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Chimeric antigen receptor containing C5aR intracellular domain, lentiviral vector, expression cell and medicine

A technology of chimeric antigen receptor and intracellular domain, applied in the direction of receptor/cell surface antigen/cell surface determinant, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, virus/phage, etc. , can solve the problems that the effect of chimeric antigen receptor cannot be expected, and the clinical results have not been shown, so as to improve the killing effect, increase the killing efficiency in vitro, and eliminate the effect of immunosuppression

Active Publication Date: 2018-05-11
赖沛龙
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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
[0005] At present, there is no combination of C5aR and other co-stimulatory molecules for the construction of chimeric antigen receptors at home and abroad, and it is impossible to predict the effect of the constructed chimeric antigen receptors

Method used

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  • Chimeric antigen receptor containing C5aR intracellular domain, lentiviral vector, expression cell and medicine
  • Chimeric antigen receptor containing C5aR intracellular domain, lentiviral vector, expression cell and medicine
  • Chimeric antigen receptor containing C5aR intracellular domain, lentiviral vector, expression cell and medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of plasmid containing anti-CD19ScFv-4-1BB-CD3ζ-C5aR (CAR19C5aR)

[0034] Prepare the plasmid of the present invention carrying the chimeric antigen receptor gene containing the intracellular domain of C5aR as follows:

[0035] (1) The plasmid pUC57-CAR19 containing anti-CD19ScFv-4-1BB-CD3ζ (CAR19) was obtained by means of gene synthesis and molecular cloning, including anti-CD19 monoclonal antibody ScFv, CD28 transmembrane region and 4-1BB, CD3ζ intracellular region , namely CD19ScFv-4-1BB-CD3ζ.

[0036] (2) The resulting pUC57-CAR19 plasmid was digested with endonucleases Pmel and Sepl to obtain the CAR19 gene, and then the CAR19 gene was connected to the lentiviral vector pWPXLd-GFP to construct pWPXLd-CAR19-GFP.

[0037] (3) Digest the obtained pWPXLd-CAR19-GFP plasmid with endonucleases NotI and SpeI to obtain the intracellular fragment 4-1BB-CD3ζ of the CAR19 gene.

[0038] (4) Using the cDNA of 4-1BB-CD3ζ tandem C5aR intracellular signaling...

Embodiment 2

[0041] Lentiviral packaging of embodiment 2CAR plasmid

[0042] Three lentiviruses expressing GFP (blank control), CAR19-GFP (control), and CAR19C5aR-GFP respectively were obtained by using the CAR plasmid of the present invention prepared in Example 1 and related control plasmids through lentiviral packaging. In Examples 2 and 3, the CAR-containing plasmid is collectively described as pWPXLd-CAR-GFP plasmid, and the CAR-overexpressing lentivirus is collectively described as CAR lentivirus.

[0043] Specific steps are as follows:

[0044] (1) Cultivate 293T cells in a 10cm culture dish, the culture medium is: DMEM high glucose medium + 10% FBS (fetal bovine serum) + 1% double antibody (100 × penicillin-streptomycin mixed solution);

[0045] (2) When the 293T cell density in the 150mm culture dish reaches 80-90%, replace the medium: DMEM high glucose medium + 1% FBS + 1% double antibody;

[0046] (3) After replacing the medium and culturing for 2-6 hours, the two plasmids pWP...

Embodiment 3

[0055] Example 3 Using packaged CAR virus to infect human T cells

[0056] (1) Separation and purification of T cells: the mononuclear cells in the blood are separated by the Ficoll density gradient method, and the red blood cells are removed by lysing with the red blood cell lysate, and then the T cells are sorted by MACS Pan-T magnetic beads;

[0057] (2) The sorted T cells were diluted with culture medium (AIM-V medium+5% FBS+penicillin 100U / ml+streptomycin 0.1mg / ml) to a cell concentration of 2.5×10 6 pcs / ml for use;

[0058](3) Stimulate T cells 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 at a ratio of 1:2, and the final density of T cells should be 5 ×10 6 A / ml / cm2. After mixing, place them in a 37°C, 5% CO2 incubator to incubate and stimulate for 48 hours.

[0059] (4) Lentiviral transfection of T cells: remove the magnetic beads in the activated T cell-magn...

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Abstract

The invention provides a chimeric antigen receptor containing a C5aR intracellular domain, a lentiviral vector, an expression cell and a medicine. The chimeric antigen receptor containing the C5aR intracellular domain comprises an extracellular domain capable of combining with an antigen, a membrane spanning domain and at least one intracellular domain, wherein the at least one intracellular domain refers to the C5aR intracellular domain, or an intracellular domain of a signal transmission area in serial connection with the C5aR intracellular domain. According to the chimeric antigen receptorprovided by the invention, a T17 cell subset can be improved, an immunosuppressive function of a regulatory T cell is eliminated, the in-vitro cytotoxicity efficiency of a tumor target cell can be remarkably improved, a killing effect of a second generation of CAR T cell on tumors is remarkably improved, and a new idea and selection are provided for a CAR-T cell therapy field.

Description

technical field [0001] The invention belongs to the technical field of tumor cell immunotherapy, and specifically relates to a chimeric antigen receptor comprising a C5aR intracellular domain, and also relates to a nucleic acid and application of the chimeric antigen receptor encoding a C5aR intracellular domain. Background technique [0002] Chimeric Antigen Receptors (CAR, Chimeric Antigen Receptors) T cells are a milestone event that is expected to cure tumors. CAR-T technology uses genetic modification technology to combine a single chain fragment variable (scFv) that recognizes tumor antigens with cells. The internal activation motif recombinant gene is transfected to T lymphocytes to achieve better tumor recognition and killing effects. CAR-T molecules usually include an extracellular hinge region, a transmembrane region, and an intracellular signal region. Among them, the extracellular hinge region is formed by connecting the heavy chain and light chain variable regi...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00
CPCC12N15/86C07K14/7051C07K16/2803C07K16/2809C07K16/30C07K2319/33C12N2510/00C12N2740/15043A61K39/4611A61K2239/31A61K2239/38A61K2239/48A61K39/4631A61K39/464412
Inventor 赖沛龙
Owner 赖沛龙
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