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CD19 targeted CAR (chimeric antigen receptor)-T cell, preparation method and application

A chimeric antigen receptor and targeting technology, applied in the field of immune cells, can solve the problems of insufficient transfection efficiency and achieve effective T cell proliferation, high transduction efficiency, and good killing effect

Inactive Publication Date: 2017-10-24
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 CD19-targeted chimeric antigen receptor T cell, preparation method and application, to improve the T cell activation ability in CAR-T cell therapy, to overcome the problem of insufficient transfection efficiency, and to kill CD19 powerful

Method used

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  • CD19 targeted CAR (chimeric antigen receptor)-T cell, preparation method and application
  • CD19 targeted CAR (chimeric antigen receptor)-T cell, preparation method and application
  • CD19 targeted CAR (chimeric antigen receptor)-T cell, preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0037] Preparation of Chimeric Antigen Receptor Targeting CD19

[0038] 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.

[0039]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

[0041] Construction of CD19-CAR lentiviral vector

[0042] 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.

[0043] The enzyme digestion system is as follows:

[0044] Plasmids containing synthetic sequences

[0045] Or Plv-EF1a empty vector plasmid 4ug

[0046] BamH1 2ul

[0047] Sal1 2ul

[0048] 10XBuffer 5ul

[0049] wxya 2 O supplemented to 50ul

[0050] 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).

[0051] The recovered CAR gene fragment and the carrier fragment were ligated using a ligation kit (purchased from Takara Company), and ligated overnight ...

Embodiment 3

[0056] Preparation of Chimeric Antigen Receptor T Cells Targeting CD19

[0057] (1) Lentivirus preparation

[0058] 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;

[0059] 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.

[0060] 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 9:6:3 with LipoFilter transfection reagent and serum-free DMEM medium, and incubate for 15 minutes Afterwards, HEK293T / 17 cells were co-transfected, and fresh culture medium was replaced after 12 hours. ...

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Abstract

The invention provides a CD19 targeted CAR (chimeric antigen receptor)-T cell, a preparation method and an application and relates to the field of immune cells. The activation capacity of T cells in CAR-T cell therapy can be improved, the problem of insufficient transfection efficiency can be solved, and the CAR-T cell has a high killing capacity for CD19. The CD19 targeted CAR-T cell expresses a CD19 targeted CAR gene on surface, and the CD19 targeted CAR gene is formed by connecting a single-chain antibody CD19ScFv of CD19, a hinge region and a transmembrane region of CD8, an intracellular signal structure of CD28, an intracellular signal structure of 4-1BB and an intracellular signal structural domain of CD3zeta in series, and the base sequence of the CD19 targeted CAR gene is shown as SEQ ID NO:2.

Description

technical field [0001] The invention relates to the field of immune cells, in particular to a chimeric antigen receptor T cell targeting CD19, a preparation method and application. 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 relatively exposed, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K35/17A61P35/00
CPCC12N5/0636A61K35/17C12N15/86C12N2510/00C12N2740/15043
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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