新型核苷酸嵌合体靶向调控免疫耐受微环境治疗实体肿瘤

Reestablishment of immune surveillance holds the promise for cancer therapy. In addition to the lack of immune recognition and/or immune stimulation, the negative feedback raised by immune activation, such as the immune checkpoint pathways, indoleamine 2,3-dioxygenase 1(IDO1)-mediated immune tolerance, compose together the hurdles for effective immunotherapy in malignant solid tumors. To restore sufficient immune surveillance in tumor microenvironment, combined strategies are required which not only stimulate immune activation but also block the negative feedback within the solid tumors. The immunostimulatory 5’-triphosphate small interfering RNA (ppp-siRNA), a RNA virus mimic, potently stimulates immune activation via the cytosolic retinoic acid inducible gene I (RIG-I) pathway, induces cancer immunogenic cell death, and selectively silences target gene. The aptamer is a DNA or RNA single strand that binds to specific target with high affinity. It has been designed as the carrier for targeted-delivery of siRNAs. To achieve a comprehensive reestablishment of immune surveillance in solid tumor, for the first time in this study, we construct a chimera consisting of PD-L1 aptamer and immunostimulatory ppp-siRNA targeting Ido1 (AptPD-L1-ppp-siIdo1), which possesses multiple functions: i) PD-L1 specific binding/targeting, ii) RIG-I activation and, iii) Ido1 silencing. Thus, the chimera not only induces tumor-targeted immune activation, but also at the same time blocks the negative feedback mediated by immune checkpoint PD-1/PD-L1 and Ido1. By means of in vitro experiments and animal tumor models, we will study the antitumor efficacy of this novel chimera, and delineate the underlying mechanisms. The results will provide a more efficient agent and a new strategy for cancer immunotherapy.

免疫监视的重建是控制癌症的希望。实体肿瘤中不仅缺乏免疫识别和活化，还存在负性免疫调控，如免疫检查点通路、吲哚胺2,3-双加氧酶1（IDO1）介导的免疫耐受等，兼顾的免疫重塑策略是解决之道。5’端三磷酸小干扰RNA（ppp-siRNA）能模拟RNA病毒激活细胞浆视磺酸诱导基因-I（RIG-I）诱导免疫活化、肿瘤免疫原性细胞死亡，并沉默靶基因。适体是一种与特异靶分子有高度亲和力的单链核苷酸序列，能与siRNA杂交成嵌合体进行靶向递送。本项目首次构建PD-L1适体与沉默IDO1的ppp-siRNA的嵌合体，使其具有靶向PD-L1、激活RIG-I通路并抑制IDO1表达等多种特性，能在靶向肿瘤、诱导免疫活化的同时，阻断微环境中PD-1/PD-L1检查点通路和IDO1介导的负性调控。在体外和小鼠实体瘤模型中，研究此新型嵌合体的抗肿瘤作用及相关机制。成果将为肿瘤免疫治疗提供一个更有效的武器和新的思路。

免疫监视的重建是控制癌症的希望。为了整全地重塑抗肿瘤免疫监视，我们将特异结合PD-L1的aptamer与沉默IDO1的5’端三磷酸化的小干扰RNA构建成嵌合体（AptPD-L1-ppp-siIdo1），在靶向肿瘤诱导免疫活化的同时，又阻断肿瘤微环境中PD-1/PD-L1和IDO1介导的负性调控，在细胞和分子水平、小鼠实体瘤模型中，研究其靶向PD-L1、激活RIG-I通路并抑制IDO1表达的多种功能。其次，我们进一步尝试合成沉默促肿瘤关键分子STAT3及靶向肿瘤细胞mRNA质量监控体系关键基因SMG1的核酸嵌合体——AptPD-L1-pppSTAT3和AptPD-L1-pppSMG1，检测了其特异性基因沉默、激活RIG-I信号并活化CD8+ T细胞介导的抗肿瘤免疫应答的功能。此外，我们还探索了ppp-RNA与表达PD-L1的重组牛痘病毒联合增强抗肿瘤的作用；最后，我们发现两个小分子药物（二氯乙酸和氟达拉滨）能够抑制显著IDO1的表达，显著促进溶瘤新城疫病毒的抗肿瘤作用。我们的研究成果一方面为靶向IDO1的肿瘤免疫治疗提供几个更有效的分子，另一方面为恶性肿瘤的免疫治疗提供了多个新的联合策略和实验基础。