Preclinical Analyses Support Clinical Investigation of Combined Anti-CD19 CAR-T Cell, JCAR017 with Ibrutinib for the Treatment of Chronic Lymphocytic Leukemia

Abstract

Chronic lymphocytic leukemia (CLL) drives systemic immune suppression and T cell dysfunction in patients, highlighting an important consideration in this setting for the manufacturing and efficacy of adoptive cell therapies using autologous T cells. In clinical studies, anti-CD19 CAR-T cells produce durable and complete responses in leukemic and some lymphomatous B cell malignancies. While preconditioning with cyclophosphamide (Cy) and fludarabine (Flu) has improved CAR-T responses in CLL patients, reported complete response rates still have been below 50%; additional therapeutic strategies likely will be required. Ibrutinib, an irreversible inhibitor of BTK, has been approved as a frontline treatment option for patients with CLL. The potent off-BTK activity of ibrutinib on ITK and TEC family kinases could affect CAR T cell biology. Recent work highlighted the ability of ibrutinib to restore CLL patient T cell functionality, enhance CAR-T production and potentially improve clinical efficacy. Additional preclinical work demonstrated improved tumor clearance when anti-CD19 CAR T cells were combined with ibrutinib in several murine tumor models. A preclinical evaluation of the combination between the anti-CD19 CAR-T product, JCAR017, and ibrutinib was performed to determine feasibility for clinical use in CLL. JCAR017 is a second generation CAR-T cell product candidate that contains a 41BB costimulatory endo-domain and is currently in phase 1 trials for non-Hodgkin lymphoma (NHL). A series of in vitro studies assessed the functional activity of JCAR017 cells (derived from 3 healthy donors), in combination with ibrutinib (500-0.05nM), across a dose range covering the cMax and cMin. Cytolytic activity was monitored by co-culturing CAR-T cells with ibrutinib-resistant K562 CD19 tumor cells at an effector-to-target ratio of 2.5:1. Ibrutinib, at concentrations tested, did not inhibit the cytolytic function of JCAR017 cells. For cells derived from some donors, addition of ibrutinib appeared to increase % target killing. To address ibrutinib effects on JCAR017 activation, cell surface markers and cytokines were tracked over 4 days following stimulation with irradiated K562 CD19 cells. We observed no significant effect on JCAR017 surface expression of CD25, CD38, CD39, CD95, CD62L, CCR7, or CD45RO, or of EGFRt, a surrogate transduction marker. With addition of ibrutinib, we observed a modest decrease in the percentage of cells expressing CD69, CD107a and PD-1. With 5 and 50nM of ibrutinib, there was a 19.5% (p<0.01) average increase in IFNγ production. At supraphysiological concentrations (500nM) we observed a 20% (p<0.05) decrease in IL-2 production, suggesting ibrutinib at high concentrations may dampen T cell activation. CAR-T cell expansion after repeated antigen stimulation has been shown to be a predictor of in vivo efficacy. JCAR017 cells stimulated every 3-4 days with irradiated target cells in the presence of ibrutinib showed no inhibition of initial growth. However, after 5 rounds of stimulation, JCAR017 + ibrutinib cells from 1 donor had enhanced proliferation compared to control, untreated cells (p<0.05). Interestingly, after 5 rounds of serial stimulation, we observed an increased proportion of CD4+CXCR3+CRTh2- Th1 cells with 500nM ibrutinib treatment compared to control (p<0.01). We assessed the in vivo anti-tumor activity of JCAR017 in combination with ibrutinib using NSG mice injected with 5x105 Nalm6-luciferase cells. After tumor engraftment, a suboptimal dose (5x105) of JCAR017 cells was transferred to mice and ibrutinib (25 mg/kg qd) was administered for the duration of the study. Ibrutinib treatment alone had no effect on tumor burden compared to vehicle treatment. Mice treated with a suboptimal JCAR017 dose + ibrutinib showed decreased tumor burden (p<0.05) and increased median survival from 44 days to >80 days (p<0.001) compared to the group receiving the suboptimal JCAR017 dose + vehicle. Similar effects were seen in replicate studies using JCAR017 cells produced from multiple donors. Ex vivo evaluation for CAR-T quantitation and immunophenotyping was also performed. Taken together, the results suggest that ibrutinib enhances intrinsic JCAR017 activity and may improve outcomes in CLL patients treated with anti-CD19 CAR T therapy, irrespective of BTK mutational status. A Phase 1b study of JCAR017 in combination with ibrutinib for BTKi R/R CLL is planned. Disclosures Qin: Juno Therapeutics: Employment. Baturevych:Juno Therapeutics: Employment. Mudri:Juno Therapeutics: Employment, Equity Ownership. Salmon:Juno Therapeutics: Employment. Ports:Juno Therapeutics: Employment.

CAR T-Cell Therapy for B-Cell non-Hodgkin Lymphoma and Chronic Lymphocytic Leukemia: Clinical Trials and Real-World Experiences

“…Preclinical data have demonstrated that ibrutinib enhances intrinsic liso-cel activity in vitro and in vivo (110). Previous prolonged exposure of CLL patients to ibrutinib may favor tisa-cel expansion and CAR T-cell clinical activity, and in a mouse model, ibrutinib-based treatment improved CAR T-cell engraftment and cytotoxic efficacy (111).…”

Accelerating chimeric antigen receptor therapy in chronic lymphocytic leukemia: The development and challenges of chimeric antigen receptor T‐cell therapy for chronic lymphocytic leukemia

“…The BTK inhibitor, ibrutinib, can modulate several immune compartments independent of its well‐known inhibitory effect on B‐cell receptor signaling . Preclinical evidence demonstrates that ibrutinib, through its modulation of T cell function, enhances the ability of CTL019 cells to expand in vivo and exert antitumor effects . In addition, T cells collected from patients exposed to ibrutinib have greater ex vivo expansion and a greater fraction of T cells with a central memory phenotype compared to T cells from CLL patients not on ibrutinib.…”