Puri R K, Debinski W, Obiri N, Kreitman R, Pastan I
Laboratory of Molecular Tumor Biology, Food and Drug Administration, National Institutes of Health, Bethesda, Maryland 20892.
Cell Immunol. 1994 Apr 1;154(1):369-79. doi: 10.1006/cimm.1994.1084.
We have previously demonstrated that functional high-affinity interleukin-4 receptors (IL-4R) are expressed on human renal cell carcinoma (RCC) cells (N. I. Obiri et al., J. Clin. Invest. 91, 88, 1993). In the present study, we examined the cytotoxic effect (determined by inhibition of protein synthesis) of a chimeric protein composed of human IL-4 and Pseudomonas exotoxin (PE) on human RCC tumor samples obtained from patients undergoing nephrectomy. The chimeric gene encoding hIL4-PE4E was constructed by fusing a cDNA clone for human IL-4 to the 5' end of a mutated cDNA encoding a full-length PE molecule. This gene was expressed in Escherichia coli, and large quantities of this recombinant protein were isolated to more than 95% purity. This chimeric protein, hIL4-PE4E, was highly cytotoxic to all six RCC cell lines examined. The concentration of hIL4-PE4E at which 50% inhibition of protein synthesis was obtained ranged from < 1 ng/ml (12 pM) to 10 ng/ml (120 pM) in five of the six isolates of RCC and 40-70 ng/ml in one other. A mutant chimeric protein which can bind to IL-4R but lacks the ADP ribosylation activity of PE was not cytotoxic to the RCC cells. The cytotoxic effect of hIL4-PE4E was IL-4R mediated because a fourfold molar excess of IL-4 abrogated the cytotoxic effect of hIL4-PE4E. A neutralizing monoclonal antibody to IL-4 also abrogated the cytotoxic effect of hIL4-PE4E. hIL4-PE4E showed very little cytotoxic activity to a normal human umbilical vein endothelial cell line (ID50 = 1000 ng/ml) and a human fibroblast cell line (ID50 approximately 400 ng/ml). Nonactivated human peripheral blood lymphocytes (PBL) were also insensitive to hIL4-PE4E (ID50, approximately 500 ng/ml), whereas phytohemagglutinin-activated PBL were highly susceptible to the cytotoxic effect of hIL4-PE4E (ID50, approximately 4 ng/ml). These data indicate that hIL4-PE4E may be a useful agent for the treatment of human RCC without affecting normal and resting immune cells.
我们之前已经证明，功能性高亲和力白细胞介素-4受体（IL-4R）在人肾细胞癌（RCC）细胞上表达（N.I.奥比里等人，《临床研究杂志》91卷，88页，1993年）。在本研究中，我们检测了一种由人IL-4和绿脓杆菌外毒素（PE）组成的嵌合蛋白对从接受肾切除术患者获取的人RCC肿瘤样本的细胞毒性作用（通过抑制蛋白质合成来确定）。编码hIL4-PE4E的嵌合基因是通过将人IL-4的cDNA克隆与编码全长PE分子的突变cDNA的5'端融合构建而成。该基因在大肠杆菌中表达，并分离出大量纯度超过95%的这种重组蛋白。这种嵌合蛋白hIL4-PE4E对所检测的所有六种RCC细胞系都具有高度细胞毒性。在六种RCC分离株中的五种中，获得50%蛋白质合成抑制时hIL4-PE4E的浓度范围为<1 ng/ml（12 pM）至10 ng/ml（120 pM），在另一种中为40 - 70 ng/ml。一种能与IL-4R结合但缺乏PE的ADP核糖基化活性的突变嵌合蛋白对RCC细胞无细胞毒性。hIL4-PE4E的细胞毒性作用是由IL-4R介导的，因为四倍摩尔过量的IL-4消除了hIL4-PE4E的细胞毒性作用。一种针对IL-4的中和单克隆抗体也消除了hIL4-PE4E的细胞毒性作用。hIL4-PE4E对正常人脐静脉内皮细胞系（半数抑制浓度=1000 ng/ml）和人成纤维细胞系（半数抑制浓度约为400 ng/ml）显示出非常低的细胞毒性活性。未活化的人外周血淋巴细胞（PBL）对hIL4-PE4E也不敏感（半数抑制浓度，约为500 ng/ml），而植物血凝素活化的PBL对hIL4-PE4E的细胞毒性作用高度敏感（半数抑制浓度，约为4 ng/ml）。这些数据表明，hIL4-PE4E可能是一种治疗人RCC的有用药物，而不影响正常和静息免疫细胞。
