This paper presents a method to synthetically tune atomically precise megamolecule nanobody–enzyme conjugates for prodrug cancer therapy. Previous efforts to create heterobifunctional protein conjugates suffered from heterogeneity in domain stoichiometry, which in part led to the failure of antibody–enzyme conjugates in clinical trials. We used the megamolecule approach to synthesize anti-HER2 nanobody–cytosine deaminase conjugates with tunable numbers of nanobody and enzyme domains in a single, covalent molecule. Linking two nanobody domains to one enzyme domain improved avidity to a human cancer cell line by 4-fold but did not increase cytotoxicity significantly due to lowered enzyme activity. In contrast, a megamolecule composed of one nanobody and two enzyme domains resulted in an 8-fold improvement in the catalytic efficiency and increased the cytotoxic effect by over 5-fold in spheroid culture, indicating that the multimeric structure allowed for an increase in local drug activation. Our work demonstrates that the megamolecule strategy can be used to study structure–function relationships of protein conjugate therapeutics with synthetic control of protein domain stoichiometry.

中文翻译：

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纳米抗体-酶大分子结构域化学计量的合成调节

本文提出了一种合成调节原子级精确大分子纳米抗体-酶偶联物用于癌症前药治疗的方法。先前创建异源双功能蛋白质偶联物的努力在结构域化学计量方面存在异质性，这在一定程度上导致了抗体-酶偶联物在临床试验中的失败。我们使用大分子方法合成抗 HER2 纳米抗体 - 胞嘧啶脱氨酶偶联物，在单个共价分子中具有可调数量的纳米抗体和酶结构域。将两个纳米抗体结构域连接到一个酶结构域可将人类癌细胞系的亲和力提高 4 倍，但由于酶活性降低，细胞毒性并未显着增加。相比之下，由一个纳米抗体和两个酶域组成的大分子导致催化效率提高了 8 倍，并且在球体培养中将细胞毒性作用提高了 5 倍以上，表明多聚体结构允许增加局部药物活化。我们的工作表明，大分子策略可用于研究蛋白质缀合物疗法与蛋白质结构域化学计量的合成控制的结构 - 功能关系。