Puri R K, Leland P, Obiri N I, Husain S R, Mule J, Pastan I, Kreitman R J
Laboratory of Molecular Tumor Biology, Food and Drug Administration, National Institutes of Health, Bethesda, Maryland 20892, USA.
Cell Immunol. 1996 Jul 10;171(1):80-6. doi: 10.1006/cimm.1996.0176.
We have previously demonstrated that a chimeric protein composed of human IL-4 and Pseudomonas exotoxin, termed IL4-PE4E, is cytotoxic to primary cells derived from human renal cell carcinoma (RCC). To improve the cytotoxicity of IL4-toxins such as IL4-PE4E and IL4-PE38KDEL to IL-4 receptor (IL-4R) positive tumor cells, a circularly permuted chimeric toxin was prepared by fusing a truncated PE gene encoding PE38KDEL 3' to a circularly permuted IL-4 mutant gene encoding IL4 amino acids 38-129, the linker GGNGG, and IL4 amino acids 1-37. The resulting chimeric protein, termed IL4(38-37)-PE38KDEL, was tested on five RCC cell lines and its cytotoxicity was compared to that of the native IL4-toxins IL4-PE4E and IL4-PE38KDEL. IL4(38-37)-PE38KDEL was found to be 5 to 10 times more cytotoxic to all cell cultures tested compared to either native IL4-toxin. The cytotoxic activity of IL4(38-37)-PE38KDEL was competible by excess IL-4 and was confirmed by clonogenic assay. IL4(38-37)-PE38KDEL bound to IL-4R on RCC cells with 6- to 12-fold higher affinity than IL4-PE38KDEL or IL4-PE4E. RCC tumor cells were found to lack the common gamma chain (gamma c) of the IL-4R reported to be present on immune cells. The stable transfection of RCC cells with the gamma c chain gene did not significantly change their sensitivity to IL4(38-37)-PE38KDEL. Taken together, our results indicate that the CPIL4-toxin IL4(38-37)-PE38KDEL is highly cytotoxic to human RCC cells due to increased binding affinity to IL-4R while it is not cytotoxic or slightly cytotoxic to T and B cells, monocytic cell lines, and fresh resting or activated bone marrow-derived cells. The gamma c does not seem to increase the internalization rate and/or processing of IL4-toxins in RCC cells. CPIL4-toxin may be a useful agent for the treatment of human RCC.
我们之前已经证明，一种由人白细胞介素4（IL-4）和铜绿假单胞菌外毒素组成的嵌合蛋白，称为IL4-PE4E，对源自人肾细胞癌（RCC）的原代细胞具有细胞毒性。为了提高IL4毒素（如IL4-PE4E和IL4-PE38KDEL）对IL-4受体（IL-4R）阳性肿瘤细胞的细胞毒性，通过将编码PE38KDEL的截短PE基因融合到编码IL4氨基酸38-129、接头GGNGG和IL4氨基酸1-37的环状排列IL-4突变基因上，制备了一种环状排列的嵌合毒素。将所得的嵌合蛋白称为IL4(38-37)-PE38KDEL，在五种RCC细胞系上进行测试，并将其细胞毒性与天然IL4毒素IL4-PE4E和IL4-PE38KDEL进行比较。结果发现，与任何一种天然IL4毒素相比，IL4(38-37)-PE38KDEL对所有测试的细胞培养物的细胞毒性高5至10倍。IL4(38-37)-PE38KDEL的细胞毒性活性可被过量的IL-4竞争，并通过克隆形成试验得到证实。IL4(38-37)-PE38KDEL与RCC细胞上的IL-4R结合的亲和力比IL4-PE38KDEL或IL4-PE4E高6至12倍。发现RCC肿瘤细胞缺乏据报道存在于免疫细胞上的IL-4R的共同γ链（γc）。用γc链基因稳定转染RCC细胞并没有显著改变它们对IL4(38-37)-PE38KDEL的敏感性。综上所述，我们的结果表明，CPIL4毒素IL4(38-37)-PE38KDEL对人RCC细胞具有高度细胞毒性，这是由于其对IL-4R的结合亲和力增加，而对T细胞、B细胞、单核细胞系以及新鲜的静息或活化的骨髓来源细胞无细胞毒性或细胞毒性轻微。γc似乎并未增加RCC细胞中IL4毒素的内化率和/或加工过程。CPIL4毒素可能是治疗人RCC的一种有用药物。
