WO2024200652A1 - Fviii-vwf fusion proteins with improved pharmacokinetics - Google Patents

Abstract

- 46 - A b s t r a c t The invention relates to a fusion protein comprising: a Factor VIII (FVIII) heavy chain; an FVIII light chain; a fragment of von Willebrand Factor (VWF); and at least two copies of an extension peptide (EP); wherein the EP has at least 90 % amino acid 5 sequence identity to SEQ ID NO: 1 and contains a cluster of O-glycosylation sites, wherein the cluster contains at least two O-glycosylated amino acids. The complex shows improved pharmacokinetic properties in comparison to FVIII. The invention further relates to a polynucleotide encoding the fusion protein as well as a vector and host cell comprising the polynucleotide.10

Description

FVIII -VWF fusion proteins with improved pharmacokinetics

FIELD OF THE INVENTION

The invention relates to fusion proteins of factor VIII (FVIII) and Willebrand factor (VWF) with improved physicochemical properties and pharmacokinetics.

BACKGROUND OF THE INVENTION

Hemophilia is a group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation. In its most common form, Hemophilia A, clotting factor VIII (FVIII) is deficient. Hemophilia A occurs in about 1 in 5,000 to 10,000 male births. The FVIII protein is an essential cofactor in blood coagulation with multifunctional properties. The deficiency of FVIII can be treated with plasma-derived concentrates of FVIII or with recombinantly produced FVIII. The treatment with FVIII concentrates has led to a normalized life of the hemophilia patients.

Hemophilia A patients are treated with FVIII on demand or as a prophylactic therapy administered several times a week. For prophylactic treatment, 15-25 lU/kg bodyweight of FVIII is administered three times a week, which is necessary due to the constant need of FVIII and its short half-life in the blood system, which in humans is only about 11 hours (Ewenstein et al., 2004). The short circulatory half-life of FVIII and the associated frequent need for FVIII concentrate infusion is a major challenge in Hemophilia A therapy.

In the blood, under normal conditions, the FVIII molecule is always associated with its cofactor von Willebrand factor (VWF), which stabilizes the FVIII molecule from different forms of degeneration. The non-covalent complex of FVIII and VWF has a high binding affinity of 0.2-0.3 nM (Vlot et al., 1996).

Historically, Hemophilia A has been treated with FVIII originating from human blood plasma. Moreover, since the 1990s, different recombinantly produced FVIII proteins were marketed. However, neither the plasma-derived nor the recombinant produced FVIII proteins have optimal pharmacokinetic properties. Like many other therapeutic proteins, they are subject to metabolic turnover by peptidases, which significantly limits their in vivo half-life.

Attempts for prolonging FVIII half-life include immunoglobulin Fc-fusion (efmoroctocog alfa, Eloctate), addition of polyethylene glycol (turoctocog alfa pegol, Esperoct; damoctocog alfa pegol, Jivi; rurioctocog alfa pegol, Adynovate), and a single-chain construct (lonoctocog alfa, Afstyla). All these technologies result in approximate 1.5 times half-life prolonged FVIII (reviewed by Tiede 2015). It is well documented that the FVIII molecule circulates in complex with VWF and both molecules are cleared simultaneously, predominantly via the VWF clearance pathways. Therefore, the half-life of FVIII is mainly determined by the half-life of VWF.

VWF and VWF fragments, containing FVIII binding sites, are known to stabilize FVIII against rapid clearance, proteolytic digestion, uptake by antigen presenting cells and to facilitate higher bioavailability after subcutaneous administration. As shown by Yee et al. (2014), the human VWF D'D3 domain is sufficient to stabilize FVIII in plasma. However, a D'D3-Fc fusion protein is able to extend FVIII half-life only in VWF-/- mice. In Hemophilia A mice, the D’D3-Fc construct does not result in FVIII half-life prolongation due to ineffective competition of the protein fragments with endogenous VWF for FVIII binding.

WO 2014/011819 A2 describes successful half-life prolongation of a FVIII construct containing the D'D3 domain of VWF, the Fc domain of IgG and XTEN. Since this construct does not bind to endogenous VWF, the same half-life prolonging effect is seen in both VWF/FVI Il-double knock-out (DKO) and Hemophilia A mice. However, although fully functional in vitro, it exhibits markedly reduced activity in vivo.

EP 3476937 A1 describes a chimeric protein that comprises FVIII and VWF (at least its D’D3 domain), which is intended to be used as a therapeutic agent for hemophilia A and aims to achieve an increase in in vivo half-life. For increase in half-life, the FVIII-VWF protein according to D1 is PEGylated to inhibit the binding of FVIII to low- density lipoprotein receptor-related protein (LRP). However, PEGylation of therapeutic proteins has a variety of disadvantages. Such are known for asparginase (PEG-ASNase), which, among others, is used for the treatment of ALL (acute lymphocytic leukemia). Here, anti-PEG antibodies already present in some patients before treatment have a negative effect on the asparginase activities measured after PEG-ASNase treatment. In addition, these anti-drug antibodies (ADAs) can cause hypersensitivity reactions (Khalil et al., 2022). Furthermore, antibodies against PEG were already known to inhibit the anticoagulant effect of aptamers (Moreno et al.,

2019). Recently, it was found that ADAs against the PEG part can inhibit the procoagulant activity of PEGylated FVIII preparations (Adynovate, Jivi and Esperoct). This effect was particularly measurable in patients treated with Jivi (damoctocog alfa pegol) and Esperoct (turoctocog alfa pegol) (Pezeshkpoor et al., 2023). These therapeutics based on PEGylated FVIII have been used for some time and on a large scale to treat hemophilia A patients. So far, however, only short-term and mostly transient effects due to pre-treatment or treatment-induced anti-PEG antibodies had been reported for Jivi (Paik and Deeks, 2019) or Esperoct/N8-GP (Giangrande et al.,

2020). Furthermore, Valsecchi et al described in 2023 that immunization with Comirnaty (BNT162b2) can cause anti-PEG ADAs. In hemophilia A patients, these may include IgMs that cross-react with all three PEGylated FVIII preparations (Adynovate, Jivi and Esperoct).

Other approaches for increasing the half-life of therapeutic proteins include the genetic fusion of the therapeutic protein to a protein with naturally long half-life such as transferrin and albumin, or to protein domains such as the C-terminal peptide (CTP) of chorionic gonadotropin (CG). As reviewed in Strohl et al (2015), different fusion proteins of a therapeutic protein and CTP have been developed and are presently tested in clinical trials. The therapeutic proteins include FSH (Elonva®), FVIIa, FIX, IFN-[3 and oxyntomodulin.

WO 2017/198435 A1 describes fusion proteins comprising a main protein, which is a mammalian protein, such as human VWF, or a fragment thereof and one or more extension peptides. The extension peptide contains a cluster of O-glycosylation sites with at least two O-glycosylated amino acids and may for example be derived from human VWF. Due to the extension peptide(s), the fusion protein has an increased half-life as compared to the main protein by itself, i.e. the mammalian protein or fragment thereof. The fusion protein may be used to increase the half-life of a binding partner, e.g. FVIII. The OCTA12 molecule, falling under the definition of WO 2017/198435 A1 , is a fusion of a fragment of VWF able to bind FVIII with high affinity. It has a markedly prolonged half-life in comparison to full-length VWF (terminal halflife up to approx. 200 h after subcutaneous administration in humans vs. approx. 18 h for full-length VWF) due to the absence of certain domains that are recognized by clearance receptors (e.g. A1 and D4 domains recognized by the SR-AI receptor). In addition, it contains three repeats of extension peptides, i.e. of a VWF-derived fourfold O-glycosylated 31 amino acid-long sequence. Of note, the non-covalent complex of FVIII and OCTA12 described in WO 2017/198435 A1 and in Vollack-Hesse et al., 2021 predominantly improves FVIII pharmacokinetic (PK) properties in subcutaneous administrations. After intravenous administration, the Cmax is only slightly improved and there is no improvement in terminal half-life in comparison with the isolated FVIII molecule (described on page 1075 and demonstrated in Figure 3A of Vollack-Hesse et al., 2021 ).

SUMMARY OF THE INVENTION

The present invention is inter alia based on the finding that complexes of a Factor VIII (FVIII) protein and a VWF fragment, in particular OCTA12, show increased expression levels, are stabilized and less prone to aggregation, when highly O-glycosylated extension peptides (EPs) are inserted into the linker connecting the proteins, into the linker connecting the heavy and the light chain and/or fused to the C-terminus of the VWF fragment. Moreover, the FVIII-VWF-EP fusion proteins stabilized by the extension peptides have prolonged circulatory half-life compared to FVIII alone.

Thus, according to a first aspect, the invention provides a fusion protein comprising a FVIII heavy chain; a FVIII light chain; a fragment of VWF; and at least two copies of an EP; wherein the EP has at least 90 % amino acid sequence identity to SEQ ID NO: 1 and contains a cluster of O-glycosylation sites, wherein the cluster contains at least two O-glycosylated amino acids.

In a second aspect, the invention relates to a polynucleotide encoding a fusion protein according to the first aspect.

According to the third aspect, the invention relates to a vector containing the polynucleotide according to the second aspect. In a fourth aspect, the invention relates to a host cell containing the polynucleotide according to the second aspect or the vector according to the third aspect, wherein the host cell is a mammalian cell.

Finally, in a fifth aspect, the invention also relates to a pharmaceutical composition comprising the fusion protein according to the first aspect for use in the treatment or prevention of a bleeding disorder.

FIGURES

Fig. 1 shows a schematic representation of the FVIII-VWF fragment-EP fusion proteins.

Fig. 2 shows a column diagram indicating the FVIII activity (FVIII:C) in culture medium supernatant after transient expression of FVIII-VWF fragment-EP fusion proteins in Expi293F cells.

Fig. 3 shows a non-reducing SDS-PAGE analysis of purified fusion proteins. (A) and (B) show FVIII-VWF-EP fusion proteins after western blot with FVIII detection (A) or Coomassie staining (B). The respective protein designation is indicated above each lane. M stands for molecular weight marker, molecular weights are expressed in kDa.

Fig. 4 shows a column diagram indicating the normalized binding levels of purified FVIII-VWF fragment fusion proteins to fIVWF. fIVWF was coated on a CM5 sensor chip, followed by injection of purified FVIII--VWF-EP proteins or control proteins. The SPR signal detected 30 s after injection stop was normalized to binding of rFVIll (Nuwiq) set to 100 %. OCTA12 is a negative control which should not bind to fIVWF.

Fig. 5 shows a diagram of the FVIII activity (FVIII:C) in HemA mouse plasma after single IV administration of rFVIll (Nuwiq) and the FVIII-VWF fragment fusion protein C17 according to the invention. DETAILED DESCRIPTION OF THE INVENTION

In order to provide a clear and consistent understanding of the specification and claims, and the scope to be given such terms, the following definitions are provided.

Definitions

A "peptide" as used herein may be composed of any number of amino acids of any type, preferably naturally occurring amino acids, which, preferably, are linked by peptide bonds. In particular, a peptide comprises at least 3 amino acids, preferably at least 5, at least 7, at least 9, at least 12, or at least 15 amino acids. Furthermore, there is no upper limit for the length of a peptide. However, preferably, a peptide according to the invention does not exceed a length of 500 amino acids, more preferably it does not exceed a length of 300 amino acids; even more preferably it is not longer than 250 amino acids.

Thus, the term “peptide” includes “oligopeptides”, which usually refers to peptides with a length of 2 to 10 amino acids, and “polypeptides” which usually refers to peptides with a length of more than 10 amino acids.

A “protein” as used herein may contain one or more polypeptide chains. Proteins with more than one polypeptide chain are often expressed as one polypeptide chain from one gene and cleaved post-translationally. Thus, the terms “polypeptide” and “protein” are used interchangeably. The polypeptides and proteins as used herein include chemically synthesized proteins as well as naturally synthesized proteins which are encoded by genes. The polypeptides or proteins may be obtained from a natural source, such as human blood or produced in cell culture as recombinant proteins.

The term “fusion protein” as used herein refers to proteins that are created through the joining of two or more genes that originally coded for separate proteins or protein fragments, wherein the components of the fusion protein are linked to each other by peptide-bonds, either directly or through peptide linkers. The term “fused” as used herein refers to components that are linked by peptide bonds, either directly or via one or more peptide linkers. According to the invention “peptide linker” is a peptide connecting two protein elements of the fusion protein, in particular the FVIII heavy chain with the FVIII light chain or the FVIII light chain with the VWF portion. The peptide linkers are also referred to just as “linkers”. Peptide linkers contain structural amino acids to permit important domain interactions, reinforce stability, and reduce steric hindrance. In addition to the structural amino acids, the linkers may contain functional motifs. Extension peptides may be considered as a protein element or as a part of a linker. The linkers may have a length of 2 to 200 amino acids.

The term “therapeutic protein” as used herein relates to proteins or polypeptides with a therapeutic effect, i.e. proteins used as active pharmaceutical ingredient.

According to the invention the terms “protein precursor”, “pro-protein” or “pro-peptide”, relate to an inactive protein (or peptide) that can be turned into an active form by post- translational modification, e.g. by enzymatic cleavage of a portion of the amino acid sequence.

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et a/., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the no brief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment).

For purposes of the present invention, the degree of sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et a/., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are: gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Desoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

The term "recombinant" when used in reference to a cell, nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature.

The term “half-life” as used herein is the time required for plasma/blood concentration to decrease by 50% after pseudo-equilibrium of distribution has been reached (in accordance with the definition in Toutain et al., 2005). The term “half-life” is also referred to as “circulatory half-life”, “terminal half-life” or “elimination half-life”.

As used herein, the terms "transformed", "stably transformed", and "transgenic", used with reference to a cell, means that the cell contains a non-native (e.g. heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.

The term “fragment” as used herein refers to a polypeptide that has an amino-terminal and/or carboxy terminal deletion of one or more amino acids as compared to the native or wild-type protein, but where the remaining amino acid sequence is identical to the corresponding positions in the amino acid sequence deduced from a full-length cDNA. Fragments are typically at least 50 amino acids in length.

The term "glycosylation" as used herein refers to the attachment of glycans to molecules, for example to proteins. Glycosylation may be an enzymatic reaction. The attachment formed may be through covalent bonds. Accordingly, a glycosylated polypeptide as used herein is a polypeptide to which one or multiple glycans are attached. The phrase "highly glycosylated" refers to a molecule such as an enzyme which is glycosylated at all or nearly all of the available glycosylation sites, for instance O-linked or N-linked glycosylation sites.

The term "glycan" as used herein refers to a polysaccharide or oligosaccharide, or the carbohydrate section of a glycoprotein or glycosylated polypeptide. Glycans may be homo- or heteropolymers of monosaccharide residues. They may be linear or branched molecules. Glycans typically contain at least three sugars and can be linear or branched. A glycan may include neutral sugar residues (e.g., glucose, N-acetylglucosamine (GIcNAc), N-acetylgalactosamine (GalNAc), galactose, mannose, fucose, arabinose, ribose, xylose, etc.), charged sugars (e.g. N-acetylneuraminic acid (sialic acid, NeuAc)), and/or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6'-sulfo-N-acetylglucosamine, etc.).

The term “O-glycans” as used herein refers to glycans that are generally found covalently linked to serine and threonine residues of mammalian glycoproteins. O-glycans may be a-linked via a GalNAc moiety to the -OH of serine or threonine by an O-glycosidic bond. Other linkages include a-linked O-fucose, |3-l inked O-xylose, a- linked 0-mannose, [3-linked O-GIcNAc, a- or [3-linked O-galactose, and a- or [3-linked O-glucose glycans.

According to the invention, the terms “O-glycosylation cluster”, “O-glycan cluster” and “cluster of O-glycosylated amino acids” are used interchangeably and relate to two or more O-glycosylated amino acids in close vicinity.

The term “sialylated” as used herein refers to molecules in particular glycans that have been reacted with sialic acid or its derivatives.

The transitional term “comprising”, which is synonymous with “including”, “containing,” or “characterized by”, is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim, except for impurities ordinarily associated therewith. When the phrase “consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. “A ‘consisting essentially of’ claim occupies a middle ground between closed claims that are written in a ‘consisting of’ format and fully open claims that are drafted in a ‘comprising’ format.”

In the context of the invention for practical reasons the term “glycosylated protein” such as the fusion protein is used in the singular form. Generally, in praxis, proteins occur in a composition of protein molecules of the same type. However, in the case of glycosylated proteins, glycosylation will not be identical in every molecule of the composition. For example, not all of the individual molecules of the composition may be glycosylated to 100 %. Moreover, differences in the glycans bound to a specific O- glycosylation site may arise. Accordingly, in the present application a reference to the “fusion protein” also relates to a composition of fusion protein molecules with identical amino acid sequences but variations in the O-glycan structure.

The terms “binding affinity” or “affinity” as used herein indicate the strength of the binding between two molecules, in particular a ligand and a protein target. Binding affinities are influenced by non-covalent intermolecular interactions between the two molecules such as hydrogen bonding, electrostatic interactions, hydrophobic interactions, and van der Waals forces.

An immune response as used herein relates to adaptive or innate immune response. The innate immune response refers to nonspecific defense mechanisms that are activated immediately or within hours of an antigen's appearance in the body. These mechanisms include physical barriers such as skin, chemicals in the blood, and immune system cells that attack foreign cells in the body. The innate immune response is activated by chemical properties of the antigen. The adaptive immune response refers to antigen-specific immune response. For this, the antigen must first be processed and recognized. Once an antigen has been recognized, the adaptive immune system creates a large number of immune cells specifically designed to attack that antigen. Fusion Proteins

According to a first aspect, the invention provides a fusion protein comprising an FVIII heavy chain; an FVIII light chain; a fragment of VWF; and at least two copies of an extension peptide.

The fusion protein according to the invention provides a technical advantage for the production process of FVIII-VWF complexes. Instead of producing and purifying two molecules separately and joining them in a specified ratio, the covalent complexation allows to reduce it to a single production and purification process. In addition, the covalent complex improves the expression levels. The fusion proteins according to the invention show an increased expression level in comparison to FVIII alone. According to one embodiment, the expression levels of the fusion proteins range from 0.6 to 10.3 lll/ml. Under the same conditions, the expression level of FVIII alone has a mean FVIII:C of 0.33 lU/ml. The FVIII activity may be determined by chromogenic assay. According to one embodiment, the expression level is equal to or above 0.6 lU/ml as measured by FVIII activity in the cell culture supernatant. The expression level may be, for example, 0.6 lU/ml, 0.8 lU/ml, 1.0 lU/ml, 1.2 lU/ml, 1.4 lU/ml, 1.6 lU/ml, 1.8 lU/ml, 2.0 lU/ml, 3.0 lU/ml, 4.0 lU/ml, 5.0 lU/ml, 6.0 lU/ml, 7.0 lU/ml, 8.0 lU/ml, 9.0 lU/ml, 10.0 lU/ml, 11.0 lU/ml, 12.0 lU/ml, 13.0 lU/ml, 14.0 lU/ml, 15.0 lU/ml. According to one embodiment, the expression level is equal to or above 4.0 lU/ml. According to one embodiment, the expression level is equal to or above 6.0 lU/ml. According to one embodiment, the expression level is equal to or below 15.0 lU/ml. According to one embodiment, the expression level is equal to or below 11.0 lU/ml.

In addition to an improved expression and purification, the fusion protein according to the invention has an increased half-life in comparison to FVIII alone. According to one embodiment, the half-life prolongation of the fusion protein is at least 20 %. According to one embodiment, the half-life prolongation of the fusion protein is at least 30 %. According to one embodiment, the half-life prolongation of the fusion protein is at least 40 %. According to one embodiment, the half-life prolongation of the fusion protein is at least 50 %. According to one embodiment, the half-life prolongation of the fusion protein is at least 60 %.

The fusion protein according to the invention also shows improved PK properties, in particular an improved half-life compared to a non-covalent complex of FVIII and VWF. In case of the non-covalent complex of FVIII and 0CTA12 of WO 2017/198435 A1 after intravenous administration, there is no improvement in terminal half-life in comparison to FVIII alone (which is described on page 1075 and demonstrated in Figure 3A of Vollack-Hesse et al., 2021 ). The fusion of OCTA12 and FVIII prevents redistribution of FVIII to intrinsic fIVWF in circulation. The fusion proteins according to the invention have significantly decreased binding ability to fIVWF in vitro as presented in Figure 4 and a prolonged half-life after intravenous administration as shown in Figure 5 and Table 6 (below).

The fusion protein according to the invention shows a reduced binding to endogenous VWF upon administration to a patient. According to one embodiment, the binding is at most 11 % of the binding of FVIII alone to VWF. The binding may be determined by surface plasmon resonance (SPR) as shown in Figure 4.

The half-life (ti/2) may be calculated by linear regression analysis of the log-linear portion of the individual plasma concentration-time curves or by non-linear regression using one-phase exponential decay model. Exemplary softwares for calculation are GraphPad Prism version 6.07 (La Jolla, CA 92037 USA) and WinNonlin, version 6.4 (Pharsight Corporation, Mountain View, CA, USA).

The calculations are based on the following equations:

Kei = elimination rate constant t/₂ = elimination half-life c = concentration t = time

Factor VIII in humans is coded by the F8 gene, which comprises 187.000 base pairs in six exons. The transcribed mRNA has a length of 9.029 base pairs and is translated into a protein of 2.351 amino acids from which 19 amino acids are removed. The FVIII molecule in humans is glycosylated with 25 N-glycosylation chains and 6 O-glycans on 31 amino acids (see Kannicht et al., 2013). After translation, the amino acid chain is cleaved by specific proteases leading to the formation of a heavy chain with about 200 kDa and a light chain with about 80 kDa. The domain organization is typically characterized as A1 -A2-B-A3-C1 -C2. The light chain is a composition of domains A3-C1 -C2. The heavy chain is composed of the domains A1 -A2-B. Heavy chains found in plasma have a heterogeneous composition with molecular weights varying from 90 to 200 kDa. The reasons for this size variation are the heterogeneity in its glycosylation, the existence of splice variants and proteolytic products such as the B-domain depleted heavy chain A1 -A2. The amino acid sequence of the full-length FVIII is identified by amino acids 20 to 2.351 of P00451 of UniProtKB, sequence version 1 of July 21 , 1986.

The human FVIII heavy chain according to the invention contains at least the domains A1 and A2 and may further contain parts of the B-domain or the entire B-domain. The amino acid sequence of the human FVIII heavy chain without the B-domain is identified by SEQ ID NO: 2. The amino acid sequence of the human FVIII heavy chain including the B-domain is identified by SEQ ID NO: 3.

According to one embodiment the FVIII heavy chain does not contain the FVIII B-domain. The human FVIII heavy chain without the B domain has the sequence of SEQ ID NO: 2. The FVIII heavy chain of the fusion protein has an amino acid sequence similar or identical to SEQ ID NO: 2. The heavy chain without the FVIII B-domain may comprise an amino acid sequence with an identity of at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99% or 100 % to SEQ ID NO: 2. According to one embodiment, the heavy chain is at least 95 % identical to SEQ ID NO: 2. Example 6 shows a fusion protein with the FVIII heavy chain sequence variant of SEQ ID NO: 37 which has the following amino acid substitution: V592A. According to one embodiment the heavy chain is at least 98 % identical to SEQ ID NO: 2. According to one embodiment the FVIII heavy chain contains the FVIII B-domain. The heavy chain with the FVIII B-domain may comprise an amino acid sequence with an identity of at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99% or 100 % to SEQ ID NO: 3. According to one embodiment the heavy chain is at least 95 % identical to SEQ ID NO: 3. According to one embodiment the heavy chain is at least 98 % identical to SEQ ID NO: 3.

According to one embodiment, the FVIII light chain comprises the domain organization A3-C1 -C2. The human FVIII light chain with the domain organization A3-C1 -C2 has the sequence of SEQ ID NO: 4. The FVIII light chain of the fusion protein has an amino acid sequence similar or identical to SEQ ID NO: 4. According to one embodiment, the FVIII light chain comprises an amino acid sequence with an identity of at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99% or 100 % to SEQ ID NO: 4. According to one embodiment, the light chain is at least 95 % identical to SEQ ID NO: 4. According to one embodiment, the light chain is at least 98 % identical to SEQ ID NO: 4. Example 6 shows a fusion protein with the FVIII light chain sequence variant of SEQ ID NO: 38 which has the following amino acid substitution: S1732T.

VWF is a multimeric adhesive glycoprotein present in the plasma of mammals, which has multiple physiological functions. During primary hemostasis, VWF acts as a mediator between specific receptors on the platelet surface and components of the extracellular matrix such as collagen. Moreover, VWF serves as a carrier and stabilizing protein for pro-coagulant Factor VIII. VWF is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule.

The domain organization of VWF is typically characterized as D3-TIL4-A1 -A2-A3-D4- C1 -C2-C3-CK. The precursor polypeptide, pre-pro-VWF, consists of a 22-residue signal peptide, a 741 residue pro-peptide (domains D1 -D2) and the 2050-residue polypeptide found in mature plasma Von Willebrand Factor (Fischer et al., 1994). Full- length VWF is identified by entry P04275 of UniprotKB (entry version 224 of April 12, 2017).

The human VWF according to the present invention has an amino acid sequence of any of the sequences of UniprotKB P04275, in particular SEQ ID NO: 5 (isoform 1 ). VWF contains two clusters of O-glycosylated amino acids. The first cluster of O-glycosylated amino acids is found between amino acids 1238 to 1268 of SEQ ID NO: 5. The second cluster includes amino acids 1468 to 1487 of SEQ ID NO: 5.

Upon secretion into plasma, VWF circulates in the form of various species with different molecular sizes. These VWF molecules consist of oligo- and multimers of the mature subunit of 2050 amino acid residues. VWF is usually found in plasma as multimers ranging in size approximately from 500 to 20.000 kDa (Furlan 1996).

According to one embodiment, the VWF fragment comprises an amino acid sequence with an identity of at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99% or 100 % to a section of SEQ ID NO: 5. According to one embodiment the VWF fragment is at least 95 % identical to a section of SEQ ID NO: 5. According to one embodiment the VWF fragment is at least 98 % identical to a section of SEQ ID NO: 5.

In the fragment of human VWF, one or more of the domains A1 , A2, A3, D4, C1 , C2, C3, CK may be missing relative to the human mature VWF (TIL3-D3-TIL4-A1 -A2-A3- D4-C1 -C2-C3-CK). The VWF fragment may, for example, have a domain organization selected from the group consisting of TIL3-D3-TIL4-A1 , TIL3-D3-TIL4-A1 -A2, TIL3- D3-TIL4-A1 -A2-A3, TIL3-D3-TIL4-A1 -A2-A3-D4, TIL3-D3-TIL4-A1 -A2-A3-D4-C1 , TIL3-D3-TIL4-A1 -A2-A3-D4-C1 -C2, and TIL3-D3-TIL4-A1 -A2-A3-D4-C1 -C2-C3-CK.

In this regard, the section of SEQ ID NO: 5 is, in particular, a section starting with amino acid 764 of SEQ ID NO: 5. Amino acids 764 to 1035 of SEQ ID NO: 5 contain the FVIII binding domain of VWF. The section may, for example, be a section as defined in WO 2015/185758 A2. As shown in WO 2015/185758 A2, the complex of FVIII and the VWF fragments as defined therein exhibit a reduced binding to phospholipid membranes compared to FVIII alone as well as a reduced binding to collagen III and heparin compared to the complex of FVIII and full-length VWF. The section of SEQ ID NO: 5, preferably starting with amino acid 764 of SEQ ID NO: 5 preferably ends with an amino acid of SEQ ID NO: 5 in the range from 1905 to 2153. According to one embodiment, the VWF fragment ends with an amino acid of VWF in the range from 2030 to 2153 of SEQ ID NO: 5. According to a further embodiment, the VWF fragment ends with an amino acid of SEQ ID NO: 5 in the range from 2100 to 2153. According to one embodiment, the VWF fragment comprises an amino acid sequence with an identity of at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99% or 100 % to SEQ ID NO: 6 or a section thereof. The VWF fragment with the amino acid sequence of SEQ ID NO: 6 is based on the section of amino acids 764 to 1268 of SEQ ID NO: 5, with two amino acid substitutions, namely C1099A and C1142A. The replacement of the two cysteines by alanines abolishes the ability of the VWF fragment to form multimers. This modified fragment of VWF is used in the fusion proteins in the examples. According to one embodiment the VWF fragment is at least 98 % identical to SEQ ID NO: 6. Example 6 shows a fusion protein with the sequence variant of SEQ ID NO: 6, namely SEQ ID NO: 39 which has the following additional amino acid substitution: A1164V.

As shown in the examples, EPs with the sequence QEPGGLWPPTDAPVSPTTLYVEDISEPPLH (SEQ ID NO: 1 ), i.e. the O-glycosylation cluster 1 of VWF (amino acids 1238-1268 of SEQ ID NO: 5) confer increased expression levels, an improved stability and a reduced tendency for aggregation to the fusion protein according to the invention. Thus, the EPs according to the invention have an amino acid sequence with an identity of at least 90 % to SEQ ID NO: 1 . According to one embodiment, the sequence identity to SEQ ID NO: 1 is preferably at least 95 %. According to one embodiment, the sequence identity of the EP to SEQ ID NO: 1 is at least 98 %. Example 6 shows a fusion protein with the sequence variant of SEQ ID NO: 1 , namely SEQ ID NO: 40 which has the following amino acid substitution: G5A. According to one embodiment, the two or more EPs have a sequence identity to SEQ ID NO: 1 of 100 %.

According to one embodiment, at least one copy of the EP is fused directly to the C-terminus of the VWF fragment. One, two, three, four, five, or six copies of the EP may be fused to the C-terminus of the VWF fragment. The FVIII-VWF-EP fusion proteins shown in the examples have three EPs at the C-terminus of the VWF- fragment, one as part of the fragment and two fused to it. The VWF protein with the amino acid sequence of SEQ ID NO: 6 joined with two extension peptide copies with the amino acid sequence of SEQ ID NO: 1 added to the C-terminus is a sequence modified derivative of OCTA12 described in WO 2017/198435 A1 . According to one embodiment, the C-terminus of the FVIII heavy chain is fused to the N-terminus of the FVIII light chain by a first linker (Linker 1 in Fig. 1 ). Linkers connecting the heavy and the light chain are known in the art. One example is the linker in NUWIQ®, i.e. SFSQNSRHQAYRYRRG (SEQ ID NO: 21 ). This linker comprises a sequence derived from the B-domain of FVIII. According to one embodiment, the first linker preferably comprises a sequence derived from the B- domain of FVIII.

The first linker may be a flexible linker or a rigid linker. Preferably, the first linker is a flexible linker. According to one embodiment, the first linker comprises a motif selected from (GGS)n, (GGGS)n, and (GGGGS)n. In this embodiment, n is an integer in the range of 1 to 10. n may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. In agreement with the one letter amino acid code, G represents glycine and S represents serine. These motifs give the first linker flexibility to allow for sufficient interactions between the FVIII heavy and light chain.

According to one embodiment, the first linker is a cleavable linker, i.e. it contains a protease cleavage site. The advantage of the presence of a protease cleavage site is the possibility of intracellular processing of FVIII to its native two-chain composition. According to one embodiment, the first linker comprises a furin cleavage site. A furin cleavage site is chosen because it is a naturally occurring cleavage site in wild type FVIII. The furin cleavage site may have the amino acid sequence of SEQ ID NO: 20.

Exemplary first linkers (with the corresponding sequence IDs) are Linker 1 -1 (SEQ ID NO: 12), Linker 1 -2 (SEQ ID NO: 13), Linker 1 -3 (SEQ ID NO: 14) , Linker 1 -4 (SEQ ID NO: 15), Linker 1 -5 (SEQ ID NO: 16), Linker 1 -6 (SEQ ID NO: 17), and Linker 1 -7 (SEQ ID NO: 18). The amino acid sequences of these linkers are shown in Table 1 (below). According to one embodiment, the amino acid sequence of the first linker has an identity of at least 90%, at least 95 %, or at least 98 % to a sequence selected from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 , SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18. According to one embodiment, the amino acid sequence of the first linker is identical to a sequence selected from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18. According to one embodiment, the first linker comprises at least one copy of the EP. The first linker may for example contain, one, two, three, four, five, six, seven, or eight copies of the EP. The FVIII-VWF-EP fusion proteins shown in the examples have three EPs in the first linker. Thus, according to one embodiment, the first linker comprises at least two copies of the EP. According to one embodiment, the first linker comprises at least three copies of the EP. The EPs may be distributed over the length of the linker, with structural amino acids of the linker or other elements between them. Alternatively, two or more of the EPs may be assembled adjacently, i.e. in a consecutive order. Two or more EPs in a row are referred to as an EP assembly. According to one embodiment, all EPs in the first linker are assembled in a consecutive order.

According to one embodiment, the C-terminus of the FVIII light chain is fused to the N-terminus of the VWF fragment by a second linker.

The second linker may be a flexible linker or a rigid linker. Preferably, the second linker is a flexible linker. According to one embodiment, the second linker comprises a motif selected from (GGS)n, (GGGS)n, and (GGGGS)n. In this embodiment, n is an integer in the range of 1 to 10. n may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. In agreement with the one letter amino acid code, G represents glycine and S represents serine. These motifs give the second linker flexibility to allow sufficient interactions and in particular binding to the VWF and FVIII binding domains. According to one embodiment, the second linker contains a (GGGGS)2, a (GGGGS)4 and/or a (GGGGS)e motif.

According to one embodiment, two consecutive copies of the GGGGS motif are located at the N-terminus and/or the C-terminus of the second linker. According to one embodiment, a (GGGGS)2 motif is located at the N-terminus. According to one embodiment, a (GGGGS)2 motif is located at the C-terminus. According to one embodiment, a (GGGGS)n motif with n > 2 is located at the C-terminus. According to one embodiment, (GGGGS)2, (GGGGS)4, (GGGGS)e or (GGGGS)s is located at the C-terminus of the second linker. As shown in Example 4, FVIII-VWF-EP-fusion proteins with a higher number of glycines in the C-terminal part of the linker show a higher binding to endogenous VWF. The reason could be that a longer flexible linker allows better interactions between the VWF fragment of the fusion protein with the FVIII portion of the fusion protein, therefore preventing an interaction with endogenous VWF to a higher extent. Thus, (GGGGS)n linkers with n > 2 are preferred. According to one embodiment, a (GGGGS)2 motif is located at each of the N-terminus and the C-terminus of the second linker.

According to one embodiment, the second linker is cleavable. According to one embodiment, the second linker comprises a thrombin cleavage site. The advantage of the presence of a protease cleavage site is the possibility of separating the VWF fragment from the FVIII heavy and light chain after FVIII activation. The thrombin cleavage site may be defined by the sequence SEQ ID NO: 19. A thrombin cleavage site is chosen because it is also part of the native FVIII sequence.

According to one embodiment, the second linker comprises at least one copy of the EP. As shown in Example 2, the FVIII-VWF-EP fusion proteins with EPs in the second linker, i.e. C4 and C17, show an even stronger increase in expression than the other FVIII-VWF-EP fusion proteins. Moreover, negatively charged EPs inserted in the second linker may reduce the tendency of the fusion protein to form higher molecular weight species. This effect is shown by C17, which contains three EPs in the linker and a low percentage of high molecular weight species (10.53%). The second linker may for example contain, one, two, three, four, five, six, seven, or eight copies of the EP. Thus, according to one embodiment, the second linker comprises at least two copies of the EP. According to one embodiment, the second linker comprises at least three copies of the EP. The EPs may be distributed over the length of the linker, with structural amino acids of the linker or other elements between them. Alternatively, two or more of the EPs may be assembled adjacently, i.e. in a consecutive order. According to one embodiment, all EPs in the second linker are assembled in a consecutive order. Exemplary second linkers (with the corresponding sequence IDs) are Linker 2-3 (SEQ ID NO: 9) and Linker 2-5 (SEQ ID NO: 11 ). The amino acid sequences of these linkers are shown in Table 2 (below). According to one embodiment, the amino acid sequence of the second linker has an identity of at least 90%, at least 95 %, or at least 98 % to a sequence selected from SEQ ID NO: 9 and SEQ ID NO: 11. According to one embodiment, the amino acid sequence of the second linker is selected SEQ ID NO: 9 and SEQ ID NO: 11 . According to one embodiment the fusion protein contains at least one half-life prolonging moiety. There is a variety of half-life prolonging moieties known in the art. The half-life prolonging moiety may be selected from an immunoglobulin Fc-domain, serum albumin or parts thereof, an albumin binding antibody, an albumin binding antibody domain, or an albumin binding protein domain. The Fc-domain is the fragment crystallizable (Fc) region of the tail region of an antibody that interacts with cell surface receptors called Fc receptors. This interaction as well as the slower renal clearance of larger sized molecules increases the half-life of the protein to which it is attached. An exemplary Fc domain is the Fc domain of IgG 1 . Several fusion proteins containing albumin to increase the half-life of the therapeutic protein have been described, including Factor VII, FVIII and Factor IX fusions with albumin. According to one embodiment, a full-length human serum albumin (HSA) is added to the fusion protein. The half-life of albumin is also regulated by a member of the Fc receptor family, i.e. the neonatal Fc Receptor (FcRn). The HSA moiety added to the fusion proteins preferably has the sequence according to Uniprot entry P02768.

VHH fragments are single-domain antibodies engineered from heavy-chain antibodies found in camelids. According to one embodiment, the half-life prolonging moiety is an albumin binding VHH domain. Albumin binding VHH domains are known in the art. One example of a cross-reactive albumin binding VHH domain is MSA21 described in EP 2316852 B1. According to one embodiment the albumin binding VHH domain is the albumin binding nanobody (ABN) shown in the examples with the SEQ ID NO: 41 .

A half-life prolonging moiety may be fused to the C-terminus of the protein. The halflife prolonging moiety may be directly fused to the C-terminus of the VWF fragment or to the C-terminus of an EP. Alternatively, the half-life prolonging moiety is fused to the C-terminus, i.e. the C-terminus of the VWF fragment or the C-terminus of an EP, by a third linker. Another option is that the half-life prolonging moiety forms part of the first linker.

According to one embodiment of the fusion protein, the C-terminus and/or the N- terminus of an EP assembly in the first and/or the second linker is directly connected to at least one copy, preferably at least two copies, of GGS, GGGS, or GGGGS. According to one embodiment of the fusion protein, the C-terminus and/or the N- terminus of a half-life prolonging moiety in the first and/or the second linker is directly connected to at least one copy, preferably at least two copies, of GGS, GGGS, or GGGGS.

According to one embodiment of the fusion protein, the first and/or the second linker contain(s) at least two copies of the GGGGS motif on either side of an extension peptide assembly and/or on either side of the half-life prolonging moiety.

Exemplary are the fusion proteins (with the corresponding sequence IDs): C1 (SEQ ID NO: 22), C2 (SEQ ID NO: 23), C3 (SEQ ID NO: 24), C4 (SEQ ID NO: 25), C5 (SEQ ID NO: 26), C6 (SEQ ID NO: 27), C7 (SEQ ID NO: 28), C8 (SEQ ID NO: 29), C16 (SEQ ID NO: 30), C17 (SEQ ID NO: 31 ), C18 (SEQ ID NO: 32), C21 (SEQ ID NO: 33), C22 (SEQ ID NO: 34), C23 (SEQ ID NO: 35), and C24 (SEQ ID NO: 36). Table 3 below shows the components (and their sequences) forming these fusion proteins.

According to one embodiment the amino acid sequence of the fusion protein has an identity of at least 90%, at least 95 %, or at least 98 % to a sequence selected from SEQ ID NO: 22 , SEQ ID NO: 23 , SEQ ID NO: 24 , SEQ ID NO: 25 , SEQ ID NO: 26,

SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 ,

SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO:

36. According to one embodiment the amino acid sequence of the fusion protein is identical to a sequence selected from SEQ ID NO: 22, SEQ ID NO: 23,

SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,

SEQ ID NO: 29), SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33,

SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.

According to a second aspect, the invention provides an isolated polynucleotide that comprises a nucleic acid sequence encoding a fusion protein according to the first aspect of the invention.

The isolated polynucleotide may be a DNA molecule or an RNA molecule. The isolated polynucleotide is preferably a DNA molecule, in particular a cDNA molecule. The techniques used to isolate or clone a polynucleotide encoding a peptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotides from such genomic DNA can be effected, e.g., by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features (see, e.g., Innis et al, 1990). Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used.

In particular, the sequence of the isolated polynucleotide may comprise a first part encoding the FVIII heavy chain, a second part encoding the first linker, a third part encoding the FVIII light chain portion, a fourth part encoding the second linker and a fifth part encoding the VWF fragment.

According to one embodiment, the first part encodes a FVIII heavy chain with a sequence identity to SEQ ID NO: 2 of at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 %.

According to one embodiment, the second part encodes a first linker with a sequence identity of at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % to a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

According to one embodiment, the third part encodes a FVIII light chain with a sequence identity to SEQ ID NO: 4 of at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 %.

According to one embodiment, the fourth part encodes a second linker with a sequence identity of at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % to a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: and SEQ ID NO: 11.

According to one embodiment, the fifth part encodes a VWF fragment with a sequence identity to SEQ ID NO: 6 of at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 %. According to one embodiment, the polynucleotide encodes 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: 22, SEQ ID

NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID

NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID

NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.

vector

In a third aspect the invention also relates to expression vectors comprising a polynucleotide according to the second aspect of the invention.

The expression vector further preferably comprises control elements such as a promoter, and transcriptional and translational stop signals. The polynucleotide according to the second aspect and the control elements may be joined together to produce a recombinant expression vector that may include one or more restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. The polynucleotide may be inserted into an appropriate expression vector for expression. In creating the expression vector, the coding sequence is located in the expression vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or a virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide of the fourth aspect of the invention. The choice of the expression vector will typically depend on the compatibility of the expression vector with the host cell into which the expression vector is to be introduced. The expression vectors may be a linear or closed circular plasmid.

The expression vector is preferably adapted to expression in mammalian cells. The expression vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

The vector is preferably one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. For integration into the host cell genome, the expression vector may rely on any other element of the expression vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location in the chromosome.

The vectors of the present invention preferably contain one or more (e.g., several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

The procedures used to ligate the elements described above to build the recombinant expression vectors of the present invention are well known to one skilled in the art (see Green and Sambrook 2012; Chapter 3)).

According to one embodiment the vector backbone of the vector according to the third aspect is selected from pCDNA3, pCDNA3.1 , pCDNA4, pCDNA5, pCDNA6, pCEP4, pCEP-puro, pCET1019, pCMV, pEF1 , pEF4, pEF5, pEF6, pExchange, pEXPR, pIRES, and pSCAS.

Host Cell

According to a fourth aspect the invention provides a host cell, comprising the expression vector according to the third aspect of the invention. The expression vector according to the third aspect is introduced into a host cell so that the expression vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

According to one embodiment the fusion protein is produced by expression in a mammalian host cell line. The fusion protein is preferably produced in a human host cell line. Generally, any human host cell line is suitable for expression of the fusion protein. The host cell is preferably of human origin in order to ensure that the fusion protein is properly processed during folding and receives the proper post-translational modifications (e.g. glycosylation, hydroxylation, phosphorylation and sulfation). A favourable glycosylation profile of the fusion protein is particularly obtained with human kidney cell lines. Preferred human kidney cell lines are HEK cell-lines, in particular HEK 293 cell lines.

Examples of HEK cell-lines for production of the glycosylated polypeptide are HEK 293 F, Flp-ln ^TM-293 (Invitrogen, R75007), 293 (ATCC® CRL-1573), 293 EBNA, 293 H (Thermo Scientific 11631017), 293S, 293T (ATCC® CRL-3216™), 293T/17 (ATCC® CRL11268™), 293T/17 SF (ATCC® ACS4500™), HEK 293 STF (ATCC® CRL 3249™), HEK-293.2sus (ATCC® CRL-1573™). A preferred cell line for production of the polypeptide is the HEK 293 F cell line.

Other human cell lines suitable as host cells for expression include, without limitation, cell lines derived from myeloid leukemia cells. Specific examples of host cells are K562, NM-F9, NM-D4, NM-H9D8, NM-H9D8-E6, NM H9D8-E6Q12, GT-2X, GT-5s and cells derived from anyone of said host cells. K562 is a human myeloid leukemia cell line present in the American Type Culture Collection (ATCC CCL-243). The remaining cell lines are derived from K562 cells and have been selected for specific glycosylation features.

Further mammalian host cell lines suitable for producing fusion proteins according to the invention include cell lines of hamster, mouse, and monkey origin. Suitable host cells include Chinese hamster ovary cells (CHO cells, e.g., DG44, DXB11 , and K1 [ATCC CCL-61 , including its glutamine auxotroph derivative CHOZn, SAFC CHOGS]) and baby hamster kidney (BHK) cells. Pharmaceutical

and medical use

The fusion proteins according to the first aspect are particularly useful as active ingredients for medical treatment. Preferably, they are useful for treatment or prevention of a bleeding disorder. The fusion proteins according to the first aspect described herein can be administered alone or in the form of pharmaceutical compositions.

Thus, according to a fifth aspect, the invention provides the fusion protein according to the first aspect for use in the treatment of a bleeding disorder.

According to one embodiment, the fusion protein may be formulated with at least one pharmaceutically acceptable carrier. Pharmaceutical compositions based on the fusion protein can be prepared and administered to a subject by any methods well known in the art of pharmacy. See, e. g, Goodman & Gilman's The Pharmacological Basis of Therapeutics, Hardman et al., eds., McGraw-Hill Professional (10th ed., 2001 ); Remington: The Science and Practice of Pharmacy, Gennaro, ed., Lippincott Williams & Wilkins (20th ed., 2003); and Pharmaceutical Dosage Forms and Drug Delivery Systems, Ansel et al. (eds), Lippincott Williams & Wilkins (7th ed., 1999). In addition, the pharmaceutical compositions of the embodiments may also be formulated to include other medically useful drugs or biological agents. The pharmaceutical composition typically comprises a therapeutically effective amount of the fusion protein combined with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is any carrier known or established in the art. Exemplary pharmaceutically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free saline solution. Other forms of pharmaceutically acceptable carriers that can be utilized for the present embodiments include binders, disintegrants, surfactants, absorption accelerators, moisture retention agents, cryoprotectants, absorbers, lubricants, fillers, extenders, moisture imparting agents, preservatives, stabilizers, emulsifiers, solubilising and bulking agents, salts which control osmotic pressure, diluting agents such as buffers, and excipients usually used for the use form of the formulation. These are optionally selected and used depending on the unit dosage of the resulting formulation. Thus, the invention also relates to a method of treatment or prevention of a bleeding disorder of a patient, said method comprising administering to said patient a pharmaceutical composition according to the fifth aspect.

As used herein “bleeding disorder” refers to a disease or condition that impairs normal hemostasis. The bleeding disorder can be, for example, Hemophilia A, Hemophilia B, Factor VIII deficiency, Factor XI deficiency, von Willebrand Disease, Glanzmann's Thrombasthenia, Bernard-Soulier Syndrome, idiopathic thrombocytopenic purpura, intracerebral hemorrhage, trauma, traumatic brain injury, and the like.

As used herein, “hemophilia” refers to a group of bleeding disorders associated with increased blood clot formation time as compared to blood clot formation time in healthy individuals without hemophilia. Hemophilia includes Hemophilia A, which is a disorder that leads to the production of defective Factor VIII, Hemophilia B, which is a disorder that leads to the production of defective Factor IX and acquired Hemophilia A, a rare bleeding disorder caused by an autoantibody to FVIII.

The bleeding disorder is preferably Hemophilia A or B. The treatment may for example be the hemophilia treatment of previously untreated patients (PUPS) or an immune tolerance induction (ITI) treatment and/or other related treatments of haemophilia disorders.

For in vivo applications, pharmaceutical compositions can be administered to the patient by any customary administration route, e.g., orally, parenterally or by inhalation. Parenteral administration includes intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, liquid agents, suspensions, emulsions and dripping agents. For parenteral administration the pharmaceutical composition should be an injectable agent such as a liquid agent or a suspension.

In other embodiments, the pharmaceutical composition is administered orally to a patient. In these embodiments, a form of the drug includes solid formulations such as tablets, coated tablets, powdered agents, granules, capsules and pills, liquid formulations such as liquid agents (e.g., eye drops, nose drops), suspension, emulsion and syrup, inhales such as aerosol agents, atomizers and nebulizers, and liposome inclusion agents. In still some other embodiments, the glycosylated polypeptide, protein complex or pharmaceutical composition is administered by inhalation to the respiratory tract of a patient to target the trachea and/or the lung of a subject.

According to one embodiment of the fifth aspect, the use comprises an intravenous or non-intravenous injection. The non-intravenous injection preferably is a subcutaneous injection.

EXAMPLES

Example 1 - Cloning, expression, and purification of the FVIII-VWF-EP fusion proteins

Aim of the

: Generation of cDNA encoding FVIII-VWF-EP fusion proteins.

Expression of the fusion proteins and purification to homogeneity.

Methods

Gene synthesis and cloning

For generating expression vectors encoding the fusion constructs, the Golden Gate cloning technology was used. To this end, cDNA fragments encoding the desired FVIII-VWF-EP fusion constructs were synthesized and cloned into donor vectors compatible for Golden Gate cloning at Twist Bioscience. Then, donor vectors containing the desired construct variant were used in a Golden Gate assembly reaction together with donor vectors containing regulatory elements as well as a proprietary acceptor backbone. The reaction generated vectors for mammalian expression containing a gpCMV promoter 5’ of the desired construct variant and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) 3’ of the desired construct variant.

The vector constructs were transformed into E. coli NEB5 alpha cells and single clones were selected following an overnight incubation at 37 °C on ampicillin- containing LB-agar plates. Plasmid DNA preparations were performed using the QIAprep DNA Mini kit (Qiagen) or the NucleoBond® Xtra Maxi Plus EF kit (Macherey-Nagel) according to the manufacturer's recommendations. By sequencing, the integrity of the vectors was verified, in particular the correct orientation and integrity of the gene encoding the desired construct variant.

The FVIII-VWF-EP fusion constructs were expressed transiently in Expi293F cells (Thermo Fisher Scientific) in 500-1000 mL scale according to the manufacturer's recommendations. Product-containing cell culture supernatant was harvested 4 to 5 days post transfection by centrifugation at 2000 x g for 20 min.

Protein Purification

Purification was accomplished by a 3-step process - capturing the product from cell culture supernatant, polishing via affinity chromatography, and rebuffering into the final matrix.

Briefly, the harvested cell culture supernatant was spiked with 0.3 M NaCI to increase the sample conductivity, filtered through a 0.2 pm PES filter, and captured by Capto MMC resin (Cytiva). To this end, a Capto MMC column was equilibrated with 0.3 M NaCI, 0.01 M CaCl2, 0.01 M L-Histidine, 0.02% polysorbate 80 at pH 6.5, and eluted with 0.3 M NaCI, 0.02 M CaCl2, 0.02 M L-Histidine, 0.8 M L-Arginine, 10% ethylene glycol. 0.02 % polysorbate 80 at pH 6.5. The column eluate was diluted 1 :2 with equilibration buffer (0.05 M Tris, 0.1 M NaCI, 0.02% polysorbate 80 pH 7.0), before loading it onto a VOLTselect affinity resin (Thermo Fisher Scienitific, custom made VWF affinity resin). The product was eluted from the column using 0.05 M Tris, 0.1 M NaCI, 1 M MgCL pH 7.0, and rebuffered into the final formulation buffer (171.1 mM NaCI, 7.1 mM L-Arginine, 26.3 mM Sucrose, 3.4 mM tri-sodium citrate, 1 .7 mM CaCL, 0.1 mM Poloxamer 188 pH 7.0), using a Sephadex G-25 desalting column (Cytiva). Results

The gene products encoded by the cloned cDNA constructs are shown in Tables 1 -3.

Table 1 - First linkers used for FVIII-VWF-EP fusion proteins.

EP stands for the extension peptide with the SEQ ID NO: 1 . The lower-case numbers stand for a number of repetitions of sequence elements to which they are allocated. ABN is the albumin binding nanobody with the sequence QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGISS LGDSTLYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQGT QVTVSS (SEQ ID NO: 41 ).

Table 2 - Second linkers used for FVIII-VWF-EP fusion proteins.

EP stands for the extension peptide with the SEQ ID NO: 1 . The lower-case numbers stand for a number of repetitions of sequence elements to which they are allocated.

Table 3 - Overview of the fusion proteins and their components.

EP stands for the extension peptide with the SEQ ID NO: 1 . The lower-case numbers stand for a number of repetitions of sequence elements to which they are allocated. ABN is the albumin binding nanobody with the amino acid sequence SEQ ID NO: 41 .

Example 2 - Chromogenic factor VIII activity (FVIII :C) of the FVIII-VWF-EP fusion proteins in the cell culture supernatant

Aim of the experiment

Characterization of the fusion proteins by chromogenic factor VIII activity (FVIII:C) analysis. Assessing the impact of linker length and of the presence of the extension peptides on the FVIII activity in the expression supernatant.

Method

The FVIII-VWF-EP fusion proteins were expressed transiently in Expi293F cells (Thermo Fisher Scientific) in 3 mL scale volume in triplicates; cell culture supernatant was harvested 4 days post transfection by centrifugation at 4800 x g for 30 min. FVI 11 : C activity was assessed by the FVIII chromogenic assay kit (Siemens) on a BCS XP system (Siemens).

Results

All FVIII-VWF-EP fusion proteins showed higher expression levels (ranging from 0.66 to 10.34 lU/ml) when compared to rFVIll (Simoctocog alfa, NUWIQ, mean FVIII:C of 0.33 lll/ml). Within the group with the same linker length and harboring the same furin cleavage site, constructs with the EPs inserted in the linker connecting the FVIII and the VWF portion showed the highest expression levels: C4 vs. C2 and C5; C17, C21 and C22 vs. C16 and C18. Molecules with EPs inserted in both linkers (C23 and C24) resulted in the highest activity levels in the supernatant (Figure 2).

In summary, the combination of optimal linker length and EPs inserted in both linker regions of the fusion proteins results in the highest expression levels, which implicates their efficient translation, highest protein stability and correct folding.

Example 3 - Characterization of the fusion proteins by size exclusion chromatography and SDS-PAGE

Aim of the

To control the size, purity and integrity of the expressed constructs. SDS-PAGE analysis was performed to control homogeneity of the expressed proteins. The size distribution and potential presence of high molecular weight species (HMWS) in the purified FVIII-VWF-EP fusion protein preparations was analysed by SEC-HPLC- analysis. Methods

SDS-PAGE:

Samples were qualitatively analyzed via non-reducing SDS-PAGE. Samples were denatured by incubation with LDS sample buffer. Gels were run on a 4-12 % BisTris gel (Invitrogen, NuPage) for 70 min at 175 V.

Coomassie staining was conducted using a ready-to-use Coomassie stain (Thermo scientific, Page Blue Protein staining), stained for 3h at RT, washed and subsequently destained in MilliQ water until the background had cleared.

SEC-HPLC:

All samples were analyzed on a Superdex 200 Increase 10/300 column (Cytiva) coupled to an ULTIMATE3000 HPLC system (Thermo Scientific). The running buffer was 171.1 mM NaCI, 7.1 mM L-Arg hydrochloride, 26.3 mM sucrose, 3.4 mM trisodium citrate dihydrate, 1.7 mM CaCl2, 0.1 mM Poloxamer 188, pH 7.0. The buffer was used with an isocratic flow rate of 0.56 mL/min.

For the analysis, samples were injected onto the Superdex column and the corresponding elution profile was recorded with UV at 280 nm. After 45 min, the analytical run was completed. Chromatograms were manually integrated and the relative area of the high molecular weight species (HMWS) in the sample was calculated.

Results

SDS-PAGE analysis of purified FVIII-VWF-EP fusion proteins showed a major band >250 kDa for all constructs, corresponding to the FVIII-VWF-EP fusion proteins (Figure 3). Residual minor protein bands between 75-170 kDa represent furin cleavage products. In all samples some portion of HMWS was detected, and the amount varied significantly. The results of the analyses are summarized in Table 4. The amount of HMWS differed depending on the presence and positioning of the EPs. These data demonstrate that, for FVIII-VWF-EP fusion proteins, the presence of EPs in the linker regions is beneficial for the correct protein folding. Moreover, the presence of EPs specifically in the FVIII-VWF linker (e.g. in C17, C23 and C24) prevents the generation of HMWS to a very high degree, and therefore has a beneficial effect on the stability of the fusion protein.

Table 4 - Percentage of HMWS in the purified FVIII-VWF-EP fusion proteins

Example 4 - Binding to full-length VWF

Aim of the experiment

Assessing the capacity of FVIII-VWF-EP fusion constructs to bind to full-length VWF (fIVWF).

Method

The binding of the FVIII-VWF-EP fusion proteins to fIVWF was tested by surface plasmon resonance (SPR) on a T200 instrument (Cytiva). Purified human fIVWF (Sekisui) was coated on a CM5 Chip via amine coupling using an amine coupling kit (Cytiva) according to the manufacturer’s instructions. FIVWF was immobilized in three different flow cells at approximately 1000 response units (RU). The running buffer was 20 mM HEPES, 150 mM NaCI, 5 mM CaCl2, 0.05% Tween 20. After each analyte injection, the surface was regenerated with a regeneration buffer (20 mM HEPES, 600 mM NaCI, 350 mM CaCl2, 0.05% Tween 20). The FVIII-VWF-EP fusion proteins were injected over three different flow cells in triplicates in a random order at a fixed concentration of 8.5 lll/ml FVIII:C. The binding level, measured 30 sec after end of the analyte injection, was normalized by dividing the RUs by the molecular weight of the respective protein and expressed in % binding of rFVIll set to 100 %.

Results

The results are shown in Figure 4. For all FVIII-VWF-EP fusion proteins a binding level of <11 % of FVIII was measured. The highest binding was registered for C16 and the lowest binding for the C17 construct.

Example 5 - Pharmacokinetics of the FVIII-VWF-EP fusion proteins

Aim of the experiment

To explore the impact of the covalent fusion of the VWF fragment with two extension peptides (VWF-EP) to the C-terminus of FVIII on its pharmacokinetics (PK). The effects of VWF-EP in combination with the impact of the presence of three additional EPs in the second linker (in construct C17) were studied in Hemophilia A (HemA) mice.

Methods

5 to 8-week-old male B6;129S-F8tm1 Kaz/J (F8-/-) mice were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA). The animals were treated by a tail vein injection of the test compound or rFVIll control at a dose of 200 lU/kg b.w. based on FVIII:C activity. The study outline is summarized in Table 5. The blood samples were collected at the indicated time points. Five animals were used for blood sampling at each time point of each group. Each mouse was used for two sampling points. Blood was collected in tubes with 3.8% Na-citrate solution. Blood samples were placed on crushed ice immediately after collecting and plasma was separated within 1 hour of sampling by centrifugation at 3350 x g (4000 rpm), 4°C for 15 min. Plasma samples were stored at -80°C until analysis by FVIII:C assay (Coamatic Factor VIII Assay Kit; Chromogenix, Bedford, MA, USA).

FVIIkAg in mouse plasma was determined by an in-house ELISA assay. In a first step, the maxisorp microtiter plates (Thermo Fisher Scientific 439454) were coated overnight with an anti-human FVIII monoclonal antibody recognizing the A2 domain (GMA8023, Green Mountain Antibodies, Burlington, USA). Following blocking and washing, diluted mouse plasma was applied on the plate and incubated for 2h at 37°C. After subsequent washing steps, the bound molecules were detected by a biotinylated anti-FVIll nanobody (Capture select Biotin anti FVIII conjugate; Thermo Fisher Scientific 7102862500) and Neutravidin-HRP (Thermo Fisher Scientific 31001 ). The colorimetric readout was obtained by using tetramethylbenzidine substrate (Sigma-Aldrich T4444) measured at a wavelength of 450 nm.

Table 5 - Study outline

Results

The results of the study are presented in Table 6 and Figure 5. rFVIll showed a halflife of 7.61 h, while the FVIII-VWF-EP fusion protein construct C17 could be detected significantly longer in the mouse plasma, due to a 1.6-fold longer T1/2 of 12.42 h. C17 also showed a higher Cmax and AUC as a result of an increased recovery and improved half-life.

Table 6 - PK analysis of FVIII :C data measured in HemA mouse plasma

This demonstrates that the covalent linking of FVIII to a VWF fragment and EPs by genetic fusion results in the improvement of its PK parameters.

For determination of the specific activity, mouse plasma FVIIkAg and FVI I l:C values were measured for rFVIll (NUWIQ) and C17, using an in-house ELISA assay and the Coamatic Factor VIII Assay Kit as described above.

Determination of the average specific FVIII activity values in 30 mice revealed a significantly higher specific activity of C17 compared to unmodified rFVIll (Table 7).

Table 7 - Average specific activities

* the FVI II :Ag Assay recognizes the FVIII chain only (in contrast to the BCA assay used in D1). When corrected for the molecular weight difference, the C17 results in a specific activity of 9811 ,7 lU/mg, which is still higher than in D1 .

This activity is higher than the specific activities of the molecules of the state-of-the- art document EP 3476937 A1. In Table 4 of EP 3476937 A1 (page 18), the specific activities of the three molecules scFVIII/D’D3-60, scFVIII/D’D3-90, and scFVIII/D’D3- 120 are listed. The respective values are 9304.3 lll/mg, 8474.5 lll/mg and 9367.2 lll/mg. Thus, the present invention provides FVIII-VWF fusion proteins with significantly higher specific activities.

Example 6 - Confirmation of the results with sequence variants

To confirm that the minor sequence variants of the FVIII heavy chain, the FVIII light chain, the VWF fragment and the EP have no impact on the properties of the constructs, additional fusion proteins based on C17 with sequence variations in one or all of the four functional elements as shown in Table 8 are produced as described in Example 1. Furthermore, the experiments according to Examples 2 to 4 are repeated.

Table 8 - Additional fusion proteins with sequence variants

Many modifications and other embodiments of the invention set forth herein will come to mind of the one skilled in the art to which the invention pertains, having the benefit of the teachings presented in the foregoing description and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Table 9 - Sequence ID overview

REFERENCES

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Claims

C l a i m s

1 . A fusion protein comprising:

• A Factor VIII (FVIII) heavy chain;

• an FVIII light chain;

• a fragment of von Willebrand Factor (VWF); and

• at least two copies of an extension peptide (EP); wherein the EP has at least 90 % amino acid sequence identity to SEQ ID NO: 1 and contains a cluster of O-glycosylation sites, wherein the cluster contains at least two O-glycosylated amino acids.

2. The fusion protein according to claim 1 , wherein

• the FVIII heavy chain does not contain the FVIII B-domain and preferably comprises an amino acid sequence with an identity of at least 90 %, more preferably at least 95 %, most preferably at least 98 % to SEQ ID NO: 2;

• the FVIII light chain comprises an amino acid sequence with an identity of at least 90 %, preferably at least 95 %, more preferably at least 98 % to SEQ ID NO: 3; and/or

• the fragment of VWF comprises an amino acid sequence with an identity of at least 90 %, preferably at least 95 %, more preferably at least 98 % to SEQ ID NO: 4.

3. The fusion protein according to claim 1 or 2, wherein

• the C-terminus of the FVIII heavy chain is fused to the N-terminus of the FVIII light chain by a first linker, wherein the first linker preferably comprises a sequence derived from the B-domain of FVIII; and/or

• the C-terminus of the FVIII light chain is fused to the N-terminus of the VWF fragment by a second linker.

4. The fusion protein according to claim 3, wherein the first linker and/or the second linker comprise(s) at least one copy, preferably at least two copies, more preferably at least three copies of the EP, wherein the EPs are preferably assembled in a consecutive order.

5. The fusion protein according to claims 1 to 4, further containing at least one half-life prolonging moiety, preferably selected from an immunoglobulin Fc-domain, serum albumin or parts thereof, an albumin binding antibody, an albumin binding protein domain, the most preferred half-life prolonging moiety being an albumin binding VHH domain.

6. The fusion protein according to claims 1 to 5, wherein the half-life prolonging moiety is either a) fused to the C-terminus of the protein by a third linker or b) forms part of the first linker.

7. The fusion protein according to claims 3 to 6, wherein the first, second and/or third linker are flexible and comprise (GGS)n, (GGGS)n, or (GGGGS)n, wherein n is an integer in the range of 1 and 10 and wherein G represents glycine and S represents serine.

8. The fusion protein according to any one of claims 3 to 7, wherein the second linker comprises a thrombin cleavage site, preferably wherein the thrombin cleavage site is defined by SEQ ID NO: 19.

9. The fusion protein according to claim 8, wherein two consecutive copies of the GGGGS motif are located at the N-terminus and/or the C-terminus of the second linker.

10. The fusion protein according to any of claims 3 to 9, wherein the amino acid sequence of the second linker is at least 95 %, more preferably at least 98 % to a sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

11. The fusion protein according to any of claims 3 to 10, wherein the first linker comprises a furin cleavage site, wherein the furin cleavage site preferably has the amino acid sequence of SEQ ID NO: 20.

12. The fusion protein according to any of claims 3 to 11 , wherein the amino acid sequence of the first linker is at least 95 %, more preferably at least 98 % to a sequence selected from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

13. The fusion protein according to any of claims 5 to 12, wherein the first and/or the second linker contain(s) at least two copies of the GGGGS motif on either side of an EP assembly and/or on either side of the half-life prolonging moiety.

14. The fusion protein according to any one of the previous claims, wherein at least two copies of the EPs are fused to the C-terminus of the VWF fragment.

15. The fusion protein according to any one of the previous claims, wherein the amino acid sequence of the fusion protein is at least 95 %, more preferably at least 98 % identical to a sequence selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.

16. A fusion protein for use in the treatment of a bleeding disorder, wherein the fusion protein is defined according to any of the preceding claims.

17. A polynucleotide encoding a fusion protein according to any of claims 1 to 16.

18. The polynucleotide according to claim 17, encoding 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: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.

19. A vector containing the polynucleotide according to claim 16 or 17, 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.

20. A host cell containing the polynucleotide according to claim 17 or 18 or the vector according to claim 19, wherein the host cell is a cell of 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.