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Enhanced immune cell and application thereof

A technology of immune cells and chimeric antigen receptors, applied in the field of biomedicine, can solve the problem of tumor cells not expressing specific and specific tumor antigens, obstacles, etc., and achieve the effect of significant effect and killing effect.

Inactive Publication Date: 2020-11-03
GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although CAR-T cells can target tumor cells expressing specific antigens, many tumor cells do not express specific and specific tumor antigens. Tumor heterogeneity is a major obstacle for cell therapy in the treatment of solid tumors

Method used

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  • Enhanced immune cell and application thereof
  • Enhanced immune cell and application thereof
  • Enhanced immune cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of CAR Molecular Carrier

[0045] In this example, first, the gene encoding the dual-target CAR molecule (amino acid sequence shown in SEQ ID NO: 3) combined with tumor antigen MUC1 scFv and endogenous NKG2D was synthesized, and the C-terminus and N-terminus of the encoding gene were respectively added Restriction endonuclease Pme1 restriction site and its protective base and restriction endonuclease Spe1 restriction site and its protection base;

[0046] The coding gene was double-digested with restriction endonucleases Pme1 and Spe1, and the digested product containing cohesive ends was recovered by agar gel electrophoresis, and then ligated into the linearized pWPXLd-eGFP plasmid (containing sticky end), the ligation reaction was carried out with the participation of T4 DNA polymerase (Invitrogent), and the CAR molecule with the antigen-binding domain as anti-MUC1 scFv and NKG2D was obtained. The structural diagram is as follows figure 1 shown...

Embodiment 2

[0049] Example 2 lentiviral packaging

[0050] In this example, the lentiviral vector constructed in Example 1 is packaged with lentivirus, and the steps are as follows:

[0051] (1) Culture 293T cells in a 10cm petri dish, the culture medium is DMEM high glucose medium+10% FBS (fetal bovine serum)+1% double antibody (100×penicillin-streptomycin mixed solution);

[0052] (2) When the 293T cell density in the culture dish reaches 80%, replace the medium with DMEM high glucose medium + 1% FBS + 1% double antibody;

[0053] (3) After replacing the medium and culturing for 2 hours, prepare a transfection reagent, take 500 μL opti-DMEM into a 15 mL centrifuge tube, add 7.2 μL of PEI (linear polyethyleneimine) with a concentration of 10 μg / μL, and mix slightly. Stand still for 5 minutes;

[0054] (4) Put 500μL opti-DMEM into a 1.5mL centrifuge tube, take 9μg of recombinant lentiviral vector, 3μg of pMD2.G helper plasmid and 12μg of psPAX, add them to the centrifuge tube, mix well,...

Embodiment 3

[0061] Example 3 T cell activation and lentiviral transfection

[0062] (1) After sorting Pan T cells from umbilical cord blood, count the cells and adjust the concentration to 1×10 6 cells / mL, then add 10 μL of Miltenyi TransAct T cell reagent to each ml of cell suspension, and replace it with fresh medium (IMDM medium + 5% FBS (fetal bovine serum) + 1% double antibody ( 100×penicillin-streptomycin mixed solution)+IL-2);

[0063] (2) After T cells were activated for 48 h, demagnetize the beads, centrifuge at 300 g for 5 min, remove the supernatant, resuspend T cells with fresh medium, add CAR-expressing recombinant lentivirus or blank control lentivirus (MOI=10), and Add 8μg / mL polybrene and 300IU / mL IL-2, place at 37°C, 5% CO 2 incubator cultivation;

[0064] (3) After 24 hours, centrifuge at 300 g for 5 minutes, remove the supernatant, and resuspend the T cells in fresh medium containing 300 IU / mL IL-2 to obtain CAR-T cells.

[0065] The CAR-T cells constructed in this ...

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Abstract

The invention provides an enhanced immune cell and an application thereof. The immune cell expresses a chimeric antigen receptor, and the chimeric antigen receptor comprises an antigen binding structural domain, a transmembrane structural domain and a signal transduction structural domain, and the antigen binding structural domain comprises an anti-MUC1 single-chain antibody and NKG2D. The chimeric antigen receptor provided by the invention has a targeting effect on MUC1 specific tumor cells and heterogeneous tumor cells, and the constructed immune cell expressing the chimeric antigen receptoralso mobilizes the function of endogenous NKG2D while targeting tumor cells expressing specific antigens, targets tumor cells with heterogeneity, and realizes clearing and killing effects on specifictumor cells and heterogeneous tumor cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to an enhanced immune cell and its application. Background technique [0002] In recent years, with the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) has become one of the most promising tumor immunotherapies. At present, CAR-T cell therapy has been widely used in the treatment of B-cell malignancies. CAR-T cells targeting CD19 are the pioneers of CAR-T therapy in the treatment of B-cell malignancies, providing an effective solution for the treatment of B-cell malignancies. [0003] Although CAR-T cells can target tumor cells expressing specific antigens, many tumor cells do not express specific and specific tumor antigens. Tumor heterogeneity is a major obstacle for cell therapy in the treatment of solid tumors. Increasing the ability of chimeric antigen receptor molecules to recognize different targets is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61P35/00C12R1/93
CPCA61P35/00A61K39/00117C07K14/7051C07K14/7056C07K16/3092C07K2317/622C07K2319/02C07K2319/03C07K2319/32C07K2319/33C12N5/0636C12N5/0646C12N7/00C12N15/86C12N2510/00C12N2740/15021C12N2740/15043
Inventor 汤朝阳秦乐吴迪邓殷健吴海鹏王翠花冯世忠冯嘉昆其他发明人请求不公开姓名
Owner GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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