Fviii-vwf fusion proteins with improved pharmacokinetics

Abstract

The present invention relates to a fusion protein comprising: a Factor VIII (FVIII) heavy chain; a 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. The complex shows improved pharmacokinetic properties compared to FVIII. The present invention further relates to a polynucleotide encoding the fusion protein as well as a vector and a host cell comprising the polynucleotide.

Description

Technical Field

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

[0002] Hemophilia is a group of inherited genetic disorders that impair the body's ability to control blood clotting. In its most common form, hemophilia A, there is a deficiency of clotting factor VIII (FVIII). Hemophilia A occurs in approximately 1 in 5,000 to 10,000 male births. FVIII protein is an essential cofactor with multifunctional properties in blood clotting. FVIII deficiency can be treated with plasma-derived FVIII concentrates or with recombinant FVIII. Treatment with FVIII concentrates can restore normal life for people with hemophilia.

[0003] Patients with hemophilia A may use FVIII for treatment or as a preventative therapy, administered several times a week. In preventative therapy, due to the continuous need for FVIII and its short half-life in the bloodstream (approximately 11 hours in humans), administration of 15-25 IU / kg FVIII three times weekly by body weight is necessary (Ewenstein et al., 2004). The short circulating half-life of FVIII and the associated need for frequent infusions of FVIII concentrate are major challenges in the treatment of hemophilia A.

[0004] In blood, under normal conditions, FVIII molecules are always associated with their cofactor von Willebrand factor (VWF), which stabilizes FVIII molecules and protects them from various forms of degradation. The non-covalent complex of FVIII and VWF has a high binding affinity of 0.2 nM–0.3 nM (Vlot et al., 1996).

[0005] Historically, hemophilia A has been treated with FVIII derived from human plasma. Furthermore, since the 1990s, various recombinant FVIII proteins have entered the market. However, neither plasma-derived nor recombinant FVIII proteins possess optimal pharmacokinetic properties. Like many other therapeutic proteins, they are subject to peptidase metabolism, which significantly limits their in vivo half-life.

[0006] Attempts to extend the half-life of FVIII have included immunoglobulin Fc fusions (efmoroctocogalfa, Eloctate), the addition of polyethylene glycols (turoctocog alfa pegol, Esperoct; damoctocog alfa pegol, Jivi; rurioctocog alfa pegol, Adynovate), and single-chain constructs (lonoctocog alfa, Afstyla). All of these techniques have resulted in an approximately 1.5-fold increase in the half-life of FVIII (as reviewed in Tiede 2015). Sufficient literature has demonstrated that FVIII molecules form complexes with VWF during cycling, and both molecules are simultaneously eliminated primarily through the VWF scavenging pathway. Therefore, the half-life of FVIII is mainly determined by the half-life of VWF.

[0007] VWF and VWF fragments containing FVIII binding sites are known to stabilize FVIII, thereby preventing its rapid clearance, proteolytic digestion, uptake by antigen-presenting cells, and promoting 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, the D'D3-Fc fusion protein only prolonged the half-life of FVIII in VWF- / - mice. In hemophilia A mice, the D'D3-Fc construct failed to prolong the half-life of FVIII because the protein fragment did not compete with endogenous VWF for FVIII binding.

[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. Because this construct does not bind to endogenous VWF, the same half-life extension was observed in both VWF / FVIII double knockout (DKO) mice and hemophilia A mice. However, despite being fully functional in vitro, it exhibited significantly reduced activity in vivo.

[0009] EP 3476937 A1 describes a chimeric protein comprising FVIII and VWF (at least their D'D3 domains) intended for use as a therapeutic agent for hemophilia A, with the aim of achieving an increased half-life in vivo. To increase the half-life, the FVIII-VWF protein according to D1 is PEGylated to inhibit the binding of FVIII to low-density lipoprotein receptor-associated protein (LRP). However, PEGylation of therapeutic proteins has various drawbacks. Such drawbacks are known in asparaginases (PEG-ASNases), used to treat conditions such as ALL (acute lymphoblastic leukemia). Here, some patients have pre-existing anti-PEG antibodies that negatively impact asparaginase activity measured after PEG-ASNase treatment. Additionally, these anti-drug antibodies (ADAs) can induce hypersensitivity reactions (Khalil et al., 2022). Furthermore, the anticoagulant effects of antibody inhibitors against PEG are known (Moreno et al., 2019). Recent findings suggest that ADA targeting the PEG portion can inhibit the procoagulant activity of PEGylated FVIII formulations (Anaconitine, Javitin, and Excipient). This effect is particularly measurable in patients treated with Javitin (damoxetine alpha polyethylene glycol) and Excipient (torsenoside alpha polyethylene glycol) (Pezeshkpoor et al., 2023). These PEGylated FVIII-based therapeutics have been used to treat hemophilia A patients for some time and on a large scale. However, to date, only short-term and mostly transient effects have been reported with Javitin (Paik and Deeks, 2019) or Excipient / N8-GP (Giangrande et al., 2020) due to pretreatment or treatment-induced anti-PEG antibodies. Furthermore, Valsecchi et al. (2023) described the potential for anti-PEG ADA to be induced by immunization with Comirnaty (BNT162b2). In patients with hemophilia A, these may include IgM that cross-react with all three PEGylated FVIII formulations (Anivel, Javitin and Esperion).

[0010] Other methods to increase the half-life of therapeutic proteins include gene fusion with proteins that have longer natural half-lives, such as transferrin and albumin, or gene fusion with protein domains, such as the C-terminal peptide (CTP) of human chorionic gonadotropin (CG). As reviewed by Strohl et al. (2015), various fusion proteins of therapeutic proteins and CTP have been developed and are currently being tested in clinical trials. Therapeutic proteins include FSH. FVIIa, FIX, IFN-β, and gastrin.

[0011] WO 2017 / 198435 A1 describes a fusion protein comprising a major protein, such as a mammalian protein (e.g., human VWF) or a fragment thereof, and one or more extended peptides. The extended peptides contain clusters of O-glycosylation sites having at least two O-glycosylated amino acids and may be derived, for example, from human VWF. Due to the presence of the extended peptides, the fusion protein has an increased half-life compared to the individual major protein, i.e., the mammalian protein or a fragment thereof. Fusion proteins can be used to increase the half-life of binding partner molecules, such as FVIII. The OCTA12 molecule, falling under the definition of WO 2017 / 198435 A1, is a fusion capable of binding a VWF fragment of FVIII with high affinity. Due to the absence of certain domains recognized by scavenging receptors (e.g., the A1 and D4 domains recognized by the SR-AI receptor), it has a significantly prolonged half-life compared to full-length VWF (a terminal half-life of up to approximately 200 hours after subcutaneous administration in humans, compared to approximately 18 hours for full-length VWF). Additionally, it contains three extended peptide repeat sequences, namely, a 31-amino acid-long sequence of quadruple O-glycosylation derived from VWF. Notably, the non-covalent complex of FVIII with OCTA12 described in WO2017 / 198435 A1 and Vollack-Hesse et al., 2021 primarily improves the pharmacokinetic (PK) properties of FVIII upon subcutaneous administration. Following intravenous administration, C... max Only a slight improvement was made, and the terminal half-life was not improved compared to the FVIII molecule alone (described in Vollack-Hesse et al., 2021, page 1075). Figure 3 (as shown in section A). Summary of the Invention

[0012] This invention is based, among other things, on the discovery that when a highly O-glycosylated extended peptide (EP) is inserted into a linker connecting proteins, a linker connecting heavy and light chains, and / or a linker fused to the C-terminus of a VWF fragment, the complex of factor VIII (FVIII) protein with the VWF fragment, particularly OCTA12, exhibits increased expression levels, is stabilized, and is less prone to aggregation. Furthermore, the FVIII-VWF-EP fusion protein stabilized by the extended peptide has a prolonged circulating half-life compared to FVIII alone.

[0013] Therefore, according to a first aspect, the present 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 EP; wherein the EP has at least 90% amino acid sequence identity with SEQ ID NO:1 and contains a cluster of O-glycosylation sites, wherein the cluster contains at least two O-glycosylated amino acids.

[0014] In a second aspect, the present invention relates to a polynucleotide that encodes the fusion protein according to the first aspect.

[0015] According to a third aspect, the present invention relates to a carrier containing the polynucleotides described in the second aspect.

[0016] In a fourth aspect, the present invention relates to a host cell containing a polynucleotide according to the second aspect or a carrier according to the third aspect, wherein the host cell is a mammalian cell.

[0017] Finally, in a fifth aspect, the present invention also relates to a pharmaceutical composition comprising the fusion protein according to the first aspect, said pharmaceutical composition for treating or preventing hemorrhagic conditions. Attached Figure Description

[0018] Figure 1 A schematic representation of the FVIII-VWF fragment-EP fusion protein is shown.

[0019] Figure 2 The bar chart shows the FVIII activity (FVIII:C) in the culture supernatant after temporary expression of the FVIII-VWF fragment-EP fusion protein in Expi293F cells.

[0020] Figure 3 The non-reducing SDS-PAGE analysis of the purified fusion protein is shown. (A) and (B) show the FVIII-VWF-EP fusion protein after protein blotting (A) and Coomassie staining (B) following FVIII detection. The protein name is indicated above each lane. M indicates the molecular weight marker, with molecular weight expressed in kDa.

[0021] Figure 4 A bar graph showing the normalized binding level of flVWF to the purified FVIII-VWF fragment fusion protein is presented. flVWF was coated on a CM5 sensor chip, followed by injection of either purified FVIII-VWF-EP protein or a control protein. The SPR signal detected 30 seconds after injection cessation is normalized relative to the binding of rFVIII (Nuwiq) set to 100%. OCTA12 is a negative control that should not bind to flVWF.

[0022] Figure 5 A graph showing the FVIII activity (FVIII:C) in HemA mouse plasma after a single IV administration of rFVIII (Nuwiq) and the FVIII-VWF fragment fusion protein C17, according to the present invention, is illustrated. Detailed Implementation

[0023] To provide a clear and consistent understanding of the specification and claims, as well as the scope of such terms, the following definitions are provided.

[0024] definition

[0025] As used herein, a "peptide" can consist of any number and type of amino acids, preferably naturally occurring amino acids linked by peptide bonds. Specifically, a peptide contains at least 3 amino acids, preferably at least 5, 7, 9, 12, or 15 amino acids. Furthermore, there is no upper limit to the length of the peptide. However, preferably, the peptide according to the invention has a length not exceeding 500 amino acids, more preferably not exceeding 300 amino acids, and even more preferably not exceeding 250 amino acids.

[0026] Therefore, the term "peptide" includes "oligopeptide," which typically refers to peptides with a length of 2 to 10 amino acids, and "polypeptide," which typically refers to peptides with a length of more than 10 amino acids.

[0027] As used herein, a "protein" can contain one or more polypeptide chains. Proteins with more than one polypeptide chain are typically expressed as a single polypeptide chain by a single gene and are cleaved post-translational. Therefore, the terms "polypeptide" and "protein" are used interchangeably. As used herein, polypeptides and proteins include chemically synthesized proteins as well as naturally synthesized proteins encoded by genes. Polypeptides or proteins can be obtained from natural sources such as human blood or produced as recombinant proteins in cell culture.

[0028] As used herein, the term "fusion protein" refers to a protein produced by linking two or more genes that originally encode independent proteins or protein fragments, wherein 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 "fusion" refers to components linked by peptide bonds, either directly or via one or more peptide linkers.

[0029] According to the present invention, a "peptide linker" is a peptide that connects two protein elements of a fusion protein, particularly connecting the FVIII heavy chain to the FVIII light chain or the FVIII light chain to the VWF moiety. Peptide linkers are also simply referred to as "linkers." Peptide linkers contain structural amino acids, thereby allowing important domain interactions, enhancing stability, and reducing steric hindrance. In addition to structural amino acids, linkers may also contain functional motifs. Extended peptides can be considered as part of a protein element or linker. The length of a linker can range from 2 to 200 amino acids.

[0030] As used herein, the term "therapeutic protein" refers to proteins or polypeptides that have therapeutic effects, i.e., proteins used as active ingredients in pharmaceuticals.

[0031] According to the present invention, the terms "protein precursor", "preprotein" or "prepeptide" refer to an inactive protein (or peptide) that can be converted into an active form through post-translational modifications, such as by removing a portion of the amino acid sequence through enzymatic cleavage.

[0032] The correlation between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity". For the purposes of this invention, the Needleman-Wunsch algorithm is used to determine the degree of sequence identity between two amino acid sequences (Needleman and Wunsch, 1970, J.Mol.Biol. 48:443-453), implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 3.0.0 or later. Optional parameters used are: a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 substitution matrix (BLOSUM62 in the EMBOSS version). The Needle output marked "Longest Identity" (obtained using the no brief option) is used as the percentage identity, and calculated as follows:

[0033] (Number of identical residues x 100) / (Alignment length - total number of gaps in alignment).

[0034] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) is used to determine the degree of sequence identity between two nucleotide sequences. This algorithm is implemented in the Needle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.), preferably version 3.0.0 or later. Optional parameters used are: a nick opening penalty of 10, a nick extension penalty of 0.5, and an EDNAFULL (EMBOSS version NCBINUC4.4) substitution matrix. The Needle output labeled "Longest Identity" (obtained using the -nobrief option) is used as the percentage identity and calculated as follows:

[0035] (Number of identical deoxyribonucleotides x 100) / (Alignment length - total number of gaps in alignment).

[0036] When the term "recombinant" is used to refer to cells, nucleic acids, proteins, or vectors, it means that the cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins, or by altering native nucleic acids or proteins, or that the cells are derived from cells that have been so modified. Thus, for example, recombinant cells express genes that are not present in the natural (non-recombinant) form of the cell, or express naturally occurring genes at different levels or under different conditions.

[0037] As used herein, the term "half-life" is the time required for plasma / blood concentration to decrease by 50% after reaching quasi-equilibrium of distribution (as defined in Toutain et al., 2005). The term "half-life" is also referred to as "circulating half-life," "terminal half-life," or "elimination half-life."

[0038] As used herein, when the terms “transformation,” “stable transformation,” and “transgenic” are used to refer to a cell, it means that the cell contains a non-natural (e.g., heterologous) nucleic acid sequence that is integrated into its genome or carried as an episome and maintained across multiple generations.

[0039] As used herein, the term "fragment" refers to a polypeptide that, compared to a native or wild-type protein, lacks one or more amino acids at its N-terminus and / or C-terminus, but whose remaining amino acid sequence is identical to the corresponding position in the amino acid sequence deduced from full-length cDNA. Fragments are typically at least 50 amino acids in length.

[0040] As used herein, the term "glycosylation" refers to the attachment of a glycan to a molecule, such as a protein. Glycosylation can be an enzymatic reaction. Attachment can also be formed via covalent bonds. Therefore, as used herein, a "glycosylated polypeptide" is a polypeptide with one or more glycans attached. The phrase "highly glycosylated" means that molecules such as enzymes have undergone glycosylation at all or almost all available glycosylation sites (e.g., O-linked or N-linked glycosylation sites).

[0041] As used herein, the term "glycan" refers to a carbohydrate segment of a polysaccharide or oligosaccharide, or a glycoprotein or glycosylated polypeptide. A glycan can be a homopolymer or heteropolymer of monosaccharide residues. It can be a linear or branched molecule. A glycan typically contains at least three sugars and can be linear or branched. A glycan can include 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'-fluororibose, 2'-deoxyribose, mannose phosphate, 6'-sulfonyl-N-acetylglucosamine, etc.).

[0042] As used herein, the term “O-glycan” refers to a glycan that is typically covalently linked to serine and threonine residues of mammalian glycoproteins.

[0043] O-glycans can be linked via an α-linked O-glycosidic bond to the -OH group of serine or threonine 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-glucan.

[0044] According to the present invention, the terms “O-glycosylated cluster”, “O-glycan cluster” and “cluster of O-glycosylated amino acids” are used interchangeably and refer to two or more closely adjacent O-glycosylated amino acids.

[0045] As used herein, the term “sialylation” refers to the reaction of molecules, particularly polysaccharides, with sialic acid or its derivatives.

[0046] The transitional term "comprising," synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional unlisted elements or method steps. The transitional phrase "consisting of," except for impurities usually associated with it, excludes any element, step, or component not specified in the claim. When the phrase "consisting of" appears in the body clause of a claim rather than immediately following the preamble, it limits only to the elements set forth in that clause; other elements are not excluded as a whole from the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps, and those materials or steps that do not materially affect the basis and novelty of the claimed invention. Claims "consisting essentially of" occupy an intermediate position between closed claims drafted in the "consisting of" format and fully open claims drafted in the "comprising" format.

[0047] In the context of this invention, the term "glycosylated protein," such as fusion protein, is used in the singular for practical reasons. Typically, in practice, proteins exist as compositions of protein molecules of the same type. However, in the case of glycosylated proteins, the glycosylation in each molecule of the composition is not uniform. For example, not all individual molecules in a complex can achieve 100% glycosylation. Furthermore, differences may occur in the binding of glycans to specific O-glycosylation sites. Therefore, in this application, the reference to "fusion protein" also refers to compositions of fusion protein molecules having the same amino acid sequence but different O-glycan structures.

[0048] As used herein, the term “binding affinity” or “affinity” refers to the strength of binding between two molecules, particularly the strength of binding between 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.

[0049] As used herein, the immune response involves either adaptive or innate immune responses. An innate immune response is a non-specific defense mechanism activated immediately or within hours of the presence of an antigen in the body. These mechanisms include physical barriers such as the skin, chemicals in the blood, and immune system cells that attack foreign cells within the body. Innate immune responses are activated by the chemical properties of the antigen. An adaptive immune response, on the other hand, is an antigen-specific immune response. 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 specifically designed to attack that antigen.

[0050] Fusion protein

[0051] According to a first aspect, the present 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 extended peptide.

[0052] The fusion protein according to the invention provides a technical advantage in the generation of the FVIII-VWF complex. Unlike generating and purifying two molecules separately and combining them in a specific ratio, covalent complexation allows for simplification into a single generation and purification process. Furthermore, the covalent complex improves expression levels. The fusion protein according to the invention exhibits increased expression levels compared to FVIII alone. According to one embodiment, the expression level of the fusion protein ranges from 0.6 IU / ml to 10.3 IU / ml. Under the same conditions, the average FVIII:C of the expression level of FVIII alone is 0.33 IU / ml. FVIII activity can be determined by colorimetric assay. According to one embodiment, the expression level is equal to or greater than 0.6 IU / ml, as determined by FVIII activity in cell culture supernatant. Expression levels can 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, and 15.0 IU / ml. According to one embodiment, the expression level is equal to or higher than 4.0 IU / ml. According to one embodiment, the expression level is equal to or higher than 6.0 IU / ml. According to one embodiment, the expression level is equal to or lower than 15.0 IU / ml. According to one embodiment, the expression level is equal to or less than 11.0 IU / ml.

[0053] In addition to improved expression and purification, the fusion protein according to the invention has an extended half-life compared to FVIII alone. According to one embodiment, the half-life of the fusion protein is extended by at least 20%. According to one embodiment, the half-life of the fusion protein is extended by at least 30%. According to one embodiment, the half-life of the fusion protein is extended by at least 40%. According to one embodiment, the half-life of the fusion protein is extended by at least 50%. According to one embodiment, the half-life of the fusion protein is extended by at least 60%.

[0054] The fusion protein according to the 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 intravenous administration of the non-covalent complex of FVIII and OCTA12 of WO 2017 / 198435 A1, there was no improvement in terminal half-life compared to FVIII alone (described in Vollack-Hesse et al., 2021, page 1075). Figure 3 (As shown in A). The fusion of OCTA12 and FVIII prevents the redistribution of FVIII into endogenous flVWF during circulation. The fusion protein according to the invention exhibits significantly reduced binding affinity for flVWF in vitro, such as... Figure 4 It exhibits, and has a prolonged half-life after intravenous administration, such as Figure 5 As shown in Table 6 (below).

[0055] The fusion protein according to the invention showed reduced binding to endogenous VWF after administration to patients. According to one embodiment, the highest binding was 11% of the binding of FVIII alone to VWF. Binding can be determined by surface plasmon resonance (SPR), such as... Figure 4 As shown.

[0056] The half-life (t) can be calculated by performing linear regression analysis on the log-linear portion of a single plasma concentration-time curve or by using nonlinear regression of a single-phase exponential decay model. 1 / 2 Exemplary software used for computation includes GraphPad Prism version 6.07 (La Jolla, CA 92037, USA) and WinNonlin version 6.4 (Pharsight Corporation, Mountain View, CA, USA).

[0057] The calculation is based on the following equation:

[0058]

[0059] K el= Elimination rate constant

[0060] t 1 / 2 =Elimination half-life

[0061] c = concentration

[0062] t = time

[0063] In humans, factor VIII is encoded by the F8 gene, which contains 187,000 base pairs across six exons. The transcribed mRNA is 9.029 base pairs long and is translated into a protein of 2,351 amino acids, with 19 amino acids removed. In humans, the FVIII molecule is glycosylated at 31 amino acids, possessing 25 N-glycosylated chains and 6 O-glycosylated chains (see Kannicht et al., 2013).

[0064] Following translation, the amino acid chain is cleaved by a specific protease, resulting in a heavy chain of approximately 200 kDa and a light chain of approximately 80 kDa. The organization of the domains is typically characterized as A1-A2-B-A3-C1-C2. The light chain is a combination of domains A3-C1-C2. The heavy chain consists of domains A1-A2-B. Heavy chains found in plasma exhibit heterogeneous compositions, with molecular weights varying between 90 kDa and 200 kDa. This size variation is attributed to heterogeneity in glycosylation, splicing variants, and the presence of proteolytic products, such as the depleted B-domain heavy chain A1-A2. The full-length FVIII amino acid sequence is identified by amino acids 20 to 2.351 in UniProtKB P00451 (Sequence Version 1, July 21, 1986).

[0065] According to the present invention, the human FVIII heavy chain contains at least domains A1 and A2, and may further contain a portion 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.

[0066] According to one embodiment, the FVIII heavy chain does not contain the FVIIIB 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 to or identical to SEQ ID NO:2. The heavy chain without the FVIII B domain may contain 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:2. According to one embodiment, the heavy chain is at least 95% identical to SEQ ID NO:2. Example 6 illustrates a fusion protein whose FVIII heavy chain sequence variant SEQ ID NO:37 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 FVIIIB domain. The heavy chain having the FVIIIB 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.

[0067] According to one embodiment, the FVIII light chain comprises the domain organization A3-C1-C2. The human FVIII light chain having 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 to or identical to SEQ ID NO:4. According to one embodiment, the FVIII light chain comprises 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: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 illustrates a fusion protein whose FVIII light chain sequence variant SEQ ID NO:38 has the following amino acid substitution: S1732T.

[0068] VWF is a multimeric adhesion glycoprotein found in mammalian plasma with multiple physiological functions. During primary hemostasis, VWF acts as a mediator between specific receptors on the platelet surface and extracellular matrix components such as collagen. Furthermore, VWF serves as a carrier and stabilizing protein for procoagulant factor VIII. VWF is synthesized in endothelial cells and megakaryocytes as a 2813-amino acid precursor molecule.

[0069] The domain organization of VWF is typically characterized as D3-TIL4-A1-A2-A3-D4-C1-C2-C3-CK. The 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). The full-length VWF is identified by UniprotKB entry P04275 (version 224, April 12, 2017).

[0070] The human VWF according to the invention has an amino acid sequence of any sequence in UniprotKB P04275, particularly SEQ ID NO:5 (isotype 1). The VWF contains a cluster of two O-glycosylated amino acids. The first O-glycosylated amino acid cluster is located between amino acids 1238 and 1268 of SEQ ID NO:5. The second cluster includes amino acids 1468 to 1487 of SEQ ID NO:5.

[0071] Once secreted into the plasma, VWF circulates in various species with different molecular sizes. These VWF molecules consist of oligomers and polymers of mature subunits of 2050 amino acid residues. VWF is usually present in plasma in polymeric form, ranging in size from approximately 500 kDa to 20,000 kDa (Furlan 1996).

[0072] According to one embodiment, the VWF fragment comprises 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 the segment of SEQ ID NO:5. According to one embodiment, the VWF fragment is at least 95% identical to the segment of SEQ ID NO:5. According to one embodiment, the VWF fragment is at least 98% identical to the segment of SEQ ID NO:5.

[0073] In human VWF segments, relative to mature human VWF

[0074] (TIL3-D3-TIL4-A1-A2-A3-D4-C1-C2-C3-CK), where one or more of the structural domains A1, A2, A3, D4, C1, C2, C3, and CK may be missing. For example, VWF fragments can have domain organization selected from the following groups: 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.

[0075] In this regard, the segment of SEQ ID NO:5 is particularly the segment beginning 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 segment can be, for example, the segment defined in WO 2015 / 185758A2. As shown in WO 2015 / 185758A2, the complex of FVIII with the VWF fragment as defined therein exhibits reduced binding to the phospholipid membrane compared to FVIII alone, and reduced binding to collagen III and heparin compared to the complex of FVIII with full-length VWF. The segment of SEQ ID NO:5 preferably begins with amino acid 764 of SEQ ID NO:5 and preferably ends with amino acids in the range of 1905 to 2153 of SEQ ID NO:5. According to one embodiment, the VWF fragment ends with amino acids in the range of 2030 to 2153 of SEQ ID NO:5. According to another embodiment, the VWF fragment ends with amino acids in the range of 2100 to 2153 of SEQ ID NO:5.

[0076] According to one embodiment, the VWF fragment comprises 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 segment thereof. The VWF fragment having the amino acid sequence of SEQ ID NO:6 is based on the segment of amino acids 764 to 1268 of SEQ ID NO:5, with two amino acid substitutions, namely C1099A and C1142A. Replacing these two cysteine ​​residues with alanine eliminates the ability of the VWF fragment to form a multimer. 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 illustrates a fusion protein whose sequence variant SEQ ID NO:6, namely SEQ ID NO:39, has the following additional amino acid substitution: A1164V.

[0077] As illustrated in the examples, an EP having the sequence QEPGGLVVPPTDAPVSPTTLYVEDISEPPLH (SEQ ID NO:1), i.e., the O-glycosylation cluster 1 of VWF (amino acids 1238 to 1268 of SEQ ID NO:5), according to the present invention, confers increased expression levels, improved stability, and reduced aggregation tendency to the fusion protein. Therefore, the EP according to the present invention has an amino acid sequence with at least 90% identity to SEQ ID NO:1. According to one embodiment, the sequence identity with SEQ ID NO:1 is preferably at least 95%. According to one embodiment, the EP has a sequence identity with SEQ ID NO:1 of at least 98%. Example 6 shows a fusion protein whose sequence variant SEQ ID NO:1, i.e., SEQ ID NO:40, has the following amino acid substitution: G5A. According to one embodiment, two or more EPs have 100% sequence identity to SEQ ID NO:1.

[0078] 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 protein shown in the example has three EPs at the C-terminus of the VWF fragment, one of which is part of the fragment, and the other two EPs are fused to said part. A VWF protein having the amino acid sequence of SEQ ID NO:6 is bound to two copies of an extended peptide of the amino acid sequence of SEQ ID NO:1 and added to the C-terminus, which is a sequence-modified derivative of OCTA12 described in WO 2017 / 198435A1.

[0079] According to one embodiment, the C-terminus of the FVIII heavy chain is connected via a first connector ( Figure 1 Linker 1) is fused to the N-terminus of the FVIII light chain. Linkers connecting heavy and light chains are known in the art. One example is... The first linker, namely SFSQNSRHQAYRYRRG (SEQ ID NO:21), contains a sequence of B domains derived from FVIII. According to one embodiment, the first linker preferably contains a sequence of B domains derived from FVIII.

[0080] The first connector can be a flexible connector or a rigid connector. Preferably, the first connector is a flexible connector. According to one embodiment, the first connector comprises components selected from (GGS). n (GGGS) n and (GGGGS) n The motifs. In this embodiment, n is an integer in the range of 1 to 10. n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Consistent with the single-letter amino acid codes, G represents glycine, and S represents serine. These motifs give the first linker flexibility, thereby allowing sufficient interaction between the FVIII heavy chain and the light chain.

[0081] According to one embodiment, the first linker is a cleavable linker, i.e., it contains a protease cleavage site. The advantage of having a protease cleavage site is that FVIII can be processed intracellularly into its native double-stranded composition. According to one embodiment, the first linker contains a furin cleavage site. The 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.

[0082] Exemplary first linkers (and their 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). Table 1 (hereinafter) shows the amino acid sequences of these linkers. 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 sequences 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 the sequences 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.

[0083] According to one embodiment, the first linker contains 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 protein shown in the example has three EPs in the first linker. Therefore, according to one embodiment, the first linker contains at least two copies of the EP. According to one embodiment, the first linker contains at least three copies of the EP. The EPs may be distributed along the length of the linker, with structural amino acids or other elements of the linker interposed therebetween. Alternatively, two or more EPs may be assembled adjacently, i.e., in a consecutive sequence. Two or more consecutive EPs are referred to as an EP assembly. According to one embodiment, all EPs in the first linker are assembled in a consecutive sequence.

[0084] According to one embodiment, the C-terminus of the FVIII light chain is fused to the N-terminus of the VWF segment via a second linker.

[0085] The second connector can be a flexible connector or a rigid connector. Preferably, the second connector is a flexible connector. According to one embodiment, the second connector comprises components selected from (GGS). n (GGGS) n and (GGGGS) nThe motifs. In this embodiment, n is an integer in the range of 1 to 10. n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Consistent with the single-letter amino acid codes, G represents glycine and S represents serine. These motifs give the second linker flexibility, thereby allowing sufficient interaction, particularly with the binding domains of VWF and FVIII. According to one embodiment, the second linker contains (GGGGS)2, (GGGGS)4, and / or (GGGGS)6 motifs.

[0086] 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... n The motif (n≥2) 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, the FVIII-VWF-EP fusion protein with a larger number of glycine residues at the C-terminal portion of the linker exhibits higher binding to endogenous VWF. This may be because the longer, more flexible linker allows for better interaction between the VWF fragment of the fusion protein and the FVIII portion of the fusion protein, thus preventing interaction with endogenous VWF to a greater extent. Therefore, (GGGGS) with n≥2... n Linkers are preferred. According to one embodiment, the (GGGGS)2 motif is located at the N-terminus and C-terminus of the second linker, respectively.

[0087] 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, upon FVIII activation, the VWF fragment may be separable from the FVIII heavy and light chains. The thrombin cleavage site may be defined by the sequence SEQ ID NO:19. The thrombin cleavage site was chosen because it is also part of the natural FVIII sequence.

[0088] According to one embodiment, the second linker contains at least one copy of the EP. As shown in Example 2, the FVIII-VWF-EP fusion proteins, namely C4 and C17, in the second linker exhibit a stronger increase in expression levels than other FVIII-VWF-EP fusion proteins. Furthermore, the insertion of a negatively charged EP into the second linker can reduce the tendency of the fusion protein to form a higher molecular weight species. This effect is demonstrated by C17, which contains three EPs in the linker and has a lower proportion 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 contains at least two copies of the EP. According to one embodiment, the second linker contains at least three copies of the EP. The EPs may be distributed along the length of the linker, with structural amino acids or other elements of the linker interposed therebetween. Alternatively, two or more EPs may be assembled adjacently, i.e., in a continuous sequence. According to one embodiment, all EPs in the second linker are assembled in a continuous sequence. Exemplary second linkers (and corresponding sequence IDs) are linkers 2-3 (SEQ ID NO: 9) and linkers 2-5 (SEQ ID NO: 11). Table 2 (hereinafter) shows the amino acid sequences of these linkers. 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 the sequences 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 from SEQ ID NO: 9 and SEQ ID NO: 11.

[0089] According to one embodiment, the fusion protein contains at least one half-life extension portion. Various half-life extension portions are known in the art. The half-life extension portion may be selected from immunoglobulin Fc domains, serum albumin or portions thereof, albumin-binding antibodies, albumin-binding antibody domains, or albumin-binding protein domains. The Fc domain is a crystallizable fragment (Fc) region of the antibody tail that interacts with a cell surface receptor called an Fc receptor. This interaction, along with the slower renal clearance of larger molecules, increases the half-life of the protein to which it is attached. An exemplary Fc domain is the Fc domain of IgG1. Several fusion proteins containing albumin to increase the half-life of therapeutic proteins have been described, including factors VII, FVIII, and IX fused to 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 a member of the Fc receptor family, namely the neonatal Fc receptor (FcRn). The HSA portion added to the fusion protein preferably has a sequence according to Uniprot entry P02768.

[0090] V H Fragment H is a single-domain antibody engineered from a heavy-chain antibody discovered in camels. According to one embodiment, the extended half-life portion is the V-fraction that binds to albumin. H H domain. Binds albumin's V... H The H domain is known in this art. A cross-reactive binding protein V to albumin H An example of the H domain is MSA21 described in EP 2316852 B1. According to one embodiment, the V domain binds to albumin. H The H domain is the albumin-binding nanobody (ABN) shown in the example, namely SEQ ID NO:41.

[0091] The extended half-life portion can fuse with the C-terminus of the protein. The extended half-life portion can fuse directly with the C-terminus of the VWF fragment or with the C-terminus of the EP. Alternatively, the extended half-life portion can fuse with the C-terminus, i.e., the C-terminus of the VWF fragment or the C-terminus of the EP, via a third linker. Another option is that the extended half-life portion forms part of the first linker.

[0092] 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 linked to at least one copy of GGS, GGGS, or GGGGS, preferably at least two copies. According to another 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 linked to at least one copy of GGS, GGGS, or GGGGS, preferably at least two copies.

[0093] According to one embodiment of the fusion protein, the first and / or second linkers contain at least two copies of the GGGGS motif on either side of the extended peptide assembly and / or on either side of the half-life extension portion.

[0094] Exemplary fusion proteins (and their corresponding sequence IDs) are: 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) that form these fusion proteins.

[0095] 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 sequences selected from the following: 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 that selected from the following sequences: 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.

[0096] Polynucleotides

[0097] 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.

[0098] The isolated polynucleotide can be a DNA molecule or an RNA molecule. The isolated polynucleotide is preferably a DNA molecule, especially a cDNA molecule. Techniques for isolating or cloning polynucleotides encoding peptides are known in the art and include isolation from genomic DNA, preparation from cDNA, or combinations thereof. Polynucleotides can be cloned from such genomic DNA, for example, by using well-known polymerase chain reaction (PCR), or by screening expression libraries with antibodies to detect cloned DNA fragments with common structural features (see, for example, Innis et al., 1990). Other nucleic acid amplification procedures can be used, such as ligase chain reaction (LCR), ligation-activated transcription (LAT), and polynucleotide-based amplification (NASBA).

[0099] Specifically, the sequence of the isolated polynucleotide may include a first part encoding the FVIII heavy chain, a second part encoding the first linker, a third part encoding the FVIII light chain, a fourth part encoding the second linker, and a fifth part encoding the VWF fragment.

[0100] According to one embodiment, the FVIII heavy chain encoded in the first part has 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:2.

[0101] According to one embodiment, the first connector encoded in the second part has 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 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.

[0102] According to one embodiment, the FVIII light chain encoded in the third part has 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.

[0103] According to one embodiment, the second connector encoded in the fourth part has 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 NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

[0104] According to one embodiment, the VWF fragment encoded in the fifth part has 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:6.

[0105] According to one embodiment, the amino acid sequence encoded by the polynucleotide has at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% identity with sequences selected from the following: 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.

[0106] expression carrier

[0107] In a third aspect, the present invention also relates to expression vectors comprising polynucleotides according to a second aspect of the present invention.

[0108] The expression vector further preferably includes control elements, such as promoters, transcription and translation termination signals. The polynucleotides and control elements according to the second aspect can be linked together to produce a recombinant expression vector, which may include one or more restriction sites, thereby allowing the insertion or substitution of polynucleotides encoding polypeptides at such sites. The polynucleotides can be inserted into a suitable expression vector for expression. In producing the expression vector, the coding sequence is located within the expression vector such that the coding sequence is operatively linked to a suitable control sequence for expression.

[0109] The recombinant expression vector can be any vector (e.g., plasmid or virus) that allows for convenient recombinant DNA procedures and enables the expression of polynucleotides according to the fourth aspect of this invention. The choice of expression vector typically depends on its compatibility with the host cell to which it will be introduced. The expression vector can be a linear or closed circular plasmid.

[0110] Expression vectors are preferably suitable for expression in mammalian cells. Expression vectors can be autonomously replicating vectors, i.e., vectors existing as extrachromosomal entities whose replication is independent of chromosome replication, such as plasmids, extrachromosomal elements, small chromosomes, or artificial chromosomes. To achieve autonomous replication, the vector may further include an origin of replication, thereby enabling the vector to replicate autonomously in the appropriate host cell. An origin of replication can be any plasmid replicon that functions intracellularly to mediate autonomous replication. The terms "origin of replication" or "plasmid replicon" refer to a polynucleotide that enables the plasmid or vector to replicate in vivo.

[0111] The vector is preferably one that integrates into the genome upon introduction into the host cell and replicates along with the chromosome into which it is integrated. For integration into the host cell genome, the expression vector can rely on any other elements of the expression vector to achieve genome integration via homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to guide integration into the precise location of the host cell genome within the chromosome via homologous recombination.

[0112] The vector of the present invention preferably contains one or more (e.g., several) selectable markers, thereby allowing convenient selection of transformed, transfected, transduced, etc., cells. A selectable marker is a gene whose product provides resistance to microbial agents or viruses, resistance to heavy metals, or protrophic nutrition for auxotrophs, etc.

[0113] The procedures for connecting the above-described elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see Green and Sambrook 2012; Chapter 3).

[0114] According to one embodiment, according to a third aspect, the vector backbone of the vector is selected from pCDNA3, pCDNA3.1, pCDNA4, pCDNA5, pCDNA6, pCEP4, pCEP-puro, pCET1019, pCMV, pEF1, pEF4, pEF5, pEF6, pExchange, pEXPR, pIRES, and pSCAS.

[0115] host cells

[0116] According to a fourth aspect, the present invention provides a host cell comprising an expression vector according to a third aspect of the 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 integrator or as a self-replicating extrachromosomal vector as described above. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations occurring during replication. The selection of the host cell depends largely on the gene encoding the polypeptide and its origin.

[0117] According to one embodiment, a fusion protein is generated by expression in a mammalian host cell line. The fusion protein is preferably generated in a human host cell line. Generally, any human host cell line is suitable for the expression of the fusion protein. The host cell is preferably of human origin to ensure that the fusion protein is properly processed during folding and receives appropriate post-translational modifications (e.g., glycosylation, hydroxylation, phosphorylation, and sulfation). Specifically, a favorable glycosylation profile of the fusion protein is obtained using a human kidney cell line. Preferred human kidney cell lines are HEK cell lines, particularly the HEK 293 cell line.

[0118] Examples of HEK cell lines used to generate glycosylated peptides include HEK 293F and Flp-In. TM -293 (Invitrogen, R75007), 293 ( CRL-1573), 293EBNA, 293H (Thermo Scientific, 11631017), 293S, 293T ( CRL-3216 TM ), 293T / 17 ( CRL11268 TM ), 293T / 17SF ( ACS4500 TM HEK 293STF CRL 3249 TM HEK-293.2sus CRL-1573 TM The preferred cell line for producing polypeptides is the HEK 293F cell line.

[0119] Other suitable human cell lines as expression host cells 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 these host cells. K562 is a human myeloid leukemia cell line residing at the American Type Culture Collection (ATCC CCL-243). The other cell lines are derived from K562 cells and were selected due to specific glycosylation characteristics.

[0120] Other mammalian host cell lines suitable for producing the fusion protein according to the invention include cell lines derived from hamsters, mice, and monkeys. Suitable host cells include Chinese hamster ovary cells (CHO cells, such as DG44, DXB11, and K1 [ATCC CCL-61, including its glutamine auxotroph derivatives CHOZn and SAFC CHOGS]) and neonatal hamster kidney (BHK) cells.

[0121] Pharmaceutical Compositions and Medical Uses

[0122] The fusion protein according to the first aspect is particularly effective as an active ingredient for medical treatment. Preferably, it is particularly effective for treating or preventing hemorrhagic conditions. The fusion protein according to the first aspect described herein can be administered alone or in the form of a pharmaceutical composition.

[0123] Therefore, according to a fifth aspect, the present invention provides a fusion protein according to a first aspect, said fusion protein for treating hemorrhagic conditions.

[0124] According to one embodiment, the fusion protein can 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 known in the pharmaceutical field. See, for example, Goodman and Gilman, *The Pharmacological Basis of Therapeutics*, edited by Hardman et al., McGraw-Hill Professional (10th ed., 2001); *Remington: The Science and Practice of Pharmacy*, edited by Gennaro, Lippincott Williams & Wilkins (20th ed., 2003); and *Pharmaceutical Dosage Forms and Drug Delivery Systems*, edited by Ansel et al., Lippincott Williams & Wilkins (7th ed., 1999). Additionally, the pharmaceutical compositions according to the embodiments can also be formulated to include other medically useful pharmaceutical or biological agents. Pharmaceutical compositions typically comprise a combination of a therapeutically effective amount of a fusion protein and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is any carrier known or established in the art. Exemplary pharmaceutically acceptable carriers include pyrogen-free sterile water and pyrogen-free sterile saline solutions. Other forms of pharmaceutically acceptable carriers that can be used in embodiments of the invention include: binders, disintegrants, surfactants, absorption enhancers, humectants, cryoprotectants, adsorbents, lubricants, fillers, spreaders, humectants, preservatives, stabilizers, emulsifiers, solubilizers and thickeners, salts for controlling osmotic pressure, diluents such as buffers, and excipients typically used in formulations. These pharmaceutically acceptable carriers are optionally selected and used based on the unit dose of the final formulation.

[0125] Therefore, the present invention also relates to a method for treating or preventing a patient’s bleeding condition, the method comprising administering to the patient a pharmaceutical composition according to the fifth aspect.

[0126] As used in this article, "hemorrhagic disorders" refer to diseases or conditions that impair normal hemostasis. Examples of hemorrhagic disorders include, for instance, hemophilia A, hemophilia B, factor VIII deficiency, factor XI deficiency, von Willebrand disease, Glanzmann's thromboasthenia, Bernard-Soulier syndrome, idiopathic thrombocytopenic purpura, intracerebral hemorrhage, trauma, and traumatic brain injury.

[0127] As used in this article, "hemophilia" refers to a group of bleeding disorders associated with an increased clot formation time compared to that of healthy individuals without hemophilia. Hemophilia includes hemophilia A, a condition that causes the production of defective factor VIII; hemophilia B, a condition that causes the production of defective factor IX; and acquired hemophilia A, a rare bleeding disorder caused by autoantibodies against FVIII.

[0128] The bleeding disorder is preferably hemophilia A or hemophilia B. Treatment may be, for example, treatment of hemophilia in previously untreated patients (PUPS), or immune tolerance induction (ITI) therapy and / or other treatments related to hemophilia.

[0129] For internal use, the pharmaceutical composition may be administered to the patient via any conventional route of administration, such as oral, parenteral, or inhalation. Parenteral administration includes intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, liquid formulations, suspensions, emulsions, and drops. For parenteral administration, the pharmaceutical composition should be an injectable formulation, such as a liquid or suspension.

[0130] In other embodiments, the pharmaceutical composition is administered orally to a patient. In these embodiments, the form of the drug includes solid formulations (such as tablets, coated tablets, powders, granules, capsules, and pills), liquid formulations (such as liquids, e.g., eye drops, nasal drops), suspensions, emulsions, and syrups, inhalers (such as aerosols, nebulizers, and nebulizers), and liposome encapsulations. In still some other embodiments, glycosylated peptides, protein complexes, or pharmaceutical compositions are administered by inhalation to a patient's airway, thereby targeting the patient's trachea and / or lungs.

[0131] According to one embodiment of the fifth aspect, the use includes intravenous injection or non-intravenous injection. Non-intravenous injection is preferably subcutaneous injection.

[0132] Example

[0133] Example 1: Cloning, expression, and purification of the FVIII-VWF-EP fusion protein

[0134] Experimental Objective Generate cDNA encoding the FVIII-VWF-EP fusion protein. Express the fusion protein and purify it to homogeneity.

[0135] method

[0136] Gene synthesis and cloning

[0137] To generate the expression vector encoding the fusion construct, Golden Gate cloning technology was used. For this purpose, a cDNA fragment encoding the desired FVIII-VWF-EP fusion construct was synthesized at Twist Bioscience and cloned into a Golden Gate-compatible donor vector. Subsequently, the donor vector containing the desired construct variant, along with a donor vector containing the regulatory element and a proprietary receptor backbone, were used in a Golden Gate assembly reaction. The reaction produced a vector for mammalian expression containing the gpCMV promoter at the 5' end of the desired construct variant and the marmot hepatitis virus post-transcriptional regulatory element (WPRE) at the 3' end of the desired construct variant.

[0138] The vector construct was transformed into E. coli NEB5α cells and single clones were selected after incubation overnight at 37°C on LB agar plates containing ampicillin.

[0139] Plasmid DNA preparation was performed according to the manufacturer's recommendations using the QIAprep DNA Mini Kit (Qiagen) or... The Xtra Maxi Plus EF kit (Macherey-Nagel) was used. Sequencing validated the integrity of the vector, particularly the correct orientation and integrity of the genes encoding the desired construct variant.

[0140] Protein expression

[0141] The FVIII-VWF-EP fusion construct was transiently expressed in Expi293F cells (Thermo Fisher Scientific) according to the manufacturer's recommendations at a scale of 500-1000 mL. Cell culture supernatant containing the product was collected 4-5 days post-transfection by centrifugation at 2000 x g for 20 minutes.

[0142] Protein purification

[0143] Purification is accomplished through a three-step process: capturing the product from the cell culture supernatant, purifying it by affinity chromatography, and rebuffering it into the final matrix.

[0144] In short, the collected cell culture supernatant was added to 0.3M NaCl to improve sample conductivity, filtered through a 0.2μm PES filter, and then captured using Capto MMC resin (Cytiva). For this purpose, the Capto MMC column was equilibrated at pH 6.5 with 0.3M NaCl, 0.01M CaCl2, 0.01M L-histidine, and 0.02% polysorbate 80, and eluted with 0.3M NaCl, 0.02M CaCl2, 0.02M L-histidine, 0.8M L-arginine, 10% ethylene glycol, and 0.02% polysorbate 80 at pH 6.5. The column eluent was diluted 1:2 with equilibration buffer (0.05M Tris, 0.1M NaCl, 0.02% polysorbate 80, pH 7.0) and then loaded onto VOLTselect affinity resin (Thermo Fisher Scientific, custom VWF affinity resin). The product was eluted from the column using 0.05M Tris, 0.1M NaCl, 1M MgCl2, pH 7.0 and reburied using a Sephadex G-25 desalting column (Stopvan), to the final formulation buffer (171.1mM NaCl, 7.1mM M-arginine, 26.3mM sucrose, 3.4mM trisodium citrate, 1.7mM CaCl2, 0.1mM poloxamer 188, pH 7.0).

[0145] result

[0146] Table 1-3 shows the gene products encoded by the cloned cDNA constructs.

[0147] Table 1: First linker for FVIII-VWF-EP fusion protein.

[0148] EP represents the extended peptide having SEQ ID NO:1. Lowercase numbers indicate the number of repetitions of the sequence element to which it is assigned. number. ABN is a nanobody that binds to albumin, and its sequence is as follows:

[0149] QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGISSLGDST LYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQGTQVTVSS (SEQ ID NO: 41).

[0150] Connection subname Connector 1 sequence Serial ID 1-1 SFSQNSRHQAYRYRRG SEQ ID NO:12 1-2 SFSQNPPVLKRHQR SEQ ID NO:13 1-3 <![CDATA[SFSQNSRH(GGGGS)2 EP3 (GGGGS)2QAYRYRRG]]> SEQ ID NO:14 1-4 <![CDATA[SFSQN(GGGGS)2 EP3 (GGGGS)2PPVLKRHQR]]> SEQ ID NO:15 1-5 SFSQNPPVLK SEQ ID NO:16 1-6 <![CDATA[SFSQN(GGGGS)2 EP3 (GGGGS)2PPVLK]]> SEQ ID NO:17 1-7 <![CDATA[SFSQNSRH(GGGGS)2 ABN (GGGGS)2QAYRYRRG]]> SEQ ID NO:18

[0151] Table 2: Second linkers for the FVIII-VWF-EP fusion protein.

[0152] EP represents the extended peptide with SEQ ID NO:1. Lowercase numbers indicate the number of repetitions of the assigned sequence elements.

[0153] Connection subname Linker 2 sequence Serial ID 2-1 <![CDATA[(GGGGS)2IEPRSFS(GGGGS)2]]> SEQ ID NO:7 2-2 <![CDATA[(GGGGS)2IEPRSFS(GGGGS)6]]> SEQ ID NO:8 2-3 <![CDATA[(GGGGS)2IEPRSFS(GGGGS)2EP3(GGGGS)4]]> SEQ ID NO:9 2-4 <![CDATA[(GGGGS)2IEPRSFS(GGGGS)4]]> SEQ ID NO:10 2-5 <![CDATA[(GGGGS)2IEPRSFS(GGGGS)2EP3(GGGGS)2]]> SEQ ID NO:11

[0154] Table 3: Overview of fusion proteins and their components.

[0155] EP represents the extended peptide having SEQ ID NO:1. Lowercase numbers indicate the number of repetitions of the assigned sequence elements. ABN is an albumin-bound nanobody with the amino acid sequence SEQ ID NO:41.

[0156]

[0157] Example 2: Chromogenic factor VIII activity (FVIII:C) of the FVIII-VWF-EP fusion protein in cell culture supernatant.

[0158] Experimental Objective

[0159] The fusion protein was characterized by chromogenic factor VIII activity (FVIII:C) analysis. The effects of linker length and the presence of extended peptides on FVIII activity in the expression supernatant were assessed.

[0160] method

[0161] The FVIII-VWF-EP fusion protein was transiently expressed in triplicate at 3 mL volumes in Expi293F cells (Thermo Fisher Scientific); the cell culture supernatant was harvested on day 4 post-transfection by centrifugation at 4800 x g for 30 min. FVIII:C activity was assessed using the FVIII Chromogenic Assay Kit (Siemens) on a BCS XP system (Siemens).

[0162] result

[0163] Compared to rFVIII (simococog alfa, NUWIQ, mean FVIII:C 0.33 IU / ml), all FVIII-VWF-EP fusion proteins showed higher expression levels (ranging from 0.66 IU / ml to 10.34 IU / ml). In groups with the same linker length and containing the same furin cleavage site, constructs with EP inserted into the linker connecting the FVIII and VWF portions showed the highest expression levels: C4, relative to C2 and C5; C17, C21, and C22, relative to C16 and C18. Molecules with EP inserted into both linkers (C23 and C24) exhibited the highest activity levels in the supernatant. Figure 2 ).

[0164] In summary, inserting EPs into both linker regions of the fusion protein and combining them with the optimal linker length resulted in the highest expression levels, indicating efficient translation, the highest protein stability, and correct folding.

[0165] Example 3: Characterization of fusion proteins by size exclusion chromatography and SDS-PAGE

[0166] Experimental Objective

[0167] The size, purity, and integrity of the expressed construct were controlled. SDS-PAGE analysis was performed to control the homogeneity of the expressed protein. SEC-HPLC analysis was used to analyze the size distribution and the presence of potential high molecular weight species (HMWS) in the purified FVIII-VWF-EP fusion protein formulation.

[0168] method

[0169] SDS-PAGE:

[0170] Samples were qualitatively analyzed by non-reducing SDS-PAGE. Samples were denatured by incubation with LDS sample buffer. Gels were run at 175V for 70 minutes on 4%–12% BisTris gels (NuPage).

[0171] Coomassie staining was performed using ready-to-use Coomassie staining solution (Thermo Scientific, Page Blue protein staining), stained for 3 hours at room temperature, washed, and then destained in MilliQ water until the background was clear.

[0172] SEC-HPLC:

[0173] All samples were analyzed on a Superdex 200 Increase 10 / 300 column (Stenford Corporation) coupled to a ULTIMATE 3000 HPLC system (Thermo Scientific). The flow buffer consisted of 171.1 mM NaCl, 7.1 mM L-arginine 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.

[0174] For the analysis, the sample was injected into a Superdex column, and the corresponding elution curve was recorded using 280 nm UV. The analytical run was completed after 45 minutes. The chromatograms were manually integrated, and the relative areas of high molecular weight species (HMWS) in the sample were calculated.

[0175] result

[0176] SDS-PAGE analysis of the purified FVIII-VWF-EP fusion protein revealed a major band >250 kDa in all constructs, corresponding to the FVIII-VWF-EP fusion protein ( Figure 3 The remaining minor protein bands, ranging from 75 kDa to 170 kDa, represent furin cleavage products. Some portions of HMWS were detected in all samples, and the amounts varied significantly. The analytical results are summarized in Table 4. The varying amounts of HMWS depended on the presence and location of EP. These data indicate that for the FVIII-VWF-EP fusion protein, the presence of EP in the linker region is beneficial for proper protein folding. Moreover, the presence of EP, particularly in the FVIII-VWF linker (e.g., in C17, C23, and C24), largely prevents HMWS formation and thus has a beneficial effect on the stability of the fusion protein.

[0177] Table 4: Percentage of HMWS in purified FVIII-VWF-EP fusion protein

[0178] Fusion protein HMWS[%] C1 25.99 C2 32.92 C5 24.69 C7 24.82 C8 31.29 C16 46.48 C17 10.53 C18 19.88 C23 11.69 C24 8.07

[0179] Example 4: Combination with full-length VWF

[0180] Experimental Objective

[0181] Evaluate the compatibility of the FVIII-VWF-EP fusion construct with a full-length VWF (flVWF).

[0182] method

[0183] The binding of FVIII-VWF-EP fusion protein to flVWF was tested using a T200 instrument (Stopvan) via surface plasmon resonance (SPR). Purified human flVWF (Sekisui) was coated onto a CM5 chip via amine conjugation using an amine conjugation kit (Stopvan) according to the manufacturer's recommendations. FlVWF was immobilized at approximately 1000 response units (RU) in three different flow cells. The run buffer consisted of 20 mM HEPES, 150 mM NaCl, 5 mM CaCl2, and 0.05% Tween 20. After each analyte injection, the surface was regenerated using regeneration buffer (20 mM HEPES, 600 mM NaCl, 350 mM CaCl2, and 0.05% Tween 20). The FVIII-VWF-EP fusion protein was injected in triplicate at a fixed concentration of 8.5 IU / ml FVIII:C into three different flow cells in randomized order. The binding level, measured 30 seconds after the analyte injection, was normalized by dividing RU by the molecular weight of the corresponding protein and expressed as rFVIII as 100% binding percentage.

[0184] result

[0185] Figure 4 The results are shown. The binding levels of all FVIII-VWF-EP fusion proteins were ≤11% of FVIII. C16 showed the highest binding, while the C17 construct showed the lowest binding.

[0186] Example 5: Pharmacokinetics of the FVIII-VWF-EP fusion protein

[0187] Experimental Objective

[0188] The aim was to investigate the effect of covalent fusion of the VWF fragment with two extended peptides (VWF-EP) to the C-terminus of FVIII on its pharmacokinetics (PK). The effects of the presence of three additional EPs in the second linker (in construct C17) and the VWF-EP were investigated in hemophilia A (HemA) mice.

[0189] method

[0190] Male B6;129S-F8tm1Kaz / J (F8- / -) mice aged 5 to 8 weeks were obtained from Jackson Laboratory (Bar Harbor, Maine, USA). Animals were treated by tail vein injection of either the test compound or the rFVIII control at a dose based on FVIII:C activity of 200 IU / kg body weight. Table 5 summarizes the study. Blood samples were collected at designated time points. Blood samples were taken from five animals per group at each time point. Each mouse was used for two sampling points. Blood was collected in tubes containing 3.8% sodium citrate solution. Immediately after collection, blood samples were placed on crushed ice, and plasma was separated within 1 hour of collection by centrifugation at 3350 x g (4000 rpm), 4°C for 15 minutes. Plasma samples were stored at -80°C until analysis was performed using the FVIII:C assessment (Coamatic Factor VIII Assessment Kit; Chromogenix, Bedford, MA, USA).

[0191] FVIII:Ag in mouse plasma was determined by internal ELISA assessment. In the first step, maxisorp microplates (Thermo Fisher Scientific 439454) were coated overnight with an anti-human FVIII monoclonal antibody recognizing the A2 domain (GMA8023, Green Mountain Antibodies, Burlington, USA). After blocking and washing, diluted mouse plasma was applied to the plates and incubated at 37°C for 2 hours. Following a subsequent washing step, the binding molecule was detected by biotinylated anti-FVIII nanobody (capture-selective biotinylate anti-FVIII conjugate; Thermo Fisher Scientific 7102862500) and neutral avidin-HRP (Thermo Fisher Scientific 31001). Colorimetric readings were obtained at 450 nm using a tetramethylbenzidine substrate (Sigma-Aldrich T4444).

[0192] Table 5: Research Summary

[0193]

[0194] result

[0195] Table 6 and Figure 5 The results were presented. rFVIII showed a half-life of 7.61 hours, while T was 1.6 times longer due to a T of 12.42 hours. 1 / 2The FVIII-VWF-EP fusion protein construct C17 was detected to have a significantly longer duration of activity in mouse plasma. C17 also showed higher C... max The increase in recovery rate and AUC is due to the improved recovery rate and improved half-life.

[0196] Table 6: PK analysis of FVIII:C data measured in HemA mouse plasma

[0197] parameter rFVIII(NUWIQ) C17 unit λ_z 0.09 0.06 1 / hour t1 / 2 7.61 12.42 Hour Tmax 1.00 1.00 Hour Cmax 294.67 327.70 ％ C0 356.38 371.56 ％ AUC 0-t 3491.00 4847.00 %*Hour AUC 0-inf_obs 3531.47 4903.32 %*Hour MRT 0-inf_obs 8.89 13.64 Hour

[0198] This indicates that covalently linking FVIII with the VWF fragment and EP via gene fusion leads to an improvement in its PK parameters.

[0199] To determine specific activity, mouse plasma FVIII:Ag and FVIII:C values ​​were measured for rFVIII (NUWIQ) and C17 using the internal ELISA assessment and Coamatic Factor VIII assessment kits as described above.

[0200] The mean FVIII specific activity values ​​determined in 30 mice showed that C17 had a significantly higher specific activity compared to unmodified rFVIII (Table 7).

[0201] Table 7: Average Specific Activity

[0202] rFVIII(NUWIQ) C17 FVIII specific activity [IU / mg] 12357,5±1582 14227±2702,8*

[0203] *FVIII:Ag assessment only identifies the FVIII chain (compared to the BCA assessment used in D1). After adjusting for molecular weight differences, the specific activity of C17 is 9811.7 IU / mg, still higher than in D1.

[0204] This activity is higher than the specific activity of the molecule in prior art document 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, with values ​​of 9304.3 IU / mg, 8474.5 IU / mg, and 9367.2 IU / mg, respectively. Therefore, this invention provides an FVIII-VWF fusion protein with significantly higher specific activity.

[0205] Example 6: Confirmation results of sequence variants

[0206] To confirm that minor sequence variants of the FVIII heavy chain, FVIII light chain, VWF fragment, and EP do not affect the properties of the construct, additional fusion proteins based on C17 with sequence variations in one or all of the four functional elements shown in Table 8 were generated, as described in Example 1. Furthermore, experiments according to Examples 2 through 4 were repeated.

[0207] Table 8: Other fusion proteins containing sequence variants

[0208]

[0209]

[0210] Benefiting from the teachings presented in the foregoing description and the associated drawings, those skilled in the art will conceive of many modifications and other embodiments of the invention set forth herein. Therefore, it will be understood that the invention should not 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. While specific terminology is used herein, such terminology is used only in a general and descriptive sense and is not intended for limiting purposes.

[0211] Table 9: Overview of Sequence IDs

[0212]

[0213]

[0214] References

[0215] 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, pp. 1-16.

[0216] Fischer B, Mitterer A, Schlokat U, DenBouwmeester R, Dorner F. “Structural analysis of recombinant von Willebrand factor: identification of hetero- and homo-dimers” FEBS Letters, 1994; 351(3):345-8. Errata: FEBS Letters, 1994; 353(3):337.

[0217] Furlan M. "Von Willebrand factor: molecular size and functional activity" Ann Hematol. 1996; 72(6):341-348.

[0218] 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. Journal of Thrombosis and Haemost. September 2020; 18 Supplement 1 (Supplement 1): 5-14.

[0219] Innis et al. (1990), PCR: A Guide to Methods and Application, Academic Press, New York.

[0220] Kannicht C, M, Kohla G et al., Characteristics of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII. Thromb Research, 2013; 131(1):78-88.

[0221] Khalil et al., 2022. Khalil A, Würthwein G, Golitsch J, Hempel G, Fobker M, Gerss J. A,Zimmermann M,Smisek P,Zucchetti M,Nath C,Attarbaschi A,VonStackelberg A, 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 lymphoblastic leukemia. Haematologica. Jan 1, 2022; 107(1):49-57.

[0222] Needleman SB, Wunsch CD. A general method applicable to the search for similarities in the aminoacid sequence of two proteins. Journal of Molecular Biology, 1970; Vol. 48(3); 443-453.

[0223] M.R. Green and J. Sambrook (2012), Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0224] Paik J, Deeks ED. Damoclot Alfa Pegol: A Review in Haemophilia A. Drugs. July 2019; 79(10):1147-1156. (Corrected in: Drugs. August 23, 2019)

[0225] Pezeshkpoor B, Sereda N, Berkemeier AC, Matuschek I, Schwarz N, Turecek PL, Horneff S, Klein C, Goldmann G, Marquardt N, Albert T, Müller J, Oldenburg J. Antidrug antibodies against the polyethylene glycol moiety inhibit the procoagulant activity of therapeutic polyethylene glycolated factor VIII. Journal of Thrombosis and Hemostasis, June 2023; 21(6):1503-1514. doi:10.1016 / j.jtha.2023.03.011. Electronic version March 18, 2023.

[0226] Strohl WR. Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters. BioDrugs. 2015; Vol. 29(4), 215-239.

[0227] Tiede A. Half-life extended factor VIII for the treatment of hemophilia A. Journal of Thrombosis and Hemostasis. 2015; Vol. 13, Supplement 1; S176-S179

[0228] 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

[0229] 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: ● Factor VIII (FVIII) heavy chain; ●FVIII light chain; ● Fragments of the von Willebrand factor (VWF); and ● At least two copies of the extended peptide (EP); The EP has at least 90% amino acid sequence identity with 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 FVIIIB domain and preferably contains an amino acid sequence that has at least 90%, more preferably at least 95%, and most preferably at least 98% identity with 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 that is at least 90%, preferably at least 95%, more preferably at least 98% identical 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 via a first linker, wherein the first linker preferably comprises a sequence derived from the B domain of the FVIII; and / or ●The C-terminus of the FVIII light chain is fused to the N-terminus of the VWF segment via a second connector.

4. The fusion protein of claim 3, wherein the first linker and / or the second linker comprises at least one copy of the EP, preferably at least two copies, more preferably at least three copies, wherein the EP is preferably assembled in a sequential order.

5. The fusion protein according to claims 1 to 4, further comprising at least one extended half-life portion, wherein the at least one extended half-life portion is preferably selected from the immunoglobulin Fc domain, serum albumin or a portion thereof, albumin-binding antibody, albumin-binding protein domain, and most preferably the extended half-life portion is albumin-binding V H H-structure domain.

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

7. The fusion protein according to claims 3 to 6, wherein the first linker, the second linker, and / or the third linker are flexible and comprise (GGS). n (GGGS) n Or (GGGGS) n , where n is an integer in the range of 1 to 10, and where 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 of claim 8, wherein two consecutive copies of the GGGGS motif are located at the N-terminus and / or C-terminus of the 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 the 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 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 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 the 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 contains at least two copies of the GGGGS motif on either side of the EP assembly and / or on either side of the extended half-life portion.

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

15. The fusion protein according to any one of the preceding 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 the group consisting of: 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 treating hemorrhagic conditions, wherein the fusion protein is defined according to any one of the preceding claims.

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

18. The polynucleotide of claim 17, wherein the amino acid sequence encoding the sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% identity with the sequence selected from the following: 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 cell of a human cell line, more preferably a cell of a human kidney cell line, most preferably a cell of a human embryonic kidney cell line, particularly a cell of the HEK293 cell line, such as HEK293F.

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