Batra J K, Fitzgerald D J, Chaudhary V K, Pastan I
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Mol Cell Biol. 1991 Apr;11(4):2200-5. doi: 10.1128/mcb.11.4.2200-2205.1991.
Two single-chain immunotoxins directed at the human transferrin receptor have been constructed by using polymerase chain reaction-based methods. Anti-TFR(Fv)-PE40 is encoded by a gene fusion between the DNA sequence encoding the antigen-binding portion (Fv) of a monoclonal antibody directed at the human transferrin receptor and that encoding a 40,000-molecular-weight fragment of Pseudomonas exotoxin (PE40). The other fusion protein, DT388-anti-TFR(Fv), is encoded by a gene fusion between the DNA encoding a truncated form of diphtheria toxin and that encoding the antigen-binding portion of antibody to human transferrin receptor. These gene fusions were expressed in Escherichia coli, and fusion proteins were purified by conventional chromatography techniques to near homogeneity. In anti-TFR(Fv)-PE40, the antigen-binding portion is placed at the amino terminus of the toxin, while in DT388-anti-TFR(Fv), it is at the carboxyl end of the toxin. Both these single-chain immunotoxins kill cells bearing the human transferrin receptors. However, anti-TFR(Fv)-PE40 was usually more active than DT388-anti-TFR(Fv), and in some cases it was several-hundred-fold more active. Anti-TFR(Fv)-PE40 was also more active on cell lines than a conjugate made by chemically coupling the native antibody to PE40, and in some cases it was more than 100-fold more active.
利用基于聚合酶链反应的方法构建了两种针对人转铁蛋白受体的单链免疫毒素。抗TFR（Fv）-PE40由编码针对人转铁蛋白受体的单克隆抗体的抗原结合部分（Fv）的DNA序列与编码绿脓杆菌外毒素40000分子量片段（PE40）的DNA序列之间的基因融合体编码。另一种融合蛋白DT388-抗TFR（Fv）由编码截短形式白喉毒素的DNA与编码抗人转铁蛋白受体抗体的抗原结合部分的DNA之间的基因融合体编码。这些基因融合体在大肠杆菌中表达，融合蛋白通过常规色谱技术纯化至接近均一。在抗TFR（Fv）-PE40中，抗原结合部分位于毒素的氨基末端，而在DT388-抗TFR（Fv）中，它位于毒素的羧基末端。这两种单链免疫毒素均能杀死表达人转铁蛋白受体的细胞。然而，抗TFR（Fv）-PE40通常比DT388-抗TFR（Fv）更具活性，在某些情况下其活性要高出数百倍。抗TFR（Fv）-PE对细胞系的活性也高于通过将天然抗体与PE40化学偶联制成的偶联物，在某些情况下其活性高出100倍以上。
