Preclinical Evaluation of [225Ac]Ac-Sibrotuzumab Employing a PEG4-Macropa Site-Specific Chelation Strategy for Targeted Ablation of Cancer-Associated Fibroblasts in Desmoplastic Tumors

Fibroblast activation protein is highly expressed in tumor-associated fibroblasts and represents a promising stromal target for Cancer therapy. Actinium-225 (²²⁵Ac) 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 [²²⁵Ac]Ac-Macropa-PEG₄-Sibrotuzumab. Sibrotuzumab was site-specifically modified with Macropa-PEG₄ and radiolabeled with ²²⁵Ac 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, [²²⁵Ac]Ac-Macropa-PEG₄-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.