Replacement of non-canonical interloop disulfide bond of alpaca VHH by hydrophobic amino acids

Non-canonical disulfide bonds are a hallmark of the VHH domain of heavy-chain antibodies, with predominant bonds between CDRs 1 and 3 and between framework FR2 and CDR3. The former interloop disulfide bond is advantageous for stability and antigen-binding affinity, albeit not indispensable. Importantly, mutations in the cysteine of the interloop disulfide bond of CDRs 1 and 3 can sometimes preserve antigen-binding activity. In contrast, the functional role of the interloop disulfide bond between FR2 and CDR3 remains largely understudied. In this study, we investigated the replacement of the disulfide bond between FR2 and CDR3 with cysteine mutations in aliphatic amino acids (alanine, valine, and isoleucine). These results indicate that this interloop disulfide bond contributes to the structural stabilization of VHH but is not required for antigen binding, consistent with the findings for the disulfide bond between CDRs 1 and 3. Furthermore, the disulfide bond between FR2 and CDR3 was not critical for maintaining reversibility following heat-induced unfolding. Given the prevalence of FR2-CDR3 interloop disulfide bonds in VHHs from llamas and alpacas, these findings provide valuable insights into VHH engineering and applications.