Preparation And Analysis In Vitro Of Recombinant Immunotoxin DAB389-4D5 ScFv And Its Derivatives

Third generation recombinant immunotoxin is the hotspot in targeted antitumor therapy.Compared with radiotherapy and chemotherapy,immunotoxin exhibits specific targeted capacity and strong cytotoxicity,so it has massive application prospect.In the late 1970s,diphtheria toxin was found to pose high apoptotic activity to animal cells,leading to being deeply studied.After more than 30-year exploration,the apoptosis-induced mechanism and spatial conformation of diphtheria toxin have been clearly explored,and it has been widely used in the preparation of recombinant immunotoxins.In the current study,the N-terminal 389 amino acids of diphtheria toxin was fused to anti-HER2/neu single-chain variable fragment(scFv)of humanized 4D5 with flexible linker(G4S)3,yielding the recombinant immunotoxin DAB389-4D5 scFv.Because of its low expression level in Escherichia coli,synonymous codon optimization was conducted in the secondary structure of the translation initiation region(TIR)of the DAB389 gene,increasing the minimal folding free energy from-16.4 to-8.4 kcal/mol.As a result,the expression level was raised from<5%to 36.7%of total soluble proteins.After purification with hydrophobic interaction and ion exchange chromatographies,the yield of purified target protein was 30 mg/L.In summary,this study provides a new option for the treatment of HER2-positive cancer cells.As MTT cytotoxicity results showed,the IC50 values of DAB389 and DAB389-4D5 scFv against HER2-positive ovarian SK-OV-3 cells were 15 and 0.37 nM,respectively,proving that 4D5 scFv exerted targeted binding to SK-OV-3 cells.However,the DAB389-4D5 scFv(<5 nM)exhibited no obvious cytotoxicity towards HER2/neu-negative human embryonic kidney HEK-293 cells.Laser confocal fluorescence immunoassay showed that DAB389-4D5 scFv posed targeted binding to SK-OV-3 cells rather than HEK-293 cells.Moreover,flow cytometry results quantitatively proved DAB389-4D5 scFv could induce strong apoptosis and necrosis of SK-OV-3 cells but not HEK-293 cells.In order to further increase the targeted binding efficiency of DAB389 towards HER2-positive carcinoma cells,DAB389 was fused with two 4D5 scFvs to construct immunotoxin DAB389-4D5 scFv-4D5 scFv.MTT results demonstrated its IC50 value towards SK-OV-3 was 0.35 nM,illustrating solo 4D5 scFv was sufficient to maintain targeted binding.Moreover,the cancer cell-targeted penetrating peptide BR2 was fused to the C-terminus of 4D5 scFv to construct DAB389-4D5 scFv-BR2,whose IC50 value towards SK-OV-3 was 0.13 nM.Compared with DAB389-4D5 scFv,BR2 promoted cytotoxicity by 3 times.At last,in order to achieve optical regulation of the immunotoxin activity,the blue light-controlled intein InN-LOV2-InC was inserted into the specific sites of DAB389-4D5 scFv,and performing cytotoxicity of toxin through self-splicing was preliminarily explored.