Chimeric clotting factors
Chimeric clotting factors which localize the therapeutic to sites of coagulation (e.g., by being targeted to platelets or being activatable at sites of coagulation), have reduced clearance rates, have improved manufacturability, have reduced thrombogenicity, have enhanced activity, or have more than one of these characteristics are described as are methods for making chimeric clotting factors and methods for improving hemostasis using these clotting factors.
1. A chimeric clotting factor, comprising:
i) a clotting factor comprising a FVII polypeptide,
ii) a targeting moiety which binds to platelets, and
iii) a scaffold moiety comprising an FcRn binding moiety;
wherein the scaffold moiety is a dimeric Fc region comprising a first Fc moiety (F1) and a second Fc moiety (F2); wherein the clotting factor is covalently bonded to the N-terminus of F1 either directly or via a first spacer moiety; and
wherein the targeting moiety is linked to F2 either directly or via a second spacer moiety.
2. The chimeric clotting factor of claim 1 , wherein the clotting factor is fused to F1 via the first spacer moiety or the targeting moiety is fused to F2 via the second spacer moiety.
3. The chimeric clotting factor of claim 2 , which comprises two polypeptides having:
(i) a first polypeptide comprising the clotting factor, the first spacer moiety, and F1, and a second polypeptide comprising the targeting moiety and F2;
(ii) a first polypeptide comprising the clotting factor, the first spacer moiety, and F1, and a second polypeptide comprising the targeting moiety, the second spacer moiety, and F2;
(iii) a first polypeptide comprising the clotting factor and F1, and a second polypeptide comprising the targeting moiety and F2; or
(iv) a first polypeptide comprising the clotting factor and F1, and a second polypeptide comprising the targeting moiety, the second spacer moiety, and F2;
wherein the two polypeptides are linked by a covalent bond.
4. The chimeric clotting factor of claim 1 , wherein the targeting moiety is fused to F2 via the second spacer moiety.
5. The chimeric clotting factor of claim 1 , wherein the second spacer moiety comprises a cleavable linker, and wherein the targeting moiety is fused to F2 via the cleavable linker.
6. The chimeric clotting factor of claim 1 , wherein the targeting moiety is selected from the group consisting of: an antibody molecule, an antigen binding fragment of an antibody molecule, an scFv molecule, a receptor binding portion of a receptor, and a peptide.
7. The chimeric clotting factor of claim 1 wherein the targeting moiety binds to resting platelets or activated platelets.
8. The chimeric clotting factor of claim 7 , wherein the targeting moiety selectively binds to a target selected from the group consisting of: GPIba, GPVI, and the nonactive form of GPIIb/IIIa.
9. The chimeric clotting factor of claim 7 , wherein the targeting moiety selectively binds to a target selected from the group consisting of: the active form of GPIIb/IIIa, P selectin, GMP-33, LAMP-1, LAMP-2, CD40L, and LOX-1.
10. The chimeric clotting factor of claim 7 wherein the targeting moiety is a peptide selected from the group consisting of PS4, OS1, and OS2.
11. The chimeric clotting factor of claim 7 , wherein the targeting moiety comprises an antibody variable region from an antibody selected from the group consisting of SCE5, MB9, and AP3.
12. The chimeric clotting factor of claim 1 , wherein the FVII polypeptide comprises a FVII light chain and a FVII heavy chain, wherein the FVII light chain is directly covalently bonded to the N-terminus of the FVII heavy chain, wherein the C-terminus of the FVII heavy chain is covalently bonded to the N-terminus of F1 by a linker, wherein the targeting moiety is covalently bonded to the N-terminus of F2 by a linker, and wherein the targeting moiety is an antibody variable region from an AP3 antibody.
13. The chimeric clotting factor of claim 12 , wherein F1 and F2 are linked by a disulfide bond.
14. The chimeric clotting factor of claim 1 , wherein the FVII polypeptide comprises a FVII light chain and a FVII heavy chain, wherein the FVII light chain is directly covalently bonded to the N-terminus of the FVII heavy chain, wherein the C-terminus of the FVII heavy chain is covalently bonded to the N-terminus of F1 by a linker, wherein the C-terminus of F1 is covalently bonded to the N-terminus of the targeting moiety by a cleavable single chain Fc (cscFc) linker, wherein the targeting moiety is covalently bonded to the N-terminus of F2 by a linker, and wherein the targeting moiety is a peptide selected from the group consisting of PS4, OS1, and OS2.
15. The chimeric clotting factor of claim 14 , wherein F1 and F2 are linked by a disulfide bond.
16. The chimeric clotting factor of claim 1 , wherein the FVII polypeptide comprises a FVII light chain and a FVII heavy chain, wherein the FVII light chain is directly covalently bonded to the N-terminus of the FVII heavy chain, wherein the C-terminus of the FVII heavy chain is covalently bonded to the N-terminus of F1 by a linker, wherein the C-terminus of F1 is covalently bonded to the N-terminus of F2 by a cleavable single chain Fc (cscFc) linker, wherein the targeting moiety is covalently bonded to the C-terminus of F2 by a linker, and wherein the targeting moiety is a peptide selected from the group consisting of PS4, OS1, and OS2.
17. The chimeric clotting factor of claim 16 , wherein F1 and F2 are linked by a disulfide bond.
18. A composition comprising the chimeric clotting factor of claim 1 and a pharmaceutically acceptable carrier.
19. A method for improving hemostasis in a subject, comprising administering to a subject in need thereof an effective amount of the composition of claim 18 .
20. A nucleic acid molecule encoding the chimeric clotting factor of claim 1 .
21. An expression vector comprising the nucleic acid molecule of claim 20 .
22. A host cell comprising the expression vector of claim 21 .
23. A method for producing a chimeric clotting factor comprising culturing the host cell of claim 22 in culture medium and recovering the chimeric clotting factor from the medium.