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Anti-CII chimeric antigen receptor coding gene, slow virus plasmid, Treg immune cell, and applications thereof

A technology of chimeric antigen receptors and coding genes, applied in blood/immune system cells, receptors/cell surface antigens/cell surface determinants, anti-animal/human immunoglobulins, etc., can solve the problem of no preparation method To achieve the effect of terminating the cytokine storm, ensuring the safety of clinical use, and ensuring safety

Active Publication Date: 2019-04-30
上海兴瑞一达生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been many research reports and patents on RA, but most of them use new drugs or new dosage forms or RNA interference technology to treat RA. There is no new patch or new composition, but there is no cell preparation using immune cells to treat RA at present, so there is no preparation method

Method used

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  • Anti-CII chimeric antigen receptor coding gene, slow virus plasmid, Treg immune cell, and applications thereof
  • Anti-CII chimeric antigen receptor coding gene, slow virus plasmid, Treg immune cell, and applications thereof
  • Anti-CII chimeric antigen receptor coding gene, slow virus plasmid, Treg immune cell, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Anti-CII chimeric antigen receptor coding gene, including Linker nucleic acid artificial sequence, CII single-chain antibody nucleic acid artificial sequence, CD8 hinge region nucleic acid artificial sequence, CD8 transmembrane region nucleic acid artificial sequence, 2B4 co-stimulatory region nucleic acid artificial sequence, CD3ζ signal The nucleic acid artificial sequence of the conducting region, the artificial nucleic acid sequence of the T2A self-cleaving region, the artificial nucleic acid sequence of CTLA4 and the artificial nucleic acid sequence of the RQR8 molecular switch region.

[0038] Such as figure 1As shown, the anti-CII chimeric antigen receptor coding gene of the present embodiment includes the Leader nucleic acid artificial sequence (SEQ ID NO.2) connected sequentially, the CII binding region nucleic acid artificial sequence (SEQ ID NO.3), and the CD8Hinge region nucleic acid artificial sequence. sequence (SEQ ID NO.4), CD8 transmembrane region nucle...

Embodiment 2

[0040] Example of plasmid preparation of anti-CII chimeric antigen receptor encoding gene.

[0041] The plasmid preparation method of the anti-CII chimeric antigen receptor encoding gene of the present embodiment comprises the following steps:

[0042] (1) According to the order of Leader-scFv(CII)-CD8-2B4-CD3ζ-T2A-CTLA4-T2A-RQR8, entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the entire expression cassette and insert the lentiviral plasmid pLent-C Between the AsiSI and NotI restriction sites of -GFP, the plasmid pLent-CII-CTLA4 was obtained. Finally, the correctly sequenced E.coli Top10 containing the lentiviral plasmid pLent-CII-CTLA4 was obtained. The lentiviral plasmid pLent-CII-CTLA4 was extracted using an endotoxin-free plasmid extraction and purification kit. After the concentration of the plasmid was determined, it was stored in a -20°C refrigerator for later use.

[0043] (2) Use the endotoxin-free plasmid extraction and purification kit (purchased...

Embodiment 3

[0045] The present invention provides an example of Treg immune cells having anti-CII chimeric antigen receptor coding genes. The preparation method comprises: first packaging the pLent-CII-CTLA4 plasmid with lentivirus, and then using the recombinant lentivirus to infect Tregs immune cells.

[0046] (1) Lentiviral packaging, titer detection

[0047] The Lentiviral Packaging Kit lentiviral packaging kit was used, and the specific method was as follows: Inoculate the lentiviral packaging cell line 293T in a 10 cm culture dish containing DMEM+10% FBS, culture at 37°C, 5% CO2, and the adherence rate was 70%. %-80% ready for transfection. Take a sterile 1.5ml EP tube or 15ml centrifuge tube, and prepare the reaction system according to the following components: serum-free DMEM: 4ml; pLent-CII-CTLA4 plasmid: 10μg; GM easyTM Lentiviral Mix: 10μl (10μg); HG TransgeneTM Reagent: 60 μl. After mixing, place it at room temperature for 20 minutes, evenly drop it into the culture dish c...

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Abstract

The invention discloses an anti-CII chimeric antigen receptor coding gene, which comprises a Linker nucleic acid artificial sequence, a CII single chain antibody nucleic acid artificial sequence, a CD8 hinge region nucleic acid artificial sequence, a CD8 trans-membrane domain nucleic acid artificial sequence, a 2B4 co-stimulation area nucleic acid artificial sequence, a CD3 zeta signal transduction area nucleic acid artificial sequence, a T2A self-cleavable region nucleic acid artificial sequence, a CTLA4 nucleic acid artificial sequence, and a RQR8 molecular switch area nucleic acid artificial sequence. The invention also discloses a slow virus plasmid containing the anti-CII chimeric antigen receptor coding gene, a Treg immune cell, and applications thereof. On the premise that the curative effect is guaranteed, the immuno-suppression activity of CAR-Tregs cells is enhanced, and at the same time, the safety of CAR-Tregs cells is guaranteed.

Description

technical field [0001] The invention relates to the field of biological genes, more specifically, anti-CII chimeric antigen receptor coding genes, lentiviral plasmids, Treg immune cells and applications thereof. Background technique [0002] Rheumatoid arthritis (Rheumatoid arthritis, RA) is a common autoimmune disease that is disabling and affects the quality of life in the world. %~4%. RA is a disease in which the joints of the hands and feet are swollen and damaged due to its own immune system invading the joints of the hands and feet. It can also be regarded as a chronic syndrome, manifested as non-specific inflammation of peripheral joints. At this time, the diseased joint and its surrounding tissues show progressive destruction, and cause dysfunction of the damaged joint. Autoreactive T lymphocytes play an important role in its abnormal immune response. [0003] At present, in the treatment of RA, the development of the disease is mainly controlled by drug therapy su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/867C12N5/10A61K35/17A61P37/02A61P29/00A61P19/02
CPCA61P19/02A61P29/00A61P37/02A61K35/17C12N5/0637C12N15/86C07K14/70503C07K14/7051C07K14/70517C07K14/70521C07K16/18C12N2740/15043C12N2510/00C12N2800/107C07K2319/00C07K2317/622C07K2319/03
Inventor 刘明录王立新刘敏金海锋万磊冯建海卢永灿张传鹏强邦明
Owner 上海兴瑞一达生物科技有限公司
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