title: PET imaging of DNA damage using (89)Zr-labelled anti-γH2AX-TAT immunoconjugates.
abstract: OBJECTIVE
  The efficacy of most anticancer treatments, including radiotherapy, depends on an ability to cause DNA double-strand breaks (DSBs). Very early during the DNA damage signalling process, the histone isoform H2AX is phosphorylated to form γH2AX. With the aim of positron emission tomography (PET) imaging of DSBs, we synthesized a (89)Zr-labelled anti-γH2AX antibody, modified with the cell-penetrating peptide, TAT, which includes a nuclear localization sequence.
  
  
  METHODS
  (89)Zr-anti-γH2AX-TAT was synthesized using EDC/NHS chemistry for TAT peptide linkage. Desferrioxamine conjugation allowed labelling with (89)Zr. Uptake and retention of (89)Zr-anti-γH2AX-TAT was evaluated in the breast adenocarcinoma cell line MDA-MB-468 in vitro or as xenografts in athymic mice. External beam irradiation was used to induce DSBs and expression of γH2AX. Since (89)Zr emits ionizing radiation, detailed radiobiological measurements were included to ensure (89)Zr-anti-γH2AX-TAT itself does not cause any additional DSBs.
  
  
  RESULTS
  Uptake of (89)Zr-anti-γH2AX-TAT was similar to previous results using (111)In-anti-γH2AX-TAT. Retention of (89)Zr-anti-γH2AX-TAT was eightfold higher at 1 h post irradiation, in cells expressing γH2AX, compared to non-irradiated cells or to non-specific IgG control. PET imaging of mice showed higher uptake of (89)Zr-anti-γH2AX-TAT in irradiated xenografts, compared to non-irradiated or non-specific controls (12.1 ± 1.6 vs 5.2 ± 1.9 and 5.1 ± 0.8%ID/g, respectively; p < 0.0001). The mean absorbed dose to the nucleus of cells taking up (89)Zr-anti-γH2AX-TAT was twofold lower compared to (111)In-anti-γH2AX-TAT. Additional exposure of neither irradiated nor non-irradiated cells nor tissues to (89)Zr-anti-γH2AX-TAT resulted in any significant changes in the number of observable DNA DSBs, γH2AX foci or clonogenic survival.
  
  
  CONCLUSIONS
  (89)Zr-anti-γH2AX-TAT allows PET imaging of DNA DSBs in a tumour xenograft mouse model.
authors.affiliation: CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7LJ, UK.
authors.forename: James C
authors.initials: JC
authors.lastname: Knight
authors.affiliation: CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7LJ, UK.
authors.forename: Caitríona
authors.initials: C
authors.lastname: Topping
authors.affiliation: CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7LJ, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Mosley
authors.affiliation: CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7LJ, UK.
authors.forename: Veerle
authors.initials: V
authors.lastname: Kersemans
authors.affiliation: CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7LJ, UK.|Royal Marsden Hospital, Sutton, Surrey, UK.
authors.forename: Nadia
authors.initials: N
authors.lastname: Falzone
authors.affiliation: Facultat de Fisica (ECM and ICC), Universitat de Barcelona, Barcelona, Spain.
authors.forename: José M
authors.initials: JM
authors.lastname: Fernández-Varea
authors.affiliation: CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7LJ, UK. bart.cornelissen@oncology.ox.ac.uk.
authors.forename: Bart
authors.initials: B
authors.lastname: Cornelissen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001692:Biological Transport; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D053903:DNA Breaks, Double-Stranded; D005260:Female; D015696:Gene Products, tat / Q000378:metabolism*; D006657:Histones / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D018796:Immunoconjugates / Q000378:metabolism / Q000493:pharmacokinetics; D051379:Mice; D049268:Positron-Emission Tomography / Q000379:methods*; D011868:Radioisotopes; D014018:Tissue Distribution; D015040:Zirconium
chemical_list: D000911:Antibodies, Monoclonal; D015696:Gene Products, tat; D006657:Histones; D018796:Immunoconjugates; D011868:Radioisotopes; D015040:Zirconium