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Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region

CONCEPT Lab, Italian Institute of Technology (IIT), 16152 Genova, Italy
Department of Chemical and Pharmaceutical Sciences, University of Trieste, 34127 Trieste, Italy
Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, China
Lab of Environmental and Life Sciences, University of Nova Gorica, 5000 Rožna Dolina-Nova Gorica, Slovenia
Department of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia
Authors to whom correspondence should be addressed.
Academic Editors: Nick Devoogdt, Serge Muyldermans and Sophie Hernot
Biomolecules 2021, 11(2), 163;
Received: 21 December 2020 / Revised: 21 January 2021 / Accepted: 24 January 2021 / Published: 26 January 2021
(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)
In vivo clinical applications of nanobodies (VHHs) require molecules that induce minimal immunoresponse and therefore possess sequences as similar as possible to the human VH domain. Although the relative sequence variability in llama nanobodies has been used to identify scaffolds with partially humanized signature, the transformation of the Camelidae hallmarks in the framework2 still represents a major problem. We assessed a set of mutants in silico and experimentally to elucidate what is the contribution of single residues to the VHH stability and how their combinations affect the mutant nanobody stability. We described at molecular level how the interaction among residues belonging to different structural elements enabled a model llama nanobody (C8WT, isolated from a naïve library) to be functional and maintain its stability, despite the analysis of its primary sequence would classify it as aggregation-prone. Five chimeras formed by grafting CDRs isolated from different nanobodies into C8WT scaffold were successfully expressed as soluble proteins and both tested clones preserved their antigen binding specificity. We identified a nanobody with human hallmarks that seems suitable for humanizing selected camelid VHHs by grafting heterologous CDRs in its scaffold and could serve for the preparation of a synthetic library of human-like single domains. View Full-Text
Keywords: nanobody framework; modeling; nanobody humanization; CDR grafting nanobody framework; modeling; nanobody humanization; CDR grafting
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MDPI and ACS Style

Soler, M.A.; Medagli, B.; Wang, J.; Oloketuyi, S.; Bajc, G.; Huang, H.; Fortuna, S.; de Marco, A. Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region. Biomolecules 2021, 11, 163.

AMA Style

Soler MA, Medagli B, Wang J, Oloketuyi S, Bajc G, Huang H, Fortuna S, de Marco A. Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region. Biomolecules. 2021; 11(2):163.

Chicago/Turabian Style

Soler, Miguel A., Barbara Medagli, Jiewen Wang, Sandra Oloketuyi, Gregor Bajc, He Huang, Sara Fortuna, and Ario de Marco. 2021. "Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region" Biomolecules 11, no. 2: 163.

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