**Hybrid Imaging Agents for Pretargeting Applications Based on Fusarinine C—Proof of Concept**

**Dominik Summer <sup>1</sup> , Milos Petrik <sup>2</sup> , Sonja Mayr <sup>1</sup> , Martin Hermann <sup>3</sup> , Piriya Kaeopookum <sup>1</sup> , Joachim Pfister <sup>1</sup> , Maximilian Klingler <sup>1</sup> , Christine Rangger <sup>1</sup> , Hubertus Haas <sup>4</sup> and Clemens Decristoforo 1,\***


Academic Editors: Alessandra Boschi and Petra Martini Received: 17 April 2020; Accepted: 29 April 2020; Published: 1 May 2020

**Abstract:** 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. <sup>68</sup>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.

**Keywords:** fusarinine C; click chemistry; fluorescence; optical imaging; PET; gallium-68
