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 immunos

Method used

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

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0044] Example 1 Construction of CAR molecular vector

[0045] This example firstly synthesizes the coding genes of CD19 and PD-L1 dual target CAR molecules (amino acid sequence is shown in SEQ ID NO: 3), and adds HindIII and BamHI restriction sites and their protective bases at both ends. base;

[0046] The coding gene was double digested with restriction enzymes 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 as shown in the table. 1 shown;

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

[0048] Table 1

[0049] Reagent Dosage Linearized pWPXLd vector 200ng CAR molecule coding gene 80ng 5×Exnase Buffer 4μL Exnase 2μL ddH 2 O

[0050] In this ex...

Example Embodiment

[0051] Example 2 Lentivirus packaging

[0052] In this example, lentivirus packaging was performed on the lentiviral vector constructed in Example 1. The steps are as follows:

[0053] (1) Cultivate 293T cells in a 10cm petri dish, the medium is DMEM high glucose medium + 10% FBS (fetal bovine serum) + 1% double antibody (100× penicillin-streptomycin mixed solution);

[0054] (2) When the density of 293T cells in the culture dish reaches 80%, change the medium to DMEM high glucose medium + 1% FBS + 1% double antibody;

[0055] (3) After changing the medium and incubating for 2 hours, prepare the transfection reagent, take 500μL opti-DMEM into a 15mL centrifuge tube, add 7.2μL PEI (linear polyethyleneimine) at a concentration of 10μg / μL, mix slightly, Let stand for 5 minutes;

[0056] (4) Take 500μL opti-DMEM into a 1.5mL centrifuge tube, take 9μg of recombinant lentiviral vector, pMD2.G helper plasmid 3μg and psPAX 12μg, add to the centrifuge tube, mix well, add to the transfection rea...

Example Embodiment

[0063] Example 3 T cell activation and lentiviral transfection

[0064] (1) After sorting out Pan T cells from umbilical cord blood, count the cells and adjust the concentration to 1×10 6 Cells / mL, then add 10μL Miltenyi TransAct T cell reagent to each milliliter of cell suspension. After 48 hours of activation, change to fresh medium (IMDM medium + 5% FBS (fetal bovine serum) + 1% double antibody ( 100× penicillin-streptomycin mixed solution)+IL-2);

[0065] (2) After T cell activation for 48 hours, demagnetize the beads, centrifuge at 300g for 5 min, remove the supernatant, resuspend the T cells in fresh medium, add recombinant lentivirus expressing CAR or blank control lentivirus (MOI = 10), and Add 8μg / mL polybrene and 300IU / mL IL-2, place at 37℃, 5% CO 2 Incubator culture

[0066] (3) After 24h, centrifuge at 300g for 5min, remove the supernatant, and resuspend the T cells in a fresh medium containing 300IU / mL IL-2 to obtain CAR-T cells.

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

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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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