Immunotoxins are proteins containing a cell-targeting element linked to a toxin that are under investigation for next-generation cancer treatment. However, these agents are difficult to synthesize, chemically heterogeneous, expensive, and show toxicity toward healthy cells. In this work, we describe the synthesis and characterization of a new type of immunotoxin that showed exquisite selectivity toward targeted cells. In our construct, targeting molecules were covalently attached or genetically fused to oligomeric pore-forming toxins. The activity of the immunotoxin was then caged by fusing a soluble protein to the transmembrane domain and activated via cleavage with furin, which is a protease that is overexpressed in many cancer cells. During the several coupling steps, directed evolution allowed the efficient synthesis of the molecules in E. coli cells, as well as selection for further specificity toward targeted cells. The final construct showed no off-target activity, while acquiring an additional degree of specificity toward the targeted cells upon activation. The pore-forming toxins described here do not require internalization to operate, while the many protomeric subunits can be individually modified to refine target specificity.

中文翻译：

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通过定向进化定制的具有笼状细胞毒性的模块化孔形成免疫毒素。

免疫毒素是含有与毒素连接的细胞靶向元素的蛋白质，目前正在研究中，用于下一代癌症治疗。然而，这些试剂难以合成，化学上异质，昂贵并且对健康细胞显示毒性。在这项工作中，我们描述了新型的免疫毒素的合成和表征，这种免疫毒素对目标细胞表现出出色的选择性。在我们的构建体中，靶向分子与寡聚成孔毒素共价连接或遗传融合。然后，通过将可溶性蛋白融合到跨膜结构域中，从而保持免疫毒素的活性，并通过用弗林蛋白酶裂解来激活，弗林蛋白酶是在许多癌细胞中过表达的一种蛋白酶。在几个偶联步骤中，定向进化使得E中的分子得以有效合成。大肠杆菌细胞，以及针对靶细胞的进一步特异性的选择。最终的构建体没有显示出脱靶活性，同时在活化后获得了对靶细胞的额外特异性。本文所述的成孔毒素不需要内部化即可操作，而许多原蛋白亚基可以单独修饰以改善靶标特异性。