Background 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.
Objectives To characterise a) whether the RA synovial anti-NETs antibodies had undergone antigen-driven affinity maturation and b) the importance of somatic hypermutations (SHM) within the VH and VL chain for their binding.
Methods 66 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 silico prediction 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.
Results 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.
Conclusions 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.
Lyskov S, et al. Serverification of molecular modeling applications: the Rosetta Online Server that Includes Everyone (ROSIE). PloS one. 2013;8(5):e63906.
Disclosure of Interest None declared