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Modified vitamin k-dependent polypeptides

a technology of vitamin k and polypeptides, which is applied in the direction of drug compositions, peptide/protein ingredients, extracellular fluid disorders, etc., can solve the problems of limited viii replacement therapy, and achieve enhanced activity, lowering the dosage of viia, and enhancing membrane binding affinity

Inactive Publication Date: 2009-11-05
NELSESTUEN GARY L
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The patent text describes modifications to vitamin K-dependent polypeptides that enhance their membrane-binding affinity and activity. These modifications include the addition or substitution of certain amino acids in the GLA domain of the polypeptide. The modified polypeptides have improved functions such as lower dosage requirements, reduced frequency of administration, and better treatment of deficiency states. The invention also includes pharmaceutical compositions and host cells that include the modified polypeptides. Overall, the patent text provides new ways to improve the function and effectiveness of vitamin K-dependent polypeptides."

Problems solved by technology

Factor VIII replacement therapy is limited due to development of high-titer inhibitory factor VIII antibodies in some patients.

Method used

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  • Modified vitamin k-dependent polypeptides
  • Modified vitamin k-dependent polypeptides
  • Modified vitamin k-dependent polypeptides

Examples

Experimental program
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example 1

[0081]Factor VII with Enhanced Membrane Affinity and Activity: It has been found that the membrane binding affinity of human blood clotting factor VII can be increased by site-directed mutagenesis. The properties of a P11Q,K33E mutant (referred to herein as Factor VIIQ11E33 or mutant factor VII) have been characterized. Membrane affinity was increased over wild type protein by about 20-fold. Autoactivation by the mutant was increased by at least 100-fold over that of wild type factor VII. The activated form of VIIQ11E33 (referred to as VIIaQ11E33) displayed about 10-fold higher activity toward factor X. The coagulation activity of VIIaQ11E33 with soluble tissue factor in normal plasma was about 10-fold higher than that of wild type VIIa. Coagulation activity of the zymogen, VIIQ11E33, with normal tissue factor (supplied as a 1:100 dilution of thromboplastin-HS), was 20-fold higher than wild type Factor VII. The degree to which activity was enhanced was dependent on conditions, with ...

example 2

[0102]Purification of Factor VII: Factor VII (wild-type or mutant) was purified by Concanavalin A (Con A), DEAE, and affinity chromatography. Crude media of transfected 293 cells were incubated with Con A resin (Pharmacia) for four hours at 4° C. The resin then was washed with a solution containing 50 mM Tris, pH 7.5, 10 mM benzamidine, 1 mM CaCl2, and 1 mM MgCl2, and factor VII was eluted with 0.2 M D-methyl mannoside, 0.5 M NaCl in 50 mM Tris buffer, pH 7.5. Factor VII was dialyzed against 50 mM Tris, pH 8.0, 50 mM NaCl, 10 mM benzamidine, and 25 mM D-methyl mannoside overnight.

[0103]Dialyzed factor VII then was incubated with DEAE resin (Pharmacia) for one hour and the mixture was packed into a column. The DEAE column was washed with 50 mM Tris, pH 8.0, 10 mM Benzamidine, and 50 mM NaCl, and factor VII was eluted with a gradient from 50 mM to 500 mM NaCl in 50 mM Tris buffer, pH 8.0 at a flow rate of 2 mL / min. Fractions containing factor VII activity were pooled and dialyzed agai...

example 3

[0112]Circulatory Time of Factor VIIQ11E33 in the Rat: Two anesthetized (sodium nembutol) Sprague Dawley rats (325-350 g) were injected with 36 μg of factor VIIQ11E33 at time zero. Injection was through the juggler vein, into which a cannula had been placed. At the times shown in FIG. 7, blood was withdrawn from the carotid artery, into which a cannula had been inserted by surgery. The amount of factor VIIQ11E33 in the circulation was estimated from the clotting time of human factor VII-deficient plasma, to which 1 μL of a 1:10 dilution of the rat plasma was added. A 1:100 dilution of rabbit brain thromboplastin-HS (Sigma Chemical Co.) was used. Coagulation was assessed by the manual tube tilt method as described in Example 1. The amount of factor VII activity in the plasma before injection of VIIQ11E33 was determined and was subtracted as a blank. The concentration of factor VIIQ11E33 in the circulation is given as log nM. A sham experiment in which a third animal received the oper...

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Abstract

The invention provides vitamin K-dependent polypeptides with enhanced membrane binding affinity. These polypeptides can be used to modulate clot formation in mammals. Methods of modulating clot formation in mammals are also described.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. Ser. No. 10 / 828,901, filed on Apr. 21, 2004, which is a continuation of U.S. Ser. No. 09 / 497,591, filed on Feb. 3, 2000, now U.S. Pat. No. 6,747,003, which is a continuation-in-part of U.S. Ser. No. 09 / 302,239, filed on Apr. 29, 1999, now U.S. Pat. No. 6,693,075, which is a continuation-in-part of U.S. Ser. No. 08 / 955,636, filed on Oct. 23, 1997, now U.S. Pat. No. 6,017,882.STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH[0002]Funding for work described herein was provided by grant no. HL15728, awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION[0003]Vitamin K-dependent proteins contain 9 to 13 gamma-carboxyglutamic acid residues (Gla) in their amino terminal 45 residues. The Gla residues are produced by enzymes in the liver that utilize vitamin K to carboxylate the side chains of glutamic acid residues in protein precursors. Vi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/06A61K38/00C07K14/745C12N9/64C12N15/57
CPCA61K38/00Y10S514/843C12N9/647C07K14/745A61P7/02
Inventor NELSESTUEN, GARY L.
Owner NELSESTUEN GARY L
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