Mesothelin-targeted alpha therapy in PDAC with [²²⁵Ac]Ac-Macropa-PEG₆-Amatuximab

Pancreatic ductal adenocarcinoma (PDAC) continues to be deadly and resistant to traditional treatments. Overexpressed in >80% of PDACs, mesothelin is an ideal target for antibody-based α-therapy. Actinium-225 (225Ac) produces high-LET α-particles leading to irreparable DNA damage, but its utility has been compromised by unstable chelation with traditional ligands. Here, we engineered a Macropa-enabled, site-specifically [²²⁵Ac]Ac-Macropa-PEG₆-Amatuximab, a radioimmunoconjugate against mesothelin. Conjugation and labeling were characterized by MALDI-TOF and SEC-HPLC. In vitro stability, immunoreactivity, and kinetics of binding were tested in mesothelin-positive AsPC-1 cells and subsequently in vivo biodistribution, dosimetry, and therapy in AsPC-1 xenograft-bearing nude mice. Conjugation had an average ratio of 3.6 ± 0.1 for chelator per antibody, radiolabeling efficiency of 96.3 ± 1.1%, and radiochemical purity ≥98%. The radioconjugate was >92% stable after 168 h in serum, with immunoreactivity (82.2 ± 2.8%) and affinity (Kd = 4.3 ± 0.9 nM). It exhibited specific, time-dependent internalization in AsPC-1 cells and minimal nonspecific uptake. In vivo, [²²⁵Ac]Ac-Macropa-PEG₆-Amatuximab exhibited prolonged circulation, specific tumor localization (3.9 ± 0.5 to 16.3 ± 2.1% ID/g, 1-168 h), and enhanced tumor-to-blood ratios (0.21-3.40). Blocking with unlabeled Amatuximab decreased tumor uptake by >60%. The tumor absorbed dose (1.82 ± 0.14 Gy/MBq) was 4-20-fold greater than doses to normal organs. Therapeutically, it caused dose-dependent tumor regression (TGI: 58% at 50 kBq; 92% at 150 kBq) and prolonged survival (>60 days vs. 0-1% in controls, p < 0.001). [²²⁵Ac]Ac-Macropa-PEG₆-Amatuximab is stable, selective, and therapeutically effective, demonstrating Macropa-based 225Ac chelation as a stable platform for targeted α-therapy of PDAC.