(T1330-05-30) Validation of a Colorimetric Enzyme Cell-Based Assay for the Detection of Neutralizing Antibodies to IL-2 in Human Serum

Purpose: To develop and validate a colorimetric enzyme cell-based assay to detect neutralizing antibodies to IL-2 in human serum. This assay provides an efficient and less labor-intensive method to test for neutralizing antibodies to IL-2 or IL-2 muteins.

Methods: This colorimetric enzyme cell-based assay consists of (i) a neutralizing antibody (NAb) screening assay and (ii) a specificity assay. The screening assay detects inhibition of IL-2 activity present in test serum samples and the specificity assay tests for IL-15 activity. The specificity assay is conducted to determine that inhibition of IL-2 activity observed in the screening assay is not due to serum cytotoxicity and thereby confirm the presence of neutralizing antibodies specific to IL-2 and not to IL-15. Sample serum is used to treat HEK-Blue^TM IL-2 receptor cells designed with a fully functional IL- 2 signaling cascade and a STAT5-inducible secreted embryonic alkaline phosphatase (SEAP) reported gene. Upon activation by human IL-2 or human IL-15, IL-2 receptor couples to JAK tyrosine kinases, which activates STAT5 and induces secretion of SEAP. Levels of SEAP are quantified by using detection media QUANTI-Blue^TM and measuring optical density by spectrophotometer. In the presence of anti-IL-2 neutralizing antibodies, IL-2 activity is neutralized, and the expression levels of SEAP are reduced. Anti-IL-2 neutralizing antibodies are not expected to neutralize IL-15 induced SEAP activation in the specificity assay. An anti-IL-2 mAb was used as the positive control (PC). Assay parameters evaluated during validation included cut point determination, sensitivity, hook effect, precision, control suitability, specificity, selectivity and stability.

Results: The validated assay cut point for IL-2 screening assay value cut point (SAV CP) was established as SAV = 0.86 and for the IL-15 specificity assay value cut point (SpAV CP) was established as SpAV = 0.88. The screening assay sensitivity was calculated to be 246 ng/mL for anti-IL-2 mAb in the IL-2 assay. Anti-IL-2 mAb did not demonstrate a hook effect at concentrations as high as 100000 ng/mL in the IL-2 screening assays. Assay controls demonstrated acceptable intra- and inter -assay precision for both the IL-2 screening and IL-15 specificity assays with a %CV less than 30%. The presence of soluble IL-2 Rα monomer did not cross-react with IL-2, IL-15 or positive control antibodies for both the IL-2 screening and the IL-15 specificity assay. The presence of hemoglobin at levels up to ~1100 mg/dL did not interfere with IL-2 and IL-15 activity. All donors at each medium and high levels of lipemia did not produce false positives. All stability samples at all tested conditions produced the expected results for IL-2 and IL-15. Positive control samples were stable for at least 6 freeze/thaw cycles, storage at ambient room temperature for 24 hours or at 2° to 8 °C for 3 days.

Conclusion: A screening and specificity assay to determine neutralizing antibodies was validated. The assay provided an efficient and high throughput method to test clinical samples against IL-2 and IL-2 muteins.