Preparation, Activity In Vitro, Linker Based Optimization Of Recombinant Immunotoxin Gigantoxin-4-4D5 ScFv

Imminotoxin is a new study hotspot in targeting anticancer drugs, exhibiting good application prospect. Traditional immunotoxins killing cancer cells need to be internalized. They are not ideal in treating solid tumor and they have some limitations.In this study, we combined a sea anemone cytolysin Gigantoxin-4 which can destroy cell membrane with a single chain antibody 4D5 scFv targeting HER2/neu by a flexible linker (G4S)3. The SUMO tag was fused to the N terminus of Gigantoxin-4-4D5 scFv and then inserted into pET-28a(+). The soluble expression of Gigantoxin-4-4D5 scFv was achieved in Escherichia coli BL21(DE3), accounting for 9.8% of the supernatant proteins. After the purification by Ni column and CM column, the purity of Gigantoxin-4-4D5 scFv reached 96% and the yield was 14.3 mg/L.The results of MTT assay indicated that Gigantoxin-4-4D5 scFv exerted high cytotoxicity against HER2 overexpressing ovarian cancer cell SK-OV-3 and breast cancer cell SK-BR-3 and the IC50 were 0.171 nM and 0.158 nM, respectively. However, no obvious effect could be observed on HER2 negative ovarian cancer cell OVCAR-3 and human embryonic kidney cell 293. In addition, the HC50 of Gigantoxin-4-4D5 scFv was 18.9 nM. Immunofluorescence analysis showed that this novel immunotoxin could specifically bind to SK-OV-3 cells with no recognition to HEK-293 cells. Scanning electron microscope observations and extracellular lactate dehydrogenase activity indicated that it could induce necrosis in SK-OV-3 cells by disrupting the cell membrane, and exhibited similar effect on SK-BR-3 cells. Moreover, it could also mediate apoptosis of SK-OV-3 and SK-BR-3 cells, but had no impact on OVCAR-3 and HEK-293 cells. The results of activity in vitro demonstrated that Gigantoxin-4-4D5 scFv could specifically integrate with the HER2 overexpressing cancer cells and killed them by apoptosis and necrosis, but not normal cells and HER2 negative cancer cells. It is a great potential immunotoxin that can target HER2 overexpressing cancer cells.Linker in fusion proteins plays an important role in the structure and activity. Therefore, we optimized the linker of Gigantoxin-4-4D5 scFv from the length and flexicity to improve its anticancer ability. The linkers designed were as follows:(D(G4S)2, (2)(G4S)4, ③KPSTPPGGS, ④(PKPSTPPGGS)2, ⑤GGSPKPSTPPGGGS ⑥GGGS(PKPSTPP)2GGGS. After purification, the activities were measured by hemolytic test and MTT assay. The results indicated that the new immunotoxin constructed by the forth linker had the greatest potential because its hemolysis decreased markedly and the cytotoxicity on SK-OV-3 cells kept unchanged.