Be0004

To establish a syngeneic mouse model, 3× 10⁵ B16F10 cells resuspended in PBS were mixed 1:1 by volume with Matrigel (354262, Corning) and subcutaneously injected into the right flank of seven-week-old wild-type female C57BL/6J mice on day 0. Treatments were started on day 7 when the tumor volume reached approximately 100 mm³. Antibodies were administered in 100μL volumes and injected intraperitoneally every three days for the following: anti-PD1 (100 μg, BE0146, BioXcell) and anti-CD8 (500 μg, BE0004, BioXcell). The RIPK1 degrader LD4172 and RIPK1 kinase inhibitor T2I were delivered in 200 μL of vehicle solution (30% PEG400, 5% Tween-80, 5% DMSO) once daily (20 mg/kg, i.p. injection). T-cell blocking or depletion was confirmed by flow cytometry in tumor-bearing mice. Tumors were measured every three days beginning on day 7 after the challenge until death. Death was defined as the point at which a progressively growing tumor reached 1.5 cm in the longest dimension, or severe tumor necrosis was observed. Measurements were performed manually by collecting the longest dimension (length) and the longest perpendicular dimension (width). Tumor volume was estimated using the following formula: (L×W²) / 2. CO₂ inhalation was used to euthanize mice on the day of euthanasia.