Activation of a Latent Epitope Causing Differential Binding of Antineutrophil Cytoplasmic Antibodies to Proteinase 3

Abstract

Objective Proteinase 3 (PR3) is the major antigen for antineutrophil cytoplasmic antibodies (ANCAs) in the systemic autoimmune vasculitis, granulomatosis with polyangiitis (GPA). PR3‐targeting ANCAs (PR3‐ANCAs) recognize different epitopes on PR3. This study was undertaken to study the effect of mutations on PR3 antigenicity. Methods The recombinant PR3 variants, iPR3 (clinically used to detect PR3‐ANCAs) and iHm5 (containing 3 point mutations in epitopes 1 and 5 generated for epitope mapping studies) immunoassays and serum samples from patients enrolled in ANCA‐associated vasculitis (AAV) trials were used to screen for differential PR3‐ANCA binding. A patient‐derived monoclonal ANCA 518 (moANCA518) that selectively binds to iHm5 within the mutation‐free epitope 3 and is distant from the point mutations of iHm5 was used as a gauge for remote epitope activation. Selective binding was determined using inhibition experiments. Results Rather than reduced binding of PR3‐ANCAs to iHm5, we found substantially increased binding of the majority of PR3‐ANCAs to iHm5 compared to iPR3. This differential binding of PR3‐ANCA to iHm5 is similar to the selective moANCA518 binding to iHm5. Binding of iPR3 to monoclonal antibody MCPR3‐2 also induced recognition by moANCA518. Conclusion The preferential binding of PR3‐ANCAs from patients, such as the selective binding of moANCA518 to iHm5, is conferred by increased antigenicity of epitope 3 on iHm5. This can also be induced on iPR3 when captured by monoclonal antibody MCPR2. This previously unrecognized characteristic of PR3‐ANCA interactions with its target antigen has implications for studying antibody‐mediated autoimmune diseases, understanding variable performance characteristics of immunoassays, and design of potential novel treatment approaches.

Risk of relapse of ANCA-associated vasculitis among patients homozygous for the proteinase 3 gene Val119Ile polymorphism

“…We previously showed that remote mutations and engagement of distal epitopes by an anti-PR3 monoclonal antibody can increase the local mobility and expose latent PR3 epitopes, leading to conformational adaptation facilitating or necessary for antibody binding 16 17. This type of epitope activation—achieved in vitro by remote mutations, or in vivo either by polymorphisms or by binding of another autoantibody or protein ligand—may be an important contributor of PR3-AAV pathogenesis 16 17. Our observation that homozygosity for the PRTN3-Val 119 Ile polymorphism is associated with a different risk for severe relapses is consistent with the hypothesis that the two different PR3 antigen variants may interact differently with pathogenic PR3-ANCA.…”

“…Consequently, homozygosity for this polymorphism, which is the only one resulting in a change of the amino acid sequence of PR3, may represent an additional identifiable risk factor for severe relapse. Moreover, our data suggest that the quantity of PR3 expressed in patients with AAV compared with healthy controls and conformational changes of the autoantigen may modulate its interactions with pathogenic PR3-ANCA promoting disease activity 17 18. Further studies are necessary to validate and better understand the association of this observation with the risk of severe relapse and its possible molecular mechanisms.…”

“…Moreover, our data suggest that the quantity of PR3 expressed in patients with AAV compared with healthy controls and conformational changes of the autoantigen may modulate its interactions with pathogenic PR3-ANCA promoting disease activity. 17 18 Further studies are necessary to validate and better understand the association of this observation with the risk of severe relapse and its possible molecular mechanisms.…”

“…We hypothesise that, in vivo, the presence of polymorphisms in proteins might modulate their antigenicity particularly during states of severe inflammation, similarly to what was found in vitro. We previously showed that remote mutations and engagement of distal epitopes by an anti-PR3 monoclonal antibody can increase the local mobility and expose latent PR3 epitopes, leading to conformational adaptation facilitating or necessary for antibody binding 16 17. This type of epitope activation—achieved in vitro by remote mutations, or in vivo either by polymorphisms or by binding of another autoantibody or protein ligand—may be an important contributor of PR3-AAV pathogenesis 16 17.…”