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Pretargeting of receptors is a useful approach in molecular imaging and therapy to reduce background noise or toxicity and enhance selectivity. In this study a three-step pretargeting approach that includes a biotinylated antibody, avidin/streptavidin, and a biotinylated imaging agent is described. A PAMAM dendrimer generation 4 (G4D)-based MRI T 1 agent biotin-G4D-DTPA-Gd (bG4D-Gd) and its sister compound with remaining free surface amine groups blocked by succinic anhydride to reduce positive charges (bG4D-Gd-SA) were synthesized. Limited selective enhancement in MRI was observed in a Her-2/neu mouse tumor xenograft by this three-step pretargeting approach that includes biotinylated trastuzumab, avidin and bG4D-Gd, or bG4D-Gd-SA. However, these dendrimer-based MRI agents with molecular weight around 29 kD reached and remained in the tumor through the enhanced permeability and retention effect. Prolonged and extensive accumulation of both bG4D-Gd and b-G4-Gd-SA in the kidneys was also observed. (1) about 20 years ago using biotin-avidin systems still holds promise in increasing the tumor/nontumor ratio compared to a direct targeting approach (2). While more popular in the delivery of radionuclides for imaging and therapy, the low concentration of receptors and the intrinsic low sensitivity of MRI have limited applications of pretargeting in MRI. Her-2/neu overexpressing breast tumors are a good model system for an MRI pretargeting approach due to the large number of receptors expressed uniformly on the cancer cell surface and the availability of trastuzumab, an FDA-approved antibody against the Her-2/neu receptors (3,4). Success in delivering MRI agents by pretargeting can also be extended to therapy, which is important as the amplification and overexpression of Her-2/neu, a member of the epidermal growth factor receptor (EGFR) family of tyrosine kinases that regulates cell proliferation and differentiation, has been linked to poor prognosis in several human cancers such as breast, prostate, lung, ovary, and colon cancers.Because of its low intrinsic sensitivity, the success of MRI depends on the development of highly efficient contrast agents. For example, it was demonstrated that conjugating of Gd(III) chelates to high molecular weight carriers is highly beneficial for in vivo MR imaging as it provides 1) prolonged intravascular circulation time that results in improved pharmacokinetics of the agent at the target site, and 2) slows down molecular rotation that translates to increased T 1 relaxivity at high magnetic field typically used in preclinical MRI studies (5). Macromolecular MRI contrast agents based on PAMAM dendrimers conjugated with a diethylenetriamimepentaacetic acid (DTPA) derivative, 2-(p-isothiocyanatobenzyl)-6 -methyl-diethylenetriaminepentaacetic acid (1B4M), to chelate gadolinium metal ions have been proven to be versatile in MRI applications due to their mono-disperse molecular size and the ability to finetune molecular weights by using dendrimers of different generations (6 -8). In our stu...

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.

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