基于三维结构信息的抗FZD/EGFR多特异性抗体开发及其抗肿瘤机理研究

The cancer stem cell (CSC) model posits that tumors are hierarchically organized and, at the apex of the hierarchy, are cells that display stem cell properties. CSCs play significant roles in cancer cellular heterogeneity, tumor evolution, and therapeutic resistance. The use of rational combinations of agents to target both CSCs and bulk cancer populations holds great promise for improved therapeutics in cancer. Wnt/FZD signaling and EGFR signaling are key signaling pathways in CSCs and bulk cancer populations, respectively. Moreover, cross-talking of the two pathways has been elaborated. Previously, we used a phage display library to isolate a multispecific antibody, WTJ-47, that recognize FZD receptors and EGFR. In this study, a step-by-step development method based on the crystallographic analysis determined the three-dimensional structure of the WTJ-47/FZD8 and WTJ-47/EGFR was employed to yield multispecific antibodies with max binding affinity (nM) to both FZD and EGFR receptors. We will next evaluate the anti-tumor effect of multispecific antibodies as well as their combination with other related targeted drugs or chemotherapeutics in both in vitro and in patient-derived solid tissue human tumors xenograft models. Moreover, multiple level functional evaluations such as cell signaling pathways, cancer stem cell frequency and epithelial- to-mesenchymal (EMT) gene expression will be used to investigate the mechanism of the anti-cancer effect of the multispecific antibodies. Overall, our study will establish theoretical and experimental basis to achieve co-targeting CSC and bulk cancer populations using a single antibody.

肿瘤干细胞（CSC）在肿瘤异质性产生、肿瘤进化、肿瘤治疗耐受等过程中发挥重要作用。同时靶向CSC和肿瘤实体细胞的治疗策略是新一代肿瘤治疗的关键。Wnt/FZD信号通路及EGFR信号通路分别为CSC及肿瘤实体细胞中的关键信号通路，且存在多个层次上的交叉激活。前期，我们利用全人源噬菌体抗体库成功获得了能同时结合FZD家族受体和EGFR的多特异性抗体WTJ-47。在本课题中，课题组首先利用WTJ-47与FZD8和EGFR的复合物晶体三维结构信息，开发具有nM级结合力的抗FZD/EGFR多特异性抗体。并在体外实验及临床结肠癌标本来源的免疫缺陷小鼠移植模型中评估其抗肿瘤效果，并拟与其它相关靶向药物及化疗药物联合应用明确其治疗应用前景。最后，在信号通路、肿瘤干细胞比率、EMT相关基因表达等多个层次上对多特异性抗体的抗肿瘤机理进行研究，为开发以干预肿瘤干细胞为主导的新型靶向疗法联提供了理论及实验依据。

表皮生长因子阻断剂和放射治疗是常见的抗瘤手段，但临床应用过程中的耐药问题限制了治疗的手段的应用。在本研究中，课题组依托抗体工程平台探索基于三维结构的双特异性抗体的抗瘤作用及其机理。其中，EGFR/Notch双特异性抗体CT16显示出优势的抗瘤作用，并远强于课题组预研究中的WTJ-47抗体。因此课题组将CT16抗体作为进一步的研究目标。研究发现，双特异性抗体可以有效的阻断EGFR和Notch信号，抑制其下游信号通路。并在体内外模型中显示出显著的抗干细胞作用。进一步的研究显示，双特异性抗体可以预防非小细胞肺癌模型和患者来源的异种种植模型中的肿瘤组织对EGFR阻断剂和放射治疗的耐药。与EGFR抑制剂相比，双特异性抗体具有优秀的放射增敏作用。双特异性抗体与单独应用放射治疗及放射治疗抗体药物联合用药相比均显示出更为优秀的抗瘤作用。在机理方面，双特异性抗体可以抑制间质标志物干细胞亚群的比率，降低DNA修复，降低干细胞比率。这些研究结果显示EGFR和Notch双信号联合阻断可以增强放射治疗的临床有效率，提示在非小细胞肺癌中双特异性抗体和放射治疗可能是一种潜在的治疗方案。