Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada.
Faculty of Arts and Sciences, University of Toronto, Toronto, Ontario M5S 3G3, Canada.
Mol Pharm. 2022 Oct 3;19(10):3652-3663. doi: 10.1021/acs.molpharmaceut.2c00457. Epub 2022 Aug 4.
Epidermal growth factor receptors (EGFR) are overexpressed in triple-negative breast cancer (TNBC) and are an attractive target for the development of theranostic radiopharmaceuticals. We studied anti-EGFR panitumumab labeled with In (panitumumab-DOTA-In) for SPECT/CT imaging and Meitner-Auger electron (MAE) radioimmunotherapy (RIT) of TNBC. Panitumumab-DOTA-In was bound, internalized, and routed to the nucleus in MCF7, MDA-MB-231/Luc, and MDA-MB-468 human breast cancer (BC) cells dependent on the EGFR expression level (1.5 × 10, 1.7 × 10, or 1.3 × 10 EGFR/cell, respectively). The absorbed dose in the nuclei of MCF7, MDA-MB-231/Luc, and MDA-MB-468 cells incubated with 4.4 MBq of panitumumab-DOTA-In (20 nM) was 1.20 ± 0.02, 2.2 ± 0.1, and 25 ± 2 Gy, respectively. The surviving fraction (SF) of MDA-MB-231/Luc cells treated with panitumumab-DOTA-In (10-300 nM; 1.5 MBq/μg) was reduced as the absorbed dose in the cell increased, with clonogenic survival reduced to an SF = 0.12 ± 0.05 at 300 nM corresponding to 12.7 Gy. The SFs of MDA-MB-468, MDA-MB-231/Luc, and MCF7 cells treated with panitumumab-DOTA-In (20 nM; 1.7 MBq/μg) were <0.01, 0.56 ± 0.05, and 0.67 ± 0.04, respectively. Unlabeled panitumumab had no effect on SF, and irrelevant IgG-DOTA-In only modestly reduced the SF of MDA-MB-231/Luc cells but not MCF7 or MDA-MB-468 cells. The cytotoxicity of panitumumab-DOTA-In was mediated by increased DNA double-strand breaks (DSB), cell cycle arrest at G2/M-phase and apoptosis measured by immunofluorescence detection by flow cytometry. MDA-MB-231/Luc tumors in the mammary fat pad (MFP) of NRG mice were clearly imaged with panitumumab-DOTA-In by microSPECT/CT at 4 days postinjection (p.i.), and biodistribution studies revealed high tumor uptake [18 ± 2% injected dose/g (% ID/g] and lower normal tissue uptake (<10% ID/g). Administration of up to 24 MBq (15 μg) of panitumumab-DOTA-In to healthy NRG mice caused no major hematological, renal, or hepatic toxicity with no decrease in body weight. Treatment of NOD SCID mice with MDA-MB-231 tumors with panitumumab-DOTA-In (22 MBq; 15 μg) slowed tumor growth. The mean time for tumors to reach a volume of ≥500 mm was 61 ± 5 days for RIT with panitumumab-DOTA-In compared to 42 ± 6 days for mice treated with irrelevant IgG-DOTA-In ( < 0.0001) and 35 ± 3 days for mice receiving 0.9% NaCl ( < 0.0001). However, tumors regrew at later time points. The median survival of mice treated with panitumumab-DOTA-In was 70 days versus 46 days for IgG-DOTA-In ( < 0.0001) or 40 days for 0.9% NaCl ( < 0.0001). We conclude that panitumumab-DOTA-In is a promising theranostic agent for TNBC. Increasing the administered amount of panitumumab-DOTA-In and/or combination with radiosensitizing PARP inhibitors used for treatment of patients with TNBC may provide a more durable response to RIT.
表皮生长因子受体（EGFR）在三阴性乳腺癌（TNBC）中过表达，是开发治疗性放射性药物的有吸引力的靶标。我们研究了抗 EGFR 帕尼单抗标记的铟（panitumumab-DOTA-In）用于 SPECT/CT 成像和 MEITNER-Auger 电子（MAE）放射免疫治疗（RIT）的 TNBC。panitumumab-DOTA-In 在 MCF7、MDA-MB-231/Luc 和 MDA-MB-468 人乳腺癌（BC）细胞中结合、内化并靶向细胞核，这取决于 EGFR 的表达水平（分别为 1.5×10、1.7×10 和 1.3×10 EGFR/细胞）。在 MCF7、MDA-MB-231/Luc 和 MDA-MB-468 细胞中孵育 4.4 MBq panitumumab-DOTA-In（20 nM）的细胞核中的吸收剂量分别为 1.20±0.02、2.2±0.1 和 25±2 Gy。用 panitumumab-DOTA-In（10-300 nM；1.5 MBq/μg）处理 MDA-MB-231/Luc 细胞的存活分数（SF）随着细胞内吸收剂量的增加而降低，当 300 nM 时，克隆存活分数降低至 SF=0.12±0.05，对应于 12.7 Gy。MDA-MB-468、MDA-MB-231/Luc 和 MCF7 细胞用 panitumumab-DOTA-In（20 nM；1.7 MBq/μg）处理的 SF 分别为<0.01、0.56±0.05 和 0.67±0.04。未标记的 panitumumab 对 SF 没有影响，而无关 IgG-DOTA-In 仅适度降低 MDA-MB-231/Luc 细胞的 SF，但不降低 MCF7 或 MDA-MB-468 细胞的 SF。panitumumab-DOTA-In 的细胞毒性是通过增加 DNA 双链断裂（DSB）、细胞周期阻滞在 G2/M 期和通过流式细胞术免疫荧光检测到的细胞凋亡来介导的。NRG 小鼠乳腺脂肪垫（MFP）中的 MDA-MB-231/Luc 肿瘤在注射后 4 天（p.i.）用 panitumumab-DOTA-In 进行 microSPECT/CT 清晰成像，生物分布研究显示高肿瘤摄取[18±2%注入剂量/克（% ID/g）]和较低的正常组织摄取（<10% ID/g）。向健康 NRG 小鼠给予高达 24 MBq（15 μg）的 panitumumab-DOTA-In 不会引起主要的血液学、肾脏或肝脏毒性，体重也没有下降。用 panitumumab-DOTA-In 治疗 MDA-MB-231 肿瘤的 NOD SCID 小鼠，肿瘤生长速度减慢。与用无关 IgG-DOTA-In 治疗的小鼠（<0.0001）和接受 0.9%NaCl 治疗的小鼠（<0.0001）相比，用 panitumumab-DOTA-In 治疗的小鼠肿瘤达到≥500 mm3 体积的时间中位数分别为 61±5 天和 42±6 天。然而，肿瘤在后期重新生长。用 panitumumab-DOTA-In 治疗的小鼠的中位生存期为 70 天，而用 IgG-DOTA-In 治疗的小鼠为 46 天（<0.0001）或用 0.9%NaCl 治疗的小鼠为 40 天（<0.0001）。我们得出结论，panitumumab-DOTA-In 是一种有前途的 TNBC 治疗性放射性药物。增加 panitumumab-DOTA-In 的给药量和/或与用于治疗 TNBC 患者的放射增敏性 PARP 抑制剂联合使用可能会提供对 RIT 的更持久反应。
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