Supplementary Material for: Improved FcγRIIb Targeting Functionally Translates into Enhanced Inhibition of Basophil Activation

<b><i>Background:</i></b> Mast cells and basophils express the high-affinity IgE receptor, FcεRI, as well as the low-affinity IgG receptor, FcγRIIb. While FcεRI is responsible for IgE-dependent degranulation upon coaggregation with allergens, FcγRIIb has been shown to downregulate degranulation through cross-linking with FcεRI. A previously developed fusion protein consisting of an anti-IgE DARPin linked to the human IgG<sub>1</sub>-Fc part (DE53-Fc) has been shown to simultaneously target FcεRI and FcγRIIb with low affinity and to thereby prevent basophil activation. The affinity of a ligand for its receptor is known to be critical for the functional consequences of the binding. So we generated two mutated DE53-Fc molecules with either an improved (DE53-Fc mut+) or a reduced (DE53-Fc mut-) binding to FcγRIIb and assessed their potential to inhibit IgE-dependent basophil activation. <b><i>Methods:</i></b> DE53-Fc was modified by introducing single site-directed point mutations in the Fc part. The mutated constructs were used to assess kinetic parameters as well as the inhibitory capacity on basophil activation and the production of leukotriene C<sub>4</sub> (LTC<sub>4</sub>) and IL-13. <b><i>Results:</i></b> DE53-Fc mut+ showed increased affinity for FcγRIIb as well as an enhanced potential to inhibit IgG<sub>1</sub> binding to FcγRIIb, resulting in improved efficacy in functional assays. Furthermore, DE53-Fc mut+ decreased de novo-synthesized LTC<sub>4</sub> as well as the cytokine IL-13, suggesting that it might be an inhibitor of the allergic late-phase reaction. <b><i>Conclusion: </i></b>Our data suggest that improved binding to FcγRIIb at constant low-affinity binding to IgE leads to more efficient coaggregation of FcεRI-FcγRIIb and results in the enhanced inhibition of basophil activation.