Introduction: Checkpoint molecules like programmed death-1 (PD-1) and T cell immunoglobulin mucin-3 (TIM-3) act as negative regulators of the immune system and can be upregulated in the setting of glioblastoma multiforme (GBM). Combined PD-1 blockade and stereotactic radiosurgery (SRS) have been shown to improve antitumor immunity and produce long-term survivors in a murine glioma model [1]. However, tumor infiltrating lymphocytes can express multiple checkpoints (including TIM-3), and expression of two or more checkpoints corresponds to a more exhausted T cell phenotype [2]. Here, we hypothesized that the addition of a second checkpoint-blocking antibody could achieve additive or synergistic antitumor effects.

Methods: C57BL/6 mice were implanted with mouse glioma cell line GL261 transfected with luciferase and randomized into 8 treatment arms: (1) control, (2) SRS, (3) anti-PD-1 antibody, (4) anti-TIM-3 antibody, (5) anti-PD-1+SRS, (6) anti-TIM-3+SRS, (7) anti-PD-1+anti-TIM-3, and (8) anti-PD-1+anti-TIM-3+SRS. Overall survival was measured. Brain, cervical lymph nodes, and peripheral blood were harvested on day 21 to assess immune activation.

Results: Survival benefits were demonstrated with combined anti-TIM-3 antibody+SRS compared to anti-TIM-3 antibody alone with a median survival (MS) of 92 vs. 25 days and overall survival (OS) of 50% vs. 0%, respectively (p<0.001 by log-rank Mantel-Cox). Dual blockade with anti-TIM-3+anti-PD-1 antibody also improved survival compared to TIM-3 blockade alone, (MS of 146 vs. 25 days, OS 60% vs. 0%, respectively, p<.05). Notably, the triple-modality treatment (anti-PD-1+anti-TIM-3+SRS) provided a significant improvement in survival compared to all other treatment arms with an OS of 100% by day 146 (p<0.05). Flow cytometry of organs harvested on day 21 showed that compared to dual therapy groups, mice treated with the triple-modality treatment had increased tumor infiltration by interferon-gamma+ (IFN?) and tumor necrosis factor-alpha+ (TNFa)-producing CD4+ T cells, as well as IFN?+ CD8+ lymphocytes.

Conclusions: Combining anti-TIM-3 with anti-PD-1 and radiation was synergistic and conferred a significant survival benefit.

Patient Care: Conventional treatment modalities have shown limited efficacy against human GBM. The addition of local radiation to dual immunotherapy with PD-1 and TIM-3 blockade is a novel treatment approach to induce antitumor immunity against malignant gliomas. Our results provide strong preclinical evidence to support combination trials in patients with primary GBM.

Learning Objectives: By the conclusion of this session, participants should be able to 1) recognize the survival benefits of triple therapy with anti-PD-1, anti-TIM-3 and stereotactic radiation and 2) describe the effects that each treatment modality has on the tumor immune microenvironment.

References: 1. Zeng J, See AP, Phallen J, Jackson CM, Belcaid Z, Ruzevick J, Durham N, Meyer C, Harris T, Alebsiano E, Pradilla G, Ford E, Wong J, Hammer H, Mathios D, Tyler B, Brem H, Tran PT, Pardoll DM, Drake CG, Lim M. Anti-PD-1 Blockade and stereotactic radiation produce long-term survival in mice with intracranial gliomas. Rad Onc Bio 2013;86:343–349. 2. Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK, Anderson AC. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J Exp Med 2010;207:2187-2194.