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-αscFv (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-Bcells,which makes directed evolution in mammalian cells easier and more efficient.