ABSTRACT Dysregulated pericellular proteolysis is a major driver of the malignant transformation of normal epithelial cells to cancer cells. The increased proteolytic activity in the tumor microenvironment has been exploited for drug development purposes. For example, antibody-drug conjugates (ADCs) consist of a highly potent anti-cancer payload conjugated to a tumor-targeting antibody. This coupling renders the cargo inactive until it is released by proteolytic cleavage in the pericellular space or upon degradation of the antibody within the target cell. ADCs are still handicapped by dose-limited toxicities, most likely due to off-target cell binding or premature drug release by proteases in circulation. The off-target binding can be overcome by engineering protease-activated pro- antibodies (pADCs) that can bind only after being activated at the site of disease by a protease while the premature drug release can be improved by utilizing peptide linker sequences that are stable in the presence of blood proteases but are efficiently cleaved by proteases at the site of disease. Our hypothesis is that pADCs that require two proteolytic activation steps, may improve our ability to selectively target anti-cancer drugs to the tumor microenvironment. The pADC will be converted into an ADC by a pericellular protease and subsequently bind to the tumor cell. The payload will be released by either an extracellular protease or an intracellular (endosomal or lysosomal) protease. The objectives of this proposal are to design ADCs and pADCs that have superior plasma stability and improved release kinetics by cancer-associated proteases. Our long-term objectives are to develop pADCs with enhanced anti-cancer efficacy compared to current therapeutics. In Aim 1, we will generate a substrate specificity profile for proteases secreted by patient-derived organoids of breast cancer. In addition, we will isolate lysosomes and endosomes and characterize the proteolytic activity in these organelles. Finally, we will evaluate protease activity in serum and plasma to understand the circulating proteolytic activity. In Aim 2, we will develop ADCs and pADCs that are selectively activated by cancer proteases and stable in plasma and serum.