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CAR-T cell specifically targeting CD19 antigen and stably expressing PD-1 antibodies at high levels as well as application of CAR-T cell

A PD-1, chimeric antigen receptor technology, applied in the field of CART cells, can solve the problems of high drug cost, drug toxicity and side effects, and high treatment cost

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

On the one hand, since PD-1 monoclonal antibody is administered intravenously, most patients who receive PD-1 antibody blocking therapy will experience varying degrees of drug toxicity
On the other hand, the production of PD-1 monoclonal antibody involves complex production, preparation and purification processes, which are costly and result in high treatment costs.
[0007] In summary, CAR-T cells have the ability to kill tumor cells and can effectively enter tumor tissues, but their activity is easily inhibited in the tumor microenvironment; while PD-1 antibodies can reactivate the anti-tumor activity of T cells , but the penetration of macromolecular antibodies to solid tumors is insufficient, systemic administration has relatively large toxic and side effects, and the drug cost is high

Method used

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  • CAR-T cell specifically targeting CD19 antigen and stably expressing PD-1 antibodies at high levels as well as application of CAR-T cell
  • CAR-T cell specifically targeting CD19 antigen and stably expressing PD-1 antibodies at high levels as well as application of CAR-T cell
  • CAR-T cell specifically targeting CD19 antigen and stably expressing PD-1 antibodies at high levels as well as application of CAR-T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1: Construction of recombinant plasmids pNB328-CD19CAR, pS328-m279V, pS328-CD19CAR, pNB328-m279V, pNB328-CD19CAR-2A-m279V, pNB328-m279V-IRES-CD19CAR and acquisition of various types of pluripotent T cells.

[0112] 1. Entrust Shanghai Jierui Biological Company to synthesize CD19CAR exogenous gene (including CD8 signal peptide, scFv, CD8 transmembrane region, CD8 hinge region, 4-1BB and CD3ζ), and introduce a polyclonal restriction site (BglII -XbaI-EcoRI-BamHI), insert a restriction site (SalI-NheI-HindIII-SpeI) downstream of it, and load it into the pNB328 vector or pS328-EF1α vector that was cut with EcoR1+SalI (the structure of pNB328 and sequence see CN 201510638974.7, which is incorporated herein by reference in its entirety; compared with pNB328, pS328 lacks the transposase coding sequence) to form recombinant plasmids, named pNB328-CD19CAR and pS328-CD19CAR, respectively.

[0113] Entrust Shanghai Jereh Biological Company to synthesize the exogenous gene ...

Embodiment 2

[0121] Example 2: Determination of positive rate of CD19CAR gene expression and expression of PD-1 antibody after PBMCs were modified and activated in different combinations of CD19CAR gene and PD-1 antibody gene.

[0122] Activated T cells constructed in Example 1 were divided into 2×10 6 Cell number The cells were collected and divided into 2×10 6 Cells / well were spread in a 6-well plate with 3ml of AIM-V medium, cultured in a 5% CO2 incubator at 37°C, and the cell supernatant was collected after 24 hours of culture, and stored at -20°C for future use.

[0123] Double-antibody sandwich ELISA method (using human PD-1 recombinant protein coated microtiter plate, HRP-labeled mouse anti-human IgG4 mAb) was used to detect, commercialized PD-1 antibody was used as a standard, and the sample to be tested was 5 times After dilution, the expression level of PD-1 antibody in the genetically modified T cells was quantitatively detected. Collect 1×10 at the same time 6 Cell pellet, w...

Embodiment 3

[0128] Example 3: Determination of the positive rate of CD19CAR gene expression and the expression of PD-1 antibody in PBMCs modified with different mass ratios of pNB328-CD19CAR and pS328-m279V plasmids.

[0129] The recombinant plasmids pNB328-CD19CAR and pS328-m279V constructed in Example 1 were electroporated into PBMCs cells (1:1, 3:5, 1:3, 1:7) at different mass ratios to obtain multiple simultaneous expression of CD19CAR gene and PD -1 antibody T cells. These T cells were divided into 2 × 10 6 Cell number The cells were collected and divided into 2×10 6 Cells / well were spread in a 6-well plate with 3ml of AIM-V medium, cultured in a 5% CO2 incubator at 37°C, and the cell supernatant was collected after 24 hours of culture, and stored at -20°C for future use.

[0130] Double-antibody sandwich ELISA method (using human PD-1 recombinant protein coated microtiter plate, HRP-labeled mouse anti-human IgG4 mAb) was used to detect, commercialized PD-1 antibody was used as a s...

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PUM

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Abstract

The invention relates to a CAR-T cell specifically targeting CD19 antigen and stably expressing PD-1 antibodies at high levels as well as an application of the CAR-T cell. Specifically, the T cell ofthe invention contains and expresses a coding sequence of a CAR (chimeric antigen receptor) recognizing CD19 antigen and a coding sequence of the PD-1 antibodies; and / or expresses the CAR recognizingCD19 antigen and the PD-1 antibodies. The T cell can overcome immune microenvironment suppression, promotes apoptosis of tumor cells, develops an anti-tumor immune response, and can be applied to treatment of multiple types of malignant tumors.

Description

technical field [0001] The present invention relates to CAR T cells that specifically target CD19 antigen and stably express high-level anti-PD-1 antibodies and their anti-tumor effects. Background technique [0002] 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 was first proposed in 1989 by the Eshhar research...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/13C12N15/62C12N15/63C07K19/00C07K16/28A61K39/395A61P35/00A61P35/02
CPCC07K16/2803C07K16/2818C07K14/7051A61K39/3955C07K2319/33C07K2319/02C07K2317/622C07K2317/73A61K2239/31A61K39/464412A61K39/4636A61K2239/48A61K2239/38A61K39/4611A61K2300/00A61K39/395A61P35/00A61P35/02C07K16/28C07K19/00C12N5/10C12N15/62C12N15/63
Inventor 钱其军金华君何周刘祥箴李林芳崔连振
Owner SHANGHAI CELL THERAPY RES INST
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