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

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.

Figure 1

Design, expression and characterization of mutated PR3. (a) Design of mutated PR3. Schematic representation of the PR3 variants. The signal sequence, prosequences, and C-terminal sequence are shown as grey, black and speckled boxes, respectively. Exons are indicated with the exon number in the box. The amino acids H⁴⁴, D⁹¹ and S¹⁷⁶ forming the catalytic triad and the two glycosylation sites on N¹⁰² and N¹⁴⁷ are marked at the wild-type PR3. PR3 variants are listed below, the introduced point mutations are written in parenthesis. (b) Enzymatic activity of rPR3; 500 ng PR3 variants were incubated with 500 ng PR3H44Q-Ang fusion protein as substrate for 1, 2, 4 and 20 hr at room temperature. Then, protein decomposition was measured by SDS–PAGE analysis. Proteins were visualized by Coomassie staining. (c) Affinity of rPR3 variants to anti-PR3 mAb. The rPR3 constructs were tested by direct ELISA using WGH1, WGM2, or WGM3 mAb. Bound antibody was detected by horseradish peroxidase-conjugated goat anti-mouse antibody. Therefore rPR3 was coated with a final concentration of 100 ng. The anti-CD30 scFv fragment was used as a non-specific control. Error bars represent SD values of triplicates.

Figure 2

Expression and purification of PR3 fusion proteins. Western blot analysis of PR3H44Q, PR3H44Q-Ang and NAng- PR3H44Q. The proteins were expressed in 293T cells and purified by Ni²⁺-NTA metal affinity chromatography. Proteins were analysed by SDS–PAGE under non-reducing conditions and visualized by immunostaining of the Western blot using mouse anti-penta-His mAb and a horseradish peroxidase-conjugated secondary donkey anti-mouse antibody, followed by ECL mediated chemiluminescence reaction.

Figure 3

Binding of anti-PR3 mAb to PR3 fusion proteins by direct ELISA. Cell culture supernatant of WGM2 and WGM3 cells or mouse anti-angiogenin mAb (1 : 2000) was used for binding analysis; bound antibody was detected by horseradish peroxidase-conjugated goat anti-mouse antibody. Error bars represent SD values of triplicates.

Figure 4

Detection of PR3-toxin-induced apoptosis by TUNEL technology, staining apoptotic cells red. To preserve fluorescence, cytospin preparations were coverslipped with mounting medium containing DAPI. WGM2 cells treated with PR3H44Q (a), PR3H44Q-Ang (b) and NAng-PR3H44Q (c); WGM3 cells treated with PR3H44Q (d), PR3H44Q-Ang (e) and NAng-PR3H44Q (f).

Figure 5

Flow cytometric analysis of PR3-toxin-induced apoptosis by annexin V binding assay. Cells were incubated with CD293 cell culture supernatant of 293T cells containing PR3H44Q, PR3H44Q-Ang or NAng-PR3H44Q (approx. 50 μg/ml), respectively. Cells were incubated for 6 hr at 37°, 5% CO₂. Apoptotic cell death was measured by flow cytometry using fluorescein-conjugated annexin V as described in the Materials and methods section. The error bars represent the standard deviation of three independent experiments.