Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Activated human coagulation factor VII fusion protein and preparation method and application thereof

A technology of human blood coagulation factor and fusion protein, which is applied in the field of activated human blood coagulation factor VII fusion protein and its preparation, and preparation of drugs for the treatment of various blood coagulation related diseases. Limited and other issues

Active Publication Date: 2017-01-04
AMPSOURCE BIOPHARMA (SHANGHAI) INC +3
View PDF22 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The inventors found that the long-acting rFVIIa disclosed in the prior art has the problems of limited half-life extension, significantly reduced biological activity or strong immunogenicity, mainly because the C-terminus of FVIIa contains a serine protease domain (residues 153-406 base), so the C-terminal fusion of other molecules will affect the catalytic activity and function of FVIIa
In addition, the present inventors found that only a relatively long conventional flexible peptide linker (eg (GGGGS)n) is connected between FVIIa and the half-life-extending moiety (eg, Fc fragment of immunoglobulin), which instead enables the fusion protein to Proteases are more sensitive, and what's worse, such a long peptide linker also makes most fusion proteins expressed as aggregates with little biological activity

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Activated human coagulation factor VII fusion protein and preparation method and application thereof
  • Activated human coagulation factor VII fusion protein and preparation method and application thereof
  • Activated human coagulation factor VII fusion protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1. Construction of expression plasmids encoding FVII fusion proteins

[0115] The gene sequences encoding FVII leader peptide, mature protein, flexible peptide linker, CTP rigid unit and human IgG vFc variant are artificially optimized codons preferred by CHO cells and obtained by artificial synthesis. The 5' and 3' ends of the full-length DNA fragment of the synthesized fusion protein each have a restriction enzyme endonuclease site, respectively SpeI and EcoRI, and the full-length DNA fragment is inserted between the corresponding restriction enzyme sites of the pUC57 transfer vector. DNA sequencing verified the sequence. Then transfer the full-length gene fragment of the fusion protein obtained above from the intermediate vector to between the SpeI (5') and EcoRI (3') sites of the transformed expression plasmid PTY1A1 using PCDNA3.1 as a template to obtain a fusion protein with a high expression plasmid. The PTY1A1 plasmid includes but is not limited to the...

Embodiment 2

[0119] Example 2. Transient expression of each fusion protein and activity assay

[0120] With 8 kinds of expression plasmids that embodiment 1 obtains, use DNAFect LT reagent in the shaking flask of 30ml TM (ATGCell company) transfection 3×10 7 In CHO-K1 cells, the transfected cells were grown for 5 days in a serum-free growth medium containing 1000ng / ml vitamin K1, and the concentration of the fusion protein in the supernatant was determined, and the method described in Example 7 or 8 was used to determine its concentration. active. The results of ELISA showed that the transient expression levels (amounts of substances) of the eight plasmids were similar under this condition, but their blood coagulation activities showed great differences. Among them, we defined the molar specific activity of FP-A as 100%. The fusion protein supernatants expressed by FP-F, FP-G and FP-H plasmids had low activities, only 29.4%, 26.3% and 41.2% of FP-A, and the purified proteins were analyz...

Embodiment 3

[0121] Example 3. Screening for stable transfected cell lines expressing fusion proteins

[0122]The above expression plasmids of FP-A, FP-B, FP-C, FP-D and FP-E were transfected into a mammalian host cell line to express the FVII fusion protein. In order to maintain stable high-level expression, the preferred host cells are DHFR-deficient CHO cells (US Patent No. 4818679). A preferred method of transfection is electroporation, although other methods including calcium phosphate co-sedimentation, lipofection, and microinjection can also be used. The electroporation method uses a GenePulser Electroporator (Bio-Rad Laboratories Company) set at a voltage of 300V and a capacitance of 1050μFd, and places 2 to 3×10 cells in a cuvette. 7 50 μg PvuI linearized expression plasmid was added to each cell, and the electroporated cells were transferred to a shake flask containing 30 ml growth medium. Two days after transfection, the medium was replaced with a growth medium containing 0.6m...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a hyperglycosylated activated human coagulation factor VII (FVIIa) fusion protein, and a preparation method and application thereof. The fusion protein comprises human FVIIa, a flexible peptide joint, at least one human chorionic gonadotropin beta-subunit carboxyl terminal peptide rigid unit and a half-life period prolonging part (a human IgG Fc variant preferred). The fusion protein has bioactivity similar to that of the natural human FVIIa and has a longer in-vivo active half-life period, so that the pharmacokinetics and the medicinal effect are improved.

Description

technical field [0001] The present invention relates to a fusion protein, more specifically to an activated human blood coagulation factor VII (FVIIa) fusion protein and its preparation method and use, especially the use in the preparation of drugs for treating various blood coagulation related diseases. Background technique [0002] FVII is a vitamin K-dependent plasma glycoprotein that is synthesized in the liver and secreted into the blood as a single-chain protease with a molecular weight of about 53KDa (Broze et al., J Biol Chem, 1980, 255:1242-1247) . The FVII zymogen is hydrolyzed by protease at a single site Arg152-Ile153 to generate a double chain connected by a disulfide bond, thereby converting to its active form FVIIa. Activated FVIIa comprises an NH2-terminally derived light chain (about 20 KDa) and a COOH-terminally derived heavy chain (about 30 KDa) linked via a single disulfide bond (Cys135 to Cys262). The light chain contains a cell membrane bound Gla doma...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K19/00C12N9/64C12N15/62A61K38/48A61K47/48A61P7/04
CPCA61K38/48A61P7/04C07K19/00C12N9/64C12N15/62C12N9/6437A61K38/00C07K2319/30C07K2319/31C12Y304/21021
Inventor 李强朱文臣高永娟朱成功李媛丽王晓山孙乃超刘宾王文文李智刘婷婷朱鹿燕李亦清任子甲朱松林肖春峰苏鸿声
Owner AMPSOURCE BIOPHARMA (SHANGHAI) INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com