H HUTTON1,2, J OOI1, S HOLDSWORTH1,2, A.R KITCHING1,2
1Centre for Inflammatory diseases, Monash University, Clayton, VIC; 2Dept Nephrology, Monash Health, Clayton, VIC
Aim: To develop and characterise anti-MPO monoclonal antibodies (mAbs) for use in a model of ANCA associated vasculitis (AAV).
Background: Currently available mouse models of anti-MPO vasculitis vary between centres, and are resource intensive, often necessitating the generation of polyclonal anti-MPO. Human studies suggest that epitope specificity is important in the pathogenicity of anti-MPO antibodies. MPO447-459 (human sequence numbering) is a B cell epitope, conserved in mice, that was exclusively associated with disease in humans.
Methods: Anti-MPO hybridoma cells were produced by Monash Antibody Technology Facility. 2000 single cells were cultured, and supernatants tested against mouse and human MPO by ELISA. Nine clones were selected for further assessment: ELISAs assessed binding to mouse and human MPO, and MPO447-459. Assessment of mAb induction of neutrophil reactive oxygen species (ROS) was performed using a flow cytometric dihydrorhodamine assay. Neutrophil recruitment at 4 hours and 7 days was assessed by injecting mice with 10ug LPS followed by 1mg mAb, polyclonal MPO-ANCA, or control IgG2a; immunohistochemistry on kidney sections was performed.
Results: Of the nine selected, two mAbs (1D1 and 2H1) bound to all three of human, mouse, and MPO447-459. These two mAbs induced neutrophil ROS comparable to polyclonal ANCA (ANCA 11.2, 1D1 mAb 17.4, 2H1 mAb 8.3% neutrophils ROS+, control IgG2a mAb 1.7, all other anti-MPO mAbs<2). At 4 hours, 1D1 administration resulted in significantly increased glomerular neutrophil recruitment compared to control IgG2a (1.50± 0.08 vs 0.42±0.12 neutrophils per glomerular cross section, p=0.036) but, as with models using polyclonal anti-MPO antibodies, prominent neutrophil recruitment was not seen after 7 days.
Conclusion: mAbs 1D1 and 2H1 show promise for in vitro assays, and in a mouse model of AAV.