Clone pk136

Cellular subsets were depleted by administering depleting antibody i.p. twice weekly beginning 1 day prior to therapy, as follows: CD8 T cells with anti-CD8α (200 µg/mouse, clone 2.43, BioXCell) and NK cells with anti-NK1.1 (250 µg/mouse, clone PK136, BioXCell). For ICAM1 blockade, mice were injected intraperitoneally with α-ICAM1 (200 µg/mouse; clone YN1/1.7.4, BioXcell) twice per week.

Monocyte depletion in marmoset monkeys was initiated 14 days after immunization by twice weekly i.v. injections of 5 mg/kg DOC-2 Fr-2 (marmoset IgG1-chimeric humanized mouse anti-human CCR2 antibody). Controls received 5 mg/kg marmoset IgG1-chimeric isotype control antibody. The administration frequency was reduced to once weekly from day 28 to the end of the experiment. In mice, all depletion and blocking experiments started at disease onset. NK cells were depleted in Th/+ mice by daily i.p. injections of 300 µg of the mouse monoclonal anti-NK1.1 antibody (Clone PK136, Bio X Cell, BE0036). Control Th/+ mice received 300 µg i.p. of the isotype control antibody C1.18.4 (Clone C1.18.4, Bio X Cell, BE0085). To block the formation of the membrane attack complex (MAC) 2 µg of the BB5.1 monoclonal antibody against mouse complement component C5 (Hycult biotech, HM1073) [23 (link)] or a mouse IgG1 control antibody (BioLegend, Clone MOPC-21) was injected intracerebrally at the time point of stereotactic cytokine injection.

To establish syngeneic tumor models, B16F10 (1 × 10⁶ cells to C57BL/6), CT26 (5 × 10⁵ cells to BALB/c), and EG7-OVA (1 × 10⁶ cells to C57BL/6) were injected subcutaneously to the right flank of mice. For lung metastasis, B16F10 (1 × 10⁵ cells) were injected intravenously to B16F10 tumor bearing mice, 4 days after subcutaneous injection. For dual flank model, 1 × 10⁶ and 5 × 10⁵ B16F10 cells were injected subcutaneously to the right and left flank of mice, respectively. When tumor reaches an average volume of 50–100 mm³, the mice were randomized into several groups according to the experimental protocol. Tumor volume (mm³) was calculated as (width)² × (length) × 0.5. IT dosing on the right tumors was performed three times with 3 days interval. To establish a mouse orthotopic hepatocarcinoma model, Hepa1-6 cells (1.5×10⁶) in HBSS medium were injected into spleen of C57BL/6 mice and the spleen was excised subsequently. IV dosing was performed three times with 3 days interval from Day 4 after cell inoculation. Isotype control antibody (10 mg/kg, BioLegend), Anti-CD8 (10 mg/kg, BioLegend, Clone 53–6.7), Anti-NK1.1 (10 mg/kg, BioLegend, Clone PK136), Anti-CSF1R (10 mg/kg, BioXcell, Clone AFS98), Anti-PD1 (10 mg/kg, BioLegend, Clone RMP1–14), and Anti-CTLA4 (5 mg/kg, BioLegend, Clone 9H10) were administered intraperitoneally.

For the in vivo depletion of CD4⁺ T, CD8⁺ T, or NK cells, anti-CD8 antibody (400 μg; clone YTS 169.4, BioXcell), anti-CD4 antibody (400 μg; clone GK1.5, BioXcell), or anti-NK1.1 antibody (400 μg; clone PK136, BioXcell) was administered twice weekly via intraperitoneal injection into MC38 tumor–bearing C57BL/6 mice 1 day before the first treatment with anti–PD-1 antibody (clone RMP1-14, BioXcell; 5 doses of 200 μg every 3 days).