Pirtobrutinib, a non-covalent BTK inhibitor, enhances T-cell anti-tumor immunity in chronic lymphocytic leukemia (CLL)

Abstract INTRODUCTION Covalent Bruton tyrosine kinase inhibitors (cBTKi) transformed the treatment of CLL and mantle cell lymphoma (MCL). cBTKi demonstrated direct anti-neoplastic effects and have clear immunomodulatory effects. Unfortunately, BTK mutations (predominantly in the C481 residue) lead to resistance and poor prognosis. Pirtobrutinib is a highly selective, reversible, non-covalent BTKi, that has been FDA approved for treatment of CLL patients after 2 lines of therapy including a BTKi and a BCL2 inhibitor. In this study we focused on the impact of pirtobrutinib on T-cells functionality. METHODS PBMCs were isolated from CLL patients. CD3+ T-cells were isolated using DynabeadsTM FlowCompTM (ThermoFisher). T-cells were then stimulated up to 72 hours with αCD3/CD28 in the presence or absence of BTKi. Apoptosis and cell cycle were measured by flow cytometry. Naïve T cells were studied under TH1/ TH2/Treg-polarizing conditions. For immunological synapse assays, T-cells and CLL cells were treated with drugs, combined at a 1:1 ratio, and imaged using confocal microscopy. RESULTS Treatment with pirtobrutinib abrogated B-cell receptor signaling in primary CLL cells, as evidenced by reduced phosphorylation of BTK, AKT and ERK. Furthermore, pirtobrutinib slowed cell cycle progression and induced apoptosis of MCL cell lines and primary CLL cells cultured in CD40L-expressing stromal conditions mimicking the lymph node microenvironment. In vivo experiments using ibrutinib-resistant PDX MCL cells showed that pirtobrutinib treatment slowed disease progression and extended survival by 1.5 and 2.5 weeks vs. ibrutinib or vehicle, respectively. Next, we conducted RNA-Seq analysis of sorted naïve T-cells from healthy donors treated with pirtobrutinib or ibrutinib for 24 hours. We observed ~80 differentially expressed genes following pirtobrutinib treatment (~330 for ibrutinib). MYC and E2F2 targets, OxPhos, unfolded protein response and DNA repair were the most significantly downregulated pathways following treatment with pirtobrutinib. Then, we analyzed the effects of pirtobrutinib on T-cell apoptosis, cell cycle and activation using ibrutinib as control. Treatment with pirtobrutinib did not induce apoptosis or alter cell cycle progresion in CD3+ T-cells. In addition, no changes in the exhaustion marker PD-1 were observed with either BTKi. Ibrutinib but not pirtobrutinib downregulated several T-cell activation markers (CD69, CD25 and CD38). Under Th1 polarizing conditions using sorted naïve CLL-derived T-cells, pirtobrutinib significantly upregulated IFN-γ and IL-2 secretion, to a greater extent than ibrutinib. Neither BTKi altered secretion of IL-4 and GATA3 under Th2 polarization conditions. Finally, although to a lesser degree compared to ibrutinib, pirtobrutinib significantly reduced differentiation of Tregs. We next quantified immunological synapse formation. Interestingly, pirtobrutinib (but not ibrutinib) significantly enhanced immunological synapse formation. This increase correlated with upregulation of CD54 (ICAM-1) expression in both T-cell (T-cell activation and antigen uptake) and B-cell (cell-cell adhesion) populations. We performed cytotoxicity experiments using SU-DHL4, Raji and murine A20 cell lines (E:T ratio 1:10). Aligned with synapse data, pirtobrutinib but not ibrutinib enhanced T-cell mediated killing of neoplastic B-cells, accompanied by a significant increase of Granzyme B, Perforin and TNFα secretion. CONCLUSIONS Pirtobrutinib exerted both direct anti-tumor and immunomodulatory effects. Pirtobrutinib partially rescued the CLL immunosuppressive phenotype by promoting Th1 polarization and reducing Treg differentiation, without compromising T-cell activation and expansion. Pirtobrutinib also facilitated immunologic synapse formation and enhanced T-cell cytotoxicity. These results provide justification for continued investigation of pirtobrutinib for treatment of lymphoid malignancies alone and in combination with T-cell enabling therapies.