TGN1412 is a "superagonistic" CD28 monoclonal antibody (IgG4) that caused serious adverse events at its first time in human clinical trial. In the present study, different in vitro methods for detecting and quantifying unwanted pro-inflammatory activity of therapeutic monoclonal antibodies (mAbs) such as TGN1412 are described. The antibody of interest is immobilised by wet-coating or air-drying onto polypropylene or polystyrene 96-well plates prior to the addition of human peripheral blood mononuclear cells (PBMCs). The cells are incubated for 16-24h with the immobilised antibody which allows the accumulation of pro-inflammatory cytokines, quantified by enzyme-linked immunoabsorbent assay (ELISA), in response to the antibody. Cytokine responses stimulated by TGN1412 immobilised by air-drying onto polypropylene and polystyrene plates were much larger than responses to TGN1412 wet-coated onto polypropylene and polystyrene plates, respectively. In additional experiments with other mAbs associated with clinical reactions, air-dried mAbs stimulated larger tumour necrosis factor-alpha (TNF) responses than antibodies added in aqueous phase. Also, TGN1412 air-dried onto plastic plates stimulated large proliferative responses of 3-day cultures of lymphocytes. It was concluded that immobilising mAbs by air-drying offers a useful in vitro method for detecting and quantifying pro-inflammatory activities of therapeutic mAbs.
