# US 2019/0169268 A1 · Glycosylated vwf fusion proteins with improved pharmacokinetics

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- Number: US 2019/0169268 A1
- Document ID: US20190169268A1
- Status: Published application
- Kind: A1
- Application number: 16/302,942
- Filed: 2017-04-26
- Published: 2019-06-06
- Earliest claimed: 2016-05-20
- Assignees: Octapharma AG
- Applicants: Octapharma AG
- Inventors: Christoph KANNICHT; Barbara SOLECKA-WITULSKA; Stefan WINGE; Tilo SCHWIENTEK
- Web page: https://patentgrep.com/patent/US20190169268A1

## CPC

- A61K 38/37: Factors VIII
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- C07K 14/745: Blood coagulation or fibrinolysis factors
- C07K 14/755: Factors VIII {, e.g. factor VIII C (AHF), factor VIII Ag (VWF)}
- C12N 15/62: DNA sequences coding for fusion proteins
- 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 2319/02: containing a signal sequence
- C07K 2319/20: containing a tag with affinity for a non-protein ligand
- C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- C07K 2319/35: containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
- 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
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention relates to a fusion protein comprising a main protein and one or more extension peptides, wherein the amino acid sequence of the main protein is identical or similar to the amino acid sequence of a mammalian protein or a fragment thereof, and said extension peptide comprises a cluster of O-glycosylated amino acids. The extension peptide is identical to a non-repeated sequence of the mammalian protein and/or identical or similar to SEQ ID NO: 1. The main protein is preferably VWF. The fusion protein has an increased half life as compared to the main protein and may be used to increase the half-life of a binding partner, e.g. FVIII. The invention further relates to the complex formed by the fusion protein, a polynucleotide encoding the fusion protein as well as a vector and host cell comprising the polynucleotide.

## Claims

### Claim 1

A fusion protein comprising a main protein and one or more extension peptides, wherein the amino acid sequence of the main protein is identical or similar to the amino acid sequence of a mammalian protein or a fragment thereof, and said extension peptide comprises a cluster of O-glycosylated amino acids.

### Claim 2 (depends on 1)

The fusion protein according to claim 1, wherein the fusion protein has an increased half-life compared to the main protein without extension peptide.

### Claim 3 (depends on 1)

The fusion protein according to claim 1, wherein the amino acid sequence of the one or more extension peptide is identical or similar to a non-repeated amino acid sequence section of said main protein or fragment.

### Claim 4 (depends on 1)

The fusion protein according to claim 1, provided that the one or more extension peptides are not derived from chorionic gonadotropin β-subunit (CG-β), in particular are not identical or similar to the C-terminal peptide (CTP) of CG-β.

### Claim 5 (depends on 1)

The fusion protein according to claim 1, wherein the cluster(s) of O-glycosylated amino acids of the extension peptides contain(s) at least two, preferably at least three, more preferably at least four O-glycosylated amino acids.

### Claim 6 (depends on 1)

The fusion protein according to claim 1, wherein the cluster of O-glycosylation sites contain at least one threonine as O-glycosylated amino acids, preferably said clusters contain both threonine and serine as O-glycosylated amino acids.

### Claim 7 (depends on 1)

The fusion protein according to claim 1, wherein the one or more extension peptides contain at least one O-glycosylation site in ten amino acids, preferably at least one O-glycosylation site in eight amino acids.

### Claim 8 (depends on 1)

The fusion protein according to claim 1, wherein the one or more extension peptides have a length in the range from 20 to 60 amino acids, preferably 22 to 40 amino acids, more preferably 26 to 36 amino acids.

### Claim 9 (depends on 1)

The fusion protein according to claim 1, wherein the one or more extension peptides have a sequence identity of at least 90% to an O-glycosylated peptide of von Willebrand Factor (VWF).

### Claim 10 (depends on 1)

The fusion protein according to claim 1, wherein the one or more extension peptides have a sequence identity of at least 90%, preferably at least 95%, more preferably at least 98%, most preferably at least 100% to an O-glycosylated peptide of VWF, in particular to SEQ ID NO: 1.

### Claim 11

The fusion protein according to comprising at least two copies, preferably at least four copies of the extension peptide.

### Claim 12 (depends on 1)

The fusion protein according to claim 1, wherein an extension peptide is directly or indirectly fused to the C-terminus of the main protein.

### Claim 13 (depends on 1)

The fusion protein according to claim 1, wherein an extension peptide forms the C-terminus of the fusion protein, wherein the C-terminal amino acid of said extension peptide is optionally linked to an affinity tag fusion peptide.

### Claim 14 (depends on 1)

The fusion protein according to claim 1, wherein the main protein comprises at least one cluster of O-glycosylated amino acids.

### Claim 15 (depends on 1)

The fusion protein according to claim 1, wherein the fusion protein comprises a dimerization domain, in particular the main protein comprises a dimerization domain.

### Claim 16 (depends on 1)

The fusion protein according to claim 1, wherein the mammalian protein is a human blood protein, preferably a coagulation factor or a protease inhibitor, more preferably selected from the group consisting of VWF, prothrombin, fibrinogen, FIII, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII, ADAMTS13, antithrombin, alpha-1 antitrypsin, C1-inhibitor, antichymotrypsin, PAI-1, PAI-3, 2-macroglobulin, TFPI, heparin cofactor II Protein Z, Protein C, and Protein S.

### Claim 17 (depends on 1)

The fusion protein according to claim 1, wherein the main protein has a sequence identity of at least 90%, preferably at least 95%, more preferably at least 98%, most preferably 100% identity to amino acids 764 to 1268 of SEQ ID NO: 2.

### Claim 18 (depends on 1)

The fusion protein according to claim 1, wherein the fusion protein comprises at least 4, preferably at least 8, more preferably at least 12 additional O-glycans compared to the main protein.

### Claim 19 (depends on 1)

The fusion protein according to claim 1, wherein the amino acid sequence of the fusion protein has an identity of at least 90%, preferably at least 95%, more preferably at least 98%, most preferably 100% to a sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

### Claim 20 (depends on 1)

The fusion protein according to claim 1, wherein the fusion protein is produced by expression in a mammalian cell line, preferably a human cell line, more preferably a human kidney cell line, most preferably a human embryonic kidney cell line, in particular a HEK293 cell line such as HEK293F.

### Claim 21 (depends on 1)

A composition of a first protein and a second protein, wherein said first protein is a fusion protein according to claim 1 is capable of binding said second protein, and said second protein is a therapeutic protein comprising an amino acid sequence which is identical or similar to the amino acid sequence of a second mammalian protein or fragment thereof.

### Claim 22 (depends on 21)

The composition according to claim 21, wherein the half-life of the second protein bound to the first protein is increased as compared to the free form of said second protein, preferably wherein half-life of the second protein bound to the first protein is increased as compared to said second protein bound to the native mammalian protein or the main protein without fusion peptide.

### Claim 23 (depends on 21)

The composition according to claim 21, wherein the molar ratio of the first protein to the second protein is in the range from 0.1 to 250, preferably in the range from 0.5 to 50 more preferably in the range from 1 to 25, most preferably in the range from 2 to 10.

### Claim 24 (depends on 21)

The composition according to claim 21, wherein the binding affinity of the first protein to the second is in the range from 0.05 to 3 nM.

### Claim 25 (depends on 21)

The composition according to claim 21, wherein the second mammalian protein is a blood protein, more preferably said second mammalian protein is a plasma protein, most preferably said second protein is a coagulation factor.

### Claim 26 (depends on 25)

The composition according to claim 25, wherein the second mammalian protein is a human protein.

### Claim 27 (depends on 5, 6, 7, 8, 9, 10, 21)

The composition according to claim 21, wherein the second protein is an FVIII protein, selected from full length FVIII, an FVIII protein in which at least a part of the B-domain is missing, and an FVIII protein in which at least a part of the B-domain is replaced by an extension peptide, wherein the extension peptide is defined according to any of claims 5 to 10.

### Claim 28 (depends on 21)

The composition according to claim 21, wherein the one or more extension peptides are located in a position in the folded state of the first protein that does not interfere with the binding of the first protein to the second protein.

### Claim 29 (depends on 1)

A complex of a first protein and a second protein, wherein said first protein is a fusion protein according to claim 1, and said second protein has an amino acid sequence which is identical or similar to the amino acid sequence of a second mammalian protein or fragment thereof.

### Claim 30 (depends on 29)

The complex according to claim 29, wherein the first protein is non-covalently bound to the second protein.

### Claim 31 (depends on 29)

The complex according to claim 29, containing two copies of the first and the second protein, wherein the two copies of the first protein form a dimer, preferably a non-covalently bound dimer.

### Claim 32 (depends on 29)

The complex according to claim 29, wherein the first protein is covalently bound to the second protein, wherein the linker is selected from a peptide bond, a chemical linker, or a glycosidic bond.

### Claim 33 (depends on 1)

A polynucleotide encoding a fusion protein according to claim 1.

### Claim 34 (depends on 1)

The polynucleotide encoding a fusion protein according to claim 1 and having an amino acid sequence with an identity of at least 90%, preferably at least 95%, more preferably at least 98%, most preferably 100% to a sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 14.

### Claim 35 (depends on 33)

A vector containing the polynucleotide according to claim 33, wherein the vector backbone is preferably selected from pCDNA3, pCDNA3.1, pCDNA4, pCDNA5, pCDNA6, pCEP4, pCEP-puro, pCET1019, pCMV, pEF1, pEF4, pEF5, pEF6, pExchange, pEXPR, pIRES, and pSCAS.

### Claim 36 (depends on 35)

A host cell containing the vector according to claim 35, wherein the host cell is preferably a mammalian cell, preferably a human cell, more preferably a human kidney cell, most preferably a human embryonic kidney cell line, in particular a HEK293 cell line such as HEK293F.

### Claim 37

(canceled)

### Claim 38 (depends on 1)

A pharmaceutical composition comprising the fusion protein according to according to claim 1.

### Claim 39

(canceled)