Authors: Karolina Woroniecka; Cosette Dechant; Kristen Rhodin; Pakawat Chongsathidkiet, MD; Daniel Wilkerson, PhD; Xiuyu Cui, MS; Peter Fecci, MD, PhD (Durham, NC)

Background: The success of checkpoint monotherapy against glioblastoma (GBM) has been disappointing. Anti-PD-1 strategies may be hampered by poor baseline T-cell activation, as well as by severe T-cell exhaustion, both characterized by our group. Identifying high levels of 41BB on GBM-infiltrating T-cells (TIL), we combined PD-1 blockade with a 4-1BB agonist, with the aim of pre-activating T-cells and licensing efficacy for checkpoint blockade. Methods: 4-1BB expression was characterized on TIL isolated from human and murine brain tumors. Mice were implanted intracranially or subcutaneously with glioma (CT2A), lung carcinoma (LLC), melanoma (B16F10), or breast carcinoma (E0771). Subsequently, mice received intraperitoneal injections with 4-1BB agonist antibody, PD-1 antagonist antibody, or the combination. Effects of a CD8 depleting antibody were also assessed, and survival was compared. Flow cytometry was used to characterize TIL number and, function, as well as expression of exhaustion-associated alternative checkpoints following treatment. Results: Our data revealed that 4-1BB is a viable target on GBM TILs. 4-1BB is frequently expressed on CD8+ TIL in human GBM, and highly expressed on TIL within murine glioma when compared to other tumors. In murine glioma, 4-1BB agonism and PD-1 blockade demonstrated an additive survival benefit in a CD8 T-cell-dependent manner. The combination decreased T-cell exhaustion and improved T-cell functionality. This strategy, surprisingly, proved most successful against glioma, in a manner correlating with 4-1BB expression on TILs. Conclusions: Poor T-cell activation and severe T-cell exhaustion both prove to be limiting factors for checkpoint blockade in the intracranial environment. 4-1BB agonism obviates these limitations and produces long-term survival in murine glioma. These results may guide personalized treatment strategies employing 4-1BB agonism to improve responses to checkpoint blockade in patients with GBM.