Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrating drugs at the site-of-action, a method embodied by the seven current FDA approved antibody-drug conjugates (ADCs). However, a variety of pharmacokinetic challenges result in relatively narrow therapeutic windows for these agents, hampering the development of new drugs. Here, we use a series of Prostate-Specific Membrane Antigen (PSMA)-binding single-domain (Humabody®) ADC constructs to demonstrate that tissue penetration of protein-drug conjugates plays a major role in therapeutic efficacy. Counterintuitively, a construct with lower in vitro potency resulted in higher in vivo efficacy than other protein-drug conjugates. Biodistribution data, tumor histology images, spheroid experiments, in vivo single-cell measurements, and computational results demonstrate that a smaller size and slower internalization rate enabled higher tissue penetration and more cell killing. The results also illustrate the benefits of linking an albumin binding domain to the single-domain ADCs. A construct lacking an albumin binding domain was rapidly cleared leading to lower tumor uptake (%ID/g) and decreased in vivo efficacy. In conclusion, these results provide evidence that reaching the maximum number of cells with a lethal payload dose correlates more strongly with in vivo efficacy than total tumor uptake or in vitro potency alone for these protein-drug conjugates. Computational modeling and protein engineering can be used to custom design an optimal framework for controlling internalization, clearance, and tissue penetration to maximize cell killing.

中文翻译：

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单域抗体-药物缀合物的增加的肿瘤渗透性改善了前列腺癌模型的体内功效。

靶向化疗药物的目的是通过将药物集中在作用部位上来提高功效和降低毒性，这是目前由FDA批准的七种抗体-药物结合物（ADC）所体现的一种方法。但是，各种药代动力学挑战导致这些药物的治疗窗口相对狭窄，从而阻碍了新药的开发。在这里，我们使用一系列前列腺特异性膜抗原（PSMA）结合单域（Humabody®）ADC构建体来证明蛋白质-药物结合物的组织渗透在治疗功效中起主要作用。与直觉相反，与其他蛋白质-药物缀合物相比，具有较低体外效能的构建体具有更高的体内功效。生物分布数据，肿瘤组织学图像，球体实验，体内单细胞测量，计算结果表明，较小的尺寸和较慢的内部化速率可实现更高的组织穿透力和更多的细胞杀伤力。结果还说明了将白蛋白结合域链接到单域ADC的好处。缺乏白蛋白结合结构域的构建体被快速清除，导致更低的肿瘤吸收率（％ID / g）和体内功效降低。总之，这些结果提供了证据，即对于这些蛋白质-药物偶联物，与致命的总肿瘤吸收量或单独的体外效力相比，以致死有效载荷剂量达到最大细胞数量与体内功效的相关性更高。计算建模和蛋白质工程可用于定制设计控制内部化，清除和组织穿透的最佳框架，以最大程度地杀死细胞。