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.

1 - 7 . (canceled) 
     
     
         8 . A composition of Factor VII polypeptides, wherein the Factor VII polypeptides comprise the amino acid sequence of SEQ ID NO: 16 and the ratio of moles of conjugated sialic acid to moles of N-linked glycan in the composition is within a range selected from the group consisting of (a) from 1 to 5; (b) from 1 to 4; (c) from 1 to 3; (d) from 1 to 2; and (e) from 0.5 to 1. 
     
     
         9 - 13 . (canceled) 
     
     
         14 . A method of preparing an isolated desialylated Factor VII polypeptide, said method comprising
 (1) producing a Factor VII polypeptide 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 selected from the group consisting of a) less than 0.05, b) less than 0.1, c) less than 1.0, d) less than 2.0, e) less than 3.0, f) less than 4.0, g) less than 5.0; and h) less than 6.0; and   (2) isolating the desialylated Factor VII polypeptide thereby produced.   
     
     
         15 . The method of  claim 14 , wherein the Factor VII polypeptide comprises the wild-type Factor VII sequence of SEQ ID NO: 16. 
     
     
         16 . The method of  claim 14 , wherein the Factor VII polypeptide comprises a variant sequence comprising two or more sequence alterations relative to SEQ ID NO: 16. 
     
     
         17 - 18 . (canceled) 
     
     
         19 . A method of preparing an isolated desialylated Factor VII polypeptide, said method comprising
 (1) generating a recombinant cell line that coexpresses a recombinant Factor VII polypeptide and 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 selected from the group consisting of a) less than 0.05, b) less than 0.1, c) less than 1.0, d) less than 2.0, e) less than 3.0, f ) less than 4.0, g) less than 5.0; and h) less than 6.0; and   (3) isolating the desialylated Factor VII polypeptide thereby produced.   
     
     
         20 . The method of  claim 19 , wherein the Factor VII polypeptide comprises the wild-type Factor VII sequence of SEQ ID NO: 16. 
     
     
         21 . The method of  claim 19 , wherein the Factor VII polypeptide comprises a variant sequence comprising two or more sequence alterations relative to SEQ ID NO: 16. 
     
     
         22 - 26 . (canceled) 
     
     
         27 . A method for treating a mammal having a disease or a disorder wherein blood clot formation is desirable, said method comprising administering to the mammal an effective amount of the composition of Factor VII polypeptides of  claim 8 . 
     
     
         28 . (canceled) 
     
     
         29 . A method for treating a mammal having 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 a composition comprising isolated desialylated Factor VII polypeptides prepared by the method of  claim 14 . 
     
     
         30 . A method for treating a mammal having 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 a composition comprising isolated desialylated Factor VII polypeptides prepared by the method of  claim 19 . 
     
     
         31 - 44 . (canceled) 
     
     
         45 . A pharmaceutical composition comprising the composition of Factor VII polypeptides of  claim 8  and a pharmaceutically acceptable excipient. 
     
     
         46 . (canceled) 
     
     
         47 . A pharmaceutical composition comprising isolated desialylated Factor VII polypeptides prepared by the method of  claim 14  and a pharmaceutically acceptable excipient. 
     
     
         48 . A pharmaceutical composition comprising isolated desialylated Factor VII polypeptides prepared by the method of  claim 19  and a pharmaceutically acceptable excipient. 
     
     
         49 - 50 . (canceled) 
     
     
         51 . The method of  claim 27 , 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, and over administration of an anticoagulant. 
     
     
         52 . The method of  claim 29 , 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, and over administration of an anticoagulant. 
     
     
         53 . The method of  claim 30 , 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, and over administration of an anticoagulant.