Tolinapant promotes antigen-specific responses via induction of necroptosis in vivo. (A) Western blots of BW5147 syngeneic tumor lysates prepared 2, 6, or 24 hours after a single dose of 25 mg/kg tolinapant (compared with vehicle control) to determine necroptosis biomarker pharmacodynamics. (B) As for panel A, except western blots of immunogenic cell death biomarkers. (C) HMGB1 measured by enzyme-linked immunosorbent assay in plasma of BW5147 tumor-bearing animals after 5 days of treatment with 25 mg/kg tolinapant. *P < .05 (Unpaired t test with Welch’s correction). (D) Kaplan-Meier curve showing the evolution of tumor incidence (as percentage of tumor-free animals) over time in the BW5147 vaccination experiment. Cells killed with mitoxantrone (MITO) were used as a positive control. Mice injected with phosphate-buffered saline only (PBS) or cells killed by tolinapant + TNF-α (tolinapant + TNF-α) cohorts, n = 10 per group. MITO, n = 8 per group.