TY - JOUR T1 - Nuclear Localizing Sequences Promote Nuclear Translocation and Enhance the Radiotoxicity of the Anti-CD33 Monoclonal Antibody HuM195 Labeled with <sup>111</sup>In in Human Myeloid Leukemia Cells JF - Journal of Nuclear Medicine JO - J Nucl Med SP - 827 LP - 836 VL - 47 IS - 5 AU - Paul Chen AU - Judy Wang AU - Kristin Hope AU - Liqing Jin AU - John Dick AU - Ross Cameron AU - Joseph Brandwein AU - Mark Minden AU - Raymond M. Reilly Y1 - 2006/05/01 UR - http://jnm.snmjournals.org/content/47/5/827.abstract N2 - 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 [Kd] = 4.3 ± 1.7 × 10−9 mol/L to 6.9 ± 1.3 × 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 &gt;2-fold in 7 of 9 AML specimens treated with an excess of 111In-NLS-HuM195 and &gt;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. ER -