Endotoxin-Free Outer Membrane Vesicles for Safe and Modular Anticancer Immunotherapy.

Bacterial outer membrane vesicles (OMVs) have emerged as promising vehicles for anticancer drug delivery due to their inherent tumor tropism, immune-stimulatory properties, and potential for functionalization with therapeutic proteins. Despite their advantages, the high lipopolysaccharide (LPS) endotoxin content in the OMVs raises significant safety and regulatory challenges. In this work, we produce LPS-attenuated and LPS-free OMVs and systematically assess the effects of LPS modification on OMVs' physicochemical characteristics, membrane protein content, immune-stimulatory capacity, tolerability, and anticancer efficacy. Our findings reveal that LPS removal increased the maximal tolerated dose of the OMVs by over 25-fold. When adjusted for comparable safety profiles, LPS-free OMVs exhibit superior anticancer effects compared with wild-type OMVs. Mechanistic investigations indicate that the LPS removal obviates immune cell death caused by LPS and reduces the negatory effects of wild type of OMVs on tumor immune cell infiltrates. We further show the functionality of the LPS-free OMV through the incorporation of an IL-2 variant protein (Neo-2/15). This functionalization augments OMV's ability of the OMV to inhibit tumor growth and promote lymphocyte infiltration into the tumor microenvironment. This study presents a safe and functionalizable OMV with improved translational prospect.