Checkpoint inhibitors have revolutionized cancer therapy but only work in a subset of patients and can lead to a multitude of toxicities, suggesting the need for more targeted delivery systems. Because of their preferential colonization of tumors, microbes are a natural platform for the local delivery of cancer therapeutics. Here, we engineer a probiotic bacteria system for the controlled production and intratumoral release of nanobodies targeting programmed cell death-ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated protein-4 (CTLA-4) using a stabilized lysing release mechanism. We used computational modeling coupled with experimental validation of lysis circuit dynamics to determine the optimal genetic circuit parameters for maximal therapeutic efficacy. A single injection of this engineered system demonstrated an enhanced therapeutic response compared to analogous clinically relevant antibodies, resulting in tumor regression in syngeneic mouse models. Supporting the potentiation of a systemic immune response, we observed a relative increase in activated T cells, an abscopal effect, and corresponding increases in systemic T cell memory populations in mice treated with probiotically delivered checkpoint inhibitors. Last, we leveraged the modularity of our platform to achieve enhanced therapeutic efficacy in a poorly immunogenic syngeneic mouse model through effective combinations with a probiotically produced cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). Together, these results demonstrate that our engineered probiotic system bridges synthetic biology and immunology to improve upon checkpoint blockade delivery.

中文翻译：

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经工程改造的益生菌，用于检查点封锁纳米抗体的局部肿瘤递送。

检查点抑制剂已经彻底改变了癌症治疗方法，但仅在部分患者中起作用，并且可能导致多种毒性，这表明需要更具针对性的递送系统。由于它们优先定植在肿瘤中，因此微生物是局部递送癌症治疗剂的天然平台。在这里，我们设计了一种益生菌细菌系统，用于通过稳定的裂解释放机制控制靶向程序性细胞死亡配体1（PD-L1）和细胞毒性T淋巴细胞相关蛋白4（CTLA-4）的纳米抗体的生产和瘤内释放。我们使用计算模型与裂解电路动力学的实验验证相结合，以确定最佳的遗传电路参数，以实现最佳治疗效果。与类似的临床相关抗体相比，该工程系统的单次注射显示出增强的治疗反应，导致同源小鼠模型中的肿瘤消退。支持全身免疫应答的增强，我们观察到用益生菌递送的检查点抑制剂治疗的小鼠中活化的T细胞的相对增加，绝对的作用以及全身T细胞记忆种群的相应增加。最后，我们通过与益生菌产生的细胞因子粒细胞巨噬细胞集落刺激因子（GM-CSF）的有效结合，利用平台的模块化在免疫原性差的同系小鼠模型中实现了增强的治疗效果。一起，