Physicochemical differences between camelid single-domain antibodies and mammalian antibodies.

Background/aim: In recent years, single-domain antibodies, also known as nanobodies, have emerged as an alternative to full immunoglobulin Gs (IgGs), due to their various advantages, including increased solubility, faster clearance, and cheaper production. Nanobodies are generally derived from the variable domain of the camelid heavy-chain-only immunoglobulin Gs (hcIgGs). Due to the high sequence homology between variable heavy chains of camelids (VHs) and humans (Vs), hcIgGs are ideal candidates for nanobody development. However, further examination is needed to understand the structural differences between Vs and VHs. This analysis is essential for nanobody engineering to mitigate potential immunogenicity, while preserving stability, functionality, and antigen specificity.

Materials And Methods: We obtained the V and VH sequences of various camelid and non-camelid mammalian antibodies from public databases and used multiple sequence alignment based on the Chothia numbering scheme. Aligned sequences were subjected to diverse analyses encompassing paratope length, binding prediction, motif, disulfide bridge, salt bridge profiling, and physicochemical characteristic distribution. Logistic Regression coupled with the Boruta - Random Forest algorithm facilitated the comprehensive examination of physicochemical properties.

Results: Our findings revealed longer, less variable paratope sequences in VHs, along with specific antigen binding residues with increased binding potential compared to Vs. Although the Vs showed more heterogeneous noncanonical disulfide bond patterns, the VHs had a higher number of noncanonical disulfide bridges. Intriguingly, a typical salt bridge between the 94th and 101st positions in the Vs had a very low encounter rate in the VHs. Surprisingly, we also identified notable differences in the physicochemical patterns of mostly conserved frameworks (FWs), especially the FW2 and FW3 regions, between Vs and VHs.

Conclusion: Our findings point to possible key sites in VHs as candidate residues for nanobody engineering efforts.