57 High-throughput quantitative characterization of cytotoxic antibody-drug conjugates using spheroid models reveals important considerations in potential molecular mechanisms of ADC resistance

Background Antibody-drug conjugates (ADCs) are a class of cancer therapeutics comprised of a linker-payload conjugated to a monoclonal antibody targeting a tumor-associated antigen (TAA), to enable the delivery of the cytotoxic payload to cancer cells. In the last few years, while several ADCs have dramatically improved treatment options and demonstrated significant improvements in both progression-free survival (PFS) and overall survival (OS) in various cancer types, many patients still progress while on these agents, and more research is needed into why this resistance occurs. Resistance to ADCs can arise through several mechanisms, including antigen downregulation, impaired ADC trafficking, and payload resistance. With this in mind, there is a need for improved in vitro models that better recapitulate in vivo tumor tissue complexity to aid in the screening and evaluation of novel ADCs during preclinical development especially to address potential mechanisms of resistance.Methods We have developed in vitro 3D models from ovarian cancer cell lines yielding spheroids in a rapid, robust and uniform manner. Specifically, spheroids were generated by seeding ovarian cancer cell lines into microtiter plates treated with ultra-low attachment coating, using automated liquid-handling robots, followed by short-term incubation under standard culturing conditions for spheroid formation. Using these spheroid models, we have developed cell-based assays to functionally evaluate the cytotoxic activity of ADCs in vitro. For further comprehensive characterization of ADC activity in our 3D cell line models, we utilized the nCounter® ADC Development Panel, a specialized gene expression tool for molecular characterization of biological function, including gene content which addresses complex questions related to mechanisms of resistance.Results and Conclusions The nCounter® ADC Development Panel directly profiles 770 genes addressing essential biological questions relevant to ADC activity, including tumor targeting and antigen expression; ADC internalization; payload release; drug mechanisms of action; target cell death; immunogenic cell death; and mechanisms of resistance. Our data highlights the combination workflows of high-throughput cell culture-based spheroid assays with comprehensive transcriptomic analysis to aid in the screening and selection of therapeutic cytotoxic ADC candidates for downstream treatment of solid tumors. Further understanding of resistance mechanisms with this approach provides direction for future research to elucidate new targets for drug development that can expand the efficacy of antibody-drug conjugates.