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

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...

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Bibliographic Details
Published inFrontiers in immunology Vol. 14; p. 1285923
Main Authors Declerck, Noemi B, Huygen, Celine, Mateusiak, Lukasz, Stroet, Marcus C M, Hernot, Sophie
Format Journal Article
LanguageEnglish
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
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Summary: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.
ISSN:1664-3224