title: Domain structure of growth signalobodies critically affects the outcome of antibody library selection.
abstract: Wide applications of antibodies have demanded rapid and easy methods for isolating high-affinity antibodies. We recently developed an antibody screening system in mammalian cells using a growth signalobody, which is a single-chain Fv (scFv) library/cytokine receptor chimera that can transduce a growth signal in response to a target oligomeric antigen. However, we have never investigated how the domain structure of signalobodies affects the outcome of library screening. In this study, we screened naïve scFv library-inserted signalobodies having distinct extracellular and transmembrane (TM) domains. Although the previously constructed signalobody with the extracellular D1/D2 domains of erythropoietin receptor had recovered the clones with high affinity against a target antigen and with low background cell growth, its D1/D2-deficient variant which was tested in this study recovered the clones with low affinity against a target antigen and with considerable background cell growth. In addition, mutagenesis in the TM domain lowered the level of the background cell growth. These results suggest that the D1/D2 domains increase a threshold to activate signalobodies, thereby selecting clones with high affinity against a target antigen and that the TM domain could be engineered to minimize background growth signalling.
authors.affiliation: Department of Bioengineering and 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: Rie
authors.initials: R
authors.lastname: Yoshida
authors.affiliation: Department of Bioengineering and 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
authors.affiliation: Department of Bioengineering and Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Department of Bioengineering and 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
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000276:immunology; D002455:Cell Division / Q000276:immunology; D002460:Cell Line; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000276:immunology*
chemical_list: D000941:Antigens; D057127:Single-Chain Antibodies
keywords: antibody selection; chimeric protein; cytokine receptor; growth signalobody; single-chain Fv