Abstracts of the 1st International Online Conference by Antibodies

Introduction: N-linked carbohydrate structures in the Fc-region of human IgG fine tune effector functions such as antibody-dependent complement activation and Fcreceptormediated phagocytosis.The Nordenfelt laboratory recently identified and characterized a human monoclonal (mAb) IgG antibody directed towards the M protein, a surface protein and virulence factor of Streptococcus pyogenes (GAS).These were isolated from memory B cells from an individual who had recovered from a GAS infection.One of the mAbs, Ab25, not only binds M protein with high affinity, but also promotes efficient phagocytosis of the bacteria in vitro.This is attributed to a natural bi-specificity towards two different M protein epitopes.Methods: Chemoenzymatic engineering was used to remove all Fc glycans on Ab25, Ab49 (monospecific mAb against M protein), and omalizumab (IgG mAb against IgE) as the control, and then generate homogenous glycoforms (G0, G0-afuc, G2, G2S2) through click chemistry.Validation of glycosylation pre-and post engineering was performed using LC-MS.These were subsequently tested using flow cytometry-based bacterial binding, phagocytosis using THP-1 cells, and complement factor C1q deposition assays.Results: Original mAb glycosylation and generated glycoforms were validated using LC-MC.Binding experiments revealed that only deglycosylation had any major effects of binding to bacteria; the phagocytosis experiment revealed that the internalization, but not association, of bacteria to THP-1 was influenced by antibody glycoform; and C1q deposition was gradually decreasing in correlation with the size and complexity of the glycans.Conclusions: Human IgG mMAbs against GAS, as well as omalizumab, could be converted to homogenous glycoforms.mAb glycoform clearly influences internalization into phagocytes as well as complement binding.This has important implications for the development of anti-infective mAbs, and highlights mAb glycan engineering as a modality when developing IgG-based therapeutic antibodies.