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CAR (chimeric antigen receptor) secreting IL-7, virus vector, expression cell, preparation method and drug

A chimeric antigen receptor, IL-7 technology, applied in receptors/cell surface antigens/cell surface determinants, viruses/phages, botanical equipment and methods, etc.

Active Publication Date: 2018-06-29
GUANGDONG GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, the following problems have not been solved: CAR-T cells play an immune killing role, relying on the mutual contact between CAR-T cells and tumor cells, and the need to overcome the local immunosuppressive microenvironment of the tumor

Method used

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  • CAR (chimeric antigen receptor) secreting IL-7, virus vector, expression cell, preparation method and drug
  • CAR (chimeric antigen receptor) secreting IL-7, virus vector, expression cell, preparation method and drug
  • CAR (chimeric antigen receptor) secreting IL-7, virus vector, expression cell, preparation method and drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Preparation of plasmids containing anti-CD19 ScFv-4-1BB-CD3ζ (CAR19) and anti-CD19 ScFv-4-1BB-CD3ζ-IL-7 (IL-7 CAR19)

[0029] Prepare the plasmid of the present invention carrying the chimeric antigen receptor gene that secretes IL-7 cytokines as follows:

[0030] (1) The plasmid pUC57-CAR19 containing anti-CD19 ScFv-4-1BB-CD3ζ (CAR19) was obtained by means of gene synthesis, molecular cloning, etc., and its gene contained anti-CD19 monoclonal antibody ScFv, CD28 transmembrane region and 4-1BB, CD3ζ The intracellular region, namely CD19ScFv-4-1BB-CD3ζ, is shown in FIG. 1 .

[0031] (2) The plasmid pUC57-IL-7 CAR19 containing anti-CD19 ScFv-4-1BB-CD3ζ-IL-7 (IL-7CAR19) was obtained by means of gene synthesis, molecular cloning, etc., and its gene contained anti-CD19 monoclonal antibody ScFv, CD28 Transmembrane region and 4-1BB, CD3ζ intracellular region and IL-7 cytokine nucleic acid series, namely CD19 ScFv-4-1BB-CD3ζ-IL-7.

[0032] (3) The resulting pUC57-C...

Embodiment 2

[0033] Example 2 Lentiviral Packaging of CAR Plasmids

[0034] Three lentiviruses expressing GFP (blank control), CAR19-GFP (control), and IL-7 CAR19-GFP 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.

[0035] Specific steps are as follows:

[0036] (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);

[0037] (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;

[0038] (3) After replacing the medium and culturing for 2-6 hours, the two plasmids pWPXLd-CAR-GFP (...

Embodiment 3

[0047] Example 3 Using packaged CAR virus to infect human T cells

[0048] (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;

[0049] (2) Dilute the sorted T cells with culture medium (AIM-V medium+5% FBS+penicillin 100U / ml+streptomycin 0.1mg / ml) to a cell concentration of 2.5×106 cells / ml for later use;

[0050] (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.

[0051] (4) Lentiviral transfection of T cells: remove the magnetic beads in the activated T cell-mag...

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PUM

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Abstract

The invention provides a CAR (chimeric antigen receptor) secreting IL-7, a virus vector, an expression cell, a preparation method and a drug. The CAR secreting IL-7 contains an extracellular domain capable of binding antigen, a membrane spanning domain, an intracellular domain and an IL-7 cytokine domain, wherein the IL-7 cytokine domain is in non-fusion connection with the intracellular domain through self-cleaving peptides. The self-cleaving peptides (2A peptides) and an IL-7 gene sequence are added to an original CAR expression vector, so that the designed CAR T can express and secrete IL-7, the tumor killing effect is enhanced, and the tumor microenvironment is improved. The CAR T cells remarkably improve tumor killing functions in vivo and in vitro.

Description

Background technique [0001] 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 peptide; the transmembrane region mainly comes from the transmembrane region of molecules such as CD8, CD28 or 4-1BB; The intracellular signaling region mainly comes from the intracellular mosaic of T cell transduction signaling molecules such as CD28, 4-1BB, CD3zeta, CD27 or...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00A61P35/02
CPCA61K35/17C07K14/5418C07K14/7051C07K2319/33C12N15/86C12N2740/15043
Inventor 翁建宇陈晓梅赖沛龙杜欣王玉连王惊华耿素霞
Owner GUANGDONG GENERAL HOSPITAL
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