Daniel Ortiz

High-dose intravenous immunoglobulin (IVIG) is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the fragment crystallizable (Fc) portion. Further, recent data indicate that recombinant multimeric Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of different recombinant human IgG1 Fc molecules with increasing valency and avidity, combined with mutations to increase binding affinity to complement protein C1q. These molecules were investigated for their potential dual antagonism: to antagonize Fcγ receptor (FcγR) effector functions (Ab-dependent cellular phagocytosis) in vitro, and to inhibit the activation of the classical complement pathway. C1q-binding mutants demonstrated an exponential increase in potency to inhibit the classical pathway in correlation with increasing multimerization. Importantly, in contrast to other multimeric Fc constructs such as Fc hexamers, no generation of complement C4a was observed. Reducing the binding affinity to FcγRIIB resulted in a half-life extension of the trivalent hIgG1-Fc molecules in human neonatal Fc receptor transgenic (hFcRn Tg) mice. Our data demonstrate a potent anti-inflammatory effect of recombinant human IgG1-Fc C1q-binding mutants in vitro and in vivo, mediated by blockade of FcγRs and inhibition of complement activation.

Immunoglobulin (IgG) Fc glycosylation has been shown to be important for the biological activity of antibodies. Fc sialylation is important for the anti-inflammatory activity of IgGs. However, evaluating the structure–activity relationship (SAR) of antibody Fc glycosylation has been hindered using simplified in vitro models in which antibodies are often displayed in monomeric forms. Presenting antibodies in monomeric forms may not accurately replicate the natural environment of the antibodies when binding their antigen in vivo. To address these limitations, we used different Fc-containing molecules, displaying their Fc domains in monovalent and multivalent fashion. Given the inhibitory role of Fc gamma receptor IIb (FcγRIIb) in autoimmune and inflammatory diseases, we focused on evaluating the impact of Fc sialylation on the activation of FcγRIIb. We report for the first time that in human cellular systems, sialic acid mediates the induction of FcγRIIb phosphorylation by IgG-Fc when the IgG-Fc is displayed in a multivalent fashion. This effect was observed with different types of therapeutic agents such as sialylated anti-TNFα antibodies, sialylated IVIg and sialylated recombinant multivalent Fc products. These studies represent the first report of the specific effects of Fc sialylation on FcγRIIb signaling on human immune cells and may help in the characterization of the anti-inflammatory activity of Fc-containing therapeutic candidates.