Development of a Genetically Engineered Bacterial Outer-Membrane Vesicle Immunotherapy for the Treatment of Cancer

Background
Composed exclusively of the gram-negative bacterial outer-membrane but unable to replicate, outer-membrane vesicles (OMVs) display many of the pathogen-associated molecular patterns (PAMPs) as the bacteria from which they originate. Consequently, OMVs potently activate the host immune response but with a much-reduced risk of toxicity, leading to their interest as a new modality of cancer immuno-therapy. As the severe side-effects associated with cytokine therapy limit widespread clinical use, there is a significant unmet medical need for immuno-therapies able to stimulate an anti-tumour response in a tolerable manner. Since OMVs activate a wide array of immune cells both directly and indirectly, it is thought that their presence will recruit cells to the tumour site, improve their recognition of the malignant cells and stimulate cytotoxic T-cells to destroy the tumour. Investigating OMVs in this capacity will further our understanding of the mechanisms underlying innate cell stimulation causing T-cell tumour killing; knowledge which can then be leveraged to create more effective immuno-therapies. Furthermore, building on experiences in the research of anti-microbial OMV vaccines, we propose the development of OMV constructs displaying fusion-proteins which target the vesicle to the tumour. These constructs aim to provide a safer alternative for clinical use by limiting the risk of off-target inflammation. In doing so, OMVs have the potential to become a new immuno-therapeutic modality able to provide a viable alternative to the many patients that do not respond to current treatments.

Aims:
1. Investigate the mechanism of OMV-mediated T-cell activation and subsequent cancer-killing.
2. Develop a genetically engineered OMV construct displaying tumour-targeting proteins for use in cancer immunotherapy.

Objectives:
1. Detail the immunological mechanism involved in OMV-mediated cancer killing
2. Understand the different dynamics of cancer killing between different tumour types
3. Investigate the targeting capabilities of OMVs expressing tumour-targeting ligands
4. Evaluate the tumour killing capability of an OMV fusion-protein construct

Research Methodology
OMV constructs will be developed using standard protocols within the field of synthetic biology (plasmid construction, PCR, bacterial transformation).
Vesicle isolation and purification will adopt commonly used techniques (ultracentrifugation) as well as investigating novel methods using the unique constructs developed (affinity purification). These novel bio-processing techniques, only possible with specifically designed OMV constructs, will be premised on the adaptation of validated protein-purification methods.
Co-culture models will be developed to investigate both the immunological response to OMVs and their cancer-killing capabilities in vitro, cytokine release and cell-surface markers will be quantified via ELISA and flow cytometry. OMV tumour-targeting shall be analysed after in vitro co-culture by immuno-fluorescence confocal microscopy.

Alignment to EPSRC's strategies and research areas
In developing and validating a novel tumour-targeting OMV via a genetically engineered bacterial strain, this project sits primarily within EPSRC's synthetic biology research area. Application of the research outcomes falls under the EPSRC healthcare technologies theme, more specifically the development of future therapies. Since OMVs are clinically available as anti-microbial vaccines their therapeutic capabilities have been well-validated. However, as this project seeks to develop innovative OMV constructs as a cancer immuno-therapy, the outcomes will help establish OMVs as a future multi-indication therapeutic modality.

Collaborators
The project is currently in collaboration with Professor Huang at Hong Kong University who is providing engineered bacterial strains for the isolation of modified OMV constructs.