Screening of a combinatorial random peptide library on EGFR ligands EGF, TGFα, and cetuximab. (A) EGF panning. VEGF and BSA were used as negative control proteins in A and B. (B) TGFα panning. (C) M225 monoclonal antibody (the original murine version of cetuximab) was immobilized onto microtiter wells at a concentration of 2 μg. The CX₇C phage library was incubated with each of the target proteins. Shown are the relative transducing units (TU) obtained from each well coated with M225, mIgG, or BSA after three rounds of selection (RI–RIII). (D) Specificity of the peptides recovered from RIII targeting. M225 was recapitulated upon binding to cetuximab on a fourth round of selection (RIV). Results are expressed as mean ± SEM of triplicate wells.

Mapping candidate epitopes within the EGFR. (A) Amino acid sequence corresponding to the extracellular domain of the EGFR (accession no. NP_005219). Leu1 is the first residue after the signal peptide. The arrow designates the signal peptide cleavage site. Yellow highlights indicate five consensus regions to which peptides derived from library screenings (on the ligands EGF, TGFα, and cetuximab) were clustered. Green and red boxes pinpoint the reciprocal residues in the two EGFR molecules involved in dimerization. (B) Location of a cetuximab-binding region within the EGFR structure. Light green and light red ribbons indicate the backbone of each EGFR homodimer. Purple designates the TGFα ligand bound to the EGFR. (Inset) Red and green indicate residues involved in EGFR dimerization (see A). Yellow ribbon shows the location of CVRAC within the EGFR homodimer (residues 283–287). (C) CVRAC-displaying phage binds specifically to cetuximab, EGF, and TGF-α. VEGF and BSA served as negative controls for binding. Recombinant proteins were coated onto microtiter wells at 10 μg/mL, and wells were incubated with either CVRAC-phage or CVAAC-phage (alanine scanning control). An insertless phage was an additional negative control. Phage input was 10⁹ TU per well. Results are expressed as mean ± SEM of triplicate wells.

Molecular interaction of CVRAC, cetuximab, and EGFR. (A) Synthetic peptides (CVRACGAD or CVRAC), compared to an unrelated control peptide (SDNRYIGSW), specifically bind to cetuximab. BSA served as an additional negative control, and the EGFR as a positive control. (B) Concentration-dependent inhibition of binding of cetuximab to the EGFR by the synthetic peptides CVRACGAD and CVRAC, in comparison with negative controls: an EGFR sequence-derived peptide (CQKCDPSC) and an unrelated negative control peptide. (C) Phage displaying alanine scanning versions of the CVRAC peptide (CARAC and CVAAC) were used to identify critical residues on the basis of their capacity to bind to cetuximab. Insertless phage served as a negative control. (D) Polyclonal antibody against CVRAC recognized the EGFR. Bars represent mean ± SEM.

The retro-inverso peptidomimetic of the CVRAC motif is recognized by cetuximab and inhibits binding of cetuximab to the EGFR. (A) Human HN5 tumor cells were treated with increasing concentrations of cetuximab (black line). Cells were also exposed to either 60 μM (red line) or 180 μM (blue line) CVRAC. Unrelated control peptide (purple line) or EGFR-related control peptide (green line) had no effect on cetuximab activity. A representative experiment is depicted. Experiments were repeated four times with similar results. Bars represent mean ± SEM. (B) Binding of retro-inverso D-form peptides (plated at 10 μg/mL) to cetuximab in an ELISA-based assay. Equivalent amounts of IgGs (cetuximab, anti-CVRAC, or h-IgG) were analyzed for binding to CVRAC or to its retro-inverso peptidomimetic _D(CARVC). (C) Effect of the synthetic peptides on HN5 tumor cells. Cells were incubated with increasing concentrations (up to 250 mM) of the peptide CVRAC, the retro-inverso peptidomimetic _D(CARVC), or a negative control peptide. Viability in the absence of peptide was set to 100%. (D) Inhibition of EGFR:cetuximab association, monitored by SPR in the presence of synthetic peptides or peptidomimetic _D(CARVC). Bars represent mean ± SEM. (E) Analysis of receptor autophosphorylation in cells stimulated with EGF or control media for 15 min, after which cetuximab or synthetic peptides were added with the growth factor to evaluate inhibition. Receptors were immunoprecipitated with antibodies against phosphorylated (p)EGFR and were immunoblotted with anti-phosphotyrosine IgG. This representative experiment shows that _D(CARVC) specifically inhibits the phosphorylation of the EGFR in human HN5 tumor cells.

CVRAC-targeted phage homes to tumors. (A) Phage displaying the peptide CVRAC or CVAAC, or insertless negative control phage, were administered i.v. into mice bearing EF43.fgf-4-derived tumors. An anti-phage antibody was used for staining. H&E staining, with the corresponding fluorescence-based immunostaining, is shown in tumors. Tumor-bearing mice received CVRAC phage, CVAAC phage, or insertless control phage as indicated. Cohorts of tumor-bearing mice (n = 5 mice/group) were used. A representative experiment is shown. (Scale bar, 100 μm.) (B) Treatment of tumor-bearing mice with peptides and peptidomimetics. BALB/c mice bearing EF43.fgf-4-derived tumors were divided into size-matched cohorts (n = 7 mice/group); individual tumor volumes are represented before (black circles) and after (white circles) treatment. Peptides and peptidomimetics were administered at 750 μg/mouse/dose for 5 days. Shown are mean tumor volumes ± SEM.

The prototype peptidomimetic drug _D(CARVC) functions through an EGFR-decoy mechanism. (A) _D(CARVC) displaces EGF from the EGFR. The EGFR was coated onto 96-well plates at decreasing concentrations. Increasing molar concentrations of the synthetic peptidomimetic _D(CARVC) were used to evaluate competitive inhibition of EGF binding (squares). _D(CAAVC) was used as a negative peptidomimetic control at the same concentrations (circles). Cetuximab (12 nM) served as a positive control for the displacement of EGF from the EGFR. (B) _D(CARVC) displaces the binding of TGFα from the EGFR. Evaluation is shown of the competitive inhibition of the binding of TGFα to the EGFR by increasing molar concentrations (as indicated) of the synthetic peptidomimetic _D(CARVC). Bars represent mean ± SEM.