Factor VIII-XTEN fusions and uses thereof
The present invention provides chimeric proteins comprising a Factor VIII (FVIII) polypeptide and XTEN polypeptides. The FVIII polypeptide can have a reduced affinity for von Willebrand Factor (VWF). XTEN polypeptides of appropriate sizes are inserted into the FVIII polypeptide at particular locations in order to extend the half-life of the FVIII polypeptide. The invention also includes nucleotides, vectors, host cells, and methods of using the chimeric proteins, e.g., to treat bleeding diseases and disorders.
1. A chimeric protein comprising (i) a Factor VIII (FVIII) polypeptide and (ii) four XTENs wherein the FVIII polypeptide has reduced affinity for von Willebrand Factor (VWF), and wherein the FVIII polypeptide lacks amino acids 746-1685 corresponding to the mature human FVIII polypeptide (SEQ ID NO:4).
2. The chimeric protein of claim 1 , wherein at least three of the four XTENs are inserted immediately downstream of residues selected from the group consisting of:
(a) residues 18, 745, and 2332 corresponding to the mature FVIII polypeptide (SEQ ID NO: 4);
(b) residues 745, 1720 and 2332 corresponding to the mature FVIII polypeptide (SEQ ID NO: 4); and
(c) residues 745, 1900, and 2332 corresponding to the mature FVIII polypeptide (SEQ ID NO: 4).
3. The chimeric protein of claim 1 , wherein at least two of the four XTENs are inserted immediately downstream of residues 745 and 2332 corresponding to the mature FVIII polypeptide (SEQ ID NO: 4).
4. The chimeric protein of claim 1 , wherein at least one of the four XTENs is 36 to 72 amino acids in length or 42 to 78 amino acids in length.
5. The chimeric protein of claim 1 , wherein at least one of the four XTENs is selected from the group consisting of SEQ ID NO:22 (36AE), SEQ ID NO:36 (42AE), SEQ ID NO: 23 (72AE), and SEQ ID NO: 24 (78AE).
6. The chimeric protein of claim 1 , wherein at least one of the four XTENs is selected from the group consisting of SEQ ID NO: 37 (144AE), SEQ ID NO: 38 (144AG), SEQ ID NO: 98 (150AE), SEQ ID NO: 99 (150 AG), and any combination thereof.
7. The chimeric protein of claim 1 , wherein:
(a) an XTEN of SEQ ID NO: 22 (36AE) is inserted immediately downstream of residue 18, an XTEN of SEQ ID NO: 37 (144AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO: 37 (144 AE) is inserted immediately downstream of residue 1900, and an XTEN of SEQ ID NO: 39 (288 AE) is inserted immediately downstream of residue 2332;
(b) an XTEN of SEQ ID NO: 36 (42AE) is inserted immediately downstream of residue 18, an XTEN of SEQ ID NO: 98 (150AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO: 98 (150 AE) is inserted immediately downstream of residue 1900, and an XTEN of SEQ ID NO: 100 (294 AE) is inserted immediately downstream of residue 2332;
(c) an XTEN of SEQ ID NO: 37 (144AE) or SEQ ID NO: 39 (288 AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO: 22 (36AE) is inserted immediately downstream of residue 1720, an XTEN of SEQ ID NO: 37 (144 AE) is inserted immediately downstream of residue 1900, and an XTEN of SEQ ID NO: 39 (288 AE) is inserted immediately downstream of residue 2332;
(d) an XTEN of SEQ ID NO: 98 (150AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO: 36 (42AE) is inserted immediately downstream of residue 1720, an XTEN of SEQ ID NO: 98 (150 AE) is inserted immediately downstream of residue 1900, and an XTEN of SEQ ID NO: 100 (294AE) is inserted immediately downstream of residue 2332;
(e) an XTEN of SEQ ID NO: 22 (36AE) is inserted immediately downstream of residue 18, an XTEN of SEQ ID NO: 37 (144AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO: 22 (36 AE) is inserted immediately downstream of residue 1720, and an XTEN of SEQ ID NO: 39 (288 AE) is inserted immediately downstream of residue 2332; or
(f) an XTEN of SEQ ID NO: 36 (42AE) is inserted immediately downstream of residue 18, an XTEN of SEQ ID NO: 98 (150AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO: 36 (42 AE) is inserted immediately downstream of residue 1720, and an XTEN of SEQ ID NO: 100 (294 AE) is inserted immediately downstream of residue 2332.
8. A polynucleotide or a set of polynucleotides encoding the chimeric protein of claim 1 .
9. A vector or a set of vectors comprising the polynucleotide or the set of polynucleotides of claim 8 and one or more promoter operably linked to the polynucleotide or the set of polynucleotides.
10. A host cell comprising the polynucleotide of claim 8 .
11. A pharmaceutical composition comprising the chimeric protein of claim 1 and a pharmaceutically acceptable carrier.
12. A method of treating a bleeding disease or disorder in a subject in need thereof comprising administering an effective amount of the chimeric protein of claim 1 .
13. A method of making a chimeric protein, comprising transfecting one or more host cells with the polynucleotide of claim 8 and expressing the chimeric protein in the host cell.
14. A method of extending or increasing the half-life of a FVIII protein, wherein the method comprises administering an effective amount of the chimeric protein of claim 1 to a subject in need thereof, wherein the XTEN sequence or sequences extend or increase the half-life of the FVIII protein.
15. The chimeric protein claim 1 , wherein the four XTENs comprise an XTEN of SEQ ID NO: 22 (36AE) is inserted immediately downstream of residue 18, an XTEN of SEQ ID NO: 37 (144AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO: 37 (144 AE) is inserted immediately downstream of residue 1900, and an XTEN of SEQ ID NO: 39 (288 AE) is inserted immediately downstream of residue 2332.
16. The chimeric protein claim 1 , wherein the four XTENs comprise an XTEN of SEQ ID NO: 37 (144AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO: 22 (36AE) is inserted immediately downstream of residue 1720, an XTEN of SEQ ID NO: 37 (144 AE) is inserted immediately downstream of residue 1900, and an XTEN of SEQ ID NO: 39 (288 AE) is inserted immediately downstream of residue 2332.
17. The chimeric protein of claim 1 , wherein the four XTENs comprise an XTEN of SEQ ID NO: 22 (36AE) is inserted immediately downstream of residue 18, an XTEN of SEQ ID NO: 37 (144AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO: 22 (36 AE) is inserted immediately downstream of residue 1720, and an XTEN of SEQ ID NO: 39 (288 AE) is inserted immediately downstream of residue 2332.