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

CD19 and CD22 dual-target chimeric antigen receptor and its application

A technology of chimeric antigen receptor and single-chain antibody, which is applied in the field of biomedicine, can solve the problems of tumor recurrence and poor effect in patients, and achieve the effect of high targeting activity, high killing effect and avoiding immune escape phenomenon

Active Publication Date: 2022-05-20
GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, medical diagnosis shows that tumor cells in some hematological tumors do not express CD19 molecules, but express CD22 molecules. Only CAR-T cells targeting CD19 molecules are not effective, and some patients have tumor recurrence after a period of time Phenomenon

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
  • CD19 and CD22 dual-target chimeric antigen receptor and its application
  • CD19 and CD22 dual-target chimeric antigen receptor and its application
  • CD19 and CD22 dual-target chimeric antigen receptor and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1 Construction of CAR Molecular Carrier

[0106] In this example, firstly, the coding gene of anti-CD19 and CD22 dual-target chimeric antigen receptor was synthesized, and the restriction endonuclease Pme1 restriction site and its protective base were added to the C-terminal and N-terminal of the coding gene respectively. Restriction endonuclease Spe1 restriction site and its protective base;

[0107] The coding gene was double-digested with restriction endonucleases Pme1 and Spe1, and the digested product containing cohesive ends was recovered by agar gel electrophoresis, and then ligated into the linearized pWPXLd-eGFP plasmid (containing sticky end), the ligation reaction was carried out with the participation of T4 DNA polymerase (Invitrogent), and a lentiviral vector containing the coding gene of CAR targeting CD19 and CD22 dual targets was obtained, and the map is as follows figure 1 shown.

[0108] In this example, the antigen-binding domains were const...

Embodiment 2

[0109] Example 2 lentiviral packaging

[0110] In order to introduce CAR molecules into T cells, 293T cells were used to prepare recombinant lentiviruses, and when 293T cells were spread to 80-90% of the bottom of a 100mm culture dish, the lentiviruses were packaged:

[0111] 2 hours before virus packaging, replace the medium with DMEM containing 1% fetal bovine serum, and add 6mL / 100mm culture dish;

[0112] Prepare the plasmid mixture shown in Table 1, the pWPXLd-expression plasmid includes the lentiviral vector containing the coding gene of CAR targeting CD19 and CD22 dual targets, and the lentivirus containing the coding gene of CAR targeting CD19 single target Vector, lentiviral vector containing the coding gene of the CAR targeting CD22 single target, and the pWPXLd-eGFP plasmid is an empty vector that does not contain the CAR coding gene;

[0113] Table 1

[0114]

[0115] Add 36 μg PEI to another 500 μL opti-MEM medium, mix well, and let stand at room temperature ...

Embodiment 3

[0121] Example 3 T cell activation and lentiviral transfection

[0122] Peripheral blood mononuclear cells (PBMC) were separated from whole blood using Ficoll density gradient centrifugation kit (GE Company), and after red blood cells were removed, T cells were sorted out using MACS Pan-T magnetic beads;

[0123] The sorted T cells were diluted with medium (AIM-V medium + 5% FBS + penicillin 100 U / mL + streptomycin 0.1 mg / mL) to a cell concentration of 2.5×10 6 pcs / mL for use;

[0124] CD2 / CD3 / CD28 T cell activation expansion kit (Miltenyi Company) was used to activate T cells, that is, the coated magnetic beads were mixed with T cells at a ratio of 1:2, and the final density of T cells was 5×10 6 piece / mL / cm 2 , after mixing, place at 37°C, 5% CO 2 The incubator was stimulated for 48 hours;

[0125] After T cells were activated for 48 hours, demagnetize the beads, centrifuge at 300g for 5 minutes, remove the supernatant, resuspend T cells with fresh medium, add recombinan...

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 present invention provides CD19 and CD22 dual-target chimeric antigen receptors and applications thereof. The chimeric antigen receptors include antigen binding domains, transmembrane domains and signal transduction domains; the antigen binding domains include The light chain variable region of an anti-CD19 single chain antibody, the heavy chain variable region of an anti-CD19 single chain antibody, the light chain variable region of an anti-CD22 single chain antibody, and the heavy chain variable region of an anti-CD22 single chain antibody. The anti-CD19 and CD22 dual-target chimeric antigen receptor of the present invention has targeting activity on CD19-positive and / or CD22-positive cells, and the T cells expressing anti-CD19 and CD22 dual-target chimeric antigen receptor have CD19 antigen expression Tumor cells with little or no expression and tumor cells with little or no expression of CD22 antigen all have a killing effect, which is beneficial to avoid immune escape and reduce the possibility of disease recurrence.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a CD19 and CD22 dual-target chimeric antigen receptor and an application thereof. Background technique [0002] Chimeric antigen receptor (chimeric antigen receptor, CAR) is composed of tumor-associated antigen binding region, extracellular hinge region, transmembrane region and intracellular signal transduction region. Usually, the antibody single-chain fragment variable (single chain fragment variable, scFv) contained in the CAR molecule has a specific binding effect on the tumor-associated antigen (tumor associated antigen, TAA). Cytoplasmic domain coupling. [0003] In recent years, with the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) has become one of the most promising tumor immunotherapies. At present, CAR-T cell therapy has been widely used in the treatment of B-cell malignancies. CAR-T cells t...

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 Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61P35/00A61P35/02
CPCC07K16/2803C07K14/7051C12N15/86C12N7/00A61K39/0011A61P35/00A61P35/02C07K2319/02C07K2319/03C07K2319/33C12N2740/15021C12N2740/15043
Inventor 秦乐汤朝阳邓殷建魏志辉其他发明人请求不公开姓名
Owner GUANGDONG ZHAOTAI INVIVO BIOMEDICINE 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