CD40 dual costimulatory activated receptor (DCR) and application thereof

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

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...

Method used

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  • CD40 dual costimulatory activated receptor (DCR) and application thereof
  • CD40 dual costimulatory activated receptor (DCR) and application thereof
  • CD40 dual costimulatory activated receptor (DCR) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] Example 1: Five recombinant plasmids, namely pNB328-EGFR G1 CAR, pNB328-EGFR G2 CAR, PS328b Construction of 40DCR1, PS328b 40DCR2 and PS328b 40DCR3

[0153] 1. Artificially synthesized 40DCR1 gene (SEQ ID NO:15), 40DCR2 gene (SEQ ID NO:16), 40DCR3 gene (SEQ ID NO:17), EGFR G1 CAR gene (SEQ ID NO:24) and EGFR G2 CAR gene (SEQ ID NO: 26), the schematic diagram of its structure is as follows: Figure 1A , Figure 1B , Figure 1C , Figure 1F and Figure 1D shown. The 5 synthetic 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 cells EGFR G1CAR, EGFR G2 CAR, EGFR G1 CAR-40DCR1, EGFR G1 CAR-40DCR2, EGFR G1 CAR-40DCR3, 40DCR1, 40DCR2, 40DCR3 and the construction and identification of 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, add 5×106 cells of the above-mentioned cells in each tube, numbers are a, b, c, d, e, f, g, h, centrifuge at 1200rmp for 3min, discard the supernatant, and take the electropo...

Embodiment 3

[0192] Example 3: Cell Proliferation Vitality Kit Detecting Cell Technology Detecting Cell Proliferation Vitality

[0193] 1. Experimental samples and reagents

[0194] The recombinant cells EGFR G1 CAR, EGFR G2 CAR, EGFR G1 CAR-40DCR2, 40DCR1, 40DCR2, 40DCR3 and Mock T prepared in Example 2.

[0195] Luminescent Cell Viability Assay, Promega, Cat. #G7570.

[0196] 2. Experimental method

[0197](1) Prepare a 96-well white plate, take the above-mentioned cells on the 8th day of culture, and add 100 μL of cell-containing AIM-V medium to each well.

[0198] (2) Prepare a blank control without cells to obtain the background fluorescence value.

[0199] (3) Add the compound to be tested into the well plate and incubate in the incubator for 30 min.

[0200] (4) Add 100 μL CellTiter-Glo reagent, mix on a shaker for 2 minutes, incubate at room temperature for 10 minutes, and read.

[0201] (5) On the 8th, 9th, and 10th days of culture, the above-mentioned steps were tested e...

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Abstract

The invention belongs to the field of cytobiology and immunology and relates to a CD40 dual costimulatory activated receptor (DCR) and an application thereof. Particularly, the CD40 DCR sequentially comprises the following elements from N end to C end: optional signal peptide, a CD40 activated single-chain antibody, an extracellular hinge region, a transmembrane region and an intracellular costimulatory signal molecule. The CD40 DCR and first-generation CAR-T containing a first signal can produce a strong cluster effect and kill tumor cells when co-modifying T cells. meanwhile, the dual activation action is only limited among contacted T cells and cannot cause strong T cell immunity and cause potential severe toxic and side effects like injection of a CD40 activated antibody.

Description

technical field [0001] The invention belongs to the fields of cell biology and immunology, and relates to a CD40 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.2016 Oct6; 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 relat...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10A61P35/00
CPCC07K16/2863C07K14/7051A61K35/17C07K2319/02C07K2319/33C12N2510/00A61P35/00C07K19/00C12N5/10C12N15/62C12N15/63
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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