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Chimeric antigen receptor T cell targeting BCMA and application of chimeric antigen receptor T cell

A technology of chimeric antigen receptors and cells, applied in the field of biomedicine, can solve problems such as cytokine storms, achieve the effects of low incidence of side effects, high killing efficiency, and good clinical application prospects

Active Publication Date: 2021-01-22
汤朝阳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing technology uses BCMA antigen as the extracellular antigen binding region to construct CAR molecules for the treatment of B cell-related diseases such as multiple myeloma, but there are problems such as cytokine storms

Method used

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  • Chimeric antigen receptor T cell targeting BCMA and application of chimeric antigen receptor T cell
  • Chimeric antigen receptor T cell targeting BCMA and application of chimeric antigen receptor T cell
  • Chimeric antigen receptor T cell targeting BCMA and application of chimeric antigen receptor T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The preparation of embodiment 1CAR carrier

[0038] The schematic diagram of the structure of the chimeric antigen receptor targeting BCMA constructed in this example is as follows figure 1 Shown, the amino acid sequence is shown in SEQ ID NO: 1, and the coding gene is shown in SEQ ID NO: 2;

[0039] Add restriction endonuclease Pme1 digestion site and its protective base and restriction endonuclease Spe1 restriction endonuclease Spe1 restriction site and its protection base to the C-terminal and N-terminal of the above coding gene respectively;

[0040] The coding gene was double-digested with restriction endonucleases Pme1 and Spe1, and the digested product containing cohesive ends was recovered by agar gel electrophoresis, and then ligated into the linearized pWPXLd-eGFP plasmid (containing sticky ends), the ligation reaction was carried out with the participation of T4 DNA polymerase (Invitrogent) to obtain a lentiviral vector containing the gene encoding αBCMAscFv...

Embodiment 2

[0042] Example 2 Recombinant lentiviral packaging

[0043] In this example, 293T cells were used to prepare recombinant lentiviruses, and when the 293T cells spread to 80-90% of the bottom of a 100mm culture dish, the lentiviruses were packaged:

[0044] 2 hours before virus packaging, replace the medium with DMEM containing 1% fetal bovine serum, and add 6mL / 100mm culture dish;

[0045] Prepare the plasmid mixture shown in Table 1, the pWPXLd-expression plasmid is a lentiviral vector expressing the CAR molecule, and the pWPXLd-eGFP plasmid is an empty vector that does not contain the gene encoding the CAR molecule;

[0046] Table 1

[0047]

[0048] Add 36 μg PEI to another 500 μL opti-MEM medium, mix well, and let stand at room temperature for 5 minutes;

[0049] Mix the plasmid mixture shown in Table 1 with PEI, mix well by pipetting, and let stand at room temperature for 25-30 minutes;

[0050] Add the above mixture dropwise to the 293T cells cultured in a 100mm cult...

Embodiment 3

[0054] Example 3 T cell activation and lentiviral transfection

[0055] 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;

[0056] 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;

[0057] 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;

[0058] After T cells were activated for 48 hours, the beads were demagnetic, centrifuged at 300 g for 5 min, and the supernatant was removed. T cells were resuspended in fresh medium...

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Abstract

The invention provides a chimeric antigen receptor T cell targeting BCMA and application of the chimeric antigen receptor T cell. A chimeric antigen receptor comprises a signal peptide, an antigen binding structural domain, a transmembrane structural domain and a signal transduction structural domain, Wherein the antigen binding domain comprises an anti-BCMA single-chain antibody; and the signal transduction structural domain comprises 4-1BB, CD3 [zeta] and TLR2. According to the chimeric antigen receptor T cell, 4-1BB, CD3[zeta] and TLR2 which are connected in series are used as signal transduction structural domains of the chimeric antigen receptor, and are matched with a specific antigen binding structural domain anti-BCMA single-chain antibody, so that the constructed chimeric antigenreceptor T cell has remarkably enhanced capability of specifically recognizing and killing BCMA positive tumor cells, and is high in safety, and bright clinical application prospects are realized in the field of B cell malignant tumor treatment.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a chimeric antigen receptor T cell targeting BCMA and an application thereof. Background technique [0002] Chimeric antigen receptor (CAR) is a recombinant receptor targeting cell surface antigens, and its structure mainly includes three parts: extracellular antigen binding region, transmembrane region and intracellular signal region. Among them, the extracellular antigen-binding region is responsible for recognizing antigens, the transmembrane region connects the extracellular antigen-binding region and the intracellular signal region, which affects the expression ability of the imported CAR gene, and the intracellular signal region is responsible for signal transmission. [0003] Chimeric antigen receptor T cells (CAR-T) use CAR molecules to recognize and bind to tumor antigens, thereby exerting the specific killing effect of T cells on tumor cells, and has now developed int...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62C12N15/867A61K39/00A61P35/00
CPCC12N5/0636C07K16/2878C07K14/7051C12N15/86A61K39/001111A61P35/00A61K2039/5158C12N2510/00C07K2319/02C12N2740/15043
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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