The Reversible BTK Inhibitor Nemtabrutinib Demonstrates Favorable Antitumor Efficacy and Enhances the Function of CAR T Cells in Mantle Cell Lymphoma

Abstract

Background Covalent Bruton's tyrosine kinase (BTK) inhibitors have revolutionized the therapy of mantle cell lymphoma (MCL). In January 2023, the reversible BTK inhibitor pirtobrutinib was approved by the FDA for the treatment of relapsed or refractory MCL. Nemtabrutinib is another reversible BTK inhibitor which also targets other kinases. The objective of this study was to evaluate the efficacy of nemtabrutinib alone and in combination with anti-CD19 CAR T cells against MCL. Methods Cell viability was measured following a 72-hour treatment with nemtabrutinib in a panel of MCL cell lines using CellTiter-Glo luminescent cell viability assay (Promega). Annexin V/PI staining was utilized to determine whether nemtabrutinib induces cell death through apoptosis. Patient-derived organoids (PDOs) were generated by culturing patient primary cells in 50% Matrigel (Corning) and cultured in cytokine-containing medium. The PDOs were treated for 72 hours, and viability assays were performed. Western blotting was employed to investigate the drug impact on kinase signaling pathways. Nemtabrutinib treated Mino cells were subjected to bulk RNA sequencing to interrogate the transcriptome profiling. The anti-MCL efficacy of nemtabrutinib was tested in an ibrutinib-resistant patient derived xenograft (PDX) mouse model in vivo. Additionally, the potential synergistic effects of nemtabrutinib and anti-CD19 CAR T cells were examined using the luciferase-expressing MCL cells. Results Nemtabrutinib demonstrated comparable growth inhibitory activity to ibrutinib in MCL cell lines with IC 50 values at micromolar concentrations (IC 50 = 0.7-10.1 μM). Remarkably, in the 3D PDO screening system that mimics the tumor microenvironment for MCL, nemtabrutinib displayed superior anti-MCL efficacy compared to ibrutinib (p < 0.01). The apoptotic potential of nemtabrutinib was further investigated using Annexin V/PI staining, revealing a dose-dependent induction of apoptosis, and demonstrated a similar sensitivity profile across cell lines observed with ibrutinib. Western blotting revealed that nemtabrutinib effectively inhibited phosphorylation at both Tyr223 and Tyr551 sites of BTK, and also dramatically suppressed the activation of Src family kinases, Syk and ERK. Bulk RNA-seq was performed to examine the transcriptome profile following nemtabrutinib treatment. 449 genes were identified to be significantly upregulated and 460 genes were downregulated. Gene set enrichment analysis uncovered a significant decrease in TNFα signaling via NF-κB, inflammatory response, and IFNγ response signalings (FDR < 0.05). In an ibrutinib-resistant PDX mouse model, treatment with nemtabrutinib resulted in a significant reduction in tumor burden and effectively attenuated the tumor involvement in spleen, liver and bone marrow. Furthermore, to enhance efficacy, we investigated the combination of nemtabrutinib with anti-CD19 CAR T cells in luciferase-expressing MCL cells. The result demonstrated that nemtabrutinib enhanced the effector function and anti-MCL activity of anti-CD19 CAR T cells. Conclusion Nemtabrutinib demonstrated favorable anti-MCL efficacy in both in vitro and in vivo studies, the promising synergistic effects observed in combination with CAR T cells warrants further investigation in both preclinical and clinical settings.