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Chimeric antigen receptor (CAR) and application thereof

A chimeric antigen receptor and antibody technology, applied in the direction of hybrid peptide, receptor/cell surface antigen/cell surface determinant, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve Issues such as increased labor and cost

Active Publication Date: 2017-06-06
广东昭泰细胞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, 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 positively correlated with the therapeutic effect of CAR T cells. To obtain a large number of CD4 CAR T cells, it is necessary to first sort out CD4 and CD8 CAR T cells during in vitro culture, and then expand CD4 and CD8 CAR T cells through different cytokine combinations, which undoubtedly increases the huge labor and cost

Method used

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  • Chimeric antigen receptor (CAR) and application thereof
  • Chimeric antigen receptor (CAR) and application thereof
  • Chimeric antigen receptor (CAR) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Construction of Chimeric Antigen Receptor

[0082] (1) Synthesize 19-H.28z, Meso-H.28z, PSCA-H.28z containing IgG4-CH3 hinge region and 19.28z, Meso.28z, PSCA.28z CAR molecular sequence without hinge region through whole gene synthesis , the C-terminus of the synthetic gene contains a restriction endonuclease PmeI restriction site, and the N-terminus contains a restriction endonuclease SpeI restriction site. The full-length gene map of the CAR molecule is as follows: figure 1 shown;

[0083] The nucleic acid sequence of the IgG4-CH3 hinge region is shown in SEQ ID NO.1:

[0084] Ggcggaggtagctctggcggtggatccggcgggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

[0085] The nucleic acid sequen...

Embodiment 2

[0099] Example 2: Lentiviral packaging

[0100] When the cells cover 80-90% of the bottom of the 100mm culture dish, perform lentiviral packaging:

[0101] (1) 2 hours before virus packaging, the medium was replaced with DMEM containing 1% fetal bovine serum, 5mL / 100mm per culture dish;

[0102] (2) The packaging plasmids to be added to each 100mm culture dish of 293T cells are as follows:

[0103] Reagent dose pWPXLd-CAR-eGFP plasmid 4.5μg pMD2.G helper plasmid 1.5μg psPAX2 6μg

[0104] Add the plasmid to 500 μL opti-MEM medium and mix well;

[0105] Among them, pWPXLd-CAR-eGFP plasmids include: pwpxld-19.28z-eGFP, pwpxld-19-H.28z-eGFP, pwpxld-Meso.28z-eGFP, pwpxld-Meso-H.28z-eGFP, pwpxld-PSCA.28z -eGFP, pwpxld-PSCA-H.28z-eGFP;

[0106] (3) Add 36 μg PEI to another 500 μL opti-MEM medium, mix well, and let stand at room temperature for 5 minutes;

[0107] (4) Mix the plasmid with PEI, mix well by pipetting, and let stand at room tempe...

Embodiment 3

[0112] Example 3: Construction of CAR T cells

[0113] (1) Separation and purification of human T cells: Peripheral blood mononuclear cells (PBMC) were separated from whole blood by Ficoll (GE Company) density gradient centrifugation; ) to sort out T cells; T cells were activated for 72 hours using a T cell activation kit (Miltenyi);

[0114] (2) Lentiviral transfection of T cells: 72h after T cell activation, demagnetize the beads, resuspend the cells in 1640 medium containing 10% fetal bovine serum, 300IU / mLIL2, add the virus supernatant prepared in Example 2, every 10 6 Add 1mL virus supernatant to cells, polybrene 8μg / mL, at 37°C, 5% CO 2 After culturing in the incubator for 12 hours, resuspend with fresh medium containing 300IU / mL IL-2, every 10 6 Add 1 mL of culture medium to each cell.

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Abstract

The invention relates to the field of tumor cellular immunotherapy, in particular to a chimeric antigen receptor (CAR) and application thereof. The CAR comprises a hinge region, and the hinge region is IgG4-CH3. The IgG4-CH3 hinge region is introduced into a CAR molecular structure, so that the amplification efficiency of CAR T cells can be specifically increased in an in-vitro culture process, the amplification of CD4+CAR T cells is mainly promoted, CD4 cells and CD8 T cells do not need to be cultured in a separated way, the process is completed in a mixing system, and the amplification efficiency of the CD4+CAR T cells is increased.

Description

technical field [0001] The present invention relates to the field of tumor cell immunotherapy, in particular to a chimeric antigen receptor and its application, specifically a method for expanding CAR T cells by using a chimeric antigen receptor, a new CD4+CAR T cell expansion method method of increasing. Background technique [0002] In the past five years, adoptive immunotherapy such as chimeric antigen receptor T cells (Chimeric antigen receptor, CAR) has rapidly transformed from the basic scientific research stage to clinical trials, and has demonstrated strong tumor killing effects in clinical trials. CAR is a recombinant receptor that can specifically target tumor antigens, and generally consists of a single chain antibody variable region gene fragment (Singlechain fragment variable, scFv), hinge region (Hinge, H), transmembrane region, costimulatory molecule, CD3ζ chain and other components. [0003] So far, the fourth generation of CAR molecules has been developed....

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N7/01C12N5/10
CPCC07K14/7051C07K14/70521C07K16/00C07K16/28C07K16/2803C07K2319/02C07K2319/03C12N5/0636C12N7/00C12N2740/15043
Inventor 不公告发明人
Owner 广东昭泰细胞生物科技有限公司
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