PC5 as a factor IX propeptide processing enzyme
View Patent ↗Compositions and methods for preparing Factor IX, Factor IX-containing fusion proteins, and Factor IX-containing conjugates with processing of Factor IX propeptide by PC5, are provided. In one embodiment PC5 is used to process a precursor polypeptide for a Factor IX-Fc monomer-dimer hybrid.
1. An isolated host cell comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a proprotein of Factor IX (proFIX) or a fusion protein comprising said proFIX, and the second polynucleotide encodes a functional PC5.
2. The host cell of claim 1 , wherein the functional PC5 comprises SEQ ID NO: 1.
3. The host cell of claim 1 , wherein the fusion protein comprises proFIX fused to a heterologous polypeptide.
4. The host cell of claim 3 , wherein the fusion protein further comprises a linker connecting the proFIX and the heterologous polypeptide.
5. The host cell of claim 4 , wherein said linker is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
6. The host cell of claim 3 , wherein the heterologous polypeptide is selected from the group consisting of an FcRn binding partner, albumin, and transferrin.
7. The host cell of claim 6 , wherein said FcRn binding partner comprises Fc.
8. The host cell of claim 7 , wherein said Fc is dimerized to a second Fc, forming proFIX-Fc monomer-dimer hybrid.
9. The host cell of claim 1 , wherein the proFIX or fusion protein comprising said proFIX is modified, wherein said modification is selected from the group consisting of glycosylation, acetylation, phosphorylation, PEGylation, addition of a lipid moiety, and addition of any organic or inorganic molecule.
10. The host cell of claim 1 , wherein the first polynucleotide and the second polynucleotide are in separate vectors.
11. The host cell of claim 1 , wherein the first polynucleotide and the second polynucleotide are in a single vector.
12. The host cell of claim 1 , which is a prokaryotic cell.
13. The host cell of claim 12 , wherein the prokaryotic cell is E. coli.
14. The host cell of claim 1 , which is an eukaryotic cell.
15. The host cell of claim 14 , wherein said cell is selected from the group consisting of a HEK293 cell, a CHO cell, a HeLa cell, and a BHK cell.
16. A method of producing mature Factor IX or a fusion protein comprising mature Factor IX comprising culturing the host cell of claim 1 under conditions that allow expression of the proFIX or the fusion protein comprising said proFIX and the functional PC5 such that, upon expression, said PC5 cleaves proFIX or the fusion protein comprising said proFIX.
17. The method of claim 16 , wherein said method increases yield of the mature Factor IX or fusion protein comprising mature Factor IX compared to the yield of mature Factor IX or a fusion protein comprising mature Factor IX produced under similar conditions without processing by the functional PC5.
18. The method of claim 17 , wherein the functional PC5 comprises SEQ ID NO: 1.
19. The method of claim 17 , wherein the fusion protein comprises a proFIX fused to a heterologous polypeptide.
20. The method of claim 19 , wherein the heterologous polypeptide is selected from the group consisting of an FcRn binding partner, albumin, and transferrin.
21. The method of claim 20 , wherein said FcRn binding partner comprises Fc.
22. The method of claim 21 , wherein said Fc is dimerized to a second Fc, forming proFIX-Fc monomer-dimer hybrid.
23. The method of claim 17 , wherein the proFIX or fusion protein comprising proFIX is modified, wherein said modification is selected from the group consisting of glycosylation, acetylation, phosphorylation, PEGylation, addition of a lipid moiety, and addition of any organic or inorganic molecule.