RRC ID 47287
Author Hashimoto K, Kurosawa K, Murayama A, Seo H, Ohta K.
Title B Cell-Based Seamless Engineering of Antibody Fc Domains.
Journal PLoS ONE
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.
Volume 11(12)
Pages e0167232
Published 2016
DOI 10.1371/journal.pone.0167232
PII PONE-D-16-22603
PMID 27907066
PMC PMC5131995
MeSH Animals Antibodies, Monoclonal / biosynthesis* Antibodies, Monoclonal / chemistry Antibodies, Monoclonal / genetics* Antibodies, Monoclonal / isolation & purification Antibody Affinity / immunology B-Lymphocytes / immunology* Base Sequence Cell Line Chickens Gene Expression Gene Order Humans Immunoglobulin Fc Fragments / genetics* Immunoglobulin G / biosynthesis Immunoglobulin G / genetics Immunoglobulin M / biosynthesis Immunoglobulin M / genetics Plasmids / genetics Protein Binding / immunology Protein Engineering* Protein Interaction Domains and Motifs Receptors, Fc / metabolism Recombinant Fusion Proteins / biosynthesis Recombinant Fusion Proteins / chemistry Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / isolation & purification Terminator Regions, Genetic
IF 2.766
Times Cited 0
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
DNA material Genome Network Project Human cDNA IRAK049H17 (HGX019785)