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

Female BALB/c mice (8 weeks old, 4 mice/group) were dosed intraperitoneally (IP) with CC1 at 10 mg/kg, 30 mg/kg or 45 mg/kg. A previous described serial micro-sampling method was used to obtain plasma from each mouse at hours 1, 6, 12, 24, 48, 72 and 96 following dosing [33] . Plasma CC1 concentration were determined with an electrochemiluminescence (ECL) method. Briefly, recombinant mouse CEACAM-1 (SinoBiological) was used as a capture reagent and sulfoTAG AffiniPure goat anti-mouse IgG was used as a detection reagent. Twenty-five microliter of the capture reagent was added to each well of MA6000 96 Small Spot plate (Meso Scale Discovery) and incubated overnight at 4° C with shaking. After washing with 0.05%Tween20 in PBS three times, the plate was blocked with 5% bovine serum albumin BSA at 150 µL per well and incubated at room temperature for 1 hour with shaking. After additional washings, Twentyfive µL of calibration standard, quality control or sample was added to each well of the washed plate and incubated at room temperature for 1 hour with shaking. After washes, the plate was incubated with 0.5 µg/mL of sulfoTAG AffiniPure goat anti-mouse IgG for 1 hour at room temperature. Then the plate was washed three times and 150 µL of 1× Reading Buffer T (Meso Scale Discovery) was added to each well of the plate followed by reading on a Meso Sector s600 Model 1201 (Meso Scale Discovery). CC1 concentration data were analyzed and key PK parameters calculated using non-compartmental methods with Phoenix ® 32 WinNonlin ® 6.3 software. Data were also analyzed with a WinNonlin ® 2 compartmental model which was used to simulate multi-dose PK profiles.