ADAMTS13 protein variants and uses thereof

Abstract

The present invention relates to ADAMTS13 protein variants comprising residues 1-685 of ADAMTS13, wherein said ADAMTS13 protein variants have one or more additional N-linked glycosylation sites compared to wild-type ADAMTS 13 and / or one or more existing N-linked glycosylation sites have been displaced compared to wild-type ADAMTS 13. The present invention further relates to compositions comprising such ADAMTS13 variants, methods for their preparation, and their use, inter alia, as antithrombotic agents and in the treatment of thrombotic diseases, thrombotic microangiopathy, thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), ischemic stroke, systemic thrombosis, COVID-19, antiphospholipid syndrome, preeclampsia / HELLP syndrome, sepsis, and sickle cell disease.

Description

[Technical Field]

[0001] This invention was made with support from the European Union's Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement no. 675746 (project PROFILE).

[0002] FIELD OF THE INVENTION The present invention relates to the field of therapy. More particularly, the present invention relates to the field of therapy for disorders involving von Willebrand factor (VWF). The present invention relates to modified proteases involved in maintaining hemostasis, in particular modified proteases that exhibit strongly reduced binding of autoantibodies while maintaining activity, and their use in the treatment of diseases. [Background technology]

[0003] BACKGROUND OF THE INVENTION Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare but life-threatening autoimmune disease caused by the development of autoantibodies against ADAMTS13 (A disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13). ADAMTS13 is a metalloprotease that proteolytically cleaves the Tyr1605-Met1606 bond within the A2 domain of von Willebrand factor (VWF). VWF is a multimeric protein that mediates platelet adhesion to damaged blood vessels. VWF multimer size is directly proportional to its biological activity; larger multimers are more active in promoting platelet adhesion to damaged vessel walls. In normal individuals, VWF multimer size is regulated by the VWF-cleaving protease ADAMTS13. VWF multimer processing is impaired in patients with iTTP due to the presence of pathogenic autoantibodies against ADAMTS13. The prolonged presence of high-molecular-weight VWF multimers in iTTP patients with autoantibodies against ADAMTS13 is associated with excessive thrombus formation in the microvasculature, resulting in life-threatening microvascular thrombosis.

[0004] Current management of iTTP includes plasmapheresis (PEX) and immunosuppression with high-dose glucocorticoids. Plasmapheresis provides a source of exogenous ADAMTS13, but its effects are short-lived due to the persistent presence of circulating pathogenic antibodies targeting ADAMTS13, including exogenous ADAMTS13. In addition to plasmapheresis, rituximab, a B-cell-depleting anti-CD20 therapeutic monoclonal antibody, is used to treat iTTP. Rituximab has also been used to prevent relapse in patients with iTTP. Recently, caplacizumab, a humanized anti-VWF nanobody that blocks platelet binding to VWF, has been shown to accelerate platelet count normalization by 1.55-fold (Scully et al., 2019). Bleeding is a defining side effect of caplacizumab treatment (Mazepa et al., 2019). Mazepa et al. reported that bleeding was the major adverse effect of caplacizumab therapy, occurring in 65% of patients in the HERCULES clinical trial (48% in the placebo arm). Mucocutaneous bleeding, including epistaxis and gingival bleeding, was the most common event, with most bleeding events being mild to moderate in severity and resolving without intervention. Three subjects who developed severe bleeding due to caplacizumab were given VWF concentrate (for severe epistaxis), tranexamic acid (for gingival bleeding), and red blood cell transfusions (for upper gastrointestinal bleeding). Overall, most caplacizumab-related bleeding resolves without intervention (although withholding the drug may be necessary), although topical vasoconstrictors and antifibrinolytics may be effective, and VWF concentrates are restricted to patients with severe, refractory bleeding.

[0005] Despite advances in the treatment of patients with iTTP, current treatment regimens are thus still suboptimal. There is a clear need for therapies that rapidly re-establish ADAMTS13 activity, which would help accelerate the normalization of platelet counts in patients with iTTP.

[0006] An autoantibody-resistant ADAMTS13 variant has been reported in the literature (Jian et al., 2012). The spacer domain of ADAMTS13 is the primary site for binding of pathogenic autoantibodies. Conservative mutations at five residues within the spacer domain create a so-called gain-of-function (GoF) variant, which is claimed to be resistant to binding of pathogenic autoantibodies occurring in iTTP patients (Jian et al., 2012). This ADAMTS13 GoF variant is also described in U.S. Patent No. US9 / 546,360. However, follow-up experiments revealed that this ADAMTS13 GoF variant is still targeted by patient-derived autoantibodies and does not resist their inhibitory effects (Graca et al., 2019). However, pathogenic autoantibodies that bind to other domains of ADAMTS13 have also been reported.

[0007] Therefore, there remains a need for ADAMTS13 variants that retain ADAMTS13 activity while effectively evading autoantibody binding. Such ADAMTS13 variants may be of great therapeutic interest based on their potential to rapidly reverse clinical symptoms in patients with iTTP and other related diseases, compared with currently available treatment options. Summary of the Invention

[0008] (Summary of the Invention) It is an object of the present invention to provide improved ADAMTS13 variants that are less susceptible to binding by autoantibodies and that retain proteolytic activity.

[0009] Accordingly, the present invention provides an ADAMTS13 protein variant comprising residues 1-1427 of ADAMTS13 or a truncated form of ADAMTS13 containing at least residues 1-685, in which one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites have been shifted compared to wild-type ADAMTS13. Preferably, the one or more N-linked glycosylation sites are not added or the one or more existing N-linked glycosylation sites are not shifted at amino acid residues 464-466, 469-471, 476-478, 493-495, 511-513, and 539-541 of the ADAMTS13 protein variant. In another preferred embodiment, the N-linked glycosylation sites are absent from the ADAMTS13 protein variant at amino acid residues 464-466, 469-471, 476-478, 493-495, 511-513, and 539-541. In a preferred embodiment, the ADAMTS13 protein variant is a full-length ADAMTS13 variant, i.e., comprises residues 1-1427 of ADAMTS13 in which one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13, and optionally contains additional mutations as described herein.

[0010] In a further aspect, the present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding an ADAMTS13 protein variant according to the present invention.

[0011] In a further aspect, the present invention provides a pharmaceutical composition comprising an ADAMTS13 protein variant according to the present invention or a nucleic acid construct according to the present invention, and one or more pharmaceutically acceptable carriers, adjuvants, excipients, and / or diluents.

[0012] In a further aspect, the present invention provides an ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, in which one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13, for use in therapy, or a nucleic acid construct comprising a nucleic acid sequence encoding said ADAMTS13 variant, preferably an ADAMTS13 protein variant or nucleic acid construct according to the invention.

[0013] In a further aspect, the present invention provides an ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, in which one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13, for use as an antithrombotic agent, or a nucleic acid construct comprising a nucleic acid sequence encoding said ADAMTS13 variant, preferably an ADAMTS13 protein variant or nucleic acid construct according to the present invention.

[0014] In a further aspect, the present invention provides an ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, in which one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13, or a nucleic acid construct comprising a nucleic acid sequence encoding said ADAMTS13 variant, preferably an ADAMTS13 protein variant or nucleic acid construct according to the invention, for use in the treatment of a disorder characterized by abnormal von Willebrand factor (VWF) activity and / or VWF processing.

[0015] In a further aspect, the present invention provides a method for treating a disorder characterized by abnormal von Willebrand factor (VWF) activity and / or VWF processing, comprising administering to a subject in need thereof a therapeutically effective amount of an ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, wherein one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13, or a nucleic acid construct comprising a nucleic acid sequence encoding said ADAMTS13 variant, preferably an ADAMTS13 protein variant or nucleic acid construct according to the present invention.

[0016] In a further aspect, the present invention provides use of an ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, in which one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13, or a nucleic acid construct comprising a nucleic acid sequence encoding said ADAMTS13 variant, preferably an ADAMTS13 protein variant or nucleic acid construct according to the invention, in the production of a medicament for the treatment of a disorder characterized by abnormal von Willebrand factor (VWF) activity and / or VWF processing.

[0017] In a further aspect, the present invention provides an ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, in which one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13, or a nucleic acid construct comprising a nucleic acid sequence encoding said ADAMTS13 variant, preferably an ADAMTS13 protein variant or nucleic acid construct according to the invention, for use in the treatment of thrombotic diseases.

[0018] In a further aspect, the present invention provides a method for the treatment of thrombotic diseases, both acquired and congenital, comprising administering to a subject in need thereof a therapeutically effective amount of an ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, which has one or more additional N-linked glycosylation sites compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites displaced compared to wild-type ADAMTS13, or a nucleic acid construct comprising a nucleic acid sequence encoding said ADAMTS13 variant, preferably an ADAMTS13 protein variant or nucleic acid construct according to the present invention.

[0019] In a further aspect, the present invention provides the use of an ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, or a nucleic acid construct comprising a nucleic acid sequence encoding said ADAMTS13 variant, preferably an ADAMTS13 protein variant or nucleic acid construct according to the invention, in the manufacture of a medicament for the treatment of thrombotic disorders, both acquired and congenital thrombotic disorders.

[0020] In a further aspect, the present invention provides a method for producing an ADAMTS13 protein variant according to the present invention, the method comprising introducing a nucleic acid molecule according to the present invention into a host cell, preferably a eukaryotic host cell, capable of N-linked glycosylation, and culturing the host cell under conditions allowing expression of the ADAMTS13 protein variant. DETAILED DESCRIPTION OF THE INVENTION

[0021] (Detailed explanation) The present inventors have identified a new strategy for developing ADAMTS13 variants with reduced binding to pathogenic autoantibodies, based on the insertion of a new consensus site for the addition of N-glycans.Such N-glycan consensus sites are preferably inserted into immunogenic regions within ADAMTS13.As shown in the examples, amino acid mutations were introduced into wild-type ADAMTS13 protein to introduce N-glycan sites at various positions.Furthermore, it has been demonstrated that this technology can be used to significantly reduce the binding of pathogenic ADAMTS13 autoantibodies that occur in iTTP patients.Without wishing to be bound by theory, it is assumed that the introduced N-glycans can mask the epitopes to which autoantibodies bind. More specifically, the introduced N-glycans mask epitopes of autoantibodies targeting ADAMTS13, including those in the spacer domain, TSP-1 (thrombospondin type 1) repeats, and CUB (complement components Clr / Cls, Uegf, and bone morphogenetic protein 1), metalloprotease domain, disintegrin domain, cystine-rich domain, and other domains of ADAMTS13 that provide binding sites for anti-ADAMTS13 autoantibodies, as well as epitopes in exposed regions within other domains. In particular, masking of key epitopes within the spacer domain of ADAMTS13 is believed to significantly reduce autoantibody reactivity. These epitopes within the spacer domain are believed to be centered around amino acid residues R568, F592, R660, Y661, and Y665 within the ADAMTS13 sequence. Mutations at these five residues can effectively reduce autoantibody binding to ADAMTS13, but they also usually result in reduced activity (Graca et al., 2019). In the present invention, we demonstrate that N-glycans can be introduced into and / or outside the classical epitope residues (R568, F592, R660, Y661, and Y665) to reduce the reactivity of ADAMTS13 with autoantibodies without losing proteolytic activity, i.e., while maintaining at least a portion of the VWF-cleaving activity of ADAMTS13.Such ADAMTS13 N-glycan variants that resist ADAMTS13-targeting autoantibodies and maintain binding to the spacer domain of VWF are of great interest for the treatment of disorders associated with abnormal von Willebrand factor (VWF) activity and / or von Willebrand factor processing.

[0022] Thus, in a first aspect, the present invention provides an ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, in which one or more N-glycosylation sites have been added and / or one or more existing N-glycosylation sites have been moved compared to wild-type ADAMTS13, with the proviso that the one or more N-linked glycosylation sites are not added and the one or more existing N-linked glycosylation sites are not moved at amino acid residues 464 to 466, 469 to 471, 476 to 478, 493 to 495, 511 to 513, and 539 to 541 of the ADAMTS13 protein variant.

[0023] In a further aspect, the present invention provides an ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, in which one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13, for use in therapy, or a nucleic acid construct comprising a nucleic acid sequence encoding said ADAMTS13 variant.

[0024] In a further aspect, the present invention provides an ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, in which one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13, for use as an antithrombotic agent, or a nucleic acid construct comprising a nucleic acid sequence encoding said ADAMTS13 variant.

[0025] In a further aspect, the present invention provides an ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, in which one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13, or a nucleic acid construct comprising a nucleic acid sequence encoding said ADAMTS13 variant, for use in the treatment of a disorder characterized by abnormal von Willebrand factor (VWF) activity and / or VWF processing.

[0026] As used herein, the term "protein" refers to a compound comprising amino acids linked via peptide bonds. A protein encoded by a gene is not limited to the amino acid sequence encoded by the gene and may include post-translational modifications of the protein.

[0027] As used herein, the terms "ADAMTS13" and "ADAMTS13 protein" refer to the protein encoded by the ADAMTS13 gene. ADAMTS13 is a member of the metalloproteinase gene family ADAM (a disintegrin and metalloproteinase), which consists of membrane-bound proteases with diverse functions. Members of the ADAMTS family are further characterized by the presence of one or more thrombospondin 1-like (TSP1) domains at the C-terminus and the absence of EGF repeats, transmembrane domains, and cytoplasmic tails present in ADAM metalloproteinases. ADAMTS13 is the only member of the ADAMTS family that has two C-terminal CUB domains (representing complement C1r / C1s proteases, sea urchin, and bone morphogenic protein) and possesses VWF (von Willebrand factor)-cleaving protease activity (Kelwick et al., 2015). The terms "wild-type ADAMTS13" and "wild-type ADAMTS13 protein" refer to naturally occurring human ADAMTS13. Figure 1 shows the amino acid sequence of full-length wild-type ADAMTS13.

[0028] As used herein, the term "ADAMTS13 protein variant" refers to a variant of ADAMTS13 having an amino acid sequence that differs from that of wild-type ADAMTS13 in that it has at least one N-glycosylation site that is not present in wild-type ADAMTS13. Additionally, the ADAMTS13 protein variant according to the present invention comprises at least amino acids 1-685 of the ADAMTS13 amino acid sequence shown in Figure 1. It has been shown that a truncated ADAMTS13 protein consisting of amino acids 1-685 shown in Figure 1 has proteolytic activity against VWF (Tao et al., 2005). In another preferred embodiment, the ADAMTS13 protein variant according to the present invention is a full-length ADAMTS13 protein variant. This means that the variant comprises the complete amino acid sequence having residues 1 to 1427 of ADAMTS13 as shown in Figure 1, in which one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13, and optionally, the variant comprises additional mutations as described herein.

[0029] In the amino acid sequences or protein variants defined herein, amino acids are represented by one-letter or three-letter abbreviations. These one-letter and three-letter abbreviations are well known to those skilled in the art and have the following meanings: 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 (Ile) is isoleucine, and K (Lys) is lysine. L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine.

[0030] All amino acid residue positions indicated herein refer to the numbering of the amino acid residues in the sequence of wild-type ADAMTS13 shown in FIG.

[0031] Mutations, particularly substitutions of one amino acid for another, are designated herein in a manner standard in the art, i.e., by indicating the amino acid present in the wild-type ADAMTS13 sequence, its position within that sequence, and the amino acid introduced at that position. For example, "R568K" ​​indicates that the arginine at position 568 has been replaced with a lysine. "568REY570 to 568NET570" indicates that the sequence arginine-glutamic acid-tyrosine at positions 568-570 has been replaced with the sequence asparagine-glutamic acid-threonine.

[0032] "N-linked glycosylation" refers to the attachment of an oligosaccharide moiety to a nitrogen atom, usually the N4 of an asparagine residue. The terms "N-linked glycosylation site" and "N-glycosylation site" are used interchangeably and refer to a site within an ADAMTS13 protein variant that is capable of N-linked glycosylation. Such a site has the amino acid sequence NXT or NXS, where X is any amino acid except P. N-linked glycosylation is a post-translational modification, and N-linked glycans on proteins can regulate protein folding, cell adhesion, and / or function. N-linked glycans can have various combinations of mannose, N-acetylglucosamine (GlcNAc), galactose, fucose, and sialic acid residues. Several N-linked glycans attached to asparagine residues 142, 146, 552, 579, 614, 667, 707, 828, 1235, and 1354 (see Figure 2) have been identified on ADAMTS13 (Verbij et al., 2016), and several other types of glycosylation have also been identified, including O-glycosylation and S- and C-mannosylation. Figure 2 also shows the most common and other structures of N-glycans identified in ADAMTS13.

[0033] The introduction of N-linked glycosylation sites at residues 464–466, 469–471, 476–478, 493–495, 511–513, and 539–541 within the cysteine-rich domain of full-length ADAMTS13 has been described by De Groot et al.. Insertion of such sites, as well as sequence exchanges and single-point mutations within this domain, were performed to investigate the functional role of the cysteine-rich domain of ADAMTS13 in VWF binding and proteolysis.

[0034] In an ADAMTS13 protein variant according to the present invention or for use in accordance with the present invention, one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13, and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13. This means that an N-glycosylation site (NXT or NXS, where X is any amino acid except P) is present at an amino acid residue not present in wild-type ADAMTS13. This can be achieved by either adding one or more additional N-linked glycosylation sites, moving one or more N-linked glycosylation sites, or a combination thereof. Preferably, one to five, more preferably one to three, and more preferably one or two N-linked glycosylation sites are added and / or moved.

[0035] In a further preferred embodiment, a proline (P) present in the ADAMTS13 sequence near the N-linked glycosylation site NXS or NXT introduced or transferred according to the present invention is replaced with another amino acid, preferably alanine. Replacement of the proline at such a position facilitates attachment of an N-glycan to the N-linked glycosylation site. As used herein, "near" refers to one or two amino acids before or after the N-linked glycosylation site. Preferably, the proline immediately following the N-linked glycosylation site is replaced with another amino acid, preferably alanine.

[0036] The addition of an N-linked glycosylation site means that an N-linked glycosylation site not present in wild-type ADAMTS13 is introduced into the ADAMTS13 protein variant of the present invention without removing the naturally occurring N-linked glycosylation site present in wild-type ADAMTS13. The addition of an N-linked glycosylation site can be achieved by introducing one or more mutations into the amino acid sequence compared to wild-type ADAMTS13 so that an N-linked glycosylation site is introduced. Such mutations can be one or more substitutions of amino acid residues with other amino acid residues, insertions of one or more amino acid residues, deletions of one or more amino acid residues, or a combination thereof, such that an N-linked glycosylation site not present in wild-type ADAMTS13 is introduced into the ADAMTS13 sequence. In a preferred embodiment, an N-linked glycosylation site is added by one or more substitutions of an amino acid residue with another amino acid residue. In particular, any amino acid can be substituted with an asparagine residue to introduce asparagine as the first residue within an N-linked glycosylation site NXS or NXT as defined herein, proline can be substituted with any other amino acid to eliminate the possibility of proline as the second residue within an N-linked glycosylation site NXS or NXT as defined herein, any amino acid other than serine and threonine can be substituted with serine or threonine to introduce serine or threonine as the third residue within an N-linked glycosylation site NXS or NXT as defined herein, or a combination thereof. Both NXS and NXT sites can be introduced into ADAMTS13 variants according to the present invention, although NXT appears to result in more efficient N-glycan addition compared to NXS. For example, in the ADAMTS13 variant NGLY3 described in the Examples herein, an N-linked glycosylation site (NMS) is added at amino acid residues 608-610 by substituting the lysine at position 608 with asparagine (K608N).As another example, in the ADAMTS13 variant NGLY4 described in the Examples herein, an N-linked glycosylation site (NST) is added at amino acid residues 609 to 611 by substituting asparagine for methionine at position 609 and threonine for isoleucine at position 611 (609MSI611 to 609NST611). Those skilled in the art are well competent to design appropriate mutations within the ADAMTS13 sequence to add one or more N-linked glycosylation sites.

[0037] Shifting an N-linked glycosylation site refers to shifting an N-linked glycosylation site present at a specific amino acid residue in wild-type ADAMTS13, particularly an asparagine residue at amino acid position 142, 146, 552, 579, 614, 667, 707, 828, 1235, or 1354, to another amino acid residue in the ADAMTS13 amino acid sequence. That is, the total number of N-linked glycosylation sites in the ADAMTS13 protein variant with shifted N-linked glycosylation is the same as the number of N-linked glycosylation sites in wild-type ADAMTS13. Preferably, the N-linked glycosylation site is shifted by between 1 and 10 amino acid residues. The shift may be downstream or upstream compared to the location of the N-linked glycosylation site in the wild-type ADAMTS13 sequence. More preferably, the N-linked glycosylation site is shifted by between 1 and 7 amino acids, more preferably between 1 and 5 amino acids, more preferably between 1 and 4 amino acids, and more preferably between 1 and 3 amino acids, for example, by 1, 2, or 3 amino acid residues either upstream or downstream compared to the location of the N-linked glycosylation site in the wild-type ADAMTS13 sequence. In a preferred embodiment, the N-linked glycosylation site is shifted by 1 or 2 amino acid residues, most preferably by 1 amino acid residue. Shifting the N-linked glycosylation site can be achieved by introducing one or more mutations into the amino acid sequence compared to wild-type ADAMTS13 such that the N-linked glycosylation site is shifted. Such mutations can be one or more substitutions of an amino acid residue with another amino acid residue, insertion of one or more amino acid residues, deletion of one or more amino acid residues, or a combination thereof, such that the location of the N-linked glycosylation site is shifted compared to the location of the N-linked glycosylation site in the wild-type ADAMTS13 sequence. In a preferred embodiment, the N-linked glycosylation site is transferred by one or more substitutions of an amino acid residue with another amino acid residue.In particular, any amino acid can be substituted with an asparagine residue to introduce asparagine as the first residue within an N-linked glycosylation site NXS or NXT as defined herein, or proline can be substituted with any other amino acid to eliminate the possibility of proline as the second residue within an N-linked glycosylation site NXS or NXT as defined herein, or any amino acid other than serine and threonine can be substituted with serine or threonine to introduce serine or threonine as the third residue within an N-linked glycosylation site NXS or NXT as defined herein, or a combination thereof. Alternatively, or in addition, asparagine can be substituted with any other amino acid to eliminate an N-linked glycosylation site present in wild-type ADAMTS13. As an example, in the ADAMTS13 variant NGLY8 described in the Examples herein, the N-linked glycosylation site was shifted from amino acid residues 667-669 to amino acid residues 668-670 by substituting asparagine at position 667 with leucine, leucine at position 668 with asparagine, threonine at position 669 with valine, and arginine at position 670 with threonine (667NLTR670 to 667LNVT670). In addition, proline at position 671 was replaced with alanine, thereby significantly increasing the possibility of N-glycan attachment (667NLTRP671 to 667LNVTA671). Those skilled in the art are well versed in the ability to design appropriate mutations within the ADAMTS13 sequence to shift one or more N-linked glycosylation sites.

[0038] In a preferred embodiment, within a domain selected from the group consisting of a metalloprotease domain, a disintegrin-like domain, a TSP type 1 domain, a TSP (thrombospondin) type 1 domain, a TSP type 1 2 domain, a TSP type 1 3 domain, a TSP type 1 4 domain, a TSP type 1 5 domain, a TSP type 1 6 domain, a TSP type 1 7 domain, a TSP type 1 8 domain, a cysteine-rich domain, a spacer domain, a CUB (complement components Clr / Cls, Uegf, and bone morphogenetic protein 1) 1 domain, a CUB2 domain, a region between two of said domains, and combinations thereof, and combinations thereof, more preferably within a domain selected from the group consisting of a metalloprotease domain, a disintegrin-like domain, a TSP type 1 domain, a TSP type 1 2 domain, a TSP type 1 3 domain, a TSP type 1 4 domain, a TSP type 1 5 domain, a TSP type 1 6 domain, a TSP type 1 7 domain, a TSP type 1 One or more N-linked glycosylation sites in an ADAMTS13 protein variant according to the present invention or for use in accordance with the present invention have been added and / or one or more existing N-linked glycosylation sites have been moved relative to wild-type ADAMTS13, within a domain selected from the group consisting of the CUB1 domain, the CUB2 domain, the region between two of said domains, and combinations thereof. The amino acid residues for each domain are shown in the first column of Table 5, whereby the numbering refers to the numbering of the amino acid residues in the sequence of wild-type ADAMTS13 shown in Figure 1. "The region between two of the domains" refers to the region between two adjacent domains, preferably between the metalloprotease domain and the disintegrin-like domain, between the disintegrin-like domain and the TSP 1 type 1 domain, between the TSP 1 type 2 domain and the TSP 1 type 3 domain, between the TSP 1 type 3 domain and the TSP 1 type 4 domain, between the TSP 1 type 4 domain and the TSP 1 type 5 domain, between the TSP 1 type 7 domain and the TSP 1 type 8 domain, and between the TSP 1 type 8 domain and the CUB1 domain.

[0039] In the ADAMTS13 protein variants provided by the present invention, the one or more N-linked glycosylation sites are not added, and the one or more existing N-linked glycosylation sites are not moved, at amino acid residues 464 to 466, 469 to 471, 476 to 478, 493 to 495, 511 to 513, and 539 to 541 of the ADAMTS13 protein variant. Preferably, the one or more N-linked glycosylation sites are not added, and the one or more existing N-linked glycosylation sites are not moved, at amino acid residues 440 to 553 or 464 to 539 of the ADAMTS13 protein variant. More preferably, the one or more N-linked glycosylation sites are not added, and the one or more existing N-linked glycosylation sites are not moved, in the cysteine-rich domain consisting of amino acid residues 440 to 556 of ADAMTS13.

[0040] In a further preferred embodiment, one or more N-linked glycosylation sites have been added or moved in an ADAMTS13 protein variant according to the present invention or for use in accordance with the present invention compared to wild-type ADAMTS13 by introducing an N-glycosylation site (NXT or NXS, where X is any amino acid except P) into a spacer domain comprising residues S556 to A685 of the ADAMTS13 sequence shown in Figure 1 and / or any of the amino acid sequences shown in Table 5, or by moving an N-glycosylation site to any of the amino acid sequences shown in Table 5. In the ADAMTS13 protein variants provided by the present invention, the one or more N-linked glycosylation sites are not added or the one or more existing N-linked glycosylation sites are not moved at amino acid residues 464 to 466, 469 to 471, 476 to 478, 493 to 495, 511 to 513, and 539 to 541 of the ADAMTS13 protein variant.

[0041] In a preferred embodiment, one or more N-linked glycosylation sites are added or moved within an ADAMTS13 protein variant of the invention or for use in accordance with the invention, wherein said one or more N-linked glycosylation sites are added and / or said one or more existing N-linked glycosylation sites are moved within a metalloprotease domain, preferably a metalloprotease domain comprising residues L80 to P226 of the ADAMTS13 sequence shown in Figure 1. The one or more N-linked glycosylation sites within the metalloprotease domain are added or moved, preferably moved, within the domains numbered 1 to 9 in Table 5.

[0042] In a preferred embodiment, in an ADAMTS13 protein variant according to or for use in accordance with the invention, one or more N-linked glycosylation sites are added or moved, and / or one or more existing N-linked glycosylation sites are moved, in the CUB domain, preferably the CUB domain comprising residues C1192 to T1427 of the ADAMTS13 sequence shown in Figure 1. In the domains numbered 59 to 89 in Table 5, the one or more N-linked glycosylation sites in the CUB domain are added or moved, preferably moved.

[0043] In a preferred embodiment, one or more N-linked glycosylation sites are added or moved within an ADAMTS13 protein variant of the invention or for use in accordance with the invention, wherein the one or more N-linked glycosylation sites are added and / or the one or more existing N-linked glycosylation sites are moved within the TSP1-2-8 domain, preferably the TSP1-2-8 domain comprising residues P682 to P1131 of the ADAMTS13 sequence shown in Figure 1. Preferably, the one or more N-linked glycosylation sites within the TSP1-2-8 domain are added or moved, preferably moved, within the domains numbered 32 to 56 in Table 5.

[0044] In a further preferred embodiment, one or more N-linked glycosylation sites are added or moved within an ADAMTS13 protein variant according to the present invention or for use in accordance with the present invention, and the one or more N-linked glycosylation sites are added and / or the one or more existing N-linked glycosylation sites are moved within a spacer domain comprising residues S556 to A685 of the ADAMTS13 sequence shown in Figure 1. The spacer domain is 130 amino acids in length and is known to mediate several critical interactions required for the protease activity of ADAMTS13. Furthermore, truncated ADAMTS13 variants including the amino acids up to and including the spacer domain, i.e., amino acids 1 to 685, have been shown to exhibit proteolytic activity (e.g., Xiao et al., 2011; De Maeyer et al., 2010). Therefore, a preferred ADAMTS13 protein variant according to the present invention comprises at least residues 1-685 of ADAMTS13, in which, compared to wild-type ADAMTS13, one or more N-glycosylation sites have been added and / or one or more existing N-glycosylation sites have been displaced, and further optional mutations as described herein have been introduced. The spacer domain contains surface-exposed residues that form the major epitope recognized by anti-ADAMTS13 autoantibodies. These residues are also referred to as the exosite-3 domain. This domain includes amino acid residues R568, F592, R660, Y661, and Y665. Alanine mutations at R660, Y661, and Y665 impair ADAMTS13 recognition by VWF (Pos et al., 2010; Pos et al., 2011). However, conservative amino acid substitutions at exosite-3 residues have been shown to result in a gain-of-function ADAMTS13 variant (Jian et al. 2012). Initially, this variant was also thought to be resistant to autoantibodies, but follow-up experiments have revealed that it is still targeted by patient-derived autoantibodies (Graca et al. 2019).Without wishing to be bound by theory, it is believed that N-glycans attached to asparagine residues within the spacer domain can shield the exosite-3 domain, thereby reducing or preventing autoantibody binding to this site.

[0045] In a particularly preferred embodiment, an ADAMTS13 protein variant according to or for use in accordance with the present invention comprises one or more additional N-linked glycosylation sites and / or one or more existing N-linked glycosylation sites moved within a spacer domain comprising residues S556 to A685 of the ADAMTS13 sequence shown in Figure 1.

[0046] It is further preferred that the one or more N-linked glycosylation sites are added and / or the one or more existing N-linked glycosylation sites are moved in a portion of the spacer domain comprising residues R568 to R670 of the ADAMTS13 sequence shown in Figure 1.

[0047] In a further preferred embodiment, the ADAMTS13 protein variant according to the present invention or used in accordance with the present invention has a proteolytic activity against von Willebrand factor (VWF) that is at least 10% of the proteolytic activity of wild-type ADAMTS13 protein against VWF. The cutoff for clinically significant levels is set at at least 10% (e.g., Hie et al., 2014). As used herein, "proteolytic activity against VWF" refers to the ability of ADAMTS13 or an ADAMTS13 protein variant to cleave VWF. As used herein, "proteolytic activity against VWF that is x% of that of wild-type ADAMTS13 protein" refers to x% of the proteolytic activity against VWF compared to recombinant wild-type ADAMTS13 under the same conditions. That is, the proteolytic activity against VWF of the ADAMTS13 protein variant according to the present invention or used in accordance with the present invention is compared to the proteolytic activity against VWF of recombinant wild-type ADAMTS13 protein under the same conditions, including, for example, by using the same assay, over the same period of time, and with the same protein concentration. Those skilled in the art are sufficiently competent to evaluate the proteolytic activity of wild-type ADAMTS13 and the ADAMTS13 protein variants according to the present invention or used in accordance with the present invention under the same conditions so that the activities are comparable. Proteolytic activity against VWF can be determined, for example, using assays such as those described in the Examples using FRETS-VWF73 (described in Kokame et al., 2005) and the Examples using VWF multimer assay (described in Graca et al., 2019) herein. Proteolytic activity against VWF can be measured, for example, using a commonly available FRETS-VWF73 substrate (AnaSpec, Fremont, CA, USA) according to the FRETS-VWF73 substrate assay protocol, for example, as described in Example 2.

[0048] Preferably, the ADAMTS13 protein variant according to the invention or for use according to the invention has a proteolytic activity towards VWF that is at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60% of the proteolytic activity towards VWF of the wild-type recombinant ADAMTS13 protein. In a particularly preferred embodiment, the ADAMTS13 protein variant according to the invention or for use according to the invention has a proteolytic activity towards VWF that is at least 70% of the proteolytic activity towards VWF of the wild-type ADAMTS13 protein. The ADAMTS13 protein variant according to the invention or for use according to the invention may also have a proteolytic activity towards VWF that is higher than the proteolytic activity of wild-type ADAMTS13, i.e., a proteolytic activity towards VWF that is more than 100% of the proteolytic activity towards VWF of the wild-type ADAMTS13 protein.

[0049] In a further preferred embodiment, the ADAMTS13 protein variant of the present invention or used in accordance with the present invention exhibits reduced binding by autoantibodies compared to wild-type ADAMTS13. As used herein, "exhibiting reduced binding by autoantibodies" means that autoantibodies specific for ADAMTS13 exhibit reduced binding to the ADAMTS13 protein variant of the present invention or used in accordance with the present invention compared to wild-type ADAMTS13. As used herein, "reduced" preferably means that binding is reduced by at least 10%, preferably at least 15%, more preferably at least 20%, more preferably at least 25%, more preferably at least 50%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%. Thus, "reduced autoantibody binding compared to wild-type ADAMTS13" preferably means that autoantibody binding, as evidenced, for example, by the reactivity of the ADAMTS13 protein variant according to the present invention with sera from iTTP patients as detailed herein below, is reduced by at least 10%, more preferably at least 15%, 20%, 25%, 50%, 75%, 80%, 85%, 90%, or 95% compared to the binding of the wild-type ADAMTS13 protein. Preferably, the autoantibody is an autoantibody that binds to an epitope located within the exosite-3 domain, more preferably an epitope comprising, but not limited to, residues F592, R568, R660, Y661, and / or Y665. Preferably, the autoantibody is an autoantibody present in the sera of patients suffering from immune-mediated thrombotic thrombocytopenic purpura (iTTP). Reduced binding by autoantibodies in iTTP patient sera can be determined, for example, by measuring the reactivity of ADAMTS13 protein variants with a serum or sera of iTTP patients in an assay such as that described in the Examples herein.Briefly, binding of autoantibodies to ADAMTS13 can be detected by immobilizing ADAMTS13 directly on a surface or indirectly by immobilizing monoclonal or polyclonal antibodies against ADAMTS13 (or V5-tag, His-tag, or any other tag) on ​​the surface. The immobilized ADAMTS13 is then incubated with a patient-derived body fluid, preferably plasma or serum, to allow binding of anti-ADAMTS13 immunoglobulins to the immobilized ADAMTS13. Bound patient-derived immunoglobulins reactive with ADAMTS13 can then be detected using a conjugated or labeled antibody that specifically recognizes human immunoglobulins. An example of such an assay is shown in Example 3. Other methods for detecting antigen-specific antibodies in body fluids from patients and normal individuals have been comprehensively described in the literature and can be applied to the detection of antibodies against ADAMTS13 (e.g., Burbelo PD and O'Hanlon TP, 2014). Because autoantibodies in the sera of iTTP patients are heterogeneous, it is preferred that the binding of autoantibodies to the ADAMTS13 protein variants of the present invention be determined in the sera of multiple iTTP patients, for example, in serum or plasma samples from at least five different iTTP patients.

[0050] In a further preferred embodiment, the ADAMTS13 protein variant according to the present invention or for use in accordance with the present invention comprises at least an N-linked glycosylation site at an amino acid residue that is located near or contributes to an autoantibody-binding site on ADAMTS13. That is, the one or more N-linked glycosylation sites that have been added and / or moved compared to wild-type ADAMTS13 are located at amino acid residues that are located near or contribute to an autoantibody-binding site on ADAMTS13. More preferably, the ADAMTS13 protein variant according to the present invention or for use in accordance with the present invention comprises an N-linked glycosylation site at an amino acid residue selected from the group consisting of R568, L591, V604, V605, A606, G607, K608, M609, R636, L637, P638, R639, Y665, L668, and combinations thereof. More preferably, the ADAMTS13 protein variant according to or for use in accordance with the present invention comprises an N-glycan at an amino acid residue mutation selected from the group consisting of R568N, L591N, V604N, V605N, A606N, G607N, K608N, M609N, R636N, L637N, P638N, R639N, Y665N, L668N, and combinations thereof, i.e., one or more N-linked glycosylation sites are added compared to wild-type ADAMTS13 by introducing a mutation selected from this group.

[0051] More preferably, an ADAMTS13 protein variant according to or for use in accordance with the present invention comprises an N-linked glycosylation site at an amino acid position selected from the group consisting of 568, 591, 608, 609, 636, 637, 665, 668 as shown in Figure 1, and a combination thereof, more preferably at an amino acid position selected from the group consisting of 591, 608, 609, 636, 665, 668 as shown in Figure 1, and a combination thereof, more preferably at an amino acid position selected from the group consisting of 608, 609, 665 as shown in Figure 1, and a combination thereof.

[0052] In one embodiment, the ADAMTS13 protein variant according to or for use in accordance with the present invention comprises a mutation selected from the group consisting of 568REY570 to 568NET570 (NGLY1), 591LFT593 to 591NFT593 (NGLY2), 608KMSI611 to 608NMSI611 (NGLY3), 608KMSI611 to 608KNST611 (NGLY4), 636RLPR639 to 636NLSR639 (NGLY5), 636RLPR639 to 636RNAS639 (NGLY6), 665YGNL668 to 665NVTL668 (NGLY7), 667NLTRP671 to 667LNVTA671 (NGLY8), and combinations thereof. That is, one or more N-linked glycosylation sites are added and / or moved compared to wild-type ADAMTS13 by introducing a mutation selected from this group. The designations in brackets, such as "NGLY1", "NGLY2", etc., refer to the variants shown in Tables 1 and 3. The designations "N-glyx" and "NGLYx", such as "N-gly1" and "NGLY1" or "N-gly2" and "NGLY2", are used interchangeably herein.

[0053] In a further preferred embodiment, the ADAMTS13 protein variants according to or for use in accordance with the present invention are 591LFT593 to 591NFT593 (NGLY2), 608KMSI611 to 608NMSI611 (NGLY3), 608KMSI611 to 608KNST611 (NGLY4), 665YGNL668 to 665NVTL668 (NGLY7), and 667NLTRP671 to 667L and NVTA671 (NGLY8), more preferably comprising a mutation selected from the group consisting of 608KMSI611 to 608NMSI611 (NGLY3), 608KMSI611 to 608KNST611 (NGLY4), and 665YGNL668 to 665NVTL668 (NGLY7), and most preferably comprising mutations 608KMSI611 to 608NMSI611 (NGLY3). That is, by introducing a mutation selected from this group, one or more N-linked glycosylation sites are added and / or moved compared to wild-type ADAMTS13. As shown in the Examples herein, these variants exhibit particularly strong reductions in binding by autoantibodies present in the sera of iTTP patients, while maintaining proteolytic activity against VWF.

[0054] In addition to the one or more added and / or moved N-linked glycosylation sites, the ADAMTS13 protein variant according to the present invention or for use in accordance with the present invention may contain further mutations at one or more amino acid residues, preferably mutation(s) that do not introduce a glycosylation site into the protein variant. The mutation(s) preferably are mutation(s) in the amino acid sequence of the protein variant compared to the amino acid sequence of the wild-type ADAMTS13 protein. For example, one or more mutations can be introduced that further reduce binding of the ADAMTS13 protein variant by autoantibodies, that result in increased proteolytic activity against VWF, and / or that increase the stability of the ADAMTS13 protein variant.

[0055] The mutations can be substitutions of one amino acid with another, insertions of one or more amino acids, or deletions of one or more amino acids. Preferably, such additional mutation(s) is / are substitutions of one or more amino acids with another amino acid. The mutations can be introduced throughout the sequence of the protein variant. In a preferred embodiment, one or more mutations, preferably substitutions, are introduced at sites within various domains of ADAMTS13 targeted by autoantibodies. In a more preferred embodiment, mutations, preferably substitutions, are introduced at one or more amino acid residues within the spacer domain comprising residues S556 to A685 as shown in FIG. 1. More preferably, the ADAMTS13 protein variant according to the present invention or for use in accordance with the present invention comprises a mutation, preferably a substitution, at an amino acid residue selected from the group consisting of R568, L591, F592, R636, L637, L668, L591, F592, R636, L637, R660, Y661, Y665, L668, and combinations thereof. For example, one or more amino acids (e.g., R660, Y661, and Y665, or R568, F592, R660, and Y661, or R568, F592, R660, Y661, and Y665) that are mutated in a known gain-of-function mutant (i.e., R568, F592, R660, Y661, and Y665) are mutated in the ADAMTS13 protein variant according to the present invention.

[0056] In a preferred embodiment, the ADAMTS13 protein variant of the invention or for use in accordance with the invention comprises a mutation selected from the group consisting of R568K, R568A, R568N, L591A, F592Y, F592A, F592N, R636A, L637A, R660K, R660A, R660N, Y661F, Y661A, Y661N, Y665F, Y665A, Y665N, L668A, and combinations thereof. As shown in the Examples herein and, for example, in Jian et al., 2012, Pos et al., 2010, and Graca et al., 2019, such mutations either preserve or increase the proteolytic activity of ADAMTS13.

[0057] In one preferred embodiment, the ADAMTS13 protein variant according to or for use in accordance with the present invention comprises the mutations R568A and Y665A.

[0058] In one preferred embodiment, the ADAMTS13 protein variant according to or for use in accordance with the present invention comprises the mutations L591A, R636A, L637A, and L668A.

[0059] In one preferred embodiment, the ADAMTS13 protein variant according to or for use in accordance with the present invention comprises the mutations R568A and Y665A or the mutations L591A, R636A, L637A, and L668A.

[0060] In a further preferred embodiment, the ADAMTS13 protein variant according to or for use in accordance with the present invention comprises the mutations R660K, R660A, or R660N; Y661F, Y661A, or Y661N; and Y665F, Y665A, or Y665N.

[0061] In a further preferred embodiment, the ADAMTS13 protein variant according to the invention comprises the mutations R568K, R568A, or R568N; F592Y, F592A, or F592N; R660K, R660A, or R660N; and Y661F, Y661A, or Y661N.

[0062] In a further preferred embodiment, the ADAMTS13 protein variant according to or for use in accordance with the present invention comprises the mutations R568K, R568A, or R568N; F592Y, F592A, or F592N; R660K, R660A, or R660N; Y661F, Y661A, or Y661N; and Y665F, Y665A, or Y665N.

[0063] In a preferred embodiment, an ADAMTS13 protein variant according to the invention or for use in accordance with the invention comprising an N-glycosylation site at amino acid residue 608 of the ADAMTS13 sequence shown in Figure 1, preferably an ADAMTS13 protein variant comprising the sequence 607GNMSI611, further comprises one or more of the above further mutations.

[0064] Mutations introduced into an ADAMTS13 protein variant according to the present invention or used in accordance with the present invention, including both mutations made to add and / or move one or more N-linked glycosylation sites and any additional mutations described herein, preferably result in an ADAMTS13 protein variant containing an amino acid sequence at least 90% identical to the corresponding amino acid sequence in wild-type ADAMTS13. The term "% sequence identity" is defined herein as the percentage of amino acids in an amino acid sequence that are identical to those in a reference or subject amino acid sequence, after aligning the sequences to achieve the highest percent sequence identity and optionally introducing gaps, if necessary. Methods and computer programs for alignment are well known in the art. Those skilled in the art will understand that consecutive amino acid residues in one amino acid sequence are compared to consecutive amino acid residues in another amino acid sequence. Sequence identity is calculated across the entire sequence of the ADAMTS13 protein variant and the corresponding sequence of wild-type ADAMTS13. That is, an ADAMTS13 protein variant consisting of residues 1 to 685 of ADAMTS13 preferably has an amino acid sequence at least 90% identical to the amino acid sequence of residues 1 to 685 of wild-type ADAMTS13; a full-length ADAMTS13 protein variant preferably has an amino acid sequence at least 90% identical to the full-length amino acid sequence of wild-type ADAMTS13; an ADAMTS13 protein variant consisting of residues 1 to 900 of ADAMTS13 preferably has an amino acid sequence at least 90% identical to the amino acid sequence of residues 1 to 900 of wild-type ADAMTS13, etc. The sequence identity is preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, and more preferably at least 98%.

[0065] The present invention further provides an ADAMTS13 protein variant according to the present invention or for use in accordance with the present invention, wherein one or more added N-linked glycosylation sites comprise an N-linked glycan and / or one or more transferred existing N-linked glycosylation sites comprise an N-linked glycan. The protein variant preferably has a proteolytic activity against von Willebrand factor (VWF) that is at least 10% of the proteolytic activity of the wild-type ADAMTS13 protein against VWF, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, and more preferably at least 50% of the proteolytic activity of the wild-type ADAMTS13 protein against VWF. As used herein, the term "N-linked glycan" refers to a carbohydrate moiety attached to a protein or protein variant via a nitrogen bond at an N-glycosylation site. While various N-linked glycans exist, the N-linked glycan may be any glycan that can be attached to an N-linked glycosylation site as defined herein. Those skilled in the art are familiar with glycans that can be attached to N-linked glycosylation sites. Figure 2 shows the structures of preferred common N-linked glycans and other N-linked glycans. In a preferred embodiment, the N-linked glycan is an N-linked glycan selected from the N-linked glycans shown in Figure 2. Attaching an N-linked glycan to an ADAMTS13 protein variant as described herein can be achieved by methods known in the art, including recombinantly producing the protein variant in a suitable host cell capable of producing glycoproteins containing N-linked glycans. Suitable host cells include eukaryotic host cells, particularly mammalian cells, such as CHO cells, NS0 cells, SP2 / 0 cells, PERC.6 cells, or HEK293 cells. Alternatively, in vitro modification of glycosylation patterns is possible.

[0066] In another preferred embodiment, the ADAMTS13 protein variant according to the invention or for use in accordance with the present invention has a proteolytic activity against von Willebrand factor (VWF) that is at least 10% of the proteolytic activity against VWF of the wild-type ADAMTS13 protein. In a further preferred embodiment, the variant is a full-length ADAMTS13 variant, i.e., having amino acids 1 to 1427 as shown in Figure 1. Preferably, the ADAMTS13 protein variant comprises an N-linked glycan at the one or more added N-linked glycosylation sites and / or the one or more transferred existing N-linked glycosylation sites comprises an N-linked glycan, and has a proteolytic activity against VWF that is at least 10% of the proteolytic activity against VWF of the wild-type ADAMTS13 protein. In a further preferred embodiment, the variant comprises additional mutations at one or more amino acid residues within the spacer domain comprising residues S556 to A685, more preferably at amino acid residues selected from the group consisting of R568, L591, F592, R636, L637, L668, L591, F592, R636, L637, R660, Y661, Y665, L668, and combinations thereof, and even more preferably at R568K, R568A, R568N, L591A, F592Y, F592A, F592N, R636A, L637A, R660K, R660A, R660N, Y661F, Y661A, Y661N, Y665F, Y665A, Y665N, L668A, and combinations thereof.

[0067] In another preferred embodiment, an ADAMTS13 protein variant according to or for use in accordance with the present invention comprises one or more additional N-linked glycosylation sites and / or one or more relocated existing N-linked glycosylation sites in a spacer domain comprising residues S556 to A685, compared to wild-type ADAMTS13, and the ADAMTS13 protein variant has proteolytic activity against von Willebrand factor (VWF) that is at least 10% of the proteolytic activity of wild-type ADAMTS13 protein against VWF. Preferably, the ADAMTS13 protein variant comprises an N-linked glycan at the one or more added N-linked glycosylation sites and / or at the one or more relocated existing N-linked glycosylation sites. In a further preferred embodiment, the variant is a full-length ADAMTS13 variant, i.e., having amino acids 1 to 1427 as shown in Figure 1. In a further preferred embodiment, the variant comprises additional mutations at one or more amino acid residues within the spacer domain comprising residues S556 to A685, more preferably at an amino acid residue selected from the group consisting of R568, L591, F592, R636, L637, L668, L591, F592, R636, L637, R660, Y661, Y665, L668, and combinations thereof, and even more preferably at an amino acid residue selected from the group consisting of R568K, R568A, R568N, L591A, F592Y, F592A, F592N, R636A, L637A, R660K, R660A, R660N, Y661F, Y661A, Y661N, Y665F, Y665A, Y665N, L668A, and combinations thereof.

[0068] In another preferred embodiment, the ADAMTS13 protein variants according to or for use in accordance with the present invention include 568REY570 to 568NET570 (NGLY1), 591LFT593 to 591NFT593 (NGLY2), 608KMSI611 to 608NMSI611 (NGLY3), 608KMSI611 to 608KNST611 (NGLY4), 636RLPR639 to 636NLSR639 (NGLY5), 636RLPL639 to 636RNAS639 (NGLY6), 665YGNL668 to 665NVTL668 (NGLY7), 665YGNL668 to 665NVTL668 (NGLY8), 665YGNL668 to 665NVTL668 (NGLY9), 665YGNL668 to 665NVTL668 (NGLY10), 665YGNL668 to 665NVTL668 (NGLY11), 665YGNL668 to 665NVTL668 (NGLY12), 665YGNL668 to 665NVTL668 (NGLY13), 665YGNL668 to 665NVTL668 (NGLY14), 665YGNL668 to 665NVTL668 (NGLY15), 665YGNL668 to 665NVTL668 (NGLY16), 665YGNL668 to 665NVTL668 (NGLY17), 665YGNL668 to 665NVTL668 (NGLY18), 665YGNL668 to 665NVTL668 (NGLY19), 665 One or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13 by introducing mutations selected from the group consisting of NLTRP (NGLY7), NLTRP to LNVTA (NGLY8), and combinations thereof, more preferably NFT to NFT (NGLY2), KMSI to NMSI (NGLY3), KMSI to KNST (NGLY4), and RLPR to NLSR (NGLY5). Preferably, the ADAMTS13 protein variant comprises an N-linked glycan at the one or more added N-linked glycosylation sites and / or the one or more transferred existing N-linked glycosylation sites comprise an N-linked glycan. In a further preferred embodiment, said variant is a full-length ADAMTS13 variant, ie having amino acids 1 to 1427 as shown in FIG.In a further preferred embodiment, the variant comprises further mutations at one or more amino acid residues within the spacer domain comprising residues S556 to A685, more preferably mutations at amino acid residues selected from the group consisting of R568, L591, F592, R636, L637, L668, L591, F592, R636, L637, R660, Y661, Y665, L668, and combinations thereof, even more preferably mutations selected from the group consisting of R568K, R568A, R568N, L591A, F592Y, F592A, F592N, R636A, L637A, R660K, R660A, R660N, Y661F, Y661A,Y661N, Y665F, Y665A, Y665N, L668A, and combinations thereof.

[0069] In another preferred embodiment, an ADAMTS13 protein variant according to or for use in accordance with the present invention has one or more N-linked glycosylation sites added or moved compared to wild-type ADAMTS13 by introducing an N-glycosylation site (NXT or NXS, where X is any amino acid except P) into any of the amino acid sequences shown in Table 5, or by moving an N-glycosylation site to any of the amino acid sequences shown in Table 5, with the proviso that the one or more N-linked glycosylation sites are not added or moved at amino acid residues 464-466, 469-471, 476-478, 493-495, 511-513, and 539-541 of the ADAMTS13 protein variant. Preferably, the ADAMTS13 protein variant comprises an N-linked glycan at one or more of the added N-linked glycosylation sites and / or the moved one or more existing N-linked glycosylation sites comprise an N-linked glycan. In a further preferred embodiment, the variant is a full-length ADAMTS13 variant, i.e., having amino acids 1-1427 as shown in Figure 1. In a further preferred embodiment, the variant comprises additional mutations at one or more amino acid residues within ADAMTS13. In a further preferred embodiment, the variant comprises further mutations at one or more amino acid residues within the spacer domain comprising residues S556 to A685, more preferably mutations at amino acid residues selected from the group consisting of R568, L591, F592, R636, L637, L668, L591, F592, R636, L637, R660, Y661, Y665, L668, and combinations thereof, even more preferably mutations selected from the group consisting of R568K, R568A, R568N, L591A, F592Y, F592A, F592N, R636A, L637A, R660K, R660A, R660N, Y661F, Y661A,Y661N, Y665F, Y665A, Y665N, L668A, and combinations thereof.

[0070] In another preferred embodiment, the ADAMTS13 protein variant according to the present invention or for use according to the present invention has an additional N-linked glycosylation site compared to wild-type ADAMTS13 by introducing a mutation selected from the group consisting of 591LFT593 to 591NFT593 (NGLY2), 608KMSI611 to 608NMSI611 (NGLY3), 608KMSI611 to 608KNST611 (NGLY4), and 636RLPR639 to 636NLSR639 (NGLY5), and the ADAMTS13 protein variant has an N-linked glycosylation site at the additional N-linked glycosylation site. and Y665F, Y665A, or Y665N.

[0071] Particularly preferred ADAMTS13 protein variants are NGLY1, NGLY2, NGLY3, NGLY4, NGLY5, NGLY6, NGLY7, NGLY8, NGLY3+NGLY7, NGLY+NGLY8, NGLY3+L591A / R636A / L637A / L668A, NGLY3+R568A / Y665A, and NGLY3+R568A / Y665A+L591A / R636A / L and NGLY3+R568A / Y665A, more preferably the variants described herein designated as NGLY2, NGLY3, NGLY4, NGLY5, NGLY7, NGLY8, NGLY3+NGLY7, NGLY+NGLY8, NGLY3+L591A / R636A / L637A / L668A, and NGLY3+R568A / Y665A.

[0072] The present invention also provides nucleic acids encoding the ADAMTS13 protein variants according to the present invention. Nucleic acid constructs comprising nucleic acid sequences encoding the ADAMTS13 protein variants according to the present invention are further provided. Both the nucleic acid sequences and constructs according to the present invention are useful for therapeutic applications and for preparing the ADAMTS13 protein variants according to the present invention.

[0073] As used herein, the term "nucleic acid" refers to DNA and RNA, including mRNA or cDNA, and synthetic variants thereof. The nucleic acid may be recombinant or synthetic.

[0074] The nucleic acid construct according to the invention is preferably present in a vector, such as an expression vector. The expression vector can be a viral or non-viral vector. Non-limiting examples of suitable expression vectors include retroviral vectors, adenoviral vectors, adeno-associated vectors, herpes simplex vectors, and lentiviral vectors, non-viral vectors, and engineered vectors. Non-viral expression vectors include nude DNA and nucleic acids packaged in synthetic or engineered compositions, such as liposomes, polymers, nanoparticles, and molecular conjugates. Methods for generating such non-viral expression vectors are well known in the art. The expression vector preferably contains a strong promoter / enhancer, such as a CMV or SV40 promoter, an optimal translation initiation sequence, such as a ribosome binding site and start codon, and / or a transcription termination sequence, including a poly(A) signal, if the protein is expressed in a eukaryotic cell. Those skilled in the art will understand that the expression vector to be used depends on the host cell used to express the ADAMTS13 protein variant, preferably an N-linked glycan-containing protein variant, according to the present invention. The expression vector is preferably suitable for expression of the nucleic acid molecules of the invention in eukaryotic host cells, more preferably mammalian host cells, more preferably CHO cells, NS0 cells, SP2 / 0 cells, PERC.6 cells, and / or HEK293 cells.

[0075] Alternatively, the nucleic acid sequences used in accordance with the present invention may be provided to a subject by gene editing techniques, including CRISPR / Cas, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs), to insert a receptor transgene into a specific genomic locus, with or without an exogenous promoter. Preferred genomic loci include the AAVS1 locus and the PD-1 locus, as known to those skilled in the art.

[0076] Also provided are pharmaceutical compositions comprising the ADAMTS13 protein variants of the present invention and one or more pharmaceutically acceptable carriers, adjuvants, excipients, and / or diluents. By "pharmaceutically acceptable," it is meant that the auxiliary carrier, diluent, or excipient must be compatible with the other ingredients of the formulation and not harmful, e.g., toxic, to the recipient. Generally, any pharmaceutically acceptable additive that does not interfere with the function of the active compound can be used. The pharmaceutical compositions of the present invention are preferably suitable for human use.

[0077] Examples of suitable carriers include aqueous solutions, lactose, starch, cellulose derivatives, etc., or mixtures thereof. In a preferred embodiment, the suitable carrier is aqueous solution, for example, physiological saline. To prepare dosage units, for example, tablets, it is envisioned to use conventional additives such as fillers, colorants, polymer binders, etc. Examples of excipients that can be incorporated into tablets, capsules, etc. include binders such as tragacanth gum, gum arabic, corn starch, or gelatin; excipients such as microcrystalline cellulose; disintegrants such as corn starch, pregelatinized starch, alginic acid, etc.; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin.

[0078] The pharmaceutical composition according to the present invention is preferably suitable or adapted for parenteral administration. The administration is preferably intravenous, intraarterial, subcutaneous, and / or intramuscular. The administration can be by injection or infusion. An injectable composition, e.g., for intravenous administration, can be, for example, a sterile aqueous solution, oily solution, dispersion, emulsion, and / or suspension, such as an isotonic aqueous buffer solution, preferably a solution containing the ADAMTS13 protein variant of the present invention in an aqueous solution. The injectable composition, e.g., for intravenous administration, can contain, for example, a solubilizing agent, a stabilizer, and / or a local anesthetic to ease pain at the site of injection or infusion.

[0079] It is within the skill of one in the art to determine appropriate dosing regimens, i.e., dosage amounts and administration intervals, depending on the condition to be treated and the desired effect (e.g., short-term action or long-term treatment). The exact dosage and regimen of these compounds and compositions thereof will further depend on the biological activity of the ADAMTS13 protein variant, the age, weight, and sex of the subject, the needs, pain, or severity of the individual subject receiving the medication, and the judgment of the physician. Examples of suitable dosages include doses in the range of 0.1 mg to 15 g, e.g., 1 to 10 g, of the ADAMTS13 protein variant of the present invention.

[0080] In an embodiment of the present invention, a pharmaceutical kit or kit of parts is provided that includes one or more containers containing one or more pharmaceutical compositions of the present invention. Various documents can be associated with such containers, such as instructions for use or notices in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical products, which notice represents agency approval for manufacture, use, or sale for administration to humans or animals. Preferably, the pharmaceutical kit or kit of parts includes instructions for use.

[0081] The ADAMTS13 protein variant used according to the present invention can be administered to a subject by various routes. For example, the protein variant can be administered by any suitable parenteral or oral route, including, for example, topical administration (e.g., cream, ointment, eye drop) or intranasal administration (e.g., solution, suspension). Parenteral administration can include, for example, intraarticular administration, intramuscular administration, intravenous administration, intraventricular administration, intraarterial administration, intrathecal administration, subcutaneous administration, or intraperitoneal administration. Intravenous administration and subcutaneous administration may be most advantageous. Furthermore, the protein variant may be administered to a subject by infusion or injection by a medical professional in a hospital.

[0082] The ADAMTS13 protein variants according to the present invention can be prepared by methods generally known and available in the art.For example, those skilled in the art will know how to prepare DNA sequences encoding the amino acid sequences of the protein variants according to the present invention using commonly known recombinant DNA techniques, and how to prepare and isolate nucleic acid molecules having said DNA sequences.The sequence of the nucleic acid molecule can be codon-optimized for expression in a suitable host cell.

[0083] The nucleic acid molecule is preferably introduced into an expression vector as described herein above using recombinant DNA techniques known to those skilled in the art.The expression vector in the context of the present invention directs the expression of the protein variant according to the present invention in a suitable host cell as described herein.As an alternative, the nucleic acid molecule can be inserted into the genome of the host cell using a suitable gene editing technique as described herein.The insertion is preferably within a locus or region that ensures the expression of the nucleic acid molecule of the present invention in the host cell.

[0084] The term "host cell," as used herein, refers to any cell capable of expressing a heterologous protein, polypeptide, or peptide. In a preferred embodiment, the host cell is capable of attaching N-linked glycans to a protein, peptide, or polypeptide. In a further preferred embodiment, the host cell is a eukaryotic host cell, more preferably a mammalian cell, more preferably selected from the group consisting of CHO cells, NS0 cells, SP2 / 0 cells, PERC.6 cells, and HEK293 cells. Suitable transfection techniques are known in the art, for example, from Green and Sambrook, 2012. Molecular Cloning: A Laboratory Manual, 4th ed., CSHL Press; Cold Spring Harbor Protocols, www.cshprotocols.cshlp.org.

[0085] The ADAMTS13 protein variants described herein have VWF proteolytic activity and are therefore particularly useful for treating disorders and / or thrombotic diseases characterized by aberrant VWF activity and / or VWF processing.

[0086] Thus, the present invention provides an ADAMTS13 protein variant comprising residues 1-685 of ADAMTS13, in which one or more N-linked glycosylation sites have been added compared to wild-type ADAMTS13, and / or one or more existing N-linked glycosylation sites have been moved compared to wild-type ADAMTS13, or a nucleic acid construct encoding such an ADAMTS13 protein variant, for use in therapy. Also provided is such an ADAMTS13 protein variant or a nucleic acid construct encoding such an ADAMTS13 protein variant, for use as an antithrombotic agent. As used herein, the term "antithrombotic agent" refers to a compound that prevents the formation of blood clots, reduces or slows the formation of blood clots, and / or combats existing blood clots.

[0087] The present invention also provides an ADAMTS13 protein variant comprising residues 1-685 of ADAMTS13, which has one or more additional N-linked glycosylation sites compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites shifted compared to wild-type ADAMTS13, or a nucleic acid construct encoding such an ADAMTS13 protein variant, for use in treating a disorder characterized by aberrant von Willebrand factor (VWF) activity and / or VWF processing. Also provided is a method for treating a disorder characterized by aberrant von Willebrand factor (VWF) activity and / or VWF processing, comprising administering to a subject in need thereof an ADAMTS13 protein variant comprising residues 1-685 of ADAMTS13, which has one or more additional N-linked glycosylation sites compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites shifted compared to wild-type ADAMTS13, or a nucleic acid construct encoding such an ADAMTS13 protein variant. As used herein, the term "von Willebrand factor" or "VWF" refers to a plasma glycoprotein that mediates platelet adhesion and aggregation. VWF is synthesized by endothelial cells and megakaryocytes as long multimers with molecular weights up to more than 20,000 kDa. The majority of circulating VWF is synthesized by endothelial cells. The majority of secreted VWF consists of prothrombotic ultra-large VWF (ULVWF) multimers. As described hereinabove, the prothrombotic activity of VWF is regulated by limited cleavage by ADAMTS13 during normal hemostasis. As used herein, "abnormal VWF activity" means that active VWF activity, in particular the prothrombotic activity of VWF, deviates from, and preferably is increased compared to, VWF activity in healthy subjects. This deviation or increase is particularly such that it results in adverse health effects, i.e., a disease or disorder.As used herein, "abnormal VWF processing" means that VWF processing, particularly VWF cleavage, particularly VWF multimer cleavage, deviates from VWF processing in healthy subjects, particularly is reduced compared to VWF processing in healthy subjects. As will be recognized by those skilled in the art, ADAMTS13 protein variants can be used to correct ADAMTS13 deficiency in a subject. Thus, in principle, any disorder in which VWF activity or processing is abnormal can be treated with the ADAMTS13 protein variants described herein. As used herein, the term "ADAMTS13 deficiency" refers to ADAMTS13 not exhibiting its role in hemostasis (controlling the size of VWF multimers by cleavage) as in healthy subjects. This can be caused by low ADAMTS13 protein levels, an excess of its substrate VWF, or the presence of autoantibodies against ADAMTS13. Preferably, the ADAMTS13 deficiency is due to the presence of autoantibodies in the subject.

[0088] In a preferred embodiment, there is provided an ADAMTS13 protein variant comprising residues 1-685 of ADAMTS13, wherein one or more additional N-linked glycosylation sites have been added compared to wild-type ADAMTS13, and / or one or more existing N-linked glycosylation sites have been relocated compared to wild-type ADAMTS13, or a nucleic acid construct encoding such an ADAMTS13 protein variant, for use in the treatment of thrombotic disorders, both acquired and congenital. Also provided is a method for the treatment of thrombotic disorders, both acquired and congenital, comprising administering to a subject in need thereof an ADAMTS13 protein variant comprising residues 1-685 of ADAMTS13, wherein one or more additional N-linked glycosylation sites have been added compared to wild-type ADAMTS13, and / or one or more existing N-linked glycosylation sites have been relocated compared to wild-type ADAMTS13, or a nucleic acid construct encoding such an ADAMTS13 protein variant. Such ADAMTS13 protein variants are able to cleave VWF, thereby reducing the activity of VWF, thereby reducing the prothrombotic activity of VWF.

[0089] In a further preferred embodiment, the disorder is thrombotic microangiopathy. More preferably, the disorder is selected from the group consisting of thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), ischemic stroke, systemic thrombosis, COVID-19, antiphospholipid syndrome, preeclampsia / HELLP syndrome, sepsis, and sickle cell disease.

[0090] ADAMTS13 is, for example, Adamts13 - / - ADAMTS13 is known to have systemic antithrombotic effects, as described by Chauhan et al. (2006), who demonstrated spontaneous thrombus formation in mice and concluded that ADAMTS13 has potent natural antithrombotic activity and that recombinant ADAMTS13 could be used as an antithrombotic agent. Therefore, the ADAMTS13 protein variants described herein can be advantageously used to treat systemic thrombosis.

[0091] Thrombotic microangiopathy includes thrombotic thrombocytopenic purpura (TTP). TTP includes both immune-mediated TTP (iTTP) and congenital TTP (cTTP). In a preferred embodiment, the thrombotic microangiopathy is TTP. In a more preferred embodiment, the TTP is iTTP. In both iTTP and congenital TTP, ADAMTS13 levels are significantly reduced. Currently, recombinant wild-type ADAMTS13 is being tested in clinical trials for the treatment of both cTTP and iTTP (Scully et al., 2019; clinical trial identification number: NCT03922308). Therefore, the ADAMTS13 protein variant according to the present invention can be used for the treatment of both iTTP and congenital TTP. Autoantibodies present in patients with iTTP limit the effectiveness of treatment with wild-type ADAMTS13, as present in human plasma or produced as wild-type recombinant ADAMTS13 protein in eukaryotic expression systems. Autoantibody-resistant ADAMTS13 variants allow for the immediate restoration of functional ADAMTS13 levels, thereby alleviating the severe thrombotic complications and other thrombotic disorders observed in patients with iTTP.

[0092] HUS is characterized by hemolytic anemia, thrombocytopenia, systemic thrombotic microangiopathy (TMA), and renal failure. Partial ADAMTS13 deficiency may be present in HUS patients. Therefore, the ADAMTS13 protein variants described herein can be advantageously used to treat HUS, particularly HUS associated with partial ADAMTS13 deficiency.

[0093] Thrombosis is the primary underlying mechanism of acute ischemic stroke (AIS). Several studies have found that ADAMTS13 levels are significantly decreased in patients with ischemic stroke, with the lowest ADAMTS13 levels observed in patients with acute stroke. As detailed in the review by Chen et al. (2019), available evidence indicates that ADAMTS13 is closely related to the occurrence, progression, and prognosis of ischemic stroke and protects the brain from ischemia-reperfusion injury. The VWF:ADAMTS13 ratio is strongly correlated with stroke risk. ADAMTS13 activity and levels have good predictive value for the occurrence and prognosis of ischemic stroke. In addition, animal studies on ADAMTS13 in the treatment of AIS have shown significant progress: injection of recombinant ADAMTS13 into wild-type mice 7 days after stroke onset increased the formation of neovasculature and vascular repair, significantly improving prognosis 14 days after stroke. It is concluded that ADAMTS13 is expected to be a novel therapeutic agent for ischemic stroke. Therefore, the ADAMTS13 protein variants described herein can be advantageously used in the treatment of ischemic stroke.

[0094] Sepsis is a coagulation disorder, and thrombotic microangiopathy may be a component of it. Thrombotic microangiopathy in sepsis is associated with low levels of ADAMTS-13. Ramsi and Al Ali (2018) described a case of sepsis-associated thrombocytopenia-associated multiple organ failure (TAMOF) that dramatically improved with plasma exchange, which restored ADAMTS13 activity and halted the pathological process and organ failure. Therefore, the ADAMTS13 protein variants described herein can be advantageously used to treat sepsis, particularly thrombotic microangiopathy in subjects with sepsis.

[0095] A low ADAMTS13 / VWF ratio has been observed in sickle cell disease, and ADAMTS13 activity is low in patients with acute chest syndrome, suggesting a quantitative decrease in ADAMTS-13 levels and suggesting that administration of recombinant ADAMTS-13 may have beneficial effects (Sins et al., 2017). This indicates that the ADAMTS13 protein variants according to the present invention can be advantageously used in the treatment of sickle cell disease.

[0096] Thrombosis affecting the pulmonary and systemic vasculature is common during severe COVID-19 (coronavirus disease 2019) caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Turecek et al. (2020) demonstrated that significantly increased plasma VWF levels were accompanied by a partial reduction in the VWF-regulatory protease ADAMTS13. Incubation of plasma samples from patients with severe COVID-19 with recombinant ADAMTS13 (rADAMTS13) significantly reduced abnormally high VWF activity, reduced overall multimer size, and depleted UHMW VWF multimers in a time- and concentration-dependent manner, suggesting that rADAMTS13 may have a therapeutic role in helping to restore hemostatic balance in COVID-19 patients. This indicates that the ADAMTS13 protein variants according to the present invention can therefore be advantageously used in the treatment of COVID-19 and / or SARS-CoV-2 infections.

[0097] Antiphospholipid syndrome and preeclampsia (PEcl) have been associated with reduced ADAMTS13 levels, high ADAMTS13 antibodies, and low ADAMTS13 activity and activity:antigen ratio (Bitsatze et al., 2021). Furthermore, thrombocytopenia and microangiopathic hemolytic anemia (TMA) are observed in HELLP syndrome. Furthermore, Austin et al. (2008) demonstrated that ADAMTS13 autoantibodies and ADAMTS13 dysfunction can occur in antiphospholipid syndrome. This indicates that the ADAMTS13 protein variants according to the present invention can be advantageously used to treat antiphospholipid syndrome and preeclampsia / HELLP syndrome, particularly antiphospholipid syndrome and preeclampsia / HELLP syndrome associated with ADAMTS13 dysfunction.

[0098] The term "subject" as used herein means the recipient of an ADAMTS13 protein variant or encoding nucleic acid according to the present invention, and includes humans and animals. The subject is preferably a mammal, more preferably a human.

[0099] As used herein, the term "therapeutically effective amount" means the amount of ADAMTS13 protein variant being administered sufficient to relieve to some extent one or more of the symptoms of the disease or disorder being treated, which may be reduction or alleviation of symptoms, reduction or alleviation of the cause of the disease or disorder, or any other desired therapeutic effect.

[0100] As used herein, the term "treating" means inhibiting a disorder, i.e., arresting or reducing its progression or at least one clinical symptom of the disease or condition, and / or alleviating the symptoms of the disease or condition.

[0101] For purposes of clarity and concise description, features may be described herein as part of the same or separate aspects or embodiments of the invention. It will be understood by those skilled in the art that the scope of the invention may include embodiments having all or any combination of the features described herein as part of the same or separate embodiments.

[0102] The invention will now be described in more detail in the following non-limiting examples. [Brief explanation of the drawings]

[0103] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: Amino acid sequence of ADAMTS13; UniProt accession number Q76LX8. [Figure 2] Figure 2: N-linked glycans identified on ADAMTS13 (adapted from Verbij et al., 2016). [ka] . [Figure 3] Figure 3: ADAMTS13 model showing ADAMTS13 exosite-3, which includes residues R568, F592, R660, Y661, and Y665 within the spacer domain. Residues L591, R636, L637, K608, and M609 surrounding exosite-3 are also shown. [Figure 4] Figure 4: Activity of ADAMTS13 variants compared to wild-type ADAMTS13 as determined by the FRETS-VWF73 assay. [Figure 5] Figure 5: Ability of wild-type ADAMTS13 and ADAMTS13 variants to cleave VWF in a VWF multimer assay. [Figure 6] Figure 6: Heatmap showing the reactivity of wild-type ADATMS13 and ADAMTS13 variants to TTP patient autoantibodies. [Figure 7] Figure 7: Activity of ADAMTS13 variants in the presence of TTP patient serum measured using FRETS-VWF73. [Figure 8] Figure 8: Proteolytic activity of WT- and NGLY3 ADAMTS13 under flow conditions. VWF string length was measured manually between both time points, and the reduction in length was calculated. Results are presented as relative activity compared to WT-ADAMTS13 (left) and based on the reduction in VWF string size for each variant (right). Both WT-ADAMTS13 and the NGLY3 variant were shown to be active in this assay. [Figure 9]Figure 9A: Activity of proteolytic ADAMTS13 variants on VWF multimers under turbulent flow, measured in the so-called vortex assay (Zhang et al., 2007). VWF processing by wild-type ADAMTS13 and ADAMTS13 variants is assessed by degradation of high molecular weight multimers. Lanes 1 and 11: VWF only; Lanes 2 and 12: wild-type ADAMTS13 + EDTA; Lanes 3 and 13: wild-type ADAMTS13; Lane 4: MDCTS domain ADAMTS13 + EDTA; Lane 5: MDCTS wild-type; Lane 6: MDCTS 5x Ala + EDTA; Lane 7: MDTCS 5x Ala; Lane 8: ADAMTS13 5x Ala + EDTA; Lane 9: ADAMTS13 5x Ala; Lanes 10 and 20: ExpiCHO supernatant (no ADAMTS13); Lane 14: NGLY3 + EDTA; Lane 15: NGLY3; Lane 16: ADAMTS13 Multi-Ala + EDTA; Lane 17: ADAMTS13 Multi-Ala; Lane 18: NGLY3 + Multi-Ala + EDTA; Lane 19: NGLY3 + Multi-Ala. MDCTS: truncated ADAMTS13 variant (residues 1-685). 5xAla corresponds to R568A / F592A / R660A / Y661A / Y665A. MultiAla corresponds to L591A / R636A / L637A / L668A. Figure 9B: Activity of proteolytic ADAMTS13 variants against VWF multimers under turbulent flow, measured in the so-called vortex assay (Zhang et al., 2007). VWF processing by wild-type ADAMTS13 and ADAMTS13 variants is assessed by degradation of high molecular weight multimers.Lanes 1 and 11: VWF only; Lanes 2 and 12: wild-type ADAMTS13 + EDTA; Lanes 3 and 13: wild-type ADAMTS13; Lane 4: ADAMTS13 + AA + EDTA; Lane 5: ADAMTS13 + AA; Lane 6: NGLY3 + AA + EDTA; Lane 7: NGLY3 + AA; Lane 8: NGLY7 + EDTA; Lane 9: NGLY7; Lanes 10 and 20: ExpiCHO supernatant (no ADAMTS13); Lane 14: NGLY3 + NGLY7 + EDTA; Lane 15: NGLY3 + NGLY7; Lane 16: NGLY8 + EDTA; Lane 17: NGLY8; Lane 18: NGLY3 + NGLY8 + EDTA; Lane 19: NGLY3 + NGLY8. AA corresponds to R568A / Y665A. [Figure 10] Figure 10: Relative activity of NGLY3 variants to wild-type ADAMTS13 (WT) measured using FRETS-VWF73 as a substrate in the presence of 10 μl of patient serum or plasma. For four patients (TTP-007, TTP-041, TTP-042, and TTP-076), a 20 μl addition was also evaluated. The activity obtained for wild-type ADAMTS13 in the absence of patient serum or plasma was set to 100%. Black bars indicate the reactivity of wild-type ADAMTS13 in the presence of various patient plasma and serum; gray bars indicate the activity of NGLY3 variants in the presence of patient plasma and serum. [Figure 11]Figure 11: Autoantibody resistance observed for NGLY3 in patients with high-titer inhibitors. A subset of patient sera and plasma samples that inhibited wild-type ADAMTS13 by at least 50% was selected for this figure. The relative activity of NGLY3 variants to wild-type ADAMTS13 (WT) measured using FRETS-VWF73 as a substrate in the presence of 10 μl of patient serum or plasma is shown. The activity obtained for wild-type ADAMTS13 in the absence of patient serum or plasma was set to 100%. Black bars indicate the reactivity of wild-type ADAMTS13 in the presence of various patient plasma and serum; gray bars indicate the activity of NGLY3 variants in the presence of patient plasma and serum. Asterisks indicate patient samples in which NGLY3 was at least 5-fold more active than wild-type ADAMTS13. [Figure 12] Figure 12: Autoantibody resistance observed for NGLY3, NGLY7, NGLY3+NGLY6, NGLY8, and NGLY3+8 in plasma from two high-titer inhibitor patients. Two patient plasma samples that inhibited wild-type ADAMTS13 by at least 95% were selected for this figure. The relative activity of the NGLY3, NGLY7, NGLY3+NGLY7, NGLY8, and NGLY3+8 variants to wild-type ADAMTS13 (WT) measured using FRETS-VWF73 as substrate in the presence of 10 μl of patient serum or plasma is shown. The activity obtained for wild-type ADAMTS13 in the absence of patient serum or plasma was set to 100%. [Figure 13] Figure 13: Relative activity of the NGLY3+NGLY7 variant to wild-type ADAMTS13 (WT) measured using FRETS-VWF73 as a substrate in the presence of 10 μl of patient serum or plasma. A total of 23 patient samples were analyzed. The activity obtained for wild-type ADAMTS13 in the absence of patient serum or plasma was set to 100%. Black bars indicate the reactivity of wild-type ADAMTS13 in the presence of various patient plasma and serum; gray bars indicate the activity of the NGLY3+NGLY7 variant in the presence of patient plasma and serum. [Figure 14]Figure 14: Autoantibody resistance observed for NGLY3, NGLY3 + Multi-Ala, and NGLY3 + AA in patient samples containing strong inhibitors. The relative activity of NGLY3, NGLY3 + Multi-Ala, and NGLY3-AA variants to wild-type ADAMTS13 (WT) was measured using FRETS-VWF73 as a substrate in the presence of 10 μl of patient serum or plasma. The activity obtained with wild-type ADAMTS13 in the absence of patient serum or plasma was set to 100%. NGLY3 + Multi-Ala corresponds to NGLY3 in the L591A / R636A / L637A / L668A combination; NGLY3 + AA corresponds to NGLY3 in the R568A / Y665A combination. [Figure 15] Figure 15: Mass spectrometry-based identification of N-glycan-modified ADAMTS13. [Example]

[0104] (Example) Example 1. Design of N-glycan variants of ADAMTS13 Autoantibodies arising in patients with immune TTP (iTTP) are frequently directed against an immunodominant region within the spacer domain consisting of residues R568, F592, R660, Y661, and Y665 (see Figure 3). In previous studies, we identified a conserved [ka] semi-conservative [ka] We generated a number of ADAMTS13 variants containing nonconservative (Y / F → N; no additional putative N-glycosylation sites) or alanine (Y / F / R → A) substitutions. A previous gain-of-function variant was also included, in which F568, R592, R660, Y661, and Y665 were all replaced with conservative residues (RFRYY → KYKFF) (Graca et al., 2019). The resulting panel of variants was tested for reactivity with autoantibodies present in the sera of 18 patients with iTTP. Our results show that while nonconservative or alanine mutations within the spacer domain result in significantly reduced autoantibody binding, autoantibody binding to ADAMTS13 spacer domain variants containing semiconservative or conservative mutations was not significantly affected compared to nonconservative variants (Graca et al., 2019). Residues R568, F592, R660, Y661, and Y665 are crucial for optimal VWF cleavage activity of ADAMTS13 (Pos et al., 2010; Jian et al., 2012). Consistent with these data, we confirmed that non-conservative or alanine substitutions resulted in reduced activity, while conservative and semi-conservative substitutions tended to have more maintained or even normal activity. Collectively, our results suggested a "trade-off" between resistance to patient autoantibody binding and proteolytic activity. The results of this study indicated that designing autoantibody-resistant ADAMTS13 variants that maintain significant proteolytic activity is not feasible through systematic substitution of residues R568, F592, R660, Y661, and Y665 in combination, particularly residues F592, R660, and Y661 (Graca et al., 2019). Therefore, we adopted an innovative approach in which residues R568, F592, R660, Y661, and Y665 themselves were left unchanged, thereby allowing binding of the unfolded VWF A2 domain to this region in the ADAMTS13 spacer domain (Pos et al., 2010; Crawley et al., 2011; Ercig et al., 2018a).Based on currently available data, we constructed a model showing the binding of residues E1660-R1668 of the unfolded A2 domain to ADAMTS13 exosite-3, which includes R568, F592, R660, Y661, and Y665 within the spacer domain (Figure 3). Based on this model, we selected several residues just within or surrounding ADAMTS13 exosite-3 within the spacer domain for insertion of additional N-glycans by selectively introducing consensus sites for N-glycan attachment (NXS or NXT) within the spacer domain. Based on the model shown in Figure 3, we inserted N-glycans at amino acid positions 568 (NGLY1), 591 (NGLY2), 608 (NGLY3), 609 (NGLY4), 636 (NGLY5), and 637 (NGLY6). The amino acid substitutions required for N-glycan introduction at these sites are listed in Table 1. Table 1: List of full-length ADAMTS13 NGLY variants generated in Example 1 [Table 1]

[0105] Example 2: Expression and functional evaluation of N-glycan variants The N-glycan variants listed in Table 1 were expressed in CHO cells using QMCF technology (described in European Patent No. EP1851319B1; www.icosagen.com). Full-length wild-type ADAMTS13 (1427 amino acids) and a full-length ADAMTS13 variant containing the substitutions R568A / F592A / R660A / Y661A / Y665A (designated ADAMTS13-AAAAA) were used as controls. A wild-type ADAMTS13 variant truncated beyond the spacer domain (amino acid sequence 1-685) was used as an additional control; this ADAMTS13 variant was designated MDTCS. Another MDCTS variant with the substitutions R568A / F592A / R660 / Y661A / Y665A was also used as a control in our studies; this variant was designated MDTCS-AAAAA. These constructs have been previously described and all were cloned into the plasmid expression vector pQMCF3 (Icosagen Cell Factory OU) (Graca et al., 2019). All cDNAs contained a carboxy-terminal V5 epitope followed by a 6xHis tag (Graca et al., 2019).

[0106] The NGLY variant was constructed as follows: a synthetic DNA fragment encoding residues M509 to W688 (540 bp) into which the NGLY substitution was introduced was designed and ordered by Genewiz (Leipzig, Germany). The synthetic fragment was flanked by an XmaI site at the 5' end and a HindIII site at the 3' end of the fragment. The XmaI site is native to the wild-type ADAMTS13 cDNA sequence. The HindIII site is [ka] The ADAMTS13 fragment was introduced by silent mutations in the nucleotide sequence encoding Q684 (CAG to CAA) and A685 (GCC to GCT), resulting in a global change to . Plasmid pUC57_mut1.1 was specially designed and obtained through Genewiz (Leipzig, Germany). In this plasmid, a larger ADAMTS13 fragment (1245 bp) encoding F494 to C908 and flanked by the 5' native PagI site and the 3' native Esp3I site was cloned. In pUC57_mut1.1, the ADAMTS13 fragment was introduced by silent mutations in the nucleotide sequence encoding L621 (CTG to CTC), resulting in a global change to . [ka] This resulted in a global change to the NGLY1-NGLY6 variant, introducing an additional artificial XhoI site (Graca et al., 2019). First, synthetic DNA fragments (540 bp) encoding NGLY1–NGLY6 were used to replace the corresponding XmaI-HindIII fragments in pUC57_mut1.1. These were then separately embedded to create pUC57_NGLY1–6. Subsequently, larger 1245 bp fragments flanked by the native PagI-Esp3I sites in each pUC57_NGLY were used to replace each wild-type fragment of ADAMTS13 in pQMCF3. The resulting pQMCF3_ADAMTS13-NGLY1–6 variants were then expressed in CHO cells as previously described (Graca et al., 2019). Supernatants were collected 10–12 days after transfection, clarified by centrifugation, and stored at -30 °C until further use.

[0107] ADAMTS13 levels in the culture supernatant were quantified by ELISA using a previously established assay (Alwan et al., 2017; Graca et al., 2019). ADAMTS13 antigen levels measured against various proteins ranged from approximately 1.0 to 2.5 μg / ml (see Table 2), similar to wild-type ADAMTS13 levels in the culture supernatant. Consistent with previous findings, MDCTS and MDCTS-AAAAA were expressed at higher levels (Table 2). These data indicate that ADAMTS13-NGLY1-6 are secreted from transfected CHO cells at levels similar to those of wild-type ADAMTS13. Table 2 - Antigen levels of ADAMTS13 NGLY variants and controls [Table 2]

[0108] We also tested whether various ADAMTS13-NGLY variants could process a small fluorogenic substrate, designated FRETS-VWF73, a minimal peptide representing the A2 domain of VWF containing the scissile bond between Tyr1605 and Met1606, and whether it could be used to assess ADAMTS13 activity (Kokame et al., 2005). Diluted culture supernatant containing ADAMTS13-NGLY variants at a concentration of 1.05 nM (0.2 μg / ml) was used in these assays. ADAMTS13 was first diluted to 2.10 nM in a volume of 100 μl in an activity buffer consisting of 20 mM HEPES, 20 mM Bis-Tris, 20 mM Tris-HCl, and 25 mM CaCl2 (pH 6.0) supplemented with 0.005% Tween 20. The reaction was then initiated by adding FRETS-VWF73 substrate (100 μl, 4 μM) and further diluting ADAMTS13 to 1.05 nM. In parallel, a standard curve was generated in a similar manner using wild-type ADAMTS13 diluted over a concentration range of 0.13125 to 2.10 nM. Activity levels were interpolated and compared to those of wild-type ADAMTS13 with a final concentration of 1.05 nM, which was set at 100%. The results of this analysis are shown in Figure 4. NGLY6 showed reduced activity compared to wild-type ADAMTS13. The activity of NGLY5 was slightly reduced compared to wild-type ADAMTS13. The potencies of NGLY1, 2, 3, and 4 were similar or even higher than those of wild-type ADAMTS13 (Figure 4).

[0109] We subsequently tested the activity of the ADAMTS13-NGLY1-6 variants in a more physiological VWF multimer assay essentially as previously described (Graca et al., 2019). ADAMTS13 variants were incubated at a concentration of 0.2 μg for 30 min at 37°C in activation buffer consisting of 20 mM Bis-Tris, 20 mM Tris-HCl, 20 mM HEPES, 25 mM CaCl2 (pH 7.5), 0.005% Tween 20, and 2% bovine serum albumin fraction V (Merck). In parallel, human recombinant VWF produced in HEK293 cells was incubated with 3 M urea at a final concentration of 80 nM for 30 min at 37°C. Denatured recombinant VWF multimers were then added to an ADAMTS13-containing mixture at a 1:1 ratio (final ADAMTS13 = 1.9 nM; final VWF = 40 nM). The ability to cleave VWF under these conditions was visualized by the disappearance of high-molecular-weight (HMW) multimers from the top of the gel and the accumulation of cleavage products, as evidenced by the appearance of high-intensity bands and satellite bands in the lower part of the gel. Samples were collected and quenched at 0, 30, and 24 hours with 4x loading buffer (composition: 9.6 M urea, 4% SDS m / v, 0.035 M Tris base, 25 mM EDTA, 7.5 μM bromophenol blue, no pH adjustment) to assess the ability of the various ADAMTS13-NGLY1-6 variants to process VWF. The results are shown in Figure 5. Under these experimental conditions, reduced VWF processing activity was observed for NGLY2 and NGLY5. The VWF processing activity of NGLY3 and NGLY4 was similar to that of wild-type ADAMTS13 (Fig. 5).

[0110] Taken together, these results demonstrate that NGLY2, 3, 4, and 5 exhibit activity in both the multimer and FRETSVWF73 assays.

[0111] Example 3. Binding of pathogenic autoantibodies from TTP patients to ADAMTS13 NGLY variants Binding of autoantibodies present in collected samples from patients with iTTPA previously developed ELISA was used to assess binding in a series of 13 iTTP patient samples (kindly provided by Professors Paul Coppo and Agnes Veyradier (Centre de Reference des Microangiopathies Thrombotiques - CNR-MAT, AP-HP, Paris, France)). A protocol for assessing patient-derived autoantibodies has been previously published (Graca et al., 2019). Plates were coated overnight with 100 μl of monoclonal antibody 3H9 (kindly provided by Professor Vanhoorelbeke, KU Leuven, Belgium) at a concentration of 1 μg / ml. Plates were then blocked with phosphate-buffered saline (PBS) supplemented with 2% BSA. Plates were then incubated with 1.05 nmol / well of ADAMTS13 (200 ng / well for full-length ADAMTS13 and 78.75 ng / well for MDTCS variants). Subsequently, various dilutions of 100 μl of each patient sample were tested for reactivity with each ADAMTS13 variant. Next, binding of patient IgG to the immobilized ADAMTS13 variants was assessed by incubating 100 μl of a pool of monoclonal antibodies against human IgG1, IgG2, IgG3, and IgG4 (Sanquin, The Netherlands), each conjugated with horseradish peroxidase and diluted 1:10,000, essentially as previously described (Graca et al., 2019). The dilutions used for the various patient samples ranged from 30× to 400×, depending on the amount and affinity of anti-ADAMTS13 antibodies present in the patient sample. Patient sample dilutions were adjusted to match an optimal target optical density of 1.6 at 450 nm (using 540 nm as a reference). A dilution curve of the human monoclonal anti-ADAMTS13 antibody II-1 was included in all experiments to correct for potential inter-assay variability, as previously outlined for data interpolation (Graca et al., 2019).The reactivity of autoantibodies in each patient sample with the ADAMTS13 NGLY variant was compared with that observed with wild-type ADAMTS13. Patient autoantibody binding was expressed as a percentage of the patient autoantibody binding to wild-type ADAMTS13. The results of this analysis are shown in Figure 6. The reactivity of autoantibodies present in the patient samples was displayed as a heat map, with reactivity with wild-type ADAMTS13 set at 100%. We also determined the reactivity of various patient samples with truncated MDTCS variants. Reduced signals were observed for three of the 13 samples (TTP-049, TTP-080, and TTP-085) compared to wild-type ADAMTS13. These data indicate that antibodies against the proximal TSP2-8 and CUB1 / 2 domains were also present in these samples. Strongly reduced reactivity with ADAMTS13-AAAAA was observed, as indicated by the green color code. Autoantibodies present in 10 of 13 patient samples did not bind to ADAMTS13-AAAAA. Residual binding to ADAMTS13-AAAAA was still observed for TTP-049, TTP-080, and TTP-085. Autoantibody reactivity in patient samples to MDTCS-AAAAA was strongly reduced in 9 of 13 patients. Residual binding to MDTCS was still observed in 4 patient samples (TTP-017, TTP-042, TTP-079, and TTP-080) (Figure 6; lane MDTCS AAAAA). Collectively, these findings indicate that autoantibodies target residues in the immunodominant epitope, consisting of R568, F592, R660, Y661, and Y665, within the spacer domain in the majority of patient samples included in this study. We subsequently addressed the reactivity of autoantibodies present in our panel of patient antibodies with ADAMTS13 NGLY1-6 (Figure 6). A slight decrease in binding was observed for NGLY1, NGLY2, and NGLY5. A more pronounced decrease in binding was observed for NGLY4 and NGLY6. Interestingly, autoantibody binding was strongly reduced for NGLY3 in 11 of 13 patients.Autoantibodies present in patient samples TTP-079 and TTP-085 still reacted strongly with NLGY3; this is most likely due to the presence of autoantibodies that bind outside the spacer domain of ADAMTS13. The lack of reactivity of the majority of patient-derived autoantibodies with NLGY3 indicates that the introduction of an N-linked glycan at amino acid position 608 abolishes autoantibody binding to the immunodominant B cell epitope within the spacer domain of ADAMTS13.

[0112] Example 4: N-glycan variants of ADAMTS13 maintain activity in the presence of pathogenic autoantibodies from patients with iTTP In Example 2, we demonstrated that NGLY2, NGLY3, NGLY4, and NGLY5 are capable of proteolytic processing of VWF multimers and the small peptide substrate FRETS-VWF73. In Example 3, we demonstrated that NGLY3 is poorly recognized by pathogenic autoantibodies present in samples from iTTP patients. This prompted us to evaluate whether NGLY3 still possesses the ability to process FRETS-VWF73 in the presence of plasma samples from iTTP patients. To test this, we selected two patient samples based on the data shown in Figure 7: sample TTP-008, which showed limited reactivity with NGLY3; and sample TTP-085, which still showed 98% reactivity with NGLY3 (likely due to autoantibodies against the carboxy-terminal TSP2-8 and / or CUB1 / 2 domains of ADAMTS13). To test whether NGLY3 maintains activity in the presence of these autoantibodies, we assessed activity using the FRETS-VWF73 assay described in Example 2. Prior to the addition of FRETS-VWF73 substrate, ADAMTS13 variants (2.10 nM) were incubated in the presence of 10 μl of patient sample or PBS at 37°C for 30 minutes. The final volume was 210 μl, of which 10 μl was added patient plasma or PBS (control). The activity level of 1.05 nM wild-type ADAMTS13 incubated in the absence of patient sample was set to 100%. The results of these experiments are illustrated in Figure 7. Incubation of wild-type ADAMTS13 with sample TTP-008 resulted in an approximately 50% decrease in activity. Incubation of wild-type ADAMTS13 with sample TTP-085 resulted in an approximately 75% decrease in activity. These results indicate that the autoantibodies present in samples TTP-008 and TTP-085 can inhibit the processing activity of ADAMTS13. We next evaluated whether these samples could also inhibit the activity of NGLY3 variants.Incubation with both TTP-008 and TTP-085 resulted in a decrease in activity from 125% to approximately 75% for both (Figure 7).

[0113] Furthermore, we analyzed the ability of NGLY3 to process VWF in various flow or shear rate assays. First, we evaluated the ability of NGLY3 to process VWF strings under flow on the surface of endothelial cells. Endothelial cells were grown in Ibidi μ-Slide VI channels coated with 1% gelatin and then seeded. HUVECs (Promocell, passage 3) were seeded at 50,000 cells per channel. The channel medium was refreshed twice daily with EGM-18 medium (Promocell) containing Supplement Mix (2% v / v) (Promocell) and penicillin / streptomycin (1% v / v) (Sigma). Measurements were performed on day 4 of confluence. Flow experiments were performed using a flow rate of 2 mL / min, corresponding to a shear stress of approximately 2.5 dynes / cm². Prior to measurement, cells were starved for 5 minutes using M199 medium (Gibco) supplemented with 0.2% BSA. Subsequently, cells were stimulated with 100 μM histamine in M199 medium supplemented with 0.2% BSA for 10 minutes. Next, VWF strings were stained for 5 minutes using an AlexaFluor-488-labeled anti-VWF polyclonal antibody (DAKO) at a dilution of 1:2000. ADAMTS13 variants were diluted to a final concentration of 0.1 μg / mL in M199 medium supplemented with 0.2% BSA. ADAMTS13-containing medium was allowed to flow over the cells for 10 minutes, during which time images were taken at three separate locations at 10-second intervals using a Zeiss Axio Observer Z1 microscope. The first and last images at each location were analyzed using ImageJ. The length of each VWF string was measured manually, and the difference in total length before and after ADAMTS13 incubation was used to determine the activity of the protein. For control, we used medium that had been in contact with ExpiCHO cells, which do not produce any ADAMTS13 (FIG. 8).

[0114] We also employed a so-called vortex assay (Zhang et al., 2007) to determine the ability of NGLY3 to process VWF multimers under turbulent flow. Incubation of 40 nM VWF with 1.0 μg / ml recADAMTS13 for 30 minutes under turbulent flow at 3000 rpm in a reaction buffer consisting of 5 mM CaCl2, 20 mM Bis-Tris, 20 mM HEPES, 20 mM Tris-HCl, and 0.005% v / v Tween 20 (final reaction volume = 200 μL) resulted in the disappearance of high-molecular-weight multimers from the sample (Figure 9A: lanes 3 and 13). Addition of 50 mM EDTA prevented the cleavage of high-molecular-weight multimers by wild-type ADAMTS13 (Figure 9A: lanes 2 and 128). We then used this vortex-based assay to test the ability of NGLY3 to process VWF multimers under turbulent flow. Similar to wild-type ADAMTS13, NGLY3 was able to efficiently process VWF multimers under turbulent flow (Fig. 9A: lane 15). Similar to wild-type ADAMTS13, the addition of 50 mM EDTA impaired the processing of VWF multimers by NGLY3 (Fig. 9A: lane 14). Collectively, these results indicate that the ability of NGLY3 to cleave VWF substrates under various conditions is comparable to that of wild-type ADAMTS13.

[0115] These results indicate that NGLY3 variants maintain significant proteolytic activity in the presence of autoantibodies against ADAMTS13.

[0116] Next, we tested NGLY3 variants on an expanded panel of 28 patient plasma samples (Figure 10). Overall, NGLY3 variants were highly active in 18 of the 28 patient samples analyzed. In a limited number of patient samples containing low-titer inhibitors, NGLY3 appeared to be slightly more active than wild-type ADAMTS13. In eight patient samples, the level of inhibition observed for NGLY3 was similar to that observed for wild-type ADAMTS13. In two of these patient samples, there was a small but clearly significant difference in favor of NGLY3. Importantly, NGLY3 variants were consistently more or equally effective than wild-type ADAMTS13.

[0117] We analyzed a subset of plasma samples containing high-titer inhibitors. High-titer inhibitors were defined as inhibitor levels that resulted in at least 50% inhibition of wild-type ADAMTS13 (Figure 11). In 13 of the 17 selected patients, NGLY3 variants were superior to wild-type ADAMTS13. In four patients, NGLY3 variants were similarly effective compared to wild-type ADAMTS13. Notably, in seven of the 17 patients, NGLY3 variants appeared to be more than five times more effective than wild-type ADAMTS13 (Figure 11; samples indicated by an asterisk).

[0118] These observations suggest that the therapeutic administration of "glycan-shielded" ADAMTS13 variants may constitute a more efficient treatment option for the treatment of patients with iTTP compared with the administration of wild-type ADAMTS13, either in purified form or as administered by plasma exchange, either as recombinant or plasma-derived ADAMTS13.

[0119] Example 5: Autoantibody-Resistance of an Expanded Panel of N-Glycan Variants As evident from the heat map shown in Figure 6, in Example 3, NGLY3 exhibited low reactivity with the patient's autoantibodies. Furthermore, as we show in Figure 7 and Example 4, NGLY3 maintains proteolytic activity in the presence of autoantibodies. In this example, we present additional NGLY variants in which the native N-glycan inserted at N667 was moved to Y665 (yielding NGLY7) or L668 (yielding NGLY8). Combinations of one or more N-glycan variants may be more efficient. Therefore, we designed combinations of NGLY3 and NGLY7 and NGLY3 and NGLY8 (see Table 3). These variants were constructed in a manner similar to that outlined in Example 1.

[0120] N-glycan-shielded ADAMTS13 variants, including at least one newly introduced or transferred N-glycan, can also be combined with individual amino acid substitutions that reduce pathogenic autoantibody binding. Therefore, we aim to create additional combinations of NGLY3 with alanine mutations (mostly outside exosite-3) and other NGLY modifications within the vicinity of exosite-3 (Table 3). Due to the lack of other promising N-glycosylation sequons within this region, we are exploring strategies to shift the naturally occurring N-glycan present at N667 within the spacer domain of ADAMTS13 by one or two residues in either direction (to the N- or C-terminus). Table 3. ADAMTS13 NGLY3 variants with additional mutations [Table 3]

[0121] These variants were designed as outlined in Example 1. A synthetic DNA fragment encoding residues M509 to W688 (540 bp) into which the new NGLY variants were introduced was designed and ordered by Genewiz (Leipzig, Germany). The synthetic fragment was flanked by an XmaI site at its 5' end and a HindIII site at its 3' end. The synthetic DNA was first cloned into the XmaI-HindIII sites of the plasmid pUC57_mut1.1 (see Example 1), embedded in a larger fragment flanked by PagI-Esp3I sites. This larger fragment was then used to replace the corresponding fragment in wild-type ADAMTS13 present in pcDNA3.1ADAMTS13.

[0122] The resulting NGLY-variants were expressed in Expi-CHO cells according to the manufacturer's instructions (Thermo Fisher Scientific). Supernatants were harvested 4 days after transfection, clarified by centrifugation, supplemented with 10 mM benzamidine, and stored at -30°C until further use. ADAMTS13 levels present in the culture supernatants were quantified by ELISA using a previously established assay (Alwan et al., 2017; GraCa et al., 2019). ADAMTS13 antigen levels ranged from 1.43 to 5.27 μg / ml (see Table 4).

[0123] Subsequently, we employed the FRETS-VWF73 fluorogenic substrate to measure the activity of the novel NGLY variants (Table 4). NGLY7 was 125% more active than wild-type, NGLY8 was 75% more active than wild-type ADAMTS13; NGLY3 + NGLY7 was 125% more active than wild-type; and NGLY3 + NGLY8 was 75% more active than wild-type ADAMTS13. R568A / Y665A was 120% more active than wild-type; and L591A / R636A / L637A / L668A was 90% more active than wild-type. NGLY3+R568A / Y665A was 120% as active as wild-type, NGLY3+L591A / R636A / L637A / L668A was 95% as active as wild-type ADAMTS13, and NGLY3+R568A / Y665A+L591A / R636A / L637A / L668A was 40% as active as wild-type ADAMTS13.

[0124] Taken together, these results indicate that the combinations of NGLY3 and NGLY7, NGLY3 and NGLY8, and NGLY7 and NGLY8 maintain their ability to convert FRETS-VWF73 substrates. The combinations of NGLY3 and R568A / Y665A and / or L591A / R636A / L637A / L668A also maintain their ability to convert FRETS-VWF73 substrates.

[0125] We also evaluated the ability of the novel variants to process VWF multimers under shear stress using a vortex assay. Under these conditions, NGLY3 was fully active; NGLY7 also clearly possessed the ability to process large VWF multimers (Fig. 9B). NGLY8 exhibited a reduced ability to process VWF multimers compared with NGLY3 and NGLY7 (Fig. 9B). The NGLY3 / NGLY8 combination also demonstrated a reduced ability to process VWF multimers under these experimental conditions, whereas the NGLY3 / NGLY7 combination maintained proteolytic activity against multimeric VWF (Fig. 9B). The NGLY3+L591A / R636A / L637A / L668A and NGLY3+R568A / Y665A combinations efficiently processed VWF multimers under these conditions (Fig. 9A). Consistent with these observations, the proteolytic activity of the R568A / Y665A and L591A / R636A / L637A / L668A variants was also reduced compared to wild-type ADAMTS13 (FIG. 9A).

[0126] We also evaluated the ability of the novel variants to process VWF multimers using denaturing conditions. Under these conditions, NGLY3 was fully active, whereas NGLY7 and NGLY8 also showed reduced activity (Table 4). The NGLY3 / NGLY7 and NGLY3 / NGLY8 combinations also showed reduced ability to process VWF multimers under these experimental conditions. The NGLY3+L591A / R636A / L637A / L668A and NGLY3+R568A / Y665A combinations were also relatively inefficient at processing VWF multimers using these specific conditions (Table 4). Table 4 - Antigen levels and activity of ADAMTS13 NGLY variants and controls [Table 4]

[0127] Next, we evaluated whether the newly designed ADAMTS13 variants could neutralize pathogenic autoantibodies generated or derived from patients with immune TTP.

[0128] First, we evaluated the antibody resistance of NGLY7, NGLY8, and the combinations of NGLY3 + NGLY7 and NGLY3 + NGLY8 against two patient samples containing high-titer inhibitors (Figure 12). NGLY7 and NGLY8 were inhibited by patient autoantibodies in a manner similar to the inhibition observed for wild-type ADAMTS13 by autoantibodies present in patient plasma. NGLY3 was only slightly inhibited compared to wild-type ADAMTS13. The combination of NGLY3 and NGLY7 was more autoantibody-resistant than NGLY3 alone. The combination of NGLY3 and NGLY8 was equally resistant compared to NGLY3 alone. Next, we tested the combination of NGLY3 and NGLY7 against 23 patient samples (Figure 13). The combination of NGLY3 and NGLY7 was shown to be autoantibody-resistant in a large number of patient samples (Figure 13).

[0129] Subsequently, we tested the NGLY3 + L591A / R636A / L637A / L668A combination and the NGLY3 + R568A / Y665A combination for their efficacy in ameliorating autoantibody resistance in patient samples. In eight out of eight samples, NGLY3 + R568A / Y665A had slightly higher activity than wild-type ADAMTS13 (Figure 14). The activity level of NGLY3 + R568A / Y665A was slightly higher than that of NGLY3 alone, suggesting an additional benefit of including an additional Ala-substitution in ADAMTS13. The NGLY3 + L591A / R636A / L637A / L668A combination was also evaluated for autoantibody resistance. In eight of eight patient samples tested, NGLY3+L591A / R636A / L637A / L668A had significantly higher activity than wild-type ADAMTS13 (Figure 14). The activity level of NGLY3+L591A / R636A / L637A / L668A in the presence of patient plasma or serum was comparable to that of NGLY3. These findings indicate that substitution of these specific amino acids with alanine has little effect on autoantibody resistance.

[0130] Example 6. Additional N-glycan variants of ADAMTS13 The previous examples focused on the spacer domain, which contains the primary binding site for pathogenic autoantibodies generated in patients with immune TTP. It is well known that autoantibodies can also target other domains on ADAMTS13 (Klaus et al., 2004; Thomas et al., 2015; Pos et al., 2011). Similar to the methods described in Examples 1-4, N-glycan-shielded ADAMTS13 variants can be designed to prevent binding of autoantibodies targeting metalloproteases, disintegrin domains, TSP-1 repeats, Cys-rich domains, epitopes outside R568, F592, R660, Y661, and R665 in the spacer domain, TSP2-8 repeats, and antibody binding sites present within the CUB1 / 2 domain. 5. Furthermore, NGLY3 and / or other N-glycan-shielded ADAMTS13 variants can be combined with individual or multiple amino acid substitutions within the above-mentioned domains that result in reduced autoantibody binding while maintaining at least partial proteolytic activity.

[0131] The 3D structure of ADAMTS13 was used to select surface residues of ADAMTS13 for potential N-glycosylation sites. The crystal structure of ADAMTS13 (PDB: 6qig) was used for the following domains: metalloprotease, disintegrin-like domain, thrombospondin type 1 repeat 1 (TSP1-1), cysteine-rich domain, and spacer domain. The remaining structure was constructed by homology modeling from the TSP1-2 to CUB2 domains as previously described (Ercig et al., 2018b). The 3D structure of ADAMTS13 was examined using the SwissPDB Viewer, and surface residues were manually selected.

[0132] Table 5: Exposed regions on ADAMTS13 that allow insertion or movement of N-glycans that interfere with binding of pathogenic autoantibodies. Bold residues are (part of) the native glycosylation sites of ADAMTS13. Bold and underlined residues are indicated to contain O-glycans. Bold and italicized residues are modified by O-fucosylation of Ser (S) residues or C-mannosylation of Trp (W) residues. [Table 5] TIFF0007748455000011.tif223170TIFF0007748455000012.tif231170TIFF0007748455000013.tif102170

[0133] Example 7. Mass spectrometry-based identification of N-glycan-modified ADAMTS13 In this example, we employed mass spectrometry to provide proof of principle for the successful N-glycosylation of the newly engineered consensus site within ADAMTS13. We selected NGLY3 variants for this analysis. As outlined in the previous example, in NGLY3, K608 is replaced by N608, thereby introducing a consensus site for N-glycan addition (see Figure 15). We also included a variant in which K608 is replaced by alanine (K608A), which does not introduce a consensus site for N-glycosylation (Figure 15). Both NGLY3, K608A, and wild-type ADAMTS13 were expressed in CHO cells as described in the previous example. Each of these three ADAMTS13 variants was purified by immunoprecipitation using mouse anti-V5 antibody coupled to magnetic Dynabeads. Before mass spectrometry analysis, these mutants were subjected to PNGaseF digestion (or no PNGaseF digestion as a control), followed by trypsin digestion of the beads. PNGaseF treatment leads to deamidation of asparagine residues when N-glycans are attached to these residues. For trypsin digestion of ADAMTS13 before mass spectrometry analysis, we employed trypsin, which cleaves after lysine and arginine.

[0134] First, we analyzed which peptide sequences were recovered after mass spectrometry analysis of wild-type ADAMTS13. The overall coverage of trypsin-digested purified wild-type ADAMTS13 was 66%. Only peptides corresponding to amino acid sequence 599-629 are shown in Figure 15. After trypsin digestion of wild-type ADAMTS13, a peptide corresponding to I599-K608 (peptide 1) was identified. A peptide containing M609-R629 (peptide 2) was not identified because the presence of a heterogeneous glycan at N614 prevented mass-based identification of this peptide. Subsequently, we treated wild-type ADAMTS13 with PGNaseF. PNGaseF treatment removes N-glycans and also deamidates asparagine, resulting in a mass increase of 1 dalton. Analysis of ADAMTS13 treated with PGNaseF allowed the identification of peptide I599-K608 and peptide M609-R629. Due to deamidation of N614, the mass of peptide M609-R629 increased by 1 Dalton. These observations indicate that peptide M609-R629 contains an N-glycan inserted at position N614 (shown by the box in Figure 15). This is consistent with the previous identification of the N-glycan at N614 (see Figure 2).

[0135] Next, we analyzed NGLY3 in a similar manner. After trypsin digestion, no peptides corresponding to the region I599-R629 were recovered. This observation suggests that one or more N-glycans may be present in this portion of NGLY3. Treatment with PGNaseF identified one peptide, Y603-R629 (peptide 3; Figure 15 ); consistent with the presence of N-glycans at N608 and N614, this confirmed that N608 and N614 were deamidated. Due to the replacement of K608 by N in NGLY3, this variant can no longer be cleaved at amino acid position 608 by trypsin (shown by the box in Figure 15 ). Together, these results indicate that an N-glycan has been successfully introduced at amino acid position 608 due to the replacement of K608 by N608.

[0136] As an additional control, we analyzed an ADAMTS13 variant in which K608 was replaced by alanine at this position. Consistent with the presence of N-linked glycans in this region, identification of trypsin-cleaved peptides corresponding to this region was not observed in the absence of PGNaseF treatment. After digestion with PGNaseF, a single Y603-R629 peptide containing a deamidated N at position 614 was identified (shown by the box in Figure 15). This analysis indicated that A608 did not contain an N-linked glycan attached to A608; only the N-glycan normally present at N614 was identified in this variant.

[0137] In summary, the approach outlined in this proposal demonstrates that introduction of a consensus site for N-glycan addition at amino acid position 608 of NGLY3 results in the attachment of an N-glycan at this position. Similarly, the presence of other N-linked glycans in NGLY1, 2, 4, 5, 6, 7, 8, and other glycan-modified ADAMTS13 variants, including those listed in Example 7, can be successfully determined using the protocol outlined in this example.

[0138] (References) [Table 6] TIFF0007748455000015.tif232170TIFF0007748455000016.tif225170TIFF0007748455000017.tif136170

Claims

1. 1. An ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13, wherein a spacer domain comprising amino acid residues S556 to A685 of ADAMTS13 has one or more additional N-linked glycosylation sites compared to wild-type ADAMTS13 and / or one or more existing N-linked glycosylation sites shifted compared to wild-type ADAMT S13, wherein said ADAMTS13 has the amino acid sequence: MHQRHPRARCPPLCVAGILACGFLLGCWGPSHFQQSCLQALEPQAVSSYLSPGAPLKGRPPSPGFQRQRQRQRRAAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPKPRQAWVWAAVRGPCSVSCGAGLRWVNYSCLDQARKELVETVQCQGSQQPPAWPEACVLEPCPPYWAVGDFGPCSASCGGGLRERPVRCVEAQGSLLKTLPPARCRAGAQQPAVALETCNPQPCPARWEVSEPSSCTSAGGAGLALENETCVPGADGLEAPVTEGPGSVDEKLPAPEPCVGMSCPPGWGHLDATSAGEKAPSPWGSIRTGAQAAHVWTPAAGSCSVSCGRGLMELRFLCMDSALRVPVQEELCGLASKPGSRREVCQAVPCPARWQYKLAACSVSCGRGVVRRILYCARAHGEDDGEEILLDTQCQGLPRPEPQEACSLEPCPPRWKVMSLGPCSASCGLGTARRSVACVQLDQGQDVEVDEAACAALVRPEASVPCLIADCTYRWHVGTWMECSVSCGDGIQRRRDTCLGPQAQAPVPADFCQHLPKPVTVRGCWAGPCVGQGTPSLVPHEEAAAPGRTTATPAGASLEWSQARGLLFSPAPQPRRLLPGPQENSVQSSACGRQHLEPTGTIDMRGPGQADCAVAIGRPLGEVVTLRVLESSLNCSAGDMLLLWGRLTWRKMCRKLLDMTFSSKTNTLVVRQRCGRPGGGVLLRYGSQLAPETFYRECDMQLFGPWGEIVSPSLSPATSNAGGCRLFINVAPHARIAIHALATNMGAGTEGANASYILIRDTHSLRTTAFHGQ wherein the N-linked glycosylation site comprises one or more amino acids at a site selected from the group consisting of residues 568, 591, 608, 609, 636, 637, 665, 668, and combinations thereof.

2. The ADAMTS13 protein variant of claim 1, which is a full-length ADAMTS13 protein variant.

3. 3. The ADAMTS13 protein variant of claim 1 or 2, comprising an N-glycan at an amino acid residue mutation selected from the group consisting of residues R568N, L591N, K608N, M609N, R636N, L637N, Y665N, L668N, and combinations thereof.

4. The ADAMTS13 protein variant according to any one of claims 1 to 3, comprising an N-linked glycosylation site at amino acid residue 608 of ADAMTS13.

5. 5. The ADAMTS13 protein variant according to any one of claims 1 to 4, having a proteolytic activity against von Willebrand factor (VWF) that is at least 10% of the proteolytic activity against VWF of the wild-type ADAMTS13 protein.

6. 6. The ADAMTS13 protein variant of any one of claims 1 to 5, comprising a mutation selected from the group consisting of 568REY570 to 568NET570 (NGLY1), 591LFT593 to 591NFT593 (NGLY2), 608KMSI611 to 608NMSI611 (NGLY3), 608KMSI611 to 608KNST611 (NGLY4), 636RLPR639 to 636NLSR639 (NGLY5), 636RLPL639 to 636RNAS639 (NGLY6), 665YGNL668 to 665NVTL668 (NGLY7), 667NLTRP671 to 667LNVTA671 (NGLY8), and combinations thereof.

7. 7. The ADAMTS13 protein variant of any one of claims 1 to 6, comprising a mutation selected from the group consisting of 608KMSI611 to 608NMSI611 (NGLY3), 608KMSI611 to 608KNST611 (NGLY4), and 665YGNL668 to 665NVTL668 (NGLY7).

8. 8. The ADAMTS13 protein variant according to any one of claims 1 to 7, comprising the mutations 608KMSI611 to 608NMSI611 (NGLY3).

9. The ADAMTS13 protein variant according to any one of claims 1 to 8, further comprising additional mutations in one or more amino acid residues.

10. 10. The ADAMTS13 protein variant of claim 9, wherein the mutation in one or more amino acid residues is within the spacer domain comprising residues S556 to A685 of ADAMTS13.

11. 11. The ADAMTS13 protein variant of any one of claims 1 to 10, further comprising a mutation at an amino acid residue selected from the group consisting of R568, L591, F592, R636, L637, R660, Y661, Y665, L668, and a combination thereof.

12. 12. The ADAMTS13 protein variant of any one of claims 1 to 11, comprising a mutation selected from the group consisting of R568K, R568A, R568N, L591A, F592Y, F592A, F592N, R636A, L637A, R660K, R660A, R660N, Y661F, Y661A, Y661N, Y665F, Y665A, Y665N, L668A, and combinations thereof.

13. 13. The ADAMTS13 protein variant according to any one of claims 1 to 12, comprising the mutations R568A and Y665A or the mutations L591A, R636A, L637A, and L668A.

14. 14. The ADAMTS13 protein variant of any one of claims 1 to 13, wherein the one or more added N-linked glycosylation sites comprise an N-glycan, and / or the one or more transferred existing N-linked glycosylation sites comprise an N-linked glycan.

15. A nucleic acid construct comprising a nucleic acid sequence encoding the ADAMTS13 protein variant according to any one of claims 1 to 14.

16. A pharmaceutical composition comprising an ADAMTS13 protein variant described in any one of claims 1 to 14 or a nucleic acid construct described in claim 15, and one or more pharmaceutically acceptable carriers, adjuvants, excipients, and / or diluents.

17. A pharmaceutical comprising the ADAMTS13 protein variant of any one of claims 1 to 14 or the nucleic acid construct of claim 15.

18. An antithrombotic agent comprising the ADAMTS13 protein variant of any one of claims 1 to 14 or the nucleic acid construct of claim 15.

19. A pharmaceutical for the treatment of a disorder characterized by abnormal von Willebrand factor (VWF) activity and / or VWF processing, comprising an ADAMTS13 protein variant according to any one of claims 1 to 14 or a nucleic acid construct according to claim 15.

20. 15. Use of an ADAMTS13 protein variant according to any one of claims 1 to 14 in the manufacture of a medicament for the treatment of a disorder characterized by abnormal von Willebrand factor (VWF) activity and / or VWF processing.

21. The method of claim 19, wherein the disorder is a thrombotic disease.

22. The pharmaceutical composition according to claim 21, wherein the thrombotic disease is thrombotic microangiopathy.

23. 23. The pharmaceutical composition of claim 21 or 22, wherein the thrombotic disease is a disorder selected from the group consisting of thrombotic thrombocytopenic purpura (TTP) (including immune-mediated TTP (iTTP)), hemolytic uremic syndrome (HUS), ischemic stroke, systemic thrombosis, COVID-19, antiphospholipid syndrome, preeclampsia / HELLP syndrome, sepsis, and sickle cell disease.

24. A method for producing an ADAMTS13 protein variant described in any one of claims 1 to 14, comprising introducing the nucleic acid construct described in claim 15 into a host cell capable of N-linked glycosylation, and culturing the host cell under conditions that allow expression of the ADAMTS13 protein variant.

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