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¹¹¹In-Labeled Trastuzumab (Herceptin) Modified with Nuclear Localization Sequences (NLS): An Auger Electron-Emitting Radiotherapeutic Agent for HER2/neu-Amplified Breast Cancer

¹ Departments of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada; ² Department of Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom; ³ Department of Medical Imaging, University of Toronto, Toronto, Ontario, Canada; and ⁴ Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada

View larger version (73K): [in this window] [in a new window]   FIGURE 1.  (A) SDS–PAGE and (B) Western blot of unmodified trastuzumab (lane 1) or DTPA-trastuzumab (lane 2) reacted with a 5-, 15-, 25-, or 50-fold molar excess of sulfo-SMCC followed by conjugation to a 60-fold molar excess of NLS-peptides (lanes 3–6, respectively). The y-axis indicates Mr (kDa). (C) Representative size-exclusion chromatograms of purification of 123I-NLS-peptides bound to trastuzumab from free peptides. Elution curves were obtained by measuring ultraviolet absorbance (OD280nm) (solid line) and radioactivity (hatched bars) in each fraction. The peak in fractions 7–10 represents trastuzumab-bound 123I-NLS-peptides. (D) 123I-NLS-peptide coupling efficiency to trastuzumab increases as the SMCC-to-IgG molar ratio increases. Values shown are mean ± SEM of triplicate determinations.

View larger version (14K): [in this window] [in a new window]   FIGURE 2.  Competition binding curve shows effect of increasing concentrations of unlabeled trastuzumab on the displacement of binding of 111In-Trastuzumab or 111In-NLS-Trastuzumab to SK-BR-3 cells. B = radioligand bound in presence of competitors; Bo = radioligand bound without competitor. Each point represents mean ± SEM of 3 assays performed in triplicate.

View larger version (18K): [in this window] [in a new window]   FIGURE 3.  Internalization of 111In-NLS-Trastuzumab and 111In-Trastuzumab by SK-BR-3 (A), MDA-MB-361 (B), and MDA-MB-231 (C) BC cells (solid bars) with respect to the total amount of radioactivity added to incubation media. Internalization was measured in presence or absence of an excess of unlabeled trastuzumab (100 nmol/L) in incubation medium to block HER2/neu receptors. (D) Internalized fraction of cell-bound radioactivity. Values shown are mean ± SEM of triplicate determinations. *P < 0.05.

View larger version (40K): [in this window] [in a new window]   FIGURE 4.  Confocal immunofluorescence microscopy of SK-BR-3 (A), MDA-MB-361 (B), and MDA-MB-231 (C) BC cells incubated with DTPA-trastuzumab with (+) or without (–) NLS-peptides. Trastuzumab was detected using an AlexaFluor-564 antihuman IgG secondary antibody (red), and the nucleus was visualized with DAPI (blue). Merging of the 2 signals is shown and images are 1-µm slices through the cells.

View larger version (42K): [in this window] [in a new window]   FIGURE 5.  Cell survival curves measured in clonogenic assays for SK-BR-3 (A), MDA-MB-361 (B), and MDA-MB-231 (C) BC cells treated with increasing concentrations of 111In-Trastuzumab, 111In-NLS3-Trastuzumab, or 111In-NLS6-Trastuzumab. Controls consisted of cells treated with unlabeled trastuzumab or irrelevant 111In-NLS3-IgG. Each point represents mean ± SEM of 3 experiments performed in triplicate. (D) Percentage of cells positive for DNA damage (>10 H2AX foci/cell) after treatment with PBS (pH 7.5), unlabeled trastuzumab, 111In-Trastuzumab, or 111In-NLS6-Trastuzumab. Significant differences are *P < 0.05, 111In-Trastuzumab compared with unlabeled trastuzumab and **P < 0.05, 111In-NLS6-Trastuzumab compared with 111In-Trastuzumab. (E–G) Induction of H2AX foci (green) in SK-BR-3 (E), MDA-MB-361(F), and MDA-MB-231 (G) cells after 111In-NLS6-Trastuzumab treatment. Nuclear DNA was stained with DAPI (blue).