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A kind of recombinant lentivirus and its application

A technology of recombinant lentivirus and viral vector, which is applied in the field of tumor cell immunotherapy, can solve the problems of unsatisfactory targeting effect of chimeric antigen and affect the treatment effect, etc., and achieve weakening of tumor immunosuppression, good curative effect, and small degree of damage Effect

Active Publication Date: 2022-05-27
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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  • A kind of recombinant lentivirus and its application
  • A kind of recombinant lentivirus and its application
  • A kind of recombinant lentivirus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0052] (1) Whole gene synthesis of human IgM signal peptide, anti-GPC3 antibody extracellular segment, human CD28 transmembrane domain, human 41BB intracellular domain, human TLR2 intracellular domain and human CD3ζ signaling domain, namely CAR-GPC3-41BB- CD3ξ-TLR2 (CAR-GTBBξ), a total gene synthesis of human IgM signal peptide, anti-GPC3 antibody extracellular segment, human CD28 transmembrane domain and intracellular domain, human TLR2 intracellular domain and human CD3ζ signaling domain, namely CAR-GPC3 -CD28-CD3ξ-TLR2(CAR-GT28ξ), whose sequence is shown in SEQ ID NO.4, and then the whole gene synthesis CAR-CD19-CD28-CD3ξ-TLR2 (Mock) nucleic acid sequence as a negative control, CAR-GPC3-41BB -CD28-CD3ξ (CAR-G28BBξ) nucleic acid sequence as a positive control, its gene sequence map is as follows figure 1 As shown, the C-terminal of the synthesized gene contains the restriction endonuclease Pme1...

Embodiment 2

[0055] Example 2: GT28ξCAR lentiviral packaging

[0056] (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);

[0057] (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;

[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 and pWPXLd-Mock-2A-EGFP) were co-transfected into 293T cells with lentiviral packaging helper plasmids pMD2.G and psPAX2, respectively. The reagents and doses added 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 supernatant of ...

Embodiment 3

[0065] Example 3: Construction of CAR T cells

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

[0067] (2) Dilute the sorted medium for T cells: 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;

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

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

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Abstract

The present invention relates to the field of tumor cell immunotherapy, and in particular to a recombinant lentivirus and its application. The recombinant lentivirus contains a chimeric antigen receptor, and the chimeric antigen receptor mainly includes a signal peptide, an antigen recognition region, a transgenic A membrane region, an intracellular co-stimulatory signal transduction domain and a CD3ζ signal transduction domain are formed in series; wherein, the intracellular co-stimulatory signal transduction domain mainly includes the intracellular region of human TLR2. The GPC3 CAT T cells prepared by the present invention have a strong cell killing effect on liver cancer cells, and highly express Th1 cytokines, which can greatly stimulate the tumor killing effect caused by non-CAR T cells, and can effectively prevent the escape and potential of GPC3 tumor cells. The threat of recurrence, and T cells expressing this chimeric antigen receptor can kill tumor cells to the greatest extent, with little damage to normal tissues, and can break through the tumor immunosuppressive microenvironment, thus having better curative effect on solid tumors.

Description

technical field [0001] The invention relates to the field of cellular immunotherapy of tumors, in particular to a recombinant lentivirus and applications thereof, in particular to a method for constructing a chimeric antigen receptor T (CAR-T) cell technology based on a tumor-specific target GPC3 and the same Application in antitumor therapy. Background technique [0002] 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 response to growth factors. The function of GPC3 is expected to be closely related to the distribution in the organism. In the embryonic stage 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., and only has a very low level of expression in the ovary, lung and kidney (Hsu HC et al., 1997 Cancer R...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 姚瑶其他发明人请求不公开姓名
Owner SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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