ADAMTS13 variants

Abstract

The present disclosure provides improved ADAMTS13 variants with amino acid substitutions in the linker 3 region. The present disclosure also provides methods for producing and using the variants.

Description

[Technical field]

[0001] The present disclosure relates to the fields of molecular biology and enzymology. The present disclosure also relates to methods of medical treatment and prophylaxis. [Background technology]

[0002] ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif 13) is a protease that regulates blood homeostasis by cleaving von Willebrand factor (VWF). Specifically, ADAMTS13 is a zinc-containing metalloprotease enzyme that cleaves and degrades VWF.

[0003] VWF is an adhesive plasma glycoprotein that circulates as large globular multimers (Sadler et al., 2009). In this large globular multimeric conformation, VWF is unable to capture platelets. The conformation of VWF changes upon binding to sites of vascular injury. This conformational change is triggered by the shear forces of flowing blood and allows platelet capture.

[0004] The biological degradation of VWF is mainly mediated by ADAMTS13, which cleaves VWF between the tyrosine at position 842 and the methionine at position 843 (or 1605–1606 of the gene) in the A2 domain, which breaks VWF down into smaller units that can be degraded by other peptidases.

[0005] VWF is required for normal platelet adhesion, but excessive VWF adhesive activity has been proposed to cause thrombotic thrombocytopenic purpura (TTP) (Moake et al., 1982). Furthermore, ADAMTS13 and VWF are thought to be important factors in other thrombotic diseases, such as stroke (De Meyer et al., 2012).

[0006] The formation of thrombi is central to the development of acute ischemic stroke. Current antithrombotic drugs used to treat or prevent cerebral ischemia are only moderately effective or carry an increased risk of severe bleeding (De Meyer et al. 2012). Only one thrombolytic therapy, recombinant tissue plasminogen activator (rt-PA), is approved for the treatment of acute ischemic stroke, but it is not widely administered due to the associated risk of hemorrhagic transformation (Wang et al. 2014). Furthermore, resistance to rt-PA thrombolysis has been observed in 40–50% of patients, which is believed to be due to changes in thrombus composition.

[0007] Administration of rt-PA has been the standard of care for 25 years (National Institute of Neurological and Stroke 1995). Retrospective analysis of CT angiograms has shown that rt-PA treatment has minimal effect on large proximal occlusions located in the internal carotid or basilar arteries (Bhatia et al. 2010). Furthermore, recent data from the DIRECT-MT clinical trial have shown that endovascular thrombectomy alone is non-inferior to rt-PA administration combined with endovascular thrombectomy (Yang et al. 2020). Moreover, the evolving landscape of associated risks over the years has led to significant refinement of guidelines for rt-PA use, prescribing complex eligibility criteria and a narrow time window for safe administration (0–4.5 h), limiting access for many (Whiteley et al. 2016; Emberson et al. 2014). Thus, the uncertainty in the safety and efficacy of rt-PA has led to an increased demand for novel thrombolytic agents in AIS.

[0008] A proportion of stroke patients present with thrombi that are particularly rich in VWF (Denorme et al. 2016), and a high VWF:ADAMTS13 ratio not only predisposes to AIS but is also consistent with worse outcomes and increased mortality (Taylor et al. 2020; Sonneveld et al. 2015). Interestingly, in a mouse model of middle cerebral artery (MCA) ischemic stroke, administration of recombinant ADAMTS13 was shown to be effective in dissolving VWF-rich occlusions that were resistant to rt-PA treatment (Denorme et al. 2016). This result is consistent with previous studies reporting that ADAMTS13 deletion is detrimental to infarct size and neurological outcomes after stroke, whereas VWF- / - animals are protected (Fujioka et al. 2010; Kleinschnitz et al. 2009).

[0009] The VWF-ADAMTS13 axis is also known to play a central role in thromboinflammation, a process increasingly recognized as exacerbating infarct development and now a therapeutic target in its own right (Stoll and Nieswandt 2019). In non-inflammatory conditions, ADAMTS13 deficiency increases VWF-dependent endothelial activation, P-selectin upregulation, and leukocyte rolling. In inflamed vessels, increased leukocyte transmigration and adhesion is observed (Chauhan et al. 2008). Similarly, using a mouse filament model of ischemic stroke, ADAMTS13 deficiency enhances immune cell infiltration, neutrophil extravasation, and proinflammatory cytokine release in the ipsilateral brain hemisphere (Khan et al. 2012). Furthermore, VWF has been shown to colocalize with neutrophil extracellular traps (NETs), known promoters of platelet recruitment and thrombus formation (Martinod and Wagner 2014). In a mouse model of MRSA-induced liver injury, it was demonstrated that ADAMTS13 administration released VWF-dependent NET adhesion to the inflamed vascular wall (Kolaczkowska et al. 2015).

[0010] From a safety perspective, the use of rt-PA increases the risk of intracerebral hemorrhage in up to 7% of AIS patients (Yaghi et al. 2017). The risk of hemorrhage increases proportionally to the severity of the stroke and the delay in rt-PA administration (Whiteley et al. 2016). Before administration of rt-PA, risk factors that increase the likelihood of hemorrhagic transformation must first be excluded, during which the efficacy of rt-PA decreases. In a mouse model of hemorrhagic stroke, administration of ADAMTS13 does not affect hemorrhage and may, in fact, have therapeutic potential in this condition (Zhao et al. 2009).

[0011] Recombinant ADAMTS13 (rADAMTS13) is a recombinant ADAMTS13 - / - It has systemic antithrombotic activity in mice (De Meyer et al., 2012b). Moreover, rADAMTS-13 has been shown to be protective in a mouse stroke model without the risk of intracerebral hemorrhage associated with rt-PA (Nakano et al., 2015).

[0012] Although early results of ADAMTS13 therapy have been promising, the need for very high doses has hindered progress. This high-dose requirement may be explained by a recently discovered mechanism of ADAMTS13 activation. Because substrate-induced conformational activation is required for wild-type ADAMTS13 to achieve full activity, high doses are required to achieve effective concentrations of active ADAMTS13. Studies have found that ADAMTS13 circulates in a resting conformation that is maintained by a self-alienating interaction between its N-terminal spacer domain and its C-terminal CUB domain (South et al., 2014).

[0013] Three linker regions in the distal domain of ADAMTS13 were found to be important for plasticity, allowing interactions between the proximal and T8-CUB2 domains during the inactive state (Deforche et al., 2015). In a more recent study, binding and functional studies were performed on a panel of truncated ADAMTS13 variants to develop a model for conformational activation of conformationally quiescent ADAMTS13 by VWF (South et al., 2017). Results showed that both isolated CUB1 and CUB2 domains in ADAMTS13 bind to the spacer domain exosite of truncated ADAMTS13 variants. However, only the CUB1 domain inhibited the proteolytic activity of truncated ADAMTS13 variants. The combined findings of this study support a model of ADAMTS13 activation in which VWF D4-CK engages the TSP8-CUB2 domain and induces a conformational change that disrupts the CUB1-spacer domain interaction, thereby activating ADAMTS13. Thus, this study suggests that the most important domains for conformational activation of ADAMTS13 are the two CUB domains and the spacer region.

[0014] ADAMTS13 variants have been previously generated with amino acid substitutions in the spacer region to prevent the need for conformational activation and overcome the problems of wild-type ADAMTS13 therapy. A gain-of-function (GoF) ADAMTS13 variant (R568K / F592Y / R660K / Y661F / Y665F) was shown to disrupt autoinhibition and enhance proteolytic activity (Jian et al., 2012). Furthermore, this GoF variant restored cerebral blood flow at lower doses than wild-type ADAMTS-13 and retained some ability to recanalize blood vessels even when administration was delayed by 1 h in a mouse stroke model (South et al., 2018). However, the reduced dose requirement exhibited by the GoF variant may not be sufficient to completely overcome the problems associated with wild-type ADAMTS13. Furthermore, the efficacy of GoF variants is reduced when administration is delayed. The development of variants with significantly reduced dose requirements and improved efficacy after delayed administration may lead to more clinically relevant therapies.

[0015] The present disclosure has been made in consideration of the above considerations. The present inventors have identified that the linker 3 region of ADAMTS13 protein is the key to the conformational activation mechanism, and have made a number of improved ADAMTS13 variants having amino acid substitutions in the linker 3 region for the first time. Summary of the Invention

[0016] In a first aspect, the disclosure provides an ADAMTS13 variant having an amino acid sequence that includes one or more amino acid substitutions in the region corresponding to SEQ ID NO:48, compared to the amino acid sequence of wild-type human ADAMTS13.

[0017] In some embodiments, the ADAMTS13 variant comprises a substitution at one or more of the following positions relative to the amino acid sequence of wild-type human ADAMTS13: A1144, A1145, A1146, P1147, P1154, P1171, P1173, P1175, P1180, and P1182.

[0018] In some embodiments, the ADAMTS13 variant comprises a substitution of an alanine residue with a valine, isoleucine, lysine, leucine, or methionine residue, as compared to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution of an alanine residue with a valine, isoleucine, or lysine residue, as compared to the amino acid sequence of wild-type human ADAMTS13.

[0019] In some embodiments, the ADAMTS13 variant comprises a substitution of a proline residue with a valine, isoleucine, lysine, leucine, or methionine residue, relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution of a proline residue with a valine, isoleucine, or lysine residue, relative to the amino acid sequence of wild-type human ADAMTS13.

[0020] In some embodiments, the ADAMTS13 variant comprises an amino acid sequence according to SEQ ID NO:50 or 156.

[0021] In some embodiments, the ADAMTS13 variant comprises a substitution with valine, isoleucine or lysine at one or more of the following positions relative to the amino acid sequence of wild-type human ADAMTS13: A1144, A1145, A1146, P1147, P1154, P1171, P1173, P1175, P1180, and P1182.

[0022] In some embodiments, the ADAMTS13 variant is: (i) the amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60; or (ii) the amino acid sequence of SEQ ID NO: 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145; or (iii) comprising the amino acid sequence of SEQ ID NO: 146, 147, 148, 149, 150, 151, 152, 153, 154, or 155.

[0023] In some embodiments, the ADAMTS13 variant comprises: (i) the amino acid sequence of SEQ ID NO:51; or (ii) the amino acid sequence of SEQ ID NO:52; or (iii) the amino acid sequence of SEQ ID NO:53; or (iv) the amino acid sequence of SEQ ID NO:54; or (v) the amino acid sequence of SEQ ID NO:55; or (vi) the amino acid sequence of SEQ ID NO:56; or (vii) the amino acid sequence of SEQ ID NO:57; or (viii) the amino acid sequence of SEQ ID NO:58; or (ix) the amino acid sequence of SEQ ID NO:59; or (x) the amino acid sequence of SEQ ID NO:60.

[0024] In some embodiments, the ADAMTS13 variant comprises: (i) the amino acid sequence of SEQ ID NO: 136; or (ii) the amino acid sequence of SEQ ID NO: 137; or (iii) the amino acid sequence of SEQ ID NO: 138; or (iv) the amino acid sequence of SEQ ID NO: 139; or (v) the amino acid sequence of SEQ ID NO: 140; or (vi) the amino acid sequence of SEQ ID NO: 141; or (vii) the amino acid sequence of SEQ ID NO: 142; or (viii) the amino acid sequence of SEQ ID NO: 143; or (ix) the amino acid sequence of SEQ ID NO: 144; or (x) the amino acid sequence of SEQ ID NO: 145.

[0025] In some embodiments, the ADAMTS13 variant comprises: (i) the amino acid sequence of SEQ ID NO: 146; or (ii) the amino acid sequence of SEQ ID NO: 147; or (iii) the amino acid sequence of SEQ ID NO: 148; or (iv) the amino acid sequence of SEQ ID NO: 149; or (v) the amino acid sequence of SEQ ID NO: 150; or (vi) the amino acid sequence of SEQ ID NO: 151; or (vii) the amino acid sequence of SEQ ID NO: 152; or (viii) the amino acid sequence of SEQ ID NO: 153; or (ix) the amino acid sequence of SEQ ID NO: 154; or (x) the amino acid sequence of SEQ ID NO: 155.

[0026] In some embodiments, the ADAMTS13 variant comprises a substitution at one or both of positions P1180 and / or P1182 with valine, isoleucine, or lysine relative to the amino acid sequence of wild-type human ADAMTS13.

[0027] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO: 59, 60, 144, 145, 154, or 155.

[0028] In some embodiments, the ADAMTS13 variant has: (i) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 61; or (ii) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 62; or (iii) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 63; or (iv) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 64; or (v) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 65. or (vi) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 66; or (vii) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 67; or (viii) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 68; or (ix) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 69; or (x) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 70.

[0029] In some embodiments, the ADAMTS13 variant exhibits increased proteolytic activity compared to wild-type human ADAMTS13.

[0030] The present disclosure also provides nucleic acids encoding ADAMTS13 variants according to the present disclosure.

[0031] The disclosure also provides an expression vector comprising a nucleic acid according to the disclosure.

[0032] The present disclosure also provides a cell comprising an ADAMTS13 variant, a nucleic acid, or an expression vector according to the present disclosure.

[0033] The present disclosure also provides a method of producing an ADAMTS13 variant, comprising culturing a cell containing a nucleic acid or expression vector according to the present disclosure under conditions suitable for expression of the ADAMTS13 variant by the cell.

[0034] The present disclosure also provides a pharmaceutical composition comprising an ADAMTS13 variant, a nucleic acid, an expression vector, or a cell according to the present disclosure, and a pharma- ceutically acceptable carrier, diluent, excipient or adjuvant.

[0035] The present disclosure also provides an ADAMTS13 variant, a nucleic acid, an expression vector, a cell, or a composition according to the present disclosure for use in a method of medical treatment or prophylaxis.

[0036] The present disclosure also provides an ADAMTS13 variant, nucleic acid, expression vector, cell, or composition according to the present disclosure for use in a method for treating or preventing a disease or condition characterized by one or more of: increased levels and / or activity of VWF or a complex containing VWF; decreased levels of ADAMTS13; decreased levels of ADAMTS13 proteolytic activity; thrombosis; and inflammation.

[0037] The present disclosure also provides the use of an ADAMTS13 variant, nucleic acid, expression vector, cell, or composition according to the present disclosure in the manufacture of a medicament for use in a method for treating or preventing a disease or condition characterized by one or more of: increased levels and / or activity of VWF or a complex containing VWF; reduced levels of ADAMTS13; reduced levels of ADAMTS13 proteolytic activity; thrombosis; and inflammation.

[0038] The present disclosure also provides a method for treating or preventing a disease or condition characterized by one or more of increased levels and / or activity of VWF or a complex containing VWF; decreased levels of ADAMTS13, decreased levels of ADAMTS13 proteolytic activity; thrombosis; and inflammation, comprising administering to a subject a therapeutically or prophylactically effective amount of an ADAMTS13 variant, nucleic acid, expression vector, cell or composition according to the present disclosure.

[0039] In some embodiments, the disease or condition is one of the following: diseases / conditions characterized by thrombosis, diseases / conditions characterized by inflammation, thrombotic thrombocytopenic purpura (TTP), ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage (SAH), intracerebral hemorrhage (ICH), chronic thromboembolic pulmonary hypotension (CTEPH), myocardial infarction (MI), ST-segment elevation myocardial infarction (STEMI), unstable angina (UA), ischemia, reperfusion, deep vein thrombosis, pulmonary The disease is selected from embolism, intravascular coagulation (DIC), hemolytic-uremic syndrome (HUS), cerebral infarction, systemic lupus erythematosus (SLE), disease caused by infection with SARS-CoV (e.g., SARS-CoV-2, e.g., COVID-19), acute respiratory distress syndrome (ARDS), pneumonia, kidney damage, nephropathy, microvascular disease, dementia, Crohn's disease, inflammatory bowel disease, ulcerative colitis, and bacterial diarrhea.

[0040] The present disclosure also provides a method of cleaving VWF, the method comprising contacting VWF or a complex containing VWF with an ADAMTS13 variant according to the present disclosure.

[0041] The present disclosure includes combinations of the aspects and preferred features described herein unless such combinations are expressly not permitted or are explicitly avoided. [Table 1(1)] [Table 1(2)] [Table 1(3)]

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[0042] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying drawings. [Brief description of the drawings]

[0043] [Figure 1A] In vitro activity of ADAMTS13 linker 3 variants. The proteolytic activities of wild-type (wt) ADAMTS13, gain-of-function (GoF) ADAMTS13 variants, and linker 3 variants were determined by FRETS-VWF73 assay both in the absence (-) and presence (+) of activating VWF-D4CK domain fragment. All activities are expressed relative to the basal activity of wtADAMTS13 (100%). [Figure 1B] In vitro activity of further ADAMTS13 linker 3 variants. The proteolytic activity of wild-type (wt) ADAMTS13 and the indicated linker 3 variants was determined by FRETS-VWF73 assay both in the absence (open bars) and presence (textured bars) of activating VWF-D4CK domain fragments. All activities are expressed relative to the basal activity of wtADAMTS13 (100%), as indicated by the dotted line. [Diagram 2]VWF-mediated platelet capture under arterial shear stress was measured in the presence of either wtADAMTS13, GoF ADAMTS13, or the linker 3 variant A1144V ADAMTS13 at various concentrations. For comparison, results from a VWF negative control are also shown. [Diagram 3] Restoration of rCBF to the MCA territory by A1144V ADAMTS13 variant (caADAMTS13) administered 1 h after MCA occlusion. A, Raw laser speckle contrast images acquired 0, 30, and 60 min after injection of either vehicle control, N-acetyl-cysteine ​​(NAC), wtADAMTS13, or caADAMTS13. Regions of interest (ROIs) within the MCA territory of the contralateral (contra) and ipsilateral (ipsi) hemispheres were used for quantification of rCBF. [Figure 4] Flux values ​​in contralateral and ipsilateral ROIs were measured at 1 min intervals for 5 min before and 60 min after injection. Traces shown are representative measurements from one animal in each treatment group. [Diagram 5] Changes in the mean flux ratio (ipsilateral (ipsi) / contralateral (contra)) over the course of the experiment were measured for each treatment group. An arbitrary flux ratio value of 0.5 was used to confirm successful MCA occlusion and as the point at which recanalization was achieved in treated animals (*). For comparison, flux ratios from sham (i.e., seizure-free) animals are also shown. [Figure 6] The effect of treatment on lesion volume was measured 24 hours after occlusion. Cresyl violet stained brain sections were used to measure lesion volume. For comparison, sections from sham animals in which FeCl3 was not applied to the MCA are also shown. [Figure 7] Lesion volumes for each treatment group were compared to the vehicle-treated group using unpaired t-tests with Welch's correction (**p<0.01). In this case, sham animals were animals that were excluded from the study due to lack of stable MCA occlusion. The % increase in flux ratio between ipsilateral and contralateral ROIs between 0 and 60 min post-injection was also compared between the vehicle and each treatment group (**p<0.01). [Figure 8]A significant negative correlation was observed between increased flux ratio and lesion volume across all groups. Animals treated with caADAMTS13 are highlighted in red. [Figure 9] Brain sections stained with hematoxylin and eosin were used to identify evidence of hemorrhagic transformation 24 hours after treatment. [Figure 10] Effect of treatment on residual thrombus content at the site of FeCl3 application 24 hours after occlusion. Whole brain sections were stained by immunofluorescence to visualize VWF and fibrin(ogen). High magnification of marked ROI. Areas of microvascular VWF-platelet deposition are indicated by white arrows. [Figure 11] Total deposition of VWF and fibrin in all areas adjacent to the MCA was quantified and compared between vehicle and treatment groups using unpaired t-tests with Welch's correction (*<0.05, **p<0.01). Sham animals were animals in which FeCl3 was not applied to the MCA. [Figure 12]Male CD1 mice, 13–14 weeks of age, were infected with Streptococcus pneumoniae using an ascending infection challenge over a 6-day period. On day 8, they were treated with either vehicle (no treatment) or A1144V ADAMTS13 (caADAMTS13) by tail vein injection. This was done in mice with ongoing infection (A) and in mice in which the infection was resolved by a single subcutaneous injection of amoxicillin (B). On day 18, 3D MGE data were acquired 2 hours after intravenous administration of α-von Willebrand factor (VWF)-conjugated MPIO particles. MGE data were fitted using a monoexponential model to estimate R2* maps. A whole-brain mask was applied and total R2* values ​​were derived from the whole brain volume (C). VWF (VWF:CBA) reactivity in the plasma of these experimental animals (relative to the reactivity of mock-infected control animals) was also measured (D). Untreated and amoxicillin-treated mice had higher levels of bound α-VWFMPIO particles (higher R2*) compared to mock-infected animals, indicating increased endothelial activation in the brain vasculature. This was accompanied by increased levels of reactive VWF species in plasma. Administration of caADAMTS13 significantly reduced the levels of VWF detected in the brain vasculature and plasma of animals with resolved or ongoing infection. Treatment groups were compared as indicated using unpaired t-tests with Welch's correction (*<0.05, **p<0.01, ****p<0.001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Aspects and embodiments of the present disclosure will now be discussed with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0045] The present disclosure is based on the present inventors' identification that the linker 3 region of ADAMTS13 can be modified to improve the therapeutic properties of the protein. The present inventors have created several ADAMTS13 variants with amino acid substitutions in the linker 3 region and improved properties compared to wild-type ADAMTS13.

[0046] The variants of the present disclosure have many advantages over wild-type ADAMTS13, recombinant wild-type ADAMTS13, and known GoF ADAMTS13 variants, including lack of substrate-induced activation requirement, broad substrate specificity, and higher enzymatic activity. The variants of the present disclosure have higher proteolytic activity compared to wild-type ADAMTS13 and known GoF ADAMTS13 variants. The variants have been shown to be effective at lower doses compared to wild-type ADAMTS13 and known GoF ADAMTS13 variants. Furthermore, unlike known GoF ADAMTS13 variants, the variants of the present disclosure have fully functional spacer exosites and remain optimally activated.

[0047] The only approved treatment for acute ischemic stroke (AIS) is recombinant tissue plasminogen activator (rt-PA), but the use of rt-PA increases the risk of intracerebral hemorrhage in up to 7% of AIS patients (Yaghi et al. 2017). The risk of hemorrhage increases in proportion to stroke severity and delay in administration (Whiteley et al. 2016). Importantly, we have found no evidence of hemorrhagic transformation after treatment with either wild-type ADAMTS13 or ADAMTS13 variants in mouse models of AIS (Denorme et al. 2016; Nakano et al. 2015). In mouse models of hemorrhagic stroke, administration of ADAMTS13 does not affect hemorrhage and may, in fact, be therapeutic in this condition (Zhao et al. 2009).

[0048] General definition As used herein, a "fragment," "variant," or "homolog" of a given protein may optionally be characterized as having one of at least 40%, preferably 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein. Fragments, variants, isoforms, and homologs of a reference protein may be characterized by their ability to perform a function performed by the reference protein.

[0049] As used herein, "sequence identity" refers to the percentage of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity between the sequences. Pairwise and multiple sequence alignments for the purpose of determining percent identity between two or more amino acid or nucleic acid sequences can be accomplished in a variety of ways known to those skilled in the art, for example using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6 (298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30 (4) 772-780) software. When using such software, it is preferred that default parameters, such as gap penalties and extension penalties, are used.

[0050] A "variant" generally refers to a protein having an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications relative to the amino acid sequence of the reference protein, but retains a significant degree of sequence identity (e.g., at least 40%) to the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein that is expressed by the same species as the species of the reference protein. A "homolog" generally refers to a variant of a reference protein that is produced by a different species compared to the species of the reference protein.

[0051] A "fragment" of a reference protein may be of any length (by number of amino acids), but optionally be at least 25% of the length of the reference protein (i.e., the protein from which the fragment is derived), and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.

[0052] Regions and positions of a given polypeptide / amino acid sequence that "correspond" to regions and positions of a reference amino acid sequence can be identified by sequence alignment, which can be performed using sequence alignment software such as, for example, ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960). A region of a given polypeptide / amino acid sequence that corresponds to a region of a reference amino acid sequence can have at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with its corresponding region of the reference amino acid sequence. As an example, the amino acid sequence shown in SEQ ID NO: 48 corresponds to positions 1131 to 1190 of SEQ ID NO: 1. As a further example, the alanine residue at position 14 of SEQ ID NO: 48 corresponds to position 1144 of SEQ ID NO: 1.

[0053] ADAMTS13 and von Willebrand factor (VWF) ADAMTS13 (a disintegrin-like metalloprotease with thrombospondin type 1 motif 13) is identified by UniProtKB Q76LX8. Alternative splicing of the mRNA encoded by the human ADAMTS13 gene results in four isoforms: isoform 1 (SEQ ID NO: 1); isoform 2 (SEQ ID NO: 2), which lacks positions 1135-1190 compared to isoform 1; isoform 3 (SEQ ID NO: 3), which lacks positions 275-305 and 1135-1190 relative to isoform 1; and isoform 4 (SEQ ID NO: 4), which lacks positions 2-329 and 693-1427 compared to isoform 1 and has a variant sequence at positions compared to isoform 1 positions 258-692.

[0054] The structure and function of ADAMTS13 are reviewed, for example, in Chen et al., Front Neurol. (2019) 10:772, the entire contents of which are incorporated herein by reference. ADAMTS13 is a 1,427 amino acid metalloprotease that contains a signal peptide (SEQ ID NO:5), a short propeptide domain (SEQ ID NO:6), a metalloprotease domain (SEQ ID NO:13), a disintegrin-like domain (SEQ ID NO:14), a thrombospondin type 1 (TSP1) repeat domain (SEQ ID NO:15), a cysteine-rich domain (SEQ ID NO:16), a spacer domain (SEQ ID NO:17), seven further TSP1 repeat domains (SEQ ID NOs:18-24), and two CUB domains (SEQ ID NOs:25 and 26).

[0055] ADAMTS13 is a protease that cleaves von Willebrand factor (VWF), a key factor in thrombosis. The biological degradation (catabolism) of VWF (e.g., within intravascular platelet aggregates) is primarily mediated by the enzyme ADAMTS13.

[0056] VWF is identified by UniProtKB P04275. Alternative splicing of the mRNA encoded by the human VWF gene generates two isoforms: isoform 1 (SEQ ID NO: 27); isoform 2 (SEQ ID NO: 28) which has variant sequences at positions corresponding to positions 1-18 and 220-314 of isoform 1 and lacks positions 315-2813 relative to isoform 1.

[0057] The structure and function of VWF are described, for example, in Bharati and Prashanth, Indian J Pharm Sci. (2011) 73(1):7-16 and Zhou et al., Blood. (2012) 120(2):449-458, both of which are incorporated by reference in their entirety. The protein encoded by VWF contains a signal peptide (SEQ ID NO:29), von Willebrand antigen 2 (SEQ ID NO:31; propeptide) and mature von Willebrand factor (SEQ ID NO:32).

[0058] Von Willebrand antigen 2 contains the VWFD1, TIL1, VWFD2, and TIL2 domains (SEQ ID NOs: 33 to 36). Mature VWF contains the TIL3, VWFD3, TIL4, VWFA1, VWFA2, VWFA3, VWFD4, VWFC1, VWFC2, VWFC3, and CTCK domains (SEQ ID NOs: 37 to 47).

[0059] VWF is normally expressed as a large multiglycoprotein complex that is released by endothelial cells in the form of ultra-large multimers (UL-VWF) of various sizes with molecular weights up to 20,000 kDa.

[0060] The metalloprotease domain of ADAMTS13 is involved in the recognition and cleavage of VWF at the peptide bond formed between positions Y1605 and M1606 (numbering with reference to SEQ ID NO: 27) in the VWFA2 domain of VWF. The region of ADAMTS13 containing the disintegrin-like domain, TSP1 repeat 1, the cysteine-rich domain and the spacer domain is involved in the binding of ADAMTS13 to the VWFA2 domain of VWF. The region of ADAMTS13 containing TSP1 repeats 5-8 and CUB domains 1 and 2 is involved in the binding of ADAMTS13 to the region of VWF containing the VWFD4, VWFC1, VWFC2, VWFC3 and CTCK domains.

[0061] ADAMTS13-mediated cleavage of VWF is important for blood homeostasis. VWF plays a major role in blood clotting. It binds to other proteins, especially factor VIII, and is thought to be involved in platelet adhesion at wound sites. High levels of VWF can lead to diseases such as stroke and other thrombotic disorders.

[0062] Dysfunction of ADAMTS13-mediated VWF cleavage leads to VWF accumulation and platelet adhesion, resulting in thrombosis and inflammation. In pathological conditions such as thrombotic thrombocytopenic purpura (TTP) and other thrombotic microangiopathy (TMA), VWF binds to the endothelium to form large multimers. As the anchored VWF chains grow, they provide a larger surface area for binding to circulating platelets (PLTs), generating the unique thrombus characteristic of TTP. This results in microvascular thrombosis, impaired blood flow, and ultimately tissue and organ damage. TTP and TMA can occur, for example, as a result of an autoimmune response to ADAMTS13 or as a result of ADAMTS13 deficiency.

[0063] As used herein, "ADAMTS13" refers to ADAMTS13 from any species, including ADAMTS13 isoforms, fragments, variants, or homologs from any species.

[0064] In some embodiments, the ADAMTS13 is from a mammal (e.g., a primate (rhesus monkey, cynomolgus monkey, or human) and / or rodent (e.g., rat or mouse) ADAMTS13). An isoform, fragment, variant, or homolog of ADAMTS13 can optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature ADAMTS13 isoform from a given species, e.g., human. In a preferred embodiment, the ADAMTS13 is a human ADAMTS13 isoform (e.g., isoform 1, 2, 3, or 4).

[0065] An isoform, fragment, variant or homologue may optionally be a functional isoform, fragment, variant or homologue, e.g., has a functional property / activity of a reference ADAMTS13 (e.g., human ADAMTS13 isoform 1), as determined by analysis of a suitable assay for the functional property activity. For example, an isoform, fragment, variant or homologue of ADAMTS13 may exhibit binding to and / or cleavage of human VWF.

[0066] In some embodiments, ADAMTS13 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:1, 2, 3, 4, 7, 8, 9, 10, 11 or 12.

[0067] As used herein, "VWF" refers to VWF from any species, including VWF isoforms, fragments, variants or homologs from any species.

[0068] In some embodiments, the VWF is from a mammal (e.g., a primate (rhesus, cynomolgus, or human) and / or rodent (e.g., rat or mouse) VWF). Isoforms, fragments, variants, or homologs of VWF may optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature VWF isoform from a given species, e.g., human. In a preferred embodiment, the VWF is a human VWF isoform (e.g., isoform 1 or 2).

[0069] The isoform, fragment, variant or homologue may optionally be a functional isoform, fragment, variant or homologue, e.g., having a functional property / activity of a reference VWF (e.g., human VEF isoform 1), as determined by analysis with a suitable assay for the functional property / activity. For example, an isoform, fragment, variant or homologue of VWF may exhibit association with human ADAMTS13 and / or may be susceptible to cleavage by human ADAMTS13.

[0070] In some embodiments, the VWF comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 27, 28, 30 or 32.

[0071] ADAMTS13 variants Aspects and embodiments of the present disclosure relate to variants of ADAMTS13.

[0072] As used herein, "ADAMTS13 variant" refers to a polypeptide comprising or consisting of an amino acid sequence that has at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to the amino acid sequence of wild-type human ADAMTS13 and that contains one or more amino acid substitutions compared to the amino acid sequence of wild-type human ADAMTS13.

[0073] In some embodiments, an ADAMTS13 variant according to the present disclosure is a polypeptide comprising or consisting of an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO:1, 2, 3, 4, 7, 8, 9, 10, 11 or 12, and comprising one or more amino acid substitutions compared to a reference sequence.

[0074] In a preferred embodiment, an ADAMTS13 variant according to the present disclosure is a polypeptide comprising or consisting of an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 1, 7 or 8 and comprising one or more amino acid substitutions compared to the reference sequence.

[0075] In some embodiments, the ADAMTS13 variant comprises or consists of an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO:1 and containing one or more amino acid substitutions relative to SEQ ID NO:1.

[0076] In some embodiments, the ADAMTS13 variant comprises or consists of an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO:7 and containing one or more amino acid substitutions relative to SEQ ID NO:7.

[0077] In some embodiments, the ADAMTS13 variant comprises or consists of an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO:8 and containing one or more amino acid substitutions relative to SEQ ID NO:8.

[0078] In some embodiments, the ADAMTS13 variant according to the present disclosure comprises two or more amino acid substitutions compared to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions compared to the amino acid sequence of wild-type human ADAMTS13.

[0079] In some embodiments, the ADAMTS13 variant comprises or consists of an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO:1 and containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions relative to SEQ ID NO:1.

[0080] In some embodiments, the ADAMTS13 variant comprises or consists of an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO:8 and containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions to SEQ ID NO:7.

[0081] In some embodiments, the ADAMTS13 variant comprises or consists of an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO:8 and containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions relative to SEQ ID NO:8.

[0082] The present inventors have identified an ADAMTS13 variant containing a substitution in the linker 3 (L3) region of ADAMTS13 that has a higher proteolytic activity than wild-type human ADAMTS13. The L3 region of ADAMTS13 consists of positions 1131 to 1190 of SEQ ID NO: 1 and is shown in SEQ ID NO: 48.

[0083] In some embodiments, an ADAMTS13 variant according to the present disclosure comprises one or more amino acid substitutions compared to the amino acid sequence of wild-type human ADAMTS13 in a region corresponding to SEQ ID NO: 48. In some embodiments, an ADAMTS13 variant according to the present disclosure comprises one or more amino acid substitutions compared to the amino acid sequence of wild-type human ADAMTS13 in a region corresponding to SEQ ID NO: 49.

[0084] In a preferred embodiment, one or more amino acid substitutions relative to the amino acid sequence of wild-type human ADAMTS13 are associated with greater proteolytic activity of the ADAMTS13 variant compared to the proteolytic activity of wild-type human ADAMTS13.

[0085] In some embodiments, ADAMTS13 variants according to the present disclosure include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) substitutions relative to the amino acid sequence of wild-type human ADAMTS13 at position(s) corresponding to the following positions (numbered relative to SEQ ID NO:1): A1144, A1145, A1146, P1147, P1154, P1171, P1173, P1175, P1180, P1182. In some embodiments, ADAMTS13 variants according to the present disclosure include one or more (e.g., 1, 2, 3 or 4) substitutions relative to the amino acid sequence of wild-type human ADAMTS13 at position(s) corresponding to the following positions (numbered based on SEQ ID NO:1): A1144, A1146, P1180, P1182.

[0086] In some embodiments, the ADAMTS13 variant comprises a substitution to the amino acid sequence of wild-type human ADAMTS13 at a position corresponding to A1144. In some embodiments, the ADAMTS13 variant comprises a substitution to the amino acid sequence of wild-type human ADAMTS13 at a position corresponding to A1145. In some embodiments, the ADAMTS13 variant comprises a substitution to the amino acid sequence of wild-type human ADAMTS13 at a position corresponding to A1146. In some embodiments, the ADAMTS13 variant comprises a substitution to the amino acid sequence of wild-type human ADAMTS13 at a position corresponding to P1147. In some embodiments, the ADAMTS13 variant comprises a substitution to the amino acid sequence of wild-type human ADAMTS13 at a position corresponding to P1154. In some embodiments, the ADAMTS13 variant comprises a substitution to the amino acid sequence of wild-type human ADAMTS13 at a position corresponding to P1171. In some embodiments, the ADAMTS13 variant comprises a substitution to the amino acid sequence of wild-type human ADAMTS13 at a position corresponding to P1173. In some embodiments, the ADAMTS13 variant comprises a substitution relative to the amino acid sequence of wild-type human ADAMTS13 at a position corresponding to P1175. In some embodiments, the ADAMTS13 variant comprises a substitution relative to the amino acid sequence of wild-type human ADAMTS13 at a position corresponding to P1180. In some embodiments, the ADAMTS13 variant comprises a substitution relative to the amino acid sequence of wild-type human ADAMTS13 at a position corresponding to P1182.

[0087] In some embodiments, the ADAMTS13 variant comprises a substitution of an alanine residue with another hydrophobic amino acid residue compared to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution of a proline residue with another hydrophobic amino acid residue compared to the amino acid sequence of wild-type human ADAMTS13.

[0088] In some embodiments, the ADAMTS13 variant comprises a substitution of an alanine residue with a valine, isoleucine, lysine, leucine, or methionine residue, relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution of an alanine residue with a valine, isoleucine, or lysine residue, relative to the amino acid sequence of wild-type human ADAMTS13.

[0089] In some embodiments, the ADAMTS13 variant comprises a substitution of a proline residue with a valine, isoleucine, lysine, leucine, or methionine residue, relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution of a proline residue with a valine, isoleucine, or lysine residue, relative to the amino acid sequence of wild-type human ADAMTS13.

[0090] In some embodiments, the ADAMTS13 variant according to the present disclosure comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) of the following substitutions at the corresponding position(s) (numbered relative to SEQ ID NO: 1) relative to the amino acid sequence of wild-type human ADAMTS13: A1144V, A1145V, A1146V, P1147V, P1154V, P1171V, P1173V, P1175V, P1180V, P1182V. In some embodiments, the ADAMTS13 variant according to the present disclosure comprises one or more (e.g., 1, 2, 3 or 4) of the following substitutions at the corresponding position(s) (numbered relative to SEQ ID NO: 1) relative to the amino acid sequence of wild-type human ADAMTS13: A1144V, A1146V, P1180V, P1182V. In some embodiments, the ADAMTS13 variant comprises the substitution A1144V relative to the amino acid sequence of wild-type human ADAMTS13.

[0091] In some embodiments, the ADAMTS13 variant according to the present disclosure comprises one or more of the following substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) at the corresponding position(s) (numbered relative to SEQ ID NO: 1) relative to the amino acid sequence of wild-type human ADAMTS13: A1144K, A1145K, A1146K, P1147K, P1154K, P1171K, P1173K, P1175K, P1180K, P1182K. In some embodiments, the ADAMTS13 variant according to the present disclosure comprises one or more of the following substitutions (e.g., 1, 2, 3 or 4) at the corresponding position(s) (numbered relative to SEQ ID NO: 1) relative to the amino acid sequence of wild-type human ADAMTS13: A1144K, A1146K, P1180K, P1182K. In some embodiments, the ADAMTS13 variant comprises the substitution A1144K relative to the amino acid sequence of wild-type human ADAMTS13.

[0092] In some embodiments, the ADAMTS13 variant according to the present disclosure comprises one or more of the following substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) at the corresponding position(s) (numbered relative to SEQ ID NO: 1) relative to the amino acid sequence of wild-type human ADAMTS13: A1144I, A1145I, A1146I, P1147I, P1154I, P1171I, P1173I, P1175I, P1180I, P1182I. In some embodiments, the ADAMTS13 variant according to the present disclosure comprises one or more of the following substitutions (e.g., 1, 2, 3 or 4) at the corresponding position(s) (numbered relative to SEQ ID NO: 1) relative to the amino acid sequence of wild-type human ADAMTS13: A1144I, A1146I, P1180I, P1182I. In some embodiments, the ADAMTS13 variant comprises the substitution A1144I relative to the amino acid sequence of wild-type human ADAMTS13.

[0093] In some embodiments, the ADAMTS13 variant comprises a substitution A1144V at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution A1146V at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1147V at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1154V at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1171V at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1173V at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1175V at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1180V at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1182V at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13.

[0094] In some embodiments, the ADAMTS13 variant comprises a substitution A1144K at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution A1146K at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1147K at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1154K at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1171K at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1173K at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1175K at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1180K at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1182K at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13.

[0095] In some embodiments, the ADAMTS13 variant comprises a substitution A1144I at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution A1146I at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1147I at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1154I at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1171I at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1173I at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1175I at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1180I at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13. In some embodiments, the ADAMTS13 variant comprises a substitution P1182I at the corresponding position relative to the amino acid sequence of wild-type human ADAMTS13.

[0096] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence set forth in SEQ ID NO:50, which is not identical to SEQ ID NO:49.

[0097] In some embodiments, the ADAMTS13 variant comprises an amino acid sequence of SEQ ID NO: 51, 136, or 146, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 51, 136, or 146 (including a V, K, or I at the position corresponding to position 1144, respectively).

[0098] In some embodiments, the ADAMTS13 variant comprises an amino acid sequence of SEQ ID NO: 52, 137, or 147, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 52, 137, or 147 (including a V, K, or I at the position corresponding to position 1145, respectively).

[0099] In some embodiments, the ADAMTS13 variant comprises an amino acid sequence of SEQ ID NO: 53, 138, or 148, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 53, 138, or 148 (including a V, K, or I at the position corresponding to position 1146, respectively).

[0100] In some embodiments, the ADAMTS13 variant comprises an amino acid sequence of SEQ ID NO: 54, 139, or 149, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 54, 139, or 149 (including a V, K, or I at the position corresponding to position 1147, respectively).

[0101] In some embodiments, the ADAMTS13 variant comprises an amino acid sequence of SEQ ID NO: 55, 140, or 150, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 55, 140, or 150 (including a V, K, or I at the position corresponding to position 1154, respectively).

[0102] In some embodiments, the ADAMTS13 variant comprises an amino acid sequence of SEQ ID NO: 56, 141, or 151, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 56, 141, or 151 (including a V, K, or I at the position corresponding to position 1171, respectively).

[0103] In some embodiments, the ADAMTS13 variant comprises an amino acid sequence of SEQ ID NO: 57, 142, or 152, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 57, 142, or 152 (including a V, K, or I at the position corresponding to position 1173, respectively).

[0104] In some embodiments, the ADAMTS13 variant comprises an amino acid sequence of SEQ ID NO: 58, 143, or 153, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 58, 143, or 153 (including a V, K, or I at the position corresponding to position 1175, respectively).

[0105] In some embodiments, the ADAMTS13 variant comprises an amino acid sequence of SEQ ID NO: 59, 144, or 154, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 59, 144, or 154 (including a V, K, or I at the position corresponding to position 1180, respectively).

[0106] In some embodiments, the ADAMTS13 variant comprises an amino acid sequence of SEQ ID NO: 60, 145, or 155, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 60, 145, or 155 (including a V, K, or I at the position corresponding to position 1182, respectively).

[0107] One of the known ADAMTS13 variants, known as gain-of-function (GoF) ADAMTS13 variant (R568K / F592Y / R660K / Y661F / Y665F), has previously been shown to disrupt autoinhibition and enhance proteolytic activity compared to wild-type human ADAMTS13, and is described in Jian et al., Blood (2012) 119:3836-3843 (incorporated herein by reference in its entirety). The amino acid substitutions of the R568K / F592Y / R660K / Y661F / Y665F variant are provided in the spacer region of ADAMTS13. This known GoF variant has higher activity than wild-type ADAMTS13, but lower proteolytic activity than the variants disclosed herein.

[0108] In some embodiments, the ADAMTS13 variants of the present disclosure are not identical to the ADAMTS13 variants disclosed in Jian et al., Blood (2012) 119:3836-3843. In some embodiments, the ADAMTS13 variants of the present disclosure do not comprise or consist of the amino acid sequence of SEQ ID NO:71.

[0109] In some embodiments, the ADAMTS13 variant includes one or more (e.g., 1, 2, 3, 4 or 5) substitutions at positions corresponding to the following positions (numbered based on SEQ ID NO: 1) relative to the amino acid sequence of wild-type human ADAMTS13: R568, F592, R660, Y661, Y665.

[0110] In some embodiments, the ADAMTS13 variant includes one or more (e.g., 1, 2, 3, 4 or 5) of the following substitutions relative to the amino acid sequence of wild-type human ADAMTS13 at the corresponding position(s) (numbered based on SEQ ID NO:1): R568K, F592Y, R660K, Y661F, Y665F.

[0111] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:13, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:13.

[0112] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:14, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:14.

[0113] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:15, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:15.

[0114] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:16, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:16.

[0115] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 17.

[0116] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:18, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:18.

[0117] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:19, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:19.

[0118] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:20.

[0119] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:21, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:21.

[0120] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:22 or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:22.

[0121] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:23, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:23.

[0122] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:24.

[0123] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:25.

[0124] In some embodiments, the ADAMTS13 variant comprises the amino acid sequence of SEQ ID NO:26, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:26.

[0125] In some embodiments, the ADAMTS13 variant comprises or consists of the amino acid sequence of SEQ ID NO:61, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:61.

[0126] In some embodiments, the ADAMTS13 variant comprises or consists of the amino acid sequence of SEQ ID NO:62, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:62.

[0127] In some embodiments, the ADAMTS13 variant comprises or consists of the amino acid sequence of SEQ ID NO:63, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:63.

[0128] In some embodiments, the ADAMTS13 variant comprises or consists of the amino acid sequence of SEQ ID NO:64, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:64.

[0129] In some embodiments, the ADAMTS13 variant comprises or consists of the amino acid sequence of SEQ ID NO:65, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:65.

[0130] In some embodiments, the ADAMTS13 variant comprises or consists of the amino acid sequence of SEQ ID NO:66, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:66.

[0131] In some embodiments, the ADAMTS13 variant comprises or consists of the amino acid sequence of SEQ ID NO:67, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:67.

[0132] In some embodiments, the ADAMTS13 variant comprises or consists of the amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:68.

[0133] In some embodiments, the ADAMTS13 variant comprises or consists of the amino acid sequence of SEQ ID NO:69, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:69.

[0134] In some embodiments, the ADAMTS13 variant comprises or consists of the amino acid sequence of SEQ ID NO:70, or an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:70.

[0135] Functional characterization of ADAMTS13 variants ADAMTS13 variants of the present disclosure may be characterized by reference to particular functional properties.

[0136] In some embodiments, the ADAMTS13 variants described herein may exhibit one or more of the following properties: Proteolytic activity, e.g., VWF cleavage activity; Increased proteolytic activity compared to wild-type human ADAMTS13; increased proteolytic activity compared with the R568K / F592Y / R660K / Y661F / Y665F ADAMTS13 variants; For example, inhibition of VWF-mediated platelet sequestration under arterial shear stress; enhanced inhibition of VWF-mediated platelet capture compared to wild-type human ADAMTS13; increased inhibition of VWF-mediated platelet capture compared with the R568K / F592Y / R660K / Y661F / Y665F ADAMTS13 variants; Inhibition of thrombosis; enhanced inhibition of thrombosis compared to wild-type human ADAMTS13; increased inhibition of thrombosis compared to the R568K / F592Y / R660K / Y661F / Y665F ADAMTS13 variant; Inhibition of blood clot formation / clotting; Enhanced inhibition of blood thrombosis / clotting compared to wild-type human ADAMTS13; Increased inhibition of blood thrombosis / clotting compared to the ADAMTS13 variant R568K / F592Y / R660K / Y661F / Y665F; For example, inhibition of inflammation in in vivo models of inflammation; increased inhibition of inflammation compared to wild-type human ADAMTS13; Increased inhibition of inflammation compared to the R568K / F592Y / R660K / Y661F / Y665F ADAMTS13 variant.

[0137] As explained above, ADAMTS13 cleaves VWF at the peptide bond formed between positions Y1605 and M1606 (numbering relative to SEQ ID NO: 27) in the VWFA2 domain of VWF. The present disclosure particularly relates to variants of ADAMTS13 (e.g., polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO: 1, 7, or 8) that have improved / increased proteolytic activity compared to wild-type human ADAMTS13.

[0138] Proteolytic activity refers to the dissolution of a peptide / polypeptide to produce smaller peptides / polypeptides and / or their constituent amino acids. Proteolysis may include hydrolysis of peptide bonds between amino acids of a peptide / polypeptide. Proteolytic activity is the enzymatic activity of a proteolytic enzyme. Proteolytic activity is determined by the amount of substrate degraded (or amount of substrate hydrolysis) by a given amount of protease over a given period of time. Proteolytic activity may be measured using any method known in the art.

[0139] An ADAMTS13 variant having improved proteolytic activity compared to wild-type human ADAMTS13 may be described as having greater / higher / improved / increased metalloprotease activity or as having greater / higher / improved / increased VWF cleavage activity compared to wild-type human ADAMTS13.

[0140] A given variant of ADAMTS13 can be assessed for proteolytic activity in a suitable in vitro assay, which may involve contacting a substrate for cleavage by ADAMTS13 with an ADAMTS13 variant under conditions suitable for cleavage of the substrate by ADAMTS13, and analyzing samples for cleavage products and / or uncleaved substrate after a sufficient time for cleavage of the substrate to occur.

[0141] An example of a suitable assay for such activity is an assay that assesses cleavage of the FRETS-VWF73 molecule, as described in South et al. Proc Natl Acad Sci US A. (2014) 111(52):18578-18583, which is incorporated herein by reference in its entirety. FRETS-VWF73 is a VWF fragment that includes VWFA2 domain residues 1596-1668, which contains the ADAMTS13 cleavage site.

[0142] Briefly, ADAMTS13 variants were incubated in white 96-well plates (Nunc) with 5 mM Bis-Tris pH 6.0, 25 mM CaCl 2 , and may be diluted to a concentration of 0.3 nM in 0.005% Tween-20. Purified VWF D4-CK fragment may be added to a final concentration of 20-60 nM before preincubation for 45 min at 37 °C, or the assay may be performed without adding purified VWF D4-CK fragment. The reaction may be initiated by adding an equal volume of 4 μM FRETS-VWF73 substrate (Peptanova). Fluorescence (excitation, 340 nm; emission, 460 nm) may be measured at 1 min intervals for 1 h at 30 °C using a suitable plate reader (e.g., Fluostar Omega plate reader (BMG Labtech)). Fluorescence measurements may be compared to values ​​obtained for wild-type human ADAMTS13.

[0143] In some embodiments, an ADAMTS13 variant according to the disclosure has a proteolytic activity that is greater than 1-fold, e.g., 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, greater than the level of proteolytic activity measured for wild-type human ADAMTS13 (e.g., a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1, 7, or 8) in the same assay. and above, indicates a level of proteolytic activity that is one of: 2-fold or more, 2.1-fold or more, 2.2-fold or more, 2.3-fold, 2.4-fold or more, 2.5-fold or more, 2.6-fold or more, 2.7-fold or more, 2.8-fold or more, 2.9-fold or more, 3-fold or more, 3.1-fold or more, 3.2-fold or more, 3.3-fold or more, 3.4-fold or more, 3.5-fold or more, 3.6-fold or more, 3.7-fold or more, 3.8-fold or more, 3.9-fold or more, 4-fold or more, 4.1-fold or more, 4.2-fold or more, 4.3-fold or more, 4.4-fold or more, 4.5-fold or more, 4.6-fold or more, 4.7-fold or more, 4.8-fold, 4.9-fold or more, or 5-fold or more.

[0144] In a preferred embodiment, an ADAMTS13 variant according to the present disclosure may exhibit a level of proteolytic activity that is more than 2.5-fold, such as one of 2.6-fold or more, 2.7-fold or more, 2.8-fold or more, 2.9-fold or more, 3-fold or more, 3.1-fold, 3.2-fold or more, 3.3-fold or more, 3.4-fold or more, 3.5-fold or more, 3.6-fold or more, 3.7-fold or more, 3.8-fold or more, 3.9-fold or more, 4-fold or more, 4.1-fold or more, 4.2-fold or more, 4.3-fold or more, 4.4-fold or more, 4.5-fold or more, 4.6-fold or more, 4.7-fold or more, 4.8-fold or more, 4.9-fold or more, or 5-fold or more, of the level of proteolytic activity determined for wild-type human ADAMTS13 (e.g., a polypeptide comprising or consisting of the amino acids of sequence SEQ ID NO: 1, 7 or 8) in the same assay.

[0145] In some embodiments, the ADAMTS13 variant according to the present disclosure has improved / enhanced proteolytic activity compared to the ADAMTS13 R568K / F592Y / R660K / Y661F / Y665F variant described in Jian et al., Blood (2012) 119: 3836-3843. In some embodiments, the ADAMTS13 variant has improved proteolytic activity compared to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 71.

[0146] In some embodiments, an ADAMTS13 variant according to the disclosure has a proteolytic activity that is greater than 1-fold, e.g., 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 1.10-fold or more, 1.12-fold or more, 1.14-fold or more, 1.16-fold or more, 1.18 ... The antibody may exhibit a level of proteolytic activity that is one of: .8 fold or more, 1.9 fold or more, 2 fold or more, 2.1 fold or more, 2.2 fold or more, 2.3 fold, 2.4 fold or more, 2.5 fold or more, 2.6 fold or more, 2.7 fold or more, 2.8 fold or more, 2.9 fold or more, 3 fold or more, 3.1 fold or more, 3.2 fold or more, 3.3 fold or more, 3.4 fold or more, 3.5 fold or more, 3.6 fold or more, 3.7 fold or more, 3.8 fold or more, 3.9 fold or more, 4 fold or more, 4.1 fold or more, 4.2 fold or more, 4.3 fold or more, 4.4 fold or more, 4.5 fold or more, 4.6 fold or more, 4.7 fold or more, 4.8 fold, 4.9 fold or more, or 5 fold or more.

[0147] In a preferred embodiment, the ADAMTS13 variant according to the disclosure has a proteolytic activity that is more than twice the level determined for the ADAMTS13 R568K / F592Y / R660K / Y661F / Y665F variant (e.g., a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 71) in the same assay, e.g., 2.1-fold or more, 2.2-fold or more, 2.3-fold or more, 2.4-fold or more, 2.5-fold or more, 2.6-fold or more, The antibody may exhibit a level of proteolytic activity that is one of: 2.7-fold or more, 2.8-fold or more, 2.9-fold or more, 3-fold or more, 3.1-fold or more, 3.2-fold or more, 3.3-fold or more, 3.4-fold or more, 3.5-fold or more, 3.6-fold or more, 3.7-fold or more, 3.8-fold or more, 3.9-fold or more, 4-fold or more, 4.1-fold or more, 4.2-fold or more, 4.3-fold or more, 4.4-fold or more, 4.5-fold or more, 4.6-fold or more, 4.7-fold or more, 4.8-fold or more, 4.9-fold or more, or 5-fold or more.

[0148] In some embodiments, the ADAMTS13 variants according to the present disclosure exhibit improved / increased inhibition of VWF-mediated platelet capture compared to wild-type human ADAMTS13 and / or the ADAMTS13 R568K / F592Y / R660K / Y661F / Y665F variants described in Jian et al., Blood (2012) 119:3836-3843.

[0149] A given variant of ADAMTS13 can be assessed for its ability to inhibit VWF-mediated platelet sequestration under arterial shear stress and / or the extent to which it inhibits VWF-mediated platelet sequestration under arterial shear stress in a suitable in vitro assay, an example of such an assay is described in Example 1 herein.

[0150] The ability of ADAMTS13 variants to inhibit VWF-mediated platelet capture under arterial shear stress can be assessed by the FRETS-VWF73 assay. The FRETS-VWF73 assay for ADAMTS13-mediated VWF proteolysis is described in South et al., 2018.

[0151] One way to perform the assay is as follows: Vena8 Fluoro+ biochips (Cellix) are coated with 200 μg / ml collagen III (Southern Biotech) and blocked with 1% BSA, 1 mg / ml glucose in HEPES buffer. Washed platelets are mixed with red blood cells and treated with 100 nM PGE1 and 75 mU / ml apyrase to prevent platelet activation before labeling the platelets with 10 μM DiOC6. Platelets are supplemented with 10 μg / ml multimeric plasma VWF and perfused over the collagen surface for 5 min at a constant shear rate of 1500 s-1 (a rate at which platelet capture is dependent on VWF). Adhesion of labeled platelets may be visualized by fluorescent imaging at 250 ms intervals using a 20x objective and analyzed using Slidebook software to determine platelet coverage (%) at 270 s. To measure the effect of ADAMTS13 on platelet capture, assays may be performed in the presence of various concentrations of WT or variant ADAMTS13. EC50 values ​​may be determined by dose-response curves.

[0152] In some embodiments, an ADAMTS13 variant according to the present disclosure inhibits VWF-mediated platelet capture to a level less than 1-fold the level at which VWF-mediated platelet capture is inhibited by wild-type human ADAMTS13 (e.g., a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:1, 7, or 8) in the same assay, for example, to one of 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less.

[0153] In some embodiments, an ADAMTS13 variant according to the present disclosure inhibits VWF-mediated platelet capture to a level less than 1-fold the level at which VWF-mediated platelet capture is inhibited by an R568K / F592Y / R660K / Y661F / Y665F variant of ADAMTS13 (e.g., a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 71) in the same assay, for example, to one of 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less.

[0154] In some embodiments, ADAMTS13 variants according to the present disclosure exhibit increased amelioration / inhibition of thrombosis compared to wild-type human ADAMTS13 and / or the ADAMTS13 R568K / F592Y / R660K / Y661F / Y665F variants described in Jian et al., Blood (2012) 119:3836-3843.

[0155] A given variant of ADAMTS13 can be assessed for its ability to inhibit thrombosis, and / or the extent to which it inhibits thrombosis, in a suitable in vitro or in vivo assay.

[0156] One such suitable in vitro assay is performed as follows: platelet-rich plasma or whole blood can be perfused over a prothrombotic surface (coated with collagen and / or tissue factor) in a microfluidic perfusion chamber. The use of fluorescently labeled donor platelets and / or fluorescently labeled fibrinogen allows for real-time monitoring of clot formation by microscopy. Other methods are known in the art.

[0157] One such suitable in vivo assay is performed as follows: FeCl is injected into the exposed middle cerebral or mesenteric arteries of the cremaster muscle of mice or rats.3 The application of .can be performed in the presence of fluorescently labeled donor platelets and leukocytes, and / or labeled fibrinogen. Thrombus formation can be visualized in situ using two-photon microscopy. Other methods are known in the art.

[0158] In some embodiments, an ADAMTS13 variant according to the present disclosure inhibits thrombosis to a level that is less than 1-fold the level at which thrombosis is inhibited by wild-type human ADAMTS13 (e.g., a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:1, 7 or 8) in the same assay, for example, one of 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less.

[0159] In some embodiments, an ADAMTS13 variant according to the present disclosure inhibits thrombosis to a level that is less than 1-fold the level at which thrombosis is inhibited by an R568K / F592Y / R660K / Y661F / Y665F variant of ADAMTS13 (e.g., a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 71) in the same assay, e.g., 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less.

[0160] In some embodiments, the ADAMTS13 variants according to the present disclosure exhibit increased improved / inhibited blood thrombosis / clotting compared to wild-type human ADAMTS13 and / or the ADAMTS13 R568K / F592Y / R660K / Y661F / Y665F variants described in Jian et al., Blood (2012) 119:3836-3843. A given variant of ADAMTS13 may be assessed for its ability to inhibit blood clot formation / clotting and / or the extent to which it inhibits blood clot formation / clotting in a suitable in vitro or in vivo assay.

[0161] One such suitable in vitro assay is performed as follows: Blood clot formation can be easily measured using a turbidity assay. The absorbance of blood (or often platelet-rich or platelet-poor plasma) is measured at 405 nm and increases upon addition of thrombin or tissue factor as fibrinogen is converted to fibrin to form clots. Other methods are known in the art.

[0162] The time it takes for bleeding to stop after transverse cutting of a rodent's tail may be used as an in vivo assay, although other methods are known in the art.

[0163] In some embodiments, the ADAMTS13 variant according to the present disclosure inhibits blood thrombosis / clotting to less than 1-fold the level at which blood thrombosis / clotting is inhibited by wild-type human ADAMTS13 (e.g., a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:1, 7 or 8) in the same assay, for example, to one of 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less.

[0164] In some embodiments, the ADAMTS13 variants according to the present disclosure inhibit blood thrombosis / clotting to a level that is less than 1-fold, e.g., 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less, that is the level at which blood thrombosis / clotting is inhibited by the R568K / F592Y / R660K / Y661F / Y665F variant of ADAMTS13 (e.g., a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 71) in the same assay.

[0165] In some embodiments, ADAMTS13 variants according to the present disclosure exhibit increased amelioration / inhibition of inflammation compared to wild-type human ADAMTS13 and / or the R568K / F592Y / R660K / Y661F / Y665F variants of ADAMTS13 described in Jian et al., Blood (2012) 119:3836-3843.

[0166] A given variant of ADAMTS13 can be assessed for its ability to inhibit inflammation, and / or the extent to which it inhibits inflammation, in a suitable in vitro or in vivo assay, examples of such assays are described in Example 1 herein.

[0167] As described in the Examples, magnetic resonance imaging (MRI) scans may be used to assess the level of inflammation. Other methods of assessing inflammation are known in the art. In the examples, efficacy of the ADAMTS13 variant is defined as a decrease in R2* on MGE MRI scans (indicating a decrease in vascular inflammation in the brain) and / or a decrease in reactive VWF species in plasma.

[0168] In some embodiments, an ADAMTS13 variant according to the present disclosure inhibits inflammation to a level that is less than 1-fold the level at which inflammation is inhibited by wild-type human ADAMTS13 (e.g., a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:1, 7 or 8) in the same assay, e.g., 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less.

[0169] In some embodiments, an ADAMTS13 variant according to the present disclosure inhibits inflammation to less than 1-fold the level at which inflammation is inhibited by an R568K / F592Y / R660K / Y661F / Y665F variant of ADAMTS13 (e.g., a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 71) in the same assay, e.g., one of 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less.

[0170] Additional Sequences and Linkers In some embodiments, an ADAMTS13 variant according to the present disclosure may further comprise one or more additional amino acids or amino acid sequences.

[0171] In some embodiments, the ADAMTS13 variants according to the present disclosure include one or more linker sequences, for example between the amino acid sequences of the ADAMTS13 variants. The linker sequences may be provided at one or both ends of one or more specific amino acid sequences of the ADAMTS13 variant.

[0172] Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, the entirety of which is incorporated herein by reference. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows the relative movement of the amino acid sequences linked by the linker sequence. Flexible linkers are known to those skilled in the art and some are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often contain a high proportion of glycine and / or serine residues.

[0173] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence consists of the sequence motif G 4 In some embodiments, the linker sequence has a length of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, or 1 to 30 amino acids.

[0174] ADAMTS13 variants may include amino acid sequence(s) for facilitating expression, folding, transport, processing, purification or detection of the polypeptide. For example, ADAMTS13 variants may include sequences encoding His, (e.g., 6XHis), Myc, GST, MBP, FLAG, HA, E, or biotin tags, optionally at the N-terminus or C-terminus of the polypeptide. Illustratively, the ADAMTS13 variants exemplified herein include a C-terminal Myc / 6XHis tag.

[0175] In some embodiments, the ADAMTS13 variant further comprises a detectable moiety, eg, a fluorescent, luminescent, immunodetectable, radioactive, chemical, nucleic acid, or enzymatic label.

[0176] In some embodiments, the ADAMTS13 variant further comprises a signal peptide (also known as a leader sequence or signal sequence). A signal peptide is usually composed of a sequence of 5-30 hydrophobic amino acids that forms a single alpha helix. Secreted proteins and proteins expressed on the cell surface often contain a signal peptide.

[0177] A signal peptide may be present at the N-terminus of a polypeptide or in a newly synthesized polypeptide. The signal peptide provides for efficient transport and secretion of the polypeptide. The signal peptide is often removed by cleavage and is therefore not included in the mature polypeptide secreted from cells expressing the polypeptide.

[0178] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as, for example, SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176). In some embodiments, the signal peptide comprises or consists of an amino acid sequence having at least 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more amino acid sequence identity to SEQ ID NO:5.

[0179] Labels and Conjugates In some embodiments, the ADAMTS13 variant according to the present disclosure further comprises a label or conjugate.

[0180] In some embodiments, the detectable moiety may be a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radioactive label, a chemical label, a nucleic acid label, or an enzyme label. The ADAMTS13 variant may be covalently or non-covalently labeled with a detectable moiety.

[0181] Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin and NDB, green fluorescent protein (GFP), rare earth chelates such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Radioactive labels include radioisotopes such as iodine. 123 , iodine 125 , iodine 126 , iodine 131 , iodine 133 ,bromine 77 ,technetium 99m ,indium 111 ,indium 113m ,gallium 67 ,gallium 68 ,ruthenium 95 ,ruthenium 97 ,ruthenium 103 ,ruthenium 105 ,mercury 207 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47 ,tellurium 121m ,tellurium 122m ,tellurium 125m ,thulium 165 ,thulium 167 ,thulium 168 ,copper 67 , Fluorine 18 ,yttrium 90 ,palladium 100 , Bismuth 217 and antimony 211Luminescent labels include radioluminescent, chemiluminescent (e.g., acridinium ester, luminol, isoluminol) and bioluminescent labels. Immunodetectable labels include haptens, peptides / polypeptides, antibodies, receptors and ligands, such as biotin, avidin, streptavidin or digoxigenin. Nucleic acid labels include aptamers. Enzymatic labels include, for example, peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase and luciferase.

[0182] In some embodiments, the ADAMTS13 variant is conjugated to a chemical moiety. The chemical moiety may be a moiety for providing a therapeutic effect. Antibody-drug conjugates are reviewed, for example, in Parslow et al., Biomedicines. 2016 Sep;4(3):14. In some embodiments, the chemical moiety may be a drug moiety (e.g., a cytotoxic agent). In some embodiments, the drug moiety may be a chemotherapeutic agent. In some embodiments, the drug moiety is selected from calicheamicin, DM1, DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5, and PBD.

[0183] Nucleic acids, cells, compositions and kits The present disclosure provides nucleic acids encoding a polypeptide according to the present disclosure. In some embodiments, the nucleic acid comprises or consists of DNA and / or RNA.

[0184] The present disclosure also provides a vector comprising a nucleic acid according to the present disclosure.

[0185] The nucleic acids and vectors according to the present disclosure can be provided in purified or isolated form, i.e., from other nucleic acids or naturally occurring biological materials. The nucleotide sequence may be included in a vector, for example an expression vector. As used herein, a "vector" is a nucleic acid molecule used as a vehicle to transfer an exogenous nucleic acid into a cell. The vector may be a vector for expressing a nucleic acid in a cell. Such a vector may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0186] The term "operably linked" may include the situation where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a way that expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if it is capable of affecting the transcription of the nucleic acid sequence. The resulting transcript(s) may then be translated into a desired peptide / polypeptide.

[0187] The nucleic acid and / or vector according to the present disclosure is preferably provided for introduction into cells.Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), as described, for example, in Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines 2016 4,9, both of which are incorporated herein by reference in their entirety.

[0188] In some embodiments, the vector may be a eukaryotic vector, e.g., a vector that contains elements necessary for expression of a protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g., a cytomegalovirus (CMV) or SV40 promoter driving protein expression. In some embodiments, the viral vector may be a lentivirus, retrovirus, adenovirus, or herpes simplex virus vector. In some embodiments, the lentiviral vector may be pELNS or may be derived from pELNS.

[0189] In some embodiments, a nucleic acid according to the disclosure comprises or consists of a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95, or to a nucleic acid sequence that encodes the same amino acid sequence as one of SEQ ID NOs: 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 as a result of codon degeneracy.

[0190] Cells containing / expressing ADAMTS13 variants The present disclosure also provides a cell that comprises or expresses the ADAMTS13 variant according to the present disclosure. Also provides a cell that comprises or expresses the nucleic acid or vector according to the present disclosure. The cell that comprises or expresses the ADAMTS13 variant, nucleic acid or vector according to the present disclosure can secrete the ADAMTS13 variant according to the present disclosure. That is, the expression of the ADAMTS13 variant, nucleic acid or vector can result in the soluble production of the ADAMTS13 variant according to the present disclosure from the cell.

[0191] The cell may be a eukaryotic cell, for example a mammalian cell, which may be a primate (such as a rhesus monkey, a cynomolgus monkey, a non-human primate or a human) or a non-human mammal (such as a rabbit, a guinea pig, a rat, a mouse or other rodent (including rodents), a cat, a dog, a pig, a sheep, a goat, a cow (including cattle such as dairy cows, or any animal in the order Equidae), a horse (including any animal in the family Equidae), a donkey, and a non-human primate).

[0192] In some embodiments, the cells are or are derived from cell types commonly used for expression of polypeptides for therapeutic use in humans. Exemplary cells are described, for example, in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461, which is incorporated herein by reference in its entirety, including, for example, CHO, HEK293, PER.C6, NS0 and BHK cells.

[0193] The present disclosure also provides a method for producing a cell comprising a nucleic acid(s) or vector(s) according to the present disclosure, comprising introducing a nucleic acid or vector according to the present disclosure into a cell. In some embodiments, the introduction of the isolated nucleic acid(s) or vector(s) according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g., retroviral transduction).

[0194] The present disclosure also provides a method for producing a cell that expresses / comprises an ADAMTS13 variant according to the present disclosure, comprising introducing a nucleic acid or vector according to the present disclosure into the cell. In some embodiments, the method further comprises culturing the cell under conditions suitable for expression of the nucleic acid(s) or vector(s) by the cell. In some embodiments, the method is performed in vitro.

[0195] The present disclosure also provides cells obtained or obtainable by a method according to the present disclosure.

[0196] Generation of ADAMTS13 variants ADAMTS13 variants according to the present disclosure may be prepared according to methods for producing polypeptides known to those skilled in the art. Polypeptides can be prepared by chemical synthesis, such as liquid phase synthesis or solid phase synthesis. For example, peptides / polypeptides can be synthesized using the methods described in, for example, Chandrudu et al., Molecules (2013), 18:4373-4388, the entire contents of which are incorporated herein by reference.

[0197] Alternatively, the polypeptide can be produced by recombinant expression.Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5 (2): 135-146, both of which are incorporated herein by reference in their entirety.

[0198] Any cell suitable for expression of a polypeptide may be used for recombinant production according to the present disclosure. The cell may be prokaryotic or eukaryotic. In some embodiments, the cell is a prokaryotic cell, such as an archaeal or bacterial cell. In some embodiments, the bacterium may be a gram-negative bacterium, such as a bacterium of the Enterobacteriaceae family, e.g., Escherichia coli. In some embodiments, the cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, e.g., a cell as described herein above.

[0199] In some cases, the cells are not prokaryotic because some prokaryotic cells are not capable of the same folding or post-translational modifications as eukaryotic cells. Additionally, very high expression levels are possible in eukaryotes, and purification of the protein from eukaryotes can be facilitated with the use of appropriate tags. Specific plasmids that facilitate secretion of the protein into the medium may also be utilized.

[0200] Production may involve the culture or fermentation of eukaryotic cells modified to express the polypeptide(s) of interest. The culture or fermentation may be carried out in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Secreted proteins may be collected by partitioning the medium / fermentation broth from the cells, extracting the protein content and separating the individual proteins to isolate the secreted polypeptide(s). Culture, fermentation and separation techniques are well known to those of skill in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition, incorporated herein by reference above).

[0201] A bioreactor contains one or more vessels in which cells can be cultured. Cultivation in a bioreactor may be continuous, with a continuous flow of reactants into the reactor and a continuous flow of cultured cells out of the reactor. Alternatively, the cultivation may be in batches. Bioreactors monitor and control environmental conditions such as pH, oxygen, flow rates into and out of the vessel, and agitation within the vessel to provide optimal conditions for the cells being cultured.

[0202] After culturing, cells expressing the polypeptide(s) of interest may be isolated. Any suitable method for isolating proteins from cells known in the art may be used. Isolating the polypeptide may require separating the cells from the nutrient medium. If the polypeptide(s) are secreted from the cells, the cells may be separated from the culture medium containing the secreted polypeptide(s) of interest by centrifugation. If the polypeptide(s) of interest are collected intracellularly, isolating the protein may include centrifugation to separate the cells from the cell culture medium, treating the cell pellet with a lysis buffer, and disrupting the cells, for example, by sonication, rapid freeze-thawing or osmotic lysis.

[0203] It may then be desirable to isolate the polypeptide(s) of interest from the supernatant or medium, which may contain other proteins and non-protein components. A common approach to separate protein components from the supernatant or medium is by precipitation. Proteins of different solubilities are precipitated with different concentrations of precipitant, such as ammonium sulfate. For example, at low concentrations of precipitant, water-soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitant, proteins of different solubilities may be differentiated. Dialysis may then be used to remove the ammonium sulfate from the separated proteins.

[0204] Other methods for isolating / purifying polypeptides are known in the art, such as ion exchange chromatography and size chromatography.Polypeptides may also be affinity purified using suitable binding partners for molecular tags on the polypeptide (e.g., His, FLAG, Myc, GST, MBP, HA, E, or biotin tags).These techniques may be used as an alternative to precipitation or may be performed after precipitation.In some cases, it may be further desirable to treat the polypeptide, for example to remove amino acid sequences, molecular tags, portions, etc.

[0205] In some embodiments, the treatment is with an endopeptidase suitable for cleavage and removal of amino acid sequences. In some embodiments, the treatment is with an enzyme to remove a moiety of interest. In some embodiments, the polypeptide is treated to remove glycans (i.e., the polypeptide is deglycosylated), for example, by treatment with a glycosidase, such as peptide:N-glycosidase (PNGase).

[0206] Other methods for distinguishing different proteins are known in the art, such as ion exchange chromatography and size chromatography, which may be used as an alternative to precipitation or may be performed after precipitation.

[0207] Once the polypeptide(s) of interest have been isolated from the culture, it may be desirable or necessary to concentrate the polypeptide(s). Many methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization.

[0208] In some embodiments, production of the polypeptide occurs in vivo, for example, after introduction of a cell containing a nucleic acid or vector encoding an ADAMTS13 variant of the present disclosure into a host, or after introduction of a nucleic acid or vector encoding an ADAMTS13 variant of the present disclosure into a cell of a host. In such embodiments, the polypeptide is transcribed, translated, and post-translationally processed into a mature polypeptide. In some embodiments, the polypeptide is produced in situ at a desired location within the host.

[0209] composition The disclosure also provides compositions comprising the ADAMTS13 variants, nucleic acids, expression vectors and cells described herein.

[0210] The ADAMTS13 variants, nucleic acids, expression vectors and cells described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may include pharma- ceutical acceptable carriers, diluents, excipients or adjuvants. The compositions may be formulated for local, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration, which may include injection or infusion.

[0211] Suitable formulations may include ADAMTS13 variants in a sterile or isotonic medium. Drugs and pharmaceutical compositions may be formulated in fluid form, including gels. Fluid formulations may be formulated for administration by injection or infusion (e.g., via catheter) into selected areas of the human or animal body.

[0212] In some embodiments, the compositions are formulated for injection or infusion, for example, into a blood vessel or tissue / organ of interest.

[0213] The present disclosure also provides methods for producing pharma- ceutically useful compositions, which may include one or more steps selected from the following: producing an ADAMTS13 variant, nucleic acid, expression vector or cell described herein; isolating an ADAMTS13 variant, nucleic acid, expression vector or cell described herein; and / or mixing an ADAMTS13 variant, nucleic acid, expression vector or cell described herein with a pharma- ceutically acceptable carrier, adjuvant, excipient or diluent.

[0214] For example, a further aspect of the disclosure provides a method of formulating or manufacturing a medicament or pharmaceutical composition for use in treating a disease / condition (e.g., a disease described herein below), the method comprising formulating a pharmaceutical composition or agent by mixing an ADAMTS13 variant, nucleic acid, expression vector or cell described herein with a pharma- ceutically acceptable carrier, adjuvant, excipient or diluent.

[0215] In aspects and embodiments of the present disclosure, the ADAMTS13 variant may be provided as a composition comprising specific chemical components at specific concentrations / proportions.

[0216] In some embodiments, the ADAMTS13 variant is provided in a buffer. As used herein, "buffer" refers to a buffer solution that resists changes in pH due to the action of its acid-base conjugate components. The buffer of the present disclosure preferably has a pH in the range of about 4.5 to about 7.0, preferably about 5.0 to about 6.5. Examples of buffers that control the pH in this range include acetate, histidine, histidine-arginine, histidine-methionine, and other organic acid buffers.

[0217] kit In some aspects of the present disclosure, a kit of parts is provided. In some embodiments, the kit may have at least one container with a predetermined amount of the ADAMTS13 variant, nucleic acid, expression vector, cell, or composition described herein.

[0218] In some embodiments, the kit may include materials for producing the ADAMTS13 variants, nucleic acids, expression vectors, cells, or compositions described herein. For example, the kit may include materials for modifying cells to express or contain an ADAMTS13 variant, nucleic acid, or expression vector according to the present disclosure, or materials for introducing a nucleic acid or expression vector according to the present disclosure into a cell.

[0219] The kit may be provided with instructions for administering the ADAMTS13 variant, nucleic acid, expression vector, cell or composition to a patient to treat or prevent a particular disease / condition, such as those described herein below.

[0220] In some embodiments, the kit may further comprise at least one container with a predetermined amount of another therapeutic / prophylactic agent (e.g., a therapeutic / prophylactic agent for the treatment / prevention of a disease / condition described herein). In such embodiments, the kit may also include a second medicament or pharmaceutical composition such that the two medicaments or pharmaceutical compositions can be administered simultaneously or separately to provide a combined treatment / prevention for a particular disease or condition.

[0221] Therapeutic and prophylactic applications The ADAMTS13 variants, nucleic acids, vectors, cells and pharmaceutical compositions described herein find use in therapeutic and prophylactic methods.

[0222] The present disclosure provides an ADAMTS13 variant, a nucleic acid, a vector, a cell, or a pharmaceutical composition according to the present disclosure for use in a method of medical treatment or prevention.The present disclosure also provides the use of an ADAMTS13 variant, a nucleic acid, a vector, a cell, or a pharmaceutical composition according to the present disclosure in the manufacture of a medicament for treating or preventing a disease or condition.The present disclosure also provides a method for treating or preventing a disease or condition, comprising administering to a subject a therapeutically or prophylactically effective amount of an ADAMTS13 variant, a nucleic acid, a vector, a cell, or a pharmaceutical composition according to the present disclosure.

[0223] The method may be effective in reducing the onset or progression of a disease / condition, alleviating the symptoms of a disease / condition, or reducing the pathology of a disease / condition. The method may be effective in preventing the progression of a disease / condition, for example, preventing the disease / condition from worsening or slowing the rate of progression of the disease / condition. In some embodiments, the method may result in an improvement of a disease / condition, for example, a reduction in the symptoms of the disease / condition, or a reduction in some other correlate of the severity / activity of the disease / condition. In some embodiments, the method may prevent the onset of the disease / condition at a later stage (e.g., chronic stage).

[0224] It is understood that the articles of the present disclosure may be used to treat / prevent any disease / condition that results in a therapeutic or prophylactic benefit from a reduction in the level and / or activity of VWF and / or VWF-comprising complexes (e.g., multimeric complexes, e.g., UL-VWF multimers). For example, the disease / condition may be one in which VWF and / or VWF-comprising complexes are pathologically implicated, such as a disease / condition in which an increased level of VWF and / or VWF-comprising complexes is positively associated with the onset, development or progression of the disease / condition and / or the severity of one or more symptoms of the disease / condition, or a disease / condition in which an increased level of VWF and / or VWF-comprising complexes is a risk factor for the onset, development or progression of the disease / condition. In particular, the articles of the present disclosure may be used to treat / prevent any disease / condition that would benefit therapeutically or prophylactically from the inhibition of thrombosis. For example, the disease / condition may be one in which thrombosis is pathologically implicated, such as one in which thrombosis is positively associated with the onset, development or progression of the disease / condition and / or one or more of the severity of the disease / condition, or one in which thrombosis is a risk factor for the onset, development, or progression of the disease / condition.

[0225] It will also be understood that the articles of the present disclosure may be used to treat / prevent any disease / condition that derives therapeutic or prophylactic benefit from an increased level of ADAMTS13 or ADAMTS13 proteolytic activity, for example, a disease / condition in which ADAMTS13 deficiency / deficiency or ADAMTS13 proteolytic activity is pathologically implicated, such as a disease / condition in which a reduced level of ADAMTS13 and / or a reduced level of ADAMTS13 proteolytic activity is positively associated with the onset, development or progression of the disease / condition and / or the severity of one or more symptoms of the disease / condition, or a disease / condition in which a reduced level of ADAMTS13 and / or a reduced level of ADAMTS13 proteolytic activity is a risk factor for the onset, development or progression of the disease / condition.

[0226] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is one that is characterized, e.g., by an increased level of VWF and / or complexes comprising VWF, as compared to, e.g., the level of VWF and / or complexes comprising VWF in the absence of the disease / condition.

[0227] In particular, the ADAMTS13 variants, nucleic acids, vectors, cells and pharmaceutical compositions according to the present disclosure find use for treating or preventing diseases / conditions associated with VWF and / or complexes containing VWF, such as diseases associated with elevated levels or activity of VWF and / or complexes containing VWF.

[0228] According to various embodiments of the present disclosure, methods of treating and / or preventing the diseases / conditions described herein may include one or more of the following: reducing the level or activity of VWF, reducing the level or activity of complexes containing VWF; reducing the level of a correlation of the level or activity of VWF, reducing the level of a correlation of the level or activity of complexes containing VWF, reducing or inhibiting thrombosis; reducing or inhibiting blood clot formation / clotting; reducing or inhibiting inflammation; increasing the level of ADAMTS13 proteolytic activity and increasing the proteolysis of VWF and / or complexes containing VWF.

[0229] Thrombotic thrombocytopenic purpura (TTP) is characterized by a severe deficiency of ADAMTS13, either due to acquired autoantibodies that inhibit function or enhance clearance (idiopathic) or due to loss of function mutations in the ADAMTS13 gene (congenital). Acute episodes are characterized by disseminated UL-VWF-rich microthrombi that cause multiorgan ischemia and death in 20% of patients, with survivors at high risk of relapse. Current treatments include time-consuming plasma infusions that replenish functional ADAMTS13 levels, but carry the risk of further complications such as severe allergic reactions, volume overload, and adverse blood-borne diseases (Sadler 2008; Kremer Hovinga et al. 2017). Recombinant human ADAMTS13 (Bax930-NCT03393975) has been shown to have a half-life and pharmacokinetic profile comparable to plasma-derived ADAMTS13 and is currently in phase 3 clinical trials in patients with congenital TTP (Scully et al. 2017). However, the use of improved ADAMTS13 variants in this setting, where the variants are not recognized by wild-type ADAMTS13 autoantibodies, may further reduce dosage and hospital stays, expanding treatment options suitable for idiopathic TTP.

[0230] Subarachnoid hemorrhage (SAH) accounts for 5% of all strokes and is associated with a very poor outcome, with a 35% mortality rate in the first 3 months and only about 55% of patients able to recover independent function (Macdonald and Schweizer 2017). Following the initial stage of brain injury, one-third of patients experience delayed cerebral ischemia caused by microthrombosis 3–14 days after hemorrhage, leading to further inflammation and neurological deterioration. Studies of SAH patients reveal elevated VWF and reduced ADAMTS13 levels after hemorrhage compared to healthy controls (Kumar et al. 2017). Protection against microglial activation and neuronal injury was observed in VWF-deficient mice after SAH (Wan et al. 2018), and systemic administration of ADAMTS13 improved microthrombosis, apoptosis, and neuroinflammation, leading to improved neuronal function (Muroi et al. 2014). Evidence also suggests a role for the ADAMTS13-VWF axis in intracerebral hemorrhage (ICH): recombinant ADAMTS13 was shown to reduce microglial activation, neutrophil accumulation, protect against blood-brain barrier disruption, and improve neurological outcomes in a mouse ICH model (Cai et al. 2015).

[0231] Chronic thromboembolic pulmonary hypotension (CTEPH) is a progressive disease caused by disseminated thrombi within the pulmonary vasculature that, if left untreated, can lead to right heart failure. Newnham and coworkers demonstrated that CTEPH patients have decreased ADAMTS13 and increased VWF plasma levels compared to healthy controls (Newnham et al. 2019). Currently, surgical removal of the pulmonary obstruction is the definitive treatment option for CTEPH, but the use of potentially thrombolytic ADAMTS13 may circumvent the existing problems of operability.

[0232] The importance of VWF-ADAMTS13 in a mouse model of myocardial infarction (MI) has been clearly demonstrated (Witsch et al. 2018). A meta-analysis of patient data described that low levels of ADAMTS13 are associated with an increased risk of MI, but this study did not find a synergistic effect with high VWF levels, which contradicts the relationship seen in ischemic stroke (Maino et al. 2015). In patients with ST-elevation myocardial infarction (STEMI), MI is associated with elevated VWF and reduced ADAMTS13 activity, but administration of recombinant ADAMTS13 in a porcine model of acute MI showed no effect on infarct size (Eerenberg et al. 2016). In patients with unstable angina (UA), an unfavorable VWF:ADAMTS13 ratio persists for up to 6 months after the acute phase, indicating a prolonged thrombogenic period and an increased likelihood of platelet clot formation in the chronic phase (Fuchigami et al. 2008). ADAMTS13 deficiency has also been associated with other ischemia / reperfusion pathologies. Using data from the Rotterdam Study, a Dutch general population-based study, Sedaghat et al. found that elevated VWF:ADAMTS13 ratios and reduced ADAMTS13 levels were independently associated with a rapid decline in renal function (Sedaghat et al. 2016). More recently, two parallel studies using mouse models of renal ischemia / reperfusion injury demonstrated a protective effect of recombinant ADAMTS13 in reducing immune cell infiltration and kidney damage (Zhou et al. 2019; Ono et al. 2019). These results are supported in VWF- / - mice, which have less kidney damage than wild-type controls (Ono et al. 2019). Furthermore, Urisono et al. reported partial protection against hepatic ischemia / reperfusion injury in VWF− / − mice compared to wild-type controls (Urisono et al. 2018).

[0233] In diabetic patients, clinical reports suggest an association between low levels of ADAMTS13 and microvascular complications such as nephropathy (kidney disease) (Taniguchi et al. 2010). Interestingly, one study demonstrated that a single nucleotide polymorphism in ADAMTS13 [Pro618Ala] reduces the proteolytic activity of ADAMTS13 and is associated with an increased incidence of renal and cardiovascular events in type 2 diabetic patients (Rurali et al. 2013). Animal models have shown that the decline in renal function is exacerbated by ADAMTS13 deficiency in wild-type mice and ameliorated in ADAMTS13- / -VWF- / - double knockout mice, demonstrating a VWF-dependent effect (Dhanesha et al. 2017). These results indicate that the VWF-ADAMTS13 axis is at least partially responsible for the endothelial dysfunction that occurs in diabetic vascular pathology and that patients may benefit from ADAMTS13 therapy.

[0234] Nearly 30 years ago, the BMJ reported that patients with Crohn's disease, ulcerative colitis, and bacterial diarrhea have high circulating VWF levels (Stevens et al. 1992). More recently, a mouse model of colitis demonstrated enhanced VWF-rich thrombi in the colon and propagation of intestinal inflammation in ADAMTS13-deficient mice, which was reduced upon administration of recombinant human ADAMTS13. The same paper also identified areas of prominent VWF staining in human colitis colon tissue compared to healthy controls, providing a strong basis for a VWF-ADAMTS13 imbalance in exacerbating inflammatory bowel disease (Zitomersky et al. 2017). The proposed mechanism of increased VWF release from the colonic endothelium, thrombocytosis, ischemia, and further VWF release provides an opportunity for ADAMTS13 intervention aimed at breaking this vicious cycle and limiting further damaging inflammation.

[0235] Given that many of the studies discussed here have only been conducted in the past 5 years, there may still be many undocumented indications for therapeutic use of ADAMTS13. Analysis of population data, such as that provided by the Rotterdam Study, may help identify specific diseases in which abnormal VWF or ADAMTS13 activity may be involved in the pathology. Indeed, analysis of data collected in the same study highlighted an association between reduced ADAMTS13 levels and risk of dementia (Wolters et al. 2018). As research continues to identify pathological inflammation as a trigger for various diseases, such applications may prove invaluable in multiple contexts.

[0236] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is characterized by an increased level and / or activity of VWF and / or an increased level and / or activity of complexes containing VWF (e.g., compared to levels in a healthy state (i.e., in the absence of the disease / condition)). In some embodiments, the disease / condition to be treated / prevented is characterized by a decreased level of ADAMTS13 and / or a decreased level of ADAMTS13 proteolytic activity (e.g., compared to levels in a healthy state (i.e., in the absence of the disease / condition)). A decreased level of ADAMTS13 and / or a decreased level of ADAMTS13 proteolytic activity may be referred to herein as "ADAMTS13 deficiency."

[0237] In some embodiments, the articles of the present disclosure are provided for use in the treatment / prevention of thrombosis or diseases / conditions characterized by thrombosis. The articles of the present disclosure may be used as antithrombogenic / anticoagulant agents.

[0238] In some embodiments, articles of the present disclosure are provided for use in the treatment / prevention of inflammation, or diseases / conditions characterized by inflammation.

[0239] In some embodiments, the disease or condition treated / prevented by the present disclosure is one of the following: diseases / conditions characterized by thrombosis, diseases / conditions characterized by inflammation, thrombotic thrombocytopenic purpura (TTP), ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage (SAH), intracerebral hemorrhage (ICH), chronic thromboembolic pulmonary hypotension (CTEPH), myocardial infarction (MI), ST-segment elevation myocardial infarction (STEMI), unstable angina (UA), ischemia, reperfusion, deep and selected from peripheral vein thrombosis, pulmonary embolism, disseminated intravascular coagulation (DIC), hemolytic-uremic syndrome (HUS), cerebral infarction, systemic lupus erythematosus (SLE), disease caused by infection with SARS-CoV (e.g., SARS-CoV-2, e.g., COVID-19), acute respiratory distress syndrome (ARDS), pneumonia, kidney damage, nephropathy, microvascular disease, dementia, Crohn's disease, inflammatory bowel disease, ulcerative colitis, and bacterial diarrhea.

[0240] The administration of the articles of the present disclosure is preferably in a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to provide a therapeutic or prophylactic effect in a subject. The actual amount administered, the rate of administration, and the duration of administration will vary depending on the nature and severity of the disease / condition, and the specific article administered. The determination of the prescription of treatment, such as dosage, is within the responsibility of general practitioners and other physicians, and will usually take into account the disease / disorder being treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to the expert. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0241] Administration can be alone or in combination with other treatments, either simultaneously or sequentially depending on the condition to be treated. The articles of the present disclosure and the therapeutic / prophylactic agents may be administered simultaneously or sequentially.

[0242] Concurrent administration refers to administration of the ADAMTS13 variant, nucleic acid, vector, cell or composition of the present disclosure together with another therapeutic / prophylactic agent, for example as a pharmaceutical composition (combined preparation) containing both agents, or immediately after each other, optionally via the same administration route, for example into the same artery, vein, or other blood vessel. Sequential administration refers to administration of (i) one of the ADAMTS13 variant, nucleic acid, vector, cell or composition of the present disclosure and (ii) another therapeutic / prophylactic agent, and then administration of the other agent separately after a certain time interval. Although in some embodiments, it is not necessary that the two agents are administered by the same route. The time interval can be any time interval.

[0243] Multiple doses of an ADAMTS13 variant, nucleic acid, vector, cell or composition of the disclosure may be provided. One or more, or each dose may be accompanied by simultaneous or sequential administration of another therapeutic / prophylactic agent.

[0244] The multiple administrations may be separated by a predetermined time interval, which may be selected as one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. By way of example, doses may be given once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0245] subject The subject according to the embodiments of the present disclosure may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female. A "subject" may be a "patient." The subject may have been diagnosed with a disease or condition (e.g., cancer) requiring treatment, may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition.

[0246] In embodiments according to the present disclosure, the subject is preferably a human subject.In some embodiments, the subject treated according to the therapeutic or preventive method of the present disclosure is a subject who has a disease described herein or is at risk of developing a disease.In embodiments according to the present disclosure, the subject can be selected for treatment according to the method based on the characterization of the specific marker of such disease / condition.

[0247] *** The features disclosed in the foregoing description, the following claims, or the accompanying drawings may, where appropriate, be expressed in a specific form thereof, or in a means for performing a disclosed function, or in terms of a method or process for obtaining a disclosed result, and such features may be utilized individually or in any combination to realize the disclosure in various forms thereof.

[0248] While the present disclosure has been described in conjunction with the exemplary embodiments set forth above, many equivalent modifications and variations will become apparent to those skilled in the art given this disclosure. Accordingly, the exemplary embodiments of the present disclosure set forth above are considered to be illustrative and not limiting. Various modifications to the described embodiments may be made without departing from the spirit and scope of the disclosure.

[0249] For the avoidance of doubt, the theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0250] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0251] Throughout this specification, the words "comprise" and "include", as well as the inclusion of claims following variations such as "comprises", "comprising" and "including", will be understood to mean the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps, unless the context requires otherwise.

[0252] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, by use of the antecedent "about," when values ​​are expressed as approximations, it will be understood that the particular value forms another embodiment. The term "about" with respect to numerical values ​​is arbitrary and may mean, for example, + / - 10%.

[0253] When a nucleic acid sequence is disclosed herein, the reverse complement thereof is also specifically contemplated.

[0254] The methods described herein may preferably be carried out in vitro. The term "in vitro" is intended to encompass procedures performed with cells in culture, while the term "in vivo" is intended to encompass procedures with / on an intact multi-cellular organism. EXAMPLES

[0255] Example 1 - Materials and Methods Protein expression and purification The pCDNA3.1 28 construct encoding recombinant human ADAMTS13 with a C-terminal Myc / His6 tag was used to generate variants by site-directed mutagenesis. Constructs were generated that encode ADAMTS13 variants containing the following substitutions in the linker 3 region of the ADAMTS13 protein: A1144V, A1145V, A1146V, P1147V, P1154V, P1171V, P1173V, P1175V, P1180V, and P1182V. Constructs were also generated that encode the known R568K / F592Y / R660K / Y661F / Y665F variants of ADAMTS13.

[0256] The amino acid sequences of the Myc / His6-tagged human ADAMTS13 and ADAMTS13 variants featured in this example are shown in SEQ ID NOs: 72-83, and the nucleotide sequences encoding the recombinant polypeptides are shown in SEQ ID NOs: 84-95.

[0257] Myc / His6-tagged human ADAMTS13 and ADAMTS13 variants were expressed from CHO-K1 cell lines stably expressing the protein-encoding constructs and then purified by fast protein liquid chromatography (FPLC) using a zinc-binding HiTrap chelating column (GE Healthcare, Chicago, IL, USA). For use in mouse stroke models, CHO-expressed ADAMTS13 was passed through a hydroxyapatite column to remove contaminating proteins, and purified ADAMTS13 protein was quantified by ELISA and dialyzed into 150 mm NaCl, 20 mm histidine, 2% sucrose, and 0.05% Tween-80 (pH 7.4).

[0258] Corresponding constructs were also generated encoding ADAMTS13 variants containing the following substitutions in the linker 3 region of the ADAMTS13 protein: A1144K, A1144I, A1145K, A1145I, A1146K, A1146I, P1147K, P1147I, P1154K, P1154I, P1171K, P1171I, P1173K, P1173I, P1175K, P1175I, P1180K, P1180I, P1182K, and P1182I. The amino acid sequences of these ADAMTS13 variants are shown in SEQ ID NOs: 96-115, and the nucleotide sequences encoding the recombinant polypeptides are shown in SEQ ID NOs: 116-135. These ADAMTS13 variants were transiently expressed in HEK293S cells and collected in concentrated conditioned medium. Protein levels were quantified by an in-house ADAMTS13 ELISA and confirmed by Western blot.

[0259] FRETS-VWF73 assay FRETS-VWF73 assay of ADAMTS13-mediated VWF proteolysis was performed as described in South et al., 2018 .

[0260] Briefly, purified Myc / His6-tagged human ADAMTS13 and ADAMTS13 variants were incubated in white 96-well plates (Nunc) with 5 mM Bis-Tris pH 6.0, 25 mM CaCl 2 , and diluted with 0.005% Tween-20 to a concentration of 0.3 nM.

[0261] In some experiments, purified VWF D4-CK fragments may be added to a final concentration of 20-60 nM, followed by preincubation for 45 min at 37° C. In other experiments, no VWF D4-CK fragments were added.

[0262] Subsequently, proteolysis was initiated by adding an equal volume of 4 μM FRETS-VWF73 substrate (Peptanova). Fluorescence (excitation, 340 nm; emission, 460 nm) was measured at 1 min intervals for 1 h at 30 °C using a Fluostar Omega plate reader (BMG Labtech). Fluorescence measurements were normalized to values ​​obtained for wild-type human ADAMTS13.

[0263] Vena8 Fluoro+ biochips (Cellix) were coated with 200 μg / ml type III collagen (Southern Biotech) and blocked with 1% BSA, 1 mg / ml glucose in HEPES buffer. Washed platelets were mixed with red blood cells, and platelets were blocked with 10 μM DiOCt 6 Prior to labeling with ADAMTS13, platelets were treated with 100 nM PGE1 and 75 mU / ml apyrase to prevent platelet activation. Platelets were supplemented with 10 μg / ml multimeric plasma VWF and perfused over the collagen surface for 5 min at a constant shear rate of 1500 s-1, a rate at which platelet capture is VWF-dependent. Adhesion of labeled platelets was visualized by fluorescent imaging at 250 ms intervals using a 20x objective and analyzed using Slidebook software to determine percent platelet coverage at 270 s. To determine the effect of ADAMTS13 on platelet capture, assays were performed in the presence of WT or variant ADAMTS13 at a range of concentrations and EC 50 Values ​​were determined by dose-response curves.

[0264] Mouse focal ischemic stroke model ADAMTS13 was administered as a bolus via tail vein catheter 1 h after MCA occlusion. The dose was 4 μl / g of a 1.5 mg / ml preparation, resulting in a final dose of 6 mg / kg. FeCl3-induced occlusion of the middle cerebral artery (MCA) was performed using a surgical technique previously described (Denorme et al., 2016). Regional cerebral blood flow (rCBF) of the MCA territory was measured by laser speckle contrast imaging, and infarct size was measured by staining brain sections (10 μm PFA-fixed frozen sections) with cresyl violet 24 h after MCA occlusion. Brain sections were also stained with hematoxylin and eosin and by immunofluorescence using antibodies against VWF (Dako, rabbit polyclonal A0082) and fibrinogen (Thermo-Fisher, sheep polyclonal PA1-85429).

[0265] Treatment of Respiratory Tract Infections Mice were infected with a mildly virulent strain of Streptococcus pneumoniae (ascending inoculation over days 1–6) and administered human ADAMTS13, ADAMTS13 variants or vehicle (as a negative control) on day 8. Inflammation in the cerebrovasculature was assessed on day 18 using gradient-echo MRI and VWF-specific MPIO contrast agent.

[0266] On day 8, ADAMTS13 was administered as a bolus via tail vein catheter. The dose was 4 μl / g of a 1.5 mg / ml preparation for a final dose of 6 mg / kg. When amoxicillin was used, it was also administered by single subcutaneous injection at a final dose of 400 mg / kg on day 8. Efficacy of ADAMTS13 variants in this model was defined as a reduction in R2* on MGE MRI scans (indicating reduced vascular inflammation in the brain) and / or a reduction in reactive VWF species in plasma.

[0267] FeCl3-induced middle cerebral artery (MCA) occlusion Mice are treated using a previously described surgical technique (Denorme et al., 2016) to induce occlusion of the MCA. Vehicle or caADAMTS13 (6 mg / kg) is administered as a bolus via the tail vein catheter 4 h after MCA occlusion. Regional cerebral blood flow (rCBF) in the MCA territory is measured by laser speckle contrast imaging 1 h after ADAMTS13 administration, and infarct size is measured by staining brain sections (10 μm PFA-fixed frozen sections) with cresyl violet 24 h after MCA occlusion. Brain sections are stained with hematoxylin and eosin and by immunofluorescence using antibodies against VWF (Dako, rabbit polyclonal A0082) and fibrinogen (Thermo-Fisher, sheep polyclonal PA1-85429).

[0268] Example 2 - Results in vitro activity The proteolytic activity of wild-type ADAMTS13, the known GoF ADAMTS13 R568K / F592Y / R660K / Y661F / Y665F variants, and the linker 3 variants was assessed by the FRETS-VWF73 assay both in the absence (−) and presence (+) of activating VWF-D4CK domain fragments ( Fig. 1A ).

[0269] The residues targeted in the ADAMTS13 variants were selected based on those most likely to significantly affect the secondary structure and therefore the activity of the protein. Specifically, the cluster of alanine residues (Ala1144Val, Ala1145Val, and Ala1146Val) was selected based on the possibility that these residues may confer plasticity to the protein structure in this region. The subsequent proline residues (Pro1147Val, Pro1154Val, Pro1171Val, Pro1173Val, Pro1175Val, Pro1180Val, and Pro1182Val) were selected based on the possibility that these residues may constrain and have a stronger influence on the secondary structure of the protein.

[0270] Indeed, the nature of the amino acid residues selected for introduction into ADAMTS13 variants was not determined based on the typical approach of chemical similarity (e.g., degree of charge and structural conservation, etc.), but on the degree of plasticity provided by these residues. Specifically, it has been found that, compared to alanine, residues such as lysine, valine, and isoleucine significantly reduce plasticity, approaching that of proline.

[0271] A1144V, A1146V, P1180V, and P1182V showed significantly higher proteolytic activity compared to wild-type ADAMTS13 and the R568K / F592Y / R660K / Y661F / Y665F variants (Figure 1A). This significant difference was observed both in the absence (-) and presence (+) of the activating VWF-D4CK domain fragment. In the absence of the activating VWF-D4CK domain fragment, the proteolytic activity of the A1144V, A1146V, P1180V, and P1182V variants was more than four-fold higher than that of wild-type ADAMTS13.

[0272] Substitution of alanine at positions 1144 and 1146 with isoleucine, an amino acid with similar rigidity to valine, induces constitutive activity similar to that seen with the A1144V and A1146V variants (Figure 1B). Substitution of valine with lysine or isoleucine at positions 1180 and 1182 increases activity by more than 10-fold, higher than that of the P1180V and P1182V variants and higher than that of A1144V.

[0273] VWF-mediated platelet capture under arterial shear stress was measured for wild-type ADAMTS13, the R568K / F592Y / R660K / Y661F / Y665F variant, and the A1144V variant. The results show that the A1144V variant was more effective at reducing platelet coverage than wild-type ADAMTS13 or the R568K / F592Y / R660K / Y661F / Y665F variant. The A1144V variant was able to reduce platelet coverage to a level comparable to the negative control condition at a concentration of 2.5 nM. The A1144V variant was found to be much more potent in reducing platelet coverage in this assay than either wild-type ADAMTS13 or the R568K / F592Y / R660K / Y661F / Y665F variant.

[0274] Results of laser speckle contrast analysis Laser speckle contrast imaging provides quantitative maps of regional cerebral blood flow in mouse brains. Flux values ​​are derived from regions of interest in the ipsilateral (stroke-affected) hemisphere (corresponding to tissue supplied by the middle cerebral artery) and from identical control regions in the contralateral hemisphere. A decrease in the ratio of these flux values ​​indicates reduced blood flow in the stroke-affected hemisphere compared to the control region. Typically, the flux ratio is reduced to approximately 0.4 in animals with MCAo compared to a value of 1 in sham animals. An increase in the flux ratio over time observed in animals treated with caADAMTS13 indicates that the occluding thrombus has been removed and blood flow has been restored to the MCA territory.

[0275] Lesion volume Figures 6-9 show the effect of treatment with wild-type ADAMTS13 or the A1144V variant on lesion volume in an ischemic stroke model, 24 hours after occlusion.

[0276] Figure 7 shows that treatment with the A1144V variant (referred to as "caADAMTS13" in Figure 7) reduced lesion volume compared to treatment with vehicle control, N-acetyl-cysteine ​​(NAC), or wild-type ASAMTS13 (wtADAMTS13).

[0277] We also found that treatment with the A1144V variant increased the flux ratio between ipsilateral and contralateral ROIs to a greater extent between 0 and 60 min post-injection than treatment with vehicle control, N-acetyl-cysteine ​​(NAC), or wild-type ASAMTS13 (wtADAMTS13) (Figure 7).

[0278] A significant negative correlation was observed between increased flux ratio and lesion volume across all treatment groups (Figure 8).

[0279] An increase in flux ratio over time indicates that the occlusive thrombus has been removed and blood flow has been restored to the MCA territory, thereby indicating that viable tissue has been salvaged, as indicated by a correlating decrease in lesion volume.

[0280] Residual thrombus contents Figures 10 and 11 show that VWF and fibrin(ogen) content at the site of FeCl3 application was lower in brain sections obtained from mice treated with the A1144V variant (referred to as "caADAMTS13") than with the vehicle control, N-acetyl-cysteine ​​(NAC), or wild-type ASAMTS13 (wtADAMTS13).

[0281] Thrombi are primarily composed of a combination of fibrin and VWF-platelet aggregates. Because human thrombus composition can vary due to differential contributions of these two components, thrombolytic agents acting only on fibrin (rt-PA) or only on VWF (NAC, WT ADAMTS13) may not completely dissolve occluded thrombi. The reduction of both VWF and fibrin observed here supports in vitro data showing that caADAMTS13 can proteolyze both VWF and fibrinogen, thereby circumventing the issue of thrombus composition.

[0282] Treatment of Respiratory Tract Infections FIG. 12 shows the effect of treatment with wild-type ADAMTS13 or the A1144V variant on brain inflammation in mice infected with Streptococcus pneumoniae.

[0283] Animals treated with the A1144V variant (caADAMTS13), either alone or in combination with amoxicillin, showed approximately a 50% reduction in brain inflammation.

[0284] In response to localized airway infections (RTI), large VWF multimers are released from the pulmonary vasculature and circulate in the plasma. Infection also leads to increased plasma concentrations of proinflammatory cytokines (IL-6, IL-1, etc.), which can lead to vascular inflammation in other organs, including the brain (Figures 12A and 12C). These changes are believed to be the primary cause of the increased risk of stroke observed in patients prior to RTI, which may account for as much as 10% of all strokes.

[0285] This response persists long after the infection is resolved by antibiotics, with both plasma and brain VWF levels remaining elevated (Figures S12B, S12C, and S12D). By attenuating this response, caADAMTS13 may be a potential prophylactic option to prevent stroke caused by RTIs.

[0286] FeCl3-induced middle cerebral artery (MCA) occlusion Treatment with caADAMTS13 induced an increase in flux ratio over time (compared to vehicle controls), indicating that the occlusive thrombus was cleared and blood flow was restored to the MCA territory, thereby salvaging viable tissue as indicated by a correlated reduction in lesion volume, without hemorrhagic transformation, even with delayed administration, as is the case with rT-PA.

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[0288] For standard molecular biology techniques, see Sambrook, J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

Claims

1. having an amino acid sequence that includes one or more amino acid substitutions in the region corresponding to SEQ ID NO:48, compared to the amino acid sequence of wild-type human ADAMTS13; An ADAMTS13 variant, wherein the ADAMTS13 variant exhibits increased proteolytic activity compared to wild-type human ADAMTS13.

2. The ADAMTS13 variant of claim 1, wherein the ADAMTS13 variant comprises a substitution at one or more of the following positions relative to the amino acid sequence of wild-type human ADAMTS13: A1144, A1145, A1146, P1147, P1154, P1171, P1173, P1175, P1180, and P1182.

3. 3. The ADAMTS13 variant of claim 1 or claim 2, comprising the amino acid sequence set forth in SEQ ID NO: 50 or 156.

4. The ADAMTS13 variant is (i) a substitution to valine, isoleucine, or lysine at one or more of the following positions relative to the amino acid sequence of wild-type human ADAMTS13: A1144, A1145, A1146, P1147, P1154, P1171, P1173, P1175, P1180, and P1182; or (ii) the amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60; or (iii) the amino acid sequence of SEQ ID NO: 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145; or (vi) the amino acid sequence of SEQ ID NO: 146, 147, 148, 149, 150, 151, 152, 153, 154, or 155; The ADAMTS13 variant according to any one of claims 1 to 3, comprising:

5. The ADAMTS13 variant is (i) a substitution at one or both of positions P1180 and / or P1182 with valine, isoleucine, or lysine relative to the amino acid sequence of wild-type human ADAMTS13; or (ii) An ADAMTS13 variant according to any one of claims 1 to 4, comprising the amino acid sequence of SEQ ID NO: 59, 60, 144, 145, 154, or 155.

6. The ADAMTS13 variant is selected from the group consisting of: (i) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 61; or (ii) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 62; or (iii) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 63; or (iv) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 64; or (v) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 65; or (vi) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:

66. or (vii) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 67; or (viii) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 68; or (ix) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 69; or (x) an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:

70.

7. A nucleic acid encoding the ADAMTS13 variant according to any one of claims 1 to 6.

8. An expression vector comprising the nucleic acid of claim 7.

9. A cell comprising an ADAMTS13 variant according to any one of claims 1 to 6, a nucleic acid according to claim 7, or an expression vector according to claim 8.

10. A method for producing an ADAMTS13 variant, comprising culturing a cell containing a nucleic acid described in claim 7 or an expression vector described in claim 8 under conditions suitable for expression of the ADAMTS13 variant by the cell.

11. A pharmaceutical composition comprising an ADAMTS13 variant described in any one of claims 1 to 6, a nucleic acid described in claim 7, an expression vector described in claim 8, or a cell described in claim 9, and a pharma- ceutically acceptable carrier, diluent, excipient or adjuvant.

12. 12. An ADAMTS13 variant according to any one of claims 1 to 6, a nucleic acid according to claim 7, an expression vector according to claim 8, a cell according to claim 9, or a composition according to claim 11, for use in a method of medical treatment or prevention.

13. for use in a method for the treatment or prevention of a disease or condition characterized by one or more of: increased levels and / or activity of VWF or a complex containing VWF; decreased levels of ADAMTS13; decreased levels of ADAMTS13 proteolytic activity; thrombosis; and inflammation, Optionally, said disease or condition is a disease / condition characterized by thrombosis, a disease / condition characterized by inflammation, thrombotic thrombocytopenic purpura (TTP), ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage (SAH), intracerebral hemorrhage (ICH), chronic thromboembolic pulmonary hypotension (CTEPH), myocardial infarction (MI), ST-elevation myocardial infarction (STEMI), unstable angina (UA), ischemia, reperfusion, deep vein thrombosis, pulmonary embolism. , intravascular coagulation (DIC), hemolytic-uremic syndrome (HUS), cerebral infarction, systemic lupus erythematosus (SLE), disease caused by SARSr-CoV infection (e.g., SARS-CoV-2, e.g., COVID-19), acute respiratory distress syndrome (ARDS), pneumonia, kidney damage, nephropathy, microvascular disease, dementia, Crohn's disease, inflammatory bowel disease, ulcerative colitis, and bacterial diarrhea; An ADAMTS13 variant according to any one of claims 1 to 6, a nucleic acid according to claim 7, an expression vector according to claim 8, a cell according to claim 9, or a composition according to claim 11.

14. A method for cleaving VWF, comprising contacting VWF or a complex containing VWF with an ADAMTS13 variant according to any one of claims 1 to 6.

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