Physicochemical differences between camelid single-domain antibodies and mammalian antibodies

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 (V_HHs) and humans (V_Hs), hcIgGs are ideal candidates for nanobody development. However, further examination is needed to understand the structural differences between V_Hs and V_HHs. This analysis is essential for nanobody engineering to mitigate potential immunogenicity, while preserving stability, functionality, and antigen specificity.

We obtained the V_H and V_HH 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.

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

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