Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells.
10.1007/s13238-012-2024-7
- Author:
Shaopeng CHEN
1
;
Junkang QIU
;
Chuan CHEN
;
Chunchun LIU
;
Yuheng LIU
;
Lili AN
;
Junying JIA
;
Jie TANG
;
Lijun WU
;
Haiying HANG
Author Information
1. Key Laboratory for Protein and Peptide Pharmaceuticals, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antibody Affinity;
Cells, Cultured;
Cytidine Deaminase;
genetics;
metabolism;
HEK293 Cells;
Humans;
Immunoglobulin Variable Region;
genetics;
immunology;
Mice;
Mutation;
Single-Chain Antibodies;
chemistry;
genetics;
immunology;
Somatic Hypermutation, Immunoglobulin;
genetics;
immunology;
Tumor Necrosis Factor-alpha;
immunology
- From:
Protein & Cell
2012;3(6):460-469
- CountryChina
- Language:English
-
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.
