背景：

肿瘤免疫治疗的目标在于将肿瘤进程控制于免疫系统可调控的“平衡期”乃至“清除期”，阻止免疫逃逸的发生。肿瘤发生早期是免疫系统与肿瘤细胞激烈博弈的关键阶段，但过度激活的T细胞易发生激活诱导性细胞死亡（AICD），从而导致 CD8⁺ T 细胞功能耗竭，削弱抗肿瘤免疫应答。因此，早期给予适当的免疫调节干预，有望维持 T 细胞功能，提升抗肿瘤免疫效率。

Interleukin-10（IL-10）传统上被认为是一种促肿瘤的免疫抑制性细胞因子，其在肿瘤进展后期水平显著升高，并参与形成免疫抑制微环境。然而，近年来研究发现 IL-10 亦具激活 CD8⁺ T 细胞、抑制肿瘤生长等潜在抗肿瘤作用，提示其功能具有双重性。若能通过合理的递送系统将 IL-10 精准作用于免疫激活相关细胞，或可释放其抗肿瘤潜力。本课题利用科室前期构建好的“即插即用”的诺如 S 蛋白病毒样颗粒（Nov-S-catcher），该递送平台可高效呈递蛋白抗原或细胞因子，具有良好的稳定性与靶向性。基于此，构建 IL-10 负载的病毒样颗粒（VLPs）纳米疫苗，旨在提升抗原递呈效率，增强 T 细胞免疫应答，为肿瘤疫苗疗效提升提供新方向。

此外，髓源性抑制细胞（Myeloid-Derived Suppressor Cells, MDSCs）是肿瘤微环境中关键的免疫抑制细胞类型，能够显著抑制效应性 T 细胞功能、促进免疫逃逸。已有研究表明，一些化疗药物如吉西他滨等可选择性清除 MDSCs，间接增强免疫治疗效果。6-Thio-2'-Deoxyguanosine（THIO）是一种新型端粒酶靶向小分子药物，能选择性作用于端粒酶阳性的肿瘤细胞，且对正常细胞影响有限。近期研究提示 THIO 还可直接杀伤 MDSCs，打破其构建的免疫抑制屏障，重塑肿瘤免疫微环境，从而增强疫苗和其他免疫疗法的疗效。因此，分别开展基于 VLP 的 IL-10 纳米疫苗与 THIO 抑制 MDSCs 的研究，为进一步开展两者的联合干预提供了基础，为构建高效肿瘤免疫治疗体系提供理论与策略支持。

目的：

本研究围绕两项免疫治疗策略分别展开，旨在从不同角度提高肿瘤免疫治疗的有效性。其一，基于 VLPs 构建 IL-10 递送平台，通过 Spycatcher/SpyTag 系统实现 IL-10 的高效定向呈递，以探究其在激活抗肿瘤免疫反应中的潜在应用价值，尤其是在调控 T 细胞功能、减轻 AICD 效应及增强疫苗免疫原性方面的作用。其二，探索端粒酶靶向药物 THIO 在肿瘤免疫微环境中对 MDSCs 的抑制作用，评估其在解除免疫抑制、重塑微环境方面的机制和效果。两部分研究分别从免疫激活和解除免疫抑制两个关键环节切入，为进一步开展两者的联合干预提供了基础，为肿瘤免疫治疗提供了新的策略。

方法：

使用镍柱亲和层析及凝胶过滤层析获得均一的蛋白纳米颗粒，Nov-S-catcher（以下简称为 NC）。

使用镍柱亲和层析获得工程化修饰的蛋白 TF-Spytag-IL10（以下简称为 IL10）。

利用 SpyCatcher/SpyTag 系统将 TF-Spytag-IL10 呈递在病毒样颗粒 Nov-S-catcher 表面，得到 NC-IL10，透射电子显微镜观察呈递 IL-10 的病毒样颗粒的形态。

在 TC-1 肿瘤模型中，探索 NC-IL10 抑制肿瘤的能力。

利用活体成像技术，观察 NC-IL10 驻留淋巴结和靶向肿瘤的能力。

分别在 TC-1 肿瘤模型和 B16-F10 肿瘤模型中，探索 NC-IL10 分别呈递不同肿瘤特异性抗原，抑制肿瘤生长、对 T 细胞及肿瘤微环境的影响。

检测多种不同细胞系的端粒酶活性，及不同浓度的 THIO 对正常细胞系 PBMC 是否有杀伤。

在 TC-1 肿瘤模型中，THIO 治疗后对小鼠微环境的影响，分别使用流式细胞术检测脾脏、淋巴结和肿瘤组织中免疫细胞的变化。

从小鼠腿骨中分离得到骨髓前体细胞，使用流式细胞术检测，加入不同浓度 THIO 对骨髓前体细胞向 MDSCs 分化的影响。

分别使用 THIO 和 PBS 对健康小鼠给予腹腔注射，通过对小鼠体温、体重进行监测，部分重要脏器的病理切片分析及血液血清生化检测，简单对 THIO 进行安全性评估。

结果：

NC 经分子筛纯化后，呈现出颗粒均一、直径约 20 nm 的环状病毒样颗粒形态，并且可以呈递工程化修饰的蛋白 TF-Spytag-IL10。呈递了蛋白的病毒样颗粒 NC-IL10，形态结构未被破坏，且表面刺突明显。

在 TC-1 肿瘤模型中，NC-IL10 有较强的抑制 TC-1 肿瘤生长和肿瘤杀伤的能力。肿瘤组织的免疫荧光结果发现，皮下注射 NC-IL10 的实验组中，肿瘤浸润的 CD8⁺ T 细胞密度显著提升。

在活体成像实验中，相较于游离的 IL-10，NC-IL10 能够更高效地靶向肿瘤组织，并且更长时间的驻留在淋巴结中。

进一步探索抗原协同呈递的作用机制发现，NC-E7-IL10 在 TC-1 模型中展现出更为卓越的抗肿瘤效果。与单独呈递 IL-10 或 E7 抗原肽的组别相比，NC-E7-IL10 不仅能显著抑制肿瘤生长，还能有效抑制 CD8⁺ T 细胞耗竭，上调抗原特异性 CTLs 比例，下调 MDSCs 比例。免疫荧光共聚焦实验进一步证实，NC-E7-IL10 能够激活 CD8 和 p-STAT3 信号通路。

在 B16 “免疫冷肿瘤” 模型中，NC-Trp2-IL10 同样表现出显著的肿瘤抑制能力。NC-Trp2-IL10 依然能够激活淋巴细胞分泌 IFN-γ，有效遏制肿瘤生长。RNA-seq 分析表明，其处理后肿瘤组织 mRNA 改变，差异表达基因涉及 p53、MAPK 等免疫相关通路。然而，研究也发现其在抑制脾肿大及下调 MDSCs 比例方面存在一定局限性。值得关注的是，NC-Trp2-IL10 对 CD8⁺ T 细胞耗竭的抑制效果与 NC-E7-IL10 相当，这一结果充分证实了肿瘤特异性抗原与 IL-10 协同呈递策略的普适性。

检测不同来源肿瘤细胞及免疫相关细胞的端粒酶活性，发现人肿瘤细胞系端粒酶活性显著高于小鼠肿瘤细胞系，MDSCs 具备一定端粒酶活性，而小鼠 PBMC 仅呈现微弱端粒酶活性。基于此，CCK8 实验证实 THIO 对正常 PBMC 无明显杀伤作用，表明其对正常细胞安全性良好。

在肿瘤微环境重塑方面，以 TC-1 荷瘤小鼠为模型，给予不同针次 THIO 治疗后，小鼠脾脏及肿瘤组织内 CTLs、NK 细胞和 Th1 细胞比例显著增加，MDSCs 比例显著降低；淋巴结组织分析显示，THIO 治疗能显著降低 MDSCs 比例。

安全性评价研究中，健康小鼠腹腔注射 THIO 后，对其心脏、肺和肾组织进行 H&E 染色及病理分析，未发现明显病理损伤；监测小鼠体温与体重变化，虽体重有轻微下降但仍在正常范围，体温无明显变化。结果显示 THIO 处理组与对照组相比，肝、肾、心脏功能相关指标以及血液系统各项指标均无显著差异，且均处于正常生理区间。

结论：

本研究围绕肿瘤免疫治疗中面临的免疫抑制挑战开展了两部分研究工作。首先，基于 SpyTag/SpyCatcher 技术构建 Nov-S-catcher（NC）病毒样颗粒，实现 IL-10 与肿瘤抗原的纳米化共递送，在“热”肿瘤 TC-1 和“冷”肿瘤 B16 小鼠模型中，NC-IL10 及相关纳米疫苗通过激活免疫细胞、调控信号通路，展现出显著的抗肿瘤效果。此外，基于肿瘤与正常细胞端粒酶活性差异，证实端粒酶靶向药物 THIO 可特异杀伤肿瘤细胞，而对正常细胞安全性良好；THIO 治疗的荷瘤小鼠表现出增加的 CTLs、NK 细胞等效应细胞比例及降低的免疫抑制细胞 MDSCs 水平；进一步实验发现，THIO 可直接影响 MDSCs 分化/增殖，重塑肿瘤免疫微环境。两部分研究分别从免疫激活和解除免疫抑制两个关键环节切入，为进一步开展两者的联合干预提供了基础，为肿瘤免疫治疗提供了新的策略。

Background:

The goal of tumor immunotherapy is to control the tumor process in the “equilibrium phase” or even the “clearance phase” that can be regulated by the immune system, and to prevent the occurrence of immune escape. The early stage of tumorigenesis is the key stage for the immune system to fight with tumor cells, but over-activated T cells are prone to activation-induced cell death, which leads to the depletion of CD8⁺ T cells and weakening the anti-tumor immune response. Therefore, appropriate immunomodulatory interventions at an early stage are expected to maintain T cell function and enhance anti-tumor immune efficiency.

IL-10 has traditionally been regarded as a pro-tumor immunosuppressive cytokine, whose level is significantly elevated in the late stage of tumor progression and is involved in the formation of an immunosuppressive microenvironment. However, recent studies have revealed that IL-10 also has potential anti-tumor effects such as activating CD8⁺ T cells and inhibiting tumor growth, suggesting that its function is dual. If IL-10 can be precisely applied to immune-activated cells through a reasonable delivery system, its anti-tumor potential can be released. In this study, we utilized the “plug-and-play” Nov-S-catcher, a delivery platform that can efficiently deliver protein antigens or cytokines with good stability and targeting properties. Based on this, the construction of IL-10-loaded VLP nanovaccines aims to enhance antigen presentation efficiency and T cell immune response, providing a new direction for tumor vaccine efficacy enhancement.

In addition, MDSCs are key immunosuppressive cell types in the tumor microenvironment, which can significantly inhibit the function of effector T cells and promote immune escape. It has been shown that some chemotherapeutic agents such as gemcitabine can selectively remove MDSCs and indirectly enhance the immunotherapeutic effect. THIO is a novel telomerase-targeting small molecule drug that can selectively act on telomerase-positive tumor cells with limited effect on normal cells. Recent studies have suggested that THIO can also directly kill MDSCs, break the immunosuppressive barrier constructed by them, and remodel the tumor immune microenvironment, thus enhancing the efficacy of vaccines and other immunotherapies. Therefore, the studies on IL-10 nanovaccines based on VLPs and THIO-mediated inhibition of MDSCs were conducted respectively, providing a foundation for further combined interventions and offering theoretical and strategic support for constructing an efficient cancer immunotherapy system.

Objective:

The present study revolves around two immunotherapy strategies respectively, aiming to improve the effectiveness of tumor immunotherapy from different perspectives. First, to construct an IL-10 delivery platform based on VLPs and achieve efficient targeted presentation of IL-10 through the Spycatcher/SpyTag system, in order to explore its potential application in activating anti-tumor immune responses, especially in regulating T cell function, attenuating the effects of AICD and enhancing the immunogenicity of vaccines. Second, to explore the direct killing effect of the telomerase-targeting drug THIO on MDSCs in the tumor immune microenvironment, and to assess its mechanism and effect in deregulating immunosuppression and remodeling the microenvironment. The two parts of the study respectively intervene from two key aspects of immune activation and immunosuppression relief, providing a foundation for further combined interventions and offering new strategies for cancer immunotherapy.

Method:

Utilize nickel column affinity chromatography and gel filtration chromatography to obtain homogeneous protein nanoparticles, Nov-S-catcher (hereinafter referred to as NC).

Use nickel column affinity chromatography to obtain the engineered modified protein TF-Spytag-IL10 (hereinafter referred to as IL10).

Employ the SpyCatcher/SpyTag system to present TF-Spytag-IL10 on the surface of the virus-like particle Nov-S-catcher to obtain NC-IL10, and observe the morphology of the virus-like particle presenting IL-10 using a transmission electron microscope.

In the TC-1 tumor model, explore the tumor-inhibiting ability of NC-IL10.

Utilize in vivo imaging technology to observe the ability of NC-IL10 to reside in the lymph nodes and target the tumor.

Respectively in the TC-1 tumor model and the B16-F10 tumor model, explore the effects of NC-IL10 presenting different tumor-specific antigens on inhibiting tumor growth, as well as on T cells and the tumor microenvironment.

Detect the telomerase activity of various different cell lines, and whether different concentrations of THIO have a killing effect on the normal cell line PBMC.

In the TC-1 tumor model, study the impact of THIO treatment on the mouse microenvironment. Use flow cytometry to detect the changes of immune cells in the spleen, lymph nodes and tumor tissues respectively.

Isolate bone marrow precursor cells from the mouse leg bone, use flow cytometry to detect, and add different concentrations of THIO to observe the impact on the differentiation of bone marrow precursor cells into MDSCs.

Intraperitoneally inject healthy mice with THIO and PBS respectively. Through monitoring the body temperature and body weight of the mice, analyzing the pathological sections of some important organs and detecting the blood serum biochemistry, simply evaluate the safety of THIO.

Results:

After purification by molecular sieve, Nov-S-catcher presents the morphology of virus-like particles with uniform particles and a diameter of about 20 nm in a ring shape, and can present the engineered modified protein TF-Spytag-IL10. For the virus-like particle NC-IL10 presenting the protein, its morphological structure is not damaged, and the surface spikes are obvious.

In the TC-1 tumor model, NC-IL10 has a strong ability to inhibit the growth and kill the TC-1 tumor. The immunofluorescence results of the tumor tissues show that in the experimental group with subcutaneous injection of NC-IL10, the density of tumor-infiltrating CD8⁺ T cells is significantly increased.

In the in vivo imaging experiment, compared with the free IL-10, NC-IL10 can more efficiently target the tumor tissue and stay in the lymph nodes for a longer time.

Further exploration of the mechanism of action of antigen co-presentation reveals that NC-E7-IL10 shows more excellent anti-tumor effects in the TC-1 model. Compared with the groups presenting IL-10 or E7 antigen peptide alone, NC-E7-IL10 can not only significantly inhibit tumor growth, but also effectively inhibit the exhaustion of CD8⁺ T cells, up-regulate the proportion of antigen-specific CTLs, and down-regulate the proportion of MDSCs. The immunofluorescence confocal experiment further confirms that NC-E7-IL10 can activate the CD8 and p-STAT3 signaling pathways.

In the B16 "immune cold tumor" model, NC-Trp2-IL10 also shows significant tumor-inhibiting ability. NC-Trp2-IL10 can still activate lymphocytes to secrete IFN-γ and effectively suppress tumor growth. RNA-seq analysis shows that the mRNA of the tumor tissue changes after its treatment, and the differentially expressed genes are involved in immune-related pathways such as p53 and MAPK. However, the study also finds that it has certain limitations in inhibiting splenomegaly and down-regulating the proportion of MDSCs. It is worth noting that the inhibitory effect of NC-Trp2-IL10 on the exhaustion of CD8⁺ T cells is comparable to that of NC-E7-IL10, and this result fully confirms the universality of the strategy of co-presenting tumor-specific antigens and IL-10.

First, detect the telomerase activity of tumor cells from different sources and immune-related cells. It is found that the telomerase activity of human tumor cell lines is significantly higher than that of mouse tumor cell lines. MDSCs have a certain telomerase activity, while mouse PBMC only shows weak telomerase activity. Based on this, the CCK8 experiment confirms that THIO has no obvious killing effect on normal PBMC, indicating that it has good safety for normal cells.

In terms of remodeling the tumor microenvironment, using TC-1 tumor-bearing mice as the model, after different doses of THIO treatment, the proportions of CTLs, NK cells and Th1 cells in the spleen and tumor tissues of the mice are significantly increased, and the proportion of MDSCs is significantly decreased. The analysis of the lymph node tissues shows that THIO treatment can significantly reduce the proportion of MDSCs.

In the safety evaluation study, after intraperitoneal injection of THIO into healthy mice, H&E staining and pathological analysis of the heart, lung and kidney tissues are carried out, and no obvious pathological damage is found. By monitoring the changes in body temperature and body weight of the mice, although the body weight slightly decreases, it is still within the normal range, and there is no obvious change in body temperature. The results show that compared with the control group, there are no significant differences in the indicators related to the functions of the liver, kidney and heart, as well as the various indicators of the blood system in the THIO treatment group, and they are all within the normal physiological range.

Conclusion:

This study addresses the challenges of immunosuppression in cancer immunotherapy through two parts of research work. Firstly, based on the SpyTag/SpyCatcher technology, a Nov-S-catcher (NC) virus-like particle was constructed to achieve nanoscale co-delivery of IL-10 and tumor antigens. In mouse models of "hot" tumors (TC-1) and "cold" tumors (B16), NC-IL10 and related nanovaccines demonstrated significant antitumor effects by activating immune cells and regulating signaling pathways. Additionally, based on the difference in telomerase activity between tumor and normal cells, it was confirmed that the telomerase-targeted drug THIO can specifically kill tumor cells while showing good safety for normal cells. Tumor-bearing mice treated with THIO exhibited increased proportions of effector cells such as CTLs and NK cells, and reduced levels of immunosuppressive cells (MDSCs). Further experiments revealed that THIO can directly affect the differentiation/proliferation of MDSCs and reshape the tumor immune microenvironment. The two parts of the study respectively address the key links of immune activation and relief of immunosuppression, providing a foundation for further combined interventions and offering new strategies for cancer immunotherapy.