The influence of variable-heavy chain families on IgG(2), (3), (4), FcγRs and B-cell superantigens protein G and L binding using biolayer interferometry.

As the most abundant immunoglobulin in blood and the most common human isotype used for therapeutic monoclonal antibodies, the engagement and activation of its Fc receptors by IgGs are crucial for antibody function. Assumed to be relatively constant within subtypes, recent studies reveal that antibody variable regions exert distal effects of modulating antibody-receptor interactions on antibody isotypes. These variable (V)-region distal effects are also expected for the IgG subtypes. With an in-depth understanding of the V-region effects, researchers can make a more informed antibody engineering approach and antibody purification strategy accounting for the functions of microbial immune evasion . 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 complementary determining regions of pertumuzab and measured their interactions with FcγRIa, FcγRIIa(H167), FcγRIIa(R167), FcγRIIb/c, FcγRIIIa(F176), FcγRIIIa(V176), FcγRIIIb(NA1) and FcγRIIIb(NA2) receptors alongside B-cell superantigens Protein L and G using biolayer interferometry. The panel of 21 IgGs demonstrated that the V(H) frameworks influenced receptor binding sites on the constant region in a non-canonical manner. However, there was minimal influence on the binding of bacterial B-cell superantigens Proteins L and Protein G on the IgGs, showing their robustness against V-region effects. These results demonstrate the role of V-regions during the humanization of therapeutic antibodies that can influence FcR-dependent immune responses while retaining binding by bacterial B-cell superantigens for antibody purification. These in vitro measurements provide a clue to detailed antibody engineering and understanding of antibody superantigen functions that would be relevant with in vivo validation.