title: Auger electron radioimmunotherapeutic agent specific for the CD123+/CD131- phenotype of the leukemia stem cell population.
abstract: Our aim was to construct and characterize (111)In-nuclear translocation sequence (NLS)-7G3, an Auger electron-emitting radioimmunotherapeutic agent that preferentially recognizes the expression of CD123 (interleukin-3 receptor [IL-3R] α-subchain) in the absence of CD131 (IL-3R β-subchain) displayed by leukemia stem cells.
  
  
  METHODS
  Monoclonal antibody 7G3 was modified with 13-mer peptides [CGYGPKKKRKVGG] harboring the NLS of SV-40 large T-antigen and with diethylenetriaminepentaacetic acid for labeling with (111)In. Immunoreactivity was evaluated in a competition radioligand binding assay and by flow cytometry. Nuclear localization of (111)In-NLS-7G3 was studied by cell fractionation in CD123(+)/CD131(-) acute myelogenous leukemia (AML)-3, -4, and -5 cells or in primary AML or normal leukocytes. Micro-SPECT was performed in nonobese diabetic (NOD)/severe combined immune deficient (SCID) mice engrafted subcutaneously with Raji-CD123 tumors or with disseminated AML-3 or -5 cells. The cytotoxicity of (111)In-NLS-7G3 on AML-5 cells was studied after 7 d in culture by trypan blue dye exclusion. DNA damage was assessed using the γ-H2AX assay.
  
  
  RESULTS
  NLS-7G3 exhibited preserved CD123 immunoreactivity (affinity, 4.6 nmol/L). Nuclear importation of (111)In-NLS-7G3 in AML-3, -4, or -5 cells was specific and significantly higher than unmodified (111)In-7G3 and was greater in primary AML cells than in normal leukocytes. Rapid elimination of (111)In-NLS-7G3 in NOD/SCID mice prevented imaging of subcutaneous Raji-CD123 tumors. This phenomenon was Fc-dependent and IgG(2a) isotype-specific and was overcome by the preadministration of excess IgG(2a) or using (111)In-NLS-7G3 F(ab')(2) fragments. AML-3 and -5 cells were engrafted into the bone marrow or spleen or at extramedullary sites in NOD/SCID mice. Micro-SPECT/CT with (111)In-NLS-7G3 F(ab')(2) showed splenic involvement, whereas foci of disease were seen in the spine or femur or at extramedullary sites in the brain and lymph nodes using (111)In-NLS-7G3 IgG(2a). The viability of AML-5 cells was reduced by exposure in vitro to (111)In-NLS-7G3; this reduction was associated with an increase in unrepaired DNA double-strand breaks.
  
  
  CONCLUSIONS
  (111)In-NLS-7G3 is a promising novel Auger electron-emitting radioimmunotherapeutic agent for AML aimed at the leukemia stem cell population. Micro-SPECT/CT was useful for visualizing the engraftment of leukemia in NOD/SCID mice.
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Jeffrey Victor
authors.initials: JV
authors.lastname: Leyton
authors.forename: Meiduo
authors.initials: M
authors.lastname: Hu
authors.forename: Catherine
authors.initials: C
authors.lastname: Gao
authors.forename: Patricia V
authors.initials: PV
authors.lastname: Turner
authors.forename: John E
authors.initials: JE
authors.lastname: Dick
authors.forename: Mark
authors.initials: M
authors.lastname: Minden
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Reilly
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D001853:Bone Marrow / Q000000981:diagnostic imaging; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D056831:Coordination Complexes / Q000627:therapeutic use*; D053648:Cytokine Receptor Common beta Subunit / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004583:Electrons; D018796:Immunoconjugates; D007205:Indium Radioisotopes; D053650:Interleukin-3 Receptor alpha Subunit / Q000235:genetics / Q000378:metabolism*; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis* / Q000532:radiotherapy*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D010641:Phenotype; D016499:Radioimmunotherapy / Q000379:methods*; D019275:Radiopharmaceuticals / Q000627:therapeutic use*; D014018:Tissue Distribution; D015899:Tomography, Emission-Computed, Single-Photon
chemical_list: C562074:111In-nuclear translocation sequence-7G3; D000911:Antibodies, Monoclonal; D056831:Coordination Complexes; D053648:Cytokine Receptor Common beta Subunit; D018796:Immunoconjugates; D007205:Indium Radioisotopes; D053650:Interleukin-3 Receptor alpha Subunit; D019275:Radiopharmaceuticals