Bacteriophage-guided cancer immunotherapy

Abstract

Over the past decades, gene delivery has become a powerful tool for treating many diseases; therefore, a wide range of delivery systems, simply categorised into viral and non-viral vectors, have been developed and applied in both research and clinical translation. In spite of high transduction efficacy, viral vectors encounter a number of limitations regarding their native entities. A hybrid bacteriophage vector has been successfully developed by our Phage Virotherapy group by integrating AAV elements into the filamentous bacteriophage genome. In doing so, the new vector, named adeno-associated phagemid-based vector (PAAV), is able to overcome limitations associated with AAV native tropism by using advantages of bacteriophages that lack native tropism for mammalian cells and tissues. PAAV vector has been applied in a few research areas including cancer therapy. To systemically target solid tumours, the vector is displayed cyclic RGD4C (CDCRGDCFC) ligands on the pIII capsids allowing it to specifically target the αVβ3 integrin receptors overexpressed on tumour cell surface and tumour blood vessels.

In this study, PAAV vector has been further refined to display the endosomal escape peptide (H5WYG) on the recombinant pVIII capsids to enhance endosomal escape and transgene expression. The PAAV displaying H5WYG peptide showed great buffering capacity at mild acidic pH relating to the situation that occurs during endosomal maturation and subsequently enhanced luciferase reporter gene expression in numerous human cancer cell lines, including lung carcinoma (A549), melanoma (M21) and meduloblastoma (UW228). Importantly, the modified vector remained safe for normal cells. H5WYG peptide facilitated endosomal escape through a proton sponge effect mechanism as PAAV-mediated gene expression decreased when applying the vascuolar ATPase inhibitor (bafilomycin A1) during vector transduction. Furthermore, displaying H5WYG peptide on the PAAV capsid augmented the secreted TNFα gene expression resulting in a greater cell death of A549, M21 and UW228 cells.

The PAAV gene delivery vector was tested in various applications in this thesis. Firstly, the vector was applied as a delivery tool to express the tumour associated antigens (MUC1 and PSMA) which are common target antigens for CAR T cell therapy. PAAV vector augmented MUC1 and PSMA expression in A549, Suit2 and UW228 cells. Although CAR T cells were not applied here, the strategy based on PAAV vector to enhance antigen expression can make the tumours more visible for CAR T cells and might be useful for CAR T cell therapy in solid tumours. Secondly, PAAV was applied on cancer vaccination in combination with a malaria vaccine. The vector was used to deliver a malarial epitope, Pb9, to present on tumour cell surfaces via restricted H2-Kd MHC class I molecules while adenoviral vector encoding a malaria sequence (ME.TRAP) was used to induce an immune response against Pb9. It is shown here that PAAV vector mediated Pb9 expression in EF43.fgf4 breast tumours. The ME.TRAP vaccine stimulated immune response according to ex vivo investigations of specific effector cell proliferation, activation and function. Pb9-specific tumour cell killing was also found both ex vivo and in vivo. The tumour cell death was mediated through caspase-3 dependent apoptosis. All these findings indicate the efficiency and numerous applications of PAAV-mediated gene delivery for cancer treatment.