Trastuzumab-conjugated oxine-based ligand for [⁸⁹Zr]Zr⁴⁺ immunoPET

A new, bifunctional chelating ligand for immuno-Positron Emission Tomography (PET) was designed, synthesized, and conjugated to Trastuzumab for a proof-of-concept study with ⁸⁹Zr. H₄neunox was synthesized from the tris(2-aminoethyl)amine backbone, decorated with 8-hydroxyquinoline moieties, and utilizes a primary amine for functionalization. A maleimide moiety extends the chelator to create H₄neunox-mal for antibody conjugation via maleimide-thiol click chemistry. Preliminary ⁸⁹Zr radiolabeling of H₄neunox indicated quantitative radiolabeling at 1 × 10^-5 M, but improved inertness towards human serum (96% intact at 7 d) and Fe³⁺ (92% intact at 24 h) compared to the previously synthesized H₅decaox. The chelator was successfully conjugated to the monoclonal antibody, Trastuzumab, and used in preliminary radiolabeling reactions (37 °C, 2 h) with ⁸⁹Zr. Radiochemical assessments of the new H₄neunox-Trastuzumab conjugate include ⁸⁹Zr radiolabeling, spin filter purification, cell-binding immunoreactivity, and in vivo PET imaging and biodistribution in SKOV-3 tumour bearing nude mice, performed in comparison with the desferrioxamine B analog, DFO-Trastuzumab. The [⁸⁹Zr]Zr(neunox-Trastuzumab) showed lowered inertness towards serum (76% intact at 24 h) as well as demetallation in vivo through bone uptake (21% ID/g) in PET imaging and biodistribution studies when compared to [⁸⁹Zr]Zr(DFO-Trastuzumab). Although the combination of the chelator and antibody had detrimental effects on their intended purposes, nonetheless, the primary amine platform of H₄neunox developed here provides an oxine-based bifunctional ligand for further derivatizations with other targeting vectors.