H and L Chain Affinity Maturation and/or Fab N-Glycosylation Influence Immunoreactivity toward Neutrophil Extracellular Trap Antigens in Rheumatoid Arthritis Synovial B Cell Clones

Abstract

We previously showed that anti-neutrophil extracellular trap (NET) rheumatoid arthritis (RA)-rmAbs derived from CD19 + B cells within RA human synovial tissues frequently react against NETs. In this study, we aimed to characterize the importance of affinity maturation via somatic hypermutation (SHM) within the Ig variable H (VH) and variable L (VL) chains and Fab-N-linked glycosylation in RA synovial B cell clones reactive to NETs and NET-derived Ags such as citrullinated histones. Selected anti-NET RA-rmAbs derived from synovial RA CD19 + B cells were subjected to overlap-PCR to generate germline (GL; VH and VL reverted into GL), hybrid clones (VH/VL region reverted into GL), and N-glycosylation mutants (N→Q) and analyzed for anti-NETs and citrullinated histones (cit-H2B) immunoreactivity. Anti-NET/cit-H2B immunoreactivity of selected RA-rmAbs was abrogated in the VH and VL GL counterpart. In RA B cell hybrid clone RA015/11.88 and RA056/11.23.2, NET and/or cit-H2B immunoreactivity was solely dependent on SHM in the IgVH region whereas RA B cell hybrid clone RA015/11.91 required affinity maturation of both VH and VL for efficient binding to cit-H2B. In 7/80 RA-rmAb, SHM resulted in ex novo N-glycosylation sites in VH and/or VL regions. Removal of Fab-linked glycans in RA056/11.23.2 in the N-mutant counterpart resulted in 90% reduction in immunoreactivity to cit-H2B. Thus, SHM in the IgVH and/or VL regions of RA synovial B cells is necessary for the immunoreactivity to NET-Ags. Fab-N-linked-glycosylation introduction sites are observed in a minority of anti-NET B cell clones but can strongly influence NET-Ag binding.

Sweetening the Deal: Glycosylation and its Clinical Applications

“…In another study, Corsiero et al has shown that N-linked glycosylation in the Fab region of Anti-NET RA-Antibodies were largely responsible for their immunoreactivity. Removing the glycans from the antibodies resulted in a 90% decrease in its reactivity towards citrullinated histone H2B [29]. Findings like these may have major implications on future therapies for autoimmune diseases like rheumatoid arthritis.…”

Impact of N-Linked Glycosylation on Therapeutic Proteins

“…Results from previous studies have provided initial evidence that N-glycans can influence many properties of Fab. For example, it was found that the N-linked glycosylation, introduced by somatic hypermutation (SHM) in the V H /V L regions of the autoantibodies isolated from patients with rheumatoid arthritis, could modulate the binding of Fab to the antigen citrullinated histone (cit-H2B) [54]. In another example, the antigen binding was tested using several anti-adalimumab and anti-infliximab antibody mutants, in which the naturally occurring Fab glycans were removed.…”

Autoantibodies in Rheumatoid Arthritis: Historical Background and Novel Findings

“…This non-conserved glycosylation has been shown to negatively influence the avidity profile of ACPA and seems to be the result of the introduction of new N-glycosylation sites during the extensive somatic hypermutation process [ 116 , 117 ]. In a recent study, the importance of Fab-glycosylation for the recognition of NET-antigens and citrullinated histones was demonstrated [ 118 ]. Interestingly, IgM ACPA do not appear to have this characteristic Fab-glycosylation [ 119 ].…”