Background In immune‐mediated thrombotic thrombocytopenic purpura (iTTP), patients develop an immune response against the multidomain enzyme ADAMTS13. ADAMTS13 consists of a metalloprotease (M) and disintegrin‐like (D) domain, 8 thrombospondin type 1 repeats (T1‐T8), a cysteine‐rich (C), a spacer (S), and 2 CUB domains (CUB1‐2). Previous epitope mapping studies have used relatively large overlapping ADAMTS13 fragments. Objectives We aimed at developing small nonoverlapping ADAMTS13 fragments to fine map anti‐ADAMTS13 autoantibodies in iTTP patients. Methods A library of 16 ADAMTS13 fragments, comprising several small (M, DT, C, S, T2‐T5, T6‐T8, CUB1, CUB2), and some larger fragments with overlapping domains (MDT, MDTC, DTC, CS, T2‐T8, CUB1‐2, MDTCS, T2‐C2), were generated. All fragments, and ADAMTS13, were expressed as a fusion protein with albumin domain 1, and purified. The folding of the fragments was tested using 17 anti‐ADAMTS13 monoclonal antibodies with known epitopes. An epitope mapping assay using small ADAMTS13 fragments was set up, and validated by analyzing 18 iTTP patient samples. Results Validation with the monoclonal antibodies demonstrated that single S and CUB1 were not correctly folded, and therefore CS and CUB1‐2 fragments were selected instead of single C, S, CUB1, and CUB2 fragments. Epitope mapping of antibodies of patients with iTTP confirmed that 6 nonoverlapping ADAMTS13 fragments M, DT, CS, T2‐T5, T6‐T8, and CUB1‐2 were sufficient to accurately determine the antibody‐binding sites. Conclusion We have developed a tool to profile patients with iTTP according to their anti‐ADAMTS13 antibodies for a better insight in their immune response.

Table of contentsWORKSHOP 4: Challenging clinical scenarios (CS01–CS06)CS01 Bullous lesions in two children: solitary mastocytomaS. Tolga Yavuz, Ozan Koc, Ali Gungor, Faysal GokCS02 Multi-System Allergy (MSA) of cystic fibrosis: our institutional experienceJessica Hawley, Christopher O’Brien, Matthew Thomas, Malcolm Brodlie, Louise MichaelisCS03 Cold urticaria in pediatric age: an invisible cause for severe reactionsInês Mota, Ângela Gaspar, Susana Piedade, Graça Sampaio, José Geraldo Dias, Miguel Paiva, Mário Morais-AlmeidaCS04 Angioedema with C1 inhibitor deficiency in a girl: a challenge diagnosisCristina Madureira, Tânia Lopes, Susana Lopes, Filipa Almeida, Alexandra Sequeira, Fernanda Carvalho, José OliveiraCS05 A child with unusual multiple organ allergy disease: what is the primer?Fabienne Gay-CrosierCS06 A case of uncontrolled asthma in a 6-year-old patientIoana-Valentina Nenciu, Andreia Florina Nita, Alexandru Ulmeanu, Dumitru Oraseanu, Carmen ZapucioiuORAL ABSTRACT SESSION 1: Food allergy (OP01–OP06)OP01 Food protein-induced enterocolitis syndrome: oral food challenge outcomes for tolerance evaluation in a Pediatric HospitalAdrianna Machinena, Olga Domínguez Sánchez, Montserrat Alvaro Lozano, Rosa Jimenez Feijoo, Jaime Lozano Blasco, Mònica Piquer Gibert, Mª Teresa Giner Muñoz, Marcia Dias da Costa, Ana Maria Plaza MartínOP02 Characteristics of infants with food protein-induced enterocolitis syndrome and allergic proctocolitisEbru Arik Yilmaz, Özlem Cavkaytar, Betul Buyuktiryaki, Ozge Soyer, Cansin SackesenOP03 The clinical and immunological outcomes after consumption of baked egg by 1–5 year old egg allergic children: results of a randomised controlled trialMerrynNetting, Adaweyah El-Merhibi, Michael Gold, PatrickQuinn, IrmeliPenttila, Maria MakridesOP04 Oral immunotherapy for treatment of egg allergy using low allergenic, hydrolysed eggStavroula Giavi, Antonella Muraro, Roger Lauener, Annick Mercenier, Eugen Bersuch, Isabella M. Montagner, Maria Passioti, Nicolò Celegato, Selina Summermatter, Sophie Nutten, Tristan Bourdeau, Yvonne M. Vissers, Nikolaos G. PapadopoulosOP05 Chemical modification of a peanut extract results in an increased safety profile while maintaining efficacyHanneke van der Kleij, Hans Warmenhoven, Ronald van Ree, Raymond Pieters, Dirk Jan Opstelten, Hans van Schijndel, Joost SmitOP06 Administration of the yellow fever vaccine in egg allergic childrenRoisin Fitzsimons, Victoria Timms, George Du ToitORAL ABSTRACT SESSION 2: Asthma (OP07–OP12)OP07 Previous exacerbation is the most important risk factor for future exacerbations in school-age children with asthmaS. Tolga Yavuz, Guven Kaya, Mustafa Gulec, Mehmet Saldir, Osman Sener, Faysal GokOP08 Comparative study of degree of severity and laboratory changes between asthmatic children using different acupuncture modalitiesNagwa Hassan, Hala Shaaban, Hazem El-Hariri, Ahmed Kamel Inas E. MahfouzOP09 The concentration of exhaled carbon monoxide in asthmatic children with different controlled stadiumPapp Gabor, Biro Gabor, Kovacs CsabaOP10 ...

Background. Deficient ADAMTS13 activity (TS13:act <10%) induced by anti-ADAMTS13 autoantibodies (autoAbs) causes immune-mediated thrombotic thrombocytopenic purpura (iTTP). Recently we showed that an open ADAMTS13 conformation is characteristic for acute iTTP patients, while folded ADAMTS13 was found in 78% of iTTP patients in remission with an TS13:act >50%. However, also iTTP patients in remission with a persistent (<10%) or moderately restored (10-50%) TS13:act have been described, but their ADAMTS13 conformation is unknown. Intriguingly, the factor responsible for inducing open ADAMTS13 in iTTP patients remains elusive. Identifying the cause of open ADAMTS13 in iTTP will help better understand the pathophysiology of iTTP and could help appreciate the prognosis and better manage the prevention of subsequent relapses. Aim. Determine ADAMTS13 conformation in plasma of iTTP patients during acute TTP and remission when TS13:act is <10%, moderately restored (10-50%) or >50% and investigate if anti-ADAMTS13 autoAbs induce conformational changes in ADAMTS13. Methods. TS13:act was determined in 120 iTTP plasma samples from 4 different centers (Marseille, Milan, Budapest, Utrecht). Samples were categorized according to the presence of clinical symptoms (acute versusremission) and their TS13:act in remission (>50%, 10-50%,<10%). Next, ADAMTS13 conformation was determined in all samples using our ADAMTS13 conformation ELISA. Additionally, presence of anti-ADAMTS13 autoAbs was also determined via ELISA. Finally, IgG's from 18 acute iTTP plasma samples were purified and added to folded ADAMTS13 from healthy donor (HD) plasma to test whether iTTP IgG's are able to induce the open HD ADAMTS13 conformation. Results. Of the 120 iTTP plasma samples, 46 were obtained during the acute (clinical signs present) and 74 during the remission phase (clinical signs absent). Further subdividing remission samples showed that TS13:act was >50% in 41, 10-50% in 14 and <10% in 19 samples. ADAMTS13 was open in 98% (45/46) of the acute samples and folded in 71% (29/41) of the remission samples with TS13:act >50%, confirming our previous results. Interestingly, ADAMTS13 was open in 93% and 89% of remission samples with TS13:act 10-50% and <10%, respectively (chi square, P<0.0001). Since anti-ADAMTS13 autoAbs influence TS13:act in iTTP patients, we next could demonstrate that open ADAMTS13 conformation was linked with presence of anti-ADAMTS13 autoAbs (chi square, P<0.0001) suggesting that anti-ADAMTS13 autoAbs could be a factor able to induce an open ADAMTS13 conformation in iTTP patients. To further test this hypothesis, we purified IgG's from 18 acute iTTP plasma's with open ADAMTS13 and added them to HD plasma containing closed ADAMTS13, where 14 of the 18 patient IgG pools (78%) did induce the open conformation in HD ADAMTS13, indicating that patient anti-ADAMTS13 autoAbs indeed can induce conformational changes in ADAMTS13. Conclusion. We show that ADAMTS13 is not only in the open conformation in iTTP patient plasma during the acute phase but also in remission when TS13:act is <10% or 10-50%. Hence, the presence of open ADAMTS13 in those remission patients indicates that the underlying pathophysiology is still ongoing, emphasizing the need for a close monitoring of those patients. In addition, anti-ADAMTS13 autoAbs were identified as a factor responsible for the change in conformation in ADAMTS13 in iTTP. Disclosures Peyvandi: Octapharma US: Honoraria; Kedrion: Consultancy; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Sobi: Speakers Bureau; Shire: Speakers Bureau; Novo Nordisk: Speakers Bureau; Octapharma US: Honoraria; Octapharma US: Honoraria; Novo Nordisk: Speakers Bureau; Kedrion: Consultancy; Roche: Speakers Bureau; Novo Nordisk: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Roche: Speakers Bureau; Roche: Speakers Bureau; Shire: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Octapharma US: Honoraria; Roche: Speakers Bureau; Roche: Speakers Bureau; Grifols: Speakers Bureau; Sobi: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Sobi: Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Novo Nordisk: Speakers Bureau; Shire: Speakers Bureau; Kedrion: Consultancy; Novo Nordisk: Speakers Bureau; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Kedrion: Consultancy; Kedrion: Consultancy; Grifols: Speakers Bureau; Octapharma US: Honoraria. Coppo:Ablynx: Consultancy. Veyradier:LFB: Other: Investigator. Vanhoorelbeke:Shire: Consultancy; Ablynx: Consultancy.

Background ADAMTS13 circulates in a folded conformation, which is mediated by interactions between the C-terminal CUB domains and its central Spacer domain. Binding of ADAMTS13 to the VWF D4-CK domains disrupts the CUB-Spacer interaction, inducing a structural change that extends ADAMTS13 into an open conformation that enhances catalytic efficiency ~2-fold. This mechanism supports a model in which ADAMTS13 unfolding induces exposure of an exosite in the Spacer domain that interacts with the VWF A2 domain, increasing the affinity between the two molecules, and, therefore, the rate of proteolysis. The D4-CK-mediated conformational activation of ADAMTS13 can be mimicked in vitro with the use of antibodies that disrupt the CUB-Spacer interaction, such as the previously published anti-CUB antibody, Ab17G2. We recently generated a novel, activating antibody against the Spacer domain (Ab3E4). Aim To characterize the mechanism by which the Ab17G2 and Ab3E4 enhance the catalytic efficiency of ADAMTS13. Methods The effects of the Ab17G2 and Ab3E4 on the activity of ADAMTS13 were studied using FRETS-VWF73. The effects of the Ab17G2 and Ab3E4 on the kinetics of VWF96 (VWF G1573-R1668) proteolysis were characterized using an in-house assay. ELISA was used to investigate conformational changes in ADAMTS13 induced by the Ab17G2 and Ab3E4. Results Both Ab17G2 and Ab3E4 enhanced FRETS-VWF73 proteolysis by ~1.7-fold. This result was reproduced using the VWF96 substrate; the Ab17G2 and Ab3E4 enhanced the catalytic efficiency (kcat/Km) of ADAMTS13 by ~1.8- and ~2.0-fold, respectively. The activation was dependent on the conformational extension of ADAMTS13, since the antibodies could not enhance the activity of an ADAMTS13 variant that lacks the TSP2-CUB2 domains (MDTCS). Surprisingly, ADAMTS13 activation was not mediated through exposure of the Spacer or Cys-rich domain exosites as previously proposed, as the Ab17G2 and Ab3E4 efficiently enhanced proteolysis of VWF96 variants in which the Spacer/Cys-rich exosite binding sites were disrupted. Kinetic analysis of VWF96 proteolysis showed that the Ab17G2- and Ab3E4-induced activation of ADAMTS13 is primarily manifest through a ~1.5- to ~2-fold increase in enzyme turnover (kcat). Thus, contrary to the current model, this suggests that the conformational extension of ADAMTS13 influences the functionality of the active site, and not substrate binding affinity (Km). Incubating ADAMTS13 with either Ab17G2 or Ab3E4 exposed a cryptic epitope in the metalloprotease domain that was specifically detected by ELISA, further corroborating that the antibodies induce a conformational change in ADAMTS13 affecting the M domain. Conclusion Antibodies can be used as tools for understanding the structure/function of enzymes. Using activating antibodies against the Spacer and CUB1 domains of ADAMTS13, we show for the first time that the activation of ADAMTS13 following its unfolding is not a result of exposure of a functional exosite in Spacer/Cys-rich domain that increases affinity to VWF. Rather, our data are consistent with an allosteric activation mechanism upon the metalloprotease domain. We propose that ADAMTS13 unfolding causes a conformational change in the active site that further activates the enzyme. We are currently investigating whether the D4-CK-induced enhancement of ADAMTS13 proteolytic activity is also mediated by conformational changes in the active site. Disclosures Vanhoorelbeke: Ablynx: Consultancy; Shire: Consultancy.