[DOT 2021] Quantify Antibody-Dependent Cell Phagocytosis (ADCP): Application of a Robust, Non-Radioactive KILR Cytotoxicity Platform

File Name/Number:: DOT 2021
Year:: 2021

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Cell-based assays play an important role in determination of mechanism of action (MOA) for therapeutic antibodies, particularly when evaluating their potential for effector functions. Antibody-dependent cellular phagocytosis (ADCP) has gained prominence as an important MOA to evaluate, especially for IND-enabling studies of antibodies with modified Fc regions. ADCP is mediated by a variety of effector cells, namely monocytes, macrophages, dendritic cells, and neutrophils, through multiple FcγRs. While the Fab region of the antibody binds to a specific antigen on the surface of target cells, the Fc region of the antibody binds and activates various Fcγ receptors on effector cells. Activation of specific Fcγ receptors, like FcγRIIa, FcγRI, and FcγRIIIa, leads to activation of a complex pathway that results in phagocytosis and destruction of the target cells in the lysosome of the effector cells.

ADCP assays can be particularly difficult to standardize due to the variability of primary human Fcγ-expressing effector cells and the lengthy and difficult protocols required for immune cell differentiation. We have applied our industry-validated KILR® cytotoxicity assay platform, which specifically measures killing of target cells in co-culture with immune cells, to quantitation of ADCP in a homogeneous plate-based assay format, serving as an inexpensive alternative to laborious and expensive FACS or imaging approaches. Measurement of ADCP activity with KILR cells is a simple, reproducible and scalable method that directly measures the physiologically relevant endpoint: target cell destruction inside the lysosomes of effector macrophages. KILR ADCP assays are fast, robust, and reproducible, supporting screening and characterization of antibody drugs during early phase bio-comparability as well as in late stage characterization work. The broad application of KILR assays along the drug development pipeline, together with the ability of each stable KILR model to quantify CDC, ADCC, as well as ADCP allows this assay platform to support the development of almost any antibody therapeutic.
We discuss examples where the same engineered target cell line is used to determine ADCC, CDC, and ADCP capabilities of an antibody drug. Generating a very low background, these assays produce robust assay windows with excellent precision, and are applicable to various stages of drug development, ranging from screening to use in QC lot release of complex biologic drugs. KILR Cytotoxicity Assays have been tested using multiple primary effector cells (PBMCs, Macrophages, and NKs) or engineered cells lines (NK-92). Further, these assays can be used to evaluate other immunotherapy drugs such as T-cell redirecting bi-specific antibodies and
chimeric antigen receptor T-cells (CAR-T), and tumor-infiltrating lymphocytes (TILs).