title: Domain order of a bispecific diabody dramatically enhances its antitumor activity beyond structural format conversion: the case of the hEx3 diabody.
abstract: The domains of bispecific diabodies (BsDbs) can be ordered in four different ways; however, the influence of domain order on the cytotoxicity of BsDbs that retarget immune cells against tumor cells had not been addressed. We previously reported the marked antitumor effects of a humanized BsDb that targets epidermal growth factor receptor and CD3 (hEx3-Db). Here, we rearranged the domains of hEx3-Db to examine the influence of domain order on the function of BsDbs. We successfully prepared homogenous dimers of hEx3-Db in all four domain configurations. Interestingly, all three rearranged hEx3s inhibited cancer growth more effectively than did the original hEx3-Db, in which both components were in variable heavy domain (VH)-variable light domain (VL) order (redesignated as hEx3-HL), and the highest effects were observed with hEx3-LH (hEx3-Db with both components in VL-VH order). In addition, hEx3-LH had comparable in vitro growth inhibitory effects to those of the tandem single-chain variable fragment (scFv) format of hEx3-Db (hEx3-tandem scFv (taFv)), which we previously showed to have greater cytotoxicity than does hEx3-HL. Flow cytometry suggested that the enhanced cytotoxicity of hEx3-LH is attributable to structural superiority for cross-linking, similar to that of hEx3-taFv. Furthermore, hEx3-LH inhibited cancer growth in mice more effectively than did hEx3-taFv; this difference may be due to differences in antibody stability. Our results show that merely rearranging the domain order of BsDbs can enhance their effects beyond those with structural format conversion.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba 6-6-11, Aramaki, Aoba-ku, Sendai 980-8579, Japan.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Asano
authors.forename: Takashi
authors.initials: T
authors.lastname: Kumagai
authors.forename: Keisuke
authors.initials: K
authors.lastname: Nagai
authors.forename: Shintaro
authors.initials: S
authors.lastname: Taki
authors.forename: Ippei
authors.initials: I
authors.lastname: Shimomura
authors.forename: Kyoko
authors.initials: K
authors.lastname: Arai
authors.forename: Hiromi
authors.initials: H
authors.lastname: Ogata
authors.forename: Mai
authors.initials: M
authors.lastname: Okada
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Hayasaka
authors.forename: Hideaki
authors.initials: H
authors.lastname: Sanada
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nakanishi
authors.forename: Teemu
authors.initials: T
authors.lastname: Karvonen
authors.forename: Hiroki
authors.initials: H
authors.lastname: Hayashi
authors.forename: Yu
authors.initials: Y
authors.lastname: Katayose
authors.forename: Michiaki
authors.initials: M
authors.lastname: Unno
authors.forename: Toshio
authors.initials: T
authors.lastname: Kudo
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Umetsu
authors.forename: Izumi
authors.initials: I
authors.lastname: Kumagai
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000276:immunology / Q000627:therapeutic use*; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000276:immunology / Q000627:therapeutic use*; D017252:CD3 Complex / Q000276:immunology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D066246:ErbB Receptors / Q000276:immunology*; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000276:immunology / Q000628:therapy*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
chemical_list: D018033:Antibodies, Bispecific; D000970:Antineoplastic Agents; D017252:CD3 Complex; D066246:ErbB Receptors