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 language | English |
|---|---|
| Pages (from-to) | 351-358 |
| Number of pages | 8 |
| Journal | Journal of Bioscience and Bioengineering |
| Volume | 110 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - Sep 2010 |
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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 journal › Article
}
TY - JOUR
T1 - Efficient affinity maturation of antibodies in an engineered chicken B cell line DT40-SW by increasing point mutation
AU - Kajita, Masamichi
AU - Okazawa, Takahiro
AU - Ikeda, Mika
AU - Todo, Kagefumi
AU - Magari, Masaki
AU - Kanayama, Naoki
AU - Ohmori, Hitoshi
PY - 2010/9
Y1 - 2010/9
N2 - 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.
AB - 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.
KW - Affinity maturation
KW - Antibody
KW - Chicken B cell line DT40
KW - Gene conversion
KW - Point mutation
KW - XRCC3
UR - http://www.scopus.com/inward/record.url?scp=77955321545&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77955321545&partnerID=8YFLogxK
U2 - 10.1016/j.jbiosc.2010.03.006
DO - 10.1016/j.jbiosc.2010.03.006
M3 - Article
VL - 110
SP - 351
EP - 358
JO - Journal of Bioscience and Bioengineering
JF - Journal of Bioscience and Bioengineering
SN - 1389-1723
IS - 3
ER -