Synthesis and evaluation of HOPO-O8-Methyl-tetrazine as a bifunctional chelator for use in [89Zr]Zr4+, [161Tb]Tb3+ and [227Th]Th4+ radioimmunoconjugates

The growing availability of new radiometals with favorable decay properties for cancer diagnosis and therapy highlights the need for chelators that can stably bind a variety of metal ions. 1-Hydroxy-2(1H)-pyridinones (1,2-HOPOs) are effective bidentate ligands with strong affinity for trivalent and tetravalent metals. In this study, we developed a bifunctional octadentate 1,2-HOPO chelator with a methyl tetrazine (Me-Tz) moiety, HOPO-O₈-Me-Tz, and evaluated its coordination to [⁸⁹Zr]Zr⁴⁺, [¹⁶¹Tb]Tb³⁺, and [²²⁷Th]Th⁴⁺ for theranostic applications. HOPO-O₈-Me-Tz was conjugated to HER2/neu targeting antibody Trastuzumab (Tmab), modified with transcyclooctene (TCO), using the inverse electron demand Diels-Alder (IEDDA) click reaction to yield HOPO-O₈-Tmab. Radiolabeled conjugates were synthesized under mild conditions (30 min, ambient temperature) and evaluated in vitro and in vivo in SKOV-3 tumour-bearing nude mice. All radiometal complexes demonstrated high stability in serum over 7 days. In vivo, [⁸⁹Zr]Zr-, [¹⁶¹Tb]Tb- and [²²⁷Th]Th-HOPO-O₈-Tmab showed high tumour uptake (9.87 ± 3.57, 11.29 ± 4.14 and 19.40 ± 5.40 %ID/g, respectively). Notably, [¹⁶¹Tb]Tb- and [²²⁷Th]Th-conjugates exhibited low bone uptake at 96 h post-injection, indicating excellent in vivo stability. The potential redistribution of the alpha-emitting daughter nuclide [²²³Ra]Ra²⁺ from [²²⁷Th]Th-HOPO-O₈-Tmab was assessed, revealing elevated ²²³Ra in the bone and joint. These findings underscore the promise of HOPO-O₈-Me-Tz as a versatile bifunctional chelator for next-generation theranostic radioimmunoconjugates, while also highlighting the importance of managing daughter radionuclide redistribution in alpha therapy.