WO2020214033A1 - Papain-like cysteine peptidase medicaments and uses thereof - Google Patents

Abstract

The invention relates to a papain-like cysteine peptidase that exhibits peptidase activity towards von Willebrand Factor (VWF); wherein said peptidase is for use as a medicament.

Description

PAPAIN-LIKE CYSTEINE PEPTIDASE MEDICAMENTS AND USES THEREOF

FIELD OF THE INVENTION

This invention is in the field of medicine. More specifically, the invention is in the field of medicaments against disorders in which von Willebrand Factor (VWF) plays an important role. The invention relates to medical methods employing a new class of proteins exhibiting VWF -cleaving peptidase activity, to the proteins themselves and uses thereof and to pharmaceutical compositions comprising such proteins. The proteins identified herein can be used as an antithrombotic.

STATE OF THE ART

Von Willebrand Factor (VWF) is a plasma glycoprotein that mediates adhesion and aggregation of platelets. The protein is synthesized by endothelial cells and megakaryocytes as long multimers with a molecular weight of up to more than 20,000 kDa (Kannicht et al., Biologicals, 43: 117— 22 (2015)). The majority of circulating VWF is synthesized by endothelial cells (Lenting et al., Thromb Haemost 104: 449-55 (2010)). Endothelial cells secrete part of the produced VWF via the constitutive pathway, but a substantial part is stored in Weibel-Palade bodies and is released upon stimulation of the endothelium with, for example, the inflammatory cytokines IL-8 and TNF-a (Hulstein et al., J Thromb Haemost., 4: 2569-75 (2006); Bernardo et al., Blood, 104: 100-6 (2004)).

Most of the secreted VWF consists of ultra-large VWF (ULVWF) multimers that are highly prothrombotic and are formed by disulfide hnkage of VWF monomers during synthesis. Prothrombotic activity of VWF is regulated during normal hemostasis through limited cleavage by the metalloproteinase AD AMTS 13 (A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13) (Kannicht et al., Biologicals, 43: 117-22 (2015)). ULVWF multimers will adopt a globular conformation in plasma (Crawley et al., Blood, 118: 3212-21 (2011)). High shear rates upon passage through the microvasculature result in (partial) unravelling of globular ULVWF, exposing the cryptic binding and cleavage sites for AD AMTS 13, resulting in the cleavage of VWF by AD AMTS 13 within its A2 domain (Crawley et al., Blood, 118: 3212-21 (2011)).

A deficiency in the metalloproteinase AD AMTS 13 is associated with thrombotic thrombocytopenic purpura (TTP), a rare but severe form of a group of hematologic disorders referred to as thrombotic micro angiopathy (TMA). In TTP, clumping of platelets by ULVWF results in unwanted thrombosis and organ damage (Crawley et al., Blood, 118: 3212-21 (2011); Clark et al., Thromb Haemost., 118: 471-9 (2018)). In acquired TTP, patients produce autoantibodies against AD AMTS 13. The current first-line treatment for patients with acquired TTP is a plasma exchange (PEX) procedure, in which the patient’s plasma is removed by apheresis and replaced with donor plasma. The aim of PEX is to replace AD AMTS 13 and remove the autoantibodies that inhibit AD AMTS 13 activity from the blood circulation, as well as any residual ULVWF multimers therefrom. This way of correcting AD AMTS 13 deficiency restores proper cleavage of ULVWF multimers, prevents microvascular thrombosis, and reverses symptoms of organ damage. However, as auto-antibodies will continuously be produced, the beneficial effect of PEX is only temporary.

Other thrombotic disorders in which accumulation of VWF plays an essential role include haemolytic uremic syndrome (HUS), HE LLP syndrome and catastrophic antiphospholipid syndrome, as well as ischaemic stroke which benefits from thrombolytic therapy.

There is a need in the art for medicaments that counteract unwanted prothrombotic effects mediated by accumulation of VWF. This is especially the case for disorders associated with AD AMTS 13 deficiency, in which regulation of VWF function is disturbed. One of such disorders is TTP, where the current therapy involves invasive and demanding PEX procedures.

It is an aim of the present invention to provide for such a medicament.

SUMMARY OF THE INVENTION

The present invention solves these problems by providing papain-like cysteine peptidases that exhibit peptidase activity towards von Willebrand Factor (VWF). The VWF-cleaving peptidases can be used in methods of treatment, for instance in treating a variety of hematologic disorders as described herein. The peptidase of this invention is for use as a medicament.

It was previously documented that the human parasitic helminth Schistosoma mansoni (Sm) possesses a tegumental cathepsin B, referred to as SmCB2, which belongs to the class of papain-like cysteine peptidases.

The class of papain-like cysteine peptidases is well-documented (see for instance the MEROPS database, wherein papain-like cysteine peptidases are identified by clan CA - family C1 - subfamily C1A).

It was discovered by the present inventors that SmCB2 acts proteolytically on VWF. The present inventors characterized SmCB2 as a VWF-cleaving peptidase. SmCB2 occurs both in the form of a proenzyme, whose amino acid sequence is provided in SEQ ID NO: 1, and in a mature and catalytically active form, whose amino acid sequence is provided in SEQ ID NO:2. The inventors discovered inter alia that SmCB2 cleaves VWF in the same amino acid region as AD AMTS 13 (i.e. the A2 domain).

It is envisaged that SmCB2 is not the only peptidase from the class of papain-like cysteine peptidases capable of cleaving VWF. The class of papain -like cysteine peptidases with VWF-cleaving activity differentiate themself from the known peptidases that cleave VWF, which are either metalloproteinases (such as AD AMTS 13, MMP-8 and MMP-9), serine peptidases (such as plasmin, leukocyte-derived proteinase 3, neutrophil elastase and cathepsin G), or peptidases that require divalent cations for their catalytic activity (such as calpain).

In a first aspect, the present invention provides a papain-like cysteine peptidase that exhibits peptidase activity towards von Willebrand Factor (VWF) for use as a medicament, preferably for use in treatments including thrombolytic therapy.

In preferred embodiments of this aspect, the papain-like cysteine peptidase that exhibits peptidase activity towards von Willebrand Factor (VWF), is for use in treating hematologic disorders, preferably disorders selected from (i) thrombotic micro angiopathy (TMA), preferably thrombotic thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), HELLP syndrome or catastrophic antiphospholipid syndrome, more preferably acquired TTP; (ii) myocardial infarction; or (iii) stroke, preferably ischemic stroke.

In a preferred embodiment of this aspect, the peptidase is a divalent metal ion-independent cysteine peptidase.

In another preferred embodiment of this aspect, the peptidase is a cathepsin B.

In another preferred embodiment of this aspect, the peptidase acts proteolytically on VWF.

In another preferred embodiment of this aspect, the peptidase has:

- a cysteine active site comprising the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 50% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2 and exhibiting VWF cleaving activity; or

- a histidine active site comprising the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 50% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2 and exhibiting VWF cleaving activity, or combinations thereof.

In yet another preferred embodiment of this aspect, the peptidase comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 50% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 and exhibiting VWF cleaving activity.

In yet another preferred embodiment of this aspect, the peptidase is the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 50% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 and exhibiting VWF cleaving activity.

In aspects of this invention, the peptidase can be used in the form of its mature protein or a VWF-cleaving part or sequence identity analog thereof as indicated above. However, in alternative embodiments, the peptidase can be used in the form of its pro-enzyme or pro-peptide or parts thereof, which parts preferably comprise the cysteine active site or histidine active site, or combinations thereof as indicated herein. The pro-enzyme or pro-peptide or part thereof may then be activated in vivo following its administration to a subject in need thereof. Such activation may include activation of the active sites or removal of inhibitory factors.

In a preferred embodiment of this aspect, the peptidase is from Schistosoma mansoni. Preferably, the peptidase is SmCB2.

In an alternative preferred embodiment, the peptidase is a recombinant protein.

In another aspect, the present invention provides a nucleic acid encoding the peptidase of the present invention as described above, for use as a medicament, preferably for use in treatments including thrombolytic therapy. In another aspect, the invention provides a peptidase or nucleic acid as described herein (i) for use in the treatment of a disorder associated with, or mediated by, VWF, (ii) for use in the treatment of a disorder associated with, or mediated by, the presence of VWF (in a subject), (iii) for use in the treatment of a disorder associated with, or mediated by,

(pathogenic) accumulation of VWF (in a subject) or (iv) for use in a method of counteracting (unwanted or pathogenic) prothrombotic effects mediated by accumulation of VWF (in a subject).

In preferred embodiments of this aspect, the nucleic acid, is for use in treating hematologic disorders, preferably disorders selected from (i) thrombotic microangiopathy (TMA), preferably thrombotic

thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), HELLP syndrome or catastrophic antiphospholipid syndrome, more preferably acquired TTP; (h) myocardial infarction; or (iii) stroke, preferably ischemic stroke.

In preferred embodiments of the above aspects, the peptidase or the nucleic acid are used as an antithrombotic.

In another aspect, the present invention provides a pharmaceutical composition comprising a peptidase and/or nucleic acid as defined herein above, and one or more pharmaceutically-acceptable carriers, adjuvants, excipients and/or diluents.

In a preferred embodiment of this aspect, the peptidase comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2 and exhibiting peptidase activity towards von Willebrand Factor (VWF).

In another aspect, the present invention provides a method of treating a hematologic disorder in a subject, comprising administering to a subject in need thereof a therapeuticahy effective amount of a peptidase or nucleic acid according to the invention as described above. In preferred embodiments of this aspect, the disorder is selected from (i) thrombotic micro angiopathy (TMA), preferably thrombotic thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), HELLP syndrome or catastrophic antiphospholipid syndrome, more preferably acquired TTP; (h) myocardial infarction; or (iii) stroke, preferably ischemic stroke.

DETAILED DESCRIPTION OF THE INVENTION

The term“cysteine peptidase”, as used herein, refers to peptidases which are distinguished by the presence of a cysteine residue which plays a critical role in the catalytic process. The terms“peptidase” and“protease” can be used interchangeably. The term“cysteine peptidase” also

encompasses proteins that comprise a cysteine peptidase as described herein, for instance as an inactive zymogen or proenzyme. The MEROPS database classifies proteases in different clans, families, subfamilies, etc. According to the well-established MEROPS database (release 12.0), cysteine (C) proteases are classified in different clans (CA,CD, CE, CF, CL, CM, CN, CO, CP, CQ, and CR), different families (C1-C118) and different

subfamihes. For instance, cathepsin B peptidases belong to clan CA, family C1, subfamily CIA (papain -like peptidases) and are identified by MEROPS identifier CO 1.060, preferably according to MEROPS database release 12.0. Further as an example, calpain peptidases belong to clan CA, family C2 and subfamily C2A. Therefore, calpain is not a papain-like cysteine peptidase.

The term“papain-like cysteine peptidase”, as used herein, refers to cysteine peptidases that include clan CA, family C1, subfamily CIA according to the MEROPS database, preferably the MEROPS database release 12.0. The skihed person understands that the terminology“papain- like” refers to a grouping or family of peptidases that is characterized by a type of enzyme employed, which in the present case is the papain enzyme type ( Carica papaya), which is a family-type peptidase. Thus, the cysteine peptidase as described herein is a cysteine peptidase of the papain family- type of cysteine peptidases. The term includes naturally occurring

peptidases, peptidases that are variants of naturally occurring peptidases such as peptidases that comprise mutations such as substitution, deletion and/or insertion mutations, or peptidases that are completely synthetic. Preferably, the cysteine peptidase as described herein is an endopeptidase.

It is within routine experimentation to determine whether a cysteine protease is a papain-like cysteine protease, such as for instance described by Caffrey et al., Mol Biochem Parasitol. 121:49-61 (2002), who first described the peptidase referred to as SmCB2 and classified it as a cathepsin B that belongs to the family of papain-like cysteine peptidases. Preferably, a papain-like cysteine peptidase as described herein comprises the conserved catalytic active site residues of papain-like cysteine peptidases of, or corresponding to, Cys122 and His291 of SEQ ID NO: 1 or SEQ ID NO:2 (see also Turk et al., Biochim Biophys Acta. 1824:68-88 (2012); Novinec & Lenarcic, BioMol Concepts 4:287-308 (2013)). In addition, preferably, a papain-like cysteine peptidase as described herein contains one or more amino acid residues of, or corresponding to, Asn311, Gin 116,

Glyl65, Glyl66, Trp313 of SEQ ID NO: 1 or SEQ ID NO:2. These residues are characteristic of papain-like cysteine peptidases (Turk et al., Biochim Biophys Acta. 1824:68-88 (2012; Novinec & Lenarcic, BioMol Concepts 4:287-308 (2013)).

The term“corresponding to”, as used herein in relation to an amino acid residue that corresponds to an amino acid residue at a certain position, refers to the fact that a conserved amino acid residue in one peptidase may have a different residue number compared to the same conserved amino acid residue in a different peptidase. One of skill in the art would readily identify corresponding amino acid positions of conserved residues, for instance when performing alignment of the amino acid sequence of multiple peptidases, for instance through Protein BLAST®. As an example, an alignment of cathepsin B peptidases of different species is provided in Figure 5.

Preferably, the peptidase as described herein is a metal ion- independent, more preferably a divalent metal ion-independent, cysteine peptidase.

Alternatively, the invention provides a metal ion-independent cysteine peptidase, preferably divalent metal ion-independent cysteine peptidase, that exhibits peptidase activity towards von Willebrand F actor (VWF); wherein said peptidase is for use as a medicament.

The term“metal ion-independent”, as used herein, refers to any cysteine peptidase whose catalytic mechanism or active site does not involve or require a metal ion for peptidase activity. This is in contrast to

metallopeptidases such as AD AMTS 13 or other peptidases that involve or require metal ions for peptidase activity including calpain. Divalent metal ions are for instance Ca²⁺, Fe²⁺, Zn²⁺, Co²⁺ and Mn²⁺. Preferably, the cysteine peptidase as described herein is a Ca²⁺ -independent cysteine peptidase.

The cysteine peptidases described herein exhibit peptidase activity towards von Willebrand Factor (VWF).

The term“exhibits peptidase activity towards von Willebrand Factor (VWF)”, is used herein to refer to a cysteine peptidase that is a VWF- cleaving peptidase. In other words, the term refers to a cysteine peptidase that exhibits von Willebrand Factor (VWF)-cleaving peptidase activity. The term“peptidase activity”, as used herein, refers to cleavage of peptide bonds in proteins (including peptides) by hydrolysis.

Preferably, the peptidase activity is an endopeptidase activity. More preferably, the cysteine peptidase exhibits peptidase activity towards, or is a VWF -cleaving peptidase that cleaves in, the A2 domain of VWF.

Peptidase activity towards the A2 domain of VWF can for instance be measured using the generally available FRETS-VWF73 substrate (AnaSpec, Fremont, Ca, USA), preferably according to the FRETS-VWF73 substrate assay protocol as described in Example 1. The FRETS-VWF73 substrate is a synthetic 73 amino acid peptide based on the amino acid sequence of the A2 domain of VWF. This fluorogenic substrate is currently used in a diagnostic assay (FRETS-VWF73 assay) to determine activity of AD AMTS 13, the peptidase that normally controls VWF multimer size through limited cleavage.

The term“von Willebrand Factor” or“VWF”, as used herein, refers to a plasma glycoprotein that mediates adhesion and aggregation of platelets. VWF is synthesized by endothelial cells and megakaryocytes as long multimers with a molecular weight of up to more than 20,000 kDa. The majority of circulating VWF is synthesized by endothehal cells. Most of the secreted VWF consists of ultra-large VWF (ULVWF) multimers (also referred to as“VWF multimers” or“ multimeric VWF” herein) that are prothrombotic. Prothrombotic activity of VWF is regulated during normal hemostasis through limited cleavage by the metalloproteinase AD AMTS 13 (A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13).

Preferably, the VWF is a mammalian VWF, more preferably a human VWF. VWF as described herein is preferably a VWF multimer (multimeric VWF), which is also referred to as ULVWF multimer or multimeric ULVWF.

Preferably, the cysteine peptidase described herein is a cathepsin B.

The term“cathepsin B”, as used herein, refers to a group of peptidases that include clan CA, family C1, subfamily C1A (papain -like peptidases) and are identified by MEROPS identifier CO 1.060, preferably according to the MEROPS database release 12.0. Preferably, cathepsin B comprises an occluding loop exhibiting dipeptidyl peptidase activity. More preferably cathepsin B comprises an occluding loop that exhibits dipeptidyl peptidase activity comprising an amino acid sequence indicated by the amino acid region of Cys200 - Cys211 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence corresponding to the amino acid sequence indicated by amino acid region Cys200 - Cys211 of SEQ ID NO: 1 or SEQ ID NO:2. The term includes reference to naturally occurring cathepsin B peptidases, cathepsin B peptidases that comprise one or more mutations such as substitution, deletion and/or insertion mutations, and cathepsin B

peptidases that are partially or completely synthetic. It is within the metes and bounds of the skilled person to classify peptidases as cathepsin B peptidases, as is for instance evidenced by Caffrey et al., Mol Biochem Parasitol. 121:49-61 (2002). Figure 5 shows sequence homology between cathepsin B peptidases of different species, which all belong to the family of papain-like cysteine peptidases. The sequence alignment shows inter alia conserved active sites around cysteine and histidine residues.

A peptidase as described herein preferably has - a cysteine active site comprising the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence having at least 50%, 60%, 70%, more preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, even more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2; and/or - a histidine active site comprising the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence having at least 50%, 60%, 70%, more preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, even more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%, sequence identity to the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2.

The skilled person understands that when reference is made to an active site having an amino acid sequence having a certain percentage of sequence identity to SEQ ID NO: 1 or SEQ ID NO:2, the active site is to exhibit an activity corresponding to the reference active site in SEQ ID NO: 1 or SEQ ID NO:2.

A peptidase as described herein preferably has - a cysteine active site comprising the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of amino acid region 113- 137 of SEQ ID NO: 1 or SEQ ID NO:2; and - a histidine active site

comprising the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2.

The term“% sequence identity” is defined herein as the percentage of amino acids in an amino acid sequence that is identical with the amino acids in a reference amino acid sequence or an amino acid sequence of interest, after ahgning the sequences and optionally introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignments are well known in the art. Sequence identity is calculated over substantially the whole length, preferably the whole (full) length, of a reference amino acid sequence or amino acid sequence interest. The skilled person understands that consecutive amino acid residues in one amino acid sequence are compared to consecutive amino acid residues in another amino acid sequence. The reference amino acid sequence or amino acid sequence of interest is SEQ ID NO: 1 or SEQ ID NO:2, preferably SEQ ID NO:2, or regions thereof when defined as reference amino acid sequence or amino acid sequence interest.

More preferably, a peptidase as described herein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence having at least 50%, 60%, 70%, more preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, even more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2.

A peptidase as described herein preferably comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2.

Even more preferably, a peptidase as described herein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, preferably SEQ ID NO:2.

A peptidase as described herein preferably is from Schistosoma mansoni, more preferably is SmCB2. SmCB2 is a cathepsin B cysteine peptidase (UniProtKB - Q95PM1; last modified: March 1, 2002 - v2), the amino acid sequence of which is provided in SEQ ID NO: 1 (proenzyme) and SEQ ID NO:2 (mature protein). The term“SmCB2”, as used herein, includes naturally occurring variants and other variants that have for instance a substitution, deletion or insertion of an amino acid residue in SEQ ID NO: 1 or SEQ ID NO:2, preferably wherein the substitution, deletion or insertion is 1-80, more preferably 1-50, even more preferably 1-20, most preferably 1- 10 amino acid residues, and the naturally occurring variants and other variants exhibit peptidase activity towards von Willebrand Factor (VWF). The peptidase as described herein may comprise the pro-domain of SmCB2, or is in the form of the proenzyme of SmCB2, preferably wherein the peptidase has the amino acid sequence of amino acid region 1-93 or 24-93 of SEQ ID NO: 1.

The use of SmCB2 as a therapeutic protein as foreseen herein may entail improving the pharmacokinetic properties of the protein, in particular hmiting its metabolic turnover by peptidases or glomerular filtration by the kidney to thereby increase its in vivo half-life. One typical modification to improve the pharmacokinetics of the protein is through conjugation to either hnear or branched-chain monomethoxy poly-ethylene glycol (PEG) or derivatives thereof (m-PEG), resulting in increases in the molecular mass through a process referred to as PEGylation. The molecular weight of the PEG molecule may vary, and is typically in the range of from about 100 Da to about 160,000 Da, such as e.g. from about 5,000 Da to about 100,000 Da. Methods for PEGylation are well known in the art and involve chemical conjugation of a PEG molecule of appropriate mass to the protein, followed by repurification of the conjugate. Another approach to improve

pharmacokinetics of the protein is by modification of glycosylation pattern, resulting in reduced clearance and extension of half-life. In addition to these chemical modifications, improving the pharmacokinetic properties of the protein may occur by providing a fusion protein, for instance by fusion of the protein to human serum albumin (HSA), to the constant fragment (Fc) domain of a human immunoglobulin (Ig) G, or to non -structured

polypeptides such as XTEN. Fusion proteins of SmCB2 are therefore also foreseen in aspects of this invention, and may include a linker or spacer sequence to separate protease and albumin roles in the fusion protein.

SmCB2 protein according to the present invention may thus have a modified circulatory half life compared to the wild type SmCB2 protein, preferably a circulatory half life that is increased at least 10-100%, more preferably at least 200-500%.

The invention also provides a peptidase as defined herein, preferably a peptidase that is pegylated or a peptidase that is fused to HSA, to the constant fragment (Fc) domain of a human immunoglobulin (Ig) G, or to a non-structured polypeptides such as XTEN. The peptidase can be another peptidase than SmCB2.

The invention also provides a nucleic acid encoding the peptidase as described herein, wherein said nucleic acid is for use a medicament.

The term“nucleic acid”, as used herein, refers to DNA and RNA including mRNA or cDNA, as well as synthetic congeners thereof. The nucleic acid can be a recombinant or synthetic nucleic acid. The skilled person is aware of appropriate methods and means for administration of nucleic acids for therapeutic purposes.

The invention further provides a peptidase or nucleic acid as described herein for use as an antithrombotic. Since the peptidase as described herein exhibits peptidase activity towards von Willebrand Factor (VWF), antithrombotic effects are provided for upon administration to a subject.

The term“antithrombotic”, as used herein, refers to a medicament that prevents the formation of blood clots or counteracts existing blood clots. Preferably, the antithrombotic is an anticoagulant or a thrombolytic agent. An anticoagulant limits the ability of the blood to clot, whereas a

thrombolytic agent provides for breakdown (lysis) of blood clots formed in blood vessels.

In other words, the peptidase or nucleic acid as described herein is for use in the treatment of thrombosis, preferably for use in thrombolysis (thrombolytic therapy).

The invention also provides a peptidase or nucleic acid as described herein for use in the treatment of a disorder associated with VWF. It is estabhshed in the present invention that peptidases as described herein exhibit peptidase activity towards von Willebrand Factor (VWF). This allows beneficial use of such peptidases (including nucleic acids encoding such peptidases) in the treatment of disorders that are associated with VWF. Such disorders are preferably hematologic disorders including coagulopathies and hemostatic disorders, more preferably thrombotic disorders. Such disorders can be acquired or inherited.

The skilled person understands that in principle any disorder in which control of VWF function is disturbed can be treated with a peptidase as described herein, especially disorders associated with, or mediated by, the presence of VWF and/or VWF accumulation. A disorder associated with VWF is preferably (i) a disorder associated with, or mediated by, the presence of VWF (in a subject), (ii) a disorder associated with, or mediated by, (pathogenic) accumulation of VWF (in a subject) and/or (iii) for counteracting (unwanted or pathogenic) prothrombotic effects mediated by accumulation of VWF (in a subject). The peptidase or nucleic acid as described herein may also be for use in correcting AD AMTS 13 deficiency in a subject.

The term“AD AMTS 13 deficiency”, as used herein, refers to AD AMTS 13 not exhibiting its normal role in hemostasis (controlling VWF multimer size through limited cleavage), regardless of the cause, which could be ADAMTS13 protein levels that are too low or autoantibodies against AD AMTS 13 resulting in functional neutralization of AD AMTS 13.

Multiple disorders are known to be associated with VWF. Preferably, the disorder associated with VWF is (i) a thrombotic

microangiopathy (TMA), preferably thrombotic thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), HELLP syndrome or

catastrophic antiphospholipid syndrome, more preferably acquired TTP; (ii) myocardial infarction; or (iii) stroke, preferably ischemic stroke.

The term“thrombotic micro angiopathy (TMA)”, as used herein, refers to a pathology that results in thrombosis in capillaries and arterioles.

The present inventors established that the peptidase as described herein is not sensitive to plasma components specific for acquired TTP patients, which is an indication that the peptidase is not inhibited by auto antibodies against AD AMTS 13. This renders the peptidase as described herein an advantageous peptidase in the treatment of a disorder associated with VWF, especially acquired TTP, where auto-antibodies against

AD AMTS 13 dysregulate hemostasis.

The invention also provides for a use of a peptidase or nucleic acid as described herein for the manufacture of a medicament, preferably an antithrombotic. The invention also provides for use of a peptidase or nucleic acid as described herein for the manufacture of a medicament for (i) the treatment of a disorder associated with, or mediated by, VWF, (ii) the treatment of a disorder associated with, or mediated by, the presence of VWF (in a subject), (iii) the treatment of a disorder associated with, or mediated by, (pathogenic) accumulation of VWF (in a subject) or (iv) counteracting (unwanted or pathogenic) prothrombotic effects mediated by accumulation of VWF (in a subject); preferably including (i) a thrombotic microangiopathy (TMA), preferably thrombotic thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), HE UUP syndrome or

catastrophic antiphospholipid syndrome, more preferably acquired TTP; (ii) myocardial infarction; or (iii) stroke, preferably ischemic stroke.

Embodiments described herein relating to the peptidase, nucleic acid and medical use are explicitly envisaged in combination with a use of a peptidase according to the invention.

The invention also provides a method of treating a subject suffering from, or suspected of suffering from, a disorder associated with, or mediated by, VWF, comprising the step of: - administering to said subject a therapeutically effective amount of a peptidase or nucleic acid as described herein. Preferably, the disorder associated with VWF is (i) a thrombotic microangiopathy (TMA), preferably thrombotic thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), HE UUP syndrome or

catastrophic antiphospholipid syndrome, more preferably acquired TTP; (ii) myocardial infarction; or (iii) stroke, preferably ischemic stroke. In the same manner, the invention provides a method of treating a subject suffering from, or suspected of suffering from, (i) a disorder associated with, or mediated by, the presence of VWF (in a subject) or (ii) a disorder associated with, or mediated by, (pathogenic) accumulation of VWF (in a subject). In the same manner, the invention also provides a method of counteracting (unwanted or pathogenic) prothrombotic effects mediated by accumulation of VWF (in a subject). Embodiments described herein relating to the peptidase, nucleic acid and medical use are explicitly envisaged in combination with a method of treating a subject, or a method of

counteracting, according to the invention.

The term“subject”, as used herein, refers to the recipient of a peptidase or nucleic acid as described herein and is a mammal, preferably a human. The terms“patient” and“subject” may be used interchangeable.

The term“therapeutically effective amount” as used herein, means that the amount of the active ingredient is of sufficient quantity to achieve the intended purpose, such as, inter alia in this case, to cleave appropriate levels of multimeric VWF. The amount of active ingredient, i.e. the amount of peptidase as described herein, preferably is in the range of about 5 mg to 10 gram in a pharmaceutical composition as described herein.

Alternatively, the invention also provides a cysteine peptidase that exhibits peptidase activity towards von Willebrand F actor (VWF); wherein said peptidase is for use as a medicament. Embodiments described herein relating to the peptidase, nucleic acid and medical use are explicitly envisaged in combination herewith.

A peptidase as described herein can be produced by methods and means generally available in the art. For instance, the person skilled in the art will understand how to generate a DNA sequence that encodes an amino acid sequence of a peptidase as described herein and how to manufacture and isolate a nucleic acid molecule with said DNA sequence using generally known recombinant DNA techniques. The sequence of the nucleic acid molecule is preferably codon-optimized for expression in a host cell. In this way codons are used that are favored for high level expression in a specific host cell.

Nucleic acid molecules are preferably inserted in an expression vector using recombinant DNA techniques known by the person skilled in the art. Expression vectors in the context of the invention direct the expression of a peptidase as described herein in a host cell. These expression vectors are preferably replicable in a host cell, either as episomes or as part of the chromosomal DNA. Further, the expression vector preferably comprises (i) a strong promoter/enhancer, such as the CMV or SV40 promoter, (ii) an optimal translation initiation sequence, such as a

ribosomal binding site and start codon, preferably a KOZAK consensus sequence and (iii) a transcription termination sequence, including a poly(A) signal when the protein is expressed in eukaryotic cells. Suitable expression vectors include plasmids and viral vectors such as adenoviruses, adeno- associated viruses and retroviruses. The person skilled in the art will understand that the expression vector to be used is dependent on the host cell that is used for expression of a recombinant protein. An expression vector is preferably suited for expression of a nucleic acid molecule of the invention in a prokaryotic cell including a bacterial cell, or, more preferred, in a eukaryotic host cell, such as a yeast cell and a mammalian cell. A suitable example is mammalian expression vector pCMV4.

As an alternative, a nucleic acid molecule may be inserted in the genome of a host cell. Said insertion preferably is at a locus or within a region that ensures expression of a nucleic acid molecule of the invention in the host cell.

Suitable host cells include prokaryotic and eukaryotic cells, such as bacterial cells, yeast cells, insect cells, animal cells, mammalian cells, murine cells, rat cells, sheep cells, simian cells and human cells. Examples of suitable eukaryotic host cells include, but are not limited to P. pastoris cells, the hamster cell line CHO and BHK-21; the murine host cells NIH3T3, NSO and C127; the simian host cells COS and Vero; and the human host cells HeLa, PER.C6, U-937, HEK 293 and Hep G2. Suitable cells are available from public sources such as ATCC and Life Technologies. A number of transfection techniques are known in the art, see, e.g., Green & Sambrook., 2012.“Molecular Cloning: A Laboratory Manual”, 4^th Edition, CSHL Press; Cold Spring Harbor Protocols, www.cshprotocols.cshlp.org). The person skilled in the art preferably employs techniques as described in these references to introduce one or more exogenous nucleic acid molecules into suitable host cells. An example of a host cell for the production of a peptidase as described herein is a P. pastoris cell or a HEK 293 cell.

The peptidase or nucleic acid as described herein are for administration in the form of a pharmaceutical composition.

Therefore, the invention also provides a pharmaceutical composition comprising a peptidase or nucleic acid as described herein, and one or more pharmaceutically-acceptable carriers, adjuvants, excipients and/or diluents. Embodiments described herein relating to the peptidase, nucleic acid and medical use are explicitly envisaged in combination with a pharmaceutical composition according to the invention. As an example, and preferably, in the pharmaceutical composition as described herein, the peptidase comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2 and that exhibits peptidase activity towards von Willebrand Factor (VWF). For the purpose of clarity and a concise description, not all of the embodiments of the peptidase, nucleic acid and medical use are repeated here.

A pharmaceutical composition as described herein preferably comprises one or more of diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art. The characteristics of the carrier will depend on the route of administration, as is known to the skilled person.

The term“pharmaceutically acceptable”, as used herein, refers to a nontoxic material that is compatible with the physical and chemical characteristics of a peptidase as described herein and does not interfere with the effectiveness of the biological activity of said peptidase. A pharmaceutical composition as described herein is preferably adapted for parenteral administration, wherein the composition is for intravenous, intra-arterial, subcutaneous, and/or intramuscular

administration. Parenteral administration involves the injection or infusion of a pharmaceutical composition as described herein into a body tissue or body fluid, whereby preferably a syringe, needle, or catheter is used. As an alternative, needle-less high-pressure administration may be used as means for parenteral administration.

For injectable compositions (e.g., intravenous compositions), the carrier may be an aqueous or oily solution, dispersions, emulsions and/or suspension. Preferably, the carrier is an aqueous solution, preferably distilled sterile water, saline, buffered saline, or another pharmaceutically acceptable excipient for injection.

It is noted that the pharmaceutical composition as described herein preferably is for use as medicament, more preferably for use as an

antithrombotic, even more preferably for use in the treatment of a disorder associated with VWF, including (i) thrombosis, (ii) a thrombotic

microangiopathy (TMA), preferably thrombotic thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), HELLP syndrome or

catastrophic antiphospholipid syndrome, more preferably acquired TTP; (iii) myocardial infarction; or (iv) stroke, preferably ischemic stroke.

It is within the metes and bounds of the skilled person to devise appropriate dosing regimens. For instance, when a short-lived

antithrombotic effect suffices to counteract a thrombotic disorder and to restore hemostasis, a composition of the invention can be administration in a single dosage form once, preferably by parenteral administration, more preferably by intravenous administration. A suitable dose can be selected from the dose range of 5 mg to 10 gram. Alternatively, for example, when the disorder to be treated is chronic and multiple administrations are necessary to achieve a positive treatment result, a composition of the invention can be administered in a single dosage form once or twice a day, weekly, biweekly (such as every other week or every 14 days) or monthly. Such administration can be a parenteral administration, such as an intravenous administration, for instance in a dose of 5 mg to 10 gram.

For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the disclosure includes embodiments having combinations of all or some of the features described.

The content of the documents referred to herein is incorporated by reference.

FIGURE LEGENDS

Figure 1. S. mansoni extracts contain a distinct VWF cleaving activity.

Cleavage of FRETS-VWF73 substrate was examined for different protein concentrations of S. mansoni worm homogenate (WH) (A) and S. mansoni soluble worm fraction (SWF) (B) in the presence (open circles) and absence (closed circles) of 5 mM EDTA. Initial rates of increase in fluorescence are represented as mean ± SD. All values represent triplicate independent experiments. C) Cleavage of native VWF by S. mansoni WH and pooled normal platelet-poor plasma (PNP) was examined with and without 5 mM EDTA. VWF multimers were incubated for 8 hours and separated by gel electrophoresis. Cleavage of VWF, characterized by appearance of the third triplet band, is indicated by black arrows (representative image of three independent experiments).

Figure 2. Characterization of the VWF-cleaving peptidase.

A) Cleavage of FRETS-VWF73 substrate by S. mansoni soluble worm fraction (SWF) (500 mg/mL) was studied in the presence or absence of various peptidase inhibitors (NEM 10 mM, leupeptin (Leu) 100 pM, pepstatin A (PepA) 5 mM, phenylmethylsulfonyl fluoride (PMSF) 1 mM, EDTA 5 mM, L-cysteine (L-cys) 50 mM). Initial rates of increase in fluorescence are represented as mean ± SD. All values represent three replicates of duplo measurements. B) Cleavage of FRETS-VWF73 substrate by S. mansoni soluble worm fraction (SWF) (500 mg/mL) was studied in the presence or absence of pooled normal platelet-poor plasma (PNP) and/or EDTA in order to examine inhibition of S. mansoni cleavage of FRETS- VWF73 by plasma components. Closed circles (●) represent PNP, open circles (o) represent PNP with addition of EDTA, closed squares (_■) represent S. mansoni soluble worm fraction (SWF) plus PNP, open squares (_□) represent S. mansoni soluble worm fraction (SWF) plus PNP in the presence of EDTA. Initial rates of increase in fluorescence are represented as mean ± SD. All values represent three replicates of duplo measurements. C) Cleavage of native VWF by S. mansoni WH in the presence of PNP was examined with and without 5 mM EDTA. VWF multimers were incubated for 4 hours and separated by gel electrophoresis. Cleavage of VWF, characterized by appearance of the third triplet band, is indicated by black arrows.

Figure 3. The S. mansoni VWF-cleaving peptidase retains partial activity in TTP patient plasma.

A) Cleavage of FRETS-VWF73 substrate by S. mansoni soluble worm fraction (SWF) (500 mg/mL) in the presence of 5 mM EDTA was studied in the presence or absence of 2% TTP patient platelet-poor plasma that has 0% ADAMTS13 activity, as determined before by FRETS-VWF73 substrate assay. Initial rates of increase in fluorescence are represented as mean ±

SD. All values represent three replicates of measurements performed in duplicate. Statistical analysis was performed with one-way ANOVA and Differences were considered statistically significant when p < 0.05. B) Cleavage of native VWF by S. mansoni WH in the presence of TTP patient platelet-poor plasma was examined with and without 5 mM EDTA. VWF multimers were incubated for 2 hours and separated by gel electrophoresis. Cleavage of VWF, characterized by appearance of the third triplet band, is indicated by black arrows. Very high multimers that are characteristic for TTP patient plasma are indicated by the grey arrow.

Figure 4. Proteolytic activity towards FRET-VWF73 substrate of purified recombinant SmCB2.

Purified recombinant SmCB2 (rSmCB2) was examined for proteolytic activity towards two fluorescent substrates Z-Phe-Arg-N AMC (panel A) and FRETS-VWF73 with (upper line) and without (lower line) 5 mM EDTA (panel B). Shown are the results of triplicate experiment in which a serial dilution of rSmCB2 was examined. The dashed line in panel B represents the VWF -cleavage activity of normal pooled plasma at a 2% (v/v)

concentration.

Figure 5. Sequence alignment of cathepsin B peptidases of different species.

Sequence similarity between cathepsin B peptidases of different species is shown, with an emphasis on the cysteine active site and histidine active site.

SEQ ID NO: 1: Amino acid sequence of SmCB2 (Uniprot Acc. No. Q95PM1-1, last modified March 1, 2002 - v2)

1 MNQYSCYLLQ LYIIILLSYG TLNEIDARRH KRMYQPLSME LINFINYEAN TTWKAAPTTR

61 FRTVSDIRRM LGALPDPNGE QLETLCTGYI SDELPKSFDA

RVEWPHCPSI SEIRDQSSCG

121 SCWAFGAVEA MSDRICIKSK GKHKPFLSAE NLVSCCSSCG MGCNGGFPHS AWLYWKNQGI

181 VTGDLYNTTN GCQPYEFPPC EHHVIGPLPS CDGDVETPSC KTNCQPGYNI P YEKDKWY GE

241 KVYRIHSNPE AIMLELMRNG PVEVDFEVYA DFPNYKSGVY QHVSGALLGG HAVRLLGWGE

301 ENNVPYWLIA NSWNSDWGDK GYFKIVRGKN ECGIESDVNA GIPKIKN

SEQ ID NO:2: Mature SmCB2 (a.a. 94-347 of SEQ ID NO: 1)

94 LPKSFDA RVEWPHCPSI SEIRDQSSCG SCWAFGAVEA

MSDRICIKSK GKHKPFLSAE

151 NLVSCCSSCG MGCNGGFPHS AWLYWKNQGI VTGDLYNTTN GCQPYEFPPC EHHVIGPLPS

211 CDGDVETPSC KTNCQPGYNI P YEKDKWY GE KVYRIHSNPE AIMLELMRNG PVEVDFEVYA

271 DFPNYKSGVY QHVSGALLGG HAVRLLGWGE ENNVPYWLIA NSWNSDWGDK GYFKIVRGKN

331 ECGIESDVNA GIPKIKN EXAMPLES

Example 1. Materials and methods

Plasma samples

Pooled platelet-poor citrated plasma was obtained from blood of 50 healthy volunteers (after informed consent) and was prepared by centrifugation (twice 10 min, 2000x g) (Sanquin, Amsterdam, the Netherlands). Blood samples from TTP patients were obtained with informed consent from TTP patients present at the annual TTP patient day (Sanquin 2016, Amsterdam, the Netherlands) and platelet-poor citrated plasma was prepared by centrifugation (twice 10 min, 2000x g). Plasma samples were stored at -80 °C.

Parasites

Intact adult S. mansoni pairs were isolated from Golden hamsters seven weeks after infection with 600 cercariae per hamster. Hamsters were anesthetized with isoflurane and perfused with 0.9% NaCl solution. Isolated worms were rinsed in 0.9% NaCl and frozen in 0.9% NaCl for storage at -20 °C. Worm homogenate (WH) was prepared in HEPES buffered saline (HBS; 10 mM HEPES, 150 mM NaCl, pH 7.4) with a Teflon potter, after which nuclei and unlysed cells were removed by centrifugation at 500x g for 5 minutes at 4°C. Soluble worm fraction was prepared by subsequent centrifugation of the WH at 14000x g for 30 minutes at 4°C. Protein concentration in the schistosomal samples were determined according to the method of Lowry et al., J Biol Chem., 193: 265-75 (1951). Anion exchange chromatography fractionation Anion exchange chromatography fractionation was performed on Q

Sepharose fast flow resin (GE Healthcare Life Sciences, Boston, MA, USA) in a Tricorn 10/50 column (bed volume 4 ml, GE Healthcare Life Sciences, Boston, MA, USA). The column was run on an ÄKTA pure system (GE Healthcare Life Sciences, Boston, MA, USA) at 1 ml/min with running buffer (20 mM triethanolamine, pH 8.0). S. mansoni soluble protein extract was prepared in running buffer as described above and 9 ml was loaded onto the column using a 10 ml Superloop (GE Healthcare Life Sciences, Boston, MA, USA).

Fractions (500 pi per fraction) were step-wise eluted with 250 mM, 500 mM, and 1 M NaCl in running buffer and kept at 4 °C. Peptidase activity was tested in 40 ml fraction with the FRETS-VWF73 substrate assay. The three fractions with the highest activity in the FRETS-VWF73 substrate assay were pooled. Anion exchange chromatography was performed in triplicate.

Mass spectrometry

Pooled active fractions and flow-through from anion exchange

chromatography (performed in triplicate) were separated by SDS-PAGE.

The lanes of the SDS-PAGE were subsequently divided into 4 bands approximately equal in size (only including proteins with molecular weights smaller than ~80 kDa) and proteins were subjected to in-gel tryptic digestion as described by Shevchenko et al., Nat Protoc. 1: 2856-60 (2006). After digestion, samples were analyzed on an Orbitrap Q-Exactive (Thermo Fisher Scientific, Waltham, MA, USA) connected to a UHPLC Proxeon Easy-nLC 1000 (Thermo Fisher Scientific, Waltham, MA, USA). Peptides were trapped on a double-fritted trap column (Dr. Maisch Reprosil C18, 3 pm, 2 cm x 100 pm (Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany)) and separated on an analytical column (Agilent Zorbax SB-C18, 1.8 pm, 40 cm x 75 pm (Agilent, Santa Clara, CA, USA)). Solvent A consisted of 0.1 M acetic acid, solvent B of 0.1 M acetic acid in 80%

acetonitrile. Samples were loaded at a pressure of 800 bar with 100% solvent A. Peptides were separated by a 30 min gradient of 10-30% buffer B followed by 30-100% B in 2 min, 100% B for 2.5 min at a flow rate of 150 nL/min. Full scan MS spectra were acquired in the Orbitrap (350-1500 m/z, resolution 35,000, AGC target 3e6, maximum injection time 250 ms). The 20 most intense precursors were selected for HCD fragmentation (isolation window 1.2 Da, resolution 17,500, AGC target 5e4, maximum injection time 120 ms, first m/z 100, NCE 33%, dynamic exclusion 60 s). The results were filtered using Percolator (Kail et al., J Proteome Res. 7: 29-34 (2008) and Spivak et al., J Proteome Res. 8: 3737-45 (2009) to a false discovery rate (FDR) below 1%. We further only accepted peptides with at least six amino acid residues, a Mascot ion score of at least 20, and search engine rank of 1 and at least 2 identified peptides for protein identification. Scores (Mascot ion score) and peptide spectrum matches (PSM; total number of identified peptide spectra matched for the protein) were used to compare the samples. Only proteins that showed a minimum PSM score of 4 and at least a two times enrichment in PSM were considered as enriched proteins.

Analysis of the data was performed with Proteome Discoverer using the S. mansoni database (Schistosoma_mansoni_v5.2.fa, Wellcome Trust Sanger Institute, Hinxton, UK, accessible

fromftp://ftp. sanger. ac.uk/pub/pathogens/Schistosoma/mansoni/Archive/Late st_assembly_annotation_others/) for annotation of the peptides. Peptidases were identified based upon the presence of a peptidase KEGG domain and cysteine peptidases were further identified by the presence of“G0:0004197” or the term“Cathepsin” or present on AmiGO 2 as“cysteine-type

endopeptidase activity”

(http://amigo.geneontology.Org/amigo/term/GO:0004197).

FRETS-VWF73 substrate assay Cleavage of the FRETS-VWF73 substrate (AnaSpec, Fremont, Ca, USA) was measured as published with some modifications (Kokame et al., Br J

Haematol. 129: 93-100 (2005)). FRETS-VWF73 substrate was dissolved at a concentration of 100 mM in 25% (v/v) dimethyl sulfoxide (DMSO) and diluted to 4 mM in assay buffer (5 mM Bis-Tris, 25 mM CaC12, 0.005% Tween-20, pH 6.0). Plasma samples or schistosomal fractions, where indicated supplemented with peptidase inhibitors, were diluted in assay buffer and FRETS-VWF73 substrate (2 pM) was added to initiate the assay. Peptidase inhibitors used were N-ethylmaleimide (NEM; 10 mM), leupeptin (Leu; 100 pM), pepstatin A (PepA; 5 mM), phenylmethylsulfonyl fluoride (PMSF; 1 mM), ethylenediaminetetraacetic acid (EDTA; 5 mM), and L-cysteine (L-cys; 50 mM). Fluorescence was monitored for 1 hour at 30°C on a Spectromax M2 fluorescence microplate reader (MTX Lab Systems, Bradenton, FL,

USA) equipped with a 340 nm excitation filter and 450 nm emission filter. Initial rates of increase in fluorescence are represented as mean ± SD.

Cleavage of native VWF

Purified VWF (Biotest AG, Dreieich, Germany, 227 pg/ml) in HBS was incubated with 1 mg/ml WH and/or 3.6% platelet-poor citrated plasma in 1.5 M urea buffer (1.5M urea, 2.5 mM Tris, pH = 8) containing 4.5 mM BaCl in a final volume of 110 pL at 37°C for 2, 4, or 8 hours. Reactions were terminated by addition of 4 volumes (440 pL) of denaturing buffer (9M Urea, 0.1% bromophenol blue, 2 mM ethylenediaminetetraacetic acid (EDTA), 35 mM Tris, 1% SDS, pH 6.7) and incubation for 30 minutes at 56°C.

Samples were separated by gel electrophoresis on a stacking gel of 1% HGT agarose (Lonza, Basel, Switzerland) in stacking gel buffer (4 mM EDTA, 70 mM Tris, 0.4% SDS, pH 6.7) and a running gel of 2.5% HGT agarose in running gel buffer (100 mM Glycine, 200 mM Tris, 0.4% SDS, pH 9.0) on a SE 600 Ruby gel electrophoresis unit (Amersham Biosciences,

Little Chalfont, UK) for 20 to 24 hours at 4°C and constant amperage of 40 mA. Used gel electrophoresis running buffers were upper chamber buffer (150 mM glycine, 100 mM Tris, 0.1% SDS, pH 8.45) and lower chamber buffer (75 mM glycine, 50 mM tris, pH 8.45). Gels were fixed in fixative (1: 10:9 acetic acid: isopropanol: water) for 1 hour at room temperature, and subsequently washed 3 times with water. In gel staining was performed by incubation with 1:5,000 diluted polyclonal rabbit-anti-human VWF antibody (A0082, DAKO, Carpinteria, CA, USA), followed by incubation with 1: 10,000 diluted goat-anti-rabbit-Alexa680 (A21076, Invitrogen, Carlsbad, CA, USA). Before use, both antibodies were diluted in 1: 1 odyssey blocking buffer (LI- COR, Lincoln, NB, USA) and phosphate buffered saline (PBS)-0.1% Tween- 20 (137 mM NaCl, 2.7 mM KC1, 9.2 mM Na₂HP0₄ H₂O, 1.76 mM

KH₂HPO₄, 0.1% Tween-20, pH 7.4). Incubations were performed for 4 hours at room temperature or overnight at 4°C and between the incubations the gels were washed twice with PBS-0.1% Tween-20. Gels were visualized on a near -infrared scanner (Odyssey, LI-COR, Lincoln, NB, USA) with Odyssey V3.0 software (LI-COR, Lincoln, NB, USA).

Statistical Analysis

Statistical analysis was performed with PRISM software (version 7.02;

GraphPad) using a one-way ANOVA to test for differences between groups.

Results

Schistosoma mansoni preparations contain proteolytic activity towards FRETS-VWF73 substrate and multimeric VWF

FRETS-VWF73 substrate is a synthetic 73 amino acid peptide based on the amino acid sequence of the A2 domain of von Willebrand Factor (VWF). This fluorogenic substrate is used in a diagnostic assay (FRETS-VWF73 assay) to determine activity of AD AMTS 13 (Kokame et al., Br J Haematol, 129: 93- 100(2005), the peptidase that normally controls VWF multimer size through limited cleavage. The FRETS-VWF73 peptide forms the minimal substrate for AD AMTS 13, containing the AD AMTS 13 cleavage site and the minimal sequence required for binding of AD AMTS 13. We used the FRETS-VWF73 substrate as a starting point to determine whether Schistosoma mansoni contains peptidases that can cleave VWF, and could thereby mimic the controlling effects of AD AMTS 13 on VWF. Different protein concentrations of S. mansoni worm homogenate (WH) (Figure 1A closed circles) or S.

mansoni soluble worm fraction (SWF) (Figure IB closed circles) were tested for their proteolytic activity on FRETS-VWF73 substrate. Both fractions of S. mansoni showed a dose-dependent proteolytic cleavage of FRETS-VWF73 substrate, indicating the presence of one or more VWF-cleaving peptidases in the fractions. The S. mansoni worms were isolated from the blood of infected hamsters, therefore, in order to rule out contamination of the S. mansoni fractions with host AD AMTS 13 from the hamster, both S. mansoni fractions were also tested in the presence of the peptidase inhibitor ethylenediaminetetraacetic acid (EDTA) (Figure 1A and B, open circles). EDTA inhibits a range of metallopeptidases, including AD AMTS 13 (Tsai et al., Blood, 87: 4235-44 (1996) (see also Figure 2B), through chelation of the metal ion required for proteolytic activity. Interestingly, analysis of the S. mansoni fractions in the presence of EDTA resulted in increased proteolytic activity towards FRETS-VWF73, thereby ruling out contaminating

AD AMTS 13 as the peptidase responsible for the observed cleavage of FRETS-VWF73.

Next, we examined whether S. mansoni fractions also have proteolytic activity towards native multimeric VWF. To allow proteolytic cleavage by AD AMTS 13, VWF needs to be (partially) unraveled, either through sheer stress or through the addition of a denaturing agent, which is done in this experimental setup by the addition of urea (Tsai et al., Blood, 87: 4235-44 (1996)). Purified VWF was incubated with S. mansoni WH or pooled normal platelet -poor plasma (PNP) for 8 hours. Incubations were performed both in the presence and absence of EDTA, to exclude effects of possible contamination of the S. mansoni fractions with host AD AMTS 13. Separation of the VWF multimers by gel electrophoresis under non-reducing conditions reveals the multimeric characteristic of VWF (Figure 1C).

Cleavage of VWF results in the appearance of the slower and faster migrating satellite bands flanking the major VWF-multimer bands to form the so-called VWF triplets (Fischer et al., Biochem J., 331: 483-8 (1998). These satellite bands result from variation in the number of N-terminal fragments that the VWF -multimers contain after cleavage. Uncleaved, purified VWF shows two bands after gel electrophoresis, corresponding to the slower migrating and intermediate triplet band. Incubation of VWF with PNP results in cleavage of VWF by AD AMTS 13, which can be observed as the appearance of the faster migrating triplet band (black arrow).

Incubation with PNP in the presence of EDTA fully inhibits AD AMTS 13 activity, and as a result only the slower migrating and intermediate triplet band can be observed. After incubation of VWF with S. mansoni WH, the faster migrating triplet band appears faintly. However, after incubation with WH in the presence of EDTA, the faster migrating triplet band appears strongly, indicating efficient cleavage of VWF by WH in the presence of EDTA. This efficient cleavage is also reflected by the loss of high molecular weight multimer bands of VWF. These results show that S. mansoni fractions contain a peptidase that cleaves multimeric VWF. Furthermore, cleavage of the FRETS-VWF73 substrate by S. mansoni fractions indicates that (at least one of) the cleavage site(s) of the peptidase is located close to the AD AMTS 13 cleavage site in the A2-domain of VWF.

The VWF-cleaving peptidase belongs to the class of cysteine peptidases Next, we characterized the VWF-cleaving peptidase of S. mansoni further to determine the class of peptidases it belongs to. Cleavage of the FRETS- VWF73 substrate was therefore examined in the presence of various peptidase inhibitors, each inhibiting a specific class of peptidases. N- ethylmaleimide (NEM) is an irreversible small-molecule inhibitor of cysteine peptidases. Leupeptin (Leu) is a reversible small-molecule inhibitor of a broad range of cysteine, serine, and threonine peptidase classes.

Pepstatin A (PepA) is a reversible small-molecule inhibitor of aspartic peptidases. Phenylmethane sulfonylfluoride (PMSF) is an irreversible small- molecule inhibitor of serine peptidases and ethylenediaminetetraacetic acid (EDTA) is a chelating agent that inhibits metallopeptidases through chelation of the metal ion required for proteolytic activity. Proteolytic cleavage of FRETS-VWF73 by S. mansoni soluble worm fraction (SWF) was strongly inhibited by both NEM and leupeptin, but was unaffected by the addition of pepstatin A or PMSF (Figure 2A). This indicates that the VWF- cleaving peptidase most likely belongs to the class of cysteine peptidases. Additionally, it was verified whether addition of L-cysteine (L-cys) could enhance proteolytic cleavage. This is characteristic for members of the cysteine peptidase family as their proteolytic activity is dependent on reduction of the active site cysteine, which can be accomplished by addition of thiol-reducing agents, such as dithiothreitol or free cysteine. The indication that the VWF-cleaving peptidase belongs to the class of cysteine peptidases is indeed further strengthened by the observation that both EDTA and L-cysteine enhance proteolytic activity, which is characteristic behaviour for (papain-like) cysteine peptidases.

Proteolytic activity of the VWF cleaving peptidase is partially inhibited by plasma components

As plasma contains many peptidase inhibitors, we next examined whether the schistosomal VWF-cleaving peptidase retained functionality in the presence of plasma components. Cleavage of FRETS-VWF73 substrate by S. mansoni soluble worm fraction (SWF) was examined in the presence of different amounts of PNP with or without EDTA (Figure 2B). PNP containing AD AMTS 13 showed a dose-dependent cleavage of FRETS- VWF73 substrate (closed circles), which can be fully inhibited through the addition of EDTA (open circles). Measurement of a fixed concentration of S. mansoni soluble worm fraction (SWF) in the presence of increasing concentrations of PNP showed a large additional proteolytic effect of the S. mansoni peptidase at low plasma concentrations (closed squares). However, at plasma concentrations above 8% PNP there was no additional proteolytic effect of the S. mansoni peptidase on FRETS-VWF73. This does not reflect substrate depletion, as a further increase in the PNP concentration from 10 to 20% resulted in increased substrate conversion. Similarly, in the presence of EDTA the S. mansoni peptidase showed strong proteolytic cleavage of FRETS-VWF73 at low plasma concentrations, but not at higher plasma concentrations (open squares). These results indicate that human plasma contains components that can inhibit the VWF-cleaving peptidase of S. mansoni at higher plasma concentrations.

We next verified whether the S. mansoni peptidase is also able to cleave native VWF in the presence of human plasma components. Purified VWF was incubated with both S. mansoni WH and PNP (3.6%) in the presence or absence of EDTA, followed by analysis of VWF multimers through gel electrophoresis (Figure 2C). The occurrence of cleavage of native VWF by the S. mansoni peptidase at low plasma concentrations (3.6%), with and without addition of EDTA, was consistent with the observed cleavage of FRETS-VWF73 at low plasma concentrations. Also in this experiment plasma components seemed to partially inhibit the S. mansoni peptidase, as cleavage of multimeric VWF in the presence of EDTA occurs more efficiently in the absence of PNP, which is visible by the increased disappearance of high multimer bands in the absence of PNP.

The VWF cleaving peptidase retains activity in TTP patient plasma After we discovered that S. mansoni soluble worm fraction contains a VWF- cleaving peptidase, we next examined whether this peptidase would be functional in TTP patient plasma, or whether it would be affected by specific components of the TTP patient plasma such as autoantibodies. Cleavage of FRETS-VWF73 substrate by S. mansoni soluble worm fraction in the presence of EDTA was therefore examined with addition of 2% PNP or platelet-poor plasma obtained from 4 different TTP patients that had 0%

AD AMTS 13 activity as previously determined by FRETS-VWF73 assay (patient data not shown) (Figure 3A). AD AMTS 13 activity in PNP was strongly inhibited after addition of plasma from TTP patient 1. This would indicate the presence of autoantibodies against ADAMTS13 in plasma from this TTP patient, which interferes with the AD AMTS 13 activity from PNP. In contrast, no significant differences in VWF cleavage of FRETS-VWF73 by S. mansoni soluble worm fraction were observed upon addition of TTP patient plasma, indicating that the VWF -cleaving S. mansoni peptidase is not sensitive to components specific for TTP patient plasma at low plasma concentrations.

We next examined whether the S. mansoni peptidase could cleave multimeric VWF in TTP patient plasma. Purified VWF was incubated with both S. mansoni WH and TTP patient plasma (3.6%) in the presence or absence of EDTA, followed by analysis of VWF multimers through gel electrophoresis (Figure 3B). The occurrence of cleavage of native VWF by the S. mansoni peptidase in the presence of TTP patient plasma (3.6%) with and without addition of EDTA, is consistent with the observed cleavage of FRETS-VWF73 in the presence of TTP patient plasma. Also in this experiment plasma components seemed to partially inhibit the S. mansoni peptidase, as cleavage of multimeric VWF in the presence of EDTA occurs more efficiently in the absence of TTP patient plasma, which is visible by the increased disappearance of high multimer bands in the absence of TTP patient plasma. Anion exchange chromatography fractionation combined with mass spectrometry identifies SmCB2 as a candidate gene for the S. mansoni VWF- cleaving cysteine peptidase

Anion exchange chromatography fractionation was used to separate proteins of S. mansoni in order to obtain fractions with high proteolytic activity towards a fluorogenic substrate, i.e. a FRETS-VWF73 substrate. The three fractions with the highest activity in the FRETS-VWF73 substrate assay were pooled and subsequently analyzed by mass

spectrometry. Proteins present in the active fraction were compared to the protein content of the flow-through fraction (control fraction) containing low VWF -cleaving activity at equal protein concentrations. Five cysteine peptidases were identified in the active fraction (Table 1, see below)), however, only one of these cysteine peptidases was consistently enriched in both mascot ion score and peptide spectrum matches (PSM) in the active fraction compared to the control fraction throughout three independent experiments. This cysteine peptidase, Schistosoma mansoni cathepsin B (Smp_141610), showed 17.2 ± 2.3 fold enrichment in score and 12.4 ± 3.1 fold enrichment in PSM compared to the control fraction. This identified the S. mansoni cathepsin B gene as the prime candidate to encode the S.

mansoni VWF-cleaving peptidase.

S. mansoni cathepsin B (Smp_141610) was first isolated and characterized by Caffrey et al., Mol Biochem Parasitol. 121:49-61 (2002). This cathepsin B peptidase was named S. mansoni cathepsin B2 (SmCB2, Uniprot Q95PM1) to distinguish it from the first recorded S. mansoni cathepsin, SmCBl

[SmCB] Consistent with this nomenclature, we will here refer to the candidate gene S. mansoni cathepsin B (Smp_141610) as SmCB2.

Table 1: Cysteine peptidases present in the schistosomal fraction with proteolytic activity towards FRETS-VWF73 substrate obtained after anion exchange chromatography of S. mansoni soluble worm fraction. Score: mascot ion score; PSM: peptide spectrum matches (total number of identified peptide spectra matched for the protein); A, B, and C indicate three independent experiments.

Purified recombinant expressed SmCB2 has proteolytic activity towards FRETS-VWF73

Recombinant expressed SmCB2 (rSmCB2) in Pichia pastoris was purified and examined for its proteolytic activity towards its known fluorogenic substrate Z-FR-AMC as described before (Caffrey et al., Mol Biochem

Parasitol. 121:49-61 (2002)), and towards the FRETS-VWF73 substrate, as described above. Recombinant SmCB2 was produced according to the protocol described in Caffrey et al., 2002. As shown in Figure 4, purified rSmCB2 is an active peptidase that cleaves the FRETS-VWF73 substrate in a dose dependent manner, which demonstrates that SmCB2 is a

schistosomal peptidase that can cleave the same VWF domain as

AD AMTS 13. Interestingly, the proteolytic activity of rSmCB2 towards the FRETS-VWF73 substrate was not enhanced by the presence of EDTA, which could suggest that SmCB2 is not the only schistosomal peptidase that can cleave human VWF or that addition of EDTA to soluble worm fraction results in activation of SmCB2 in crude homogenates by for instance induction of cleavage of inhibiting pro-peptides or conformational changes that remove other inhibitory factors.

Claims

Claims

1. A papain -like cysteine peptidase that exhibits peptidase activity towards von Willebrand Factor (VWF); wherein said peptidase is for use as a medicament.

2. The peptidase for use according to claim 1, wherein said peptidase is a divalent metal ion-independent cysteine peptidase.

3. The peptidase for use according to claim 1 or claim 2, wherein said peptidase is a cathepsin B.

4. The peptidase for use according to any one of the preceding claims, wherein said peptidase has

- a cysteine active site comprising the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 50% sequence identity to the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2; and/or

- a histidine active site comprising the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 50% sequence identity to the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2.

5. The peptidase for use according to any one of the preceding claims, wherein said peptidase has

- a cysteine active site comprising the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2; and - a histidine active site comprising the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2.

6. The peptidase for use according to any one of the preceding claims, wherein said peptidase comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence having at least 50% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2.

7. The peptidase for use according to any one of the preceding claims, wherein said peptidase comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2.

8. The peptidase for use according to any one of the preceding claims, wherein said peptidase is a SmCB2.

9. The peptidase for use according to any one of the preceding claims, wherein said peptidase is the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2.

10. A nucleic acid encoding the peptidase as defined in any one of the preceding claims, wherein said nucleic acid is for use as a medicament.

11. The peptidase for use according to any one of claims 1-9 or the nucleic acid for use according to claim 10, wherein said peptidase or nucleic acid is for use as an antithrombotic.

12. The peptidase for use according to any one of claims 1-9 or the nucleic acid for use according to claim 10, wherein said peptidase or nucleic acid is for use in the treatment of a disorder associated with VWF.

13. The peptidase for use according to any one of claims 1-9 or the nucleic acid for use according to claim 10, wherein said peptidase or nucleic acid is (i) for use in the treatment of a disorder associated with, or mediated by, the presence of VWF, (ii) for use in the treatment of a disorder associated with, or mediated by, accumulation of VWF and/or (iii) for use in a method of counteracting prothrombotic effects mediated by accumulation of VWF.

14. The peptidase or nucleic acid for use according to claim 12 or claim 13, wherein said disorder or said method of counteracting is:

(i) a thrombotic micro angiopathy (TMA), preferably thrombotic

thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), HELLP syndrome or catastrophic antiphospholipid syndrome, more preferably acquired TTP;

(ii) myocardial infarction; or

(iii) stroke, preferably ischemic stroke.

15. A pharmaceutical composition comprising a peptidase as defined in any one of claims 1-9 or a nucleic acid as defined in claim 10, and one or more pharmaceutically-acceptable carriers, adjuvants, excipients and/or diluents.

16. The pharmaceutical composition according to claim 15, wherein the peptidase comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2 and that exhibits peptidase activity towards von Willebrand Factor (VWF).