In vivo and in vitro studies on renal uptake of radiolabeled affibody molecules for imaging of HER2 expression in tumors

Affibody molecules (6-7 kDa) are a new class of small robust three-helical scaffold proteins. Radiolabeled subnanomolar anti-HER2 affibody Z _HER2:342 was developed for imaging of HER2 expression in tumors, and a clinical study has demonstrated that the ¹¹¹In- and ⁶⁸Ga-labeled affibody molecules can efficiently detect HER2 expressing metastases in breast cancer patients. However, a significant renal accumulation of radioactivity after systemic injection of a radiolabeled anti-HER2 affibody conjugate is observed. The aim of this study was to investigate the mechanism of renal reabsorption of anti-HER2 affibody at the molecular level. Renal accumulation of radiolabeled anti-HER2 affibody molecules was studied in a murine model and in vitro using opossum-derived proximal tubule (OK) cells. It was found that kidney reabsorption of affibody molecule was not driven by megalin/cubilin. Amino acids in the target-binding side of affibody molecule were involved in binding to OK cells. On OK cells, two types of receptors for anti-HER2 affibody molecule were found: K_D1=0.8 nM, B_max1=71,500 and K_D2=9.2 nM, B_max2=367,000. The results of the present study indicate that affibody molecule and other scaffold-based targeting proteins with a relatively low kidney uptake can be selected using in vitro studies with tubular kidney cells.