Mark Roest | CSO
Mithridate bv

Festival of Biologics Day 2 @ 14:50

Creating customized enzymes through molecular fusion of single domain antibodies with catalytic enzyme sequences.

Single-domain antibodies (sdAbs) have several advantages over conventional antibodies for therapeutic use. These include a smaller size, better tissue penetration, lower immunogenicity, easier and higher-yield production, and greater thermodynamic stability. Thrombotic Thrombocytopenic Purpura (TTP) is a life-threatening disorder characterized by impaired or deficient ADAMTS13 activity, resulting in the inability to cleave von Willebrand factor (VWF) and a subsequent loss of platelets and life threatening thrombosis over the whole body. While congenital TTP arises from genetic ADAMTS13 deficiency, the majority of TTP cases are acquired and mediated by autoantibodies targeting ADAMTS13, leading to its functional depletion. Recombinant ADAMTS13 (rADAMTS13) is an effective treatment for congenital TTP; however, it is ineffective in acquired TTP due to rapid clearance by patient autoantibodies.

This study describes the preclinical development of MIT-2001, a fusion protein of an anti-VWF single-domain antibody (sdAb) with the catalytic domain of ADAMTS13, designed to evade recognition by TTP patient autoantibodies. By replacing all non-catalytic domains of ADAMTS13 with a high-affinity anti-VWF sdAb, we generated a customized enzyme retaining proteolytic activity while minimizing immunogenic epitopes.

The anti-VWF sdAb selectively bound active VWF and inhibited platelet-VWF interactions in solution without impairing platelet adhesion to collagen-bound VWF. The replacement of multiple ADAMTS13 domains with a single sdAb significantly reduced autoantibody recognition, which was further diminished by introducing three point mutations in the catalytic domain. These modifications improved production yields while preserving proteolytic activity against VWF, comparable to full-length ADAMTS13.

The combination of low autoantibody recognition, high production yield, and stable proteolytic activity renders MIT-2001 a promising therapeutic candidate for the treatment of autoimmune-mediated TTP.

Mark Roest, CSO, Mithridate bv

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Mark Roest / Creating customized enzymes through molecular fusion of single domain antibodies with catalytic enzyme sequences.

Mark Roest, CSO,
Mithridate bv

Mark Roest is a biomedical scientist and entrepreneur with more than 25 years of experience in thrombosis, haemostasis, platelet biology, coagulation and fibrinolysis. He studied Biomedical Sciences at Leiden University and obtained his PhD from Utrecht University in 1999 with a thesis on genetic...

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