Instantaneous Drug Release Visualization Through the Novel Conjugated-Antibody Specificity Assay

Antibody-drug conjugates (ADCs) have transformed the field of targeted drug delivery systems; these systems use monoclonal antibodies for targeting, conjugated to cytotoxic drugs through a chemical linker. ADC success requires binding to the target receptor, internalizing the target cell, trafficking to the lysosome, cleavage of the linker, and finally, drug release. Failure at any of these steps results in an ineffective therapeutic. Currently, only confocal microscopy is used to visualize the internalization of ADCs and lysosomal trafficking; however, this method cannot investigate linker robustness, lysosomal enzyme-cleavage activity, or drug release rates, all of which are essential for the efficacy of an ADC. To fill this knowledge gap, we developed the Conjugated-Antibody Specificity Assay (CASA) to examine the internalization and drug release of ADCs using standard ADC synthesis techniques and a fluorescent plate reader. CASA can track the mechanism of antibody-drug conjugates in vitro with enzymes and in cellular environments.