Postmus Tim, Schilder Nelly, Ferreira de Santana Juliana, Langerhorst Pieter, Kaijen Paul, Coppo Paul, Joly Bérangère S, Veyradier Agnès, Vanhoorelbeke Karen, Voorberg Jan
Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands.
Service d'Hématologie biologique, Hôpital Lariboisière, Assistance Publique-Hôpitaux de Paris Nord, Université Paris Cité, Paris, France.
Blood Adv. 2025 Apr 8;9(7):1728-1737. doi: 10.1182/bloodadvances.2024014298.
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.
在免疫介导的血栓性血小板减少性紫癜（iTTP）中，患者会产生针对ADAMTS13的抗体。大多数患者表现出抑制性抗间隔区抗体。有人提出，与羧基末端CUB结构域结合的非抑制性抗体可增强iTTP中ADAMTS13的清除。此外，抗CUB抗体可诱导一种开放构象，该构象已被证明是疾病严重程度和复发风险的重要生物标志物。我们探讨了在ADAMTS13的CUB结构域中引入N-聚糖是否能减少致病性抗CUB自身抗体的结合。通过酶联免疫吸附测定评估了一组来自iTTP患者的抗CUB单克隆抗体与新设计的N-聚糖修饰的ADAMTS13 CUB结构域变体的结合情况。此外，针对这些变体的一个子集，对iTTP患者的血浆样本进行了筛选，这些样本主要含有针对ADAMTS13羧基末端结构域的抗体。在ADAMTS13的CUB1/2结构域的第1251、1255和1368位氨基酸位置引入N-聚糖可有效减少7种源自iTTP患者的抗CUB抗体中的6种的结合。在9个患者样本中的8个中观察到与CUB N-聚糖变体的结合减少。对于NGLY3 + CUB - NGLY变体，结合率从81%降至47%，对于5ALA + CUB - NGLY变体，结合率从60%降至28%。我们的研究结果共同表明，在ADAMTS13的CUB结构域内引入N-聚糖可防止iTTP患者中抗CUB抗体的结合。基于这些发现，我们提出CUB - NGLY修饰的ADAMTS13变体可用于改善iTTP患者的治疗。
