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Chimeric antigen receptor containing c3ar intracellular domain, lentiviral vector, expressing cell and drug

A chimeric antigen receptor, intracellular domain technology, applied in receptor/cell surface antigen/cell surface determinant, virus/phage, retroRNA virus, etc., can solve the unpredictable effect of chimeric antigen receptor How to improve the killing effect, improve the killing efficiency in vitro, and eliminate the effects of immunosuppression

Active Publication Date: 2020-06-30
GUANGDONG GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The third generation CAR-T has been used in clinical trials of mantle cell lymphoma (MCL) and follicular non-Hodgkin lymphoma (NHL), but it has not shown better clinical results than the second generation
Combining C3aR with other co-stimulatory molecules is used for the construction of chimeric antigen receptors, and it is impossible to predict the effect of the constructed chimeric antigen receptors

Method used

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  • Chimeric antigen receptor containing c3ar intracellular domain, lentiviral vector, expressing cell and drug
  • Chimeric antigen receptor containing c3ar intracellular domain, lentiviral vector, expressing cell and drug
  • Chimeric antigen receptor containing c3ar intracellular domain, lentiviral vector, expressing cell and drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of Plasmids Containing Anti-CD19 ScFv-4-1BB-CD3ζ-C3aR (CAR19C3aR)

[0032] The plasmid carrying the chimeric antigen receptor gene containing the C3aR intracellular domain of the present invention is prepared according to the following steps:

[0033] (1) Plasmid pUC57-CAR19 containing anti-CD19ScFv-4-1BB-CD3ζ(CAR19) was obtained by means of gene synthesis, molecular cloning and other means , namely CD19ScFv-4-1BB-CD3ζ.

[0034] (2) The obtained pUC57-CAR19 plasmid was digested by endonuclease Pmel and Sep1 to obtain the CAR19 gene, and then the CAR19 gene was connected to the lentiviral vector pWPXLd-GFP to construct pWPXLd-CAR19-GFP.

[0035] (3) The obtained pWPXLd-CAR19-GFP plasmid was digested with endonucleases NotI and SpeI to obtain the intracellular fragment 4-1BB-CD3ζ of the CAR19 gene.

[0036] (4) Using the cDNA of the tandem C3aR intracellular signaling domain of 4-1BB-CD3ζ as a template, four primers were constructed, and 4-1BB-CD3ζ...

Embodiment 2

[0039] Example 2 Lentiviral Packaging of CAR Plasmids

[0040] Three lentiviruses expressing GFP (blank control), CAR19-GFP (control) and CAR19C3aR-GFP were obtained by lentivirus packaging using the CAR plasmid of the present invention prepared in Example 1 and related control plasmids. In Examples 2 and 3, the CAR-containing plasmid is uniformly described as pWPXLd-CAR-GFP plasmid, and the CAR-overexpressing lentivirus is uniformly described as CAR lentivirus.

[0041] Specific steps are as follows:

[0042] (1) Cultivate 293T cells in a 10cm petri dish, the medium is: DMEM high glucose medium + 10% FBS (fetal bovine serum) + 1% double antibody (100 × penicillin-streptomycin mixed solution);

[0043] (2) When the density of 293T cells in the 150mm culture dish reaches 80-90%, replace the medium: DMEM high glucose medium + 1% FBS + 1% double antibody;

[0044] (3) After replacing the medium and culturing for 2-6 hours, the two plasmids pWPXLd-CAR-GFP (that is, containing CA...

Embodiment 3

[0052] Example 3 Infection of human T cells using packaged CAR virus

[0053] (1) Separation and purification of T cells: Mononuclear cells in blood were separated by Ficoll density gradient method, and red blood cells were removed by lysing with red blood cell lysate, and then T cells were sorted by MACS Pan-T magnetic beads;

[0054] (2) Dilute the sorted T cells with medium (AIM-V medium + 5% FBS + penicillin 100U / ml + streptomycin 0.1 mg / ml) to a cell concentration of 2.5×10 6 pcs / ml for use;

[0055] (3) T cells are stimulated by magnetic beads coated with CD2, CD3 and CD28 antibodies (product source: Miltenyi, Germany), that is, the coated magnetic beads and T cells are mixed in a ratio of 1:2, and the final density of T cells should be 5 ×10 6 pcs / ml / cm2. After mixing, the cells were placed in a 37°C, 5% CO2 incubator for stimulation for 48 hours.

[0056] (4) Lentivirus-transfected T cells: remove the magnetic beads in the activated T cell-magnetic bead mixture by ma...

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PUM

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Abstract

Provided are a chimeric antigen receptor comprising C3aR intracellular domain, a lentiviral vector, an expressing cell, and a drug. The chimeric antigen receptor comprises an extracellular domain capable of binding an antigen, a transmembrane domain, and at least one intracellular domain. The intracellular domain is selected from C3aR intracellular domain and an intracellular domain of a signaling domain in series with C3aR intracellular domain. The chimeric antigen receptor can increase T17 cell subset, eliminate immunosuppression of regulatory T cells, and increase killing efficiency for tumor target cells in vitro.

Description

technical field [0001] The invention belongs to the technical field of cellular immunotherapy of tumors, in particular to a chimeric antigen receptor comprising a C3aR intracellular domain, and also to nucleic acids and applications comprising a chimeric antigen receptor encoding the C3aR intracellular domain. Background technique [0002] Chimeric Antigen Receptor (CAR, Chimeric Antigen Receptors) T cells are a milestone event expected to cure tumors. CAR-T technology is the application of genetic modification technology to combine a single chain fragment variable (scFv) that recognizes tumor antigens. The internal activation motif recombinant gene is transfected into T lymphocytes to achieve better recognition and killing of tumors. CAR-T molecules usually include extracellular hinge region, transmembrane region and intracellular signal region. The extracellular hinge region is formed by connecting the heavy chain and light chain variable regions of a single-chain antibod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00A61P35/02
CPCA61K35/17C07K14/7051C07K2319/33C12N15/86C12N2740/15043
Inventor 杜欣翁建宇赖沛龙陈晓梅
Owner GUANGDONG GENERAL HOSPITAL
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