Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

c-Met-targeted single-chain antibody, chimeric antigen receptor, recombinant vector, CAR-T cell and application

一种嵌合抗原受体、单链抗体的技术，应用在肿瘤治疗领域，能够解决影响CAR-T应用效果等问题，达到好抑癌效果、能力增强、延长生存期的效果

Active Publication Date: 2021-03-26
BEIJING DCTY BIOTECH CO LTD +1
View PDF3 Cites 1 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • c-Met-targeted single-chain antibody, chimeric antigen receptor, recombinant vector, CAR-T cell and application
  • c-Met-targeted single-chain antibody, chimeric antigen receptor, recombinant vector, CAR-T cell and application
  • c-Met-targeted single-chain antibody, chimeric antigen receptor, recombinant vector, CAR-T cell and application

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0074] 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 preparation method of the 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 conventional T cell separation methods 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 carbo...

Embodiment 1

[0078] Phage Display Screening for Variable Region Sequences

[0079] 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

[0081] CAR plasmid construction

[0082] 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,

[0083] 2A45-F: taaggaattcgatgttgtgatgacc (SEQ ID No. 32)

[0084] 2A45-R: ggtggatcctgaggagacggtgac (SEQ ID No. 33)

[0085] 2D81-F: taaggaattccagattgttctctctc (SEQ ID No. 34)

[0086] 2D81-R: ggtggatcctgaggagactgtgag (SEQ ID No. 35)

[0087] 3D88-F: aaggaattcgacgttgtgatgacc (SEQ ID No. 36)

[0088] 3D88-R: gtggatcctgaggagactgtgag (SEQ ID No. 37)

[0089] 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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a c-Met-targeted single-chain antibody, a chimeric antigen receptor, a recombinant vector, a CAR-T cell and application, and belongs to the technical field of tumor treatment. The amino acid sequence of the single-chain antibody is shown as SEQ ID No.2; and 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 c-Met-targeted chimeric antigen receptor can be highly and specifically combined with a c-Met antigen, T cells modified by the c-Met-targeted chimeric antigen receptor can efficiently kill c-Met high-expression tumor cells in a targeting manner, and relatively ideal curative effects are achieved in vivo and in vitro.

Description

[0001] The application number of the present invention is CN202010697872.3, and the title of the invention is "a c-Met-targeting single-chain antibody, chimeric antigen receptor, recombinant vector, CAR-T cell and application", and the application date is July 2020 Divisional application for invention patent on the 20th. technical field [0002] 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 [0003] 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 extracel...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com