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

T cell for expressing immunosuppressive checkpoint receptor molecule and application of T cell

A technology of immunosuppression and receptor molecules, which is applied in the field of biomedicine, can solve the problems of clinical application uncertainty, cytokine storm, accidental injury of normal cells, etc., and achieve the effect of enhancing anti-tumor ability, strengthening ability, and avoiding excessive activation

Active Publication Date: 2021-01-15
汤朝阳
View PDF7 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, antibodies need to infiltrate into tumor tissue to be effective, and there are uncertainties in the clinical application of PD-1 antibodies and CTLA-4 antibodies
[0005] The prior art has also reported CAR-T co-expressing the diabody PD-1 and CD19 / Mesothelin / PSCA, although it can target tumor cells expressing PD-1 or CD19 / Mesothelin / PSCA or both at the same time, it may cause Cytokine storm, accidentally injuring normal cells

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
  • T cell for expressing immunosuppressive checkpoint receptor molecule and application of T cell
  • T cell for expressing immunosuppressive checkpoint receptor molecule and application of T cell
  • T cell for expressing immunosuppressive checkpoint receptor molecule and application of T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The preparation of embodiment 1 lentiviral vector

[0056] In this embodiment, at first the following nucleic acid molecules are genetically synthesized:

[0057] ①A receptor molecule formed in series by the nucleic acid sequences of CTLA-4, CD28 intracellular segment, CD3ζ and TLR2 (schematic diagram as shown in figure 1 shown);

[0058] ②A receptor molecule formed by tandem nucleic acid sequences of CTLA-4, CD28 intracellular segment, TLR2 and CD3ζ;

[0059] Wherein, the CTLA-4 immunosuppressive checkpoint coding gene is shown in SEQ ID NO: 3, the CD28 intracellular segment coding gene is shown in SEQ ID NO: 4, the CD3ζ coding gene is shown in SEQ ID NO: 5, and the TLR2 coding gene As shown in SEQ ID NO:6.

[0060] Adding a restriction endonuclease Pme1 restriction site and its protective bases and a restriction endonuclease Spe1 restriction site and its protective bases to the C-terminus and N-terminal of the nucleic acid molecule;

[0061] The coding gene was do...

Embodiment 2

[0062] Example 2 lentiviral packaging

[0063] In order to introduce receptor molecules into T cells, 293T cells were used to prepare recombinant lentiviruses, and when 293T cells spread 80-90% of the bottom of a 100mm culture dish, the lentiviruses were packaged:

[0064] 2 hours before virus packaging, replace the medium with DMEM containing 1% fetal bovine serum, and add 6mL / 100mm culture dish;

[0065] Prepare the plasmid mixture shown in Table 1, the pWPXLd-expression plasmid includes the lentiviral vector containing the coding gene of CTLA-4-CD28-CD3ζ-TLR2, or the lentiviral vector containing the coding gene of CTLA-4-CD28-TLR2-CD3ζ Viral vector, the pWPXLd-eGFP plasmid is an empty vector that does not contain the gene encoding the receptor molecule;

[0066] Table 1

[0067]

[0068] Add 36 μg PEI to another 500 μL opti-MEM medium, mix well, and let stand at room temperature for 5 minutes;

[0069] Mix the plasmid mixture shown in Table 1 with PEI, mix well by pip...

Embodiment 3

[0074] Example 3 T cell activation and lentiviral transfection

[0075] 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;

[0076] 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;

[0077] CD2 / CD3 / CD28 T cell activation expansion kit (Miltenyi) was used to activate T cells and placed at 37°C, 5% CO 2 The incubator was stimulated for 48 hours;

[0078]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 different lentiviruses (MOI=10), and add 8μg / mL polybrene and 300IU / mL IL -2, placed at 37°C, 5% CO 2 incubator cultivation;

[0079] After 24 hours, centrifuge at 30...

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 T cell for expressing an immunosuppressive checkpoint receptor molecule and an application of the T cell. The immunosuppressive checkpoint receptor molecule is expressed on aT cell membrane in a chimeric manner and comprises an immunosuppressive checkpoint and an immune activation signal structural domain, wherein the immunosuppressive checkpoint is a signal peptide, an extracellular domain and a transmembrane domain of the immunosuppressive checkpoint receptor molecule; the immune activation signal structural domain comprises a combination of a CD28 intracellular segment, CD3 zeta and TLR2; and the CD28 is located at the N end of the CD3 zeta, and the TLR2 is located at the C end of the CD3 zeta. Compared with wild type T cells, the T cell disclosed by the invention has a remarkably enhanced anti-immunosuppression effect, has a stronger killing effect on tumor cells expressing immunosuppression checkpoint ligands, does not cause cytokine storm, is higher in safety, and has a wider application prospect in the field of tumor treatment.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a T cell expressing an immunosuppressive checkpoint receptor molecule and an application thereof. Background technique [0002] Chimeric antigen receptor T cell (CAR-T) immunotherapy is a cellular immunotherapy method based on chimeric antigen receptors. Transducing the gene sequence of CAR (CAR) into T cells to generate tumor-specific T cells that can bind to target antigens has become a powerful means of treating malignant hematological tumors in recent years. [0003] However, CAR-T immunotherapy targeting solid tumors is facing a huge challenge. CAR-T cells have problems such as being unable to break through the barriers of solid tumor tissues, unable to resist immunosuppression in tumor tissues, and having weak competitive viability in hypoxic microenvironments. . [0004] Researchers conduct tumor immunotherapy by constructing PD-1 antibodies (Pembrolizumab, Nivolumab) ...

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): C12N5/10C07K19/00C12N15/62C12N15/867A61K39/00A61P35/00
CPCC12N5/0636C07K14/7051C07K14/70521C07K14/70596C12N15/86A61K39/001111A61K39/001129A61P35/00C12N2510/00C07K2319/02C07K2319/03C07K2319/00C12N2740/15043C12N2800/107A61K2039/5156
Inventor 汤朝阳秦乐吴迪魏志辉王翠花王艳艳其他发明人请求不公开姓名
Owner 汤朝阳
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