Department of Urology, University Hospital Freiburg, Breisacher Strasse 117, 79106 Freiburg, Germany.
Anticancer Res. 2010 Sep;30(9):3373-9.
In this study, different variants of the anti-PSMA single-chain antibody fragment (scFv) D7 were cloned, varying in linker type, position of hexahistidine tags and VH-VL orientation. From these scFv, Pseudomonas exotoxin A-based immunotoxins were constructed and their biological activities against prostate cancer cells were compared.
Binding of the constructs to PSMA-expressing prostate cancer cells was determined by flow cytometry. ADP-ribosyltransferase activity was analysed and cytotoxicity was measured with WST-1 assays.
Different linker types did not influence the characteristics of the scFv or immunotoxins. The addition of an N-terminal hexahistidine-tag, however, resulted in decreased expression, binding and cytotoxicity. scFv in VH-VL orientation showed the highest expression and binding, whereas immunotoxins in VL-VH orientation exhibited the best binding and cytotoxicity.
The present study showed how structure influences the characteristics of scFv and immunotoxins. It is therefore suggested that for each individual construct the optimal structure has to be determined separately.
在这项研究中，克隆了不同变体的抗 PSMA 单链抗体片段 (scFv) D7，其连接类型、六组氨酸标签的位置和 VH-VL 方向不同。从这些 scFv 中构建了基于假单胞菌外毒素 A 的免疫毒素，并比较了它们对前列腺癌细胞的生物学活性。
通过流式细胞术测定构建物与表达 PSMA 的前列腺癌细胞的结合。分析 ADP-核糖基转移酶活性，并用 WST-1 测定法测量细胞毒性。
不同的连接类型不会影响 scFv 或免疫毒素的特性。然而，添加 N 端六组氨酸标签会导致表达、结合和细胞毒性降低。以 VH-VL 方向排列的 scFv 表现出最高的表达和结合，而以 VL-VH 方向排列的免疫毒素则表现出最佳的结合和细胞毒性。
本研究表明结构如何影响 scFv 和免疫毒素的特性。因此，建议对于每个单独的构建体，都必须单独确定最佳结构。
