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Chimeric antigen receptor targeting MSLN and application of chimeric antigen receptor

A chimeric antigen receptor and antigen technology, applied in the field of biomedicine, can solve problems such as high cost and cumbersome process

Inactive 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

Recent studies have found that the ratio of CD4+ and CD8+ T cells in CAR-T cells has a great influence on the anti-tumor activity of CAR-T cells, and the ratio of CD4+ T cells in CAR-T cells is related to the therapeutic effect of CAR-T cells It is positively correlated, so in order to obtain a large number of CD4+CAR-T cells, it is necessary to sort out CD4+CAR-T cells and CD8+CAR-T cells first, and then expand CD4+CAR-T cells through different cytokine combinations and CD8+ CAR-T cells, the process is cumbersome and the cost is huge

Method used

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  • Chimeric antigen receptor targeting MSLN and application of chimeric antigen receptor
  • Chimeric antigen receptor targeting MSLN and application of chimeric antigen receptor
  • Chimeric antigen receptor targeting MSLN and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of CAR Molecular Carrier

[0044] In this example, first, Meso-H-CAR (SEQ ID NO: 4) whose hinge region is IgG4-CH3 and Meso-CAR whose hinge region is IgG4 were synthesized from the whole gene, and PmeI and SpeI enzyme cleavage sites were added at both ends, respectively. The schematic diagram of Meso-H-CAR is shown in figure 1 shown;

[0045] The encoding gene of the CAR molecule synthesized by PmeI and SpeI double digestion and the lentiviral expression vector pwpxld-eGFP were recovered by agarose gel electrophoresis and ligated;

[0046] 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.

Embodiment 2

[0047] Example 2 lentiviral packaging

[0048] In this example, the lentiviral vector constructed in Example 1 is packaged with lentivirus, and the steps are as follows:

[0049] (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);

[0050] (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;

[0051] (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;

[0052] (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

[0059] Example 3 T cell activation and lentiviral transfection

[0060] (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);

[0061] (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;

[0062] (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.

[0063] The CAR-T cells constructed in this ...

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Abstract

The invention provides a chimeric antigen receptor targeting MSLN and an application of the chimeric antigen receptor. The chimeric antigen receptor comprises an antigen binding structural domain, a hinge region, a transmembrane structural domain and a signal transduction structural domain, wherein the antigen binding structural domain comprises an anti-Mesothelin single-chain antibody, and the hinge region is IgG4-CH3. The chimeric antigen receptor targeting MSLN is constructed by introducing an IgG4-CH3 hinge region into a CAR molecule, so that the in-vitro amplification efficiency of CAR-Tcells is remarkably improved, and the chimeric antigen receptor targeting MSLN has important significance in the field of solid tumor treatment.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a chimeric antigen receptor targeting MSLN 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 cell therapy has achieved amazing efficacy, there are still many obstacles. For example, the expansion efficiency of CAR-T cells, especially CD4+ CAR-T cells, needs to be improved. In the conventional in vitro culture system, CD8+ T cells have a more obvious growth advantage than CD4+ T cells. I...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61P35/00
CPCA61P35/00C07K14/7051C07K16/2821C07K16/30C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
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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