A method for determining antibody-dependent cellular phagocytosis

Abstract Antibody-based therapeutics are a powerful tool to treat diseases. While their mechanism of action (MOA) always involves binding to a specific target via the Fab region of the antibody, the induction of effector functions through the Fc region of the antibody is equally important for antibody therapeutics designed to deplete tumor cells. By binding of the Fc region to FcγRs on the surface of immune cells, antibody therapeutics exert effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), both of which induce target cell death and aid in the efficacy of the treatment. There are many in vitro assays designed to measure ADCC and CDC activity of therapeutics. However, another major Fc effector function is antibody-dependent cellular phagocytosis (ADCP). ADCP is the mechanism by which antibody-opsonized target cells activate the FcγRs on the surface of macrophages to induce phagocytosis, resulting in the internalization and degradation of the target cell through acidification of the phagosome. ADCP has been implicated as a major MOA of several biologics, but this activity is difficult to measure in an in vitro assay. Most assays measure the association between target cells and macrophages, however co-localization does not indicate phagocytosis. In this presentation, we describe the development of a robust method for measuring ADCP activity. By labeling target cells with a pH sensitive dye that only fluoresces in mature phagosomes, it is possible to quantitate the ADCP activity of different antibody therapeutics. This method was developed on parallel platforms, utilizing both flow cytometry as well as a novel method quantitating the ADCP activity on a cell imaging system.