Purpose: Checkpoint molecules like programmed death-1 (PD-1) and T-cell immunoglobulin mucin-3 (TIM-3) are negative immune regulators that may be upregulated in the setting of glioblastoma multiforme. 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. However, tumor-infiltrating lymphocytes (TIL) can express multiple checkpoints, and expression of ≥2 checkpoints corresponds to a more exhausted T-cell phenotype. We investigate TIM-3 expression in a glioma model and the antitumor efficacy of TIM-3 blockade alone and in combination with anti-PD-1 and SRS. Experimental Design: C57BL/6 mice were implanted with murine glioma cell line GL261-luc2 and randomized into 8 treatment arms: (i) control, (ii) SRS, (iii) anti-PD-1 antibody, (iv) anti-TIM-3 antibody, (v) anti-PD-1 + SRS, (vi) anti-TIM-3 + SRS, (vii) anti-PD-1 + anti-TIM-3, and (viii) anti-PD-1 + anti-TIM-3 + SRS. Survival and immune activation were assessed. Results: Dual therapy with anti-TIM-3 antibody + SRS or anti-TIM-3 + anti-PD-1 improved survival compared with anti-TIM-3 antibody alone. Triple therapy resulted in 100% overall survival (P < 0.05), a significant improvement compared with other arms. Long-term survivors demonstrated increased immune cell infiltration and activity and immune memory. Finally, positive staining for TIM-3 was detected in 7 of 8 human GBM samples. Conclusions: This is the first preclinical investigation on the effects of dual PD-1 and TIM-3 blockade with radiation. We also demonstrate the presence of TIM-3 in human glioblastoma multiforme and provide preclinical evidence for a novel treatment combination that can potentially result in long-term glioma survival and constitutes a novel immunotherapeutic strategy for the treatment of glioblastoma multiforme.
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