Cell line expressing single chain factor viii polypeptides and uses thereof

Abstract

The present invention provides cell lines for producing single chain FVIII polypeptides, e.g., chimeric single chain FVIII polypeptides, methods of producing single chain FVIII polypeptides, single chain FVIII polypeptides, and methods of treating Hemophilia A with a single chain Factor VIII polypeptide.

1 . An isolated cell line comprising a recombinant vector encoding a single chain FVIII polypeptide, which comprises at least two amino acid substitutions at R1645 and R1648 corresponding to full-length Factor VIII. 
     
     
         2 . The cell line of  claim 1 , wherein the at least two amino acid substitutions correspond to R1645A and R1648A. 
     
     
         3 - 5 . (canceled) 
     
     
         6 . The cell line of  claim 1 , wherein the single chain FVIII polypeptide comprises a full or partial deletion of the B domain. 
     
     
         7 . The cell line of  claim 1 , wherein the single chain FVIII polypeptide comprises: Formula I: (A1)-(A2)-[B]-(A3)-(C1)-(C2);
 wherein, a) A1 is an A1 domain of FVIII; b) A2 is an A2 domain of FVIII; c) [B] is a B domain of FVIII, a fragment thereof, or is deleted; d) A3 is an A3 domain of FVIII; e) C1 is a C1 domain of FVIII; and f) C2 is a C2 domain of FVIII; and   wherein the single chain FVIII polypeptide exhibits procoagulation activity.   
     
     
         8 . (canceled) 
     
     
         9 . The cell line of  claim 1 , wherein the single chain FVIII polypeptide comprises a sequence at least 90% or 95% identical to a Factor VIII amino acid sequence comprising SEQ ID NO:8. 
     
     
         10 . The cell line of  claim 1 , wherein the single chain FVIII polypeptide further comprises a heterologous moiety. 
     
     
         11 - 19 . (canceled) 
     
     
         20 . A single chain FVIII polypeptide comprising at least two amino acid substitutions in the processing domain which inhibits cleavage by a FVIII processing enzyme, wherein the at least two amino acid substitutions are at R1645 and R1648 corresponding to full-length Factor VIII. 
     
     
         21 - 23 . (canceled) 
     
     
         24 . The single chain FVIII polypeptide of  claim 20 , comprising a full or partial deletion of the B domain. 
     
     
         25 . The single chain FVIII polypeptide of  claim 20 , comprising Formula I: (A1)-(A2)-[B]-(A3)-(C1)-(C2);
 wherein, a) A1 is an A1 domain of FVIII; b) A2 is an A2 domain of FVIII; c) [B] is a B domain of FVIII, a fragment thereof, or is deleted; d) A3 is an A3 domain of FVIII; e) C1 is a C1 domain of FVIII; and f) C2 is a C2 domain of FVIII; and   wherein the single chain FVIII polypeptide exhibits procoagulation activity.   
     
     
         26 . The single chain FVIII polypeptide of  claim 25 , wherein the A1 domain and the A3 domain are connected by a metal ion mediated interaction. 
     
     
         27 . The single chain FVIII polypeptide of  claim 20 , wherein the single chain FVIII polypeptide comprises a sequence at least 90% or 95% identical to a Factor VIII amino acid sequence comprising SEQ ID NO:8. 
     
     
         28 . The single chain FVIII polypeptide of  claim 20 , wherein the single chain FVIII polypeptide further comprises a heterologous moiety. 
     
     
         29 - 32 . (canceled) 
     
     
         33 . A method for producing a single chain FVIII polypeptide comprising culturing the cell line of  claim 1  under conditions sufficient for production of the single chain FVIII polypeptide. 
     
     
         34 - 35 . (canceled) 
     
     
         36 . The method of  claim 33 , wherein the cell line is a mammalian cell line. 
     
     
         37 . The method according to  claim 36  wherein the mammalian cell line is selected from the group consisting of HEK293 cells, CHO cells, BHK cells, and HeLa cells. 
     
     
         38 . A polynucleotide encoding the single chain FVIII polypeptide of  claim 20 . 
     
     
         39 . A vector comprising the polynucleotide of  claim 38 . 
     
     
         40 . A composition comprising the single chain FVIII polypeptide of  claim 20  and a pharmaceutically acceptable carrier. 
     
     
         41 . A method of preventing, treating, ameliorating, or managing a clotting disease or condition in a patient in need thereof by administering an effective amount of the composition of  claim 40 . 
     
     
         42 . A method for diagnosing or imaging a clotting disease or condition in a patient with the composition of  claim 40 .