Session Type: ACR Concurrent Abstract Session
Session Time: 4:30PM-6:00PM
We previously showed that anti-citrullinated peptide/protein antibodies (ACPA) targeting citrullinated antigens contained in neutrophils extracellular traps (NETs) can be manufactured within ectopic germinal center-like structure (GC-LS) in the rheumatoid arthritis (RA) synovium. Here, we aimed to characterise a) whether the RA synovial anti-NETs antibodies had undergone antigen-driven affinity maturation, b) the importance of somatic hypermutations (SHM) within the VH and VL chain for their binding and c) whether the presence of N-glycosylation sites in the variable domains introduced by SHM can modulate the reactivity towards NETs.
82 full recombinant monoclonal antibodies (RA-syn-rmAbs) were generated from single CD19+ B cells FACS-sorted from fresh GC-LS+ synovial cell suspensions following IgVH+VL genes cloning. RA-syn-rmAbs for reversion experiments (n=9) were chosen according to their level of reactivity towards NETs. Reversion of the IgV genes into germ-line (GL) and generation of hybrid clones (where VH, VL or selected CDRs/FRs were reverted into GL, n=5) was performed by overlap-PCR. Further characterization of the mature vs GL RA-syn-rmAbs sequence was performed by structural alignment via an in silicoprediction model using the root-mean-square-deviation (RMSD) score to measure the similarity between the mature and GL structures (1). Anti-NETs immunoreactivity was detected using cell-based immunoassays with activated peripheral blood or RA synovial fluid neutrophils.
Structural alignment showed some differences in the 3D structure between mature vs GL sequences in 3 RA-syn-rmAbs suggesting that SHM is critical for antigen binding. Accordingly, the anti-NETs immunoreactivity of the RA-syn-rmAbs was dependent on affinity maturation within GC-LS and was completely abrogated when the full IgVH+VL genes were reverted to GL. Conversely, when only the IgVL gene was reverted to GL, 2 out of 5 RA-syn-rmAbs displayed a residual reactivity toward NETs suggesting that in some clones the VH chain plays a dominant role in conferring the reactivity towards NETs. Preliminary data also showed that the increased molecular weight observed in selected anti-NET antibodies, likely due to the presence of Fab-linked N-glycans sites (2), was lost in the GL counterpart.
Importantly, our data showed that SHM is necessary for the development of high-affinity NETs-binding antibodies in synovial GC-LS. The importance of defining the contribution of individual CDRs and FRs to the affinity of antigen-binding sites may help to engineer new therapeutic Abs and design of CDRs/FRs-specific peptides for tolerogenic strategy.
1. Lyskov S, et al. Serverification of molecular modeling applications: the Rosetta Online Server that Includes Everyone (ROSIE). PloS one. 2013;8(5):e63906.
2. Rombouts Y, et al. Extensive glycosylation of ACPA-IgG variable domains modulates binding to citrullinated antigens in rheumatoid arthritis. Ann Rheum Dis. 2015.
To cite this abstract in AMA style:Corsiero E, Carlotti E, Pitzalis C, Bombardieri M. The Importance of Somatic Hypermutation for the Immunoreactivity Towards Neutrophil Extracellular Traps (NETs)-Citrullinated Autoantigens of RA Synovial Monoclonal Antibodies [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/the-importance-of-somatic-hypermutation-for-the-immunoreactivity-towards-neutrophil-extracellular-traps-nets-citrullinated-autoantigens-of-ra-synovial-monoclonal-antibodies/. Accessed August 11, 2020.
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