Application Note 177: Integrated evaluation of payload-, Fc-, and target-mediated mechanisms of ADCs using streamlined complementary platforms

The poster presented by Alpana Prasad of Eurofins DiscoverX during the AACR annual meeting 2026; now available for download.

Authors: Alpana Prasad¹, Surekha Bonasu¹, Radhika Venkatnarayanan¹, Jennifer Lin-Jones¹, Jane E. Lamerdin¹, Gaurav Agrawal¹, Nguyen Ly², Jesus A. Diaz de Leon², Venkatesh Chari¹

1 Eurofins DiscoverX, Fremont, CA,
2 Biosensing Instrument, Tempe, AZ

Abstract: 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.