Integrated evaluation of payload-, Fc-, and target-mediated mechanisms of ADCs using streamlined complementary platforms

This study demonstrates an integrated approach for comprehensive characterization of antibody-drug conjugates (ADCs) using complementary bioanalytical platforms. The objective was to evaluate target binding, internalization, and both payload- and Fc-mediated cytotoxic mechanisms to generate a holistic understanding of ADC mode of action.ADC binding affinity and kinetic parameters were characterized by surface plasmon resonance microscopy (SPRm) to assess cell-surface receptor engagement. Internalization was measured in engineered tumor cell lines expressing relevant antigens (e.g., BCMA, CD33) using enzyme-fragment complementation (EFC)-based PathHunter® internalization assays. Functional cytotoxicity was quantified using the KILR® Cytotoxicity platform, providing direct readouts of target-cell death. Fc-effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), were evaluated using effector cell models to distinguish immune-mediated from payload-driven mechanisms.Here we show characterization data for 2 different ADCs which demonstrated high-affinity binding by SPRm and efficient internalization by PathHunter assays. In KILR assays, the ADC produced a concentration-dependent cytotoxic response distinct from its unconjugated parental antibody. Comparative analysis revealed that payload-mediated killing dominated overall cytotoxicity, while measurable ADCC and ADCP activities reflected retained Fc function. Integration of biophysical and cell-based results provided mechanistic resolution of ADC function at multiple levels. Combining SPRm, PathHunter internalization assays, and KILR cytotoxicity enables comprehensive ADC characterization encompassing binding, uptake, and multi-mechanistic cytotoxicity. This integrated workflow supports discovery, optimization, and lot-release testing by linking molecular properties to functional outcomes, enhancing mechanistic understanding critical for ADC development.