In order to generate an antibody directed enzyme prodrug therapy, here we designed a chimeric protein by fusing the F8 antibody that recognizes the EDA of fibronectin (expressed on the tumor neovasculature) and an evolved variant of the ROS-generating enzyme D-amino acid oxidase (DAAO). The F8(scFv)-DAAO-Q144R recombinant protein is expressed by both CHO-S and E. coli cells. The F8(scFv)-DAAO-Q144R from E. coli cells is fully soluble, shows a high specific activity, is more thermostable in blood than the native DAAO, possesses a binding affinity for EDA well suited for efficient tumor accumulation, and localizes in tumor tissues. Notably, the F8(scFv)-DAAO-Q144R conjugate generates a stronger cytotoxicity to tumor cells than the native enzyme, especially when an inhibitor of heme oxygenase-1 (HO-1) is used, making it a promising candidate for a selective antitumor oxidative therapy controlled by the substrate addition, in the so called “activity on demand”, thus sparing normal tissue from damage.

为了产生抗体导向的酶前药疗法，我们在此设计了一种嵌合蛋白，通过融合识别纤连蛋白的 EDA（在肿瘤新血管系统上表达）的 F8 抗体和产生 ROS 的酶 D-氨基酸氧化酶的进化变体（大奥）。F8(scFv)-DAAO-Q144R 重组蛋白由 CHO-S 和大肠杆菌细胞表达。来自大肠杆菌的 F8(scFv)-DAAO-Q144R细胞完全可溶，显示出高比活性，在血液中比天然 DAAO 更热稳定，对 EDA 具有结合亲和力，非常适合有效的肿瘤积累，并定位于肿瘤组织中。值得注意的是，与天然酶相比，F8(scFv)-DAAO-Q144R 偶联物对肿瘤细胞产生更强的细胞毒性，尤其是在使用血红素加氧酶-1 (HO-1) 抑制剂时，使其成为选择性抗肿瘤的有希望的候选物由底物添加控制的氧化疗法，即所谓的“按需活性”，从而使正常组织免受损伤。