Preparation method for CAR-T cell targeted to EGFRvIII and application

A cell-targeted technology, applied in the field of CAR-T cell preparation, to achieve the effects of enhanced loading, simple operation, and simple preparation process

Inactive Publication Date: 2018-11-30
SUZHOU MAXIMUM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, using the plasmid system, the preparation process is relatively simple, and the exogenous gene is integrated into the genome through a transposase, which is simple to operate and relatively high in integration efficiency. Importantly, it is safer than the retrovirus system, and EGFRvIII is only available in tumors. It appears in cells but is not expressed in normal tissue cells, which makes EGFRvIII a good target for tumor therapy, so CAR-T immunotherapy that chooses EGFRvIII protein as the target antigen may have fewer side effects than other CAR-T cell therapies

Method used

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  • Preparation method for CAR-T cell targeted to EGFRvIII and application
  • Preparation method for CAR-T cell targeted to EGFRvIII and application
  • Preparation method for CAR-T cell targeted to EGFRvIII and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Codon optimization

[0042] (1) Prepare the EGFRvIII chimeric antigen receptor (CAR), namely 139CAR, and send the ScFv fragment to the company for codon optimization to make it easier to express in human cells. The sequence after codon optimization is SEQ ID NO.1 Nucleotide sequence shown.

[0043] (2) Synthesize the target fragment, and then replace the ScFv fragment in 139CAR, as attached figure 1 The circular plasmid map of PB-EGFRvⅢ-CAR-BBZ-puro shown and attached figure 2 Linear vector map of PB-EGFRvIII-CAR-BBZ-puro shown.

Embodiment 2

[0044] Example 2 PBMC cell preparation

[0045] Take 15mL of human peripheral blood, treat with anticoagulation (EATA or heparin), centrifuge at room temperature for 15 minutes at 350*g for 5 minutes, and suck out the plasma for use;

[0046] Add 1 times the volume of PBS buffer to the blood cells to dilute and mix well, take a 15mL centrifuge tube, add 5mL of lymphocyte separation medium Ficoll, and slowly add 5mL of diluted blood cells to the upper layer. This step must be slow to prevent Ficoll from mixing with blood cells.

[0047] Rising slowly, centrifuging at 450*g for 25 minutes at room temperature, the blood cells are divided into platelet layer, white blood cell layer (buffy coat), polysucrose (Ficoll) layer and red blood cell layer from top to bottom, suck platelet layer (2mL), and white blood cell layer (buffy coat, about 3mL) was transferred to a new 15mL centrifuge tube.

[0048] Add 10mL PBS buffer, centrifuge at 300*g for 10 minutes, discard the supernatant, r...

Embodiment 3

[0049] Example 3 Preparation of CAR-T cells targeting EGFRvIII

[0050] (1) After slightly culturing PBMC with RPMI 1640 medium containing 10% FBS for 1 hour, centrifuge at 300*g for 8 minutes, and completely remove the supernatant; configure the electroporation system: the plasmid PB-EGFRvⅢ CAR-BBZ-puro (sequence is the nucleotide sequence shown in SEQ ID NO.2) 5 μ g, transposase plasmid Transposase (can adopt

[0051] http: / / www.biofeng.com / zaiti / buru / Super-PiggyBac-Transposase.html published Transposase) 5μg, 82μL electroporation buffer and 18μL supplement 1 and mix well;

[0052] (2) Resuspend the cells in the plasmid electrotransfer buffer mixture prepared in step (1), and transfer them to the electroporation cup, and use the instrument Lonza 2B, U-014 program for electroporation, and the cells after electroporation are quickly transferred to the preheated cultured in a 37°C incubator for two hours, digested with 5μg / mL DNase 1 to remove the DNA released by dead cells du...

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Abstract

The invention discloses a preparation method for a CAR-T cell targeted to EGFRvIII and modified by a gene and application. The method comprises the following steps: optimizing an amino acid sequence codon targeted to the EGFRvIII, enabling the amino acid sequence codon to be more suitably expressed in a human source host body, and preparing the CAR-T cell; knocking out a PD-1 gene in the CAR-T cell by using a single base editing method; or combining a secretion interleukin 12 (IL-12), and using for promoting proliferation of the activated T cell; or combining a secretion anti-PD1 antibody, andusing for combining with the PD1 on other T cells, and relieving the inhibiting effect of a tumor tissue to the T cell. The method is capable of improving transfection efficiency and expression efficiency of the imported gene through selecting a PiggyBac transposon system, shortening operating time, and simplifying a preparation process of the CAR-T. In addition, the method avoids using of a virus vector while knocking out the PD-1 gene, so the safety is higher.

Description

technical field [0001] The present invention relates to the field of immunology, in particular to the field of immunotherapy, in particular to a preparation method and application of CAR-T cells targeting EGFR vIII. Background technique [0002] EGFR (Epidermal Growth Factor Receptor) is a glycoprotein, which belongs to the tyrosine kinase receptor, and is the product encoded and expressed by the proto-oncogene C-erb-1 (HER-1), with a molecular weight of 170KDa. EGFR is located on the surface of the cell membrane. Activated by binding to ligand, it is the receptor of epithelial growth factor (EGF) cell proliferation and signal transduction, EGFR binds and activates its corresponding ligand, as a signal transduction complex, and then activates the downstream signal transduction pathway to cause a series of cell Response: Promote signal transduction and cell growth and differentiation, accelerate cell cycle, promote angiogenesis, and promote tumor growth and metastasis. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/62C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/2863C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/85C12N2501/515C12N2510/00C12N2800/107C12N2800/22
Inventor 刘希尚小云
Owner SUZHOU MAXIMUM BIO TECH CO LTD
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