Influence of structural variations on biological activity of anti-PSMA scFv and immunotoxins targeting prostate cancer.

BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.