The Influence of Variable-Heavy (VH) 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 IgG 2 /IgG 3 /IgG 4 antibodies by changing the V H family (V H 1-7) frameworks while retaining the complementarity determining regions of Pertuzumab and measured the interaction of the IgGs with FcγRIa, FcγRIIa H167 , FcγRIIa R167 , FcγRIIb/c, FcγRIIIa F176 , FcγRIIIa V176 , FcγRIIIb NA1 , and FcγRIIIb NA2 receptors alongside antibody superantigens proteins L and G using biolayer interferometry. Results The library of 21 IgGs demonstrated that the V H 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. STATEMENT OF SIGNIFICANCE IgGs are the predominant isotype for clinical and research applications. Despite the vast amount of research to study it, particularly on IgG1, there remains a gap in understanding how the variable regions and the receptor binding sites can influence one another in the other IgG subtypes, across the IgG subtypes with different hinges and makeup. This study investigates the effect of these variable regions on the engagement of receptors and also how bacterial antibody superantigens present in microflora and used in antibody purification can exert distal effects.