Multi-Angle Effector Function Analysis of Human Monoclonal IgG Glycovariants.

Tilman Schlothauer,Johannes Buchner,Sophia N Karagiannis,Marco Thomann,Alexander Knaupp,Tetyana Dashivets,Petra Rueger

doi:10.1371/journal.pone.0143520

Tilman Schlothauer, Johannes Buchner + Show 5 more

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https://doi.org/10.1371/journal.pone.0143520

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Abstract

Therapeutic performance of recombinant antibodies relies on two independent mechanisms: antigen recognition and Fc-mediated antibody effector functions. Interaction of Fc-fragment with different FcR triggers antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity and determines longevity of the antibody in serum. In context of therapeutic antibodies FcγRs play the most important role. It has been demonstrated that the Fc-attached sugar moiety is essential for IgG effector functionality, dictates its affinity to individual FcγRs and determines binding to different receptor classes: activating or inhibitory. In this study, we systematically analyze effector functions of monoclonal IgG1 and its eight enzymatically engineered glycosylation variants. The analysis of interaction of glycovariants with FcRs was performed for single, as well as for antigen-bound antibodies and IgGs in a form of immune complex. In addition to functional properties we addressed impact of glycosylation on the structural properties of the tested glycovariants. We demonstrate a clear impact of glycosylation pattern on antibody stability and interaction with different FcγRs. Consistent with previous reports, deglycosylated antibodies failed to bind all Fcγ-receptors, with the exception of high affinity FcγRI. The FcγRII and FcγRIIIa binding activity of IgG1 was observed to depend on the galactosylation level, and hypergalactosylated antibodies demonstrated increased receptor interaction. Sialylation did not decrease the FcγR binding of the tested IgGs; in contrast, sialylation of antibodies improved binding to FcγRIIa and IIb. We demonstrate that glycosylation influences to some extent IgG1 interaction with FcRn. However, independent of glycosylation pattern the interaction of IgG1 with a soluble monomeric target surprisingly resulted in an impaired receptor binding. Here, we demonstrate, that immune complexes (IC), induced by multimeric ligand, compensated for the decreased affinity of target bound antibody towards FcRs, showing the importance of the IC-formation for the FcR- mediated effector functions.

Highlights

Over the past several years, therapeutic antibodies for the treatment of various diseases have become a considerable part of the biopharmaceutical industry
As glycosylation has been reported to be important for the structural integrity and stability of antibodies[47,48,49,50], we assessed the thermal stability of the samples by applying the Thermofluor Stability Assay (TSA) using the fluorescent dye SYPRO orange
Consistent with previous reports, the deglycosylated antibody failed to bind all Fc gamma receptors (FcγRs) except the high affinity FcγRI

Summary

Introduction

Over the past several years, therapeutic antibodies for the treatment of various diseases have become a considerable part of the biopharmaceutical industry. More than 40 therapeutic mAbs and mAb fragments are approved and prescribed today [1,2], the majority being of the IgG isotype.[3] The IgG molecule consists of two light chains (two domains each) and two heavy chains (four domains each). Each light chain together with two domains of a heavy chain forms the Fab (fragment antigen binding) region. The clinical efficacy of therapeutic antibodies rely on two functional properties: first, the ability of the Fab regions to recognize and bind the target; and second, the ability to induce different immune system effector mechanisms through interaction of the Fc region with Fc gamma receptors (FcγRs), the C1q component of complement and the neonatal receptor (FcRn)

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