Brinkmann U, Pai L H, FitzGerald D J, Willingham M, Pastan I
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8616-20. doi: 10.1073/pnas.88.19.8616.
The genes encoding the heavy- and light-chain Fv regions of the monoclonal murine antibody B3, which recognizes a carbohydrate antigen on the surface of many human carcinomas, were cloned by PCR techniques and used to generate single-chain immunotoxins containing Pseudomonas exotoxin (PE). The light and heavy chains were connected by a flexible linker to form a single-chain antigen-binding protein, B3(Fv), which was in turn fused to truncated forms of PE lacking the cell-binding domain. The single-chain Fv and two different B3(Fv) immunotoxins, B3(Fv)-PE40 and B3(Fv)-PE38KDEL, were expressed in Escherichia coli and the single-chain immunotoxins were purified to near homogeneity. Both recombinant immunotoxins were shown to be cytotoxic specifically to carcinoma cell lines that express the B3 antigen on their surface; B3(Fv)-PE38KDEL was significantly more active. Furthermore, intravenous administration of B3(Fv)-PE38KDEL caused complete regression of human epidermoid carcinomas growing subcutaneously in immunodeficient mice.
单克隆鼠抗体B3可识别多种人类癌表面的碳水化合物抗原，编码其重链和轻链Fv区的基因通过聚合酶链反应（PCR）技术克隆出来，并用于生成含有绿脓杆菌外毒素（PE）的单链免疫毒素。轻链和重链通过一个柔性接头连接，形成单链抗原结合蛋白B3(Fv)，该蛋白又与缺失细胞结合域的截短形式的PE融合。单链Fv及两种不同的B3(Fv)免疫毒素B3(Fv)-PE40和B3(Fv)-PE38KDEL在大肠杆菌中表达，且单链免疫毒素被纯化至近乎同质。两种重组免疫毒素均显示对表面表达B3抗原的癌细胞系具有特异性细胞毒性；B3(Fv)-PE38KDEL的活性明显更高。此外，静脉注射B3(Fv)-PE38KDEL可使免疫缺陷小鼠皮下生长的人表皮样癌完全消退。
