Autoimmune responses against posttranslationally modified antigens are a hallmark of several autoimmune diseases. For example, antibodies against citrullinated protein antigens (ACPA) have shown their relevance for the prognosis and diagnosis of rheumatoid arthritis (RA), and have been implicated in disease pathogenesis. It is conceivable that other autoantibody systems, recognizing other posttranslationally modified proteins, are also present in RA. Here, we describe the presence of an autoantibody system that discriminates between citrulline- and homocitrulline-containing antigens in the sera of RA-patients. IgG antibodies recognizing carbamylated (homocitrulline-containing) antigens were present in sera of over 45% of RA-patients. Likewise, anticarbamylated protein (anti-CarP) IgA antibodies were observed in 43% of RA-sera. ACPA and anti-CarP antibodies are distinct autoantibodies because, in selected double-positive patients, the anti-CarP antibody binding to carbamylated antigens could be inhibited by carbamylated antigens, but not by control or citrullinated antigens. Similarly, ACPA-binding to citrullinated antigens could only be inhibited by citrullinated antigens. In line with this observation, 16% of ACPA-negative RA-patients, as measured by a standard ACPA assay, harbored IgG anti-CarP antibodies, whereas 30% of these patients tested positive for IgA anti-CarP antibodies. The presence of anti-CarP antibodies was predictive for a more severe disease course in ACPA-negative patients as measured by radiological progression. Taken together, these data show the presence of a unique autoantibody system recognizing carbamylated, but not citrullinated, protein antigens. These antibodies are predictive for a more severe clinical course in ACPA-negative RA-patients, indicating that anti-CarP antibodies are a unique and relevant serological marker for ACPA-negative RA.

The vast majority of ACPA-IgG harbour N-glycans in their variable domains. As N-linked glycosylation requires glycosylation consensus sites in the protein sequence and as these are lacking in the 'germline-counterparts' of identified variable domains, our data indicate that the N-glycosylation sites in ACPA variable domains have been introduced by somatic hypermutation. This finding also suggests that ACPA-hyperglycosylation confers a selective advantage to ACPA-producing B cells. This unique and completely novel feature of the citrulline-specific immune response in RA elucidates our understanding of the underlying B cell response.

In addition to ACPA and IgM-RF, also the newly identified anti-CarP antibodies appear many years before the diagnosis of RA.

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Objective. Recently, we discovered a new autoantibody system in rheumatoid arthritis (RA): anticarbamylated protein (anti-CarP) antibodies. These antibodies have value in predicting joint destruction; however, it is not clear whether they are present before the diagnosis of RA and whether they have value as predictors of RA development. Therefore, we studied whether anti-CarP antibodies are present in patients with arthralgia and whether their presence is associated with the development of RA. Conclusion. Anti-CarP antibodies are present in patients with arthralgia, and their presence predicts the development of RA independent of anti-CCP-2 antibodies.

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Objective Anti-carbamylated protein (anti-CarP) antibodies could further elucidate early RA pathogenesis and predict clinical disease. We compared diagnostic accuracy of anti-CarP antibodies for future RA to other RA-related antibodies in military personnel. Methods Stored pre-RA diagnosis serum samples from 76 RA cases were tested for anti-CarP Fetal Calf Serum (FCS), anti-CarP Fibrinogen (Fib), anti-CCP2, RF-Neph, and RF-isotypes (IgM, IgG, and IgA). Positivity for all antibodies was determined as ≥2SD of log-transformed means from controls. Relationships between autoantibodies and future RA were assessed in prediagnosis serum for all RA cases compared to controls using sensitivity, specificity, and logistic regression. Differences in diagnostic accuracy between antibody combinations were assessed using comparisons of area under the curves (AUCs). Results Anti-CarP-FCS was 26% sensitive and 95% specific for future RA, where anti-CarP-Fib was 16% sensitive and 95% specific for future RA. Anti-CarP-FCS positivity was associated with future RA, while anti-CarP-Fib trended towards association. The antibody combination of anti-CCP2 and/or ≥2 RFs (RF-Neph and/or RF-isotypes) resulted in an AUC of 0.72 for future RA, where the AUC was 0.71 with the addition of anti-CarP-FCS to this prior combination. Conclusion Adding anti-CarP-FCS to antibody combinations did not improve AUC. However, anti-CarP-FCS was associated with future onset of RA, and was present in prediagnosis serum in ~10% of RA cases negative for anti-CCP2, but positive for RF.

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