Characterization of murine CEACAM1 in vivo reveals low expression on CD8(+) T cells and no tumor growth modulating activity by anti-CEACAM1 mAb CC1

McLeod, Robbie L.; Angagaw, Minilik H.; Baral, Toya Nath; Liu, Liming; Moniz, Raymond Joseph; Laskey, Jason; Hsieh, SuChun; Lee, Mike; Han, Jin-Hwan; Issafras, Hassan; Javaid, Sarah; Loboda, Andrey; Sadekova, Svetlana; O'Connor, Joann A.; Tse, Archie; Punnonen, Juha

Evaluation of the homophilic interaction of CEACAM1 or interaction of CEACAM1 to TIM-3 was performed by protein ELISA or cell ELISA (cELISA). For cELISA, microtiter plates were seeded with mCEACAM1 or mTIM-3 transfected CHO cells 1 or 2 days prior so that on the assay day the cells were ~80% confluent. On the assay day, the cell culture supernatant was aspirated and 50 µl of serially diluted mCEACAM-hFc (Sino Biologics) or control protein with hFc were added to the microtiter plates. Dilution was done in cell culture medium (DMEM (Gibco) supplemented with 10% fetal bovine serum. After 30 minutes of incubation at room temperature, plates were washed three times with washing buffer (PBS containing 0.05% Tween 20) . Afterwards, 50 µl of horseradish peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch) diluted 1:2000 in cell culture medium was added, and incubated at room temperature for 30 min. Finally, plates were washed five times, and 50 µl of 1-Step Nitro TMB-ELISA substrate (ThermoFischer) per well was added for 5 min. Reactions were stopped by adding 50 µl of TMB Stop solution (KPL), and the absorbance was measured at 450-620 nm. Protein ELISA is performed very similar to cELISA with few difference. For protein ELISA, microtiter plates were coated with 50 µl of mCEACAM-His (Sino Biologics) 1 µg/ml in PBS at 4° C over-night. Next day, plates are washed three times with wash buffer and blocked with 200 µl Supeblock (Thermo Scientific) for one hour at room temperature. After washing the plates, addition of mCEACAM1-hFc or control hFc, addition of anti-human HRP and incubation steps were same as in cELISA. Finally, plates were washed five times, and 50 µl of ABTS peroxidase substrate (KPL) per well was added. After 5 min. the absorbance was measured at 405 nm.