Recombinant factor viii proteins

Patent metadata

Field	Value
Publication number	US-2016355568-A1
Application number	US-201314379196-A
Country	US
Kind code	A1
Filing date	Feb 15, 2013
Priority date	Feb 15, 2012
Publication date	Dec 8, 2016
Grant date	—

Abstract

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.

First claim

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 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.

2 - 5 . (canceled)

6 . The recombinant FVIII protein of claim 1 , wherein the A1-1 region corresponds to a region in native mature human FVIII from about amino acid 15 to about amino acid 45 or from about amino acid 18 to about amino acid 41 of SEQ ID NO:1.

7 . (canceled)

8 . The recombinant FVIII protein of claim 1 , wherein the A1-2 region corresponds to a region in native mature human FVIII from about amino acid 201 to about amino acid 232 or from about amino acid 218 to about amino acid 229 of SEQ ID NO:1.

9 . (canceled)

10 . The recombinant FVIII protein of claim 1 , wherein the A2-1 region corresponds to a region in native mature human FVIII from about amino acid 395 to about amino acid 421 or from about amino acid 397 to about amino acid 418 of SEQ ID NO:1.

11 . (canceled)

12 . The recombinant FVIII protein of claim 1 , wherein the A2-2 region corresponds to a region in native mature human FVIII from about amino acid 577 to about amino acid 635 or from about amino acid 595 to about amino acid 607 of SEQ ID NO:1.

13 . (canceled)

14 . The recombinant FVIII protein of claim 1 , wherein the A3-1 region corresponds to a region in native mature human FVIII from about amino acid 1705 to about amino acid 1732 or from about amino acid 1711 to about amino acid 1725 of SEQ ID NO:1.

15 . (canceled)

16 . The recombinant FVIII protein of claim 1 , wherein the A3-2 region corresponds to a region in native mature human FVIII from about amino acid 1884 to about amino acid 1917 or from about amino acid 1899 to about amino acid 1911 of SEQ ID NO:1.

17 - 18 . (canceled)

19 . The recombinant FVIII protein of claim 1 , wherein the 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 188 of SEQ ID NO:1, amino acid 216 of SEQ ID NO:1, amino acid 220 of SEQ ID NO:1, amino acid 221 of SEQ ID NO:1, amino acid 224 of SEQ ID NO:1, amino acid 333 of SEQ ID NO:1, amino acid 336 of SEQ ID NO:1, amino acid 339 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 416 of SEQ ID NO:1, amino acid 442 of SEQ ID NO:1, amino acid 490 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 1796 of SEQ ID NO:1, amino acid 1802 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 1 , wherein the heterologous moiety is inserted into a3.

21 . The recombinant FVIII protein of claim 20 , wherein the heterologous moiety is inserted into a3 immediately downstream of an amino acid which corresponds to amino acid 1656 of SEQ ID NO:1.

22 . The recombinant FVIII protein of claim 1 , further comprising two, three, four, five, six, seven, eight, or nine additional heterologous moieties.

23 - 25 . (canceled)

26 . The recombinant FVIII protein of claim 1 , wherein the heterologous moiety comprises an element which increases the in vivo half-life of the protein, wherein the element is selected from:
(a) albumin; (b) albumin-binding polypeptide; (c) Fc; (d) PAS; (e) the C-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin; (f) polyethylene glycol (PEG); (g) hydroxyethyl starch (HES); (h) albumin-binding small molecules; (i) a clearance receptor, or fragment thereof, wherein the clearance receptor blocks binding of the recombinant FVIII protein to FVIII clearance receptors; (j) a low-density lipoprotein receptor-related protein 1 (LRP1) or a FVIII-binding fragment thereof; and (k) any combination of (a)-(j).

27 - 29 . (canceled)

30 . The recombinant FVIII protein of claim 1 , wherein the heterologous moiety comprises a peptide or polypeptide which enables visualization or localization of the recombinant FVIII protein, wherein the peptide or polypeptide which enables visualization or localization is selected from:
(a) a biotin acceptor peptide; (b) a lipoic acid acceptor peptide; (c) a fluorescent protein; (d) GFP; (e) RFP; (f) YFP; (g) EGFP; (h) EYFP; (i) a cysteine-containing peptide for ligation of a biarsenical dye or for conjugating metastable technetium; (j) 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH); (k) a peptide for conjugating europium clathrates for fluorescence resonance energy transfer (FRET)-based proximity assays; and (l) any combination of (a)-(k).

31 - 39 . (canceled)

40 . An isolated nucleic acid comprising a sequence encoding the recombinant FVIII protein of claim 1 .

41 . An expression vector comprising the nucleic acid of claim 40 .

42 . A host cell comprising the isolated nucleic acid of claim 40 .

43 - 44 . (canceled)

45 . A method of producing a recombinant FVIII protein comprising culturing the host cell of claim 42 under conditions in which the recombinant FVIII protein is expressed.

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 - 110 . (canceled)

111 . The expression vector of claim 41 , wherein the vector is selected from;
(a) a plasmid; (b) a phagemid; (c) a virus; (d) a retrovirus vector; (e) an adenovirus vector; (f) an adeno-associated virus vector; (g) a SV40-type virus vector; (h) a polyomavirus vector; (i) an Epstein-Barr virus vector; (j) a papilloma virus vector; (k) a herpes virus vector; (l) a vaccinia virus vector; (m) a polio virus vector; (n) a Moloney murine leukemia virus vector; (o) a Harvey murine sarcoma virus vector; (p) a murine mammary tumor virus vector; (q) a Rous sarcoma virus vector; and (r) any derivative thereof.

Assignees

• Biogen Idec Inc

Inventors

• Kulman John

Classifications

• A61P7/04

  Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents · CPC title

• C07K14/79

  Transferrins, e.g. lactoferrins, ovotransferrins · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title

• C07K2319/31

  fusions, other than Fc, for prolonged plasma life, e.g. albumin · CPC title