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Hybrid imaging combining the beneficial properties of radioactivity and optical imaging within one imaging probe has gained increasing interest in radiopharmaceutical research. In this study, we modified the macrocyclic gallium-68 chelator fusarinine C (FSC) by conjugating a fluorescent moiety and tetrazine (Tz) moieties. The resulting hybrid imaging agents were used for pretargeting applications utilizing click reactions with a trans -cyclooctene (TCO) tagged targeting vector for a proof of principle both in vitro and in vivo. Starting from FSC, the fluorophores Sulfocyanine-5, Sulfocyanine-7, or IRDye800CW were conjugated, followed by introduction of one or two Tz motifs, resulting in mono and dimeric Tz conjugates. Evaluation included fluorescence microscopy, binding studies, logD, protein binding, in vivo biodistribution, µPET (micro-positron emission tomography), and optical imaging (OI) studies. 68 Ga-labeled conjugates showed suitable hydrophilicity, high stability, and specific targeting properties towards Rituximab-TCO pre-treated CD20 expressing Raji cells. Biodistribution studies showed fast clearance and low accumulation in non-targeted organs for both SulfoCy5- and IRDye800CW-conjugates. In an alendronate-TCO based bone targeting model the dimeric IRDye800CW-conjugate resulted in specific targeting using PET and OI, superior to the monomer. This proof of concept study showed that the preparation of FSC-Tz hybrid imaging agents for pretargeting applications is feasible, making such compounds suitable for hybrid imaging applications.

Fusarinine C (FSC) has recently been shown to be a promising and novel chelator for 89 Zr. Here, FSC has been further derivatized to optimize the complexation properties of FSC-based chelators for 89 Zr-labeling by introducing additional carboxylic groups. These were expected to improve the stability of 89 Zr-complexes by saturating the 8-coordination sphere of [ 89 Zr] Zr 4+ , and also to introduce functionalities suitable for conjugation to targeting vectors such as monoclonal antibodies. For proof of concept, succinic acid derivatization at the amine groups of FSC was carried out, resulting in FSC(succ)₂ and FSC(succ)₃. FSC(succ)₂ was further derivatized to FSC(succ)₂ AA by reacting with acetic anhydride (AA). The Zr 4+ complexation properties of these chelators were studied by reacting with ZrCl₄. Partition coefficient, protein binding, serum stability, acid dissociation, and transchelation studies of 89 Zr-complexes were carried out in vitro and the results were compared with those for 89 Zr-desferrioxamine B ([ 89 Zr]Zr-DFO) and 89 Zr-triacetylfusarinine C ([ 89 Zr]Zr-TAFC). The in vivo properties of [ 89 Zr]Zr-FSC(succ)₃ were further compared with [ 89 Zr]Zr-TAFC in BALB/c mice using micro-positron emission tomography/computer tomography (microPET/CT) imaging. Fusarinine C (succ)₂AA and FSC(succ)₃ were synthesized with satisfactory yields. Complexation with ZrCl₄ was achieved using a simple strategy resulting in high-purity Zr-FSC(succ)₂AA and Zr-FSC(succ)₃ with 1:1 stoichiometry. Distribution coefficients of 89 Zr-complexes revealed increased hydrophilic character compared to [ 89 Zr]Zr-TAFC. All radioligands showed high stability in phosphate buffered saline (PBS) and human serum and low protein-bound activity over a period of seven days. Acid dissociation and transchelation studies exhibited a range of in vitro stabilities following the order: [ 89 Zr]Zr-FSC(succ)₃ > [ 89 Zr]Zr-TAFC > [ 89 Zr]Zr-FSC(succ)₂AA >> [ 89 Zr]Zr-DFO. Biodistribution studies of [ 89 Zr]Zr-FSC(succ)₃ revealed a slower excretion pattern compared to [ 89 Zr]Zr-TAFC. In conclusion, [ 89 Zr]Zr-FSC(succ)₃ showed the best stability and inertness. The promising results obtained with [ 89 Zr]Zr-FSC(succ)₂AA highlight the potential of FSC(succ)₂ as a monovalent chelator for conjugation to targeted biomolecules, in particular, monoclonal antibodies.