Article Text

PDF
FRI0007 The Importance of Somatic Hypermutation for the Immunoreactivity Towards Neutrophil Extracellular Traps (NETS)-Citrullinated Autoantigens of Ra Synovial Monoclonal Antibodies
  1. E. Corsiero,
  2. E. Carlotti,
  3. C. Pitzalis,
  4. M. Bombardieri
  1. Centre of Experimental Medicine and Rheumatology, William Harvey Research Institute, London, United Kingdom

Abstract

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.

References

  1. 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

Statistics from Altmetric.com

Request permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.