Preclinical Evaluation of [ 225 Ac]Ac–Sibrotuzumab Employing a PEG 4 –Macropa Site‐Specific Chelation Strategy for Targeted Ablation of Cancer‐Associated Fibroblasts in Desmoplastic Tumors

ABSTRACT Fibroblast activation protein is highly expressed in tumor‐associated fibroblasts and represents a promising stromal target for cancer therapy. Actinium‐225 ( 225 Ac) offers potent α‐emissions for targeted radiotherapy but requires exceptionally stable chelation. We report the synthesis and preclinical evaluation of the novel FAP‐targeted α‐immunoconjugate [ 225 Ac]Ac–Macropa–PEG 4 –Sibrotuzumab. Sibrotuzumab was site‐specifically modified with Macropa–PEG 4 and radiolabeled with 225 Ac under mild conditions. Radiochemical purity, serum stability, and immunoreactivity were assessed by radio‐iTLC, SEC–HPLC, and FAP + /FAP − cell assays. In vivo biodistribution and therapeutic efficacy were evaluated in NIH/3T3‐FAP + xenograft–bearing mice. The radioconjugate was obtained in 78.5% ± 2.2% yield with > 98% radiochemical purity and > 90% stability over 7 days. The immunoreactive fraction was 88.7%, and the construct demonstrated strong FAP‐specific uptake with ~30% internalization at 24 h. In vivo, [ 225 Ac]Ac–Macropa–PEG 4 –Sibrotuzumab showed high tumor accumulation (24.1 ± 1.6 %ID/g at 168 h), rapid clearance from normal tissues, and minimal bone uptake. Treatment achieved ~62% tumor growth inhibition, prolonged survival beyond 28 days, and induced partial (50%) or complete (17%) responses without systemic toxicity. These findings highlight Macropa as a robust chelation strategy and support Sibrotuzumab‐based α‐immunoconjugates for precise targeting of FAP‐positive desmoplastic tumors.