Recombinant immunotoxins (RIT) are chimeric proteins containing an Fv that binds to tumor cells, fused to a fragment of Pseudomonas exotoxin (PE) that kills the cell. Their efficacy is limited by their short half-life in the circulation. Chemical modification with polyethylene glycol (PEG) is a well-established method to extend the half-lives of biologics. Our goal was to engineer RITs with an increase in half-life and high cytotoxic activity. We introduced single cysteines at different locations in five anti-mesothelin RITs and employed site-specific PEGylation to conjugate them to 20-kDa PEG. Because our previous PEGylation method using β-mercaptoethanol reduction gave poor yields of PEG-modified protein, we employed a new method using tris(2-carboxyethyl)phosphine to reduce the protein and could PEGylate RITs at approximately 90% efficiency. The new proteins retained 19% to 65% of cytotoxic activity. Although all proteins are modified with the same PEG, the radius of hydration varies from 5.2 to 7.1, showing PEG location has a large effect on protein shape. The RIT with the smallest radius of hydration has the highest cytotoxic activity. The PEGylated RITs have a 10- to 30-fold increase in half-life that is related to the increase in hydrodynamic size. Biodistribution experiments indicate that the long half-life is due to delayed uptake by the kidney. Antitumor experiments show that several PEG-RITs are much more active than unmodified RIT, and the PEG location greatly affects antitumor activity. We conclude that PEGylation is a useful approach to improve the half-life and antitumor activity of RITs.

中文翻译：

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抗间皮素重组免疫毒素的位点特异性聚乙二醇化可延长半衰期和抗肿瘤活性


重组免疫毒素 (RIT) 是一种嵌合蛋白，含有与肿瘤细胞结合的 Fv，并与能够杀死细胞的假单胞菌外毒素 (PE) 片段融合。它们的功效因其在循环中的半衰期短而受到限制。聚乙二醇 (PEG) 化学修饰是延长生物制剂半衰期的成熟方法。我们的目标是设计具有更长半衰期和高细胞毒活性的 RIT。我们在五个抗间皮素 RIT 的不同位置引入了单个半胱氨酸，并采用位点特异性聚乙二醇化将它们与 20-kDa PEG 缀合。由于我们之前使用 β-巯基乙醇还原的 PEG 化方法得到的 PEG 修饰蛋白收率较差，因此我们采用了一种使用三（2-羧乙基）膦还原蛋白的新方法，并且可以以约 90% 的效率对 RIT 进行 PEG 化。新蛋白质保留了 19% 至 65% 的细胞毒活性。尽管所有蛋白质都用相同的 PEG 修饰，但水合半径从 5.2 到 7.1 不等，表明 PEG 位置对蛋白质形状有很大影响。水合半径最小的 RIT 具有最高的细胞毒活性。聚乙二醇化 RIT 的半衰期延长了 10 至 30 倍，这与流体动力学尺寸的增加有关。生物分布实验表明，半衰期长是由于肾脏延迟吸收所致。抗肿瘤实验表明，几种PEG-RIT比未修饰的RIT活性高得多，并且PEG位置极大地影响抗肿瘤活性。我们的结论是，聚乙二醇化是改善 RIT 半衰期和抗肿瘤活性的有效方法。