(176q) Engineered Fn3 Proteins Have Therapeutic Effect on Mesothelin-Expressing Cancer Cells

Mesothelin (MSLN) is a cell surface protein overexpressed in numerous cancers, including breast, ovarian, lung, liver, and pancreatic. Aberrant expression of MSLN promotes tumor progression and metastasis through interaction with tumor biomarker CA125. Molecules that bind MSLN have potential therapeutic and diagnostic applications. However, MSLN remains an underdeveloped tumor biomarker, and no MSLN-targeting molecules are currently approved for routine clinical use.

We engineered variants of the fibronectin type III domain (Fn3) non-antibody protein scaffold to bind to MSLN with moderate affinity (100s nM K_D). Lead candidates were recombinantly produced in bacterial culture. Fn3 variants specifically bound to MSLN on human cancer cells, were internalized, and co-localized to early endosomes, indicating their promise for drug delivery applications.

Following additional rounds of directed evolution, we have identified further evolved Fn3 variants with an order of magnitude enhanced binding affinity for MSLN. We are currently evaluating the in vitro therapeutic activity of the improved variants in several cancer cell lines. Results demonstrate that treatment with the engineered MSLN-binding proteins have cytotoxic effect on KB-3-1 cells, an MSLN-expressing cell line, but not on cells that do not express MSLN. Further, treatment of KB-3-1 cells with the engineered Fn3 variants causes apoptosis, as demonstrated by PI and annexin V staining. Meanwhile, treatment of cells with mitomycin C, a currently approved chemotherapeutic, non-specifically kills both MSLN-positive and MSLN-negative cell lines. Ongoing research aims to elucidate the mechanism of induced cell death, towards providing insights on a novel mechanism for targeted therapy for MSLN-positive tumors.