Abstract Text: Identification of robust intracellular pharmacodynamic effects in global clinical trials remains a challenge in drug development. Rapid cell preservation reagents have reduced the costs and complexity of performing FACS-based immune studies in the global trial setting. However, a complication can be chemical alteration of antigen-antibody epitopes that antagonize their association, limiting the identification of immune subsets and cell surface proteins linked to function. Additionally, shipment of samples may take more than 24 hours, at which time, sensitive signaling molecules like pSTAT5 are no longer viable to use as biomarkers. In order to circumvent these logistical challenges, we compared three whole blood fixation reagents to determine their viability in the global trial setting. To test these reagents, healthy cynomolgus and human whole blood was stimulated with either IL-2 or IL-15 and preserved using either Proteomic Stabilizer (SmartTube inc.), Whole Blood cell stabilizer (Cytodelics Inc.), or Cyto-Chex (Streck Inc.) to determine the stability of phosphorylated signaling molecules across various immune cell populations. Impact of sample fixation on checkpoint and functional related proteins was evaluated. We observe the preservation of cell surface antigens utilizing these reagents to identify major immune cell subsets while preserving measurements of transient pharmacodynamic effects. These studies provide an attractive solution to determine test article effects on transiently expressed biomarker activities. Whole blood fixation protocols enable the ability to monitor discrete changes in checkpoint and functional markers via flow cytometry in the global clinical trial setting.
