title: Site-specific antibody-drug conjugation through glycoengineering.
abstract: Antibody-drug conjugates (ADCs) have been proven clinically to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prepare ADCs by targeting primary amines or hinge disulfides have a number of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymatically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidation of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatography and LC-MS analyses revealed the incorporation of ~1.6 cytotoxic agents per antibody molecule, approximating the number of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-positive tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-positive tumor xenograft model. These findings suggest that enzymatic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chemical process to ensure reproducible drug loading.
authors.affiliation: Sanofi-Genzyme R&D Center, Genzyme Corporation, A Sanofi Company , Framingham, Massachusetts 01701, United States.
authors.forename: Qun
authors.initials: Q
authors.lastname: Zhou
authors.forename: James E
authors.initials: JE
authors.lastname: Stefano
authors.forename: Charlene
authors.initials: C
authors.lastname: Manning
authors.forename: Josephine
authors.initials: J
authors.lastname: Kyazike
authors.forename: Bo
authors.initials: B
authors.lastname: Chen
authors.forename: Diego A
authors.initials: DA
authors.lastname: Gianolio
authors.forename: Anna
authors.initials: A
authors.lastname: Park
authors.forename: Michelle
authors.initials: M
authors.lastname: Busch
authors.forename: Julie
authors.initials: J
authors.lastname: Bird
authors.forename: Xiaoyang
authors.initials: X
authors.lastname: Zheng
authors.forename: Helene
authors.initials: H
authors.lastname: Simonds-Mannes
authors.forename: Jennifer
authors.initials: J
authors.lastname: Kim
authors.forename: Rick C
authors.initials: RC
authors.lastname: Gregory
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Miller
authors.forename: William H
authors.initials: WH
authors.lastname: Brondyk
authors.forename: Pradeep K
authors.initials: PK
authors.lastname: Dhal
authors.forename: Clark Q
authors.initials: CQ
authors.lastname: Pan
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D006031:Glycosylation; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D015394:Molecular Structure; D009374:Neoplasms, Experimental / Q000188:drug therapy* / Q000473:pathology; D011134:Polysaccharides / Q000737:chemistry; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D000068878:Trastuzumab
chemical_list: D000906:Antibodies; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D011134:Polysaccharides; D012794:Sialic Acids; D012799:Sialyltransferases; D000068878:Trastuzumab