Tac (CD25) is expressed on multiple hematologic malignancies and is a target for cancer therapies. LMB-2 is an extremely active anti-Tac recombinant immunotoxin composed of an Fv that binds to Tac and a 38-kDa fragment of Pseudomonas exotoxin A (PE38). Although LMB-2 has shown high cytotoxicity toward Tac-expressing cancer cells in clinical trials, its efficacy was hampered by the formation of anti-drug antibodies against the immunogenic bacterial toxin and by dose-limiting off-target toxicity. To reduce toxin immunogenicity and nonspecific toxicity, we introduced six point mutations into domain III that were previously shown to reduce T-cell immunogenicity and deleted domain II from the toxin, leaving only the 11aa furin cleavage site, which is required for cytotoxic activity. Although this strategy has been successfully implemented for mesothelin and CD22-targeting immunotoxins, we found that removal of domain II significantly lowered the cytotoxic activity of anti-Tac immunotoxins. To restore cytotoxic activity in the absence of PE domain II, we implemented a combined rational design and screening approach to isolate highly active domain II–deleted toxin variants. The domain II–deleted variant with the highest activity contained an engineered disulfide-bridged furin cleavage site designed to mimic its native conformation within domain II. We found that this approach restored 5-fold of the cytotoxic activity and dramatically improved the MTD. Both of these improvements led to significantly increased antitumor efficacy in vivo. We conclude that the next-generation anti-Tac immunotoxin is an improved candidate for targeting Tac-expressing malignancies. Mol Cancer Ther; 17(7); 1486–93. ©2018 AACR.

中文翻译：

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通过假单胞菌外毒素 A 结构域 II 工程提高抗 Tac (CD25) 免疫毒素的体内功效


Tac (CD25) 在多种血液恶性肿瘤中表达，是癌症治疗的靶点。 LMB-2 是一种活性极高的抗 Tac 重组免疫毒素，由结合 Tac 的 Fv 和假单胞菌外毒素 A (PE38) 的 38 kDa 片段组成。尽管 LMB-2 在临床试验中对表达 Tac 的癌细胞表现出高细胞毒性，但其功效受到针对免疫原性细菌毒素的抗药物抗体的形成和剂量限制性脱靶毒性的阻碍。为了降低毒素的免疫原性和非特异性毒性，我们在结构域 III 中引入了 6 个点突变，这些点突变先前已被证明可以降低 T 细胞免疫原性，并从毒素中删除了结构域 II，仅留下细胞毒活性所需的 11aa 弗林蛋白酶切割位点。尽管这一策略已成功应用于间皮素和 CD22 靶向免疫毒素，但我们发现结构域 II 的去除显着降低了抗 Tac 免疫毒素的细胞毒活性。为了在缺乏 PE 结构域 II 的情况下恢复细胞毒活性，我们实施了一种组合的合理设计和筛选方法来分离高活性结构域 II 缺失的毒素变体。具有最高活性的结构域 II 缺失变体包含工程化的二硫键桥弗林蛋白酶切割位点，旨在模拟其在结构域 II 内的天然构象。我们发现这种方法使细胞毒活性恢复了 5 倍并显着改善了 MTD。这两项改进都显着提高了体内抗肿瘤功效。我们的结论是，下一代抗 Tac 免疫毒素是针对表达 Tac 的恶性肿瘤的改进候选药物。摩尔癌症治疗； 17(7)； 1486–93。 ©2018 AACR。