Bacterial Membrane Vesicles as Heteroantigen Reservoirs for Potentiated Colorectal Cancer Immunotherapy.

Cancer immunotherapy, including immune checkpoint blockade (ICB), often has limited efficacy due to inadequate neoantigen exposure. Although neoantigen vaccines can enhance immune sensitization, their clinical application is hindered by high identification costs and tumor heterogeneity. Alternatively, heteroantigen presentation by cancer cells offers a promising strategy to improve immunotherapy. Herein, hyaluronic acid (HA)-modified bacterial membrane vesicles were developed as heteroantigen reservoirs (HLGV) and then further loaded with PD-L1 siRNA (siPD-L1) for immune checkpoint blockade. Intracellularly, HLGV released heteroantigens and siPD-L1 into the cytoplasm via early endosomal membrane fusion. The heteroantigens were presented by MHC class I molecules, while siPD-L1 escaped lysosomal degradation and effectively silenced PD-L1 expression. When administered peritumorally, the synergistic therapeutic effect of heteroantigen presentation and ICB significantly inhibited both subcutaneous CT-26 tumors and distant metastases. For colorectal cancer immunotherapy, HLGV@siPD-L1 was encapsuled into chondroitin sulfate hydrogel microspheres (HEBV@siPD-L1) and administered through oral gavage. Ultimately, HEBV@siPD-L1 increased the infiltration of macrophages and CD8+ T cells and effectively suppressed the growth of orthotopic colorectal cancer, with the tumor inhibition rate reaching 91.6%. These findings demonstrated the potential of bacterial membrane vesicles as heteroantigen reservoirs to activate antitumor immune responses, which could further synergize with ICB for promoted immune efficacy.