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CD19 and CD30 dual-target chimeric antigen receptor and its application

A chimeric antigen receptor and single-chain antibody technology, applied in the field of biomedicine, can solve the problems of tumor recurrence and poor effect in patients, and achieve the effect of high-efficiency targeting, high-efficiency killing effect, and avoiding the phenomenon of immune escape.

Active Publication Date: 2022-02-15
GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, medical diagnosis shows that tumor cells in some hematological tumors do not express CD19 molecules, but express CD30 molecules. Only CAR-T cells targeting CD19 molecules are not effective, and some patients have tumor recurrence after a period of time Phenomenon

Method used

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  • CD19 and CD30 dual-target chimeric antigen receptor and its application
  • CD19 and CD30 dual-target chimeric antigen receptor and its application
  • CD19 and CD30 dual-target chimeric antigen receptor and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Construction of CAR Molecular Carrier

[0066] In this example, firstly, the coding gene (SEQ ID NO: 10) of the anti-CD19 and CD30 dual-target chimeric antigen receptor is genetically synthesized, and restriction endonuclease Pme1 digestion sites are added to the C-terminal and N-terminal of the coding gene respectively and its protective base and restriction endonuclease Spe1 restriction site and its protective base;

[0067] The coding gene was double-digested with restriction endonucleases Pme1 and Spe1, and the digested product containing cohesive ends was recovered by agar gel electrophoresis, and then ligated into the linearized pWPXLd-eGFP plasmid (containing Sticky ends), the ligation reaction was carried out with the participation of T4 DNA polymerase (Invitrogent), and a lentiviral vector containing the coding gene of CAR targeting CD19 and CD30 dual targets was obtained.

[0068] In this example, the antigen-binding domains were constructed simulta...

Embodiment 2

[0069] Example 2 lentiviral packaging

[0070] In order to introduce CAR molecules into T cells, 293T cells were used to prepare recombinant lentiviruses, and when 293T cells were spread to 80-90% of the bottom of a 100mm culture dish, the lentiviruses were packaged:

[0071] 2 hours before virus packaging, replace the medium with DMEM containing 1% fetal bovine serum, and add 6mL / 100mm culture dish;

[0072] Prepare the plasmid mixture shown in Table 1, the pWPXLd-expression plasmid includes the lentiviral vector containing the coding gene of CAR targeting CD19 and CD30 dual targets, and the lentivirus containing the coding gene of CAR targeting CD19 single target Vector, a lentiviral vector containing a gene encoding a CAR targeting a CD30 single target, and the pWPXLd-eGFP plasmid is an empty vector that does not contain a gene encoding a CAR;

[0073] Table 1

[0074]

[0075] Add 36 μg PEI to another 500 μL opti-MEM medium, mix well, and let stand at room temperature...

Embodiment 3

[0081] Example 3 T cell activation and lentiviral transfection

[0082] Peripheral blood mononuclear cells (PBMC) were separated from whole blood using Ficoll density gradient centrifugation kit (GE Company), and after red blood cells were removed, T cells were sorted out using MACS Pan-T magnetic beads;

[0083] The sorted T cells were diluted with medium (AIM-V medium + 5% FBS + penicillin 100 U / mL + streptomycin 0.1 mg / mL) to a cell concentration of 2.5×10 6 pcs / mL for use;

[0084] CD2 / CD3 / CD28 T cell activation expansion kit (Miltenyi Company) was used to activate T cells, that is, the coated magnetic beads were mixed with T cells at a ratio of 1:2, and the final density of T cells was 5×10 6 piece / mL / cm 2 , after mixing, place at 37°C, 5% CO 2 The incubator was stimulated for 48 hours;

[0085] After T cells were activated for 48 hours, demagnetize the beads, centrifuge at 300g for 5 minutes, remove the supernatant, resuspend T cells with fresh medium, add recombinan...

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Abstract

The present invention provides CD19 and CD30 dual-target chimeric antigen receptors and applications thereof. The chimeric antigen receptors include antigen binding domains, transmembrane domains and signal transduction domains; the antigen binding domains include Anti-CD19 single-chain antibody and anti-CD30 single-chain antibody. The anti-CD19 and CD30 dual-target chimeric antigen receptor of the present invention has targeting activity on CD19-positive and / or CD30-positive cells, and the T cells expressing anti-CD19 and CD30 dual-target chimeric antigen receptor have the expression level of CD19 antigen Tumor cells with little or no expression and tumor cells with little or no expression of CD30 antigen all have a killing effect, which is beneficial to avoid immune escape and reduce the possibility of disease recurrence.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a CD19 and CD30 dual-target chimeric antigen receptor and an application thereof. Background technique [0002] Chimeric antigen receptor (chimeric antigen receptor, CAR) is composed of tumor-associated antigen binding region, extracellular hinge region, transmembrane region and intracellular signal transduction region. Usually, the antibody single chain fragment variable (single chain fragment variable, scFv) contained in the CAR molecule has a specific binding effect on tumor-associated antigen (tumor associated antigen, TAA). Plasma domain coupling. [0003] In recent years, with the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) has become one of the most promising tumor immunotherapies. At present, CAR-T cell therapy has been widely used in the treatment of B-cell malignancies. CAR-T cells targeting ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61P35/02C12R1/93
CPCC07K16/2803C07K16/2878C07K14/7051C12N15/86C12N7/00C12N5/0636A61K39/001117A61K39/001112A61P35/02C07K2317/622C07K2319/03C07K2319/02C07K2319/33C12N2740/15043C12N2740/15021C12N2510/00A61K2039/804
Inventor 汤朝阳秦乐吴迪邓殷健吴海鹏王翠花冯世忠冯嘉昆其他发明人请求不公开姓名
Owner GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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