Engineering microproteins targeting the interface of tumor biomarkers Mesothelin and MUC16

Mesothelin (MSLN) is a cell surface protein overexpressed in ovarian, pancreatic, liver, lung, and triple-negative breast cancer cells, with very limited expression in healthy tissues (Chang et al., 1996). This expression makes MSLN a promising biomarker for tumor detection and targeted therapy. Our research aims to engineer and characterize highly stable mesothelin-targeting agents for use as a molecular diagnostic and targeted therapeutic. MUC16, also known as CA125, is a recognized tumor biomarker that binds to the extracellular domain of MSLN (Chang et al., 1992). The interaction between MUC16 and MSLN has been shown to enable tumor metastasis, and our research looks to engineer a high-affinity protein to obstruct this interface as a viable strategy to reduce cancer progression and metastasis (Chang et al., 1996). In order to have therapeutic benefits, the engineered protein must bind to the native MSLN receptor with higher binding affinity than native MUC16. Using molecular cloning techniques, the mesothelin minimal binding domain (MSLN) has been cloned into a yeast surface display plasmid, and a yeast soluble secretion plasmid. With the yeast surface display plasmid, MSLN was successfully expressed on the surface of yeast, using yeast surface display techniques. The yeast displayed MSLN will be utilized as a means to rapidly measure the binding affinity of engineered protein variants that are solubly expressed and purified. Furthermore, MSLN was solubly expressed and purified using a hexahistidine tag with nickel affinity resin, followed by size exclusion chromatography. The soluble, purified MSLN will be used as the target against which engineered protein variants will be tested for their ability to bind to MSLN. In addition, mammalian cell lines expressing MSLN were identified and curated, in order to test the engineered fibronectin for biophysical properties and their potential as in vivo therapeutics and diagnostics via cell binding, cytotoxicity and cell migration assays. Further work will involve testing the binding affinity of the soluble MSLN towards the surface of cancer cell lines previously reported to express MSLN on their surface.