Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells
Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells
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DOI：
10.1007/s13238-012-2024-7
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发表时间：
2012-06-01
期刊：
影响因子：
21.1
通讯作者：
Hang, Haiying
中科院分区：
文献类型：
--
作者：
Chen, Shaopeng;Qiu, Junkang;Hang, Haiying
激活诱导的胞苷脱氨酶（AID）是通过启动体细胞超突变（SHM）和类别转换重组产生抗体多样性所必需的。一些研究小组已经成功地利用AID的特性在体外B细胞中产生靶蛋白定向进化的突变体库。悬浮培养的B细胞不便于转染和克隆。在这项研究中，我们建立了一个基于艾滋病的突变积累和分选系统在贴壁的人细胞。首先将小鼠AID基因转染人非小细胞肺癌H1299细胞，筛选出稳定的细胞克隆（H1299-AID）。然后将抗hTNF-α单链抗体（ATscFv）转染H1299-AID细胞，ATscFv在H1299-AID细胞表面展示。通过4轮扩增/流式细胞术分选对hTNF-α具有最高亲和力的细胞，分离出两个ATscFv突变体基因克隆。与野生型ATscFv相比，两种突变体在中和hTNF-α的细胞毒性方面更加有效。结果表明，体细胞超突变定向进化可以在贴壁非B细胞中进行，这使得哺乳动物细胞中的定向进化更容易和更有效。
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-alpha 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.