Preclinical characterization of 3p-C-DEPA(-NCS) and 3p-C-DEPA(-TFP)-PEG(4) as potential Actinium-225 bifunctional chelators using DOTA(-NCS) and macropa(-NCS) as benchmarks.

BACKGROUND: Actinium-225 ((225)Ac) based targeted alpha therapies (TAT) have emerged as a promising strategy for the treatment of several cancer types due to its favourable decay properties, including high linear energy transfer and short particle range, which enable precise tumour targeting. However, there are limited bifunctional chelators (BFCs) available for (225)Ac. In this study, we aim to evaluate the potential of DEPA-based chelators for (225)Ac-labelling. RESULTS: The BFCs 3p-C-DEPA-(NO2), 3p-C-DEPA-(NCS), and 3p-C-DEPA(-TFP)-PEG(4) were synthesized with high yield (≥ 86%) and purity (> 96%). Excellent radiochemical conversions (RCCs) were achieved for [(225)Ac]Ac-3p-C-DEPA-(NO2) across a range of concentrations (0.7- 13.4 µg) with high RCC's (93.7 to 96.8%) after 1 h at room temperature. Stability studies demonstrated that over 95% of this (225)Ac-labelled complex remained intact after 6 days in human serum. The HPLC and bioanalyzer analysis of the immunoconjugates 3p-C-DEPA(-TFP)-PEG(4)-trastuzumab, DOTA-trastuzumab, 3p-C-DEPA-trastuzumab and macropa-trastuzumab showed 98% purity with less than 2% impurities. A RCC of 94.6% was obtained for [(225)Ac]Ac-3p-C-DEPA-trastuzumab, 93.5% for [(225)Ac]Ac-3p-C-DEPA(-TFP)-PEG(4)-trastuzumab, 80.9% for [(225)Ac]Ac-DOTA-trastuzumab, and 96.5% for [(225)Ac]Ac-macropa-trastuzumab after 2 h incubation at 37 °C. In PBS, high stability of [(225)Ac]Ac-3p-C-DEPA(-TFP)-PEG(4)-trastuzumab was observed (91.3 ± 4.3%), which is comparable to that of [(225)Ac]Ac-macropa-trastuzumab (81.9 ± 5.6%). In contrast, [(225)Ac]Ac-3p-C-DEPA-trastuzumab and [(225)Ac]Ac-DOTA-trastuzumab were less stable in PBS with only 48.3 ± 1.2% and 60.1 ± 0.6% intact tracer left after 10 d. There were no major significant differences between the biodistribution profile of [(225)Ac]Ac-3p-C-DEPA-trastuzumab, [(225)Ac]Ac-3p-C-DEPA(-TFP)-PEG(4)-trastuzumab, [(225)Ac]Ac-DOTA-trastuzumab and [(225)Ac]Ac-macropa-trastuzumab in all organs of interest (p > 0.05 for all organs). CONCLUSIONS: 3p-C-DEPA(-TFP)-PEG₄ demonstrated excellent potential as a bifunctional chelator for (225)Ac, showing high radiolabelling efficiency under mild conditions and outstanding in vitro stability of the resulting (225)Ac-labelled bioconjugate. Further preclinical studies are warranted to validate its therapeutic potential.