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

• Published in: Advanced science Vol. 11; no. 30; pp. e2400700 - n/a
• Main Authors: Mateusiak, Łukasz, Floru, Sam, De Groof, Timo W. M., Wouters, Janne, Declerck, Noemi B., Debie, Pieterjan, Janssen, Simone, Zeven, Katty, Puttemans, Janik, Vincke, Cécile, Breckpot, Karine, Devoogdt, Nick, Hernot, Sophie
• Format: Journal Article
• Language: English
• Published: Germany John Wiley & Sons, Inc 01.08.2024; John Wiley and Sons Inc; Wiley
• Subjects: Animals; Biodistribution; Breast cancer; Camelids, New World; Cancer therapies; Cell Line, Tumor; Clinical trials; Cloning; Colorectal cancer; Contrast agents; Disease Models, Animal; fluorescence molecular imaging; fluorescence‐guided surgery; Fluorescent Dyes; Glioma; Humans; Lungs; Mice; Neoplasms - diagnostic imaging; Neoplasms - immunology; Neoplasms - surgery; Optical Imaging - methods; Receptors, Urokinase Plasminogen Activator - metabolism; Single-Domain Antibodies - immunology; single‐domain antibodies; Surgery; Surgery, Computer-Assisted - methods; Tumors; urokinase plasminogen activator receptor; single‐domain antibodies; fluorescence‐guided surgery; fluorescence molecular imaging; urokinase plasminogen activator receptor
• ISSN: 2198-3844; 2198-3844
• DOI: 10.1002/advs.202400700

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. This study details the generation and characterization of a panel of high‐affinity anti‐uPAR Nanobodies specific for various cancer types. The selected lead fluorescently‐labeled Nanobody demonstrates rapid and precise tumor localization and demarcation in an orthotopic human glioma model. These outcomes indicate the potential of these Nanobodies for applications in preclinical and clinical settings, providing real‐time guidance during surgical procedures.