title: Construction and humanization of a functional bispecific EGFR × CD16 diabody using a refolding system.
abstract: We previously reported the construction and activity of a humanized, bispecific diabody (hEx3) that recruited T cells towards an epidermal growth factor receptor (EGFR) positive tumor. Herein, we describe the construction of a second functional, fully humanized, anti-EGFR bispecific diabody that recruits another subset of lymphocyte effectors, the natural killer cells, to EGFR-expressing tumor cells. After we confirmed that an anti-EGFR × anti-CD16 bispecific diabody (Ex16) consisting of a previously humanized anti-EGFR variable fragment (Fv) and a mouse anti-CD16 Fv had growth inhibitory activity, we designed a humanized anti-CD16 Fv to construct the fully humanized Ex16 (hEx16). However, the humanized form had lower activity for inhibition of cancer growth. To restore its growth inhibitory activity, we introduced mutations into the Vernier zone, which is located near the complementarity-determining regions and is involved in their binding activity. We efficiently prepared 15 different hEx16 mutants by expressing each chimeric single-chain component for hEx16 separately. We then used our in vitro refolding system to select the most functional mutant, which had a growth inhibitory effect comparable with that of the commercially available chimeric anti-EGFR antibody, cetuximab. Our refolding system could aid in the efficient optimization of other proteins with heterodimeric structure.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Asano
authors.forename: Makoto
authors.initials: M
authors.lastname: Nakayama
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kawaguchi
authors.forename: Tsuguo
authors.initials: T
authors.lastname: Kubota
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nakanishi
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Umetsu
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: Izumi
authors.initials: I
authors.lastname: Kumagai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D015195:Drug Design; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D058851:GPI-Linked Proteins / Q000037:antagonists & inhibitors; D006801:Humans; D006825:Hybridomas; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D007155:Immunologic Factors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015979:Killer Cells, Lymphokine-Activated / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000096:biosynthesis / Q000737:chemistry; D009369:Neoplasms / Q000276:immunology / Q000378:metabolism / Q000628:therapy; D058849:Protein Refolding; D017452:Receptors, IgG / Q000037:antagonists & inhibitors*; D016415:Sequence Alignment
chemical_list: D018033:Antibodies, Bispecific; D061067:Antibodies, Monoclonal, Humanized; C428200:FCGR3B protein, human; D058851:GPI-Linked Proteins; D007135:Immunoglobulin Variable Region; D007155:Immunologic Factors; D050506:Mutant Chimeric Proteins; D017452:Receptors, IgG; C512478:EGFR protein, human; D066246:ErbB Receptors