Allosteric Effects between the Antibody Constant and Variable Regions: A Study of IgA Fc Mutations on Antigen Binding

Abstract

Therapeutic antibodies have shifted the paradigm of disease treatments from small molecules to biologics, especially in cancer therapy. Despite the increasing number of antibody candidates, much remains unknown about the antibody and how its various regions interact. Recent findings showed that the antibody constant region can govern localization effects that are useful in reducing side effects due to systemic circulation by the commonly used IgG isotypes. Given their localized mucosal effects, IgA antibodies are increasingly promising therapeutic biologics. While the antibody Fc effector cell activity has been a focus point, recent research showed that the Fc could also influence antigen binding, challenging the conventional idea of region-specific antibody functions. To investigate this, we analysed the IgA antibody constant region and its distal effects on the antigen binding regions using recombinant Pertuzumab IgA1 and IgA2 variants. We found that mutations in the C-region reduced Her2 binding experimentally, and computational structural analysis showed that allosteric communications were highly dependent on the antibody hinge, providing strong evidence that we should consider antibodies as whole proteins rather than a sum of functional regions.

Keywords: Pertuzumab; allosteric; antibody; biologics; constant region; isotype IgA; variable region.

Figure 1

Synergistic allosteric effects by the two IgA1 and IgA2 constant region mutations on the Her2-binding variable regions. (A) Binding kinetics analysis of the isotype variants IgA1 and IgA2 to Her2, using the antibodies at 200 nM to 25 nM to pre-loaded Her2 on NTA biosensors. The binding kinetics was measured using Blitz^®. All experiments were performed in triplicates independently. The binding kinetics values of the wild-type IgA1 and IgA2 shown were obtained from our previous work [25]. (B) Surface presentations of the quantified allosteric communications (presented by residual allosteric free energy change Δg_residue) demonstrate destabilizing effects on the Her2 binding region caused by the mutations (black dots) in both the mutant constant region variants. (C) The quantified allosteric effects shown were based on the event of Her2 bindings. In (B,C), the effects were estimated using the minimized structures of both the wild-type IgA subtypes for perturbations with respect to mutating or binding events.

Figure 2

Residue couplings and hinge-dependent domain proximity of the two variants IgA1 (in A) and IgA2 (in B). The sequence alignments of the mutation regions were performed using the EVcoupling server [39] and the coupling values were mapped back to the minimized structures of the variants. Distances were estimated between the centre of mass of the Fc region and that of the Her2-binding sites. Note that in the distance distributions of the IgA2 mutant (in B) showed the plotted values obtained from two replicates that successfully reached equilibrium in the given time scales.