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

Coagulation Factor VII polypeptides

a technology of coagulation factor and polypeptide, which is applied in the field of human coagulation factor vii polypeptides, can solve the problems of fibrin clot formation and bleeding, and achieve the effect of increasing tf independent activity and increasing activity

Inactive Publication Date: 2006-09-14
NOVO NORDISK AS
View PDF10 Cites 33 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0168] In a further aspect the present invention relates to a use of a composition comprising wild type human FVIIa and a Factor VII polypeptide with a Tissue Factor binding affinity lower than recombinant wild type human Factor VIIa and substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa, for the preparation of a medicament for the treatment of bleeding episodes in a subject or for the enhancement of the normal haemostatic system.

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

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
  • Coagulation Factor VII polypeptides
  • Coagulation Factor VII polypeptides
  • Coagulation Factor VII polypeptides

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0396] Construction of DNA Encoding FVII-(S43N), FVII-(K62E), FVII-(Q64E), FVII-(I69F), FVII-(K62E / I69A), FVII-(Q64E / I69A), FVII-(Q62E / Q64E / I69A), FVII-(K62E / I69F), FVII-(Q64E / I69F), FVII-(K62E / Q64E / I69F), FVII-(S43C), FVII-(I69C), FVII-(Q64C), FVII-(M306C), FVII-(R277C), FVII-(I69N / F71T), FVII-(F71N / L73T), FVII-(R277N), and FVII-(D309N / L311T):

[0397] DNA constructs encoding FVII-(S43N), FVII-(K62E), FVII-(Q64E), FVII-(I69F), FVII-(K62E / I69A), FVII-(Q64E / I69A), FVII-(Q62E / Q64E / I69A), FVII-(K62E / I69F), FVII-(Q64E / I69F), FVII-(K62E / Q64E / I69F), FVII-(S43C), FVII-(I69C), FVII-(Q64C), FVII-(M306C), FVII-(R277C), FVII-(I69N / F71T), FVII-(F71N / L73T), FVII-(R277N), and FVII-(D309N / L311T) were prepared by site-directed mutagenesis using a supercoiled, double stranded DNA vector with insert of human FVII and two synthetic primers containing the desired mutation. The following primers were used:

For FVII-(S43N):5′-GGACGAAGCTGTTCTGGATTAACTACAGTGATGGGGACCAG-3′(SEQ ID NO:2)5′-CTGGTCCCCATCACTGTAGTTA...

example 2

[0400] Preparation of FVII-(S43N), FVII-(K62E), FVII-(Q64E), FVII-(I69F), FVII-(K62E / I69A), FVII-(Q64E / I69A), FVII-(Q62E / Q64E / I69A), FVII-(K62E / I69F), FVII-(Q64E / I69F), FVII-(K62E / Q64E / I69F), FVII-(S43C), FVII-(I69C), FVII-(Q64C), FVII-(M306C), FVII-(R277C), FVII-(I69N / F71T), FVII-(F71N / L73T), FVII-(R277N), and FVII-(D309N / L311T).

[0401] 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 variant FVII polypeptide. The Factor VII polypeptide was purified as follows:

[0402] Conditioned medium was loaded onto a 50-ml column of Q Sepharose Fast Flow (Pharmacia Biotech) after addition of 5 mM EDTA, and 10 mM Tris, adjustment of pH to 8.0 and adjustment of the conductivity to 10-11 mS / cm by adding water. Elution of the protein was accomplished by a gradient from 10 mM Tris, 50 mM NaCl, pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl2, pH 8.0. The fraction...

example 3

[0403] In Vitro Hydrolysis Assay

[0404] Native (wild-type) Factor VIIa and Factor VIIa variant (both hereafter referred to as “Factor VIIa”) are assayed in parallel to directly compare their specific activities. The assay is 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, is 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 is 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, is used to calculate the ratio between the activities of variant and wild-type Factor VIIa:

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

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

PropertyMeasurementUnit
dissociation constant Kdaaaaaaaaaa
dissociation constant Kdaaaaaaaaaa
dissociation constant Kdaaaaaaaaaa
Login to View More

Abstract

The present invention relates to novel coagulation Factor VII polypeptides, polynucleotide constructs encoding such polypeptides, as well as vectors and host cells comprising and expressing the polynucleotide, pharmaceutical compositions, uses and methods of treatment.

Description

FIELD OF THE INVENTION [0001] The present invention relates to novel human coagulation Factor VII polypeptides, as well as polynucleotide constructs encoding such polypeptides, vectors and host cells comprising and expressing the polynucleotide, pharmaceutical compositions comprising Factor VII polypeptides, uses and methods of treatment. BACKGROUND OF THE INVENTION [0002] Blood coagulation is a process consisting of a complex interaction of various blood components (or factors) that eventually gives rise to a fibrin clot. Generally, the blood components, which participate in what has been referred to as the coagulation “cascade”, are enzymatically inactive proteins (proenzymes or zymogens) that are converted to proteolytic enzymes by the action of an activator (which itself is an activated clotting factor). Coagulation factors that have undergone such a conversion are generally referred to as “active factors”, and are designated by the addition of the letter “a” to the name of the ...

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
Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/04C07H21/04A61K38/36C07K14/745C12N9/64
CPCA61K38/00C07K14/745C12N9/6437C12N9/647C12Y304/21021A61P7/00A61P7/04A61P9/00
Inventor OSTERGAARD, HENRIKBJORN, SORENPERSSON, EGON
Owner NOVO NORDISK AS
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