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

Objective To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor [alpha] (PDGFR[alpha]) in systemic sclerosis (SSc) and develop novel assays for detection of serum anti-PDGFR[alpha] autoantibodies. Methods Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFR[alpha] and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFR[alpha] 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[alpha] recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFR[alpha] extracellular domains, and expression analyses of alanine-scanned PDGFR[alpha] mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFR[alpha] autoantibodies or, selectively, the agonistic ones. Results Three types of anti-PDGFR[alpha] recombinant human mAb, with the same VH but distinct VL chains, were generated. Nonagonistic VHPAM-V[kappa]13B8 recognized 1 linear epitope, whereas agonistic VHPAM-Vλ16F4 and VHPAM-V[kappa]16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFR[alpha] 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 VHPAM-V[kappa]16F4-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 VHPAM-V[kappa]16F4 epitope inhibited PDGFR[alpha] signaling triggered by serum IgG from SSc patients. Conclusion Agonistic anti-PDGFR[alpha] autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFR[alpha] signaling in patients with SSc.