METHOD OF PRODUCING FACTOR VIII PROTEINS BY RECOMBINANT METHODS
Provided herein are methods and compositions for producing Factor VIII proteins. Such methods include introducing into a cell a nucleic acid molecule encoding a Factor VIII protein operably linked to a promoter, wherein the promoter is characterized by the ability to produce commercially viable Factor VIII protein; and incubating the cell under conditions for producing commercially viable Factor VIII protein. Also provided are nucleic acid molecules which encode a Factor VIII protein operably linked to a Chinese hamster elongation factor 1-a (CHEF1) promoter, which may be used in the methods provided herein.
1 . A method for producing a recombinant Factor VIII protein, comprising the steps of:
a. introducing into a cell a nucleic acid molecule encoding a modified human Factor VIII protein operably linked to a Chinese hamster elongation factor 1-alpha promoter, wherein said promoter is characterized by the ability to produce commercially viable Factor VIII protein and wherein said modification comprises a truncated or deleted B domain, a SQ linker, and a Pace/Furin cleavage site, characterized in that upon Pace/Furin cleavage and secretion the protein becomes a two-chain form comprising an A1-A2 chain and an A3-C1-C2 chain; and
b. incubating the cell under appropriate conditions for producing commercially viable Factor VIII protein.
2 . The method of claim 1 , wherein the cell is a mammalian cell.
3 . The method of claim 1 , wherein said modification further comprises a substitution corresponding to the Phenylalanine residue at 309 with a Serine residue.
4 . The method of claim 1 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites.
5 . A method for identifying a cell expressing commercially viable Factor VIII protein, comprising:
a. introducing into a cell a nucleic acid molecule encoding a modified human Factor VIII protein operably linked to a Chinese hamster elongation factor 1-alpha promoter, wherein said promoter is characterized by the ability to produce commercially viable Factor VIII protein and wherein said modification comprises a truncated or deleted B domain, a SQ linker, and a Pace/Furin cleavage site, characterized in that upon Pace/Furin cleavage and secretion the protein becomes a two-chain form comprising an A1-A2 chain and an A3-C1-C2 chain;
b. incubating the cells under conditions for producing commercially viable Factor VIII protein;
c. selecting clones expressing high levels of FVIII relative to the other clones;
d. amplifying the cells selected from step c.; and
e. identifying at least one subclone expressing a higher level of FVIII relative to those selected in step c.
6 . The method of claim 5 , wherein the cells are mammalian cells.
7 . The method of claim 5 , wherein said modification further comprises a substitution corresponding to the Phenylalanine residue at 309 with a Serine residue.
8 . The method of claim 5 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites.
9 . An isolated nucleic acid comprising a nucleotide sequence encoding a modified human FVIII polypeptide operably linked to a Chinese hamster elongation factor 1-alpha promoter, wherein said promoter is characterized by the ability to produce commercially viable Factor VIII protein and wherein said modification comprises a truncated or deleted B domain, a SQ linker, and a Pace/Furin cleavage site, characterized in that upon Pace/Furin cleavage and secretion the protein becomes a two-chain form comprising an A1-A2 chain and an A3-C1-C2 chain.
10 . The nucleic acid of claim 9 , wherein said modification further comprises a substitution corresponding to the Phenylalanine residue at 309 with a Serine residue.
11 . The nucleic acid of claim 9 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites.
12 . A vector comprising the nucleic acid of claim 9 .
13 . A host cell comprising the vector of claim 12 .
14 . A composition comprising the nucleic acid of claim 9 and a parenterally acceptable vehicle or excipient.
15 . An isolated modified human FVIII polypeptide produced by the method of claim 1 .
16 . A composition comprising the polypeptide of claim 15 and a parenterally acceptable vehicle or excipient.
17 . A method of treating a patient for hemophilia comprising the step of administering to the patient a therapeutically effective amount of the isolated modified human FVIII polypeptide of claim 16 .
18 . A method of treating a patient for hemophilia comprising the step of administering to the patient a therapeutically effective amount of a nucleic acid comprising a nucleotide sequence encoding a modified human FVIII polypeptide operably linked to a Chinese hamster elongation factor 1-alpha promoter, wherein said promoter is characterized by the ability to produce commercially viable Factor VIII protein and wherein said modification comprises a truncated or deleted B domain, a SQ linker, and a Pace/Furin cleavage site, characterized in that upon Pace/Furin cleavage and secretion the protein becomes a two-chain form comprising an A1-A2 chain and an A3-C1-C2 chain.
19 . The method of claim 18 , wherein said modification further comprises a substitution corresponding to the Phenylalanine residue at 309 with a Serine residue.
20 . The method of claim 18 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites.