在哺乳动物中，模式识别受体在免疫监视中起到关键作用，是免疫调控网络的重要组成部分。近期越来越多研究表明，STING信号通路在诱导T细胞抗肿瘤中扮演了重要角色，STING通路及其激动剂环二核苷酸(CDNs)已成为肿瘤免疫治疗的研究热点。然而，仅用CDNs很难在免疫抑制的肿瘤微环境中起到足够强的免疫刺激作用。为了解决上述问题，本论文以CDNs为基础，设计并评价了两种纳米抗肿瘤体系，主要研究成果如下：为了进一步提高CDNs的免疫疗效，我们尝试用自噬抑制剂-氯喹(CQ)组合环二鸟苷酸(CDG)对抗肿瘤。研究发现CQ+CDG不仅能够上调胰腺癌细胞MHC I表达，而且还能激活APC。在此基础上，我们利用聚合物PC7A构筑了基于CQ和CDG的纳米联合治疗体系CQ CDG@PC7A。体外实验发现CQ CDG@PC7A能够促进CQ和CDG的跨膜能力。通过小鼠活体成像，进一步证明CQ CDG@PC7A有效改善了CQ和CDG的肿瘤靶向性和半衰期。最后在荷瘤小鼠模型研究中发现，CQ CDG@PC7A显著提高了小鼠胰腺癌细胞的MHC I表达，逆转了肿瘤微环境中的免疫抑制网络，明显提升了肿瘤浸润CD8+T细胞群的比例。同时活化了周围次级淋巴组织，建立了有效的抗肿瘤免疫网路。上述结果验证了肿瘤自噬抑制剂与免疫刺激剂在肿瘤免疫治疗中存在协同效应，可应用于肿瘤免疫治疗。CDG诱导的细胞因子种类有限，影响了自身的免疫刺激活性。我们通过筛选发现STING激动剂CDGSF与TLR7/8激动剂522存在明显的免疫协同效应。522能够有效扩展CDGSF引发的细胞因子种类，获得更强的免疫刺激效果。同时发现该协同免疫效果可能通过上调NF-κB、P65和TBK-1信号通路实现。将CDGSF-522联合佐剂应用于抗肿瘤疫苗，与PC7A构筑了MCNVs纳米体系。发现该体系能够显著提升CDGSF、522和抗原的跨膜效率以及淋巴结靶向性，通过协同免疫效应和尺寸效应引发了有效的肿瘤抗原特异性免疫应答，为小鼠提供了免疫保护，使其在25天内的存活率达到100%。本论文基于CDNs构筑了两种纳米联合治疗体系，通过协同免疫效应和尺寸效应提升了CDNs抗肿瘤免疫力，为未来的肿瘤联合疗法提供了新的思路。
In mammals, pattern recognition receptors are responsible for immune surveillance, which is an important component of the immune regulatory network. Recent studies have shown that the STING signaling pathway plays an important role in the induction of T cells against tumors. Cyclic dinucleotides (CDNs), agonists of the STING pathway, became research hotspot of cancer immune therapy. However, it is difficult to reverse the immunosuppressive condition in the tumor microenvironment with CDNs alone. To address the above-mentioned problems, two nanoantitumor systems based on CDNs were designed and evaluated in this thesis.We attempted to improve the immunotherapeutic effects of CDNs by combining the autophagy inhibitor chloroquine (CQ) with cyclic diguanylate (CDG). Subsequently, we found that CQ+CDG not only upregulate MHC I expression in pancreatic cancer cells, but also activates APCs. To improve their transmembrane ability, we constructed CQ CDG@PC7A nanosystem with CQ, CDG and the polymer PC7A. After immunizing mice with CQ CDG@PC7A, it was found that the nanosystem effectively improved the tumor targeting ability and half-life of CQ+CDG. Finally, in a tumor-bearing mouse model, CQ CDG@PC7A significantly increased MHC I expression in pancreatic cancer cells, which reversed the immunosuppressive network in TME and significantly elevated the proportion of tumor-infiltrating CD8+ T cells compared to other controls. In addition, the treatment similarly activated the surrounding secondary lymphoid tissues and established a strong anti-tumor immune network. The above results validate the synergistic effects of tumor autophagy inhibitors and immune adjuvants in tumor immunotherapy.CDG induces limited variety of cytokines, which restricts its immunostimulatory application. It was found that the STING agonist CDGSF had a significant immune synergistic effects with TLR7/8 agonist 522, which can effectively expand the CDGSF-induced cytokine species and thus obtain a stronger immunostimulatory effects. Our study revealed that this synergistic immune effects may be achieved through upregulation of NF-κB P65 and TBK-1 signaling pathways. Based on the above results, we constructed multicomponent nanovaccines (MCNVs) by encapsulating CDGSF, 522 and OVA antigens with polymer PC7A. We found that MCNVs significantly increased the transmembrane efficiency and lymph node targeting ability of immunostimulants and antigen, which triggered potent anti-tumor immunity. Furthermore, MCNVs amplified the therapeutic effects of CDGSF through synergistic immune effects and size effects, providing immune protection in mice with a 100% survival rate within 25 days.In this thesis, two therapeutic nanosystems were constructed based on CDNs. The anti-tumor immunity of CDNs was enhanced by synergistic immune effects and size effects, which provided new vision for future tumor combination therapy.
