Humanising a nanobody isn't just a sequence identity exercise. Get it wrong and you can quietly wreck the very properties that made the molecule worth developing in the first place. Cambridge-based antibody discovery CRO Isogenica has published a case study showing how a structure-guided, iterative approach to VHH humanisation can substantially increase human-likeness while holding onto binding affinity, expression yield, and thermal stability.
Working from an alpaca-derived VHH, Isogenica's team combined bioinformatic germline comparison with expert knowledge of nanobody architecture, accounting for the hallmark camelid residues and disulphide bonds that keep these small, single-domain antibodies stable, to design successive panels of humanised variants. Across two rounds of optimisation and 40+ constructs, most variants retained expression, monomeric purity, and antigen binding comparable to the parental molecule, with most second-generation variants exceeding 80% human-likeness and top candidates combining high humanness scores (up to 83%) with binding within two-to-three-fold of the original.
The result: a practical demonstration that rational, iterative humanisation can deliver clinic-ready human-likeness without starting the discovery process over, and a useful reference point for anyone weighing humanisation risk against a promising camelid lead.
Read the full case study, including the mutation panels, biophysical data and kinetics: Retaining binding characteristics and strong biophysics during VHH humanisation
