降87.82％，具体结果见图9。
实施例10、WTE-CH12介导的表达嵌合抗原受体T淋巴细胞的荷瘤小鼠体内抗肿瘤活性
1、WTE-CH12介导的CART细胞对荷瘤小鼠(U87MG-EGFRvIII)体内抗肿瘤活性
6-10周龄的免疫缺陷的NOD/SCID小鼠(由上海斯莱克实验动物有限责任公司提供)用于构建人EGFR相关肿瘤的异种移植模型，其遗传学特征是缺乏T细胞，B细胞，NK细胞以及巨噬细胞功能。实验中U87MG-EGRFRvIII接种细胞数为5×105/只，CAR+T细胞5×106/只，WTE-CH12抗体50μg/只，实验分组如下：
1：U87MGEGFRVIII+PBS
2：U87MGEGFRVIII+PBS+CARTcell
3：U87MGEGFRVIII+WTE-CH12(50μg)
4：U87MGEGFRVIII+CARTcell+WTE-CH12(50μg)
实验步骤具体而言，6～8周小鼠按照以上所示分组(6只/组)，接种细胞前一日腹腔给环磷酰胺100mg/Kg(工作液为20mg/ml，工作剂量为5μl/g小鼠)。次日，1、3组小鼠右侧皮下接种U87MG-EGFRvIII细胞悬液(2.5×106/ml，200μl)，2、4、5组右侧皮下接 种U87MG-EGFRvIII与CART细胞混合悬液，该悬液由细胞浓度为5×106/ml的U87MG-EGFRvIII 100μl和细胞浓度为5×107/ml CAR+T细胞100μl 1:1体积比例混合所得。在上述细胞接种后1h，1、2组小鼠被分别尾静脉注射PBS(100μl)，3、4组小鼠被分别尾静脉注射50μgWTE-CH12抗体(0.5mg/ml，100μl)。
在指定日用游标卡尺测量肿瘤的大小，肿瘤体积根据下列公式计算：
Figure PCTCN2015082460-appb-000003
小鼠模型中肿瘤体积的减小被设定为WTE-CH12介导的CAR+T细胞对肿瘤抑制效果的依据。肿瘤抑制率计算公式如下：
Figure PCTCN2015082460-appb-000004
结果如图10所示，在U87MG-EGFRvIII荷瘤小鼠模型中，在细胞接种后的第25天，对照组2(只注射U87MG-EGFRvIII肿瘤细胞及CAR T效应细胞组)中的小鼠与对照组1(只注射肿瘤细胞组)相比，没有观察到有显著干预U87MG-EGFRvIII肿瘤的生长，抑制率为21.2％。对照组3(只注射肿瘤细胞及WTE-CH12抗体组)与对照组2相比虽有一定的抑瘤效果，抑制率为33.5％，但效果和实验组4(注射肿瘤细胞、效应细胞及WTE-CH12抗体组)抑瘤效果67.6％相比差异较为显著，说明在WTE-CH12抗体的介导下表达抗WTE多肽单链抗体的CART细胞表现出强烈抑制U87MG-EGFRvIII生长的能力。
2、WTE-CH12介导的CART细胞对荷瘤小鼠(Huh-7-EGFRVIII)体内抗肿瘤活性
实验用小鼠同上所述，实验中Huh-7-EGRFRvIII接种细胞数为3×106/只，CAR+T细胞分别为3×106/只(效应细胞、靶细胞比例为1:1)、9×106/只(效应细胞、靶细胞比例为3:1)，WTE-CH12抗体50μg/只，实验分组如下：
1：Huh-7-EGFRvIII+PBS；
2：Huh-7-EGFRvIII+CAR T+PBS(效应细胞、靶细胞比例1:1)；
3：Huh-7-EGFRvIII+CAR T+WTE-CH12(50μg)(效应细胞、靶细胞比例1:1)；
4:Huh-7-EGFRvIII+CAR T+PBS(效应细胞、靶细胞比例3:1)；
5：Huh-7-EGFRvIII+CAR T+WTE-CH12(50μg)(效应细胞、靶细胞比例3:1)。
实验步骤具体而言，6～8周小鼠按照以上所示分组(5只/组)，接种细胞前一日腹腔给环磷酰胺100mg/Kg(工作液为20mg/ml，工作剂量为5μl/g小鼠)。次日，第1组小鼠右侧皮下接种Huh-7-EGFRvIII细胞悬液(1.5×107/ml，200μl)，2、3组右侧皮下接种Huh-7-EGFRvIII与CART细胞混合悬液，该悬液由细胞浓度为1.5×107/ml的Huh-7-EGFRvIII和细胞浓度为1.5×107/ml CAR+T细胞各100μl以1:1体积比例混合所 得。4、5组右侧皮下接种Huh-7-EGFRvIII与CART细胞混合悬液，该悬液由细胞浓度为1.5×107/ml的Huh-7-EGFRvIII和细胞浓度为4.5×107/ml CAR+T细胞各100μl以1:1体积比例混合所得。在上述细胞接种后1h，1、2、4组小鼠被分别尾静脉注射PBS(100μl)，3、5组小鼠被分别尾静脉注射50μgWTE-CH12抗体(0.5mg/ml，100μl)。肿瘤体积测量及肿瘤抑制率计算公式同实施例10-1中所述。
结果如图11所示，在Huh-7-EGFRvIII荷瘤小鼠模型中，1:1实验组3(注射肿瘤细胞、效应细胞及WTE-CH12抗体)相对于1:1对照组2(只注射肿瘤细胞和效应细胞)，有显著干预Huh-7-EGFRvIII肿瘤生长的能力，抑制率达到87.5％。3:1实验组5(注射肿瘤细胞、效应细胞及WTE-CH12抗体)相对于3:1对照组4(只注射肿瘤细胞和效应细胞)，具有更为显著地抑制Huh-7-EGFRvIII肿瘤生长的能力，抑制率达到100％。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 用于抑制病理性靶细胞的系统，其特征在于，其中包括：
    (1)融合蛋白，包括多肽标签和特异性识别病理性靶细胞的结合分子；和
    (2)嵌合抗原受体免疫效应细胞，其表达特异性识别所述多肽标签的结合分子。
  2. 如权利要求1所述的系统，其特征在于，所述的多肽标签为具有低免疫原性的无关抗原，其在非肿瘤组织中低表达或不表达；较佳地，所述的多肽标签是WTE标签。
  3. 如权利要求1所述的系统，其特征在于，所述的病理性靶细胞是肿瘤细胞，所述的特异性识别病理性靶细胞的结合分子结合于肿瘤细胞上的肿瘤相关抗原；较佳地，所述的肿瘤相关抗原选自：
    EGFR，EGFRvIII，de4 EGFR，EpCAM，CD19，CD20，CD33，HER2，EphA2，IL13R，GD2，LMP1，Claudin 18.A2，PLAC1，NY-ESO-1，MAGE4，MUC1，MUC16，LeY，CEA，GPC3，Mesothelin，CAIX，CD123，IL13R，EphA2。
  4. 如权利要求1所述的系统，其特征在于，所述的免疫效应细胞包括：T淋巴细胞，NK细胞。
  5. 如权利要求1所述的系统，其特征在于，所述的病理性靶细胞是表达EGFRvIII的肿瘤细胞；且
    所述的特异性识别病理性靶细胞的结合分子是特异性结合EGFRvIII的抗体。
  6. 如权利要求1所述的系统，其特征在于，所述的嵌合抗原受体免疫效应细胞重组表达CD28，CD137，CD3ζ，CD27，CD8，CD19，CD134，CD20，FcRγ中的一种或多种。
  7. 权利要求1-6任一所述的系统的用途，用于制备抑制病理性靶细胞的药盒。
  8. 一种用于制备权利要求7所述的药盒的试剂盒，其特征在于，所述的试剂盒中包括：
    (a)表达构建物a，其包括融合蛋白的表达盒，所述融合蛋白包括多肽标签和特异性识别病理性靶细胞的结合分子；
    (b)表达构建物b，其包括表达特异性识别所述多肽标签的结合分子的表达盒；和
    (c)免疫效应细胞。
  9. 一种分离的多肽，其特征在于，所述的多肽的氨基酸序列由SEQ ID NO:38所示的核苷酸序列所编码。
  10. 一种特异性结合权利要求9所述的多肽的单链抗体，其特征在于，所述的单链抗体由SEQ ID NO:35所示的核苷酸序列所编码。
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