Be0087

Doxorubicin (DOX, MedChem Express, HY-15142) and Cyclophosphamide (CTX, MedChem Express, HY-17420) were used in the chemotherapy cocktail. The clinically used doses of DOX and CTX were converted from human to mouse equivalents using FDA-approved formula, HED (mg/kg) = Animal Dose (mg/kg) × 0.08. Chemotherapy cocktail was administered intravenously either once a week (MTD) or thrice a week (once every two and a half days) (MCT) for a period of 4 weeks. The dosage of DOX used was 2 mg/kg body weight and CTX 20 mg/kg body weight for the MTD cohort and a third of the MTD dose for the MCT cohort. For a PD-1 inhibitor treatment trial, both PD-1 inhibitor (BioXcell, BE0146) and IgG (BioXcell, BE0087) antibodies were injected intraperitoneally once a week at a dose of 150 µg per mouse. The PD-1 inhibitor was dosed at 6 mg/kg body weight, which is a mouse equivalent dose of PD-1 inhibitor clinical human dose. Stat1 inhibitor (MedChem Express, HY-B0028) was given at 8.4 mg/kg body weight (derived from clinically used human doses and calculated as per mouse equivalents) dose in MCT strategy intravenously along with MCT as a cocktail prepared right before administration.

At the age of four months, a combination of anti-mouse PD1 (Bio X Cell, BE0146) and anti-mouse CTLA-4 (Bio X Cell, BE0131) was administered at a dose of 5 μg of each antibody per gram of mouse, both antibodies in 100 μL. To eliminate CD8 T cells, anti-mouse CD8α (Bio X Cell, BE0061) was administered at a dose of 100 μg per mouse. As control treatment, a combination of rat IgG2a isotype control (Bio X Cell, BE0089) and polyclonal Syrian hamster IgG (Bio X Cell, BE0087) was used as a control at the same dose. All treatments were administered by intraperitoneal injection every three days for eighteen days. Mice were weighed every three days to discard treatment toxicity. The in vivo response to immune checkpoint blockade or CD8 T cells inhibition was evaluated by measuring each prostate lobe volume one week after the treatment regime was completed.

All experiments were approved by the NCI-Bethesda Animal Care and Use Committee. Six to 8 week-old female albino C57BL/6 mice were purchased from Jackson laboratories (Bar Harbor, ME). For intracranial tumor implantation, mice were injected with 1*10³ GL261 cells that were stably transduced with a firefly luciferase-mCherry lentiviral vector. water-soluble dexamethasone (D2915, Sigma Aldrich) was administered at 1 mg/kg/day by oral gavage. The non-toxic solubilizer, 2-hydroxypropyl-beta-cyclodextrin (H-107, Sigma Aldrich) was dissolved with water and matched in concentration to water-soluble dexamethasone for vehicle control. InvivoMab anti-mouse CTLA-4 (BE0131, BioXCell) or InVivoMab polyclonal Syrian hamster IgG isotype antibody (BE0087, BioXCell) were administered by intraperitoneal injection. Tumor was detected by luminescence imaging and analyzed with LivingImage Software.