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Optimizing Radiolabeled Engineered Anti-p185^HER2 Antibody Fragments for In vivo Imaging

¹ Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California at Los Angeles; ² Department of Pathology, Keck School of Medicine of the University of Southern California, Hoffman Medical Research Center, Los Angeles, California; ³ Division of Molecular Biology and ⁴ Division of Immunology, Beckman Research Institute of the City of Hope; ⁵ Department of Radioimmunotherapy, ⁶ Division of Radiology, and ⁷ Division of Radiation Oncology, City of Hope National Medical Center, Duarte, California; and ⁸ Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program, Stanford University School of Medicine, Stanford, California

Requests for reprints: Tove Olafsen, Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California at Los Angeles, 700 Westwood Plaza, Los Angeles, CA 90095. Phone: 310-267-2819; E-mail: tolafsen{at}mednet.ulca.edu.

We have recently described the in vivo properties of an iodinated anti-p185^HER2 engineered antibody fragment [minibody (scFv-C_H3)₂; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target in vitro, only modest tumor uptake [5.6 ± 1.7% injected dose per gram (ID/g) of tissue] was achieved in nude mice bearing MCF7/HER2 breast cancer tumors. Here, in an attempt to improve targeting, the 10H8 minibody was conjugated to 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA), radiometal labeled, and evaluated in vivo. The tumor uptake of ¹¹¹In-DOTA 10H8 minibody was 5.7 ± 0.1% ID/g, similar to the radioiodinated 10H8 minibody. However, in addition to the expected liver clearance, the kidneys had unexpectedly high activity (34.0 ± 4.0% ID/g). A minibody derived from a second anti-p185^HER2 antibody (trastuzumab; hu4D5v8) was also made. Tumor uptakes, evaluated by quantitative microPET using ⁶⁴Cu-DOTA hu4D5v8 minibody, were 4.2 ± 0.5% ID/g. Furthermore, in non-tumor-bearing mice, ¹¹¹In-DOTA hu4D5v8 minibody exhibited similar elevated uptake in the kidneys (28.4 ± 6.5% ID/g). Immunohistochemical staining of kidneys from non-tumor-bearing mice showed strong specific staining of the proximal tubules, and Western blot analysis of kidney lysate confirmed the presence of cross-reactive antigen. To further improve tumor uptake and normal tissue distribution, a larger hu4D5v8 fragment [(scFv-C_H2-C_H3)₂; 105 kDa] was made, engineered to exhibit rapid clearance kinetics. This fragment, when evaluated by microPET, exhibited improved tumor targeting (12.2 ± 2.4% ID/g) and reduced kidney uptake (13.1 ± 1.5% ID/g). Thus, by manipulating the size and format of anti-p185^HER2 antibody fragments, the kidney activity was reduced and high or low expression of p185^HER2 in xenografts could be distinguished by microPET imaging.

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