Multiplexed evaluation of a cell-based assay for the detection of antidrug neutralizing antibodies to panitumumab in human serum using automated fluorescent microscopy.

The method described here employs a high-content cell-based assay format for the detection of neutralizing antibodies (NAbs) to panitumumab, a fully human monoclonal antagonistic antibody to the human epidermal growth factor (EGF) receptor in human serum (screening assay). A specificity assay was also developed and qualified to confirm that the neutralizing activity was attributable to the presence of NAbs and not due to serum interference (serum interference assay). The ArrayScan IV HCS reader was used for the measurement of tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and STAT-1 redistribution between the cytoplasm and nucleus in the human epidermoid carcinoma cell line A431. Assay conditions were developed by (1) optimizing the response of the A431 cells to recombinant human EGF in pooled human serum, (2) evaluating the ability of panitumumab to inhibit the EGF response, and (3) assessing the assay's sensitivity for detecting a positive control affinity purified rabbit polyclonal anti-panitumumab antibody. Panitumumab dose-dependently inhibited 4 ng/mL EGF, and the positive control antibody showed a dose-dependent neutralization of 50 ng/mL panitumumab. The experiments indicated that in comparison to STAT-1 translocation, EGFR phosphorylation was the optimal endpoint for the screening and serum interference assays. Assay cut points were derived for the screening and serum interference assays by obtaining normalized ratios of mean fluorescence intensity values obtained with EGFR phosphorylation by testing sera from healthy human donor sera. The assay sensitivity was determined to be 0.125 microg/mL for the positive control antibody.