# US 10,370,430 B2 · Recombinant factor VIII proteins

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- Number: US 10,370,430 B2
- Document ID: US10370430B2
- Status: Granted
- Kind: B2
- Application number: 14/379,196
- Filed: 2013-02-15
- Published: 2016-12-08
- Granted: 2019-08-06
- Earliest claimed: 2012-02-15
- Assignees: Bioverativ Therapeutics Inc.
- Applicants: Bioverativ Therapeutics Inc.
- Inventors: John Kulman
- Web page: https://patentgrep.com/patent/US10370430B2

## CPC

- C07K 14/755: Factors VIII {, e.g. factor VIII C (AHF), factor VIII Ag (VWF)}
- A61K 35/14: Blood; Artificial blood (perfluorocarbons A61K31/02; umbilical cord blood A61K35/51; haemoglobin A61K38/42)
- A61K 38/37: Factors VIII
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K 14/43504: {from invertebrates}
- C07K 14/59: Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/76: Albumins
- C07K 14/79: Transferrins, e.g. lactoferrins, ovotransferrins
- 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)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO 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

### 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 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 1)

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.

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 1)

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.

### Claim 11 (depends on 1)

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.

### Claim 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.

### Claim 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.

### Claim 14 (depends on 1)

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

### Claim 15 (depends on 1)

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

### Claim 16 (depends on 1)

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

### Claim 17 (depends on 12)

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.

### Claim 18 (depends on 12)

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.

### Claim 19 (depends on 12)

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.

### Claim 20 (depends on 13)

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.