Clinical progress in the field of HER2-positive breast cancer therapy has been dramatically improved by understanding of the immune regulatory mechanisms of tumor microenvironment. Passive immunotherapy utilizing recombinant monoclonal antibodies (mAbs), particularly trastuzumab and pertuzumab has proved to be an effective strategy in HER2-positive breast cancer treatment. However, resistance to mAb therapy and relapse of disease are still considered important challenges in clinical practice. There are increasing reports on the induction of cellular and humoral immune responses in HER2-positive breast cancer patients. More recently, increasing efforts are focused on using HER2-derived peptide vaccines for active immunotherapy. Here, we discuss the development of various HER2-derived vaccines tested in animal models and human clinical trials. Different formulations and strategies to improve immunogenicity of the antigens in animal studies are also discussed. Furthermore, other immunotherapeutic approaches to HER2 breast cancer including, CTLA-4 inhibitors, immune checkpoint inhibitors, anti PD-1/PD-L1 antibodies are presented.

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We have investigated the in vitro immunogenicity and in vivo prophylactic and therapeutic potential of lambda (λ) phage particles displaying the E75 peptide (derived from HER2 protein) in an implantable TUBO breast tumor model of BALB/c mice. The mice were immunized with the E75-displaying phage (λF7-gpD::E75) every 2-week intervals over a 6-week period, and the generated immune responses were studied. Results showed in vitro induction of immune responses by the λF7 (gpD::E75) construct compared to the control λF7 and buffer groups. In the in vivo prophylactic study, all the control and vaccinated mice groups developed tumors. However, in the therapeutic experiments, we observed a significant difference in tumor size at days 14-36 for mice immunized with λF7 (gpD::E75) compared to control groups (P < 0.05). Moreover, the survival time prolonged in mice immunized with λF7 (gpD::E75). The discrepancy between the results obtained from the in vitro and in vivo studies may have been a result of the induction of Foxp3 CD4CD25 which has been previously reported to hamper effective T cell functionality. In conclusion, we observed a significant immune stimulatory response in the in vitro study, while in vivo, the vaccine was not able to exert significant tumor inhibitory effects. We suggest that the presence of Foxp3 CD4CD25 cells may have impaired the anti-tumor response in mice challenged in vivo with the TUBO xenograft tumor.

Generating a protective and long-lasting immune response is the primary goal in the expanding field of immunotherapeutic research. In current study we designed an immunogenic bacteriophage- based vaccine to induce a cytotoxic T lymphocyte activity against a mice tumor model over-expressing HER2/neu. Bacteriophage λ displaying a HER2/neu derived peptide GP2 was constructed and used as an anti-cancer vaccine in a BALB/c mouse xenograft tumor model. The results of our study indicated that phage nanoparticles displaying GP2 as a fused peptide to the gpD phage capsid protein induced a robust CTL response. Furthermore, the chimeric phage nanoparticles protected mice against HER2/neu-positive tumor challenge in both prophylactic and therapeutic settings. In conclusion, we propose that λ phage nanoparticles decorated with GP2 peptide merit further investigation for the development of peptide-based vaccines against HER2/neu overexpressing tumors.

One of the challenging issues in vaccine development is peptide and adjuvant delivery into target cells. In this study, we developed a vaccine and therapeutic delivery system to increase cytotoxic T lymphocyte (CTL) response against a breast cancer model overexpressing HER2/neu.Gp2, a HER2/neu-derived peptide, was conjugated to Maleimide-mPEG2000-DSPE micelles and post inserted into liposomes composed of DMPC, DMPG phospholipids, and fusogenic lipid dioleoylphosphatidylethanolamine (DOPE) containing monophosphoryl lipid A (MPL) adjuvant (DMPC-DMPG-DOPE-MPL-Gp2). BALB/c mice were immunized with different formulations and the immune response was evaluated in vitro and in vivo. ELISpot and intracellular cytokine analysis by flow cytometry showed that the mice vaccinated with Lip-DOPE-MPL-GP2 incited the highest number of IFN-γ+ in CD8+ cells and CTL response. The immunization led to lower tumor sizes and longer survival time compared to the other groups of mice immunized and treated with the Lip-DOPE-MPL-GP2 formulation in both prophylactic and therapeutic experiments. These results showed that co-formulation of DOPE and MPL conjugated with GP2 peptide not only induces high antitumor immunity but also enhances therapeutic efficacy in TUBO mice model. Lip-DOPE-MPL-GP2 formulation could be a promising vaccine and a therapeutic delivery system against HER2 positive cancers and merits further investigation.

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Phage display technique has been increasingly researched for vaccine design and delivery strategies in recent years. In this study, the AE37 (Ii-Key/HER-2/neu 776-790) peptide derived from HER2 (human epidermal growth factor receptor protein) was used as a fused peptide to the lambda phage (λF7) coat protein gpD, and the phage nanoparticles were used to induce antitumor immunogenicity in a TUBO model of breast cancer in mice. Mice were immunized with the AE37 peptide displaying phage, λF7 (gpD::AE37) every 2-week intervals over 6-weeks, then the generated immune responses were evaluated. An induction of CTL immune response by the λF7 (gpD::AE37) construct compared to the control λF7 and buffer groups was observed in vitro. Moreover, in the in vivo studies, the vaccine candidate showed promising prophylactic and therapeutic effects against the HER2 overexpressing cancer in BALB/c mice.

Despite years of intensive research, breast cancer remains the leading cause of death in women worldwide. New technologies including oncolytic virus therapies, virus, and phage display are among the most powerful and advanced methods that have emerged in recent years with potential applications in cancer prevention and treatment. Oncolytic virus therapy is an interesting strategy for cancer treatment. Presently, a number of viruses from different virus families are under laboratory and clinical investigation as oncolytic therapeutics. Oncolytic viruses (OVs) have been shown to be able to induce and initiate a systemic antitumor immune response. The possibility of application of a multimodal therapy using a combination of the OV therapy with immune checkpoint inhibitors and cancer antigen vaccination holds a great promise in the future of cancer immunotherapy. Display of immunologic peptides on bacterial viruses (bacteriophages) is also increasingly being considered as a new and strong cancer vaccine delivery strategy. In phage display immunotherapy, a peptide or protein antigen is presented by genetic fusions to the phage coat proteins, and the phage construct formulation acts as a protective or preventive vaccine against cancer. In our laboratory, we have recently tested a few peptides (E75, AE37, and GP2) derived from HER2/neu proto-oncogene as vaccine delivery modalities for the treatment of TUBO breast cancer xenograft tumors of BALB/c mice. Here, in this paper, we discuss the latest advancements in the applications of OVs and bacterial viruses display systems as new and advanced modalities in cancer immune therapeutics.