Small-Animal SPECT/CT of HER2 and HER3 Expression in Tumor Xenografts in Athymic Mice Using Trastuzumab Fab–Heregulin Bispecific Radioimmunoconjugates

Abstract

Heterodimerization of human epidermal growth factor receptor 2 (HER2) with HER3 initiates aberrant downstream growth-signaling pathways in tumors. Our objective was to construct bispecific radioimmunoconjugates (bsRICs) that recognize HER2 and HER3 and evaluate their ability to image tumors in athymic mice that express one or both receptors using small-animal SPECT/CT. bsRICs were constructed by reacting the maleimide-derivatized trastuzumab Fab fragments that bind HER2 with a thiolated form of the HER3-binding peptide of heregulin-β1 (HRG) with or without a 12- or 24 mer polyethylene glycol (PEG) spacer. bsRICs were derivatized with diethylenetriaminepentaacetic acid for labeling with (111)In. The ability of (111)In-bsRICs to bind HER2 or HER3 was determined in competition assays with unlabeled Fab or HRG on cells expressing one or both receptors. Tumor and normal-tissue uptake were examined in CD1 athymic mice bearing subcutaneous tumor xenografts that expressed HER2, HER3, or both receptors, with or without the preadministration of unlabeled Fab or HRG to determine the specificity of uptake. Conjugation of Fab to HRG was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis-Western blot and size-exclusion high-performance liquid chromatography. Improved HER2 and HER3 binding and greater displacement of binding by competitors was found for (111)In-bsRICs that incorporated a PEG spacer, with the PEG(24) spacer being optimal. The highest uptake of (111)In-bsRICs (7.8% ± 2.1% injected dose per gram [%ID/g]) in BT-474 human breast cancer xenografts (HER2-positive/HER3-positive) occurred at 48 h after injection. The preadministration of trastuzumab Fab decreased uptake in SK-OV-3 (HER2-positive/HER3-negative) human ovarian cancer xenografts from 7.0 ± 1.2 to 2.6 ± 1.5 %ID/g (P < 0.001). The preadministration of an excess of HRG decreased uptake in MDA-MB-468 (HER2-negative/HER3-positive) human breast cancer xenografts from 4.4 ± 0.9 to 2.6 ± 0.5 %ID/g (P < 0.05). All tumors were imaged by small-animal SPECT/CT. (111)In-bsRICs composed of trastuzumab Fab and HRG exhibited specific binding in vitro to tumor cells displaying HER2 or HER3 and were taken up specifically in vivo in tumors expressing one or both receptors, permitting tumor visualization by small-animal SPECT/CT. These agents could be useful for imaging heterodimerized HER2 and HER3 receptors because their bivalent properties may result in preferential binding to the heterodimerized forms. The approach may also be extended to constructing bsRICs for visualizing other peptide growth factor receptors.