Reprogramming the tumor immune microenvironment using engineered dual-drug loaded Salmonella

In the present study, we asked whether engineering of attenuated Salmonella with dual payloads of ClyA and FlaB (Salmonella-based Armed Microbe, SAM) might potentiate antitumor activity in primary and metastatic tumor models by inducing ICD and improving macrophage-activating capacity. ClyA is a potentially immunogenic pore-forming protein that can trigger ICD, which is characterized by release of damage-associated molecular patterns (DAMPs) and neoantigens. FlaB is a promising adjuvant that activates tumor-associated macrophages (TAMs)* that may have the potential to inhibit metastasis.

Our Major Findings

•Bacteria expressing ClyA and FlaB produced strong anti-tumor effects in diverse tumor-bearing mouse models.
•Localized secretion of ClyA from bacteria promoted release of TSAs/TAAs and DAMPs by inducing ICD of cancer cells, resulting in the establishment of long-term anti-tumor memory.
•Bacteria expressing FlaB promoted M2-to-M1 polarization in the tumor microenvironment via TLR4 and TLR5 signaling pathways, which markedly inhibited tumor metastasis.
•Intratumoral injection of engineered SAM exerted a tumor-suppressive effect in both SAM-treated and untreated tumors, which was similar to that elicited by intravenous injection of bacteria.

Novelty, Advantages, and Translational Potential

•To the best of our knowledge, this is the first report to describe the rational design of ClyA- and FlaB-secreting bacteria as immunoadjuvants (by increasing DAMPs/TSA levels and TAM activation) for the boosting of antitumor T cell and TAM responses, leading to the prevention of tumor recurrence and distant metastasis. We verified the role of SAM-FC in HMGB1 release and cross-presentation of TSAs in various models in vitro and in vivo.
•Taking advantages of bacteria’s inherent immunostimulatory nature, secretion of ClyA and FlaB complementarily elicited a very effective anticancer immune response.
•SAM-FC exhibited high translational potential with excellent and long-lasting antitumor immunity and low systemic toxicity.

Overall, our results indicate that the engineered SAM can activate both the innate and adaptive branches of immunity and could be used in a rational design for the targeted delivery of multiple immunotherapeutic payloads for the induction of potent and long-lasting antitumor immunity.