title: Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar.
abstract: Conjugation of small molecule drugs to specific sites on the antibody molecule has been increasingly used for the generation of relatively homogenous preparations of antibody-drug conjugates (ADCs) with physicochemical properties similar or identical to those of the naked antibody. Previously a method for conjugation of small molecules to glycoproteins through existing glycans by using an engineered glycotransferase and a chemically reactive sugar as a handle was developed. Here, for the first time, we report the use of this method with some modifications to generate an ADC from a monoclonal antibody, m860, which we identified from a human naïve phage display Fab library by panning against the extracellular domain of human HER2. M860 bound to cell surface-associated HER2 with affinity comparable to that of Trastuzumab (Herceptin), but to a different epitope. The m860ADC was generated by enzymatically adding a reactive keto-galactose to m860 using an engineered glycotransferase and conjugating the reactive m860 to aminooxy auristatin F. It exhibited potent and specific cell-killing activity against HER2 positive cancer cells, including trastuzumab-resistant breast cancer cells. This unique ADC may have utility as a potential therapeutic for HER2 positive cancers alone or in combination with other drugs. Our results also validate the keto-galactose/engineered glycotransferase method for generation of functional ADCs, which could potentially also be used for preparation of ADCs targeting other disease markers.
authors.affiliation: a Protein Interactions Group; Laboratory of Experimental Immunology; Cancer and Inflammation Program; Center for Cancer Research; National Cancer Institute; National Institutes of Health ; Frederick , MD , USA.
authors.forename: Zhongyu
authors.initials: Z
authors.lastname: Zhu
authors.forename: Boopathy
authors.initials: B
authors.lastname: Ramakrishnan
authors.forename: Jinyu
authors.initials: J
authors.lastname: Li
authors.forename: Yanping
authors.initials: Y
authors.lastname: Wang
authors.forename: Yang
authors.initials: Y
authors.lastname: Feng
authors.forename: Ponraj
authors.initials: P
authors.lastname: Prabakaran
authors.forename: Simona
authors.initials: S
authors.lastname: Colantonio
authors.forename: Marzena A
authors.initials: MA
authors.lastname: Dyba
authors.forename: Pradman K
authors.initials: PK
authors.lastname: Qasba
authors.forename: Dimiter S
authors.initials: DS
authors.lastname: Dimitrov
mesh_terms: D062365:Aminobenzoates / Q000737:chemistry; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002241:Carbohydrates / Q000737:chemistry*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D061986:MCF-7 Cells; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D011485:Protein Binding / Q000276:immunology; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D062365:Aminobenzoates; D000906:Antibodies; D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D016695:Glycosyltransferases; D018796:Immunoconjugates; D009842:Oligopeptides; D004364:Pharmaceutical Preparations; D018719:Erb-b2 Receptor Tyrosine Kinases; C543533:auristatin
keywords: ADC; anti-HER2; antibody-drug conjugates; glycoengineering; site-specific ADC