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Controlled internalization of Her-2/neu receptors by cross-linking for targeted delivery

Receptor mediated internalization is a crucial step for targeted intracellular delivery of therapeutic and imaging agents. It was recently demonstrated that trastuzumab, an FDA approved humanized monoclonal antibody against Her-2/neu tyrosine kinase receptor, did not induce endocytosis of the internalization resistant Her-2/neu receptor. Here we report that accelerated internalization of trastuzumab can be induced by cross-linking the cell membrane bound antibody-receptor complex with an avidin/streptavidin-biotin system. We demonstrated that internalization was achieved both in vitro and in vivo in Her-2/neu expressing human breast cancer cell lines (BT-474, SK-BR-3, and AU-565) and that repetitive labeling cycles further amplified the loading of cargo molecules within the targeted cells. No trastuzumab binding and internalization was observed in Her-2/neu negative MDA-MB-231 cells, whereas weak membrane binding and negligible internalization were detected in MCF-7 cells with low expression level of Her-2/neu receptor. The method was used to noninvasively image Her-2/neu receptors in isolated cells and in a preclinical breast cancer model with MRI. The controlled internalization of Her-2/neu receptors can potentially enhance intracellular delivery of drugs and imaging probes, and improve imaging sensitivity and selectivity as well as therapeutic efficacy, through antibody-directed binding and internalization using a pretargeting approach.