Micro-SPECT/CT with 111In-DTPA-pertuzumab sensitively detects trastuzumab-mediated HER2 downregulation and tumor response in athymic mice bearing MDA-MB-361 human breast cancer xenografts.

UNLABELLED: 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 microg/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 microg/mL), compared with untreated controls (62% +/- 2%, P < 0.0001; 32% +/- 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 +/- 2.6 percentage injected dose per gram [%ID/g] vs. 28.5 +/- 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 = 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 +/- 0.4 vs. 34.6 +/- 9.9 %ID/g, respectively; P < 0.001); this decrease was associated with an almost-completed eradication of HER2-positive tumor cells determined immunohistochemically. CONCLUSION: (111)In-DTPA-pertuzumab sensitively imaged HER2 downregulation after 3 d of treatment with trastuzumab and detected a reduction in viable HER2-positive tumor cells after 3 wk of therapy in MDA-MB-361 human breast cancer xenografts.

Associations between the uptake of 111In-DTPA-trastuzumab, HER2 density and response to trastuzumab (Herceptin) in athymic mice bearing subcutaneous human tumour xenografts.

PURPOSE: The purpose of the study was to investigate the associations between uptake of (111)In-DTPA-trastuzumab, tumour HER2 density and response to trastuzumab (Herceptin) of human breast cancer (BC) xenografts in athymic mice. MATERIALS AND METHODS: The tumour uptake of (111)In-DTPA-trastuzumab in athymic mice bearing BC xenografts with increasing HER2 density (0 to 3+) was evaluated. Specific uptake ratios were established in biodistribution (SUR) and imaging studies (ROI-SUR) using (111)In-labeled mouse IgG ((111)In-DTPA-mIgG). Further corrections were made for circulating radioactivity using tumour-to-blood ratios defined as a localization index (LI) and region-of-interest localization index (ROI-LI), respectively. Mice were treated with trastuzumab (Herceptin). A tumour growth inhibition index (TGI) was calculated and relative TGIs calculated by dividing the TGI of control by that of trastuzumab-treated mice. RESULTS: Strong, nonlinear associations with HER2 density were obtained if the uptake of (111)In-DTPA-trastuzumab was corrected for nonspecific IgG localization (i.e., SUR; r (2) = 0.99) and circulating radioactivity (i.e., LI; r (2) = 0.87), but without these corrections, the association between HER2 density and tumour uptake was poor (r (2) = 0.22). There was a strong association between ROI-SUR and ROI-LI values and HER2 expression (r (2) = 0.90 and r (2) = 0.95, respectively. All tumours were imaged. Relative TGI values were associated with increasing uncorrected tumour uptake of (111)In-DTPA-trastuzumab but not always with HER2 density (i.e., MCF-HER2-18 cells with trastuzumab-resistance). CONCLUSION: HER2 expression (0 to 3+) can be differentiated using (111)In-DTPA-trastuzumab, but requires correction of tumour uptake for nonspecific IgG localization and circulating radioactivity. The uncorrected uptake of (111)In-DTPA-trastuzumab was associated with tumour response to trastuzumab.

Trastuzumab-resistant breast cancer cells remain sensitive to the auger electron-emitting radiotherapeutic agent 111In-NLS-trastuzumab and are radiosensitized by methotrexate.

UNLABELLED: 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-(N-maleimidomethyl)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)In-trastuzumab 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)In-trastuzumab, 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 x 10(5) receptors/cell), compared with 231-H2N and TrR1 cells (6.1 x 10(5) and 5.1 x 10(5) receptors/cell, respectively), and lowest in MDA-MB-231 and TrR2 cells (0.4 x 10(5) and 0.6 x 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%+/-0.01%, 2.5%+/-0.2%, 2.8%+/-0.7%, and 0.5%+/-0.1% to 0.5%+/-0.1%, 4.6%+/-0.1%, 5.2%+/-0.6%, and 1.5%+/-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)In-trastuzumab and 16- to 77-fold lower than for trastuzumab. MDA-MB-231 and TrR2 cells were less sensitive to (111)In-NLS-trastuzumab 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.

The level of insulin growth factor-1 receptor expression is directly correlated with the tumor uptake of (111)In-IGF-1(E3R) in vivo and the clonogenic survival of breast cancer cells exposed in vitro to trastuzumab (Herceptin).

INTRODUCTION: Our objective was to define the relationships between tumor uptake of [(111)In]-IGF-1 and [(111)In]-IGF-1(E3R), an analogue which does not bind insulin growth factor-1 (IGF-1) binding proteins (i.e., IGFBP-3), and the level of IGF-1 receptor (IGF-1R) expression on human breast cancer (BC) xenografts in athymic mice, as well as the feasibility for tumor imaging. A second objective was to correlate IGF-1R (and HER2 density) with the cytotoxicity of trastuzumab in the absence/presence of IGFBP-3 or the IGF-1R tyrosine kinase inhibitor, AG1024. METHODS: The tumor and normal tissue uptake of [(111)In]-IGF-1 and [(111)In]-IGF-1(E3R) were determined at 4 h postinjection in mice implanted subcutaneously with MDA-MB-231, H2N, HR2 or MCF-7/HER2-18 human BC xenografts (8.5x10(4), 1.4x10(4), 4.0x10(4) and 1.0x10(5) IGF-1R/cell, respectively). The effect of co-injection of IGF-1 (50 microg) or IGFBP-3 (2 or 25 microg) was studied. The relationship between tumor uptake of [(111)In]-IGF-1(E3R) and IGF-1R density was examined. MicroSPECT/CT imaging was performed on mice with MCF-7/HER2-18 tumors injected with [(111)In]-IGF-1(E3R). The surviving fraction of BC cells exposed to trastuzumab (67.5 mug/ml) in the absence/presence of IGFBP-3 (1 microg/ml) or the IGF-1R kinase inhibitor, AG1024 (1 or 5 microg/ml), was determined. RESULTS: [(111)In]-IGF-1 was specifically taken up by MCF-7/HER2-18 xenografts; tumor uptake was decreased twofold when co-injected with IGF-1 (1.9+/-0.1 vs. 1.0+/-0.1 %ID/g). Co-injection of IGBP-3 decreased kidney uptake of [(111)In]-IGF-1 up to twofold and increased circulating radioactivity threefold. There was a strong linear correlation (r(2)=0.99) between the tumor uptake of (111)In-IGF-1(E3R) and IGF-1R density. Tumor uptake ranged from 0.4+/-0.05 %ID/g for H2N to 2.5+/-0.5 %ID/g for MCF-7/HER2-18 xenografts. MCF-7/HER2-18 tumors were visualized by microSPECT/CT. Resistance of BC cells to trastuzumab was directly associated with IGF-1R expression, despite co-expression of HER2. The resistance of HR2 cells could be partially reversed by IGFBP-3 or AG1024. CONCLUSION: Imaging of IGF-1R expression using [(111)In]-IGF-1(E3R) may be useful for identifying HER2-positive tumors in BC patients that are resistant to trastuzumab through this mechanism.

Properties of [(111)In]-labeled HIV-1 tat peptide radioimmunoconjugates in tumor-bearing mice following intravenous or intratumoral injection.

INTRODUCTION: Our objective was to evaluate the tumor and normal tissue distribution and nuclear importation properties of [(111)In]-mouse IgG (mIgG) conjugated to tat peptides (GRKKRRQRRRPPQGYG) in athymic mice with subcutaneous BT-474 human breast cancer xenografts. METHODS: Tumor and normal tissue uptake was compared after intravenous (iv) or intratumoral injection of [(111)In]-mIgG-tat and [(111)In]-mIgG. Area under the curve (AUC) was estimated for blood, liver, spleen, kidneys and tumor. Nuclear localization was measured by subcellular fractionation and estimated by microdosimetry. Imaging studies were performed with a gamma-camera. RESULTS: [(111)In]-mIgG-tat was eliminated from the blood and normal tissues two- to threefold more rapidly after iv injection than [(111)In]-mIgG. Tumor uptake was 4-5% injected dose per gram (%ID/g). Tumor radioactivity after intratumoral injection was initially very high (146-154 %ID/g), but declined 12- to 14-fold by 144 h postinjection. There was greater retention of [(111)In]-mIgG-tat in BT-474 tumors after intratumoral than iv injection, and the AUC (610+/-157 %ID h) was threefold greater than for intratumorally injected [(111)In]-mIgG (200+/-37 %ID h). Tat peptides increased nuclear localization of [(111)In]-mIgG after iv injection in tumor, kidney and liver cells, but only in tumor cells after intratumoral injection. Tumors were not imaged after iv administration but were predominant with intratumorally injected [(111)In]-mIgG and [(111)In]-mIgG-tat. Estimated radiation doses to the nucleus of tumor cells from intratumoral [(111)In]-mIgG-tat were 2.8x10(3) mGy/MBq and were 15-fold higher than for iv injection. CONCLUSION: [(111)In]-labeled tat immunoconjugates may have potential for imaging intracellular epitopes or localized Auger electron radiotherapy of tumors.

Cellular penetration and nuclear importation properties of 111In-labeled and 123I-labeled HIV-1 tat peptide immunoconjugates in BT-474 human breast cancer cells.

INTRODUCTION: Our objective was to compare the cell penetration and nuclear importation properties of 111In-labeled and 123I-labeled immunoconjugates (ICs) composed of 16-mer peptides (GRKKRRQRRRPPQGYG) derived from HIV-1 transactivator of transcription (tat) protein and anti-mouse IgG (mIgG) in BT-474 breast cancer (BC) cells. METHODS: [111In]tat ICs were constructed by site-specific conjugation of tat peptides to NaIO4(-)-oxidized carbohydrates in the Fc domain of diethylenetriaminepentaacetic-acid-modified anti-mIgG antibodies. Immunoreactivity against mIgG was assessed in a competition assay. The kinetics of the accumulation of [111In]anti-mIgG-tat IC and [123I]anti-mIgG-tat ICs in BT-474 cells and the elimination of radioactivity from cells, cytoplasm or nuclei were determined. The effects of excess tat peptides or NH4Cl (an inhibitor of endosomal acidification) on cellular uptake and nuclear importation of [111In]anti-mIgG-tat were measured. RESULTS: [111In]anti-mIgG-tat was >97% radiochemically pure and exhibited preserved immunoreactivity with mIgG epitopes. [123I]Anti-mIgG-tat penetrated BT-474 cells more rapidly than [111In]anti-mIgG-tat ICs and achieved a 1.5-fold to a 2-fold higher uptake in cells and nuclei. Cell penetration and nuclear uptake of [111In]anti-mIgG-tat were inhibited by excess tat peptides and NH4Cl. Elimination of radioactivity from BT-474 cells and nuclei was more rapid and complete for 123I-labeled than for 111In-labeled anti-mIgG-tat ICs. CONCLUSION: Tat peptides derived from HIV-1 tat protein promoted the penetration and nuclear uptake of radioactivity following the incubation of 111In-labeled and 123I-labeled anti-mIgG antibodies with BT-474 human BC cells. 111In-labeled tat ICs are feasible for inserting radionuclides into cancer cells with potential for targeting intracellular and, particularly, nuclear epitopes for imaging and/or radiotherapeutic applications.

Positron-emission tomography imaging of the TSPO with [(18)F]FEPPA in a preclinical breast cancer model.

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.

A comparison of 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging subcutaneous HER2-positive tumor xenografts in athymic mice using microSPECT/CT or microPET/CT.

BACKGROUND: Our objective was to compare 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging small or large s.c. tumor xenografts in athymic mice that display a wide range of human epidermal growth factor receptor-2 (HER2) expression using microSPECT/CT or microPET/CT. METHODS: Trastuzumab Fab were labeled with 111In or 64Cu by conjugation to 1,4,7,10-tetraazacyclododecane N, N', N'', N'''-tetraacetic acid (DOTA). The purity of 111In- and 64Cu-DOTA-trastuzumab Fab was measured by SDS-PAGE and HPLC. HER2 binding affinity was determined in saturation radioligand binding assays using SKBR-3 cells (1.3 × 106 HER2/cell). MicroSPECT/CT and microPET/CT were performed in athymic mice bearing s.c. BT-20 and MDA-MB-231 xenografts with low (0.5 to 1.6 × 105 receptors/cell), MDA-MB-361 tumors with intermediate (5.1 × 105 receptors/cell) or SKOV-3 xenografts with high HER2 expression (1.2 × 106 receptors/cell) at 24 h p.i. of 70 MBq (10 µg) of 111In-DOTA-trastuzumab Fab or 22 MBq (10 µg) of 64Cu-DOTA-trastuzumab Fab or irrelevant 111In- or 64Cu-DOTA-rituximab Fab. Tumor and normal tissue uptake were quantified in biodistribution studies. RESULTS: 111In- and 64Cu-DOTA-trastuzumab were > 98% radiochemically pure and bound HER2 with high affinity (Kd = 20.4 ± 2.5 nM and 40.8 ± 3.5 nM, respectively). MDA-MB-361 and SKOV-3 tumors were most clearly imaged using 111In- and 64Cu-DOTA-trastuzumab Fab. Significantly higher tumor/blood (T/B) ratios were found for 111In-DOTA-trastuzumab Fab than 111In-DOTA-rituximab Fab for BT-20, MDA-MB-231 and MDA-MB-361 xenografts, and there was a direct association between T/B ratios and HER2 expression. In contrast, tumor uptake of 64Cu-DOTA-trastuzumab Fab was significantly higher than 64Cu-DOTA-rituximab Fab in MDA-MB-361 tumors but no direct association with HER2 expression was found. Both 111In- and 64Cu-DOTA-trastuzumab Fab imaged small (5 to 10 mm) or larger (10 to 15 mm) MDA-MB-361 tumors. Higher blood, liver, and spleen radioactivity were observed for 64Cu-DOTA-trastuzumab Fab than 111In-DOTA-trastuzumab Fab. CONCLUSIONS: We conclude that 111In-DOTA-trastuzumab Fab was more specific than 64Cu-DOTA-trastuzumab Fab for imaging HER2-positive tumors, especially those with low receptor density. This was due to higher levels of circulating radioactivity for 64Cu-DOTA-trastuzumab Fab which disrupted the relationship between HER2 density and T/B ratios. Use of alternative chelators that more stably bind 64Cu may improve the association between T/B ratios and HER2 density for 64Cu-labeled trastuzumab Fab.

Comparisons of [18F]-1-deoxy-1-fluoro-scyllo-inositol with [18F]-FDG for PET imaging of inflammation, breast and brain cancer xenografts in athymic mice.

INTRODUCTION: The aim of the study was to evaluate the uptake of [(18)F]-1-deoxy-1-fluoro-scyllo-inositol ([(18)F]-scyllo-inositol) in human breast cancer (BC) and glioma xenografts, as well as in inflammatory tissue, in immunocompromised mice. Studies of [(18)F]-2-fluoro-2-deoxy-d-glucose ([(18)F]-FDG) under the same conditions were also performed. METHODS: Radiosynthesis of [(18)F]-scyllo-inositol was automated using a commercial synthesis module. Tumour, inflammation and normal tissue uptakes were evaluated by biodistribution studies and positron emission tomography (PET) imaging using [(18)F]-scyllo-inositol and [(18)F]-FDG in mice bearing subcutaneous MDA-MB-231, MCF-7 and MDA-MB-361 human BC xenografts, intracranial U-87 MG glioma xenografts and turpentine-induced inflammation. RESULTS: The radiosynthesis of [(18)F]-scyllo-inositol was automated with good radiochemical yields (24.6%±3.3%, uncorrected for decay, 65±2 min, n=5) and high specific activities (≥195 GBq/µmol at end of synthesis). Uptake of [(18)F]-scyllo-inositol was greatest in MDA-MB-231 BC tumours and was comparable to that of [(18)F]-FDG (4.6±0.5 vs. 5.5±2.1 %ID/g, respectively; P=.40), but was marginally lower in MDA-MB-361 and MCF-7 xenografts. Uptake of [(18)F]-scyllo-inositol in inflammation was lower than [(18)F]-FDG. While uptake of [(18)F]-scyllo-inositol in intracranial U-87 MG xenografts was significantly lower than [(18)F]-FDG, the tumour-to-brain ratio was significantly higher (10.6±2.5 vs. 2.1±0.6; P=.001). CONCLUSIONS: Consistent with biodistribution studies, uptake of [(18)F]-scyllo-inositol was successfully visualized by PET imaging in human BC and glioma xenografts, with lower accumulation in inflammatory tissue than [(18)F]-FDG. The tumour-to-brain ratio of [(18)F]-scyllo-inositol was also significantly higher than that of [(18)F]-FDG for visualizing intracranial glioma xenografts in NOD SCID mice, giving a better contrast.

Antitumor effects and normal-tissue toxicity of 111In-nuclear localization sequence-trastuzumab in athymic mice bearing HER-positive human breast cancer xenografts.

UNLABELLED: (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 x 10(5) or 0.5 x 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. A single dose of (111)In-NLS-trastuzumab (9.25 MBq)-compared with mice receiving (111)In-trastuzumab, trastuzumab, (111)In-NLS-hIgG, or normal saline-significantly slowed the rate of growth of MDA-MB-361 tumors over 60 d (0.014 d(-1) vs. 0.033 d(-1), 0.046 d(-1), 0.030 d(-1), and 0.061 d(-1), respectively; P < 0.05). (111)In-NLS-trastuzumab had no effect on the growth of MDA-MB-231 tumors. Two doses of (111)In-NLS-trastuzumab (9.25 MBq; 4 mg/kg) separated by 2 wk increased the survival of mice with MDA-MB-361 tumors, compared with mice treated with trastuzumab or normal saline (>140 d vs. 96 and 84 d, respectively; P < 0.001 or 0.027, respectively). CONCLUSION: (111)In-NLS-trastuzumab is a promising radioimmunotherapeutic agent that could be effective for treatment of HER2-overexpressing breast cancer in humans.

111In-labeled immunoconjugates (ICs) bispecific for the epidermal growth factor receptor (EGFR) and cyclin-dependent kinase inhibitor, p27Kip1.

OBJECTIVE: Probing intranuclear proteins in breast cancer (BC) cells by using radiolabeled antibodies is restricted by delivery barriers presented by cell and nuclear membranes. Our aim was to construct immunoconjugates (ICs) bispecific for epidermal growth factor receptors (EGFRs) and the intranuclear cyclin-dependent kinase inhibitor (CDKI) p27(Kip1) modified with nuclear-localizing sequences (NLSs) to facilitate their nuclear uptake following EGFR-mediated internalization. METHODS: Bispecific ICs were constructed by first modifying EGF with peptides [GGPKKKRKVGYGCG] harboring NLS from SV-40 large T-antigen (underlined), then conjugating NLS-EGF to anti-p27(Kip1) antibodies through an extended PEO(12)-maleimide linker (Compound 1). Analogous ICs were constructed by using mouse IgG (Compound 2), a disrupted NLS (Compound 3) or omission of the EGF moiety (Compound 4). Binding to EGFR on MDA-MB-468, H2N, or HR2 BC cells and to p27(Kip1) in HELA cell lysate was measured. Internalization and nuclear importation were evaluated. Retention of the ICs in H2N or trastuzumab (Herceptin)-resistant HR2 cells exposed to trastuzumab to modulate p27(Kip1) expression with/without coexposure to the IGF-1 receptor kinase inhibitor, AG1024, was determined. RESULTS: Trastuzumab (10 microg/mL) unexpectedly decreased p27(Kip1) expression by 1.7-2.4-fold in H2N or HR2 cells. Conjugation of EGF to anti-p27(Kip1) antibodies (Compound 1) decreased the binding affinity of the ICs 7-fold toward EGFR and p27(Kip1). All ICs bound EGFR on MDA-MB-468 cells except Compound 4. Compound 1 was internalized into H2N cells over 48 hours and Compound 2 exhibited 1.6-fold greater nuclear importation in H2N or MDA-MB-468 cells than Compound 3. There was a significantly lower retention of Compound 1 in H2N cells exposed to trastuzumab, compared to unexposed cells, corresponding to decreased p27(Kip1), but in HR2 cells, diminished retention was observed only when these cells were coexposed to trastuzumab and AG]024. CONCLUSION: We conclude that (111)In-labeled bispecific ICs were constructed that specifically bound EGFR and p27(Kip1). These ICs were internalized into BC cells expressing EGFR and HER2 and imported into the nucleus. Their decreased retention by cells with trastuzumab-modulated p27(Kip1) suggests that they may be useful for probing this CDKI by imaging.

In vivo monitoring of intranuclear p27(kip1) protein expression in breast cancer cells during trastuzumab (Herceptin) therapy.

INTRODUCTION: Trastuzumab, a humanized antibody directed against the Her2 receptor, induces the expression of p27(kip1), an intranuclear cyclin-dependent kinase inhibitor in some breast cancer cells. The aim of this study was to develop a radioimmunoconjugate (RIC) to monitor trastuzumab-induced p27(kip1) protein up-regulation in vivo. MATERIALS AND METHODS: Anti-p27(kip1) IgG was purified, and conjugated to diethylenetriaminopentaacetate, to allow radiolabeling with (111)In for in vivo detection. Then tat peptide (GRKKRRQRRRPPQGYG), containing a nuclear localization sequence (underlined), was conjugated to the Fc-domain of IgG, using NaIO(4) oxidation of carbohydrates and the resulting Schiff base stabilized with NaCNBH(3). The conjugate was radiolabeled with (111)In, yielding [(111)In]-anti-p27(kip1)-tat. (111)In labeling efficiency, purity and p27(kip1) binding were measured. Trastuzumab-induced p27(kip1) up-regulation was assessed in a panel of breast cancer cell lines by Western blot analysis. Uptake and retention of [(111)In]-anti-p27(kip1)-tat were measured in MDA-MB-361 and SKBr3 cells after exposure to trastuzumab. Uptake of [(111)In]-anti-p27(kip1)-tat was determined at 72 h postintravenous injection in MDA-MB-361 xenografts in athymic mice treated with trastuzumab or saline. RESULTS: [(111)In]-anti-p27(kip1)-tat was synthesized to 97% purity. The RIC was able to bind to p27(kip1) protein and internalized in the cells and was transported to the nuclei of MDA-MB-361 cells. The level of p27(kip1) protein in MDA-MB-361 cells was increased after exposure to clinically relevant doses of trastuzumab for 3 days. Trastuzumab-mediated induction of p27(kip1) was not associated with increased cellular uptake or nuclear localization of [(111)In]-anti-p27(kip1)-tat (6.53+/-0.61% vs. 6.98+/-1.36% internalized into trastuzumab-treated vs. control cells, respectively). However, retention of [(111)In]-anti-p27(kip1)-tat at 72 h was increased approximately twofold (13.5+/-1.3% vs. 6.6+/-0.6% of internalized [(111)In]-anti-p27(kip1)-tat was retained in trastuzumab-treated vs. control cells, respectively; P=.016). Immunohistochemistry showed up-regulation of p27(kip1) in trastuzumab-treated xenografts. Tumour uptake of [(111)In]-anti-p27(kip1)-tat was significantly higher in trastuzumab-treated compared to control animals (6.5+/-0.9 vs. 4.8+/-0.1 %ID/g at 72 h postinjection, respectively; P=.0065). CONCLUSION: [(111)In]-Anti-p27(kip1)-tat may be useful for monitoring changes in the expression of the intranuclear protein, p27(kip1). Up-regulation of p27(kip1) resulted in increased retention of [(111)In]-anti-p27(kip1)-tat in cells treated with trastuzumab. Modest, but statistically significantly higher, retention was also observed in tumours in mice treated with trastuzumab. Since responsiveness to trastuzumab correlated to up-regulation of p27(kip1), it may be possible to use [(111)In]-anti-p27(kip1)-tat to guide treatment with Herceptin and other drugs which alter p27(kip1) expression.

18F-FDG small-animal PET/CT differentiates trastuzumab-responsive from unresponsive human breast cancer xenografts in athymic mice.

UNLABELLED: Breast cancers (BCs) with high human epidermal growth factor receptor type 2 (HER2) expression are most likely to respond to trastuzumab; however, the mechanisms of action of trastuzumab are complex and there are no established biomarkers to accurately monitor treatment outcome in individual patients. Therefore, our aim was to determine, in human BC xenografts in athymic mice treated with trastuzumab, whether there were any changes in (18)F-FDG uptake that were associated with response to the drug and that could have utility in monitoring response in patients. METHODS: Baseline tumor uptake of (18)F-FDG was measured in mice with MDA-MB-361 HER2-overexpressing xenografts and MDA-MB-231 xenografts with low HER2 expression by small-animal PET imaging on day 0. Mice were treated with phosphate-buffered saline (PBS) or trastuzumab (4 mg/kg), and small-animal PET was repeated 2 d after treatment. Maintenance doses of trastuzumab (2 mg/kg) or PBS were administered on days 7 and 14, and mice were imaged again on days 9 and 16. Tumor uptake was measured as percentage injected dose per gram (%ID/g) by volume-of-interest analysis on days 0 (baseline), 2, 9, and 16, followed by biodistribution studies on day 16. Tumor growth was measured, and a tumor growth index was calculated. RESULTS: The treatment of mice with trastuzumab, compared with control mice treated with PBS, resulted in a significant decrease in tumor uptake of (18)F-FDG in HER2-overexpressing MDA-MB-361 xenografts after 16 d of treatment (2.6 +/- 0.8 %ID/g vs. 4.6 +/- 1.8 %ID/g, respectively; P < 0.03) but not after 2 or 9 d of treatment (P = 0.28-0.32). In contrast, there was no significant change in the tumor uptake of MDA-MB-231 xenografts with low HER2 expression during the entire course of therapy (4.4 +/- 1.7 %ID/g vs. 3.6 +/- 1.1 %ID/g, respectively; P = 0.31). Trastuzumab treatment, compared with PBS treatment of controls, resulted in significant growth inhibition of MDA-MB-361 xenografts as early as 10 d from the initiation of treatment (tumor growth index, 0.7 +/- 0.2 vs. 1.7 +/- 0.3, respectively; P < 0.0005), whereas no tumor growth inhibition was observed for MDA-MB-231 xenografts (5.3 +/- 2.7 and 5.2 +/- 3.0; P = 0.95). CONCLUSION: Changes in the tumor uptake of (18)F-FDG after therapy accurately identified responding and nonresponding human BC xenografts in athymic mice treated with trastuzumab; however, diminished glucose utilization did not precede changes in tumor volume.