Alexa Fluor 750-ZHER2:342 Affibody (2011)

Epidermal growth factor (EGF) is a 53-amino acid growth factor (6.2 kDa) secreted by ectodermic cells, monocytes, kidneys and duodenal glands (1). EGF stimulates growth of epidermal and epithelial cells. EGF with at least seven other growth factors and their transmembrane receptor kinases play important roles in cell proliferation, survival, adhesion, migration and differentiation. The EGF receptor (EGFR) family consists of four transmembrane receptors, including EGFR (HER1/erbB-1), HER2 (erbB-2/neu), HER3 (erbB-3) and HER4 (erbB-4) (2). HER1, HER3 and HER4 comprise three major functional domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase domain. No ligand has been clearly identified for HER2. However, HER2 can be activated as a result of ligand binding to other HER receptors with the formation of receptor homodimers and/or heterodimers (3). HER1 as well as HER2 are overexpressed on many solid tumor cells such as breast, non-small-cell lung, head and neck, and colon cancer (4-6). The high levels of HER1 and HER2 expression on cancer cells are associated with a poor prognosis (7-10).Optical fluorescence imaging is increasingly being used to monitor biological functions of specific targets (11-13). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–700 nm) are used. Near-infrared (NIR) fluorescence (700–1,000 nm) detection avoids the natural background fluorescence interference of biomolecules, providing a high contrast between target and background tissues in small animals. NIR fluorophores have a wider dynamic range and minimal background fluorescence as a result of reduced scattering compared with visible fluorescence detection. NIR fluorophores also have high sensitivity, attributable to low background fluorescence, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is a non-invasive alternative to radionuclide imaging in small animals.Trastuzumab is a humanized IgG1 monoclonal antibody (mAb) against the extracellular domain of recombinant HER2 with an affinity constant (Kd) of 0.1 nM (14). 111In-Trastuzumab, Cy5.5-trastuzumab, and 68Ga-trastuzumab-F(ab')2 have been developed for imaging of human breast cancer (15-19). However, the pharmacokinetics of intact radiolabeled mAb, with high liver uptake and slow blood elimination, are generally not ideal for imaging. Smaller antibody fragments, such as Fab or F(ab´)2, have better imaging pharmacokinetics because they are rapidly excreted by the kidneys. A novel class of recombinant affinity ligands (Affibody molecules) for HER2 was constructed based on a 58-amino-acid Z-domain residues from one of the IgG-binding domains of staphylococcal protein A (20). Affibody molecules exhibit high binding affinity to HER2 with Kd values of PubMed ]. A synthetic Affibody molecule His6-ZHER2:342-Cys Affibody was labeled with Alexa Fluor 750 (Alexa750) (21). Alexa750-ZHER2:342 has been evaluated in nude mice bearing human SKBR-3 breast adenocarcinoma tumor.