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Interferon mutant, interferon mutant fusion antibody, and preparation method and application thereof

A technology for fusing antibodies and mutants, applied in the field of bioengineering, can solve the problems of strong systemic side effects and short half-life of interferon, achieve strong tumor killing effect and immune regulation effect, inhibit the formation of tumor angiogenesis, and improve affinity Effect

Active Publication Date: 2018-12-14
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the shortcomings of interferon, such as short half-life and strong systemic side effects, limit its clinical application.

Method used

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  • Interferon mutant, interferon mutant fusion antibody, and preparation method and application thereof
  • Interferon mutant, interferon mutant fusion antibody, and preparation method and application thereof
  • Interferon mutant, interferon mutant fusion antibody, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of the recombinant plasmid of anti-VEGFR2-IFNαmut

[0038] According to the existing anti-VEGFR2 fusion antibody anti-VEGFR2-IFNα (refer to literature Li Z, Zhu Y, Li C, et al. Anti-VEGFR2-interferon-α2 regulates the tumor microenvironment and exhibits potent antitumor efficacy against colorectal cancer[J].Oncoimmunology , 2017,6(3):e1290038.doi:10.1080 / 2162402X.2017.1290038.) to prepare a recombinant plasmid for constructing anti-VEGFR2-IFNαmut; the anti-VEGFR2-IFNα is coupled at the C-terminus of the Fc segment of the antibody through G4S Linker For the fusion antibody linked with IFNα, in its heavy chain expression plasmid, the two ends of the part expressing IFNα contain restriction sites BamHI and PmeI. Based on this, we first obtained the expression sequence of IFNα by double enzyme digestion of the plasmid, and then, using IFNα as a template, designed mutation primers, obtained IFNαmut by overlapping PCR, and then based on the obtained IFNα...

Embodiment 2

[0061] Example 2 Expression, purification and SDS identification of anti-VEGFR2-IFNαmut

[0062] The light and heavy chains were transiently transfected into CHO cells, and stable and high-yielding monoclonal cell lines were selected by pressure selection and two limited dilution methods. The stable high-yielding cell line was expanded and cultivated, the supernatant was collected, centrifuged at 6000rpm, 4°C for 30min, filtered with a 0.22μm filter membrane, purified with a Protein A affinity chromatography column, and the eluent was used to elute the protein. The purified anti-VEGFR2-IFNαmut and anti-VEGFR2-IFNα were mixed with non-reduced SDS-PAGE loading buffer at a ratio of 4:1 (20μl:5μl) in an Eppendorf tube, placed in a water bath at 100°C for 5 minutes and then 3000rpm After centrifugation for 5 minutes, the protein sample was ready for use. Configure 10% SDS-PAGE gel for electrophoresis, stop electrophoresis after 80mA constant current for 30min, 120min constant curr...

Embodiment 3

[0063] Example 3 Western Blot identification of anti-VEGFR2-IFNαmut

[0064] The purified anti-VEGFR2-IFNαmut and anti-VEGFR2-IFNα were subjected to SDS-PAGE electrophoresis, 80V constant voltage for 30min, 120V constant voltage for 30min, transfer for 2h, and the protein was transferred to PVDF membrane (purchased from Millipore); the transfer was completed , block the membrane in 5% MTBS (TBS containing 5% skimmed milk) at room temperature for 2h; dilute the primary antibody with 5% MTBS at 1:2000 (A&B picture is HRP-coupled goat anti-human Fc antibody, without Anti-antibody; C&D picture is rabbit anti-human κ chain antibody; E&F picture is mouse anti-human IFNα antibody) (purchased from abcam), incubated at room temperature for 1.5h, washed three times with TBST and three times with TBS, each time for 10min; Dilute the secondary antibody with % MTBS at 1:5000 (C&D picture is the HRP-coupled goat anti-rabbit secondary antibody; E&F picture is the goat anti-mouse secondary an...

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Abstract

The invention belongs to the technical field of gene-engineering antibodies, and particularly discloses a fusion antibody (anti-VEGFR2-IFN[alpha]mut) composed of an antibody targeted to human vascularendothelial growth factor (VEGFR, aka KDR) and an interferon alpha (IFN-alpha) mutant, and a preparation method and an application of the fusion antibody. The invention also discloses the amino acidsequences of heavy-chain and light-chain immune globulin molecules of the fusion antibody (anti-VEGFR2-IFN[alpha]mut). The fusion antibody, in vitro, can inhibit reproduction of human umbilical vein endothelial cells (HUVEC) and a part of tumor cells, wherein the inhibition degree is higher than a fusion antibody anti-VEGFR2-IFN[alpha] of wild-type IFN[alpha] and anti-VEGFR2.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a high-affinity fully human fusion antibody that can specifically bind to human VEGFR2. Background technique [0002] The occurrence and development of tumor cells is inseparable from the nutrient supply of new blood vessels, and the growth of new blood vessels is inseparable from the interaction of various cytokines and their receptors. Existing studies have shown that the vascular endothelial growth factor / vascular endothelial growth factor receptor (VEGF / VEGFR) signaling pathway plays a key regulatory role in the formation of new blood vessels. As the main receptor of VEGF, VEGFR2 regulates the proliferation, survival, migration and permeability of vascular endothelial cells. [0003] Human vascular endothelial growth factor receptor 2 (vascular endothelial growth factor receptor2, also known as VEGFR2 or KDR, Flk-1, referred to herein as "VEGFR2") is a 230kDa transmembrane glyc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/56C07K19/00C12N15/62C12N15/13C12N15/85A61K39/395A61K38/21A61P35/00
CPCA61K2039/505A61P35/00C07K14/56C07K16/2863C07K2319/00C12N15/85
Inventor 张娟尚鹏钊高瑞王旻朱怡佳王斐
Owner CHINA PHARM UNIV
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