经核定位肽序列修饰的¹¹¹铟标记抗CD33单克隆抗体可杀死耐药性急性髓系白血病细胞和原发性急性髓系白血病标本。

Drug-resistant AML cells and primary AML specimens are killed by 111In-anti-CD33 monoclonal antibodies modified with nuclear localizing peptide sequences.

作者信息

Kersemans Veerle, Cornelissen Bart, Minden Mark D, Brandwein Joseph, Reilly Raymond M

机构信息

Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.

出版信息

J Nucl Med. 2008 Sep;49(9):1546-54. doi: 10.2967/jnumed.107.047399. Epub 2008 Aug 14.

DOI:10.2967/jnumed.107.047399

PMID:18703602

Abstract

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.

摘要

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。