尽管来源于革兰氏阴性和革兰氏阳性细菌的细胞外囊泡（EVs）具有诱导强效抗肿瘤反应的能力，但大规模生产细菌EVs仍然是作为新一代癌症免疫治疗药物的发展障碍。本文中，我们开发了用于大规模生产大肠杆菌EVs的制造工艺，即外膜囊泡（OMVs）。通过结合金属沉淀和尺寸排阻色谱，我们从160升培养基中分离出357毫克总蛋白质量的大肠杆菌OMVs，相当于3.93×1015个颗粒（OMVs总蛋白质质量为1.10×1010个/微克）。我们发现这些大规模生产的大肠杆菌OMVs导致了两个小鼠同种移植瘤模型的完全缓解。进一步分析表达新抗原的肿瘤模型中的肿瘤微环境，我们发现大肠杆菌OMV治疗导致CD8+ T细胞的浸润和活化增加，特别是表达TCF-1和PD-1的癌症抗原特异性CD8+ T细胞。此外，大肠杆菌OMVs与抗-PD-1抗体免疫疗法表现出协同的抗肿瘤活性，导致明显的肿瘤生长抑制和激活的癌症抗原特异性干细胞样CD8+ T细胞浸润到肿瘤微环境。这些数据凸显了大规模生产的大肠杆菌OMVs作为开发新一代癌症免疫治疗药物的新候选品的强效抗肿瘤活性。© 2023年作者。《细胞外囊泡杂志》由Wiley Periodicals, LLC代表国际细胞外囊泡学会出版。

Despite the capability of extracellular vesicles (EVs) derived from Gram-negative and Gram-positive bacteria to induce potent anti-tumour responses, large-scale production of bacterial EVs remains as a hurdle for their development as novel cancer immunotherapeutic agents. Here, we developed manufacturing processes for mass production of Escherichia coli EVs, namely, outer membrane vesicles (OMVs). By combining metal precipitation and size-exclusion chromatography, we isolated 357 mg in total protein amount of E. coli OMVs, which was equivalent to 3.93 × 1015 particles (1.10 × 1010 particles/μg in total protein amounts of OMVs) from 160 L of the conditioned medium. We show that these mass-produced E. coli OMVs led to complete remission of two mouse syngeneic tumour models. Further analysis of tumour microenvironment in neoantigen-expressing tumour models revealed that E. coli OMV treatment causes increased infiltration and activation of CD8+ T cells, especially those of cancer antigen-specific CD8+ T cells with high expression of TCF-1 and PD-1. Furthermore, E. coli OMVs showed synergistic anti-tumour activity with anti-PD-1 antibody immunotherapy, inducing substantial tumour growth inhibition and infiltration of activated cancer antigen-specific stem-like CD8+ T cells into the tumour microenvironment. These data highlight the potent anti-tumour activities of mass-produced E. coli OMVs as a novel candidate for developing next-generation cancer immunotherapeutic agents.© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.