Influence of plate type on PBMC-based in vitro cytokine release assays for pre-clinical assessment of cytokine release syndrome.

Following the incidence of life-threatening cytokine release syndrome (CRS) in healthy volunteers in the 2006 first-time-in-human (FTIH) trial of a CD28 mAb (TGN1412), there is now a regulatory expectation to investigate immuno-stimulatory mAbs for CRS hazard using in vitro cytokine release assays (CRAs) prior to FTIH trials, and CRAs incorporating more than one test article may be used prior to combination clinical trials. To this end, many organisations stimulate healthy donor human peripheral blood mononuclear cells (PBMCs) using immobilised (plate-bound) mAbs, however methodology is not standardized, and differences in methods used throughout the pharmaceutical / biotechnology industry could influence assay outcomes. This study investigated the impact of micro-titre plate type on mAb immobilisation and cytokine release. We observed superior assay performance when stimulating PBMCs with OKT3 anti-CD3 mAb immobilised onto polystyrene plates of the Costar type. In contrast, Falcon polystyrene plates and polypropylene plates elicited reduced cytokine induction, and lower apparent potency, reflecting slower association of mAbs onto Falcon polystyrene, and higher background stimulation of PBMCs by polypropylene plates. We observed essentially no difference between Costar polystyrene plates with flat- and round-bottom wells. Costar polystyrene plates were saturated by OKT3 mAb solutions of 10 μg/mL (2 μg/well) or higher. We therefore recommend using Costar polystyrene plates for CRAs supporting FTIH clinical studies for immuno-stimulatory mAbs, similar biologics, and their combinations. These observations should aid the design and optimisation of CRAs, hence helping to inform clinical development.