title: Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells.
abstract: Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.
authors.affiliation: Key Laboratory for Protein and Peptide Pharmaceuticals, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Shaopeng
authors.initials: S
authors.lastname: Chen
authors.forename: Junkang
authors.initials: J
authors.lastname: Qiu
authors.forename: Chuan
authors.initials: C
authors.lastname: Chen
authors.forename: Chunchun
authors.initials: C
authors.lastname: Liu
authors.forename: Yuheng
authors.initials: Y
authors.lastname: Liu
authors.forename: Lili
authors.initials: L
authors.lastname: An
authors.forename: Junying
authors.initials: J
authors.lastname: Jia
authors.forename: Jie
authors.initials: J
authors.lastname: Tang
authors.forename: Lijun
authors.initials: L
authors.lastname: Wu
authors.forename: Haiying
authors.initials: H
authors.lastname: Hang
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D002478:Cells, Cultured; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D009154:Mutation; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D027041:Somatic Hypermutation, Immunoglobulin / Q000235:genetics* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D007135:Immunoglobulin Variable Region; D057127:Single-Chain Antibodies; D014409:Tumor Necrosis Factor-alpha; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase