Preclinical Analyses Support Clinical Investigation of Combined Anti-CD19 CAR-T Cell, JCAR017 with Ibrutinib for the Treatment of Chronic Lymphocytic Leukemia
Authors: Qin Jim , Baturevych Alex , Mudri Sherri , Salmon Ruth , Ports Michael 
Published: October 2019

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.