General type CART/TCRT cells with antibody drug resistance as well as construction method of general type CART/TCRT cells

An antibody drug and construction method technology, applied in the fields of genetic engineering and synthetic biology, can solve the problems of difficult to achieve treatment, unable to generate universal CART cells, etc., and achieve low-cost effects

Active Publication Date: 2018-03-23
NANJING BIOHENG BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0018] Knocking out the TCRα and β chains alone cannot generate universal CART cells, because this kind of CART cells still have HLA molecules expressed...

Method used

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  • General type CART/TCRT cells with antibody drug resistance as well as construction method of general type CART/TCRT cells
  • General type CART/TCRT cells with antibody drug resistance as well as construction method of general type CART/TCRT cells
  • General type CART/TCRT cells with antibody drug resistance as well as construction method of general type CART/TCRT cells

Examples

Experimental program
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Embodiment 1

[0095] Primary T cell expansion. Primary human T cells were cultured in RPMI 1640 containing 10% FBS, 100U / ml penicillin, 100μg / ml streptomycin sulfate, 10mM Hepes, and used CD3 / CD28 antibody-coupled magnetic beads at a ratio of 1:3 Perform an activation stimulus. Count the cells, add medium every 2 days, collect T cells at the right time according to the cell size and growth parameters, and perform functional testing or cryopreservation.

[0096] Design and Implementation of CRISPR

[0097] The sgRNA and crRNA targeted sequences within exon 1 of the TCRα constant region, the consensus sequence of exon 1 and 2 of the TCRβ constant region, and the exon 1 sequence of CD52, and were cloned into vectors containing the T7 promoter. These plasmids were linearized with restriction enzymes. sgRNA / crRNA were transcribed using the MEGAscript T7Transcription kit (Life Technologies, Carlsbad, CA), and the RNA was stored at -80°C.

[0098] The sgRNA and crRNA sequences used in the scre...

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Abstract

The invention belongs to the fields of genetic engineering and synthetic biology, and in particular relates to general type CART/TCRT cells with antibody drug resistance as well as a construction method of the general type CART/TCRT cells. The general type CART/TCRT cells are allogeneic T cells having chimeric antigen receptors and T cell receptors, and alpha and beta chains and CD52 molecules ofthe T cell receptors are knocked out; a technology for knocking out the alpha and beta chains and the CD52 molecules of the T cell receptors is a CRISPR technology; the general type CART/TCRT cells not only retain the targeting of tumor specific antigen, but also eliminate the problems of GvHD and rejection, and weaken the sensitivity to an antibody drug Alemtuzumab; the general type CART/TCRT cells can be used as a general type simple, low-cost and high-activity CART/TCRT cell preparation.

Description

technical field [0001] The invention belongs to the field of genetic engineering and synthetic biology, and specifically relates to a universal CART / TCRT cell with antibody drug resistance and a construction method thereof. Background technique [0002] Chimeric antigen receptor T cells (CART) are currently one of the most promising tumor immunotherapies. The basic principle is to extract the patient's own T cells and express specific chimeric antigens through genetic and cell engineering methods. Receptors enable it to recognize and bind to tumor cell surface antigens, thereby targeting and killing tumor cells. CAR-T has achieved remarkable curative effect in the treatment of leukemia and lymphoma. On August 30, 2017, the US Food and Health Safety Committee FDA announced the approval of Novartis CAR-T cell therapy for the treatment of relapsed or refractory children, Juvenile B-cell acute lymphoblastic leukemia (rALL), available under the trade name Kymriah (tisagenlecleuc...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/113A61K35/17A61P35/00A61P35/02
CPCA61K35/17C07K14/7051C07K14/70592C07K2319/33C12N5/0636C12N15/113C12N2310/10
Inventor 贺小宏王延宾袁鹏
Owner NANJING BIOHENG BIOTECH CO LTD
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