Department of Anatomy, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, California 94143, United States.
Department of Biomedical Engineering, Northwestern University, Evanston, Illinois 60208, United States.
Bioconjug Chem. 2021 Jan 20;32(1):143-152. doi: 10.1021/acs.bioconjchem.0c00578. Epub 2020 Dec 10.
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.
本文提出了一种方法，用于综合调节原子精度的巨型分子纳米抗体-酶缀合物，以进行前药癌症治疗。以前在创建异双功能蛋白缀合物方面的努力存在结构域化学计量异质性的问题，这在一定程度上导致抗体-酶缀合物在临床试验中的失败。我们使用巨型分子方法合成了抗 HER2 纳米抗体-胞嘧啶脱氨酶缀合物，在单个共价分子中具有可调节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上，将对人癌细胞系的亲和力提高了 4 倍，但由于酶活性降低，细胞毒性没有显著增加。相比之下，由一个纳米抗体和两个酶结构域组成的巨型分子将催化效率提高了 8 倍，并在球体培养物中使细胞毒性增加了 5 倍以上，表明多聚体结构允许局部药物激活增加。我们的工作表明，巨型分子策略可用于研究具有合成控制蛋白结构域化学计量的蛋白缀合物治疗剂的结构-功能关系。
