Coagulation factor vii polypeptides

A factor, polypeptide technology, applied in the field of modified coagulation factor VII polypeptide, to achieve low dose, improved hemostatic protection, long duration effect

Inactive Publication Date: 2015-06-10
NOVO NORDISK HEALTH CARE AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, recombinant Factor Vila therapy still leaves a significant unmet medical need, and there is a need for recombinant Factor Vila polypeptides with improved pharmaceutical properties, such as increased functional half-life in vivo and improved activity

Method used

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  • Coagulation factor vii polypeptides
  • Coagulation factor vii polypeptides
  • Coagulation factor vii polypeptides

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Experimental program
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Effect test

Embodiment 1

[0159] To map the FVIIa-antithrombin interaction, a library of FVIIa variants was designed in silico based on a complex structural model of the FVIIa-antithrombin complex. Using the published X-ray structure of the FXa-antithrombin Michaelis complex (Johnson et al. 2006) as a template, construct figure 1 The model shown in . Mutagenesis was performed on FVIIa residues in close proximity to antithrombin in the model (maximum distance between FVIIa and antithrombin side chain is 12 Å). The first library was designed to explore the effect of conservative changes on the binding of human FVIIa to antithrombin. An alignment of FVII sequences from multiple species (chimpanzee, dog, pig, cow, mouse, rat, and rabbit) is shown at figure 2 middle. The side chain in human FVIIa in close proximity to antithrombin, which has a different side chain in other species, was mutated to that of the corresponding species. An example is that the residue in position 286 is Gln in human FVII and...

Embodiment 2

[0161] Use KOD Xtreme™ Hot Start DNA Polymerase from Novagen or QuickChange from Stratagene ? The site-directed mutagenesis kit uses a site-directed mutagenesis PCR-based method to introduce mutations into mammalian expression vectors encoding FVII cDNA. The following expression vectors were used: pTT5 (Durocher et al. (2002) Nucleic Acid Res. 30(2):e9) for transfection of HEK293F and HKB11 cells; pQMCF from Icosagen (Estonia) for transfection of CHOEBNALT85; pZEM219b (Busby (1991) J.Biol.Chem., 266:15286-15292) and pMPSVHE (Artelt et al. (1988) Gene 62:213-219) for transfection of CHO-K1; pLN174 (Persson et al. (1996) FEBS Lett., 385:241-243) were used to transfect BHK cells. Primers were designed according to the manufacturer's recommendations. Introduction of desired mutations was verified by DNA sequencing (MWG Biotech, Germany).

Embodiment 3

[0163] FVII variants in baby hamster kidney (BHK) cells, Freestyle TM 293-F human embryonic kidney cells (HEK293F; Gibco by Life Technologies, Naerum, DK), HKB11 (hybrid cell line of HEK293 and human B cell line) cells (ATCC, LGC Standards AB, Boras, Sweden), Chinese hamster ovary ( CHOK1) cells or CHO-EBNALT85 cells from Icosagen Cell Factory, Estonia.

[0164] BHK adherent cells were transfected with the FVII variant constructs using GeneJuice® from Merck Millipore (Hellerup, Denmark) according to the manufacturer's instructions for generating stable cell lines. Methotrexate (Sigma-Aldrich) was used as the selection reagent. Stable cell lines were grown in medium to large scale, giving a total of 5 - 10 liters of cell supernatant. Cells were incubated in an incubator at 37°C, 5 or 8% CO 2 Under culture in DMEM (Gibco by Life Technologies, Naerum, DK), described DMEM is supplemented with 2% (V / V) fetal bovine serum (Gibco by Life Technologies, Naerum, DK), 1% (v / v) Penic...

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Abstract

The present invention relates to modified coagulation Factor VII (Factor VII) polypeptides having coagulant activity as well as polynucleotide constructs encoding such polypeptides, vectors and host cells comprising and expressing such polynucleotides, pharmaceutical compositions, uses and methods of treatment.

Description

technical field [0001] The present invention relates to modified coagulation Factor VII (Factor VII) polypeptides having procoagulant activity. It also relates to polynucleotide constructs encoding such polypeptides, vectors and host cells comprising and expressing such polynucleotides, pharmaceutical compositions comprising such polypeptides, and uses and methods of treatment of such polypeptides. [0002] Sequence listing incorporated herein by reference [0003] SEQ ID NO: 1: Wild-type human coagulation factor VII. Background of the invention [0004] Injury to blood vessels activates the hemostatic system, which involves complex interactions between cellular and molecular components. The process that eventually causes the bleeding to stop is called hemostasis. An important part of hemostasis is blood coagulation and clot formation at the site of injury. The coagulation process is highly dependent on the function of several protein molecules. These are called clotti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64A61K38/48A61K47/48
CPCA61K38/4846C12N9/6437C12Y304/21021A61K47/60A61K47/61A61K47/64A61K47/643A61K47/644A61P7/04
Inventor H.R.斯坦尼克O.H.奧森H.奧斯特加亚尔德
Owner NOVO NORDISK HEALTH CARE AG
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