A convenient labeling strategy for the preparation of fluorescent/radioactive sdab-based tracers carrying 99mTc and IRDye800CW for intra-operative decision making

Noemi Declerck, Celine Marina Huygen, Lukasz Mateusiak, Marcus Stroet, Sophie Hernot

Onderzoeksoutput: Unpublished abstract

Samenvatting

Cancer surgery is applied in the treatment of solid tumors and aims for the total removal of tumor lesions and complete remission of the patient. Yet, intra-operative identification of tumor margins remains difficult, frequently resulting in positive resection margins and a necessity for adjuvant therapies. Intra-operative guidance using fluorescence molecular imaging has been proven to improve the identification of tumor margins and thus support complete resections. However, insufficient tissue penetration of the fluorescent signal hinders the identification of deep or occult tumor lesions. The combination of fluorescence imaging with intra-operative gamma-probing provides an ideal balance of the deep signal penetration of gamma rays for tumor localization and a fluorescent signal for precise tumor resection. Preferentially, both modalities are combined in a site-specifically labeled bimodal single-label tracer. In this study we propose a convenient way to label targeting moieties, such as single-domain antibodies (sdab), with 99mTc and IRDye800CW. 99mTc is a gamma-emitting radioisotope which can be used pre- and intra-operatively for tumor detection using SPECT/CT or gamma-probing respectively. The near infra-red dye IRDye800CW shows high in vivo signals enabling accurate identification of tumor margins.
An anti-uPAR and a control sdab were engineered to carry a carboxy-terminal hexahistidine- and cysteine-tag separated by a 14 amino acid linker. First, sdab were site-specifically labeled with IRDye800CW-maleimide on the cysteine-tag and subsequently with 99mTc[Tc](CO)3 on the hexahistidine-tag. Quality controls were conducted for each step via size-exclusion chromatography and hydrophobic interaction chromatography. The influence of the bimodal label on in vivo biodistribution and tumor targeting was evaluated. Tumor-bearing mice were imaged using SPECT/CT and fluorescence imaging after intravenous injection of 2 nmol of labeled compound. Ex vivo analysis on organs of interest was performed using fluorescence imaging and gamma-counting.
The site-specific labeling of the sdab with IRDye800CW and subsequently 99mTc[Tc](CO)3 was successful. The labeling strategy was shown to be influenced by the amount of activity used in the second labeling step. Labeling of 25 µg of the sdab-IRDye800CW construct with up to 74 MBq 99mTc[Tc](CO)3 resulted in good preservation of the fluorescent signal. Higher activities led to a decrease in or the loss of the fluorescent signal. Up-scalability of the labeling was proven to be possible via labeling 200 µg of the sdab-IRDye800CW with 220 MBq 99mTc[Tc](CO)3 for the in vivo experiments without any consequences for the fluorescent signal. The IRDye800CW/99mTc tracers showed an in vivo and ex vivo biodistribution profile with rapid blood clearance, renal clearance, no unexpected background signals, and specific tumor uptake for the anti-uPAR sdab as compared to a non-targeting control sdab. Thus, the bimodal label shows no signs of influencing the in vivo biodistribution of the tracer negatively.
The bimodal labeling was successfully optimized and could be implemented as a standard bimodal labeling strategy for other sdab. Furthermore, the in vivo biodistribution of the tracer is not negatively influenced by the addition of the bimodal label. Therefore, this new bimodal fluorescent/radioactive sdab-based tracer has the potential to be translated and implemented into clinical practice.
Originele taal-2English
StatusUnpublished - 18 jan 2023

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