Confirming the applicability of the anti-EGFR Nanobodies for fluorescence-guided surgery in canine and human head and neck cancer

For the majority of solid cancers, surgical removal remains the mainstay of anti-cancer therapy with the aim to completely remove all tumoural tissue. An important outcome measure is therefore the absence of residual cancer cells after surgery. The use of a fluorescent contrast agent that highlights the tumour cells in real-time may facilitate resection of solid cancers. The epidermal growth factor receptor (EGFR), overexpressed in various types of malignancies, in particular in head and neck cancer such as squamous cell carcinoma (SCC), has been demonstrated to be a relevant biomarker to distinguish malignant from healthy cells, resulting in high tumour-to-background contrasted images during real-time fluorescence- guided surgery (FGS). In this project, we will use Nanobodies (Nb’s) against EGFR to serve as ligands because the much smaller Nb’s have better kinetics and tissue penetration as compared to monoclonal antibodies, which are currently used for FGS. Furthermore, we will perform clinical trials in companion dogs with naturally occurring cancer, a relevant but still severely underexploited preclinical model for advancing human patient anti-cancer strategies. When fluorescent contrast agents can successfully identify malignant cells in dogs with cancer, it is likely that these agents will succeed in clinical trials in human patients with a similar tumour type.

The proposed project aims to assess whether anti-EGFR Nb’s have potential to allow for early diagnosis in cases of head and neck cancer, to improve the surgical outcome by achieving negative surgical margins because of intra-operative margin delineation and disseminated tumour cell identification. Consequently, FGS using anti-EGFR Nb’s might have the potential to reduce the need for adjuvant therapy and associated morbidity, and to increase survival time in patients with head and neck cancer. To this end, three strategies will be tested. In a first step, topically applied fluorescence-labelled anti- EGFR Nb’s will be applied on fresh tissue biopsies of human and equivalent canine head and neck cancers to demonstrate whether topical ex vivo application of anti-EGFR fluorescent-labelled Nb’s can successfully identify EGFR-positive head and neck cancer at the time of diagnosis in dogs and humans. In addition, the fluorescence-labelled anti-EGFR Nb’s will be topically applied to the wound bed in dogs after surgical removal of a head and neck malignancy to screen for remaining fluorescence, which would be indicative of residual cancer cells. Furthermore, dogs with head and neck SCC will receive intravenous anti-EGFR Nb 1 hour before the planned surgical removal of the malignancy. Fluorescence-guided surgery will be performed in which fluorescence intensities of tumour and adjacent tissues will be compared in vivo during surgery as well as after surgical removal of the tumour. In all three steps studies, biopsies will be taken and processed for immunohistochemistry (IHC) and the expression and distribution of EGFR will be compared to the fluorescence data. This proof-of-concept project will reveal preliminary data on sensitivity and specificity of the novel contrast agent to identify malignant tissue that overexpresses EGFR. This information is of paramount importance: if the anti-EGFR Nb’s can indeed differentiate between malignant and normal tissue, the investments to produce the Nb’s under Good Manufacturing Practice (GMP) are justified and clinical trials in humans can be designed.