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Nuclear Localizing Sequences Promote Nuclear Translocation and Enhance the Radiotoxicity of the Anti-CD33 Monoclonal Antibody HuM195 Labeled with ¹¹¹In in Human Myeloid Leukemia Cells

¹ Division of Nuclear Medicine, University Health Network, Toronto, Ontario, Canada; ² Department of Hematology/Oncology, University Health Network, Toronto, Ontario, Canada; ³ Department of Cell and Molecular Biology and Department of Medical Oncology, University Health Network, Toronto, Ontario, Canada; ⁴ Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada; ⁵ Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; ⁶ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; ⁷ Department of Medical Imaging, University of Toronto, Toronto, Ontario, Canada; and ⁸ Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada

Correspondence: For correspondence or reprints contact: Raymond M. Reilly, PhD, Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, Ontario, M5S 2S2, Canada. E-mail: raymond.reilly{at}utoronto.ca

Our objective was to evaluate the toxicity of the anti-CD33 monoclonal antibody HuM195 modified with peptides (CGYGPKKKRKVGG) harboring the nuclear localizing sequence (NLS; underlined) of simian virus 40 large T antigen and labeled with ¹¹¹In against acute myeloid leukemia (AML) cells. Methods: HuM195 was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC) to introduce maleimide groups for reaction with NLS-peptides and then conjugated with diethylenetriaminepentaacetic acid for labeling with ¹¹¹In. The immunoreactivity of NLS-HuM195 was evaluated by its ability to displace the binding of ¹¹¹In-HuM195 to HL-60 leukemia cells. Nuclear localization was measured in HL-60 cells by subcellular fractionation. The antiproliferative effects of ¹¹¹In-NLS-HuM195 and ¹¹¹In-HuM195 on HL-60, U937, or K562 cells with high, intermediate, or minimal CD33 expression, respectively, were studied. The survival of HL-60 cells or patient AML specimens treated with ¹¹¹In-NLS-HuM195 or ¹¹¹In-HuM195 was studied. Normal tissue toxicity was evaluated in BALB/c mice injected intravenously with of 3.7 MBq (22 µg) of ¹¹¹In-NLS-HuM195 or ¹¹¹In-HuM195. Results: NLS-HuM195 exhibited relatively preserved CD33 binding affinity (dissociation constant [K_d] = 4.3 ± 1.7 x 10^–9 mol/L to 6.9 ± 1.3 x 10^–9 mol/L). Nuclear uptake increased from 10.5% ± 0.5% for ¹¹¹In-HuM195 to 28.5% ± 4.1% or 65.9% ± 1.5% for ¹¹¹In-HuM195 substituted with 4 or 8 NLS-peptides, respectively. The inhibitory concentrations of 50% (IC₅₀) and 90% (IC₉₀) for HL-60 cells treated with ¹¹¹In-NLS-HuM195 were 37 kBq per 10³ cells and 77–81 kBq per 10³ cells, respectively. The IC₅₀ and IC₉₀ values for ¹¹¹In-HuM195 were 92 kBq per 10³ cells and 203 kBq per 10³ cells. Growth inhibition was correlated with the level of CD33 expression. The survival of HL-60 cells was reduced from 232 ± 22 colonies (control) to 7 ± 1 colonies with 1.48 mBq per cell of ¹¹¹In-NLS-HuM195; no colonies were found at 3.33 mBq per cell. The surviving fraction decreased >2-fold in 7 of 9 AML specimens treated with an excess of ¹¹¹In-NLS-HuM195 and >10-fold in 2 of these specimens. There were no decreases in body weight or hematologic parameters or increases in alanine aminotransferase or creatinine in mice administered 3.7 MBq (22 µg) of ¹¹¹In-NLS-HuM195 or ¹¹¹In-HuM195. There was no morphologic damage to the liver or kidneys. Conclusion: We conclude that NLS-peptides routed ¹¹¹In-HuM195 to the nucleus of AML cells, where the emitted Auger electrons were lethal. ¹¹¹In-NLS-HuM195 is a promising targeted radiotherapeutic agent for AML.

Key Words: acute myelogenous leukemia • ¹¹¹In • nuclear localization sequences • simian virus 40 large T antigen • Auger electrons

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