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Human coagulation factor VII polypeptides

a human coagulation factor and polypeptide technology, applied in the field of new drugs, can solve the problems of fibrin clot formation and major bleeding, and achieve the effect of improving the normal haemostatic system

Inactive Publication Date: 2008-01-10
NOVO NORDISK HEALTH CARE AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides Factor VII polypeptides with various substitutions relative to the amino acid sequence of SEQ ID NO:1. These substitutions include replacement of F374 with any other amino acid, and replacement of one or more amino acids selected from the group consisting of L305, S3 14, K157, K337, D334, S3 36, V158, E296, and M298. The invention also provides Factor VII polypeptides with two or more of the above substitutions. The invention further provides Factor VII polypeptides with different numbers of substitutions, ranging from two to eight. The invention further provides Factor VII polypeptides with different combinations of the above substitutions. The invention also provides Factor VII polypeptides with different amino acids at specific positions in the protease domain. The invention further provides Factor VII polypeptides with different residues in the protease domain. The invention also provides Factor VII polypeptides with different residues in the amino acid sequence of SEQ ID NO:1. The invention also provides Factor VII polypeptides with different residues in the amino acid sequence of SEQ ID NO:1. The invention provides Factor VII polypeptides with improved function and stability.

Problems solved by technology

Thrombin finally converts fibrinogen to fibrin resulting in formation of a fibrin clot.
Bleeding is also a major problem in connection with surgery and other forms of tissue damage.

Method used

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  • Human coagulation factor VII polypeptides
  • Human coagulation factor VII polypeptides
  • Human coagulation factor VII polypeptides

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0294] DNA encoding L305V / S314E / F374Y-FVII, L305V / K337A / F374Y-FVII, L305V / S314E / K337A / F374Y-FVII, and V158D / E296V / M298Q / L305V / S314E / K337A / F374Y-FVII.

[0295] DNA constructs encoding L305V / S314E / F374Y-FVII, L305V / K337A / F374Y-FVII, L305V / S314E / K337A / F374Y-FVII were prepared by site-directed mutagenesis using a supercoiled, double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation. The following primers were used (in subsequent PCR reactions if necessary to obtain the desired FVII variant):

For L305V-FVII:(SEQ ID NO:2)5′-CGT GCC CCG GGT GAT GAC CCA GGA C-3′(SEQ ID NQ:3)5′-GTC CTG GGT CAT CAC CCG GGG CAC G-3′For K337A-FVII:(SEQ ID NO:4)5′-CGG ATG GCA GCG CGG ACT CCT GCA AGG G-3′(SEQ ID NO:5)5′-CCC TTG CAG GAG TCC GCG CTG CCA TCC G-3′For V158D-FVII:(SEQ ID NO:6)5′-GTG GGG GGC AAG GAC TGC CCC AAA GGG G-3′(SEQ ID NO:7)5′-CCC CTT TGG GGC AGT CCT TGC CCC CCA C-3′For E296V / M298Q-FVII:(SEQ ID NO:8)5′-GCC ACG GCC CTG GTG CTC CAG GTC CTC AAC ...

example 2

Preparation of L305V / S314E / F374Y-FVII, L305V / K337A / F374Y-FVII, and L305V / S314E / K337A / F374Y-FVII

[0299] BHK cells were transfected essentially as previously described (Thim et al. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBS Lett. 385, 241-243) to obtain expression of the specific FVII variant. The Factor VII variant was purified as follows:

[0300] Conditioned medium was loaded onto a 25-ml column of Q Sepharose Fast Flow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100 and 10 mM Tris, adjustment of pH to 8.0 and adjustment of the conductivity to 10-11 mS / cm by adding water.

[0301] Elution of the protein was accomplished by stepping from 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100, pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl2, 0.1% Triton X-100, pH 8.0. The fractions containing the specific FVII variant were pooled and applied to a 25-ml column containing monoclonal antibody F1A2 (Novo Nordisk, BagsvaErd, Denmark) coupled to CNBr-activated Sepha...

example 3

In Vitro Hydrolysis Assay

[0303] Native (wild-type) Factor Vila and Factor VIIa variant (both hereafter referred to as “Factor VIIa”) were assayed in parallel to directly compare their specific activities. The assay was carried out in a microtiter plate (MaxiSorp, Nunc, Denmark). The chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), final concentration 1 mM, was added to Factor VIIa (final concentration 100 nM) in 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 5 mM CaCl2 and 1 mg / ml bovine serum albumin. The absorbance at 405 nm was measured continuously in a SpectraMax™ 340 plate reader (Molecular Devices, USA). The absorbance developed during a 20-minute incubation, after subtraction of the absorbance in a blank well containing no enzyme, was used to calculate the ratio between the activities of variant and wild-type Factor VIIa:

Ratio=(A405 nm Factor VIIa variant) / (A405 nm Factor VIIa wild-type).

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Abstract

The present invention relates to novel human coagulation Factor VIIa variants having coagulant activity as well as polynucleotide constructs encoding such variants, vectors and host cells comprising and expressing the polynucleotide, pharmaceutical compositions, uses and methods of treatment.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of Ser. No. 10 / 669,537 filed Sep. 24, 2003 and claims benefit of priority under 35 U.S.C. 119 of Danish application no. PA 2002 01423 filed Sep. 25, 2002 and U.S. provisional application No. 60 / 417,927 filed Oct. 11, 2002, the contents of which are fully incorporated herein by reference.FIELD OF THE INVENTION [0002] The present invention relates to novel human coagulation Factor VIIa polypeptides having coagulant activity as well as polynucleotide constructs encoding such polypeptides, vectors and host cells comprising and expressing the polynucleotide, pharmaceutical compositions, uses and methods of treatment. BACKGROUND OF THE INVENTION [0003] Blood coagulation is a process consisting of a complex interaction of various blood components (or factors) that eventually gives raise to a fibrin clot. Generally, the blood components, which participate in what has been referred to as the coagulation “cascad...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027A01H5/00C07H21/00C12N15/63C12N15/82C12N5/06C12N5/08C12N5/10C12P21/00A61K38/00C07K14/745
CPCC07K14/745A61K38/00
Inventor PERSSON, EGONOLSEN, OLE HVILSTED
Owner NOVO NORDISK HEALTH CARE AG
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