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PLoS One. 2016 Dec 1;11(12):e0167232. doi: 10.1371/journal.pone.0167232. eCollection 2016.

B Cell-Based Seamless Engineering of Antibody Fc Domains.

Author information

1
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.

Abstract

Engineering of monoclonal antibodies (mAbs) enables us to obtain mAbs with additional functions. In particular, modifications in antibody's Fc (fragment, crystallizable) region can provide multiple benefits such as added toxicity by drug conjugation, higher affinity to Fc receptors on immunocytes, or the addition of functional modules. However, the generation of recombinant antibodies requires multiple laborious bioengineering steps. We previously developed a technology that enables rapid in vitro screening and isolation of specific mAb-expressing cells from the libraries constructed with chicken B-cell line DT40 (referred to as the 'ADLib system'). To upgrade this ADLib system with the ability to generate customized mAbs, we developed a novel and rapid engineering technology that enables seamless exchanges of mAbs' Fc domains after initial selections of mAb-producing clones by the ADLib system, using a gene-replacement unit for recombinase-mediated cassette exchange (RMCE). In this system, Cre-recombinase recognition sites were inserted into the Fc region of the active DT40 IgM allele, allowing the replacement of the Fc domain by the sequences of interest upon co-transfection of a Cre recombinase and a donor DNA, enabling the rapid exchange of Fc regions. Combining this method with the ADLib system, we demonstrate rapid Fc engineering to generate fluorescent antibodies and to enhance affinity to Fc receptors.

PMID:
27907066
PMCID:
PMC5131995
DOI:
10.1371/journal.pone.0167232
[Indexed for MEDLINE]
Free PMC Article

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