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CD19 and PD-L1 double-target chimeric antigen receptor and application thereof

A technology of chimeric antigen receptor and PD-L1, which is applied in the field of biomedicine to alleviate the problem of drug resistance, overcome the mechanism of immune escape, and improve the effect of anti-tumor effect

Active Publication Date: 2020-10-09
GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, after some patients received CD19 CAR-T therapy, the tumor relapsed again. The reason may be related to the immunosuppressive microenvironment. The tumor may escape immune surveillance by stimulating the immunosuppressive receptor PD-1 on T cells

Method used

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  • CD19 and PD-L1 double-target chimeric antigen receptor and application thereof
  • CD19 and PD-L1 double-target chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of CAR Molecular Carrier

[0045] In this example, the gene encoding the CD19 and PD-L1 dual-target CAR molecule (the amino acid sequence is shown in SEQ ID NO: 3) was firstly synthesized, and HindIII and BamHI enzyme cleavage sites and their protective bases were added to both ends respectively. base;

[0046] The coding gene was double-digested with restriction endonucleases HindIII and BamHI, and the digested product containing sticky ends was recovered by 1.5% agarose gel electrophoresis, and ligated into the linearized lentiviral expression vector pWPXLd-eGFP. The system is shown in the table 1 shown;

[0047] After incubating at 37°C for 30 minutes, place it quickly on ice for 5 minutes, then add 20 μL of Trans1-T1 competent, let stand for 30 minutes, heat shock at 42°C for 90 seconds, and smear the plate to obtain the recombinant lentiviral vector.

[0048] Table 1

[0049] Reagent Dosage Linearized pWPXLd vector 200ng...

Embodiment 2

[0051] Example 2 lentiviral packaging

[0052] In this example, the lentiviral vector constructed in Example 1 is packaged with lentivirus, and the steps are as follows:

[0053] (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);

[0054] (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;

[0055] (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;

[0056] (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

[0063] Example 3 T cell activation and lentiviral transfection

[0064] (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);

[0065] (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;

[0066] (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.

[0067] The CAR-T cells constructed in this ...

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Abstract

The invention provides a CD19 and PD-L1 double-target chimeric antigen receptor and an application thereof. The chimeric antigen receptor comprises an antigen binding structural domain, a transmembrane structural domain and a signal transduction structural domain, wherein the antigen binding structural domain comprises an anti-CD19 single-chain antibody and an anti-PD-1 antibody. The anti-CD19 andPD-L1 double-target chimeric antigen receptor disclosed by the invention not only can recognize CD19 positive tumor cells in a targeting manner, but also can relieve the immunosuppression effect of PD-L1 on the surfaces of the tumor cells on T cells, overcomes the immune escape mechanism of the tumor cells, and alleviates the drug resistance problem of CAR-T treatment.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a CD19 and PD-L1 dual-target chimeric antigen receptor 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] However, after some patients received CD19 CAR-T therapy, the tumor relapsed again. The reason may be related to the immunosuppressive microenvironment. The tumor may escape immune surveillance by stimulating the immunosuppressive receptor PD-1 on T cells. The overexpression of PD-L1 in tumor cells ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61P35/00
CPCC07K16/2803C07K16/2827C07K14/7051C12N15/86C12N7/00C12N5/0636A61K39/001102A61K39/001112A61P35/00C07K2317/622C07K2319/03C07K2319/02C07K2319/33C12N2740/15021C12N2740/15043C12N2800/107C12N2510/00A61K2039/804A61K2039/5158
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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