Preparation And Antitumor Activity Of Recombinant Immunotoxin RTA-4D5-KDEL

With the maturation of antibody technology,immunotoxins have become a research hotspot for antitumor drugs.The immunotoxin antibody portion enables selective targeting of immunotoxins and can avoid damage to normal cells.The ricin A chain(RTA)has N-glycosidase activity and catalyzes the 28S rRNA to remove one adenine,thereby inhibiting protein synthesis and resulting in cell death.RTA is extremely toxic and an RTA molecule can inactivate thousands of ribosomes within 1 min.In this study,RTA was fused with single chain antibody 4D5 scFv targeting cell surface HER2 receptor and endoplasmic reticulum targeting peptide KDEL respectively.The fusion tag SUMO is fused at the N-terminus of the recombinant immunotoxin to facilitate soluble expression of the immunotoxin in the Escherichia coli system.The recombinant immunotoxin RTA-4D5 and RTA-KDEL was successfully constructed.KDEL was fused to the C-terminus of RTA-4D5 to construct targeted and localized immunotoxin RTA-4D5-KDEL.The results of CCK-8 assay showed that RTA-4D5 had a strong inhibitory effect on SK-OV-3 highly expressing HER2 with an IC50 of 0.7 ?g/mL.However,the inhibition of normal cells HEK 293 and H460 was reduced relative to RTA.The results indicated that 4D5 scFv targeted SK-OV-3 cells that express high levels of HER2.The IC50 of RTA-4D5-KDEL against SK-OV-3 was 25 ng/mL,a 28-fold and 440-fold increase relative to RTA-4D5 and RTA respectively.However,there was little effect on H460 and HEK 293.Confocal laser scanning experiments showed that RTA-4D5 can accumulate on the surface of SK-OV-3 cells efficiently after 1 h of dosing,while almost no fluorescence was observed on the surface of HEK 293 cells.After 24 h of dosing,most of RTA-4D5-KDEL was located on the endoplasmic reticulum.Flow cytometry experiments demonstrated that RTA-4D5-KDEL caused cell death through apoptosis and necrosis,and as the concentration of immunotoxins increased,the cell's apoptosis was stronger.In order to further reduce the side effects of recombinant immunotoxins on normal cells,RTA-KDEL was split into two parts by protein structure analysis and connected to the N-terminus and C-terminus of the light-controlled splicing protein InN-LOV-InC to initially construct a light-controlled immunotoxin.Through in vitro light experiments,four light-controlled splicing chimeric proteins with shearing effects were found.The recombinant immunotoxin RTA-4D5-KDEL,which has a strong inhibitory effect on SK-OV-3 highly expressing HER2,and almost no effect on normal cells HEK 293,was obtained.