title: Improved stability and yield of Fv targeted superantigen by introducing both linker and disulfide bond into the targeting moiety.
abstract: 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.
authors.affiliation: 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.
authors.forename: Huai-Jie
authors.initials: HJ
authors.lastname: Hao
authors.forename: Yong-Qiang
authors.initials: YQ
authors.lastname: Jiang
authors.forename: Yu-Ling
authors.initials: YL
authors.lastname: Zheng
authors.forename: Ru
authors.initials: R
authors.lastname: Ma
authors.forename: Duo-Wei
authors.initials: DW
authors.lastname: Yu
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D004220:Disulfides; D004768:Enterotoxins; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D011994:Recombinant Proteins; D018089:Superantigens; C055966:immunoglobulin Fv; C026066:enterotoxin A, Staphylococcal