Oncode Institute and Department of Cell Biology, Center for Molecular Medicine, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
Crystal and Structural Chemistry, Department of Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, 3584 CH, The Netherlands.
Nat Commun. 2019 Jan 21;10(1):365. doi: 10.1038/s41467-018-08172-z.
Wnt-induced β-catenin-mediated transcription is a driving force for stem cell self-renewal during adult tissue homeostasis. Enhanced Wnt receptor expression due to mutational inactivation of the ubiquitin ligases RNF43/ZNRF3 recently emerged as a leading cause for cancer development. Consequently, targeting canonical Wnt receptors such as LRP5/6 holds great promise for treatment of such cancer subsets. Here, we employ CIS display technology to identify single-domain antibody fragments (VHH) that bind the LRP6 P3E3P4E4 region with nanomolar affinity and strongly inhibit Wnt3/3a-induced β-catenin-mediated transcription in cells, while leaving Wnt1 responses unaffected. Structural analysis reveal that individual VHHs variably employ divergent antigen-binding regions to bind a similar surface in the third β-propeller of LRP5/6, sterically interfering with Wnt3/3a binding. Importantly, anti-LRP5/6 VHHs block the growth of Wnt-hypersensitive Rnf43/Znrf3-mutant intestinal organoids through stem cell exhaustion and collective terminal differentiation. Thus, VHH-mediated targeting of LRP5/6 provides a promising differentiation-inducing strategy for treatment of Wnt-hypersensitive tumors.
Wnt 诱导的 β-连环蛋白介导的转录是成体组织稳态中干细胞自我更新的驱动力。由于泛素连接酶 RNF43/ZNRF3 的突变失活导致 Wnt 受体表达增强，最近成为癌症发展的主要原因。因此，靶向经典 Wnt 受体，如 LRP5/6，为治疗此类癌症亚群提供了巨大的希望。在这里，我们采用 CIS 展示技术来鉴定与 LRP6 P3E3P4E4 区域具有纳摩尔亲和力的单域抗体片段（VHH），并强烈抑制细胞中 Wnt3/3a 诱导的 β-连环蛋白介导的转录，同时不影响 Wnt1 反应。结构分析表明，单个 VHH 可变地使用不同的抗原结合区来结合 LRP5/6 第三β-螺旋桨上相似的表面，在空间上干扰 Wnt3/3a 的结合。重要的是，抗 LRP5/6 VHH 通过干细胞耗竭和集体终末分化阻断 Wnt 超敏性 Rnf43/Znrf3 突变肠道类器官的生长。因此，VHH 介导的 LRP5/6 靶向为治疗 Wnt 超敏性肿瘤提供了一种有前途的诱导分化策略。
