BODIPY-FL-neutravidin-biotin-Cetuximab

Epidermal growth factor (EGF) is a cytokine (6.2 kDa) composed of 53 Amino acids 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: 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 and 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 used to monitor biological functions of specific targets (11-13). Target-specific optical activation has been studied on the basis of protease activation of quenched fluorophore-labeled enzyme substrates (14, 15). Another approach is based on the binding of biotin to BODIPY-FL–conjugated neutravidin (nAv) leading to an ~10-fold increase in fluorescence signal (16). The biotin-nAv binding inhibited the photon-induced electron transfer from aromatic Amino acids to the excited state of BODIPY-FL, which led to dequenching of the BODIPY-FL. Cetuximab is a humanized IgG₁ monoclonal antibody (mAb) against the extracellular domain of recombinant HER1 with an affinity constant of 0.1 nM. Cetuximab is available commercially in the United States and has been Cancer.gov/cancertopics/druginfo/cetuximab"> approved by the U.S. Food and Drug Administration for the treatment of metastasized colorectal Cancer and advanced squamous cell carcinoma of the head and neck. Cetuximab is currently being evaluated in several Cancer.gov/search/ResultsClinicalTrialsAdvanced.aspx?protocolsearchid=2740900"> clinical trials for the treatment of a variety of cancers. Hama et al. (16) pretargeted peritoneal tumor metastases with biotin-cetuximab and then administered nAv-conjugated BODIPY-FL (nAv-BDPfl), which increased in fluorescence upon binding to biotin-cetuximab. BODIPY-FL is a green Fluorescent Dye with an absorbance maximum at 505 nm, an emission maximum at 513 nm, and a high extinction coefficient of 80,000 M^-1cm^-1.