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 (FcγRs) on effector cells by eliciting antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP, respectively). Because (i) the IgG Fc domain binds to multiple FcγRs with varying affinities; (ii) even low Fc:FcγRs affinity interactions can play a significant role when antibodies are engaged in high avidity immune complexes and (iii) most effector cells express multiple FcγRs, the clearance mechanisms that can be mediated by individual FcγR are not well-understood. Human FcγRIIIa (hFcγRIIIa; CD16a), which exists as two polymorphic variants at position 158, hFcγRIIIa V158 and hFcγRIIIa F158 , is widely considered to only trigger ADCC, especially with natural killer (NK) cells as effectors. To evaluate the role of hFcγRIIIa ligation in myeloid-derived effector cells, and in particular on macrophages and monocytes which express multiple FcγRs, we engineered an aglycosylated engineered human Fc (hFc) variant, Fc3aV, which binds exclusively to hFcγRIIIa V158 . Antibodies formatted with the Fc3aV variant bind to the hFcγRIIIa V158 allotype with a somewhat lower K D than their wild type IgG1 counterparts, but not to any other hFcγR. The exceptional selectivity for hFcγRIIIa V158 was demonstrated by SPR using increased avidity, dimerized GST-fused versions of the ectodomains of hFcγRs and from the absence of binding of large immune complex (IC) to CHO cells expressing each of the hFcγRs, including notably, the FcγRIIIa F158 variant or the highly homologous FcγRIIIb. We show that even though monocyte-derived GM-CSF differentiated macrophages express hFcγRIIIa at substantially lower levels than the other two major activating receptors, namely hFcγRI or hFcγRIIa, 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 hFcγRIIIa 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 hFcγRs as tools for the detailed molecular understanding of hFcγR biology.

Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life

“…Overall, strengthening or disrupting Fc interactions with its binding partners as measured by in vitro affinity has translated to the desired outcome in vivo . The optimization of Fc for specific functions not only can improve in vivo functions, but it also provides a means to dissect the importance of specific Fc receptors (172) and downstream CDC, ADCC, phagocytosis, or circulation half-life in treating specific diseases (119, 173). While antibody-based biologics have been successful in the treatment of disease, new opportunities exist for antibody biologics as durable prevention strategies for infectious diseases (174–176).…”

Boosting therapeutic potency of antibodies by taming Fc domain functions

“…1b) [20][21][22] , which has been shown by glycoengineered antibodies to exhibit enhanced affinity toward FcγRIIa, resulting in increased ADCP activity 21,23 . However, the existence of other human Fc-glycoengineered antibodies that do not affect FcγRIIa binding 24 but improve ADCP 20 also indicates the possibility that FcγRIIIa contributes downstream signaling to phagocytic activity 25 .…”

Reprogramming the Constant Region of Immunoglobulin G Subclasses for Enhanced Therapeutic Potency against Cancer

“…Together with well-known functions of macrophages in tumor phagocytic activity [86], these results indicate that ADCP activity is critical in therapeutic efficacy against cancer. The ADCP activity of macrophages is triggered by FcγRIIa intracellular signaling [87][88][89]; however, anti-CD20 or anti-Her2 antibodies with engineered Fcs that only bind to FcγRIIIa triggered not only ADCC, but also ADCP using human NK cells and macrophages in vitro, which is an unknown function of FcγRIIIa [90]. This study again highlights a clinical ramification of FcγRIIIa in cancer therapeutics.…”

“…Engineering of FcγR-selective Fc may be challenging, as the amino acid sequences of FcγR ecto-domains are highly homologous (FcγRIIa and FcγRIIb exhibit 96% identity in amino acid sequence) [105]. With the precedent of FcγRI-and FcγRIIIa-selective Fcs [90,106], FcγRIIa-and FcγRIIb-selective Fcs should be developed to dissect antibody-mediated effector mechanisms for understanding human immunobiology and providing future antibody therapeutics. These selective Fcs are designed for i) tumor cell destruction, such as anti-CD20 antibodies [5], ii) immune cell activation, such as agonistic anti-CD40 [107,108] or anti-TNFR antibodies [109], which elicit antigen presentation and subsequent adaptive immune response by engaging FcγRIIb, and iii) anti-tumor vaccine effect [92].…”