Recombinant factor VIII proteins

• US 10,370,430 B2
• Filed: 02/15/2013
• Issued: 08/06/2019
• Est. Priority Date: 02/15/2012
• Status: Active Grant

• Current Assignee: Bioverativ Therapeutics Inc. (Sanofi )
• Original Assignee: Bioverativ Therapeutics Inc. (Sanofi )
• Inventors: Kulman, John
• Primary Examiner(s): Szperka, Michael
• Application Number:
  US14/379,196
• Publication Number:
  US 20160355568A1
• Time in Patent Office: 2,363 Days
• Field of Search: None
• CPC Class Codes: A61K 35/14 Blood; Artificial blood perfluorocarbons A61K31/02; umbilical cord blood A61K35/51; haemoglobin A61K38/42
• CPC Class Codes: A61K 38/00 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 Class Codes: A61K 38/37 Factors VIII
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/43504 from invertebrates
• CPC Class Codes: C07K 14/59 Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/755 Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• CPC Class Codes: C07K 14/76 Albumins
• CPC Class Codes: C07K 14/79 Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.

Claims

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 first half-life extending and a second half-life extending moiety are inserted within the FVIII protein immediately downstream of amino acids which correspond to amino acids in mature native human FVIII selected from the group consisting of;

a amino acids 18 and 1656 of SEQ ID NO;

1;

b amino acids 40 and 1656 of SEQ ID NO;

1;

c amino acids 399 and 1656 of SEQ ID NO;

1;

d amino acids 403 and 1656 of SEQ ID NO;

1;

e amino acids 1656 and 1720 of SEQ ID NO;

1;

f amino acids 1656 and 1900 of SEQ ID NO;

1;

g amino acids 18 and 399 of SEQ ID NO;

1;

h amino acids 26 and 399 of SEQ ID NO;

1; and

i amino acids 40 and 399 of SEQ ID NO;

1;

wherein the first half-life extending moiety or the second half-life extending moiety 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;

(k) an XTEN; and

(l) any combination of (a)-(j); and

wherein the recombinant FVIII protein exhibits procoagulant activity.

2. A composition comprising the recombinant FVIII protein of claim 1 and a pharmaceutically acceptable excipient.

3. The recombinant FVIII protein of claim 1, wherein the first half-life extending moiety is inserted immediately downstream of amino acid 399 of SEQ ID NO:

1, corresponding to mature native human FVIII; and

the second half-life extending moiety is inserted immediately downstream of amino acid 1656 of SEQ ID NO;

1, corresponding to mature native human FVIII.

4. The recombinant FVIII protein of claim 1, wherein the first half-life extending moiety is inserted immediately downstream of amino acid 18 of SEQ ID NO:

1, corresponding to mature native human FVIII; and

the second half-life extending moiety is inserted immediately downstream of amino acid 399 of SEQ ID NO;

1, corresponding to mature native human FVIII.

5. The recombinant FVIII protein of claim 1, wherein the first half-life extending moiety is inserted immediately downstream of amino acid 26 of SEQ ID NO:

1, corresponding to mature native human FVIII; and

the second half-life extending moiety is inserted immediately downstream of amino acid 399 of SEQ ID NO;

1, corresponding to mature native human FVIII.

6. The recombinant FVIII protein of claim 1, wherein the first half-life extending moiety is inserted immediately downstream of amino acid 40 of SEQ ID NO:

1, corresponding to mature native human FVIII; and

the second half-life extending moiety is inserted immediately downstream of amino acid 399 of SEQ ID NO;

1, corresponding to mature native human FVIII.

7. The recombinant FVIII protein of claim 1, wherein the first half-life extending moiety is inserted immediately downstream of amino acid 18 of SEQ ID NO:

1, corresponding to mature native human FVIII; and

the second half-life extending moiety is inserted immediately downstream of amino acid 1656 of SEQ ID NO;

1, corresponding to mature native human FVIII.

8. The recombinant FVIII protein of claim 1, wherein the first half-life extending moiety is inserted immediately downstream of amino acid 40 of SEQ ID NO:

1, corresponding to mature native human FVIII; and

the second half-life extending moiety is inserted immediately downstream of amino acid 1656 of SEQ ID NO;

1, corresponding to mature native human FVIII.

9. The recombinant FVIII protein of claim 1, wherein the first half-life extending moiety is inserted immediately downstream of amino acid 1656 of SEQ ID NO:

1, corresponding to mature native human FVIII; and

the second half-life extending moiety is inserted immediately downstream of amino acid 1720 of SEQ ID NO;

1, corresponding to mature native human FVIII.

10. The recombinant FVIII protein of claim 1, wherein the first half-life extending moiety is inserted immediately downstream of amino acid 403 of SEQ ID NO:

1, corresponding to mature native human FVIII; and

the second half-life extending moiety is inserted immediately downstream of amino acid 1656 of SEQ ID NO;

1, corresponding to mature native human FVIII.

11. The recombinant FVIII protein of claim 1, wherein the first half-life extending moiety is inserted immediately downstream of amino acid 1656 of SEQ ID NO:

1, corresponding to mature native human FVIII; and

the second half-life extending moiety is inserted immediately downstream of amino acid 1900 of SEQ ID NO;

1, corresponding to mature native human FVIII.

14. The recombinant FVIII protein of claim 1, wherein the first half-life extending moiety or the second half-life extending moiety is an albumin.

15. The recombinant FVIII protein of claim 1, wherein the first half-life extending moiety or the second half-life extending moiety is an Fc.

16. The recombinant FVIII protein of claim 1, wherein the first half-life extending moiety or the second half-life extending moiety is an XTEN.

12. 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 first half-life extending moiety is inserted within the FVIII protein immediately downstream of amino acid 26 of SEQ ID NO;

1, corresponding to mature native human FVIII;

wherein a second half-life extending moiety is inserted within the FVIII protein immediately downstream of amino acid 403 of SEQ ID NO;

1, corresponding to mature native human FVIII;

wherein a third half-life extending moiety is inserted within the FVIII protein immediately downstream of amino acid 1656 of SEQ ID NO;

1, corresponding to mature native human FVIII;

wherein the first half-life extending moiety, the second half-life extending moiety, or the third half-life extending moiety 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;

(k) an XTEN; and

(l) any combination of (a)-(j); and

wherein the recombinant FVIII protein exhibits procoagulant activity.

17. The recombinant FVIII protein of claim 12, wherein the first half-life extending moiety, the second half-life extending moiety, or the third half-life extending moiety is an albumin.

18. The recombinant FVIII protein of claim 12, wherein the first half-life extending moiety, the second half-life extending moiety, or the third half-life extending moiety is an Fc.

19. The recombinant FVIII protein of claim 12, wherein the first half-life extending moiety, the second half-life extending moiety, or the third half-life extending moiety is an XTEN.

13. 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 first half-life extending moiety is inserted within the FVIII protein immediately downstream of amino acid 26 of SEQ ID NO;

1, corresponding to mature native human FVIII;

wherein a second half-life extending moiety is inserted within the FVIII protein immediately downstream of amino acid 1656 of SEQ ID NO;

1, corresponding to mature native human FVIII;

wherein a third half-life extending moiety is inserted within the FVIII protein immediately downstream of amino acid 1720 of SEQ ID NO;

1, corresponding to mature native human FVIII;

wherein the first half-life extending moiety, the second half-life extending moiety, or the third half-life extending moiety 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;

(k) an XTEN; and

(l) any combination of (a)-(j); and

wherein the recombinant FVIII protein exhibits procoagulant activity.

20. The recombinant FVIII protein of claim 13, wherein the first half-life extending moiety, the second half-life extending moiety, or the third half-life extending moiety is an XTEN.