MR imaging of the her2/neu and 9.2.27 tumor antigens using immunospecific contrast agents Martin A. Funovics a, *, Barbara Kapeller b , Christoph Hoeller c , Henry S. Su d , Rainer Kunstfeld c , Stephan Puig a , Karin Macfelda b a Department of Radiology, Medical University Vienna, Austria b Department of Biomedical Research, Medical University Vienna, Austria c Department of Dermatology, Medical University Vienna, Austria d Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Received 22 July 2003; accepted 27 January 2004 Abstract Molecular imaging of tumor antigens using immunospecific magnetic resonance (MR) contrast agents is a rapidly evolving field, which can potentially aid in early disease detection, monitoring of treatment efficacy, and drug development. In this study, we designed, synthetized, and tested in vitro two novel monocrystalline iron oxide nanoparticles (MION) conjugated to antibodies against the her2/neu tyrosine kinase receptor and the 9.2.27 proteoglycane sulfate. MION was synthetized by coprecipitation of iron II and iron III salts in 12-kD dextran solution; antibody coupling was performed by reductive amination. The relaxivity of the conjugates was 24.1–29.1 mM -1 s -1 , with 1.8 to 2.1 antibody molecules per nanoparticle. A panel of cultured melanoma and mammary cell lines was used for testing. The cells were incubated with the particles at 16 –32 g Fe/ml in culture medium for 3 h at 37°C, and investigated with immune fluorescence, transmission electron microscopy (TEM), MRI of cell suspensions in gelatine, and spectrophotometric iron determination. All receptor-positive cell lines, but not the controls, showed receptor-specific immune fluorescence, and strong changes in T 2 signal intensity at 1.5 T. The changes in 1/T 2 were between 1.5 and 4.6 s -1 and correlated with the amount of cell-bound iron (R = 0.92). The relaxivity of cell-bound MION increased to 55.9  10.4 mM -1 s -1 . TEM showed anti-9.2.27 conjugates binding to the plasma membrane, while the anti-her2/neu conjugates underwent receptor-mediated endocytosis. In conclusion, we obtained receptor-specific T 2 MR contrast with novel covalently bound, multivalent MION conjugates with anti-9.2.27 and anti-her2/neu to image tumor surface antigens. This concept can potentially be expanded to a large number of targets and to in vivo applications. © 2004 Elsevier Inc. All rights reserved. Keywords: Iron oxide nanoparticles; Molecular imaging; MRI; Tumor surface antigens 1. Introduction Non-invasive in vivo imaging of antigen expression can provide an avenue of diagnostic and therapeutic approaches for a broad range of diseases. Diagnostic imaging of tumors using monoclonal antibodies (Mabs) has previously been investigated [1]. Recently, targeted [2] and activatible [3] imaging probes have been designed to assess distinct mo- lecular markers (i.e., antigen expression or enzyme activity) in diseased tissue for either biological or biochemical char- acterization or evaluation of treatment efficacy [4]. Due to the relatively low expression levels of many of these target molecules, imaging modalities for such investigations are limited to those with the highest detection sensitivities. Nuclear imaging techniques such as PET (positron emission tomography) or SPECT (single-photon emission tomogra- phy) allow for detection of minute amounts [5]; however, major disadvantages are limited spatial and temporal reso- lutions. Optical imaging methods, such as epifluorescent imaging [6] or fluorescence-mediated tomography [7], have recently gained importance with the development of acti- vatible fluorescent probes. These new probes are optically silent in their native, quenched state and become fluorescent only upon specific enzymatic cleavage within the target tissue, thus providing significantly reduced background sig- nals [8]. However, to date, optical imaging methods still suffer from limited depth penetration, which limits them to small animal models and precludes their application in a * Corresponding author. Tel.: +43-1-40400-5802; fax: +43-1-40400- 5830. E-mail address: martin_funovics@yahoo.com (M. Funovics). Magnetic Resonance Imaging 22 (2004) 843– 850 0730-725X/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mri.2004.01.050