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Universal chimeric antigen receptor T cell preparation technology

A chimeric antigen receptor, general-purpose technology, applied in the direction of receptor/cell surface antigen/cell surface determinant, animal cells, cancer antigen components, etc., can solve the problems of high time and money cost for separating and transforming cells

Inactive Publication Date: 2019-03-12
GRACELL BIOTECH SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this treatment method is severely restricted by the individual condition of the patient, and the cost of time and money caused by the isolation and transformation of cells is also very high

Method used

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  • Universal chimeric antigen receptor T cell preparation technology
  • Universal chimeric antigen receptor T cell preparation technology
  • Universal chimeric antigen receptor T cell preparation technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0210] Embodiment 1 Structure design of CAR

[0211] Taking CAR19 as an example, construct CAR. Among them, the CAR19 structure uses the second-generation CD19 CARs, including the scFv from FMC63, the hinge and transmembrane region from CD28, and the intracellular segment is CD28 and CD3ζ, as shown below:

[0212]

[0213] The DNA sequence of CAR19 is as follows (SEQ ID NO.:1):

[0214]

[0215] The amino acid sequence of CAR19 is as follows (SEQ ID NO.:2):

[0216]

Embodiment 2

[0217] Example 2 Construction of CAR19-HLA-Cw1 (hereinafter referred to as 19.C1) structure

[0218] The α1 and α2 structural sequences in the HLA-Cw1 molecule are followed by a CD8hinge and transmembrane region, and then integrated into the back of the CAR19 molecule, connected by T2A, and jointly constructed into the lentiviral vector backbone, referred to as 19.C1.1. As follows:

[0219]

[0220] Alternatively, the scFv sequence of the KIR antibody is followed by a CD8hinge and transmembrane region, and then integrated into the back of the CAR19 molecule, connected by T2A, and jointly constructed into the lentiviral vector backbone, referred to as 19.C1.2. As follows:

[0221]

[0222] 1-7F9VL amino acid sequence (SEQ ID NO.:7):

[0223] EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRT

[0224] 1-7F9VH amino acid sequence (SEQ ID NO.:8):

[0225] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAA...

Embodiment 3

[0226] Example 3 lentiviral vector construction

[0227] The CD19 CAR was cloned into the backbone of the FUW lentiviral vector and placed under the promoter of EF1α to form Fuw-EF1α-CD19CAR; the three plasmids of Fuw-EF1α-CD19CAR, pMD2.G and psPAX2 (addgene) were transferred into 293T using Lipofectamine2000 (ATCC, CRL-3216) to prepare the lentiviral expression vector, collect the virus supernatant on the second day and the third day, and obtain the virus after supercentrifugation and concentration (Merck Millipore).

[0228]Similarly, the 19.C1 CAR was cloned into the backbone of the FUW lentiviral vector and placed under the EF1α promoter to form Fuw-EF1α-19.C1 CAR; the Fuw-EF1α-19.C1 CAR, pMD2.G and psPAX2 (addgene ) The three plasmids were transferred into 293T (ATCC, CRL-3216) using Lipofectamine2000 to prepare lentiviral expression vectors, and the virus supernatant was collected on the second and third days, and the virus was obtained after supercentrifugation and conc...

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Abstract

The invention relates to a universal chimeric antigen receptor T cell, a preparation method thereof and an application of the cell, and particularly provides a CAR (chimeric antigen receptor)-T cell.Binding of the CAR and cells HLA-A / HLA-B and TCR is inhibited, and TCR (T cell receptor) gene expression of the cell is silenced. The universal CAR-T cell can be used for treating allogenic tumors, GVHD (graft-versus-host disease) and HVG (host versus graft) reaction are avoided in allogenic infusion, and survival and anti-tumor effects of the allogenic CAR-T cell in a receptor are improved.

Description

technical field [0001] The invention relates to the field of immune cell therapy, and more specifically relates to a general chimeric antigen receptor T cell and its preparation method and application. Background technique [0002] Cellular immunotherapy is an emerging tumor treatment mode with significant curative effect, and it is a new type of autoimmune anti-cancer treatment. It is a method of using biotechnology and biological agents to culture and expand immune cells collected from patients in vitro and then infuse them back into the patient's body to stimulate and enhance the body's autoimmune function, so as to achieve the purpose of treating tumors. [0003] Chimeric immune antigen receptors (Chimeric antigen receptors, CARs) are composed of extracellular antigen recognition region, usually scFv (single-chain variable fragment), transmembrane region and intracellular co-stimulatory signal region. The extracellular segment of CARs can recognize a specific antigen, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/90C12N9/22A61P35/00
CPCC12N5/0636C12N9/22C12N15/907C12N2510/00A61K2239/48A61K39/46434A61K39/4621A61K39/464412A61K39/4611A61K39/4631A61K2239/38A61P35/00A61K48/005C07K14/7051C07K2317/622C07K16/2803C07K14/70539C07K2319/03C07K2317/56C12N2740/16043A61P37/06C12N15/1138C12N2310/20C07K2317/53
Inventor 曹卫刘丽萍蔡松柏
Owner GRACELL BIOTECH SHANGHAI CO LTD
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