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Radioimmunotherapy using 225Ac, a highly cytotoxic α-particle emitter, has potential for treating advanced breast cancer, especially human epidermal growth factor receptor 2 (HER2)–positive cases. We use a pretargeted radioimmunotherapy (PRIT) approach consisting of a 3-step intravenous regimen (step 1: bispecific anti-HER2/anti-DOTA antibody; step 2: clearing agent; step 3: 225Ac-radiolabeled Proteus DOTA, or [225Ac]Ac-Pr). Our goal was to establish curative 225Ac-PRIT with high therapeutic indices. Methods: The impact of [225Ac]Ac-Pr specific activity was evaluated in the BT-474 breast xenograft model. We tested the effects of [225Ac]Ac-Pr dosing during PRIT on tumor-targeting efficiency and tissue biodistribution. Using a 225Ac-PRIT regimen consisting of a ratio of 1.19 nmol of bispecific antibody to 0.60–0.66 nmol of [225Ac]Ac-Pr, we evaluated therapy in the BT-474 model and a patient-derived xenograft model. BT-474-tumor–bearing mice were treated with 1 or 2 cycles of 225Ac-PRIT (37 kBq/cycle) separated by 1 wk. A dose escalation study was performed on the BT-474 model to establish an absorbed radiation dose of approximately 40 Gy (relative biological effectiveness [RBE], 5) as a nephrotoxic dose, as no such histologic findings were observed in prior studies at the 20.7-Gy (RBE, 5) renal dose level. Results: In the BT-474 model, 100% (20/20) achieved complete responses and histologic cure in 17 of 20 (85%) of the treated animals. One-cycle and 2-cycle treatments were equally effective. Treatments were well tolerated, with no chronic radiation toxicity documented during necropsy at 175 d. Dosimetry estimates (RBE, 5) per 37 kBq administered for tumors and kidneys were 210 and 3.5 Gy, respectively. In the patient-derived xenograft model, a single 225Ac-PRIT treatment led to 60% (3/5) complete response and prolonged survival (>93 d) versus no treatment (30 d; P = 0.0185). Lastly, a 225Ac-PRIT regimen was identified that induces severe chronic nephrotoxicity (41.4

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