Background/Purpose:

The presence of serum IgG anti-citrullinated protein autoantibodies (ACPA) in rheumatoid arthritis (RA), is associated with more aggressive disease progression. Although ACPA have been suggested to be important in the pathogenesis, the mechanisms and pathogenic targets are still poorly described. ACPA multi-reactivity to synthetically citrullinated peptides have been reported, yet how this corresponds to ACPA full-length protein targeting have not previously been explored. Here we used a novel protein array approach to investigate the citrulline reactivity broadness of three single-cell derived ACPA monoclonal antibodies (mAbs) compared to polyclonal IgG anti-CCP2.

Methods:

The macroarray platform (hEXselect, Engine) consists of 20 776 E.coli on-array expressed protein fragments from 6 909 genes with between 1-37 fragments for each gene. All originating from human fetal brain cDNA, cloned into an IPTG inducible vector with N-terminal RGS-His6-Tag as expression control. The array was enzymatically citrullinated with rabbit PAD. Duplicated spotting and alkaline phosphatase conjugated anti-human IgG (Fc) with AttoPhos substrate enabled reliable spectral fluorescent intensity detection that were scored from 0-3 using a grid based analyzing software. Binding of two recombinant ACPA mAbs derived from RA synovial plasma cells (1325:01B09 and 1325:04C03) and one mAb derived from tetramer sorted blood memory cells (37CEPT2C04) were evaluated. The technology was validated with ELISA and Western blot using individually expressed selected protein fragments.

Results:

Purified polyclonal IgG anti-CCP2 from ACPA+ RA patients contained, as expected, reactivity to a range of citrullinated full-length proteins and protein fragments (674 total hits, 154 with visual scoring 3). However, interestingly, the individually analyzed monoclonal ACPA also displayed reactivity to a large number of citrullinated targets. While some shared targets were identified, also unique mAb-specific antibody targets were detected. The ACPA mAb 1325:01B09 reacted to in total 1790 citrullinated protein fragments (1328 mAb-unique; 190 with visual scoring 3), 1325:04C03 reacted to 408 (115 mAb-unique; 42 with visual scoring 3), and 37CEPT2C04 reacted to 792 (487 mAb-unique; 118 with visual scoring 3). Reactivity to native unmodified proteins were limited (53, 19, or 22 hits, respectively). Among the identified proteins were known targets e.g. Cit-vimentin, Cit-hnRNPs, and Cit-histones, as well as less studied RA autoimmunity targets e.g. Cit-40S ribosomal proteins.

Conclusion:

All investigated monoclonal ACPA were multi-reactive to citrullinated protein targets to a much larger extent than previously described and bound hundreds of full-length proteins and protein fragments. Nevertheless, ACPA mAbs demonstrate individual distinct binding patterns. Further studies on how ACPA bind to proteins in cells during physiological citrullination and the clinical relevance of ACPA multi-reactivity will be of importance.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/individual-ra-derived-monoclonal-anti-citrullinated-protein-autoantibodies-acpa-have-extensive-citrulline-multi-reactivity-demonstrated-by-a-large-scale-protein-array-platform/