肿瘤相关抗原ROR1的结构及其与功能性抗体和配体的作用机制

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed in a variety of human cancers, but not in most normal adult tissues. It plays important roles in promoting tumor cell growth, cancer progression and metastasis. Anti-ROR1 monoclonal antibodies and ROR1 specific inhibitors are capable of inducing the apoptosis of tumor cells. ROR1 serves as a potential new target for cancer therapy due to its biological and clinical significance. However, the 3D structures of the extracellular domains and the functional antigenic epitope of ROR1 are not available and their functions are not clear, limiting the development of diagnostic and therapeutic antibodies. This proposal aims to utilize the nuclear magnetic resonance (NMR) technique to determine the solution structures of the extracellular Frizzled domain and Kringle domain of ROR1. The interactions between each domain of ROR1 and the functional antibodies R11 and R12 which can induce the apoptosis of tumor cells will be further studied. The functional domain and the antigenic epitope of ROR1 will be identified. Moreover, this study will verify the in vitro interaction between ROR1 and its ligand Wnt5a, and show the active binding site on ROR1 which will serve as the potential target for ROR1 inhibitors. Taken together, these structural data will provide important guidance for designing specific antibodies against the functional antigenic epitope and small-molecule inhibitors against the Wnt5a active binding site.

受体酪氨酸激酶样孤儿受体ROR1异常高表达于多种肿瘤细胞，并不表达于绝大多数正常的成人细胞中。其在促进肿瘤细胞生长、迁移及侵袭的过程中发挥着重要作用，靶向ROR1的特异性抗体和抑制剂能够诱导肿瘤细胞凋亡，是近几年癌症治疗研究的热点靶标分子。但是，目前缺乏ROR1活性区域功能性抗原表位的三维空间结构和功能信息，很大程度上限制了功能性抗体和抑制剂的研发。本项目拟应用核磁共振NMR技术解析ROR1胞外Frizzled和Kringle结构域的三维结构，检测ROR1各结构域与诱导肿瘤细胞凋亡的功能性抗体R11和R12的相互作用，揭示ROR1功能性抗原表位的精确结构。同时探索ROR1特异性识别配体Wnt5a的分子基础及结合位点，发现潜在的小分子抑制剂的作用靶点。这些结构信息将为进一步研发靶向功能性抗原表位的抗体和针对配体Wnt5a识别位点的小分子药物设计提供重要的理论依据。

受体酪氨酸激酶样孤儿受体ROR1异常高表达于多种肿瘤细胞，并不表达于绝大多数正常的成人细胞。其严格的肿瘤组织分布，且在促进肿瘤细胞生长、迁移及侵袭的过程中发挥着重要作用，作为肿瘤治疗的潜在热点靶标已引起日益关注。抑制ROR1的功能可以阻断肿瘤细胞存活和增殖的信号通路，激活细胞凋亡信号通路，诱导肿瘤细胞凋亡，是近几年癌症治疗研究的热点靶标分子。目前缺乏ROR1活性区域功能性抗原表位的三维空间结构和功能信息，很大程度上限制了功能性抗体和抑制剂的研发。本项目充分摸索蛋白质正确表达的三大因素：表达载体、宿主菌株、表达诱导条件，成功表达二硫键正确折叠的ROR1胞外Frizzled和Kringle结构域；应用核磁共振NMR技术解析Kringle结构域的三维结构,结合同源建模方法获得Frizzled结构域结构，通过表面等离子共振SPR技术未检测到Kringle结构域与潜在配体Wnt5a间的直接相互作用。通过ELISA确认ROR1 Kringle结构域与诱导肿瘤细胞凋亡的功能性抗体R11间直接相互作用，揭示ROR1功能性抗原为胞外Kringle结构域。利用核磁共振滴定方法确定相互作用界面信息，Kringle-R11抗体复合物分子量较大，大部分的Kringle结构域HSQC谱锋消失，获得部分相互作用界面信息。ROR1功能性抗原结构信息为进一步研发靶向功能性抗原表位的抗体和小分子药物设计提供重要的理论依据。本项目部分研究成果已经发表在国际杂志Scientific reports、Biomolecular NMR Assignments上，一篇文章正在撰写过程中。