Background

[PubMed]

The epidermal growth factor (EGF) and its receptor (EGFR) are known to play a role in the neoplastic transformation of cells (1). The EGFR is a tyrosine kinase receptor family that includes four members, EGFR and three other human EGF receptors (HERs) designated as HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4. The four receptors constitute the HER-kinase axis and regulate cellular responses through complex receptor–ligand interactions. The various signal transduction pathways that respond to EGFR activation and mediate cell responses have been described by Brandt et al. (2). The various EGFRs are known to be amplified, mutated, or overexpressed in several cancers and are a target for the development of pharmaceutical agents that inhibit either the receptor or the signal transduction pathway (3). It has also been reported that the detection of circulating ErbB2-positive tumor cells indicates a poor prognosis for the cancer patient (4). A variety of inhibitors, including small molecules, antibodies, vaccines, and gene therapies, have been developed and tested against the EGFRs for the treatment of cancer (5, 6). However, to achieve therapeutic effects the receptor or signal transduction pathway must be involved in maintenance of the malignant phenotype because HER inhibitor therapy does not necessarily result in treatment of a cancer (7).

The use of antibodies alone against EGFR (e.g., cetuximab) and ErbB2 (e.g., trastuzumab) for the treatment of cancer has yielded only moderate results, and it has been observed that antibodies are most useful when used in combination with chemotherapy (8). In some cases cardiotoxicity has been reported in individuals treated with trastuzumab in combination with some chemotherapeutic agents (9). In an effort to develop better and safer anti-cancer drugs, investigators have attempted to use peptides that bind to receptors on tumors to treat the disease (10, 11). The development of peptide pharmaceuticals has also been pursued because, compared to the larger biomolecules such as antibodies, these molecules are easier to synthesize and have low immunogenicity, rapid blood clearance, and a higher uptake in tumor tissue (12). Using bacteriophage technology, a peptide of six amino acids, lys-cys-cys-tyr-ser-leu (KCCYSL), that specifically recognizes and binds the extracellular domain of ErbB2 was identified, and the investigators hypothesized that the peptide could be developed into an imaging and therapeutic agent against cancer cells that overexpress ErbB2 (13). As an extension to the earlier study, the tumor imaging and targeting ability of KCCYSL was evaluated after labeling with radioactive indium (¹¹¹In). Using the labeled peptide, Kumar et al. performed in vitro and in vivo studies in human breast carcinoma cells that overexpress ErbB2 and in xenografted mice (14).

Synthesis

[PubMed]

The procedure used to identify and characterize KCCYSL from a phage display library as an ErbB2 extracellular domain binding peptide was described in detail by Karasseva et al. (13). The synthesis of ¹¹¹In-labeled KCCYSL was described by Kumar et al. (14). The peptide was synthesized using solid-phase Fmoc technology on a peptide synthesizer. The bifunctional chelator 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) was linked with KCCYSL through a gly-ser-gly (GSG) spacer placed between the amino terminus of DOTA and the hexapeptide to obtain DOTA-(GSG)-KCCYSL. For use as control, a scrambled peptide version (KYLCSC) was also prepared similarly. The peptides were purified by reverse-phase high-pressure liquid chromatography (RP-HPLC) on a Vydac C₁₈ column. The peptides were freeze-dried and stored at -20°C until required. Electrospray ionization-mass spectrometery was used to confirm the identity of the peptides. Details of how this technique was used to confirm the identity of the peptides were not provided in the publication.

For radiolabeling, the respective peptides were mixed with radioactive [¹¹¹In]indium chloride in ammonium acetate buffer (pH 5.5) (14). The mixture was incubated at 85°C for 60 min, and the reaction was terminated by the addition of ethylenediamine tetraacetic acid to remove unreacted ¹¹¹In. The labeled conjugates were then purified by RP-HPLC and flush-dried with nitrogen gas, and the pH was adjusted to 8.0 with sodium phosphate buffer in saline. Tris(2-carboxyethyl)phosphine hydrochloride was then added to the peptide stock solution to prevent oxidation of the cysteine thiols. Although the radiolabeling efficiency of the reaction was reported to be 40–50%, the specific activity, radiochemical yield, and purity of the product was not provided by the investigators (14). The labeled peptide was reported to be stable for 12 h in phosphate-buffered saline and for at least 1 h in mouse serum.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The in vitro binding of ¹¹¹In-DOTA(GSG)-KCCYSL was investigated in MDA-MB-435 cells that express ErbB2 receptors (14). For the same study, K-562 cells, which do not express ErbB2 receptors, were used as negative controls. The cells were incubated with the radiolabeled peptide for various times at 37°C, and binding of the label with MDA-MB-435 cells increased gradually and reached saturation by 2 h. Under the same conditions the K-562 cells bound little radioactivity. Neither cell line bound the scrambled version of the labeled peptide. In competition experiments with MDA-MB-435 cells, using an increasing concentration of cold DOTA(GSG)-KCCYSL, the binding of radioactivity decreased in a concentration-dependent manner, indicating a specificity of the labeled peptide for ErbB2 receptors (14). The 50% inhibitory concentration of the labeled peptide with these cells was determined to be 42.5 ± 2.67 nM.

To determine internalization of bound radioactivity, the MDA-MB-435 cells were incubated with the labeled peptide for different time periods and washed with ice-cold cell-binding medium to remove any unbound radioactivity. The cells were then incubated on ice with acetic acid in saline for 5 min and pelleted by centrifugation. Radioactivity was counted to determine the internalized (cell pellet) and the surface bound fraction (supernatant). The cell surface–associated label increased up to 2 h and ~11% of the total radioactivity was determined to be internalized during this period (P < 0.0001).

Animal Studies

Human Studies

[PubMed]

No references are currently available.

NIH Support

The imaging study reported in this chapter was funded by an NIH P50 Grant #CA103130.

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