RECOMBINANT FACTOR VIII PROTEINS
Provided are recombinant Factor VIII proteins, e.g., human Factor VIII proteins with heterologous moieties inserted into flexible permissive loops located in the Factor VIII A domains, while retaining the procoagulant activity of Factor VIII.
1 . A recombinant FVIII protein comprising: a first polypeptide comprising Formula I: (A1)-a1-(A2)-a2-[B]; and a second polypeptide comprising Formula II: a3-(A3)-(C1);
wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer;
wherein, a) A1 is an A1 domain of FVIII; b) A2 is an A2 domain of FVIII; c) [B] is a B domain of FVIII, a fragment thereof, or is deleted; d) A3 is an A3 domain of FVIII; e) C1 is a C1 domain of FVIII; f) a1, a2, and a3 are acidic spacer regions;
wherein the A1 domain comprises a permissive loop-1 (A1-1) region and a permissive loop-2 (A1-2) region;
wherein the A2 domain comprises a permissive loop-1 (A2-1) region and a permissive loop-2 (A2-2) region;
wherein the A3 domain comprises a permissive loop-1 (A3-1) region and a permissive loop-2 (A3-2) region;
wherein a heterologous moiety is inserted into at least one of the regions A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2; and
wherein the recombinant FVIII protein exhibits procoagulant activity.
2 . A recombinant FVIII protein comprising: a first polypeptide comprising Formula I: (A1)-a1-(A2)-a2-[B]; and a second polypeptide comprising Formula II: a3-(A3)-(C1);
wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer;
wherein, a) A1 is an A1 domain of FVIII; b) A2 is an A2 domain of FVIII; c) [B] is a B domain of FVIII, a fragment thereof, or is deleted or optionally not present; d) A3 is an A3 domain of FVIII; e) C1 is a C1 domain of FVIII; f) a1, a2, and a3 are acidic spacer regions;
wherein a heterologous moiety is inserted into a3; and
wherein the recombinant FVIII protein exhibits procoagulant activity.
3 . The recombinant FVIII protein of claim 1 , wherein the first polypeptide and the second polypeptide form a single polypeptide chain comprising the formula (A1)-a1-(A2)-a2-[B]-[a3]-(A3)-(C1).
4 . The recombinant FVIII protein of claim 1 , wherein the second polypeptide comprises the formula [a3]-(A3)-(C1)-(C2), wherein (C2) is a C2 domain of FVIII).
5 - 17 . (canceled)
18 . The recombinant FVIII protein of claim 1 , wherein a heterologous moiety is inserted into at least two of the regions A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2.
19 . The recombinant FVIII protein of claim 1 , wherein a heterologous moiety is inserted immediately downstream of an amino acid which corresponds to an amino acid in mature native human FVIII selected from the group consisting of: amino acid 18 of SEQ ID NO:1, amino acid 22 of SEQ ID NO:1, amino acid 26 of SEQ ID NO:1, amino acid 40 of SEQ ID NO:1, amino acid 216 of SEQ ID NO:1, amino acid 220 of SEQ ID NO:1, amino acid 224 of SEQ ID NO:1, amino acid 399 of SEQ ID NO:1, amino acid 403 of SEQ ID NO:1, amino acid 409 of SEQ ID NO:1, amino acid 599 of SEQ ID NO:1, amino acid 603 of SEQ ID NO:1, amino acid 1711 of SEQ ID NO:1, amino acid 1720 of SEQ ID NO:1, amino acid 1725 of SEQ ID NO:1, amino acid 1900 of SEQ ID NO:1, amino acid 1905 of SEQ ID NO:1, amino acid 1910 of SEQ ID NO:1, and any combination thereof.
20 . The recombinant FVIII protein of claim 2 , wherein an additional heterologous moiety is inserted into a3.
21 . (canceled)
22 . The recombinant FVIII protein of claim 1 , further comprising two, three, four, five, six, seven, eight, or nine additional heterologous moieties.
23 . (canceled)
24 . The recombinant FVIII protein of claim 2 , wherein an additional heterologous moiety is inserted immediately downstream of an amino acid which corresponds to an amino acid in mature native human FVIII selected from the group consisting of: amino acid 18 of SEQ ID NO:1, amino acid 22 of SEQ ID NO:1, amino acid 26 of SEQ ID NO:1, amino acid 40 of SEQ ID NO:1, amino acid 216 of SEQ ID NO:1, amino acid 220 of SEQ ID NO:1, amino acid 224 of SEQ ID NO:1, amino acid 399 of SEQ ID NO:1, amino acid 403 of SEQ ID NO:1, amino acid 409 of SEQ ID NO:1, amino acid 599 of SEQ ID NO:1, amino acid 603 of SEQ ID NO:1, amino acid 1711 of SEQ ID NO:1, amino acid 1720 of SEQ ID NO:1, amino acid 1725 of SEQ ID NO:1, amino acid 1900 of SEQ ID NO:1, amino acid 1905 of SEQ ID NO:1, amino acid 1910 of SEQ ID NO:1, and any combination thereof.
25 . (canceled)
26 . The recombinant FVIII protein of claim 1 , wherein at least one heterologous moiety comprises an element which increases the in vivo half-life of the protein.
27 - 37 . (canceled)
38 . The recombinant FVIII protein of claim 1 , wherein the recombinant protein has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the procoagulant activity of native FVIII.
39 . The recombinant FVIII protein of claim 1 , wherein procoagulant activity is measured by a chromogenic substrate assay, a one stage clotting assay or both.
40 . An isolated nucleic acid comprising a sequence encoding the recombinant FVIII protein of claim 1 .
41 - 45 . (canceled)
46 . A composition comprising the recombinant FVIII protein of claim 1 and a pharmaceutically acceptable excipient.
47 . A method of preventing, treating, ameliorating, or managing a clotting disease or condition in a patient in need thereof by administering an effective amount of the composition of claim 46 .
48 - 69 . (canceled)
70 . The recombinant FVIII protein of claim 1 , wherein the first polypeptide and the second polypeptide are fused to each other, thereby forming a single chain polypeptide.
71 . The recombinant FVIII protein of claim 70 , wherein the single chain polypeptide contains one or more substitutions, deletions, or mutations at residues 1645 and 1648 corresponding to mature full-length FVIII sequence.
72 - 90 . (canceled)
91 . The recombinant FVIII protein of claim 1 , wherein the recombinant FVIII protein comprises a heterologous moiety at an insertion site which corresponds to an amino acid in mature native human FVIII selected from the group consisting of: amino acid 18 of SEQ ID NO:1, amino acid 22 of SEQ ID NO:1, amino acid 26 of SEQ ID NO:1, amino acid 40 of SEQ ID NO:1, amino acid 216 of SEQ ID NO:1, amino acid 220 of SEQ ID NO:1, amino acid 224 of SEQ ID NO:1, amino acid 399 of SEQ ID NO:1, amino acid 403 of SEQ ID NO:1, amino acid 409 of SEQ ID NO:1, amino acid 599 of SEQ ID NO:1, amino acid 603 of SEQ ID NO:1, amino acid 1711 of SEQ ID NO:1, amino acid 1720 of SEQ ID NO:1, amino acid 1725 of SEQ ID NO:1, amino acid 1900 of SEQ ID NO: 1, amino acid 1905 of SEQ ID NO:1, amino acid 1910 of SEQ ID NO:1, and any combination thereof.
92 . The recombinant FVIII protein of claim 91 , wherein a3 comprises an additional heterologous moiety.
93 - 107 . (canceled)
108 . A method of constructing a recombinant FVIII protein comprising designing a polynucleotide sequence encoding the recombinant FVIII protein,
wherein the recombinant FVIII protein comprises: a first polypeptide comprising Formula I: (A1)-a1-(A2)-a2-[B]; and a second polypeptide comprising Formula II: a3-(A3)-(C1);
wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer;
wherein, a) A1 is an A1 domain of FVIII; b) A2 is an A2 domain of FVIII; c) [B] is a B domain of FVIII, a fragment thereof, or is deleted; d) A3 is an A3 domain of FVIII; e) C1 is a C1 domain of FVIII; f) a1, a2, and a3 are acidic spacer regions;
wherein the A1 domain comprises a permissive loop-1 (A1-1) region and a permissive loop-2 (A1-2) region;
wherein the A2 domain comprises a permissive loop-1 (A2-1) region and a permissive loop-2 (A2-2) region;
wherein the A3 domain comprises a permissive loop-1 (A3-1) region and a permissive loop-2 (A3-2) region;
wherein at least one of the regions A1-1, A1-2, A2-1, A2-2, A3-1, A3-2, or a3 comprises a heterologous moiety; and
wherein the recombinant FVIII protein exhibits procoagulant activity.
109 - 110 . (canceled)