A comparison of the binding sites of antibodies and single-domain antibodies

Abstract

Antibodies are the largest class of biotherapeutics. However, in recent years, single-domain antibodies have gained traction due to their smaller size and comparable binding affinity. Antibodies (Abs) and single-domain antibodies (sdAbs) differ in the structures of their binding sites: most significantly, single-domain antibodies lack a light chain and so have just three CDR loops. Given this inherent structural difference, it is important to understand whether Abs and sdAbs are distinguishable in how they engage a binding partner and thus, whether they are suited to different types of epitopes. In this study, we use non-redundant sequence and structural datasets to compare the paratopes, epitopes and antigen interactions of Abs and sdAbs. We demonstrate that even though sdAbs have smaller paratopes, they target epitopes of equal size to those targeted by Abs. To achieve this, the paratopes of sdAbs contribute more interactions per residue than the paratopes of Abs. Additionally, we find that conserved framework residues are of increased importance in the paratopes of sdAbs, suggesting that they include non-specific interactions to achieve comparable affinity. Furthermore, the epitopes of sdAbs are only marginally less accessible than those of Abs: we posit that this may be explained by differences in the orientation and compaction of sdAb and Ab CDR-H3 loops. Overall, our results have important implications for the engineering and humanization of sdAbs, as well as the selection of the best modality for targeting a particular epitope.

Applications and challenges in designing VHH-based bispecific antibodies: leveraging machine learning solutions

“… 7 , 8 A comparison of CDR3 lengths in human VH sequences and VHH sequences within the Observed Antibody Space (OAS) 9 revealed that, on average, VHH domains have CDR3 loops that are 1.4 residues longer. 10 Due to the long CDR3s, VHH domains can exist with structural conformations beyond those seen in conventional antibodies. 11 Indeed, camel and llama VHHs may possess an additional disulfide bond between CDR1 and CDR3 or CDR2 and CDR3, respectively, thereby increasing conformational stability of these long CDR3 loop structures.…”

Prospects for the computational humanization of antibodies and nanobodies

“…In addition to their distinct hallmark residues, nanobodies more often incorporate framework residues into their paratopes (the antigen binding site) ( 12 , 14 , 79 , 80 ). This is critical to consider in developing computational methods based on protocols such as CDR grafting, where it may be assumed that only the CDRs contribute to antigen-binding.…”

“…Their smaller size facilitates expression, improves tumor penetration, and increases solubility, while maintaining comparable binding affinity to conventional antibodies (8)(9)(10)(11)(12)(13). However, structural differences between antibodies and nanobodies affect how they interact with their antigens (14). Therefore, it is likely that many humanization protocols designed for conventional antibodies, particularly computational tools, are not immediately applicable to nanobodies.…”

PLAbDab-nano: a database of camelid and shark nanobodies from patents and literature

“…Within the VHH structure, the binding site (paratope) is concentrated primarily in three hypervariable loops, known as the complementarity-determining regions (CDRs). The CDR3, which is longer in nanobodies compared to conventional antibodies (7 -12), contributes most to the binding site (12). Framework residues also play a significant role in nanobody binding, being incorporated into the paratope more frequently than is observed for conventional antibodies (12)(13)(14).…”

“…The CDR3, which is longer in nanobodies compared to conventional antibodies (7 -12), contributes most to the binding site (12). Framework residues also play a significant role in nanobody binding, being incorporated into the paratope more frequently than is observed for conventional antibodies (12)(13)(14). VNARs possess a structurally analogous scaffold to VHHs, but differ in that they feature only two CDR loops (CDR1 and CDR3), alongside two additional hypervariable regions (HV2 and HV4) (15,16).…”