Background

[PubMed]

Growth factors such as the epidermal growth factor (EGF), the vascular endothelial growth factor (VEGF), and the tumor necrosis factor are known to have important roles in the development and progression of cancers (1-3). The EGF mediates its activity through a group of tyrosine kinase (TK)–dependent EGF receptors (EGFR) that promote not only cell proliferation but also cell survival and the motility phenotype that is necessary for the development of an invasive cancer (4). As a consequence, the EGFRs and their associated TKs are the targets of several anti-EGFR antibodies and TK inhibitors that are either approved by the United Stated Food and Drug Administration or are being evaluated in clinical trials for the treatment of various neoplasms. Before treatment, the patients are screened to determine the extent of EGFR expression in the tumors with the use of immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) because these techniques help predict the probable therapeutic response and outcome for the individual (5, 6). However, only 10–25% of the patients who are EGFR-positive according to IHC or FISH analysis respond to the anti-EGFR therapy, which indicates that a poor correlation exists between the EGFR expression analysis and the treatment (7, 8). This is probably because only the active receptors are important to predict efficacy of the anti-EGFR agents for the treatment of cancers, and ICH and FISH do not distinguish between the active and inactive EGFR (9).

On binding the ligand, a characteristic feature of a functional EGFR is that it is internalized by the cell. Levashova et al. (10) envisioned that perhaps the natural ligand EGF itself could be used as an imaging agent to detect the active EGFRs in tumors. The investigators used an Escherichia coli (E. coli)–expressed EGF containing an N-terminal cysteine fusion tag of 15 amino acids (Cys-EGF) that can be used to conjugate therapeutic or imaging probes as done earlier with VEGF (11). In addition, because an active EGF/EGFR complex is composed of two molecules each of EGF and EGFR, the investigators also bioengineered an N-terminal cysteine fusion tag containing a dimeric EGF (dEGF) molecule, with the second EGF molecule linked to the first through a linker of 9 amino acids (11). The EGF and the dEGF were then labeled with radioactive copper (⁶⁴Cu) to obtain [⁶⁴Cu]Cys-EGF and [⁶⁴Cu]Cys-dEGF. The biodistribution and imaging characteristics of the two molecules were then investigated in mice bearing xenograft tumors. It is pertinent to mention here that the imaging and biodistribution studies of only [⁶⁴Cu]Cys-EGF are described in this chapter. Investigations performed with [⁶⁴Cu]Cys-dEGF are described separately in MICAD (www.micad.nih.gov).

Synthesis

[PubMed]

The Cys-EGF was expressed in and purified from E. coli as described elsewhere to obtain Cys-tagged single-chain VEGF (Cys-scVEGF) (12). Purified Cys-EGF was labeled with ⁶⁴Cu as described elsewhere (12) with a procedure developed to label scVEGF using pegylated-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (PEG-DOTA). The EGF-PEG-DOTA complex was purified with reverse-phase high-performance liquid chromatography, and purity of the product was reported to be >90% (10). Unconjugated ⁶⁴Cu was removed by the addition of ethylenediamine tetraacetic acid, and the radiolabeled Cys-EGF (EGF-PEG-DOTA/Cu) was purified with size-exclusion chromatography on a NP-5 column (10). The radiochemical purity of EGF-PEG-DOTA/Cu was not reported. Specific activity of EGF-PEG-DOTA/Cu was reported to be ~4.6 MBq/μg (~125 μCi/μg) (10). The stability of EGF-PEG-DOTA/Cu was reported to be at least 3 h at room temperature in phosphate-buffered saline and at least 75 min at 37°C in plasma (10).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Using F98/EGFR(F) rat glioma cells that overexpress the human EGFR, the receptor affinity constant for EGF-PEG-DOTA/Cu was determined with Scatchard’s analysis to be 3.0 nM (10). In another study, EGF-PEG-DOTA was reported to induce EGFR TK phosphorylation in these cells in a dose-dependent manner (10). EGF-PEG-DOTA/Cu was also reported to compete with an EGF-Shiga-like toxin subunit A conjugate (EGF-SLT) for binding to the EGFR (10). EGF-SLT is extremely toxic to MDA23 1luc human breast cancer cells, and the ability of proteins to compete for binding with the EGFR was measured by counting the number of cells surviving after a 72-h exposure to EGF-SLT (1 nM) in the presence of an increasing concentration of Cys-EGF (0–25 nM). It was reported that cell survival increased with an increasing EGF-PEG-DOTA/Cu concentration under these experimental conditions, which indicates that EGF-PEG-DOTA/Cu protected the cells from EGF-SLT toxicity.

Animal Studies

Human Studies

[PubMed]

No references are currently available.

Supplemental Information

[Disclaimers]

No information is currently available.

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