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Single-chain antibody targeting c-Met, chimeric antigen receptor, recombinant vector, CAR-T cell and application thereof

A chimeric antigen receptor and single-chain antibody technology, applied in the field of tumor treatment, can solve the problems affecting the application effect of CAR-T, and achieve the effect of good tumor suppression effect, prolonging survival period and enhancing ability.

Active Publication Date: 2020-10-30
BEIJING DCTY BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for solid tumors, due to the limitations of tumor microenvironment and other factors, the application effect of CAR-T is affected, and scientists from all over the world are trying to solve this problem

Method used

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  • Single-chain antibody targeting c-Met, chimeric antigen receptor, recombinant vector, CAR-T cell and application thereof
  • Single-chain antibody targeting c-Met, chimeric antigen receptor, recombinant vector, CAR-T cell and application thereof
  • Single-chain antibody targeting c-Met, chimeric antigen receptor, recombinant vector, CAR-T cell and application thereof

Examples

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preparation example Construction

[0072] The invention provides a chimeric antigen receptor T cell targeting c-Met, which is obtained by transfecting T cells with the recombinant vector targeting c-Met. In the present invention, the method for preparing chimeric antigen receptor T cells targeting c-Met preferably includes the following steps: 1) separating and culturing T cells from the peripheral blood of healthy people; The virus vector and the cultured T cells described in step 1) are mixed and transfected to obtain the chimeric antigen receptor T cells targeting c-Met. In the present invention, there is no special limitation on the separation method of the T cells, and the conventional T cell separation method in the field can be used; in the present invention, after the separation, the T cells are placed in X-VIVO+100U IL2 complete culture medium and add CD3 / CD28 magnetic beads to culture; the culture is preferably carried out in an incubator, the temperature of the culture is preferably 37°C, the carbon ...

Embodiment 1

[0076] Phage Display Screening for Variable Region Sequences

[0077] Total RNA was extracted from the spleen and bone marrow of mice immunized with c-Met extracellular antigen using the trizol method, and the mouse antibody variable region gene was amplified using the mouse antibody scFv gene amplification kit (Cat. No.: P001Z), and a segment composed of multiple The peptide linker Linker (hinge region sequence: SSGGGGSGGGGGGSSRSS) composed of glycine (Gly) and serine (Ser) connects the heavy chain and light chain variable regions of the antibody to form a single-chain antibody gene fragment scFv, and clone the scFv single-chain antibody gene fragment into In the phagemid vector pCANTAB5E, a scFv phage display library was constructed, so that the single-chain antibody scFv could be displayed and expressed in the phage display library. After plasmid extraction, the phage plasmids were electrotransformed to build a library with a storage capacity of ≥10 8 . Subsequently, the ...

Embodiment 2

[0079] CAR plasmid construction

[0080] The idea of ​​CAR sequence construction is as follows: figure 2 shown. Design the following amplification primers according to the sequence of the 3 c-Met-targeting scFv sequences obtained by sequencing,

[0081] 2A45-F: taaggaattcgatgttgtgatgacc (SEQ ID No. 32)

[0082] 2A45-R: ggtggatcctgaggagacggtgac (SEQ ID No. 33)

[0083] 2D81-F: taaggaattccagattgttctctctc (SEQ ID No. 34)

[0084] 2D81-R: ggtggatcctgaggagactgtgag (SEQ ID No. 35)

[0085] 3D88-F: aaggaattcgacgttgtgatgacc (SEQ ID No. 36)

[0086] 3D88-R: gtggatcctgaggagactgtgag (SEQ ID No. 37)

[0087] 2A45, 2D81, 3D88 scFv gene fragments carrying restriction sites were respectively obtained by PCR amplification. The above gene fragments were respectively combined with the PZX-CAR vector (the map of the PZX-CAR vector is as follows: figure 2 shown) were subjected to EcoRI and BamHI double enzyme digestion treatment respectively, after agarose gel electrophoresis, the gel w...

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Abstract

The invention provides a single-chain antibody targeting c-Met, a chimeric antigen receptor, a recombinant vector, a CAR-T cell and an application thereof, and belongs to the technical field of tumortherapy, and the amino acid sequence of the single-chain antibody is shown as one of SEQ ID No. 1 to SEQ ID No. 3; wherein the chimeric antigen receptor comprises a CD8a signal peptide, a Flag label,the single-chain antibody, a CD8a hinge region-transmembrane region, a 4-1BB functional region and a CD3 functional region which are connected in sequence; the chimeric antigen receptor targeting c-Met can be highly specifically combined with a c-Met antigen, T cells modified by the chimeric antigen receptor targeting c-Met can efficiently kill c-Met high-expression tumor cells in a targeting manner, and ideal curative effects are achieved in vivo and in vitro.

Description

technical field [0001] The invention belongs to the technical field of tumor treatment, and in particular relates to a single-chain antibody targeting c-Met, a chimeric antigen receptor, a recombinant vector, CAR-T cells and applications. Background technique [0002] The c-Met gene is located on chromosome 7 (7q21-q31), consists of 21 exons and 20 introns, and is about 120kDa in length. The c-Met primary transcript produces a 150 kDa polypeptide, which is then partially glycosylated to form a 170 kDa single-chain precursor protein. The precursor protein is further glycosylated to form a heterodimer of about 190 kDa, which is composed of a 50 kDa extracellular chain (α chain) and a 145 kDa transmembrane chain (β chain) (Maulik G, Shrikhande A, Kijima T, et al. et al.Role of the hepatocyte growth factor receptor,c-Met,in oncogenesis and potential for therapeutic inhibition.Cytokine Growth Factor Rev,2002,13(1):41-59.; SattlerM,Ma PC,Salgia R.Therapeutic targeting of the rec...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K16/2863C07K14/7051C12N15/86A61P35/00C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/43C12N2740/15043C12N5/0636A61K39/00111C07K2319/74C12N2800/107C12N2510/00A61K2039/5156
Inventor 焦顺昌陈静远梁皓
Owner BEIJING DCTY BIOTECH CO LTD
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