Affinity-stability trade-off mechanism of residue 35 in framework region 2 of VHH antibodies with β-hairpin CDR3

Abstract

Single-domain VHH antibodies are promising therapeutic and diagnostic tools. The third complementarity-determining region (CDR3) is usually the most critical region for antigen recognition by VHH 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 VHH 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.