Scalable cultured gut microbiota-derived nanovesicles for oral augmentation of checkpoint blockade therapy

Abstract

Microbiota-targeted strategies, such as fecal microbiota transplantation (FMT), hold promise for enhancing immunotherapy but face clinical challenges including safety risks, batch-to-batch variability, and dependence on substantial quantities of donor fecal material. To overcome these limitations, we established a standardized in vitro expansion platform for responder patient-derived gut microbiota. Through optimization of culture conditions, we achieved stable viability and retention of key immunotherapy-enhancing strains over serial passages, enabling a scalable 6.4 × 10⁷-fold expansion. Leveraging this platform, we engineered hybrid nanovesicles derived from bacterial protoplasts, followed by silica surface coating (HNVs@Silica) for oral delivery. The silica shell significantly improved nanovesicle stability under gastrointestinal conditions and conferred superior antitumor efficacy compared with uncoated counterparts. Notably, HNVs@Silica produced from the 2nd, 4th, and 6th passages retained comparable potency in augmenting anti-PD-1 (αPD-1) therapy. Mechanistically, HNVs@Silica remodeled intestinal immunity, particularly within Peyer's patches, by activating and expanding effector CD8⁺ T cells. This local response enhanced the priming and systemic dissemination of tumor-specific T cells, thereby improving the efficacy of αPD-1. Collectively, our findings establish a safe, scalable, and orally bioavailable alternative to FMT for enhancing cancer immunotherapy.