ArticleProtein science : a publication of the Protein Society2024
On the humanization of VHHs: Prospective case studies, experimental and computational characterization of structural determinants for functionality.
Article in Protein science : a publication of the Protein Society, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed.
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- A side-by-side biophysical comparison of five single-domain antibody scaffolds used for synthetic display libraries.The Journal of biological chemistry · 2026Article
- Disulphide and sequence-encoded conformational priors guide nanobody structure prediction.bioRxiv : the preprint server for biology · 2026Article
- Mechanisms and applications of camelid variable heavy-chain nanobodies against bacterial and parasitic protozoal pathogens.Frontiers in immunology · 2026Review
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- J3ExoA: A Novel Anti-HIV Immunotoxin Fusion of Anti-Gp120 J3VHH and PE38 Fragment ofPharmaceuticals (Basel, Switzerland) · 2025Article
- Computational Prediction of Single-Domain Immunoglobulin Aggregation Propensities Facilitates Discovery and Humanization of Recombinant Nanobodies.Antibodies (Basel, Switzerland) · 2025Article
- Optimization of synthetic human VProtein science : a publication of the Protein Society · 2025Article
- Design of nanobody targeting SARS-CoV-2 spike glycoprotein using CDR-grafting assisted by molecular simulation and machine learning.PLoS computational biology · 2025Article
- Beyond monoclonal antibodies: constraints and the case for alternative PD-1/PD-L1-targeting formats.Frontiers in immunology · 2025Review
- On the humanization of VHHs: Prospective case studies, experimental and computational characterization of structural determinants for functionality.Protein science : a publication of the Protein Society · 2024Article
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16 authors.
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Abstract
The humanization of camelid-derived variable domain heavy chain antibodies (VHHs) poses challenges including immunogenicity, stability, and potential reduction of affinity. Critical to this process are complementarity-determining regions (CDRs), Vernier and Hallmark residues, shaping the three-dimensional fold and influencing VHH structure and function. Additionally, the presence of non-canonical disulfide bonds further contributes to conformational stability and antigen binding. In this study, we systematically humanized two camelid-derived VHHs targeting the natural cytotoxicity receptor NKp30. Key structural positions in Vernier and Hallmark regions were exchanged with residues from the most similar human germline sequences. The resulting variants were characterized for binding affinities, yield, and purity. Structural binding modes were elucidated through crystal structure determination and AlphaFold2 predictions, providing insights into differences in binding affinity. Comparative structural and molecular dynamics characterizations of selected variants were performed to rationalize their functional properties and elucidate the role of specific sequence motifs in antigen binding. Furthermore, systematic analyses of next-generation sequencing (NGS) and Protein Data Bank (PDB) data was conducted, shedding light on the functional significance of Hallmark motifs and non-canonical disulfide bonds in VHHs in general. Overall, this study provides valuable insights into the structural determinants governing the functional properties of VHHs, offering a roadmap for their rational design, humanization, and optimization for therapeutic applications.
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