biostudies-literatureSang ZNCI NIH HHSNIGMS NIH HHS418-429.e3https://www.ebi.ac.uk/biostudies/studies/S-EPMC11698024biostudies-literatureUnknown30(3)Nanobodies (Nbs) have emerged as a promising class of biologics. Despite having marked physicochemical properties, Nbs are derived from camelids and may require humanization to improve translational potentials. By systematically analyzing the sequence and structural properties of Nbs, we found substantial framework diversities and revealed the key differences between Nbs and human immunoglobulin G antibodies. We identified conserved residues that may contribute to enhanced solubility, structural stability, and antigen binding, providing insights into Nb humanization. Based on big data analysis, we developed "Llamanade," an open-source software to facilitate rational humanization of Nbs. Using sequence as input, Llamanade can rapidly extract sequence features, model structures, and optimizeStructure (London, England : 1993)Llamanade: An open-source computational pipeline for robust nanobody humanization.PMC11698024R01 GM139297R35 GM137905R21 CA261737Xiang YBahar ISang ZShi YfalseLlamanade: An open-source computational pipeline for robust nanobody humanization.Nanobodies (Nbs) have emerged as a promising class of biologics. Despite having marked physicochemical properties, Nbs are derived from camelids and may require humanization to improve translational potentials. By systematically analyzing the sequence and structural properties of Nbs, we found substantial framework diversities and revealed the key differences between Nbs and human immunoglobulin G antibodies. We identified conserved residues that may contribute to enhanced solubility, structural stability, and antigen binding, providing insights into Nb humanization. Based on big data analysis, we developed "Llamanade," an open-source software to facilitate rational humanization of Nbs. Using sequence as input, Llamanade can rapidly extract sequence features, model structures, and optimize2022-01-01T00:00:00Z2022 Mar2026-06-02T07:59:36.186Z2025-04-04T13:42:03.67ZS-EPMC116980243489547110.1016/j.str.2021.11.006