Development of a Mesothelin-Binding Engineered Scaffold Protein as a Theranostic for Pleural Mesothelioma

Mesothelin (MSLN) is a tumor biomarker expressed at high levels on the surface of numerous cancers with extremely limited expression in healthy tissues. MSLN-targeting agents developed for diagnosis and therapy could have a significant impact on the management of MSLN-expressing cancers. Pleural mesothelioma (PM) is a deadly cancer that arises from mesothelial cells lining the pleura and is predominantly linked to asbestos exposure. There are currently no effective treatments, and diagnosis occurs in late stages of disease due to the lack of clinical symptoms in the early stages. Recent efforts to diagnose and treat PM have focused on identifying and targeting relevant biomarkers, including MSLN. We engineered proteins based on the nonantibody fibronectin type III (Fn3) protein scaffold that bind MSLN with high affinity and specificity, using yeast-surface display and directed evolution. Previous work with Fn3 scaffold proteins has demonstrated tissue distribution desirable for applications in molecular imaging and targeted radiotherapy, which may overcome limitations encountered thus far with antibody-based approaches to treat PM. The MSLN-targeting Fn3 was further developed for bioconjugation with the 1,4,7,10-tetraazacyclododecane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA) radiometal chelator. MSLN-binding Fn3 specifically binds to the MSLN-expressing PM lines, colocalizes with MSLN, and internalizes upon binding. Fn3-DOTAGA was further coupled with cold metal gallium-69, and the resulting conjugate maintained binding with high affinity to MSLN-expressing PM cells. MSLN-binding Fn3-DOTAGA-⁶⁹Ga is a promising molecule with diagnostic and therapeutic relevance, toward applications in molecular imaging and targeted radiotherapy.