原位肿瘤疫苗由于无需事先抗原鉴定而能实现个体化肿瘤疗法的优势，已逐渐成为研究热点。各种原位肿瘤疫苗方案在临床前和临床研究中表现出可观的抗肿瘤功效。然而，原位肿瘤疫苗的设计仍需要进一步优化，其潜在的免疫机制也有待深入研究。本研究建立了一种新颖的三联原位疫苗策略，将局部放射治疗与肿瘤内注射TLR9激动剂CpG和OX40激动剂相结合。通过在B16F10黑素瘤的双侧肿瘤和肺转移模型中评估局部和外周抗肿瘤效力以及生存益处，进一步使用多参数流式细胞术和RNA测序研究了原位疫苗诱导的免疫反应和与免疫相关的肿瘤环境变化。基于分析结果，RT + CpG + αOX40三联原位疫苗与免疫检查点阻滞疗法相结合，以探索潜在的协同抗肿瘤功效。在接受RT + CpG + αOX40三联疫苗后，治疗和未治疗的外周抗肿瘤疗效明显增强，毒性最小。局部放射治疗和OX40激动剂的引入分别有益于局部和外周病灶的抑制，这可能部分归因于肿瘤微环境中效应器记忆T细胞的增加。进一步分析表明，三联原位疫苗不仅激活了治疗肿瘤的微环境，上调了多个与免疫相关的途径，还增强了全身抗肿瘤反应，因此实现了优越的全身肿瘤控制和生存益处。此外，三联原位疫苗与免疫检查点阻滞疗法协同作用，显著提高了抗编程性细胞凋亡蛋白（PD-1）抗体的治疗效果。这种三联原位疫苗诱导了强烈的抗肿瘤反应，实现了有效的全身肿瘤控制和生存益处，并与免疫检查点阻滞疗法展现出显著的协同抗肿瘤效果。这些数据初步证实了三联原位疫苗的疗效、可行性和安全性，表明它在临床情境中作为单独治疗和综合免疫治疗方案的一部分具有广泛应用潜力。© 2023. 版权归BioMed Central Ltd.所有，属于Springer Nature的一部分。

In situ tumor vaccine has been gradually becoming a hot research field for its advantage of achieving personalized tumor therapy without prior antigen identification. Various in situ tumor vaccine regimens have been reported to exert considerable antitumor efficacy in preclinical and clinical studies. However, the design of in situ tumor vaccines still needs further optimization and the underlying immune mechanism also waits for deeper investigation.A novel triple in situ vaccine strategy that combining local radiation with intratumoral injection of TLR9 agonist CpG and OX40 agonist was established in this sturdy. Local and abscopal antitumor efficacy as well as survival benefit were evaluated in the bilateral tumors and pulmonary metastasis model of B16F10 melanoma. In situ vaccine-induced immune responses and immune-associated variation in tumor environment were further investigated using multiparameter flow cytometry and RNA sequencing. Base on the analysis, the RT + CpG + αOX40 triple in situ vaccine was combined with checkpoint blockade therapy to explore the potential synergistic antitumor efficacy.Enhanced tumor suppression was observed with minimal toxicity in both treated and untreated abscopal tumors after receiving RT + CpG + αOX40 triple vaccine. The introduction of local radiation and OX40 agonist benefit more to the inhibition of local and abscopal lesions respectively, which might be partially attributed to the increase of effector memory T cells in the tumor microenvironment. Further analysis implied that the triple in situ vaccine did not only activate the microenvironment of treated tumors, with the upregulation of multiple immune-associated pathways, but also enhanced systemic antitumor responses, thus achieved superior systemic tumor control and survival benefit. Moreover, the triple in situ vaccine synergized with checkpoint blockade therapy, and significantly improved the therapeutic effect of anti-programmed cell death protein (PD)-1 antibody.This triple combining in situ vaccine induced intensive antitumor responses, mediated effective systemic tumor control and survival benefit, and displayed impressive synergistic antitumor effect with checkpoint blockade therapy. These data preliminary confirmed the efficacy, feasibility and safety of the triple combining in situ vaccine, suggesting its great application potential as both monotherapy and a part of combined immunotherapeutic regimens in clinical scenario.© 2023. BioMed Central Ltd., part of Springer Nature.