Background

[PubMed]

Epidermal growth factor (EGF) is a 53-amino acid cytokine (6.2 kDa) 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 (7-10).

Trastuzumab (a humanized immunoglobulin G₁ (IgG₁) monoclonal antibody against the extracellular domain of recombinant HER2) (11) and C225 (an anti-EGFR, chimeric, monoclonal antibody) have been labeled as ¹¹¹In-trastuzumab (12-14) and ^99mTc-EC-C225 (15, 16) for imaging EGFR expression on solid tumors using single-photon emission computed tomography (SPECT). However, antibodies that are approximately 25-fold larger than EGF may not be easily transported to cells within solid tumors. Therefore, ¹¹¹In-EGF and ^99mTc-HYNIC-EGF have been developed for SPECT imaging studies of tumors (17, 18). However, positron emission tomography (PET) offers better sensitivity, resolution, and quantification than SPECT (19). ⁶⁸Ga (t_1/2 = 68 min, 89% β⁺ decay) is an attractive radionuclide for labeling EGF and is readily available from a commercial ⁶⁸Ge/⁶⁸Ga generator. Cells retain ⁶⁸Ga well, providing a good signal/background ratio. For evaluation as a PET imaging agent, ⁶⁸Ga has been attached to EGF via 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) to form ⁶⁸Ga-DOTA-EGF (20).

Synthesis

[PubMed]

Commercially available monofunctional N-hydroxysuccinimide ester of DOTA was used to conjugate human EGF to form DOTA-EGF, which was purified by column chromatography (20). ⁶⁸Ga was conjugated to DOTA-EGF under microwave heating for 1 min to 95°C. ⁶⁸Ga-DOTA-EGF was purified by column chromatography. The yield of ⁶⁸Ga-DOTA-EGF was 60–77% with a radiochemical purity >95% and specific activities of 12–20 MBq/nmol (0.32–0.54 mCi/nmol).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Velikyan et al. (20) performed cell-binding assays with ⁶⁸Ga-DOTA-EGF using EGFR-expressing human cervical carcinoma A431 and glioma U343 cell lines. Binding of ⁶⁸Ga-DOTA-EGF (approximately 0.01 μM) to both cell lines was completely blocked by EGF (approximately 1 μM). Saturation binding experiments determined that dissociation constant (K_d) values were 2.0 and 2.3 nM for A431 and U343 cells, respectively. The number of binding sites per cells was 1.9 × 10⁶ per A431 cell and 7.8 × 10⁵ per U343 cell. Both cell lines rapidly internalized ⁶⁸Ga-DOTA-EGF at 37°C.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

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