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Purpose  Amplification of the HER2/neu gene and/or overexpression of the corresponding protein have been identified in approximately 20% of invasive breast carcinomas. Assessment of HER2 expression  in vivo  would advance development of new HER2-targeted therapeutic agents and, potentially, facilitate choice of the proper treatment strategy offered to the individual patient. We present novel HER2-specific probes for  in vivo  evaluation of the receptor status by near-infrared (NIR) optical imaging.    Experimental Design  Affibody molecules were expressed, purified, and labeled with NIR-fluorescent dyes. The binding affinity and specificity of the obtained probe were tested  in vitro . For  in vivo  validation, the relationship of the measured NIR signal and HER2 expression was characterized in four breast cancer xenograft models, expressing different levels of HER2. Accumulation of Affibody molecules in tumor tissue was further confirmed by  ex vivo  analysis.    Results  Affibody-DyLight conjugates showed high affinity to HER2 (K D  = 3.66±0.26). No acute toxicity resulted from injection of the probes (up to 0.5 mg/kg) into mice. Pharmacokinetic studies revealed a relatively short (37.53±2.8 min) half-life of the tracer in blood. Fluorescence accumulation in HER2-positive BT-474 xenografts was evident as soon as a few minutes post injection and reached its maximum at 90 minutes. On the other hand, no signal retention was observed in HER2-negative MDA-MB-468 xenografts. Immunostaining of extracted tumor tissue confirmed penetration of the tracer into tumor tissue.    Conclusions  The results of our studies suggest that Affibody-DyLight-750 conjugate is a powerful tool to monitor HER2 status in a preclinical setting. Following clinical validation, it might provide complementary means for assessment of HER2 expression in breast cancer patients (assuming availability of proper NIR scanners) and/or be used to facilitate detection of HER2-positive metastatic lesions during NIR-assisted surgery.

Affibody-DyLight Conjugates for In Vivo Assessment of HER2 Expression by Near-Infrared Optical Imaging