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Expression vector for expressing IL-7 and CAR and immune cell

An expression vector and cell technology, applied in the field of CAR and IL-7 expressing T cells, can solve the problem of low survival efficiency of CAR-T cells, and achieve the effect of strong cancer treatment effect

Inactive Publication Date: 2020-12-15
南京艾德免疫治疗研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this regard, considering the low survival efficiency of the transferred CAR-T cells in vivo, or the activation of endogenous immunocompetent cells induced by the transferred CAR-T cells, it is necessary to develop technologies to solve the above problems

Method used

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  • Expression vector for expressing IL-7 and CAR and immune cell
  • Expression vector for expressing IL-7 and CAR and immune cell
  • Expression vector for expressing IL-7 and CAR and immune cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Construction of Lentivirus Containing CAR Gene

[0037] In this example, the single-chain antibody targeting human mesothelin (MSLN) is used as the unified extracellular recognition antigen structure, and the following two chimeric antigen receptors need to be constructed respectively ( figure 1 ):

[0038] DAP12-T2A-MSLN(scfv)-TREM1 cut (MSLN1)

[0039] DAP12-T2A-MSLN(scfv)-TREM1 cut -F2A-IL-7 (MSLN2)

[0040] 1. Synthesize the gene sequence containing the chimeric antigen receptor (CAR) targeting human mesothelin

[0041]Artificially synthesized DNA fragments encoding MSLN1 and MSLN2: including natural killer activating receptor (DAP12 for short), T2A, anti-human mesothelin single-chain antibody scfv (MSLN (scfv) for short), truncated myeloid cell-triggered receptor Body (referred to as TREM1 cut ), F2A, DNA fragment of IL-7, its structure is as follows figure 1 shown. Wherein the nucleotide sequence of DAP12 is shown in SEQ ID NO.1, the amino acid ...

Embodiment 2

[0060] 5) Add 400 μl of Opti-MEM to each centrifuge tube, pipette with a 200 μl pipette to dissolve the precipitate, and minimize the generation of foam; divide the virus into 25 to 50 μl tubes, freeze them in a -80°C refrigerator for long-term Preservation; Example 2: Titer determination

[0061] 1) Take a 24-well plate and inoculate 293T cells. 5×10 cells per well 4 One, the volume of the medium added is 500ul, the growth rate of different types of cells is different, and the cell fusion rate during virus infection is 40%-60%;

[0062] 2) Prepare 3 sterile EP tubes, add 90ul of fresh complete medium (high sugar DMEM + 10% FBS) to each tube to inoculate cells 24 hours later, take the cells in two wells to count, and determine the number of cells at the time of infection The actual number of , denoted as N;

[0063] 3) Take 10ul of the virus stock solution to be determined and add it to the first tube, mix gently, then take 10ul and add it to the second tube, and then opera...

Embodiment 3

[0074] Embodiment 3, virus infection T cell

[0075] 1. Isolation and activation of T cells and virus infection

[0076] (1) Isolation of human peripheral blood mononuclear cells

[0077] Use a blood collection tube containing anticoagulant to collect about 10ml of peripheral blood, settle naturally at room temperature (18-25°C) for about 30min, collect the upper layer of plasma, and centrifuge the collected upper layer of plasma at 5000r / min for 10min at a volume ratio of 1:1 Add to the lymphocyte separation medium (purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.), gradient centrifugation, 3000r / min, centrifugation for 30min, after centrifugation, the centrifuge tube is layered from top to bottom: the first layer is the plasma layer ; The second layer is the buffy coat layer of lymphocytes; the third layer is the transparent separation liquid layer; the fourth layer is the red blood cell layer. Aspirate the buffy coat of lymphocytes, wash twice with ...

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Abstract

The invention aims to provide a CAR expressing T cell which expresses both a chimeric antigen receptor (CAR) and an immune function-promoting factor of the T cell in the T cell, and a CAR expressing vector for preparing the CAR expressing T cell, wherein the CAR expressing T cell is a T cell having proliferative ability, survivability and killing ability. In the invention, according to the CAR expressing T cell that the CAR expression vector is manufactured, and the CAR expression T cell is guided, the CAR expression vector contains a nucleic acid encoding a chimeric antigen receptor (CAR) anda nucleic acid encoding an immune function promoting factor of the T cell, and the nucleic acid encoding the immune function promoting factor is nucleic acid encoding interleukin 7.

Description

technical field [0001] The present invention relates to a CAR and interleukin 7 (IL-7) expression vector, and CAR and IL-7 expressing T cells introduced with the CAR and interleukin 7 (IL-7) expression vector. Background technique [0002] Chimeric antigen receptor (CAR) is the core component of CAR-T. Using the ligand-binding domain properties, CAR can redirect the specificity and reactivity of selected immune cells, thus endowing T cells with an HLA-independent manner. The ability to recognize tumor antigens, which allows CAR-engineered T cells to recognize a wider range of targets than natural T-cell surface receptors TCR. The basic design of CAR includes a tumor-associated antigen (TAA) binding region (usually derived from the scFV segment of the antigen-binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region and an intracellular signal Area. [0003] In recent years, various studies on CAR-T cells have been conducted. For examp...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10
CPCC12N15/86C07K16/30C07K14/5418C12N5/0636C07K2317/622C12N2510/00C12N2740/15043C07K2319/33
Inventor 王恩秀张海汪晨
Owner 南京艾德免疫治疗研究院有限公司
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