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Recombinant lentivirus and application thereof

A technology of recombinant lentivirus and viral vector, applied in the field of cellular immunotherapy of tumors, can solve the problems of affecting the therapeutic effect and unsatisfactory chimeric antigen targeting effect.

Active Publication Date: 2017-05-31
SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The targeting effect of the chimeric antigen is not very ideal, and the tumor microenvironment will affect its therapeutic effect

Method used

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  • Recombinant lentivirus and application thereof
  • Recombinant lentivirus and application thereof
  • Recombinant lentivirus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of Chimeric Antigen Receptor Lentiviral Vector

[0052] (1) Synthesize human IgM signal peptide, anti-GPC3 antibody extracellular region, human CD28 transmembrane region, human 41BB intracellular region, human TLR2 intracellular region and human CD3ζ signaling domain through whole gene synthesis, namely CAR-GPC3-41BB- CD3ξ-TLR2 (CAR-GTBBξ), the whole gene synthetic human IgM signal peptide, anti-GPC3 antibody extracellular domain, human CD28 transmembrane region and intracellular region, human TLR2 intracellular region and human CD3ζ signaling domain, namely CAR-GPC3 - CD28-CD3ξ-TLR2 (CAR-GT28ξ), its sequence is shown in SEQ ID NO.4, and then the whole gene synthesis CAR-CD19-CD28-CD3ξ-TLR2 (Mock) nucleic acid sequence is used as a negative control, CAR-GPC3-41BB -CD28-CD3ξ (CAR-G28BBξ) nucleic acid sequence is used as a positive control and its gene sequence map is as follows figure 1 As shown, the C-terminal of the synthesized gene contains a ...

Embodiment 2

[0055] Example 2: GT28ξCAR lentiviral packaging

[0056] (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);

[0057] (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;

[0058] (3) After replacing the medium and culturing for 2-6 hours, the pWPXLd-CAR-EGFP plasmids (pWPXLd-CAR-GTBBξ-2A-EGFP, pWPXLd-CAR-GT28ξ-2A-EGFP, pWPXLd-CAR-G28BBξ- 2A-EGFP, pWPXLd-Mock-2A-EGFP) were co-transduced into 293T cells with lentiviral packaging helper plasmids pMD2.G and psPAX2 respectively, and the reagents and dosages were as follows:

[0059] Reagent dose pWPXLd-CAR-EGFP plasmid 9μg pMD2.G helper plasmid 3μg psPAX2 12μg PEI 72μg

[0060] (4) 24, 48 and 72 hours after transformation, collect the culture supern...

Embodiment 3

[0065] Example 3: Construction of CAR T cells

[0066] (1) Separation and purification of human T cells: the mononuclear cells in human blood are separated by the Ficoll density gradient method, and the red blood cells are removed by red blood cell lysate, and then the T cells are sorted by MACS Pan-T magnetic beads;

[0067] (2) Dilute the sorted T cells with 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;

[0068] (3) Stimulate T cells by magnetic beads coated with CD2, CD3, and CD28 antibodies (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 piece / mL / cm 2 . After mixing, place at 37°C, 5% CO 2 Incubator culture stimulation for 48 hours;

[0069] (4) Lentiviral transfection of T cells: remove the magnetic beads in the activated T cell-magnetic bead mixture by magnetic field, centrifuge at 300g for 5min...

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Abstract

The invention relates to the field of tumor cellular immunotherapy, and in particular relates to a recombinant lentivirus and application thereof. The recombinant lentivirus comprises a chimeric antigen receptor, wherein the chimeric antigen receptor mainly comprises signal peptide, an antigen recognition domain, a transmembrane domain, an intracellular co-stimulation signal transduction domain and a CD3 zeta signal transduction domain which are serially connected; the intracellular co-stimulation signal transduction domain mainly comprises a human TLR2 (Toll Like Receptor 2) intracellular domain. A GPC3 CAT T (Glypican 3 CAT T) cell prepared from the recombinant lentivirus has an intense cell killing effect on liver cancer cells, a Th1 cell factor can be highly expressed, a tumor killing effect caused by non-CAR T (Chimeric Antigen Receptor T) cell can be stimulated to the maximum extent, escape and potential reoccurrence risk of GPC 3-tumor cells can be effectively prevented, the tumor cells can be killed by T cells expressing the chimeric antigen receptor, normal tissue can be slightly damaged, a tumor immunosuppression micro environment can be broken through, and thus a relatively good treatment effect on solid tumor can be achieved.

Description

technical field [0001] The present invention relates to the field of tumor cell immunotherapy, in particular to a recombinant lentivirus and its application, specifically to the construction method of chimeric antigen receptor T (CAR-T) cell technology based on tumor-specific target GPC3 and its application in antitumor therapy. Background technique [0002] The GPC3 (Glypican-3, glypican 3) gene is located on human chromosome X26.10 and is involved in life processes such as cell division, growth, development, and cell responses to growth factors. The function of GPC3 is expected to be closely related to the distribution in the organism. During the embryonic period of the human body, GPC3 is expressed in the placenta, embryonic liver, embryonic lung and embryonic kidney. In adult normal tissues, it is not expressed in the brain, liver, spleen, stomach, intestine, etc., but has very low levels of expression in ovary, lung, and kidney (Hsu HC et al., 1997 Cancer Research). I...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N15/861C12N7/01A61K35/17A61K39/00A61P35/00
CPCA61K39/0011A61K35/17C12N5/0636C12N7/00C12N15/86C07K14/705C07K14/70507C07K14/7051C07K14/70596C07K16/303C07K2319/03C07K2319/02C12N2740/15043C12N2740/15021C12N2710/10043C12N2710/10021C12N2740/10043C12N2740/10021
Inventor 不公告发明人
Owner SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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