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A chimeric antigen receptor modified T cell that can autocrine tlr4 scFv and targets cMet and its application

A technology of chimeric antigen receptors and chimeric receptors, which is applied in the field of T lymphocytes and its preparation, can solve problems such as the lack of research reports on T cell immunotherapy, so as to overcome the problem of tumor immune evasion, reduce the probability of escape, Effects that promote killing

Active Publication Date: 2021-02-09
苏州福济达细胞工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no research report on c-Met as the target, chimeric antigen receptor and related modified T cell immunotherapy integrated with autocrine and neutralizing anti-TLR4 antibody at the same time

Method used

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  • A chimeric antigen receptor modified T cell that can autocrine tlr4 scFv and targets cMet and its application
  • A chimeric antigen receptor modified T cell that can autocrine tlr4 scFv and targets cMet and its application
  • A chimeric antigen receptor modified T cell that can autocrine tlr4 scFv and targets cMet and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Synthesis of CAR-expressed genes

[0062] The bispecific chimeric antigen receptor provided by the present invention is composed of CD8α signal peptide, c-Met scFv, CD8 transmembrane region, CD137 intracellular signal region, CD3ζ intracellular signal region, T2A self-cleavage sequence, antibody secretion signal peptide VH3 and TLR4 scFv are formed in series, the structure is as follows figure 1 The gene sequence of anti-c-Met and TLR4 single-chain antibody (anti-c-Met scFv and TLR4 scFv) is derived from the monoclonal antibody sequence constructed in this study, and it is codon-optimized to ensure that the coding amino acid sequence remains unchanged. In this case, it is more suitable for the expression of T cells, and the sequence information of each gene is shown in SEQUENCE LISTING (SEQ ID NO. 1~18 of the sequence table).

Embodiment 2

[0063] Example 2: Construction of CAR plasmid

[0064] Use overlap PCR to complete the splicing of each structure of the CAR in Example 1, respectively, to obtain a CAR c-Met , secreted CAR1 c-Met / TLR4 , and the control CAR CD19 (Unrelated CAR).

[0065] And add the Xba I restriction site at the 5' end, the Not I restriction site at the 3' end, and the EcoR I restriction site at the 5' end of T2A. The vector pCDH-CMV-MCS-EF1a-CopGFP was digested with Xba I / Not I double enzyme, and then the CAR was separated by In-fusion PCR method. c-Met Fragmented, secreted CAR c-Met t / TLR4 Fragment and control CAR CD19 The fragment was ligated with the pCDH-CMV-MCS-EF1a-CopGFP vector (see figure 2 ).

[0066] The product obtained from the ligation was transformed into E.coli (DH5α) competent, and after picking a single clone for cultivation, the plasmid was extracted and sequenced to obtain pCDH-CAR c-Met , pCDH-secreted CAR c-Met t / TLR4 and pCDH-CAR CD19 plasmid.

Embodiment 3

[0067] Example 3: Secretory-specific CAR binding ability and identification of exogenous CD3ζ expression

[0068] X-293T cells in logarithmic growth phase were collected 1D before transfection, and X-293T cells were seeded in 10cm cell culture dishes at a seeding volume of 10 7 , the cells were grown in DMEM medium containing 10% FBS, placed in a 37°C, 5% CO2 cell incubator and cultured in a cell incubator with a microscope to observe that the cell density reached 60-80% before transfection.

[0069] pCDH-CAR c-Met , pCDH-secreted CAR c-Met t / TLR4 and pCDH-CAR CD19 Plasmids were transfected into X-293T cells.

[0070] After transfection 1D and 2D, the fluorescent microscope was used to observe the GFP fluorescence expression after transfection, and X-293T cells were collected after transfection for 2D, and the total cell protein was extracted. The expression of exogenous CD3ζ was detected by Western Blot. like image 3 As shown, each CAR plasmid expressed exogenous CD3ζ....

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Abstract

The invention provides a chimeric antigen receptor (CAR) modified T cell capable of secreting TLR4 scFv and targeting cMet as well as application of the T cell. The T cell contains a coding sequence of a CAR recognizing cMet and a coding sequence of a TLR4 scFv antibody secreting a signal peptide. The CAR modified T cell can be activated while specifically targeting tumor cells with high expression of cMet, secret the TLR4 scFv antibody, weaken the immunosuppressive action of a tumor microenvironment, reduce expression of Treg cells and promote the killing effect of CD8<+>T cells on tumor.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a specific chimeric antigen receptor-modified T lymphocyte with autocrine antibody function and a preparation method and application thereof. Background technique [0002] With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) (represented by chimeric antigen receptor T-cell immunotherapy, CAR-T) (represented by tumor immune cell therapy has attracted extensive attention in the academic circles in recent years. It is expected to become a major breakthrough in immunotherapy.CAR-T cells combine the high affinity of antibodies and the killing effect of T cells by constructing specific chimeric antigen receptor carriers, and the genetically modified antibodies are combined with the corresponding tumor antigens. After binding, T cells can be activated in a non-restrictive manner by the major histocompatibility comple...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCA61K39/0011A61P35/00A61K2039/812A61K2039/82A61K2039/836A61K2039/844A61K2039/852A61K2039/86A61K2039/892C07K14/7051C07K16/2863C07K16/2896C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 唐奇朱进郭娇娇彭燕冯振卿毛圆常新霞
Owner 苏州福济达细胞工程有限公司
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