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.

Role of the IgE variable heavy chain in FcεRIα and superantigen binding in allergy and immunotherapy

“…Because we previously demonstrated allosteric communications between the V-and C-regions on IgAs 30 and such an effect was also reported on IgGs by many other research groups, [26][27][28][29] we next sought to study whether such allosteric effects explained the differences in FcεRIa binding present in our pertuzumab IgE variants. To do this, we computationally modeled the full-length structures of all the pertuzumab IgE variants and quantified the allosteric communications between the V-and C-regions by using the AlloSigMA server (Fig 3, B).…”

“…Pertuzumab and trastuzumab VH1-VH7 IgE variants were produced by means of cotransfection, as previously performed. [30][31][32]36 Affinity purification was carried out with the Protein L column, followed by size exclusion chromatography fractionation with the Superdex 200-pg 16/600 column (GE Healthcare) to extract monomeric fractions, which were further concentrated by using a 100-kDa Amicon Ultra protein concentrator (catalog no. ACS510024; Merck-Millipore).…”

“…Measurements of pertuzumab IgE variants to Her2 were carried out by loading Her2 on an Ni-NTA biosensor, followed by IgE binding, as previously described. [30][31][32]36,37 Measurements toward FcεRIa were performed in the same manner, as previously described. 32 Measurements of association and dissociation rates of omalizumab to pertuzumab variants were performed with omalizumab preloaded to Anti-hIgG-Fc Capture biosensors (catalog no.…”

“…As a possible mechanism for superantigen activation of FcεRIa-bound IgE, recent investigations on antibodies have demonstrated that antigen binding at antibody V-regions can elicit structural changes that facilitate cell signaling 25 and that even VH families can elicit distal effects on antibody constant regions (C-regions) for IgGs [26][27][28][29] and IgA. 30 This allosteric effect was also shown when the changing of antibody C-regions affected antigen binding. 31 To investigate the effects of different VH family FWRs on FcεRIa and superantigen binding, as well as for anti-IgE therapeutics, we investigated 7 VH families using pertuzumab antibody as a model for grafting onto the IgE C-region 31 and measured interactions with FcεRIa, superantigen spA, anti-IgE (omalizumab), and Her2 using biolayer interferometry.…”

The Determination of HIV-1 RT Mutation Rate, Its Possible Allosteric Effects, and Its Implications on Drug Resistance

“…Following experimental validation via recombinant methods to establish the level and type of effect (detrimental or augmented catalytic activity), the site can then be targeted for intervention, especially if they are able to elicit drastic protein structural changes to affect functionality. One such example is in IgA1 and IgA2, which had varying allosteric communications due to different intermediate protein regions [84].…”