Cytokine release assays for the prediction of therapeutic mAb safety in first-in man trials — Whole blood cytokine release assays are poorly predictive for TGN1412 cytokine storm

Abstract

The therapeutic monoclonal antibody (mAb) TGN1412 (anti-CD28 superagonist) caused near-fatal cytokine release syndrome (CRS) in all six volunteers during a phase-I clinical trial. Several cytokine release assays (CRAs) with reported predictivity for TGN1412-induced CRS have since been developed for the preclinical safety testing of new therapeutic mAbs. The whole blood (WB) CRA is the most widely used, but its sensitivity for TGN1412-like cytokine release was recently criticized. In a comparative study, using group size required for 90% power with 5% significance as a measure of sensitivity, we found that WB and 10% (v/v) WB CRAs were the least sensitive for TGN1412 as these required the largest group sizes (n = 52 and 79, respectively). In contrast, the peripheral blood mononuclear cell (PBMC) solid phase (SP) CRA was the most sensitive for TGN1412 as it required the smallest group size (n = 4). Similarly, the PBMC SP CRA was more sensitive than the WB CRA for muromonab-CD3 (anti-CD3) which stimulates TGN1412-like cytokine release (n = 4 and 4519, respectively). Conversely, the WB CRA was far more sensitive than the PBMC SP CRA for alemtuzumab (anti-CD52) which stimulates FcγRI-mediated cytokine release (n = 8 and 180, respectively). Investigation of potential factors contributing to the different sensitivities revealed that removal of red blood cells (RBCs) from WB permitted PBMC-like TGN1412 responses in a SP CRA, which in turn could be inhibited by the addition of the RBC membrane protein glycophorin A (GYPA); this observation likely underlies, at least in part, the poor sensitivity of WB CRA for TGN1412. The use of PBMC SP CRA for the detection of TGN1412-like cytokine release is recommended in conjunction with adequately powered group sizes for dependable preclinical safety testing of new therapeutic mAbs.

Is an <i>in vitro</i> whole blood cytokine assay useful to detect the potential risk of severe infusion reaction of monoclonal antibody pharmaceuticals?

“…A significant increase of IL-8, IL-10 and IL-6 can be detected with n = 9, 14, and 28 sample sizes at 90% statistical power after treatment with CD28SA. This was inconsistent with the previous finding by Vessillier et al (2015) that a sample size of at least n = 52 was needed to detect a positive response in whole blood and that IL-8 was not significantly increased by TGN1412. They reported that the background level of IL-8 in WBCA was 2,538 pg/mL with the IgG4 control and 2,474 pg/mL with TGN1412 at 5 μg/mL of treatment (Vessillier et al, 2015).…”

“…This was inconsistent with the previous finding by Vessillier et al (2015) that a sample size of at least n = 52 was needed to detect a positive response in whole blood and that IL-8 was not significantly increased by TGN1412. They reported that the background level of IL-8 in WBCA was 2,538 pg/mL with the IgG4 control and 2,474 pg/mL with TGN1412 at 5 μg/mL of treatment (Vessillier et al, 2015). In the present study, medians of IL-8 measurements were 608 pg/mL when treated with panitumumab at 100 μg/mL and 375 pg/mL with PBS, which significantly increased to 1,270, 1,074, and 2,196 pg/mL when concentrations of CD28SA were 1, 10 and 100 μg/mL, respectively (Fig.…”

“…Wolf et al (2012Wolf et al ( , 2013 and Bailey et al (2013) detected positive cytokine responses to anti-CD28 mAbs in WBCA. However, the low sensitivity of WBCA raises the question of how practical a tool it is to identify hazards (Thorpe et al, 2013;Vessillier et al, 2015). The biggest criticism is that the sample size required for the WBCA to detect a positive response with anti-CD28 mAb may be too big.…”

Mechanisms and Barriers in Cancer Nanomedicine: Addressing Challenges, Looking for Solutions

“…Some conventional preclinical models (rats and non-human primates) may be relatively insensitive to endotoxin and to developing a cytokine storm. 38 In such cases, supplementing in vivo studies with in vitro assays utilizing human blood should be considered. Another consideration for selecting an animal model is related to the variable sensitivity of animal strains to a particular type of immunotoxicity.…”

Deletion of CD28 Co-stimulatory Signals Exacerbates Left Ventricular Remodeling and Increases Cardiac Rupture After Myocardial Infarction

“…6 However, the use of a superagonistic anti-CD28 antibody may not be realistic because of the many serious side-effects, such as multi-organ failure and near-fatal cytokine release syndrome, which were found in another clinical study. 34 Further studies are necessary to confirm the effects of CD28 modification on LV remodeling after MI and elucidate the detailed mechanism of it.…”