title: B Cell-Based Seamless Engineering of Antibody Fc Domains.
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.
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Hashimoto
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Kurosawa
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Akiho
authors.initials: A
authors.lastname: Murayama
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Hidetaka
authors.identifier: http://orcid.org/0000-0002-8676-8955
authors.initials: H
authors.lastname: Seo
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Ohta
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000915:Antibody Affinity / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D015870:Gene Expression; D023061:Gene Order; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics*; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics; D007075:Immunoglobulin M / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding / Q000276:immunology; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D011961:Receptors, Fc / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013728:Terminator Regions, Genetic
chemical_list: D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins