De Novo Computational Design of VHH Nanobodies Against LGR5

VHH discovery traditionally relies on animal immunization or large-scale library screening, methods that are slow, costly, and often ineffective for challenging targets such as GPCRs. We present a fully de novo computational pipeline for epitope-directed VHH design, integrating generative backbone modeling, deep learning–based sequence optimization, and iterative experimental feedback. Using LGR5 as a model, we progressed from in silico design to functional binders without structural templates. Across three design–test–learn cycles, millions of candidates were reduced to epitope-specific binders with nanomolar affinity and high thermal stability (melting temperature [Tm] > 65 °C). Cryogenic electron microscopy (cryo-EM) confirmed atomic-level agreement (RMSD ≈ 2.2 Å). This structure-validated approach accelerates timelines, reduces cost, and is broadly applicable to GPCRs and other membrane proteins, enabling “on-demand” therapeutic antibody generation.