Selective killing of B-cell hybridomas targeting proteinase 3, Wegener's autoantigen

Immunology. 2004 Jun;112(2):228-36. doi: 10.1111/j.1365-2567.2004.01875.x.

Abstract

Wegener's granulomatosis (WG) is a rare disease characterized by granulomatous lesions, small vessel vasculitis and the presence of anti-neutrophil cytoplasmic autoantibodies (C-ANCAs) in the sera of affected patients. Their main target antigen is proteinase 3 (PR3), a neutrophil and monocyte-derived neutral serine protease. Since the standard treatment of this severe autoimmune disease, with cyclophosphamide and corticosteroids, is associated with potential side-effects, the development of a more specific immunotherapeutic agent is warranted. The key role of ANCA in the pathogenesis of vasculitis and the effectiveness of anti-CD20 antibodies in patients with refractory WG points towards the importance of B cells in WG. We thus evaluated a new approach to selectively eliminate PR3-specific autoreactive B cells by targeting the B-cell receptor. For this purpose we used a bifunctional recombinant fusion protein consisting of the antigen PR3 and a toxin. The cytotoxic component of this novel fusion protein was the ribonuclease angiogenin, a human toxin with low immunogenicity. The toxin was stabilized by exchanging the catalytically relevant histidine in position 44 with glutamine to eliminate the autoproteolytic activity. PR3H44Q was fused either to the N terminus or to the C terminus of angiogenin. The recombinant proteins were expressed in 293T cells. Binding assays demonstrated the appropriate size and recognition by anti-PR3 antibodies. Using TUNEL technology, we demonstrated that these autoantigen toxins kill proteinase 3-specific B-cell hybridomas selectively by inducing apoptosis. The data indicate that autoantigen-toxins are promising tools in the treatment or co-treatment of autoimmune diseases in which the antigen is known.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / immunology
  • Apoptosis / immunology
  • Autoantigens / immunology*
  • Autoimmune Diseases / immunology
  • B-Lymphocytes / immunology*
  • Cells, Cultured
  • Granulomatosis with Polyangiitis / immunology*
  • Humans
  • Hybridomas / immunology
  • Immunotoxins / immunology*
  • In Situ Nick-End Labeling
  • Mutagenesis, Insertional
  • Myeloblastin
  • Recombinant Fusion Proteins / immunology
  • Ribonuclease, Pancreatic / immunology
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / immunology*
  • Tumor Cells, Cultured

Substances

  • Antibodies, Monoclonal
  • Autoantigens
  • Immunotoxins
  • Recombinant Fusion Proteins
  • angiogenin
  • Ribonuclease, Pancreatic
  • Serine Endopeptidases
  • Myeloblastin