Epidermal growth factor (EGF) is a growth factor composed of 53 amino acids (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: 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 poor patient prognosis because high levels are related to increased proliferation (7-10).
Trastuzumab is a humanized IgG1 monoclonal antibody (mAb) against the extracellular domain of recombinant HER2 with an affinity constant (Kd) of 0.1 nM (11). Trastuzumab is approved for clinical use for anti-cancer therapies in both Europe and North America. 111In-Trastuzumab, Cy5.5-trastuzumab, and 68Ga-trastuzumab-F(ab')2 have been developed for imaging human breast cancer (12-16). However, the pharmacokinetics of the 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, based on the Z-domain residues (58 amino acids) from one of the IgG-binding domains of staphylococcal protein A, was constructed (17). Affibody molecules exhibit high binding affinity to HER2 with Kd values <100 pM. Various radiolabeled Affibody molecules have been studied in terms of their ability to image HER2 in tumors [PubMed]. The EGFR-specific Affibody ZEGFR:1907 (6.7 kDa) was identified to have good affinity (Kd = 5.4 nM) for EGFR (HER1) and was labeled with 111In via isothiocyanate-benzoyl-diethylenetriamine pentaacetic acid (DTPA) to form 111In-Bz-DTPA-ZEGFR:1907 for use with single-photon emission computed tomography (18) imaging in nude mice bearing human tumors (19). To facilitate site-specific conjugation, Ac-Cys-ZEGFR:1907 (Cys at the N terminal) was synthesized and conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidethylacetamide (maleimido-mono-amide-DOTA) to form Ac-Cys(DOTA)-ZEGFR:1907 (DOTA-ZEGFR:1907), and the product was labeled with 64Cu (t1/2, 12.7 h) to form 64Cu-DOTA-ZEGFR:1907 for positron emission tomography imaging studies in tumor-bearing mice (20). Miao et al. (21) reported the site-specific labeling of Ac-Cys-ZEGFR:1907 with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM) to form [18F]FBEM-Cys-ZEGFR:1907 for use with in vivo imaging of EGFR-positive tumors.
Ac-Cys-ZEGFR:1907 was prepared with an automated solid-phase peptide synthesizer (21). [18F]FBEM was prepared by reaction of N-succinimidyl-4-[18F]fluorobenzoate with amino-ethyl-maleimide in dimethyl sulfoxide for 30 min at 50°C. [18F]FBEM was isolated with high-performance liquid chromatography (HPLC). [18F]FBEM-Cys-ZEGFR:1907 was prepared by reaction of [18F]FBEM with Cys-ZEGFR:1907 (15 nmol) in sodium acetate buffer (pH 7) for 40 min at room temperature. [18F]FBEM-Cys-ZEGFR:1907 was purified with PD-10 column chromatography, with a radiochemical purity of >95% as measured with HPLC. The specific activity of [18F]FBEM-Cys-ZEGFR:1907 was 10 MBq/nmol (0.27 mCi/nmol) at the end of synthesis. Total synthesis and purification time was ~3 h. [18F]FBEM-Cys-ZEGFR:1907 remained >90% intact in mouse serum for 3 h at 37°C.
In Vitro Studies: Testing in Cells and Tissues
In vitro binding specificity tests showed that binding of [18F]FBEM-Cys-ZEGFR:1907 to A431 human epidermoid carcinoma cells expressing EGFR was mediated by the receptors because saturation of receptors by incubation with non-labeled Ac-Cys-ZEGFR:1907 significantly decreased binding [18F]FBEM-Cys-ZEGFR:1907 (21). [18F]FBEM-Cys-ZEGFR:1907 exhibited a dissociation constant (Kd) value of 37 ± 3 nM for the EGFR of A431 cells as measured with saturation assay at 4°C.
Miao et al. (21) performed ex vivo biodistribution studies of 1.9–2.6 MBq (0.05–0.07 mCi, ~0.4 nmol) [18F]FBEM-Cys-ZEGFR:1907 in nude mice (n = 3/group) bearing A431 xenografts at 3 h after injection. Tumor accumulation of radioactivity was 3.94 ± 0.39% injected dose per gram (ID/g). Kidney, liver, and blood accumulation was 3.78%, 9.28%, and 3.75% ID/g, respectively. Accumulation in most other organs was <1.6% ID/g. Co-injection of 6.9 and 77 nmol unlabeled Ac-Cys-ZEGFR:1907 resulted in tumor accumulation of 8.06 ± 1.44% ID/g and 0.97 ± 0.42% ID/g, respectively. The high blocking dose inhibited tumor accumulation by 75% (P < 0.05), whereas the low blocking dose enhanced tumor accumulation by 105% (P < 0.05). The high blocking dose decreased radioactivity in most normal organs except the kidney. On the other hand, the low blocking dose exhibited a slight decrease of radioactivity in most normal organs except the lung, kidney, skin, pancreas, and bone.
PET imaging analysis was performed in nude mice bearing A431 tumors at 1 h and 3 h after intravenous injection of 1.9–2.6 MBq (0.05–0.07 mCi, ~0.4 nmol) [18F]FBEM-Cys-ZEGFR:1907 with or without co-injection of 6.9 and 77 nmol unlabeled Ac-Cys-ZEGFR:1907 (21). With no blocking dose, the tumor was clearly visualized at 1 h and 3 h after injection. With the 6.9-nmol blocking dose, the tumor exhibited higher tumor/background contrast than with no blocking dose. With the 77-nmol blocking dose, the tumor was not visualized at 1 and 3 h.
Other Non-Primate Mammals
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Created: July 31, 2012; Last Update: November 1, 2012.
National Center for Biotechnology Information (US), Bethesda (MD)
Leung K. N-[2-(4-[18F]Fluorobenzamido)ethyl]maleimide-Cys-ZEGFR:1907. 2012 Jul 31 [Updated 2012 Nov 1]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.