The GEM-handle as convenient labeling strategy for bimodal single-domain antibody-based tracers carrying 99m Tc and a near-infrared fluorescent dye for intra-operative decision-making

• Published in: Frontiers in immunology Vol. 14; p. 1285923
• Main Authors: Declerck, Noemi B, Huygen, Celine, Mateusiak, Lukasz, Stroet, Marcus C M, Hernot, Sophie
• Format: Journal Article
• Language: English
• Published: Switzerland 2023
• Subjects: Animals; Fluorescent Dyes; Mice; Neoplasms - diagnostic imaging; Single-Domain Antibodies; Tissue Distribution; Tomography, Emission-Computed, Single-Photon - methods; intraoperative imaging; cancer surgery; fluorescence imaging; hybrid tracer; bimodal tracer; gamma-probing; nanobody

Intra-operative fluorescence imaging has demonstrated its ability to improve tumor lesion identification. However, the limited tissue penetration of the fluorescent signals hinders the detection of deep-lying or occult lesions. Integrating fluorescence imaging with SPECT and/or intra-operative gamma-probing synergistically combines the deep tissue penetration of gamma rays for tumor localization with the precision of fluorescence imaging for precise tumor resection. In this study, we detail the use of a genetically encoded multifunctional handle, henceforth referred to as a GEM-handle, for the development of fluorescent/radioactive bimodal single-domain antibody (sdAb)-based tracers. A sdAb that targets the urokinase plasminogen activator receptor (uPAR) was engineered to carry a GEM-handle containing a carboxy-terminal hexahistidine-tag and cysteine-tag. A two-step labeling strategy was optimized and applied to site-specifically label IRDye800CW and Tc to the sdAb. Bimodal labeling of the sdAbs proved straightforward and successful. Tc activity was however restricted to 18.5 MBq per nmol fluorescently-labeled sdAb to prevent radiobleaching of IRDye800CW without impeding SPECT/CT imaging. Subsequently, the biodistribution and tumor-targeting capacity of the bimodal tracer were evaluated in uPAR-positive tumor-bearing mice using SPECT/CT and fluorescence imaging. The bimodal sdAb showed expected renal background signals due to tracer clearance, along with slightly elevated non-specific liver signals. Four hours post-injection, both SPECT/CT and fluorescent images achieved satisfactory tumor uptake and contrast, with significantly higher values observed for the anti-uPAR bimodal sdAb compared to a control non-targeting sdAb. In conclusion, the GEM-handle is a convenient method for designing and producing bimodal sdAb-based tracers with adequate characteristics.