Immune Thrombotic Thrombocytopenic Purpura (iTTP) is a rare life-threatening bleeding disorder with an annual incidence of 2-6 cases per million per year. Patients with iTTP suffer from an accumulation of platelets on uncleaved von Willebrand Factor (VWF) multimers. Normally these multimers get cleaved by a protease called ADAMTS13. However, in iTTP, antibodies target ADAMTS13, inhibiting the protease and promoting its clearance from the bloodstream. ADAMTS13 consists of many different domains: the metallo-protease domain, the disintegrin domain, the cysteine-rich domain, the spacer domain, eight TSP domains, and two CUB domains. The antibodies in iTTP target multiple domains, but most patients (>95%) have antibodies directed against the spacer domain. Much research has been done against these antibodies, revealing a clear epitope consisting of five key residues (R568, F592, R660, Y661, Y665). A smaller group also has antibodies against the CUB domains of ADAMTS13 (20-40%), but no detailed epitope is available yet. Here we set out to learn more about these antibodies, where they bind to the CUB-domains, and if we could diminish antibody binding to both the spacer as well as the CUB-domains by modifying ADAMTS13. We found that anti-CUB domain antibodies bound both the CUB1 as well as the CUB2 domains. Interestingly, these antibodies specifically bound residues that are important for the regulation of ADAMTS13 activity. Once we had a clearly defined epitope for both the anti-spacer as well as the anti-CUB antibodies, we made modified at these locations by introduction of N-glycans. N-glycans are large sugar chains which can give additional functionality to proteins. Here, our approach was to attach this large sugar moiety to block the antibodies from binding. After doing this for both the spacer and the CUB domains of ADAMTS13, we found that reduced binding of patients antibodies to the modified ADAMTS13. The autoantibody resistant ADAMTS13 we designed can potentially be used for more effective treatment of acute TTP when compared to currently available treatment strategies.

THESIS 2026: Towards novel therapies of ITTP, defining and shielding of antibody epitopes on ADAMTS13 - Hedzer Tjerk (Tim) Postmus