Dual-labeled trastuzumab-based imaging agent for the detection of human epidermal growth factor receptor 2 overexpression in breast cancer

Abstract

Overexpression of the human epidermal growth factor receptor (HER) family has been implicated in cancer because of its participation in signaling pathways regulating cellular proliferation, differentiation, motility, and survival. In this work, we exploited the extracellular binding property of trastuzumab, a clinically therapeutic monoclonal antibody to the second member of the HER family (HER2), to design a diagnostic imaging agent, (111In-DTPA)n-trastuzumab-(IRDye 800CW)m, that is dual labeled with 111In, a γ-emitter, and a near-infrared (NIR) fluorescent dye, IRDye 800CW, to detect HER2 overexpression in breast cancer cells. The stoichiometric ratios “n” and “m” refer to the number of diethylenetriaminepentaacetic acid dianhydride (DTPA) and IRDye 800CW molecules bound per trastuzumab molecule, respectively. Methods: Fluorescence microscopy and confocal microscopy were used to determine the molecular specificity of (DTPA)n-trastuzumab-(IRDye800)m in vitro in SKBr3 (HER2-positive) and MDA-MB-231 (HER2-negative) breast cancer cells. SKBr3 cells were incubated with (DTPA)n-trastuzumab-(IRDye800)m or IRDye800CW or pretreated with trastuzumab or human IgG followed by (DTPA)n-trastuzumab-(IRDye800)m and examined under a fluorescence microscope. For in vivo characterization, athymic nude mice bearing HER2-overexpressing SKBr3-luc subcutaneous xenografts were injected intravenously with (111In-DTPA)n-trastuzumab-(IRDye800)m and imaged with SPECT and NIR fluorescence imaging at 48 h. Tumor-bearing mice were also injected intravenously with trastuzumab 24 h before administration of (111In-DTPA)n-trastuzumab-(IRDye800)m. Nonspecific uptake in the SKBr3-luc tumors was analyzed by injecting the mice with IRDye 800CW and (111In-DTPA)p-IgG-(IRDye800)q, where “p” and “q” are the stoichiometric ratios of DTPA and IRDye 800CW bound per IgG antibody, respectively. Results: (DTPA)n-trastuzumab-(IRDye800)m showed significantly greater binding to SKBr3 cells than to MDA-MB-231 cells. Confocal imaging revealed that this binding occurred predominantly around the cell membrane. Competitive binding studies with excess trastuzumab before incubation with (DTPA)n-trastuzumab-(IRDye800)m abolished this binding affinity, but pretreatment with nonspecific IgG did not alter binding. In vivo nuclear and optical imaging of SKBr3-luc xenografts injected with (111In-DTPA)n-trastuzumab-(IRDye800)m revealed significantly more uptake in the tumor region than in the contralateral muscle region. The tumor-to-muscle ratio decreased in mice pretreated with trastuzumab and in mice injected with IRDye 800CW and (111In-DTPA)p-IgG-(IRDye800)q. Ex vivo imaging of dissected organs confirmed these results. Finally, coregistration of histologic hematoxylin–eosin stains with autoradiography signals from tumor and muscle tissue slices indicated that (111In-DTPA)n-trastuzumab-(IRDye800)m bound only in tumor tissue and not to muscle. Conclusion: Dual-labeled (111In-DTPA)n-trastuzumab-(IRDye800)m may be an effective diagnostic biomarker capable of tracking HER2 overexpression in breast cancer patients.