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Bifunctional fusion protein targeting VEGF and EGFR and application of bifunctional fusion protein

A fusion protein, dual-function technology, applied in the fields of genetic engineering and biomedicine, can solve problems such as poor curative effect, and achieve the effects of inhibiting angiogenesis or cell growth, high stability and definite curative effect.

Inactive Publication Date: 2019-12-31
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Existing antibodies or antibody-drug conjugates are not effective in clinical trials of PDAC treatment, and there is an urgent need to explore more effective and personalized strategies to deal with complex genetic heterogeneity and unique tissue characteristics to achieve better PDAC Molecular Imaging and Therapeutics

Method used

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  • Bifunctional fusion protein targeting VEGF and EGFR and application of bifunctional fusion protein
  • Bifunctional fusion protein targeting VEGF and EGFR and application of bifunctional fusion protein
  • Bifunctional fusion protein targeting VEGF and EGFR and application of bifunctional fusion protein

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Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1 fusion protein construction and expression

[0041]Human non-immune ScFv (variable domain single-chain fragment: VH-linker-VL) antibody library constructed from healthy donor peripheral blood mononuclear cells (stock capacity: 2×10 11 ), to screen for VEGF165 and EGFR. Then with library preparation 2 x 10 13 Phage-scfv particles were incubated. After washing, three rounds of elutriation were performed. Then 2000 single clones were randomly selected, and the specific clones specifically binding to VEGF165 or EGFR were screened. Finally, clones with high optical density were selected for antibody gene sequencing. The VH CDR3 domains of these two scFvs are located at both ends of the sequence respectively. From N-terminus to C-terminus, the tandem CDRs are anti-vegf VHCDR3, CDR2, CDR1, linker, VL CDR3, CDR2, CDR1, linker, anti-egfr VL CDR1, CDR2, CDR3, linker VH CDR1, CDR2, CDR3. Synthetic bispecific ScFv genes were cloned into expression vector pET30a a...

Embodiment 2

[0043] The affinity evaluation of embodiment 2 fusion protein

[0044] Surface plasmon resonance imaging (SPRi) analysis was performed on a Plexera PlexArray HT system (Plexera LLC, Bothell, WA) using a bare gold SPRi chip (nano-gold chip with a gold layer thickness of 47.5 nm). All purified peptides were printed on the gold surface with cysteine ​​residues sulfhydryl groups. The printed chip was then incubated overnight in a humid chamber at 4°C. The SPRi chip was washed with 5% (m / v) skimmed milk in PBS and blocked overnight before use. The SPRi analysis program was as follows: running buffer (PBST, baseline stable); sample (five concentrations of protein, bound); running buffer (PBST, wash); HO in deionized water (regeneration). 3 PO 4 The content is 0.5% (v / v). Bi50 protein was diluted with PBST to a concentration of 10μg / mL, 5μg / mL, 2.5μg / mL, 1.25μg / mL, 0.625μg / mL. Record and analyze the SPRi signal.

[0045] Experimental results show that Bi50 can not only combine ...

Embodiment 3

[0046] Example 3 Targeting effect of fusion protein Bi50 and pancreatic cancer double-positive cell Bxpc3

[0047] The human pancreatic cell line Bxpc3 cells were cultured in DMEM medium containing 100 mL / L fetal bovine serum at 1×10 5 / mL cell concentration into a round glass-bottom Petri dish at 37°C, 5% CO 2 After culturing in the cell incubator for 24 hours, the culture medium was discarded, and FITC-Bi50 (50 μg / L) dissolved in DMEM medium was added; the control group was added with the same molar concentration of ScFv-FITC dissolved in DMEM medium; After incubation for 40 min, the solutions were discarded and washed 3 times with pre-cooled PBS.

[0048] The fluorescence distribution in the cells was detected with a laser scanning confocal microscope (Olympus FV1000-IX81).

[0049] The result is as image 3 As shown, strong green fluorescence was observed in Bxpc3 cells added with Bi50, while Bxpc3 cells in the control group ScFv had only weak green fluorescence signals...

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Abstract

The present invention provides a bifunctional fusion protein targeting VEGF and EGFR and an application of the bifunctional fusion protein. The provided fusion protein can specifically bind epidermalgrowth factor receptor (EGFR) and vascular endothelial growth factor (VEGF). The provided fusion protein provides targeting with exact curative effect and high stability, and synergistically inhibitsEGFR / VEGF(R) pathway. The fusion protein blocks phosphorylation of Akt / Stat3 signaling pathway, thus inhibits angiogenesis or cell growth and has a significant targeting imaging effect and a significant anti-PDAC tumor effect. The provided fusion protein has very strong targeting specificity to induce tumor cell apoptosis and can be used for treatment of malignant tumors.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biomedicine, in particular to a bifunctional fusion protein targeting VEGF and EGFR and its application. Background technique [0002] Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant and aggressive solid tumor that is difficult to detect early and has a poor prognosis due to its complex genetic heterogeneity. So far, no effective drug for the treatment of PDAC has been found, whether it is chemotherapy, targeted drugs or immunotherapy. Despite the rapid development of a large number of treatments for PDAC in recent years, the 5-year survival rate remains below 5%. PDAC consists of rather complex tissue elements and is characterized by a dense tissue background, high pressure, and poor interstitial vascularization. These endogenous properties also lead to a series of deadlocked therapies, such as poor penetration, embarrassing drug resistance, rapid recurrence and complex tum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/62A61K45/06A61K39/395A61P35/00
CPCA61K45/06A61K2039/505A61P35/00C07K16/2863C07K16/30C07K16/32C07K2317/31C07K2317/92
Inventor 王倩王子华赵心明
Owner CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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