Nanobodies versus canonical antibodies: an updated comparison of their binding modes

Heavy-chain–only antibodies, produced by the adaptive immune systems of camelids and cartilaginous fish, complement canonical antibodies that contain variable domains from both heavy and light chains. We refine previous studies by providing a detailed analysis of the binding modes of VHHs versus canonical antibodies, using a dataset with a ∼ 20-fold increase in the number of cases. We show that VHHs exhibit a larger buried surface area despite relying on a single variable domain than double domain antibodies. This property can be attributed to contributions from both framework regions and CDR3. We further demonstrate that the binding modes of VHHs, characterized by the number of FR and CDR regions contacting the antigen, are more diverse than previously reported. In addition, we find that VHH and canonical antibody interfaces display similar solvation properties, although VHH interfaces are more tightly packed.

Finally, we discuss the thermodynamic and kinetic implications of these findings for the design of high-affinity VHHs, an issue of particular importance in protein engineering and design.