An engineered mycobacterium smegmatis expressing anti-PD-L1/IL-15 immunocytokine induces and activates specific antitumor immunity

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, which can sustainably deliver drugs locally without causing severe systemic risks. Here, we have innovatively engineered a nonpathogenic, adjuvant-acting Mycobacterium smegmatis (M. smegmatis) that coexpresses a PD-L1 inhibitor and an IL-15 cytokine complex containing the IL-15Ra sushi domain (Ms-PDL1scfv-IL15). We demonstrate that PD-L1 inhibitor and IL-15 secretion 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 anti-tumor polarization of macrophages, increases the proliferation and activation of NK cells and tumor-infiltrating CD8+ T cells, inhibits the Tregs, 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 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. In summary, this engineered M. smegmatis to 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. Overall design: To further explore the molecular mechanisms underlying Ms-PDL1scfv-IL15 which induce long-term and specific anti-tumor protection, we performed bioinformatics analysis of mRNA transcriptomes towards Ms-PDL1scfv-IL15 treated tumor