全人单克隆抗核抗体入胞/核机制及其抗肿瘤作用研究

The abundant antinuclear antibodies (ANA) is a characteristic index of immune imbalance, which is closely related to pathogenesis of tumors. However, the cellular penetration of ANA and the relevant regulation to tumor cell still remains unclear. We have prepared 5 kinds of fully human monoclonal ANA. Through in vivo and in vitro experiments, we found these ANA could penetrate into the living tumor cells, and suppress the tumor cell proliferation. Therefore, we speculate that ANA could exert an antitumor effect by the penetration into tumor cell directly, or by the immune regulation indirectly. In this study, we will use these ANA to do follow experiments: ① observe the cellular/nuclear penetration of ANA; ② analyze the characteristic sequence (nuclear localization signal, Fc receptor binding sequence and so on), and further, knockout the characteristic sequence and clarify its cellular/nuclear penetration mechanism; ③ to identify the target antigens of ANA, and confirm its function by the mutation and truncation; ④ to detect the regulatory effects of ANA on the immune cells and tumor cells. and the biological safety assess of ANA will be detected by in vivo experiments. We will evaluate the potential application of ANA by two aspects, including the direct penetration and indirect regulation of immune microenvironment. This study will provide reliable evidence for cellular/nuclear penetration mechanism, and provide molecular targets for tumor treatment.

抗核抗体（ANA）是免疫失衡的特征性指标，免疫失衡与肿瘤发生发展密切相关，但是ANA能否自主进入活细胞及其对肿瘤的调控作用仍无报道。我们已筛选并制备出5种全人单克隆ANA，通过与活细胞共孵育发现其能够进入活细胞内，且体内外实验均发现这些ANA可抑制多种肿瘤细胞增殖。因此我们推测ANA可能通过自主入胞/核直接发挥抗肿瘤作用，或通过调节免疫间接发挥抗肿瘤作用。本课题用上述ANA拟进行：① 实时监测ANA入胞/核活动；② 分析ANA核定位信号、Fc受体等特征性序列，通过序列敲除等方法明确ANA入胞/核机制；③ 免疫学方法鉴定ANA靶点蛋白，突变或截短靶蛋白验证其功能；④ 体内外验证ANA对免疫细胞和肿瘤细胞的调控作用，并评价ANA的生物安全性。本研究将从直接入胞/核、间接免疫调控两方面评价ANA在抗肿瘤方面的潜在应用，为明确ANA入胞/核及机制提供可靠依据，为肿瘤靶向精准治疗提供新的分子靶点。

抗核抗体（ANA）是免疫失衡的特征性指标，免疫失衡与自身免疫病和肿瘤发生发展密切相关，但是ANA能否自主进入活细胞及其对肿瘤的调控作用仍无报道。本项目使用已筛选并制备出的2种全人单克隆ANA，通过荧光标记并与活细胞共孵育发现其能够进入活细胞内，但对于不同的肿瘤细胞系，进入时间和浓度都有差异；抑制剂阻断实验结果表明，ANA通过小窝蛋白介导的胞吞作用进入细胞。体外实验表明ANA可通过抑制靶点蛋白核转运、降解靶点蛋白、调控其下游通路活性，抑制多种肿瘤细胞增殖与分化。阐明了以DDX5为靶点的全人源ANA对肿瘤抑制作用的机制。另外，ANA可以通过调控免疫细胞和体细胞的天然免疫分子参与自身免疫性疾病的致病过程，3B5抗体可以通过抑制IFN活化抑制通路相关蛋白表达，上调IFN系统活性，活化免疫细胞；通过对3B5抗体处理后小鼠模型的肝脏进行测序分析发现，ANA广泛影响了自身免疫性疾病相关基因的表达，这些调控作用没有明确的引起组织病理损伤，表明3B5抗体主要通过细胞内功能调控发挥作用，这一发现有助于我们深入了解ANA在自身免疫性疾病发生、发展过程中的作用机制。本研究从两个方面①ANA直接入胞调控靶点蛋白功能；②调控天然免疫参与自身免疫；阐明了ANA在抗肿瘤和自身免疫病诊疗方面的潜在应用价值，为明确ANA入胞/核及机制提供可靠依据，为肿瘤靶向精准治疗和自身免疫调控提供新的分子靶点。