Superantigens are unconventional antigens which recognise immune receptors outside their usual recognition sites e.g. complementary determining regions (CDRs), to elicit a response within the target cell. T-cell superantigens crosslink T-cell receptors and MHC Class II molecules on antigen-presenting cells, leading to lymphocyte recruitment, induction of cytokine storms and T-cell anergy or apoptosis among many other effects. B-cell superantigens, on the other hand, bind immunoglobulins on B-cells, affecting opsonisation, IgG-mediated phagocytosis, and driving apoptosis. Here, through a review of the structural basis for recognition of immune receptors by superantigens, we show that their binding interfaces share specific physicochemical characteristics when compared with other protein-protein interaction complexes. Given that antibody-binding superantigens have been exploited extensively in industrial antibody purification, these observations could facilitate further protein engineering to optimize the use of superantigens in this and other areas of biotechnology.

Superantigens are unconventional antigens which recognise immune receptors outside the usual binding sites e.g. complementary determining regions (CDRs), to elicit a response within the target cell. T-cell superantigens crosslink T-cell receptors and MHC Class II molecules on antigen-presenting cells, leading to lymphocyte recruitment, induction of cytokine storms and T-cell anergy or apoptosis among many other effects. B-cell superantigens, on the other hand, bind immunoglobulin receptors on B-cells affecting opsonisation, IgG-mediated phagocytosis, and drive B-cells into apoptosis. Here, through a review of the structural basis for recognition of immune receptors by superantigens, we show that their binding interfaces share specific physicochemical characteristics when compared with other protein-protein interaction complexes. Given that antibody-binding superantigens have been exploited extensively in industrial antibody purification, these observations could facilitate further protein engineering to optimize the use of superantigens in this and other areas of biotechnology.

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.

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 reveal that antibody variable regions can exert distal effects of modulating antibody–receptor interactions on many antibody isotypes. These 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, antibody purification strategy, and better investigate the functions of microbial immune evasion proteins. Methods In this study, we created a panel of IgG2/IgG3/IgG4 antibodies by changing the VH family (VH1–7) frameworks while retaining the complementary determining regions of Pertuzumab and measured their interactions with FcγRIa, FcγRIIaH167, FcγRIIaR167, FcγRIIb/c, FcγRIIIaF176, FcγRIIIaV176, FcγRIIIbNA1, and FcγRIIIbNA2 receptors alongside B-cell superantigens Protein L and G using biolayer interferometry. Results The panel 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-interaction. 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. Conclusions These results demonstrate the role of V-regions during the humanisation of therapeutic antibodies that can confer or diminish 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.