SAT0675B ANTI-MODIFIED PROTEIN AUTOANTIBODIES IN RA DISPLAY IMPORTANT PEPTIDE CROSS-REACTIVITY BUT YET PROTEIN RECOGNITION SELECTIVITY
Authors: Peter Sahlström, Johanna Steen, Björn Forsström, Philip Titcombe, Ragnhild Stålesen, Ute Nonhoff, Zoltán Konthur, Luca Piccoli, Karin Lundberg, Holger Bang, Daniel Mueller, Anca Catrina, Lars Klareskog, Karl Skriner, Vivianne Malmström, Caroline Grönwall
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> The continuing increase of anti-citrullinated protein autoantibodies (ACPA) titers together with epitope spreading close to onset of disease, suggests that antibody responses to different citrullinated antigens may be critical in rheumatoid arthritis (RA) pathogenesis. Interestingly, monoclonal antibodies demonstrate reactivity to multiple cit-antigens that can even expand to other protein modifications. The width of this cross-reactivity is still not understood. <h3>Objectives:</h3> To characterize the targets of monoclonal ACPA in relation to amino acid motif recognition, cross-reactivity with others post-translational modifications, and cellular localization. <h3>Methods:</h3> A peptide array (NimbleGen, Roche) containing 16aa arginine- or lysine in pairs with citrulline- or homocitrulline peptides (53019 and 49211 cognate peptide pairs, respectively) derived from 1610 extracellular proteins and known RA cit-targets was used to screen 12 monoclonal ACPA with CCP2 reactivity. In addition, these ACPA were also screened for reactivity to acetylated-histone peptides and for reactivity to acetylated HeLa cell-extracts from cytosol, membrane, nuclear and cytoskeleton fractions. Three of the described mAbs together with polyclonal anti-CCP2 IgG were further evaluated on a macroarray platform (HEXselect, Engine) consisting of 20776 <i>E.coli</i> on-array expressed His-tagged protein fragments from 6909 genes originating from a human cDNA library. The array was enzymatically citrullinated with rabbit PAD and mAb-reactivity was scored from 0-3. <h3>Results:</h3> On the peptide arrays, all 12 ACPA displayed low reactivity to unmodified peptides (&lt;0.06%), while reacting to 1,000s of synthetically citrullinated peptides (&gt;3,4% of the peptides). Based on the sequence from the positive peptides, consensus amino acids motifs were created, identifying aa-patterns with only a few critical citrulline-flanking residues (e.g. Cit-Gly; Gly-Cit; Arg-Cit-Asp). Intriguingly, five of the antibodies also reacted with the carbamylated peptides (&gt;2.2%) and the recognition of certain homocitrulline-motifs also correlated with cross-reactivity to acetylated peptides. Interestingly, these AMPA reacted with acetylated-histones in NETs and apoptotic cells and in the nuclear fraction of <i>in vitro</i> acetylated cell-extracts. Three of the 12 ACPA were further screened on the macroarray and displayed multiple binding to citrullinated proteins and protein fragments identifying primarily previously unknown autoantibody targets (96, 210 or 917 positive hits for the mAbs, scoring 2-3), while limited binding was seen to native proteins. <h3>Conclusion:</h3> ACPA display multi-reactivity to citrullinated peptides and proteins to a much greater extent than previously appreciated. Additionally, some ACPA, but not all, show distinct cross-reactivity to other post-translational modifications. Importantly, different autoreactive clones display modified protein recognition patterns dominated by proteins from different cellular structures. These reactivity profiles are likely to have impact on functionality and pathogenesis. <h3>Disclosure of Interests:</h3> Peter Sahlström: None declared, Johanna Steen: None declared, Björn Forsström: None declared, Philip Titcombe: None declared, Ragnhild Stålesen: None declared, Ute Nonhoff: None declared, Zoltán Konthur Shareholder of: Engine GmbH, Luca Piccoli: None declared, Karin Lundberg: None declared, Holger Bang Shareholder of: Orgentec GmbH, Daniel Mueller: None declared, Anca Catrina Grant/research support from: Yes, but not for the presented study., Lars Klareskog Grant/research support from: Yes, but not for the presented study., Karl Skriner Shareholder of: Engine GmbH, Vivianne Malmström: None declared, Caroline Grönwall: None declared