[摘 要] 目的：探究CpG寡核苷酸和OX40激动剂抗体原位疫苗（CpG + OX40）联合抗血管生成药物安罗替尼（anlotinib）治疗 小鼠卵巢癌的全身抗肿瘤效应及免疫机制。方法：建立双侧（原发侧和转移侧）ID8细胞小鼠卵巢癌模型，分组给予安罗替尼、 CpG + OX40或CpG + OX40 + 安罗替尼（三联疗法）治疗。通过监测肿瘤体积和记录生存期评估各治疗组的抗肿瘤效果。采用 流式细胞术和ELISA法检测肿瘤微环境中的免疫细胞和细胞因子变化，qRT-PCR法检测移植瘤组织中反映血管密度和成熟度的 分子的相对表达量。结果：与其他治疗组相比，三联疗法显著抑制治疗侧（原发侧）和未治疗侧（转移侧）肿瘤的生长（P < 0.01）， 延长荷瘤鼠生存期（P < 0.05）。流式术检测结果显示 ，三联疗法显著提高原发侧和转移侧肿瘤内CD4+ T和CD8+ T细胞浸 润比例（P < 0.05）。免疫细胞耗竭实验表明，单独耗竭CD4+ T、CD8+ T或NK细胞时，三联疗法对原发侧肿瘤的抑制作用无明显 变化，而转移侧的抗肿瘤作用显著减弱但仍强于PBS组（P < 0.01）。仅当3 种免疫细胞同时耗竭时，肿瘤抑制效应与 PBS 组 差异无统计学意义（P > 0.05 ）。ELISA 法检测结果显示，与各治疗组相比，三联疗法组原发侧和转移侧肿瘤内Th1细胞相 关细胞因子明显增加（P < 0.05），Th2细胞相关细胞因子的表达显著降低（P < 0.05）。qRT-PCR法结果显示，与对照组相比，三联 疗法组双侧移植瘤组织内的CD31表达显著降低（P < 0.000 1），血管生成素（Ang）-1/Ang-2的比值显著升高（P < 0.001）。结论： CpG + OX40原位疫苗联合安罗替尼具有更强的全身抗肿瘤效应，适应性免疫和固有免疫及血管密度调控在其中发挥关键作用， 为晚期肿瘤患者提供潜在治疗选择。

[Abstract] Objective: To investigate the systemic anti-tumor effects and immune mechanisms of CpG oligonucleotide and OX40 agonist antibody in situ vaccine (CpG + OX40) combined with anti-angiogenic drug anlotinib in the treatment of mouse ovarian cancer. Methods: A bilateral (primary and metastatic) ID8 cell mouse ovarian cancer model was established. And tumor-bearing mice were treated with anlotinib, CpG + OX40, or CpG + OX40 + anlotinib (the triple treatment), respectively. The anti-tumor effects of different treatment groups were evaluated by monitoring tumor volume and survival time. The changes of immune cells and cytokines in the tumor microenvironment were detected using flow cytometry and ELISA. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative expression levels of molecules reflecting vascular density and maturity in tumor tissues. Results: Compared with other treatment groups, the CpG + OX40 + anlotinib treatment significantly inhibited tumors on the treatment side (primary side) and the untreated side (metastatic side) (P < 0.01), and significantly improved the survival time of tumor-bearing mice (P < 0.05). The flow cytometry analyses showed that the CpG + OX40 + anlotinib treatment significantly increased the proportion of tumor infiltrating CD4+ T and CD8+ T cells in tumors at both the treated and untreated sides (P < 0.05). Immune cell depletion confirmed that when CD4+ T, CD8+ T, or NK cells were depleted alone, there was no significant difference in the inhibitory effects of the triple treatment on primary-side tumors, while the anti-tumor effects on metastatic-side tumors were significantly weakened but were still stronger than those of the PBS group (P < 0.01). When all three types of immune cells were depleted simultaneously, there was no statistically significant difference in their tumor suppressive effects compared with the PBS group (P > 0.05). The ELISA results showed that compared with other treatment groups, the triple treatment group showed a significant increase in Th1-related cytokines in the primary-side and metastatic-side tumors (P < 0.05), and a significant decrease in the expression of Th2-related cytokines (P < 0.05). The qRT-PCR results showed that, compared with the control group, the triple treatment group exhibited significantly lower CD31 expression (P < 0.0001) and a significantly higher Ang-1/Ang-2 ratio (P < 0.001) in tumor tissues on both sides. Conclusion: The combination of CpG + OX40 in situ vaccine and anlotinib has stronger systemic anti-tumor effects. Adaptive immunity, innate immunity and vascular density regulation play a crucial role in its anti-tumor effects, which provide potential treatment options for advanced cancer patients.

国家自然科学基金青年项目（No. 82072927）；青岛市2022年度医药卫生科研指导项目（No. 2022-WJZD065）