In vitro activity: In the in vitro signaling assay on primary human CLL cells, acalabrutinib inhibits tyrosine phosphorylation of downstream targets of ERK, IKB, and AKT. Acalabrutinib demonstrates higher selectivity for BTK with IC50 determinations on nine kinases with a cysteine residue in the same position as BTK. Importantly, unlike ibrutinib, acalabrutinib does not inhibit EGFR, ITK, or TEC. acalabrutinib has no effect on EGFR phosphorylation on tyrosine residues Y1068 and Y1173. Compared with ibrutinib, acalabrutinib has much higher IC50(>1000 nM) or virtually no inhibition on kinase activities of ITK, EGFR, ERBB2, ERBB4, JAK3, BLK, FGR, FYN, HCK, LCK, LYN, SRC, and YES1.

Kinase Assay: Previous study showed that ACP-196 had high selectivity for Btk when tested against a panel of 395 non-mutant kinases, which was associated with the reduced intrinsic reactivity of ACP-196's electrophile. Moreover, ACP-196 could not inhibit EGFR, Itk or Txk, which is unlike ibrutinib. In addition, the phosphoflow assays on EGFR expressing cell lines furthre confirmed ibrutinib's EGFR inhibition without inhibition observed for ACP-196.

Cell Assay: the phosphoflow assays on EGFR expressing cell lines furthre confirmed ibrutinib's EGFR inhibition without inhibition observed for ACP-196.