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Immune cell for heterogeneous tumor treatment and application thereof

A technology of immune cells and chimeric antigen receptors, applied in the field of biomedicine, can solve the problems of not expressing specific tumor antigens, tumor heterogeneity obstacles, etc., and achieve a significant effect of clearing and killing

Inactive Publication Date: 2020-11-03
GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many tumor cells do not express specific and specific tumor antigens, and tumor heterogeneity is a major obstacle for cell therapy in the treatment of solid tumors

Method used

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  • Immune cell for heterogeneous tumor treatment and application thereof
  • Immune cell for heterogeneous tumor treatment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of CAR Molecular Carrier

[0046] In this example, the NKG2D CAR molecule was constructed, and the structural diagram is as follows figure 1 Shown, the amino acid sequence is shown in SEQ ID NO: 2, and the nucleic acid sequence is shown in SEQ ID NO: 3;

[0047] Firstly, the coding gene of NKG2D chimeric antigen receptor (SEQ ID NO: 3) is genetically synthesized, and the restriction endonuclease Pme1 restriction site and its protective base and Restriction endonuclease Spe1 restriction site and its protective base;

[0048] 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), to obtain a lentiviral vector containing the gene encoding the CAR molecule...

Embodiment 2

[0050] Example 2 lentiviral packaging

[0051] The lentiviral vector constructed in Example 1 was transferred into Escherichia coli, and positive single clones were selected for overnight culture, and the lentiviral vector was extracted using a plasmid extraction kit for virus packaging;

[0052] Use 293T cells for virus packaging and prepare the packaging system. The pWPXLd-expression plasmid includes a lentiviral vector containing the gene encoding NKG2DCAR and a lentiviral vector containing the gene encoding antiCD19 CAR, and the pWPXLd-eGFP plasmid is an empty vector that does not contain the gene encoding CAR ;

[0053] The helper plasmid and the lentiviral vector with CAR molecule were simultaneously transfected into 293T cells, and the first and second viruses were harvested after 48h and 72h respectively, filtered and frozen at -80°C for use in immune cell transduction.

Embodiment 3

[0054] Example 3 T cell activation and lentiviral transfection

[0055] Mononuclear cells (PBMC) were isolated from adult peripheral blood, and total T cells were sorted out using a T cell sorting kit. After being stimulated in vitro by CD3 and CD28 antibodies for 24 hours, the overexpressed CAR molecule prepared in Example 2 was added. recombinant lentivirus;

[0056] After 12 hours of transduction, the T cells were centrifuged to change the medium; three days after the transduction, the content of GFP-positive cells was measured by flow cytometry, and the proportion of CAR-T was evaluated;

[0057] According to culture medium per milliliter 2 × 10 6 Cell density culture and expansion of CAR-T cells to obtain NKG2D-CAR-T, antiCD19-CAR-T and WT (transfection blank control eGFP lentivirus) cells, and freeze the cells ten days later until use.

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PUM

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Abstract

The invention provides an immune cell for heterogeneous tumor treatment and an application thereof. The immune cell expresses a chimeric antigen receptor, and the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain and a signal transduction domain, wherein the antigen binding domain comprises an NKG2D, and the signal transduction structural domain comprises 4-1BB,CD3 [zeta] and TLR2. The chimeric antigen receptor provided by the invention has a targeting effect on heterogeneous tumor cells, and the constructed immune cell expressing the chimeric antigen receptor realizes the clearing and killing effects on the heterogeneous tumor cells by mobilizing the function of endogenous NKG2D.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to immune cells for treating heterogeneous tumors and applications 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 targeting ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61P35/00C12R1/93
CPCA61K2039/5158A61P35/00A61K39/001102C07K14/7051C07K14/7056C07K14/70578C07K14/70596C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N5/0646C12N7/00C12N15/86C12N2510/00C12N2740/15021C12N2740/15043
Inventor 汤朝阳秦乐吴迪邓殷健吴海鹏王翠花冯世忠冯嘉昆其他发明人请求不公开姓名
Owner GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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