Engineering M13 bacteriophage to display HER2 mimotopes on pVIII for vaccine development

Abstract

HER2-positive breast cancer is a subtype characterized by overexpression of the human epidermal growth factor receptor 2 (HER2), which is associated with aggressive tumor growth and poor prognosis. Peptide mimics of HER2 epitopes, recognized by tumor growth-inhibitory antibodies like trastuzumab, have the potential to be developed into effective vaccines. Active immunization with these mimotopes could induce tumor-inhibitory antibodies, offering a cost-effective alternative to trastuzumab therapy. This study aimed to design high-affinity mimotopes to trastuzumab using bioinformatics tools and to evaluate the potential of M13 bacteriophages as platforms for displaying these mimotopes. Using the Peptiderive server, peptide sequences involved in binding HER2 to trastuzumab were identified and refined. Docking studies validated their enhanced binding affinity to trastuzumab. The optimized mimotopes were then displayed on the pVIII coat protein of M13 bacteriophages using the PAK8 phagemid vector. ELISA assays confirmed the binding of these recombinant phages to trastuzumab.This study highlights the effectiveness of using bioinformatics tools to enhance mimotope design for therapeutic applications. The M13 bacteriophage displaying optimized mimotopes shows potential as a vaccine candidate for HER2-positive breast cancer, offering a promising approach to overcoming current limitations of trastuzumab therapy.

Keywords: Bacteriophage; HER2; Mimotope; Phage display; Trastuzumab.

Fig. 1

HPEPDOCK docking results were visualized using PyMol. The sequences of the mimotopes; (a) QMWAPQWGPD (P10), (b) QLGPYELWELSH (P12), (c) QMGPYKDWEF (D10) and (d) QMGPYQDWEFSH (D12), were examined for their binding across the complementarity-determining regions (CDRs) of trastuzumab. CDRs and important residues of the Fab have been labeled. Bonds between residues are shown in purple dots. (a) and (b) represent experimentally mimotopes, while (c) and (d) show newly designed sequences.

Fig. 2

Simulated immune responses to the D12 mimotope fused with M13 phage pVIII protein. (a) B lymphocyte population dynamics by functional state; (b) Antigen (Ag) clearance and corresponding antibody production; (c) TH cell counts over time, distinguishing between memory and non-memory subsets; (d) TH cell counts by functional state over time; (e) TH cell subset distribution: Th0, Th1, Th2, Th17, and regulatory T cells (TR), shown as absolute counts (top) and relative percentages (bottom) and (f) cytokine concentration profiles over time.

Fig. 3

Colony PCR screening using backbone-specific primers to identify positive clones. The expected amplicon size is 348 bp. In some wells, a non-specific insert band is due to self-ligation of partially digested phagemid vectors in which the original insert was not excised. A 1 kb DNA ladder (SinaClon BioScience Co. Iran) was used as a molecular weight size marker.

Fig. 4

Nucleotide sequence alignment of recombinant phagemid (pAK + D12) and sequencing results (recN_M13F). The specific regions are annotated in color: the M13 reverse primer binding site is shown in purple, the N-terminal signal peptide sequence is highlighted in yellow, and the gene encoding the pVIII coat protein is marked in green. The SfiI and NotI restriction sites are indicated, marking the insertion points for the mimotope sequence QMGPYQDWEFSH, cloned at the N-terminus of the M13 pVIII coat protein. The chromatogram trace data below each alignment provides a visual confirmation of sequence accuracy, supporting the integrity of the recombinant construct.

Fig. 5

ELISA results showing binding affinity of HER2 mimotopes to trastuzumab. Asterisks (** and ***) denote statistically significant differences (P < 0.01 and P < 0.001, respectively) between phage-displayed mimotopes and the VCSM13 helper phage. Hashtags (# and ##) indicate statistically significant differences (P < 0.05 and P < 0.01, respectively) between mimotopes and M13 phages displaying an unrelated sequence (NS-M13). Individual data points are shown alongside the mean values. Error bars represent the mean ± standard deviation.