Safety assessment of fluorescence-labeled anti-EGFR Nanobody in healthy dogs

Introduction Fluorescence-guided surgery (FGS) in oncology consists of perioperative visualization of tumoral tissue using fluorescent contrast agents. A promising target is the epidermal growth factor receptor (EGFR) as it is overexpressed in several types of cancer. The anti-EGFR Nanobody (Nb) is a unique agent in terms of stability, specificity, and pharmacokinetics. When conjugated to a fluorescent dye, it enables rapid and high contrast molecular imaging and could help achieve clean surgical margins in human and canine cancer patients, the latter being an important translational cancer model. Methods In preparation for a clinical trial in canine patients, a preclinical safety assessment was performed in purpose-bred dogs. First, anti-EGFR Nb 7D12 was produced and conjugated to the near-infrared dye s775z under GLP conditions. The purity and functionality were further assessed. Then, three experimental Beagles received the bioconjugate intravenously at escalating doses: 5.83, 11.66, and 19.47mg/m2, separated by a 14-day wash out period. Physical examinations, including heart and respiratory rate, temperature and blood pressure; and fluorescence imaging of the oral mucosa and auricular cartilage were conducted at fixed timepoints. Blood and urine analyses were performed before and 24h after intravenous Nb administration and the animals were continuously monitored between injections. Results/Discussion 7D12 was successfully labeled with s775z. Purity was confirmed by size-exclusion chromatography and SDS-PAGE, and endotoxin level was <0.22EU/mg. Identity was assessed by mass-spectrometry and showed that Nbs with a degree-of-labeling of 1 were most prevalent. The binding affinity, as well as the association and dissociation kinetics, were determined using surface plasmon resonance. Following IV injection of the tracer, no signs of hypersensitivity were observed. Vital parameters stayed within the normal range; blood and urine collected pre- and 24h p.i. did not present immune-related alterations or signs of organ toxicity, despite administration of dosages far above the intended clinical dose. Mean fluorescence intensity on oral mucosa and auricular cartilage reached its maximum between 1 and 2h, decreasing subsequently until 8h after injection. Considering this time frame was never determined in canine patients, we expect to confirm the imaging window in a clinical setting. Conclusions This study is the first to report intravenous administration of Nbs in dogs and the results indicate that 7D12-s775z is well tolerated in this species, also after repeated administration. Successful completion of this study paves the way to conduct a clinical trial in canine patients with EGFR-overexpressing spontaneous tumors, which in turn will progress the clinical translation of this novel tracer in human cancer patients.