ADAMTS13 and Anti-ADAMTS13 Autoantibodies in Thrombotic Thrombocytopenic Purpura – Current Perspectives and New Treatment Strategies

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Expert Review of Hematology

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ADAMTS13 and anti-ADAMTS13 autoantibodies in thrombotic thrombocytopenic purpura – current perspectives and new treatment strategies

Claudia Tersteeg, Sebastien Verhenne, Elien Roose, An-Sofie Schelpe, Hans Deckmyn, Simon F. De Meyer & Karen Vanhoorelbeke

To cite this article: Claudia Tersteeg, Sebastien Verhenne, Elien Roose, An-Sofie Schelpe, Hans Deckmyn, Simon F. De Meyer & Karen Vanhoorelbeke (2016) ADAMTS13 and anti-ADAMTS13 autoantibodies in thrombotic thrombocytopenic purpura – current perspectives and new treatment strategies, Expert Review of Hematology, 9:2, 209-221, DOI: 10.1586/17474086.2016.1122515

To link to this article: http://dx.doi.org/10.1586/17474086.2016.1122515

Accepted author version posted online: 18 Nov 2015. Published online: 08 Dec 2015.

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Download by: [T&F Internal Users], [Priti Nagda] Date: 25 April 2016, At: 07:19 EXPERT REVIEW OF HEMATOLOGY, 2016 VOL. 9, NO. 2, 209–221 http://dx.doi.org/10.1586/17474086.2016.1122515

REVIEW ADAMTS13 and anti-ADAMTS13 autoantibodies in thrombotic thrombocytopenic purpura – current perspectives and new treatment strategies Claudia Tersteeg, Sebastien Verhenne, Elien Roose, An-Sofie Schelpe, Hans Deckmyn, Simon F. De Meyer and Karen Vanhoorelbeke Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium

ABSTRACT ARTICLE HISTORY A deficiency in ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type-1 Received 29 September 2015 repeats, member 13) is associated with thrombotic thrombocytopenic purpura (TTP). Congenital Accepted 17 November 2015 TTP is caused by a defect in the ADAMTS13 gene resulting in decreased or absent enzyme activity; KEYWORDS acquired TTP results from autoantibodies that either inhibit the activity or increase the clearance ADAMTS13; thrombotic of ADAMTS13. Despite major progress in recent years in our understanding of the disease, many thrombocytopenic purpura aspects around the pathophysiology of TTP are still unclear. Newer studies expanded the TTP (TTP); von Willebrand factor; field from ADAMTS13 and inhibitory antibodies to immune complexes, cloned autoantibodies, congenital TTP; acquired and a possible involvement of other proteases. Additionally, several new treatment strategies TTP; immune complexes; supplementing plasma-exchange and infusion are under investigation for a better and more autoantibodies; proteases specific treatment of TTP patients. In this review, we discuss the recent insights in TTP pathophysiology and describe upcoming therapeutic opportunities.

Introduction ADAMTS13 The multimeric glycoprotein von Willebrand factor ADAMTS13 is a multi-domain enzyme consisting of a (VWF) plays an important role in maintaining normal metalloprotease (M) and disintegrin-like (D) domain, hemostasis by mediating platelet adhesion to collagen a thrombospondin type-1 repeat (T), a cysteine-rich (C) during vascular injury.[1] Endothelial cells and platelets and spacer (S) domain, seven additional thrombospon- secrete VWF as ultra-large (UL) multimers. As a conse- din type-1 repeats, and two CUB domains (visualized in quence of the shear conditions in the flowing blood, Figure 1). ADAMTS13 circulates as a constitutively active these UL-VWF multimers unfold, thereby exposing their enzyme; however, cleavage of its substrate only occurs cryptic platelet binding sites turning them into a upon unfolding of VWF. Indeed, unfolding of the UL- prothrombotic state. Under normal physiological VWF multimers under normal shear conditions exposes conditions, UL-VWF multimers are digested by the not only the platelet binding sites but also the cryptic metalloprotease ADAMTS13 (A Disintegrin And ADAMTS13 cleavage site, which is situated in the VWF Metalloprotease with ThromboSpondin type-1 repeats, A2 domain. Upon unfolding of the A2 domain, the member 13) into smaller, folded, and quiescent multi- proximal MDTCS domains of ADAMTS13 bind the cryp- Downloaded by [T&F Internal Users], [Priti Nagda] at 07:19 25 April 2016 mers. A deficiency in ADAMTS13 is associated with the tic domains in VWF like a molecular zipper, thereby life-threatening microangiopathic disorder thrombotic positioning the proteolytically active metalloprotease thrombocytopenic purpura (TTP), where during acute domain of ADAMTS13 toward the cleavage site at posi- episodes of TTP, VWF-rich platelet thrombi block the tion Tyr1605-Met1606 in VWF.[4] The role of the microcirculation. Acute episodes of TTP are suspected remaining distal thrombospondin and CUB domains is by thrombocytopenia and hemolytic anemia.[2] less clear. These distal domains were recently shown to Patients suffer from symptoms such as neurologic regulate the proteolytic activity of ADAMTS13 by shield- abnormalities and organ failure, which without treating the proximal domains.[5,6] The distal domains are ment can have fatal consequences. The exact molecular crucial for docking full-length ADAMTS13 onto VWF trigger for acute TTP episodes is not known; however, under high shear conditions,[7] which results in disease onset is often related to pregnancy, infections, unshielding of the MDTCS domains and allosteric acti- or the use of certain drugs.[3] vation of ADAMTS13.[6] In line with this, C-terminally

14% (n=7)[9] 0% (n=15)[9] 17% (n=48)[10] 35% (n=48)[10] 100% (n=48)[10] 28% (n=24)[11] 64% (n=24)[11] 97% (n=67)[12] 36% (n=67)[12] 31% (n=67)[12] 97% (n=92)[13] 28% (n=92)[13] 31% (n=92)[13]

D T1 C S T2 T3 T4 T5 T6 T7 T8 CUB1 CUB2

0% (n=15)[9] 100% (n=15)[9] 46% (n=67)[12] 100% (n=24)[11]

56% (n=24)[11] 12% (n=67)[12]

Figure 1. Epitope mapping of anti-ADAMTS13 autoantibodies demonstrated that almost all acquired TTP patients have anti-spacer domain antibodies. Anti-ADAMTS13 autoantibodies in plasma from acquired TTP patients were epitope mapped against the different domains of ADAMTS13: metalloprotease (M) and disintegrin-like (D) domain, a thrombospondin type-1 repeat (T1), a cysteine-rich (C) and spacer (S) domain, seven additional thrombospondin type-1 repeats (T2–T8) and two CUB domains (CUB1-2). The percentage of antibodies binding to the specific domain with the corresponding number of patients included in the study is given in the figure.[9–13]

truncated ADAMTS13 showed reduced activity, result- distributed throughout the ADAMTS13 gene in more ing in faster thrombus formation in mice.[8] than 150 patients.[18]MorethanhalfofADAMTS13 Hence, ADAMTS13 is an essential protease required mutations are single amino acid substitutions, for proteolysis of VWF and the maintenance of normal whereas the rest include frame-shifts, splice-site hemostasis. A deficiency in ADAMTS13 is observed in mutations, and nonsense mutations.[18]Mostmuta- patients with TTP, either due to a congenital defect tions cause secretion defects; however, truncated resulting in decreased or absent activity, or due to the forms of ADAMTS13 with decreased enzymatic activ- presence of autoantibodies inhibiting the activity or ity and/or substrate binding have been reported.[19] increasing the clearance of ADAMTS13. Furthermore, combinations of mutations and single nucleotide polymorphisms (SNPs) may result in a more severe ADAMTS13 deficiency.[20]Ithasbeen Congenital TTP hypothesized that specific SNPs and ADAMTS13 The congenital form of TTP is caused by mutations mutations are typical for specific ethnic sub- in the ADAMTS13 gene, which is present on chromo- groups.[17] Downloaded by [T&F Internal Users], [Priti Nagda] at 07:19 25 April 2016 some 9q34.[14]CongenitalorhereditaryTTPisalso Often a distinction is made between early- and known as the Upshaw–Schulman syndrome and has late-onset congenital TTP, depending on the patient’s aprevalenceoflessthan1case per million per year age at which the first TTP symptoms occur. Early- and accounts for 2–4% of all TTP cases.[15]The onset TTP is characterized by repeated episodes of prevalence of congenital TTP is probably underesti- thrombocytopenia and hemolytic anemia during mated due to the heterogeneity of the clinical childhood, which is easily detected by neonatal course and symptoms common to other diseases. Coombs-negative jaundice. In contrast, late-onset [16]. The autosomal recessive inheritance pattern of TTP, appearing only in adults, is not well character- ADAMTS13 mutations was unraveled in 2001 by link- ized because of its highly variable clinical onset.[21]It age analysis in four pedigrees.[14]Heterozygous was suggested that certain ADAMTS13 mutations can mutations result in a reduced ADAMTS13 activity indicate the time of TTP presentation. For example, (40–70%) and a normal phenotype, while TTP symp- the mutation R1060W was initially found in 10 toms are present in over 90% of individuals with patients with adult-onset TTP only.[22]However,a compound heterozygous or homozygous mutations. child was then identified with the same mutation. [17]Over140mutationshavebeenfound [22]Thissamemutationwasfoundinahigherthan EXPERT REVIEW OF HEMATOLOGY 211

expected proportion of patients suffering from preg- Acquired TTP nancy-onset TTP, and might thus be a risk factor Acquired TTP is characterized by a heterogeneous and during pregnancy.[23,24] Additionally, suggestions polyclonal autoimmune response against ADAMTS13. have been made that the position of the mutation [29] The antibody repertoire of acquired TTP patients in ADAMTS13 can be linked to disease severity. It was consists of autoantibodies that interfere with the pro- suggested that different mutations in the proximal teolytic activity of ADAMTS13, or increase its clearance metalloprotease domain and distal thrombospondin from the circulation.[29,30] Remarkably, approximately repeats are associated with a less-aggressive 5% of healthy individuals also possess low-affinity and phenotype in childhood TTP, compared to neonatal non-neutralizing anti-ADAMTS13 autoantibodies.[31] TTP presentation, which is associated with mutations The exact mechanisms of developing an autoimmune located in the first proximal thrombospondin domain. response against ADAMTS13 are still poorly under- [22]However,siblingswiththesameADAMTS13 stood. Although acquired TTP is mainly thought to be mutations but different clinical course have been a B-cell-mediated autoimmune disease, evidence for a reported,[25]emphasizingthedifficultandunpredict- significant involvement of T-cells is increasing.[32] In able nature of TTP. this respect, HLA DRB1*11 was identified as being a Long-term follow-up data of TTP patients are scarce, risk factor for the development of TTP as a result of hampering our understanding of the impact of single or molecular mimicry.[33] An HLA-DRB1*11-presented multiple TTP attacks on overall quality of life. An Arab CUB2-derived peptide (FINVAPHAR) demonstrated family, with two compound heterozygote family substantial homology with peptide sequences derived members having severe early-onset Upshaw–Schulman from different bacterial proteins.[33,34] It was syndrome, has been under the longest observation suggested that an infection, resulting in the presenta- (44 years) ever.[26] The main long-term symptoms tion by antigen-presenting cells of pathogen-derived reported are deep psychological problems and short- peptides that share homology with the CUB2-derived term memory loss, with an enormous impact on daily peptide, might activate an autoimmune response life. More understanding of the long-term effects of TTP against ADAMTS13.[33,34] is essential and centralized data registries with long- term patient follow-up are necessary to acquire this information. International registries are making Epitope mapping and (sub)class distribution of attempts to unify and centralize all congenital TTP autoantibodies patients. The main goal is to obtain insight into this rare disease, eventually leading to improved health care The anti-ADAMTS13 autoantibodies present in the by constructing evidence-based guidelines.[27] In 2006, plasma of acquired TTP patients have been intensively the International Hereditary TTP registry (www.ttpregis investigated in terms of antibody titer, ADAMTS13 try.net; ClinicalTrial.gov identifier: NCT01257269) was domain specificity, and Ig (sub)class distribution to established, which in the meantime contains 145 unravel a possible link between these parameters and patients and family members from 16 nationalities. the severity of the disease. Interestingly, almost all Broader registries exist, including also patients with acquired TTP patients (85–100%) have autoantibodies acquired TTP or other thrombotic microangiopathies against the cysteine/spacer domain of ADAMTS13, Downloaded by [T&F Internal Users], [Priti Nagda] at 07:19 25 April 2016 such as hemolytic uremic syndrome. The United although autoantibodies against the other proximal Kingdom TTP (UK TTP) registry is actively recruiting domains (MDT) have also been identified (Figure 1). patients until 2018 (http://public.ukcrn.org.uk/search/ [9–13,35]Inaddition,in14–61% of these patients, StudyDetail.aspx?StudyID=8435). Also in other countries besides the anti-MDTCS autoantibodies, autoantibo- such as Italy (www.ttpdatabase.org), France (www.cnr- dies against the distal thrombospondin and CUB mat.fr), Japan [28], and Australia (https://ttp.registry.org. domains were found, illustrating the heterogeneous au/) patients are being, or have been, recruited. anti-ADAMTS13 immune response (Figure 1).[9–13,35] Despite abundant case reports and research on sev- Anti-ADAMTS13 autoantibodies have been identified eral mutations, many aspects of TTP still remain that (partially) neutralize ADAMTS13 activity, thereby unknown. The trigger for an acute episode, the differ- providing an explanation for the observed ADAMTS13 ent recurrence rates as well as the long-term conse- deficiency in these patients.[9,13]Alltheseneutraliz- quences of TTP, correlated to specific ADAMTS13 ing autoantibodies have an epitope in the ADAMTS13 mutations, are important aspects that will hopefully spacer domain, a domain known to contain the major be unraveled by the data gathered within the different exosite for the binding of ADAMTS13 to the unfolded registries. VWF A2 domain.[9,13]Neutralizinganti-ADAMTS13 212 C. TERSTEEG ET AL.

autoantibodies against other functional important IgM-complexes can form large aggregates with their ADAMTS13 domains like the metalloprotease and corresponding antigens and can easily activate comple- CUB domains have not been identified yet. Recently ment via the classical pathway.[45] IgG-complexes are however, it was reported that autoantibodies directed usually smaller in size; IgG3 gives the highest comple- against the distal ADAMTS13 domains induce ment activation via the classical pathway, followed by ADAMTS13 deficiency predominantly by clearing the IgG1 and IgG2, whereas IgG4 is not able to activate enzyme instead of neutralizing its activity.[13,36] complement and furthermore has a low affinity for Levels of antibody titers seem to be correlated with cellular Fc-receptors.[46] IgA-complexes do not activate disease severity. Indeed, a high inhibitory titer at the complement via the classical pathway, but can initiate presentation of acute symptoms is associated with activation via the alternative pathway, which is not the persistence of an undetectable ADAMTS13 activity dependent on activation by immune complexes.[47] in remission, leading to a three times higher probabil- IgG4, the main antibody subclass found in TTP patients, ity of recurrence.[37–39]Moreover,anADAMTS13 is expected to form small immune complexes with inhibitor titer of 2 or more Bethesda units/mL is asso- ADAMTS13 as IgG4 antibodies are involved in the half- ciated with a lower survival in patients with an antibody exchange reaction, resulting in random com- ADAMTS13 activity of <10%.[40]Furthermore,low binations of half-antibody fragments that are monova- ADAMTS13 antigen levels (<13.5 ng/mL) at presenta- lent for a specific antigen.[48] As these small complexes tion are associated with a higher mortality risk.[13] are not efficiently cleared by the reticulo-endothelial Also, the isotype distribution of anti-ADAMTS13 auto- system or via Fc-receptors, it was suggested that this antibodies is a possible prognostic factor for disease might contribute to a longer circulating half-life and progression. IgGs were found in all patients, with IgG4 therefore an increased detection level.[43] It remains (69–90%) as the most prevalent, followed by IgG1 however unclear whether these IgG4 complexes, or (73–52%), IgG2 (15–50%), and IgG3 (8–33%), as well complexes with antibodies from another (sub)class, as IgM (7–10%) and IgA (19–21%).[12,30] High levels are able to activate complement or form immune com- of IgG4 could suggestively be explained by chronic plex deposits in the vascular wall. Nevertheless, com- antigen stimulation.[41] Patients with high levels of plement activation is reported during acute TTP IgG4 and undetectable levels of IgG1 seem to have an episodes. Increased levels of complement factors C3a, increased relapsing rate compared to patients with C5a, C5b-9, and Bb are detected during the acute phase low IgG4 and detectable IgG1.[30]Additionally,during of the disease compared to patients in remission, the acute phase, a high IgA titer is associated with a [49–51] and decrease during PEX treatment.[49] lower platelet count, while a high IgG titer seems to However, this increase in complement activation mar- correlate with the requirement of a higher number of kers is significantly lower compared to the highly ele- plasma exchanges (PEXs) to achieve remission.[39,42] vated markers measured in patients with hemolytic However, large patient studies are needed to identify uremic syndrome.[50] In renal tissue from TTP patients, strong prognostic factors for disease severity and C3 and C5b-9 deposition was observed in and around relapse. glomerular endothelial capillaries and within thrombi. Additionally, C3 deposition was demonstrated in the glomeruli and tubuli of kidneys from mice suffering

Downloaded by [T&F Internal Users], [Priti Nagda] at 07:19 25 April 2016 Pathological consequences of circulating immune from TTP after exposure to Shigatoxin.[52] It however complexes remains to be investigated whether immune complexes Antibody binding to ADAMTS13 results in the formation are responsible for the increased complement activa- of circulating immune complexes. In patients with tion and what role immune complexes play in the acquired TTP, immune complexes were indeed pathophysiology of TTP (Figure 2). detected during both the acute event and remission. [43,44] Whereas free anti-ADAMTS13 antibodies were Cloning of autoantibodies to unravel the mainly IgG1 and IgG4 subclasses,[30] anti-ADAMTS13 pathophysiology of acquired TTP antibodies present in immune complexes were predominantly of the IgG4 subclass. Ninety-two per cent of The predominant isotype of autoantibodies (either the immune complexes during acute and remission unbound, or circulating in an immune complex) was phase consisted of IgG4 anti-ADAMTS13 antibodies, in demonstrated to contain possible predictive evidence line with the high prevalence of IgG4 anti-ADAMTS13 on the progress of acquired TTP episodes. However, at autoantibodies in the plasma of acquired TTP present, a fundamental understanding of how exactly patients.[43] different isotypes influence the pathophysiology of EXPERT REVIEW OF HEMATOLOGY 213

Immune complex deposition?

Neutrophil NET release activation? NETs in thrombi? Complement activation? UL-VWF Elastase (Anti-)ADAMTS13 immune complex VWF Plasmin proteolysis? activation? Platelet Plasmin uPA/uPAR VWF-rich microthrombus

Figure 2. Other processes with a possible role in TTP: immune complexes, activated neutrophils, and plasmin. Circulating immune complexes of anti-ADAMTS13 autoantibodies bound to ADAMTS13 have been demonstrated in plasma from TTP patients. Whether these immune complexes are deposited in underlying tissues or activate complement, and subsequently play a role in the underlying pathophysiology is currently not known. Additionally, possible activation of neutrophils during acute TTP episodes has been demonstrated by the presence of elevated plasma levels of neutrophil extracellular traps (NET) markers and neutrophil elastase. Elevated NET markers indicate a possible role of NETs in the progression of TTP symptoms, as NETs have the ability to bind platelets and promote thrombus formation. Elastase on the other hand is able to cleave VWF in vitro and could support the degradation of UL-VWF multimers. Also plasmin has the capacity to proteolyze VWF, when activated via the uPA/uPAR pathway. Elevated plasmin levels were demonstrated during the acute phase of TTP and could play a role in the size modulation of VWF.

acquired TTP is still largely lacking. Epitope mapping of was used to clone over 130 unique anti-ADAMTS13 autoantibodies might provide additional new insights antibodies from the immune repertoire of four unre- into the pathophysiology of acquired TTP although it lated acquired TTP patients.[54] In the cloned antibody remains difficult to characterize the effect of a single libraries of both studies, it was observed that the major- antibody in a patient’s antibody repertoire on ity of antibodies were derived from the VH1-69 germ- ADAMTS13 function, with such a heterogeneous and line and that almost all were able to inhibit the polyclonal immune response. Recent advances in the proteolytic activity of ADAMTS13.[54,55] Recently, cloning of autoantibodies, however, open new possibi- three additional scFv fragments with high affinity, lities to characterize in detail anti-ADAMTS13 antibodies high inhibitory capacity, and spacer domain specificity and to gain more insight into the pathophysiology of were selected through phage display.[56] With these acquired TTP. scFv fragments, it was demonstrated that antibody Luken et al. were the first to clone anti-ADAMTS13 binding to the five loops within the spacer domain, autoantibodies from mononuclear cells of an acquired which is necessary for the correct binding of TTP patient in remission.[53] Using phage display, sin- ADAMTS13 to VWF, inhibits the proteolysis of VWF by gle-chain variable (scFv) fragments were expressed as physical blockage of the ADAMTS13-VWF interaction, combined variable heavy (VH) and variable light seg- [56] thereby confirming and extending previous pub-

Downloaded by [T&F Internal Users], [Priti Nagda] at 07:19 25 April 2016 ments and were selected against the cysteine/spacer lications.[53,55] Also spleens of acquired TTP patients domain. Seven scFv fragments were identified and were found to be a rich source of antibody-producing divided into four groups based on their VH germline cells. The group of Kremer-Hovinga was able to isolate gene segments: VH1-69, VH1-01, VH3-09, and VH3-07. 29 clones from spleens of two acquired TTP patients All these different germlines produced anti-cysteine/ through phage display and Epstein Barr Virus transfor- spacer antibodies, demonstrating that multiple B-cell mation.[57] Of these antibodies, 93% were inhibitory clones develop antibodies against the same domain. and 79% were directed against the spacer domain. The scFv fragments and full-length IgGs were able to Only a limited amount of VH genes were found: VH1- partially inhibit ADAMTS13 activity, indicating that one 3, VH1-69, VH3-30, and VH4-28, as well as a limited single inhibitory antibody against the spacer domain number of CDR3 motifs, of which four were shared by might not be enough to explain the pathogenicity of the two patients.[57] These recent developments have autoantibodies. Competition experiments performed demonstrated that the spleen contains a limited auto- with one of the scFv furthermore indicated that anti- immune repertoire against ADAMTS13. Most cloned bodies with a similar epitope are present in the plasma autoantibodies share common CDR3 motifs, inhibit of other acquired TTP patients.[53] The same technique ADAMTS13 activity, and are directed against the spacer 214 C. TERSTEEG ET AL.

domain. As the pathogenic relevance of shared CDR3 Recently, rituximab, a humanized anti-CD20 monoclo- motives was demonstrated in other autoimmune dis- nal antibody, has been shown to be a safe and effective eases such as chronic lymphocytic leukemia,[58] shared adjunct to PEX, especially in patients with refractory or CDR3 motifs might also be crucial for the pathogenicity relapsing TTP.[62] Standard intravenous rituximab treat- of anti-ADAMTS13 autoantibodies. Characterization of ment (375 mg/m2 weekly for 4 weeks) results in B-cell novel cloned autoantibodies will provide us with more depletion, reduced anti-ADAMTS13 antibody titers, and information on the polyclonal immune response in increased plasma ADAMTS13 activity in most patients. acquired TTP patients. In addition, it would especially [62] Early administration, preferably within the first 3 be interesting to unravel whether acquired TTP patients days, is associated with faster attainment of remission, have anti-distal autoantibodies that inhibit ADAMTS13 fewer number of PEX, shorter hospitalization, and a function given the functional role of distal domains in reduced relapsing risk.[63] These benefits have stimu- the docking of ADAMST13 to VWF. On the other hand, lated the use of rituximab as prophylactic therapy in such autoantibodies might not be present in acquired patients with a history of TTP and deficient ADAMTS13 TTP patients as it was recently described that autoanti- activity in remission.[64] Although preventive rituximab bodies directed against the distal domain are mainly infusions maintained a sustained remission in these clearance-inducing autoantibodies.[13] Future studies patients,[64] concerns regarding overtreatment and will definitely help us better understand the role of increased susceptibility for infections are raised and the immune system, and more specifically the role of therefore a risk/benefit ratio on an individual patient B- and T-cells, in the formation of autoantibodies. level has yet to be made. Since the introduction of PEX in clinical practice, survival rates did not further increase during the last Current and future treatment opportunities for 20 years. However, due to a better insight into the TTP pathophysiology of TTP, different novel adjuvant treat- Acute TTP episodes require prompt treatment. For ment strategies have been investigated (Table 1). more than 20 years, the use of PEX or plasma infusion has been crucial in the management of TTP episodes, Blocking the VWF–GPIb interaction reducing mortality rates from 90% to 10–20%.[59,60] PEX is superior to plasma infusion for acquired TTP One novel strategy encompasses the blockade of the and should be initiated once a presumptive diagnosis VWF–platelet glycoprotein (GP)Ib interaction, thereby of TTP is made.[59] PEX not only replaces the deficient preventing the formation of VWF-rich platelet thrombi. or inhibited ADAMTS13 by active ADAMTS13 from ARC1779, an aptamer blocking the GPIb-binding VWF donor plasma, but also removes potential disease-caus- A1 domain, inhibits the formation of occlusive thrombi ing factors such as autoantibodies, immune complexes, in a carotid artery thrombosis model in cynomolgus or bacterial toxins from the circulation. Plasma infusion monkeys.[65] This aptamer was the first targeted ther- only replenishes ADAMTS13 activity in acquired TTP apy tested in humans and provided proof of principle patient’s plasma and can provide temporary benefit for this approach in the treatment of TTP in a small until PEX can be initiated.[61] The number of PEX cohort of patients.[66] More comprehensive data required to achieve remission depends on the severity regarding the efficacy of inhibiting the VWF–GPIb inter- Downloaded by [T&F Internal Users], [Priti Nagda] at 07:19 25 April 2016 of initial disease or progression of the symptoms during action were obtained from other preclinical and clinical admission. In general, patients with acquired TTP studies. In a baboon model of acquired TTP, the anti- require a higher number of PEX and longer treatment body GBR600 [67] and the nanobody Caplacizumab,[68] to normalize platelet counts compared to congenital both targeting the VWF A1 domain, were capable of TTP patients. Due to the lack of circulating antibodies treating and preventing TTP symptoms. Caplacizumab in patients with congenital TTP, regular plasma infu- was subsequently tested in a multicenter phase II ran- sions alone are generally sufficient to achieve remission domized, placebo-controlled trial, in which acquired or prevent TTP relapses. In patients with acquired TTP, TTP patients were given Caplacizumab as an adjunct immunosuppressive drugs are used as adjuvant therapy to daily PEX.[69] Caplacizumab-treated patients to PEX. Immunosuppressive agents tone down achieved faster platelet normalization and developed the immune response and suppress the production of less recurrent thrombocytopenia that required initiation new autoantibodies. Consistent use of corticosteroids in of PEX <30 days after the last PEX therapy.[70] addition to PEX seems to further increase survival and Interestingly, only minor bleeding episodes and man- reduce the frequency of relapses, especially in patients ageable side effects were observed in patients in the with severe acquired ADAMTS13 deficiency.[62] active drug treatment arm. These data suggest that by EXPERT REVIEW OF HEMATOLOGY 215

Table 1. New treatment strategies for the treatment of congenital and acquired TTP. Strategy Drugs Experimental phase Outcome Blocking VWF-platelet ARC1779 Cynomolgus monkeys [65] Inhibition of formation of occlusive thrombi GPIb interaction Placebo-controlled clinical study Supported safety (n =9)[66] GBR600 Baboons [67] Prevention and treatment of TTP symptoms Caplacizumab Baboons [68] Prevention and treatment of TTP symptoms Phase II placebo-controlled clinical Faster platelet normalization, less recurrent trial [69,70] thrombocytopenia Replenish ADAMTS13 rADAMTS13 (BAX930) Mouse model for congenital TTP [71] Reduced thrombocytopenia and severe organ damage activity by recombinant Rat model for acquired TTP [72] Overriding inhibitory antibodies, reduction of severe TTP enzyme symptoms Phase I dose-escalation study Ongoing, assess safety and pharmacokinetics in congenital TTP patients Autoantibody-resistant In vitro study [73] Reduced binding of autoantibodies, VWF-cleaving rADAMTS13 activity preserved Platelet-delivered Mouse model for congenital and Prevention of TTP symptoms rADAMTS13 acquired TTP [74] Reducing VWF N-acetylcysteine Adamts13-/- mice [75] Reduction of circulating UL-VWF multimers, faster multimer size mesenteric thrombus resolution Pilot clinical study in TTP patients Ongoing, assess platelet count, number of plasma exchange required and microvascular thrombotic complications Thrombolytic agents Mouse model for congenital TTP [76] Activation of plasmin cleaved VWF and reduced TTP symptoms Gene therapy Viral vectors Adamts13-/- mice [77–80] Restoration of ADAMTS13 antigen and activity Mouse model for congenital TTP [80] Prevention of TTP symptoms Nonviral vectors Mouse model for congenital TTP [81] Reduced thrombocytopenia and severe organ damage

preventing the interaction between VWF and platelets, these ADAMTS13 variants was reduced while the VWF- the standard of care of TTP patients might improve. cleaving activity was preserved or even enhanced.[73] Furthermore, transgene expression of ADAMTS13 from platelets has recently been shown to prevent both rADAMTS13 congenital and acquired TTP in mice.[74] Despite the Another potential future therapy is the use of recombi- fact that translation into clinic is challenging, this nant ADAMTS13 (rADAMTS13) as substitute for human approach is promising as the platelet-derived plasma. Preclinical animal studies have demonstrated rADAMTS13 is less susceptible to inhibition by inhibi- that the administration of rADAMTS13 before or after tory anti-ADAMTS13 antibodies. induction of TTP in Adamts13-/- mice reduced the inci- dence of severe thrombocytopenia and organ damage. [71] A phase I clinical trial was recently initiated to Reducing the size of VWF multimers assess rADAMTS13 in the treatment and prophylaxis of congenital TTP. Although the approach indeed Recently, Chen and colleagues demonstrated that VWF seems more applicable to congenital TTP patients, in multimer size can be pharmacologically decreased with Downloaded by [T&F Internal Users], [Priti Nagda] at 07:19 25 April 2016 vitro studies nevertheless have shown that rADAMTS13 N-acetylcysteine (NAC).[75] Although this FDA- is able to restore ADAMTS13 activity in the plasma of approved drug is currently used to treat acetamino- acquired TTP patients.[82] This was confirmed in an in phen toxicity and chronic obstructive lung disease, it vivo rat model of acquired TTP. Following the adminis- has been shown that NAC can reduce intrachain dis- tration of anti-ADAMTS13 antibodies, infusion of ulfide bonds, thereby reducing circulating UL-VWF mul- rADAMTS13 did override the effect of the inhibitory timers in the plasma of mice.[75] Hence, NAC could antibodies, resulting in the reduction of severe TTP reduce the size of UL-VWF multimers in TTP patients. symptoms such as thrombocytopenia and hemolytic Human case reports on TTP treatment with NAC how- anemia, demonstrating promising value for future treat- ever demonstrated varying outcomes [83–85] and a ment also in acquired TTP.[72] An elegant way to cir- clinical trial is currently ongoing to test its efficacy cumvent autoantibody inhibition of rADAMTS13 may under controlled conditions. Finally, activation of plas- be the administration of autoantibody-resistant minogen using thrombolytic agents could be a possible ADAMTS13 variants. Jian and colleagues have demon- new treatment strategy. Plasmin was shown to cleave strated that, by modifying an exosite in the spacer VWF, resulting in a reduction of TTP symptoms in a domain of ADAMTS13, binding of autoantibodies to mouse model of congenital TTP.[76] 216 C. TERSTEEG ET AL.

Gene therapy Val1607) and PMNE and PR3 two amino acids distal (Val1607-Thr1608).[89] This suggests that neutrophils Lifelong treatment with plasma products, mainly in trapped in VWF-rich microthrombi during acute TTP patients with congenital TTP, is inconvenient, expen- episodes might be able to modulate the cleavage of sive, and can result in complications (e.g. fever, urti- VWF in the absence of ADAMTS13 (Figure 2). However, caria). Gene therapy for TTP offers a long-term evidence that this might be the case is still largely treatment that would improve the patient’s comfort lacking and recent studies on neutrophil-derived NETs and quality of life. Different gene therapeutic actually suggest a role of neutrophils in TTP pathology. approaches have already been investigated in mouse NETs are extracellular DNA traps released from neutro- models. Using viral [77–80] and nonviral [81] vectors phils that undergo ‘netosis’ and have recently been that encode full-length ADAMTS13 or the MDTCS frag- shown to promote thrombus formation.[90] ment, long-term expression of transgene enzyme in Interestingly, during acute TTP episodes, increased Adamts13-/- mice was achieved and resulted in the amounts of NET markers such as extracellular DNA elimination of circulating UL-VWF multimers and the and histones are observed in patient plasma and even prevention of TTP symptoms after challenge with correlate with disease activity.[91] In addition, TTP shigatoxin [80] or recombinant human VWF.[81] patients were shown to have decreased levels of Although more research is needed to progress from DNaseI, which could impair NET degradation.[92] This preclinical phase to clinical trial, recent advances in suggestively implies that NET formation is either trig- gene therapy for other monogenetic disorders such as gered by acute episodes of TTP, or plays a role in the hemophilia B [86] and X-linked severe combined immu- onset of acute symptoms (Figure 2). Clearly, further nodeficiency [87] are encouraging to further develop studies are needed to elucidate the role of neutrophils, gene therapy for the treatment of congenital TTP the release of leukocyte proteases, and the formation of patients. NETs in TTP.

Other proteases beyond ADAMTS13 in TTP pathology Plasmin ADAMTS13 levels do not always predict TTP occur- Next to leukocyte proteases, plasmin also has the rence. Patients with undetectable ADAMTS13 levels ability to degrade VWF.[76,88,93]Theexactcleavage can remain asymptomatic for years, which have led site(s) within VWF are yet unknown; however, a to the concept of a triggering event that induces the lysine-dependent binding of plasminogen to the onset of acute TTP symptoms. Alternatively, in the VWF A1 domain has been demonstrated.[76]The absence of ADAMTS13 it is possible that other pro- zymogen plasminogen circulates in the plasma, and teases might help regulate VWF size and hence con- can be activated by tissue plasminogen activator tribute to the pathophysiology of TTP. A variety of (tPA) or urokinase plasminogen activator (uPA) into enzymes, such as plasmin, thrombin, and different plasmin. In vitro cleavage of VWF by plasmin was leukocyte proteases, can cleave VWF in vitro [88,89]. demonstrated to be dependent on uPA and its recep- Whereas thrombin has a relatively weak capacity to tor (uPAR) for plasminogen activation and subse-

Downloaded by [T&F Internal Users], [Priti Nagda] at 07:19 25 April 2016 cleave VWF,[88]leukocyteproteasesandplasminon quent proteolytic activity (Figure 2).[76]Plasmin the other hand seem to play a more important role in activity is tightly regulated by several inhibitors. VWF size regulation in vitro. Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of uPA and tPA, whereas α2-antiplasmin and α2-macroglobulin directly inhibit active plasmin. The Leukocytes role of the plasminogen activation system in TTP is Leukocyte proteases secreted by mainly neutrophils still unclear, but several reports indicated a possible including PMNE, proteinase 3 (PR3), cathepsin G, and role in TTP pathology. Elevated plasmin-α2-antiplas- matrix metalloprotease 9 (MMP-9) are all able to pro- min complexes, indicative for increased plasmin activ- teolyse VWF.[89] By performing mass spectrometry on ity, have been found in plasma samples from patients cleaved FRETS-VWF73 substrate, it was demonstrated with acute TTP (Figure 2).[76]Furthermore,increased that cathepsin G has the same cleavage site as uPAR-expression was demonstrated in kidney biop- ADAMTS13 (Tyr1605-Met1606), whereas MMP-9 cleaves sies of patients with TTP.[94]Thisindicatesthatacti- one amino acid distal from this cleavage site (Met1606- vation of several components of the plasminogen EXPERT REVIEW OF HEMATOLOGY 217

activation pathway occurs during the acute phase of management. It is furthermore very important to seek TTP. However, despite the higher plasmin activity, for new treatment strategies, as TTP patients suffer patients still develop TTP symptoms. One explanation from devastating symptoms and not always respond for this might be an increased inhibition of plasmin, to PEX treatment or rituximab. Current new as elevated PAI-1 levels have been measured during developments regarding TTP treatment hold promis- the acute phase of TTP compared to patients in ing value for future therapy. With Caplacizumab and remission.[95] rADAMTS13 in clinical trials, better and safer treat- However, it remains unclear whether plasmin acti- ment strategies supplementing or completely repla- vation might not also aggravate acute TTP, as plas- cing current PEX are coming closer toward clinical min also has the ability to cleave ADAMTS13, thereby use. The value of new experimental drugs and stra- reducing its activity.[96]Notonlyplasmin,butalso tegies such as autoantibody-resistant ADAMTS13, thrombin and factor Xa were able to cleave NAC, plasmin, and (non-)viral gene therapy still have ADAMTS13 into non-active fragments. Indeed, in a to demonstrate their potential in more appropriate patient with acute TTP, ADAMTS13 was found to be animal models or in clinical trials. However, we proteolytically degraded by plasmin.[97]Itwasthere- believe that within a few years, better and safer fore suggested that cleavage of ADAMTS13 by plas- treatment strategies will be available for this severe min was the cause of TTP in this patient. Therefore, and life-threatening disease. plasmin might regulate TTP episodes or contribute to the onset of TTP by either cleaving VWF or cleaving plasma ADAMTS13, respectively. However, the exact Five-year view contribution of both pathways in the development of We believe that within the next five years, different TTP remains to be determined. congenital TTP registries will combine their data to increase the patient population. With a larger number Discussion and conclusion of patients, it will be easier to find the trigger for acute episodes, indications for recurrence of acute With the trigger for disease onset or autoantibody episodes, and long-term consequences of TTP. We formation still unknown, the etiology around TTP is furthermore think that in the coming years, the role still not clear. TTP registries in different countries of anti-ADAMTS13 autoantibodies in the pathophy- made it possible to unify patient data, aiming to siology of acquired TTP will be unraveled. Cloning increase the number of patients available to obtain of autoantibodies is currently ongoing in several more insights into this rare disease. Research is research groups and will further unravel the complete currently focusing on the formation of autoantibo- autoantibody repertoire in acquired TTP patients. For dies, demonstrating that cloned autoantibodies both congenital as well as acquired TTP, strong prog- can provide us with more information on the func- nostic factors to predict disease severity and relapse tional aspects of the antibodies and more informa- are expected to be established in the coming years. tion on the autoimmune repertoire. New therapeutic And last, better treatment opportunities will be avail- strategies for the treatment of congenital or able soon once proven successful in current ongoing acquired TTP currently in clinical trials include the

Downloaded by [T&F Internal Users], [Priti Nagda] at 07:19 25 April 2016 clinical trials. anti-VWF A1 domain nanobody Caplacizumab and recombinant ADAMTS13. These new strategies hold promising value for future treatment of this severe disease. Financial & competing interests disclosure CTersteegisaPostdoctoralFellow supported by the Research Expert commentary Foundation – Flanders (Fonds voor Wetenschappelijk Onderzoek, FWO), Belgium (12N0715N). A Schelpe is sup- Many efforts have been made to link antibody titer, ported by a PhD grant from the Agency for Innovation by ADAMTS13 domain specificity, and Ig (sub)class dis- Science and Technology (IWT, www.iwt.be)Flanders,Belgium tribution of anti-ADAMTS13 autoantibodies with dis- (IWT-SB/141136). The work was supported by FWO grants ease severity and relapse. A better understanding of G.0584.11 and G.0298.12 and the KU Leuven grant OT/14/ the role of anti-ADAMTS13 autoantibodies in the 07. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial pathophysiology TTP together with larger patient stu- interest in or financial conflict with the subject matter or dies is however required to provide physicians with materials discussed in the manuscript apart from those strong prognostic factors that are helpful in disease disclosed. 218 C. TERSTEEG ET AL.

Key issues ● TTP is a heterogeneous disease with many aspects around the etiology still unknown. ● TTP registries are unifying patient data all over the world to obtain more insights into the onset and presentation of congenital TTP. ● Acquired TTP is both B- and T-cell driven and the literature indicates a role for molecular mimicry in the initiation of autoantibody formation. ● Acquired TTP patients have autoantibodies of mainly the IgG4 isotype. Anti-spacer antibodies inhibit the activity of ADAMTS13, whereas antibodies against the distal thrombospondin or CUB-domains seem to be more involved in clearance. ● Immune complexes can activate complement and leukocytes and increased activation levels of both are measured in TTP plasma samples. However, the role of immune complexes in the pathophysiology of TTP is currently not known. ● Cloning of autoantibodies provided us with more information on the functional aspects of the antibodies and demonstrated a limited autoimmune repertoire. ● New treatment strategies currently enrolled in clinical trials are the anti-VWF A1 domain nanobody Caplacizumab and recombinant forms of ADAMTS13. Furthermore, NAC, plasmin, and (non-)viral gene therapy demonstrated promising therapeutic opportunities in pre-clinical in vivo studies. ● There is increasing evidence that other proteases next to ADAMTS13 play a role in the presentation of TTP symptoms.

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