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Our goals in this study were to determine whether 111 In-trastuzumab coupled to peptides harboring nuclear localizing sequences (NLSs) could kill trastuzumab-resistant breast cancer cell lines through the emission of Auger electrons and whether the combination of radiosensitization with methotrexate (MTX) would augment the cytotoxicity of this radiopharmaceutical. Methods: Trastuzumab was derivatized with sulfosuccinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate for reaction with NLS peptides and then conjugated with diethylenetriaminepentaacetic acid for labeling with 111 In. HER2 expression was determined by Western blot and by radioligand binding assay using 111 In-trastuzumab in a panel of breast cancer cell lines, including SK-BR-3, MDA-MB-231 and its HER2-transfected subclone (231-H2N), and 2 trastuzumab-resistant variants (TrR1 and TrR2). Nuclear importation of 111 In-NLS-trastuzumab and 111 Intrastuzumab in breast cancer cells was measured by subcellular fractionation, and the clonogenic survival of these cells was determined after incubation with 111 In-NLS-trastuzumab, 111 Intrastuzumab, or trastuzumab (combined with or without MTX). Survival curves were analyzed according to the dose-response model, and the radiation-enhancement ratio was calculated from the survival curve parameters. Results: The expression of HER2 was highest in SK-BR-3 cells (12.6 · 10 5 receptors/cell), compared with 231-H2N and TrR1 cells (6.1 · 10 5 and 5.1 · 10 5 receptors/cell, respectively), and lowest in MDA-MB-231 and TrR2 cells (0.4 · 10 5 and 0.6 · 10 5 receptors/cell, respectively). NLS peptides increased the nuclear uptake of 111 In-trastuzumab in MDA-MB-231, 231-H2N, TrR1, and TrR2 cells from 0.1% 6 0.01%, 2.5% 6 0.2%, 2.8% 6 0.7%, and 0.5% 6 0.1% to 0.5% 6 0.1%, 4.6% 6 0.1%, 5.2% 6 0.6%, and 1.5% 6 0.2%, respectively. The cytotoxicity of 111 In-NLS-trastuzumab on breast cancer cells was directly correlated with the HER2 expression densities of the cells. On a molar concentration basis, the effective concentration required to kill 50% of 231-H2N and TrR1 cells for 111 In-NLS-trastuzumab was 9-to 12-fold lower than for 111 Intrastuzumab and 16-to 77-fold lower than for trastuzumab.MDA-MB-231 and TrR2 cells were less sensitive to 111 In-NLStrastuzumab or 111 In-trastuzumab, and both cell lines were completely insensitive to trastuzumab. The radiation-enhancement ratio induced by MTX for 231-H2N and TrR1 cells after exposure to 111 In-NLS-trastuzumab was 1.42 and 1.68, respectively. Conclusion: Targeted Auger electron radioimmunotherapy with 111 In-NLS-trastuzumab can overcome resistance to trastuzumab, and MTX can potently enhance the sensitivity of HER2-overexpressing breast cancer cells to the lethal Auger electrons emitted by this radiopharmaceutical.

Pertuzumab is a HER2 dimerization inhibitor that binds to an epitope unique from that of trastuzumab. Our objective was to determine whether SPECT with 111 In-diethylenetriaminepentaacetic acid-pertuzumab ( 111 In-DTPA-pertuzumab) could sensitively detect an early molecular response to trastuzumab manifested by HER2 downregulation and a later tumor response revealed by a decreased number of HER2-positive viable tumor cells. Methods: Changes in HER2 density in SKBr-3 and MDA-MB-361 BC cells exposed to trastuzumab (14 mg/mL) in vitro were measured by saturation binding assays using 111 In-DTPA-pertuzumab and by confocal immunofluorescence microscopy and flow cytometry with fluorescein isothiocyanate-labeled HER2/neu antibodies. Imaging of HER2 downregulation was studied in vivo in athymic mice with subcutaneous MDA-MB-361 tumors treated for 3 d with trastuzumab (4 mg/kg) or nonspecific human IgG (hIgG) or phosphate-buffered saline (PBS). Imaging of tumor response to trastuzumab was studied in mice bearing subcutaneous MDA-MB-361 xenografts treated with trastuzumab (4 mg/kg), followed by weekly doses of nonspecific hIgG or rituximab or PBS (2 mg/ kg). Mice were imaged on a micro-SPECT/CT system at 72 h after injection of 111 In-DTPA-pertuzumab. Tumor and normal-tissue biodistribution was determined. Results: 111 In-DTPA-pertuzumab saturation binding to SKBr-3 and MDA-MB-361 cells was significantly decreased at 72 h after exposure in vitro to trastuzumab (14 mg/mL), compared with untreated controls (62% 6 2%, P , 0.0001; 32% 6 9%, P , 0.0002, respectively). After 3 d of trastuzumab, in vivo tumor uptake of 111 In-DTPA-pertuzumab decreased 2-fold in trastuzumab-versus PBS-treated mice (13.5 6 2.6 percentage injected dose per gram [%ID/g] vs. 28.5 6 9.1 %ID/g, respectively; P , 0.05). There was also a 2-fold decreased tumor uptake in trastuzumab-versus PBS-treated mice by image volume-of-interest analysis (P 5 0.05), suggesting trastuzumab-mediated HER2 downregulation. After 3 wk of trastuzumab, tumor uptake of 111 In-DTPA-pertuzumab decreased 4.5-fold, compared with PBS-treated mice (7.6 6 0.4 vs. 34.6 6 9.9 %ID/g, respectively; P , 0.001); this decrease was associated with an almost-completed eradication of HER2-positive tumor cells determined immunohistochemically. Mol ecular imaging is a powerful new tool that has great potential for aiding in the optimal use of novel targeted cancer therapies by revealing the expression of target receptors in situ on lesions throughout the body; probing downstream treatment-induced molecular events, thus providing early mechanistic evidence of tumor response; and monitoring the prior existence or emergence of resistance pathways implicated in treatment failure (1). Trastuzumab (Herceptin; Roche Pharmaceuticals) is a humanized IgG 1 monoclonal antibody (mAb) approved for the treatment of early and advanced breast cancer (BC) which overexpresses the HER2 transmembrane tyrosine kinase (2,3). HER2 positivity is evaluated in a primary BC biopsy by immunohistochemical staining for HER...

111 In-nuclear localization sequence-trastuzumab is a radioimmunotherapeutic agent consisting of trastuzumab modified with NLS peptides (CGYGPKKKRKVGG) and labeled with the Auger electron emitter 111 In. Our objectives were to evaluate the tumor growth-inhibitory properties and normal-tissue toxicity of 111 In-NLS-trastuzumab in mice after intraperitoneal administration. Methods: The pharmacokinetics of 111 In-NLS-trastuzumab after intravenous (tail vein) or intraperitoneal injection in BALB/c mice were compared. Normal-tissue toxicity was determined in BALB/c mice at 2 wk after intraperitoneal injection of 3.7-18.5 MBq (4 mg/kg) of 111 In-NLS-trastuzumab by monitoring body weight, histopathologic examination of tissues, and hematology (white blood cell, platelet, red blood cell, and hemoglobin) or clinical biochemistry (alanine transaminase and creatinine) parameters. A no-observable-adverse-effect-level (NOAEL) dose was defined. Athymic mice bearing subcutaneous MDA-MB-361 or MDA-MB-231 human breast cancer xenografts (5.0 · 10 5 or 0.5 · 10 5 HER2/cell, respectively) were treated with a single NOAEL dose or 2 doses administered intraperitoneally and separated by 2 wk. Control groups were administered 111 In-trastuzumab, trastuzumab, nonspecific 111 In-NLS-human IgG (hIgG), or normal saline. Results: The bioavailability of 111 In-NLS-trastuzumab after intraperitoneal injection was 0.7. The NOAEL dose was 9.25 MBq (4 mg/kg); doses greater than or equal to 18.5 MBq decreased white blood cell or platelet counts, and doses of 27.7 MBq decreased red blood cell counts. There was no increase in alanine transaminase or creatinine at any doses tested. There were no morphologic changes to the liver, kidneys, heart, or spleen or loss of body weight.

The present study aims to image the 18-kDa translocator protein (TSPO; formerly known as the peripheral benzodiazepine receptor) in a preclinical human breast cancer (BC) xenograft mouse model with positron-emission tomography (PET). An automated radiosynthesis of [(18)F]-N-(2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide ([(18)F]FEPPA) was validated for human use using a commercial synthesis module and resulted in a high radiochemical yield (30%±8%, uncorrected; n=54) and specific activity (6±4 Ci/μmol). Tumor uptake of [(18)F]FEPPA in mice bearing subcutaneous MDA-MB-231 BC xenografts was evaluated by PET-computed tomography imaging and ex vivo biodistribution studies. Although the tumor was successfully visualized, ex vivo biodistribution studies revealed low tumor uptake (0.7%ID/g), with the majority of radioactivity distributed in the spleen, muscle, and heart despite high TSPO expression in this cell line. Our laboratory routinely prepares [(18)F]FEPPA for human-imaging studies in the central nervous system, and we envision that radiopharmaceuticals that target the TSPO have the potential for imaging macrophages in the tumor microenvironment.