Engineered <i>Mycobacterium smegmatis</i> expressing anti-PD-L1/IL-15 immunocytokine induces and activates specific antitumor immunity

Background Immune checkpoint inhibitors and cytokines have revolutionized tumor treatment but are still limited by dose-dependent toxicity and efficacy. In situ vaccine platforms based on intelligent microbes are promising therapeutic strategies that sustainably deliver drugs locally without causing severe systemic risks. Methods In this study, we have innovatively engineered a non-pathogenic, adjuvant-acting Mycobacterium smegmatis ( M. smegmatis ) that co-expresses a programmed cell death-ligand 1 (PD-L1) inhibitor and an interleukin-15 (IL-15) cytokine complex containing the interleukin-15 receptor alpha (IL-15Rα) sushi domain (Ms-PDL1scfv-IL15). Results We demonstrate that the fusion protein of PD-L1 inhibitor and IL-15 cytokine systemically binds mouse or human PD-L1 and maintains IL-15 stimulatory activity. The bifunctional Ms-PDL1scfv-IL15 overcomes resistance to PD-L1 blockade, recruits numerous immune cells in situ, induces dendritic cells (DCs) maturation, initiates the M1 antitumor polarization of macrophages, increases the proliferation and activation of natural killer cells and tumor-infiltrating CD8 + T cells, inhibits regulatory T cells, elicits abscopal effects, stimulates rapid tumor regression, prevents metastasis, and leads to long-term survival in several syngeneic tumor mouse models. We also found that the combination of Ms-PDL1scfv-IL15 with granulocyte-macrophage colony-stimulating factor (GM-CSF) synergistically stunted the tumor progress and stasis. Moreover, intratumoral administration of Ms-PDL1scfv-IL15 can capture tumor antigen fragments, and boost DCs presentation of antigens, which remarkably initiates tumor antigen-specific immune response, leading to durable tumor regression and specific antitumor immunity. Conclusion In summary, the engineered M. smegmatis can recruit and activate innate and adaptive antitumor immune responses, offering a potent cancer immunotherapy strategy to treat patients with cold tumors or resistance to checkpoint blockade.