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. 2012 Apr 1;3(6):460–469. doi: 10.1007/s13238-012-2024-7

Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells

Shaopeng Chen 1,2, Junkang Qiu 1, Chuan Chen 1, Chunchun Liu 1,3, Yuheng Liu 1, Lili An 1, Junying Jia 3, Jie Tang 1, Lijun Wu 2,✉, Haiying Hang 1,✉
PMCID: PMC4875482  PMID: 22467272

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.

Keywords: antibody, activation-induced cytidine deaminase (AID), somatic hypermutation, affinity maturation, TNF-alpha

Footnotes

These authors contributed equally to the work.

Contributor Information

Lijun Wu, Email: ljw@ipp.ac.cn.

Haiying Hang, Email: hh91@ibp.ac.cn.

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