Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


PDGF Receptor as Therapeutic and Diagnostic Target in Systemic Sclerosis.

Moroncini4,  Gianluca, Grieco5,  Antonella, Paolini5,  Chiara, Nacci3,  Giulia, Cuccioloni2,  Massimiliano, Mozzicafreddo2,  Matteo, Tonnini5,  Cecilia

Dipartimento di Biologia e Patologia Molecolare e Cellulare, Università Federico II, Napoli, Italy
Dipartimento di Biologia Molecolare, Cellulare e Animale, Università di Camerino, Italy
Dipartimento di Genetica, Biologia e Biochimica, Università di Torino, Italy
Dipartimento di Scienze Mediche e Chirurgiche, Clinica Medica, Università Politecnica delle Marche, Ancona, Italy
Dipartimento di Scienze Mediche e Chirurgiche, Clinica Medica, Università Politecnica delle Marche, Ancona, Italy

Background:

Systemic sclerosis or scleroderma (SSc) is a disease characterized by fibrosis of skin and visceral organs. We have provided evidence that the serum of SSc patients contains stimulatory auto-antibodies (auto-abs) directed to the PDGF receptor (PDGFR) that elicit Ha Ras-ERK 1/2 signaling and collagen production in normal human fibroblasts in vitro (Baroni SS et al, NEJM 2006). A recent study (Olson LE, Soriano P, Dev Cell 2009) has confirmed the central role of increased PDGFR activation and signaling in driving systemic fibrosis in vivo in transgenic mice.

Objectives:

To identify the epitopes of PDGFR extracellular domains bound by stimulatory auto-abs. This information will be used to generate i) PDGFR selective inhibitors and ii) binding assays for detection of functional auto-abs in serum.

Methods and Results:

IgG-positive memory B cells obtained from peripheral blood of SSc patients were immortalized by EBV infection. Supernatants of individual lymphocyte clones were screened by immunofluorescence and flow cytometry for the ability to react selectively with F alpha cells (mouse fibroblasts expressing the human PDGFR alpha), but not with F-/- cells (mock-transfected mouse fibroblasts). Positive clones were further screened for the production of antibodies stimulating reactive oxygen species (ROS) generation in normal human fibroblasts in vitro. Positive clones were expanded in serum-free medium, IgG were purified from supernatants and tested to confirm both binding and biological activity on fibroblasts. mRNA was obtained from such positive B cell clones for sequencing and cloning of antibody variable regions into a human IgG expression vector. Human IgG constructs were expressed in CHO cells, recombinant (rec.) monoclonal antibodies (mAbs) were purified from medium and tested by immunoprecipitation and stimulation experiments. These rec. monoclonal human IgG showed different extents of PDGFR binding and stimulation. Few mAbs displayed the properties identified in total IgG pools purified from serum of SSc patients, since they bound to PDGFR and induced ROS, p-ERK and type I collagen gene expression in normal human fibroblasts. Molecular docking simulation indicated that the PDGFR epitopes bound by stimulatory mAbs differ from those of non-biologically active mAbs. Epitope mapping was confirmed by binding competition experiments using a rec. PDGFR immobilized onto a biosensor. A peptide library is under construction to further define the map of PDGFR epitopes targeted by the different mAbs.

Conclusions:

We identified the specific epitopes bound by functional PDGFR auto-abs that trigger PDGFR signaling. This information is shedding light on the structure of active PDGFR domains, on SSc pathogenesis, and can be used to devise new therapeutic strategies to block PDGFR signaling and, specifically, the progression of SSc. Moreover, synthetic polypeptides corresponding to these functional PDGFR domains will be employed to selectively detect stimulatory auto-abs in serum, possibly implicated in the early phases of SSc pathogenesis.

To cite this abstract, please use the following information:
Moroncini, Gianluca, Grieco, Antonella, Paolini, Chiara, Nacci, Giulia, Cuccioloni, Massimiliano, Mozzicafreddo, Matteo, et al; PDGF Receptor as Therapeutic and Diagnostic Target in Systemic Sclerosis. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :2001
DOI: 10.1002/art.29766

Abstract Supplement

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