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Drug-Resistant AML Cells and Primary AML Specimens Are Killed by ¹¹¹In-Anti-CD33 Monoclonal Antibodies Modified with Nuclear Localizing Peptide Sequences

¹ Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada; ² Department of Hematology and Oncology, University Health Network, University of Toronto, Toronto, Ontario, Canada; ³ Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Ontario, Canada; ⁴ Department of Medical Imaging, University of Toronto, Toronto, Ontario, Canada; and ⁵ Division of Pathophysiology and Clinical Investigation, Toronto General Research Institute, Toronto, Ontario, Canada

Correspondence: For correspondence or reprints contact: Raymond M. Reilly, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College St., Toronto, Ontario M5S 3M2. E-mail: Raymond.Reilly{at}utoronto.ca

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.

Key Words: acute myelogenous leukemia • ¹¹¹In • Auger electrons • CD33 • multidrug resistance

COPYRIGHT © 2008 by the Society of Nuclear Medicine, Inc.

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