Identification of PD1 and TIM3 As Checkpoints That Limit Chimeric Antigen Receptor T Cell Efficacy in Leukemia (2016)

Chimeric antigen receptor T cell (CART) therapy has developed as a powerful therapy in hematological malignancies. CD19 directed CART cells have resulted in complete response rates (RR) of ∼90% in acute lymphoblastic leukemia. However, the overall RR in other malignancies such as chronic lymphocytic leukemia is around 50%. This could be related to CART exhaustion induced by leukemia cells. Here, we aim to evaluate the role of inhibitory receptors/pathways in inducing CART cell dysfunction As a tumor model, we used an acute myeloid leukemia (AML) cell line (MOLM14) and primary AML samples and treated them with CD33 or CD123 CART cells (2nd generation CARs with 4-1BB and CD3z domains) Incubation of primary AML samples with CD33 or CD123 directed CARTs resulted in a significant up-regulation of PD-L1 on tumor cells after 24 hours of incubation (0% on day 0 vs 80% on day 1, P <0.001), and up-regulation of PD-1 and TIM-3 on T cells 3-7 days post co-culture (8% of T cells expressed PD-1 on day 0 vs 43% on day 3, P=0.03 and 13% of T cells expressed TIM-3 on day 0 vs 71% on day 3, p=0.001, Figure 1). For in vivo experiments, we used NSG (NOD-SCID-g-/-) mice and engrafted them with the MOLM14 cell line. Treatment of these AML xenografts with suboptimal doses of CD33 or CD123 CARTs resulted in initial anti-tumor responses, followed by relapses in 40-60% of the mice (Figure 2A). There was a significant up-regulation of TIM-3 on T cells isolated from mice with relapsed disease compared with T cells isolated from mice in remission after CART cell therapy (Figure 2B) Next, we investigated the role of adding checkpoint inhibitors to improve T cell function ex vivo after CART cell therapy. Marrows of mice that relapsed after CART cell therapy (containing CART cells and leukemia) were cultured with PD-1 or TIM-3 blocking antibodies or both. CART cell effector functions such as cytokine production and Ki-67 proliferation marker improved in the presence of checkpoint antagonists especially when both PD-1 and TIM-3 blocking antibodies were combined (figure 2C) Finally, we tested the combination of checkpoint blockade with CARTs in AML xenografts. MOLM14 xenografts were treated with suboptimal doses of CD33 or CD123 CARTs with or without checkpoint blockade (PD-1 blockade, TIM-3 blockade or the combination of both on days 3, 6, 9 and 12 post T cell therapy). The addition of PD-1 or TIM-3 blockade to CART cell therapy resulted in a synergistic anti-tumor activity as shown in Figure 3. The durable complete RR was: 45% for treatment with CART123 alone, 80% for treatment with CART123+PD-1 blockade, 100% for treatment with CART123+TIM-3 blockade, and 80% for treatment with CART123+ both PD-1 and TIM-3 blockade) Our preclinical results indicate that PD-1 and TIM-3 pathways are involved in CART exhaustion and dysfunction in AML. Combination of checkpoint inhibitors with CART cells may lead to enhanced efficacy in AML and other hematological malignanciesFigure 2A) NSG mice were injected with MOLM14 AML cell line (1×108 IV) Engraftment was confirmed on Day 6 by bioluminescence imaging AML xenografts were then treated with suboptimal doses CART123 (0.5×106 IV) and followed with serial imaging. Relapsed disease was observed in 60% of AML xenografts B) T cells isolated from bone marrows of relapsed mice after CART cell therapy express higher levels of TIM3 compared to T cells isolated from bone marrows of mice in remission. C) Co-culture of T cells isolated From relapsed mice with TIM3 and PD1 inhibitors (10 ug/ml for 72 hours), in the presence of the antigen target results in improvement of cytokine production and Ki-67 proliferation markerView Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Combination of CARTs and checkpoint inhibitors in AML xenografts. MOLM14 engrafted xenografts were treated with suboptimal doses of CART 123 in combination with PD1 inhibitors, TIM3 inhibitors or the combination of both antibodies. Antibodies were given on day 3,6,9, and 12 post T cells at a dose of 10mg/kg/dose. The combination of checkpoint blockade and CARTs resulted in synergistic anti-leukemic effect.View Large Image Figure ViewerDownload Hi-res image Download (PPT)