Optimization of the Isolation Method for Large-Scale Production of Synthetic Bacterial Vesicles for Cancer Immunotherapy

Abstract

Bacterial vesicles have emerged as therapeutic drug candidates to treat a wide range of diseases, including cancer. However, critical concerns remain regarding their safety, in view of inducing systemic inflammation. To address this, semi-synthetic bacterial vesicles (SyBV) have recently been developed, directly derived from bacterial cell membranes. These vesicles have reduced toxicity but retained immunomodulatory ability, which is important for immuno-oncology purposes. In this study, the manufacturing process of SyBV has been further refined to meet Good Manufacturing Practice (GMP) standards. Multiple steps are reconsidered in the optimized method, including adaptation of centrifugation steps, benzonase treatment, and elimination of sonication steps, thereby producing a new set of optimized SyBV, designated as SyBV^Opti. Similarly to SyBV isolated using the previous protocol, SyBV^Opti do not activate macrophages but stimulate dendritic cells to produce IL-12 in a dose-dependent manner—moreover, these vesicles attenuate tumor growth in vivo in a model of malignant melanoma. Further, the optimized process applies to a GMP-compatible Escherichia coli source for producing SyBV^Opti under GMP conditions. Collectively, these findings describe a reliable technique for large-scale production of SyBV with preserved therapeutic potential, allowing GMP manufacturing and translation of these bioactive vesicles into clinical practice.