Affinity‐stability trade‐off mechanism of residue 35 in framework region 2 of <scp>V<sub>H</sub>H</scp> antibodies with β‐hairpin <scp>CDR3</scp>
Single-domain V<sub>H</sub>H antibodies are promising therapeutic and diagnostic tools. The third complementarity-determining region (CDR3) is usually the most critical region for antigen recognition by V<sub>H</sub>H antibodies. When CDR3 adopts a short and extended β-hairpin conformation, framework region 2 (FR2) often interacts directly with the antigen. However, the importance of these interactions in antigen recognition remains unclear. In this research, we investigated the role of FR2 residues in V<sub>H</sub>H antibodies with β-hairpin CDR3s. We found that several FR2 residues, particularly at positions 35 and 37, are critical for high-affinity antigen binding. Notably, a trade-off was observed: introducing a charged residue at position 35 enhanced binding affinity but reduced thermal stability. These findings provide insights into optimizing FR2 in single-domain antibodies to improve their functionality for diagnostic and therapeutic applications.
Authors
- Makoto Nakakido (ORCID: https://orcid.org/0000-0003-0328-9914)
- Ryo Matsunaga (ORCID: https://orcid.org/0000-0001-7702-9176)
- Satoru Nagatoishi (ORCID: https://orcid.org/0000-0002-0794-3963)
- Kouhei Tsumoto (ORCID: https://orcid.org/0000-0001-7643-5164)
- Daisuke Kuroda (ORCID: https://orcid.org/0000-0003-2390-4785)
- Koichi Yamamoto
Institutions
- National Institute of Infectious Diseases (JP)
- The University of Tokyo (JP)
Publication Details
- Journal
- Protein Science
- Published
- 2025-03-18
- DOI
- https://doi.org/10.1002/pro.70095
- Citations
- 5
- Primary Topic
- Monoclonal and Polyclonal Antibodies Research
- Type
- article
- Field-Weighted Citation Impact
- 6.80
Funders
- Japan Agency for Medical Research and Development
- Japan Society for the Promotion of Science