Enhancing the expansion of Naïve, potent T-cells via BTK inhibition reinvigorates CD19-CAR T-cell immunity and boosts antitumor efficacy in relapsed Mantle Cell Lymphoma

Abstract Background Emerging evidence indicates that inhibition of BTK and tyrosine kinases enhances immune responses and potentiates CAR T-cell effector functions, potentially contributing to durable clinical benefit. Preclinical and clinical data suggest that concomitant administration of a BTK inhibitor and CAR T cells yields superior therapeutic outcomes compared to either agent alone. Given the potential of BTK inhibition to enhance CAR-T cell efficacy in mantle cell lymphoma (MCL), this rationale has driven efforts to explore whether combining BTK inhibitors (BTKi) ibrutinib, and pirtobrutinib, or pan-tyrosine kinases inhibitor (TKi), dasatinib, with CD19-CAR T-cell therapy could improve CAR T-cell effector functions and antitumor potency by enhancing T-cell fitness and persistence. The preclinical data would support a proof-of-concept for employing BTK inhibitors as a bridging or combinatorial strategy to augment the efficacy of CAR T-cell therapy in MCL. Methods CD19-CAR T cells were generated using human T cells isolated from healthy donors via a lentiviral transduction, with optional inclusion of BTKi (ibrutinib, pirtobrutinib) and TKi (dasatinib). The competence of CAR T cells was assessed by examining T-cell activation, proliferation, and metabolic activity. CAR T-cell effector functions were evaluated by cytokine secretion and T-cell-mediated cytotoxicity assays in a standard co-culture and ex vivo TME-mimicking model composed of tumor cells and immune cells obtained from MCL patient specimens on top of lymph node stromal YK6 cells. In vivo antitumor efficacy was evaluated in both cell line-derived (CDX) and patient-derived (PDX) xenograft models established in-house. Patient samples were collected after obtaining informed consent and approval from the Institutional Review Board at the University of Texas MD Anderson Cancer Center. Results CD19-directed CAR T cells were generated in the presence of ibrutinib and/or the tyrosine kinase inhibitor, dasatinib. Immune phenotyping of the allogeneic CAR T cells revealed that treatment with BTKi (ibrutinib), and TKi (dasatinib), preserved naïve-like features and enhanced metabolic fitness in allogeneic CD19-CAR T cells as evidenced by: (1) over 50% of CAR T cells expressing central memory and stem cell memory markers (CD45RA, CD62L, and CCR7), compared to inhibitor-free controls; and (2) enhanced mitochondrial respiratory capacity and glycolytic activity. For functional analysis, we employed either a standard co-culture system with CD19⁺ MCL cell lines or a newly developed TME-mimicking model. The latter consisted of tumor and immune cells isolated from MCL patient specimens, co-cultured atop of CD40L/IL-21-expressing lymph node stromal cell line YK6 cells, supplemented with additional key cytokines. Upon activation, CAR T cells displayed robust target-specific secretion of type I cytokines, including interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα), along with a substantial, target-specific cytotoxic response within 24 hours of co-culture. These enhanced effector responses were constantly observed in a co-culture with CD19-expressing MCL cell lines, including ibrutinib-resistant Maver-1, harboring ATM deletions and gain-of-function TP53 mutations. Importantly, treatment with a sub-cytotoxic dose (25mg/kg) of ibrutinib in combination with CD19-CAR T cells significantly enhanced tumor clearance, prolonged survival, and sustained long-term remission in a CD19⁺ tumor-bearing CDX model. Notably, the pronounced antitumor efficacy of ibrutinib or pirtobrutinib in combination with CD19-CAR T cells was consistently demonstrated in PDX models derived from MCL patients with prior relapse following CD19-directed CAR T-cell therapy. Both combinations yielded comparable therapeutic outcomes. These findings suggest that the BTK inhibition may potentiate CD19-CAR T-cell therapy across a broad spectrum of MCL, irrespective of the patient's sensitivity to BTK inhibitors. ConclusionBTK inhibition enhances the metabolic fitness, effector functions, and anti-lymphoma activity of CD19-CAR T cells. Combination treatment with either ibrutinib or pirtobrutinib and CD19-CAR T cells demonstrates comparable and pronounced antitumor efficacy in preclinical PDX models of MCL that relapsed following CAR T-cell therapy.