Background In a previous study, we have identified endogeneously citrullinated sites in fibrinogen from RA synovial tissue¹. Within the alpha chain, Arg573 and Arg591 were found citrullinated with an occupancy rate in the range of 1-2% and in the β-chain, Arg72 and Arg74 were also found citrullinated.

Objectives In the present study, we demonstrate that citrullinated fibrinogen peptides are autoantigenic and can be used as probes for development of ACPA neutralizing compounds opening new opportunities for treatment of CCP positive RA patients.

Methods The autoantigenic potential was investigated using the Phadia´s ImmunoCAP ISAC® system. Citrullinated and unmodified fibrinogen peptides were immobilized onto a glass slide in an arrayed fashion and serum from 404 CCP positive and 532 CCP negative RA patients and 461 healthy controls from the EIRA cohort were tested. We also assayed the identified citrulline fibrinogen peptides for their ability to prevent purified ACPA² to bind to CCP (CCPlus® ELISA, Euro-Diagnostica AB). Peptides were individually or in combinations incubated with different ACPA pools and the blocking efficiency was expressed as percent of inhibition and IC50. Corresponding arginine peptides were used as controls.

Results We found that 31% (87% are CCP positive) of patients were positive to Cit573 peptide. For the Cit591 peptide, the corresponding numbers were 10% (65%), for the Cit74 peptide 28% (68%) and for the Cit72 peptide 20% (68%). Interestingly, citrullinated 573 and Cit591 peptides revealed a maximum of 77% and 48% ACPA inhibition, respectively. When equally mixed, these peptides displayed an additive higher degree of ACPA neutralization (84%). In contrast, Cit74 and Cit72 peptides reached a more modest maximum inhibition of 26% and 30%, respectively. This experiment was repeated using a different set of ACPA pool and then the efficiencies were lower for Cit573 (47%) but similar for Cit591 (51%). Logically, the efficiency of specific citrullinated compounds will depend on the individual ACPA specificities.

Conclusions Here we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes found in RA synovial membranes. These peptides can now be used as additional biomarkers for studies of ACPA sub-specificity profiles as recently reported³. We also demonstrate that these citrullinated peptides can be used as neutralizing agents blocking a significant portion of ACPA binding to CCP. These results open novel possibilities for the design of personalized ACPA blockers preventing for instance the osteoclastogenesis and bone loss induced by ACPA⁴.

References

1. Hermansson M. et al. Proteomics: Clinical Applications., 2010, May; 4(5):511-8;

2. Ossipova, E., Cerqueira, C., et al 2013, submitted;

3. Brink, M., et al., Arthritis Rheum. 2013 Jan 10, in press;

4. Harre, U. et al., J Clin Invest. 2012 May 1;122(5):1791-802

Disclosure of Interest None Declared