A simple whole blood bioassay detects cytokine responses to anti-CD28SA and anti-CD52 antibodies

article i nfo Article history: Received 27 September 2011 Accepted 18 December 2012 Introduction: In 2006 the anti-CD28 superagonistic IgG4 TGN1412, having passed pre-clinical safety screens, caused a severe 'cytokine storm' in 6 healthy volunteers. Others have shown that for TGN1412 to in- duce an inflammatory signal in human peripheral blood mononuclear cells (PBMCs) or in human diluted blood, endothelial cells or bound monoclonal antibody (mAb) is required as part of a bioassay complex. These types of protocols rely on different donor cells and therefore have limitations as bioassays for pre-clinical testing. Methods: We performed studies using human PBMC/endothelial cell co-cultures, whole blood/endothelial cell co-cultures and human whole blood alone. We bracketed responses of a CD28 superagonist antibody with mAbs against CD52 (alemtuzumab, MabCampath-1H) or epidermal growth fac- tor receptor (cetuximab, Erbitux) and with the immunostimulant lipopolysaccharide. We detected cytokine responses at the level of protein release (using ELISAs and Luminex assays) and gene induction (using real-time PCR arrays). Results: Here we confirm that IL-8 release was induced in a mixed endothelial cell-PBMC system by the anti-CD28 mAb. We go on to show that an alemtuzumab and an anti-CD28 mAb, but not cetuximab induced the release of a range of cytokines including IL-8, IL-6, IFNγ, IL-2 and IL10 after 24 h and induced cytokine gene induction after 1 h. Co-cultures of whole blood and HUVECS showed larger variability but no superiority over whole blood alone at a range of time points (0.5-48 h). Discussion: We suggest that, whilst limitations exist, human blood-based in vitro assays may prove useful in assessing the potential of mAbs and other biotherapeutics to cause release of cytokines in humans.