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

Abstract

Multidrug resistance (MDR) is a major challenge to the successful treatment of acute myeloid leukemia (AML). Our purpose was to determine whether 111In-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 111In by diethylenetriaminepentaacetic acid substitution to a specific activity of 1-8 MBq/μg. The binding affinity of HuM195 and M195 was determined for HL-60 and mitoxantrone-resistant HL-60-MX-1 cells. Nuclear localization of 111In-NLS-HuM195, 111In-NLS-M195, 111In-HuM195, and 111In-M195 was measured by subcellular fractionation. The antiproliferative effects of 111In-NLS-HuM195, 111In-NLS- M195, 111In-HuM195, and 111In-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°C to 2.5-250 mBq/cell of 111In-NLS-HuM195, 111In-HuM195, or 111In-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 111In-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: 111In-NLS-HuM195 was more effective at killing HL-60 and HL-60-MX-1 cells than was 111In-HuM195, a strong correlation between the specific activity of the 111In-labeled radioimmunoconjugates and their cytotoxicity toward AML cells existed, and leukemic cells from patients were killed by 111In-NLS-M195 or 111In-M195, but the cytotoxic response among specimens was heterogeneous. Conclusion: NLS conjugation enhanced the nuclear uptake and cytotoxicity of 111In-HuM195 and 111In-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 111In-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. Copyright © 2008 by the Society of Nuclear Medicine, Inc.