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Bridged bispecific fusion protein

A fusion protein and bispecific technology, applied in the direction of hybrid immunoglobulin, fusion polypeptide, immunoglobulin, etc.

Active Publication Date: 2016-11-09
REMEGEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although the prior art has made the above-mentioned progress in this field, there is still a need for angiogenesis inhibitors with higher stability and stronger affinity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1 Construction of recombinant expression plasmids for dual-target fusion proteins

[0097] 1. Preparation of DNA fragments of VEGFR and FGFR

[0098] VEGF receptor and FGF receptor cDNA fragments were obtained by synthesis (synthesized by Nanjing GenScript Biotechnology Co., Ltd.), and the sequences are shown in SEQ ID NO: 14 and SEQ IN NO: 16. The synthesized cDNA fragment was directly synthesized onto the PUC57-vector (the vector can be purchased from Nanjing GenScript Biotechnology Co., Ltd.).

[0099] 2. Preparation of DNA Fragment of Long Adapter Fc

[0100] Fc-cDNA sequence was synthesized, and the synthesized Fc had adapters (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) before and after, which was synthesized on the vector HX1 (the vector can be purchased from Nanjing GenScript Biotechnology Co., Ltd.) , thereby obtaining an Fc DNA fragment with a long linker, also referred to as Fc long herein. Specifically, the front and back joints are:...

Embodiment 2

[0121] Example 2 Transient expression of fusion protein

[0122]Use the MAX plasmid purification kit (QIAGEN) to purify the corresponding fusion protein plasmid DNA, determine the concentration of the plasmid DNA with an ultraviolet spectrophotometer, and culture 1 μg of the recombinant plasmid and 6 μL of liposomes (FuGENE 6 Transfection Reagent, Roche Company) in 100 μL of fresh IMDM solution (GIBCO company), mix well, let it stand for 15 minutes, then add 3×10 cells according to cell density 5 / mL inoculated in CHO cells (ATCC) cultured overnight in a 6-well plate, the complete culture medium of the cells contained 88% IMDM, 10% FBS, 1% HT and 1% glutamine (both products of GIBCO Company), at 37 °C, 5% CO 2 After culturing in the incubator for 48 hours, the supernatant was collected, and the relative content of the fusion protein secreted and expressed by CHO was determined with a human IgG ELISA protein quantification kit (BETHYL Company).

Embodiment 3

[0123] Example 3 Determination of binding affinity of fusion protein

[0124] The binding ability of the fusion protein constructed above to VEGF and FGF-2 was detected by ELISA method. Coat the plate with 20ng / well VEGF or 50ng / well FGF-2, 100μl / well, overnight at 2-8°C. Wash the plate 3 times in a plate washer. 3% BSA-PBST solution blocked, 37 degrees 2h. Wash the plate 3 times in a plate washer. Loading: use PBST solution to dilute the marked line from 10000ng / ml (for VEGF-coated plates) or 50000ng / ml (for FGF-coated plates) to 9 points of serial dilution, 100μl / well, 37 degrees for 1h. Wash the plate 3 times in a plate washer. Dilute the secondary antibody (Goat anti-human IgG-Fc-HRP) 5000 times with PBST solution. Add TMB color developing solution to develop color, and keep away from light for 5 min at room temperature. with 2M H 2 SO 4 The test was terminated, and the optical density absorbance value was read with a 450nm microplate reader. Wherein, the VEGFR-FG...

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Abstract

The invention relates to a bridged bispecific fusion protein, specifically, relates to an improved fusion protein for antagonizing an angiogenesis inducible factor and a use thereof, more specifically, relates to the fusion protein of a VEGF receptor and an FGF receptor and the use thereof in treatment of angiogenesis-related diseases.

Description

technical field [0001] The present invention relates to a bridged bispecific fusion protein, specifically, the present invention relates to an improved fusion protein antagonizing angiogenesis-inducing factors and its use, more specifically, the present invention relates to a fusion protein of VEGF receptor and FGF receptor and Its use in the treatment of diseases associated with angiogenesis. Background technique [0002] Angiogenesis is one of the basic elements leading to the growth and metastasis of malignant tumors [1]. The angiogenesis process is regulated by a variety of factors, some factors promote angiogenesis, some factors inhibit angiogenesis, and a considerable number of angiogenesis stimulating factors are known, such as vascular endothelial growth factor (VEGF), fibroblast Growth factor (Fibroblast growth factor, FGF), hepatocyte growth factor (Hepatocyte growth factor, HGF), DDR1, EphA1, EphA2, EphA8, EphB1, EphB4, EGFR, HER-2, ErbB3, MET, RON, CSF1R, KIT, ...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/62A61K39/395A61P35/00A61P27/02
CPCC07K16/468C07K2317/31C07K2317/92C07K2317/94C07K2319/30
Inventor 房健民姜静李伟伟
Owner REMEGEN CO LTD
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