Abstract 2780: Unique pharmacodynamic properties conferred by differential binding to BTK, pirtobrutinib vs covalent inhibitors

Covalent Bruton tyrosine kinase inhibitors (cBTKi) have transformed the treatment of B cell malignancies. Despite the efficacy of cBTKi, treatment failure often occurs through development of resistance or intolerance. Pirtobrutinib, a highly selective, non-covalent BTKi, potently inhibits both WT and C481 mutant BTK. Pirtobrutinib has favorable oral pharmacology, is well tolerated, and has shown promising efficacy in patients with poor prognosis B cell malignancies following prior therapy, including prior cBTKi. Here we report in vitro BTK binding, conformation, and activation differences between cBTKi and pirtobrutinib. Cellular studies showed pirtobrutinib inhibited BTK phosphorylation at both Y223 and Y551 in REC-1 and Ramos A1 human lymphoma cell lines. In contrast, despite inhibition of Y223 phosphorylation, both cell lines treated with cBTKi, ibrutinib, acalabrutinib or zanubrutinib, retained Y551 phosphorylation. The differential effects on Y551 were also observed in PBMC isolated from treatment naïve human CLL donors treated in vitro with pirtobrutinib or ibrutinib. To investigate the binding effects of cBTKi and pirtobrutinib on BTK stability and conformation, a series of biophysical and structural studies were performed. In a hydrogen/deuterium exchange mass spectrometry study using full length BTK, all compounds significantly inhibited exchange in regions surrounding the ATP binding site in the kinase domain. While cBTKi induced significantly increased exchange for peptides in the SH3 and SH2 domains, pirtobrutinib significantly reduced exchange in a portion of the SH3 domain, nearly the entire SH2 domain, and SH2 linker, suggesting that pirtobrutinib uniquely stabilizes BTK in a closed/inactive conformation. Consistent with this proposed model, crosslinking mass spectrometry results of inhibitor bound BTK showed that pirtobrutinib promoted different interactions between the SH2 domain and kinase domain than cBTKi. To confirm that pirtobrutinib promotes greater stabilization of BTK, a mass spectrometry based cellular thermal shift assay was performed in both Ramos and BTK overexpressing HEK293 cells. In both cell lines, pirtobrutinib showed significantly greater stabilization of BTK compared to cBTKi. In addition, crystallographic studies of BTK in complex with pirtobrutinib revealed domain organization consistent with stabilized SH2-SH3 interactions with the kinase domain. These data suggest pirtobrutinib may differentially impact BTK stability