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. A eukaryotic cell comprising a first expression vector encoding a proprotein of Factor IX (proFIX), or a fusion protein of proFIX, and a second expression vector encoding a functional PC5.
2. The eukaryotic cell of claim 1 , wherein the functional PC5 comprises an amino acid sequence provided by SEQ ID NO:1.
3. The eukaryotic cell of claim 1 , wherein the first expression vector encodes proFIX.
4. The eukaryotic cell of claim 1 , wherein the fusion protein is a proFIX-FcRn binding partner fusion protein.
5. The eukaryotic cell of claim 4 , wherein the proFIX-FcRn binding partner fusion protein comprises a linker connecting proFIX to FcRn binding partner.
6. The eukaryotic cell of claim 4 , wherein the proFIX-FcRn binding partner fusion protein is a proFIX-Fc fusion protein.
7. The eukaryotic cell of claim 6 , wherein the proFIX-Fc fusion protein comprises a human Fc gamma.
8. The eukaryotic cell of claim 6 , wherein the proFIX-FcRn binding partner fusion protein comprises a linker connecting proFIX to Fc.
9. The eukaryotic cell of claim 6 , wherein the proFIX-Fc fusion protein is a proFIX-Fc homodimer.
10. The eukaryotic cell of claim 6 , wherein the proFIX-Fc fusion protein is a proFIX-Fc monomer-dimer hybrid.
11. The eukaryotic cell of claim 1 , wherein the fusion protein is a proFIX-albumin fusion protein.
12. The eukaryotic cell of claim 11 , wherein the proFIX-albumin fusion protein comprises a linker connecting proFIX to albumin.
13. The eukaryotic cell of claim 1 , wherein the fusion protein is a proFIX-transferrin fusion protein.
14. The eukaryotic cell of claim 13 , wherein the proFIX-transferrin fusion protein comprises a linker connecting proFIX to transferrin.
15. The eukaryotic cell of claim 1 , wherein the eukaryotic cell is a mammalian cell.
16. The eukaryotic cell of claim 1 , wherein the eukaryotic cell is a HEK293 cell.
17. The eukaryotic cell of claim 1 , wherein the eukaryotic cell is a CHO cell.
18. An expression vector comprising
a first polynucleotide sequence encoding a proprotein of Factor IX (proFIX), or a fusion protein thereof, operably linked to an expression control sequence permitting expression of the proFIX or the fusion protein thereof, and
a second polynucleotide sequence encoding a functional PC5, operably linked to an expression control sequence permitting expression of the functional PC5.
19. A eukaryotic cell comprising the expression vector of claim 18 .
20. A method for producing a mature Factor IX-containing polypeptide from a proprotein of Factor IX (proFIX), or a fusion protein thereof, the method comprising
culturing the eukaryotic cell of claim 1 under conditions that allow:
expression of both the proFIX or the fusion protein thereof and the functional PC5, and
processing of the proFIX or the fusion protein thereof by the functional PC5.
21. A method for increasing yield of a mature Factor IX-containing polypeptide from a proprotein of Factor IX (proFIX), or a fusion protein thereof, the method comprising
culturing the eukaryotic cell of claim 1 under conditions that allow:
expression of both the proFIX or the fusion protein thereof and the functional PC5, and
processing of the proFIX or the fusion protein thereof by the functional PC5,
wherein yield of mature Factor IX-containing polypeptide is increased compared to yield of mature Factor IX-containing polypeptide produced under similar conditions without the processing by the functional PC5.
22. The eukaryotic cell of any one of claims 5 , 8 , 12 , or 14 , wherein the linker is GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:16).