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

Abstract

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

Blockade of PDGF Receptors by Crenolanib Has Therapeutic Effect in Patient Fibroblasts and in Preclinical Models of Systemic Sclerosis

“…Increased PDGF-A has also been reported in the dermal interstitial blister fluid of SSc patients (Clark et al, 2015). In addition, agonistic anti-PDGFRα autoantibodies have been detected in the serum of SSc patients (Moroncini et al, 2015). …”

Induction of Scleroderma Fibrosis in Skin‐Humanized Mice by Administration of Anti−Platelet‐Derived Growth Factor Receptor Agonistic Autoantibodies

“…Furthermore, the chemical proteomic profile of nilotinib reveals that a nonkinase target, such as the oxidoreductase quinone reductase 2, which contributes to superoxide generation (28), is also inhibited by the drug. Thus, to confirm that the activation of fibrosis in vivo by autoantibodies occurs through PDGFR, healthy control skin-humanized mice were injected with human anti-PDGFR monoclonal antibodies generated from SSc B cells, as previously described by us (14). Human anti-PDGFR monoclonal antibody V H PAM-V k 16F4, which, upon binding and stimulating the receptor, induces the scleroderma phenotype in normal human fibroblasts in vitro (14), induced fibrosis in vivo when compared to V H PAM2V k 13B8, which binds but does not stimulate the receptor in vitro (14).…”

“…Thus, to confirm that the activation of fibrosis in vivo by autoantibodies occurs through PDGFR, healthy control skin-humanized mice were injected with human anti-PDGFR monoclonal antibodies generated from SSc B cells, as previously described by us (14). Human anti-PDGFR monoclonal antibody V H PAM-V k 16F4, which, upon binding and stimulating the receptor, induces the scleroderma phenotype in normal human fibroblasts in vitro (14), induced fibrosis in vivo when compared to V H PAM2V k 13B8, which binds but does not stimulate the receptor in vitro (14). This was demonstrated in the current study by morphometric analysis of picrosirius staining of the healthy control skin grafts (area stained as a percentage [mean 6 SD] of the area stained in PBS-treated healthy control skin grafts [set at 100] 139 6 20 and 97 6 15 with V H PAM-V k 16F4 and V H PAM2V k 13B8 treatment, respectively [P , 0.01]) ( Figure 6A) and COL1A2 gene expression in the skin grafts (fold increased expression 3.6 6 0.5 with V H PAM-V k 16F4 versus 1.05 6 0.25 with V H PAM2V k 13B8 and 1 6 0.3 with PBS [P , 0.01]) ( Figure 6B).…”

Peptide-Based Vaccination Therapy for Rheumatic Diseases

“…A careful screening of epitope peptides is a critical prerequisite for the efficacy and safety of peptide-based vaccination therapy in autoimmune diseases including rheumatic 7 Journal of Immunology Research diseases [214,215]. During the development of rheumatic diseases, epitope specificity exists in the pathogenic roles of antigens or cytokines, and different epitope peptides thus can exert obviously distinct roles in modulating immune response [216,217]. Some epitope peptides can precipitate but not inhibit disease progression.…”