Fc Galactosylation Promotes Hexamerization of Human IgG1, Leading to Enhanced Classical Complement Activation

Abstract

Human IgG contains one evolutionarily conserved N-linked glycan in its Fc region at position 297. This glycan is crucial for Fc-mediated functions, including its induction of the classical complement cascade. This is induced after target recognition through the IgG–Fab regions, allowing neighboring IgG–Fc tails to associate through Fc:Fc interaction, ultimately leading to hexamer formation. This hexamerization seems crucial for IgG to enable efficient interaction with the globular heads of the first complement component C1q and subsequent complement activation. In this study, we show that galactose incorporated in the IgG1–Fc enhances C1q binding, C4, C3 deposition, and complement-dependent cellular cytotoxicity in human erythrocytes and Raji cells. IgG1–Fc sialylation slightly enhanced binding of C1q, but had little effect on downstream complement activation. Using various mutations that decrease or increase hexamerization capacity of IgG1, we show that IgG1–Fc galactosylation has no intrinsic effect on C1q binding to IgG1, but enhances IgG1 hexamerization potential and, thereby, complement activation. These data suggest that the therapeutic potential of Abs can be amplified without introducing immunogenic mutations, by relatively simple glycoengineering.

Antibody glycosylation in COVID-19

“…While the role of IgG1 afucosylation in steering effector functions via FcγRIII interaction is receiving due attention, the role of other glycosylation features such as galactosylation, sialylation and bisection is poorly understood. For anti-SARS-CoV-2 antibody galactosylation and sialylation, further research is needed on its role in effector functions and complement activation [ 46 ] and possible contribution to disease pathology. Likewise, antibody glycosylation-dependent effects such as FcγRIIa-mediated prothrombotic platelet activation [ 27 ] must be investigated further to comprehend the glycosylation-dependent interaction of antibodies with receptors.…”

Biophysical Evaluation of Rhesus Macaque Fc Gamma Receptors Reveals Similar IgG Fc Glycoform Preferences to Human Receptors

“…Slight changes in the composition of the Fc glycan can impart changes to the Fc-FcgR dynamic that may resonate all the way to the severity of clinical presentation (19)(20)(21)(22)(23)(24)(25)(26)(27). More specifically, an increase in galactose content has been associated with increased propensity of the Fc domain to hexamerize (28), as well as with a slight (≤2 fold) increase in affinity for most of the low affinity (i.e., not FcgRI) FcgRs (29,30), while the absence of a fucose molecule branching from the asparagine-proximal N-acetylglucosamine (GlcNAc) has been credited with up to an astounding 50-fold increase in affinity for FcgRIIIa/b (31)(32)(33). This increase in affinity translates to improvement in antibody-dependent cellular cytotoxicity by FcgRIIIa-bearing natural killer (NK) cells (34,35), which has made it an attractive tool for enhancing the efficacy of therapeutic monoclonal antibodies (mAbs) (36,37).…”

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

“…Of note, galactosylation has recently been found to affect the hexamerization potential of IgG1, which may possibly increase avidity contributions to FcɣR interactions. 34 , 35 We found no indication that other multivalent interactions, such as aggregate formation exist in our analytical setup. Interestingly, the effect of galactosylation in glycoengineered mAbs was more pronounced for mAb4 than for mAb3 (G0N/G0N vs G1N/G0N).…”