FVIII-VWF fusion protein with improved pharmacokinetics

Abstract

The present invention relates to a fusion protein comprising a factor VIII (FVIII) heavy chain, an FVIII light chain, a von Willebrand factor (VWF) fragment, and at least two copies of an extended peptide (EP), wherein the EP has at least 90% amino acid sequence identity with SEQ ID NO: 1 and further comprises a cluster of O-glycosylation sites, each cluster containing at least two O-glycosylated amino acids. This complex exhibits improved pharmacokinetic properties compared to FVIII. The present invention further relates to a polynucleotide encoding the fusion protein, as well as a vector and host cell containing the polynucleotide.

Description

[Technical Field]

[0001] This invention relates to a fusion protein of factor VIII (FVIII) and Willbrand factor (VWF) with improved physicochemical properties and pharmacokinetics. [Background technology]

[0002] Hemophilia is a group of genetic disorders that impair the body's ability to clot or control blood clotting. The most common type, hemophilia A, is characterized by a deficiency in blood clotting factor VIII (FVIII). Hemophilia A occurs in approximately 1 in 5,000 to 10,000 male births. The FVIII protein is an essential cofactor for blood clotting and is multifunctional. FVIII deficiency can be treated with plasma-derived FVIII concentrate or recombinant FVIII preparations. Treatment with FVIII concentrate has normalized the lives of hemophilia patients.

[0003] Patients with hemophilia A are treated with FVIII administered on demand or prophylactically several times a week. Prophylactic treatment involves administering 15-25 IU / kg body weight of FVIII three times a week. This is necessary because FVIII is always needed and has a short half-life in the blood system, only about 11 hours in humans (Ewenstein et al., 2004). The short circulating half-life of FVIII, and the resulting need for frequent intravenous infusions of concentrated FVIII preparations, is a major challenge in the treatment of hemophilia A.

[0004] In the blood, under normal conditions, the FVIII molecule is always bound to its cofactor, von Willebrand factor (VWF), which stabilizes the FVIII molecule from various forms of denaturation. The non-covalent complex of FVIII and VWF has a high binding affinity of 0.2-0.3 nM (Vlot et al., 1996).

[0005] Historically, hemophilia A has been treated with human plasma-derived FVIII. Furthermore, since the 1990s, various recombinant FVIII proteins have become commercially available. However, neither plasma-derived nor recombinant FVIII proteins possess optimal pharmacokinetic properties. Like many other therapeutic proteins, they are affected by peptidase-mediated turnover, severely limiting their in vivo half-life.

[0006] Attempts to extend the half-life of FVIII include immunoglobulin Fc fusion (efmoroctocog alfa, Eloctate), addition of polyethylene glycol (turoctocog alfa pegol, Esperoct; damoctocog alfa pegol, Jivi; rurioctocog alfa pegol, Adynovate), and single-strand constructs (lonoctocog alfa, Afstyla). These techniques extend the half-life of FVIII by approximately 1.5 times (review by Tiede, 2015). It is well known that the FVIII molecule circulates in complex with VWF, and both molecules are cleared simultaneously, mainly via the VWF clearance pathway. Therefore, the half-life of FVIII is primarily determined by the half-life of VWF.

[0007] VWF and VWF fragments containing an FVIII binding site are known to stabilize FVIII against rapid clearance, proteolytic digestion, and uptake by antigen-presenting cells, thereby promoting high 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, the D'D3-Fc fusion protein can extend the FVIII half-life only in VWF- / - mice. In hemophilia A mice, the D'D3-Fc construct does not result in an extension of the FVIII half-life. This is because the competition between the protein fragment and endogenous VWF for FVIII binding is not effective.

[0008] WO 2014 / 011819 A2 describes the successful extension of the half-life of an 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 extension effect is observed in both VWF / FVIII double knockout mice (DKO) and hemophilia A mice. However, while it functions well in vitro, its activity is significantly reduced in vivo.

[0009] EP 3476937 A1 describes a chimeric protein containing FVIII and VWF (at least its D'D3 domain), intended for use as a therapeutic agent for hemophilia A, aiming to achieve an increased in vivo half-life. To lengthen the half-life, the FVIII-VWF protein is PEGylated by D1 to inhibit the binding of FVIII to low-density lipoprotein receptor-associated protein (LRP). However, PEGylation of therapeutic proteins has various drawbacks. Asparginase (PEG-ASNase) is one example, particularly used in the treatment of ALL (acute lymphoblastic leukemia). Here, in some patients, anti-PEG antibodies are already present before treatment, negatively affecting asparginase activity measured after PEG-ASNase treatment. Furthermore, these anti-drug antibodies (ADAs) can cause hypersensitivity reactions (Khalil et al., 2022). In addition, it was already known that antibodies against PEG inhibit the anticoagulant effect of aptamers (Moreno et al., 2019). Recently, it has been discovered that ADA targeting the PEG portion inhibits 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 PEGylated FVIII-based therapies have long been used extensively in the treatment of hemophilia A patients. However, until now, only short-term, mainly transient effects due to prior treatment or treatment-induced anti-PEG antibodies had been reported for Jivi (Paik and Deeks, 2019) and Esperoct / N8-GP (Giangrande et al., 2020). Furthermore, Valsecchi et al. reported in 2023 that immunization with Comirnaty (BNT162b2) induces anti-PEG ADA. In patients with hemophilia A, there is a possibility that IgM cells may cross-react with all three types of PEGylated FVIII preparations (Adynovate, Jivi, and Esperoct).

[0010] Other approaches to extending the half-life of therapeutic proteins include fusing them with proteins that already have long half-lives, such as transferrin and albumin, or with protein domains such as the C-terminal peptide (CTP) of chorionic gonadotropins (CG). As reviewed by Strohl et al (2015), various fusion proteins of therapeutic proteins and CTPs have been developed and are currently undergoing clinical trials. Examples of therapeutic proteins include FSH (Elonva(R)), FVIIa, FIX, IFN-β, and oxytomodulin.

[0011] WO 2017 / 198435 A1 describes a fusion protein comprising a main protein or fragment thereof, which is a mammalian protein such as human VWF, and one or more extension peptides. The extension peptides contain a cluster of O-glycosylation sites having at least two O-glycosylated amino acids, and may be derived from, for example, human VWF. Due to the extension peptides, the fusion protein has an increased half-life compared to its own main protein, i.e., the mammalian protein or fragment thereof. The fusion protein can be used to increase the half-life of a binding partner, for example, FVIII. The OCTA12 molecule that fits the definition in WO 2017 / 198435 A1 is a fusion of a VWF fragment that can bind to FVIII with high affinity. Because it lacks specific domains recognized by clearance receptors (such as the A1 and D4 domains recognized by SR-AI receptors), its half-life is significantly extended compared to full-length VWF (the terminal half-life after subcutaneous administration in humans is approximately 200 hours, compared to approximately 18 hours for full-length VWF). Furthermore, the 31-amino acid sequence derived from VWF, which is O-glycosylated four times, i.e., the extended peptide, is repeated three times. Notably, the non-covalent complex of FVIII and OCTA12, described in WO 2017 / 198435 A1 and Vollack-Hesse et al., 2021, primarily improves the pharmacokinetic (PK) properties of FVIII after subcutaneous administration. Compared to isolated FVIII molecules after intravenous administration, C maxOnly a slight improvement is observed, and no improvement in terminal half-life is seen (as described on page 1075 of Vollack-Hesse et al., 2021, and shown in Figure 3A). [Overview of the Initiative] [Means for solving the problem]

[0012] This invention is based, in particular, on the discovery that complexes of factor VIII (FVIII) protein and VWF fragments, especially OCTA12, exhibit high expression levels, are stabilized, and are less prone to aggregation.

[0013] O-glycosylated extended peptides (EPs) are inserted into linkers that connect proteins, linkers that connect heavy and light chains, or fused to the C-terminus of VWF fragments. Furthermore, FVIII-VWF-EP fusion proteins stabilized by the extended peptides have a longer circulating half-life compared to FVIII alone.

[0014] Accordingly, in a first embodiment, the present invention provides a fusion protein comprising an FVIII heavy chain; an FVIII light chain; a VWF fragment; and at least two copies of EP; wherein EP has at least 90% amino acid sequence identity with respect to SEQ ID NO: 1 and further comprises a cluster of O-glycosylation sites, the cluster comprising at least two O-glycosylated amino acids.

[0015] In a second embodiment, the present invention relates to a polynucleotide encoding a fusion protein according to the first embodiment.

[0016] In a third embodiment, the present invention relates to a vector comprising a polynucleotide according to a second embodiment.

[0017] In a fourth embodiment, the present invention relates to a host cell comprising a polynucleotide according to the second embodiment or a vector according to the third embodiment, wherein the host cell is a mammalian cell.

[0018] Finally, in a fifth aspect, the invention also relates to a pharmaceutical composition comprising a fusion protein according to the first aspect for use in the treatment or prevention of bleeding disorders.

Brief Description of the Drawings

[0019] [Figure 1] Figure 1 is a schematic diagram of the FVIII-VWF fragment-EP fusion protein. [Figure 2] Figure 2 is a bar graph showing the FVIII activity (FVIII:C) in the culture supernatant after transient expression of the FVIII-VWF fragment-EP fusion protein in Expi293F cells. [Figure 3] Figure 3 is a non-reducing SDS-PAGE analysis of the purified fusion protein. (A) and (B) show the FVIII-VWF-EP fusion protein after Western blot by FVIII detection (A) or Coomassie staining (B). The name of each protein is shown above each lane. M represents the molecular weight marker, and the molecular weight is expressed in kDa. [Figure 4] Figure 4 is a column diagram showing the normalized binding level of the purified FVIII-VWF fragment fusion protein to flVWF. flVWF was coated on a CM5 sensor chip, and then the purified FVIII-VWF-EP protein or control protein was injected. The SPR signal detected 30 seconds after injection stop was normalized with the binding of rFVIII (Nuwiq) as 100%. OCTA12 is a negative control and should not bind to flVWF. [Figure 5] Figure 5 is a diagram of the FVIII activity (FVIII:C) in the plasma of HemA mice after single intravenous administration of rFVIII (Nuwiq) and the FVIII-VWF fragment fusion protein C17 according to the invention.

Modes for Carrying Out the Invention

[0020] The following definitions are provided to give a clear and consistent understanding of the present specification and claims, and the scope given to such terms.

[0021] definition As used herein, “peptides” may consist of any number of any type of amino acids, preferably naturally occurring amino acids, which are preferably linked by peptide bonds. In particular, a peptide contains at least three amino acids, preferably at least five, at least seven, at least nine, at least twelve, or at least fifteen amino acids. Furthermore, there is no upper limit to the length of a peptide. However, preferably, the peptide according to the present invention does not exceed the length of 500 amino acids, more preferably 300 amino acids, and even more preferably 250 amino acids.

[0022] Therefore, the term "peptide" typically includes "oligopeptides," which refer to peptides with a length of 2 to 10 amino acids, and "polypeptides," which refer to peptides with a length of 10 amino acids or more.

[0023] As used herein, "protein" may contain one or more polypeptide chains. Proteins with one or more polypeptide chains are often expressed as one polypeptide chain from a single gene and cleaved post-translation. Therefore, the terms "polypeptide" and "protein" are used interchangeably. Polypeptides and proteins as used herein include not only chemically synthesized proteins but also naturally synthesized proteins encoded by genes. Polypeptides or proteins may be obtained from natural sources such as human blood or produced in cell culture as recombinant proteins.

[0024] As used herein, the term “fusion protein” refers to a protein created by the fusion of two or more genes that originally encoded separate proteins or protein fragments, where the components of the fusion protein are linked to each other by peptide bonds, either directly or via peptide linkers. As used herein, the term “fused” refers to components linked by peptide bonds, either directly or via one or more peptide linkers.

[0025] According to the present invention, a "peptide linker" is a peptide that links two protein elements of a fusion protein, particularly the FVIII heavy chain and the FVIII light chain, or the FVIII light chain and the VWF moiety. Peptide linkers are also simply called "linkers." Peptide linkers contain structural amino acids that enable important domain interactions, enhance stability, and reduce steric hindrance. In addition to structural amino acids, linkers may contain functional motifs. Extended peptides can be considered as protein elements or as part of a linker. Linkers can have lengths of 2 to 200 amino acids.

[0026] As used herein, the term “therapeutic protein” refers to a protein or polypeptide that has therapeutic effects, i.e., a protein used as a pharmaceutical active ingredient.

[0027] According to the present invention, the terms "protein precursor," "proprotein," or "propeptide" refer to an inactive protein (or peptide) that can become active through post-translational modification, such as enzymatic cleavage of a portion of its amino acid sequence.

[0028] The relationship between two amino acid sequences or two nucleotide sequences is represented by a parameter called "sequence identity." For the purposes of this 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), preferably version 3.0.0 or later, 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). The optional parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "longest identity" (obtained using the no brief option) is used as the percentage of identity and is calculated as follows: (Number of identical residues × 100) / (Length of alignment - Total number of gaps within the alignment).

[0029] For the purposes of this invention, the degree of sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, cited above), preferably version 3.0.0 or later, implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et a / ., 2000, cited above). The optional parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EDNAFULL (EMBOSS version NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percentage of identity and is calculated as follows: (100 identical deoxyribonucleotides) / (length of alignment - total number of gaps within alignment)

[0030] When used in relation to cells, nucleic acids, proteins, or vectors, the term "recombinant" indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a different nucleic acid or protein, or by alteration of the native nucleic acid or protein, or that the cell originates from such a modified cell. For example, recombinant cells may express genes not found in the cell's native (non-recombinant) form, or they may express native genes at different levels or under different conditions than in nature.

[0031] As used herein, the term “half-life” refers to the time required for the plasma / blood concentration to decrease by 50% after the distribution has reached a pseudo-equilibrium (following the definition by Toutain et al., 2005). The term “half-life” is also referred to as “circulatory half-life,” “terminal half-life,” or “excretion half-life.”

[0032] In this specification, the terms “transformed,” “stable transformed,” and “transgenic” as used with respect to cells mean that the cell contains non-native (e.g., heterologous) nucleic acid sequences incorporated into its genome or are carried as episomes that are maintained over multiple generations.

[0033] As used herein, the term “fragment” refers to a polypeptide that, compared to the native or wild-type protein, has one or more amino acid deletions at the amino-terminus and / or carboxy-terminus, but whose remaining amino acid sequence is identical to the corresponding positions of the amino acid sequence inferred from the full-length cDNA. The length of a fragment is typically at least 50 amino acids.

[0034] As used herein, the term "glycosylation" refers to the attachment of sugar chains to a molecule, such as a protein. Glycosylation can be an enzymatic reaction. The bond formed may be a covalent bond. Therefore, as used herein, a glycosylated polypeptide is a polypeptide to which one or more sugar chains are attached. "Highly glycosylated" refers to an enzyme-like molecule that is glycosylated at all or nearly all of its available glycosylation sites, such as O-linked or N-linked glycosylation sites.

[0035] As used herein, the term “glycan” refers to a polysaccharide or oligosaccharide, or the carbohydrate portion of a glycoprotein or glycosylated polypeptide. Glycans are homopolymers or heteropolymers of monosaccharide residues. They may be linear or branched. Glycans typically contain at least three sugars and are linear or branched. Glycans may contain neutral sugar residues (e.g., glucose, N-acetylglucosamine (GlcNAc), 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′-fluoribose, 2′-deoxyribose, phosphomannose, 6′-sulfo-N-acetylglucosamine, etc.).

[0036] As used herein, the term "O-glycan" generally refers to a sugar chain covalently bonded to serine and threonine residues in mammalian glycoproteins.

[0037] O-glycans may be α-linked to the -OH group of serine or threonine via an O-glycosidic bond through the GalNAc moiety. Other linkages include α-linked O-fucose, β-linked O-xylose, α-linked O-mannose, β-linked O-GlcNAc, α- or β-linked O-galactose, and α- or β-linked O-glucose sugar chains.

[0038] According to the present invention, the terms "O-glycosylation cluster," "O-glycan cluster," and "O-glycosylation amino acid cluster" are used interchangeably and relate to two or more adjacent O-glycosylation amino acids.

[0039] As used herein, the term "sialylation" refers to a molecule, particularly a sugar chain, that has been reacted with sialic acid or a derivative thereof.

[0040] The transitional term "comprising" is synonymous with "including," "containing," or "characterized by," and is comprehensive or open-ended, and does not exclude additional, unreproduced elements or method steps. The transitional expression "consisting of" excludes elements, steps, or components not explicitly stated in the claim, except for impurities that usually accompany them. If the phrase "consists of" appears in a section of the claim text rather than immediately following the preamble, the phrase limits only the elements described in that section, and does not exclude other elements from the claim as a whole. The transitional expression "consisting essentially of" limits the scope of the claim to specified materials or steps that "do not substantially affect the basic and novel features" of the invention described in the claim. Claims that state "consisting essentially of" fall between closed claims, which are written in the "consisting of" format, and fully open claims, which are written in the "comprising" format.

[0041] In the context of this invention, for practical reasons, the term “glycosylated protein,” such as fusion protein, is used in the singular form. Generally, in actual clinical practice, proteins are composed of the same type of protein molecule. However, in the case of glycosylated proteins, glycosylation is not identical in all molecules of the composition. For example, not all individual molecules in the composition are necessarily 100% glycosylated. Furthermore, there may be differences in the sugar chains that bind to specific O-glycosylation sites. Therefore, in this application, the reference to “fusion protein” also relates to compositions of fusion protein molecules that have the same amino acid sequence but have mutations in the O-glycan structure.

[0042] As used herein, the terms “binding affinity” or “affinity” refer to the strength of binding between two molecules, particularly a ligand and a protein target. Binding affinity is influenced by non-covalent intermolecular interactions between the two molecules, such as hydrogen bonds, electrostatic interactions, hydrophobic interactions, and van der Waals forces.

[0043] The immune responses used herein refer to adaptive immune responses or innate immune responses. Innate immune responses refer to nonspecific defense mechanisms that are activated immediately after or within a few hours of an antigen appearing in the body. These mechanisms include physical barriers such as the skin, chemicals in the blood, and immune system cells that attack foreign substances in the body. Innate immune responses are activated by the chemical properties of the antigen. Adaptive immune responses are antigen-specific immune responses. For this to occur, the antigen must first be processed and recognized. Once the antigen is recognized, the adaptive immune system produces a large number of immune cells that are specifically designed to attack that antigen.

[0044] Fusion protein According to a first aspect, the present invention provides a fusion protein comprising an FVIII heavy chain; an FVIII light chain; a VWF fragment; and at least two copies of an extended peptide.

[0045] The fusion protein according to the present invention offers technical advantages in the manufacturing process of the FVIII-VWF complex. Instead of separately manufacturing and purifying the two molecules and combining them in a specific ratio, covalent complexation reduces the manufacturing and purification steps to one. Furthermore, the covalent complex enhances the expression level. The fusion protein according to the present invention shows an increased expression level compared to FVIII alone. According to one embodiment, the expression level of the fusion protein is in the range of 0.6 to 10.3 IU / ml. Under the same conditions, the expression level of FVIII alone was an average FVIII:C of 0.33 IU / ml. FVIII activity can be measured by a chromogenic assay. According to one embodiment, the expression level is 0.6 IU / ml or higher, as measured by FVIII activity in the cell culture supernatant. The expression level may be, for example, 0.6 IU / ml, 0.8 IU / ml, 1.0 IU / ml, 1.2 IU / ml, 1.4 IU / ml, 1.6 IU / ml, 1.8 IU / ml, 2.0 IU / ml, 3.0 IU / ml, 4.0 IU / ml, 5.0 IU / ml, 6.0 IU / ml, 7.0 IU / ml, 8.0 IU / ml, 9.0 IU / ml, 10.0 IU / ml, 11.0 IU / ml, 12.0 IU / ml, 13.0 IU / ml, 14.0 IU / ml, or 15.0 IU / ml. According to one embodiment, the expression level is 4.0 IU / ml or higher. According to one embodiment, the expression level is 6.0 IU / ml or higher. According to one embodiment, the expression level is 15.0 IU / ml or lower. According to one embodiment, the expression level is 11.0 IU / ml or less.

[0046] In addition to improved expression and purification, the fusion protein according to the present invention exhibits an increased half-life compared to FVIII alone. According to one embodiment, the half-life extension of the fusion protein is at least 20%. According to one embodiment, the half-life extension of the fusion protein is at least 30%. According to one embodiment, the half-life extension of the fusion protein is at least 40%. According to one embodiment, the half-life extension of the fusion protein is at least 50%. According to one embodiment, the half-life extension of the fusion protein is at least 60%.

[0047] The fusion protein according to the present invention also exhibits improved PK properties, particularly an improved half-life, compared to the non-covalent complex of FVIII and VWF. In the case of the non-covalent complex of FVIII and OCTA12 of WO 2017 / 198435 A1 after intravenous administration, no improvement in terminal half-life is observed compared to FVIII alone (this is described on page 1075 of Vollack-Hesse et al., 2021, and demonstrated in Figure 3A). The fusion of OCTA12 and FVIII prevents the redistribution of circulating FVIII to endogenous flVWF. The fusion protein according to the present invention shows a significantly reduced binding ability to flVWF in vitro, as shown in Figure 4, and an extended half-life after intravenous administration, as shown in Figure 5 and Table 6 (below).

[0048] The fusion protein according to the present invention exhibits reduced binding to endogenous VWF when administered to a patient. According to one embodiment, the binding is at most 11% of that of FVIII alone to VWF. Binding can be determined by surface plasmon resonance (SPR), as shown in Figure 4.

[0049] Half-life (t 1 / 2 This can be calculated by linear regression analysis of the log-linear portion of individual plasma concentration-time curves, or by nonlinear regression using a one-phase exponential decay model. Representative software for this calculation includes GraphPad Prism version 6.07 (La Jolla, CA 92037 USA) and WinNonlin, version 6.4 (Pharsight Corporation, Mountain View, CA, USA). The calculation is based on the following formula:

number

[0050] Human blood coagulation factor VIII is encoded by the F8 gene, which consists of 187,000 base pairs and six exons. The transcribed mRNA is 9029 base pairs long and is translated into a 2351 amino acid protein with 19 amino acids removed. The human FVIII molecule is glycosylated with 25 N-glycosylation chains and 6 O-glycans over 31 amino acids (see Kannicht et al., 2013).

[0051] After translation, the amino acid chain is cleaved by specific proteases, forming a heavy chain of approximately 200 kDa and a light chain of approximately 80 kDa. The domain configuration is typically characterized as A1-A2-B-A3-C1-C2. The light chain has the configuration of domains A3-C1-C2. The heavy chain consists of domains A1-A2-B. Heavy chains present in plasma have a heterogeneous composition with molecular weights ranging from 90 to 200 kDa. The reasons for this size variation are heterogeneity of glycosylation, the presence of splice variants, and proteolytic products such as heavy chain A1-A2 lacking the B domain. The full-length amino acid sequence of FVIII is identified from amino acids 20 to 2351 in UniProtKB P00451 (sequence version 1, July 21, 1986).

[0052] The human FVIII heavy chain according to the present invention comprises at least domains A1 and A2, and may further comprise part or all of domain B. The amino acid sequence of the human FVIII heavy chain excluding domain B is shown in Sequence ID No. 2. The amino acid sequence of the human FVIII heavy chain including domain B is identified in Sequence ID No. 3.

[0053] According to one embodiment, the FVIII heavy chain does not contain the FVIII B domain.

[0054] The human FVIII heavy chain, excluding 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 to or identical to SEQ ID NO: 2. The FVIII heavy chain, excluding the B domain, may contain an amino acid sequence that is 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% identical to SEQ ID NO: 2.

[0055] According to one embodiment, the heavy chain is at least 95% identical to SEQ ID NO: 2. Example 6 shows a fusion protein with an FVIII heavy chain sequence variant of SEQ ID NO: 37 having the 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 includes an FVIII B domain. A heavy chain having an FVIII B domain may contain an amino acid sequence that is 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% identical 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.

[0056] According to one embodiment, the FVIII light chain includes the domain configuration A3-C1-C2. A human FVIII light chain having the A3-C1-C2 domain configuration has the sequence of Sequence ID No. 4.

[0057] The FVIII light chain of the fusion protein has an amino acid sequence similar to or identical to SEQ ID NO: 4. According to one embodiment, the FVIII light chain contains an amino acid sequence that is 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% identical 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 an FVIII light chain sequence variant of SEQ ID NO: 38 having the amino acid substitution S1732T.

[0058] VWF is a multimeric adhesive glycoprotein found in mammalian plasma and possesses multiple physiological functions. During primary hemostasis, VWF acts as an intermediary between specific receptors on the platelet surface and components of the extracellular matrix, such as collagen. Furthermore, VWF is both a carrier and a stabilizing protein for coagulation factor VIII, a coagulation-promoting factor. VWF is synthesized in endothelial cells and megakaryocytes as a precursor molecule of 2813 amino acids.

[0059] The domain structure 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 propeptide (domains D1-D2), and a 2050-residue polypeptide found in mature plasma von Willebrand factor (Fischer et al., 1994). Full-length VWF is identified by UniprotKB entry P04275 (entry version 224, April 12, 2017).

[0060] The human VWF according to the present invention has any amino acid sequence of UniprotKB P04275, particularly sequence number 5 (isoform 1). The VWF has two clusters of O-glycosylated amino acids. The first cluster of O-glycosylated amino acids is found between amino acids 1238 and 1268 of sequence number 5.

[0061] The second cluster contains amino acids 1468 to 1487 of sequence number 5.

[0062] When secreted into plasma, VWF circulates in various forms with different molecular sizes. These VWF molecules consist of oligomers and multimers of a mature subunit comprising 2050 amino acid residues. VWF usually exists in plasma as a multimer, with a size of approximately 500 to 20,000 kDa (Furlan 1996).

[0063] According to one embodiment, the VWF fragment contains an amino acid sequence that is 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% identical to the portion of SEQ ID NO: 5. According to one embodiment, the VWF fragment is at least 95% identical to the portion of SEQ ID NO: 5. According to one embodiment, the VWF fragment is at least 98% identical to the portion of SEQ ID NO: 5.

[0064] In human VWF fragments, one or more domains A1, A2, A3, D4, C1, C2, C3, and CK may be missing compared to mature human VWF (TIL3-D3-TIL4-A1-A2-A3-D4-C1-C2-C3-CK). The VWF fragment may have a domain configuration selected from the group consisting of, for example, 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.

[0065] In this regard, the section of SEQ ID NO: 5 is, in particular, the section of SEQ ID NO: 5 beginning with amino acid 764. Amino acids 764 to 1035 of SEQ ID NO: 5 contain the FVIII binding domain of VWF. The section may be, for example, the section defined in WO 2015 / 185758 A2. As shown in WO 2015 / 185758 A2, a complex of FVIII with a VWF fragment as defined therein shows reduced binding to the phospholipid membrane compared to FVIII alone, and also shows reduced binding to collagen III and heparin compared to a complex of FVIII with full-length VWF. Preferably, the section of SEQ ID NO: 5 beginning with amino acid 764 preferably ends with amino acids of SEQ ID NO: 5 in the range of 1905 to 2153. According to one embodiment, the VWF fragment ends with amino acids of VWF in the range of 2030 to 2153 of SEQ ID NO: 5. According to a further embodiment, the VWF fragment ends with amino acids of SEQ ID NO: 5 in the range of 2100 to 2153.

[0066] According to one embodiment, the VWF fragment contains an amino acid sequence having 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% identity with SEQ ID NO: 6 or a portion thereof. The VWF fragment having the amino acid sequence of SEQ ID NO: 6 has two amino acid substitutions, namely C1099A and C1142A, based on the portion of amino acids 764 to 1268 of SEQ ID NO: 5. When the two cysteines are substituted with alanine, the multimerization ability of the VWF fragment is lost. This modified VWF fragment is used in the fusion protein of the example. According to one embodiment, the VWF fragment is at least 98% identical to SEQ ID NO: 6. Example 6 shows a fusion protein with SEQ ID NO: 39 having a sequence variant of SEQ ID NO: 6, namely the additional amino acid substitution A1164V.

[0067] As shown in the examples, the EP having the sequence QEPGGLVVPPTDAPVSPTTLYVEDISEPPLH (Sequence ID 1), i.e., O-glycosylated cluster 1 of VWF (amino acids 1238-1268 of Sequence ID 5), gives the fusion protein according to the present invention increased expression levels, improved stability, and reduced aggregation tendency.

[0068] Therefore, the EP according to the present invention has an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. According to one embodiment, the sequence identity with respect to SEQ ID NO: 1 is preferably at least 95%. According to one embodiment, the sequence identity of the EP with respect to SEQ ID NO: 1 is at least 98%. Example 6 shows a fusion protein with a sequence variant of SEQ ID NO: 1, i.e., SEQ ID NO: 40 having the amino acid substitution G5A. According to one embodiment, two or more EPs have 100% sequence identity with SEQ ID NO: 1.

[0069] According to one embodiment, at least one copy of the EP is directly fused to the C-terminus of the VWF fragment.

[0070] One, two, three, four, five, or six copies of EP may be fused to the C-terminus of the VWF fragment. The FVIII-VWF-EP fusion protein shown in the examples has three EPs at the C-terminus of the VWF fragment, one as part of the fragment and two fused to it. The VWF protein having the amino acid sequence of SEQ ID NO: 6, with two extended peptide copies having 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.

[0071] 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 Figure 1). Linkers connecting heavy and light chains are known in the art. One example is NUWIQ(R). The linker is SFSQNSRHQAYRYRRG (SEQ ID NO: 21). This linker contains a sequence derived from the B domain of FVIII. According to one embodiment, the first linker preferably contains a sequence derived from the B domain of FVIII.

[0072] 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 is (GGS) n (GGGS) n , and (GGGGS) n It includes a motif selected from the following. 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. It corresponds to a single-letter amino acid code, where G represents glycine and S represents serine. These motifs provide flexibility to the first linker and allow for sufficient interaction between the FVIII heavy and light chains.

[0073] According to one embodiment, the first linker includes a cleavable linker, i.e., a protease cleavage site. The advantage of the presence of the protease cleavage site is that FVIII may be processed in the cell to its original double-stranded composition. According to one embodiment, the first linker includes a furin cleavage site. The furin cleavage site was chosen because it is a cleavage site that naturally exists in wild-type FVIII. The furin cleavage site may have the amino acid sequence of SEQ ID NO: 20.

[0074] Exemplary first linkers (corresponding sequence numbers) are linker 1-1 (sequence number 12), linker 1-2 (sequence number 13), linker 1-3 (sequence number 14), linker 1-4 (sequence number 15), linker 1-5 (sequence number 16), linker 1-6 (sequence number 17), and linker 1-7 (sequence number 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 at least 90%, at least 95%, or at least 98% identity with a sequence selected from sequence numbers 12, 13, 14, 15, 16, 17, and 18. According to one embodiment, the amino acid sequence of the first linker is identical to a sequence selected from sequence numbers 12, 13, 14, 15, 16, 17, and 18.

[0075] According to one embodiment, the first linker contains at least one copy of an EP. The first linker may contain, for example, one, two, three, four, five, six, seven, or eight copies of an EP. The FVIII-VWF-EP fusion protein shown in the examples has three EPs in the first linker. Therefore, according to one embodiment, the first linker contains at least two copies of an EP. According to one embodiment, the first linker contains at least three copies of an EP. The EPs may be distributed along the length of the linker, or they may be sandwiched between structural amino acids or other elements of the linker. Alternatively, two or more EPs may be assembled adjacent to each other, i.e., in a consecutive order. When two or more EPs are arranged side by side, it is called an EP assembly. According to one embodiment, all EPs of the first linker are assembled in a consecutive order.

[0076] 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.

[0077] The second linker may be a flexible (flexible) linker or a rigid linker. Preferably, the second linker is a flexible (flexible) linker. According to one embodiment, the second linker is (GGS) n , (GGGS) n , and (GGGGS) n and contains a motif selected from. 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. It corresponds to the one-letter amino acid code, where G represents glycine and S represents serine. These motifs impart flexibility to the second linker and allow for sufficient interaction, particularly binding to the VWF and FVIII binding domains. According to one embodiment, the second linker contains the (GGGGS)2, (GGGGS)4 and / or (GGGGS)6 motifs 。

[0078] According to one embodiment, two consecutive copies of the GGGGS motif are located at the N-terminus and / or C-terminus of the second linker. According to one embodiment, the (GGGGS)2 motif is located at the N-terminus. According to one embodiment, the (GGGGS)2 motif is located at the C-terminus. According to one embodiment, (GGGGS) with n≧2 n The motif is located at the C-terminus. According to one embodiment, (GGGGS)2, (GGGGS) 4、 (GGGGS)6 or (GGGGS)8 is located at the C-terminus of the second linker. As shown in Example 4, FVIII-VWF-EP fusion proteins with a large number of glycines at the C-terminal portion of the linker show higher binding to endogenous VWF. The reason may be that a longer flexible (flexible) linker allows for better interaction between the VWF fragment and the FVIII portion of the fusion protein, and as a result, prevents interaction with endogenous VWF to a higher degree. Therefore, (GGGGS) with n≧2 n The linker of is preferred. According to one embodiment, the (GGGGS)2 motif is located at each of the N-terminus and C-terminus of the second linker.

[0079] According to one embodiment, the second linker is cleavable. According to one embodiment, the second linker includes a thrombin cleavage site. The advantage of having a protease cleavage site is that it may be possible to separate the VWF fragment from the FVIII heavy and light chains after FVIII activation. The thrombin cleavage site is defined by the sequence of Sequence ID No. 19. The thrombin cleavage site was chosen because it is also part of the native FVIII sequence.

[0080] According to one embodiment, the second linker contains at least one copy of EP. As shown in Example 2, FVIII-VWF-EP fusion proteins having EP in the second linker, namely C4 and C17, show a stronger increase in expression than other FVIII-VWF-EP fusion proteins. Furthermore, negatively charged EP inserted into the second linker may reduce the tendency of the fusion protein to form high molecular weight species. This effect is demonstrated by C17, which contains three EP in the linker and has a low proportion of high molecular weight species (10.53%). The second linker can contain, for example, one, two, three, four, five, six, seven, or eight copies of EP. Thus, according to one embodiment, the second linker contains at least two copies of EP. According to one embodiment, the second linker contains at least three copies of EP. The EP may be distributed along the length of the linker, or it may be sandwiched between the structural amino acids or other elements of the linker. Alternatively, two or more EPs can be assembled adjacent to each other, i.e., in a consecutive order. According to one embodiment, the EPs of the second linker are all combined in a consecutive order. Exemplary second linkers (corresponding sequence numbers) are linker 2-3 (sequence number 9) and linker 2-5 (sequence number 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 at least 90%, at least 95%, or at least 98% identity with sequences selected from sequence numbers 9 and 11. According to one embodiment, the amino acid sequence of the second linker is selected from sequence numbers 9 and 11.

[0081] According to one embodiment, the fusion protein includes at least one half-life extension region. There are various half-life extension regions known in the art. The half-life extension region can be selected from an immunoglobulin Fc domain, serum albumin or a portion thereof, an albumin-binding antibody, an albumin-binding antibody domain, or an albumin-binding protein domain. An Fc domain is a crystallizable fragment (Fc) region of the tail region of an antibody that interacts with a cell surface receptor called the Fc receptor. In addition to this interaction, larger molecules have slower renal clearance, thus extending the half-life of the bound protein. An exemplary Fc domain is the Fc domain of IgG1. Several fusion proteins containing albumin have been reported to increase the half-life of therapeutic proteins, including fusion proteins of factor VII, factor FVIII, factor IX, and albumin. According to one embodiment, full-length human serum albumin (HSA) is added to the fusion protein. The half-life of albumin is also regulated by members of the Fc receptor family, namely the neonatal Fc receptor (FcRn). The HSA moiety added to the fusion protein preferably has a sequence according to Uniprot entry P02768.

[0082] V H The H fragment is a single-domain antibody designed from heavy chain antibodies found in camelids. According to one embodiment, the half-life extension portion is albumin-bound V H This is the H domain. Albumin-binding V H The H domain is known in this art. Cross-reactive albumin-binding V H An example of an H domain is MSA21, described in EP 2316852 B1. According to one embodiment, albumin-binding V H The H domain is an albumin-binding nanobody (ABN) as exemplified in Sequence ID No. 41.

[0083] The half-life extension portion can be fused to the C-terminus of a protein. The half-life extension portion may be directly fused to the C-terminus of the VWF fragment, or to the C-terminus of the EP. Alternatively, the half-life extension portion may be fused to the C-terminus, i.e., the C-terminus of the VWF fragment or the C-terminus of the EP, by a third linker. Another option is for the half-life extension portion to form part of the first linker.

[0084] According to one embodiment of the fusion protein, the C-terminus and / or N-terminus of the EP assembly in the first and / or second linker are directly ligated 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 N-terminus of the half-life extension portion in the first and / or second linker are directly ligated to at least one copy, preferably at least two copies, of GGS, GGGS, or GGGGS.

[0085] According to one embodiment of the fusion protein, the first and / or second linker includes copies of at least two GGGGS motifs on both sides of the extended peptide assembly and / or on both sides of the half-life extension portion.

[0086] Examples of fusion proteins (corresponding SEQ ID NOs): 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), C24 (SEQ ID NO: 36). Table 3 below shows the components (and their sequences) that make up these fusion proteins.

[0087] According to one embodiment, the amino acid sequence of the fusion protein has at least 90%, at least 95%, or at least 98% identity with a sequence selected from SEQ ID NOs. 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36. According to one embodiment, the amino acid sequence of the fusion protein is identical to a sequence selected from SEQ ID NOs. 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36.

[0088] Polynucleotides According to a second aspect, the present invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a fusion protein according to a first aspect of the present invention.

[0089] The isolated polynucleotide may be a DNA molecule or an RNA molecule. Preferably, the isolated polynucleotide is a DNA molecule, particularly a cDNA molecule. Techniques used to isolate or clone peptide-encoding polynucleotides are known in the art and include isolation from genomic DNA, preparation from cDNA, or combinations thereof. Cloning of such polynucleotides from genomic DNA can be performed, for example, by detecting cloned DNA fragments sharing structural features using well-known polymerase chain reaction (PCR) or antibody screening of expression libraries (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) can also be used.

[0090] In particular, the isolated polynucleotide sequence may include a first portion encoding the FVIII heavy chain, a second portion encoding the first linker, a third portion encoding the FVIII light chain portion, a fourth portion encoding the second linker, and a fifth portion encoding the VWF fragment.

[0091] According to one embodiment, the first portion encodes an FVIII heavy chain having 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% sequence identity with respect to SEQ ID NO: 2.

[0092] According to one embodiment, the second part codes a first linker having 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% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, 17, and 18.

[0093] According to one embodiment, the third portion encodes an FVIII light chain having 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% sequence identity with SEQ ID NO: 4.

[0094] According to one embodiment, the fourth portion encodes a second linker having 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% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, and SEQ ID NOs: 11.

[0095] According to one embodiment, the fifth portion encodes a VWF fragment having 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% sequence identity with sequence number 6.

[0096] According to one embodiment, the polynucleotide encodes an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% identity with a sequence selected from SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36.

[0097] Expression vector In a third embodiment, the present invention also relates to an expression vector comprising a polynucleotide according to a second aspect of the present invention.

[0098] The expression vector preferably further includes regulatory elements such as a promoter and transcription and translation stop signals. A recombinant expression vector can be constructed by conjugating a polynucleotide and regulatory elements together according to a second embodiment, and this vector may contain one or more restriction sites at such sites that allow for the insertion or substitution of a polynucleotide encoding a polypeptide. The polynucleotide can be inserted into an expression vector suitable for expression. When constructing the expression vector, the coding sequence is positioned in the expression vector so as to be operably linked with a regulatory sequence suitable for expression.

[0099] The recombinant expression vector may be any vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and result in the expression of polynucleotides according to the fourth aspect of the present invention. The selection of the expression vector usually depends on its compatibility with the host cell into which it is introduced. The expression vector may be a linear plasmid or a closed circular plasmid.

[0100] The expression vector is preferably adapted for expression in mammalian cells. The expression vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. For autonomous replication, the vector may further include an origin of replication that enables the vector to autonomously replicate within the host cell. The origin of replication may be any plasmid replicator that mediates autonomous replication within the cell. The terms “origin of replication” or “plasmid replicator” mean a polynucleotide that enables the plasmid or vector to replicate in vivo.

[0101] The vector is preferably one that, upon introduction into a host cell, is integrated into the genome and replicates along with the integrated chromosome. To be integrated into the host cell's genome, the expression vector may rely on any other elements of the expression vector for integration by homologous or non-homologous recombination. Alternatively, the vector may further contain polynucleotides to guide the integration into the host cell's genome by homologous recombination to the precise location of the chromosome.

[0102] The vector of the present invention preferably includes one or more (e.g., several) selection markers that allow for easy selection of transformed cells, transfected cells, transduced cells, etc. A selectable marker is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, prototrophicity to supplemental organisms, etc.

[0103] The procedure used to ligate the above elements to construct the recombinant expression vector of the present invention is well known to those skilled in the art (see Green and Sambrook 2012; Chapter 3).

[0104] According to one embodiment, the vector backbone of the vector according to the third embodiment is selected from pCDNA3, pCDNA3.1, pCDNA4, pCDNA5, pCDNA6, pCEP4, pCEP-puro, pCET1019, pCMV, pEF1, pEF4, pEF5, pEF6, pExchange, pEXPR, pIRES, and pSCAS.

[0105] host cell According to a fourth aspect, the present invention provides a host cell comprising an expression vector according to a third aspect of the present invention. The expression vector according to the third aspect is introduced into the host cell such that the expression vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector, as described above. The term “host cell” includes offspring of a parent cell that are not identical to the parent cell due to mutations that occur during replication. The selection of the host cell largely depends on the gene encoding the polypeptide and its source.

[0106] According to one embodiment, the fusion protein is produced by expression in a mammalian host cell line. Preferably, the fusion protein is produced in a human host cell line. In general, any human host cell line is suitable for the expression of the fusion protein. It is preferable that the host cell is of human origin so that the fusion protein is properly processed during folding and undergoes appropriate post-translational modifications (e.g., glycosylation, hydroxylation, phosphorylation, sulfation). A preferred glycosylation profile of the fusion protein is obtained particularly in human kidney cell lines. Preferred human kidney cell lines are HEK cell lines, particularly the HEK293 cell line.

[0107] Examples of HEK cell lines for glycosylated polypeptide production include HEK 293 F and Flp-In. TM-293(Invitrogen, R75007), 293(ATCC(R) CRL-1573), 293 EBNA, 293 H(Thermo Scientific 11631017), 293S, 293T(ATCC(R) CRL-3216 TM ), 293T / 17 (ATCC(R) CRL11268 TM ), 293T / 17 SF (ATCC(R) ACS4500 TM ), HEK 293 STF (ATCC(R) CRL 3249 TM ), HEK-293.2sus (ATCC(R) CRL-1573 TM ) is available. The preferred cell line for polypeptide production is the HEK 293 F cell line.

[0108] Other human cell lines suitable as host cells for expression include, but are not limited to, cell lines derived from myeloid leukemia cells. Specific examples of host cells include K562, NM-F9, NM-D4, NM-H9D8, NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X, GT-5s, and cells derived from any of the aforementioned host cells. K562 is a human myeloid leukemia cell line found 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 characteristics.

[0109] Mammalian host cell lines suitable for the production of fusion proteins according to the present invention include cell lines derived from hamsters, mice, and monkeys. Suitable host cells include Chinese hamster ovary cells (CHO cells, e.g., DG44, DXB11, and K1 [ATCC CCL-61, its glutamine-deficient derivative CHOZn, SAFC CHOGS]) and baby hamster kidney (BHK) cells.

[0110] Pharmaceutical compositions and medical applications The fusion protein according to the first embodiment is particularly useful as an active ingredient for medical use. Preferably, it is useful for the treatment or prevention of hemorrhagic disorders. The fusion protein according to the first embodiment described herein may be administered alone or in the form of a pharmaceutical composition.

[0111] Accordingly, according to a fifth aspect, the present invention provides a fusion protein according to the first aspect for use in the treatment of hemorrhagic disorders.

[0112] According to one embodiment, the fusion protein may be formulated with at least one pharmaceutically acceptable carrier. The pharmaceutical composition based on the fusion protein can be prepared and administered to a subject by any method well known in the pharmaceutical art. See, for example, 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, eds., Lippincott Williams & Wilkins (20th ed., 2003); and *Pharmaceutical Dosage Forms and Drug Delivery Systems*, Ansel et al. (eds), Lippincott Williams & Wilkins (7th ed., 1999). Furthermore, the pharmaceutical composition of the embodiment may also be formulated to include other medically useful drugs or biological agents. The pharmaceutical composition typically contains a therapeutically effective amount of the fusion protein in combination with a pharmaceutically acceptable carrier. A 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. Other forms of pharmaceutically acceptable carriers available in this embodiment include binders, disintegrants, surfactants, absorption enhancers, humectants, cryoprotectants, absorbents, lubricants, fillers, bulking agents, hydrating agents, preservatives, stabilizers, emulsifiers, solubilizers and bulking agents, osmotic pressure-controlling salts, diluents such as buffers, and additives commonly used in the formulation's intended use. These are optionally selected and used depending on the unit dose of the resulting formulation.

[0113] Accordingly, the present invention also relates to a method for treating or preventing a bleeding disorder in a patient, the method comprising administering to the patient a pharmaceutical composition according to a fifth embodiment.

[0114] As used herein, "hemorrhagic disorder" refers to a disease or condition in which normal hemostasis is impaired. Examples of hemorrhagic disorders include hemophilia A, hemophilia B, factor VIII deficiency, factor XI deficiency, von Willebrand disease, Glanzmann thrombasthenia, Bernard-Soulier syndrome, idiopathic thrombocytopenic purpura, intracerebral hemorrhage, trauma, and traumatic brain injury.

[0115] In this specification, “hemophilia” refers to a group of hemorrhagic disorders characterized by an increased thrombus formation time compared to that of healthy individuals without hemophilia. Hemophilia includes hemophilia A, a disorder resulting in deficiency of coagulation factor VIII production; hemophilia B, a disorder resulting in deficiency of coagulation factor IX production; and acquired hemophilia A, a rare hemorrhagic disorder caused by autoantibodies against FVIII.

[0116] The bleeding disorder is preferably hemophilia A or B. Treatment may include, for example, hemophilia therapy for untreated patients (PUPS), immune tolerance induction (ITI) therapy, and / or other related therapies for hemophilic disease.

[0117] For in vivo applications, the pharmaceutical composition can be administered to the patient by any conventional route of administration, such as orally, parenterally, or by inhalation. Parenteral administration includes intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, and solutions, suspensions, emulsions, and drops. For parenteral administration, the pharmaceutical composition is preferably an injectable preparation such as a solution or suspension.

[0118] In other embodiments, the pharmaceutical composition is administered orally to the patient. In these embodiments, the form of the drug includes solid formulations such as tablets, coated tablets, powders, granules, capsules and tablets; liquid formulations such as liquid formulations (e.g., eye drops, nasal sprays), suspensions, emulsions and syrups; aerosol formulations; inhalants such as nebulizers and sprayers; and liposome encapsulants. In several other embodiments, the glycosylated polypeptide, protein complex or pharmaceutical composition is administered by inhalation into the patient's respiratory system to target the trachea and / or lungs.

[0119] According to one embodiment of the fifth aspect, use includes intravenous or non-intravenous injection. Non-intravenous injection is preferably subcutaneous injection. [Examples]

[0120] Example 1: Cloning, expression, and purification of the FVIII-VWF-EP fusion protein Objective of the experiment Preparation of cDNA encoding the FVIII-VWF-EP fusion protein. Expression and uniform purification of the fusion protein.

[0121] method Gene synthesis and cloning Golden Gate cloning technology was used to construct the expression vector encoding the fusion construct. For this purpose, a cDNA fragment encoding the desired FVIII-VWF-EP fusion construct was synthesized and cloned into a donor vector suitable for Golden Gate cloning at Twist Bioscience. Next, the donor vector containing the desired construct variant was used in the Golden Gate assembly reaction along with a donor vector containing regulatory elements and a proprietary acceptor backbone. This reaction produced a mammalian expression vector containing the gpCMV promoter at 5' and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) at 3' of the target construct variant.

[0122] The vector construct was transformed into E. coli NEB5α cells, cultured overnight at 37°C on ampicillin-containing LB-agar plates, and then a single clone was selected.

[0123] Plasmid DNA preparation was performed using the QIAprep DNA Mini kit (Qiagen) or the NucleoBond(R) Xtra Maxi Plus EF kit (Macherey-Nagel) according to the manufacturer's recommendations. Sequencing confirmed the vector's integrity, particularly the correct orientation and completeness of the genes encoding the desired construct variants.

[0124] Protein expression The FVIII-VWF-EP fusion construct was transiently expressed in Expi293F cells (Thermo Fisher Scientific) in 500-1000 mL scales according to the manufacturer's recommendations. Cell culture supernatant containing the product was collected 4-5 days after transfection by centrifugation at 2000 xg for 20 minutes.

[0125] Protein purification Purification was achieved through a three-step process: recovery from the cell culture supernatant, purification by affinity chromatography, and reconstitution into the final matrix.

[0126] In short, 0.3 M NaCl was added to the collected cell culture supernatant to increase the conductivity of the sample, which was then filtered through a 0.2 μm PES filter and captured with Capto MMC resin (Cytiva). For this purpose, the Capto MMC column was equilibrated to pH 6.5 with 0.3 M NaCl, 0.01 M CaCl2, 0.01 M L-histidine, and 0.02% polysorbate 80, and eluted with 0.3 M NaCl, 0.02 M CaCl2, 0.02 M L-histidine, 0.8 M L-arginine, and 10% ethylene glycol. 0.02% polysorbate 80, pH 6.5. The column eluate was diluted 1:2 with equilibration buffer (0.05 M Tris, 0.1 M NaCl, 0.02% polysorbate 80 pH 7.0) and then loaded onto 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 NaCl, and 1 M MgCl2 pH 7.0, and re-buffered with final formulation buffer (171.1 mM NaCl, 7.1 mM L-arginine, 26.3 mM Sucrose, 3.4 mM trisodium citrate, 1.7 mM CaCl2, 0.1 mM Poloxamer 188 pH 7.0) using a Sephadex G-25 desalting column (Cytiva).

[0127] result Table 1-3 shows the gene products encoded by the cloned cDNA constructs.

[0128] Table 1: The first linker used in the FVIII-VWF-EP fusion protein. EP represents the extended peptide of SEQ ID NO: 1. The lowercase numbers represent the number of repetitions of the assigned sequence element. ABN is an albumin-binding nanobody with the sequence QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQGTQVTVSS (SEQ ID NO: 41). [Table 1]

[0129] Table 2: The second linker used in the FVIII-VWF-EP fusion protein. EP represents the extended peptide of sequence number 1. The lowercase number represents the number of repetitions of the assigned sequence element. [Table 2]

[0130] Table 3 - Overview of fusion proteins and their components

[0131] EP represents the extended peptide of SEQ ID NO: 1. The lowercase number represents the number of repetitions of the assigned sequence element. ABN is the albumin-bound nanobody of amino acid sequence SEQ ID NO: 41. [Table 3]

[0132] Example 2 - Characterization of fusion proteins in cell culture supernatant by chromogenic factor VIII activity (FVIII:C) analysis Objective of the experiment Characterization of fusion proteins by chromogenic factor VIII activity (FVIII:C) analysis. Evaluation of the effects of linker length and the presence of extension peptides on FVIII activity in the expression supernatant. method The FVIII-VWF-EP fusion protein was transiently expressed in 3 mL triples in Expi293F cells (Thermo Fisher Scientific); the cell culture supernatant was collected 4 days after transfection by centrifugation at 4800 × g for 30 minutes. FVIII:C activity was evaluated using the FVIII chromogenic assay kit (Siemens) with the BCS XP system (Siemens). result All FVIII-VWF-EP fusion proteins showed higher expression levels (ranging from 0.66 to 10.34 IU / ml) compared to rFVIII (simoctocog alfa, NUWIQ, mean FVIII:C was 0.33 IU / ml). Among groups with the same linker length and the same Furin cleavage site, constructs with EP inserted into the linker connecting the FVIII and VWF portions showed the highest expression levels: C4 vs. C2 and C5; C17, C21 and C22 vs. C16 and C18. Molecules with EP inserted into both linkers (C23 and C24) showed the highest activity levels in the supernatant (Figure 2).

[0133] In summary, the combination of optimal linker length and EP insertions in the linker regions of both fusion proteins results in the highest expression levels, suggesting efficient translation, maximum protein stability, and correct folding.

[0134] Example 3 - Characterization of fusion protein by size exclusion chromatography and SDS-PAGE Objective of the experiment The size, purity, and integrity of the expressed construct were controlled. SDS-PAGE analysis was performed to control the homogeneity of the expressed protein. The size distribution and potential presence of high molecular weight species (HMWS) in the purified FVIII-VWF-EP fusion protein preparation were analyzed by SEC-HPLC.

[0135] method SDS-PAGE: Samples were qualitatively analyzed by non-reducing SDS-PAGE. Samples were incubated in LDS sample buffer to denaturate them. Gels were electrophoresed on 4-12% BisTris gel (Invitrogen, NuPage) at 175 V for 70 minutes.

[0136] Coomassie staining was performed using a ready-to-use Coomassie stain (Thermo Scientific, Page Blue Protein staining). After staining with RT for 3 hours, the samples were washed, and then destained with MilliQ water until the background was clear.

[0137] SEC-HPLC: All samples were analyzed using a Superdex 200 Increase 10 / 300 column (Cytiva) connected to an ULTIMATE3000 HPLC system (Thermo Scientific). The electrophoresis buffer consisted of 171.1 mM NaCl, 7.1 mM L-Arg hydrochloride, 26.3 mM sucrose, 3.4 mM trisodium citrate dihydrate, 1.7 mM CaCl2, and 0.1 mM Poloxamer 188, at pH 7.0. The buffer was used at an isocratic flow rate of 0.56 mL / min.

[0138] For the analysis, the sample was injected into a Superdex column, and the corresponding elution profile was recorded at UV 280 nm. After 45 minutes, the electrophoresis was completed. The chromatogram was manually integrated, and the relative area of ​​high molecular weight species (HMWS) in the sample was calculated.

[0139] result SDS-PAGE analysis of purified FVIII-VWF-EP fusion proteins revealed a major band of 250 kDa or greater corresponding to the FVIII-VWF-EP fusion protein in all constructs (Figure 3). The minor protein bands remaining between 75-170 kDa are Furin cleavage products. Some HMWS was detected in all samples, with considerable variation in its amount. The analytical results are summarized in Table 4. The amount of HMWS differed depending on the presence and location of the EP. These data indicate that the presence of EP in the linker region of the FVIII-VWF-EP fusion protein is beneficial for the correct folding of the protein. Furthermore, the specific presence of EP in the FVIII-VWF linker (e.g., C17, C23, and C24) has a beneficial effect on the stability of the fusion protein by highly preventing the generation of HMWS. [Table 4]

[0140] Example 4 - Bonding to full-length VWF Objective of the experiment Evaluation of the binding ability of FVIII-VWF-EP fusion constructs to full-length VWF (flVWF). method The binding of the FVIII-VWF-EP fusion protein to flVWF was tested by surface plasmon resonance (SPR) using a T200 instrument (Cytiva). Purified human flVWF (Sekisui) was coated onto a CM5 chip by amine coupling using an amine coupling kit (Cytiva) according to the manufacturer's instructions. FlVWF was immobilized in three different flow cells at approximately 1000 reaction units (RU). The running buffer was 20 mM HEPES, 150 mM NaCl, 5 mM CaCl2, and 0.05% Tween 20. After each analyte injection, the surface was regenerated with a regeneration buffer (20 mM HEPES, 600 mM NaCl, 350 mM CaCl2, 0.05% Tween 20). The FVIII-VWF-EP fusion protein was injected into three different flow cells in a random order at a fixed concentration of 8.5 IU / ml FVIII:C. The binding levels measured 30 seconds after the end of analyte injection were normalized by dividing the RU by the molecular weight of each protein, and expressed with the binding rate of rFVIII set to 100%.

[0141] result The results are shown in Figure 4. For all FVIII-VWF-EP fusion proteins, a binding level of less than 11% of FVIII was measured. C16 showed the highest binding, while C17 showed the lowest binding.

[0142] Pharmacokinetics of Example 5-FVIII-VWF-EP Fusion Protein Objective of the experiment To investigate the effect of covalently linking a VWF fragment and two extended peptides (VWF-EP) to the C-terminus of FVIII on its pharmacokinetics (PK). In hemophilia A (HemA) mice, the effects of VWF-EP and the presence of three additional EPs in the second linker (of construct C17) were examined. method Male B6;129S-F8, 5-8 weeks old TM1Kaz / J (F8- / -) mice were obtained from Jackson Laboratory (Bar Harbor, Maine, USA). Based on FVIII:C activity, the test compound or rFVIII control was injected via tail vein at a dose of 200 IU / kg bw. The study summary is presented in Table 5. Blood samples were collected at the indicated times. Five mice were used for blood collection at each time point in each group. Each mouse was used for two sampling points. Blood was collected in a tube containing 3.8% sodium citrate solution. Immediately after collection, the blood samples were placed on crushed ice, and plasma was separated within one hour of collection by centrifugation at 3350 xg (4000 rpm), 4°C, for 15 minutes. Plasma samples were stored at -80°C until analysis using the FVIII:C assay (Coamatic Factor VIII Assay Kit; Chromogenix, Bedford, MA, USA).

[0143] FVIII:Ag in mouse plasma was measured using our proprietary ELISA method. As a first step, a maxisorp microtiter plate (Thermo Fisher Scientific 439454) was coated overnight with an anti-human FVIII monoclonal antibody (GMA8023, Green Mountain Antibodies, Burlington, USA) that recognizes the A2 domain. After blocking and washing, diluted mouse plasma was spread onto the plate and incubated at 37°C for 2 hours. Following subsequent washing, bound molecules were detected using biotinylated anti-FVIII nanobodies (Capture select Biotin anti FVIII conjugate; Thermo Fisher Scientific 7102862500) and Neutravidin-HRP (Thermo Fisher Scientific 31001). Colorimetric measurements were performed using a tetramethylbenzidine substrate (Sigma-Aldrich T4444) at a wavelength of 450 nm. Table 5 Survey Overview [Table 5]

[0144] result The results are shown in Table 6 and Figure 5. rFVIII showed a half-life of 7.61 hours, while the FVIII-VWF-EP fusion protein construct C17 had a half-life of 12.42 hours, which is 1.6 times longer. 1 / 2 This resulted in significantly longer detection in mouse plasma. C17 also showed improved recovery and half-life, leading to higher C maxと The AUC was shown. Table 6 - PK analysis of FVIII:C data measured in HemA mouse plasma [Table 6]

[0145] This indicates that the PK parameters of FVIII are improved by covalently bonding it to the VWF fragment and EP through gene fusion.

[0146] To measure specific activity, FVIII:Ag and FVIII:C levels in mouse plasma were measured for rFVIII(NUWIQ) and C17 using the in-house ELISA assay and the Coamatic factor VIII assay kit as described above.

[0147] When the average specific FVIII activity of 30 mice was measured, it was revealed that the specific activity of C17 was significantly higher compared to unmodified rFVIII (Table 7).

[0148] Table 7 Average specific activity [Table 7] * The FVIII:Ag assay recognizes only the FVIII chain (in contrast to the BCA assay used in D1). After correcting for molecular weight differences, the specific activity of C17 is 9811.7 IU / mg, which is still higher than in D1.

[0149] This activity is higher than the specific activity of the molecule described in the cutting-edge literature EP 3476937 A1. Table 4 (page 18) of EP 3476937 A1 lists the specific activities of three molecules: scFVIII / D'D3-60, scFVIII / D'D3-90, and scFVIII / D'D3-120. The values ​​are 9304.3 IU / mg, 8474.5 IU / mg, and 9367.2 IU / mg, respectively. Therefore, the present invention provides an FVIII-VWF fusion protein with remarkably high specific activity.

[0150] Example 6 - Confirmation of results using sequence variants To confirm that minor sequence mutations in the FVIII heavy chain, FVIII light chain, VWF fragment, and EP do not affect the properties of the construct, additional C17-based fusion proteins with sequence mutations in one or all of the four functional elements shown in Table 8 are prepared as described in Example 1. Furthermore, the experiments in Examples 2 to 4 are repeated. Table 8 - Other Fusion Proteins with Sequence Mutations [Table 8]

[0151] Many modifications and other embodiments of the present invention described herein will be recalled by those skilled in the art who benefit from the teachings shown in the foregoing description and the relevant drawings. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Certain terms are used herein, but these are used in a general and descriptive sense only and are not intended to be limiting. Table 9 - Overview of Sequence IDs [Table 9] JPEG2026513540000012.jpg27170

[0152] References Ewenstein BM, Collins P, Tarantino MD, Negrier C, Blanchette V, Shapiro AD, Baker D, Spotts G, Sensel M, Yi SE, Gomperts ED. Hemophilia therapy innovation development of an advanced category recombinant factor VIII by a plasma / albumin-free method Proceedings of a Special Symposium at the XIXth Congress of the International Society on Thrombosis and Haemostasis; 2004, Vol. 41, 1-16. Fischer B, Mitterer A, Schlokat U, DenBouwmeester R, Dorner F. “Structural analysis of recombinant von Willebrand factor: identification of hetero- and homo-dimers” FEBS Lett. 1994; 351(3): 345-8. Erratum in: FEBS Lett 1994; 353(3): 337. Furlan M. “Von Willebrand factor: molecular size and functional activity” Ann Hematol. 1996; 72(6): 341-348. Giangrande P, Abdul Karim F, Nemes L, You CW, Landorph A, Geybels MS, Curry N. Long-term safety and efficacy of N8-GP in previously treated adults and adolescents with hemophilia A: Final results from pathfinder2. J Thromb Haemost. 2020 Sep;18 Suppl 1(Suppl 1):5-14. Innis et al. (1990) PCR: A Guide to Methods and Application, Academic Press, New York. Kannicht C, Ramstrom M, Kohla G, et al. Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII. Thromb Res. 2013; 131(1): 78-88. Khalil et al, 2022 Khalil A, Wurthwein G, Golitsch J, Hempel G, Fobker M, Gerss J, Moricke A, Zimmermann M, Smisek P, Zucchetti M, Nath C, Attarbaschi A, Von Stackelberg A, Gokbuget N, Rizzari C, Conter V, Schrappe M, Boos J, Lanvers-Kaminsky C. Pre-existing antibodies against polyethylene glycol reduce asparaginase activities on first administration of pegylated E. coli asparaginase in children with acute lymphocytic leukemia. Haematologica. 2022 Jan 1;107(1):49-57. Needleman SB, Wunsch CD. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970; Vol. 48(3); 443-453. M.R. Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Paik J, Deeks ED. Damoctocog Alfa Pegol: A Review in Haemophilia A. Drugs. 2019 Jul;79(10):1147-1156. (Erratum in: Drugs. 2019 Aug 23) Pezeshkpoor B, Sereda N, Berkemeier AC, Matuschek I, Schwarz N, Turecek PL, Horneff S, Klein C, Goldmann G, Marquardt N, Albert T, Muller J, Oldenburg J. Antidrug antibodies against the polyethylene glycol moiety inhibit the procoagulant activity of therapeutic polyethylene glycolated factor VIII. J Thromb Haemost. 2023 Jun;21(6):1503-1514. doi: 10.1016 / j.jtha.2023.03.011. Epub 2023 Mar 18. Strohl WR. Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters. BioDrugs. 2015; Vol. 29(4), 215-239. Tiede A. Half-life extended factor VIII for the trea TM ent of hemophilia A. JThromb Haemost. 2015; Vol. 13 Suppl 1; S176-S179 Vlot AJ, Koppelman SJ, Meijers JC, Dama C, van den Berg HM, Bouma BN, Sixma JJ, Willems GM. Kinetics of factor VIII-von Willebrand factor association. Blood. 1996; Vol. 87(5); 1809-1816 Yee A, Gildersleeve RD, Gu S, Kretz CA, McGee BM, Carr KM, Pipe SW, Ginsburg D. A von Willebrand factor fragment containing the D'D3 domains is sufficient to stabilize coagulation factor VIII in mice. Blood. 2014; Vol. 124(3); 445-452.

Claims

1. A fusion protein comprising a factor VIII (FVIII) heavy chain, an FVIII light chain, a von Willebrand factor (VWF) fragment, and at least two copies of an extended peptide (EP), EP is a fusion protein having at least 90% amino acid sequence identity with SEQ ID NO: 1, and further containing a cluster of O-glycosylation sites, each cluster containing at least two O-glycosylated amino acids.

2. The FVIII heavy chain does not contain the FVIII B domain and preferably contains an amino acid sequence that is at least 90%, more preferably at least 95%, and most preferably at least 98% identical to SEQ ID NO:

2. The FVIII light chain comprises an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, identity with SEQ ID NO: 3, and / or The VWF fragment contains an amino acid sequence having at least 90%, preferably at least 95%, and more preferably at least 98% identity with SEQ ID NO:

4. The fusion protein according to claim 1.

3. The C-terminus of the FVIII heavy chain is fused to the N-terminus of the FVIII light chain by a first linker, the first linker preferably includes a sequence derived from the B-domain of FVIII, and / or The fusion protein according to claim 1 or 2, wherein 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 comprises at least one copy, preferably at least two copies, more preferably at least three copies of the EP, and the EP is assembled preferably in a sequential order.

5. Preferably, the half-life extension portion further comprises at least one selected from immunoglobulin Fc domain, serum albumin or a portion thereof, albumin-binding antibody, and albumin-binding protein domain, with the most preferred half-life extension portion being albumin-binding V H A fusion protein according to any one of claims 1 to 4, wherein the H domain is present.

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

7. The first, second and / or third linker is flexible, (GGS) n (GGGS) n , and (GGGGS) n A fusion protein according to any one of claims 3 to 6, comprising n being an integer in the range of 1 to 10, G representing glycine, and S representing serine.

8. The fusion protein according to any one of claims 3 to 7, wherein the second linker includes a thrombin cleavage site, preferably 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 C-terminus of a second linker.

10. The fusion protein according to any one of claims 3 to 9, wherein the amino acid sequence of the second linker is at least 95%, more preferably at least 98%, of 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 one of claims 3 to 10, wherein the first linker includes a furin cleavage site, and the furin cleavage site preferably has the amino acid sequence of SEQ ID NO:

20.

12. The fusion protein according to any one of claims 3 to 11, wherein the amino acid sequence of the first linker is at least 95%, more preferably at least 98%, of 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 one of claims 5 to 12, wherein the first linker and / or the second linker comprises at least two copies of the GGGGS motif on both sides of the EP assembly and / or on both sides of the half-life extension portion.

14. The fusion protein according to any one of claims 1 to 13, wherein at least two copies of EP are fused to the C-terminus of a VWF fragment.

15. The fusion protein according to any one of claims 1 to 14, 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 NOs. 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36.

16. A fusion protein for use in the treatment of hemorrhagic disorders, wherein the fusion protein is defined according to any one of claims 1 to 15.

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

18. The polynucleotide according to claim 17, comprising encoding an amino acid sequence having at least 90%, preferably 95%, more preferably 98% or more, and most preferably 100% identity to a sequence selected from SEQ ID NOs. 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36.

19. A vector comprising a 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 comprising the polynucleotide according to claim 17 or 18 or the vector according to claim 19, wherein the host cell is a mammalian cell line, preferably a human cell line, more preferably a human kidney cell line, most preferably a human embryonic kidney cell line, particularly a HEK293 cell line such as HEK293F.

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