# US 7,612,188 B2 · Modified vitamin K. dependent polypeptides

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- Number: US 7,612,188 B2
- Document ID: US7612188B2
- Status: Granted
- Kind: B2
- Application number: 11/555,769
- Filed: 2006-11-02
- Published: 2007-07-12
- Granted: 2009-11-03
- Earliest claimed: 1997-10-23
- Assignees: Regents of the University of Minnesota
- Inventors: Gary L Nelsestuen
- Web page: https://patentgrep.com/patent/US7612188B2

## CPC

- C07K 14/47: from mammals
- A61K 38/16: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof {(enzyme inhibitors A61K38/005)}
- A61K 38/4846: {Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)}
- A61K 45/00: Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K 14/745: Blood coagulation or fibrinolysis factors
- C12N 9/647: {Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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.

## Claims

### Claim 1

An isolated nucleic acid comprising a nucleic acid sequence encoding a vitamin K-dependent polypeptide that increases clot formation comprising a modified GLA domain that enhances membrane binding affinity of said polypeptide relative to a corresponding native vitamin K-dependent polypeptide, wherein said modified GLA domain comprises the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 with two to five amino acid substitutions, which include a glutamine residue substituted at amino acid position 10 and an amino acid substitution at one or more amino acid positions selected from positions 28, 32, 33, 34.

### Claim 2 (depends on 1)

A vector comprising the nucleic acid of claim 1.

### Claim 3 (depends on 2)

A host cell comprising the vector of claim 2.

### Claim 4

An isolated nucleic acid comprising a nucleic acid sequence encoding a mutant Factor VII or VIIa polypeptide that increases clot formation, wherein said polypeptide comprises a modified GLA domain that enhances membrane binding affinity of said polypeptide relative to a corresponding native Factor VII or Factor VIIa polypeptide, said modified GLA domain comprising two to five amino acid substitutions, which include a glutamine residue substituted at amino acid position 10 and an amino acid substitution at one or more amino acid positions selected from positions 28, 32, 33, and 34 of SEQ ID NO:3 or SEQ ID NO:4.

### Claim 5 (depends on 4)

A vector comprising a nucleic acid of claim 4.

### Claim 6 (depends on 5)

A host cell comprising the vector of claim 5.

### Claim 7 (depends on 3)

A method for producing a vitamin K-dependent polypeptide comprising a modified GLA domain that enhances membrane binding affinity of said polypeptide relative to a corresponding native vitamin K-dependent polypeptide, wherein said modified GLA domain comprises the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 with two to five amino acid substitutions, which include a glutamine residue substituted at amino acid position 10 and an amino acid substitution at one or more amino acid positions selected from positions 28, 32, 33, and 34 of SEQ ID NO: 3 or SEQ ID NO: 4, the method comprising (a) culturing a host cell of claim 3 under conditions which permit expression of the polypeptide, and (b) recovering the polypeptide.