AI-guided design and ex vivo validation of nanobodies targeting aggregation motifs of intrinsically disordered protein tau

Abstract Intrinsically disordered proteins (IDPs) represent major yet challenging therapeutic targets in neurodegenerative disease due to their conformational heterogeneity and aggregation-prone behavior. Tau protein is a prototypical IDP that forms pathological aggregates in Alzheimer’s disease and related tauopathies. Despite extensive clinical efforts, tau-directed monoclonal antibodies have demonstrated limited efficacy. Concurrently, single-domain antibodies (nanobodies) have been gaining importance because of their small size and membrane penetrating capabilities. New design paradigms are therefore required for nanobodies to enable precise targeting of disease-relevant conformations. Here, we describe a biophysical modelling and AI-guided nanobody discovery targeting the VQIVYK motif of tau, which constitutes the structural core of neurofibrillary tangles in Alzheimer’s Disease. Biophysical modelling-based target analysis identified low-energy conformational states of VQIVYK. These conformational insights were used to guide AI-driven nanobody design of CDR3 loops. Starting from a nanobody scaffold, we generated 145 candidate nanobodies through systematic backbone sampling and neural network-guided sequence design, followed by multi-dimensional computational prioritization. Two candidates demonstrated robust binding to synthetic full tau protein in ELISA binding assays, achieving binding indices of 148.9% and 140%, relative to reference controls. Notably, one candidate also exhibited strong reactivity in post-mortem Alzheimer’s disease human brain tissue, with a binding index of 236.1%, exceeding that of the positive control (222.9%). Structural analysis indicates that our nanobodies’ engineered CDR3 engages VQIVYK through optimized aromatic and hydrophobic interactions. Together, these findings establish a proof-of-concept for biophysics-guided, AI-guided nanobody engineering against IDPs and identifies them as a promising lead for tau-targeted single domain antibody development. Competing Interest Statement The authors have declared no competing interest.