Kreitman R J, Puri R K, Pastan I
Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Cancer Res. 1995 Aug 1;55(15):3357-63.
We reported previously that circularly permuted interleukin-4 (IL4), composed of amino acids 38-129 of IL4 connected by a linker peptide GGNGG to amino acids 1-37, is preferable to native IL4 for fusing to the amino terminus of truncated Pseudomonas exotoxin (PE) to make a recombinant toxin, because the new ligand-toxin junction results in improved IL4 receptor (IL4R)-binding (R. J. Kreitman et al., Proc. Natl. Acad. Sci. USA, 91: 6889-6893, 1994). We now report that the improved binding of circularly permuted IL4-toxin is associated with improved antitumor activity in tumor-bearing mice. For in vivo testing, we made an improved circularly permuted IL4-toxin, termed IL4(38-37)-PE38KDEL. It contains an N38D mutation at the amino terminus, allowing improved expression and large-scale production in Escherichia coli. It also contains the truncated toxin PE38KDEL, which is composed of amino acids 253-364 and 381-608 of PE, followed by KDEL. To evaluate antitumor activity, nude mice carrying s.c. tumors composed of IL4R-bearing human A431 epidermoid carcinoma cells were injected with recombinant toxins i.v. every other day for three doses. IL4(38-37)-PE38KDEL induced complete remissions in 80% of mice receiving 50 micrograms/kg x 3 and 100% of mice receiving 100 micrograms/kg x 3, while only 70% of mice receiving 200 micrograms/kg x 3 of the native IL4-toxin IL4-PE38KDEL obtained complete remission. Disease-free survival after obtaining complete remissions was higher in mice treated with IL4(38-37)-PE38KDEL 50 micrograms/Kg QOD x 3 than with IL4-PE38KDEL 200 micrograms/Kg QOD x 3 (P < 0.03). IL4(38-37)-PE38KDEL and IL4-PE38KDEL exhibited similar toxicity and pharmacokinetics in the mice, indicating that the improved antitumor activity of the circularly permuted IL4-toxin was due to its improved binding to the IL4R on the target cells.
我们之前报道过，环状排列的白细胞介素-4（IL4），由IL4的38 - 129位氨基酸通过连接肽GGNGG连接到1 - 37位氨基酸组成，与天然IL4相比，更适合融合到截短的铜绿假单胞菌外毒素（PE）的氨基末端以制备重组毒素，因为新的配体 - 毒素连接方式可改善IL4受体（IL4R）的结合（R. J. Kreitman等人，《美国国家科学院院刊》，91: 6889 - 6893, 1994）。我们现在报道，环状排列的IL4 - 毒素结合能力的提高与荷瘤小鼠抗肿瘤活性的增强相关。为了进行体内测试，我们制备了一种改良的环状排列的IL4 - 毒素，称为IL4(38 - 37)-PE38KDEL。它在氨基末端含有N38D突变，有利于在大肠杆菌中提高表达和大规模生产。它还包含截短的毒素PE38KDEL，由PE的253 - 364位氨基酸和381 - 608位氨基酸组成，后面跟着KDEL。为了评估抗肿瘤活性，给携带由表达IL4R的人A431表皮样癌细胞组成的皮下肿瘤的裸鼠静脉注射重组毒素，每隔一天注射一次，共注射三剂。IL4(38 - 37)-PE38KDEL使接受50微克/千克×3剂量的小鼠中有80%完全缓解，接受100微克/千克×3剂量的小鼠中有100%完全缓解，而接受200微克/千克×3剂量的天然IL4 - 毒素IL4 - PE38KDEL的小鼠中只有70%完全缓解。接受50微克/千克隔天一次×3剂量的IL4(38 - 37)-PE38KDEL治疗的小鼠在完全缓解后的无病生存期高于接受200微克/千克隔天一次×3剂量的IL4 - PE38KDEL治疗的小鼠（P < 0.03）。IL4(38 - 37)-PE38KDEL和IL4 - PE38KDEL在小鼠中表现出相似的毒性和药代动力学，表明环状排列 的IL4 - 毒素抗肿瘤活性的提高是由于其与靶细胞上IL4R的结合能力增强。
