An engineered human fc variant with exquisite selectivity for FcγRIIIaV158 reveals that ligation of FcγRIIIa mediates potent antibody dependent cellular phagocytosis with GM-CSF-differentiated macrophages

Abstract

IgG antibodies mediate the clearance of target cells via the engagement of Fc gamma receptors (Fc gamma Rs) on effector cells by eliciting antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP, respectively). Because (i) the IgG Fc domain binds to multiple Fc gamma Rs with varying affinities; (ii) even low Fc:Fc gamma Rs affinity interactions can play a significant role when antibodies are engaged in high avidity immune complexes and (iii) most effector cells express multiple FcgRs, the clearance mechanisms that can be mediated by individual FcgR are not well-understood. Human Fc gamma RIIIa (hFc gamma RIIIa; CD16a), which exists as two polymorphic variants at position 158, hFc gamma RIIIa(V158) and hFc gamma RIIIaF158, is widely considered to only trigger ADCC, especially with natural killer (NK) cells as effectors. To evaluate the role of hFcgRIIIa ligation in myeloid-derived effector cells, and in particular on macrophages and monocytes which express multiple FcgRs, we engineered an aglycosylated engineered human Fc (hFc) variant, Fc3aV, which binds exclusively to hFcgRIIIaV158. Antibodies formatted with the Fc3aV variant bind to the hFc gamma RIIIaV158 allotype with a somewhat lower KD than their wild type IgG1 counterparts, but not to any other hFcgR. The exceptional selectivity for hFc gamma RIIIaV158 was demonstrated by SPR using increased avidity, dimerized GST-fused versions of the ectodomains of hFc gamma Rs and from the absence of binding of large immune complex (IC) to CHO cells expressing each of the hFc gamma Rs, including notably, the FcgRIIIaF158 variant or the highly homologous Fc gamma RIIIb. We show that even though monocyte-derived GM-CSF differentiated macrophages express hFc gamma RIIIa at substantially lower levels than the other two major activating receptors, namely hFc gamma RI or hFc gamma RIIa, Fc3aV-formatted Rituximab and Herceptin perform ADCP toward CD20- and Her2-expressing cancer cells, respectively, at a level comparable to that of the respective wild-type antibodies. We further show that hFc gamma RIIIa activation plays a significant role on ADCC by human peripheral monocytes. Our data highlight the utility of Fc3aV and other similarly engineered exquisitely selective, aglycosylated Fc variants toward other hFc gamma Rs as tools for the detailed molecular understanding of hFc gamma R biology.