[225Ac]Ac–anti-DLL3 targeted alpha therapy for neuroendocrine tumors: preclinical radiosynthesis, biodistribution, and therapeutic efficacy
摘要
High-grade neuroendocrine tumors (NETs) exhibit aggressive progression and limited response to existing systemic therapies. Delta-like ligand 3 (DLL3) is selectively overexpressed on high-grade NETs, offering an attractive target for precision radiotherapeutics. Targeted alpha therapy (TAT) with actinium-225 (225Ac) enables high-LET, short-range cytotoxicity ideal for eliminating resistant tumor populations. The aim was to develop, radiolabel, and preclinically evaluate a Macropa–PEG4–anti-DLL3 (rovalpituzumab) antibody conjugate labeled with 225Ac for NET-directed alpha therapy. Rovalpituzumab was conjugated via thiol-maleimide chemistry to Macropa-PEG4 and radiolabeled with 225Ac under mild conditions. Radiochemical purity, stability, and immunoreactivity were evaluated by SEC-HPLC and iTLC. Binding, internalization, apoptosis, and clonogenic assays were conducted in DLL3-positive H727 and NCI-H69 cells. Biodistribution, dosimetry, and therapy studies were performed in H727 and NCI-H69 xenograft mice. Conjugation yielded ~ 3.7 chelators/antibody with 85% recovery, and radiolabeling achieved > 98% purity with > 88% stability over 7 days in human serum. The radioconjugate exhibited high-affinity DLL3 binding (Kd ≈ 0.5 nM) and efficient internalization (78% and 56% total cell-associated activity at 24 h in H727 and NCI-H69 cells, respectively). In vitro, [225Ac]Ac–Macropa–PEG4–rovalpituzumab induced ~ 70% apoptosis and reduced clonogenic survival to ~ 15% at 5 kBq/mL with minimal non-targeted cytotoxicity. In vivo biodistribution studies showed tumor uptake of 19.5%ID/g at 24 h in H727 xenografts, with ~ 70% reduction in blocking cohorts (p < 0.01), confirming DLL3-mediated targeting. A single 250 kBq therapeutic dose induced substantial tumor regression and significantly extended median survival from 22–24 to 46 days (p < 0.001) with acceptable acute tolerability over 60 days. [225Ac]Ac–Macropa–PEG4–rovalpituzumab demonstrates favorable radiochemical properties (> 98% purity, > 88% seven-day stability), high-affinity DLL3 binding (Kd ≈ 0.5 nM), and potent DLL3-specific cytotoxicity in vitro. In H727 subcutaneous xenografts, a single 250 kBq dose induced tumor regression and significantly extended median survival (46 vs. 22–24 days, p < 0.001) with acceptable acute tolerability. These preclinical findings support further evaluation of DLL3-targeted alpha therapy for neuroendocrine tumors, with additional studies needed to assess long-term toxicity, dose optimization, and efficacy in clinically relevant tumor models.