title: (111)In-labeled trastuzumab (Herceptin) modified with nuclear localization sequences (NLS): an Auger electron-emitting radiotherapeutic agent for HER2/neu-amplified breast cancer.
abstract: The cytotoxicity and tumor-targeting properties of the anti-HER2/neu monoclonal antibody trastuzumab modified with peptides (CGYGPKKKRKVGG) harboring the nuclear localization sequence ([NLS] italicized) of simian virus 40 large T-antigen and radiolabeled with (111)In were evaluated.
  
  
  METHODS
  Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) for reaction with NLS-peptides and labeled with (111)In using diethylenetriaminepentaacetic acid (DTPA). The immunoreactivity of (111)In-NLS-trastuzumab was determined by its ability to displace the binding of trastuzumab to SK-BR-3 human breast cancer (BC) cells. Cellular uptake and nuclear localization were evaluated in SK-BR-3, MDA-MB-361, and MDA-MB-231 BC cells, expressing high, intermediate, or very low levels of HER2/neu, respectively, by cell fractionation and confocal microscopy. Biodistribution and nuclear uptake were compared in athymic mice bearing MDA-MB-361 xenografts. The cytotoxicity of (111)In-trastuzumab and (111)In-NLS-trastuzumab was studied by clonogenic assays, and DNA damage was assessed by probing for phosphorylated histone H2AX (gammaH2AX) foci.
  
  
  RESULTS
  The dissociation constant for binding of (111)In-NLS-trastuzumab to SK-BR-3 cells was reduced <3-fold compared with that of (111)In-trastuzumab, demonstrating relatively preserved receptor-binding affinity. The receptor-mediated internalization of (111)In-trastuzumab in SK-BR-3, MDA-MB-361, and MDA-MB-231 cells increased significantly from 7.2% +/- 0.9%, 1.3% +/- 0.1%, and 0.2% +/- 0.05% to 14.4% +/- 1.8%, 6.3% +/- 0.2%, and 0.9% +/- 0.2% for (111)In-NLS-trastuzumab harboring 6 NLS-peptides, respectively. NLS-trastuzumab localized in the nuclei of BC cells, whereas unmodified trastuzumab remained surface-bound. Conjugation of (111)In-trastuzumab to NLS-peptides did not affect its tissue biodistribution but promoted specific nuclear uptake in MDA-MB-361 xenografts (2.4-2.9 %ID/g [percentage injected dose per gram] for (111)In-NLS-trastuzumab and 1.1 %ID/g for (111)In-trastuzumab). (111)In-NLS-trastuzumab was 5- and 2-fold more potent at killing SK-BR-3 and MDA-MB-361 cells than (111)In-trastuzumab, respectively, whereas toxicity toward MDA-MB-231 cells was minimal. (111)In-NLS-trastuzumab was 6-fold more effective at killing SK-BR-3 cells than unlabeled trastuzumab. Formation of gammaH2AX foci occurred in a greater proportion of BC cells after incubation with (111)In-NLS-trastuzumab compared with (111)In-trastuzumab or unlabeled trastuzumab.
  
  
  CONCLUSIONS
  NLS-peptides routed (111)In-trastuzumab to the nucleus of HER2/neu-positive human BC cells, rendering the radiopharmaceutical lethal to the cells through the emission of nanometer-micrometer range Auger electrons. The greater cytotoxic potency of (111)In-NLS-trastuzumab compared with unlabeled trastuzumab in vitro and its favorable tumor-targeting properties in vivo suggest that it could be an effective targeted radiotherapeutic agent for HER2/neu-amplified BC in humans.
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Danny L
authors.initials: DL
authors.lastname: Costantini
authors.forename: Conrad
authors.initials: C
authors.lastname: Chan
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cai
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Vallis
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Reilly
mesh_terms: D021581:Active Transport, Cell Nucleus; D000911:Antibodies, Monoclonal / Q000493:pharmacokinetics / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized; D001943:Breast Neoplasms / Q000532:radiotherapy*; D002478:Cells, Cultured; D004249:DNA Damage; D004583:Electrons; D005260:Female; D006801:Humans; D007205:Indium Radioisotopes / Q000627:therapeutic use*; D019913:Nuclear Localization Signals; D016499:Radioimmunotherapy; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors; D014018:Tissue Distribution; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D007205:Indium Radioisotopes; D019913:Nuclear Localization Signals; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab