N-glycan shielded CUB domains of ADAMTS13 prevent binding of C-terminal antibodies in patients with immune-mediated TTP.

In immune-mediated thrombotic thrombocytopenic purpura (iTTP), patients develop antibodies against ADAMTS13. Most patients exhibit inhibitory antispacer antibodies. Noninhibitory antibodies binding to the carboxy-terminal CUB domains have been suggested to enhance the clearance of ADAMTS13 in iTTP. Furthermore, anti-CUB antibodies induce an open conformation, which has been shown to be an important biomarker for disease severity and relapse risk. We explored whether the introduction of N-glycans in the CUB domains of ADAMTS13 can reduce the binding of pathogenic anti-CUB autoantibodies. The binding of a panel of anti-CUB monoclonal antibodies derived from patients with iTTP, to newly designed N-glycan modified ADAMTS13 CUB domain variants, was assessed by enzyme-linked immunosorbent assay. In addition, a subset of these variants was screened against plasma samples from patients with iTTP, which primarily contain antibodies directed toward the carboxy-terminal domains of ADAMTS13. Introduction of N-glycans at amino acid positions of 1251, 1255, and 1368 in the CUB1/2 domains of ADAMTS13 can effectively reduce the binding of 6 out of 7 anti-CUB antibodies derived from patients with iTTP. Reduced binding to CUB N-glycan variants was observed in 8 out of 9 patient samples. Binding was decreased from 81% to 47% for NGLY3+CUB-NGLY and 60% to 28% for 5ALA+CUB-NGLY variants. Collectively our findings show that the introduction of N-glycans within the CUB domain of ADAMTS13 can prevent the binding of anti-CUB antibodies in patients with iTTP. Based on these findings, we propose that CUB-NGLY modified ADAMTS13 variants can be used for improved treatment of patients with iTTP.