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Improved-type human coagulation factor FVII-Fc fusion protein as well as preparation method and application thereof

A fusion protein, hfvii-l-vfc 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, poor stability, etc. Reduced injection frequency and improved tolerability

Active Publication Date: 2013-11-20
上海日耘生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] 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-type human coagulation factor FVII-Fc fusion protein as well as preparation method and application thereof
  • Improved-type human coagulation factor FVII-Fc fusion protein as well as preparation method and application thereof
  • Improved-type human coagulation factor FVII-Fc fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Construction of an expression plasmid encoding hFVII-L-vFc fusion protein

[0051] 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 1351bp DNA fragment was inserted into the EcoRV restriction site of the 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 IgG2vFc variant (Pro331Ser mutation) is also an artificially optimized codon preferred by CHO cells, and the 5' and 3' ends of the synthesized fra...

Embodiment 2

[0053] Example 2. Transient expression of fusion proteins and activity assays of flexible peptide linkers of different lengths

[0054] 5 kinds of expression plasmids that embodiment 1 obtains use DNAFect LT reagent (ATGCeII) to transfect 3×10 in the shaking flask of 30ml 7 CHO-K1 cells, the transfected cells were grown for 5 days in the serum-free growth medium containing 100ng / ml vitamin K1, and the fusion protein concentration in the supernatant was measured by the method detailed in Example 8, and the The activity was determined by the method described in 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 activity of the FVII supernatant expressed by the PFVII-B, PFVII-C, PFVII-D and PFVII-E plasmids was 113% of that ...

Embodiment 3

[0055] Example 3. Screening for stable transfected cell lines expressing fusion proteins

[0056] 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. 4,818,679). 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 Gene Pulser Electroporator (Bio-Rad Laboratories) set at a voltage of 250V and a capacitance of 1050μFd, and places 2 to 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 days...

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Abstract

The invention discloses a recombined human coagulation factor FVII-Fc fusion protein as well as a preparation method and an application thereof. The fusion protein sequentially comprises human FVII, a flexible peptide junction and an IgG2Fc variant from an N terminal to a C terminal, wherein the Fv variant has no splitting property and shows tiny Fc-mediated adverse side effect. The fusion protein has the biological activity similar to or higher than that of the human FVII and greatly prolonged plasma half-life, thus the pharmacodynamics and the drug effect are improved.

Description

technical field [0001] 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 [0002] Coagulation is a process consisting of complex interactions between various blood components (or factors) that gradually lead to the formation of a fibrin clot. The blood components that typically participate 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 one of these coagulation factors. [0003] 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 FVI...

Claims

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Application Information

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IPC IPC(8): C12N9/50C12N15/62C12N15/63C12N5/10A61K38/48A61K47/48A61P7/04A61L27/54A61L31/16
CPCA61K38/00A61L27/54A61L31/16A61L2300/252A61P7/04C07K2319/30C12N9/6437C12Y304/21021
Inventor 李强刘长付冯维武翠孙见宇周若芸孙乃超
Owner 上海日耘生物医药有限公司
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