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Method for producing chimeric antigen receptor modified gamma delta T cell

A chimeric antigen receptor and cell technology, applied in the biological field, can solve the problems of low cell content, low purity of the final product, low transfection rate, etc.

Active Publication Date: 2018-09-28
HEBEI SENLANG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the natural activity of γδT cells makes them an excellent carrier for CAR-T cell therapy, due to the limitations of low cell content, difficulty in large-scale expansion in vitro, and low transfection rate, no CAR-modified γδT cells have been used so far. Reports of successful clinical application
Traditional γδT cell expansion techniques in vitro, including solid-phase anti-pan TCRγδ antibody expansion, or using IPP / HMBPP / ZOL to selectively expand Vγ9δ2T cells, are all expanded from peripheral mononuclear cells (PBMC). The proportion of γδT cells is low, and gene transfection is interfered by other irrelevant cells, resulting in low transfection rate
In addition, the final product expanded from PBMC has low purity and is easy to mix with αβT cells, which is not conducive to the development of general CAR-T products
Sorting and purifying γδT cells can solve the above problems, but without the assistance of other cells, the activation and expansion efficiency will be greatly reduced

Method used

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  • Method for producing chimeric antigen receptor modified gamma delta T cell
  • Method for producing chimeric antigen receptor modified gamma delta T cell
  • Method for producing chimeric antigen receptor modified gamma delta T cell

Examples

Experimental program
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Effect test

Embodiment 1

[0070] Example 1. A method for expanding γδT cells

[0071] 1. Construction of K562-shFPPS tumor cell line with reduced expression of FPPS

[0072] 1. Construction of lentiviral recombinant plasmid

[0073] (1) Preparation of recombinant plasmid

[0074] The U6-based shRNA construction system of the Vectorbuilder system of Saiye Biotechnology Co., Ltd. was used to construct the lentiviral recombinant plasmid. Specific steps are as follows:

[0075] Design and synthesize a specific shRNA coding sequence (shFPPS) targeting FPPS as the experimental group: (Sequence 1); wherein, the bold 6bp sequence in the middle is the stem-loop sequence, its left 21bp sequence is the sense sequence, and the right 21bp sequence is the antisense sequence; at the same time, the following Scramble-shRNA coding sequence (shSRB) was synthesized as a negative control Group: Among them, the bold 6bp sequence in the middle is the stem-loop sequence, the left 21bp sequence is the sense sequence, a...

Embodiment 2

[0115] Example 2. Production method of CAR-γδT cells

[0116] 1. Preparation of CAR22 lentiviral particles

[0117] In the present invention, the CAR structure is used to genetically modify γδT cells. The CAR structure from the amino terminal to the carboxyl terminal is: ScFv(CD22)-Hinge(CD8)-TM(CD8)-CD137-CD3ζ, that is, from the amino terminal to the carboxyl terminal in sequence It is: single-chain variable region, CD8a hinge region and transmembrane region, CD137 signal domain and CD3ζ chain intracellular region derived from CD22 monoclonal antibody (clone number: M971). Its amino acid sequence is shown in sequence 3 of the sequence listing, and its nucleotide sequence is shown in sequence 4 of the sequence listing. Insert the DNA molecule shown in Sequence 4 into the Senl_pLenti-EF1 vector (the Senl_pLenti-EF1 vector is a vector obtained by adding restriction sites PacI and SpeI on both sides of the original plasmid cloning site, and the original plasmid name is LV-pRRLEF...

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Abstract

The invention discloses a method for producing a chimeric antigen receptor modified gamma delta T cell. The method ensures that shFPPS targeted to FPP synthetase transfers a K562 cell through a lentiviral vector, the expression quantity of FPPS in the K562 cell is lowered, and a K562-shFPPS cell line with lowed FPPS expression quantity is constructed. According to the method, a K562-shFPPS cell line is added into a gamma delta T cell culture system to be co-cultured with gamma delta T cell, and the fact that the K562-shFPPS cell line can promote the in vitro differentiation and expansion of the gamma delta T cell is found. According to the method, the lentiviral vector expressing CAR is further added into the gamma delta T cell culture system containing the cell line, so that co-culturingis performed, and the fact that the K562-shFPPS cell line can further effectively improve the transfection efficiency of CAR gene is found. The method effectively solves the technical bottleneck of mass production of CAR-gamma delta T cell, and has an excellent application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for producing chimeric antigen receptor-modified γδT cells. Background technique [0002] Adoptive cellular immunotherapy is to infuse in vitro cultured, activated, and genetically modified autologous or allogeneic immune cells to patients to exert anti-tumor activity. Chimeric Antigen Receptor (CAR) modified T cell therapy technology (CAR-T technology) is to modify immune effector cells through genetic engineering technology, so that the modified cells can specifically recognize and kill cells expressing specific antigens. Target cells, so as to achieve the purpose of specifically eliminating tumor cells. CAR-T cells that specifically target the B lymphocyte surface marker CD19 molecule have the most significant curative effect in the treatment of B lymphocyte malignancies, and can achieve complete remission in 90% of relapsed and refractory B-lineage acute lymphoblastic l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0784C12N15/867
CPCC12N5/0639C12N15/86C12N2740/15043A61K39/464412A61K39/4611C12N5/0636A61K39/464413A61K39/4631C07K14/7051C12N2510/00C12N2502/30C12N2502/99C12N2501/2302C12N2740/16043C12N15/1137C12N2310/14C12N2310/531C12Y205/0101C07K14/70517C07K14/70578C07K14/7056C12N9/1085C07K2319/00C07K2319/03C07K2319/30C07K2319/33C12N15/115C12N15/85C12N2740/15041
Inventor 李建强王庆龙王琳
Owner HEBEI SENLANG BIOTECH CO LTD
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