title: Generation of camelid VHH bispecific constructs via in-cell intein-mediated protein trans-splicing.
abstract: Production of various combinations of bispecific variable domain of heavy chain of heavy chain-only antibody (VHH) constructs to evaluate their therapeutic potential usually requires several gene-engineering steps. Here, we present an alternative method of creating bispecific VHH constructs in vivo through protein trans-splicing (PTS) reaction; this method may reduce the number of gene manipulation steps required. As a proof-of-concept, we constructed a bispecific antibody (bsAb) containing an anti-epidermal growth factor receptor VHH and anti-green fluorescent protein VHH, and we evaluated and confirmed its bispecificity. We also tested antibody labeling by fluorescent protein tagging using the PTS reaction. Compared with the conventional gene construction method, bsAb construction via PTS is a promising alternative approach for generating multiple bsAb combinations.
authors.affiliation: Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jyonan, Yonezawa, Yamagata 992-8510, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Shibuya
authors.affiliation: Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jyonan, Yonezawa, Yamagata 992-8510, Japan.
authors.forename: Natsuki
authors.initials: N
authors.lastname: Haga
authors.affiliation: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 183-8538, Japan.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Asano
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 6-6-11-606 Aoba-yama, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Nakazawa
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 6-6-11-606 Aoba-yama, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Hattori
authors.affiliation: Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jyonan, Yonezawa, Yamagata 992-8510, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Takeda
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 6-6-11-606 Aoba-yama, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
authors.forename: Aruto
authors.initials: A
authors.lastname: Sugiyama
authors.affiliation: Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jyonan, Yonezawa, Yamagata 992-8510, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Kurotani
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 6-6-11-606 Aoba-yama, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
authors.forename: Izumi
authors.initials: I
authors.lastname: Kumagai
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 6-6-11-606 Aoba-yama, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Umetsu
authors.affiliation: Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jyonan, Yonezawa, Yamagata 992-8510, Japan makabe@yz.yamagata-u.ac.jp.
authors.forename: Koki
authors.initials: K
authors.lastname: Makabe
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D016466:CHO Cells; D002161:Camelids, New World; D003412:Cricetulus; D006801:Humans; D047668:Inteins; D015202:Protein Engineering; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D020040:Trans-Splicing
chemical_list: D018033:Antibodies, Bispecific; D061905:Single-Domain Antibodies
keywords: bispecific antibody; intein; protein splicing