title: IgG4 autoantibodies against muscle-specific kinase undergo Fab-arm exchange in myasthenia gravis patients.
abstract: Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies are pathogenic.
authors.affiliation: Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands.
authors.forename: Inga
authors.initials: I
authors.lastname: Koneczny
authors.affiliation: Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands.
authors.forename: Jo A A
authors.initials: JA
authors.lastname: Stevens
authors.affiliation: Department of Clinical and Experimental Medicine, Neurology Unit, University of Pisa, Via Paradisa 2, 56124 Pisa, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: De Rosa
authors.affiliation: Neurology Department, Nuffield Department of Clinical Neurosciences, West Wing, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom.
authors.forename: Saif
authors.initials: S
authors.lastname: Huda
authors.affiliation: Department of Neurology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands; Department of Human Genetics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Maartje G
authors.initials: MG
authors.lastname: Huijbers
authors.affiliation: Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Saxena
authors.affiliation: Department of Clinical and Experimental Medicine, Neurology Unit, University of Pisa, Via Paradisa 2, 56124 Pisa, Italy.
authors.forename: Michelangelo
authors.initials: M
authors.lastname: Maestri
authors.affiliation: Hellenic Pasteur Institute, 127 Vasilissis Sofias Avenue 115 21, Ampelokipi, Athens, Greece; Neurology Department, University Hospital, Herestraat 49, 3000 Leuven, Belgium.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Lazaridis
authors.affiliation: Hellenic Pasteur Institute, 127 Vasilissis Sofias Avenue 115 21, Ampelokipi, Athens, Greece; Neurology Department, University Hospital, Herestraat 49, 3000 Leuven, Belgium.
authors.forename: Paraskevi
authors.initials: P
authors.lastname: Zisimopoulou
authors.affiliation: Hellenic Pasteur Institute, 127 Vasilissis Sofias Avenue 115 21, Ampelokipi, Athens, Greece; Neurology Department, University Hospital, Herestraat 49, 3000 Leuven, Belgium.
authors.forename: Socrates
authors.initials: S
authors.lastname: Tzartos
authors.affiliation: Department of Neurology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: Verschuuren
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Silvère M
authors.initials: SM
authors.lastname: van der Maarel
authors.affiliation: Neurology Department, University Hospital, Herestraat 49, 3000 Leuven, Belgium; KU Leuven - University of Leuven, Department of Neurosciences, VIB - Vesalius Research Center, Experimental Neurology - Laboratory of Neurobiology, Leuven, Belgium.
authors.forename: Philip
authors.initials: P
authors.lastname: van Damme
authors.affiliation: Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands.
authors.forename: Marc H
authors.initials: MH
authors.lastname: De Baets
authors.affiliation: Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Molenaar
authors.affiliation: Neurology Department, Nuffield Department of Clinical Neurosciences, West Wing, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom.
authors.forename: Angela
authors.initials: A
authors.lastname: Vincent
authors.affiliation: Department of Clinical and Experimental Medicine, Neurology Unit, University of Pisa, Via Paradisa 2, 56124 Pisa, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Ricciardi
authors.affiliation: Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands. Electronic address: p.martinez@maastrichtuniversity.nl.
authors.forename: Pilar
authors.initials: P
authors.lastname: Martinez-Martinez
authors.affiliation: Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands. Electronic address: m.losen@maastrichtuniversity.nl.
authors.forename: Mario
authors.initials: M
authors.lastname: Losen
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D018033:Antibodies, Bispecific / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D015551:Autoimmunity / Q000276:immunology; D005260:Female; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D009157:Myasthenia Gravis / Q000175:diagnosis / Q000276:immunology*; D020794:Receptor Protein-Tyrosine Kinases / Q000276:immunology*; D011950:Receptors, Cholinergic / Q000276:immunology*; D055815:Young Adult
chemical_list: D018033:Antibodies, Bispecific; D001323:Autoantibodies; D001324:Autoantigens; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011950:Receptors, Cholinergic; C095714:MUSK protein, human; D020794:Receptor Protein-Tyrosine Kinases
keywords: Autoimmunity; Fab-arm exchange; IgG4; MuSK; Myasthenia gravis