TIGIT/CD226与其配体PVR竞争结合的分子机制研究

Tumor immunotherapy has recently emerged as a hotspot in tumor therapeutic area. The negative costimulatory molecules represented by PD-1 and CTLA-4 are the most attractive targets for anti-tumor therapy in the process of tumor immune response, and the antibodies targeting PD-1 and CTLA-4 have exihibited dramatic antitumor efficacy in clinical research. TIGIT, as a novel negative costimulatory molecule involved in immune cells’ activation, could induce the tumor immune tolerance and escape through the binding with its ligand PVR. CD226, a costimulatory molecule, could bind with the same ligand PVR by competing with TIGIT, exihibited opposite function in regulating immune response. However, the competitive binding mechanism between TIGIT and CD226 is still unclear. Identification of the molecular mechanisms between TIGIT and CD226 with PVR is of pivotal importance both theoretically and practically. In this study, by using multi-disciplinary research tools, such as homology modelling, molecular dynamics simulation, mutagenesis, ELISA and SPR, we intend to elucidate the ligand binding mechanism of TIGIT/CD226 with PVR. Morever, we expect to provide new insights into a better understanding of the differential binding mode between TIGIT and CD226, which could lay a structural foundation for drug development.

肿瘤免疫疗法是肿瘤治疗领域的研究热点，其中，以PD-1和CTLA-4为代表的负性共刺激分子是肿瘤免疫应答过程中最受关注的靶点，针对PD-1及CTLA-4为靶标的抗体取得了良好的临床效果。TIGIT是免疫细胞活化过程中的新型负性共刺激分子，其与配体PVR结合介导肿瘤的免疫耐受和免疫逃逸；CD226可与TIGIT竞争结合PVR，与TIGIT发挥相反的功能，但TIGIT与CD226的竞争结合机制尚不清楚。因此，研究TIGIT/CD226与PVR结合的分子机制具有重要的理论意义和应用价值。本课题以解析的TIGIT-PVR的晶体结构为模板，通过同源建模、分子动力学模拟等手段，结合使用点突变、ELISA及SPR等实验方法，开展对TIGIT及CD226与其配体PVR相互作用的分子机制研究，鉴定其结合模式的差异，以期深入了解其竞争结合的分子机制，为针对TIGIT的药物设计提供结构基础。

以PD-1/PD-L1为代表的靶向免疫检查点的抑制剂在癌症免疫治疗中取得了较好的临床效果。作为重要的免疫检验点分子，TIGIT与配体PVR结合介导肿瘤的免疫耐受和免疫逃逸；阻断TIGIT/PVR取得了较好的临床治疗效果。鉴定TIGIT/PVR的相互作用模式，深入了解受体配体的互作机制，为靶向TIGIT的低分子量抑制剂的设计至关重要。以TIGIT/PVR复合物晶体结构为模板，通过分子动力学模拟发现，在相互作用过程中，PVR的CC'环、C'C''环和FG环发生了较大的分子内重排，形成了更多的氢键交联。以此为基础，我们鉴定了PVR与TIGIT相互作用的潜在残基，预测得到了高亲和力PVR突变体。使用流式结合MST验证突变体与TIGIT的结合力，共培养实验验证PVR突变体对Jurkat-hTIGIT细胞功能的影响，最终得到了四个与TIGIT高亲和力的PVR突变体PVRS72W、PVRS72R、PVRG131V和PVRS132Q，突变体可以对Jurkat-hTIGIT细胞产生更强的抑制作用。此外我们依据TIGIT与PVR的相互作用筛选得到了靶向PVR的小分子liothyronine，并靶向D构型TIGIT 蛋白筛选得到了D构象多肽抑制剂DTBP-3，两者均具有较好的抗肿瘤效果。在该项目的支持下，发表SCI论文5篇（标注基金号），申请发明专利1项，协助培养取得硕士学位证的硕士研究生2名，博士研究生3名，在读博士研究生1名。