The Shraga Segal Department of Microbiology, Immunology, and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel.
Sci Rep. 2022 May 3;12(1):7169. doi: 10.1038/s41598-022-11021-1.
Cancer immunotherapies are highly potent and are gaining wide clinical usage. However, severe side effects require focusing effector immune cell activities on the tumor microenvironment (TME). We recently developed a chimeric antigen receptor tumor-induced vector (CARTIV), a synthetic promoter activated by TME factors. To improve CARTIV functions including background, activation levels, and synergism, we screened a library of promoters with variations in key positions. Here, we present a screening method involving turning ON/OFF stimulating TNFα and IFNγ cytokines, followed by sequential cell sorting. Sequencing of enriched promoters identified seventeen candidates, which were cloned and whose activities were then validated, leading to the identification of two CARTIVs with lower background and higher induction. We further combined a third hypoxia element with the two-factor CARTIV, demonstrating additional modular improvement. Our study presents a method of fine-tuning synthetic promoters for desired immunotherapy needs.
癌症免疫疗法具有很强的功效，并正在获得广泛的临床应用。然而，严重的副作用要求将效应免疫细胞的活性集中在肿瘤微环境（TME）上。我们最近开发了一种嵌合抗原受体肿瘤诱导载体（CARTIV），这是一种由 TME 因子激活的合成启动子。为了提高 CARTIV 的功能，包括背景、激活水平和协同作用，我们筛选了一个具有关键位置变异的启动子文库。在这里，我们提出了一种筛选方法，涉及开启/关闭刺激 TNFα 和 IFNγ 细胞因子，然后进行连续的细胞分选。对富集启动子的测序鉴定出了十七个候选者，然后对这些候选者进行克隆，并验证其活性，从而鉴定出两个具有更低背景和更高诱导的 CARTIV。我们进一步将第三个缺氧元件与两因素 CARTIV 结合，证明了额外的模块改进。我们的研究提出了一种用于满足所需免疫治疗需求的精细调整合成启动子的方法。
