Generation and Characterization of Novel Pan-Cancer Anti-uPAR Fluorescent Nanobodies as Tools for Image-Guided Surgery.

Fluorescence molecular imaging plays a vital role in image-guided surgery. In this context, the urokinase plasminogen activator receptor (uPAR) is an interesting biomarker enabling the detection and delineation of various tumor types due to its elevated expression on both tumor cells and the tumor microenvironment. In this study, anti-uPAR Nanobodies (Nbs) are generated through llama immunization with human and murine uPAR protein. Extensive in vitro characterization and in vivo testing with radiolabeled variants are conducted to assess their pharmacokinetics and select lead compounds. Subsequently, the selected Nbs are converted into fluorescent agents, and their application for fluorescence-guided surgery is evaluated in various subcutaneous and orthotopic tumor models. The study yields a panel of high-affinity anti-uPAR Nbs, showing specific binding across multiple types of cancer cells in vitro and in vivo. Lead fluorescently-labeled compounds exhibit high tumor uptake with high contrast at 1 h after intravenous injection across all assessed uPAR-expressing tumor models, outperforming a non-targeting control Nb. Additionally, rapid and accurate tumor localization and demarcation are demonstrated in an orthotopic human glioma model. Utilizing these Nbs can potentially enhance the precision of surgical tumor resection and, consequently, improve survival rates in the clinic.

Safety assessment of fluorescently labeled anti-EGFR Nanobodies in healthy dogs.

Introduction: Surgical resection is one of the main treatment options for several types of cancer, the desired outcome being complete removal of the primary tumor and its local metastases. Any malignant tissue that remains after surgery may lead to relapsing disease, negatively impacting the patient's quality of life and overall survival. Fluorescence imaging in surgical oncology aims to facilitate full resection of solid tumors through the visualization of malignant tissue during surgery, following the administration of a fluorescent contrast agent. An important class of targeting molecules are Nanobodies® (Nbs), small antigen-binding fragments derived from camelid heavy chain only antibodies. When coupled with a fluorophore, Nbs can bind to a specific receptor and demarcate tumor margins through a fluorescence camera, improving the accuracy of surgical intervention. A widely investigated target for fluorescence-guided surgery is the epidermal growth factor receptor (EGFR), which is overexpressed in several types of tumors. Promising results with the fluorescently labeled anti-EGFR Nb 7D12-s775z in murine models motivated a project employing the compound in a pioneering study in dogs with spontaneous cancer. Methods: To determine the safety profile of the study drug, three healthy purpose-bred dogs received an intravenous injection of the tracer at 5.83, 11.66, and 19.47 mg/m2, separated by a 14-day wash-out period. Physical examination and fluorescence imaging were performed at established time points, and the animals were closely monitored between doses. Blood and urine values were analyzed pre- and 24 h post administration. Results: No adverse effects were observed, and blood and urine values stayed within the reference range. Images of the oral mucosa, acquired with a fluorescence imaging device (Fluobeam®), suggest rapid clearance, which was in accordance with previous in vivo studies. Discussion: These are the first results to indicate that 7D12-s775z is well tolerated in dogs and paves the way to conduct clinical trials in canine patients with EGFR-overexpressing spontaneous tumors.