Data from Optimizing Radiolabeled Engineered Anti-p185<sup>HER2</sup> Antibody Fragments for <i>In vivo</i> Imaging

Abstract

<div>Abstract<p>We have recently described the <i>in vivo</i> properties of an iodinated anti-p185<sup>HER2</sup> engineered antibody fragment [minibody (scFv-C<sub>H</sub>3)<sub>2</sub>; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target <i>in vitro</i>, 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-<i>N</i>, <i>N</i>′, <i>N</i>′′, <i>N</i>′′′-tetraacetic acid (DOTA), radiometal labeled, and evaluated <i>in vivo</i>. The tumor uptake of <sup>111</sup>In-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<sup>HER2</sup> antibody (trastuzumab; hu4D5v8) was also made. Tumor uptakes, evaluated by quantitative microPET using <sup>64</sup>Cu-DOTA hu4D5v8 minibody, were 4.2 ± 0.5% ID/g. Furthermore, in non-tumor-bearing mice, <sup>111</sup>In-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<sub>H</sub>2-C<sub>H</sub>3)<sub>2</sub>; 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<sup>HER2</sup> antibody fragments, the kidney activity was reduced and high or low expression of p185<sup>HER2</sup> in xenografts could be distinguished by microPET imaging.</p></div>