Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells
Protein & Cell
; (12): 460-469, 2012.
Article
in En
| WPRIM
| ID: wpr-757281
Responsible library:
WPRO
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.
Full text:
1
Index:
WPRIM
Main subject:
Immunoglobulin Variable Region
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Cells, Cultured
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Chemistry
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Tumor Necrosis Factor-alpha
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Cytidine Deaminase
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Somatic Hypermutation, Immunoglobulin
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Allergy and Immunology
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Single-Chain Antibodies
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HEK293 Cells
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Genetics
Limits:
Animals
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Humans
Language:
En
Journal:
Protein & Cell
Year:
2012
Type:
Article