High-throughput Quantification of Antibody-Dependent Phagocytosis using Live-Cell Analysis

Abstract The uptake and clearance of tumor cells can be promoted with monoclonal antibodies (mAbs) via antibody-dependent phagocytosis (ADCP) or through the blockade of “don’t-eat-me” signals, such as CD47. These mechanisms hold immunotherapeutic promise and are studied extensively in drug development. High-throughput assays were conducted using pHrodo®, a pH-sensitive fluorophore, that enables live-cell imaging and kinetic quantification of phagocytosis. pHrodo® labeled target cells were incubated with mAbs and added to effector cells in 96-well plates. Images were acquired using the Incucyte® Live-Cell Analysis System and fluorescence automatically quantified with integrated software. In line with known pro-phagocytic effects, anti-CD47 promoted phagocytosis of CCRF-CEM tumor cells by blocking CD47 “don’t-eat-me” signals. Clinical anti-CD20 mAbs Truxima® and Rituximab both promoted ADCP of Ramos target cells by primary macrophages. In addition, the ADCP response of different Rituximab isotypes were compared. Anti-CD20-IgG1 mAb and Fc mutated anti-CD20-IgG1fut (non-fucosylated) exhibited concentration-dependent responses (EC50 values of 25.1 ng/mL and 42.8 ng/mL, respectively), however anti-CD20-IgG1NQ (non-glycosylated) showed no response. ADCP was also examined in adherent target cells with varied HER2 profiles. Anti-HER2 (Trastuzumab) induced an ADCP response in HER2-positive AU565 cells but not in HER2-low MDA-MB-231 cells, consistent with established correlations between HER2 expression and ADCP response. These data exemplify that live-cell analysis is a powerful approach that enables functional quantification of ADCP and is amenable to antibody screening for therapeutic candidates.