WO2017219936A1 - 一种高效稳定表达激活型抗体的car-t细胞及其用途 - Google Patents

一种高效稳定表达激活型抗体的car-t细胞及其用途 Download PDF

Info

Publication number
WO2017219936A1
WO2017219936A1 PCT/CN2017/088957 CN2017088957W WO2017219936A1 WO 2017219936 A1 WO2017219936 A1 WO 2017219936A1 CN 2017088957 W CN2017088957 W CN 2017088957W WO 2017219936 A1 WO2017219936 A1 WO 2017219936A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
cell
cells
nucleic acid
transgenic
Prior art date
Application number
PCT/CN2017/088957
Other languages
English (en)
French (fr)
Inventor
钱其军
金华君
胥阶英
李林芳
叶真龙
何周
江芏青
吴红平
Original Assignee
上海细胞治疗研究院
上海细胞治疗工程技术研究中心集团有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海细胞治疗研究院, 上海细胞治疗工程技术研究中心集团有限公司 filed Critical 上海细胞治疗研究院
Publication of WO2017219936A1 publication Critical patent/WO2017219936A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464404—Epidermal growth factor receptors [EGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464424—CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/51—Stomach
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element

Definitions

  • the present invention belongs to the field of cell biology and oncology, and relates to a CAR-T cell which efficiently and stably expresses an activated antibody and uses thereof.
  • Immunotherapy for malignant tumors has developed rapidly in recent years, achieving remarkable clinical results.
  • immunological checkpoints such as CTLA4, PD1/PDL1 monoclonal antibody treatment, through the activation of residual tumor-specific T cells in situ, the overall effective rate of a variety of malignant tumors reached 30%, and many patients once Long-term survival of the drug;
  • transgenic CAR-T/TCR-T cell therapy is obtained by rapidly obtaining tumor-specific T cells by means of ex vivo gene modification, and adopting therapeutic effects after repeated reinfusion for complete relapse of refractory B cell leukemia The response rate is over 90%.
  • Chimeric Antigen Receptor is generally composed of a scFv (single-chain variable Fragment) single-chain antibody (the antibody light and heavy chain variable region is formed by Linker linkage, which is responsible for binding membrane antigen), and is formed by a hinge structure (responsible for formation). The correct conformation, forming a dimer) is linked to the transmembrane region, the intracellular signal structure (responsible for transmitting T cell activation signals).
  • CAR-modified T cells are assigned to recognize tumor cell membrane antigen peptide molecules and initiate killing or proliferation.
  • CAR-T cells recognize antigens expressed on the cell membrane, rather than antigens that are presented to the cell surface after binding to MHC molecules, they can bypass T cell MHC restriction and avoid down-regulation or deletion of MHC molecules by tumor cells. Caused by immune escape.
  • the design of CAR was first proposed by Israeli scholar Eshhar and his colleagues in 1989. It can be divided into three generations according to its development stage.
  • the first generation of CAR receptors contains an svFC fragment that specifically recognizes tumor antigens, and the intracellular activation signal is transmitted by the IT3 (immunoreceptor tyrosine-based activation motifs) signal chain of CD3 ⁇ or Fc ⁇ RI ⁇ .
  • IT3 immunoglobulin-like effector activity
  • Fc ⁇ RI ⁇ Fc ⁇ RI ⁇
  • the first-generation CAR receptor lacks the costimulatory signal of T cells, which leads to the short-term effects of T cells, short-lived in vivo, and low secretion of cytokines.
  • the second generation of CAR receptors increases the intracellular domain of costimulatory signaling molecules, including, for example, CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), and inducible T-cell co-stimulator ( ICOS) and DNAX-activation protein 10 (DAP10) and other domains enhance T cell proliferation and cytokine secretion, IL-2, IFN- ⁇ and GM-CSF increase, thereby breaking the immunosuppression of tumor microenvironment AICD (activation induced cell death (AICD)).
  • the third generation of CAR receptors is based on the second generation of CAR and then a second co-stimulatory molecule such as 4-1BB.
  • multiple target CAR-T cells are undergoing clinical trials for solid tumor treatment, including GD2, FR- ⁇ , L1-CAM, HER2, EGFR, EGFRvIII, VEGFR-2, IL-13R ⁇ 2, FAP, Mesothelin, c-MET, PSMA, CEA, GPC3, EphA2, MUC1, CAIX (carbonic anhydrase IX), and the like.
  • Solid tumor tissue has immunosuppressive microenvironment, including Treg cells, tumor-associated fibroblasts, bone marrow-derived immature DC cells, M2 macrophages, etc. and their secreted cytokines such as IL-6, IL-10, IDO, VEGF, TGF ⁇ , etc., these cells and their secreted cytokines can inhibit the function of T cells either directly or indirectly.
  • immunosuppressive microenvironment including Treg cells, tumor-associated fibroblasts, bone marrow-derived immature DC cells, M2 macrophages, etc. and their secreted cytokines such as IL-6, IL-10, IDO, VEGF, TGF ⁇ , etc.
  • immunological checkpoint antibodies such as PD1/PDL1
  • immuno-negative regulators such as IDO small molecule inhibitors
  • Immune microenvironment, over-expression of immunoregulatory factors such as IL-12
  • direct targeting of tumor stromal cells such as CAR-T targeting FAP-positive tumor-associated fibroblasts
  • the microenvironment of inhibition enhances the viability and killing effect of infiltrating CAR-T cells.
  • Solid tumors are highly heterogeneous, with high differences between different patients, different lesions in the same patient, and different tumor cells in the same lesion. This high degree of heterogeneity has caused tumor targeting therapy to fall into the disadvantage of lacking an ideal universal, broad-spectrum target, limiting the efficacy of CAR-T cells in the treatment of solid tumors. Therefore, in order to expand the killing range of CAR-T cells, some researchers have proposed the design concept of TanCAR, which combines two scFvs combined with different tumor-associated antigens to form a new CAR that can simultaneously recognize and combine two targets. Effectively improve the efficacy of CAR-T cells.
  • T cells on tumor cells require a clustering effect.
  • the killing of a tumor cell requires the cooperation of several T cells, so that only when the effector cells reach a certain amount, the tumor cells can be effectively killed. Therefore, when T cells contact tumor cells of a specific target, they can rapidly proliferate and amplify the killing function by direct contact with the paracrine pathway.
  • CAR-T cell By intravenous administration, under the condition of blood tumor, it is very easy to contact with tumor cells, the number of CAR-T cells can be rapidly amplified, and even excessive amplification to form a cytokine storm, so the curative effect is relatively good; under the condition of solid tumor, blood circulation The number of tumor cells is limited, and CAR-T cells need to reach the tumor site to receive stimulation, and it is difficult to achieve an effective dose.
  • CAR-T especially CAR-T cells capable of broadly recognizing tumor membrane antigens
  • CAR-T cells capable of broadly recognizing tumor membrane antigens
  • T cell proliferation and survival it can effectively overcome solid tumor CAR-
  • T treatment has greatly improved the efficacy.
  • Retroviral or lentiviral packaging and preparation with a full-length antibody expression cassette is difficult and difficult to express antibodies efficiently (for example, using CAR-T cells to express antibodies at a concentration of only 200 ng/ml, Oncotarget.2016Apr 29.doi: 10.18632 /oncotarget.9114), generally used to express a single-chain antibody with a simple structure (lack of constant region fragment, incomplete function and short half-life).
  • CAR-T cells to express antibodies at a concentration of only 200 ng/ml
  • Oncotarget.2016Apr 29.doi: 10.18632 /oncotarget.9114 generally used to express a single-chain antibody with a simple structure (lack of constant region fragment, incomplete function and short half-life).
  • a first aspect of the present invention provides a transgenic T cell, wherein the T cell genome stably integrates an expression cassette of a nucleic acid sequence comprising an activated antibody encoding a chimeric antigen receptor, and an immunostimulatory molecule or a receptor thereof, and Both ends of the expression cassette contain the inverted terminal repeat of the transposon.
  • the activating antibody is selected from the group consisting of an antibody full length sequence or a functional fragment thereof.
  • the activating antibody is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and scFv-Fc.
  • the transposon is selected from the group consisting of: piggybac, sleeping beauty, frog prince, Tn5, and Ty; preferably, the transposon is a piggybac.
  • the activated antibody expressed by the T cell is secreted or membrane anchored; preferably, the antibody is a membrane anchored.
  • the chimeric antigen receptor is directed against one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT1, ⁇ 1,4-acetyl-aminogalactosyl) Transferase 1), FR (Flavin reductase), PSMA (prostate specific membrane antigen), PMEL promelanosome protein), CA9 (carbonic anhydrase IX), CD171/L1-CAM, IL-13R ⁇ 2, MART- 1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer/testis antigen 1B), MAGE (melanoma-associated antigen E1) family protein, BAGE (B black) Family of tumor antigens, family proteins, GAGE (growth hormone releasing factor) family proteins, AFP ( ⁇ -fetoprotein), MUC1 (mucin 1, cell surface related), CD22, CD23, CD30, CD33, CD44v7
  • the transgenic T cell further comprises a brake molecule.
  • the brake molecule is a membrane antigen that is recognized by a marketed antibody drug; preferably, the membrane antigen is selected from the group consisting of CD11a, CD15, CD19, CD20, CD25, CD44, CD47, CD52, EGFR, ERBB2, ERBB3, ERBB4, VEGFRR1, VEGFR2, EpCAM, MSLN, GPIIb/IIIa, ⁇ 4 integrin, and ⁇ 4 ⁇ 7 integrin; preferably, the membrane antigen is CD20.
  • the activating antibody is directed against one or more of the following antigens: CD28, CD137, CD134, CD40, CD40L, ICOS, HVEM, CD2, CD27, CD30, GITR, LIGHT, DR3 , SLAM, CD226, CD80, CD86; preferably, the activating antibody is an scFv of an anti-CD28 antibody.
  • the transgenic killer cell is transferred to the following nucleic acid construct C:
  • Nucleic acid construct C contains a 5' inverted terminal repeat of the transposon (5' ITR), a nucleic acid sequence encoding an optional brake molecule, a chimeric antigen receptor (CAR) and an activated antibody, and controls expression of the nucleic acid sequence Promoter, polyA tailing signal sequence, transposon 3' inverted terminal repeat (3'ITR), transposase coding sequence and promoter controlling expression of transposase coding sequence.
  • 5' ITR 5' inverted terminal repeat of the transposon
  • CAR chimeric antigen receptor
  • the nucleic acid construct is transferred into the cell using one or more of viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation, preferably employed Electric rotation.
  • a second aspect of the invention provides a pharmaceutical composition comprising a transgenic T cell as described herein and a pharmaceutically acceptable excipient.
  • a third aspect of the invention provides the use of a transgenic T cell or pharmaceutical composition as described herein, characterized in that said use is selected from the group consisting of:
  • Preparation of a medicament for inhibiting tumor cell growth preparation of a medicament for inhibiting virus growth, preparation of a medicament for treating a tumor, preparation of a medicament for treating a viral infectious disease, preparation of a medicament for treating a bacterial infectious disease, and preparation a drug for treating an autoimmune disease;
  • the tumor is selected from the group consisting of liver cancer, lung cancer, colon cancer, pancreatic cancer, gastric cancer, breast cancer, nasopharyngeal carcinoma, lymphoma, ovarian cancer, bladder cancer, prostate cancer, and head and neck cancer.
  • a fourth aspect of the invention provides a nucleic acid construct comprising a transposon 5' inverted terminal repeat (5' ITR) encoding an optional brake molecule, a chimeric antigen receptor (CAR), and Nucleic acid sequence of an activated antibody of an immunostimulatory molecule or a receptor thereof, a promoter for controlling expression of the nucleic acid sequence, a polyA tailing signal sequence, a transposon 3' inverted terminal repeat (3'ITR), a transposase A coding sequence and a promoter that controls expression of the transposase coding sequence.
  • 5' ITR transposon 5' inverted terminal repeat
  • CAR chimeric antigen receptor
  • the activating antibody is selected from the group consisting of an antibody full length sequence or a functional fragment thereof.
  • the activating antibody is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and scFv-Fc.
  • the nucleic acid construct further comprises a coding sequence for the CD28 extracellular hinge region and the transmembrane region.
  • the transposase is a transposase from a piggybac transposing system
  • the 5' inverted terminal repeat (5' ITR) and the 3' inverted terminal repeat are piggybac The 5' inverted terminal repeat and the 3' inverted terminal repeat of the cassette.
  • the brake molecule is CD20.
  • the chimeric receptor antigen is a chimeric antigen receptor directed against the EGFR family.
  • the activating antibody is directed against one or more of the following antigens: CD28, CD137, CD134, CD40, CD40L, ICOS, HVEM, CD2, CD27, CD30, GITR, LIGHT, DR3 , SLAM, CD226, CD80, CD86; preferably, the activating antibody is an scFv of an anti-CD28 antibody.
  • the nucleic acid construct comprises the nucleic acid sequence set forth in SEQ ID NO:2.
  • FIG. 1 Schematic diagram of the expression cassette of antibodies.
  • ITR is a transposon terminal repeat and HyPB is a piggybac transposase.
  • Figure 2 Flow cytometric map of CD28 membrane-anchored antibody of herinCAR-CD28 cells.
  • Figure 3 CD28 molecular flow assay of herinCAR-CD28 cells.
  • Figure 4 In vitro proliferation assay of herinCAR-CD28 cells.
  • Figure 5 Detection of the killing activity of herinCAR-CD28 cells against tumor cells in vitro.
  • Figure 6 In vivo inhibition of transplanted tumors by herinCAR-CD28 cells.
  • Figure 7 Proliferation assay of herinCAR-CD28 cells in transplanted tumors.
  • Figure 8 Functional assay of the herinCAR-CD28 cell molecular brake system.
  • expression cassette refers to the entire element required for expression of a gene, including a promoter, a gene coding sequence, and a PolyA tailing signal sequence.
  • coding sequence is defined herein as a portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (eg, brake molecule, CAR, single chain antibody, hinge region, and transmembrane region).
  • the boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame of the 5' end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame of the 3' end of the mRNA.
  • a coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • activating antibody refers to an antibody that, when present, activates a particular immune response.
  • the activating antibody can be the full length sequence of the antibody or a functional fragment thereof.
  • the activating antibody is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and scFv-Fc.
  • an activating antibody described herein is a single chain antibody.
  • Fab antigen-binding fragment
  • Fc fragment crystallizable (Fc) of an antibody
  • Fc fragment crystallizable
  • antigenic epitope also known as an antigenic determinant (AD) refers to a specific chemical group in an antigen molecule that determines the specificity of an antigen.
  • a polypeptide epitope contains an epitope of 5 to 6 amino acid residues and can be recognized by a specific antibody. The nature, number and spatial configuration of the epitope determine the specificity of the antigen. According to the amino acid continuity of the epitope, it can be divided into a linear epitope and a spatial epitope.
  • the linear epitope is an epitope composed of a sequence of adjacent amino acids, and the spatial epitope is several non-adjacent, but An epitope consisting of amino acids that are adjacent in space structure.
  • costimulatory molecule refers to a molecule that is present on the surface of an antigen presenting cell and that binds to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal.
  • the proliferation of lymphocytes requires not only the binding of antigens, but also the signals of costimulatory molecules.
  • the costimulatory signal is transmitted to the T cells mainly by binding to the co-stimulatory molecule CD80 on the surface of the antigen presenting cells, and CD86 binds to the CD28 molecule on the surface of the T cell.
  • B cells receive a costimulatory signal that can pass through a common pathogen component such as LPS, or through a complement component, or through activated antigen-specific Th cell surface CD40L.
  • linker or hinge is a polypeptide fragment that links between different proteins or polypeptides for the purpose of maintaining the spatial conformation of the linked protein or polypeptide to maintain the function or activity of the protein or polypeptide.
  • exemplary linkers include linkers containing G and/or S, as well as, for example, Furin 2A peptide.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (K D ) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • “Specific recognition” has a similar meaning.
  • pharmaceutically acceptable excipient refers to carriers and/or excipients that are compatible pharmacologically and/or physiologically to the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents include, but are not limited to, phosphate buffers
  • surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80
  • ionic strength enhancers include, but are not limited to, sodium chloride.
  • the term "effective amount” refers to a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
  • disease and/or condition refers to a physical state of the subject that is associated with the disease and/or condition described herein.
  • subject can refer to a patient or other animal that receives the pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog, a monkey, or a cow. , horses, etc.
  • nucleic acid constructs C comprising a transposon 5' inverted terminal repeat (5' ITR) encoding an optional brake molecule, embedded Nucleic acid sequence of an antigen-receptor (CAR) and an immunostimulatory molecule or its receptor-activated antibody (such as a single-chain antibody of interest) and a promoter for controlling expression of the nucleic acid sequence, polyA tailing signal sequence, transposon 3' inverted terminal repeat (3' ITR), a transposase coding sequence and a promoter that controls expression of the transposase coding sequence.
  • 5' ITR transposon 5' inverted terminal repeat
  • CAR antigen-receptor
  • immunostimulatory molecule or its receptor-activated antibody such as a single-chain antibody of interest
  • CAR Chimeric antigen receptor
  • Suitable chimeric antigen receptors for use herein can be various CARs well known in the art.
  • CAR in turn comprises a signal peptide, a polypeptide that binds to a tumor cell membrane antigen, a hinge region, a transmembrane region, and an intracellular signal region.
  • the CAR of the present invention can be constructed using signal peptides, hinge regions, transmembrane regions, and intracellular signal regions well known in the art for constructing CARs.
  • a polypeptide that binds to a tumor cell membrane antigen is capable of binding to a tumor cell to express a membrane antigen with moderate affinity.
  • the polypeptide is usually inserted with an epitope, and the inserted position is selected from any one, two or three of the following three positions:
  • the signal peptide and the polypeptide that binds to the tumor cell membrane antigen bind to the inside of the polypeptide of the tumor cell membrane antigen, and the polypeptide and the hinge region that bind to the tumor cell membrane antigen.
  • the polypeptide that binds to the tumor cell membrane antigen is a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is a single chain antibody or a Fab fragment.
  • FR Fullness reductase
  • PSMA prostate specific membrane antigen
  • PMEL pre-melanin protein CA9 (carbonic anhydrase IX), CD171/L1-CAM, IL-13R ⁇ 2, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer/testis antigen 1B), MAGE (melanoma-associated antigen E1) Family proteins, BAGE (B melanoma antigen family) family proteins, GAGE (growth hormone releasing factor) family proteins, AFP ( ⁇ -fetoprotein), MUC1 (mucin 1, cell surface related), CD22, CD23, CD30, CD33 , CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R ⁇ , EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1, ST8 ⁇ -N-acetyl-ceramide ⁇ -2,8-sialic acid converting enzyme
  • herinCAR from CN 201510812654.9 is used herein (hereby incorporated by reference in its entirety).
  • the polypeptide that binds to a tumor cell membrane antigen is a native polypeptide that is the amino acid sequence HERIN encoded by the eighth intron of the human Her2 gene; preferably, the amino acid sequence thereof is SEQ ID NO of CN 201510812654.9: 5 is shown.
  • amino acid sequence of a CAR signal peptide of the invention is set forth in SEQ ID NO: 3 of CN 201510812654.9.
  • the hinge region of a CAR of the invention is selected from any one or more of the extracellular hinge region of CD8, the extracellular hinge region of CD28, and the extracellular hinge region of CD4; preferably extracellular to CD8 Hinge area.
  • the extracellular hinge region of CD8 is set forth in SEQ ID NO: 7 of CN 201510812654.9.
  • the transmembrane region of a CAR of the invention is selected from any one or more of a transmembrane region of CD8, a transmembrane region of CD28, and a transmembrane region of CD4; preferably, a CD8 transmembrane region;
  • the amino acid sequence of the CD8 transmembrane region is set forth in SEQ ID NO: 8 of CN 201510812654.9.
  • the intracellular signal region of a CAR of the invention may be selected from the group consisting of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, DAP10, CD3 ⁇ , and Fc ⁇ RI ⁇ .
  • the signal region is preferably a 4-1BB intracellular signal region and a CD3 intracellular signaling region, or a CD28 intracellular signal region and a CD3 intracellular signaling region; preferably, the 4-1BB intracellular signal region and the CD3 intracellular signaling region
  • the amino acid sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 10 of CN 201510812654.9, respectively; preferably, the amino acid sequences of the CD28 intracellular signal region and the CD3 intracellular signaling region are respectively SEQ ID NO of CN 201510812654.9: 11 and SEQ ID NO:10.
  • the epitope is directly linked to a polypeptide that binds to a tumor cell membrane antigen or is linked by a protein linker.
  • the linker is at least 2 glycines, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 glycines.
  • the chimeric antigen receptor of the invention consists of a signal peptide, a CD20 epitope, an amino acid sequence HERIN encoded by Herrin, the eighth intron of the human Her2 gene, a CD20 epitope, a CD8 hinge region, a CD8
  • the transmembrane region and the 4-1BB co-stimulatory peptide were sequentially constructed.
  • the amino acid sequence thereof is shown as SEQ ID NO: 1 of CN 201510812654.9.
  • single-chain antibody the amino acid sequence of interest is connected via a hinge antibody light chain variable region (V region L) and the amino acid sequence of a heavy chain variable region (V H region) is made, having An antibody fragment that binds to antigenic ability.
  • the single chain antibody of interest is from an antibody of interest.
  • the nucleic acid constructs herein can encode two single chain antibodies, one single chain antibody being present in the CAR shown, and the other single chain antibody being a single chain antibody linked to the CAR.
  • the two single chain antibodies may be the same or different, and preferably, the two single chain antibodies perform different antibody functions.
  • Antibodies of interest may be human antibodies, including human murine chimeric antibodies and humanized antibodies.
  • the antibody of interest is preferably an activated antibody, particularly an activated antibody directed against an immunocostimulatory molecule and its receptor.
  • the antibody may be secreted or membrane anchored; preferably a membrane anchored.
  • the antibody is an scFv of an anti-CD28 antibody.
  • CD28 refers to human leukocyte differentiation antigen 28, which is the official name of CDBI in NCBI GeneBank, ID number 940, and has three isomers (cDNA sequence/protein sequence), respectively NM_006139.3/ NP_006130.1, NM_001243077.1/NP_001230006.1, NM_001243078.1/NP_001230007.1.
  • a single-chain antibody may comprise, or consist of, a heavy chain variable region and a light chain variable region of each of the antibodies of interest, and an optional linker.
  • the heavy chain variable region and the light chain variable region can be joined by well-known linkers, such as those containing G and/or S.
  • the linker is usually 15 to 20 amino acids in length.
  • the linker is (GGGS) n and n is an integer from 1 to 5.
  • the nucleic acid sequence encoding the activated antibody may further comprise a hinge region and a transmembrane region sequence from the same or different antibodies.
  • a hinge region and a transmembrane region sequence from the same or different antibodies.
  • an anti-CD28 antibody an scFv derived from the antibody can be used, and the sequence derived from the extracellular hinge region and the transmembrane region of the CD28 can be ligated after the scFv.
  • brake molecules may be selected from the group consisting of: CD11a, CD15, CD19, CD20, CD25, CD44, CD47, CD52, EGFR, ERBB2, ERBB3, ERBB4, VEGFR1, VEGFR2, EpCAM, MSLN (mesothelin), GPIIb/IIIa, ⁇ 4 integrin and ⁇ 4 ⁇ 7 integrin.
  • the membrane antigen is CD20.
  • a linear or spatial epitope of the membrane antigen can be used.
  • the brake molecule can be attached to the CAR via a linker sequence commonly used in the art (such as the G and S-containing linker sequences described above), or can be directly attached to the CAR.
  • CAR can be directly linked to a single-chain antibody or can be linked by a linker sequence (eg, Furin 2A peptide).
  • CD20 refers to human leukocyte differentiation antigen 20, which is officially named MS4A1 in NCBI GeneBank, ID number 931, and has two isomers (cDNA sequence/protein sequence), respectively NM_021950.3/NP_068769.2, NM_152866.2/NP_690605.1.
  • amino acid sequence of CD20 it includes a full length of the CD20 protein or a fragment of CD20 having CD20 function; a fusion protein of the full length or fragment is also included.
  • mutations or variations may be naturally occurring or artificially introduced in the amino acid sequence of CD20 without affecting its biological function.
  • a corresponding sequence fragment in its native or artificial variant is also included.
  • the corresponding promoter sequence can be selected based on the coding sequence of the selected CAR.
  • promoters include, but are not limited to, the EF1 ⁇ promoter.
  • the promoter may further include an enhancer, such as one, any two or all of the mCMV enhancer, the hCMV enhancer and the CD3e enhancer, as described in CN201510021408.1 (hereby incorporated by reference herein in its entirety). Three.
  • various promoter sequences disclosed in CN201510021408.1 are used herein, including but not limited to the mCMV enhancer-containing, hCMV enhancer, and EF1 ⁇ promoters shown in SEQ ID NO: 1 of the application.
  • CCEF promoter TEF promoter containing CD3e enhancer and EF1 ⁇ promoter shown in SEQ ID NO: 2; CD3e enhancer, mCMV enhancer, hCMV enhancer and EF1 ⁇ promoter represented by SEQ ID NO: TCEF promoter; CCEFI promoter containing the mCMV enhancer, hCMV enhancer and intron-containing EF1 ⁇ promoter shown in SEQ ID NO: 4; CD3e enhancer and inclusion contained in SEQ ID NO: The TEFI promoter of the EF1 ⁇ promoter; and the TCEFI promoter containing the CD3e enhancer, the mCMV enhancer, the hCMV enhancer, and the intron-containing EF1 ⁇ promoter shown in SEQ ID NO: 5.
  • the transposase may be a transposase from a piggybac, sleeping beauty, frog prince, Tn5 or Ty transposition system.
  • sequences of the 5' ITR and 3' ITR in the nucleic acid constructs of the invention are also correspondingly altered to sequences adapted to the transposition system, which can be readily determined by those skilled in the art. Determined.
  • the transposase is a transposase from a piggybac transposition system.
  • the 5' inverted terminal repeat and the 3' inverted terminal repeat of the transposon are the 5' inverted terminal repeat and the 3' inverted terminal repeat of the piggybac transposon, respectively.
  • the transposon 5' inverted terminal repeat is SEQ ID NO: 1 as described in CN 201510638974.7, the disclosure of which is incorporated herein by reference.
  • the 3' inverted terminal repeat of the transposon is as shown in CN 201510638974.7 SEQ ID NO: 4.
  • the piggybac transposase is a transposase comprising a c-myc nuclear localization signal coding sequence.
  • the coding sequence for the piggybac transposase is as shown in CN 201510638974.7 SEQ ID NO: 5.
  • the promoter of the transposase coding sequence can be a variety of promoters known in the art for controlling expression of the transposase coding sequence.
  • the expression of the transposase coding sequence is controlled using a CMV promoter.
  • the sequence of the CMV promoter can be as shown in CN 201510638974.7 SEQ ID NO: 6.
  • PolyA tailing signal sequences well known in the art can be used.
  • the polyA is from SV40.
  • the sequence set forth in CN 201510638974.7 SEQ ID NO: 3 can be used.
  • a suitable promoter can be selected for the selected membrane antigen to control the expression of the membrane antigen.
  • the promoter is an EF1 ⁇ promoter.
  • the sequence of the EF1 ⁇ promoter is as shown in CN 201510638974.7 SEQ ID NO: 8.
  • the nucleic acid construct C herein comprises a 5' inverted terminal repeat (5' ITR) in turn, which controls the encoding of a brake molecule, a chimeric antigen receptor (CAR), and immuno-stimulation A promoter for expression of a nucleic acid sequence of an activated antibody of a molecule or its receptor (such as a single-chain antibody of interest), an activated antibody encoding a brake molecule, a chimeric antigen receptor (CAR), and an immunostimulatory molecule or its receptor (eg, single-chain antibody of interest) nucleic acid sequence, polyA tailing signal sequence, transposon 3' inverted terminal repeat (3'ITR), transposase coding sequence and promoter controlling expression of transposase coding sequence .
  • 5' ITR 5' inverted terminal repeat
  • CAR chimeric antigen receptor
  • the nucleic acid construct C herein comprises a 5' inverted terminal repeat (5' ITR), which is ligated in turn, controls the encoding of a brake molecule, a chimeric antigen receptor (CAR), and immuno-stimulation
  • An activated antibody of a molecule or its receptor such as a single-chain antibody of interest
  • a promoter of a nucleic acid sequence expressed by an extracellular hinge region of an antigen to which the antibody is directed and a transmembrane region
  • a nucleic acid sequence of the extracellular hinge region and the transmembrane region of an antigen to which the antibody is directed a polyA tailing signal sequence, transposition
  • the 3' inverted terminal repeat (3' ITR) the transposase coding sequence and a promoter that controls expression of the transposa
  • nucleic acid construct C herein comprises a transposon 5' inverted terminal repeat (5' ITR), an EF1 ⁇ promoter, a brake molecule (CD20), a CAR, an anti-CD28 single-chain antibody , extracellular hinge region and transmembrane region of CD28, polyA tailing signal sequence, transposon 3' inverted terminal repeat (3'ITR), transposase coding sequence and promoter controlling expression of transposase coding sequence (CMV).
  • 5' ITR transposon 5' inverted terminal repeat
  • CD20 brake molecule
  • CAR an anti-CD28 single-chain antibody
  • extracellular hinge region and transmembrane region of CD28 extracellular hinge region and transmembrane region of CD28
  • polyA tailing signal sequence transposon 3' inverted terminal repeat (3'ITR)
  • transposase coding sequence and promoter controlling expression of transposase coding sequence (CMV).
  • the nucleic acid construct C comprises the nucleic acid sequence set forth in SEQ ID NO:2.
  • the nucleic acid construct C herein may be a recombinant expression vector (recombinant expression vector C) for expressing the CAR, scFv and the optional brake molecule.
  • the expression vector is a transposon vector.
  • the vector is one or more selected from the group consisting of a piggybank, sleeping beauty, frog prince, Tn5, and Ty.
  • the expression vector usually contains other elements normally contained in the vector, such as a multiple cloning site, a resistance gene, an origin of replication, and the like.
  • the recombinant expression vector uses pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vector, pUC57, pMAX or pDC315 series vectors as a backbone.
  • the recombinant expression vector uses a pCDNA3 series vector, a pCDNA4 series vector, a pCDNA5 series vector, a pCDNA6 series vector, a pRL series vector, a pUC57 vector, a pMAX vector, or a pDC315 series vector as a backbone.
  • the invention employs a pSN vector constructed using CN 201510638974.7, the vector structure of which is shown in Figure 1 of the application.
  • the nucleic acid construct C/recombinant expression vector C of the present invention can be transferred into a cell of interest.
  • Methods for transfer are routine methods in the art including, but not limited to, viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation.
  • the nucleic acid construct or recombinant expression vector is electroporated.
  • the cells of interest may be various T cells well known in the art including, but not limited to, peripheral blood T lymphocytes, cytotoxic killer T cells (CTLs), helper T cells, suppressor/regulatory T cells, ⁇ T cells, and cytokines.
  • T cells of mixed cell populations such as induced killer cells (CIK) and tumor infiltrating lymphocytes (TIL).
  • the transposon contained in the nucleic acid construct or the recombinant expression vector is contained because it contains an ITR element and a transposase required for transposition.
  • the nucleic acid sequence between the 5' inverted terminal repeat and the 3' inverted terminal repeat of the transposon is integrated into the genome of the cell of interest.
  • the nucleic acid construct C/recombinant expression vector C contains a nucleic acid sequence encoding a brake molecule, the cell will further express the brake molecule.
  • transgenic T cells in which the expression of a nucleic acid sequence comprising an activated antibody (eg, a single chain antibody of interest) encoding a CAR and an immunocostimulatory molecule or its receptor is stably integrated into the genome of the T cell. frame. Further, the T cell genome stably integrates the 5' inverted terminal repeat (5' ITR), which is sequentially linked, and controls the encoding of the brake molecule, chimeric antigen receptor (CAR), and immune co-stimulation.
  • an activated antibody eg, a single chain antibody of interest
  • an immunocostimulatory molecule or its receptor stably integrated into the genome of the T cell. frame.
  • the T cell genome stably integrates the 5' inverted terminal repeat (5' ITR), which is sequentially linked, and controls the encoding of the brake molecule, chimeric antigen receptor (CAR), and immune co-stimulation.
  • 5' ITR 5' inverted terminal repeat
  • Activator of a molecule or its receptor such as a single-chain antibody of interest
  • a hinge region of an antigen to which the antibody is directed and a promoter for expression of a nucleic acid sequence of a transmembrane region, encoding a brake molecule, a chimeric antigen receptor (CAR) , an antibody that activates an immunostimulatory molecule or its receptor (such as a single-chain antibody of interest), a nucleic acid sequence of an extracellular hinge region and a transmembrane region to which the antibody is directed, a polyA tailing signal sequence, and a transposon 3 'Inverse end repeat sequence (3' ITR).
  • the transgenic T cell expresses an activated antibody.
  • the antibody may be a secreted antibody or a membrane anchored form, preferably a membrane anchored form.
  • the antibody may be an immunostimulatory molecule-activated antibody that acts on the T cell itself. After the transgenic T cells express the antibody, the cluster effect can be enhanced.
  • a T cell herein stably expresses a CAR of interest and an scFv of interest. In certain embodiments, a T cell herein is transformed into a nucleic acid construct C/recombinant expression vector C as described herein.
  • the transgenic T cells herein can have different biological activities including, but not limited to, inhibition of tumors, inhibition of viruses, inhibition of bacteria, and the like.
  • the CAR-T cells herein can be used to inhibit tumor cell growth, inhibit viral growth, treat tumors, treat viral infectious diseases, treat bacterial infectious diseases, and treat autoimmune diseases.
  • Tumors include, but are not limited to, liver cancer, lung cancer, colon cancer, pancreatic cancer, gastric cancer, breast cancer, nasopharyngeal carcinoma, lymphoma, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer.
  • transgenic T cells described herein for the preparation of a medicament for inhibiting tumor cell growth, for preparing a medicament for inhibiting virus growth, for preparing a medicament for treating a tumor, for preparing a medicament for treating a viral infectious disease, and for preparing A medicament for treating a bacterial infectious disease, or a medicament for preparing a medicament for treating an autoimmune disease.
  • the transgenic T cell is a CAR-T cell.
  • the present invention also provides a kit comprising the recombinant expression vector described herein.
  • the kit may also contain various reagents suitable for transfecting the recombinant expression vector into a cell, and optionally instructions for those skilled in the art to transfect the recombinant expression vector into a cell.
  • the invention also relates to a composition of a recombinant expression vector, the composition of the recombinant expression vector described herein.
  • the composition may also contain a corresponding solvent or carrier.
  • the inventors have successfully constructed a pluripotent CAR-T cell that efficiently and stably expresses an activated antibody through a large number of preliminary experiments and creative labor.
  • the pluripotent CAR-T cell genome stably integrates activation through a transposon system.
  • the expression cassette of the type antibody especially the activated antibody of the costimulatory molecule and its receptor, thereby maintaining the activity of stably and efficiently expressing the activated antibody under the premise of maintaining the original CAR-T cell killing activity, so that the returned antibody
  • the cells can proliferate rapidly at the tumor site.
  • the invention overcomes the defects that the current gene transfection vector system has low T cell transfection rate and low expression level of the antibody, and the immune killer cells stably express the chimeric antigen receptor and the activated antibody at a high level, thereby overcoming the CAR-T cells are difficult to achieve an effective number of problems during solid tumor CAR-T cell therapy.
  • the immune system is activated uncontrollably, resulting in an immune overreaction.
  • the antibody can be anchored to the CAR-T cell membrane to activate adjacent cells by direct contact.
  • the cells can be quickly cleared by listed monoclonal antibodies (such as rituximab), which improves the safety of treatment.
  • the transgenic T cells produced by the present invention can be used for the treatment of various malignant tumors and viruses.
  • HerinCAR coding sequence containing CD20- rituximab molecular brakes HerinCAR coding sequence containing CD20- rituximab molecular brakes:
  • herinCAR-CD28 coding sequence (SEQ ID NO: 1) wherein the double underline indicates the cleavage site, and the wavy underlined is the CD20 epitope coding sequence recognized by the rituximab antibody.
  • FIG. 1 A schematic diagram of the pNB328-herinCAR-CD28 vector is shown in Figure 1.
  • PBMC peripheral blood mononuclear cells
  • pNB328-herinCAR-CD28 transfect 6 ⁇ g of pNB328-herinCAR-CD28 into the nucleus by Lonza 2b-Nucleofector instrument at 37 °C.
  • herinCAR-CD28 cells the gene-modified T cells targeting the EGFR family and containing the CD20- rituximab brake CAR, CD28 single-chain antibody, referred to as herinCAR-CD28 cells, can be simultaneously expressed.
  • the same source PBMC was transfected with pNB328-herinCAR plasmid to obtain herinCAR-T cells.
  • the suspended herinCAR-CD28 cells and control herin CAR-T cells were collected and counted, and then added to two 1.5 ml EP tubes at 1 ⁇ 10 6 cells/tube, washed twice with PBS, and centrifuged at 1200 rpm for 5 min. The supernatant was discarded; 2 ⁇ l of anti-human IgG Fab2' antibody (purchased from Jackson ImmunoResearch) was added, and the mixture was lightly precipitated and mixed uniformly. Incubate at room temperature for 30 min in the dark, PBS was washed once, centrifuged at 1200 rpm for 5 min, and the supernatant was added to add 400 ⁇ l of physiological The saline is transferred to the flow tube and detected by the machine. The experimental results showed that the surface of herinCAR-CD28 cells had CD28 antibody molecules on the surface relative to the control cells, as shown in Fig. 2.
  • the suspended herinCAR-CD28 cells and control herin CAR-T cells were collected and counted, and then added to two 1.5 ml EP tubes at 1 ⁇ 10 6 cells/tube, washed twice with PBS, and centrifuged at 1200 rpm for 5 min. Discard the supernatant; add 2 ⁇ l of the isotype control antibody IgG1-PE, anti-CD28-PE antibody (both purchased from BD), gently precipitate to mix well, incubate at room temperature for 30 min in the dark, wash once with PBS, centrifuge at 1200 rpm for 5 min. The supernatant was discarded and 400 ⁇ l of physiological saline was added to transfer the cells to a flow tube, which was detected by a machine.
  • the herinCAR-CD28 cells and herinCAR cells constructed in Example 2 were plated at 4 ⁇ 10 4 /well in 96-well plates, and each cell was plated with 3 replicate wells in a total volume of 200 ⁇ l at 37 ° C, 5% CO 2 . Incubate in the incubator, add 20 ⁇ l of CCK8 reagent after 24h, 48h, 72h, 96h, respectively, incubate for 6h at 37°C in the dark, and measure the OD value at 450nm on the microplate reader. The results showed that the proliferation rate of herinCAR-CD28 cells was significantly higher than that of control herinCAR cells, indicating that CD28 antibody on the surface of herinCAR-CD28 cell membrane can promote the proliferation of T cells, as shown in Figure 4.
  • the gastric cancer cell line BGC-27 and the ovarian cancer cell line SK-OV3 (purchased from the American Standard Biological Collection Center, ATCC) were plated on a RTCA cell proliferation plate (purchased from ACEA Biosciences, USA) at a ratio of 10,000 cells/well.
  • a RTCA cell proliferation plate purchased from ACEA Biosciences, USA
  • cell growth was recorded in real time (reflected by the measured cell index, the higher the value indicates the better cell status).
  • the herinCAR cells and herinCAR-CD28 cells constructed in Example 2 were added to the xCELLigence RTCA DP according to the target ratio (E:T) of 8:1, 4:1, 2:1, and 0:1, respectively.
  • E:T target ratio
  • Cell growth was measured on a multi-functional real-time label-free cell analyzer.
  • the cell index was determined according to the measured cell ratio and the target ratio of 0:1 (without effector cells). The ratio of the tumor cell lysis rate is determined.
  • Example 7 Identification of in vivo killing activity of herinCAR-CD28 cells
  • BGC-27 malignant gastric cancer cells were subcutaneously injected into NOD-SCID mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.), and 10 days later, the herinCAR cells constructed in Example 2 and herinCAR-CD28 were injected through the tail vein.
  • the growth of the transplanted tumor was measured by injecting a dose of 2 x 10 5 or PBS buffer.
  • the results showed that the inhibitory effect of herinCAR-CD28 cells on liver cancer was significantly different from that of the control group (Fig. 6), indicating that herinCAR-T cells co-expressing CD28 antibody have a good anti-tumor effect in vivo.
  • Example 8 Proliferation detection of herinCAR-CD28 cells in transplanted tumors
  • Example 7 On the 35th day after the treatment, the tumor-bearing mice were sacrificed, the transplanted tumor tissues were taken, and genomic DNA was extracted (operated by a tissue DNA extraction kit of Sigma-Aldrich Co., Ltd.). RT-PCR was used to detect the relative copy number of herinCAR gene in transplanted tumor tissues (primers are shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the reaction system and procedure are based on RealMaster Mix (SYBR Green) detection reagent. Box operation). The results showed that the copy number of herinCAR-CD28 cells in the transplanted tumor was significantly increased relative to herinCAR-T cells (Fig. 7), indicating that expression of CD28 antibody enhanced the survival time of CAR-T cells in the transplanted tumor.
  • RT-PCR was used to detect the relative copy number of herinCAR gene in transplanted tumor tissues (primers are shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the reaction system and procedure are based on RealMaster Mix (SY
  • R GAAGGGCGTCGTAGGTGTC (SEQ ID NO: 4).
  • Example 9 In vivo clearance test of herinCAR-CD28 cells (validation of molecular brake function)
  • HerinCAR-CD28 cells (injected dose 5 ⁇ 10 6 ) were injected into the tail of BABL/c nude mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.), and 100 ⁇ g of rituximab antibody or human IgG control antibody was intravenously administered 3 days later. After 12 hours, blood and bone marrow samples were taken and the ratio of herinCAR-CD28 cells (CD20 and CD3 double positive cells) was detected by flow cytometry.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Dermatology (AREA)
  • Communicable Diseases (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种转基因T细胞，其基因组中稳定整合了含编码嵌合抗原受体、免疫共刺激分子或其受体的激活型抗体的核酸序列的表达框以及两端包含转座子的反向末端重复序列。还提供了含有该转基因T细胞的药物组合物以及核酸构建体。

Description

一种高效稳定表达激活型抗体的CAR-T细胞及其用途 技术领域
本发明属于细胞生物学和肿瘤学领域，涉及一种高效稳定表达激活型抗体的CAR-T细胞及其用途。
背景技术
针对恶性肿瘤的免疫治疗近年来发展迅速，取得令人瞩目的临床疗效。其中，免疫检查点(如CTLA4、PD1/PDL1)单抗治疗，通过原位激活患者残留的肿瘤特异性T细胞发挥作用，对多种恶性肿瘤的总体有效率达到30％，且许多患者一旦起效能长期存活；转基因CAR-T/TCR-T细胞治疗，是通过离体基因修饰的手段快速获得肿瘤特异性T细胞后，过继回输进行治疗发挥作用，对复发难治性B细胞白血病的完全缓解率超过90％。
嵌合抗原受体(Chimeric Antigen Receptor,CAR)一般由一个scFv(single-chain variable Fragment)单链抗体(抗体轻重链可变区经Linker连接构成，负责结合膜抗原)，通过铰链结构(负责形成正确构象，形成二聚体)与跨膜区、胞内信号结构(负责传递T细胞活化信号)连接构成。经CAR基因修饰的T细胞(CAR-T)被赋予识别肿瘤细胞膜抗原肽分子，启动杀伤或增殖的能力。由于CAR-T细胞识别的是表达在细胞膜上的抗原，而非与MHC分子结合后被递呈到细胞表面的抗原，因而可以绕过T细胞MHC限制性，避免由于肿瘤细胞下调或缺失MHC分子引起的免疫逃逸。
CAR的设计最早由以色列学者Eshhar和其同事于1989年提出，按其发展阶段到目前为止可分为三代。第一代CAR受体包含了胞外特异识别肿瘤抗原的svFC片段，胞内激活信号由CD3ζ或FcεRIγ的ITAM(immunoreceptor tyrosine-based activation motifs)信号链来传递。但是第一代CAR受体缺乏T细胞的共刺激信号，导致T细胞只能发挥瞬间效应，在体内存在时间短、细胞因子分泌少等缺点。第二代CAR受体增加了共刺激信号分子的胞内结构域，包括如CD28，CD134/OX40，CD137/4-1BB，lymphocyte-specific protein tyrosine kinase(LCK)，inducible T-cell co-stimulator(ICOS)以及DNAX-activation protein 10(DAP10)等结构域，增强了T细胞的增殖能力及细胞因子的分泌功能，IL-2、IFN-γ以及GM-CSF增加，从而突破肿瘤微环境的免疫抑制、延长AICD(activation induced cell death,AICD)。第三代CAR受体则是在第二代CAR的基础上再融合一个二级共刺激分子如4-1BB，从而 产生一个三重信号的CAR受体，第三代CAR受体改造的T细胞具有更好的效应功能和体内存活时间。近年来，有部分学者提出第四代CAR受体概念，他们在第三代CAR的基础上增加了IL12、IL15等具有正向调节T细胞功能的细胞因子，进一步提高T细胞的增殖、杀伤、体内存活能力。
目前，已有多个靶标的CAR-T细胞正在开展实体瘤治疗的临床试验，包括GD2、FR-α、L1-CAM、HER2、EGFR、EGFRvIII、VEGFR-2、IL-13Rα2、FAP、Mesothelin、c-MET、PSMA、CEA、GPC3、EphA2、MUC1、CAIX(carbonic anhydrase IX)等。其中，部分临床试验取得相对良好的结果，例如，针对GD2的CAR-T细胞治疗高风险性神经母细胞瘤的临床试验(19例患者)，8例患者回输后肿瘤完全消退，3例未消退的患者在回输后第6周的观察时间点显示完全应答，1名完全应答患者192周仍能检测到CAR-T细胞；针对HER2的CAR-T细胞治疗HER2阳性实体瘤临床试验(共19例，其中骨肉瘤16例)，4例维持无疾病进展状态12周至14月，其中3例肿瘤消退，1例消退90％以上。然而总体而言，实体瘤的CAR-T治疗相对于血液肿瘤，疗效仍存在较大差距，究其原因，主要包括：
1、抑制免疫的微环境
实体瘤组织存在免疫抑制的微环境，包括Treg细胞、肿瘤相关成纤维细胞、骨髓来源未成熟DC细胞、M2型巨噬细胞等及其它们分泌的细胞因子，如IL-6、IL-10、IDO、VEGF、TGFβ等，这些细胞及其分泌的细胞因子可以通过直接或间接的方式抑制T细胞的功能。通过放化疗破坏肿瘤微环境，免疫检查点抗体(如PD1/PDL1)或免疫负调节因子(如IDO小分子抑制剂)特异阻断相关信号通路，表观遗传修饰相关酶类的抑制剂整体调整免疫微环境，过表达免疫正调节因子(如IL-12)，直接靶向清除肿瘤基质细胞(如靶向FAP阳性肿瘤相关成纤维细胞的CAR-T)等策略，均可以作用于实体瘤免疫抑制的微环境，提高浸润的CAR-T细胞的存活能力与杀伤效果。
2、缺乏合适的CAR-T治疗靶点
实体瘤具有高度的异质性，不同患者、同一患者不同病灶、同一病灶不同肿瘤细胞之间均存在高度的差异。这种高度的异质性致使肿瘤靶向治疗陷入缺乏理想的通用、广谱靶点的不利境地，限制了CAR-T细胞治疗实体瘤的疗效。因而，为扩大CAR-T细胞的杀伤范围，有学者提出TanCAR的设计理念，将两种结合不同肿瘤相关抗原的scFv串联在一起，构成一个新的能同时识别并结合两个靶点的CAR，有效提高了CAR-T细胞的疗效。
3、在体内难以达到有效数量
T细胞杀伤肿瘤细胞必需群集效应，杀伤一个肿瘤细胞需要数个T细胞的协作，因而只有效应细胞达到一定数量后，肿瘤细胞才可被有效杀伤。所以，当T细胞接触特定靶点的肿瘤细胞后，可快速增殖，并通过直接接触与旁分泌途径，放大杀伤功能。CAR-T细胞 通过静脉给药，在血液肿瘤条件下，非常容易接触肿瘤细胞，CAR-T细胞的数量可快速放大，甚至过量放大形成细胞因子风暴，因而疗效相对较好；而实体瘤条件下，血液循环中肿瘤细胞数量有限，CAR-T细胞需达到肿瘤部位才能接受刺激，难以达到有效剂量。
因而，如能在CAR-T，尤其是能广谱识别肿瘤膜抗原的CAR-T细胞基础上，表达具有直接或间接刺激T细胞增殖、存活等功能的抗体，则能有效克服实体瘤CAR-T治疗的难题，大幅提高疗效。
尽管已经有将外源基因转导入例如T细胞的报导，但目前常用基因转染载体系统对具有细胞杀伤毒性的效应免疫细胞转染率低，或者难以使外源基因在其细胞内高水平地表达。利用腺病毒载体(非整合型)可以介导外源基因在T细胞内较高效的短时表达，但活化的T细胞增殖速度非常快，携带的外源基因表达框在细胞传代中将快速丢失，表达难以持久；利用逆转录病毒或慢病毒可以介导外源基因在T细胞基因组中的整合，理论上可实现稳定表达，但抗体包含轻链与重链，编码序列长、分子量大，导致携带全长抗体表达框的逆转录病毒或慢病毒包装与制备存在较大困难、难以高效表达抗体(例如，利用CAR-T细胞表达抗体，浓度仅200ng/ml,Oncotarget.2016Apr 29.doi:10.18632/oncotarget.9114)，一般用于表达结构简单的单链抗体(缺乏恒定区片段，功能不全且半衰期短)。因而，在此之前，尚无CAR-T细胞高效表达抗体的报道。
发明内容
本发明第一方面提供一种转基因T细胞，所述T细胞基因组中稳定整合了含编码嵌合抗原受体，及免疫共刺激分子或其受体的激活型抗体的核酸序列的表达框，且表达框的两端包含转座子的反向末端重复序列。
在一个或多个实施方案中，所述激活型抗体选自：抗体全长序列或其功能性片段。
在一个或多个实施方案中，所述激活型抗体选自：Fab、Fab’、F(ab’)2、Fv、scFv和scFv-Fc。
在一个或多个实施方案中，所述转座子选自：piggybac、sleeping beauty、frog prince、Tn5和Ty；优选地，所述转座子为piggybac。
在一个或多个实施方案中，所述T细胞表达的激活型抗体为分泌型或膜锚定型；优选地，所述抗体为膜锚定型。
在一个或多个实施方案中，所述嵌合抗原受体针对如下抗原中的一种或多种：CD19、CD20、CEA、GD2(又称B4GALNT1，β1,4-乙酰基-氨基半乳糖基转移酶1)、FR(Flavin还原酶)、PSMA(前列腺特异性膜抗原)、PMEL前黑素小体蛋白)、CA9(碳酸酐酶IX)、CD171/L1-CAM、IL-13Rα2、MART-1(又称粘蛋白-A)、ERBB2、NY-ESO-1(又称CTAG1B，癌/睾丸抗原1B)、MAGE(黑素瘤相关抗原E1)家族蛋白、BAGE(B黑 素瘤抗原家族)家族蛋白、GAGE(生长激素释放因子)家族蛋白、AFP(α-胎蛋白)、MUC1(mucin 1，细胞表面相关)、CD22、CD23、CD30、CD33、CD44v7/8、CD70、VEGFR1、VEGFR2、IL-11Rα、EGP-2、EGP-40、FBP、GD3(又称ST8SIA1，ST8α-N-乙酰基-神经酰胺α-2,8-唾液酸转换酶1)、PSCA(前列腺干细胞抗原)、FSA(又称KIAA1109)、PSA(又称KLK3，激肽释放酶相关的肽酶3)、HMGA2、胎儿型乙酰胆碱受体、LeY(又称FUT3)、EpCAM、MSLN(间皮素)、IGFR1、EGFR、EGFRvIII、ERBB3、ERBB4、CA125(又称MUC16，mucin 16，细胞表面相关)、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、SCC(又称SERPINB3)、AFU(又称FUCA1)、EBV-VCA、POA(又称VDR，维生素D(1,25-二氢维生素D3)受体)、β2-MG(β-2-微球蛋白)和PROGRP(GRP胃泌素释放肽)；优选地，所述嵌合抗原受体为针对EGFR家族的嵌合抗原受体。
在一个或多个实施方案中，所述转基因T细胞还包含刹车分子。
在一个或多个实施方案中，所述刹车分子为可被已上市抗体药物识别的膜抗原；优选地，所述膜抗原选自CD11a、CD15、CD19、CD20、CD25、CD44、CD47、CD52、EGFR、ERBB2、ERBB3、ERBB4、VEGFR1、VEGFR2、EpCAM、MSLN、GPIIb/IIIa、α4整合素和α4β7整合素；优选地，所述膜抗原为CD20。
在一个或多个实施方案中，所述激活型抗体针对如下抗原中的一种或多种：CD28、CD137、CD134、CD40、CD40L、ICOS、HVEM、CD2、CD27、CD30、GITR、LIGHT、DR3、SLAM、CD226、CD80、CD86；优选地，所述激活型抗体为抗CD28抗体的scFv。
在一个或多个实施方案中，所述转基因杀伤性细胞转入了以下核酸构建物C：
核酸构建物C：含有转座子5’反向末端重复序列(5’ITR)，编码任选的刹车分子、嵌合抗原受体(CAR)和激活型抗体的核酸序列及控制该核酸序列表达的启动子，polyA加尾信号序列，转座子3’反向末端重复序列(3’ITR)，转座酶编码序列和控制转座酶编码序列表达的启动子。
在一个或多个实施方案中，采用病毒转导、显微注射、粒子轰击、基因枪转化和电转中的一种或多种方法将所述核酸构建物转入所述细胞中，优选地采用电转。
本发明第二方面提供一种药物组合物，所述药物组合物含有本文所述的转基因T细胞和药学上可接受的辅料。
本发明第三方面提供本文所述的转基因T细胞或药物组合物的用途，其特征在于，所述用途选自：
制备用于抑制肿瘤细胞生长的药物、制备用于抑制病毒生长的药物、制备用于治疗肿瘤的药物、制备用于治疗病毒感染性疾病的药物、制备用于治疗细菌感染性疾病的药物和制备用于治疗自身免疫疾病的药物；
其中，所述肿瘤选自：肝癌、肺癌、结肠癌、胰腺癌、胃癌、乳腺癌、鼻咽癌、淋巴瘤、卵巢癌、膀胱癌、前列腺癌和头颈部肿瘤。
本发明第四方面提供一种核酸构建物，所述核酸构建物含有转座子5’反向末端重复序列(5’ITR)，编码任选的刹车分子、嵌合抗原受体(CAR)和免疫共刺激分子或其受体的激活型抗体的核酸序列及控制该核酸序列表达的启动子，polyA加尾信号序列，转座子3’反向末端重复序列(3’ITR)，转座酶编码序列和控制转座酶编码序列表达的启动子。
在一个或多个实施方案中，所述激活型抗体选自：抗体全长序列或其功能性片段。
在一个或多个实施方案中，所述激活型抗体选自：Fab、Fab’、F(ab’)2、Fv、scFv和scFv-Fc。
在一个或多个实施方案中，所述核酸构建物还包括CD28胞外铰链区和跨膜区的编码序列。
在一个或多个实施方案中，所述转座酶为来自piggybac转座系统的转座酶，所述5’反向末端重复序列(5’ITR)和3’反向末端重复序列是piggybac转座子的5’反向末端重复序列和3’反向末端重复序列。
在一个或多个实施方案中，所述刹车分子是CD20。
在一个或多个实施方案中，所述嵌合受体抗原是针对EGFR家族的嵌合抗原受体。
在一个或多个实施方案中，所述激活型抗体针对如下抗原中的一种或多种：CD28、CD137、CD134、CD40、CD40L、ICOS、HVEM、CD2、CD27、CD30、GITR、LIGHT、DR3、SLAM、CD226、CD80、CD86；优选地，所述激活型抗体为抗CD28抗体的scFv。
在一个或多个实施方案中，所述核酸构建物含有SEQ ID NO:2所示的核酸序列。
附图说明
图1：抗体的表达框模式图。ITR为转座子末端重复序列，HyPB是piggybac转座酶。
图2：herinCAR-CD28细胞的CD28膜锚定型抗体流式检测图。
图3：herinCAR-CD28细胞的CD28分子流式检测图。
图4：herinCAR-CD28细胞的体外增殖检测。
图5：herinCAR-CD28细胞在体外对肿瘤细胞的杀伤活性检测。
图6：herinCAR-CD28细胞对移植瘤的体内抑制作用检测。
图7：herinCAR-CD28细胞在移植瘤内的增殖检测。
图8：herinCAR-CD28细胞分子刹车系统的功能检测。
具体实施方式
下面对本发明涉及的部分术语进行解释。
在本发明中，术语“表达框”是指表达一个基因所需的完整元件，包括启动子、基因编码序列、PolyA加尾信号序列。
术语“编码序列”在文中定义为核酸序列中直接确定其蛋白产物(例如刹车分子，CAR，单链抗体，铰链区和跨膜区)的氨基酸序列的部分。编码序列的边界通常是由紧邻mRNA 5’端开放读码框上游的核糖体结合位点(对于原核细胞)和紧邻mRNA 3’端开放读码框下游的转录终止序列确定。编码序列可以包括，但不限于DNA、cDNA和重组核酸序列。
术语“激活型抗体”指存在时可激活某一特定的免疫反应的抗体。所述激活型抗体可以是抗体的全长序列或其功能性片段。在一个或多个实施方案中，所述激活型抗体选自：Fab、Fab’、F(ab’)2、Fv、scFv和scFv-Fc。在某些实施方案中，本文所述的激活型抗体是单链抗体。
术语“抗原结合片段”(antigen-binding fragment,Fab)是指位于抗体分子"Y"结构两臂末端，由高变区氨基酸序列组成，决定抗体结合抗原的特异性的肽段。
术语“Fc”即抗体的可结晶段(fragment crystallizable,Fc)，是指位于抗体分子"Y"结构的柄部末端，包含抗体重链恒定区CH2和CH3结构域的肽段,是抗体与效应分子或者细胞相互作用的部位。
术语“抗原表位”又称抗原决定簇(antigenic determinant，AD)，指抗原分子中决定抗原特异性的特殊化学基团。一般情况下，一个多肽表位含5～6个氨基酸残基抗原表位，可被特定的抗体所识别。抗原表位的性质、数目和空间构型决定抗原的特异性。而根据抗原表位的氨基酸连续性的不同，可以分为线性表位与空间表位，线性表位是一段序列相邻的氨基酸组成的表位，而空间表位是数个不相邻，但在空间结构上相邻的氨基酸组成的表位。
术语“共刺激分子”是指存在于抗原提呈细胞表面，能与Th细胞上的共刺激分子受体结合，产生协同刺激信号的分子。淋巴细胞的增殖不仅需要抗原的结合，还需要接受共刺激分子的信号。共刺激信号传递给T细胞主要是通过表达在抗原呈递细胞表面的共刺激分子CD80，CD86与T细胞表面的CD28分子结合。B细胞接受共刺激信号可以通过一般的病原体成分例如LPS，或者通过补体成分，或者通过激活了的抗原特异性的Th细胞表面的CD40L。
术语“接头”或铰链是连接不同蛋白或多肽之间的多肽片段，其目的是使所连接的蛋白或多肽保持各自的空间构象，以维持蛋白或多肽的功能或活性。示例性的接头包括含有G和/或S的接头，以及例如Furin 2A肽。
术语“特异性结合”是指抗体或者抗原结合片段与其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的亲和力 (KD)结合该抗原。“特异性识别”具有类似的含义。
术语“药学上可接受的辅料”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
术语“有效量”是指可在受试者中实现治疗、预防、减轻和/或缓解本发明所述疾病或病症的剂量。
术语“疾病和/或病症”是指所述受试者的一种身体状态，该身体状态与本发明所述疾病和/或病症有关。
术语“受试者”可以指患者或者其它接受本发明药物组合物以治疗、预防、减轻和/或缓解本发明所述疾病或病症的动物，特别是哺乳动物，例如人、狗、猴、牛、马等。
本文提供一类核酸构建物(本文也称为“核酸构建物C”)，该类核酸构建物含有转座子5’反向末端重复序列(5’ITR)，编码任选的刹车分子、嵌合抗原受体(CAR)和免疫共刺激分子或其受体的激活型抗体(如感兴趣单链抗体)的核酸序列及控制该核酸序列表达的启动子，polyA加尾信号序列，转座子3’反向末端重复序列(3’ITR)，转座酶编码序列和控制转座酶编码序列表达的启动子。
“嵌合抗原受体”(CAR)是人工改造受体，能够将识别肿瘤细胞表面抗原的特异性分子(如抗体)锚定在免疫细胞(如T细胞)上，使免疫细胞识别肿瘤抗原或病毒抗原和杀死肿瘤细胞或病毒感染的细胞。
适用于本文的嵌合抗原受体可以是本领域周知的各种CAR。通常，CAR依次包含信号肽、结合肿瘤细胞膜抗原的多肽、铰链区、跨膜区和胞内信号区。可采用本领域周知的用于构建CAR的信号肽、铰链区、跨膜区和胞内信号区来构建本发明的CAR。通常，结合肿瘤细胞膜抗原的多肽能够以中等亲和力结合肿瘤细胞广泛表达膜抗原，该多肽通常插入有抗原表位，插入的位置选自如下3个位置中的任意1个、2个或3个：信号肽与结合肿瘤细胞膜抗原的多肽之间，结合肿瘤细胞膜抗原的多肽内部，以及结合肿瘤细胞膜抗原的多肽和铰链区之间。所述结合肿瘤细胞膜抗原的多肽为天然多肽或人工合成多肽；优选地，人工合成多肽为单链抗体或Fab片段。
在某些实施方案中，所述嵌合抗原受体针对如下抗原中的一种或多种：CD19、CD20、CEA、GD2(又称B4GALNT1，β1,4-乙酰基-氨基半乳糖基转移酶1)、FR(Flavin还原酶)、PSMA(前列腺特异性膜抗原)、PMEL前黑素小体蛋白)、CA9(碳酸酐酶IX)、 CD171/L1-CAM、IL-13Rα2、MART-1(又称粘蛋白-A)、ERBB2、NY-ESO-1(又称CTAG1B，癌/睾丸抗原1B)、MAGE(黑素瘤相关抗原E1)家族蛋白、BAGE(B黑素瘤抗原家族)家族蛋白、GAGE(生长激素释放因子)家族蛋白、AFP(α-胎蛋白)、MUC1(mucin 1，细胞表面相关)、CD22、CD23、CD30、CD33、CD44v7/8、CD70、VEGFR1、VEGFR2、IL-11Rα、EGP-2、EGP-40、FBP、GD3(又称ST8SIA1，ST8α-N-乙酰基-神经酰胺α-2,8-唾液酸转换酶1)、PSCA(前列腺干细胞抗原)、FSA(又称KIAA1109)、PSA(又称KLK3，激肽释放酶相关的肽酶3)、HMGA2、胎儿型乙酰胆碱受体、LeY(又称FUT3)、EpCAM、MSLN(间皮素)、IGFR1、EGFR、EGFRvIII、ERBB3、ERBB4、CA125(又称MUC16，mucin 16，细胞表面相关)、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、SCC(又称SERPINB3)、AFU(又称FUCA1)、EBV-VCA、POA(又称VDR，维生素D(1,25-二氢维生素D3)受体)、β2-MG(β-2-微球蛋白)和PROGRP(GRP胃泌素释放肽)。优选地，所述CAR为针对EGFR家族的嵌合抗原受体。
在某些实施方案中，本文使用来自CN 201510812654.9的herinCAR(本文将该申请的全部内容以引用的方式纳入本文)。
在某些实施方案中，所述结合肿瘤细胞膜抗原的多肽为天然多肽，是人类Her2基因第8个内含子Herin编码的氨基酸序列HERIN；优选地，其氨基酸序列如CN 201510812654.9的SEQ ID NO:5所示。
在某些实施方案中，本发明CAR信号肽的氨基酸序列如CN 201510812654.9的SEQ ID NO:3所示。
在某些实施方案中，本发明CAR的铰链区选自CD8的胞外铰链区、CD28的胞外铰链区和CD4的胞外铰链区的任意一种或多种；优选地为CD8的胞外铰链区。在某些实施方案中，所述CD8的胞外铰链区如CN 201510812654.9的SEQ ID NO:7所示。
在某些实施方案中，本发明CAR的跨膜区选自CD8的跨膜区、CD28的跨膜区和CD4的跨膜区的任意一种或多种；优选地，为CD8跨膜区；优选地，所述CD8跨膜区的氨基酸序列如CN 201510812654.9的SEQ ID NO:8所示。
在某些实施方案中，本发明CAR的胞内信号区可选自CD28、CD134/OX40、CD137/4-1BB、LCK、ICOS、DAP10、CD3ζ和FcεRIγ中的任意一种或多种的胞内信号区，优选为4-1BB胞内信号区和CD3ζ胞内信号区，或者CD28胞内信号区和CD3ζ胞内信号区；优选地，所述4-1BB胞内信号区和CD3ζ胞内信号区的氨基酸序列分别如CN 201510812654.9的SEQ ID NO:9和SEQ ID NO:10所示；优选地，所述CD28胞内信号区和CD3ζ胞内信号区的氨基酸序列分别如CN 201510812654.9的SEQ ID NO:11和SEQ ID NO:10所示。
在某些实施方案中，所述抗原表位与结合肿瘤细胞膜抗原的多肽直接相连或者通过蛋白接头相连。通常，所述接头为至少2个甘氨酸，例如2、3、4、5、6、7、8、9或10个甘氨酸。
在某些实施方案中，本发明的嵌合抗原受体由信号肽、CD20抗原表位、人类Her2基因第8个内含子Herin编码的氨基酸序列HERIN、CD20抗原表位、CD8铰链区、CD8跨膜区、4-1BB共刺激肽段依次构成。优选地，其氨基酸序列如CN 201510812654.9的SEQ ID NO:1所示。
本文中，感兴趣的“单链抗体”(scFv)是指由抗体轻链可变区(VL区)氨基酸序列和重链可变区(VH区)氨基酸序列经铰链连接而成，具有结合抗原能力的抗体片段。在某些实施方案中，感兴趣单链抗体(scFv)来自感兴趣的抗体。应理解的是，本文的核酸构建物可编码两种单链抗体，一种单链抗体存在于所示的CAR之中，另一种单链抗体为与CAR相连的单链抗体。这两种单链抗体可相同，也可不同，优选的是，两种单链抗体执行不同的抗体功能。
感兴趣的抗体可以是人抗体，包括人鼠嵌合抗体和人源化抗体。感兴趣的抗体优选为激活型抗体，尤其是针对免疫共刺激分子及其受体的激活型抗体。抗体可以是分泌型或膜锚定型；优选地为膜锚定型。
在某些实施方案中，所述抗体针对如下抗原中的一种或多种：CD28、CD137、CD134、CD40、CD40L、ICOS、HVEM、CD2、CD27、CD30、GITR、LIGHT、DR3、SLAM、CD226。优选地，所述抗体为抗CD28抗体的scFv。
本文中，术语“CD28”是指人白细胞分化抗原28，它在NCBI GeneBank的官方名称为CD28，ID号为940，有3个异构体(cDNA序列/蛋白序列)，分别为NM_006139.3/NP_006130.1，NM_001243077.1/NP_001230006.1，NM_001243078.1/NP_001230007.1。
单链抗体可含有各感兴趣抗体各自的重链可变区和轻链可变区，或由重链可变区和轻链可变区以及任选的接头组成。重链可变区和轻链可变区之间可通过熟知的接头连接，例如含G和/或S的接头。接头长度通常为15～20个氨基酸。在某些实施方案中，接头为(GGGS)n，n为1－5的整数。
在某些实施方案中，本发明核酸构建物C中，编码激活型抗体的核酸序列之后还可包括来自同一或不同抗体的铰链区和跨膜区序列。例如，当使用抗CD28抗体时，可使用衍生自该抗体的scFv，并在该scFv之后连接上来自该CD28胞外铰链区和跨膜区序列。
本文所用“刹车分子”和“分子刹车”可互换使用，指可被已上市抗体药物识别的膜抗原。通过加入识别该“刹车分子”的抗体药物，携带该“刹车分子”的细胞能被快速清除，从而提高治疗安全性。合适的刹车分子(膜抗原)可选自：CD11a、CD15、CD19、CD20、CD25、 CD44、CD47、CD52、EGFR、ERBB2、ERBB3、ERBB4、VEGFR1、VEGFR2、EpCAM、MSLN(间皮素)、GPIIb/IIIa、α4整合素和α4β7整合素。在某些实施方案中，膜抗原为CD20。在某些实施方案中，可使用所述膜抗原的线性表位或空间表位。
当存在刹车分子时，刹车分子可通过本领域常用的接头序列(如前文所述的含G和S的接头序列)与CAR连接，或者可直接与CAR连接。CAR与单链抗体可直接相连，也可通过接头序列(例如Furin 2A肽)进行连接。
“CD20”是指人白细胞分化抗原20，它在NCBI GeneBank的官方名称为MS4A1，ID号为931，有2个异构体(cDNA序列/蛋白序列)，分别为NM_021950.3/NP_068769.2,NM_152866.2/NP_690605.1。当提及CD20的氨基酸序列时，其包括，CD20蛋白的全长或者具有CD20功能的CD20的片段；还包括所述全长或片段的融合蛋白。并且，本领域技术人员理解，在CD20的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。并且，当描述CD20的蛋白序列片段时，还包括其天然或人工变体中的相应序列片段。
可根据所选的CAR的编码序列选择相应的启动子序列。这类启动子的例子包括但不限于EF1α启动子。如CN201510021408.1所述(本文将其全部内容以引用的方式纳入本文)，启动子上游还可包括增强子，如mCMV增强子、hCMV增强子和CD3e增强子中的一个、任意两个或全部三个。
因此，在某些实施方案中，本文使用CN201510021408.1所公布的各种启动子序列，包括但不限于该申请SEQ ID NO:1所示的含mCMV增强子、hCMV增强子和EF1α启动子的CCEF启动子；SEQ ID NO:2所示的含CD3e增强子和EF1α启动子的TEF启动子；SEQ ID NO:3所示的含CD3e增强子、mCMV增强子、hCMV增强子和EF1α启动子的TCEF启动子；SEQ ID NO:4所示的含mCMV增强子、hCMV增强子和含内含子的EF1α启动子的CCEFI启动子；SEQ ID NO:5所示的含CD3e增强子和含内含子的EF1α启动子的TEFI启动子；以及SEQ ID NO:5所示的含CD3e增强子、mCMV增强子、hCMV增强子和含内含子的EF1α启动子的TCEFI启动子。
本文中，转座酶可以是来自piggybac、sleeping beauty、frog prince、Tn5或Ty转座系统的转座酶。当使用来自不同转座系统的转座酶时，本发明核酸构建物中的5’ITR和3’ITR的序列也相应改变为与该转座系统适配的序列，这可由本领域技术人员容易地确定。
在某些实施方案中，转座酶是来自piggybac转座系统的转座酶。因此，在这些实施方案中，转座子5’反向末端重复序列和3’反向末端重复序列分别为piggybac转座子的5’反向末端重复序列和3’反向末端重复序列。在某些实施方案中，转座子5’反向末端重复序列如CN 201510638974.7(本文将其内容以引用的方式纳入本文)SEQ ID NO:1所示。在某些实 施方案中，转座子3’反向末端重复序列如CN 201510638974.7SEQ ID NO:4所示。在某些实施方案中，piggybac转座酶为含c-myc核定位信号编码序列的转座酶。在某些实施方案中，piggybac转座酶的编码序列如CN 201510638974.7SEQ ID NO:5所示。
转座酶编码序列的启动子可以是本领域已知的用于控制转座酶编码序列表达的各种启动子。在某些实施方案中，使用CMV启动子控制转座酶编码序列的表达。CMV启动子的序列可如CN 201510638974.7SEQ ID NO:6所示。
可使用本领域周知的polyA加尾信号序列。在某些实施方案中，所述polyA来自SV40。在某些实施方式中，可使用CN 201510638974.7SEQ ID NO:3所示的序列。
可针对所选膜抗原选择合适的启动子，用以控制膜抗原的表达。在某些实施方案中，启动子是EF1α启动子。在某些实施方案中，EF1α启动子的序列如CN 201510638974.7SEQ ID NO:8所示。
在某些实施方案中，本文的核酸构建物C含有依次连接的转座子5’反向末端重复序列(5’ITR)，控制编码刹车分子、嵌合抗原受体(CAR)和免疫共刺激分子或其受体的激活型抗体(如感兴趣单链抗体)的核酸序列表达的启动子，编码刹车分子、嵌合抗原受体(CAR)和免疫共刺激分子或其受体的激活型抗体(如感兴趣单链抗体)的核酸序列，polyA加尾信号序列，转座子3’反向末端重复序列(3’ITR)，转座酶编码序列和控制转座酶编码序列表达的启动子。
在某些实施方案中，本文的核酸构建物C含有依次连接的转座子5’反向末端重复序列(5’ITR)，控制编码刹车分子、嵌合抗原受体(CAR)、免疫共刺激分子或其受体的激活型抗体(如感兴趣单链抗体)、抗体所针对的抗原的胞外铰链区和跨膜区的核酸序列表达的启动子，编码刹车分子、嵌合抗原受体(CAR)、免疫共刺激分子或其受体的激活型抗体(如感兴趣单链抗体)、抗体所针对的抗原的胞外铰链区和跨膜区的核酸序列，polyA加尾信号序列，转座子3’反向末端重复序列(3’ITR)，转座酶编码序列和控制转座酶编码序列表达的启动子。
在某些实施方案中，本文的核酸构建物C含有依次连接的转座子5’反向末端重复序列(5’ITR)，EF1α启动子，刹车分子(CD20)，CAR，抗CD28单链抗体，CD28的胞外铰链区和跨膜区，polyA加尾信号序列，转座子3’反向末端重复序列(3’ITR)，转座酶编码序列和控制转座酶编码序列表达的启动子(CMV)。
在某些实施方案中，所述核酸构建物C含有SEQ ID NO:2所示的核酸序列。
本文的核酸构建物C可以是种重组表达载体(重组表达载体C)，用于表达所述CAR、scFv和所述任选的刹车分子。优选的是，表达载体是转座子载体。在某些实施方案中，载体为选自如下转座子载体中的一种或多种：piggybac、sleeping beauty、frog prince、Tn5和 Ty。除核酸构建物C所含的核酸序列外，表达载体中通常还含有载体通常所含的其它元件，例如多克隆位点、抗性基因、复制起始位点等。
在某些实施方案中，所述重组表达载体采用pUC18、pUC19、pMD18-T、pMD19-T、pGM-T载体、pUC57、pMAX或pDC315系列载体作为骨架。在其它实施方案中，所述重组表达载体采用pCDNA3系列载体、pCDNA4系列载体、pCDNA5系列载体、pCDNA6系列载体、pRL系列载体、pUC57载体、pMAX载体或pDC315系列载体作为骨架。在某些实施方案中，本发明使用CN 201510638974.7构建的pSN载体，其载体结构如该申请图1所示。
可将本发明的核酸构建物C/重组表达载体C转入感兴趣的细胞中。转入的方法为本领域常规的方法，包括但不限于：病毒转导、显微注射、粒子轰击、基因枪转化和电转等。在某些实施方案中，采用电转将所述核酸构建物或重组表达载体。
感兴趣的细胞可以是本领域周知的各种T细胞，包括但不限于外周血T淋巴细胞、细胞毒杀伤T细胞(CTL)、辅助T细胞、抑制/调节性T细胞、γδT细胞以及细胞因子诱导的杀伤细胞(CIK)、肿瘤浸润淋巴细胞(TIL)等混合细胞群体的T细胞。
当将核酸构建物C/重组表达载体C转染感兴趣的T细胞时，由于其含有转座所需的ITR元件和转座酶，因此该核酸构建物或重组表达载体所含的转座子5’反向末端重复序列和转座子3’反向末端重复序列之间的核酸序列(包括5’和3’反向末端重复序列本身)被整合到感兴趣细胞的基因组中。当核酸构建物C/重组表达载体C含有编码刹车分子的核酸序列时，细胞将进一步表达刹车分子。
因此，本文还提供一类转基因T细胞，所述T细胞的基因组中稳定整合了包含编码CAR及免疫共刺激分子或其受体的激活型抗体(如感兴趣单链抗体)的核酸序列的表达框。更进一步地，所述T细胞的基因组中稳定整合了依次连接的转座子5’反向末端重复序列(5’ITR)，控制编码刹车分子、嵌合抗原受体(CAR)、免疫共刺激分子或其受体的激活型抗体(如感兴趣单链抗体)、抗体所针对的抗原的铰链区和跨膜区的核酸序列表达的启动子，编码刹车分子、嵌合抗原受体(CAR)、免疫共刺激分子或其受体的激活型抗体(如感兴趣单链抗体)、抗体所针对的抗原胞外铰链区和跨膜区的核酸序列，polyA加尾信号序列，以及转座子3’反向末端重复序列(3’ITR)。
在某些实施方案中，所述转基因T细胞表达激活型抗体。所述抗体可以为分泌型抗体或膜锚定型，优选为膜锚定型。该抗体可以是作用于T细胞自身的免疫共刺激分子激活型抗体。在该转基因T细胞表达该抗体后，可增强集群效应。
在某些实施方案中，本文的T细胞稳定表达感兴趣的CAR及感兴趣的scFv。在某些实施方案中，本文的T细胞转入了本文所述的核酸构建物C/重组表达载体C。
根据所表达的抗体的生物学功能，本文的转基因T细胞可具有不同的生物学活性，包括但不限于抑制肿瘤、抑制病毒、抑制细菌等。因此，本文的CAR-T细胞可用于抑制肿瘤细胞的生长、抑制病毒的生长、治疗肿瘤、治疗病毒感染性疾病、治疗细菌感染性疾病、以及治疗自身免疫疾病。肿瘤包括但不限于肝癌、肺癌、结肠癌、胰腺癌、胃癌、乳腺癌、鼻咽癌、淋巴瘤、卵巢癌、膀胱癌、前列腺癌、头颈部肿瘤。
因此，本文也提供一种药物组合物，该药物组合物含有本文所述的转基因T细胞和药学上可接受的载体或赋形剂。本文还提供本文所述的转基因T细胞在制备用于抑制肿瘤细胞生长的药物、制备用于抑制病毒生长的药物、制备用于治疗肿瘤的药物、制备用于治疗病毒感染性疾病的药物、制备用于治疗细菌感染性疾病的药物、或制备用于治疗自身免疫疾病的药物中的用途。
在本文的一个或多个实施方案中，所述转基因T细胞是CAR-T细胞。
因此，本发明还提供了一种试剂盒，所述试剂盒含有本文所述的重组表达载体。试剂盒还可含有适用于将所述重组表达载体转染入细胞中的各种试剂，以及任选的指导本领域技术人员将所述重组表达载体转染入细胞的说明书。
因此，在某些实施方案中，本发明也涉及一种重组表达载体的组合物，该组合物本文所述的重组表达载体。组合物中还可含有相应的溶剂或载体。
本发明人经过大量的预实验和创造性的劳动，成功构建了一种高效稳定表达激活型抗体的多能CAR-T细胞，该多能CAR-T细胞基因组中通过转座子系统稳定整合了激活型抗体(尤其是共刺激分子及其受体的激活型抗体)表达框，从而在保持其原有CAR-T细胞杀伤活性的前提下，具备稳定高效表达激活型抗体的活性，使回输的细胞在肿瘤部位能快速增殖。
本发明克服了目前常用基因转染载体系统对T细胞转染率低、介导抗体表达水平低的缺陷，使免疫杀伤细胞高水平稳定地表达嵌合抗原受体与激活型抗体，克服了在实体瘤CAR-T细胞治疗过程中CAR-T细胞难以达到有效数量的问题。为避免激活型抗体分泌到细胞外后，不可控的激活免疫系统，造成免疫过激反应，可选择将抗体锚定在CAR-T细胞膜上，通过直接接触激活邻近细胞。同时，该细胞又能快速的被上市单抗(如美罗华)清除，提高了治疗的安全性。本发明制得的转基因T细胞能够用于多种恶性肿瘤与病毒的治疗。
下面将结合实施案例对本发明所涉及的实施方案进行详细描述。本领域技术人员将会理解，下面的实施案例仅用于说明本发明，而不应视为限定本发明的范围。实施案例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁 克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：重组质粒pNB328-herinCAR-CD28的构建
按SEQ ID NO:1所示含CD20-美罗华分子刹车的herinCAR编码序列(靶向EGFR家族的CAR，参见CN 201510812654.9)与SEQ ID NO:2所示herinCAR-CD28编码序列(靶向EGFR家族且包含CD20-美罗华分子刹车的CAR与CD28单链-膜结合型抗体，中间用2A相连)，委托上海杰瑞生物公司合成，并在其上下游分别引入EcoRI与SalI酶切位点,装入pNB328载体，分别命名为pNB328-herinCAR、pNB328-herinCAR-CD28。
含CD20-美罗华分子刹车的herinCAR编码序列：
Figure PCTCN2017088957-appb-000001
GCCACCATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGT
Figure PCTCN2017088957-appb-000002
Figure PCTCN2017088957-appb-000003
GGTGGAGGTGGAGGTGGAGGTGGAGGTGGTACCCACTCACTGCCCCCGAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCTCTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTCAGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAGTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTGATAA
Figure PCTCN2017088957-appb-000004
(SEQ ID NO:1)其中双下划线表示酶切位点，波浪下划线的为美罗华抗体识别的CD20表位编码序列。 herinCAR-CD28编码序列：
Figure PCTCN2017088957-appb-000005
GCCACCATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGT
Figure PCTCN2017088957-appb-000006
Figure PCTCN2017088957-appb-000007
GGTGGAGGTGGAGGTGGAGGTGGAGGTGGTACCCACTCACTGCCCCCGAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGACCCTCTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCCTGTCAGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAGTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCGTAAAAGGCGAGCTCCTGTTAAACA GACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTCGAGTCCAACCCTGGGCC CATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGT
Figure PCTCN2017088957-appb-000008
Figure PCTCN2017088957-appb-000009
Figure PCTCN2017088957-appb-000010
ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGTGATAA
Figure PCTCN2017088957-appb-000011
(SEQ ID NO:2)，其中双下划线表示酶切位点，单下划线表示Furin 2A的编码序列，虚线下划线的CD28单链抗体的编码序列。
pNB328-herinCAR-CD28载体的模式图见图1。
实施例2：herinCAR-CD28细胞的构建
准备1×107新鲜分离获得的外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)，通过Lonza 2b-Nucleofector仪器，将6μg的pNB328-herinCAR-CD28，质粒转染到细胞核中，置37℃、5％CO2孵箱培养；6小时后转移到含30ng/mL抗CD3抗体、3000IU/mL IL-2(购自Novoprotein公司)的6孔板中，置37℃、5％CO2孵箱培养。待细胞长满后，按1:5的比例传代培养。即得同时表达靶向EGFR家族的且含CD20-美罗华分子刹车CAR、CD28单链抗体的基因修饰T细胞，简称herinCAR-CD28细胞。同时，相同来源PBMC转染pNB328-herinCAR质粒，获得herinCAR-T细胞。
实施例3：herinCAR-CD28细胞表面CD28抗体分子的检测
收集悬浮的实施例2构建的herinCAR-CD28细胞和对照herinCAR-T细胞，计数后以1×106个细胞/管分别加入2个1.5ml的EP管中，PBS清洗两遍，1200rpm离心5min，弃上清；分别加入2μl抗人IgG Fab2’抗体(购自Jackson ImmunoResearch公司)，轻弹沉淀使其混合均匀，室温避光孵育30min，PBS清洗一遍，1200rpm离心5min，弃上清加入400μl的生理盐水将细胞转移至流式管中，上机检测。实验结果发现，相对于对照细胞，herinCAR-CD28细胞表面具有CD28抗体分子，具体如图2所示。
实施例4：herinCAR-CD28细胞表面CD28分子的检测
收集悬浮的实施例2构建的herinCAR-CD28细胞和对照herinCAR-T细胞，计数后以1×106个细胞/管分别加入2个1.5ml的EP管中，PBS清洗两遍，1200rpm离心5min， 弃上清；分别加入2μl的同型对照抗体IgG1-PE，anti-CD28-PE抗体(均购自BD公司)，轻弹沉淀使其混合均匀，室温避光孵育30min，PBS清洗一遍，1200rpm离心5min，弃上清加入400μl的生理盐水将细胞转移至流式管中，上机检测。实验结果发现，相对于herinCAR对照细胞，herinCAR-CD28细胞CD28的阳性率显著提高，表明herinCAR-CD28细胞通过其膜表面CD28抗体，可有效激活邻近T细胞的CD28信号，具体如图3所示。
实施例5：herinCAR-CD28细胞的增殖检测
将实施例2构建的herinCAR-CD28细胞和herinCAR细胞按4×104/孔铺在96孔板中，每种细胞铺3个复孔，总体积为200μl，置于37℃，5％CO2培养箱培养，分别在培养24h，48h，72h，96h后加入20μl的CCK8试剂，37℃避光孵育6h，酶标仪上450nm测OD值。结果发现，herinCAR-CD28细胞的增殖速度明显高于对照herinCAR细胞，说明herinCAR-CD28细胞膜表面的CD28抗体能够促进T细胞的增殖，具体如图4所示。
实施例6：herinCAR-CD28细胞的体外杀伤活性检测
在RTCA细胞增殖板(购自美国ACEA Biosciences公司)上按10000个/孔的比例铺胃癌细胞株BGC-27、卵巢癌细胞株SK-OV3(购自美国标准生物品收藏中心，ATCC)，置于xCELLigence RTCA DP多功能实时无标记细胞分析仪上，实时记录细胞的生长情况(以测得的细胞指数来反映，数值越高表明细胞状态越佳)。24小时后，分别按8:1、4:1、2:1、0:1效靶比(E:T)，加入实施例2构建的herinCAR细胞与herinCAR-CD28细胞，重新置于xCELLigence RTCA DP多功能实时无标记细胞分析仪上进行细胞生长情况检测，48个小时后，根据测得的各效靶比条件下细胞指数与0：1效靶比条件下(不加效应细胞)相应细胞指数的比值，确定肿瘤细胞裂解率。
结果显示，相对于不表达CD28抗体的herinCAR-T细胞，表达CD28抗体的herinCAR-T细胞，即herinCAR-CD28细胞对BGC-27与SK-OV3细胞的杀伤活性显著提高(图5)。以上结果表明，共表达CD28抗体后，能增强CAR-T细胞对肿瘤细胞的杀伤活性。
实施例7：herinCAR-CD28细胞的体内杀伤活性鉴定
在NOD-SCID小鼠(购自上海斯莱克实验动物有限公司)中皮下注射5×106的BGC-27恶性胃癌细胞，10天后经尾静脉分别注射实施例2构建的herinCAR细胞、herinCAR-CD28细胞(注射剂量2×105)或PBS缓冲液，测定移植瘤的生长状况。结果表明，相对于对照组，herinCAR-CD28细胞对肝癌的抑制作用具有显著差异(图6)，表明共表达CD28抗体的herinCAR-T细胞在体内具有良好的抗肿瘤作用。
实施例8：herinCAR-CD28细胞在移植瘤内的增殖检测
实施例7治疗后的第35天，处死荷瘤小鼠，取移植瘤组织，提取基因组DNA(按Sigma-Aldrich公司的组织DNA抽提试剂盒进行操作)。利用RT-PCR检测herinCAR基因在移植瘤组织中的相对拷贝数(引物如SEQ ID NO:3与SEQ ID NO:4所示,反应体系与程序按天根生物的RealMaster Mix(SYBR Green)检测试剂盒操作)。结果表明，相对于herinCAR-T细胞，herinCAR-CD28细胞在移植瘤内的拷贝数显著提高(图7)，表明表达CD28抗体可增强CAR-T细胞在移植瘤内的存活时间。
F：CAGTGAGATTGGGATGAAAGG(SEQ ID NO:3)；
R：GAAGGGCGTCGTAGGTGTC(SEQ ID NO:4)。
实施例9：herinCAR-CD28细胞的体内清除试验(分子刹车功能的验证)
在BABL/c裸鼠(购自上海斯莱克实验动物有限公司)尾静脉注射herinCAR-CD28细胞(注射剂量5×106)，3天后静脉注射100μg的美罗华抗体或人IgG对照抗体。12个小时后，采集血液及骨髓样品，用流式细胞仪检测herinCAR-CD28细胞的比例(CD20与CD3双阳性细胞)。
结果表明，相对于注射人IgG抗体的对照组，在注射美罗华抗体(Rituxan)后，输注的实施例2构建的herinCAR-CD28细胞在血液与骨髓中的比例显著降低(图8)。可见，CD20分子刹车在体内能有效发挥作用，可以通过ADCC及CDC效应将含有CD20表位的herinCAR-CD28细胞清除。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (11)

  1. 一种转基因T细胞，其特征在于，所述T细胞基因组中稳定整合了含编码嵌合抗原受体，及免疫共刺激分子或其受体的激活型抗体的核酸序列的表达框，且表达框的两端包含转座子的反向末端重复序列。
  2. 如权利要求1所述的转基因T细胞，其特征在于，所述转座子选自：piggybac、sleeping beauty、frog prince、Tn5和Ty；优选地，所述转座子为piggybac。
  3. 如权利要求1所述的转基因T细胞，其特征在于，所述激活型抗体选自：抗体全长序列或其功能性片段；优选地，选自：Fab、Fab’、F(ab’)2、Fv、scFv和scFv-Fc；进一步优选地，所述抗体为scFv。
  4. 如权利要求1所述的转基因T细胞，其特征在于，所述T细胞表达的激活型抗体为分泌型或膜锚定型，优选地，所述抗体为膜锚定型。
  5. 如权利要求1所述的转基因T细胞，其特征在于，所述嵌合抗原受体针对如下抗原中的一种或多种：CD19、CD20、CEA、GD2(又称B4GALNT1，β1,4-乙酰基-氨基半乳糖基转移酶1)、FR(Flavin还原酶)、PSMA(前列腺特异性膜抗原)、PMEL前黑素小体蛋白)、CA9(碳酸酐酶IX)、CD171/L1-CAM、IL-13Rα2、MART-1(又称粘蛋白-A)、ERBB2、NY-ESO-1(又称CTAG1B，癌/睾丸抗原1B)、MAGE(黑素瘤相关抗原E1)家族蛋白、BAGE(B黑素瘤抗原家族)家族蛋白、GAGE(生长激素释放因子)家族蛋白、AFP(α-胎蛋白)、MUC1(mucin 1，细胞表面相关)、CD22、CD23、CD30、CD33、CD44v7/8、CD70、VEGFR1、VEGFR2、IL-11Rα、EGP-2、EGP-40、FBP、GD3(又称ST8SIA1，ST8α-N-乙酰基-神经酰胺α-2,8-唾液酸转换酶1)、PSCA(前列腺干细胞抗原)、FSA(又称KIAA1109)、PSA(又称KLK3，激肽释放酶相关的肽酶3)、HMGA2、胎儿型乙酰胆碱受体、LeY(又称FUT3)、EpCAM、MSLN(间皮素)、IGFR1、EGFR、EGFRvIII、ERBB3、ERBB4、CA125(又称MUC16，mucin 16，细胞表面相关)、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、SCC(又称SERPINB3)、AFU(又称FUCA1)、EBV-VCA、POA(又称VDR，维生素D(1,25-二氢维生素D3)受体)、β2-MG(β-2-微球蛋白)和PROGRP(GRP胃泌素释放肽)；优选地，所述嵌合抗原受体为针对EGFR家族的嵌合抗原受体。
  6. 如权利要求1所述的转基因T细胞，其特征在于，所述转基因T细胞还包含刹车分子；优选地，该刹车分子为可被已上市抗体药物识别的膜抗原；优选地，所述膜抗原选自CD11a、CD15、CD19、CD20、CD25、CD44、CD47、CD52、EGFR、ERBB2、ERBB3、ERBB4、VEGFR1、VEGFR2、EpCAM、MSLN、GPIIb/IIIa、α4整合素和α4β7整合素；优选地，所述膜抗原为CD20。
  7. 如权利要求1所述的转基因T细胞，其特征在于，所述激活型抗体针对如下抗原中的一种或多种：CD28、CD137、CD134、CD40、CD40L、ICOS、HVEM、CD2、CD27、CD30、GITR、LIGHT、DR3、SLAM、CD226、CD80、CD86；优选地，所述激活型抗体为抗CD28抗体的scFv。
  8. 如权利要求1-7中任一项所述的转基因T细胞，其特征在于，所述转基因杀伤性细胞转入了以下核酸构建物C：
    核酸构建物C：含有转座子5’反向末端重复序列(5’ITR)，编码任选的刹车分子、嵌合抗原受体(CAR)和免疫共刺激分子或其受体的激活型抗体的核酸序列及控制该核酸序列表达的启动子，polyA加尾信号序列，转座子3’反向末端重复序列(3’ITR)，转座酶编码序列和控制转座酶编码序列表达的启动子；
    任选地，采用病毒转导、显微注射、粒子轰击、基因枪转化和电转中的一种或多种方法将所述核酸构建物转入所述细胞中，优选地采用电转。
  9. 一种药物组合物，其特征在于，所述药物组合物含有权利要求1-8中任一项所述的转基因T细胞和药学上可接受的辅料。
  10. 权利要求1-8中任一项所述的转基因T细胞或权利要求9所述的药物组合物的用途，其特征在于，所述用途选自：
    制备用于抑制肿瘤细胞生长的药物、制备用于抑制病毒生长的药物、制备用于治疗肿瘤的药物、制备用于治疗病毒感染性疾病的药物、制备用于治疗细菌感染性疾病的药物和制备用于治疗自身免疫疾病的药物；
    其中，所述肿瘤选自：肝癌、肺癌、结肠癌、胰腺癌、胃癌、乳腺癌、鼻咽癌、淋巴瘤、卵巢癌、膀胱癌、前列腺癌和头颈部肿瘤。
  11. 一种核酸构建物，其特征在于，所述核酸构建物含有转座子5’反向末端重复序列(5’ITR)，编码任选的刹车分子、嵌合抗原受体(CAR)和免疫共刺激分子或其受体的激活型抗体的核酸序列及控制该核酸序列表达的启动子，polyA加尾信号序列，转座子3’反向末端重复序列(3’ITR)，转座酶编码序列和控制转座酶编码序列表达的启动子；
    优选地，所述激活型抗体为单链抗体；
    优选地，所述转座酶为来自piggybac转座系统的转座酶，所述5’反向末端重复序列(5’ITR)和3’反向末端重复序列是piggybac转座子的5’反向末端重复序列和3’反向末端重复序列；
    优选地，所述刹车分子是CD20；
    优选地，所述嵌合受体抗原是针对EGFR家族的嵌合抗原受体；
    优选地，所述激活型抗体针对如下抗原中的一种或多种：CD28、CD137、CD134、CD40、CD40L、ICOS、HVEM、CD2、CD27、CD30、GITR、LIGHT、DR3、SLAM、CD226、CD80、CD86；优选地，所述激活型抗体为抗CD28抗体的scFv；
    优选地，所述核酸构建物含有SEQ ID NO:2所示的核酸序列。
PCT/CN2017/088957 2016-06-20 2017-06-19 一种高效稳定表达激活型抗体的car-t细胞及其用途 WO2017219936A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610445661.4 2016-06-20
CN201610445661.4A CN107523549A (zh) 2016-06-20 2016-06-20 一种高效稳定表达激活型抗体的car‑t细胞及其用途

Publications (1)

Publication Number Publication Date
WO2017219936A1 true WO2017219936A1 (zh) 2017-12-28

Family

ID=60734619

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/088957 WO2017219936A1 (zh) 2016-06-20 2017-06-19 一种高效稳定表达激活型抗体的car-t细胞及其用途

Country Status (2)

Country Link
CN (2) CN107523549A (zh)
WO (1) WO2017219936A1 (zh)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10227574B2 (en) 2016-12-16 2019-03-12 B-Mogen Biotechnologies, Inc. Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods
CN109680004A (zh) * 2019-01-09 2019-04-26 上海美丽人生医疗科技有限公司 联合EpCAM和MSLN单链抗体的双靶点CAR载体及其构建方法和在卵巢癌中的应用
CN111097043A (zh) * 2020-01-13 2020-05-05 广东昭泰体内生物医药科技有限公司 一种胃癌药物组合物及其应用
CN111560075A (zh) * 2020-04-24 2020-08-21 武汉科技大学 一种含双靶点嵌合抗原受体基因的载体、car-t细胞及其应用
US11278570B2 (en) 2016-12-16 2022-03-22 B-Mogen Biotechnologies, Inc. Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods
US11760983B2 (en) 2018-06-21 2023-09-19 B-Mogen Biotechnologies, Inc. Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods
WO2024097800A1 (en) * 2022-11-01 2024-05-10 Fate Therapeutics, Inc. Off-the-shelf therapeutic cells with multiplex genomic engineering for targeting kallikrein-2

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109337872B (zh) * 2017-07-27 2023-06-23 上海细胞治疗研究院 高效扩增car-t细胞的人工抗原递呈细胞及其用途
CN109971719B (zh) * 2017-12-28 2023-05-12 上海细胞治疗研究院 自分泌CD40抗体且靶向ErbB受体家族的CAR-T细胞及其用途
CN109971723B (zh) * 2017-12-28 2023-07-07 上海细胞治疗研究院 包含CD40抗体与muc1特异性嵌合抗原受体基因的T细胞及其用途
CN108913721A (zh) * 2018-07-23 2018-11-30 安徽古生物科技有限公司 表达cd40抗体的慢病毒载体、car-t细胞的构建方法及应用
US20220242948A1 (en) * 2018-10-19 2022-08-04 Cartexell Inc. Anti-l1cam antibody or antigen-binding fragment thereof and chimeric antigen receptor comprising same
US20220162301A1 (en) * 2019-04-12 2022-05-26 PhanesTherapeutics, Inc. Humanized anti-folate receptor 1 chimeric antigen receptors and uses thereof
CN110734929B (zh) * 2019-11-14 2022-11-22 南通大学 一种转座子介导的高效非病毒真核细胞稳定转染方法
CN114317600A (zh) * 2020-10-12 2022-04-12 上海君赛生物科技有限公司 新型PiggyBac转座子系统及其用途
CN115947869B (zh) * 2022-11-28 2023-12-12 广州佰芮慷生物科技有限公司 一种靶向人巨细胞病毒的嵌合抗原受体、car-nk细胞及用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104910278A (zh) * 2015-05-27 2015-09-16 上海吉凯基因科技有限公司 一种用于制备cart细胞的具有高效转染能力和生物学活性的慢病毒
CN105154473A (zh) * 2015-09-30 2015-12-16 上海细胞治疗研究院 一种高效安全的转座子整合系统及其用途
CN105331586A (zh) * 2015-11-20 2016-02-17 上海细胞治疗研究院 一种包含高效杀伤启动机制的肿瘤精准t细胞及其用途

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
LT2961831T (lt) * 2013-02-26 2020-11-10 Memorial Sloan Kettering Cancer Center Kompozicijos ir būdai, skirti imunoterapijai
TWI654206B (zh) * 2013-03-16 2019-03-21 諾華公司 使用人類化抗－ｃｄ１９嵌合抗原受體治療癌症
CN104140974B (zh) * 2013-05-08 2017-09-29 科济生物医药（上海）有限公司 编码gpc‑3嵌合抗原受体蛋白的核酸及表达gpc‑3嵌合抗原受体蛋白的t淋巴细胞
EP3134434A4 (en) * 2014-04-25 2017-10-25 Bluebird Bio, Inc. Kappa/lambda chimeric antigen receptors
CN105315375B (zh) * 2014-07-17 2021-04-23 恺兴生命科技(上海)有限公司 靶向cld18a2的t淋巴细胞及其制备方法和应用
CN104630146A (zh) * 2015-01-24 2015-05-20 马飞 肿瘤细胞特异性多克隆t细胞制备方法及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104910278A (zh) * 2015-05-27 2015-09-16 上海吉凯基因科技有限公司 一种用于制备cart细胞的具有高效转染能力和生物学活性的慢病毒
CN105154473A (zh) * 2015-09-30 2015-12-16 上海细胞治疗研究院 一种高效安全的转座子整合系统及其用途
CN105331586A (zh) * 2015-11-20 2016-02-17 上海细胞治疗研究院 一种包含高效杀伤启动机制的肿瘤精准t细胞及其用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LUO, FEIFEI: "Genetic engineering technology based on PD-1/PDL1 signal conversion chimeric receptor", CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, 31 July 2015 (2015-07-31), pages E072 - 32, ISSN: 1674-022X *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10227574B2 (en) 2016-12-16 2019-03-12 B-Mogen Biotechnologies, Inc. Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods
US11111483B2 (en) 2016-12-16 2021-09-07 B-Mogen Biotechnologies, Inc. Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems and methods
US11162084B2 (en) 2016-12-16 2021-11-02 B-Mogen Biotechnologies, Inc. Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods
US11278570B2 (en) 2016-12-16 2022-03-22 B-Mogen Biotechnologies, Inc. Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods
US12037611B2 (en) 2016-12-16 2024-07-16 R&D Systems, Inc. Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods
US11760983B2 (en) 2018-06-21 2023-09-19 B-Mogen Biotechnologies, Inc. Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods
CN109680004A (zh) * 2019-01-09 2019-04-26 上海美丽人生医疗科技有限公司 联合EpCAM和MSLN单链抗体的双靶点CAR载体及其构建方法和在卵巢癌中的应用
CN111097043A (zh) * 2020-01-13 2020-05-05 广东昭泰体内生物医药科技有限公司 一种胃癌药物组合物及其应用
CN111560075A (zh) * 2020-04-24 2020-08-21 武汉科技大学 一种含双靶点嵌合抗原受体基因的载体、car-t细胞及其应用
CN111560075B (zh) * 2020-04-24 2023-06-23 武汉科技大学 一种含双靶点嵌合抗原受体基因的载体、car-t细胞及其应用
WO2024097800A1 (en) * 2022-11-01 2024-05-10 Fate Therapeutics, Inc. Off-the-shelf therapeutic cells with multiplex genomic engineering for targeting kallikrein-2

Also Published As

Publication number Publication date
CN114891751A (zh) 2022-08-12
CN107523549A (zh) 2017-12-29

Similar Documents

Publication Publication Date Title
WO2017219936A1 (zh) 一种高效稳定表达激活型抗体的car-t细胞及其用途
WO2017219937A1 (zh) 一种高效稳定表达抑制性抗体的car-t细胞及其用途
WO2017219934A1 (zh) 一种高效稳定表达抗体的杀伤性细胞及其用途
JP7300763B2 (ja) Ｃａｒ発現ベクター及びｃａｒ発現ｔ細胞
CN105331586B (zh) 一种包含高效杀伤启动机制的肿瘤精准t细胞及其用途
US11034763B2 (en) Flag tagged CD19-CAR-T cells
CN110818802B (zh) 一种嵌合t细胞受体star及其应用
AU2014225788B2 (en) Engager cells for immunotherapy
CN103483452B (zh) 双信号独立的嵌合抗原受体及其用途
CN108135998B (zh) 高亲合力hpv t细胞受体
CN111629734A (zh) 用于共刺激的新型平台、新型car设计以及过继性细胞疗法的其他增强
CN108004259B (zh) 靶向b细胞成熟抗原的嵌合抗原受体及其用途
KR20190101979A (ko) 합성 면역 수용체 및 이의 사용 방법
JP7431171B2 (ja) 抗体修飾キメラ抗原受容体修飾ｔ細胞及びその使用
WO2017219933A1 (zh) 一种高效稳定表达抗体的t细胞及其用途
ES2690420T3 (es) Receptor de antígeno quimérico anti-toso y su uso
WO2021244486A1 (zh) 信号转换受体及其用途
AU2021318297B2 (en) Immune Synapse-Stabilizing Chimeric Antigen Receptor (CAR) T Cell
WO2019184886A1 (zh) 促进免疫细胞增殖的方法
WO2022214089A1 (zh) 细胞免疫治疗的应用
JP2021514188A (ja) Ｆｏｘｐ３標的因子組成物と養子細胞療法のための使用方法
JP2022526194A (ja) Ｆｌｔ３特異的キメラ抗原受容体およびその使用方法
JP7527049B2 (ja) Ｃａｒ発現ベクター及びｃａｒ発現ｔ細胞
WO2024060140A1 (zh) EGFRvIII嵌合抗原受体及其用途
TWI840351B (zh) T細胞受體及表現其之工程化細胞

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17814674

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17814674

Country of ref document: EP

Kind code of ref document: A1