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Chimeric antigen receptor-modified T cells capable of self-expressing PD-1 antibody and targeting mesothelin and uses thereof

A chimeric antigen receptor, PD-1 technology, applied in the fields of genetic engineering and immunology, can solve the problems of high production cost, low response rate, 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

PD-1 antibody blocking therapy has a certain degree of effect on advanced or refractory melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, colorectal cancer, Hodgkin's lymphoma, ovarian cancer, etc. Therapeutic effect, but its high production cost and low response rate limit its clinical application

Method used

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  • Chimeric antigen receptor-modified T cells capable of self-expressing PD-1 antibody and targeting mesothelin and uses thereof
  • Chimeric antigen receptor-modified T cells capable of self-expressing PD-1 antibody and targeting mesothelin and uses thereof
  • Chimeric antigen receptor-modified T cells capable of self-expressing PD-1 antibody and targeting mesothelin and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101]Example 1: Construction of recombinant plasmids pNB328-meso3CAR, pS328-antiPD1, pNB328-meso3CAR-2A-antiPD1 and pNS328-antiPD1-IRES-meso3CAR

[0102] Entrust commercial companies to artificially synthesize the coding sequence of meso3CAR (SEQ ID NO: 3), the coding sequence of antiPD1 (SEQ ID NO: 4), the coding sequence of meso3CAR-2A-antiPD1 (the coding sequence of 2A is shown in SEQ ID NO: 5) The amino acid sequence is shown in SEQ ID NO: 6) and the coding sequence of antiPD1-IRES-meso3CAR (the coding sequence of IRES is shown in SEQ ID NO: 7), and its structural pattern is shown in figure 1 shown. Each sequence was loaded between the EcoRI and SalI restriction sites of pNB328 and pS328 vectors (see CN 201510638974.7 for the structure and sequence of pNB328, the entire contents of which are incorporated herein by reference; compared with pNB328, pS328 lacks PB transduction transposon, other elements are the same as pNB328), and the constructed recombinant plasmids were ...

Embodiment 2

[0103] Example 2: Construction of mesothelin-targeted CAR T cells expressing PD1 antibody

[0104] Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll separation. Cultivate PBMCs for 2-4 hours, and the unattached suspension cells are initial T cells. Collect the suspension cells into a 15ml centrifuge tube, centrifuge at 1200rmp for 3min, discard the supernatant, add physiological saline, centrifuge at 1200rmp for 3min, discard the physiological saline, and repeat this step; take four 1.5ml centrifuge tubes and add 5×10 6 Cells, numbered a, b, c, centrifuged at 1200rmp for 3min, discarded the supernatant, took the electrotransfer kit (from Lonza company), a, b, c tubes were added in proportion to a total of 100ul of electrotransfer reagents, a tube was added to the constructed recombinant plasmid 4ug each of pNB328-meso3CAR and pS328-antiPD1, add 6ug of pNB328-meso3CAR-2A-antiPD1 plasmid to tube b, add 6ug of pNB328-antiPD1-IRES-meso3CAR plasmid to tube c; Se...

Embodiment 3

[0106] Example 3: Comparing the positive rate and antibody secretion of three kinds of mesothelin-targeted CAR T cells expressing PD1 antibody

[0107] 1. Positive rate of CAR T cells detected by flow cytometry

[0108] Collect the meso3CAR-antiPD1T cells, meso3CAR-2A-antiPD1T cells, and antiPD1-IRES-meso3CAR T cells obtained in the examples, and divide each into two parts, each with 1×10 6 For each cell, wash twice with normal saline, resuspend the cells in 100ul normal saline, add 1ug of mesothelin-biotin to one part, and not add to the other, and incubate at 4°C for 30 minutes. Wash twice with normal saline, resuspend the cells with 100ul normal saline again, add 1ul streptomycin-PE antibody, and incubate at 4°C for 30 minutes. Washed twice with normal saline, tested on the machine, and only added secondary antibody as a control, the results are as follows: Figure 2A .

[0109] 2. ELISA detection of antiPD1 antibody expression levels of the three T cells

[0110] ① Dil...

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Abstract

The present invention provides chimeric antigen receptor-modified T cells capable of self-expressing PD-1 antibody and targeting mesothelin and uses thereof. The T cells of the present invention self-express PD1 antibodies and target mesothelin, and preferably contain (1) a coding sequence of a chimeric antigen receptor that expresses and recognizes mesothelin and a coding sequence of a PD-1 antibody, and / or (2) the chimeric antigen receptor that expresses and recognizes mesothelin and the PD-1 antibody. The T cells of the present invention inhibit the immune checkpoint PD-1 / PD-L1 pathway during the adoptive cellular immunotherapy, and can activate the function of the residual tumor specific T cells in situ, increase the antitumor killing effect of endogenous cytotoxic T cells, and promotethe proliferation of CAR-T cells in vivo, thereby improving the curative effect of specifically killing tumors.

Description

technical field [0001] The invention belongs to genetic engineering and immunology, and relates to a chimeric antigen receptor modified T cell targeting mesothelin and its application. Background technique [0002] Cancer has now become the number one killer of human health. The fast pace of life, huge work pressure, unhealthy eating habits, and poor environment are all accomplices to the occurrence of cancer, making the high incidence and younger trend of cancer more and more obvious. The current commonly used treatment methods are very limited in effect, and a more effective treatment method still needs to be explored to improve the survival rate and quality of life of cancer patients. [0003] Immunotherapy against malignant tumors has developed rapidly in recent years and has achieved remarkable clinical efficacy. Since 2011, Nature and JCO, the top journal of clinical oncology, have published review articles with the same title "The era of tumor immunotherapy has come"...

Claims

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Application Information

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IPC IPC(8): C12N5/10C07K16/28C12N15/13C12N15/62A61K39/395A61P35/00
CPCC07K16/2818C07K14/7051A61K35/17A61K2039/505C07K2319/02C07K2319/33C07K2319/30A61K39/395A61K48/00A61P35/00C07K16/28C07K16/46C12N5/10C12N15/62C12N15/63G01N33/68
Inventor 钱其军金华君江芏青刘祥箴何周王超崔连振李林芳
Owner SHANGHAI CELL THERAPY RES INST
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