title: 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 (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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Veerle
authors.initials: V
authors.lastname: Kersemans
authors.forename: Bart
authors.initials: B
authors.lastname: Cornelissen
authors.forename: Mark D
authors.initials: MD
authors.lastname: Minden
authors.forename: Joseph
authors.initials: J
authors.lastname: Brandwein
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Reilly
mesh_terms: D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D000970:Antineoplastic Agents; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects*; D016503:Drug Delivery Systems / Q000379:methods*; D018432:Drug Resistance, Multiple / Q000187:drug effects; D006801:Humans; D007205:Indium Radioisotopes / Q000627:therapeutic use*; D015470:Leukemia, Myeloid, Acute / Q000000981:diagnostic imaging / Q000532:radiotherapy*; D055629:Natural Cytotoxicity Triggering Receptor 2; D019913:Nuclear Localization Signals / Q000627:therapeutic use*; D011877:Radionuclide Imaging; D011971:Receptors, Immunologic / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D007205:Indium Radioisotopes; C112837:NCR2 protein, human; D055629:Natural Cytotoxicity Triggering Receptor 2; D019913:Nuclear Localization Signals; D011971:Receptors, Immunologic