Synthesis and preclinical development of a novel ⁶⁸Ga/⁸⁹Zr-Labelled ανβ6-integrin targeting trimer

The αvβ6 integrin has emerged as a valuable target for theranostic applications in nuclear medicine with high applicability across a variety of cancers, including head-and-neck, lung, breast, and pancreatic carcinomas. [⁶⁸Ga]Ga-Trivehexin is a prominent example of a diagnostic tracer targeting this integrin. In this work, we aimed to expand on this concept by developing FSC(PEG4-αvβ6)₃, a novel tracer that retains the Trivehexin design, but features PEGylated spacers and replaces the TRAP chelator with Fusarinine C (FSC), enabling labelling with Zirconium-89 in addition to Gallium-68. Preclinical characterization of [⁶⁸Ga]Ga/[⁸⁹Zr]Zr-FSC(PEG4-αvβ6)₃ included affinity determination towards the αvβ6 integrin and cellular uptake studies in αvβ6-positive H2009 cells. A subcutaneously xenografted H2009 tumor model was used to assess the PET imaging potential and biodistribution at early time points with the Gallium-68 labelled compound, and at later time points (up to 6 days post-injection) with the Zirconium-89 labelled version.

While [⁶⁸Ga]Ga-FSC(PEG4-αvβ6)₃ exhibited moderate binding to αvβ6, its affinity, cellular internalization, and tumor uptake in vivo were lower compared to [⁶⁸Ga]Ga-Trivehexin. Notably, this decreased target engagement was associated with reduced nonspecific binding, which we primarily attributed to the incorporation of PEGylated linkers. Despite indication of in vivo degradation of [⁸⁹Zr]Zr-FSC(PEG4-αvβ6)₃, still a meaningful evaluation of pharmacokinetics and biodistribution at extended time points was feasible, revealing prolonged tumor persistence up to 6 days post-injection.

FSC(PEG4-αvβ6)₃ represents the first-generation tracer targeting the αvβ6 integrin based on the multifunctional chelator Fusarinine C, thereby expanding the repertoire of radionuclides applicable from Gallium-68 to Zirconium-89. Following further optimization, this novel class of compounds holds significant promise for enabling clinical translation and advancing the development of next-generation αvβ6-directed imaging agents.