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Chimeric T cell receptor STAR and application thereof

A cell receptor and cell technology, applied in the field of treatment and diagnosis of diseases, antibody-T cell receptor chimeric receptor

Active Publication Date: 2020-02-21
CHINA IMMUNOTECH BEIJING BIOTECH CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that although a pairing strategy for modifying TCR-T to optimize exogenous TCR molecules has been proposed, there are still many areas for improvement.

Method used

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  • Chimeric T cell receptor STAR and application thereof
  • Chimeric T cell receptor STAR and application thereof
  • Chimeric T cell receptor STAR and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1, construction of STAR targeting EGFR

[0101] The specific construction method is as follows:

[0102] 1. Determination of the sequence of the TCR constant region

[0103] The constant regions (C regions) of the α chain and β chain of TCR in STAR are obtained from the cDNA of human peripheral blood T cells by PCR molecular cloning; on the basis of the original TCR sequence, the α chain and β chain are separately The 48th and 57th amino acid positions of the constant region were mutated to cysteine ​​to help form an additional disulfide bond between the α chain and the β chain, increasing their mutual pairing efficiency, and named it E1-TCR.

[0104] 2. Determination of the antibody sequence targeting EGFR

[0105] The antibody heavy chain variable region (VH) and the antibody light chain variable region (VL) select cetuximab (Cetuximab, referred to as Cetux), only as an example to explain the content of the present invention, and other known antibodies can b...

Embodiment 2

[0119] Example 2. Functional verification of STAR targeting EGFR

[0120] 1. Lentiviral Packaging

[0121] The pHAGE vector carrying the target gene and the packaging plasmids pMD2.G and psPAX2 were transfected into 293T cells in proportion (using PEI transfection). Collect the supernatant of the cell culture medium at 48 hours and 72 hours, mix the supernatant with PEG8000, let it stand overnight and then centrifuge to obtain the virus pellet. Resuspend with a small volume of medium to achieve the effect of virus concentration.

[0122] 2. Lentiviral Infection of Human T Cell Lines

[0123] Infect Jurkat clone 5 cells (Jurkat subclone with endogenous TCR deletion) with lentivirus carrying the target gene. The concentrated lentivirus and the transfer agent Polybrene were added to the T cell culture medium, and centrifuged at 1500rpm at 32°C for 2 hours. After 3 days of infection, the fluorescent reporter gene can be observed and the expression of the target protein can be ...

Embodiment 3

[0133] Example 3, construction of STAR targeting CD19

[0134] The specific construction method is as follows:

[0135] 1. Determination of the sequence of the TCR constant region

[0136] The cDNA derived from human peripheral blood T cells or mouse spleen T cells is obtained by PCR molecular cloning; on the basis of the original TCR sequence, the 48th and 57th amino acid sites of the constant regions of the α chain and β chain are mutated, respectively It is cysteine ​​to help form an additional disulfide bond between the α chain and the β chain to increase their mutual pairing efficiency, and they are named E1-TCR (human source) or E11-TCR (mouse source) respectively.

[0137] 2. Determination of the antibody sequence targeting CD19

[0138] Antibody heavy chain variable region (VH) and antibody light chain variable region (VL) select the scFv fragment of CD19-specific mouse monoclonal antibody (clone number FMC63), only as an example to explain the content of the present...

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PUM

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Abstract

The invention discloses a chimeric T cell receptor STAR (Synthetic T Cell Receptor and Antigen Receptor), related preparations and drugs, and application of the chimeric T cell receptor STAR in preparation of cell drugs, and also relates to the preparation or an drug composition used for treatment of corresponding diseases such as tumors or infectious diseases.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an antibody-T cell receptor chimeric receptor, a construction method and its application, including treatment and diagnosis of diseases. Background technique [0002] Chimeric antigen receptor T-cell therapy (CAR-T) and T-cell receptor therapy (TCR-T) are two cutting-edge gene therapies that harness a patient's own T lymphocytes to treat cancer. They can express specific receptors and target and recognize specific cells such as tumor cells. They have received extensive attention and research, and have been transformed from basic immune research to clinical applications. Based on the latest advances in synthetic biology, immunology and genetic modification technologies, it is possible to synthesize engineered T cells with enhanced specific functions. [0003] CAR-T uses antibody fragments that can bind to specific antigens to recognize antigens on the surface of tumor cells. In recent...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K35/17A61P35/00A61P35/02A61P31/12A61P31/20A61P31/14A61P31/18
CPCC07K14/7051C07K16/2863C07K16/2803C07K16/2887C12N5/0636C12N5/0646C12N5/0638A61P35/00A61P35/02A61P31/12A61P31/20A61P31/14A61P31/18C07K2319/00C07K2319/03C07K2319/74C07K2319/33C12N2510/00A61K39/464412A61K39/464404A61K39/4631A61K39/4632A61K39/464424A61K39/4611C07K19/00C12N5/10C12N15/62C07K14/705A61K35/12A61K38/17
Inventor 刘玥王嘉盛刘光娜赵学强林欣
Owner CHINA IMMUNOTECH BEIJING BIOTECH CO LTD
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