Isolation and cloning of stimulatory anti-PDGF receptor auto-antibodies from the immunological repertoire of patients with systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and visceral organs. We have provided evidence that the serum of SSc patients contains stimulatory auto-antibodies directed to the PDGF receptor (PDGFR) that elicit HaRas-ERK1/2 signalling and collagen production in normal human fibroblasts in vitro. Recently, the key role of increased PDGFR activation and signalling in promoting systemic fibrosis has been confirmed in vivo in transgenic mice. To identify the epitopes of PDGFR extracellular domains bound by stimulatory auto-antibodies, B cells from patients with SSc were immortalized with Epstein Barr Virus and a small panel of cultures producing PDGFR-specific immunoglobulins were isolated. mRNA was extracted from such B cells for sequencing and cloning of immunoglobulin variable regions into a human IgG expression vector. Human IgG constructs were expressed in CHO cells, recombinant monoclonal antibodies (rmAbs) were purified from the culture medium and characterised. Selected rmAbs proved to be able to recapitulate properties identified in serum IgG from SSc patients, since they bound to PDGFR and induced reactive oxygen species, pERK and type I collagen gene expression in normal human fibroblasts. Molecular docking simulation predicted that stimulatory rmAbs and non-stimulatory rmAbs recognize different epitopes on PDGFR. This assumption was confirmed through epitope mapping performed by binding competition experiments using a recombinant PDGFR immobilized onto a biosensor. In conclusion, we isolated PDGFR-specific autoantibodies from the immunoglobulin repertoire of SSc patients and identified the specific epitopes bound by stimulatory auto-antibodies that trigger PDGFR signaling, possibly implicated in the pathogenesis of the disease.