免疫毒素 (IT) 是重组嵌合蛋白，它将蛋白质毒素与靶向部分结合起来，以促进毒素选择性递送至癌细胞。在这里，我们提出了一种新策略，通过在内吞途径的还原条件下促进 IT 与靶受体解离来增强 IT 的胞质进入。我们设计了 monobodySS，这是一种基于人纤连蛋白 III 型结构域的单体，具有含二硫键 (SS) 的互补位，靶向 EGFR、EpCAM、Her2 和 FAP 等受体。 MonobodySS 表现出 SS 依赖性靶受体结合，在还原条件下结合显着减少。然后，我们创建了基于 monobodySS 的 IT，其携带假单胞菌外毒素 A (PE25) 的 25kDa 片段，称为 monobodySS-PE25。这些IT对表达靶受体的癌细胞表现出剂量依赖性细胞毒性，并且与抑制SS减少的对照相比，由于在较低剂量下具有更高的疗效，因此具有更宽的治疗窗。 ERSS/28-PE25 对 EGFR 的 KD 为 28nM，与缺乏 SS 键的 ER/21-PE25 相比，在同等剂量和更低剂量下表现出更优异的肿瘤杀伤效力。在体内，ERSS/28-PE25 在抑制 EGFR 过表达异种移植小鼠模型中的肿瘤生长方面优于 ER/21-PE25。这项研究提出了一种利用含有 SS 的互补位来开发实体瘤靶向 IT 的策略，以增强胞质递送和抗肿瘤功效。版权所有 © 2024。由 Elsevier B.V. 出版。

Immunotoxins (ITs) are recombinant chimeric proteins that combine a protein toxin with a targeting moiety to facilitate the selective delivery of the toxin to cancer cells. Here, we present a novel strategy to enhance the cytosolic access of ITs by promoting their dissociation from target receptors under the reducing conditions of the endocytic pathway. We engineered monobodySS, a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. MonobodySS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobodySS-based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobodySS-PE25. These ITs showed dose-dependent cytotoxicity against target receptor-expressing cancer cells and a wider therapeutic window due to higher efficacy at lower doses compared to controls with SS reduction inhibited. ERSS/28-PE25, with a KD of 28 nM for EGFR, demonstrated superior tumor-killing potency compared to ER/21-PE25, which lacks an SS bond, at equivalent and lower doses. In vivo, ERSS/28-PE25 outperformed ER/21-PE25 in suppressing tumor growth in EGFR-overexpressing xenograft mouse models. This study presents a strategy for developing solid tumor-targeting ITs using SS-containing paratopes to enhance cytosolic delivery and antitumor efficacy.Copyright © 2024. Published by Elsevier B.V.