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.
Highlights
Antibodies (Ab), known as immunoglobulins (Ig), have been widely used for therapeutic, diagnostic and research purposes [1]
Development of recombinase-mediated cassette exchange (RMCE) host cell line by integrating the human IgG-Fc cassette flanked by loxP and lox2272 into the endogenous chicken IgM-Fc region
We developed a DT40 cell line expressing chicken/human chimeric IgG1 by integrating the coding sequence of hIgG1-Fc into the genomic region between exon Cμ1 and Cμ3 by disrupting Cμ2
Summary
Antibodies (Ab), known as immunoglobulins (Ig), have been widely used for therapeutic, diagnostic and research purposes [1]. Monoclonal antibodies (mAbs), which bind to a given antigen, are valuable as drugs or research reagents due to their superior homogeneity. It is difficult to generate mAbs against poorly immunogenic antigens such as auto-antigens, toxic compounds and lipids. It consists of time-consuming steps such as animal immunization, while in vitro screening systems like phage display can overcome these disadvantages [3]. Phage display has its weakness in the time-consuming recombinant DNA engineering steps, PLOS ONE | DOI:10.1371/journal.pone.0167232. Phage display has its weakness in the time-consuming recombinant DNA engineering steps, PLOS ONE | DOI:10.1371/journal.pone.0167232 December 1, 2016
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