Development and Validation of a Quasi-Quantitative Bioassay for Neutralizing Antibodies Against CP-870,893

The human monoclonal antibody CP-870,893 is a CD40 receptor agonist currently being developed for the treatment of cancer. A bioassay to measure neutralizing antibodies (Nab) to CP-870,893 in 5% human serum matrix was developed and validated utilizing the Daudi cell line and flow cytometric detection. Additionally, samples from CP-870,893 treated cynomolgus monkeys were analyzed in the bioassay and compared to results obtained using a competitive receptor-binding (CRB) Nab immunoassay to determine if the CRB assay may be used in place of the bioassay. Treatment of Daudi cells fo r2d with CP-870,893 leads to a concentration-dependent increase in CD54 cell surface expression. The presence of antidrug Nab attenuates CP-870,893 binding to CD40 and the induction of CD54. An anti-idiotype monoclonal antibody (Mab) and a monkey sera pool were identified as positive controls for neutralization of CP-870,893. During development, it was observed that the assay robustness was altered by culture media and FBS substitutions. Fo rv alidation the following parameters were established: cutpoint factors in the presence (0.779) and absence (1.282) of 50 ng/ml CP870,893, linear region of the concentration-response (1‐100 ng/ml CP-870,893), intra- and inter-assay precision (CV ≤ 25%), specificity and recovery (±25%), sensitivity (∼500 ng anti-idiotype Mab per ml serum), technician to technician ruggedness (CV ≤ 25%), and stability (positive control, CD54 labeling, and cell line). A concentration dependent increase in CP-870,893 neutralization was observed in a 3-mo toxicity study in monkeys using both the Bioassay and CRB assay (R 2 = 0.94) suggesting the CRB Nab assay may