title: Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody-Drug Conjugates.
abstract: A robust, generally applicable, nongenetic technology is presented to convert monoclonal antibodies into stable and homogeneous ADCs. Starting from a native (nonengineered) mAb, a chemoenzymatic protocol allows for the highly controlled attachment of any given payload to the N-glycan residing at asparagine-297, based on a two-stage process: first, enzymatic remodeling (trimming and tagging with azide), followed by ligation of the payload based on copper-free click chemistry. The technology, termed GlycoConnect, is applicable to any IgG isotype irrespective of glycosylation profile. Application to trastuzumab and maytansine, both components of the marketed ADC Kadcyla, demonstrate a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover, the superiority of the native glycan as attachment site was demonstrated by in vivo comparison to a range of trastuzumab-based glycosylation mutants. A side-by-side comparison of the copper-free click probes bicyclononyne (BCN) and a dibenzoannulated cyclooctyne (DBCO) showed a surprising difference in conjugation efficiency in favor of BCN, which could be even further enhanced by introduction of electron-withdrawing fluoride substitutions onto the azide. The resulting mAb-conjugates were in all cases found to be highly stable, which in combination with the demonstrated efficacy warrants ADCs with a superior therapeutic index.
authors.affiliation: SynAffix BV , Pivot Park, Molenstraat 110, 5342 CC, Oss, The Netherlands.
authors.forename: Remon
authors.initials: R
authors.lastname: van Geel
authors.affiliation: SynAffix BV , Pivot Park, Molenstraat 110, 5342 CC, Oss, The Netherlands.
authors.forename: Marloes A
authors.initials: MA
authors.lastname: Wijdeven
authors.affiliation: SynAffix BV , Pivot Park, Molenstraat 110, 5342 CC, Oss, The Netherlands.
authors.forename: Ryan
authors.initials: R
authors.lastname: Heesbeen
authors.affiliation: SynAffix BV , Pivot Park, Molenstraat 110, 5342 CC, Oss, The Netherlands.
authors.forename: Jorge M M
authors.initials: JM
authors.lastname: Verkade
authors.affiliation: SynAffix BV , Pivot Park, Molenstraat 110, 5342 CC, Oss, The Netherlands.
authors.forename: Anna A
authors.initials: AA
authors.lastname: Wasiel
authors.affiliation: SynAffix BV , Pivot Park, Molenstraat 110, 5342 CC, Oss, The Netherlands.
authors.forename: Sander S
authors.initials: SS
authors.lastname: van Berkel
authors.affiliation: SynAffix BV , Pivot Park, Molenstraat 110, 5342 CC, Oss, The Netherlands.
authors.forename: Floris L
authors.initials: FL
authors.lastname: van Delft
mesh_terms: D000080044:Ado-Trastuzumab Emtansine; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D002240:Carbohydrate Sequence; D057930:Click Chemistry; D006031:Glycosylation; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry*; D008453:Maytansine / Q000031:analogs & derivatives / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D055550:Protein Stability; D000068878:Trastuzumab / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D001386:Azides; D018796:Immunoconjugates; D011134:Polysaccharides; D008453:Maytansine; D000068878:Trastuzumab; D000080044:Ado-Trastuzumab Emtansine