title: Antibody conjugation via one and two C-terminal selenocysteines.
abstract: Conventional antibody conjugation methods generate antibody-drug conjugates that are heterogeneous mixtures with undefined stoichiometry and variable pharmacokinetic and pharmacodynamic properties. We have previously described a strategy to generate site-specific antibody conjugates by genetic engineering of an antibody with a single C-terminal selenocysteine, the 21st natural amino acid, which displays unique chemical reactivity allowing selective conjugation in the presence of all other natural amino acids. In the present work, we describe a method for expanding this technology to higher drug-to-antibody ratios by genetically engineering an antibody with two C-terminal selenocysteines. Both selenocysteines effectively conjugate to a fluorescent iodoacetamide derivative and the resulting conjugate fully retains its antigen binding capability. Our method provides a platform for creating stoichiometrically defined antibody-drug conjugates for therapeutic intervention.
authors.affiliation: Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA.
authors.forename: Xiuling
authors.initials: X
authors.lastname: Li
authors.affiliation: Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Jiahui
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA; Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA. Electronic address: crader@scripps.edu.
authors.forename: Christoph
authors.initials: C
authors.lastname: Rader
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004337:Drug Carriers / Q000737:chemistry* / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding; D015202:Protein Engineering; D017279:Selenocysteine / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D004337:Drug Carriers; D017279:Selenocysteine
keywords: Antibody conjugation; Antibody engineering; Selenocysteine