[²²⁵Ac]Ac-Macropa-PEG₄-FPA150 enables targeted alpha therapy of B7-H4-Expressing tumors

Actinium-225 (²²⁵Ac) based targeted alpha therapy (TAT) is highly promising for eliminating resistant tumor cells owing to its high linear energy transfer and short path length. However, its clinical translation remains limited by the availability of efficient tumor-targeting strategies and suitable chelation chemistry. The immune checkpoint molecule B7-H4, overexpressed in several aggressive cancers, represents an attractive therapeutic target. Herein, we report [²²⁵Ac]Ac-Macropa-PEG₄-FPA150, a radioimmunoconjugate designed for B7-H4 targeted TAT. FPA150 was conjugated to Macropa-PEG₄ and radiolabeled with ²²⁵Ac at room temperature using mild aqueous conditions. Radiochemical yield (RYC), purity (RCP), stability, and immunoreactivity were assessed, in vitro. Binding affinity and specificity were assessed using B7-H4-positive (MDA-MB-468, SKOV3) and negative (A549) cell lines. In vivo evaluation included biodistribution, blocking, dosimetry and therapeutic efficacy in tumor-bearing mice. Radiolabeling achieved a yield of 61.3 ± 1.9% with >95% purity. The radioconjugate revealed excellent stability (>95% in PBS and >92% in serum at 72 h) and high immunoreactivity (0.87). Strong and specific binding was observed (KD: 0.4-0.7 nM), with minimal uptake in B7-H4 negative cells and significant blocking (p < 0.001). Tumor accumulation peaked at 72 h (22.36 ± 1.92%ID/g), beside favorable tumor-to-organ ratios. Dosimetry revealed the highest absorbed dose in tumors (7.42 ± 0.78 Gy/MBq). A single administration resulted in dose-dependent tumor inhibition and prolonged survival without observable toxicity. These findings highlight [²²⁵Ac]Ac-Macropa-PEG₄-FPA150 as a promising candidate for B7-H4 targeted TAT and support Macropa as an effective chelator for ²²⁵Ac radiopharmaceuticals.