Intratumoral Injection of Engineered Mycobacterium smegmatis Induces Antitumor Immunity and Inhibits Tumor Growth.

Conventional type 1 dendritic cells are essential for antigen presentation and successful initiation of antitumor CD8⁺ T cells. However, their abundance and function within tumors tend to be limited. Mycobacterium smegmatis, a fast-growing, nonpathogenic mycobacterium, proves to be easily modified with synthetic biology. Herein, we construct an engineered M. smegmatis expressing a fusion protein of Fms-like tyrosine kinase 3 ligand and costimulator CD40darpin (rM-FC) since the 2 drugs are reported to have a good synergistic effect. Intratumoral delivery of rM-FC effectively recruits and activates dendritic cells (DCs), especially CD103⁺ DCs and CD80⁺CD86⁺ DCs, further inducing sufficient migration of effector memory T cells into the tumor microenvironment. This successfully converts the so-called immune-desert tumors to the "hot" phenotype. In B16F10 mouse melanoma tumor models, local injection of rM-FC into the primary tumor triggers a robust T cell immune response to restrain the growth of both the treated tumors and the distant untreated ones. The population of PDL1⁺ tumor cells increased after the in situ vaccination, and murine tumors became more responsive to programmed death ligand 1 (PDL1) blockade, prompting the combination therapy. Overall, our findings demonstrate that rM-FC acts as a strong DC agonist and remarkably enhances antitumor immunity.