Preparation method and application of chimeric antigen receptor T cell of targeted CD19

A chimeric antigen receptor, CD19-CAR technology, applied in the field of genetic engineering, can solve problems such as insufficient transfection efficiency, and achieve the effect of effectively stimulating T cell proliferation, improving killing ability, and improving killing efficiency

Active Publication Date: 2019-03-15
青岛见康华美医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a preparation method and application of chimeric antigen receptor T cells targeting CD19, to improve the activation ability of T cells in CAR-T cell therapy, to overcome the problem of insufficient transfection efficiency, and to improve the ability to kill CD19 powerful

Method used

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  • Preparation method and application of chimeric antigen receptor T cell of targeted CD19
  • Preparation method and application of chimeric antigen receptor T cell of targeted CD19
  • Preparation method and application of chimeric antigen receptor T cell of targeted CD19

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Experimental program
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Effect test

Embodiment 1

[0048] Preparation of Chimeric Antigen Receptor Targeting CD19

[0049] The chimeric antigen receptor gene targeting CD19 is composed of CD19 single-chain antibody CD19ScFv, the hinge region and transmembrane region of CD8, the intracellular signaling structure of CD28, and the intracellular signaling domain of CD3ζ. figure 1 , its base sequence is shown in SEQ ID NO: 2, and its length is 1584bp.

[0050] Among them, the single-chain antibody CD19ScFv sequence of CD19 is obtained from the heavy chain and light chain variable region sequences of the mouse anti-human CD19 antibody clone FMC63 through codon optimization, and its base sequence is shown in SEQ ID NO: 1, with a length of 726bp .

Embodiment 2

[0052] Construction of CD19-CAR lentiviral vector

[0053] The chimeric antigen receptor gene sequence targeting CD19 is added with BamH1 and Sal1 enzyme cleavage sites at both ends, and the whole sequence is synthesized. The synthesized sequence and lentiviral expression vector Plv-EF1a were double digested with restriction endonucleases BamH1 and Sal1.

[0054] The enzyme digestion system is as follows:

[0055] Plasmids containing synthetic sequences

[0056] Or Plv-EF1a empty vector plasmid 4μg

[0057] BamH1 2μl

[0058] Sal1 2μl

[0059] 10XB μffer 5 μl

[0060] wxya 2 O supplemented to 50 μl

[0061] The digested products were subjected to agarose gel electrophoresis, the fragments with the correct size were excised, and the DNA fragments were recovered using a gel extraction kit (purchased from Takara Company).

[0062] The recovered CAR gene fragment and the carrier fragment were ligated using a ligation kit (purchased from Takara Company), and ligated overnig...

Embodiment 3

[0067] Preparation of Chimeric Antigen Receptor T Cells Targeting CD19

[0068] (1) Lentivirus preparation

[0069] The lentiviral backbone plasmid Plv-EF1a, the lentiviral expression plasmid Plv-EF1a-CD19ScFv, the helper plasmids psPAX2 and pMD2.G were extracted with an endotoxin-free plasmid extraction kit (Qiangen Company), and extracted according to the operating instructions. Photometer to measure the concentration;

[0070] Take HEK293T / 17 cells in good cell state, inoculate them in culture dishes, the number should be about 80% after 24 hours, and culture them in DMEM medium containing 10% FBS.

[0071] After 24 hours, prepare the transfection system with the expression plasmid Plv-EF1a or Plv-EF1a-CD19ScFv and the helper plasmids psPAX2 and pMD2.G at a mass ratio of 8:5:3 with LipoFilter transfection reagent and serum-free medium, and after incubation for 15 minutes HEK293T / 17 cells were co-transfected, and fresh culture medium was replaced after 12 hours. The serum...

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Abstract

The invention provides a preparation method of chimeric antigen receptor T cell of targeted CD19, and relates to the technical field of gene engineering. The method comprises the following specific steps: construction of CD19-CAR lentiviral vector, preparation of lentivirus, separation of T cell and infection and proliferation of T cell. The method optimizes a transfection system and a culture medium adopted during cell culture. The method improves the transduction efficiency of T cell, solves the problem of insufficient transfection efficiency, obviously improves the kill capability of CAR-Tcell to CD19 positive cell, ensures that the kill capability reaches greater than 98 percent, and shows huge potential in cancer treatment.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a preparation method and application of chimeric antigen receptor T cells targeting CD19. Background technique [0002] B-cell malignancies are a group of malignant heterogeneous diseases of the hematological system, including acute and chronic B-lymphoblastic leukemia and some lymphoma subtypes. Because of their high recurrence rate and poor prognosis, they are clinically difficult to cure hematological malignancies. As a B cell lineage-specific cell surface differentiation antigen, CD19 is not only expressed on the surface of normal pre-B cells and mature B cells, but also widely expressed on the surface of a variety of B cell malignant tumor cells, while on the surface of hematopoietic stem cells, plasma cells and other normal tissue cells CD19 soluble protein has not been detected in the blood. In addition, the distribution of CD19 molecules on the membrane is re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N7/01C12N15/62A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K16/2803C07K2319/33C12N5/0636C12N7/00C12N15/86C12N2500/30C12N2500/34C12N2501/2302C12N2501/2307C12N2501/2315C12N2501/51C12N2501/515C12N2510/00C12N2533/52C12N2740/15021C12N2740/15043
Inventor 汝昆陈树英宋鸽蔺亚妮魏万旭
Owner 青岛见康华美医学检验有限公司
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