In recent years, there has been significant progress in developing targeted cancer diagnostics and therapeutics, allowing for increased efficacy and reduced toxicity compared to previous approaches for diagnosis and treatment. For many cancers however, such as triple-negative breast, ovarian, pancreatic, or lung, targeted options are not yet available. Thus, new biomarkers for these cancers must be identified and appropriately translated for clinical application. Mesothelin (MSLN), a cell surface protein, is expressed at high levels on these tumors, with limited expression in healthy tissue. Furthermore, MSLN has been shown to bind another known tumor biomarker MUC16 (CA125), an interaction shown to facilitate cancer progression and metastasis. The differential expression pattern of MSLN, and its association with MUC16, makes MSLN a promising tumor biomarker. We aim to engineer MSLN-targeting proteins to interrupt the MSLN-MUC16 tumor biomarker interface, with applications as both molecular diagnostics and targeted therapeutics.
Based on the non-antibody fibronectin scaffold, we are using directed evolution and yeast surface display technology to engineer high-affinity proteins that target the domain of MSLN responsible for binding MUC16. We have identified, recombinantly expressed in yeast, and purified the minimal binding domain of MSLN that binds MUC16. Using magnetic- and fluorescent-activated cell sorting, we have identified fibronectin variants that bind the MSLN minimal binding domain with high affinity. We have established in vitro assays to measure the engineered proteins’ binding to tumor cell lines expressing MSLN. Work in progress includes measuring the stability, binding affinity, and bioactivity of the candidate proteins. Future work will evaluate the engineered proteins as molecular imaging agents by injecting fluorescently labeled variants into tumor-bearing mice and measuring in vivo tumor contrast.
A broad outcome of our work is to further develop the fibronectin scaffold combined with directed evolution for the advancement of paired diagnostic/therapeutics, with implications in developing “theranostics” for other identified tumor biomarkers.