Systems for Factor VIII Processing and Methods Thereof
The present invention provides methods of reducing nonprocessed Factor VIII or a chimeric polypeptide comprising Factor VIII comprising co-transfecting in a host cell a polynucleotide encoding Factor VIII with a polynucleotide encoding a protein convertase, where the endogenous processing enzymes of the host cell are insufficient to convert all of the Factor VIII to its processed isoform; expressing a proprotein convertase from a second polynucleotide in the host cell; and reducing the nonprocessed Factor VIII by processing with said proprotein convertase.
1 . A method for decreasing nonprocessed Factor VIII in a culturing medium, comprising,
a) expressing Factor VIII from a first polynucleotide in a host cell, where the endogenous processing enzymes of the host cell are insufficient to convert all of the Factor VIII to its processed isoform;
b) contacting said Factor VIII with an exogenous proprotein convertase; and
c) decreasing the nonprocessed Factor VIII by processing with said proprotein convertase.
2 . The method of claim 1 , wherein said exogenous proprotein convertase is expressed from a second polypeptide in the host cell expressing said Factor VIII or is added to said culture medium.
3 - 4 . (canceled)
5 . The method of claim 1 , wherein said Factor VIII is processed by cleaving Arginine corresponding to amino acid residue 1648 in SEQ ID NO: 6 (full-length Factor VIII) or amino acid residue 754 in SEQ ID NO: 2 (B domain-deleted Factor VIII).
6 . The method of claim 5 , wherein said processed Factor VIII comprises a Factor VIII heavy chain and a Factor VIII light chain, wherein said heavy chain and light chain are associated by a non-covalent bond.
7 - 8 . (canceled)
9 . The method of claim 1 , wherein said proprotein convertase is selected from the group consisting of proprotein convertase subtilisin/kexin type 3, proprotein convertase subtilisin/kexin type 5, proprotein convertase subtilisin/kexin type 7, a yeast Kex 2 and a combination thereof.
10 - 19 . (canceled)
20 . The method of claim 1 , wherein said Factor VIII is expressed as full-length Factor VIII or partial or full B-domain deleted Factor VIII.
21 - 28 . (canceled)
29 . The method of claim 1 , wherein said Factor VIII is fused to an immunoglobulin constant region or a portion thereof.
30 - 34 . (canceled)
35 . The method of claim 1 , wherein said first polynucleotide and said second polynucleotide are located on the same vector or on two different vectors.
36 - 39 . (canceled)
40 . The method of claim 1 , wherein said host cell is a CHO cell, a HEK293 cell, a HKB11 cell, or a BHK cell.
41 . A host cell comprising an expression vector comprising a first polynucleotide sequence encoding Factor VIII and a second polynucleotide sequence encoding a proprotein convertase, wherein said Factor VIII is processed by said proprotein convertase.
42 . The host cell of claim 41 , wherein said first and second polynucleotides are contained in a first and second expression vectors, respectively.
43 . The host cell of claim 41 , wherein said proprotein convertase cleaves Factor VIII at Arginine located at corresponding to amino acid residue 1648 in SEQ ID NO: 6 (full-length Factor VIII) or amino acid residue 754 in SEQ ID NO: 2 (BDD Factor VIII).
44 . The host cell of claim 41 , wherein said Factor VIII contains more than 75%, 80%, 85%, 90%, 95%, and 100% of the processed Factor VIII after processing by said proprotein convertase.
45 - 47 . (canceled)
48 . The host cell of claim 41 , wherein said proprotein convertase is selected from the group consisting of proprotein convertase subtilisin/kexin type 3, proprotein convertase subtilisin/kexin type 5, proprotein convertase subtilisin/kexin type 7, a yeast Kex 2 and a combination thereof.
49 - 67 . (canceled)
68 . The host cell of claim 41 , wherein said host cell is a CHO cell, a HEK293 cell, a HKB11 cell, or a BHK cell.
69 . A method of producing Factor VIII, comprising
a) culturing the host cell of claim 41 in a culture medium that expresses said Factor VIII and said proprotein convertase, and
b) processing said Factor VIII by said protein convertase.
70 . (canceled)
71 . An expression vector comprising a first polynucleotide encoding Factor VIII and a second polynucleotide encoding a proprotein convertase, wherein said Factor VIII is processed by the proprotein convertase when said Factor VIII and said proprotein convertase are expressed in a host cell comprising said vector.
72 . The vector of claim 71 , wherein said proprotein convertase cleaves Factor VIII at Arginine corresponding to amino acid residue 1648 in SEQ ID NO: 6 (full-length Factor VIII) or amino acid residue 754 in SEQ ID NO: 2 (B-domain deleted Factor VIII) when said proprotein convertase is expressed in a host cell comprising said vector.
73 - 86 . (canceled)
87 . A method of producing Factor VIII, comprising
a) transfecting the expression vector of claim 71 in a host cell,
b) culturing said host cell in a culture medium to express said Factor VIII and said proprotein convertase, wherein said proprotein convertase processes said Factor VIII.
88 . (canceled)
89 . An isolated polypeptide comprising Factor VIII produced by the method of claim 1 .