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Chimeric antigen receptor and application thereof

A technology of chimeric antigen receptors and antigens, applied in the fields of application, antibody medical components, carriers, etc., can solve the problems of ineffective effects, etc., and achieve the goal of releasing immunosuppressive effects, enhancing anti-tumor ability, and good in vivo expansion ability Effect

Active Publication Date: 2020-10-09
SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the current situation that CAR-T cells have no obvious effect on solid tumors, the present invention provides a chimeric antigen receptor and its application. The chimeric antigen receptor prepared by the present invention targets TGFβ and can relieve the immunity of TGFβ to it. Inhibitory effect, thereby exerting anti-tumor ability

Method used

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  • Chimeric antigen receptor and application thereof
  • Chimeric antigen receptor and application thereof
  • Chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Construction of Chimeric Antigen Receptor

[0050] (1) Synthesize CAR (1928z CAR and T28z CAR) domains through the whole gene, such as figure 1 shown;

[0051] The amino acid sequence of the chimeric antigen receptor is as follows:

[0052] 1928z CAR (SEQ ID NO.2):

[0053] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQAKRKPRKAPSRNICYDAFVSYSERDAYWVENLMVQELENFNPPFKLCLHKRDFIPGKWIIDNIIDSIEKSHKTVFVLSENFVKSEWCKYELDFSHFRLFDENNDAAILILLEPIEKKAIPQRFCKLRKIMNTKTYLEWPMDEAQREGFWVNLRAAIKSTSKIVAPVKQTLNFDLLKLAGDVESNPGPASMVSKGEELFTGVVPILVE...

Embodiment 2

[0056] Example 2: TGFβ activates CAR-jurkat cells in vitro

[0057] The expression plasmids of 1928z CAR-T cells and T28z CAR-T cell vectors in Example 1 were used to prepare CAR-expressing lentiviruses from 293T cells, and finally infected Jurkat cells. The specific steps are as follows:

[0058] (1) put 10 5 CAR Jurkat cells were inoculated into 48-well plates, and TGFβ (0, 3, 10, 30 ng / ml) was added respectively, with a volume of 200 μl, and co-cultured at 37°C for 24 hours;

[0059] (2) After culturing for 24 hours, the 48-well plate was taken out, and the cell supernatant was frozen at -20°C for detection of IL2, and the cells were used for flow cytometry;

[0060] (3) After washing the cells with PBS, add an appropriate amount of APC-labeled human-CD69 antibody, incubate at room temperature for 20 minutes, wash with PBS, and then detect with a flow cytometer;

[0061] (4) Cell supernatant The expression of IL2 in the cell supernatant was detected with an ELISA kit.

...

Embodiment 3

[0063] Example 3: T28z CAR-T immunized mice have no toxic side effects

[0064] In order to verify the safety of T28z CAR-T cells, this example constructed mouse-derived CAR-T cells (since the scFv of TGFβ can be activated by both human-derived and mouse-derived TGFβ, the mouse-derived version of T28z CAR-T The scFv can be used universally), using m1928z T cells that can target mouse B cells as a positive control, and evaluating the safety of T28zT cells by observing the physiological state of mice, as follows:

[0065] (1) Prepare retrovirus by using retrovirus system, then isolate T cells from the spleen of B6 mice, and finally infect mouse T cells with retrovirus, and the infection efficiency is detected by flow cytometry;

[0066] (2) Inject the prepared CAR-T cells into the irradiated B6 mice through the tail vein, 3 mice in each group, each injected with 5×10 5 CAR-T cells, the peripheral blood of the mice was regularly drawn to detect the growth of T cells and B cells ...

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Abstract

The invention relates to a chimeric antigen receptor and application thereof. The chimeric antigen receptor is formed by connecting an antigen binding structural domain, a transmembrane structural domain and an intracellular structural domain in series. The antigen binding structural domain is combined with a tumor surface antigen, the tumor antigen is TGF beta, and the antigen binding structuraldomain is a single-chain antibody aiming at the tumor antigen TGF beta. Compared with other chimeric antigen receptors and other tumor antigens, the chimeric antigen receptor provided by the inventionhas a better effect, has safety and long-term effect, and indeed achieves a very good curative effect on solid tumors.

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 construction method of chimeric antigen receptor T (CAR-T) cell technology based on tumor antigen TGFβ and its Application in antitumor therapy. Background technique [0002] 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 immunotherapy. Generally, a chimeric antigen receptor CAR consists of a tumor-associated antigen-binding region, an extracellular hinge region, a transmembrane region, and an intracellular signal transduction region. Usually, CAR contains the single chain fragment variable (single chain fragment variable, scFv) region of the antibody or the binding domain specific to the tumor associated antigen (tumor associated antigen, TAA), which communicates with the T cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61P35/00
CPCC07K16/22C07K16/30C07K14/7051C12N15/86C12N7/00C12N5/0636A61K39/001134A61P35/00C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N2740/15021C12N2740/15043C12N2800/107C12N2510/00C07K2319/60
Inventor 不公告发明人
Owner SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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