Processable single chain molecules and polypeptides made using same
The present invention features inter alia nucleic acid molecules which encode polypeptides comprising a single chain Fc region and the polypeptides they encode. The Fc moieties of these constructs are linked by a cleavable scFc linker which is adjacent to at least one enzymatic cleavage site, e.g., an intracellular processing site. The resulting processed molecules comprise two polypeptide chains and substantially lack the extraneous amino acid sequence found in single chain Fc linker molecule. Methods of making and using these dimeric molecules are also described.
1. A polypeptide comprising a first biologically active moiety and a second biologically active moiety, wherein the polypeptide comprises a formula selected from the group consisting of: A-F1-B-P1-L-P2-F2 and A-F1-P1-L-P2-B-F2 in linear sequence from amino to carboxy terminus,
wherein A is the first biologically active moiety, which comprises a clotting factor selected from the group consisting of FVII, FVIIa, and a portion thereof, wherein the clotting factor has pro-clotting activity;
B is the second biologically active moiety, which comprises an antibody or antigen-binding fragment thereof;
P1 and P2 are the enzymatic cleavage sites in a cscFc linker;
L is the peptide linker in the cscFc linker; and
F1 and F2 are the Fc moieties, and
wherein the first biologically active moiety and the second biologically active moiety are different biologically active moieties.
2. The polypeptide of claim 1 , wherein P1 and P2 are recognized by different enzymes.
3. The polypeptide of claim 2 , wherein at least one of P1 or P2 comprises an amino acid sequence selected from: RRRR (SEQ ID NO: 40), RKRRKR (SEQ ID NO: 39), RRRRS (SEQ ID NO: 38), TQSFNDFTR (SEQ ID NO: 7), SVSQTSKLTR (SEQ ID NO: 8), DFLAEGGGVR (SEQ ID NO: 9), TTKIKPR (SEQ ID NO: 10), LVPRG (SEQ ID NO:35), and ALRPR (SEQ ID NO: 94).
4. The polypeptide of claim 1 , wherein the cscFc linker has a length of about 1 to about 50 amino acids.
5. The polypeptide of claim 1 , wherein the cscFc linker comprises a gly/ser peptide.
6. The polypeptide of claim 5 , wherein the gly/ser peptide is of the formula S(Gly 4 Ser) 6 (SEQ ID NO: 26) or S(Gly 4 Ser) 4 (SEQ ID NO: 97).
7. A polypeptide comprising two polypeptide chains, wherein the first polypeptide chain comprises a light chain of a clotting factor linked to a first Fe moiety and the second polypeptide chain comprises a heavy chain of a clotting factor linked to a second Fc moiety, wherein the light chain and the heavy chain associate to form an enzymatically active clotting factor.
8. The polypeptide of claim 7 , wherein the clotting factor is selected from the group consisting of FVII, FVIIa, FIX, FIXa, FX, and FXa.
9. A composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.
10. A composition comprising the polypeptide of claim 7 and a pharmaceutically acceptable carrier.
11. The polypeptide of claim 1 , wherein the second biologically active moiety comprises an antigen-binding fragment of an antibody.
12. The polypeptide of claim 1 , wherein the second biologically active moiety comprises a F(ab) or a scFv.
13. The polypeptide of claim 1 , wherein the second biologically active moiety comprises a scFv.
14. The polypeptide of claim 1 , wherein the cscFc linker is cleaved by a thrombin.
15. The polypeptide of claim 1 , which is present in a cell in vitro.
16. The polypeptide of claim 1 , which is produced in a cell.
17. The polypeptide of claim 1 , wherein the first biologically active moiety comprises FVIIa and the second biologically active moiety comprises a scFv.
18. A method of treating a hemostatic disorder in a subject in need thereof, comprising administering to the subject the polypeptide of claim 1 .
19. A method of treating a hemostatic disorder in a subject in need thereof, comprising administering to the subject the polypeptide of claim 7 .
20. The method of claim 18 , wherein the hemostatic disorder comprises a hemophilia.
21. The method of claim 19 , wherein the hemostatic disorder comprises a hemophilia.