Improved stability and yield of Fv targeted superantigen by introducing both linker and disulfide bond into the targeting moiety.
Bacterial superantigens (SAg) are the most potent activators of human T lymphocytes and recombinant immunotoxin using bacterial SAg shows promising clinical values. To engineer superantigen for immunotherapy of hepatocellular carcinoma, we genetically fused the superantigen staphylococcus enterotoxin A (SEA(D(227)A)) to the single-chain disulfide-stabilized Fv (scdsFv) of anti-hepatoma monoclonal antibody HAb25 through a short peptide GGGSGGS. We expressed this recombinant protein in Escherichia coli and extract it from inclusion bodies. We found purified scdsFv-targeted SAg contains equivalent binding affinity with disulfide-stabilized Fv (dsFv) targeted SAg and single-chain Fvs (scFv) targeted SAg, but more stable and more suitable for large scale production. The MTS(3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu m, inner salt) assay shows that the scdsFv-targeted SAg also shares the ability to activate a large number of T lymphocytes and has cytotoxic activity on human hepatoma cell line SMMC-7721. Therefore, this novel generation of recombinant immunotoxins using scdsFv has a high potential in hepato cancer treatment and the same strategy may also be applied to other cancer treatments.
Hao
Huai-Jie
HJ
State Key Laboratory of Pathogen and Biosecurity, The Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, No. 20 Dongda Street, Fengtai District, Beijing 100071, China.
Jiang
Yong-Qiang
YQ
Zheng
Yu-Ling
YL
Ma
Ru
R
Yu
Duo-Wei
DW
D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D006528:Carcinoma, Hepatocellular / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D004220:Disulfides / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004355:Drug Stability; D004768:Enterotoxins / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000922:Immunotoxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry; D018089:Superantigens / Q000737:chemistry*; D013601:T-Lymphocytes / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004220:Disulfides; D004768:Enterotoxins; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D011994:Recombinant Proteins; D018089:Superantigens; C055966:immunoglobulin Fv; C026066:enterotoxin A, Staphylococcal