Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
Immunobiochemistry Lab, Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Int J Biol Macromol. 2019 Aug 1;134:1120-1131. doi: 10.1016/j.ijbiomac.2019.05.137. Epub 2019 May 23.
Immunotoxins are a class of recombinant proteins which consist of an antibody and a part of a bacterial or herbal toxin. Immunotoxins containing Pseudomonas aeruginosa exotoxin A (PEA) have been found to be very applicable in clinical trials. Many obstacles such as solubility and absorbency reduce their usability in solid tumors. The current study aims to overcome the mentioned barriers by addition and removal of functional and non-functional domains with a structural approach. In the experimental section, we took advantage of molecular dynamics simulations to predict the functionality of candidate immunotoxins which target human HER2 receptors and confirmed our findings with in vitro experiments. We found out when no changes were made to domain II of PEA, addition of solubilizing domains to immunotoxins would not reduce their targeting and anti-tumor activity, while increasing the yield of expression and stability. On the other side, when we replaced domain II with eleven amino acids of furin cleavage site (FCS), the activity of the immunotoxin was mainly affected by the FCS neighboring domains and linkers. A combination of seven beneficial point mutations in domain III was also assessed and reconfirmed that the toxicity of the immunotoxin would be reduced dramatically. The obtained results indicate that the addition or removal of domains cannot depict the activity of immunotoxins and the matter should be assessed structurally in advance.
免疫毒素是一类由抗体和部分细菌或植物毒素组成的重组蛋白。已发现含有铜绿假单胞菌外毒素 A（PEA）的免疫毒素非常适用于临床试验。许多障碍，如溶解度和吸收率，降低了它们在实体瘤中的可用性。本研究旨在通过添加和去除具有结构方法的功能和非功能结构域来克服所述障碍。在实验部分，我们利用分子动力学模拟来预测靶向人 HER2 受体的候选免疫毒素的功能，并通过体外实验证实了我们的发现。我们发现当不改变 PEA 的结构域 II 时，向免疫毒素中添加增溶结构域不会降低其靶向性和抗肿瘤活性，同时提高表达产量和稳定性。另一方面，当我们用 11 个氨基酸的弗林切割位点（FCS）替换结构域 II 时，免疫毒素的活性主要受 FCS 邻近结构域和连接子的影响。还评估了结构域 III 中的七个有益点突变的组合，并再次证实免疫毒素的毒性会显著降低。所得结果表明，添加或去除结构域不能描述免疫毒素的活性，应提前进行结构评估。
