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CAR-T cell containing unstable structural domain, preparation method thereof and method for adjusting functions of CAR-T cell

A technology for stabilizing structures and cells, applied in the fields of immunology and cell biology, can solve problems such as difficulty in application, lack of adjustability, and inability to precisely regulate the expression level of cytokines in the time of cell therapy, and achieve steps that are easy to operate and process little effect

Inactive Publication Date: 2019-08-02
THE SECOND HOSPITAL OF SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, unstable domains are generally linked to immunomodulatory factors. These factors may enhance the efficacy of adoptive cell therapy, but precise control is required to optimize its therapeutic effect. Current technology cannot precisely regulate the time when cells exert therapeutic effects or the expression level of cytokines , which makes it difficult for many potential applications to be deployed safely and efficiently, and the lack of tunability also makes it difficult to safely express transiently expressed potent proteins

Method used

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  • CAR-T cell containing unstable structural domain, preparation method thereof and method for adjusting functions of CAR-T cell
  • CAR-T cell containing unstable structural domain, preparation method thereof and method for adjusting functions of CAR-T cell
  • CAR-T cell containing unstable structural domain, preparation method thereof and method for adjusting functions of CAR-T cell

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preparation example Construction

[0030] The present invention also includes a method for preparing CAR-T cells containing an unstable domain, comprising the following steps:

[0031] ①The gene encoding the unstable domain is connected to the end of the CAR gene to construct a fusion gene; specifically, the gene encoding the unstable domain is placed at the 3' end of the CAR gene and connected to form a fusion gene;

[0032] ② Insert the fusion gene clone obtained in step ① into the lentiviral plasmid;

[0033] ③Packing the virus and preparing the lentivirus carrying the fusion gene;

[0034] ④ Infect T cells with the lentivirus obtained in step ③ and amplify them.

[0035] The invention also discloses a method for regulating the function of CAR-T cells containing an unstable domain, adding a small molecule compound corresponding to the unstable domain; the amount of the small molecule compound added is 0-1000 μmol / L. Such as figure 1 as shown, figure 1 The triangle in is a small molecular compound, which ...

Embodiment 1

[0045] Example 1 Preparation of DHFR-CD19CAR-T cells

[0046] 1. Whole-gene synthesis of the fusion gene of DHFR and CD19CAR

[0047] The unstable domain DD selects the unstable domain of dihydrofolate reductase (Dihydrofolate reductase), referred to as DHFR; its small molecule compound ligand is trimethoprim (TMP)

[0048] The sequence of DHFR is SED ID NO: 1, the transmembrane region, co-stimulatory signal region, and CD3ζ sequence of CD19CAR are SED ID NO: 2, according to figure 2 The positional relationship of the ScFV sequence, the transmembrane region, the co-stimulatory signal region, and the CD3ζ sequence were connected to construct a CD19CAR, whose sequence is SEQ ID NO: 3;

[0049] The fusion gene of DHFR and CD19CAR is synthesized from the entire gene, so that the upstream restriction site of the fusion gene is XbaI, and the downstream restriction site is BamHI; the sequence of the fusion gene (DHFR-CD19CAR) is SEQ ID NO: 4; in this fusion gene, The DHFR encoding...

Embodiment 2

[0100] Using TMP to regulate the number of CAR molecules on the membrane surface of DHFR-CD19CAR-T cells prepared in Example 1

[0101] Take the DHFR-CD19CAR-T cells constructed in Example 1, place them in a 24-well plate, and add DHFR ligand-trimethoprim to the final concentrations of 0nM and 10nM, respectively, for 5*10^5 CAR-T cells per well , 100nM, 1000nM, 10000nM; 3 parallels in each group; cultivated for 24h;

[0102] Take biotin-labeled CD19 protein (Biotinylated Human CD19, Fc Tag, ultrasensitivity (primary amine labeling), ACROBiosystems, Cat. No. CD9-H8259), FITC-labeled streptavidin (FITC-SA, Biolegend, Cat. No.405201), perform flow cytometric detection according to the method in the corresponding instructions, and measure the average fluorescence intensity of the CAR-positive cell population. The more CAR molecules on the membrane surface, the stronger the fluorescence intensity.

[0103] Such as image 3 As shown, the average fluorescence intensity increases wi...

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Abstract

The invention relates to a CAR-T cell containing an unstable structural domain, a preparation method thereof and a method for adjusting functions of the CAR-T cell. An encoding gene in the unstable structural domain is contained in the CAR-T cell; a 3' end of CAR gene without a termination codon is directly connected with the encoding gene in the unstable structural domain, so as to form a fusiongene; a fusion protein encoded by the fusion gene is expressed in the CAR-T cell; a CAR molecule exists on a N end of the fusion protein; the unstable structural domain is on a C end and is located oncytomembrane. The preparation method for the CAR-T cell containing the unstable structural domain provided by the invention has few processes and easy steps and is convenient for industrialization. According to the method for adjusting the functions of the CAR-T cell containing the unstable structural domain disclosed by the invention, the quantity of CAR molecules can be adjusted by only addinga small molecule compound corresponding to the unstable structural domain. The existence of the unstable structural domain has no influence on the functions of the CAR molecules.

Description

technical field [0001] The invention relates to the technical fields of immunology and cell biology, in particular to a CAR-T cell containing an unstable domain, a preparation method thereof, and a method for regulating the function of the CAR-T cell. Background technique [0002] In the field of cellular immunotherapy, CAR-T therapy has become a research hotspot. Its main principle is that T cells modified by chimeric antigen receptors can specifically recognize tumor-associated antigens, enabling effector T cells to target and kill The activity and persistence are higher than those of conventionally applied immune cells, and can overcome the local immunosuppressive microenvironment of the tumor and break the state of host immune tolerance. At present, with the deepening of research, people are increasingly considering the safety issues in the application of CAR-T, that is, how to precisely control the antigenic activity caused by CAR-T cells (such as cytokine storm, etc.),...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62C12N15/867
CPCC07K14/47C07K2319/00C12N5/0636C12N9/003C12N15/86C12N2510/02C12N2740/15043C12Y105/01003
Inventor 张文孙娇刘江唐东起
Owner THE SECOND HOSPITAL OF SHANDONG UNIV
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