Studies on cytokine release syndrome (CRS) caused by antibody pharmaceuticals

Abstract

In a first-in-human trial in 2006, the CD28 superagonist antibody (Ab) TGN1412 caused severe cytokine release syndrome (CRS) in all 6 human volunteers. They faced life threatening conditions involving multiorgan failure. The clinical signs of CRS correlate with immune cell activation following a cascade of systemic cytokine release, which can be fatal. The first human dose of TGN1412 had a 500-fold safety margin against the no observable adverse effect level (NOAEL) established in cynomolgus monkeys, but the animal study failed to predict the CRS risk. Consequently, in vitro cytokine release assays using human cells are now used to detect the potential risk of CRS. In this work, we investigated the detection and mitigation of the CRS risk caused by Ab pharmaceuticals.

There are two major in vitro assays for detecting CRS risks: one composed of whole blood with aqueous-phase test articles (i.e., the whole blood cytokine assay [WBCA]), and the other composed of peripheral blood mononuclear cells (PBMCs) with solid-phase test articles (i.e., the PBMC assay). First, we determined an appropriate sample size and confirmed the suitability of the WBCA as a hazard identification tool for CRS. Next, we compared the cytokine concentration and cytokine-producing cells after stimulation with the TGN1412 in the WBCA and the PBMC assay. The results showed that the types of cytokines induced by TGN1412 and the cells producing them differed in the two assays. The results emphasize the need for a better understanding of the characteristics of these in vitro assays and for a test method that is optimized according to the mechanisms of therapeutic mAbs.

Although T cell engagers (TE) show promising therapeutic potential as anti-tumor antibodies, it is still difficult to manage CRS in clinical use. We reported that single bolus infusion of anti-GPC3/CD3 TE caused life-threatening CRS, but daily step-up dosing of the TE did not, even at the lethal single dose. Clinical and non-clinical studies suggest that CRS after subsequent doses of CD3 bispecific constructs is less severe than after the priming dose, and that step-up dosing reduces cytokine levels in animals and humans. However, the mechanism underlying the reduced cytokine induction after priming treatment with TE is unclear. We made an in vitro model elucidating how priming treatment decreases cytokine release from human PBMCs after repeated treatment with anti-GPC3/CD3 TE. Priming treatment with TE did not decrease CD3 expression, CD3-mediated signal transduction, T cell activation markers, and cytotoxicity, but did decrease cytokine mRNA expression. Thus, we conducted ATAC-seq to detect accessible (open) chromatin regions. As a result, the priming treatment decreased chromatin accessibility at the transcription regulation region of IL2. In addition, we found a correlation between chromatin accessibility and individual differences in cytokine induction. The measurement of epigenetic states in T cells could be used to monitor CRS tolerance and to estimate the individual difference in CRS. We would like to further advance this research to find strategies for avoiding CRS and biomarkers for measuring individual differences in the risk of CRS.