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Improved human blood coagulation factor FVII-Fc fusion protein and preparation method and application thereof

A fusion protein and application technology, applied in the field of treatment of various coagulation-related diseases, can solve the problems of no half-life FVII-Fc fusion protein, low expression amount, difficult construction, etc. The effect of improving the quality of life

Active Publication Date: 2015-07-15
AMPSOURCE BIOPHARMA (SHANGHAI) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In view of the difficulties in the preparation of homodimeric Fc fusion FVII described in the above prior art and the limitations in the clinical application process, such as low expression, short half-life and poor stability, there is an urgent need in this field to develop Long-acting, stable FVII derivatives that can be produced at a reasonable cost
Due to the inherent difficulties in the construction of hFVII-L-vFc fusion protein, no FVII-Fc fusion protein with significantly prolonged half-life and stable and high-efficiency expression has been obtained so far

Method used

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  • Improved human blood coagulation factor FVII-Fc fusion protein and preparation method and application thereof
  • Improved human blood coagulation factor FVII-Fc fusion protein and preparation method and application thereof
  • Improved human blood coagulation factor FVII-Fc fusion protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Construction of expression plasmid encoding hFVII-L-vFc fusion protein

[0052] The target gene sequence encoding hFVII leader peptide and mature protein is artificially optimized CHO cell preferred codons and obtained by artificial synthesis. In order to facilitate the insertion of the target gene fragment into the specific site of the expression vector, there is a restriction enzyme endonuclease site at the 5' and 3' ends of the synthesized fragment, respectively SpeI and BamHI. The full-length 1351 bp DNA fragment was inserted into the EcoRV restriction site of a transfer vector such as pUC57 to obtain an intermediate plasmid whose hFVII gene sequence was verified by DNA sequencing. The preferred fusion gene of the flexible peptide linker GlySer containing 2 amino acids in the present invention and the human IgG2 vFc variant (Pro331Ser mutation) is also an artificially optimized CHO cell preferred codon, and the synthesized fragment has one at the 5' and 3...

Embodiment 2

[0054] Example 2. Transient expression of fusion proteins and activity assays of flexible peptide linkers of different lengths.

[0055] The five expression plasmids obtained in Example 1 were transfected with 3X10 DNAFect LT reagent (ATGCell) in a 30 ml shake flask 7 For CHO-K1 cells, the transfected cells were grown for 5 days in serum-free growth medium containing 100 ng / ml vitamin K1, and the concentration of the fusion protein in the supernatant was determined by the method detailed in Example 8, and carried out with The activity was measured by the method described in Example 7. ELISA results showed that the transient expression levels of FVII of the five plasmids were similar under this condition, but their coagulation activities showed great differences. The activity of the FVII supernatant expressed by the PFVII-A plasmid containing 2 amino acid linkers was the lowest, and the activities of the FVII supernatant expressed by the PFVII-B, PFVII-C, PFVII-D and PFVII-E ...

Embodiment 3

[0056] Example 3. Screening of stable transfected cell lines with high expression of fusion protein

[0057] The above expression plasmid of PFVII-D (containing the peptide linker with the sequence GlySerGlyGlyGlyGlySerGlyGlyGlyGlyGlySerGlyGlyGlyGlyGlySer) was transfected into a mammalian host cell line to express hFVII-L-vFc 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 applied a Gene Pulser Electroporator (Bio-Rad Laboratories) set at 250 V voltage and 1050 μFd capacitance, placed in 2~3×10 cells in a cuvette. 7 20 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 day...

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Abstract

Disclosed are a recombinant fusion protein of human blood coagulation factor FVII-Fc, preparation method therefor and use thereof. The fusion protein comprises a human FVII, flexible peptide linker, and IgG2 Fc variant sequentially from the N-terminal to the C-terminal. The Fc variant has on lytic activity, and shows minimal Fc-mediated adverse side-effects. The fusion protein has a bioactivity similar to or higher than that of human FVII and a greatly prolonged plasma half-life, thereby improving the pharmacokinetics and pharmaceutical effect.

Description

[0001] The present invention is a divisional application, the original application number is 201310357373.X, the filing date is August 16, 2013, and the title of the invention is "Improved human blood coagulation factor FVII-Fc fusion protein and its preparation method and application". technical field [0002] The invention relates to an Fc fusion protein of improved human blood coagulation factor FVII and its preparation method and application, especially the application for treating various blood coagulation-related diseases. Background technique [0003] Coagulation is a process consisting of a complex interaction between various blood components (or factors) that gradually leads to the formation of a fibrin clot, usually the blood components involved in what is known as the coagulation "cascade" are proenzymes or enzymes Progenases, proteins that do not have enzymatic activity, are converted into active enzymes under the action of activators. Coagulation factor FVII is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/50C12N15/62C12N15/63C12N5/10A61K38/48A61P7/04
CPCC12N15/62C12N9/50A61L31/16C12N15/63C07K2319/30A61L2300/252C07K19/00A61L27/54A61K38/48C12N9/6437A61K38/00A61P7/04C12Y304/21021
Inventor 李强刘长付冯维武翠孙见宇周若芸孙乃超
Owner AMPSOURCE BIOPHARMA (SHANGHAI) INC
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