Fluorescent nanobodies: A tool to leverage surgical cancer management to a maximum patient advantage

Despite significant advancements in cancer management over the past 30 years, surgery remains the primary treatment for early-stage solid tumors. Achieving R0 resection, where no ecancer cells are detected microscopically, is crucial as residual cancerous tissue increases recurrence rates and decreases survival. Improving surgical resection completeness directly impacts survival rates, while positive margins often necessitate additional treatments that can
severely affect quality of life.

Fluorescence-guided surgery (FGS) has gained clinical interest as a cost-effective, highly sensitive, and easy-to-implement real-time technology that detects light emitted by fluorophores in situ. These fluorophores can be linked to targeting moieties that recognize specific biomarkers, facilitating cancer-related biomarker expression assessment. Targeting moieties include small molecules, peptides, protein scaffolds, antibody fragments, and whole antibodies. The challenge of delineating cancerous tissue due to microscopic extensions beyond visible tumors underscores the need for better intra-operative visualization tools. Specifically, there is an urgent need for improved contrast agents that can distinguish malignant from healthy tissue.This doctoral work focuses on evaluating camelid-derived single-domain antibodies, or Nanobodies® (Nbs), as a platform for manufacturing fluorescent tracers.

The first research chapter of this thesis focuses on the impact of small structural variations in coupled fluorophores on the performance of tracers. It examines how different versions of the Cy5 fluorescent dye, when linked with an anti-HER2 Nb, affect the biodistribution and tumortargeting capabilities of the tracer in subcutaneous tumor models. Based on these findings, the most promising fluorophore is selected for further evaluation.

The following chapter addresses the visualization of lymph nodes (LNs) during cancer surgery. It proposes using an anti-MMR Nb labeled with the dye selected in investigation described in the previous chapter to visualize all LNs within the surgical view. This tracer is compared to its counterpart labeled with the state-of-the-art IRDye800CW dye, transitioning the model from murine to bigger, more clinically relevant porcine models.

The final two research chapters elaborate on generating and validating a fluorescent anti-uPAR tracer for use across various tumor types. First, the investigation focuses on covering the generation of these new tracers, evaluating their biodistribution, tumor-targeting following radiolabeling, and initial proof-of-concept tests in subcutaneous tumor models. It culminates with studies in an orthotopic glioma model. The following chapter extends the application of the lead compounds to other orthotopic models, specifically targeting gastrointestinal tumors and cancers prone to metastasis.

In conclusion, this doctoral dissertation elaborates on the development and assessment of new Nb-based fluorescent tracers with potential for FGS. However, further research is needed to facilitate their translation into the clinical setting.