BME MS Thesis Defense: Harithi Nandasena

Title: "Development of Nanobody-based Binders for Tumor Microenvironmental Targeting"

Advisory Committee: Chair - Samagya Banskota, PhD Member 2: Mario Cabodi, PhD Member 3: Liangliang Hao, PhD

Abstract: Achieving selective tumor recognition remains a central challenge in cancer diagnostics and therapy. Nanobodies, single-domain antibody fragments (~15 kDa) derived from camelid heavy-chain antibodies, offer compelling advantages over conventional monoclonal antibodies, including high stability, deep tissue penetration, and ease of bacterial expression. Despite growing interest in nanobody-based tumor probes, target selection remains largely empirical, and no systematic framework exists to understand how the biological class of a tumor-associated antigen shapes probes' specificity and contrast. This thesis addresses that gap by developing a panel of nanobody constructs directed against five biologically distinct tumor-associated protein classes: cell-surface receptors (EGFR; 7D12), immune checkpoint proteins (PD-L1; KN035, 5DXW), extracellular matrix splice variants (FN EIIIB; NJB2), extracellular matrix glycoproteins (Tenascin-C; NJT3, NJT4, NJT6), and vascular endothelial receptors (VEGFR-2; NTV1). Two constructs, NJB2 and NJT4, were successfully expressed, purified, and fluorescently labeled via site-specific maleimide-thiol conjugation at a C-terminal cysteine. Labeled probes were applied to CT26 colorectal lung metastasis tissue sections and imaged by fluorescence slide scanning. QuPath-based quantification across n=5 tumor and n=5 peritumoral regions of interest per probe revealed that NJB2 (anti-FN EIIIB) achieved a tumor-to-peritumoral CY5 intensity ratio of 3.50 (p = 0.003) and NJT4 (anti-Tenascin-C) achieved a ratio of 4.58 (p = 0.0005), demonstrating that both ECM-class probes produced significant and reproducible tumor selectivity. These findings provide preliminary tissue-level evidence that spatially confined, stromal ECM targets support high probe contrast and establish a production and imaging pipeline for the systematic evaluation of the remaining constructs.