Antibody-drug conjugates (ADCs) are a rapidly emerging class of cancer therapeutics composed of an antibody scaffold, a cytotoxic payload, and a joining linker. They exhibit properties of both biologics and small molecule inhibitors, can exert activity through multiple mechanisms, and some are capable of inducing target-independent cell killing via a phenomenon known as the bystander effect. Due to this inherent structural and biological complexity, cell-based functional screening is an essential component of ADC development. However, the majority of preclinical in vitro studies for ADCs are limited to a small number of cell lines and thus provide minimal insights into their activity. High-throughput drug-screening platforms, such as PRISM (Profiling Relative Inhibition Simultaneously in Mixtures), that have been highly effective in characterizing small molecule inhibitors could facilitate characterization of ADCs at an unprecedented scale and provide more comprehensive insights into ADC mechanisms, target specificity, and potency. To test the suitability of the PRISM platform for ADC screening, we investigated several ADCs conjugated to the HER2-targeting antibody trastuzumab that differed in terms of linker type (cleavable and non-cleavable), payload class, and drug-to-antibody ratio. ADCs were screened in parallel with controls including unconjugated antibodies and free payloads across a panel of nearly 500 cell lines consisting of diverse cell lineages. We explored whether ADC selectivity was associated with target antigen expression and found a clear association between cell line sensitivity and ERBB2 (HER2) gene expression, with the strongest effects on viability observed in a subset of cell lines overexpressing ERBB2. Univariate biomarker analysis identified ERBB2 gene expression as a significantly correlated feature of sensitivity to all HER2-targeting ADCs. Notably, this correlation was retained across ADCs with payloads capable of exerting bystander killing of cells with low ERBB2 expression. Upon further comparison of biomarker profiles across different ADCs, established relationships between payload resistance and ABCB1 efflux pumps emerged for a subset of ADC payloads. These results indicate that PRISM pooled cell line screening is able to distinguish ADCs with different payload characteristics while maintaining selectivity of the primary target. Overall, our study highlights how PRISM can be leveraged to analyze ADC activity and uncover clinically relevant targets. This approach can aid in evaluating potential ADC candidates by providing a better understanding of ADC mechanisms of action and how their unique structural components drive cytotoxic activity.

Citation Format: Jillian N Eskra, Ellen Nguyen, Aydin Golabi, Shiker Nair, Antonella Masciotti, Anthony Fazio, Mustafa Kocak, Melissa Ronan, Matthew G Rees, Jennifer A Roth. PRISM high-throughput screening of antibody-drug conjugates uncovers clinically relevant targets [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR004.