4-1BB agonist monotherapy. For studies with a palpable model, 0.5 × 106 MC38hB7-H3 or 0.1 × 106 B16-F10hB7-H3 cells were subcutaneously implanted into the right flank of C57BL/6 and 4-1BB KO mice. CT26hB7-H3 cells (0.5 × 106) were subcutaneously implanted into the right flank of BALB/c mice. Tumor growth was monitored by digital caliper measurements three times a week, and tumor volume was calculated with the following formula: length × width × width × 0.5, where length is the largest diameter and width is the smallest diameter. When the tumors reached 2 to 4 mm in diameter, usually 6 to 8 days after inoculation, mice were randomized to receive treatment. Tumor-bearing mice were treated with two (for MC38 and CT26 tumor model) or four (for B16-F10 tumor model) intraperitoneal injections (3-day interval) of InVivoPlus hIgG1 isotype control (Bio X Cell, BP0297), 1D8, B5, or B5×1D8. Four (for the B16-F10 tumor model) or 7 (for the MC38 and CT26 tumor model) days after the last treatment, serum was prepared to analyze the ALT/AST level. The dose of antibodies is determined by their molar ratio: mAb:bsAb = 3:4. Mice were euthanized when tumor size reached a diameter of 20.0 mm length. The tumor-free status was classified as a tumor with a diameter of less than 3 mm for three or more consecutive measurements. To study the immune memory responses, surviving mice from the MC38hB7-H3 challenge and age-matched naïve mice were subcutaneously implanted with 0.5 × 106 parental MC38 tumor cells, which are not expressing hB7-H3, into the left flank. Some surviving mice were subcutaneously implanted with parental MC38 tumor cells on the left flank and irrelevant B16-F10 tumor cells on the right flank.

For studies with h4-1BB KI mice, 0.5 × 106 MC38hB7-H3 cells were implanted into the right front flank. Tumor-bearing mice were randomly enrolled into treatment groups (n = 6 or 8 per group) when the mean tumor size reaches approximately 100 mm3 (80 to 120 mm3). Tumor-bearing mice were treated a total of five or six intraperitoneal injections (every 3 days) of hIgG1 isotype (7.5 mg/kg), B5 (7.5 mg/kg), 1A10 (7.5 mg/kg), or B5×1A10 (3 mg/kg) for Fig. 6D and hIgG1 isotype (2.25 mg/kg) or B5×1A10 (3 mg/kg) for Fig. 6E. Tumor growth was monitored twice a week, and mice were euthanized when tumor volume reached 3000 mm3.

4-1BB agonist with ICB combination therapy. For the combination therapy, MC38hB7-H3 tumor–bearing mice were randomized to receive the treatment when the tumor reached an average volume of 100 to 200 mm3, usually 12 to 14 days after inoculation. Tumor-bearing mice were treated with four intraperitoneal injections (3-day interval) of hIgG1 isotype or B5×1D8 and two intraperitoneal injections (5-day interval) of InVivoMAb anti-mouse PD-1 (clone 29F.1A12), InVivoMAb anti-mouse CTLA-4 (clone 9D9), InVivoMAb anti-mouse Tim-3 (clone RMT3-23), or InVivoMAb rat IgG2a isotype control (clone 2A3) (all from Bio X Cell).

In vivo cell depletion. For a study with immune cell depletion, InVivoMAb anti-mouse CD4 (clone GK1.5), InVivoMAb anti-mouse CD8α (clone 2.43), InVivoMAb anti-mouse NK1.1 (clone PK136), and InVivoMAb rat IgG2b isotype control (clone LTF-2) (all from Bio X Cell) were intraperitoneally injected at −1, 1, 5, 8, and 11 days after the beginning of treatment at a dose of 0.2 mg per injection. The depletion efficiency was validated by flow cytometric analysis of peripheral blood.

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