Abstract 5298: Affibody – NIR conjugate for optical imaging of HER2-expressing tumors
Authors: Zielinski Rafal J. , Lyakhov Ilya , Moinuddin Hassan , Chernomordik Victor , Garcia-Glaessner Alejandra , Ardeshirpour Yasaman , Gandjbakhche Amir , Capala Jacek 
Published: June 2012

Abstract
Amplification of the HER2/neu gene and/or overexpression of the protein have been identified in approximately 20% of invasive breast carcinoma, Particularly in breast cancer, elevated HER2 status is associated with increased proliferation and survival of cancer cells and thus contributes to poor therapy outcomes and unfavorable prognosis.
Affibody molecules are unique class of artificial ligands. They are relatively small, (∼7-kDa), affinity proteins, structurally based on a 58-amino-acid scaffold derived from the Z domain of the Staphylococcus aureus- protein, and were obtained by combinatorial protein engineering.
Optical imaging is a powerful tool allowing non-invasive in vivo analysis of macroscopic distribution of fluorescent labels. Introduction of Near-Infrared fluorescent beacons significantly improved the light penetration making the Over the past several years, there has been an explosion of reports describing successful in vivo NIR fluorescence imaging using antibodies or antibodies fragment or small molecules as contrast agents.
Here we report the construction, in vitro and in vivo evaluation of a new Affibody based probe for Near Infrared imaging of HER2-positive tumors.
Unique, C-terminal cysteine residue of HER2-Affibody molecules were modified with maleimide derivatives of optical beacons. The conjugates maintained high affinity to HER2 receptors with estimated KD value 3.66 ±0.26 nM using HER2 positive BT474 cells. Competition assay showed slight affinity reduction due to labeling (IC50=24.1 nM at 50nM labeled Affibody). No dye accumulation was observed for “non-specific” Affibody molecules (raised against Taq polymerase). HER2-Affibody binding was well correlated with receptor expression in five different cell lines as determined by FACS and HER2 Elisa respectively. Receptor specificity was also confirmed by immunofluorescence imaging: Cells with high HER2 level (SKBR3) showed almost exclusive membrane retention of the probe even after long period of incubation. Neither membrane retention nor intracellular uptake of the probe was observed for HER2-negative MDA MB-468 cells.
In vivo characteristic of the probe was performed in mice subcutaneous tumor models using NIR fluorescence small-animal imager. The probe showed relatively fast clearance rate from the circulation after intravenous administration (T1/2=31.8±10.42 min). The highest accumulation was recorded for BT474 tumors 90 min post probe administration. No signal was detected in tumors injected with HER2-nonspecyfic probe or for HER2 negative tumors receiving HER2-Affibody. Interestingly, accumulation rate better correlated with HER2 expression level in the tumor than maximum uptake.
HER2-dependend probe accumulation was confirmed also by IHC analysis.
Our results suggest that optical imaging using Affibody-based probes, may become a noninvasive alternative tool for assessment of HER2 expression in breast tumors.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5298. doi:10.1158/1538-7445.AM2011-5298