Intracellular Trafficking and Apoptosis Signaling of Engineered Fn3 Protein for Mesothelin-Targeted Cancer Therapy

Cancer is a leading cause of disease-related mortality worldwide. Unlike conventional chemotherapy and surgical debulking, targeted therapy utilizes small molecule drugs or monoclonal antibodies to specifically inhibit signaling pathways that drive cancer cell growth. This approach offers a more effective treatment with fewer side effects, ultimately improving overall survival (OS). Mesothelin (MSLN) is a tumor associated antigen overexpressed on the surface of many cancer types, making it a promising biomarker for therapeutic development. The Moore lab engineered multiple variants of a small protein scaffold based on fibronectin type III (Fn3) that selectively binds to MSLN with high affinity. Specifically, we have engineered MSLN-binding Fn3 3.4.4 that could either induce apoptosis or can be coupled with chemotherapy drugs to develop protein-drug conjugates (PDC) as targeted treatments. Even though Fn3 3.4.4 has been developed as a potential targeted therapeutic, its efficacy and apoptosis-mediated signaling are not yet fully understood. Therefore, this thesis aims to further characterize Fn3 3.4.4 in MSLN-expressing cells by integrating engineering and biological principles. From an engineering perspective, intracellular trafficking has been measured towards the goal to develop bio-kinetic transport models and optimize the design of protein-drug conjugates (PDCs). Confocal studies with immunofluorescence have been conducted to analyze the colocalization of Fn3 within various cellular compartments such as endosomes and lysosomes. 6 From a biological perspective, this thesis reports the development of methods to understand the signaling pathways induced in MSLN-expressing cancer cells by Fn3 treatment. More specifically, the potential for translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus has been studied as a possible caspase-independent apoptosis pathway. Methods reported include measuring AIF activation with immunofluorescence and cellular fraction western blot studies, providing insight for future development of MSLN-targeting therapeutic strategies.