title: Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.
abstract: The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
authors.affiliation: Genentech Inc., 1 DNA Way, S. San Francisco, California, USA.
authors.forename: Ben-Quan
authors.initials: BQ
authors.lastname: Shen
authors.forename: Keyang
authors.initials: K
authors.lastname: Xu
authors.forename: Luna
authors.initials: L
authors.lastname: Liu
authors.forename: Helga
authors.initials: H
authors.lastname: Raab
authors.forename: Sunil
authors.initials: S
authors.lastname: Bhakta
authors.forename: Margaret
authors.initials: M
authors.lastname: Kenrick
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Parsons-Reponte
authors.forename: Janet
authors.initials: J
authors.lastname: Tien
authors.forename: Shang-Fan
authors.initials: SF
authors.lastname: Yu
authors.forename: Elaine
authors.initials: E
authors.lastname: Mai
authors.forename: Dongwei
authors.initials: D
authors.lastname: Li
authors.forename: Jay
authors.initials: J
authors.lastname: Tibbitts
authors.forename: Jakub
authors.initials: J
authors.lastname: Baudys
authors.forename: Ola M
authors.initials: OM
authors.lastname: Saad
authors.forename: Suzie J
authors.initials: SJ
authors.lastname: Scales
authors.forename: Paul J
authors.initials: PJ
authors.lastname: McDonald
authors.forename: Philip E
authors.initials: PE
authors.lastname: Hass
authors.forename: Charles
authors.initials: C
authors.lastname: Eigenbrot
authors.forename: Trung
authors.initials: T
authors.lastname: Nguyen
authors.forename: Willy A
authors.initials: WA
authors.lastname: Solis
authors.forename: Reina N
authors.initials: RN
authors.lastname: Fuji
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Flagella
authors.forename: Darshana
authors.initials: D
authors.lastname: Patel
authors.forename: Susan D
authors.initials: SD
authors.lastname: Spencer
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Khawli
authors.forename: Allen
authors.initials: A
authors.lastname: Ebens
authors.forename: Wai Lee
authors.initials: WL
authors.lastname: Wong
authors.forename: Richard
authors.initials: R
authors.lastname: Vandlen
authors.forename: Surinder
authors.initials: S
authors.lastname: Kaur
authors.forename: Mark X
authors.initials: MX
authors.lastname: Sliwkowski
authors.forename: Richard H
authors.initials: RH
authors.lastname: Scheller
authors.forename: Paul
authors.initials: P
authors.lastname: Polakis
authors.forename: Jagath R
authors.initials: JR
authors.lastname: Junutula
mesh_terms: D062365:Aminobenzoates / Q000737:chemistry / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000097:blood* / Q000737:chemistry / Q000276:immunology*; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000276:immunology; D001666:Binding Sites, Antibody / Q000276:immunology*; D045744:Cell Line, Tumor; D002470:Cell Survival; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000008:administration & dosage / Q000737:chemistry* / Q000276:immunology*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology; D008252:Macaca fascicularis; D008301:Maleimides / Q000737:chemistry; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy / Q000276:immunology; D008453:Maytansine / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology; D011487:Protein Conformation; D015202:Protein Engineering; D051381:Rats; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000737:chemistry; D000068878:Trastuzumab
chemical_list: D062365:Aminobenzoates; D000906:Antibodies; D061067:Antibodies, Monoclonal, Humanized; D018796:Immunoconjugates; D007074:Immunoglobulin G; D008301:Maleimides; D009842:Oligopeptides; D013438:Sulfhydryl Compounds; C543533:auristatin; D008453:Maytansine; D003545:Cysteine; D000068878:Trastuzumab