Objectives The citrullinating enzyme peptidylarginine deiminase type 4 (PAD4) is the target of a polyclonal group of autoantibodies in patients with rheumatoid arthritis (RA). A subgroup of such antibodies, initially identified by cross-reactivity with peptidylarginine deiminase type 3 (PAD3), is strongly associated with progression of radiographic joint damage and interstitial lung disease and has the unique ability to activate PAD4. The features of these antibodies in terms of their T cell-dependent origin, genetic characteristics and effect of individual antibody specificities on PAD4 function remain to be defined.
Methods We used PAD4 tagged with the monomeric fluorescent protein mWasabi to isolate PAD4-specific memory B cells from anti-PAD4 positive patients with RA and applied single cell cloning technologies to obtain monoclonal antibodies.
Results Among 44 single B cells, we cloned five antibodies with PAD4-activating properties. Sequence analysis, germline reversion experiments and antigen specificity assays suggested that autoantibodies to PAD4 are not polyreactive and arise from PAD4-reactive precursors. Somatic mutations increase the agonistic activity of these antibodies at low calcium concentrations by facilitating their interaction with structural epitopes that modulate calcium-binding site 5 in PAD4.
Conclusions PAD4-activating antibodies directly amplify a key process in disease pathogenesis, making them unique among other autoantibodies in RA. Understanding the molecular basis for their functionality may inform the design of future PAD4 inhibitors.
- rheumatoid Arthritis
- autoimmune diseases
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Handling editor Tore K Kvien
Contributors All authors contributed to the preparation of this manuscript and approved the final version for publication. JS planned the study, cloned, expressed, purified and characterised the monoclonal antibodies, analysed/interpreted the data and wrote the manuscript. ED and AR conceptualised the study and analysed data. KS performed immunoprecipitation assays. GPS and TM provide expertise in the analysis of monoclonal antibodies. COB provided RA samples. MFK generated recombinant proteins. FA conceptualised and designed the study, participated in data analysis, directed the project and wrote the manuscript.
Funding This study was sponsored by MedImmune, the global biologics R&D arm of AstraZeneca, The Jerome L. Greene Foundation and grant number P30AR053503 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS). The content is solely the responsibility of the authors and does not necessarily represent the official views of NIAMS or the National Institutes of Health.
Competing interests FA, ED and AR received a grant from Medimmune and are authors on issued Patent No. 8 975 033 entitled "Human Autoantibodies Specific for PAD3 which are Cross-reactive with PAD4 and their Use in the Diagnosis and Treatment of Rheumatoid Arthritis and Related Diseases". ED previously served on the scientific advisory board for Padlock Therapeutics, Inc. FA serves as consultant for Bristol-Myers Squibb Company. GPS is employed by MedImmune LLC, and owns stocks in Astra Zeneca. TM is employed by Gilead Science and is a former employee of MedImmune LLC. JS, ED, AR and FA submitted an invention disclosure by the Johns Hopkins University that covers the sequences and the use of the human anti-PAD4 monoclonal antibodies. The remaining authors declare that they have no competing interests.
Provenance and peer review Not commissioned; externally peer reviewed.
Author note Tomas Mustelin. Present address: Gilead Sciences, Immunology & Inflammation, Foster City, CA 94404, USA.Maximilian F Konig. Present address: Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.Kevon Sampson. Present address: National Institutes of Health, Bethesda, MD 20892, USA.
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