ArticleProtein science : a publication of the Protein Society2022
Molecular basis for thermal stability and affinity in a VHH: Contribution of the framework region and its influence in the conformation of the CDR3.
Article in Protein science : a publication of the Protein Society, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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21 citing papers in PubMed.
- Multidimensional maturation of antibody variable domains with machine-learning assistance.mAbs · 2026Article
- Combining AI Structure Prediction and Integrative Modeling for Nanobody-Antigen Complexes.Journal of chemical information and modeling · 2026Article
- Camelid VHHs as a Versatile Platform for Combating Viral Infections.Pharmaceuticals (Basel, Switzerland) · 2026Review
- Translational opportunities in aptamer and nanobody lateral flow assays within the WHO REASSURED framework.Sensors & diagnostics · 2026Review
- Efficient generation of epitope-targeted antibodies with Germinal.Nature biotechnology · 2026Article
- Characterising nanobody developability to improve therapeutic design using the Therapeutic Nanobody Profiler.Communications biology · 2026Article
- Mechanisms and applications of camelid variable heavy-chain nanobodies against bacterial and parasitic protozoal pathogens.Frontiers in immunology · 2026Review
- Article
- Development of a Chicken Immunoglobulin Heavy Chain Variable Region (VH) Single-Domain Antibody (sdAb) Against Calsequestrin (CSQ) and Its Application.Antibodies (Basel, Switzerland) · 2025Article
- Improving the solubility of single domain antibodies using VH-like hallmark residues.Protein science : a publication of the Protein Society · 2025Article
- Neutralization of the Pandemic Influenza A/H1N1 Virus withAntibodies (Basel, Switzerland) · 2025Article
- Nanobody engineering: computational modelling and design for biomedical and therapeutic applications.FEBS open bio · 2025Review
- A Simple Analysis of the Second (Extra) Disulfide Bridge of VMolecules (Basel, Switzerland) · 2024Article
- Structure-guided in vitro evolution of nanobodies targeting new viral variants.PLoS pathogens · 2024Article
- Development of novel humanized VHH synthetic libraries based on physicochemical analyses.Scientific reports · 2024Article
- Isolation and characterization of Hc-targeting chimeric heavy chain antibodies neutralizing botulinum neurotoxin type B.Frontiers in immunology · 2024Article
- Structure and Dynamics Guiding Design of Antibody Therapeutics and Vaccines.Antibodies (Basel, Switzerland) · 2023Review
- VHH CDR-H3 conformation is determined by VH germline usage.Communications biology · 2023Article
- NbThermo: a new thermostability database for nanobodies.Database : the journal of biological databases and curation · 2023Article
- Molecular basis for thermal stability and affinity in a VHH: Contribution of the framework region and its influence in the conformation of the CDR3.Protein science : a publication of the Protein Society · 2022Article
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6 authors.
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Abstract
The camelid single domain antibody, referred to VHH or Nanobody, is considered a versatile tool for various biotechnological and clinical applications because of its favorable biophysical properties. To take advantage of these characteristics and for its application in biotechnology and therapy, research on VHH engineering is currently vigorously conducted. To humanize a camelid VHH, we performed complementarity determining region (CDR) grafting using a humanized VHH currently in clinical trials, and investigated the effects of these changes on the biophysical properties of the resulting VHH. The chimeric VHH exhibited a significant decrease in affinity and thermal stability and a large conformational change in the CDR3. To elucidate the molecular basis for these changes, we performed mutational analyses on the framework regions revealing the contribution of individual residues within the framework region. It is demonstrated that the mutations resulted in the loss of affinity and lower thermal stability, revealing the significance of bulky residues in the vicinity of the CDR3, and the importance of intramolecular interactions between the CDR3 and the framework-2 region. Subsequently, we performed back-mutational analyses on the chimeric VHH. Back-mutations resulted in an increase of the thermal stability and affinity. These data suggested that back-mutations restored the intramolecular interactions, and proper positioning and/or dynamics of the CDR3, resulting in the gain of thermal stability and affinity. These observations revealed the molecular contribution of the framework region on VHHs and further designability of the framework region of VHHs without modifying the CDRs.
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