Inhibitive ability synthesis Notch, dual-target spot system and preparation method and application of dual-target spot system

An inhibitory, dual-target technology, applied in the field of genetic engineering, can solve the problem of off-target toxicity of CAR-T cells, and achieve the effect of solving the problem of off-target toxicity, quick response, and improved accuracy

Active Publication Date: 2019-10-29
深圳市思考者医疗信息咨询有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0020] The purpose of the present invention is to provide an inhibitory synthetic Notch and dual-target system and its preparation method and application, to solve the problem that CAR-T cells still have off-target toxicity in the prior art

Method used

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  • Inhibitive ability synthesis Notch, dual-target spot system and preparation method and application of dual-target spot system
  • Inhibitive ability synthesis Notch, dual-target spot system and preparation method and application of dual-target spot system
  • Inhibitive ability synthesis Notch, dual-target spot system and preparation method and application of dual-target spot system

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Embodiment 1

[0073] According to the amino acid sequence of the dual-target system, the corresponding gene sequence was synthesized, the codons were humanized and optimized, and a BamHI restriction site was added at the 5'-end, and a SalI restriction site was added at the 3'-end. Take 4μg DNA containing the gene of the dual-target system, add 2μL 10×NEBuffer 3.1, 14μL ddH 2 O, mix well, then add 1 μL BamHI and 1 μL SalI, mix well, and place in a 37°C incubator for 4 hours. Using the same method, take 2μg pWPT-GFP plasmid, add 2μL 10×NEBuffer 3.1, 14μL ddH 2 O, mix well, then add 1 μL BamHI and 1 μL SalI, mix well, and place in a 37°C incubator for 4 hours. The double-enzyme-digested DNA fragments were prestained with Sybergreen, transferred to 1% agarose gel sample wells, and electrophoresed at 80V. Under UV light, the target band is cut, recovered and purified. Take 1 μg each of the double-enzyme-digested DNA fragment of the dual-target system gene and the pWPT-GFP plasmid, add 1 μL 10...

Embodiment 2

[0075] The culture medium of 293T cells and HUH-7 cells is DMEM medium containing 10% fetal bovine serum and 100×diluted penicillin and streptomycin. The culture medium of HepG2 cells and SK-HEP-1 cells is MEM medium containing 10% fetal bovine serum and 100× diluted penicillin and streptomycin. Jurkat T cell medium is RPMI-1640 medium containing 10% fetal bovine serum and 100 μg / mL gentamicin sulfate. The above cells were placed at 37°C, 5% CO 2 Cultivated in an incubator. 293T, HepG2, HUH-7 and SK-HEP-1 cells were digested and passaged with 0.25% trypsin.

[0076] Take 5 μg each of the dual-target system pWPT-GFP expression plasmid, psPAX2 and pMD2.G plasmids, and dissolve them in 480 μL 1×HBS (HEPES buffered saline solution). Take 36 μL PEI (polyethyleneimine, 2.5 mg / mL, pH7.4), dilute it to 360 μL with 1×HBS, add it dropwise to the plasmid solution, mix well while adding, and let stand for 20 minutes. The plasmid-PEI mixture was added dropwise to the 10 cm culture dish...

Embodiment 3

[0080]Aspirate the medium of 293T, SK-HEP-1, HepG2, and HUH-7 cells, and add 1 mL of 0.25% trypsin to digest. After the cells were digested completely, the medium containing fetal bovine serum was added to stop the digestion, centrifuged at 400×g for 3 minutes, washed twice with PBS, and 500 μL PBS was added to suspend the cells. Add 5 μL of FITC-mouse anti-EGFR IgG1 antibody, FITC-mouseanti-EpCAM IgG1 antibody or FITC-mouse IgG1 isotype control antibody, and incubate at 4°C for 0.5 hours in the dark. Wash twice with PBS, suspend cells with 500 μL PBS, and perform flow cytometry detection. Test results such as Figure 5 As shown, 293T cells are EGFR-EpCAM-, SK-HEP-1 cells are EGFR+EpCAM-, HepG2 cells are EGFR-EpCAM+, HUH-7 cells are EGFR+EpCAM+, which meet the requirements for verifying dual-target systemic killing experiments.

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Abstract

The invention relates to an inhibitive ability synthesis Notch, a dual-target spot system and a preparation method and application of the dual-target spot system. The inhibitive ability synthesis Notch comprises fusion protein combined by a Notch1 sequence and CSK kinase. The inhibitive ability synthesis Notch is used for obtaining the dual-target spot system, at the same time, two kinds of tumor-associated antigen can be targeted, accuracy of T cells to kill and wound cancer cells is improved, and off-target virulence caused by targeting single tumor-associated antigen is overcome.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an inhibitory synthetic Notch and dual-target system and its preparation method and application. Background technique [0002] In recent years, tumor immunotherapy has developed rapidly and has become the fourth treatment after surgery, chemotherapy and radiotherapy. Tumor immunotherapy achieves the goal of killing tumors by activating or enhancing the function of the human immune system, including tumor vaccines, cytokines, oncolytic viruses, immune checkpoint inhibitors, and adoptive cell therapy (ACT), etc. Immune checkpoint inhibitors and chimeric antigen receptor T cells (chimeric antigen receptor T cells, CAR-T) have made significant progress in tumor treatment trials, showing the potential to cure tumors. In 2013, "Science" magazine listed tumor immunotherapy as the top ten scientific breakthroughs of the year. In 2017, the U.S. FDA approved two CD19CAR-T cel...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00
CPCC07K14/47C12N9/12C07K14/7051C12N15/86A61K35/17A61P35/00C12N2740/15043C07K2319/03C07K2319/02A61K2039/5158A61K39/0011A61K2039/5156
Inventor 曾思敬
Owner 深圳市思考者医疗信息咨询有限公司
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