Effect of humanizing mutations on the stability of the llama single-domain variable region

Soler, Miguel A. (author), Medagli, Barbara (author), Wang, Jiewen (author), Oloketuyi, Sandra (author), Bajc, Gregor (author), Huang, He (author), Fortuna, Sara (author), De Marco, Ario (author)

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.

• Language: English
• Keywords: nanobody framework, modeling, nanobody humanization, CDR grafting
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2021
• Number of pages: 13 str.
• Numbering: Vol. 11, iss. 2, art. 163
• PID: 20.500.12556/RUL-134891
• UDC: 577
• ISSN on article: 2218-273X
• DOI: 10.3390/biom11020163
• COBISS.SI-ID: 48829955
• Publication date in RUL: 10.02.2022

Record is a part of a journal

• Title: Biomolecules
• Shortened title: Biomolecules
• Publisher: MDPI
• ISSN: 2218-273X
• COBISS.SI-ID: 519952921

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.
• Licensing start date: 01.02.2021