Novel Cyclic Peptides for Targeting EGFR and EGRvIII Mutation for Drug Delivery

Abstract

The epidermal growth factor-epidermal growth factor receptor (EGF-EGFR) pathway has become the main focus of selective chemotherapeutic intervention. As a result, two classes of EGFR inhibitors have been clinically approved, namely monoclonal antibodies and small molecule kinase inhibitors. Despite an initial good response rate to these drugs, most patients develop drug resistance. Therefore, new treatment approaches are needed. In this work, we aimed to find a new EGFR-specific, short cyclic peptide, which could be used for targeted drug delivery. Phage display peptide technology and biopanning were applied to three EGFR expressing cells, including cells expressing the EGFRvIII mutation. DNA from the internalized phage was extracted and the peptide inserts were sequenced using next-generation sequencing (NGS). Eleven peptides were selected for further investigation using binding, internalization, and competition assays, and the results were confirmed by confocal microscopy and peptide docking. Among these eleven peptides, seven showed specific and selective binding and internalization into EGFR positive (EGFR+ve) cells, with two of them-P6 and P9-also demonstrating high specificity for non-small cell lung cancer (NSCLC) and glioblastoma cells, respectively. These peptides were chemically conjugated to camptothecin (CPT). The conjugates were more cytotoxic to EGFR+ve cells than free CPT. Our results describe a novel cyclic peptide, which can be used for targeted drug delivery to cells overexpressing the EGFR and EGFRvIII mutation.

Keywords: EGFR; NSCLC; glioblastoma; peptide; targeted drug delivery.

Figure 1

Detection of EGFR^WT and EGFRvIII expression in K562, H1299, H1975, and DKMG cells by flow cytometry. Cells were incubated with (A) anti-human EGFR WT-APC or (B) anti-human EGFRvIII antibodies for 1 h at 4 °C, and were washed and analyzed by flow cytometry. The gray curves are unstained cells, and the red curves indicate positive cells for EGFR^wt or EGFRvIII. For each sample, 20,000 cells were examined. The data shown are from one experiment that is representative of three repeated experiments.

Figure 2

Biological activity of the peptide–FITC conjugates. (A) Binding and (B) internalization activity of P1-P11 FITC-labeled peptides on K562, H1299, and DKMG cells measured by flow cytometry. Cells were treated with 2.5 µM peptide–FITC conjugates and incubation for 3 h at 37 °C before the FACS analysis. The experiments were repeated three times. Mean and ±SD are shown (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Competitive uptake of P3, P4, P5, P6, P9, and P11 peptides by FACS. Fluorescence was measured for H1299 cells (30,000 cells) after incubation with (2.5 µM) P3–P6, P9, and P11 by FITC-labeled peptides in the absence or presence of 80 nM of the EGF protein. The experiments were performed in duplicate. For each sample, the mean ± SD is shown (** p < 0.01, *** p < 0.001).

Figure 4

EGFR status and peptide internalization into HEK-293, MCF-10A, and MDA-MB-435 cells. (A) Flow cytometry analysis of EGFR expression in cells. Cells were treated with monoclonal antibodies against human EGFR. Expression levels were measured by the APC fluorescence intensity. (B) Cells were incubated with 2.5 µM peptides for 3 h at 37 °C, collected with trypsin, and analyzed by flow cytometry. The experiment was repeated three times; untreated cells were used as the control. The mean of the FITC positive cells ± SD is presented (n = 5).

Figure 5

Intracellular localization of FITC-labeled P6 and P9 peptides. HEK-293, H1299, and DKMG. Cells were grown in glass-bottom black plates and incubated for 0 and 3 h at 37 °C with 25 µM of FITC-labeled P6 and P9 peptides. Cell nuclei were stained with Hoechst-293. After incubation, cells were washed three times with PBS (Ca²⁺ and Mg²⁺), fixed with 4% paraformaldehyde (PFA), and then analyzed by confocal laser microscopy. Magnification ×200.

Figure 6

Docked structures of peptides P6, P9, and P11 to the EGFR extracellular region. (A) Unbiased rigid body docking of the peptides to EGFR was carried out with the HDOCK server. The top ten docked poses of each peptide are represented as spheres corresponding to their centers of mass, and colored green (P6), magenta (P9), and blue (P11). The homodimeric structure of EGFR is shown using ribbon representation, with chains A and B colored gray and gold, respectively. The vast majority of peptides were bound to the EGFR receptor in the cavity between domains I and III. (B) The docked structure of peptides P6 (green), P9 (magenta), and P11 (blue) are shown using a stick representation. The peptides were bound to the EGFR receptor in the cavity between domains I and III in vicinity to the EGF binding site. Peptides P6 and P9 show substantial overlap with EGF while peptide P11 is bound deeper within the cavity (toward domain II) and has a smaller overlap with EGF. For clarity, the EGF structure (cyan) is shown using semitransparent ribbon representation and removed from the structure during the docking simulations.

Figure 7

Synthesis of the P6-CPT and P9-CPT conjugates.

Figure 8

CPT release profiles for P6-CPT and P9-CPT peptide drug conjugates. Peptide conjugates were incubated at 37 °C in a complete growth RPMI medium for 0.5, 3, 7, 24, 48, and 72 h. (A) peptide 6 (B) peptide 9. The integrity of the conjugates was measured by mass spectrometry. Each measurement was performed in triplicate.

Figure 9

Analysis of cytotoxicity of the P6, P6-CPT, P9, P9-CPT, and CPT. (A) H1299 P6, (B) H1299 P9, and (C) DKMG P9 cells were cultured in a 96-well plate with six different concentrations of peptides, peptide conjugates, or CPT for 24, 48, and 72 h in two different protocols. The cell viability was measured by the XTT assay. Viability of the control non-treated cells was used as 100%. The viability of treated cells was represented as percentages of the control. Data are shown as Mean ± SD.