title: Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation.
abstract: Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.
authors.affiliation: a Genmab ; Utrecht , The Netherlands.
authors.forename: Ewald T J
authors.initials: ET
authors.lastname: van den Bremer
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Beurskens
authors.forename: Marleen
authors.initials: M
authors.lastname: Voorhorst
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Engelberts
authors.forename: Rob N
authors.initials: RN
authors.lastname: de Jong
authors.forename: Burt G
authors.initials: BG
authors.lastname: van der Boom
authors.forename: Erika M
authors.initials: EM
authors.lastname: Cook
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Lindorfer
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Taylor
authors.forename: Patrick Hc
authors.initials: PH
authors.lastname: van Berkel
authors.forename: Paul Whi
authors.initials: PW
authors.lastname: Parren
mesh_terms: D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D003167:Complement Activation / Q000235:genetics / Q000276:immunology*; D015922:Complement C1q / Q000235:genetics / Q000276:immunology*; D003602:Cytotoxicity, Immunologic; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology*; D008239:Lysine / Q000235:genetics / Q000276:immunology; D020125:Mutation, Missense
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D015922:Complement C1q; D008239:Lysine
keywords: ADCC, antibody-dependent cell-mediated cytotoxicity ; CDC, complement-dependent cytotoxicity; CEX, cation-exchange; cIEF, capillary isoelectric focusing; CPB, carboxy peptidase B; ESI-MS, electrospray ionization mass spectrometry; IEF, isoelectric focusin; complement activation; herapeutic antibody; post-translational control