IRDye 800-Anti–epidermal growth factor receptor Affibody

Epidermal growth factor (EGF) is a 53-amino-acid cytokine (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 cancers (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 IgG monoclonal antibody (mAb) against the extracellular domain of recombinant HER2 with an affinity constant () of 0.1 nM (14). In-Trastuzumab, Cy5.5-trastuzumab, and Ga-trastuzumab-F(ab') have been developed for imaging human breast cancer (15-19). C225 (an anti-EGFR, mouse–human chimeric, monoclonal IgG antibody, also known as erbitux and cetuximab) was labeled as Tc-EC-C225 (20, 21) to image EGFR expression in solid tumors using single-photon emission computed tomography. 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´), have better imaging pharmacokinetics because they are rapidly excreted by the kidneys. A novel class of recombinant affinity ligands (Affibody molecules) for EGFR was constructed on the basis of a 58-amino-acid Z-domain residue from one of the IgG-binding domains of staphylococcal protein A (22). Affibody molecules exhibit high binding affinity to EGFR with values of 25–50 nM. Various radiolabeled Affibody molecules have been studied in their ability for imaging of EGFR in tumors [PubMed]. A synthetic Affibody molecule (Eaff) specific to EGFR was labeled with NIR dye IRDye 800CW (Eaff800) (23). Eaff800 has been evaluated in nude mice bearing human A431 skin carcinoma tumor.