An image-based real time method to measure antibody-dependent cellular phagocytosis

Abstract Antibody-dependent cellular phagocytosis (ADCP) is an important mechanism of action of therapeutic antibodies. In vitro quantification of ADCP has been technically challenging. Fluorescence microscopy and flow cytometry based assays were traditionally used to measure ADCP activity, these methods were static measurements at certain time point, thus provide no information about engulfment kinetics. Moreover, the flow cytometry assays require lifting the macrophage cells from the labware which may affect macrophage activities. We have developed a real time ADCP assay using a live cell analysis system (IncuCyte). The target cells labeled with a pH sensitive dye only fluoresce when they are engulfed in acidic phagosomes of macrophages in the presence of specific antibodies. Our assay is a plate-based with a simple mix and read protocol without disturbing the macrophages. The new method is quantitative and robust, can be readily used for high-throughput screening for functional antibodies. By studying the ADCP activities in different subtypes of macrophages, we found M2 microphages showed the strongest ADCP activity compare to M0 and M1 macrophages, due to their different expression levels of FcgRs. The assay is highly sensitive to differentiate the antibody variants with enhanced Fc function. It can also be used to study the kinetics and mechanisms of ADCP.