CD28 bidirectional activation co-stimulatory molecular receptor and applications 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, a

Method used

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  • CD28 bidirectional activation co-stimulatory molecular receptor and applications thereof
  • CD28 bidirectional activation co-stimulatory molecular receptor and applications thereof
  • CD28 bidirectional activation co-stimulatory molecular receptor and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Example 1: 5 recombinant plasmids, namely recombinant plasmids pNB328-Muc1 G1 CAR, pNB328-Muc1 G2 CAR, PS328b 28DCR1, PS328b 28DCR2 and PS328b Construction of 28DCR3

[0152] 1. Artificially synthesized 28DCR1 gene (SEQ ID NO:15), 28DCR2 gene (SEQ ID NO:16), 28DCR3 gene (SEQ ID NO:17), Muc1 G1 CAR gene (SEQ ID NO:24) and Muc1 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

[0155] Example 2: Nine Chimeric Antigen Receptor Modified T Cells, namely Recombinant Cell Muc1 G1 CAR, Muc1 G2 CAR, Muc1 G1 CAR-28DCR1, Muc1 G1 CAR-28DCR2, Muc1 G1 CAR-28DCR3, 28DCR1, 28DCR2, 28DCR3 and the construction and identification of Mock T

[0156] (1) Construction of 9 kinds of recombinant cells

[0157] 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.

[0158] 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

[0191] Example 3: Cell Proliferation Vitality Kit Detecting Cell Technology Detecting Cell Proliferation Vitality 1. Experimental samples and reagents

[0192] The recombinant cells Muc1 G1 CAR, Muc1 G2 CAR, Muc1 G1 CAR-28DCR2, 28DCR1, 28DCR2, 28DCR3 and Mock T prepared in Example 2.

[0193] Luminescent Cell Viability Assay, Promega, Cat. #G7570.

[0194] 2. Experimental method

[0195] (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.

[0196] (2) Prepare a blank control without cells to obtain the background fluorescence value.

[0197] (3) Add the compound to be tested into the well plate and incubate in the incubator for 30 min.

[0198] (4) Add 100 μL CellTiter-Glo reagent, mix on a shaker for 2 minutes, incubate at room temperature for 10 minutes, and read.

[0199] (5) On the 8th, 9th, and 10th days of culture, the above-mentioned steps were tested every day....

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Abstract

The invention belongs to the field of cell biology and immunology, and relates to a CD28 bidirectional activation co-stimulatory molecular receptor and applications thereof. In particular, the presentinvention relates to a CD28 bidirectionally activated co-stimulatory molecular receptor which successively comprises, from the N-terminal to the C-terminal, an optional signal peptide, a CD28 activated single chain antibody, an extracellular hinge region, a transmembrane region and an intracellular co-stimulatory signal molecule. When the CD28 bidirectionally activated co-stimulatory molecular receptor modies T cells together with the first-generation of CAR-T containing the first signal, a strong clustering effect can be produced, and the tumor cells can be killed. Meanwhile, the bidirectional activation effect is only limited between the T cells in contact with each other, which is not like the CD28-injected activated antibody which triggers strong T cell immunity and causes potential serious toxic and side effects.

Description

technical field [0001] The invention belongs to the fields of cell biology and immunology, and relates to a CD28 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
CPCC07K16/3092C07K14/7051A61K35/17C07K2319/02C07K2319/33C12N2510/00A61K38/17A61P35/00C07K14/705C07K16/28C07K19/00C12N5/10C12N15/62C12N15/63
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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