Development and validation of a cell-based SEAP reporter assay for the detection of neutralizing antibodies against an anti-IL-13 therapeutic antibody

Abstract

A cell-based bioassay capable of detecting neutralizing antibodies (NAb) specific to a therapeutic anti-IL-13 monoclonal antibody was developed, validated and used to analyze normal human and asthma serum samples. At the time of this study, a neutralizing assay was unavailable for anti-IL-13 antibody therapeutics with sufficient rigor for validation. Thus, we describe here a method and considerations for validation. The assay used IL-13 responsive HEK293 cells transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene. Cells were plated at 5.4×10(4) per assay well due to 90% confluence on the subsequent day. Optimal IL-13 and anti-IL-13 concentrations were determined to be 600 pg/mL and 900 ng/mL respectively. We demonstrated the assay's cut point, sensitivity, specificity/cross reactivity, selectivity/matrix interference, and precision. Also, we demonstrated how the drug inhibitory concentration (IC(50), IC(75), and IC(90)) can affect sensitivity and dynamic range/assay window. We characterized the differences in assay response between serum samples of normal population and asthma population. Asthma samples demonstrated an elevated OD ratio in average compared to normal samples. Thus, separate cut points were needed and calculated to be 1.78 and 2.43 for normal and asthma serum, respectively. The assay sensitivity was 670 ng/mL with the positive control (affinity purified rabbit anti-drug polyclonal antibodies). Potential false positives resulting from endogenous serum cytokines including IL-13, IL-4, and Interferon alpha (INF-α) were evaluated and the results indicated that the interfering concentrations for these cytokines are much higher than the respective physiological concentrations. Based on these data, the risk of false positive by endogenous cytokines was considered to be low. In addition, irrelevant anti-drug positive control antibodies were evaluated for assay specificity and did not demonstrate neutralizing capability. Further, no matrix interference in the intended patient population was found when using a final assay serum concentration of 16.7%. The validated assay had acceptable intra- and inter- assay precision in that all %CVs were ≤25%. Overall, this assay successfully proceeded through validation and was used to determine NAb responses within serum samples.