The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery

Abstract

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

Site-Specific, Platform-Based Conjugation Strategy for the Synthesis of Dual-Labeled Immunoconjugates for Bimodal PET/NIRF Imaging of HER2-Positive Tumors

“…The strategy used for the design of this MOMIP relies on a lysine platform, which has been successfully used in the past for the synthesis of trivalent platform. ,,− Indeed, the lysine amino acid is an ideal scaffold for the synthesis of such trivalent platforms, as several lysines with orthogonal protecting groups are marketed for peptide synthesis and are easily accessible at affordable prices. The orthogonality of the different protecting groups thus allows for the sequential and selective amide couplings and deprotections to yield the final products in reasonable yield.…”

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

“…In comparison, the [ 111 In]In‐MSAP.2Rs15d sdAb- compound previously tested by Debie et al. did not show non-specific liver accumulation, most likely due to the use of a more hydrophilic Cy5-based fluorophore ( 27 ). Towards the future, exploration of alternative near-infrared fluorescent dyes possessing improved in vivo pharmacokinetic behavior, enhanced radiostability, and a structure that does not interfere with the tricarbonyl chemistry could further enhance the potential of GEM-based bimodal tracers ( 52 – 54 ).…”

“…The typical in vivo biodistribution of sdAb-based tracers is characterized by rapid renal clearance, leading to background signals mainly concentrated in the kidneys and bladder. This efficient clearance coupled with fast target recognition facilitates high-contrast imaging within 1 h p.i ( 27 , 34 , 36 , 49 – 52 ). The bimodal sdAb-GEM tracers presented in this study exhibit a comparable biodistribution profile except for a slightly elevated liver accumulation (approximately 2%ID/g).…”

“…Moreover, in comparison with peptides, sdAbs serve as a platform technology that allows straightforward generation of sdAbs targeting almost any biomarker of interest, along with predictable pharmacokinetics. Previous preclinical and clinical investigations involving sdAbbased tracers for fluorescent, nuclear or bimodal imaging have shown the potential of these targeting moieties (3,27,(32)(33)(34)(35)(36)(37)(38)(39). In this paper, we report on our GEM-handle labeling approach for achieving bimodal labeling of sdAb-based tracers, focusing on a sdAb previously designed to target the urokinase plasminogen activator receptor (uPAR) (40).…”

Emerging applications of nanobodies in cancer therapy

“…In , the anti-HER2 Nb 2Rs15d was modified with a fluorescent dye and a radioisotope, both incorporated into a single label to overcome tracer heterogeneity. Intraperitoneal ovarian cancer lesions could then succesfully be imaged via both single photon emission computed tomography (SPECT)/CT and intraoperative fluorescence imaging (Debie et al, 2021).…”