Efficient affinity maturation of antibodies in an engineered chicken B cell line DT40-SW by increasing point mutation

Masamichi Kajita, Takahiro Okazawa, Mika Ikeda, Kagefumi Todo, Masaki Magari, Naoki Kanayama, Hitoshi Ohmori

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

The chicken B cell line DT40 undergoes hypermutation of immunoglobulin variable region (IgV) genes during culture, thereby constituting an antibody (Ab) library. We previously established an in vitro Ab generation system using an engineered line DT40-SW whose hypermutation machinery can be switched on and off. Abs for various antigens (Ags) can be obtained from the DT40-SW library and the specificity of the Ag-specific clones can be stabilized by stopping hypermutation. Furthermore, the affinity of obtained monoclonal Abs (mAbs) can be improved through further mutation followed by selection, a process analogous to "affinity maturation" that occurs in vivo. Although gene conversion dominantly diversifies the IgV genes in DT40 cells, point mutation is considered to be more favorable for fine-tuning Ab properties during affinity maturation. Here, we examined whether affinity maturation occurs more efficiently when the hypermutation pattern was transformed from gene conversion into point mutation in DT40-SW cells. To this end, we disrupted the XRCC3 gene that is essential for gene conversion. It was found that hemizygous disruption of the XRCC3 gene was sufficient to increase the point mutation frequency. Since hemizygous disruption is conducted more easily, we tested whether the XRCC3 (+/-) mutant generates high-affinity Abs through affinity maturation more efficiently than the wild type. Using this affinity maturation technique, we generated an improved 4-hydroxy-3-nitrophenylacetyl-specific mAb with ~600-fold lower KD than that of the original mAb. Taken together, hemizygous disruption of the XRCC3 gene is considered to be useful for obtaining high-affinity mAbs from DT40-SW cells though affinity maturation.

Original languageEnglish
Pages (from-to)351-358
Number of pages8
JournalJournal of Bioscience and Bioengineering
Volume110
Issue number3
DOIs
Publication statusPublished - Sep 2010

Fingerprint

Antibody Affinity
Point Mutation
Antibodies
Chickens
B-Lymphocytes
Genes
Gene Conversion
Cells
Cell Line
Immunoglobulin Variable Region
Essential Genes
Mutation Rate
Cell culture
Libraries
Machinery
Clone Cells
Tuning
Antigens
Mutation

Keywords

  • Affinity maturation
  • Antibody
  • Chicken B cell line DT40
  • Gene conversion
  • Point mutation
  • XRCC3

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Bioengineering

Cite this

Efficient affinity maturation of antibodies in an engineered chicken B cell line DT40-SW by increasing point mutation. / Kajita, Masamichi; Okazawa, Takahiro; Ikeda, Mika; Todo, Kagefumi; Magari, Masaki; Kanayama, Naoki; Ohmori, Hitoshi.

In: Journal of Bioscience and Bioengineering, Vol. 110, No. 3, 09.2010, p. 351-358.

Research output: Contribution to journalArticle

Kajita, Masamichi ; Okazawa, Takahiro ; Ikeda, Mika ; Todo, Kagefumi ; Magari, Masaki ; Kanayama, Naoki ; Ohmori, Hitoshi. / Efficient affinity maturation of antibodies in an engineered chicken B cell line DT40-SW by increasing point mutation. In: Journal of Bioscience and Bioengineering. 2010 ; Vol. 110, No. 3. pp. 351-358.
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