Development of an in vitro pre-clinical cellular assay to predict induction of Cytokine Storm by therapeutic antibodies. (125.16)

Abstract

Abstract Cytokine storm (Hypercytokinemia/Cytokine Release Syndrome) is an acute immune reaction consisting of a positive feedback loop between cytokines and immune cells, resulting in severe inflammation and organ failure. A clinical trial of immunotherapeutic anti-CD28 antibody TGN1412 in 2006 demonstrated the importance of thorough pre-clinical screening and need for more sensitive assays to predict cytokine storm. In that study, 6 healthy volunteers suffered from hypercytokinemia at doses 500-fold less than the lowest dose tested in non-human primates (NHP), showing the failure of standard pre-clinical safety approaches of that time. We have developed an in vitro assay to evaluate antibodies for induction of cytokine storm. Frozen PBMC from humans and NHP are cultured with test articles bound to the cell-culture plate and assayed for cytokine production and cell proliferation. Anti-CD28 superagonist antibodies, which have been shown to stimulate human PBMCs using similar mechanisms as TGN1412, are used as a positive control. Human PBMC produced significantly more IL-2, IL-6, IFN-gamma, TNF-alpha, MIP-1-alpha and several other cytokines compared to isotype control or untreated cells, while PBMC from NHP were non-responsive. The increased production of key pro-inflammatory cytokines in response to stimulation with anti-CD28 antibodies is accompanied by increased PBMC proliferation, while no proliferation increase is detected in isotype-treated or unstimulated human PBMC.