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Enhanced immune cell and application thereof

An immune cell, chimeric antigen receptor technology, applied in the field of biomedicine, can solve problems such as obstacles, tumor cells do not express specific and specific tumor antigens, etc.

Pending Publication Date: 2020-10-30
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

[0049] Example 1 Construction of CAR Molecular Carrier

[0050] In this example, fragments with NKG2D and 4-1BB and CD3ζ intracellular signal transduction domains were first synthesized, connected to the pWPXLD-EGFP vector, and then the scFv targeting GPC3 was specifically amplified by PCR and cloned into the above expression vector , to obtain a CAR molecule whose antigen binding domain is anti-GPC3 scFv and NKG2D (the amino acid sequence is shown in SEQ ID NO: 3, and the nucleic acid sequence is shown in SEQ ID NO: 4), and the structural diagram is shown in figure 1 shown.

[0051] The constructed plasmid was transformed into Escherichia coli, a single clone with the target plasmid was selected for overnight culture, and the plasmid was extracted using a plasmid extraction kit to obtain a recombinant lentiviral vector.

[0052] In this example, CARs (antiGPC3scFv-CD8α-4-1BB-CD3ζ) and NKG2D CARs (NKG2D-CD8α-4-1BB-CD3ζ) with antigen-binding domains respectively anti-GPC3 scFv...

Embodiment 2

[0053] Example 2 lentiviral packaging

[0054] In this example, the lentiviral vector constructed in Example 1 is packaged with lentivirus, and the steps are as follows:

[0055] (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);

[0056] (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;

[0057] (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;

[0058](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

[0065] Example 3 T cell activation and lentiviral transfection

[0066] (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);

[0067] (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;

[0068] (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.

[0069] 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, the chimeric antigen receptor comprises an antigen binding structuraldomain, a transmembrane structural domain and a signal transduction structural domain, the antigen binding structural domain comprises an anti-GPC3 single-chain antibody and NKG2D, the chimeric antigen receptor disclosed by the invention has a targeting effect on both GPC3 specific tumor cells and heterogeneous tumor cells, and the constructed immune cell for expressing the chimeric antigen receptor mobilizes the function of endogenous NKG2D while targeting tumor cells for expressing specific antigens, targets tumor cells with heterogeneity, and realizes the clearing and killing effects on specific tumor cells and heterogeneity 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): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61P35/00
CPCA61P35/00A61K39/001129C07K14/7051C07K14/7056C07K16/303C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N5/0646C12N7/00C12N15/86C12N2510/00C12N2740/15021C12N2740/15043
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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