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A2.04 The role of somatic hypermutation and n-glycosylation in the anti-nets immunoreactivity of ra synovial monoclonal antibodies
  1. E Corsiero,
  2. E Carlotti,
  3. E Prediletto,
  4. L Jagemann,
  5. C Pitzalis,
  6. M Bombardieri
  1. Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, UK

Abstract

Background and objectives 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 centre-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/VL chain for their binding and c) whether the presence of N-glycosylation sites in the Fab domains introduced by SHM can modulate the reactivity towards NETs.

Materials and methods 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. N-glycosylation sites (N-x-S/T) in the Fab domains were predicted using the NetNGlyc1.0 Server and the presence of glycans was detected by total glycoprotein staining on gel. Reversion of the IgV genes into germ-line (GL), generation of hybrid clones (where VH/VL or selected CDRs/FRs were reverted into GL, n = 5) and N-glycosylation mutants (where asparagine (N) was substituted with glutamine (Q), n = 7) was performed by overlap-PCR. Anti-NETs immunoreactivity was detected using cell-based immunoassays with activated peripheral blood or RA synovial fluid neutrophils.

Results The RA-syn-rmAbs anti-NETs immunoreactivity was dependent on affinity maturation within GC-LS and was completely abrogated when the full IgVH+VL genes were reverted to GL. Similarly, when only the single IgVH/VL gene was reverted to GL, the reactivity towards NETs was lost suggesting that both VH+L chains are important in conferring the reactivity towards NETs. Moreover, the increased molecular weight observed in selected anti-NET antibodies was dependent on the presence of Fab-linked glycans and was lost in the GL counterpart.

Conclusions Importantly, our data showed that SHM is necessary for the development of high-affinity NETs-binding antibodies in synovial GC-LS and for the introduction of N-glycosylation sites in the Fab domain which could influence the NET-antigens binding. 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.

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