Epitope Specificity Determines Pathogenicity and Detectability of Anti-Platelet-Derived Growth Factor Receptor Autoantibodies in Systemic Sclerosis

ObjectiveTo identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor (PDGFR) in systemic sclerosis (SSc) and develop novel assays for detection of serum anti-PDGFR autoantibodies. MethodsEpstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFR and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFR IgG were cloned into an IgG expression vector to generate distinct recombinant human monoclonal autoantibodies (mAb), which were characterized by binding and functional assays. The epitopes of anti-PDGFR recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFR extracellular domains, and expression analyses of alanine-scanned PDGFR mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFR autoantibodies or, selectively, the agonistic ones. ResultsThree types of anti-PDGFR recombinant human mAb, with the same V-H but distinct V-L chains, were generated. Nonagonistic V(H)PAM-V13B8 recognized 1 linear epitope, whereas agonistic V(H)PAM-V16F4 and V(H)PAM-V16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFR antibodies were detected in 66 of 70 patients with SSc, 63 of 130 healthy controls, 11 of 26 patients with primary Raynaud's phenomenon (RP), and 13 of 29 patients with systemic lupus erythematosus (SLE). Serum V(H)PAM-V16F4-like antibodies were found in 24 of 34 patients with SSc, but not in healthy controls, patients with primary RP, or patients with SLE. Peptides composing the V(H)PAM-V16F4 epitope inhibited PDGFR signaling triggered by serum IgG from SSc patients. ConclusionAgonistic anti-PDGFR autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFR signaling in patients with SSc.