Affinity Improvement of a Therapeutic Antibody by Structure-Based Computational Design: Generation of Electrostatic Interactions in the Transition State Stabilizes the Antibody-Antigen Complex

Abstract

The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.

Advances in Antibody Design

“…By combining successful mutations, the affinity for type 4 was enhanced by more than two orders of magnitude without reducing affinity for other serotypes. Most of the successful mutations were charged or polar, and located at the periphery of the binding interface, which is generally consistent with findings from other studies (22–24, 26). This exciting study highlights the potential of using design methods to achieve unique binding specificities that are difficult to achieve using conventional discovery methods.…”

“…More generally, these findings are consistent with other studies revealing that the likelihood of identifying beneficial mutations is higher outside the initial binding interface (24–27), likely due to the reduced risk of disrupting existing antibody–antigen interactions. This (22) and related work (24–26, 28, 29) have demonstrated the potential of using existing methods for calculating electrostatic interactions to identify mutations that improve antibody affinity.…”

Biophysical characterization and single‐chain Fv construction of a neutralizing antibody to measles virus

“…The thermodynamic analysis suggested that the relatively moderate DH and unfavorable DS may account for the moderate binding affinity. The thermodynamic parameters were compared with those of antibodies [30][31][32][33][34] and immune cell receptors [35][36][37] ( Table 4). Among antibodies, the 2F4-scFv exhibited relatively smaller DG, but similar level of DG with those for binding of cell surface receptors to ligands (5.9-7.2 kcalÁmol À1 ) ( Table 4).…”

Sphinx: merging knowledge-based andab initioapproaches to improve protein loop prediction

“…Knowledge of H3 structures is therefore extremely useful, enabling predictions to be made about antibody binding properties and hence their suitability as drugs (Clark et al , 2006; Diskin et al , 2011; Kiyoshi et al , 2014; Kuroda et al , 2012; Krawczyk et al , 2013; Lewis et al , 2014; Lippow et al , 2007; Thakkar et al , 2014). …”