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CD137 bidirectional activation costimulatory molecule receptor and usage thereof

A chimeric antigen receptor, C-terminal technology, applied in the field of cell biology and immunology, which can solve the problems of lack of bystander function and inability to activate surrounding T cells

Active Publication Date: 2019-07-05
SHANGHAI CELL THERAPY RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, no matter what kind of CAR-T cells, they can only provide stimulating signals to the modified T cells, lack the bystander function, and cannot activate surrounding T cells, resulting in a stronger cluster effect, causing a series of activation of T cell functions. cascade reaction

Method used

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  • CD137 bidirectional activation costimulatory molecule receptor and usage thereof
  • CD137 bidirectional activation costimulatory molecule receptor and usage thereof
  • CD137 bidirectional activation costimulatory molecule receptor and usage thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] Example 1: 5 recombinant plasmids, pNB328-meso CARG1, pNB328-meso G2 CAR, PS328b Construction of 137DCR1, PS328b 137DCR2 and PS328b 137DCR3

[0153] 1. Artificially synthesized 137DCR1 gene (SEQ ID NO:15), 137DCR2 gene (SEQ ID NO:16), 137DCR3 gene (SEQ ID NO:17), meso G1 CAR gene (SEQ ID NO:24) and meso G2 CAR gene (SEQ ID NO: 26), the schematic diagram of its structure is as follows: Figure 1A , Figure 1B , Figure 1C , Figure 1E and Figure 1F shown. The 5 synthesized genes were respectively loaded into the PNB328 vector and the PS328b vector, between the EcoRI and SalI restriction sites,

Embodiment 2

[0156] Example 2: Nine Chimeric Antigen Receptor Modified T Cells, namely Recombinant Cell Meso G1 CAR, meso G2 CAR, meso G1 CAR-137DCR1, meso G1 CAR-137DCR2, meso G1 CAR-137DCR3, 137DCR1, 137DCR2, Construction and Identification of 137DCR3 and Mock T

[0157] (1) Construction of 9 kinds of recombinant cells

[0158] Peripheral blood mononuclear cells (PBMCs) were adhered and cultured for 2-4 hours, and the unattached suspension cells were naive T cells. Collect the suspended cells into a 15ml centrifuge tube, centrifuge at 1200rmp for 3min, discard the supernatant; add normal saline, centrifuge at 1200rmp for 3min, discard the normal saline, and repeat the steps of "adding normal saline, centrifuging at 1200rmp for 3min, discarding the normal saline" 3 times.

[0159] Take 8 1.5ml centrifuge tubes and add 5×10 6 The above-mentioned cells, numbered a, b, c, d, e, f, g, h, were centrifuged at 1200rmp for 3min, the supernatant was discarded, and the electrotransfer ki...

Embodiment 3

[0192] Example 3: Detection of cell proliferation activity by flow cytometry

[0193] 1. Experimental samples, reagents and instruments

[0194] The recombinant cells 137DCR1, 137DCR2, 137DCR 3 and Mock T obtained in Example 2.

[0195] Prepare PBS containing 2% FBS (1ml Hyclone FBS+49ml PBS) in a 50ml centrifuge tube; use ddH 2 O 10 x BD Perm / Wash TM The buffer was diluted 10 times and placed on ice; the stock solution of Hoechst 33342 was diluted with ddH 2 O was diluted 1:100 into a working solution (1 μl / test).

[0196] Low-temperature centrifuge (pre-cooled at 4°C).

[0197] 2. Experimental method

[0198] All reagents were kept on ice during the experiment.

[0199] Specific steps are as follows:

[0200] (1) Charge 1×10 respectively 6 -2×10 6 Add appropriate amount of PBS containing 2% FBS to each of the above cells in a 1.5ml centrifuge tube, centrifuge at 5000rpm at 4°C for 5min, and discard the supernatant;

[0201] (2) Resuspend the cells with 100 μl Fi...

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Abstract

The invention belongs to the fields of cell biology and immunology, and relates to a CD137 bidirectional activation costimulatory molecule receptor and usage thereof. Specifically, the invention relates to a CD137 bidirectional activation costimulatory molecule receptor which sequentially comprises, from N terminal to C terminal, the following elements: optional signal peptides, CD137 activated single-chain antibodies, an extracellular hinge region, a transmembrane region, and intracellular costimulatory signaling molecules. When the CD137 bidirectional activation costimulatory molecule receptor modifies T cells together with a first-generation CAR-T containing a first signal, a strong cluster effect can be produced so as to kill tumor cells. Furthermore, the bidirectional activation effect is limited only between T cells which are in contact with each other, so that, strong T cell immunity is not triggered as it does when activated antibodies to CD137 are injected; and thus, potentialserious side effects are avoided.

Description

technical field [0001] The invention belongs to the field of cell biology and immunology, and relates to a CD137 bidirectionally activating co-stimulatory molecule receptor and the use of T cells modified by the receptor to treat malignant tumors. Background technique [0002] Adoptive cell therapy (ACT) is to reinfuse processed autologous or allogeneic immune cells (mainly autologous cells) to tumor patients to directly kill tumor cells, or to kill tumor cells by stimulating the body's immune response to achieve therapeutic purposes. Adoptive cell therapy for tumors is currently developing rapidly, and has achieved very good results in the clinical treatment of various malignant tumors (Nature.2016; Jun16; 534(7607):396-401); (Cell.2016Oct6; 167(2): 405-418.e13). Tumor immune cell therapy is considered to be one of the most promising means of treating malignant tumors. [0003] T cell activation requires the stimulation of two signals, namely two T cell activation relate...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10A61P35/00
CPCC07K16/2878C07K14/70521C07K14/70578C07K14/70517C12N5/0636C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2510/00A61K39/4611A61K2239/31A61K2239/59A61K39/464468A61K39/464417A61K2239/28A61K2239/38A61K39/4631A61P35/00A61P37/02C07K19/00C12N5/10C12N15/62
Inventor 钱其军金华君许慧敏刘祥箴李林芳王超
Owner SHANGHAI CELL THERAPY RES INST
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