The influence of variable-heavy chain families on IgG2, 3, 4, FcγRs and B-cell superantigens protein G and L binding using biolayer interferometry

Anthony M Deacy; Samuel Ken-En Gan

doi:10.1093/abt/tbad016

The influence of variable-heavy chain families on IgG₂, ₃, ₄, FcγRs and B-cell superantigens protein G and L binding using biolayer interferometry

Antib Ther. 2023 Jul 5;6(3):182-193. doi: 10.1093/abt/tbad016. eCollection 2023 Jul.

Authors

Anthony M Deacy^{1

2}, Samuel Ken-En Gan^{1

2

3

4}

Affiliations

¹ Antibody& Product Development Lab, Agency for Science, Technology and Research (A*STAR), Singapore, and Wenzhou-Kean University, Wenzhou, Zhejiang, China.
² School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
³ Zhejiang Bioinformatics International Science and Technology Cooperation Centre, Wenzhou-Kean University, Wenzhou, Zhejiang Province, China.
⁴ Wenzhou Municipal Key Lab of Applied Biomedical and Biopharmaceutical Informatics, Wenzhou-Kean University, Wenzhou, Zhejiang Province, China.

Abstract

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 IgG₂/IgG₃/IgG₄ antibodies by changing the V_H family (V_H1-7) 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.

Keywords: FcγR; antibody; biolayer interferometry; superantigen.