mAbs (Jan 2021)

Fc gamma receptor IIIb binding of individual antibody proteoforms resolved by affinity chromatography–mass spectrometry

  • Steffen Lippold,
  • Alexander Knaupp,
  • Arnoud H. de Ru,
  • Rayman T. N. Tjokrodirijo,
  • Peter A. van Veelen,
  • Erwin van Puijenbroek,
  • Steven W. de Taeye,
  • Dietmar Reusch,
  • Gestur Vidarsson,
  • Manfred Wuhrer,
  • Tilman Schlothauer,
  • David Falck

DOI
https://doi.org/10.1080/19420862.2021.1982847
Journal volume & issue
Vol. 13, no. 1

Abstract

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The crystallizable fragment (Fc) of immunoglobulin G (IgG) activates key immunological responses by interacting with Fc gamma receptors (FcɣR). FcɣRIIIb contributes to neutrophil activation and is involved in antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). These processes present important mechanisms-of-actions of therapeutic antibodies. The very low affinity of IgG toward FcɣRIIIb (KD ~ 10 µM) is a technical challenge for interaction studies. Additionally, the interaction is strongly dependent on IgG glycosylation, a major contributor to proteoform heterogeneity. We developed an affinity chromatography–mass spectrometry (AC-MS) assay for analyzing IgG-FcɣRIIIb interactions in a proteoform-resolved manner. This proved to be well suited to study low-affinity interactions. The applicability and selectivity of the method were demonstrated on a panel of nine different IgG monoclonal antibodies (mAbs), including no-affinity, low-affinity and high-affinity Fc-engineered or glycoengineered mAbs. Thereby, we could reproduce reported affinity rankings of different IgG glycosylation features and IgG subclasses. Additional post-translational modifications (IgG1 Met252 oxidation, IgG3 hinge-region O-glycosylation) showed no effect on FcɣRIIIb binding. Interestingly, we observed indications of an effect of the variable domain sequence on the Fc-binding that deserves further attention. Our new AC-MS method is a powerful tool for expanding knowledge on structure–function relationships of the IgG-FcɣRIIIb interaction. Hence, this assay may substantially improve the efficiency of assessing critical quality attributes of therapeutic mAbs with respect to an important aspect of neutrophil activation.

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