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Preparation method and application of universal chimeric antigen receptor regulatory T cell

A chimeric antigen receptor and cell technology, which is applied in the field of general chimeric antigen receptor regulatory T cell preparation, can solve problems such as death and organ damage of patients, and achieve the effect of inhibiting inflammatory reactions

Pending Publication Date: 2022-02-15
郑州赛龙泰克生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, once CRS occurs, it will cause irreversible damage to the patient's organs, which can lead to death in severe cases

Method used

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  • Preparation method and application of universal chimeric antigen receptor regulatory T cell
  • Preparation method and application of universal chimeric antigen receptor regulatory T cell
  • Preparation method and application of universal chimeric antigen receptor regulatory T cell

Examples

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Effect test

Embodiment example 1

[0032] see Figure 1 to Figure 4 , the present invention provides a technical solution: a general chimeric antigen receptor regulatory T cell preparation method and application, comprising the following steps:

[0033] Isolation of S1, CD4+ regulatory T cells:

[0034] S101, selecting healthy persons to isolate mononuclear cells from peripheral blood or umbilical cord blood by gradient centrifugation;

[0035] S102. Using the magnetic bead method to isolate CD4+ T cells with a negative selection method, and then use a positive selection method to isolate CD4+CD25+ regulatory T cells;

[0036] S2. Preparation of lentivirus: use 293T cells to package the CAR expression system constructed by lentivirus, namely:

[0037] S201. After mixing the DNA of the lentiviral packaging system in proportion with a commercial transfection reagent, add the pre-cultured 293T cells together;

[0038] S202. After 48 hours, the cell supernatant is collected, and the supernatant is rich in lentiv...

Embodiment example 2

[0044] as attached Figure 1-3 As shown, a general chimeric antigen receptor regulatory T cell is applied in stem cell transplantation technology or tumor therapy.

[0045] Taking CS1 as an example, CD4+ regulatory T cells in peripheral blood were transfected with a retrovirus carrying an anti-CS1 chimeric antigen receptor gene, so that CD4+ regulatory T cells expressed anti-cs1-CAR and generated anti-CS1 -CARTreg with greater than 90% transduction (see figure 1 ); afterward, anti-CS1-CARTreg was co-cultured with multiple myeloma cells (MM1s), and after 6 hours, anti-CS1-CARTreg was detected by flow cytometry for perforin and granzyme, and combined with attached figure 1 As shown, after co-culturing anti-CS1-CARTreg with multiple myeloma, CD4+Treg expressing CAR secreted a large amount of perforin and granzyme, while wild-type CD4+Treg (CD4+Treg not expressing CAR) did not Secretion of perforin and granzyme; this result indicates that the anti-CS1 chimeric antigen receptor o...

specific Embodiment example 1

[0050] Step 1. Purchase experimental materials:

[0051] Main instruments: biological safety cabinet (Haier, HR40-IIA2), CO 2 Incubator (Thermo, 3111), flow cytometer (BD, fortesaX20), cell counter (Nexcelom, CellometerAuto2000)

[0052] Reagents: FBS (Lonsera, S711-001S), X-vivo15 (Lonza, 04-418Q), Dynabeads CD3 / CD28 (Lifetechnology, 40203D), Ficoll (Dayou, DKW-LSH-0250), Luciferase detection (Promega, E6120) , anti-human CD3 (FITC, Biolegend, 300306), anti-human CD4 (BV785, BD, 740962), CAR-specific monoclonal antibody (self-standard), anti-human GranzymeB (PE / Dazzle594, Biolegend, 372216), anti-human Perforin ( BV421, Biolegend, 308122).

[0053] Step 2, the experimental method, is divided into the following five steps:

[0054] The first step, carrier construction

[0055] The NCBI website database searched the gene sequence information of human IgG1 hinge region, human CD28 transmembrane region, CD3z intracellular region and anti-CS-1 single chain antibody;

[0056] ...

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Abstract

The invention discloses a preparation method and application of a universal chimeric antigen receptor regulatory T cell, and relates to the technical field of chimeric antigen receptor regulatory T cells, in particular to the preparation method and application of the universal chimeric antigen receptor regulatory T cell, and the preparation method comprises the following steps: S1, separating CD4 + regulatory T cells; s2, preparing lentivirus: packaging a CAR expression system constructed by the lentivirus by using 293T cells; and S3, transfecting the CD4 < + > regulatory T cells to synthesize CAR-Treg. The CD4 + regulatory T cells are used for replacing CD3 + T cells for gene recombination, so that the regulatory T cells express the chimeric antigen receptor to generate the chimeric antigen receptor regulatory T cells, and the purposes of recognizing and killing tumor cells are achieved.

Description

technical field [0001] The invention relates to the technical field of chimeric antigen receptor regulatory T cells, in particular to a general preparation method and application of chimeric antigen receptor regulatory T cells. Background technique [0002] At present, the CAR-T cell therapy that has been approved for clinical application all adopts the method of reinfusion of autologous CD3+ T cells after genetic modification in vitro; this method has the following two defects: [0003] 1. Patients who are usually treated with CAR-T cells are patients with advanced cancer who are ineffective or relapsed after chemotherapy. After a large number of chemotherapy drugs are used in such patients, most of their own CD3+ T cells will have a small number, and Function is affected by the drug; [0004] 2. Therefore, genetic modification with the patient's own CD3+ T cells will affect the anti-tumor effect of CAR-T cells, which is why patients treated with CAR-T cells still have a r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/62C12N5/10C12N5/0783A61K39/00A61P35/00A61P37/02A61P37/06
CPCC12N15/86C07K16/2803C07K14/7051C12N5/0637C12N5/0636A61K39/001111A61P35/00A61P37/02A61P37/06C12N2740/15043C12N2800/107C07K2317/622C07K2319/03C07K2319/33C07K2319/74C12N2510/00C12N2509/00A61K2039/5156
Inventor 李献生
Owner 郑州赛龙泰克生物科技有限公司
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