Polypeptide for treatment of microvascular thrombosis

Abstract

The current invention relates to a polypeptide comprising a truncated form of ADAMTS13 and an immunoglobulin targeting agent, wherein the targeting agent is able to bind to a von Willebrand factor (VWF) via a VWF-binding domain at a site of a thrombus.

Description

[0001] POLYPEPTIDE FOR TREATMENT OF MICROVASCULAR THROMBOSIS

[0002] FIELD OF THE INVENTION

[0003] This invention operates within the biopharmaceutical realm, specifically focusing on preventing or treating conditions linked to microvascular thrombosis, notably thrombotic thrombocytopenic purpura (TTP). The innovation involves fusion proteins that combine a targeting agent, primarily nanobodies against VWF, with truncated ADAMTS13. The objective is to enzymatically degrade VWF and to disrupt platelet-VWF interactions, thereby mitigating the risk of thrombosis.

[0004] BACKGROUND ART

[0005] Microvascular thrombosis (MVT) is characterized by the formation of microvascular platelet aggregates, minimally comprised of platelets and von Willebrand factor (VWF). This phenomenon is notably evident in thrombotic thrombocytopenic purpura (TTP), where microthrombi rich in platelets and VWF, yet deficient in fibrin, pose a critical threat by obstructing microvasculature, leading to potentially life-threatening consequences. MVT serves as a common feature across various disease states, including TTP, haemolytic uremic syndrome, antiphospholipid antibody syndrome, and complement-mediated thrombotic microangiopathy. In severe cases of MVT, the repercussions extend to multiorgan failure, resulting in lethal outcomes. Even in less severe instances, MVT can lead to organ damage, diminishing both the quality of life and life expectancy of affected individuals.

[0006] Recent studies suggest that microvascular disease serves as a key factor in cardiovascular events among individuals diagnosed with cardiovascular disease within the broader population, especially in instances where overt signs of macrovascular obstruction are not evident in radiological examinations. This microvascular involvement is believed to contribute significantly to heart failure, especially among female patients.

[0007] Patients with TTP endure episodes of MVT, wherein platelets form complexes with ultralarge multimers of von Willebrand Factor (VWF). This pathological state arises from a significant reduction in ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type one repeats, member 13) activity, an enzyme essential for regulating the thrombogenicity of VWF by enzymatically reducing its multimer size. To accomplish this, VWF undergoes a structural transformation, transitioning from its globular form to an unrolled conformation, exposing its A2 domain for proteolysis. The majority of TTP patients face a formidable challenge with the presence of neutralizing autoantibodies against ADAMTS13 (acquired, immune-mediated TTP, iTTP). Additionally, a smaller subgroup experiences TTP due to mutations in ADAMTS13, leading to deficiency and manifesting as Upshaw-Shulman syndrome (congenital TTP). Current therapeutic approaches encompass (1) ADAMTS13 replacement through plasma exchange or recombinant ADAMTS13, (2) anti-CD20 rituximab to induce B-cell depletion and reduce anti-ADAMTS13 autoantibodies, and (3) the nanobody Caplacizumab, which targets the VWF Al domain, impeding the interaction between VWF and platelet GPIbo. However, these treatments have significant drawbacks: plasma exchange is invasive and carries risks such as infection and thrombosis; recombinant ADAMTS13 has a short half-life and may be neutralized by autoantibodies; rituximab causes immunosuppression and has a delayed therapeutic effect; and Caplacizumab, while effective, increases bleeding risk and is costly.

[0008] The current invention aims to solve at least some of these drawbacks. By providing a polypeptide the current invention offers a solution with reduced immunogenicity, enhanced stability and half-life, high specificity and reduced side effects.

[0009] Single domain antibodies directed against the Al domain of activated VWF specifically recognizing the activated VWF conformation at the site of thrombus formation but not binding to circulating inactivated forms of VWF are part of patent (W02004 / 062551 A2 and US7939277 B2). These nanobodies can be considered as target proteins to bring truncated ADAMTS13-MDTCS in close proximity to the A2 domain of VWF. Especially All (AU / VWFa- 11), that specifically recognized the GPIbo-binding conformation is considered as a target protein.

[0010] AU / VWFa-11 binds to the Al domain of active VWF in a different region as GPIbo, and Hulstein at al. 2005 concluded that AU / VWFa-11 could not interfere with the interaction between VWF and GPIbo based on an in vitro perfusion assay. It is an object of the present intervention to provide evidence that AU / VWFa-11 inhibits binding of platelet to VWF in a GPIbo- binding conformation in solution, but not to VWF immobilized to collagen and that it may prevent excessive platelet aggregates in circulation, without affecting physiological haemostasis at sites of vascular injury, making it an ideal targeting agents for the polypeptides.

[0011] Polypeptides aimed at treating TTP are detailed in CN104926946 and WO2019185723. CN104926946 involves a fusion protein combining mutant ADAMTS13-MDTCS with human serum albumin, designed to maintain the biological activity of the original ADAMTS13 protein and extend its half-life. However, this invention lacks a targeting agent that binds to VWF, necessary for bringing ADAMTS13-MDTCS close to its cleavage site.

[0012] In contrast, the WO2019185723 describes fusion proteins incorporating a plasminogen activator (preferable uPA or tPA) capable of activating plasminogen that can cleave VWF, similar to ADAMTS13. These fusion proteins include a targeting agent to direct the plasminogen activator to thrombus sites containing VWF, platelets, or activated vascular endothelium. While this approach may avoid issues related to ADAMTS13 autoantibodies, the targeting agent solely facilitates activation at the required site, lacking additional mechanisms of action present in the targeting agent of this invention.

[0013] The invention provides fusion proteins for targeted delivery of truncated ADAMTS13 with the option of a dual mechanism of action: (1) proteolysis of VWF and (2) inhibition of platelet aggregate formation in solution. The invention further provides therapeutic methods for TTP. However, VWF and ADAMTS13 are now recognized as important contributors to a growing number of diseases and disorders in which vascular inflammation and thrombosis play a role, including cancer, atherosclerosis, sepsis, stroke, neurological conditions and liver disease. Therefore, additional therapeutic applications need to be explored.

[0014] SUMMARY OF THE INVENTION

[0015] In a first aspect, the invention relates to a polypeptide comprising a truncated form of ADAMTS13 and a targeting agent for targeting the truncated ADAMTS13 to VWF at a site of thrombus according to claim 1. Preferably, the targeting agent in the polypeptide of the invention specifically binds to VWF. Further specific positions of the polypeptide are provided in the independent claims 2 to 13.

[0016] In a second aspect, the invention relates to a pharmaceutical composition comprising the polypeptide disclosed herein, according to claim 14.

[0017] In a third aspect, the invention relates to the use of the nucleic acid or pharmaceutical composition for use as a medicament, for instance for the treatment of microvascular thrombosis .

[0018] In a last aspect, the invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide in accordance with the invention as defined above .

[0019] DESCRIPTION OF THE FIGURES

[0020] Figure 1. Interaction of Syn-VWFAl with VWF in its native or activated conformation. The binding of Syn-VWFAl to HVWF, R1306W, R1306Q, VWF in diluted NPP and VWF in vortexed diluted NPP treated with (grey) or without (black) ristocetin. Data represent the mean ± SD (n=3). Differences between the conditions with and without ristocetin were tested by unpaired Student's t-test, with *** P<0.001, ** P<0.01.

[0021] Figure 2. Syn-VWFAl inhibits binding of VWF to platelets in solution. The inhibitory effect of Syn-VWFAl on the binding of native VWF (A), HVWF (B) or R1306W VWF (C) to platelets in whole blood, in the absence or presence of ristocetin, was determined by flow cytometry. In panel A, only native VWF in WB was present in all conditions. In panels B and C, HVWF or R1306W, respectively, were added to the WB. For all experiments, WB was pre-incubated with buffer (control), 1.5 pM Syn-VWFAl, 0.5 mg / ml ristocetin or the combination thereof. Data represent mean±SD of duplicate measurements of blood from 3 healthy donors. *** P<0.001, **P<0.01,* P<0.05. (D) The inhibitory effect of Syn- VWFA1 on platelet agglutination was measured in platelet rich plasma with light transmission aggregometry. Syn-VWFAl (0-3 pM) dose-dependently inhibited ristocetin- induced (1.25 mg / ml), VWF-dependent platelet agglutination expressed as percentage inhibition of maximal platelet agglutination. (E) Similarly, Syn-VWFAl inhibits R1306W- induced platelet agglutination, but not collagen- and ADP-induced platelet aggregation. Results are expressed as maximal aggregation relative to the control condition without Syn-VWFAl, i.e. 100% represents no inhibition and 0% full inhibition by Syn-VWFAl. Data represent mean±SD (n=3).

[0022] Figure 3. Syn-VWFAl does not inhibit platelet binding to immobilized collagen. (A-D) Whole blood with DiOC6-labeled platelets was perfused over immobilized collagen type I at a shear rate of 1600 s’1. Data represent mean±SD of 10 images taken after 4 minutes perfusion with WB from 5 healthy donors. (A) Quantification of the platelet covered area (%), as a measure of platelet adhesion, in the absence and presence of 1.8 pM Syn- VWFAl. (B) The fluorescent intensity of platelet aggregates, indicative of the amount of platelets clustered together, in the absence and presence of 1.8 pM Syn-VWFAl. (C, D) Representative images of platelet aggregates (GFP) in the absence (C) and presence (D) of 1.8 pM Syn-VWFAl.

[0023] Figure 4. Syn-VWFAl does not inhibit platelet binding to immobilized collagen. HVWF activated by ristocetin (0.5 mg / mL) was pre-incubated with 3 pM Syn-VWFAl ("+ Syn-VWFAl pre-inc" condition) or saline ("- Syn-VWFAl" and "+ Syn-VWFAl" conditions). This was added to collagen type III coated wells, followed by addition of saline (negative control), GPlbo (in the "+ Syn-VWFAl pre-inc" and "- Syn-VWFAl" conditions) or a combination of GPlbo and 3 pM Syn-VWFAl ("+ Syn-VWFAl" condition). Binding of GPlba is expressed as the OD (mean±SD) at 490 nm (n=3).

[0024] Abbreviations: Neg. control; negative control; pre-inc, pre-incubated with Syn-VWFAl, i.e. Syn-VWFAl was added to ristocetin-activated HVWF before addition to collagen, in contrast to the "+ Syn-VWFAl" condition in which Syn-VWFAl was added simultaneously with GPlba to collagen-bound activated HVWF.

[0025] Figure 5. Syn-VWFAl inhibits platelet string length and platelet string number on stimulated endothelial cells. Unstimulated HUVECs (white bars) or HUVECs stimulated for 10 mins with 25 pM histamine (black bars) were seeded at 7xl06cells / mL and incubated overnight at 37°C w / 5% CO2. The next day, resuspended lyophilised platelets (2xl08cells / mL) in the presence of Syn-VWFAl (0, 0.5, 1, 5 pM final cone.) were perfused at 2.5 dyne / cm2. Formation of platelet strings (panel A) and platelet string number (lower panel B) are shown.

[0026] Figure 6. Proteolytic activity of ADAMTS13 polypeptides. HVWF was cleaved with recombinant ADAMTS13, MDTCS or MDTCS-Syn-VWFAl (panel A) or ADAMTS13, MDTCS- RARAA or MDTCS-RARAA-Syn-VWFAl (panel B) at 37°C for 6h in, in buffer containing urea. Proteolytic activity was stopped immediately (lane 1) or after 6h (lane 2-8). VWF multimer patterns were examined and the fraction of low multimers was shown.

[0027] Figure 7. Proteolytic activity of ADAMTS13 polypeptides. FRETS-VWF73 assay (FIG. 7A) and VWF73 activity ELISA (FIG. 7B) were performed using MDTCS-Syn-VWFAl, MDTCS-RARAA-Syn-VWFAl, ADAMTS13, MDTCS and MDTCS-RARAA.

[0028] Figure 8. Effect of ADAMTS13 constructs on platelet string length on stimulated endothelial cells. HUVECs stimulated for 10 mins with 25 pM histamine (right) were seeded at 7xl06cells / mL and incubated overnight at 37°C w / 5% CO2. The next day, resuspended lyophilised platelets (2xl08cells / mL) in the presence of Syn-VWFAl, recombinant ADAMTS13, MDTCS, MDTCS-Syn-VWFAl, MDTCS-RARAA and MDTCS- RARAA-Syn-VWFAl were perfused at 2.5 dyne / cm2. Formation of platelet strings (panel A) and platelet string number (lower panel B) are shown.

[0029] Figure 9. polypeptides inhibit binding of VWF to platelets in solution. The inhibitory effect of 1 pM Syn-VWFAl, recombinant ADAMTS13, MDTCS, MDTCS-Syn-VWFAl, MDTCS- RARAA and MDTCS-RARAA-Syn-VWFAl on the ristocetin-induced binding of VWF to platelets in whole blood, was determined by flow cytometry. The inhibition of the VWF binding to platelets is shown.

[0030] Figure 10. ADAMTS13 polypeptides inhibit platelet agglutination. The inhibitory effect of 1.25 pM Syn-VWFAl, MDTCS, MDTCS-Syn-VWFAl, MDTCS-RARAA, MDTCS- RARAA-Syn-VWFAl and 0.625 pM recombinant ADAMTS13, on the ristocetin-induced platelets agglutination in platelet rich plasma was determined in platelet aggregometry. The inhibition of platelet agglutination is shown.

[0031] Figure 11. Activity testing in FRETS- VWF73 assay for resistance to autoantibodies in iTTP patients. The relative activity (FIG. 11A) and remaining activity (FIG. 11B) of MDTCS-RARAA, MDTCS-RARAA-Syn-VWFAl, ADAMTS13, MDTCS and MDTCS-Syn-VWFAl were tested.

[0032] Figure 12. Proteolytic activity of MDTCS-RARAA-L-Syn-VWFAl in FRET-VWF73 assay. FRET-VWF73 assay (FIG. 12) was performed using full-length rADAMTS13, MDTCS-RARAA-L-Syn-VWFAl and MDTCS-RARAA-Syn-VWFAl. Figure 13. Proteolytic activity of MDTCS-RARAA-L-Syn-VWFAl in GST-VWF73- based ADAMTS13 activity ELISA. MDTCS-RARAA-L-Syn-VWFAl, rADAMTS13 and MDTCS-RARAA-Syn-VWFAl were tested at different concentrations (Fig 13A) and the relative activity compared to rADATMS13 was calculated for polypeptides at 8 nM (Figure 13B).

[0033] Figure 14. Proteolytic activity of MDTCS-RARAA-L-Syn-VWFAl in multimer assay. The VWF-cleaving activity of MDTCS-RARAA-Syn-VWFAl and MDTCS-RARAA-L-Syn- VWFAl was evaluated across a concentration range using a VWF multimer assay, whereas rADAMTS13 was assessed only at 8 nM (FIG. 14A). The percentage of low molecular weight multimers formed is shown in FIG. 14B.

[0034] Figure 15. Inhibition of platelet string length and platelet string number on stimulated endothelial cells by MDTCS-RARAA-L-Syn-VWFAl . The beads-on-a- string assay was used to evaluate the dual-action mechanism of Syn-VWFAl, MDTCS- RARAA-Syn-VWFAl, MDTCS-RARAA-L-Syn-VWFAl and rADAMTS13, all at 2 nM. The platelet string length is shown in FIG. 15A while the platelet string number is shown FIG. 15B.

[0035] Figure 16. Inhibition of binding of VWF to platelets by MDTCS-RARAA-L-Syn- VWFA1. A whole blood flow cytometry assay was performed to evaluate the interaction between platelets and VWF in the presence of Syn-VWFAl, MDTCS-RARAA-Syn-VWFAl, and MDTCS-RARAA-L-Syn-VWFAl. The percentage of inhibition of VWF binding to platelets is shown.

[0036] Figure 17. Inhibition of platelets agglutination by MDTCS-RARAA-L-Syn-VWFAl. The inhibitory effect of Syn-VWFAl, MDTCS-RARAA-Syn-VWFAl and MDTCS-RARAA-L- Syn-VWFAl on ristocetin-induced platelet agglutination was assessed using LTA in PRP. The percentage of inhibition of platelet agglutination is shown.

[0037] Figure 18. Resistance of fusion proteins against proteolysis by plasmin. MDTCS- Syn-VWFAl, MDTCS-RARAA-Syn-VWFAl, and MDTCS-RARAA-L-Syn-VWFAl or rADAMTS13 (400 nM) were incubated with 1 or 10 nM plasmin for 1 hour at 37°C. SDS- PAGE was performed to separate the proteins according to their size.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] DEFINITIONS Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0040] As used herein, the following terms have the following meanings:

[0041] "A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

[0042] "About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / -20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0043] "Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

[0044] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0045] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0046] The expression "% by weight", "weight percent", "%wt" or "wt%", here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

[0047] Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0048] The term "sequence identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid or nucleotide-by-nucleotide basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid or nucleotide occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[0049] A gap, i.e., a position in an alignment where a residue or nucleotide is present in one sequence but not in the other, is regarded as a position with non-identical residues or nucleotides. In global alignments, terminal gaps (regions beginning at the end of a sequence where the nucleotide or residue does not correspond to a position in the other sequence and extending for all contiguous positions) are discarded before identity is calculated, while internal gaps are counted as differences. However, in local alignments, both internal and terminal gaps are counted as differences.

[0050] Determining the percentage of sequence identity can be done manually or by using computer programs that are available in the art. Examples of useful algorithms for this purpose include PILEUP, BLAST (available through the National Center for Biotechnology Information at http: / / www.ncbi.nlm.nih.gov / ), and implementations of the Needleman- Wunsch algorithm for global alignment and the Smith-Waterman algorithm for local alignment, where in local alignments, end gaps are counted as differences.

[0051] The term "thrombus" or "site of a thrombus" as interchangeably used herein, refers in the present context to a blood clot formed within the vascular system that impedes blood flow. More specifically, "thrombus" denotes an agglutination and / or aggregation of platelets, von Willebrand factor (VWF), and / or cellular elements, such as vascular endothelium, but notably excludes fibrin, forming a solid mass within the circulatory system.

[0052] The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin.

[0053] In amino acid sequences or protein variants as defined herein, amino acids are denoted by single-letter or three-letter symbols. These single-letter and three- letter symbols are well known to the person skilled in the art and have the following meaning: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (lie) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gin) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Vai) is valine, W (Trp) is tryptophan, Y (Tyr) is tyrosine.

[0054] Mutations, in particular substitutions of an amino acid by another amino acid, are indicated herein in a way that is standard in the art, i.e. by indicating the amino acid present in wildtype ADAMTS13 sequence, the position of the amino acid in the sequence, and the amino acid that is introduced at the position. E.g. "F592A" indicates that the phenylalanine at position 592 is substituted by a alanine.

[0055] The term "signal peptide" (sometimes referred to as signal sequence) is a short peptide (usually 16-30 amino acids long) present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. At the end of the signal peptide there is usually a stretch of amino acids that is recognized and cleaved by signal peptidase either during or after completion of translocation (from the cytosol into the secretory pathway, i.e. ER) to generate a free signal peptide and a mature protein. Signal peptides are extremely heterogeneous and many prokaryotic and eukaryotic signal peptides are functionally interchangeable even between different species however the efficiency of protein secretion may depend on the signal peptide. Suitable signal peptides are generally known in the art e.g. from Kall et al. and von Heijne.

[0056] "N-linked glycosylation" refers to the attachment of an oligosaccharide moiety to a nitrogen atom, typically the N4 of an asparagine residue. The terms "N-linked glycosylation site" and "N glycosylation site" are used interchangeably and refer to a site in the ADAMTS13 protein variant where N-linked glycosylation is possible. Such site has the amino acid sequence NXT or NXS, wherein X is any amino acid except P. N-linked glycosylation is a posttranslational modification and N-linked glycans of a protein can modulate the folding, cell attachment and / or function of a protein. N-linked glycans can have different combinations of mannose, N- acetylglucosamine (GIcNAc), galactose, fucose and sialic acid residues.

[0057] Unless indicated otherwise, the terms "immunoglobulin" and "antibody" whether it used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or VH / VL domains, respectively). In addition, the term "sequence" as used herein (for example in terms like "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH sequence" or "protein sequence"), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the same, unless the context requires a more specific interpretation.

[0058] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH", or to "VHH" in case of a heavy chain antibody such as the camelid antibodies that consist of only heavy chains. The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites. The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the average 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" (HVRs) or complementarity determining regions (CDRs) that are each about 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a 0-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the p-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies.

[0059] The terms "VHH", "VHH domain" and "nanobody" are interchangeable herein and are used herein to refer to the variable domain of a heavy chain antibody, i.e. an antibody consisting only of heavy chains and devoid of light chains as are known e.g. from Camelids. The amino acid sequence and structure of a VHH can be considered without however being limited thereto to be comprised of four framework regions or "FR's", which are referred to in the art and herein below as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDRs", which are referred to in the art as "Complementarity Determining Region 1" or "CDR1"; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or"CDR3", respectively. The total number of amino acid residues in a VHH can be in the region of 110-120, is preferably 112-115, and is most preferably 113. It should however be noted that parts, fragments or analogs (as further described herein below) of a VHH are not particularly limited as to their length and / or size, as long as such parts, fragments or analogs meet the further functional requirements outlined herein below and are also preferably suitable for the purposes described herein.

[0060] The amino acid residues of a VHH (or conventional variable domain) are numbered according to the general numbering for VH domains given by Kabat et al., 1991 (Sequences of Proteins of Immunological Interest. 5th ed: U.S. Dept, of Health and Human Services) as applied to VHH domains from Camelids by Riechmann and Muyldermans et al. 1999 (1999, J. Immunol. Methods; 231 : 25-38);. According to this numbering, FR1 of a VHH comprises the amino acid residues at positions 1-30, CDR1 of a VHH comprises the amino acid residues at positions 31-36, FR2 of a VHH comprises the amino acids at positions 36- 49, CDR2 of a VHH comprises the amino acid residues at positions 50-65, FR3 of a VHH comprises the amino acid residues at positions 66-94, CDR3 of a VHH comprises the amino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113. In this respect, it should be noted that as is well known in the art for VH domains and for VHH domains the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. Generally, however, it can be said that, according to the numbering of Kabat and irrespective of the number of amino acid residues in the CDRs, position 1 according to the Kabat numbering corresponds to the start of FR1 and visa versa, position 36 according to the Kabat numbering corresponds to the start of FR2 and visa versa, position 66 according to the Kabat numbering corresponds to the start of FR3 and visa versa, and position 103 according to the Kabat numbering corresponds to the start of FR4.

[0061] Alternative methods for numbering the amino acid residues of VH domains, which methods can also be applied in an analogous manner to VHH domains from Camelids, are the method described by Chothia et al. (1989, Nature 342, 877-883), the so-called "AbM definition" and the so-called "contact definition". However, in the present description, claims and figures, the numbering according to Kabat as applied to VHH domains by Riechmann and Muyldermans will be followed, unless indicated otherwise.

[0062] Generally, it should be noted that the term "VHH" (or nanobody) as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. For example, VHHs as used in the invention can be obtained (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" (as described below) of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain; (4) by "camelization" of a naturally occurring VH domain from any animal species, in particular a species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (6) by preparing a nucleic acid encoding a VHH using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained; and / or (7) by any combination of the foregoing. Suitable methods and techniques for performing the foregoing are state of the art and therefore known to the skilled person.

[0063] One particularly preferred class of VHHs for use in the invention comprises VHHs with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being. This can be performed in a manner known per se, which will be clear to the skilled person. Again, it should be noted that such humanized VHHs of the invention can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material.

[0064] The term "thrombolytic activity" refers to the ability of a substance to dissolve thrombi or blood clots. The term "linker" refers to a molecule or set of molecules that connects two or more other molecules together.

[0065] A "blocking" antibody or an "antagonist" antibody is one which inhibits or reduces biological activity of the antigen it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. An "agonist antibody", as used herein, is an antibody which mimics at least one of the functional activities of a polypeptide of interest.

[0066] The term "pharmaceutical-acceptable carrier, excipient or diluent" refers to a substance that is considered safe and effective and is included in a drug to aid the delivery of the therapeutic substance.

[0067] The term "parenteral administration" refers to the administration of a substance by a route other than the digestive tract, such as intravenous, subcutaneous, or intramuscular routes. It may be any molecule which may improve the polypeptide's selectivity, effectiveness and / or safety of administration to a human or animal body, such as by continuous or triggered release or by allowing membrane permeation of the polypeptide. The term "thrombotic or embolic state" refers to a condition characterized by the formation of blood clots within a blood vessel or the lodging of a loose clot within a blood vessel, respectively.

[0068] The term "acute or chronic disease or condition" refers to a disease or condition that has a rapid onset and / or a short course, or a disease or condition that is persistent or long- lasting, respectively

[0069] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

[0070] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0071] Peptides and polypeptides

[0072] In a first aspect, the invention relates to a polypeptide comprising a truncated form of ADAMTS13 and a targeting agent, wherein the targeting agent is able to bind to a von Willebrand factor (VWF) via a VWF-binding domain at a site of a thrombus. The targeting agent delivers the truncated ADAMTS13 to a site of a thrombus comprising the VWF.

[0073] ADAMTS13

[0074] The term 'ADAMTS13' refers to a protein encoded by the ADAMTS13 gene. ADAMTS13 is a member of the metalloproteinase gene family, ADAM (a disintegrin and metalloproteinase), a family consisting of membrane-anchored proteases with different functions. ADAMTS family members are further characterized by the presence of one or more thrombospondin 1-like (TSP1) domain(s) at the C-terminus and the absence of an EGF repeat, a transmembrane domain and a cytoplasmatic tail, present in ADAM metalloproteinases. ADAMTS13 is the only ADAMTS member to possess two C-terminal CUB domains and possesses VWF cleaving protease activity.

[0075] ADAMTS13 is a ~190 kDa multidomain plasma glycoprotein that consists of 14 domains: a metalloprotease domain (MP) according to SEQ ID N°:23, a disintegrin-like domain (Dis) according to SEQ ID N°:24, a thrombospondin type 1 (TSP1) repeat according to SEQ ID N°:25, a cysteine-rich domain (Cys-rich) according to SEQ ID N°:26, a Spacer domain according to SEQ ID N°:27 and 7 additional thrombospondin type-1 repeats (TSP2-TSP8) according to SEQ ID N°:30 to SEQ ID N°:36, and 2 CUB domains (CUB1 and CUB2) according to SEQ ID N0:37 and SEQ ID N°:38. TSP2-TSP-8 and two CUB domains fold back and interact with the central Spacer domain. ADAMTS13 circulates in a folded conformation that is stabilized by an interaction between the central Spacer domain and the C-terminal CUB domains.

[0076] Binding of ADAMTS13 to the VWF D4(-CK) domains induces a structural change that extends ADAMTS13 into an open conformation that enhances its function. Multiple ADAMTS13 exosite interactions are involved in recognition of the unfolded A2 domain of VWF. In binding VWF, the ADAMTS13 cysteine-rich and spacer domain exosites bring enzyme and substrate into proximity. Thereafter, binding of the ADAMTS13 disintegrin-like domain exosite to VWF allosterically activates the adjacent metalloprotease domain to facilitate proteolysis. The C-terminal TSP2-8 / CUB1-2 domains of ADAMTS13 are important for full in vivo proteolytic activity because these domains allow ADAMTS13 to anchor itself onto the D4-CK domains of full-length VWF under shear flow conditions.

[0077] Preferably, the polypeptide disclosed herein comprises a truncated version of ADAMTS13. The term "truncated ADAMTS13" refers to a protein in the polypeptide of this invention with the same or similar amino acid sequence as ADAMTS13 wherein the C-terminal thrombospondin type-1 repeats (TSP2-TSP8), and 2 CUB domains (CUB1 and CUB2) are deleted and that minimally consists of the MP domain that is responsible for the proteolytical cleavage of VWF. More specifically "truncated ADATMS13" can refer to: a) M according to SEQ ID N°:23 comprising the metalloprotease domain (MP) of ADAMTS13; b) MD according to SEQ ID N°:39 comprising the MP domain and the disintegrin-like domain (Dis) according to SEQ ID N°:24; c) MDT according to SEQ ID N°:40, comprising the MP domain, the Dis domain and the thrombospondin type 1 (TSP1) domain according to SEQ ID N°:25; d) MDTC according to SEQ ID N°:41 comprising the MP domain, the Dis domain, the TSP1 domain and the cysteine-rich domain (Cys-rich) according to SEQ ID N°:26; e) MDTCS according to SEQ ID N°:42 comprising the MP domain, the Dis domain, the TSP1 domain, the Cys-rich domain and the Spacer region according to SEQ ID N° 28 or any other combination of these domains.

[0078] In an embodiment of the polypeptide, as disclosed herein, the truncated form of ADAMTS13 comprises at least one or more of: a) a metalloprotease (MP) domain or fragments thereof; b) an MP domain and a disintegrin-like domain (Dis) or fragments thereof; c) an MP domain, a Dis domain and a thrombospondin type 1 (TSP1) repeat or fragments thereof; d) an MP domain, a Dis domain, a TSP1 repeat and a cysteine-rich domain (Cys-rich) or fragments thereof; e) an MP domain, a Dis domain, a TSP1 repeat, a Cys-rich domain and a Spacer domain or fragments thereof.

[0079] In an embodiment of the polypeptide, as disclosed herein, the truncated form of ADAMTS13 comprises maximally the MDTCS domains and minimally the M domain. In an embodiment, the truncated ADAMTS13 comprises at least one domain, 2 domains, 3 domains, 4 domains or 5 domains wherein at least one domain is M. In further embodiments, the truncated ADAMTS13 comprises the domains selected from M, MD, MT, MC, MS, MDT, MDC, MDS, MTC, MTS, MCS, MDTC, MDTS, MDCS, or MTCS.

[0080] In some embodiments, the truncated ADAMTS13-MDTCS retains crucial domains for in vivo efficacy: the MP, Dis, TSP1 repeat, Cys-rich and Spacer domain. The MDTCS variant of ADAMTS13 is capable of cleaving murine VWF efficiently in vivo. Surprisingly, the polypeptide ensures the original protein ADAMTS13 biological activity of MDTCS, in turn significantly increasing the half-life of MDTCS.

[0081] In another preferred embodiment, the polypeptide of the invention preferably comprises only the MP domain that is responsible for the catalytical activity of ADAMTS13 (SEQ ID N°:23) or the smallest structure that is needed for the polypeptide to be active, the MP and Dis domain (SEQ ID N°:24) or the MP, Dis and TSP1 domain (SEQ ID N°:25) coupled to the target protein.

[0082] In an embodiment of the polypeptide as disclosed herein, the truncated ADAMTS13 has a sequence according to SEQ ID N°: 39, SEQ ID N°: 40, SEQ ID N°: 41, or SEQ ID N°: 42, wherein SEQ ID N°s: 39, 40, 41, and 42 are optionally preceded by an amino acid sequence AAGGI (SEQ ID N°: 78) corresponding to (part of) the N-terminus of the mature ADAMTS13 protein; or according to SEQ ID N° 77. In other embodiments, said truncated ADAMTS13 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID N°: 39, SEQ ID N°: 40, SEQ ID N°: 41 or SEQ ID N°: 42, wherein SEQ ID N°s 39, 40, 41, and 42 are optionally preceded by the AAGGI sequence (SEQ ID N°: 78), or to SEQ ID N°: 77.

[0083] In another embodiment, said truncated ADAMTS13 has a sequence that differs with maximally 60, more preferably maximally 55, even more preferably maximally 50, even more preferably maximally 45, even more preferably maximally 40, even more preferably maximally 35, even more preferably maximally 30, even more preferably maximally 25, even more preferably maximally 20, even more preferably maximally 10, even more preferably maximally 8, even more preferably maximally 5, even more preferably maximally 4, even more preferably maximally 3, even more preferably maximally 2 or even more preferably maximally 1 amino acid residues from one of the sequences SEQ ID N°: 39, SEQ ID N°: 40, SEQ ID N°: 41, SEQ ID N°: 42, again optionally preceded by the AAGGI sequence (SEQ ID N°: 78), or from SEQ ID N°: 77.

[0084] In an embodiment of the polypeptide, as disclosed herein, the truncated form of ADAMTS13 is not recognised by auto-antibodies against ADAMTS13 and / or said truncated ADAMTS13 comprises mutations in the amino acid sequence diminishing the recognition of autoantibodies.

[0085] ADAMTS13 mutants that evade recognition by autoantibodies are crucial in developing therapeutic strategies for patients with iTTP, where autoantibodies against ADAMTS13 contribute to the pathology. The mutants are designed to maintain or enhance the enzymatic activity of ADAMTS13 while avoiding binding by neutralizing autoantibodies. Several strategies have been explored to create such mutants.

[0086] In embodiments of the polypeptide as disclosed herein, said truncated ADAMTS13 comprises one or more added N-linked glycosylation sites, one or more shifted N-linked glycosylation sites and / or one or more non-alanine amino acids substituted to alanine.

[0087] Such amino acid mutations include but are not limited to substitution to alanine in exosite- 3 of the spacer domain (SEQ ID N°:28), or insertion or sift of a N-glycans in the spacer domain (SEQ ID N°:29).

[0088] The spacer domain of ADAMTS13 is often a target for autoantibodies in iTTP. Conservative mutations in 5 residues within the spacer domain create a so-called Gain-of-Function (GoF) variant that is resistant to the binding of pathogenic autoantibodies that develop in patients with iTTP. However, experiments revealed that this GoF variant of ADAMTS13 was still targeted by patient-derived autoantibodies and does not resist their inhibitory action. Several epitope mapping studies revealed that in the exosite-3, an epitope comprising residues R568 / F592 / R660 / Y661 / Y665 (RFRYY)) is commonly targeted in nearly 95% of patients with immune iTTP.

[0089] Alanine modifications lead to reductions in exosite-spacer autoantibody binding but also result in reductions in activity. However, the inventors observed that fusing an alanine mutant to a targeting agent significantly increases the proteolytic activity of said ADMST13 mutant without compromising the autoantibody evading mechanism. Preferably, the alanine mutant of truncated ADAMTS13 is a triple-alanine mutant that has substitutions F592A, Y661A, and Y665A, the so called RARAA ADAMTS13 mutant. In a preferred embodiment, the alanine mutant of ADAMTS13 has a sequence according to SEQ ID N°: 62. In other embodiments, said truncated ADAMTS13 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID N°: 62.

[0090] In some embodiments, the ADAMTS13 comprises one or more of the following substitutions R568A, F592A, R660A, Y661A, and Y665A.

[0091] In an embodiment of the polypeptide disclosed herein, a truncated form of ADAMTS13 with the triple-alanine mutations in the spacer domain is used as the catalytic domain protease in a polypeptide having a sequence according to SEQ ID N°:2 or SEQ ID N°: 79. Said polypeptide has stronger proteolytical activity compared to the MDTCS_RARAA mutant.

[0092] In an embodiment, the polypeptide as described herein comprises a truncated form of ADAMTS13 that is intentionally devoid of its distal C-terminal modules— namely the thrombospondin type-1 repeats (TSP1-2 through TSP1-8) and the tandem CUB1-2 domains— which have been mapped to the principal plasmin-cleavage sites. In particular, the truncated ADAMTS13 is only comprised of those domains that are crucial for ADAMTS13's function of cleaving von Willebrand Factor (VWF). Hence, in an embodiment, said truncated ADAMTS13 comprises at most the MDTCS domains or parts thereof, such as the MDTC or MDT domains, thereby ensuring that known plasmin-susceptible regions are omitted. As a result, this truncated ADAMTS13 exhibits resistance to proteolytic degradation by proteases, including but not limited to plasmin, most preferably this truncated ADAMTS13 exhibits resistance to proteolytic degradation by plasmin.

[0093] In some embodiments, the polypeptide is formed by fusing the fusion agent with ASAMTS13 with mutations in the different domains according to SEQ ID N°:4 or SEQ ID N°:5, resulting in a mutated form of the MDTCS sequence that is not recognised by autoantibodies.

[0094] In an embodiment of the polypeptide, as disclosed herein, said truncated ADAMTS13 comprises a wild-type ADAMTS13 sequence with one or more N-linked glycosylation sites added compared to said ADAMTS13 sequence and / or one or more existing N-linked glycosylation sites shifted compared to wild-type ADAMTS13, thereby shielding ADAMTS13 from auto-antibodies. N-glycan-mediated shielding of ADAMTS13 refers to the protective role played by N-glycans (glycan structures attached to asparagine residues) in the protein, which helps shield it from being targeted by autoantibodies. The shielding effect is attributed to the steric hindrance provided by the complex branched structures of N- glycans.

[0095] In a preferred embodiment of the polypeptide, N-glycans are introduced in and / or outside of the classic epitope residues R568, F592, R660, Y661 and / or Y665. These insertions reduce the reactivity of ADAMTS13 with autoantibodies, without losing proteolytic activity while retaining at least part of the VWF cleaving activity of ADAMTS13. For example, by insertion of a single N-glycan (NGLY) (K608N) in the spacer domain, the NGLY3 variant (NGLY3), interferes with the binding of pathogenic anti-spacer domain antibodies while conserving proteolytic activity toward VWF. In addition, NGLY3+7 (insertion of a N-glycan K608N and a NGLY shift from N667 to Y665) reduces both autoantibody binding and preserves the activity of the protein. In a preferred embodiment, the alanine mutant of ADAMTS13 has a sequence according to SEQ ID N°:63 or SEQ ID N°:64. In other embodiments, said truncated ADAMTS13 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID N°:63 or SEQ ID N°:64.

[0096] In an embodiment of the polypeptide, the truncated form of ADAMTS13 does not contain the 7 additional thrombospondins type-1 repeats (TSP2-TSP8) according to SEQ ID N°:30 to SEQ ID N°:36 and 2 CUB domains (CUB1 and CUB2) according to SEQ ID N°:37 and SEQ ID N°:38. The target protein that interacts with VWF takes over the function of the C- terminal TSP2-8 / CUB1-2 domains and therefore the proteolytical activity of truncated ADAMTS13 is not impaired.

[0097] In a preferred embodiment the truncated ADAMTS13 that is incorporated in the polypeptide as disclosed herein is depleted of the spacer (SEQ ID N°:27). In immune TTP, more than 90% of the autoantibodies are directed against the spacer domain.

[0098] Targeting agent

[0099] The targeting agent of the polypeptide disclosed herein is any ligand or binding molecule that specifically binds to VWF. Preferably, however, the targeting agent is a proteinaceous targeting agent. More preferably, the proteinaceous targeting agent is a part of the single amino acid chain of the polypeptide, which chain also comprises the truncated ADAMTS13- MDTCS.

[0100] A targeting agent "which binds" VWF, is an agent that binds the target with sufficient affinity such that the targeting agent is useful as a therapeutic agent in targeting a structure, e.g. an MVT, cell or tissue expressing or exposing the target, and does not significantly cross-react with other proteins or molecules. In such embodiments, the extent of binding of the targeting agent to a "non-target" molecule (e.g. protein) will be less than about 10% of the binding of the targeting agent to its particular target molecule as determined by flow cytometry analysis. With regard to the binding of a targeting agent to a target molecule, the term "specific binding" or "specifically binds to" or "binds a" or is "specific for" a particular target molecule or polypeptide, e.g. an epitope on a particular polypeptide target, means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labelled target. In one embodiment, the term "specific binding" refers to binding where a targeting agent binds to a particular target molecule, polypeptide or epitope on a particular polypeptide without substantially binding to any other molecule, polypeptide or epitope.

[0101] "Affinity" as used herein, refers to the strength of the interaction between the targeting agent of the polypeptide and its specific binding site in the VWF. Higher affinity indicates a stronger binding interaction, meaning the ligand is more likely to bind to the target molecule even at lower concentrations. Affinity is often quantified by the dissociation constant (Kd), with a lower Kd value representing higher affinity.

[0102] In an embodiment of the polypeptide as disclosed herein, the targeting agent binds to: a. an Al domain of VWF or a part of an Al domain of VWF; b. an A2 domain of VWF or a part of an A2 domain of VWF; c. an A3 domain of VWF or a part of an A3 domain of VWF; and / or d. a D4-CK domain of VWF or a part of a D4-CK domain of VWF.

[0103] The basic human VWF monomer is a 2050 amino acid protein. Every monomer contains a number of specific domains with a specific function including the D' / D3 domain, which binds to coagulation factor VIII, the Al domain, which inter alia binds to the platelet receptor GPIbo, the A2 domain with the binding and cleavage site of ADAMTS13, the A3 domain, which binds to collagen, and the C-terminal CK domains which contain a sequence (RGD) that is recognized by the activated ollb[33 receptor on platelets. Monomeric VWF dimerizes at the C-terminal CK domains and multimers assemble via the formation of N-terminal disulphide bonds between D3 domains. Multimeric VWF can be extremely large, more than 20000 kDa, and can consist of over 80 subunits of 250 kDa each. Sufficient levels of functional VWF are crucial for haemostasis, particularly in the rapid flow of the arterioles and at site of injury. VWF plays dual roles in clot formation. Firstly, VWF mediates the initiation and progression of the primary haemostatic plug, by acting as a molecular bridge between collagen of the exposed subendothelial matrix components and the platelet receptor GPIbo. This transient interaction with collagen-bound VWF is insufficient for platelet arrest, but it does slow down the platelets in the rapidly flowing blood, mediating the 'tethering and rolling' of platelets on the endothelial surface. The interaction via GPIbo triggers activation of the platelet ollb[33, o2Ibl and GPVI receptors, resulting in stable platelet adhesion. Secondly, VWF is essential for fibrin clot formation, as it assembles with procoagulant FVIII in the circulation, thereby protecting it from rapid clearance. By acting as its carrier protein, VWF also localises FVIII to the site of vascular injury, where FVIII is important to support secondary haemostasis after the formation of the initial platelet plug.

[0104] In an embodiment, the targeting agent binds the Al domain or a part thereof, facilitating the polypeptide's localization to areas where VWF interacts with platelet receptor GPIbo.

[0105] In another embodiment, the targeting agent binds the A2 domain or a part thereof ensuring the polypeptide targets the cleavage site for ADAMTS13, enhancing the precision of VWF cleavage at the site of thrombus. Preferably, ADAMTS13 proteolytically cleaves VWF in the A2 domain at position Tyrl605-Metl606.

[0106] Alternatively, the targeting agent binds to the D4-CK domain or a part thereof, which is involved in VWF multimerization and interaction with the platelet ollb[33 receptor.

[0107] In yet another embodiment, the targeting agent binds to the A3 domain or a part thereof directing the polypeptide to regions where VWF interacts with collagen, crucial for stable platelet adhesion.

[0108] By targeting specific domains of VWF, the polypeptide ensures localized action, reducing systemic side effects. The high association rates between the polypeptide and VWF at the site of thrombus formation enhance the therapeutic efficacy of the polypeptide. Additionally, by incorporating the targeting mechanism during VWF production, the need for post-production manipulations is reduced, preserving the functional integrity of VWF.

[0109] VWF can exist in different configurations depending on the physiological conditions and mechanical forces it encounters. In its globular or unactivated conformation, VWF circulates in the bloodstream as a compact, spherical structure, with key binding domains concealed, preventing premature platelet adhesion and thrombus formation. Upon exposure to high shear stress or vascular injury, VWF transitions to an unfolded or activated conformation. In this elongated state, previously hidden binding sites, such as the Al and A2 domains, become exposed, facilitating interactions with platelets and the subendothelial matrix. This conformational change is crucial for the initiation and propagation of thrombus formation at sites of vascular damage. In an embodiment of the polypeptide, as disclosed herein, said targeting agent has affinity towards an unfolded or activated conformation of VWF. The targeting agent preferably binds more strongly to the unfolded or activated conformation of VWF compared to the globular, circulating, or unactivated conformation. Higher affinity for the unfolded or activated VWF ensures that the targeting agent selectively interacts with VWF at sites of vascular injury, enhancing the therapeutic efficacy of the polypeptide.

[0110] In alternative embodiments, the targeting agent of the polypeptide of the invention has affinity towards any and all forms, conformation, domains and epitopes of VWF. The targeting agent thus specifically binds to at least one of the unfolded or activated conformation of VWF and the globular, circulating or unactivated conformation of VWF. In one embodiment, the targeting agent that binds at least unfolded VWF preferentially binds unfolded VWF over globular VWF, more preferably, said targeting agent binds to unfolded or activated conformation of VWF and does not bind to circulating or unactivated globular forms of VWF. The targeting agent ensures that the therapeutic polypeptide selectively acts at sites where VWF is active in platelet adhesion and aggregation.

[0111] In some embodiments, the targeting agent of the polypeptide disclosed herein, specifically binds to at least one of the Al domain, the Al domain of activated VWF, the VWF A2 domain, the A2 domain of activated VWF, the VWF A3 domain, the VWF A3 domain of activated VWF, the D4-CK domain, the D4-CK domain of activated VWF, the D4-CK domain, the D4-CKdomain of activated VWF the VWF D3 domain, the VWF D3 domain, the VWF D3 domain of activated VWF, and / or fragments thereof.

[0112] Preferably, the binding of said targeting agent to any of the VWF domains disclosed herein does not interfere with the proteolytic activity of the truncated ADAMTS13. The polypeptide retains its proteolytic activity while not being recognised by autoantibodies and binding to the VWF site.

[0113] In an embodiment of the polypeptide, as disclosed herein, the polypeptide comprises a plurality of targeting agents such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 targeting agents. In preferred embodiments, said targeting agent is of a proteinaceous nature.

[0114] In an embodiment of the polypeptide as disclosed herein, said targeting agent comprises an antibody variable domain and / or a protein binding domain.

[0115] A preferred antibody variable domain that is present as a targeting agent in a polypeptide of the invention is a VHH or fragment thereof, more preferably the antibody variable domain is a humanized VHH. Suitable examples of targeting agents that bind VWF are the VHH camelid antibody fragments, more specifically VHH directed against the Al domain of activated VWF. In an embodiment of the polypeptide, as disclosed herein, the targeting agent comprises at least an antibody variable domain that specifically binds to a domain of VWF, and / or a binding domain from a protein that naturally binds VWF.

[0116] The term 'protein that naturally binds VWF' refers in the present invention to any protein that inherently interacts with von Willebrand factor (VWF) under physiological conditions.

[0117] The VWF site where the targeting agent targets the polypeptide of the invention can in principle be any site of VWF in close proximity to the A2 domain (proteolytical site ADAMTS13). However, the polypeptides of the invention are also aimed at clearing the site of MVT while leaving physiological haemostasis at sites of vascular injury unaffected. Preferably, therefore, the function of the targeting agent is not limited to exclusively bringing the truncated ADAMTS13 to VWF but can also interfere with the binding of VWF to platelets.

[0118] The binding site may be identified using any method known in the art. A non-limiting method employs Hydrogen-Deuterium Exchange (HDX) mass spectrometry to investigate the interaction of Syn-VWFAl (SEQ ID N°:2 or SEQ ID N°:79) with the Al domain of VWF (SEQ ID N°: 14). It was observed that Syn-VWFAl increases deuterium uptake in the VWF segment 1261-1472, indicating that Syn-VWFAl enhances overall HDX uptake and induces conformational changes in VWF. A small protected region (1288-1293) was identified, suggesting this is the likely nanobody binding site. Additionally, enhanced exposure in the o2 and o3 helices at early time points suggests localized conformational changes, which, despite their modest scale, may have significant functional consequences.

[0119] In an embodiment, the VHH specifically binds a domain in VWF in the proximity of the ADAMTS13 proteolytic site. Any suitable examples of VHHs that bind VWF known in the art, may be used with the polypeptide as disclosed herein, as targeting agents. Non-limiting examples include AU / VWFa-11 according to SEQ ID N°: l or Syn-VWFAl according to SEQ ID N°:2 or SEQ ID N°:79; the latter being a humanized form of Syn-VWFAl according to SEQ ID N°:2.

[0120] In an embodiment, the VHH that binds to the Al domain of VWF that is present as a targeting agent in the polypeptide disclosed herein, has an amino acid sequence with at least 90% sequence identity to SEQ ID N°: l or SEQ ID N°:2 or SEQ ID N°:79. In other embodiments, said VHH had at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID N°: 1 or SEQ ID N°: 2 or SEQ ID N°:79. In another embodiment, said VHH has a sequence that differs with maximally 15, more preferably maximally 14, even more preferably maximally 13, even more preferably maximally 12, even more preferably maximally 11, even more preferably maximally 10, even more preferably maximally 9, even more preferably maximally 8, even more preferably maximally 7, even more preferably maximally 6, even more preferably maximally, even more preferably maximally 4, even more preferably maximally 2, or even more preferably maximally 1 amino acid residues from one of the sequences SEQ ID N°: 1 or SEQ ID N°:2 or SEQ ID N°:79.

[0121] The VHH according to SEQ ID N°:2 or SEQ ID N°:79, has unique characteristics, more specifically said VHH is a blocking antibody and i) recognizes VWF in a GPIb-binding conformation, ii) inhibits ristocetin-induced platelet-VWF binding and aggregation, iii) does not inhibit platelet collagen interaction via VWF nor inhibits platelet binding to immobilized collagen, and iv) inhibits platelet string length and platelet string number on stimulated endothelial cells. The present inventors have found that SEQ ID N°:2 and SEQ ID N°:79 dose-dependently block platelet-binding to activated VWF in solution, but not to (collagen- ) immobilized active VWF. This proves that SEQ ID N°:2 and SEQ ID N°:79 have the potential to prevent pathological platelet-VWF aggregate formation while leaving physiological haemostasis at sites of vascular injury unaffected.

[0122] Alternatively, the VHH of the polypeptide disclosed herein, binds to the Al domain of VWF and has a sequence according to or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID N°:43, SEQ ID N°:44, SEQ ID N°:45, SEQ ID N°:46, SEQ ID N°:47, SEQ ID N°:48, SEQ ID N°:49, SEQ ID N°:50, or ID N°:51.

[0123] In yet another alternative embodiment, the VHH binds to the A3 domain of VWF and has a sequence according to or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID N°:52, SEQ ID N°:53, SEQ ID N°:54, SEQ ID N°:55, SEQ ID N°:56, SEQ ID N°:57, or SEQ ID N°:58.

[0124] In an alternative embodiment, the VHH binds specifically to the Al domain of the active conformation of VWF, rather than binding to freely circulating unactivated VWF. In such an embodiment, the VHH has a sequence according to or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID N°:2, SEQ ID N°:79, SEQ ID N°:59, SEQ ID N°:60, or SEQ ID N°:61.

[0125] The synergy between truncated ADAMTS13 and VHH in the polypeptide enhances the treatment of thrombotic conditions by combining targeted delivery with potent enzymatic activity. The VHH specifically binds to von Willebrand factor (VWF) at the thrombus site, directing the truncated ADAMTS13 precisely where it is needed, which increases therapeutic efficacy and minimizes off-target effects. This targeted approach ensures efficient proteolytic degradation of VWF, leading to more effective thrombus dissolution. Additionally, the polypeptide design minimizes recognition by auto-antibodies, reducing immunogenicity. The VHH component also enhances the stability and circulation time of the truncated ADAMTS13, ensuring prolonged therapeutic action. The high binding affinity of the VHH ensures robust localization at the thrombus site, maximizing the therapeutic effect. This combination leads to a synergistic reduction in thrombus size and stability while minimizing systemic side effects. Furthermore, the ability to include multiple VHH targeting agents allows for a multi-target approach, enhancing the efficacy of thrombus targeting and degradation. These synergistic effects make the polypeptide a highly effective and targeted treatment option for thrombotic conditions.

[0126] In addition to mediating proteolytic cleavage of VWF, the polypeptide described herein also inhibits in some embodiments the formation of platelet agglutination in solution. This fusion protein therefore achieves dual targeting of VWF: the ADAMTS13 moiety, engineered to evade recognition by autoantibodies, ensures effective cleavage of VWF, while the VHH component prevents VWF from binding to the platelet receptor GPIbo. This dual mechanism of action results in potent antithrombotic activity. Moreover, the targeting of VWF at the thrombus site by the fusion construct is more efficient and effective than that achieved by the ADAMTS13 mutant and optionally also by the VHH component alone, resulting in enhanced therapeutic efficacy through their synergistic combination.

[0127] The polypeptide preferably comprises a linker that links the truncated form of ADAMTS13 and the targeting agent. The term "linker" refers to a molecule or set of molecules that connects two or more other molecules together. The linker serves as a bridge connecting these two functional domains. It may relate to a sequence of amino acids or other molecular structures that allow for the spatial arrangement of the two domains in a manner that optimizes their synergistic interaction. The length of the linker may vary depending on the specific requirements of the therapeutic application. It is preferably between 5 and 50 amino acids long, more preferably between 15 and 45 amino acids, even more preferably between 10 and 40 amino acids, and most preferably between 6 and 10 amino acids. The specific sequence of the linker may also be tailored to optimize the stability, solubility, and therapeutic efficacy of the polypeptide. Any linker known in the art may be used with the polypeptide disclosed herein. Non-limiting examples include linkers having sequences according to SEQ ID N°:65 to SEQ ID N°:75.

[0128] Flexible linkers are usually applied when the joined domains require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in an aqueous solution by forming hydrogen bonds with the water molecules and therefore reduces the unfavourable interaction between the linker and the protein moieties. Preferred flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues ("GS" linker). An example of a preferred flexible linker is SSSGSS (SEQ ID N° : 73). Another example of a preferred (and widely used) flexible linker has the sequence of (GGGS)n (SEQ ID N° :75). By adjusting the copy number "n", the length of this GS linker can be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions. Besides the GS linkers, many other flexible linkers have been designed for recombinant polypeptides. These flexible linkers are also rich in small or polar amino acids such as Gly and Ser, but can contain additional amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility, such as e.g. the flexible linkers KESGSVSSEQLAQFRSLD (SEQ ID N° : 67:) and EGKSSGSGSESKST (SEQ ID N° : 68), that have been applied for the construction of a bioactive scFv's.

[0129] In an embodiment, the polypeptide as disclosed herein comprises in a N- to C-terminal order a truncated ADAMTS13 as defined in any of the previous embodiments, optionally a linker amino acid sequence, and a targeting agent as disclosed in any of the previous embodiments.

[0130] Alternatively, the polypeptide disclosed herein comprises in an N- to C-terminal order a targeting agent as disclosed in any of the previous embodiments, optionally a linker amino acid sequence, and a truncated ADAMTS13 as disclosed in any of the previous embodiments.

[0131] Any sequence order in the polypeptide, type of VHH, or truncated ADAMTS13 may be utilized without departing from the scope of the invention.

[0132] Non-limiting examples of the sequence order and type of VHH and truncated ADAMTS13 are provided in Table 1.

[0133] Table 1. Examples of VHH types, linkers, and truncated ADAMTS13 domains, along with their potential sequence order in the polypeptide according to embodiments of the invention.

[0134] In an embodiment of the polypeptide as disclosed herein, said polypeptide exhibits thrombolytic activity. This fusion of a VHH with a truncated ADAMTS13 delivers a higher concentration of the enzyme to the site of a clot, leading to enhanced thrombolytic effectiveness. This thrombolytic activity of the polypeptide is indicative of the potential benefits it may offer in efficiently and specifically resolving blood clots within the body. Notably, this thrombolytic activity is not confined to a specific range but can be observed across a broad spectrum of physiological conditions. This broad-spectrum activity may enhance the versatility of the polypeptide as a therapeutic agent, as it may be effective in treating a variety of thrombotic conditions.

[0135] The therapeutic benefit arises from the synergy between the VHH and truncated ADAMTS13, which combines targeted delivery with potent enzymatic cleavage of VWF. The VHH specifically binds VWF at the thrombus, directing ADAMTS13 precisely to its substrate and minimising off-target effects. In embodiments, the VHH blocks the interaction between VWF and platelet GPIb, thereby inhibiting platelet aggregation in solution. This dual mode of action— proteolytic cleavage and platelet binding inhibition— results in robust antithrombotic activity. However, in embodiments, it is also contemplated that the targeting function of the VHH alone, without interfering with platelet binding, may be sufficient or even preferable in certain therapeutic contexts, for example to reduce the optional risk of bleeding.

[0136] The fusion protein is further optimised through design features that reduce recognition by autoantibodies, enhance plasma stability, and extend circulation time. The high binding affinity of the VHH ensures efficient localisation at the clot, while inclusion of multiple VHHs allows for multi-targeting strategies, further improving thrombus resolution. Together, these properties make the polypeptide a potent, targeted, and well-tolerated therapeutic candidate for thrombotic conditions.

[0137] Pharmaceutical compositions

[0138] The polypeptide as disclosed herein and described in the previous embodiments, may be included in a composition comprising a pharmaceutical-acceptable carrier, excipient or diluent.

[0139] A carrier is considered as being pharmaceutically acceptable, when it does not have any or not substantially adverse unwanted effects on the human or animal body, e.g. it is considered generally safe, nontoxic and / or does not cause unwanted biological side reactions. Suitable pharmaceutically acceptable carriers are well known to the person 1 skilled in the art. The choice of carrier may depend upon the route of administration and concentration of the polypeptide and the carrier may be in the form of a lyophilised composition or an aqueous solution. Generally, an appropriate amount of pharmaceutically acceptable salt is used in the carrier to render the composition isotonic. Examples of the carriers include but are not limited to saline, Ringer's solution and dextrose solution. Preferably, acceptable excipients, carriers, or stabilisers are non-toxic at the dosages and concentrations employed, including buffers such as citrate, phosphate, and other organic acids; salt-forming counter-ions, e.g. sodium and potassium; low molecular weight (> 10 amino acid residues) polypeptides; proteins, e.g. serum albumin, or gelatine; hydrophilic polymers, e.g. polyvinylpyrrolidone; amino acids such as histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates including glucose, mannose, or dextrins; monosaccharides; disaccharides; other sugars, e.g. sucrose, mannitol, trehalose or sorbitol; chelating agents, e.g. EDTA; non-ionic surfactants, e.g. Tween, Pluronics or polyethylene glycol; antioxidants including methionine, ascorbic acid and tocopherol; and / or preservatives, e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, e.g. methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol). The composition may also optionally include other components, such as buffering agents or stabilizing agents.

[0140] Preferably, the composition for parenteral administration is prepared in a sterile environment and is formulated as a solution, a suspension, an emulsion, a lyophilized powder for reconstitution, or other suitable forms for parenteral administration.

[0141] The composition comprising the polypeptide as disclosed herein is preferably formulated for parenteral administration. The administration of the peptide or composition via parenteral routes includes but is not limited to, intravenous, intramuscular, subcutaneous, or intraperitoneal routes. The parenteral administration of the composition allows for the rapid delivery of the therapeutic agent directly into the systemic circulation, bypassing the gastrointestinal tract and potential enzymatic degradation or alteration. This rapid delivery is especially advantageous in acute thrombotic events, where time is of the essence and rapid therapeutic action can improve patient outcomes significantly.

[0142] In an embodiment, the composition may be administered in a single dose or multiple doses depending on the severity of the disease or condition characterized by a thrombotic or embolic state. The dosage may be adjusted according to the weight, age, sex, and overall health condition of the subject. The exact dosage and frequency of administration may be determined by a healthcare provider based on clinical judgment and patient response to treatment. The content of the polypeptide in the pharmaceutical composition is not limited as far as it is useful for treatment, prevention, or amelioration but preferably contains 0.0000001 - 10% by weight per total composition.

[0143] Methods of treatment

[0144] The current disclosure relates to methods of treatment and prevention of various diseases using the polypeptide or a composition comprising the polypeptide of the invention, wherein the polypeptide comprises truncated ADAMTS13 and a targeting agent that specifically binds to VWF.

[0145] In an embodiment, the current disclosed related to the polypeptide or composition comprising said fusion peptide according to any of the previous embodiments, for use as a medicament. In an embodiment, the current disclosed related to the polypeptide or composition comprising said fusion peptide according to any of the previous embodiments, for use in the treatment, prevention or amelioration of microvascular thrombosis in a subject in need thereof.

[0146] The polypeptide or composition as disclosed herein, are used for the treatment, prevention or amelioration of microvascular thrombosis or the risk of microvascular thrombosis occurrence is reduced in a disease or condition selected from the group consisting of: acquired or hereditary thrombotic thrombocytopenic purpura (TTP), complement-mediated thrombotic microangiopathy, haemolytic uremic syndrome, antiphospholipid antibody syndrome, non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such as angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischemia, cardioembolism, peripheral vascular disease, restenosis, sickle cell disease and myocardial infract.

[0147] In an embodiment, the current disclosed related to the polypeptide or composition comprising said fusion peptide according to any of the previous embodiments, for use in the prevention and / or treatment of stroke in a subject in need thereof, preferably for use in the prevention and / or treatment of tissue plasminogen activator (tPA)-resistant stroke.

[0148] In another or further embodiment, the polypeptide or composition as disclosed herein is for use in emergency medicine, or wherein the disease or condition is an acute or chronic disease or condition. The polypeptide or pharmaceutical composition comprising the polypeptide as disclosed herein can be administered by any suitable route of administration including oral administration and parenteral administration such as intranasal, subcutaneous, intravenous, intraarterial, intracardial and intramuscular administration. Preferably, the polypeptide or pharmaceutical composition comprising the polypeptide as disclosed herein is administered parenterally. Therefore, in one embodiment, the polypeptide or pharmaceutical composition can be formulated for parenteral administration. In a preferred embodiment, the polypeptide or pharmaceutical composition is formulated for parenteral administration, in particular, selected from intravenous, intraarterial, intracardiac, intradermal, subcutaneous, intraembolic, intramucosal or intraarticular administration. For example, the polypeptide or pharmaceutical composition can be formulated for intravenous administration, such as intravenous infusion or injection.

[0149] In another preferred embodiment, the polypeptide or pharmaceutical composition is administered by parenteral administration, in particular, selected from intravenous, intraarterial, intracardiac, intradermal, subcutaneous, intraembolic, intramucosal or intraarticular administration. For example, the polypeptide or pharmaceutical composition is administered by intravenous administration, such as intravenous infusion or injection.

[0150] In a further embodiment the polypeptide or pharmaceutical composition as described herein is used in combination with a further thrombolytic agent. The further thrombolytic agent is preferably selected from a tPA protein, streptokinase, alteplase, reteplase, tenecteplase, urokinase, prourokinase, and anistreplase (APSAC). These thrombolytic agents are well-known in the art and are approved or used as thrombolytic agents. The further thrombolytic agent can be administered to a subject at a lower dose in the combination comprising the polypeptide of the invention and a second thrombolytic agent as compared to the administration of the second thrombolytic agent alone. For example, the dose may be lowered by 10%, 20%, 30%, 40%, 50% or 90%. For example, the further thrombolytic agent can be administered spatially separately with the polypeptide or pharmaceutical composition or together, such as a single composition, vial, container, kit or kit-of-parts. For example, the further thrombolytic agent can be administered temporally separately from the polypeptide or pharmaceutical composition, or at the same time point, and / or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 30 minutes or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24 or 48 hours.

[0151] The disclosure further relates to a method of treating, preventing or ameliorating a disease or condition characterized by a microvascular thrombosis in a subject in need thereof, comprising administering to said subject a pharmaceutically effective amount of the polypeptide or of the pharmaceutical composition. As used herein, the term "pharmaceutically effective amount" in the context of the administration of a therapy to a subject refers to the amount of a therapy that achieves a desired therapeutic, preventive / prophylactic or ameliorating effect.

[0152] A suitable amount and dosage can be determined by persons skilled in the art. For example, a polypeptide or pharmaceutical composition described herein may administered to a subject (e.g., via intravenous injection) at about 0.001 mg / kg, 0.01 mg / kg 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 6 mg / kg, or about 10 mg / kg.

[0153] Thereby, administering of the polypeptide or pharmaceutical composition of the present invention refers to any route of drug administration known to the person skilled in the art, such as parenteral, intravenous, intraperitoneal, subcutaneous, oral, intranasal or sublingual administration. Suitable dosage regimens are also well known to the person skilled in the art. Preferably, the polypeptide or pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount, i.e. in a dose or concentration causing a biological response in the body the polypeptide or pharmaceutical composition is administered to.

[0154] Alternatively, the polypeptide as disclosed herein may be used in diagnostic of thrombotic conditions.

[0155] Nucleic acids, vectors, host cells

[0156] The current disclosure also relates to nucleic acids encoding the polypeptide of the invention, to vectors comprising said nucleic acids and to host cells comprising the nucleic acids encoding the polypeptide of the invention or comprising the vectors comprising the nucleic acids encoding the polypeptide.

[0157] The term "nucleic acid" describes any form of deoxyribonucleic acid (DNA), ribonucleic acid (RIMA) or artificial nucleic acid known to the person skilled in the art.

[0158] In an embodiment, the disclosure relates to a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide as defined in any of the previous embodiments, wherein the nucleotide sequence encoding the polypeptide further preferably comprises a nucleotide sequence encoding a signal peptide operably linked to the polypeptide, and wherein nucleic acid molecule further preferably comprises regulatory elements leading to the expression of the polypeptide, which regulatory elements are operably linked to the nucleotide sequence.

[0159] Nucleotide sequences encoding the VHH, truncated ADAMTS13 or polypeptides disclosed herein, and modified versions of these are determined using methods well known in the art, i.e. nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the VHH, truncated ADAMTS13 or polypeptide. Such a polynucleotide encoding the VHH, truncated ADAMTS13 or polypeptide can be assembled from chemically synthesized oligonucleotides, which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the VHH, truncated ADAMTS13 or polypeptide, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR. A nucleic acid encoding the VHH, truncated ADAMTS13 or polypeptide can be chemically synthesized or obtained from a suitable source (e.g., cells selected to express the VHH, truncated ADAMTS13 or polypeptides described herein) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a DNA clone that encodes the VHH, truncated ADAMTS13 or polypeptide. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well-known in the art.

[0160] DNA coding for the VHH, truncated ADAMTS13 or polypeptide of the invention described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding to sequences encoding FR1 and FR4 regions. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells that do not otherwise produce the VHH, truncated ADAMTS13 or polypeptide, to obtain the synthesis of the VHH, truncated ADAMTS13 or polypeptides in the recombinant host cells.

[0161] The VHH, truncated ADAMTS13 or polypeptide are amplified using PCR primers. For example, PCR primers including VHH variable domain nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VHH sequences. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VHH domain, truncated ADAMTS13 domains or the entire polypeptide, can be cloned into vectors, which optionally express further domain(s) such as a purification tag, as in the examples, or a protein providing for an extended half-life in vivo. In certain embodiments, the expression vectors comprise a promoter, a secretion signal, a cloning site for the variable region, and a selection marker such as neomycin. The vectors are then transfected into cell lines to generate stable or transient cell lines that express the VHH, the truncated ADAMTS13 or polypeptide, using techniques known to those of skill in the art.

[0162] Site-directed or high-density mutagenesis of the variable region or other mutagenesis methods can be used to optimize the specificity, affinity, etc. of the VHH. In particular, affinity maturation strategies are known in the art and can be employed to generate high- affinity VHH. Preferably, the nucleic acid is part of a vector. Vectors are plasmids which are used to introduce a desired nucleic acid sequence, such as a gene, into a target cell, resulting in the transcription and translation of the protein encoded by the nucleic acid sequence, e.g. the VHH, the truncated ADAMTS13 or the polypeptide disclosed herein. Therefore, the expression vector in general comprises regulatory sequences, such as promoter and enhancer regions, as well as a polyadenylation site in order to direct efficient transcription of the nucleic acid sequence on the expression vector. The expression vector may further comprise additional necessary or useful regions, such as a selectable marker for selection in eukaryotic or prokaryotic cells, a purification tag for the purification of the resulting protein, a multiple cloning site or an origin of replication. The expression vector may be a viral or a non-viral vector. In general, various kinds of viral vectors, such as retroviral vectors, e.g. lentiviral or adenoviral vectors, or plasmids may be used. A vector comprising the nucleic acid encoding the VHH, truncated ADAMTS13 or polypeptide disclosed herein may further be introduced in a host cell.

[0163] Methods for introducing such a vector into a host cell are well known to the person skilled in the art, such as any known transfection method, e.g. any nonviral transfection method (e.g. chemical-based, non-chemical or particle-based) or any virus-based transfection method. Examples of suitable methods are transfection methods based on calcium phosphate precipitation, lipofection, cationic polymers, Fugene, Dendrimer, nanoparticles, microinjection, cell squeezing, electroporation, particle gun (also known as gene gun), magnet-assisted transfection, optical transfection, protoplast fusion, hydrodynamic delivery, sonoporation, transferrin-based infection, antibody-mediated transfection or virus-based transfection (e.g. based on adenoviral or lentiviral vectors).

[0164] Suitable host cells are well known to the person skilled in the art, such as mammalian cells (such as human, mouse, rat or hamster cells), insect cells, bacterial cells or yeast cells. Such host cell may comprise a nucleic acid of the present invention e.g. integrated in its genome or in a vector. Methods for introducing such nucleic acid in the host cell are described above and further well-known to the person skilled in the art.

[0165] However, the invention is not limited to this application. The proteins, compositions, and methods detailed herein are applicable across various fields where thrombus binding is relevant.

[0166] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.

[0167] EXAMPLES Example 1

[0168] Materials and methods

[0169] Production of the VHH Syn-VWFAl

[0170] HEK293E-253 cells were transfected with endotoxin-free maxiprep DNA for the desired sequence of the anti-VWF VHH. Six days post-transfection conditioned medium containing recombinant protein was harvested by low-speed centrifugation (10 minutes, 1000 g) followed by high-speed centrifugation (10 minutes, 4000g) and immobilized metal affinity chromatography (IMAC) purification. The VHH was further purified by gel filtration using a Superdex75 26 / 600 column. The resulting VHH-containing fractions were sterilized by filtration over a 0.22 pm syringe filter and stored at 4°C.

[0171] Immunosorbent assay to assess the specificity of Syn-VWFAl for VWF

[0172] Overnight at 4°C, 96 wells microtiter plates (Nunc Maxisorp, Sigma-Aldrich) were coated with 121 nM Syn-VWFAl in 50 mM NaHCOs coating buffer (pH 9.6). After blocking with 2% BSA in phosphate-buffered saline (PBS) for 45 minutes at room temperature (RT), wells were washed with 0.1% Tween-20 in PBS. Normal pooled plasma (diluted 1 :25, static or vortexed at 2,500 rpm for 10 minutes), HVWF (final concentration 170 ng / ml), R1306W VWF or R1306Q VWF (final concentrations 1.7 pg / mL), with or without ristocetin (0.5 mg / mL), were incubated for 2 hours at RT. After washing three times with 0.1% Tween- 20 in PBS, wells were incubated with HRP-conjugated anti-VWF antibodies in PBS / 1%BSA for 2 hours at RT. Plates were washed three times before measuring VWF binding to Syn- VWFAl by adding HRP substrate SIGMAFAST™ OPD. After 8 minutes, the reaction was stopped with 3 M sulfuric acid (H2SO4, Aldrich, USA). Optical densities at 490 nm were measured using an ELx808 Absorbance Microplate Reader (Biotek, USA).

[0173] Flow cytometric analysis of platelet-VWF binding

[0174] Binding of VWF to platelets was tested by adding 5 pL of 1 :4 diluted whole blood to 20 pL reaction mix consisting of 2 pg / mL HVWF or 4 pg / mL R1306W VWF (with or without 0.5 mg / ml ristocetin), 2 pl FITC-conjugated anti-VWF antibody and 0.5 pl PE-conjugated anti- CD41a (directed against GPIIb and used as a platelet marker) in HEPES-buffered saline (HBS, 10 mmol / L HEPES, 150 mmol / L NaCI, 1 mmol / L MgSO4, 5 mmol / L KCL, pH 7.4). The effect of Syn-VWFAl on the binding of VWF to platelets was studied by adding 1.5 pM Syn- VWFAl to the reaction mix. Following incubation for 20 mins at RT, the reactions were stopped by adding 250 pl of fixation solution (137 mmol / L NaCI, 2.7 mmol / L KCI, 1.12 mmol / L NaH2PO4, 1.15 mmol / L KH2PO4, 10.2 mmol / L Na2HPO4, 4 mmol / L EDTA, 0.5% formaldehyde). Flow cytometric analysis was performed on a BD Accuri flow cytometer (BD Pharmingen). A gate was placed on the CD41a positive cells to select for platelets. Binding of VWF to platelets was detected by the median fluorescent intensity (MFI) in the FL1 channel.

[0175] Light transmission aggregometry

[0176] Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared from citrated whole blood by centrifugation at 156 g for 15 min (PRP) and two times at 2500 g for 15 min (PPP). Platelet count in PRP was adjusted with autologous (PPP) to 250 x 109 / L. Light transmission aggregometry (LTA) was performed on a Chronolog aggregometer (Chronolog Corporation, Havertown, PA, USA). Optimal platelet aggregation was defined as maximal light transmission obtained with autologous PPP. Increasing concentrations of the Syn-VWFAl were added to pre-warmed (37°C, 5 min) PRP and incubated for 20 minutes. Subsequently, platelet aggregation in PRP in response to ristocetin (final concentration 1.25 mg / mL) was measured in the absence and presence of Syn-VWFAl for 15 minutes. Similarly, platelet aggregation in PRP in response to ADP (final concentration 4 mM), collagen (final concentration 4 mg / mL) and R1306W VWF (final concentration 3.4 pg / mL) in the absence and presence of 1.5 pM of Syn-VWFAl was assessed. Data were analyzed using AGGRO / LINK software (Chrono-log). Aggregation was expressed as percentage (%) maximum aggregation compared to PPP, and the % inhibition of maximum platelet aggregation by the Syn-VWFAl was calculated

[0177] Platelet adhesion to collagen and VWF under shear

[0178] Microchannels of Vena8 Fluoro Biochip (Cellix Ltd.) were coated with 12 mL of fibrillar Horm collagen type I at a concentration of 200 mg / mL for 2 hours at RT in a humidified box, blocked with 20 mL of 10% BSA in HEPES (pH 7.45) for 15 min at RT, and washed with HEPES (pH 7.45) at 2000 s1for 1 min. Citrate-anticoagulated whole blood was incubated with 1.8 pM Syn-VWFAl (or HEPES) and DiCOe (1 mg / ml) for 20 min at RT, protected from light. Blood was injected via a Kima microfluidic pump (Cellix Ltd.) into the microchannels at a fluid shear stress of 1600 s-1within 4 hours of collection. After 4 min, non-bound platelets and other blood components were washed from the channels by infusing rinse buffer (HEPES pH 7.45, 2 mM CaCI?, 1 U / mL unfractionated heparin) for 4 min at 1600 s’x. Phase contrast and fluorescent images (green fluorescent protein [GFP]) captured from 10 arbitrarily chosen microscopic fields per flow experiment were recorded with an EVOS microscope (Advanced Microscopy Group, Bothell, WA), equipped with a 60x oil ultra- transparent objective (Olympus, Hamburg, Germany), and accompanying software. The area covered with platelets (% of total image) and the density of platelets in aggregates were analyzed with Imaged software. Binding of GPlba to collagen-bound VWF

[0179] To assess binding of GPlba to collagen-bound VWF, HVWF was pre-incubated with ristocetin (0.5 mg / mL) in the absence or presence of Syn-VWFAl (3 pM, "+Syn-VWFA1 pre-inc"), and added to collagen type III coated wells of a commercial VWF-collagen binding (VWF:CB) assay (Asserachrom VWF:CB, 00239, Stago, Leiden, the Netherlands). After 2 hours of incubation at RT, wells were washed 3 times and subsequently incubated with saline (negative control) or solutions of human recombinant GPlba (0.5 pg / mL) with Syn-VWFAl (3 pM, "+Syn-VWFA1") or saline ("-Syn-VWFAl" and "+Syn-VWFA1 pre-inc") in dilution buffer (PBS / 1% BSA), again for 2 hours at RT. Following another wash step, binding of GPlba was detected with sheep anti-human GPlba antibodies (2 pg / mL) and HRP-conjugated donkey anti-sheep IgG antibodies (diluted 100-fold), both incubated 1.5 hours at RT with a washing step in between. OPD was used as a substrate, as described above for the active VWF immunosorbent assay.

[0180] Beads on a string assay

[0181] Endothelial cells were obtained from human umbilical veins (HUVECs). The umbilical cords were first washed with phosphate buffer (140 mM NaCI, 0.4 mM KCI, 1.3 mM NaH2PO4, 1.0 mM Na2HPO4, 0.2% glucose, pH 7.4) and then infused with a collagenase solution (0.02%; Invitrogen Life Technologies, Carlsbad, CA). After a 30-minute incubation at room temperature, the cords were rinsed with 100 mL phosphate buffer. Eluates containing endothelial cells were centrifuged at 250g for 10 minutes. The cell pellets were resuspended in Medium 199 (Invitrogen Life Technologies) containing 20% heat- inactivated fetal calf serum and 0.2 mM L-glutamine. Cellix Vena8+ endothelial chips were coated with 1% gelatine. Unstimulated HUVECs (white bars) or HUVECs stimulated for 10 mins with 25 pM histamine (Sigma-Aldrich, St Louis, MO, black bars) were seeded at 7xl06cells / mL and incubated overnight at 37°C w / 5% CO2.

[0182] The next day, cells were washed with warmed HEPES buffer prior to flowing resuspended lyophilised platelets (2xl08cells / mL) in the presence of Syn-VWFAl (0, 0.5, 1, 5 pM final cone.) at 2.5 dyne / cm2.

[0183] Results

[0184] Syn-VWFAl specifically recognizes VWF in a GPIba-binding conformation

[0185] The specificity of Syn-VWFAl for the active conformation of VWF was assessed by studying the binding of Syn-VWFAl to HVWF, R1306W VWF, R1306Q VWF, and VWF in plasma with an immunosorbent assay (Figure 1). Syn-VWFAl could not interact with HVWF, however, when HVWF was preincubated with ristocetin, the signal increased 33-fold. Similarly, NPP incubated with Syn-VWFAl resulted in a weak signal, while the signal increased in the presence of ristocetin. In contrast, VWF harboring mutations in the Al domain (R1306W or R1306Q), generating constitutively active VWF, showed spontaneous strong interaction with Syn-VWFAl. This interaction was further augmented by treatment with ristocetin. Finally, NPP was vortexed to simulate high shear stress. As a result, Syn-VWFAl interacted with the shear-unfolded VWF in NPP. Treatment of shear-unfolded VWF in NPP with ristocetin did not further increase binding to Syn-VWFAl.

[0186] Syn-VWFAl inhibits ristocetin-induced platelet-VWF binding and aggregation

[0187] Since activated VWF binds to Gplbo on platelets, the next step was to investigate whether Syn-VWFAl could inhibit this interaction in a whole blood flow cytometric assay. Native VWF and HVWF were only able to bind to platelets after pre-incubation with ristocetin (Figure 2A, B). R1306W VWF spontaneously bound to platelets and this binding was enhanced two-fold upon preincubation with ristocetin (Figure 2C), corroborating our observations in the immunosorbent assay. Remarkably, Syn-VWFAl abrogated both the spontaneous binding of R1306W VWF as well as the ristocetin-induced binding of native VWF, HVWF and R1306W VWF to platelets (Figure 2A-C).

[0188] The inhibitory effect of Syn-VWFAl for ristocetin-induced VWF binding to platelets was confirmed with light transmission aggregometry (LTA) in platelet-rich plasma. Syn-VWFAl dose-dependently inhibited ristocetin-induced platelet agglutination (Figure 2D), while ADP- and collagen-induced platelet aggregation was not affected by 1.5 pM of Syn-VWFAl (Figure 2E). In addition, at this concentration, Syn-VWFAl abolished platelet agglutination induced by R1306W VWF (Figure 2E).

[0189] Syn-VWFAl does not inhibit platelet collagen interaction via VWF

[0190] The effect of Syn-VWFAl on the interaction of platelets with collagen via VWF under shear stress was studied in two different assays. In a perfusion assay, whole blood with DiOC6- labeled platelets was perfused over immobilized collagen type I at a share rate of 1600 s’x. The mean surface covered with platelets was 13.2%. Pre-incubation of the blood with Syn-VWFAl at a concentration of 1.8 pM did not affect adhesion of platelets (% surface area covered by platelets) to collagen type I (13%, Figure 3A, C, D). Moreover, the mean fluorescent intensity of the platelet aggregates was comparable in the absence (15.4 AU) and presence of Syn-VWFAl (15 AU) (Figure 3B-D).

[0191] The platelet collagen interaction under shear stress was tested using the PFA-200 analyzer. The aperture closure time for both collagen-epinephrine and collagen-ADP cartridges was measured. Whole blood was incubated with Syn-VWFAl at concentrations ranging from 762 nM up till 6.1 pM. Syn-VWFAl could only prolong the closure time at considerably higher concentrations (>3 pM) than those used in the static assays described above (Figure 3E).

[0192] Syn-VWFAl does not inhibit platelet binding to immobilized collagen

[0193] To investigate the reduced effect of Syn-VWFAl on platelet collagen binding in the perfusion system and PFA-200 assay, HVWF was activated by ristocetin, followed by binding to collagen type III pre-coated to a microtiter plate (Figure 3F). Under these static conditions, Syn-VWFAl did not affect binding of human GPIba to collagen-immobilized HVWF (Figure 3F). This lack of an effect of Syn-VWFAl was observed both when Syn- VWFAl was pre-incubated with ristocetin-activated HVWF before addition to collagen, as well as when Syn-VWFAl was added together with GPIba after incubation of ristocetin- activated HVWF with collagen.

[0194] Syn-VWFAl inhibits platelet string length and platelet string number on stimulated endothelial cells

[0195] Lyophilized platelets (2xl08cells / mL) were perfused over unstimulated HUVECs and HUVECS stimulated with histamine. For stimulated HUVECs, platelet string length and platelet string number was significantly higher compared to unstimulated HUVECs. Furthermore, Syn-VWFAl could dose-dependently decrease the platelet string length and platelet string number when lyophilized platelets were perfused over stimulated HUVECs indicating that Syn-VWFAl could inhibit binding of platelets to VWF released from endothelial cells (Figure 5).

[0196] Example 2

[0197] Materials and methods

[0198] Cleavage of purified VWF with recombinant ADAMTS13

[0199] HVWF was incubated at 37°C in buffer (10 mmol / L HEPES and 6.5 mmol / L BaCI2, 1.5M urea, pH7.4) with recombinant ADAMTS13 (8 nM) or with truncated ADAMTS13 MDTCS (wild-type, RFRYY), MDTCS-Syn-VWFAl (polypeptide SEQ ID N°:3), mutant MDTCS with RARAA mutation (MDTCS-RARAA) or MDTCS-RARAA-Syn-VWFAl (polypeptide SEQ ID N°:4) at 8 nM, 16 nM, 32 nM and 64 nM. After an incubation of 6 hours, the reactions were stopped with EDTA (final concentration 10 mmol / L). Samples were treated with anionic detergent and the VWF multimer pattern was evaluated by electrophoresis on 2% agarose gels using the semi-automated Hydrasys 2 instrument. After electrophoretic separation, VWF multimers were immunofixated with a polyclonal anti-VWF-IgG antibody and a peroxidase-conjugated anti-IgG antibody and incubated in peroxidase and chromogen substrate (TTF1 / TTF2). Gels were scanned with a Hydrasys2. Beads on a string assay

[0200] Endothelial cells were obtained from human umbilical veins (HUVECs). The umbilical cords were first washed with phosphate buffer (140 mM NaCI, 0.4 mM KCI, 1.3 mM NaH2PC>4, 1.0 mM NazHPC , 0.2% glucose, pH 7.4) and then infused with a collagenase solution (0.02%; Invitrogen Life Technologies, Carlsbad, CA). After a 30-minute incubation at room temperature, the cords were rinsed with 100 mL phosphate buffer. Eluates containing endothelial cells were centrifuged at 250g for 10 minutes. The cell pellets were resuspended in Medium 199 (Invitrogen Life Technologies) containing 20% heat- inactivated fetal calf serum and 0.2 mM L-glutamine. Cellix Vena8+ endothelial chips were coated with 1% gelatine. HUVECs were stimulated for 10 mins with 25 pM histamine (Sigma-Aldrich, St Louis, MO, black bars) were seeded at 7xl06cells / mL and incubated overnight at 37°C w / 5% CO2.

[0201] The next day, cells were washed with warmed HEPES buffer prior to flowing resuspended lyophilised platelets (2xl08cells / mL) in the presence of Syn-VWFAl, recombinant ADAMTS13, MDTCS, MDTCS-Syn-VWFAl, MDTCS-RARAA and MDTCS-RARAA-Syn-VWFAl at 2.5 dyne / cm2.

[0202] Flow cytometric analysis of platelet-VWF binding

[0203] Binding of VWF to platelets was tested by adding 5 pL of 1 :4 diluted whole blood to 20 pL reaction mix consisting of 0.5 mg / ml ristocetin, 2 pl FITC-conjugated anti-VWF antibody and 0.5 pl PE-conjugated anti-CD41a (directed against GPIIb and used as a platelet marker) in HEPES-buffered saline (HBS, 10 mmol / L HEPES, 150 mmol / L NaCI, 1 mmol / L MgSO4, 5 mmol / L KCL, pH 7.4). The effect of Syn-VWFAl, recombinant ADAMTS13, MDTCS, MDTCS-Syn-VWFAl (SEQ ID N° :3), MDTCS-RARAA and MDTCS-RARAA-Syn- VWFAl (SEQ ID N° :4) on the binding of VWF to platelets was studied. Following incubation for 20 mins at RT, the reactions were stopped by adding 250 pl of fixation solution (137 mmol / L NaCI, 2.7 mmol / L KCI, 1.12 mmol / L NaH2PO4, 1.15 mmol / L KH2PO4, 10.2 mmol / L Na2HPO4, 4 mmol / L EDTA, 0.5% formaldehyde). Flow cytometric analysis was performed on a BD Accuri flow cytometer (BD Pharmingen). A gate was placed on the CD41a positive cells to select for platelets. Binding of VWF to platelets was detected by the median fluorescent intensity (MFI) in the FL1 channel.

[0204] Light transmission aggregometry

[0205] Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared from citrated whole blood by centrifugation at 156 g for 15 min (PRP) and two times at 2500 g for 15 min (PPP). Platelet count in PRP was adjusted with autologous (PPP) to 250 x 109 / L. Light transmission aggregometry (LTA) was performed on a TA-8V aggregometer (Stago, France). Optimal platelet aggregation was defined as maximal light transmission obtained with autologous PPP. Increasing concentrations of Syn-VWFAl, recombinant ADAMTS13, MDTCS, MDTCS-Syn-VWFAl (SEQ ID N° :3), MDTCS-RARAA and MDTCS-RARAA-Syn- VWFA1 (SEQ ID N° :4) were added to pre-warmed (37°C, 5 min) PRP and incubated for 20 minutes. Subsequently, platelet aggregation in PRP in response to ristocetin (final concentration 1.25 mg / mL) was measured . Agglutination was expressed as percentage (%) maximum agglutination compared to PPP, and the % inhibition of maximum platelet aggregation was calculated.

[0206] FRETS-VWF73 assay

[0207] Recombinant ADAMTS13, MDTCS, MDTCS-Syn-VWFAl (SEQ ID N° :3), MDTCS-RARAA and MDTCS-RARAA-Syn-VWFAl (SEQ ID N° :4) all at 8nM, were diluted with reaction buffer (5 mM Bis-Tris, 25 mM CaCI2, 0.005%Tween 20, pH 6.0) in a 96 well white Nunc plate (ThermoFisher). Plasma samples of two iTTP patients, INH105 and INH106 from ECAT Foundation with functional inhibitory antibody titers of ± 5.0 U / mL and ± 10.0 U / mL, respectively were diluted 20-fold and co-incubated with all constructs at 8 nM and analyzed relative to absence of patient samples. FRETS-VWF73 substrate was added to initiate reactions and fluorescence was measured kinetically at 30°C. Cleavage efficiencies were determined by calculating the linear slope of time-course fluorescence data. All activities were normalized to recombinant ADAMTS13 (defined as 100% activity).

[0208] GST- VWF73 ADAMTS13 activity ELISA

[0209] White 96-well Nunc plates (ThermoFisher) were coated overnight at 4°C with 8 pg / ml GST- VWF73. The plates were then blocked with blocking buffer (PBS, 0.05%Tween 20, 2% BSA, pH 7.4) for 1 hour. ADAMTS13, MDTCS, MDTCS-Syn-VWFAl (SEQ ID N° :3), MDTCS- RARAA and MDTCS-RARAA-Syn-VWFAl (SEQ ID N° :4) were diluted in reaction buffer (lOmM HEPES, 6.5 nM BaCI2, pH 7.4) to final concentrations of 8 nM. The diluted enzymes were added to the plates and allowed to react for 30 minutes. Reactions were stopped by the addition of EDTA to a final concentration of 10 mmol / L. To detect cleavage, a monoclonal anti-human VWF-A2 capture antibody which binds exclusively to cleaved VWF73 and not to the intact VWF73 was added at 0.5 pg / mL and incubated for 2 hours. After washing, an HRP-conjugated rat anti-mouse IgG secondary antibody (1 : 1000 dilution) was added and incubated for an additional 2 hours. A chemiluminescent substrate was then added, and luminescence was measured using a microplate reader (Synergy Hl, BioTek, USA). All incubation steps were performed at room temperature on a plate shaker. Plates were washed three times with wash buffer between each step. Cleavage efficiency was assessed by calculating the linear slope of the concentration-dependent luminescence signal. Enzymatic activity was normalized to that of recombinant ADAMTS13, which was defined as 100% activity.

[0210] Results

[0211] Proteolytic activity of ADAMTS13 polypeptides

[0212] The proteolytic activity of two polypeptides (SEQ ID N°:3 and SEQ ID N°:4) was compared with the proteolytic activity of the MDTCS variants without the target protein and with recombinant ADAMTS13. Both MDTCS and MDTCS-Syn-VWFAl showed strong proteolytic activity comparable with recombinant ADAMTS13 (Figure 6, panel A). MDTCS-RARAA did not show a significant proteolytic activity up to concentrations 8 times higher compared to recombinant ADAMTS13 (Figure 6, panel B). However, for the polypeptide MDTCS-RARAA- Syn-VWFAl, a dose-dependent increase of the cleavage of VWF multimers was observed indicating that the target protein can increase the proteolytic activity of the ADAMTS13 construct (Figure 6, panel B).

[0213] Both FRETS-VWF73 and ELISA-VWF73 assays showed that MDTCS-Syn-VWFAl (SEQ ID N°:3) and MDTCS-RARAA-Syn-VWFAl (SEQ ID N°:4) exhibited higher proteolytic activity compared to ADAMTS13 (Figure 7A and 7B). MDTCS also demonstrated strong VWF73 cleavage activity, while MDTCS-RARAA showed significantly reduced activity.

[0214] ADAMTS13 polypeptides inhibit platelet string length and platelet string number on stimulated endothelial cells

[0215] Lyophilized platelets (2xl08cells / mL) were perfused over HUVECS stimulated with histamine. MDTCS was more potent compared to MDTCS_Syn-VWFAl in decreasing string length and platelet string number. MDTCS-RARAA lost activity compared to MDTCS, however, interestingly, the MDTCS-RARAA_Syn-VWFAl polypeptide was very potent in decreasing platelet string length as well as platelet string number (Figure 8 A and B)

[0216] Effect of ADAMTS13 polypeptides on VWF platelet interaction

[0217] Since activated VWF binds to Gplba on platelets, the next step was to investigate whether the polypeptides with as target protein Syn-WFAl could inhibit this interaction in a whole blood flow cytometric assay. Native VWF was only able to bind to platelets after preincubation with ristocetin (data not shown). Remarkably, the polypeptides (SEQ ID N°:3 and SEQ ID N°:4) with Syn-VWFAl as target protein abrogated the ristocetin-induced binding of native VWF, to platelets (Figure 9) and were more potent compared to the target protein Syn-VWFAl alone. Recombinant ADAMTS13, MDTCS and MDTCS-RARAA had no effect on ristocetin-induced binding to VWF. The ADAMTS13 polypeptides inhibit ristocetin-induced platelet agglutination

[0218] The inhibitory effect of the polypeptides for ristocetin-induced VWF binding to platelets was confirmed with light transmission aggregometry (LTA) in platelet-rich plasma. The polypeptides (SEQ ID N° :3 and SEQ ID N° :4) with Syn-VWFAl as target protein dose- dependently abrogated the ristocetin-induced platelet agglutination (FigurelO) and were more potent compared to the target protein Syn-VWFAl alone. Recombinant ADAMTS13, MDTCS and MDTCS-RARAA had no effect on ristocetin-induced binding to VWF (Figure 10).

[0219] Resistance of MDTCS-RARAA and MDTCS- RARAA-Syn-VWFAl to autoantibodies in iTTP patients

[0220] To investigate whether the mutations in the spacer domain in MDTCS-RARAA and MDTCS- RARAA-Syn-VWFAl result in resistance to ADAMTS13 autoantibodies, these proteins and ADAMTS13, MDTCS and MDTCS-Syn-VWFAl were incubated with iTTP plasma samples (INH 105 and INH 106) prior to activity testing in the FRETS-VWF73 assay. Additionally, the proteins were tested without pre-incubation (non-TTP condition). In the control condition, ADAMTS13, MDTCS, MDTCS-Syn-VWFAl, and MDTCS- RARAA-Syn-VWFAl were active, while MDTCS-RARAA showed limited activity. Incubation with iTTP plasma resulted in a significant decrease in the activity of ADAMTS13, MDTCS, and MDTCS-Syn-VWFAl, with approximately 40% remaining activity. In contrast, MDTCS-RARAA and and MDTCS- RARAA-Syn-VWFAl maintained 70-90% of their activity (Figure 7).

[0221] Example 3

[0222] Materials and methods

[0223] FRETS- VWF73 assay

[0224] Recombinant ADAMTS13, MDTCS-RARAA-Syn-VWFAl (SEQ ID N° :4) and MDTCS-RARAA- L-Syn-VWFAl all at 8nM, were diluted with reaction buffer (5 mM Bis-Tris, 25 mM CaCI2, 0.005%Tween 20, pH 6.0) in a 96 well white Nunc plate (ThermoFisher). FRETS-VWF73 substrate was added to initiate reactions and fluorescence was measured kinetically at 30°C. Cleavage efficiencies were determined by calculating the linear slope of time-course fluorescence data. All activities were normalized to recombinant ADAMTS13 (defined as 100% activity).

[0225] GST-VWF73 ADAMTS13 activity ELISA

[0226] White 96-well Nunc plates (ThermoFisher) were coated overnight at 4°C with 8 pg / ml GST- VWF73. The plates were then blocked with blocking buffer (PBS, 0.05%Tween 20, 2% BSA, pH 7.4) for 1 hour. ADAMTS13, MDTCS-RARAA-Syn-VWFAl (SEQ ID N° :4) and MDTCS- RARAA-L-Syn-VWFAl were diluted in reaction buffer (lOmM HEPES, 6.5 nM BaCI2, pH 7.4) to final concentrations ranging from 0.25 to 8 nM. The diluted enzymes were added to the plates and allowed to react for 30 minutes. Reactions were stopped by the addition of EDTA to a final concentration of 10 mmol / L. To detect cleavage, a monoclonal anti-human VWF- A2 capture antibody which binds exclusively to cleaved VWF73 and not to the intact VWF73 was added at 0.5 pg / mL and incubated for 2 hours. After washing, an HRP-conjugated rat anti-mouse IgG secondary antibody (1 : 1000 dilution) was added and incubated for an additional 2 hours. A chemiluminescent substrate was then added, and luminescence was measured using a microplate reader (Synergy Hl, BioTek, USA). All incubation steps were performed at room temperature on a plate shaker. Plates were washed three times with wash buffer between each step. Cleavage efficiency was assessed by calculating the linear slope of the concentration-dependent luminescence signal. Enzymatic activity was normalized to that of recombinant ADAMTS13, which was defined as 100% activity.

[0227] Results

[0228] Proteolytic activity of MDTCS-RARAA-L-Syn-VWFAl

[0229] In the FRET-VWF73 assay, the fusion proteins exhibited approximately double the proteolytic activity of full-length rADAMTS13, while the longer linker in the fusion protein MDTCS-RARAA-L-Syn-VWFAl resulted in a 1.3 fold higher activity compared to MDTCS- RARAA-Syn-VWFAl (Figure 12).

[0230] In the GST-VWF73-based ADAMTS13 activity assay, MDTCS-RARAA-L-Syn-VWFAl reached its maximal cleavage activity at a lower concentration compared to both rADAMTS13 and MDTCS-RARAA-Syn-VWFAl (Figure 13A). Upon normalization to rADAMTS13, MDTCS- RARAA-L-Syn-VWFAl exhibited significantly enhanced activity, reaching 477%, whereas MDTCS-RARAA-Syn-VWFAl demonstrated 246% relative activity (Figure 13B).

[0231] In the multimeric assay, MDTCS-RARAA-Syn-VWFAl and MDTCS-RARAA-L-Syn-VWFAl were evaluated across a concentration range, whereas rADAMTS13 was assessed only at 8 nM. At this concentration, rADAMTS13 displayed higher cleavage activity than both fusion proteins. However, upon increasing the concentration of the fusion proteins up to 64 nM, their cleavage efficiency became comparable to that of rADAMTS13 (Figure 14A) In the figure, RARAA-VHH relates to MDTCS-RARAA-Syn-VWFAl and RARAA-L-VHH relates to MDTCS-RARAA-L-Syn-VWFAl . The percentage of low molecular weight multimers formed increased from 18% to approximately 90% (Figure 14B).

[0232] Inhibit platelet string length and platelet string number on stimulated endothelial cells

[0233] The beads-on-a-string assay was used to evaluate the dual-action mechanism of the fusion proteins. Compared to the control group, treatment with Syn-VWFAl, MDTCS-RARAA-Syn- VWFA1, MDTCS-RARAA-L-Syn-VWFAl and rADAMTS13 significantly reduced platelet string length (Figure 15). Additionally, rADAMTS13, MDTCS-RARAA-Syn-VWFAl and MDTCS- RARAA-L-Syn-VWFAl also decreased the average number of platelet strings (Figure 15).

[0234] Inhibition of binding of VWF to platelets

[0235] To evaluate the inhibitory potential of Syn-VWFAl, MDTCS-RARAA-Syn-VWFAl and MDTCS-RARAA-L-Syn-VWFAl on platelet- VWF interaction, we performed a whole blood flow cytometric assay. Syn-VWFAl, MDTCS-RARAA-Syn-VWFAl and MDTCS-RARAA-L- Syn-VWFAl each inhibited ristocetin-induced (0.5 mg / mL) VWF binding to platelets in a dose-dependent manner (Figure 16). Notably, the fusion proteins MDTCS-RARAA-Syn- VWFAl and MDTCS-RARAA-L-Syn-VWFAl exhibited markedly stronger inhibitory effects than Syn-VWFAl alone.

[0236] Inhibition of platelets agglutination

[0237] The inhibitory effect of Syn-VWFAl, MDTCS-RARAA-Syn-VWFAl and MDTCS-RARAA-L- Syn-VWFAl on ristocetin-induced platelet agglutination was assessed using LTA in PRP. All three proteins inhibited ristocetin-induced platelet agglutination in a dose-dependent manner (Figure 17). Moreover, the fusion proteins MDTCS-RARAA-Syn-VWFAl and MDTCS-RARAA-L-Syn-VWFAl demonstrated stronger inhibitory activity compared to Syn- VWFAl alone.

[0238] Example 4: ADAMTS13 fusion proteins exhibit increased resistance to plasmin-mediated proteolysis compared to native ADAMTS13

[0239] Introduction rADAMTS13 is approved for the treatment of congenital TTP and has shown therapeutic potential in preclinical models of ischemic stroke, [1, 2] myocardial infarction, [3] and other conditions, [4-9] without increased bleeding risk. However, its clinical efficacy is limited, not only due to autoantibody neutralization but also to proteolytic degradation by plasma proteases such as thrombin, plasmin, and factor Xia. These proteases predominantly target the flexible linker regions T4 (between TSP4 and TSP5) and T8 (between TSP8 and CUB1), leading to impaired ADAMTS13 stability and reduced proteolytic activity against VWF. Engineering of protease-resistant variants, including T4L / T8L-ADAMTS13 and T4L / T8L-ADAMTS13(I380G), has improved stability without compromising function

[0010] . We hypothesize that the ADAMTS13 fusion proteins that replace the TSP2-TSP8 and CUB1- 2 domains with oVWF offer similar protection against proteolysis by plasmin, while preserving therapeutic efficacy. Method

[0240] Each fusion protein according to embodiments of the current invention (MDTCS-Syn- VWFA1, MDTCS-RARAA-Syn-VWFAl, and MDTCS-RARAA-L-Syn-VWFAl) or rADAMTS13 (400 nM) were incubated with / without human recombinant plasmin (1 nM or 10 nM) in reaction buffer (Tris-HCI [pH 7.4], 150 mM NaCI, 5 mM CaCI?, 10 pM ZnCI?, 0.005% Tween 20, pH 7.4) at 37°C for 1 hour. Reactions were stopped with SDS loading dye containing 50 mM DTT and incubated in metal bath for 5 minutes. The indicated amounts of proteins were mixed 3: 1 with 10 pL of 4xLaemmli buffer (Bio-Rad Laboratories, Veenendal, the Netherlands). Samples were incubated for 5 min at 90°C before being separated on a 4- 20% Stain-Free Protein Gel (Bio-Rad). A protein marker (Bio-Rad) with a known molecular weight was run with the samples in the same gel. The run time was approximately 35 min at 200 volts. Gels were analyzed with the Gel Doc EZ gel Imager (Bio-Rad).

[0241] Results

[0242] ADAMTS13 fusion proteins exhibit increased resistance to plasmin-mediated proteolysis compared to native ADAMTS 13

[0243] To assess plasmin-mediated degradation, rADAMTS13 and three ADAMTS13 fusion proteins were incubated with increasing concentrations of plasmin. As expected, after 1 hour incubation with plasmin, rADAMTS13 showed clear dose-dependent degradation, with partial cleavage observed at 1 nM plasmin and near complete breakdown at 10 nM. In contrast, the fusion proteins (MDTCS-Syn-VWFAl, MDTCS-RARAA-Syn-VWFAl, and MDTCS-RARAA-L-Syn-VWFAl) showed only limited degradation under the same conditions, indicating substantially improved stability (Figure 18).

[0244] References

[0245] 1. Denorme, F., et al., ADAMTS13-mediated thrombolysis of t- PA- resista nt occlusions in ischemic stroke in mice. Blood, 2016. 127(19): p. 2337-45.

[0246] 2. South, K., et al., Enhanced activity of an ADAMTS-13 variant (R568K / F592Y / R660K / Y661F / Y665F) against platelet agglutination in vitro and in a murine model of acute ischemic stroke. J Thromb Haemost, 2018. 16(11): p. 2289- 2299.

[0247] 3. De Meyer, S.F., et al., Protective anti-inflammatory effect of ADAMTS13 on myocardial ischemia / reperfusion injury in mice. Blood, 2012. 120(26): p. 5217-23.

[0248] 4. Kleinveld, D.J.B., et al., Plasma and rhADAMTS13 reduce trauma-induced organ failure by restoring the ADAMTS13-VWF axis. Blood Adv, 2021. 5(17): p. 3478- 3491. 5. Erpenbeck, L., et al., ADAMTS13 Endopeptidase Protects against Vascular Endothelial Growth Factor Inhibitor-Induced Thrombotic Microangiopathy. J Am Soc Nephrol, 2016. 27(1): p. 120-31.

[0249] 6. Johnston, I., et al., Recognition of PF4-VWF complexes by heparin-induced thrombocytopenia antibodies contributes to thrombus propagation. Blood, 2020. 135(15) : p. 1270-1280.

[0250] 7. Wong, S.L., et al., Recombinant human ADAMTS13 treatment and anti-NET strategies enhance skin allograft survival in mice. Am J Transplant, 2020. 20(4) : p. 1162-1169.

[0251] 8. Zhou, S., et al., ADAMTS13 inhibits oxidative stress and ameliorates progressive chronic kidney disease following ischaemia / reperfusion injury. Acta Physiol (Oxf), 2021. 231(3) : p. el3586.

[0252] 9. Zitomersky, N.L., et al., ADAMTS13 Deficiency Worsens Colitis and Exogenous ADAMTS13 Administration Decreases Colitis Severity in Mice. TH Open, 2017. 1(1) : p. el l-e23.

[0253] 10. DeYoung, V., et al., Development of a protease-resistant ADAMTS13 to improve stability against proteolytic degradation. Blood Adv, 2025. 9(11) : p. 2695-2705.

[0254] SEQUENCES

[0255] Note: SEQ ID N° :3 to 10 are fused proteins with a linker sequence. In this sequence list, the linker is provided as SSSGSS, but in reality this linker can consist of any linker sequence from Table 1 or another linker known in the art.

[0256] SEQ ID N° : l AU / VWFa-11 (a VWFA1 domain-VHH according to an embodiment of the invention)

[0257] EVQLVESGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIGSDSSTLYTSSVRGRF

[0258] TISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSSGIYYREGSYAYWGQGTQVTVSS

[0259] SEQ ID N° :2 Syn-VWFAl (a VWFA1 domain-VHH according to an embodiment of the invention)

[0260] AVQLVESGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIGSDSSTLYTSSVRGRF

[0261] TISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSSGIYYREGSYAYWGQGTQVTVSS

[0262] SEQ ID N° :3 MDTCS-Syn-VWFAl (MDTCS ADAMTS13 MDTCS domains fused with a VWFA1 domain-VHH)

[0263] AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNI

[0264] TANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLI

[0265] TEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSA GRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDP LDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVT RRRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHW GAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGR MDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGG RYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLT RPDITFTYFQPKPRQASSSGSSAVQLVESGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKER EFVAFIGSDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSSGIYYREGSY AYWGQGTQVTVSS

[0266] SEQ ID N° :4 MDTCS-RARAA-Syn-VWFAl (ADAMTS13 MDTCS domains with triple-alanine mutations in the S region-RARAA, fused with a VWFA1 domain-VHH)

[0267] AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNI TANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLI TEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSA GRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDP LDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVT RRRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHW GAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGR MDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLATHLAVRIGG RYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRAGEEAGNLT RPDITFTYFQPKPRQASSSGSSAVQLVESGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKER EFVAFIGSDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSSGIYYREGSY AYWGQGTQVTVSS

[0268] SEQ ID N° : 5 Syn-VWFAl-MDTCS-RARAA mutant (VWFA1 domain-VHH fused with the ADAMTS13 MDTCS domains with triple-alanine mutations in the S regions- RARAA)

[0269] AVQLVESGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIGSDSSTLYTSSVRGRF TISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSSGIYYREGSYAYWGQGTQVTVSSSSSGSSA AGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNIT ANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLIT EDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAG RARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPL DQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTR RRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWG AAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRM DSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLATHLAVRIGGR YVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRAGEEAGNLTR PDITFTYFQPKPRQA SEQ ID N° : 6 MDTC-Syn-VWFAl (ADAMTS13 MDTC domains fused with a VWFA1 domain- VHH)

[0270] AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNI TANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLI TEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSA GRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDP LDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVT RRRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHW GAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGR MDSQQVWDRCQVCGGDNSTCSSSGSSAVQLVESGGRLVKAGASLRLSCAASGRTFSSLPMAWFR QAPGKEREFVAFIGSDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSSG IYYREGSYAYWGQGTQVTVSS

[0271] SEQ ID N° :7 Syn-VWFAl-MDTC (VWFA1 domain-VHH fused with the ADAMTS13 MDTC domains)

[0272] AVQLVESGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIGSDSSTLYTSSVRGRF TISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSSGIYYREGSYAYWGQGTQVTVSSSSSGSSL HLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTS SLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGF DLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARCV WDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSSC SRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQCN NPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVPHS QGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQV

[0273] WDRCQVCGGDNSTC

[0274] SEQ ID N° :8 MDT-Syn-VWFAl (ADAMTS13 MDT domains fused with a VWFA1 domain- VHH)

[0275] AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNI TANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLI TEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSA GRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDP LDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVT RRRQCNNPRPAFGGRACVGADLQAEMCNTQACESSSGSSAVQLVESGGRLVKAGASLRLSCAASG RTFSSLPMAWFRQAPGKEREFVAFIGSDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVY YCAARSSAFSSG IYYREGSYAYWGQGTQVTVSS

[0276] SEQ ID N° :9 MD-Syn-VWFAl (ADAMTS13 MD domains fused with a VWFA1 domain-VHH) AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNI TANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLI TEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSA GRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDP

[0277] LDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVSSSGSSAVQLVESGGRLVKAGASLR LSCAASGRTFSSLPMAWFRQAPGKEREFVAFIGSDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLK PEDTAVYYCAARSSAFSSGIYYREGSYAYWGQGTQVTVSS

[0278] SEQ ID N° : 10 M-Syn-VWFAl (ADAMTS13 M domain fused with a VWFA1 domain-VHH)

[0279] AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNI TANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLI TEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSA GRARCVWDPPSSSGSSAVQLVESGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAF

[0280] IGSDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSSGIYYREGSYAYWG QGTQVTVSS

[0281] SEQ ID N° : l l von Willebrand factor (VWF) full length from position 764 to 2813

[0282] SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPC FHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQ DYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSS

[0283] QCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGD CACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEP LACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDV VNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKA

[0284] FVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTL FQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPE NKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPDMAQVTVGPGLLGVSTLGPKRNSMVLDVAF VLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGDILQRVREI

[0285] RYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNA NVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFP ASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQI GDALGFAVRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQL

[0286] RILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTV TCQPDGQTLLKSHRVNCDRGLRPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKL TGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYV GGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDFMLRD

[0287] GTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHKVLAPATFYAICQQDS CHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCPPSLVYNHCEHGCPRHCDGNVSSCGDHPS EGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCPT AKAPTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHCERGLQPTLTNPGECRPNFTCACRKE ECKRVSPPSCPPHRLPTLRKTQCCDEYECACNCVNSTVSCPLGYLASTATNDCGCTTTTCLPDKVCV

[0288] HRSTIYPVGQFWEEGCDVCTCTDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPS

[0289] ACEVVTGSPRGDSQSSWKSVGSQWASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVCPSGFQL SCKTSACCPSCRCERMEACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLG YKEENNTGECCGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPF DEHKCLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSID

[0290] INDVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK

[0291] SEQ ID N° : 12 VWF TIL 3 domain from position 776 to 827

[0292] CPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERC

[0293] SEQ ID N° : 13 VWF D3 domain from position 865 to 1032

[0294] ATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEG GEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNF DGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCA

[0295] SEQ ID N° : 14 VWF Al domain from position 1277 to 1453

[0296] DLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRI ASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIV IPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEI

[0297] SEQ ID N° : 15 VWF A2 domain from position 1498 to 1665

[0298] DVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGDILQR VREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVG PNANVQELERIGWPNAPILIQDFETLPREAPDLV

[0299] SEQ ID N° : 16 VWF A3 domain from position 1691 to 1871

[0300] DVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVD VMQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPI GIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLHKL

[0301] SEQ ID N° : 17 VWF D4-CK domain from position 1948 to 2124

[0302] CVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVKHSALSV ELHSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKT YGLCGICDENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQ

[0303] SEQ ID N° : 18 VWF Cl domain from position 2255 to 2328 TQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCPTAKAPTCGLCEVARLRQNADQCC

[0304] PEYECVCD

[0305] SEQ ID N° : 19 VWF 02 domain from position 2429 to 2495

[0306] KVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGR CL

[0307] SEQ ID N° : 20 VWF C3 domain from position 2580 to 2645

[0308] EACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNTGECCGRCL

[0309] SEQ ID N° : 21 (VWF CTCK domain from position 2724 to 2812)

[0310] CNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSIDINDVQDQCSCCSPTRTEPMQVALHCT NGSVVYHEVLNAMECKCSPRKCS

[0311] SEQ ID N° : 22 ADAMTS13 full length from position 75 to 1427

[0312] AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNI

[0313] TANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLI TEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSA GRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDP LDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVT

[0314] RRRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHW

[0315] GAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGR MDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGG RYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLT RPDITFTYFQPKPRQAWVWAAVRGPCSVSCGAGLRWVNYSCLDQARKELVETVQCQGSQQPPAWP

[0316] EACVLEPCPPYWAVGDFGPCSASCGGGLRERPVRCVEAQGSLLKTLPPARCRAGAQQPAVALETCNP QPCPARWEVSEPSSCTSAGGAGLALENETCVPGADGLEAPVTEGPGSVDEKLPAPEPCVGMSCPPG WGHLDATSAGEKAPSPWGSIRTGAQAAHVWTPAAGSCSVSCGRGLMELRFLCMDSALRVPVQEEL CGLASKPGSRREVCQAVPCPARWQYKLAACSVSCGRGVVRRILYCARAHGEDDGEEILLDTQCQGL

[0317] PRPEPQEACSLEPCPPRWKVMSLGPCSASCGLGTARRSVACVQLDQGQDVEVDEAACAALVRPEAS VPCLIADCTYRWHVGTWMECSVSCGDGIQRRRDTCLGPQAQAPVPADFCQHLPKPVTVRGCWAGP CVGQGTPSLVPHEEAAAPGRTTATPAGASLEWSQARGLLFSPAPQPRRLLPGPQENSVQSSACGRQ HLEPTGTIDMRGPGQADCAVAIGRPLGEVVTLRVLESSLNCSAGDMLLLWGRLTWRKMCRKLLDMT

[0318] FSSKTNTLVVRQRCGRPGGGVLLRYGSQLAPETFYRECDMQLFGPWGEIVSPSLSPATSNAGGCRLF INVAPHARIAIHALATNMGAGTEGANASYILIRDTHSLRTTAFHGQQVLYWESESSQAEMEFSEGFLK AQASLRGQYWTLQSWVPEMQDPQSWKGKEGT

[0319] SEQ ID N° : 23 ADAMTS13 M domain (Peptidase M 12B) from position 80 to 286 LHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLT

[0320] SSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTG

[0321] FDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARC VWDPP

[0322] SEQ ID N° :24 ADAMTS13 D (Disintegrin) domain from position 287 to 383

[0323] RPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSSCSRLLV PLLDGTECGVEKWCSKGRCRSLVELTPIAAV

[0324] SEQ ID N° :25 ADAMTS13 T TSP type-1 1) domain from position 384 to 43

[0325] HGRWSSWGPRSPCSRSCGGGVVTRRRQCNNPRPAFGGRACVGADLQAEMCNTQACE

[0326] SEQ ID N° :26 ADAMTS13 C (Cysteine-rich) domain from position 440 to 555

[0327] KTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLD

[0328] GTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQVWDRCQVCGGDNSTC

[0329] SEQ ID N° :27 ADAMTS13 S (Spacer) region from position 556 to 685

[0330] SPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAGKMSISPNTTYPSLLEDG

[0331] RVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPKPRQA

[0332] SEQ ID N° :28 ADAMTS13 S (Spacer) region from position 556 to 685 with triple-alanine mutations (RARAA)

[0333] SPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLATHLAVRIGGRYVVAGKMSISPNTTYPSLLED

[0334] GRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRAGEEAGNLTRPDITFTYFQPKPRQA

[0335] SEQ ID N° :29 ADAMTS13 S (Spacer) region from position 556 to 685 with N-glycan shielding (NGLY3)

[0336] SPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAGNMSISPNTTYPSLLEDG

[0337] RVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPKPRQA

[0338] SEQ ID N° :30 ADAMTS13 region from 682 to 730 TSP type-1 2

[0339] PRQAWVWAAVRGPCSVSCGAGLRWVNYSCLDQARKELVETVQCQGSQQP

[0340] SEQ ID N° :31 (ADAMTS13 region from 742 to 805 TSP type-1 3)

[0341] CPPYWAVGDFGPCSASCGGGLRERPVRCVEAQGSLLKTLPPARCRAGAQQPAVALETCNPQPCP

[0342] SEQ ID N° :32 (ADAMTS13 region from 808 to 859 TSP type-1 4)

[0343] WEVSEPSSCTSAGGAGLALENETCVPGADGLEAPVTEGPGSVDEKLPAPEPC

[0344] SEQ ID N° :33 (ADAMTS13 region from 896 to 950 TSP type-1 5) VWTPAAGSCSVSCGRGLMELRFLCMDSALRVPVQEELCGLASKPGSRREVCQAVP

[0345] SEQ ID N° : 34 (ADAMTS13 region from 951 to 1011 TSP type-1 6)

[0346] CPARWQYKLAACSVSCGRGVVRRILYCARAHGEDDGEEILLDTQCQGLPRPEPQEACSLEP

[0347] SEQ ID N° : 35 (ADAMTS13 region from 1012 tol068 TSP type-1 7)

[0348] CPPRWKVMSLGPCSASCGLGTARRSVACVQLDQGQDVEVDEAACAALVRPEASVPCL

[0349] SEQ ID N° : 36 (ADAMTS13 region from 1072 tol l31 TSP type-1 8)

[0350] CTYRWHVGTWMECSVSCGDGIQRRRDTCLGPQAQAPVPADFCQHLPKPVTVRGCWAGPCV

[0351] SEQ ID N° : 37 (ADAMTS13 region from 1192 tol298 CUB 1)

[0352] CGRQHLEPTGTIDMRGPGQADCAVAIGRPLGEVVTLRVLESSLNCSAGDMLLLWGRLTWRKMCRKL LDMTFSSKTNTLVVRQRCGRPGGGVLLRYGSQLAPETFYRE

[0353] SEQ ID N° : 38 (ADAMTS13 region from 1299 tol427 CUB 2)

[0354] CDMQLFGPWGEIVSPSLSPATSNAGGCRLFINVAPHARIAIHALATNMGAGTEGANASYILIRDTHSL RTTAFHGQQVLYWESESSQAEMEFSEGFLKAQASLRGQYWTLQSWVPEMQDPQSWKGKEGT

[0355] SEQ ID N° : 39 (ADAMTS13 MD domains)

[0356] LHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLT

[0357] SSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTG FDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARC VWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSS CSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAV

[0358] SEQ ID N° : 40 (ADAMTS13 MDT domains)

[0359] LHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLT

[0360] SSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTG FDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARC VWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSS CSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQC

[0361] NNPRPAFGGRACVGADLQAEMCNTQACE

[0362] SEQ ID N° : 41 (ADAMTS13 MDTC domains)

[0363] LHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLT

[0364] SSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTG FDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARC VWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSS CSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQC NNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVP HSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQ VWDRCQVCGGDNSTC

[0365] SEQ ID N° : 42 (ADAMTS13 MDTCS domains)

[0366] LHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLT SSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTG FDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARC VWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSS CSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQC NNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVP HSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQ VWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAG KMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFT YFQPKPRQA

[0367] SEQ ID N° : 43 (A50 a VWFA1 domain-VHH according to an embodiment of the invention)

[0368] QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAAISRRGDNVYYADSVK GRFAISRDNAESTLYLQMNSLKPEDTAVYYCAAHVTVSA ITLSTSTYDYWGQGTQVTVSS

[0369] SEQ ID N° : 44 (153 a VWFA1 domain-VHH according to an embodiment of the invention)

[0370] QVQLQDSGGGLVQAGGSLRLSCAASGRTKDMAWFRQPPGKEREFVAVIYSSDGSTLVAASVKGRFTI SRDNAKNTVYLQMTSLKPADTAVYYCATSRGYSGTYY STSRYDYWTGGTQVTVSS

[0371] SEQ ID N° : 45 (Z29 a VWFA1 domain-VHH according to an embodiment of the invention)

[0372] QVQLQESGGGSVQAGDSLTLSCAASGRTFSMHAMGWFRQAPGKEREFVAAIS PSAFTEYADSLKGRFTVSRDNAKKLVWLQMNGLKPEDTAAYYCAARRGAFTA TTAPLYDYWGQGTQVTVSS

[0373] SEQ ID N° : 46 (M53 a VWFA1 domain-VHH according to an embodiment of the invention)

[0374] QVQLQDSGGGLVQAGESLRLSCGTSGRTFGRRAMAWFRQAPGKERQFVAWIARYDGSTLYADSVK GRFTISRDDNKNTMYLHMNNLTPEDTAVYYCAAGPRGLY YESRYEYWGQGTLVTVSS

[0375] SEQ ID N° : 47 (2A1-4L-79 a VWFA1 domain-VHH according to an embodiment of the invention)

[0376] QVQLQDSGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIGSDSSTLYTSSVRGR

[0377] FTISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSS GIYYREGSYAYWGQGTQVTVSS SEQ ID N°: 48 (2A1-4L-129 a VWFA1 domain-VHH according to an embodiment of the invention)

[0378] QVQLQESGGGLVQAGASLRLSCAASGRSFSSYPMAWFRQAPGKEREFVVFIGSDHSTLYSTSVRGR FTISRDNAKNTVYLQMMNLKPEDTAVYYCAARNSAWSSGIYYRETSYDYWGQGTQVTVSS

[0379] SEQ ID N° : 49 (2A1-4L-34 a VWFA1 domain-VHH according to an embodiment of the invention)

[0380] QVQLQDSGGGSVQAGASLRLSCAASGGTFSSYAMAWFRQAPGKEREFVGFIGSDGSTLYSSSVRG RFTISRDNAKNTVALQMMNLKPEDTAVYYCAARARYSGI YYRETDYPYWGQGTQVTVSS

[0381] SEQ ID N° : 50 (2A1-4L-78 a VWFA1 domain-VHH according to an embodiment of the invention)

[0382] QVQLQESGGGLVQAGASLRLSCTASGRSFGGFPMGWFRQAPGKEREFVSGLTRSLFTVYADSVKG RFTVSTDNTKNTVYLQMNSLKPEDTAVYYCAARPDLYAYSRDPNEYDYWGQGTQVTVSS

[0383] SEQ ID N° : 51 (2LA1-15 a VWFA1 domain-VHH according to an embodiment of the invention)

[0384] QVQLQDSGGGLVQSGGSLRLACAASGRIVSTYAMGWFRQSPGKEREFVATVKGRFTISRDNAKNTL YLQMNSLKPEDTAVYYCAKTKRTGIFTTARMVDYWGQGTQVTVSS

[0385] SEQ ID N° : 52 (C37 a VWFA3 domain-VHH according to an embodiment of the invention)

[0386] QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTISTYGEPRYADSVKG RFTISRDNANNTLYLQMNSLRPEDTAVYYCARGAGTSSY LPQRGNWDQGTQVTISS

[0387] SEQ ID N° : 53 (C37-hum a VWFA3 domain-VHH according to an embodiment of the invention)

[0388] QVQLQESGGGLVQPGGSLRLSCAASGFTFSWYPMSWVRQAPGKGLEWVSTISTYGEPRYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAGTSSYLPQRGNWDQGTQVTISS

[0389] SEQ ID N° : 54 (AM-2-75 a VWFA3 domain-VHH according to an embodiment of the invention)

[0390] QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTISTYGEPRYADSVKG RFTISRDNANNTLYLQMNSLRPEDTAVYYCARGAGTSSY LPQRGNWDQGTQVTVSS

[0391] SEQ ID N° : 55 (22-2L-34 a VWFA3 domain-VHH according to an embodiment of the invention)

[0392] QVQLQDSGGGLVQAGGSLRLSCAASVRIFTSYAMGWFRQAPGKEREFVAAINRSGKSTYYSDSVEG RFTISRDNAKNTVSLQMDSLKLEDTAVYYCAADYSGSYTSLWSRPERLDWGQGTQVTVFS SEQ ID N° : 56 (22-4L-16 a VWFA3 domain-VHH according to an embodiment of the invention)

[0393] QVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVADTGGISWIRTQGYNYWGQGTQVTVSS

[0394] SEQ ID N° : 57 (T76 a VWFA3 domain-VHH according to an embodiment of the invention)

[0395] QVQLQESGGGLVQPGESLRLSCAASGSIFSINTMGWYGQAPGKQRELVASITFGGVTNYADSVKGRF TISRDNTNDTVYLQMNSLKPEDTAVYICNAVTWGGLT NYWGQGTQVTVSS

[0396] SEQ ID N° : 58 (AM-4-15-3 a VWFA3 domain-VHH according to an embodiment of the invention)

[0397] QVQLQDSGGGLVQPGGSLRLACAASGSIFSINSMGWYRQAPGKQRELVAHALADGSASYRDSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTVPSSVTK GYWGQGTQVTVSS

[0398] SEQ ID N° : 59 (A12 a VHH binding to the active VWF conformation according to an embodiment of the invention)

[0399] QVQLVESGGGLVQAGGSLRLSCTASGRTFSTYALGWFRQVPGKGREFIAVIYWRDGSSLYSDSVKG RFTISKDNAKNTVYLQMNSLKPEDTAVYYCANRHDSRGTYYSSRGYDYWGQGTQVTVSS

[0400] SEQ ID N° : 60 (A13 a VHH binding to the active VWF conformation according to an embodiment of the invention)

[0401] QVQLVESGGGLVQAGGSLRLSCAASGRTKDMAWFRQPPGKEREFVAVIYSSDGSTLVAASVKGRFT ISRDNAKNTVYLQMTSLKPADTAVYYCATSRGYSGTYY STSRYDYWGQGTQVTVSS

[0402] SEQ ID N° : 61 (A15 a VHH binding to the active VWF conformation according to an embodiment of the invention)

[0403] QVQLVESGGGLVQAGGSLRLSCAASGRTKDMAWFRQPPGKEREFVAVIYSSDGSTLVAASVTGRFT ISRDNAKNMVYLQMTSLKPADTAVYYCASSRGYSGTYYSTSRYDYWGQGTQVTVSS

[0404] SEQ ID N° :62 (ADAMTS13 MDTCS domains with RARAA mutations)

[0405] LHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLT SSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTG FDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARC VWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSS CSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQC NNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVP HSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQ VWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLATHLAVRIGGRYVVAG KMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRAGEEAGNLTRPDITFT YFQPKPRQA

[0406] SEQ ID N° :63 (ADAMTS13 MDTCS domains with NGLY3 mutation)

[0407] LHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLT SSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTG FDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARC VWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSS CSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQC NNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVP HSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQ VWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAG NMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFT YFQPKPRQA

[0408] SEQ ID N° :64 (ADAMTS13 MDTCS domains with NGLY3+7 mutation)

[0409] LHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLT SSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTG FDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARC VWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSS CSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQC NNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVP HSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQ VWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAG NMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEENVTLTRPDITFT YFQPKPRQA

[0410] SEQ ID N° : 65 (linker according to an embodiment of the invention)

[0411] (G)8

[0412] SEQ ID N° : 66 (linker according to an embodiment of the invention)

[0413] LE

[0414] SEQ ID N° : 67 (linker according to an embodiment of the invention)

[0415] KESGSVSSEQLAQFRSLD

[0416] SEQ ID N° : 68 (linker according to an embodiment of the invention)

[0417] EGKSSGSGSESKST

[0418] SEQ ID N° : 69 (linker according to an embodiment of the invention) GSAGSAAGSGEF

[0419] SEQ ID N° : 70 (linker according to an embodiment of the invention)

[0420] (EAAAK)n

[0421] SEQ ID N° : 71 (linker according to an embodiment of the invention)

[0422] LEAGCKNFFPRJ.SFTSCGSLE

[0423] SEQ ID N° : 72 (linker according to an embodiment of the invention)

[0424] CRRRRRRJ,EAEAC

[0425] SEQ ID N° : 73 (linker according to an embodiment of the invention)

[0426] SSSGSS

[0427] SEQ ID N° : 74 (linker according to an embodiment of the invention)

[0428] (GGGGS)n

[0429] SEQ ID N° : 75

[0430] (GGGS)n

[0431] SEQ ID N° : 76 (MDTCS-RARAA-L-Syn-VWFAl)

[0432] AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNI TANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLI TEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSA GRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDP LDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVT RRRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHW

[0433] GAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGR MDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLATHLAVRIGG RYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRAGEEAGNLT RPDITFTYFQPKPRQAGGGGSGGGGSGGGGSAVQLVESGGRLVKAGASLRLSCAASGRTFSSLPM AWFRQAPGKEREFVAFIGSDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSA FSSGIYYREGSYAYWGQGTQVTVSSAAALPETGLE

[0434] SEQ ID N° : 77 (MDCTS of ADAMTS13)

[0435] AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNI TANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLI TEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSA GRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDP LDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVT RRRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHW

[0436] GAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGR

[0437] MDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGG

[0438] RYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLT RPDITFTYFQPKPRQA

[0439] SEQ ID N° : 78 (five N-terminal amino acids of the mature ADAMTS13 protein)

[0440] AAGGI

[0441] SEQ ID N°: 79 (humanized Syn-VWFAl - a VWFA1 domain-VHH according to an embodiment of the invention) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIGSDSSTLYTSSVRGR

[0442] FTISRDNGKNTVYLQMNNLKPEDTAVYYCAARSSAFSSGIYYREGSYAYWGQGTQVTVSS

Claims

CLAIMS1. A polypeptide comprising a truncated form of ADAMTS13 and a targeting agent, wherein the targeting agent is able to bind to a von Willebrand factor (VWF) via a VWF-binding domain at a site of a thrombus.

2. The polypeptide according to claim 1 wherein said targeting agent binds to: a. an Al domain of VWF or a part of an Al domain of VWF; b. an A2 domain of VWF or a part of an A2 domain of VWF; c. an A3 domain of VWF or a part of an A3 domain of VWF; and / or d. a D4-CK domain of VWF or a part of a D4-CK domain of VWF.

3. The polypeptide according to any of the previous claims, wherein said targeting agent has affinity towards an unfolded or activated conformation of VWF.

4. The polypeptide according to any one of the preceding claims, wherein said targeting agent comprises an antibody variable domain and / or a protein binding domain.

5. The polypeptide according to claim 4, wherein said targeting agent comprises an antibody variable domain, said antibody variable domain is a single variable domain on a heavy chain (VHH) domain or a fragment thereof, preferably a humanized VHH.

6. The polypeptide according to any one of the preceding claims, wherein the truncated form of ADAMTS13 comprises at least one or more of: a. a metalloprotease (MP) domain or fragments thereof; b. an MP domain and a disintegrin-like domain (Dis) or fragments thereof; c. an MP domain, a Dis domain and a thrombospondin type 1 (TSP1) repeat or fragments thereof; d. an MP domain, a Dis domain, a TSP1 repeat and a cysteine-rich domain (Cys-rich) or fragments thereof; e. an MP domain, a Dis domain, a TSP1 repeat, a Cys-rich domain and a Spacer domain or fragments thereof.

7. The polypeptide according to any one of the previous claims, wherein the truncated form of ADAMTS13 has an amino acid sequence with a sequence identity of at least 80% to SEQ ID N°: 23, SEQ ID N°: 39, SEQ ID N°:40, SEQ ID N°:41, SEQ ID N°:42 or SEQ ID N°: 77, wherein SEQ ID N°s: 23, 39, 40, 41, and 42 are optionally preceded by SEQ ID N°: 78.

8. The fusion peptide according to claim 7, wherein said truncated ADAMTS13 comprises one or more added N-linked glycosylation sites, one or more shifted N- linked glycosylation sites and / or one or more non-alanine amino acids substituted to alanine.

9. The polypeptide according to any of the previous claims, wherein the polypeptide exhibits thrombolytic activity.

10. The polypeptide according to any of the previous claims, wherein the truncated form of ADAMTS13 and the targeting agent are linked through a linker amino acid sequence.

11. The polypeptide according to any one of the previous claims, wherein polypeptide comprises in an N- to C-terminal order: a. a truncated form of ADAMTS13, a linker amino acid sequence, and a targeting agent; or b. a targeting agent, a linker amino acid sequence, and a truncated form of ADAMTS13.

12. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises a plurality of targeting agents.

13. The polypeptide according to any of the previous claims, wherein said truncated ADAMTS13 domain is resistant to proteolytic degradation, preferably by plasmin.

14. A pharmaceutical composition comprising a polypeptide according to any of the preceding claims 1 to 13 and a pharmaceutically acceptable carrier, excipient or diluent.

15. The polypeptide according to any of the preceding claims 1 to 13 or the pharmaceutical composition according to claim 14, for use as a medicament.

16. The polypeptide or the pharmaceutical composition for use according to claim 15, for use in the prevention and / or treatment of microvascular thrombosis in a subject in need thereof.

17. The polypeptide or the pharmaceutical composition for use according to any of the claims 15 or 16, for the treatment and / or prevention of disease or condition selected from the group consisting of: acquired or hereditary thrombotic thrombocytopenic purpura (TTP), complement-mediated thrombotic microangiopathy, haemolytic uremic syndrome, antiphospholipid antibody syndrome, non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such as angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischemia, cardioembolism, peripheral vascular disease, restenosis, sickle cell disease and myocardial infract.

18. The polypeptide or the pharmaceutical composition for use according to any of claim 15, for use in the prevention and / or treatment of stroke in a subject in need thereof.

19. The polypeptide or the pharmaceutical composition for use according to any of claim 15 or 18, for use in the prevention and / or treatment of tissue plasminogen activator (tPA)-resistant stroke.

20. A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide according to any one of the previous claims 1-13.

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