基于非典型性受体酪氨酸激酶ROR2跨膜信号转导结构机制的靶标确证

Receptor tyrosine kinases (RTKs) are a type of practically approved drug targets with high druggablity. ROR2, an atypical RTK, is essentially not expressed in adult tissues, but highly expressed in tumor tissues from an increasingly long list of cancers such as osteosarcoma, pancreatic ductal adenocarcinoma and cervical cancer, which suggests that ROR2 is a potentially high value drug target for developing anticancer therapeutics. Since ROR2 is a pseudokinase without intracellular kinase activity, the traditional strategy of screening small-molecule kinase inhibitors is no longer applied for ROR2 target validation. Wnt5a-ROR2 signal axis is critical for progression of tumors with high ROR2 expression, so the interaction between Wnt5a and ROR2 may be the Achilles‘s heel for anticancer drug development. In this project, we plan to elucidate structural mechanisms of ROR2 transmembrane signaling transduction and structure-function relationships of sFRPs and sizzled, a type of secreted protein called Wnt5a decoy receptors. The structural biology study will facilitate development of ROR2 mAbs and discovery of Wnt5a decoy proteins through directed evolution. At the cellular and animal levels, the resultant ROR2 mAbs and Wnt5a decoy proteins will be evaluated for their pharmacological functions of inhibiting proliferation, invasion and metastasis of cancer cells with high expression of ROR2. Also, at the structural and molecular levels, we are intended to investigate action mechanisms of these protein drug candidates and provide atomic details for interaction between them and targets. Currently, we have developed 22 anti-ROR2 mAbs and solved the crystal structure of sizzled. The crystal structure implicates that elimination of the NTR domain would be helpful for enhancing Wnt5a binding affinity to this family of decoy receptors. Our progress in this project has proved that our research plan is feasible.

非典型性受体酪氨酸激酶ROR2在正常成人组织中基本不表达，但在胰腺导管癌等癌症组织中高表达，是潜在的药物靶标。Wnt5a-ROR2信号轴对ROR2高表达的肿瘤进展十分重要，破坏Wnt5a-ROR2相互作用可以成为发现抗肿瘤药物的出发点。本项目将阐明ROR2的跨膜信号转导的结构机制以及wnt5a的捕获受体sFRPs或sizzled的结构功能关系，以此指导ROR2单克隆抗体的制备和wnt5a捕捉受体的分子定向进化，从而发现能抑制Wnt5a-ROR2信号轴的蛋白质类候选药物，并在细胞水平和动物水平上评价它们抑制ROR2高表达肿瘤细胞增殖、侵润、转移的药效，在结构和分子水平上阐明它们的作用机制。当前，我们获得了22株抗ROR2的单克隆抗体；解析了wnt5a的捕获受体sizzled的晶体结构，该结构提示去除NTR结构域将有助于获得Wnt5a高亲和力的捕获受体，这些进展说明项目具有可行性。

非典型性受体酪氨酸激酶ROR2在黑色素瘤、肾细胞癌等很多癌症组织中高表达，是潜在的药物靶标。在本项目的主要研究内容包括：（1）阐明ROR2的生物学功能与黑色素瘤等肿瘤进展之间的关系；（2）阐明ROR2配体wnt5a结合蛋白--分泌型 Frizzled相关蛋白（sFRPs）的结构和功能关系；（3）发展以针对ROR2胞外区的单克隆抗体，并以它们或sFRPs片段作为干预Wnt5a-ROR2功能的调节工具，在细胞、整体动物水平上评估它们抑制ROR2高表达肿瘤细胞的生长及转移的能力，籍以确证ROR2为抗肿瘤药物靶标。. 本项目取得的重要结果如下。其一，我们运用RNAi介导的功能失去性实验，证实了ROR2具有促进人黑色素瘤细胞A2058的EMT转变、伪足形成从而实现侵袭、转移的生物学功能。其二，我们解析了人源sFRP2的亮氨酸富集结构域（cysteine-rich domain，CRD结构域）以及sFRP家族成员非洲爪蟾sizzled的晶体结构---本家族第一个全长蛋白的结构；通过离体的双荧光素酶实验和基于结构的定点突变实验，我们发现sizzled的CRD表现出较全长蛋白更为有效的抑制Wnt3a信号的能力；而在活体实验中，人源sFRP2和其CRD结构域都可以抑制小鼠黑色素瘤细胞B16F10的肺转移。其三，我们用杆状病毒/哺乳动物细胞重组表达了了人源ROR2的胞外区，并以高纯度的重组蛋白免疫小鼠；通过传统的杂交瘤技术获得了22株ROR2单克隆抗体，并利用细胞免疫荧光实验证实了抗体可识别细胞表面的ROR2；在离体细胞实验中，我们发现单克隆抗体2E4、4C10和9A6可抑制ROR2高表达的人肾癌细胞786-0增殖。更为重要的是，我们发现8H6、9E1可抑制人黑色素瘤细胞A2058的伪足形成、侵袭和小鼠肺转移。此外，我们克隆并重组表达了抗体4C10、8H6等抗体的Fab区，获得了它们与ROR2胞外区的复合物的晶体，但尚需优化结晶条件以期获得可用于结构解析的复合物晶体。. 上述研究意义在于：确证了ROR2可以作为抗黑素瘤转移的药物靶标，这将有助于发现治疗ROR2高表达肿瘤的精准药物；也有助于利用单抗8H6等药理学工具进一步探讨wnt5a-ROR2信号轴调控肿瘤发生的分子机制。

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