title: Ligand-inducible dimeric antibody for selecting antibodies against a membrane protein based on mammalian cell proliferation.
abstract: A method for selecting antibodies against a membrane protein is important for attaining a variety of antibody-based diagnostics and therapies. In this study, we propose a novel system to select specific antibodies against a membrane protein based on mammalian cell proliferation as a readout. The system employs a chimeric membrane protein in which a target membrane protein of interest is fused to the intracellular signaling domain of a cytokine receptor. The chimeric membrane protein transduces a cell proliferation signal through dimerization when co-expressed with a specific single-chain Fv fused with a mutant of FK-binding protein 12 (scFv-Fk) that can be conditionally dimerized by a synthetic ligand AP20187. To demonstrate this system, ErbB2 and gp130 were chosen as the target membrane protein and cytokine receptor, respectively. Consequently, co-expression of the ErbB2/gp130 chimera and ErbB2-specific scFv-Fk rendered the cells proliferative in response to AP20187. The system also allowed selection of high-affinity binders from a mixture composed of dominant low-affinity binders. This system may be extended to affinity maturation of scFvs by modulating AP20187 concentration in the selection process.
authors.affiliation: Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Miura
authors.affiliation: Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.|Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan. kawahara@bio.t.u-tokyo.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kawahara
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D050822:Cytokine Receptor gp130 / Q000235:genetics*; D006801:Humans; D008024:Ligands; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology; D022061:Tacrolimus Binding Protein 1A / Q000235:genetics*; D014161:Transduction, Genetic
chemical_list: D050822:Cytokine Receptor gp130; D008024:Ligands; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D022061:Tacrolimus Binding Protein 1A
keywords: antibody engineering; cell proliferation; chimeric protein; cytokine receptor; membrane protein