IRDye 800CW-Anti-epidermal growth factor receptor nanobody 7D12

Epidermal growth factor (EGF) is a 53-amino acid growth factor (6.2 kDa) that is secreted by ectodermic cells, monocytes, kidneys, and duodenal glands (1). EGF stimulates growth of epidermal and epithelial cells. EGF and 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 because high levels are related to increased proliferation (7-10). Trastuzumab, a humanized immunoglobulin G (IgG) monoclonal antibody (mAb) against the extracellular domain of recombinant HER2 (11), was labeled as In-trastuzumab (12-14). C225, an anti-EGFR (HER1), mouse-human chimeric, monoclonal IgG antibody, also known as erbitux or cetuximab, was labeled as Tc-EC-C225 (15, 16) for imaging EGFR expression on solid tumors using single-photon emission computed tomography (SPECT). However, the pharmacokinetics of the intact radiolabeled mAb, with high liver uptake and slow blood elimination, are generally not ideal for imaging (17, 18). Smaller antibody fragments, such as scFv, Fab, or F(ab'), have better imaging pharmacokinetics because they are rapidly excreted by the kidneys. Nanobodies are the smallest intact antigen-binding fragments (15 kDa) isolated from heavy-chain camelid antibodies and exhibit efficient and specific tumor targeting (19-21). Nanobody 7D12 was labeled with Tc at its C-terminus hexahistidine tail for SPECT (22) and with Ga and Zr for positron emission tomography (PET) (23) imaging EGFR (HER1) expression in tumors in mice. Oliveira et al. (24) prepared IRDye 800CW-anti-EGFR nanobody 7D12 for near-infrared (NIR) fluorescence imaging studies in tumor-bearing mice. IRDye 800CW is an indocyanine-type NIR fluorophore with peak absorption at 785 nm and peak excitation emission at 803 nm. Fluorescence imaging has an advantage in sensitivity in small animals where the signal is not degraded by significant absorption.