SHORT-ACTING FACTOR VII POLYPEPTIDES

Abstract

Short-acting Factor VII peptides are disclosed. A shortened half-life is desirable for treatment of acute bleeding and similar disorders. Modification of the sialylation and/or glycosylation of Factor VII and variants thereof produced peptides useful in treating conditions of acute bleeding.

Description

Claims

1-50. (canceled)

51. An isolated variant Factor VII polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16 and comprising mutation(s) that result in enhanced tissue factor-independent enzymatic activity on activated platelets compared with wild type human FVIIa; wherein the variant Factor VII polypeptide has a ratio of moles of conjugated sialic acid to moles of N-linked glycan between 0 and 2.0.

52. The isolated variant Factor VII polypeptide of claim 51, wherein the variant Factor VII polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 16.

53. The isolated variant Factor VII polypeptide of claim 51, wherein the variant Factor VII polypeptide comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 16.

54. The isolated variant Factor VII polypeptide of claim 51, wherein the variant Factor VII polypeptide has a ratio of moles of conjugated sialic acid to moles of N-linked glycan that is less than 1.0.

55. The isolated variant Factor VII polypeptide of claim 51, wherein the variant Factor VII polypeptide has a ratio of moles of conjugated sialic acid to moles of N-linked glycan that is less than 0.1.

56. A method of preparing the isolated variant Factor VII polypeptide of claim 51, the method comprising (1) obtaining a sialylated Factor VII polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16 and comprising mutation(s) that result in enhanced tissue factor-independent enzymatic activity on activated platelets compared with wild type human FVIIa;(2) contacting the sialylated Factor VII polypeptide with sialidase under conditions such that sufficient amounts of covalently attached sialic acid residues are removed from the sialylated Factor VII polypeptide to produce a desialylated Factor VII polypeptide having a ratio of moles of conjugated sialic acid to moles of N-linked glycan between 0 and 2.0 and(3) isolating the variant Factor VII polypeptide thereby produced.

57. A method of preparing the isolated variant Factor VII polypeptide of claim 51, the method comprising (1) producing a Factor VII polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16 and comprising mutation(s) that result in enhanced tissue factor-independent enzymatic activity on activated platelets compared with wild type human FVIIa in a recombinant cell line that is deficient in its ability to sialylate peptides such that it produces a desialylated Factor VII polypeptide having a ratio of moles of conjugated sialic acid to moles of N-linked glycan between 0 and 2.0; and(2) isolating the variant Factor VII polypeptide thereby produced.

58. A method of preparing the isolated variant Factor VII polypeptide according to claim 51, the method comprising (1) obtaining a recombinant cell line that coexpresses (a) a recombinant Factor VII polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16 and comprising mutation(s) that result in enhanced tissue factor-independent enzymatic activity on activated platelets compared with wild type human FVIIa; and (b) a recombinant sialidase enzyme;(2) culturing said recombinant cell line to allow expression of both the recombinant Factor VII polypeptide and the recombinant sialidase enzyme, wherein said recombinant sialidase enzyme removes sufficient amounts of covalently attached sialic acid residues to produce a desialylated Factor VII polypeptide having a ratio of moles of conjugated sialic acid to moles of N-linked glycan between 0 and 2.0; and(3) isolating the variant Factor VII polypeptide thereby produced.

59. A pharmaceutical composition comprising the isolated variant Factor VII polypeptide of claim 51 and a pharmaceutically acceptable excipient.

60. The pharmaceutical composition of claim 59, wherein the ratio of moles of conjugated sialic acid per mole of N-linked glycan in the composition is from 0.1 to 1.

61. The pharmaceutical composition of claim 59, wherein the ratio of moles of conjugated sialic acid per mole of N-linked glycan in the composition is from 1 to 2.

62. A method for treating a disease or a disorder wherein blood clot formation is desirable, said method comprising administering to a mammal in need thereof an effective amount of the isolated variant Factor VII polypeptide of claim 51, wherein the disease or disorder is selected from the group consisting of a hemorrhage, gastrointestinal bleeding, uncontrolled bleeding, bleeding in a mammal undergoing transplantation or resection or surgery, variceal bleeding, thrombocytopenia, hemophilia, intracranial hemorrhage, aortic aneurysm, over administration of an anticoagulant, penetrating traumatic injury; blunt traumatic injury; bleeding in elective surgery; bleeding in cardiac surgery; bleeding in spinal surgery; orthopedic surgery; neurosurgery; oncology surgery; post-partum surgery; menorrhagia; bleeding in stem cell transplantation; bleeding in liver transplantation; gastrointestinal bleeding; active variceal bleeding in cirrhosis; non variceal bleeding in cirrhosis; diffuse alveolar hemorrhage; aortic aneurysm; intracerebral hemorrhage; traumatic brain injury; brain contusion; reversal of warfarin; reversal of heparin; reversal of anticoagulants; reversal of anti-thrombotics; Factor VII deficiency; burns; prophylaxis in hemophilia patients with inhibitors; partial hepatectomy for non-cirrhotic and cirrhotic patients; acquired hemophilia; idiopathic thrombocytopenic purpura; Glanzmann’s Thrombasthenia; Glanzmann’s Thrombasthenia refractory to platelet transfusion and Bernard-Soulier Syndrome.

63. The method of claim 62, wherein the disease or disorder is a hemorrhage.

64. The isolated variant Factor VII polypeptide of claim 51, characterized by having at least 50% of the activity to promote blood clotting as wild type Factor VII measured under the same conditions.