Zewe M, Rybak S M, Dübel S, Coy J F, Welschof M, Newton D L, Little M
Recombinant Antibody Group, Diagnostics and Experimental Therapy Program, German Cancer Research Center, Heidelberg.
Immunotechnology. 1997 Jun;3(2):127-36. doi: 10.1016/s1380-2933(97)00070-5.
Immunotoxins based on plant and bacterial proteins are usually very immunogenic. Human ribonucleases could provide an alternative basis for the construction of less immunogenic reagents. Two members of the human RNase family, angiogenin and eosinophil-derived neurotoxin (EDN), have been fused to a single chain antibody against the transferrin receptor, which is known to be internalised by endocytosis. The fusion proteins proved to be very efficient inhibitors of protein synthesis using various cell lines. It is not yet known whether the side effects of angiogenin and EDN will compromise their potential use as immunotoxins.
The goal of this work was to construct a human immunotoxin with no harmful side effects. Bovine pancreatic ribonuclease has been shown to be as potent as ricin at abolishing protein synthesis on injection into oocytes. We therefore decided to clone its human analogue, which is fairly ubiquitous and per se non-toxic. An immunofusion of human pancreatic RNase with a single chain antibody against the transferrin receptor was tested for its ability to inhibit protein synthesis in three different human tumor cell lines.
DNA coding for the human pancreatic RNase was cloned partially from a human fetal brain cDNA library and then completed by PCR using a human placental cDNA library as a template. The RNase gene was then fused with a DNA coding for an single chain antibody against the transferrin receptor (CD71). After expressing the fusion protein in E. coli, the gene product was isolated from inclusion bodies and tested for cytotoxicity.
This fusion protein inhibited the protein synthesis of three human tumor cell lines derived from a melanoma, a renal carcinoma and a breast carcinoma, with IC50s of 8, 5 and 10 nM, respectively. These values were comparable with those using a similar fusion protein constructed with eosinophil derived neurotoxin (EDN) as the toxic moiety (IC50s of 8, 1.2 and 3 nM, respectively). The slightly lower activities of the human pancreatic RNase-scFv (pancRNase-scFv) with two of the cell lines suggests that fewer molecules are reaching the cytoplasmic compartment, since it was twice as active as EDN-scFv in inhibiting the protein synthesis of a rabbit reticulocyte lysate.
These results demonstrate that the human pancreatic RNase, which is expected to have a very low immunogenic potential in humans with no inherent toxicity, may be a potent cytotoxin for tumor cells after antibody targeting.
基于植物和细菌蛋白的免疫毒素通常具有很强的免疫原性。人核糖核酸酶可为构建免疫原性较低的试剂提供另一种基础。人核糖核酸酶家族的两个成员，血管生成素和嗜酸性粒细胞衍生的神经毒素（EDN），已与一种抗转铁蛋白受体的单链抗体融合，已知该受体可通过内吞作用内化。这些融合蛋白被证明是使用各种细胞系的非常有效的蛋白质合成抑制剂。血管生成素和EDN的副作用是否会影响它们作为免疫毒素的潜在用途尚不清楚。
这项工作的目标是构建一种无有害副作用的人免疫毒素。已证明牛胰核糖核酸酶在注射到卵母细胞中时在消除蛋白质合成方面与蓖麻毒素一样有效。因此，我们决定克隆其人类类似物，它相当普遍且本身无毒。测试了人胰核糖核酸酶与抗转铁蛋白受体的单链抗体的免疫融合物在三种不同人类肿瘤细胞系中抑制蛋白质合成的能力。
编码人胰核糖核酸酶的DNA部分从人胎儿脑cDNA文库中克隆，然后以人胎盘cDNA文库为模板通过PCR完成。然后将核糖核酸酶基因与编码抗转铁蛋白受体（CD71）的单链抗体的DNA融合。在大肠杆菌中表达融合蛋白后，从包涵体中分离出基因产物并测试其细胞毒性。
这种融合蛋白抑制了源自黑色素瘤、肾癌和乳腺癌的三种人类肿瘤细胞系的蛋白质合成，IC50分别为8、5和10 nM。这些值与使用以嗜酸性粒细胞衍生的神经毒素（EDN）作为毒性部分构建的类似融合蛋白的值相当（IC50分别为8、1.2和3 nM）。人胰核糖核酸酶 - 单链抗体（pancRNase - scFv）对其中两种细胞系的活性略低，这表明到达细胞质区室的分子较少，因为它在抑制兔网织红细胞裂解物的蛋白质合成方面比EDN - scFv活跃两倍。
这些结果表明，人胰核糖核酸酶在人类中预计具有非常低的免疫原性且无内在毒性，在抗体靶向作用后可能是一种有效的肿瘤细胞细胞毒素。
