The Influence of Variable-Heavy (V_H) Chain Families on IgG₂,₃,₄on FcγRs and Antibody Superantigens Protein G and L Binding using Biolayer Interferometry

Abstract

Background: As the most abundant immunoglobulin in blood and the most common human isotype used for therapeutic monoclonal antibodies, the engagement and subsequent activation of its Fc receptors by IgGs are crucial for antibody function. While generally assumed to be relatively constant within subtypes, recent studies have shown the antibody variable regions to exert distal effects of modulating antibody receptor interactions on many antibody isotypes. Such effects are also expected for IgG and its subtypes with the in-depth understanding of these V region effects highly relevant for engineering antibodies, antibody purifications, and understanding to how robust the microbial immune evasion proteins are. Methods: In this study, we created a panel of IgG2/IgG3/IgG4 antibodies by changing the VH family (VH1 to 7) frameworks while retaining the complementarity determining regions of Pertuzumab and measured the interaction of the IgGs with FcgRIa, FcgRIIaH167, FcgRIIaR167, FcgRIIb/c, FcgRIIIaF176, FcgRIIIaV176, FcgRIIIbNA1, and FcgRIIIbNA2 receptors alongside antibody superantigens proteins L and G using biolayer interferometry. Results: The library of 21 IgGs demonstrated that the VH frameworks influenced receptor binding sites on the constant region of the subtypes significantly, providing non-canonical interactions and non-interactions. However, there was minimal influence on the binding of bacterial B-cell superantigens Proteins L and G on the IgGs, showing their robustness against V region effects. Conclusions: These results demonstrate the importance of the V-regions during humanization of therapeutic antibodies that can confer or diminish FcR dependent immune responses, while remaining both suitable and susceptible to the binding by bacterial antibody superantigens in antibody purification and be present with normal flora.