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, Fc&#947;RIIb. While FcεRI is responsible for IgE-dependent degranulation upon coaggregation with allergens, Fc&#947;RIIb 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 IgG₁-Fc part (DE53-Fc) has been shown to simultaneously target FcεRI and Fc&#947;RIIb 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 Fc&#947;RIIb 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 C₄ (LTC₄) and IL-13. <b><i>Results:</i></b> DE53-Fc mut+ showed increased affinity for Fc&#947;RIIb as well as an enhanced potential to inhibit IgG₁ binding to Fc&#947;RIIb, resulting in improved efficacy in functional assays. Furthermore, DE53-Fc mut+ decreased de novo-synthesized LTC₄ 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 Fc&#947;RIIb at constant low-affinity binding to IgE leads to more efficient coaggregation of FcεRI-Fc&#947;RIIb and results in the enhanced inhibition of basophil activation.