Bacterial extracellular vesicle applications in cancer immunotherapy

Abstract

Cancer therapy is undergoing a paradigm shift toward immunotherapy focusing on various approaches to activate the host immune system. As research to identify appropriate immune cells and activate anti-tumor immunity continues to expand, scientists are looking at microbial sources given their inherent ability to elicit an immune response. Bacterial extracellular vesicles (BEVs) are actively studied to control systemic humoral and cellular immune responses instead of using whole microorganisms or other types of extracellular vesicles (EVs). BEVs also provide the opportunity as versatile drug delivery carriers. Unlike mammalian EVs, BEVs have already made it to the clinic with the meningococcal vaccine (Bexsero®). However, there are still many unanswered questions in the use of BEVs, especially for chronic systemically administered immunotherapies. In this review, we address the opportunities and challenges in the use of BEVs for cancer immunotherapy and provide an outlook towards development of BEV products that can ultimately translate to the clinic.

Keywords: Bacterial extracellular vesicles; Cancer immunotherapy; Mammalian extracellular vesicles; Membrane vesicles; Outer membrane vesicles.

Graphical abstract

Fig. 1

Biogenesis of Bacterial Extracellular Vesicles (BEVs). BEVs secreted from Gram-positive and Gram-negative bacteria differ based on the physical characteristics of the parent cell. Gram-positive BEVs may form by the action of degradative enzymes such as endolysin. Degradative enzymes provoke thinning of the peptidoglycan resulting in release of EVs. After thinning, bubbling cell death results in formation of cytoplasmic membrane vesicles (CMV). BEVs from Gram Negative bacteria are formed by blebbing or cell lysis of the outer membrane. The EVs pinched off from the outer surface are known as Outer Membrane Vesicles (OMV). Explosive cell lysis results in formation of outer inner membrane vesicles (OIMV) and explosive outer membrane vesicles (EOMV).

Fig. 2

Outer Membrane Vesicles (OMVs) as a Versatile Drug Delivery System. OMVs' surface or internal content can be modified for different applications. The modulation of surface characteristics enables targeting via Plug and Display Technology [122,123]. Chen et al., utilized OMVs expressing arginyl-glycyl-aspartic acid (RGD) peptide on the surface as a coat for 5-fluorouracil tegafur-loaded polymeric nanoparticles called ORFTs [116]. Lipopolysaccharide (LPS) on the membrane can also be reduced to elicit controlled immunogenicity [112]. OMVs' cytosolic content can be better controlled by processing that can improve its toxicity profile further, for example high pH treatment produced spherical synthetic bacterial vesicles (SyBVs) with less cytoplasm-derived content that showed improved toxicity profile [113]. BEVs inherent nature to elicit an immune response can be combined with utilizing them as carriers of anti-tumor drugs.

Fig. 3

Industrial Production and Scale-up of Outer Membrane Vesicles (OMV). (A) Upstream Development comprises of bacterial fermenters to culture the parent bacteria for deriving and isolating OMVs. Variables affecting upstream development include aerobic conditions [128], peptidoglycan hydrolyzing enzymes [131,189], iron [77] and lactate [153] availability, genetic modulation of cell envelop lipids [91,99,190,191] (B) Downstream process for large scale production – The modified bacteria are cultured in industry grade bacterial cultivation tank. Large scale centrifugation separates bacteria from the vesicles. Cellular debris remains in the supernatant containing the vesicles. The ultrafiltration process eliminates any material larger than 0.22/0.45 μm. Further purification by TFF and SEC purges unwanted proteins/RNA/cellular material from the suspension. The ultra-purified and filtered OMV are subjected to density gradient centrifugation. Some factors that affect downstream processing include presence of debris and size [54]. (C) Modification of antigenicity – the LPS on OMV may be therapeutically useful but might act as antigens producing adverse/undesired side effects. Their antigenicity can be modified and controlled by endotoxin removal using detergent treatment or genetic engineering. (D) The functionality of OMVs can be modified by using surface peptides to translocate active molecules to OMV periplasm or lumen. Tat transporter/ClyA protein/secretory proteins can be used to achieve translocation. (E) The isolated and modified OMV must be analyzed for physico-chemical characteristics. Techniques such as Cryo-TEM and NTA can be used for OMV quantification and physical characterization. LC-MS and SDS-PAGE are used for protein identification. (F) Storage and stability studies are performed to assess the effect of storage temperature and time. Abbreviations: OMV- Outer Membrane Vesicles, TFF- Tangential Flow Filtration, SEC – Size Exclusion Chromatography, CryoTEM – Cryogenic Transmission Electron Microscopy, NTA – Nanoparticle Tracking Analysis, LC-MS – Liquid Chromatography-Mass Spectrometry, SDS-PAGE – Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis.