Tumor-specific cytokine therapy mediated by engineered Salmonella with a synthetic protein delivery system.

Cytokine treatment provides clinical benefits by stimulating the patient’s immune system to attack cancer cells. However, its effectiveness as a monotherapy is limited because of its systemic toxicity and short in vivo half-life. To address these limitations, we assessed the therapeutic impact of delivering cytokines specifically to tumors using tumor-targeting bacteria. A Salmonella strain was engineered to minimize its pathogenic traits by deleting key elements (Salmonella pathogenicity islands-1 and -2) responsible for invasion and replication within the mammalian host, generating the ΔSPI-1ΔSPI-2 strain. A plasmid encoding a synthetic type 3 secretion system (SynT3SS V3.0) was introduced into this strain using a previously reported genetic system after reorganizing the gene clusters and placed under control of the Ptet promoter. Finally, the anticancer cytokine Neoleukin-2/15 (Neo-2/15) was introduced into the strain via a separate plasmid. This plasmid encoded an N-terminal secretion signal from SptP (SptP167) and was driven by the constitutive promoter PJ23110. Upon inducing SynT3SS V3.0 using doxycycline, the recombinant protein SptP167::Neo-2/15 was secreted into the bacterial culture supernatant with a yield of 0.37 ± 0.07 mg/L. Starved cytotoxic T lymphocytes treated with the culture supernatant containing SptP167::Neo-2/15 proliferated, as observed with hIL-2, purified Neo-2/15, or purified SptP167::Neo-2/15 treatment. Administering Salmonella ΔSPI-1ΔSPI-2 strains carrying plasmids encoding SynT3SS V3.0 and SptP167::Neo-2/15 caused tumor regression and lifespan extension in CT26 tumor-bearing mice. Engineered bacteria represent a promising new modality for cancer therapy by combining the targeted delivery of therapeutic cytokines with the synergistic immunostimulatory effects of bacterial intrinsic factors. Furthermore, this strategy could be adapted for other protein-based payloads, potentially enabling the tumor-specific delivery of a diverse range of therapeutic agents.