A Simple & Powerful Cell-Based Assay to Quantify Specific Target Cell Death by Antibody-Dependent Cellular Phagocytosis (ADCP)

A Simple & Powerful Cell-Based Assay to Quantify Specific Target Cell Death by Antibody-Dependent Cellular Phagocytosis (ADCP)
File Name/Number:
KILR ADCP Application Note

Year:
2022

 

With the increasing industry focus on antibody drugs, there is an ever-greater need for functional bioassays that interrogate the therapeutic antibody's mechanisms of action (MOA). One common human immunoglobulin isotype used for therapeutic intervention is IgG1 that contains two antigen-binding Fab arms and an Fc region. The Fc domain of IgG1 can bind to Fcγ receptors expressed on immune effector cells and mediate target cell death by various mechanisms such as Complement-Dependent Cytotoxicity (CDC), Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC), and Antibody-Dependent Cell-Mediated Phagocytosis (ADCP). Regulatory authorities now commonly require data on the impact of each of these Fc-mediated effector mechanisms for the submitted antibody therapeutic. There are several available methods to measure these Fc-mediated effector MOAs, however getting a reproducible true measure of ADCP, in particular, has been quite challenging.

Antibody-Dependent Cell-Mediated Phagocytosis

ADCP is a physiologically important MOA of therapeutic antibodies that can be mediated by various immune effector cells, namely monocytes, macrophages, dendritic cells, and neutrophils through multiple FcγRs. The Fab region of the antibody binds to a specific antigen on the surface of target cells. In contrast, the Fc region of the antibody binds and activates various Fcγ receptors on immune effector cells. Activation of specific Fcγ receptors, like FcγRIIa, FcγRI, and FcγRIIIa, leads to the continued activation of a complex pathway that triggers phagocytosis and destruction of the target cells within the lysosomes of different effector cells.

 

Traditional methods for measuring ADCP require loading the target cells with a fluorescent dye or reporter protein (e.g. Green Fluorescent Protein) and then tracking the number of target cells that have been engulfed by macrophages using flow cytometry or confocal microscopy. These methods are laborious, time-consuming, and measure co-localization of macrophages and target cells rather than target cell death by phagocytosis.