Background

[PubMed]

Epidermal growth factor (EGF) is a cytokine 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 a poor prognosis (7-10).

Trastuzumab is a humanized IgG₁ monoclonal antibody (mAb) against the extracellular domain of recombinant HER2 with an affinity constant (K_d) 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 K_d values <100 pM. Various radiolabeled Affibody molecules have been studied in terms of their ability to image HER2 in tumors [PubMed]. Recently, the EGFR-specific Affibody Z_EGFR:1907 (8.1 kDa) was identified and labeled with ¹¹¹In via isothiocyanate-BZ-diethylenetriaminepentaacetic acid (DTPA) to form ¹¹¹In-BZ-DTPA-Z_EGFR:1907 for single-photon emission computed tomography (SPECT) imaging in nude mice bearing human tumors (18).

Synthesis

[PubMed]

Isothiocyanate-BZ-DTPA and Z_EGFR:1907 (1:1 molar ratio) were incubated in sodium borate buffer (pH 9) for 20 h at 37°C (18). BZ-DTPA-Z_EGFR:1907 was isolated from the incubation mixture with a NAP-5 column with 95% coupling efficiency. BZ-DOTA- Z_EGFR:1907 in 0.2 M ammonium acetate buffer (pH 5.25) was mixed with a predetermined amount of ¹¹¹InCl₃. The mixture was incubated for 1 h at room temperature with a labeling efficiency of >95%. ¹¹¹In-BZ-DTPA-Z_EGFR:1907 was purified with NAP-5 chromatography with a radiochemical purity of >95%. The specific activity of ¹¹¹In-BZ-DTPA-Z_EGFR:1907 was not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

In vitro binding specificity tests showed that binding of ¹¹¹In-BZ-DTPA-Z_EGFR:1907 to A431 cells expressing EGFR was mediated by the receptors because saturation of receptors by preincubation with non-labeled Z_EGFR:1907 significantly decreased binding of ¹¹¹In-BZ-DTPA-Z_EGFR:1907 (18). The cell-bound radioactivity remained at 35–42% of the initially bound activity for up to 24 h. AlexaFluor 488-Z_EGFR:1907 exhibited a K_d value of 2.8 nM for the EGFR of A431 cells.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

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