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De Novo Computational Design of VHH Nanobodies Against LGR5

Xu, C.; Li, Y.; Nguyen, T.; Zhou, Y.; Cong, L.; Lee, T.-H.; Lang, Y.; Shek, R.; Yi, L.; Greisen, P.

2025-12-05 bioengineering
10.64898/2025.12.02.691880 bioRxiv
Show abstract

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 {degrees}C). Cryogenic electron microscopy (cryo-EM) confirmed atomic-level agreement (RMSD {approx} 2.2 [A]). This structure-validated approach accelerates timelines, reduces cost, and is broadly applicable to GPCRs and other membrane proteins, enabling "on-demand" therapeutic antibody generation.

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