title: One-Step Conjugation Method for Site-Specific Antibody-Drug Conjugates through Reactive Cysteine-Engineered Antibodies.
abstract: Engineered cysteine residues are particularly convenient for site-specific conjugation of antibody-drug conjugates (ADC), because no cell engineering and additives are required. Usually, unpaired cysteine residues form mixed disulfides during fermentation in Chinese hamster ovarian (CHO) cells; therefore, additional reduction and oxidization steps are required prior to conjugation. In this study, we prepared light chain (Lc)-Q124C variants in IgG and examined the conjugation efficiency. Intriguingly, Lc-Q124C exhibited high thiol reactivity and directly generated site-specific ADC without any pretreatment (named active thiol antibody: Actibody). Most of the cysteine-maleimide conjugates including Lc-Q124C showed retro-Michael reaction with cysteine 34 in albumin and were decomposed over time. In order to acquire resistance to a maleimide exchange reaction, the facile procedure for succinimide hydrolysis on anion exchange resin was employed. Hydrolyzed Lc-Q124C conjugate prepared with anion exchange procedure retained high stability in plasma. Recently, various stable linkage schemes for cysteine conjugation have been reported. The combination with direct conjugation by the use of Actibody and stable linker technology could enable the generation of stable site-specific ADC through a simple method. Actibody technology with Lc-Q124C at a less exposed position opens a new path for cysteine-based conjugation, and contributes to reducing entry barriers to the preparation and evaluation of ADC.
authors.affiliation: Research Core Function Laboratories and §Innovative Technology Laboratories, Research Functions Unit, #R&D Planning Department, R&D Division, and ‡Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd. , Tokyo, 100-8185, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Shinmi
authors.affiliation: Research Core Function Laboratories and §Innovative Technology Laboratories, Research Functions Unit, #R&D Planning Department, R&D Division, and ‡Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd. , Tokyo, 100-8185, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Taguchi
authors.affiliation: Research Core Function Laboratories and §Innovative Technology Laboratories, Research Functions Unit, #R&D Planning Department, R&D Division, and ‡Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd. , Tokyo, 100-8185, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Iwano
authors.affiliation: Research Core Function Laboratories and §Innovative Technology Laboratories, Research Functions Unit, #R&D Planning Department, R&D Division, and ‡Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd. , Tokyo, 100-8185, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Research Core Function Laboratories and §Innovative Technology Laboratories, Research Functions Unit, #R&D Planning Department, R&D Division, and ‡Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd. , Tokyo, 100-8185, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Masuda
authors.affiliation: Research Core Function Laboratories and §Innovative Technology Laboratories, Research Functions Unit, #R&D Planning Department, R&D Division, and ‡Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd. , Tokyo, 100-8185, Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Enokizono
authors.affiliation: Research Core Function Laboratories and §Innovative Technology Laboratories, Research Functions Unit, #R&D Planning Department, R&D Division, and ‡Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd. , Tokyo, 100-8185, Japan.
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Shiraishi
mesh_terms: D000818:Animals; D001665:Binding Sites; D003545:Cysteine; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000235:genetics*; D007074:Immunoglobulin G / Q000235:genetics; D008297:Male; D051379:Mice; D015202:Protein Engineering; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D018796:Immunoconjugates; D007074:Immunoglobulin G; D013438:Sulfhydryl Compounds; D003545:Cysteine