Kersemans Veerle, Cornelissen Bart, Minden Mark D, Brandwein Joseph, Reilly Raymond M
Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
J Nucl Med. 2008 Sep;49(9):1546-54. doi: 10.2967/jnumed.107.047399. Epub 2008 Aug 14.
UNLABELLED: Multidrug resistance (MDR) is a major challenge to the successful treatment of acute myeloid leukemia (AML). Our purpose was to determine whether (111)In-HuM195 anti-CD33 antibodies modified with peptides harboring nuclear localizing sequences (NLS) could kill drug-resistant AML cell lines and primary AML patient specimens expressing MDR transporters through the emission of Auger electrons. METHODS: HuM195, M195, and irrelevant mouse IgG (mIgG) were conjugated to 10+/-3 NLS peptides and then labeled with (111)In by diethylenetriaminepentaacetic acid substitution to a specific activity of 1-8 MBq/microg. The binding affinity of HuM195 and M195 was determined for HL-60 and mitoxantrone-resistant HL-60-MX-1 cells. Nuclear localization of (111)In-NLS-HuM195, (111)In-NLS-M195, (111)In-HuM195, and (111)In-M195 was measured by subcellular fractionation. The antiproliferative effects of (111)In-NLS-HuM195, (111)In-NLS-M195, (111)In-HuM195, and (111)In-M195 (2.5-250 kBq/well) on HL-60 and HL-60-MX-1 were studied using the WST-1 assay. Clonogenic survival of HL-60 and HL-60-MX-1 leukemic cells and 10 primary AML specimens with MDR phenotype (assessed by flow cytometry) was determined after exposure for 3 h at 37 degrees C to 2.5-250 mBq/cell of (111)In-NLS-HuM195, (111)In-HuM195, or (111)In-NLS-mIgG. Clonogenic survival versus the amount of radioactivity incubated with the cells (mBq/cell) was plotted, and the mean lethal amount of radioactivity and the lower asymptote of the curve (plateau) were determined. RESULTS: The (111)In-labeled anti-CD33 monoclonal antibodies HuM195 and M195 modified with NLS were efficiently routed to the nucleus of HL-60 cells and their mitoxantrone-resistant clone after CD33-mediated internalization. The following are the principal findings of our study: (111)In-NLS-HuM195 was more effective at killing HL-60 and HL-60-MX-1 cells than was (111)In-HuM195, a strong correlation between the specific activity of the (111)In-labeled radioimmunoconjugates and their cytotoxicity toward AML cells existed, and leukemic cells from patients were killed by (111)In-NLS-M195 or (111)In-M195, but the cytotoxic response among specimens was heterogeneous. CONCLUSION: NLS conjugation enhanced the nuclear uptake and cytotoxicity of (111)In-HuM195 and (111)In-M195 toward drug-resistant AML cell lines as well as patient specimens expressing a diversity of MDR phenotypes, including Pgp-170, BCRP1, or MRP1 transporters. Targeted Auger electron radioimmunotherapy using (111)In-labeled anti-CD33 monoclonal antibodies modified with NLS may be able to overcome MDR and provide a means of treating chemotherapy-resistant myeloid leukemias in patients.
未标记：多药耐药（MDR）是成功治疗急性髓系白血病（AML）的主要挑战。我们的目的是确定用含有核定位序列（NLS）的肽修饰的（111）In-HuM195抗CD33抗体是否可以通过俄歇电子发射杀死表达MDR转运蛋白的耐药AML细胞系和原发性AML患者标本。 方法：将HuM195、M195和无关的小鼠IgG（mIgG）与10±3个NLS肽偶联，然后通过二乙三胺五乙酸取代用（111）In标记，比活度为1-8 MBq/μg。测定HuM195和M195对HL-60和米托蒽醌耐药的HL-60-MX-1细胞的结合亲和力。通过亚细胞分级分离测量（111）In-NLS-HuM195、（111）In-NLS-M195、（111）In-HuM195和（111）In-M195的核定位。使用WST-1试验研究（111）In-NLS-HuM195、（111）In-NLS-M195、（111）In-HuM195和（111）In-M195（2.5-250 kBq/孔）对HL-60和HL-60-MX-1的抗增殖作用。在37℃下将HL-60和HL-60-MX-1白血病细胞以及10个具有MDR表型（通过流式细胞术评估）的原发性AML标本暴露于2.5-250 mBq/细胞的（111）In-NLS-HuM195、（111）In-HuM195或（111）In-NLS-mIgG 3小时后，测定其克隆形成存活率。绘制克隆形成存活率与与细胞孵育的放射性量（mBq/细胞）的关系图，并确定放射性的平均致死量和曲线的下渐近线（平台期）。 结果：用NLS修饰的（111）In标记的抗CD33单克隆抗体HuM195和M195在CD33介导的内化后有效地进入HL-60细胞及其米托蒽醌耐药克隆的细胞核。以下是我们研究的主要发现：（111）In-NLS-HuM195在杀死HL-60和HL-60-MX-1细胞方面比（111）In-HuM195更有效，（111）In标记的放射免疫缀合物的比活度与其对AML细胞的细胞毒性之间存在强相关性，并且患者的白血病细胞被（111）In-NLS-M195或（111）In-M195杀死，但标本之间的细胞毒性反应是异质的。 结论：NLS偶联增强了（111）In-HuM195和（111）In-M195对耐药AML细胞系以及表达多种MDR表型（包括Pgp-170、BCRP1或MRP1转运蛋白）的患者标本的核摄取和细胞毒性。使用用NLS修饰的（111）In标记的抗CD33单克隆抗体进行靶向俄歇电子放射免疫治疗可能能够克服MDR，并为治疗患者的化疗耐药性髓系白血病提供一种方法。
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