A sensitive enzyme immunoassay for the quantitation of human CTLA4Ig fusion protein in mouse serum: pharmacokinetic application to optimizing cell line selection.

A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantitation of human CTLA4Ig in mouse serum was validated. The EIA method employed a technique in which a monoclonal anti-CTLA4 antibody was adsorbed onto 96-well polystyrene microtiter plates and used to capture the CTLA4Ig in mouse serum samples. The captured CTLA4Ig was then detected using a goat anti-human IgGFc antiserum conjugated to the enzyme horseradish peroxidase. The validation included assessments of method accuracy and precision, range of reliable response, lower limit of quantitation (LLQ), inter-analyst robustness, storage stability in mouse serum and assay specificity. The results indicate that this validated assay is precise, accurate, and reproducible. This EIA has a range of reliable response in 10% mouse serum of 0.14-4.58 ng ml-1 resulting in a 100% serum equivalent curve of 1.4-45.8 ng ml-1. Assessment of individual standard curve variations indicated a reproducible response with R2 values of > or = 0.995. The LLQ was established at 1.4 ng ml-1. The accuracy and precision estimates, based on the quality control values, were within 3.8% and 5.2% respectively, for CTLA4Ig. Stability of CTLA4Ig was established in mouse serum for 5 days at both 4 degrees C and room temperature, for 2 months at -70 degrees C and through five freeze-thaw cycles. This validated assay was successfully employed in the assessment of pharmacokinetic characteristics of CTLA4Ig in mice and to aid in the selection of an optimal CTLA4Ig-producing cell line.

Pharmacokinetics and pharmacodynamics of CTLA4lg (BMS-188667), a novel immunosuppressive agent, in monkeys following multiple doses.

The dose proportionality and multiple dose pharmacokinetics of CTLA4Ig, an immunosuppressive drug under development, were investigated in 12 cynomolgus monkeys in a parallel study. The activity of CTLA4Ig in suppressing the immunoresponses to sheep red blood cell (SRBC) was also assessed. Two monkeys per gender were randomly assigned to one of the three dose levels, 1.0, 2.9, and 8.7 mg/kg of CTLA4Ig. The monkeys in each dose level received the assigned intravenous dose of CTLA4Ig by a bolus injection into a saphenous vein on days 1, 4, 8, 11, 15, and 18. Immediately after the administration of CTLA4Ig on day 1, each monkey was challenged with SRBC. Serial blood samples were collected up to 720 h following administration of CTLA4Ig on day 18 (last dose). In addition, predose blood samples were obtained on days 1, 4, 8, 11, 15, and 18. Serum samples were analyzed for the concentrations of CTLA4Ig using a validated ELISA method. The data obtained were subjected to noncompartmental pharmacokinetic analyses. The serum concentrations of CTLA4Ig attained steady state by day 11 regardless of the dose levels. The mean AUC0-720 values of CTLA4Ig increased in a dose proportional manner. The mean values of Cmax increased in a ratio of 1:3:3:8:5 while the dose administered increased in a ratio of 1:3:9. Predose serum concentrations (Cmin) of CTLA4Ig increased in a dose proportional manner. The mean T1/2 values were not significantly different among the dose groups, suggesting that the elimination characteristics of CTLA4Ig were not altered as the dose increased. Dose-related immunosuppressive activity of CTLA4Ig (78-98% inhibition) was observed in monkeys immunized intravenously with SRBC. In conclusion, CTLA4Ig exhibits linear kinetics and demonstrates significant immunosuppressive activity over a dose range of 1.0-8.7 mg/kg in monkeys.