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-α (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 deletion of the 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 2 , wherein the mammalian cell is selected from the group consisting of a COS-1, CHO, and HEK 293 cell.
4 . The method of claim 1 , wherein said modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
5 . The method of claim 1 , wherein the Factor VIII protein is produced at a level selected from the group consisting of at least about 20 IU/mL, at least about 30 IU/mL, at least about 40 IU/mL, at least about 50 IU/mL, at least about 60 IU/mL, at least about 70 IU/mL, at least about 80 IU/mL, at least about 90 IU/mL, at least about 100 IU/mL, at least about 110 IU/mL, at least about 120 IU/mL, at least about 130 IU/mL, at least about 140 IU/mL, at least about 150 IU/mL, at least about 160 IU/mL, at least about 170 IU/mL, at least about 180 IU/mL, at least about 190 IU/mL, at least about 200 IU/mL, and at least about 210 IU/mL.
6 . A method for identifying a cell expressing commercially viable Factor VIII protein, comprising:
a) introducing into cells 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 deletion of the 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 in step c); and
e) identifying at least one subclone expressing a higher level of FVIII relative to those selected in step c).
7 . The method of claim 6 , wherein the cells are mammalian cells.
8 . The method of claim 7 , wherein the mammalian cell is selected from the group consisting of a COS-1, CHO, and HEK 293 cell.
9 . The method of claim 6 , wherein said modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
10 . 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 deletion of the 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.
11 . The isolated nucleic acid of claim 10 , wherein said modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
12 . A vector comprising the nucleic acid of claim 10 .
13 . A host cell comprising the vector of claim 12 .
14 . A composition comprising the nucleic acid of claim 10 .
15 . The composition of claim 14 , further comprising a parenterally acceptable vehicle or excipient.
16 . An isolated 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 deletion of the 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.
17 . The modified human FVIII polypeptide of claim 16 , wherein said modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
18 . A composition comprising the polypeptide of claim 16 .
19 . The composition of claim 18 , further comprising a parenterally acceptable vehicle or excipient.
20 . A method for treating a patient for hemophilia comprising the step of administering to the patient a therapeutically effective amount of a nucleic acid comprising the 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 deletion of the 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.
21 . The method of claim 20 , wherein said modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
22 . A method for treating a patient for hemophilia comprising the step of administering to the patient a therapeutically effective amount of a protein comprising 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 deletion of the 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.
23 . The method of claim 22 , wherein said modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
24 . 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 B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site, 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-truncated B domain chain and an A3-C1-C2 chain; and
(b) incubating the cell under conditions for producing commercially viable Factor VIII protein.
25 . The method of claim 24 , wherein the cell is a mammalian cell.
26 . The method of claim 25 , wherein the mammalian cell is selected from the group consisting of a COS-1, CHO, and HEK 293 cell.
27 . The method of claim 24 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites.
28 . The method of claim 24 , wherein said modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
29 . The method of claim 24 , wherein the Factor VIII protein is produced at a level selected from the group consisting of at least about 20 IU/mL, at least about 30 IU/mL, at least about 40 IU/mL, at least about 50 IU/mL, at least about 60 IU/mL, at least about 70 IU/mL, at least about 80 IU/mL, at least about 90 IU/mL, at least about 100 IU/mL, at least about 110 IU/mL, at least about 120 IU/mL, at least about 130 IU/mL, at least about 140 IU/mL, at least about 150 IU/mL, at least about 160 IU/mL, at least about 170 IU/mL, at least about 180 IU/mL, at least about 190 IU/mL, at least about 200 IU/mL, and at least about 210 IU/mL.
30 . A method for identifying a cell expressing commercially viable Factor VIII protein, comprising:
a) introducing into cells 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 B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site, 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-truncated B domain 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 in step c); and
e) identifying at least one subclone expressing a higher level of FVIII relative to those selected in step c).
31 . The method of claim 30 , wherein the cells are mammalian cells.
32 . The method of claim 31 , wherein the mammalian cell is selected from the group consisting of a COS-1, CHO, and HEK 293 cell.
33 . The method of claim 30 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites.
34 . The method of claim 30 , wherein said modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
35 . 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 B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site, 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-truncated B domain chain and an A3-C1-C2 chain.
36 . The isolated nucleic acid of claim 35 , wherein said modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
37 . The isolated nucleic acid of claim 35 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites.
38 . A vector comprising the nucleic acid of claim 35 .
39 . A host cell comprising the vector of claim 38 .
40 . A composition comprising the nucleic acid of claim 35 .
41 . The composition of claim 40 , further comprising a parenterally acceptable vehicle or excipient.
42 . An isolated 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 B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site, 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-truncated B domain chain and an A3-C1-C2 chain.
43 . The modified human FVIII polypeptide of claim 42 , wherein said modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
44 . The modified human FVIII polypeptide of claim 42 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites.
45 . A composition comprising the polypeptide of claim 42 .
46 . The composition of claim 45 , further comprising a parenterally acceptable vehicle or excipient.
47 . A method for treating a patient for hemophilia comprising the step of administering to the patient a therapeutically effective amount of a nucleic acid comprising the 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 B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site, 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-truncated B domain chain and an A3-C1-C2 chain.
48 . The method of claim 47 , wherein said modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
49 . The method of claim 47 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites.
50 . A method for treating a patient for hemophilia comprising the step of administering to the patient a therapeutically effective amount of a protein comprising 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 B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site, 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-truncated B domain chain and an A3-C1-C2 chain.
51 . The method of claim 50 , wherein said modification further comprises a substitution of the Phenylalanine residue at 309 with a Serine residue.
52 . The method of claim 50 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites.