title: Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics.
abstract: One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)-variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH-VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.
authors.affiliation: a Department of Biomolecular Engineering ; Graduate School of Engineering; Tohoku University ; Sendai , Japan.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Asano
authors.forename: Ippei
authors.initials: I
authors.lastname: Shimomura
authors.forename: Shota
authors.initials: S
authors.lastname: Konno
authors.forename: Akiko
authors.initials: A
authors.lastname: Ito
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Masakari
authors.forename: Ryota
authors.initials: R
authors.lastname: Orimo
authors.forename: Shintaro
authors.initials: S
authors.lastname: Taki
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: Shozo
authors.initials: S
authors.lastname: Furumoto
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Onitsuka
authors.forename: Takeshi
authors.initials: T
authors.lastname: Omasa
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 / Q000235:genetics / Q000276:immunology* / Q000493:pharmacokinetics; D000970:Antineoplastic Agents / Q000276:immunology* / Q000493:pharmacokinetics / Q000494:pharmacology; D019540:Area Under Curve; D001665:Binding Sites / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D066246:ErbB Receptors / Q000276:immunology; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D061986:MCF-7 Cells; D016513:Mice, SCID; D009369:Neoplasms / Q000188:drug therapy / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D047368:Tumor Burden / Q000187:drug effects / Q000276:immunology; D023041:Xenograft Model Antitumor Assays
chemical_list: D018033:Antibodies, Bispecific; D000970:Antineoplastic Agents; D017252:CD3 Complex; D007074:Immunoglobulin G; D007371:Interferon-gamma; D066246:ErbB Receptors
keywords: ADCC, antibody-dependent cell-mediated cytotoxicity; AUC, area-under-the-curve; CD3; EGFR, epidermal growth factor receptor; FITC-CD3ϵγ, fluorescein isothiocyanate-labeled CD3ϵγ; DVD-IgTM, dual variable domain immunoglobulin; FITC-sEGFR, FITC-labeled sEGFR; Fv, variable fragment; ICR, imprinting control region; IgG-like bispecific antibody; MTS, 3-(4, 5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SPR, surface plasmon resonance; SUV, standardized uptake value; T-LAK cells, lymphokine-activated killer cells with the T-cell phenotype; VH, variable heavy domain; VL, variable light domain; antibody engineering; bispecific diabody; bsAb, bispecific antibody; bsDb, bispecific diabody; cancer immunotherapy; effective domain order; epidermal growth factor receptor; sEGFR, soluble EGFR; scDb, single-chain diabody; scFv, single-chain Fv; taFv, tandem scFv