H₂BZmacropa-NCS: A Bifunctional Chelator for Actinium-225 Targeted Alpha Therapy

Actinium-225 (²²⁵Ac) is one of the most promising radionuclides for targeted alpha therapy (TAT). With a half-life of 9.92 days and a decay chain that emits four high-energy α particles, ²²⁵Ac is well-suited for TAT when conjugated to macromolecular targeting vectors that exhibit extended in vivo circulation times. The implementation of ²²⁵Ac in these targeted constructs, however, requires a suitable chelator that can bind and retain this radionuclide in vivo. Previous work has demonstrated the suitability of a diaza-18-crown-6 macrocyclic chelator H₂macropa for this application. Building upon these prior efforts, in this study, two rigid variants of H₂macropa, which contain either one (H₂BZmacropa) or two (H₂BZ₂macropa) benzene rings within the macrocyclic core, were synthesized and investigated for their potential use for ²²⁵Ac TAT. The coordination chemistry of these ligands with La³⁺, used as a nonradioactive model for Ac³⁺, was carried out. Both NMR spectroscopic and X-ray crystallographic studies of the La³⁺ complexes of these ligands revealed similar structural features to those found for the related complex of H₂macropa. Thermodynamic stability constants of the La³⁺ complexes, however, were found to be 1 and 2 orders of magnitude lower than those of H₂macropa for H₂BZmacropa and H₂BZ₂macropa, respectively. The decrease in thermodynamic stability was rationalized via the use of density functional theory calculations. ²²⁵Ac radiolabeling and serum stability studies with H₂BZmacropa showed that this chelator compares favorably with H₂macropa. Based on these promising results, a bifunctional version of this chelator, H₂BZmacropa-NCS, was synthesized and conjugated to the antibody codrituzumab (GC33), which targets the liver cancer biomarker glypican-3 (GPC3). The resulting GC33-BZmacropa conjugate and an analogous GC33-macropa conjugate were evaluated for their ²²⁵Ac radiolabeling efficiencies, antigen-binding affinities, and in vivo biodistribution in HepG2 liver cancer tumor-bearing mice. Although both conjugates were comparably effective in their radiolabeling efficiencies, [²²⁵Ac]Ac-GC33-BZmacropa showed slightly poorer serum stability and biodistribution than [²²⁵Ac]Ac-GC33-macropa. Together, these results establish H₂BZmacropa-NCS as a new bifunctional chelator for the preparation of ²²⁵Ac radiopharmaceuticals.