Cu-1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-epidermal growth factor receptor-binding fibronectin domain E13.4.3'

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 the 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 poor patient 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, IgG mAb, also known as erbitux and cetuximab, was labeled as Tc-EC-C225 (15, 16) and Cu-DOTA-cetuximab for imaging EGFR expression on solid tumors with the use of single-photon emission computed tomography (SPECT) and positron emission tomography (PET), respectively. 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 (15 kDa) and affibodies (50 kDa) exhibit efficient and specific tumor targeting but with high kidney accumulation (19-21). An engineered fibronectin scaffold with EGFR-binding domain (Fn, 10 kDa) was radiolabeled with Cu 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugation to form Cu-DOTA-Fn as a PET probe for imaging of EGFR in tumor-bearing nude mice (22). Fn does not contain the integrin binding sequence of Arg-Gly-Asp (RGD) (23).