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Targeting Muc1 type chimeric antigen receptor modified T cell and application thereof

A technology of chimeric antigen receptors and intracellular domains, applied in the fields of genetic engineering and oncology

Active Publication Date: 2019-02-15
SHANGHAI CELL THERAPY RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these new sugar chains and peptide epitopes can be ideal targets for CAR-T cell therapy, since different sugar chains and peptide epitopes are displayed on the surface of different tumor cells, specific targeting of a certain A glycopeptide CAR-T cell may not be used in many types of tumors

Method used

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  • Targeting Muc1 type chimeric antigen receptor modified T cell and application thereof
  • Targeting Muc1 type chimeric antigen receptor modified T cell and application thereof
  • Targeting Muc1 type chimeric antigen receptor modified T cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Construction of recombinant plasmids pNB328-Muc1CAR1, pNB328-Muc1CAR2, pNB328-Muc1CAR3 and the acquisition of Muc1CAR gene-modified T cells

[0087] 1. Artificial synthesis containing signal peptide (CD8 signal peptide or light chain signal peptide), antigen recognition single-chain antibody (anti-Muc1scFv), hinge region (CD8 hinge region or IgG4Fc CH2CH3 hinge region), CD8 transmembrane region, costimulatory signal The Muc1CAR exogenous genes of the molecular intracellular domain and the immunoreceptor tyrosine activation motif (named Muc1CAR1, Muc1CAR2 and Muc1CAR3, respectively), and introduced a multicloning restriction site (BglII-XbaI-EcoRI-BamHI ), insert a restriction site (SalI-NheI-HindIII-SpeI) downstream of it, entrust Shanghai Jierui Biology Co., Ltd. to synthesize it, and put it into the pNB328-EF1α vector (containing EF1α promoter pNB328, see CN 201510812654.9 for the pNB328 vector) to form recombinant plasmids, named pNB328-Muc1CAR1, pNB32...

Embodiment 2

[0091] Example 2: The expression of T cells after PBMCs cells derived from different patients were activated by Muc1CAR exogenous gene modification Muc1CAR gene expression identification

[0092] The activated T cells of different patients after the recombinant plasmid pNB328-Muc1CAR1 or pNB328-Muc1CAR2 or pNB328-Muc1CAR3 were electroporated by 2×10 6 Cell number Collect the cell pellet, add 80ul of 2X SDS-PAGE Loadingbuffer, boil at 100°C for 10min, and store at -20°C for later use. Western blotting (using CD3ζ antibody as the primary antibody, and HRP-goat anti-human secondary antibody) was used to detect the expression of Muc1CAR.

[0093] The result is as figure 2 shown. The results showed that T cells could stably express Muc1CAR1 and Muc1CAR3 recombinant proteins, but could not express Muc1CAR2 recombinant proteins.

Embodiment 3

[0094] Example 3: PBMCs cells from different patients were modified by Muc1CAR exogenous gene in T cell genome Detection of expression level of Muc1CAR genome

[0095] Genomic DNA of Mock T cells (T cells electroporated with pNB328 empty vector) and Muc1CAR-T cells (kit method) was extracted, and the experimental steps were determined according to the instructions attached to the kit, and the DNA concentrations of Mock T cells and Muc1CAR-T cells were measured. The expression level of the Muc1CAR genome was detected by real-time fluorescent quantitative PCR. The reaction program was: 50°C, 2min→95°C, 10min→95°C, 15s→60°C, 1min, 40 cycles.

[0096] The result is as image 3 shown. The results showed that the Muc1CAR1, Muc1CAR2 and Muc1CAR3 genomes were all integrated into the T cell genome through the PB transposase system.

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Abstract

The invention relates to a targeting Muc1 type chimeric antigen receptor modified T cell and application thereof. The targeting Muc1 type chimeric antigen receptor modified T cell specifically comprises membrane protein signal peptide, an anti-Muc1 membrane proximal single-chain antibody, a hinge region with more than 50 amino acid residue, a transmembrane domain, a co-stimulatory signal moleculenotch intracellular domain and an immune receptor tyrosine activation group sequence from N terminal to C terminal. The targeting Muc1 type chimeric antigen receptor modified T cell also comprises a Tcell in which an antigen receptor is embedded. The T cell is capable of recognizing and combining Muc1 antigen membrane proximal and is capable of secreting cytokines by efficiently activating and proliferating, so as to exterminate Muc1 positive tumor cells. The T cell is applicable to treatment of various Muc1 positive malignant tumors.

Description

technical field [0001] The invention belongs to genetic engineering and oncology, and relates to a chimeric antigen receptor modified cell specifically targeting Muc1 antigen and its anti-tumor effect. Background technique [0002] Chimeric antigen receptor T cell (chimeric antigen receptor T cell, CAR-T) therapy technology is undoubtedly a rising star in the field of tumor immune cell therapy. CAR-T technology splices the variable region gene sequence of an antibody that recognizes an antigen molecule with the intracellular region sequence of T lymphocyte immune receptors through genetic engineering technology, and uses retrovirus or lentiviral vectors, transposons or transposons to The seat enzyme system or direct mRNA transduction into lymphocytes, and express fusion protein on the cell surface, so that T lymphocytes can recognize specific antigens in a non-MHC-restricted manner, and enhance their ability to recognize and kill tumors. [0003] Since the structure of CAR ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/13C12N5/10A61P35/00
CPCA61K35/17C07K14/7051C07K16/3092C07K2317/622C07K2319/03C07K2319/02C07K2319/33A61K39/395A61P35/00A61P35/04C07K19/00C12N5/10C12N15/09
Inventor 钱其军金华君何周王超李林芳
Owner SHANGHAI CELL THERAPY RES INST
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