title: 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.
abstract: 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 (111)In 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 (111)In. The immunoreactivity of NLS-HuM195 was evaluated by its ability to displace the binding of (111)In-HuM195 to HL-60 leukemia cells. Nuclear localization was measured in HL-60 cells by subcellular fractionation. The antiproliferative effects of (111)In-NLS-HuM195 and (111)In-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 (111)In-NLS-HuM195 or (111)In-HuM195 was studied. Normal tissue toxicity was evaluated in BALB/c mice injected intravenously with of 3.7 MBq (22 microg) of (111)In-NLS-HuM195 or (111)In-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 (111)In-HuM195 to 28.5% +/- 4.1% or 65.9% +/- 1.5% for (111)In-HuM195 substituted with 4 or 8 NLS-peptides, respectively. The inhibitory concentrations of 50% (IC(50)) and 90% (IC(90)) for HL-60 cells treated with (111)In-NLS-HuM195 were 37 kBq per 10(3) cells and 77-81 kBq per 10(3) cells, respectively. The IC(50) and IC(90) values for (111)In-HuM195 were 92 kBq per 10(3) cells and 203 kBq per 10(3) 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 (111)In-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 (111)In-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 microg) of (111)In-NLS-HuM195 or (111)In-HuM195. There was no morphologic damage to the liver or kidneys.
  
  
  CONCLUSIONS
  We conclude that NLS-peptides routed (111)In-HuM195 to the nucleus of AML cells, where the emitted Auger electrons were lethal. (111)In-NLS-HuM195 is a promising targeted radiotherapeutic agent for AML.
authors.affiliation: Division of Nuclear Medicine, University Health Network, Toronto, Ontario, Canada.
authors.forename: Paul
authors.initials: P
authors.lastname: Chen
authors.forename: Judy
authors.initials: J
authors.lastname: Wang
authors.forename: Kristin
authors.initials: K
authors.lastname: Hope
authors.forename: Liqing
authors.initials: L
authors.lastname: Jin
authors.forename: John
authors.initials: J
authors.lastname: Dick
authors.forename: Ross
authors.initials: R
authors.lastname: Cameron
authors.forename: Joseph
authors.initials: J
authors.lastname: Brandwein
authors.forename: Mark
authors.initials: M
authors.lastname: Minden
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Reilly
mesh_terms: D021581:Active Transport, Cell Nucleus; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000627:therapeutic use; D015703:Antigens, CD / Q000276:immunology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology*; D049109:Cell Proliferation; D002470:Cell Survival; D018922:HL-60 Cells; D006801:Humans; D007205:Indium Radioisotopes / Q000627:therapeutic use*; D020014:K562 Cells; D007951:Leukemia, Myeloid / Q000000981:diagnostic imaging* / Q000532:radiotherapy*; D019913:Nuclear Localization Signals; D010455:Peptides / Q000737:chemistry; D011877:Radionuclide Imaging; D063268:Sialic Acid Binding Ig-like Lectin 3; D020298:U937 Cells
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; C571165:Cd33 protein, mouse; D007205:Indium Radioisotopes; D019913:Nuclear Localization Signals; D010455:Peptides; D063268:Sialic Acid Binding Ig-like Lectin 3; C101579:monoclonal antibody M195