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 hemostasia using these clotting factors.
1 . A chimeric clotting factor which comprises
i) a clotting factor selected from the group consisting of FVII, FIX and FX,
ii) a targeting moiety which binds to platelets and, optionally,
iii) a spacer moiety between the clotting factor and the targeting moiety.
2 . The chimeric clotting factor of claim 1 , which comprises a structure represented by the formula A B C, wherein A is the clotting factor; wherein B is the optional spacer moiety; and wherein C is the targeting moiety which binds to platelets.
3 . The chimeric clotting factor of claim 2 , which comprises a structure from amino terminus to carboxy terminus represented by a formula selected from the group consisting of: A B C and C B A.
4 . The chimeric clotting factor of claim 1 , wherein said chimeric clotting factor exhibits increased generation of thrombin in the presence of platelets as compared to an appropriate control lacking the targeting moiety.
5 . The chimeric clotting factor of claim 1 , further comprising a scaffold moiety and, optionally, a second spacer moiety.
6 . The chimeric clotting factor of claim 2 , further comprising D and E, wherein D is an optional second spacer moiety; and E is a scaffold moiety and wherein the chimeric clotting factor comprises a structure from amino terminus to carboxy terminus represented by a formula selected from the group consisting of: A B C D E; A D E B C; E D A B C; C B A D E; E D C B A; and C B E D A.
7 . The chimeric clotting factor of claim 6 , wherein E is a dimeric Fc region comprising a first Fc moiety, F1 and a second Fc moiety, F2.
8 . The chimeric clotting factor of claim 7 , which comprises a cleavable scFc (cscFc) linker interposed between the two Fc moieties.
9 - 17 . (canceled)
18 . The chimeric clotting factor of claim 7 , wherein the chimeric clotting factor has a structure selected from the group consisting of: A linked to F1 via a spacer moiety and C linked to F2; A linked to F1 and C linked to F2; A linked to F1 and C is linked to F2 via a spacer moiety; and A linked to F1 via a spacer moiety and C is linked to F2 via a spacer moiety.
19 . The chimeric clotting factor of claim 18 , which comprises two polypeptides wherein the first polypeptide comprises the moieties A B F1; A B F1; A B F1; or A B F1 D C and the second polypeptide comprises the moieties C F2; C D F2; F2 D C; or F2 D C, wherein the two polypeptide chains form an Fc region.
20 - 21 . (canceled)
22 . The chimeric clotting factor of claim 7 , wherein the targeting moiety is fused to at least one of the F1 and F2 moieties via a spacer moiety.
23 . The chimeric clotting factor of claim 22 , wherein the spacer moiety comprises a cleavable linker.
24 . 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.
25 . The chimeric clotting factor of claim 1 , wherein the targeting moiety binds to resting platelets or activated platelets.
26 . (canceled)
27 . The chimeric clotting factor of claim 25 , wherein the targeting moiety selectively binds to a target selected from the group consisting of: GPIba, GPVI, the nonactive form of GPIIb/IIIa, an active form of GPIIb/IIIa, P selectin, GMP-33, LAMP-1, LAMP-2, CD40L, LOX-1, and a GPIb complex.
28 - 29 . (canceled)
30 . The chimeric clotting factor of claim 25 wherein the targeting moiety is a peptide selected from the group consisting of PS4, OS1, and OS2 or an antibody variable region from an antibody selected from the group consisting of SCE5, MB9, and AP3.
31 . (canceled)
32 . The chimeric clotting factor of claim 1 wherein the clotting factor is Factor VII, or a high specific activity variant of Factor VII.
33 . (canceled)
34 . The chimeric clotting factor of claim 1 wherein the clotting factor is Factor IX or a high specific activity variant of Factor VII.
35 . (canceled)
36 . The chimeric clotting factor of claim 1 wherein the clotting factor is Factor X or a high specific activity variant of Factor X.
37 . (canceled)
38 . The chimeric clotting factor of claim 1 , wherein the clotting factor is secreted by a cell in active form or is activated in vivo.
39 . (canceled)
40 . The chimeric clotting factor of claim 1 , wherein the chimeric clotting factor comprises a heterologous enzymatic cleavage site not naturally present in the clotting factor.
41 - 43 . (canceled)
44 . A chimeric clotting factor comprising FVII and a heterologous enzymatic cleavage site activatable by a component of the clotting cascade.
45 . The chimeric clotting factor of claim 44 , which comprises a scaffold moiety and, optionally, a spacer moiety.
46 . The chimeric clotting factor of claim 44 , wherein the scaffold moiety is a dimeric Fc region comprising a first Fc moiety, F1, and a second Fc moiety, F2.
47 . (canceled)
48 . The chimeric clotting factor of claim 46 , wherein the chimeric clotting factor has a structure selected from the group consisting of: the clotting factor linked to F1 via a spacer moiety; the clotting factor linked to F2 via a spacer moiety; the clotting factor is directly linked to F1; and the clotting factor is directly linked to F2.
49 . The chimeric clotting factor of claim 44 , further comprising a targeting moiety, C.
50 . The chimeric clotting factor of claim 44 , wherein the chimeric clotting factor is synthesized as a single polypeptide chain comprising a cscFc linker.
51 . The chimeric clotting factor of claim 50 , wherein the cscFc linker is adjacent to the heterologous enzymatic cleavage site which results in cleavage of the linker.
52 . The chimeric clotting factor of claim 51 , wherein the heterologous enzymatic cleavage site is an intracellular processing site.
53 . The chimeric clotting factor of claim 50 , wherein the cscFc linker is flanked by two enzymatic cleavage sites which are recognized by the same or by different enzymes.
54 . The chimeric clotting factor of claim 51 , wherein the cscFc linker has a length of about 10 to about 50 amino acids or about 20 to about 30 amino acids.
55 - 58 . (canceled)
59 . The chimeric clotting factor of claim 44 , wherein the FVII is a high specific activity variant of Factor VII.
60 . (canceled)
61 . The chimeric clotting factor of claim 44 , wherein the heterologous enzymatic cleavage site is selected from the group consisting of: a factor XIa cleaveage site, a factor Xa cleavage site, and a thrombin cleavage site.
62 . The chimeric clotting factor of claim 44 , wherein the heterologous enzymatic cleavage site is genetically fused to the amino terminus of the heavy chain moiety of the clotting factor.
63 . The chimeric clotting factor of claim 49 , wherein the targeting moiety binds to resting platelets or activated platelets.
64 . (canceled)
65 . The chimeric clotting factor of claim 63 , wherein the targeting moiety selectively binds to a target selected from the group consisting of: GPIba, GPVI, a nonactive form of GPIIb/IIIa, an active form of GPIIb/IIIa, P selectin, GMP-33, LAMP-1, LAMP-2, CD40L, LOX-1, and a GPIb complex.
66 - 70 . (canceled)
71 . A nucleic acid molecule encoding the chimeric clotting factor of claim 1 .
72 . An expression vector comprising the nucleic acid molecule of claim 71 .
73 . (canceled)
74 . A host cell comprising the expression vector of claim 72 .
75 - 77 . (canceled)
78 . A method for producing a chimeric clotting factor comprising culturing the host cell of claim 74 in culture medium and recovering the chimeric clotting factor from the medium.
79 . A processed, heterodimeric polypeptide comprising two polypeptide chains, wherein said processed, heterodimeric polypeptide is made by expressing the vector of claim 78 in cell culture medium and isolating the mature, heterodimeric polypeptide from the culture medium.
80 . A composition comprising the chimeric clotting factor of claim 1 and a pharmaceutically acceptable carrier.
81 . (canceled)
82 . A method for improving hemostasis in a subject, comprising administering the composition of claim 80 .
83 . The chimeric clotting factor of claim 49 , wherein the targeting moiety is a peptide selected from the group consisting of PS4, OS1, and OS2, or an antibody variable region from an antibody selected from the group consisting of SCE5, MB9, and AP3.
84 . A nucleic acid molecule encoding the chimeric clotting factor of claim 44 .
85 . An expression vector comprising the nucleic acid molecule of claim 84 .
86 . A host cell comprising the expression vector of claim 85 .
87 . A composition comprising the chimeric clotting factor of claim 44 and a pharmaceutically acceptable carrier.
88 . A method for improving hemostasis in a subject, comprising administering the composition of claim 87 .