Mouse syngeneic tumor models

4T1 breast carcinoma and CT26 colon carcinoma cell lines were obtained from ATCC. Sa1/N fibrosarcoma and MC38 colon adenocarcinoma cells were obtained from Dr. J. Allison (MD Anderson Cancer Center), and B16-SIY melanoma cells were obtained from Dr. T. Gajewski (University of Chicago). Sa1/N and B16-SIY cells were cultured in DMEM with glutamine, 1X beta-mercaptoethanol and 10% FBS. MC38, CT26 and 4T1 cells were cultured in RPMI with glutamine and 10% FBS. Cells were tested and confirmed negative for Mycoplasma and viral pathogens.

For tumor cell inoculation, one vial of Sa1/N cells was thawed, washed and cultured in T150 flasks in Sa1/N culture media. Cells were expanded in 2 passages. Cells were prepared by Trypsin-EDTA (Gibco) treatment for 5min at 37˚C and washed. Cells were counted, and viability was determined by trypan blue (used if viability was > = 95%). Sa1/N cells were resuspended in PBS at 1x10⁷/mL and kept on ice. 6–8 week old female A/J mice were inoculated s.c. on the right flank with 1x10⁶ Sa1/N cells in 100uL PBS using tuberculin syringes with 27-gauge needles. Tumor growth was monitored and on day 7, animals were redistributed into new cages after normalizing the average tumor volume to 100mm³ for each treatment group. 10 mice were included in each treatment group. Mice were dosed i.p. on days 7, 10, 14 and 17 with 5.0, 0.3, or 0.25 mg/kg of 37A10, 37A10-mG2a, JTX-1011-mG2a, 37A10-mG1-agly, or mouse IgG2a (clone C1.18.4, BioXcell) as indicated. Tumor infiltrating cells were analyzed on day 12 as described below. Tumor re-challenge studies were performed by re-inoculating mice on the left flank ~45 days after the last treatment.

One vial of MC38 cells was thawed, washed and cultured in T150 flasks in culture media. Cells were expanded in 2 passages. Cells were prepared by Trypsin-EDTA treatment for 5min at 37˚C and washed. Cells were counted, and viability was determined by trypan blue (used if viability was > = 95%). MC38 cells were resuspended in PBS at 5x10⁶/mL and kept on ice. 6–8 week old female C57Bl/6 mice were inoculated s.c. on the right flank with 5x10⁵ MC38 cells in 100uL of PBS using tuberculin syringes with 27-gauge needles. On day 6, animals were randomized by tumor volume prior to assignment to a treatment group. 10 mice were included in each treatment group with an average starting volume of 100mm³. Animals were administered antibodies via i.p. injections, and dosed on days 6, 9, 13 and 16.

One vial of CT26 or 4T1 cells was thawed, washed and cultured in T150 flasks in culture media. Cells were expanded in 2 passages. Cells were prepared by Trypsin-EDTA treatment for 5min at 37˚C and washed. Cells were counted, and viability was determined by trypan blue (used if viability was > = 95%). CT26 or 4T1 cells were resuspended in PBS at 1x10⁶/mL and kept on ice. 6–8 week old female Balb/c mice were inoculated s.c. on the right flank with 1x10⁵ CT26 or 4T1 cells in 100uL of PBS using tuberculin syringes with 27-gauge needles. On day 3, animals were redistributed into new cages for randomization. 10 mice were included in each treatment group. Animals were administered antibodies via i.p. injections on days 3, 6, 10 and 13 for CT26, and on days 3 and 10 for 4T1.

For in house MC38, B16-SIY, CT26 and 4T1 models, animals were treated with 0.25 mg/kg ICOS antibody (37A10-mG2a in CT26, JTX-1011-mG2a in others) and/or 10 mg/kg anti-PD-1 (RPM1-14, BioXcell) or 0.25 mg/kg mouse IgG2a plus 10 mg/kg rat IgG2a (2A3, BioXcell) isotype controls. For the study in huCTLA-4 knock-in mice, animals were treated with a single 10 mg/kg dose of either clinical grade ipilimumab (Yervoy^®, Bristol-Meyers Squibb) or human IgG1 isotype control administered intravenously, and two weekly 0.25 mg/kg doses of ICOS antibody or mouse IgG2a isotype control administered intraperitoneally. Tumor volumes were measured using calipers and calculated using the formula [0.5 x (length x width²).