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A method for detecting the number of car-t cells

A cell number and cell technology, applied in the detection of chimeric antigen receptor T cell number, a new field of detection of chimeric antigen receptor T cell number, can solve the problem of relative quantification, incomplete detection results, and increased molecular weight of CAR-T and other problems, to achieve the effect of convenient operation, accurate measurement results, and improvement of in vitro killing efficiency

Active Publication Date: 2020-08-07
GUANGDONG GENERAL HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of the number of CAR-T cells is mainly as follows: 1. Use the PCR method to detect extracellular scFV. The disadvantage is that the detection method is time-consuming and relatively quantitative. Different scFVs need to design different PCR primers and discuss; 2. The introduction of CAR molecules Green fluorescent protein (GFP), which is currently a more accurate method for detecting CAR-T, but because GFP is found in jellyfish and does not exist in the human body, its safety requires long-term observation, and because GFP does not exist in the human body may Anti-GFP antibodies will be produced, which will affect the function of CAR-T cells; 3. Introduce IgG4 or IgG1 into the hinge region of the extracellular region, and detect the expression level of IgG4 or IgG1 by flow cytometry. The disadvantage is that after the introduction of IgG4 or IgG1, Increase the molecular weight of CAR-T, which affects the transfection rate of CAR-T, and there is also IgG4 or IgG1 in the human body, and the detection results are not completely accurate

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  • A method for detecting the number of car-t cells
  • A method for detecting the number of car-t cells
  • A method for detecting the number of car-t cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 contains CD19ScFv-4-1BB-CD3ζ (CAR19), anti-CD19ScFv-4-1BB-CD3ζ-C5aR (CAR19C5aR)

[0026] Plasmid preparation

[0027] Prepare the plasmid of the present invention carrying the chimeric antigen receptor gene containing the intracellular domain of C5aR as follows:

[0028] (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.

[0029] (2) The plasmid pUC57-CAR19 containing anti-CD19 ScFv-4-1BB-CD3ζ-C5aR (CAR19) was obtained by means of gene synthesis, molecular cloning, etc. The intracellular region of C5aR, that is, CD19ScFv-4-1BB-CD3ζ-C5aR (CAR19C5aR), C5aR is part or all of the NCBI Reference Sequence: NM_001736.3 nucleic acid series. (figure 1)

Embodiment 2

[0030] Lentiviral packaging of embodiment 2CAR plasmid

[0031] 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.

[0032] Specific steps are as follows:

[0033] (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);

[0034] (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;

[0035] (3) After replacing the medium and culturing for 2-6 hours, the two plasmids pWP...

Embodiment 3

[0043] Example 3 Using packaged CAR virus to infect human T cells

[0044] (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;

[0045] (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;

[0046] (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.

[0047] (4) Lentiviral transfection of T cells: remove the magnetic beads in the activated T cell-mag...

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Abstract

The invention provides a method for detecting number of CAR-T (chimeric antigen receptor T) cells. The method is characterized in that by introducing C5aR into the CAR-T cells, the expression rate ofC5aR is detected, so as to obtain the number of CAR-T cells. The method has the advantages that the application of C5aR intracellular domain into the detection of CAR-T cells is proofed for the firsttime through experiment; after the C5aR intracellular domain is introduced, the killing function of the CAR-T cell is not influenced, but is improved, so that the method of introducing the C5aR intracellular domain to detect the number of CAR-T cells has broad application prospect.

Description

technical field [0001] The invention belongs to the technical field of tumor cell immunotherapy, and specifically relates to a chimeric antigen receptor T cell number detection technology, especially a new method for detecting the chimeric antigen receptor T cell number. 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 regions of the single-chain antibody through a ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00G01N33/566
CPCG01N33/505G01N33/566G01N2333/7051G01N2333/70521G01N2333/70596
Inventor 赖沛龙陈晓梅翁建宇杜欣
Owner GUANGDONG GENERAL HOSPITAL
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