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

Dual-signal chimeric antigen receptor as well as preparation method and application thereof

A chimeric antigen receptor and dual-signal technology, applied in the field of biomedicine, can solve the problems of inaccurate positioning and poor targeting of antigen receptors that kill malignant tumor cells, and achieve the effect of improving accuracy and targeting

Pending Publication Date: 2020-12-11
SHENZHEN LUOHU PEOPLELS HOSPITAL
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a dual-signal chimeric antigen receptor and its preparation method and application, aiming to solve the problems of poor targeting and inaccurate positioning of antigen receptors killing malignant tumor cells in the prior art

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
  • Dual-signal chimeric antigen receptor as well as preparation method and application thereof
  • Dual-signal chimeric antigen receptor as well as preparation method and application thereof
  • Dual-signal chimeric antigen receptor as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0098] Specifically, the preparation method of the natural killer cells modified by the dual-signal chimeric antigen receptor comprises the following steps:

[0099] G01. Synthesizing the above-mentioned gene fragment for expressing the dual-signal chimeric antigen receptor, cloning it into a lentiviral expression vector, and preparing a lentiviral expression vector containing the gene fragment for expressing the dual-signal chimeric antigen receptor;

[0100] G02. Packaging the lentiviral expression vector containing the gene fragment for expressing the dual-signal chimeric antigen receptor, and obtaining the lentivirus containing the gene fragment for expressing the dual-signal chimeric antigen receptor;

[0101] G03. Using the lentivirus containing the gene fragment for expressing the dual-signal chimeric antigen receptor to infect the natural killer cells derived from peripheral blood to obtain the natural killer cell modified by the dual-signal chimeric antigen receptor. ...

Embodiment 1

[0141] NY-ESO-1 single chain antibody (NY-ESO-1 scFv) fragment and plasmid acquisition

[0142] Step 1: Obtain the total cDNA of mice immunized with NY-ESO-1 antigen. The specific steps are as follows:

[0143] (1) BALB / C mice were immunized with NY-ESO-1 antigen, and when the titer met the requirements (above 1:100000), the mice were sacrificed, the spleen was taken, and the total RNA of the spleen was extracted by the Trizol method.

[0144] (2) Synthesize the total cDNA using iScript cDNA Synthesis Kit, the reaction system is as follows:

[0145]

[0146] Reaction conditions: 25°C for 5min, 42°C for 30min, 85°C for 5min, 4°C hold,

[0147] The total cDNA of mouse spleen was prepared.

[0148] Step 2: Obtain the VH gene of the heavy chain variable region and the VL gene of the light chain variable region of the NY-ESO-1 antibody. The specific steps are as follows:

[0149] (1) Design the upstream and downstream primers for the VH gene of the heavy chain variable region...

Embodiment 2

[0168] Construction of lentiviral expression vector and packaging preparation of lentivirus

[0169] The first step: the construction of lentiviral expression vector, the specific operation method is as follows:

[0170] Entrusted Nanjing GenScript Biotechnology Co., Ltd. to synthesize two sequences for the construction of lentiviral expression vectors:

[0171] Expression vector one (PLV-Easy-T-2.1-NY-ESO-1-CAR lentiviral expression vector) includes: NY-ESO-1t single-chain antibody (scFv), CD8 hinge region and transmembrane domain, 41BB intracellular Domain and DAP12-ITAM domain;

[0172] Expression vector two (PLV-Easy-T-2.1-γδTCR / NY-ESO-1-CAR lentiviral expression vector) includes: the first signal peptide, γδTCR antigen receptor, T2A, signal peptide SP, NY-ESO-1 single Chain antibody, CD8 hinge region and transmembrane domain, 41BB intracellular domain and DAP12-ITAM domain.

[0173] as attached figure 1 as shown, figure 1 The gene fragment structure of the expression...

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 gene fragment for expressing a dual-signal chimeric antigen receptor. The gene fragment for expressing the dual-signal chimeric antigen receptor comprises a gamma delta TCR gene fragment and an NY-ESO-1 single-chain gene fragment which are sequentially connected. Wherein the gamma delta TCR antigen receptor expressed by the gamma delta TCR gene fragment can specifically recognize phosphorylated antigen small molecules on the surface of overexpressing high-metabolism malignant tumor cells as a first recognition signal; after the gamma delta TCR antigen receptor is identified, the NY-ESO-1 single-chain antibody expressed by the NY-ESO-1 single-chain gene can specifically identify a tumor antigen corresponding to the surface of a malignant tumor cell, and is used as asecond identification signal to activate downstream signal molecules to rapidly and specifically kill the tumor cell. The dual-signal chimeric antigen receptor adopts dual recognition signals, so that the dual-signal chimeric antigen receptor has high targeting property, and the accuracy and high efficiency of positioning the antigen receptor on the surface of a tumor cell are improved.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a dual-signal chimeric antigen receptor and its preparation method and application. Background technique [0002] Metabolism is the basic feature of the body's life activities, including material metabolism and accompanying energy metabolism. The release, conversion and utilization of energy in the process of material metabolism of organisms is called energy metabolism. An important feature of malignant tumors is the high metabolic capacity of tumor cells, which can specifically overproduce small tumor phosphorylated antigen molecules. [0003] In recent years, adoptive cell therapy technology, chimeric antigen receptor (chimeric antigen receptor, CAR) modified T cell technology has made a significant breakthrough in the clinical application of tumor treatment, showing good targeting in clinical trials , lethality and persistence, showing great application potential and dev...

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
IPC IPC(8): C12N15/62C12N15/867C12N5/10C07K19/00
CPCC07K14/7051C07K16/3069C12N5/0646C12N15/86C12N2740/15043C12N2510/00C07K2317/622C07K2319/03C07K2319/33Y02A50/30
Inventor 刘兵周美龄刘韬
Owner SHENZHEN LUOHU PEOPLELS HOSPITAL
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