Be0061

LM-OVA memory mice received injections of 1 mg/kg FTY720 (Sigma-Aldrich) dissolved in 2% cyclodextrin (diluted in sterile PBS) via an intraperitoneal route on days –1, 0, +1, and 2 relative to HSV-2 infection. Control mice received diluent (2% cyclodextrin in sterile PBS).
CD8⁺ T cells were depleted at days –3, –1, +1, and +3 relative to HSV-2 infection by intraperitoneal injection of 200 μg of anti-CD8 (BE0061, Bio X Cell) or isotype control antibody (BE0090, Bio X Cell) in 200 μL volume (diluted in sterile PBS).

Approximately 1 × 10⁶ B16.F10 cells suspended in 100 µL of PBS were inoculated into the rear right flank of 6–8 weeks old C57BL/6 mice (The Jackson Laboratory) via a subcutaneous injection. When tumors reached a volume of approximately 50 mm³, mice were given three 100 µL intratumoral injections administered every 3 days with treatments of either PBS, D-PDB polymer NPs, Edited Alu-NPs, or Alu-NPs [i.e., AluJb RNA (Left Arm)/D-PDB at an N/P ratio of 4] at a 2 µg RNA dose or polymer equivalent. Edited Alu-NPs utilized an inactive form of AluJB RNA (Left Arm). Tumor volume, total murine mass, and murine well-being were recorded every other day. A maximum tumor volume of approximately 1,500 mm³ was the predetermined study endpoint and used as the basis for “survival” analysis. A T-cell depletion study was employed to evaluate the T-cell dependence of treatment efficacy. Mice were inoculated and treated as described above. However, T-cell depletion via intraperitoneal injection of α-CD8 mAb (BE0061; BioXCell) was initiated 24 hours prior to NP treatment. To this end, 100 µg of α-CD8 mAb was injected on days −1, 2, 5, 8, and 11 (Fig. 5F). Tumor volume, total murine mass, and murine well-being were again recorded qod.

The CD8α-depleting antibody (2.43 mAb) (BioXCell, BE0061) was diluted in sterile saline for in vivo administration. BALB/c mice were injected with 200 μg 2.43 mAb intraperitoneally on day -3 and day -1 relative to the start of the functional assay on day 0.