Figure 2.

Orelabrutinib lacked off-target inhibition effect on cellular IL-2-inducible T cell kinase (ITK) compared with ibrutinib

(A and B) p-ITK and p-IκBα were assessed of proteins obtained from YT and NK-92 cells (two kinds of NK/T lymphoma cell lines expressing ITK) treated with ibrutinib or orelabrutinib for 2 h. PBMCs obtained from healthy adults were also used to verify the different effects of orelabrutinib and ibrutinib on ITK inhibition. After being pretreated with or without CD3/CD28-activating agent, PBMCs were treated with different concentrations of BTK inhibitors, orelabrutinib or ibrutinib. Proteins were extracted from pre-treated cells, and proteins of p-ITK and its associated downstream pathway were analyzed by western blot. These western blots were quantified from three biologically repeated tests. ∗p < 0.05 and ∗∗p < 0.01 compared with control group. For the western blots quantitation of PBMCs, control group activated with CD3/CD28 was regarded as control arm. (C) Cell viability was measured by Cell Titer-Glo luminescent cell viability assay in YT and NK-92 cells after being treated with indicated concentration of ibrutinib and Orel for 72 h. HBL-1, a DLBCL cell line, is highly sensitive to BTK inhibitors, and NK92 cell line has high ITK expression. Cell viability was compared in HBL-1, NK92, and YT cells treated with increasing concentration of Orel. The presentation of cell viability was normalized to no treatment control.