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Chimeric antigen receptor targeting GPC3 and application of chimeric antigen receptor

A technology of chimeric antigen receptor and antigen, applied in the field of biomedicine

Pending Publication Date: 2021-01-12
汤朝阳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The influence of the hinge region on the expansion of CAR-T cells has been reported, but how to further improve the targeted killing ability of CAR-T remains to be explored

Method used

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  • Chimeric antigen receptor targeting GPC3 and application of chimeric antigen receptor
  • Chimeric antigen receptor targeting GPC3 and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The preparation of embodiment 1 lentiviral vector

[0043] In this example, firstly, a GPC3-H-TLR2-CAR whose hinge region is IgG4-CH3, signal transduction domain is CD28 intracellular region, CD3ζ and TLR2 is synthesized from the whole gene, and PmeI and SpeI restriction sites are added at both ends respectively. ;

[0044] 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;

[0045] 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.

[0046] In this example, a GPC3-TLR2-CAR with IgG4 as the hinge region, CD28 intracellular region, CD3ζ and TLR2 as the signal transduction domain was simultaneously constructed, the hinge region was IgG4-CH3, and...

Embodiment 2

[0047] Example 2 Recombinant 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...

Embodiment 3

[0059] Example 3 T cell activation and lentiviral transfection

[0060] Peripheral blood mononuclear cells (PBMC) were separated from whole blood using Ficoll density gradient centrifugation kit (GE Company), and after red blood cells were removed, T cells were sorted out using MACS Pan-T magnetic beads;

[0061] The sorted T cells were diluted with medium (AIM-V medium + 5% FBS + penicillin 100 U / mL + streptomycin 0.1 mg / mL) to a cell concentration of 2.5×10 6 pcs / mL for use;

[0062] CD2 / CD3 / CD28 T cell activation expansion kit (Miltenyi Company) was used to activate T cells, that is, the coated magnetic beads were mixed with T cells at a ratio of 1:2, and the final density of T cells was 5×10 6 piece / mL / cm 2 , after mixing, place at 37°C, 5% CO 2 The incubator was stimulated for 48 hours;

[0063] After T cells were activated for 48 hours, demagnetize the beads, centrifuge at 300 g for 5 min, remove the supernatant, resuspend T cells with fresh medium, add recombinant l...

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Abstract

The invention provides a chimeric antigen receptor targeting GPC3 and an application of the chimeric antigen receptor. The chimeric antigen receptor comprises an antigen binding structural region, a hinge region, a transmembrane structural region and a signal transduction structural region, wherein the antigen binding structural region is an anti-GPC3 single-chain antibody; the hinge region is IgG4-CH3; and the signal transduction structural region is a combination of a CD28 intracellular region, CD3 zeta, and TLR2. The IgG4-CH3 is adopted as the hinge region of the chimeric antigen receptor,the CD28 intracellular region, the CD3 zeta and the TLR2 which are connected in series are adopted as the signal transduction structural region of the chimeric antigen receptor, the hinge region and the signal transduction structural region cooperate with each other, in-vitro amplification of CD4 + CAR-T cells is promoted, and cell signals transmitted by an extracellular region are expanded. The killing effect of the CAR-T cells is enhanced.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a chimeric antigen receptor targeting GPC3 and its application. Background technique [0002] Chimeric antigen receptor T cell (CAR-T) immunotherapy is a cellular immunotherapy method based on chimeric antigen receptors. The gene sequence of CAR (CAR) is transfected into T cells to generate tumor-specific T cells that can bind the target antigen. With the development of tumor immunology theory and clinical technology, CAR-T therapy 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] The hinge region of the chimeric antigen receptor is located between the scFv and the immune cell membrane, usuall...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10C12R1/93
CPCC07K14/7051C07K16/303C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N7/00C12N15/86C12N2510/00C12N2740/15021C12N2740/15043C12N2800/107
Inventor 汤朝阳秦乐吴迪冯世忠冯嘉昆王艳艳其他发明人请求不公开姓名
Owner 汤朝阳
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