Lambda bacteriophage nanoparticles displaying GP2, a HER2/neu derived peptide, induce prophylactic and therapeutic activities against TUBO tumor model in mice.

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.

The viral approach to breast cancer immunotherapy.

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.

Specific Systems for Imaging.

Microscopy allows for the characterization of small objects invisible to the naked eye, a technique that, since its conception, has played a key role in the development across nearly every field of science and technology. Given the nanometer size of the materials explored in the field of nanotechnology, the contributions of modern microscopes that can visualize these materials are indispensable, and the ever-improving technology is paramount to the future success of the field. This chapter will focus on four fundamental areas of microscopy used in the field of nanotechnology including fluorescence microscopy (Sect. 3.1), particle tracking and photoactivated localization microscopy (Sect. 3.2), quantum dots and fluorescence resonance energy transfer (Sect. 3.3), and cellular MRI and PET labeling (Sect. 3.4). The functionality, as well as the current and recommended usage of each given imaging system, will be discussed.

Phage-based Nanomedicines as New Immune Therapeutic Agents for Breast Cancer.

Despite years of investigation, breast cancer remains a major cause of death worldwide. Phage display is a powerful molecular method in which peptide and protein libraries can be displayed via genetic fusions on the surface of phages. This approach has tremendous potential for biomedical applications and has already facilitated the discovery of specific antibodies, specific antigens, and peptides with potential roles in the diagnosis and treatment of malignancies including breast cancer. In this review, we discuss the new and the latest advancements in the applications of the phage display technique in the provision of immune therapeutics for breast cancer.

Immunogenicity and antitumor activity of the superlytic λF7 phage nanoparticles displaying a HER2/neu-derived peptide AE37 in a tumor model of BALB/c mice.

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.

Lambda phage nanoparticles displaying HER2-derived E75 peptide induce effective E75-CD8+ T response.

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 CD4+CD25+ 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+ CD4+CD25+ cells may have impaired the anti-tumor response in mice challenged in vivo with the TUBO xenograft tumor.

Bacteriophage lambda display systems: developments and applications.

Bacteriophage (phage) Lambda (λ) has played a key historic role in driving our understanding of molecular genetics. The lytic nature of λ and the conformation of its major capsid protein gpD in capsid assembly offer several advantages as a phage display candidate. The unique formation of the λ capsid and the potential to exploit gpD in the design of controlled phage decoration will benefit future applications of λ display where steric hindrance and avidity are of great concern. Here, we review the recent developments in phage display technologies with phage λ and explore some key applications of this technology including vaccine delivery, gene transfer, bio-detection, and bio-control.

Construction and analysis of a genetically tuneable lytic phage display system.

The Bacteriophage λ capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by λ Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to λDam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the λDam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage λ.

Graphical analysis of flow cytometer data for characterizing controlled fluorescent protein display on λ phage.

As native virus particles typically cannot be resolved using a flow cytometer, the general practice is to use fluorescent dyes to label the particles. In this work, an attempt was made to use a common commercial flow cytometer to characterize a phage display strategy that allows for controlled levels of protein display, in this case, eGFP. To achieve this characterization, a number of data processing steps were needed to ensure that the observed phenomena were indeed capturing differences in the phages produced. Phage display of eGFP resulted in altered side scatter and fluorescence profile, and sub-populations could be identified within what would otherwise be considered uniform populations. Surprisingly, this study has found that side scatter may be used in the future to characterize the display of nonfluorescent proteins.