Optimized Nanobody Engineering for EGFR/MSLN Targeting in Triple‑Negative Breast Cancer

• Maame Agyeman, Biological Sciences, University of Delaware

• Karl Schmitz, Biological Science, University of Delaware

Approximately 15% of breast cancers are classified as triple-negative breast cancers (TNBC) because they lack three common biomarkers: estrogen receptor, progesterone receptor, and HER2. The absence of these biomarkers makes TNBC tumors more difficult to detect and treat, and consequently creates an urgent need for new therapeutics targeting TNBC-enriched biomarkers. Epidermal growth factor (EGFR) and mesothelin (MSLN) are two cell-surface proteins that become highly active in many cancers, including TNBC, with EGFR driving cell division and MSLN helping activate EGFR. We aim to use yeast surface display to develop optimized nanobodies targeting EGFR and MSLN. Yeast surface display provides a way to study and engineer binders for cancer‑related proteins by displaying a library of nanobodies on the yeast cell surface, such that each yeast cell presents one nanobody variant and carries the DNA that encodes it. To develop an optimized nanobody display platform, we engineered a panel of constructs that include a promoter to drive expression, a signal peptides (ECM14, KSH1, or OST1MF) to direct expression on the cell surface, a nanobody that recognize EGFR or MSLN, an HA tag for detection, a fluorescent reporter protein to confirm expression, and a display anchors (Aga2p, Sed1p, or the 649 stalk) to attach the nanobody to the yeast cell wall. When nanobody-displaying yeast cells are exposed to EGFR/MSLN, flow cytometry can measure how strongly each nanobody binds, allowing selection of variants with highest affinity. This approach connects receptor biology, ligand binding, and synthetic biology tools to support the discovery of nanobodies that may useful in TNBC detection, or in therapeutically slowing cancer progression by blocking EGFR/MSLN function.