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.

本研究旨在探究，搭载ClyA与FlaB双载荷的减毒沙门氏菌（即基于沙门氏菌的武装微生物，Salmonella-based Armed Microbe, SAM），能否通过诱导免疫原性细胞死亡（Immunogenic Cell Death, ICD）并增强巨噬细胞激活能力，在原发性及转移性肿瘤模型中增强抗肿瘤活性。
ClyA是一种具有免疫原性的成孔蛋白，可触发免疫原性细胞死亡，其特征为释放损伤相关分子模式（damage-associated molecular patterns, DAMPs）与新抗原。FlaB则是一种极具潜力的佐剂，可激活肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs），进而发挥抑制肿瘤转移的潜能。

我们的主要研究发现
• 表达ClyA与FlaB的工程菌在多种荷瘤小鼠模型中展现出强效抗肿瘤效应。
• 工程菌局部分泌的ClyA可通过诱导癌细胞发生免疫原性细胞死亡，促进肿瘤特异性抗原（tumor-specific antigens, TSAs）、肿瘤相关抗原（tumor-associated antigens, TAAs）及损伤相关分子模式的释放，进而建立长期抗肿瘤免疫记忆。
• 表达FlaB的工程菌可通过Toll样受体4（Toll-like receptor 4, TLR4）与Toll样受体5（Toll-like receptor 5, TLR5）信号通路，促进肿瘤微环境中巨噬细胞从M2型向M1型极化，进而显著抑制肿瘤转移。
• 瘤内注射工程化SAM不仅在给药肿瘤灶中发挥抑瘤作用，对未给药的远端肿瘤灶同样有效，其效果与静脉注射工程菌的抗肿瘤效应相当。

研究创新性、优势与转化潜力
• 据我们所知，本研究首次报道了合理设计可分泌ClyA与FlaB的工程菌作为免疫佐剂——通过提升损伤相关分子模式、肿瘤特异性抗原水平并激活肿瘤相关巨噬细胞，以增强抗肿瘤T细胞与肿瘤相关巨噬细胞应答，最终实现预防肿瘤复发与远端转移的目标。我们在多种体外与体内模型中，验证了SAM-FC在高迁移率族蛋白B1（high mobility group box 1, HMGB1）释放及肿瘤特异性抗原交叉呈递中的作用。
• 依托工程菌固有的免疫刺激特性，ClyA与FlaB的分泌表达可形成互补效应，协同诱导强效抗肿瘤免疫应答。
• SAM-FC展现出极高的转化潜力，可诱导持久且优异的抗肿瘤免疫，且全身毒性极低。

综上，本研究结果表明，工程化SAM可同时激活固有免疫与适应性免疫分支，有望通过合理设计实现多免疫治疗载荷的靶向递送，进而诱导强效且持久的抗肿瘤免疫应答。