绿原酸调节LAG3逆转肿瘤免疫耐受的分子机制研究

Lymphocyte activation gene-3 (LAG3) is identified as an important coinhibitory receptor of T cells, as same as programmed death protein-1 (PD1) and cytotoxic T lymphocyte associated antigen 4 (CTLA-4). LAG3 binds toward its ligand MHC class II to negatively regulate T cell proliferation and activation. In particular, LAG3 is regulated by extracellular cleavage to soluble LAG3 (sLAG3), the later cloud block the inhibitory effect of LAG3 on T cells surface and activate antigen presenting cells (APCs) to indirectly further activate T cells. .In our previous phaseⅠclinical trials, we found that cancer patients with a favorable prognosis after treatment with chlorogenic acid had higher serum sLAG3 levels, which is consistent with the report by Triebel, et al. In mouse tumor models, we found chlorogenic acid inhibited the tumor growth by polarization of macrophage to M1 phenotype and enhancement of LAG3-negative tumor-infiltrating lymphocytes. These findings suggested that chlorogenic acid exhibited anti-tumor immunotherapy by regulating LAG3. To date, blocking LAG-3 using monoclonal antibodies and soluble recombinant dimeric LAG-3 protein (LAG-3-Ig) has been tested in anti-tumor phaseⅠ/Ⅱ clinical trials, but no one small molecule drug targeted LAG3 has been reported. And, the regulatory mechanism of chlorogenic acid on LAG3 in antitumor immunotherapy has been still unclear. .Therefore, the aim of this study is focused on the interaction of LAG3 and MHCII, trafficking of LAG3 to surface and production of sLAG3 to elucidate the molecular mechanism of chlorogenic acid on reversion of tumor immune evasion by regulating LAG3. The study will facilitate the clinical application of chlorogenic acid on cancer therapy and provide a solid foundation and new approach for the development of small molecule candidate of LAG3-targeted drug.

淋巴细胞活化基因3(LAG3)与PD1、CTLA4并列作为重要的T细胞共抑制性受体。LAG3与MHCⅡ结合负性调节T细胞增殖与活化。此外LAG3被剪切为可溶性的LAG3（sLAG3）来抵御LAG3对T细胞的抑制作用，或活化APC激活T细胞。前期临床研究发现绿原酸注射液的疗效与肿瘤患者血清sLAG3升高密切相关。小鼠肿瘤模型中，绿原酸增强M1型巨噬细胞对肿瘤的杀伤作用，并增加LAG3阴性肿瘤浸润淋巴细胞。这提示，绿原酸可能调节LAG3发挥抗肿瘤免疫作用。目前，国际上靶向LAG3的药物唯有LAG3单抗和LAG3融合蛋白，在Ⅰ/Ⅱ期临床研究，尚无其小分子药物报道，也未检索到绿原酸调节LAG3抗肿瘤免疫的作用。本项目从LAG3-MHCII相互作用、LAG3向细胞膜迁移和sLAG3的生成等方面阐述绿原酸调节LAG3逆转肿瘤免疫耐受分子机制。该项目为推进绿原酸临床应用及靶向LAG3药物的研发奠定基础。

淋巴细胞活化基因3(LAG3)与PD1、CTLA4并列作为重要的T细胞共抑制性受体，在肿瘤免疫耐受中发挥非常关键的作用。LAG3与 MHCII 结合负性调节T细胞增殖与活化。此外LAG3被剪切为可溶性的LAG3（sLAG3）来抵御LAG3对T细胞的抑制作用，或活化APC激活T细胞。本研究发现CHA与人源和鼠源的LAG3蛋白无亲合活性，也不直接抑制LAG3与MHCⅡ相互作用。但其有降低淋巴细胞膜LAG3表达的作用，一方面，这与其增加溶酶体活性，促进早期内体胞吞——溶酶体降解相关；另一方面与抑制PKCβ1激酶介导的LAG3表达相关。此外，绿原酸可通过降低负调控分子TIMP3表达和活化p-ERK信号通路，间接促进ADAM17的成熟和活化，行使其剪切LAG3的作用。在细胞和动物水平，绿原酸也表现出降低T细胞LAG3表达，促进T细胞增殖和活化，从而抑制肿瘤生长的作用。此外，依托本项目建立的筛选平台体系，我们已经首次获得与LAG3蛋白具有亲和活性，且抑制LAG3与MHCⅡ相互作用的小分子化合物。综上，该项目的实施可推进绿原酸临床应用，并为靶向LAG3药物的研发奠定重要的基础实验数据。