Evaluating A Cd63 Assay As A Biomarker For Syk Inhibitor Activity

Background: Spleen tyrosine kinase (SYK) inhibitors have therapeutic potential for severe asthma. In rats, activity of SYK inhibitor BI 1002494 measured with a CD63 assay correlated well with bronchoconstriction and airway resistance (Lamb DJ, et al. JPET 2016; 357:554–61). Aim: To evaluate a CD63 assay as a biomarker for SYK inhibitor activity in humans. Methods: Healthy volunteer whole blood was sensitized with anti-dinitroprusside (DNP) immunoglobulin E (IgE) and challenged with DNP. SYK inhibitor activity was measured with surface expression of CD63 as an index of basophil degranulation. Results: Strong correlation between CD63 IC50 and histamine release IC50 (R2=0.998) across 14 SYK inhibitor compounds confirmed CD63 as a good surrogate for functional basophil degranulation. BI 1002494 had a median CD63 IC50 of 113 nM (95% CI 103, 135) across 153 consecutive assays, with inter-sample variability of 17–2530 nM. Individual concentration curve gradients for basophil activation by the SYK inhibitor did not correlate with IC50 (R2=0.15). The proportion of basophils activated by anti-DNP/DNP (14–100%) correlated with IC50 (R2=0.44), suggesting that the more IgE receptors (i.e. the more SYK molecules) engaged, the greater the amount of inhibitor required. Similar assay performance and IC50 values from clinical biomarker labs indicate that the assay is robust and translatable. For 1000 SYK inhibitors, poor correlation was found between enzymatic kinase activity and CD63 potency (R2=0.22), possibly due to differences in cellular permeability and plasma protein binding. Conclusion: Employing a CD63 assay in ex vivo stimulated human whole blood could be used for screening compounds for human SYK inhibitor activity.