Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells |
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Authors: | Shaopeng Chen Junkang Qiu Chuan Chen Chunchun Liu Yuheng Liu Lili An Junying Jia Jie Tang Lijun Wu Haiying Hang |
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Affiliation: | 1. Key Laboratory for Protein and Peptide Pharmaceuticals, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 2. Key Laboratory of Ion Beam Bioengineering, Institute of Technical Biology and Agriculture Engineering, Chinese Academy of Sciences, Hefei 230031, China; 3. Core Facility Center, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China |
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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-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-B cells, which makes directed evolution in mammalian cells easier and more efficient. |
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Keywords: | antibody activation-induced cytidine deaminase (AID) somatic hypermutation affinity maturation TNF-alpha |
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