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Preparation method and application of CAR (Chimeric Antigen Receptor)-T cells of targeting HER2 (Human Epidermal Growth factor receptor 2)

A cellular and targeted technology, applied in the fields of immunology and immunotherapy, which can solve the problems of low HDR efficiency and imprecise gene editing

Inactive Publication Date: 2018-12-07
SUZHOU MAXIMUM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low efficiency of HDR (integration rarely occurs), and the non-homologous end-joining mechanism is prone to random insertions and deletions (indels), new bases may be randomly introduced near the breakpoint, resulting in imprecise gene editing

Method used

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  • Preparation method and application of CAR (Chimeric Antigen Receptor)-T cells of targeting HER2 (Human Epidermal Growth factor receptor 2)
  • Preparation method and application of CAR (Chimeric Antigen Receptor)-T cells of targeting HER2 (Human Epidermal Growth factor receptor 2)
  • Preparation method and application of CAR (Chimeric Antigen Receptor)-T cells of targeting HER2 (Human Epidermal Growth factor receptor 2)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Embodiment 1 constructs BE3 plasmid

[0129] The BE3 plasmid rAPOBEC1-SpCas9-NLS-UGI-NLS was formed by fusing Cas9nickase with cytidine deaminase (APOBEC1), the structure of which is as follows figure 2 shown.

Embodiment 2

[0130] Example 2 Designing sgRNA bases

[0131] For 6 target genes: human PD1, LAG3, TIGIT, VISTA, 2B4 and CD160, this example selects the following target gene sequences to design the corresponding sgRNA, where the bold underline indicates PAM, and the italic underline indicates the candidate mutation code son:

[0132] The sgRNA against human PD1 (hPD-1) is:

[0133]

[0134] The sgRNA targeting human LAG3 (hLAG3) is:

[0135]

[0136] The sgRNA against human TIGIT (hTIGIT) is:

[0137]

[0138] The sgRNA against human VISTA (hVISTA) is:

[0139]

[0140] The human 2B4 sgRNA is:

[0141]

[0142] The sgRNA against human CD160 (hCD160) is:

[0143]

Embodiment 3

[0144] Electric transgene knockout of embodiment 3T cells

[0145] BE3-mediated base editing is performed on primary cells, and stop codons are introduced to achieve gene knockout. This example is the electrotransgenic knockout of primary cells on human T cells:

[0146] (1) Separation and purification of PBMC cells

[0147] A. Use an anticoagulant tube to collect peripheral blood, and shake it while collecting to fully mix the peripheral blood with the anticoagulant;

[0148] B. Mix equal volumes of peripheral blood cells and lymphocyte separation medium, centrifuge, and absorb buffy coat cells after centrifugation;

[0149] C. Centrifuge after mixing the obtained buffy coat cells with PBS or serum-free cell culture medium, and the precipitate is the crude product of the PBMC cells;

[0150] D. Repeat the above steps A-C three times for the crude PBMC cells obtained in step C to obtain isolated and purified PBMC cells.

[0151] (2) Enrichment of CD3 positive T cells

[01...

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PUM

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Abstract

The invention discloses a preparation method and application of gene-edited CAR (Chimeric Antigen Receptor)-T cells capable of secreting a cell factor or an antibody. The method for preparing the CAR-T cells comprises the step of transfecting plasmid vectors containing gene-edited plasmids and CAR modification to T cells. The CAR-T cells provided by the invention are used for knocking out negativeregulator genes on surfaces of the T cells through gene editing and gene modification, integrating a single-chain antibody for identifying a tumor cell surface antigen and an expression frame for secreting the cell factor or secreting the antibody into the T cells and continuously expressing in the T cells, so that the T cells returned back are prevented from being inhibited by a tumor microenvironment and a better killing effect on the tumor cells is realized.

Description

Technical field: [0001] The invention belongs to the field of immunology, in particular to the field of immunotherapy, and in particular to a preparation method and application of CAR-T cells modified and targeted to HER2 based on base editing technology. Background technique: [0002] Tumor immunotherapy uses the body's own immune system to kill and inhibit tumor cells. Unlike traditional methods, this method does not directly target tumors, but reactivates the human immune system to kill and inhibit tumors. In 2013, "Science" magazine rated tumor immunotherapy as one of the top ten major scientific breakthroughs. In recent years, tumor immunotherapy has developed rapidly and has become the fourth tumor treatment method after surgery, chemotherapy and radiotherapy. Adoptive cellular immunotherapy for tumors has undergone early lymphokine-activated killer cell (LAK) therapy, cytokine-induced killer cell (CIK, DC-CIK) therapy, NK cell therapy, and tumor-infiltrating lymphocy...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/705C07K14/70503C07K14/70521C07K14/70532C12N5/0636C12N15/85C12N15/907C12N2501/2302C12N2501/515C12N2510/00C12N2810/10
Inventor 许先进辛雨
Owner SUZHOU MAXIMUM BIO TECH CO LTD
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