Preclinical characterization of 3p-C-DEPA-NCS and 3p-C-DEPA-TFP-PEG4 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 (1–20 µM) with high RCC’s (93.7 to 96.8%) after 1 hour 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-DOTA-trastuzumab and [ ²²⁵ Ac]Ac-macropa-trastuzumab in all organs of interest (p > 0.05 for all organs). However, the liver uptake of [ ²²⁵ Ac]Ac-DOTA-trastuzumab (14.1 ± 2.9% IA/g) was higher than [ ²²⁵ Ac]Ac-3p-C-DEPA-trastuzumab (9.0 ± 3.3% IA/g) (p = 0.04). Conclusions 3p- C -DEPA _− TFP -PEG₄ demonstrated excellent potential as a bifunctional chelator for ²²⁵Ac, 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.