Complement-mediated ADCP as a distinct and finite cytotoxic mechanism of monoclonal antibodies

Introduction: Macrophage phagocytosis is a major cytotoxic mechanism for therapeutic monoclonal antibodies (mAbs) that opsonize target cells. This antibody-dependent cellular phagocytosis (ADCP) can occur via the Fcγ or complement pathways, but the relative contribution of these pathways to mAb-mediated cell clearance is not known. Here, we analyzed the kinetics, functional cooperation, and phagocytic capacities of Fcγ receptor-dependent and complement-dependent ADCP, separately and concomitantly, in primary macrophages challenged with mAb-opsonized lymphocytes. Methods: Using quantitative live-cell imaging of primary mouse macrophages, genetic disruption of Fcγ receptor signaling, and controlled modulation of complement activity, we directly compared the kinetics, capacity, and exhaustion behavior of ADCP via the Fcγ (fADCP) and complement (cADCP) pathways. Results: cADCP operates as a mechanistically independent phagocytic pathway with distinct temporal dynamics. Relative to fADCP, cADCP exhibits delayed onset but substantially greater cumulative target clearance. When both pathways are engaged simultaneously, their effects on target removal are additive, indicating functional non-redundancy. Notably, macrophages rendered refractory to further phagocytosis following fADCP retain full capacity for cADCP, demonstrating that complement receptor-mediated engulfment can bypass Fcγ receptor-associated hypophagia. However, despite its greater capacity, cADCP is also finite, as increasing target burden induces a dose-dependent state of complement-associated phagocytic exhaustion that is kinetically distinct from fADCP hypophagia and is largely reversible within 24 h. Discussion: Our mechanistic understanding of mAb-mediated cytotoxicity via ADCP is disproportionately focused on Fcγ receptor engagement and signaling, with comparatively less emphasis on the role of complement activation. These findings establish complement-mediated ADCP as a quantitatively powerful macrophage effector pathway that can be leveraged to enhance the overall cytotoxic efficacy of mAbs. Further, our work provides a functional framework for understanding how Fcγ receptor and complement pathways differentially contribute to macrophage cytotoxic capacity and highlights effector exhaustion as a shared but mechanistically distinct constraint on sustained antibody-mediated cell clearance. Effector exhaustion therefore represents a fundamental bottleneck to durable responses to ADCP-inducing therapeutic antibodies, but one that can potentially be mitigated via utilization of both ADCP pathways.