Development of analytical methods for detection of anti-drug antibodies neutralizing IL-15 and TGF-β RII activity.

To comply with regulatory guidelines and ensure that biopharmaceutical agents meet safety and efficacy, we developed an analytical method to detect anti-drug antibodies (ADAs) generated in response to HCW9218, a fusion molecule containing the human soluble TGF-β RII and IL-15/IL-15 Rα sushi domains. The method demonstrated a sensitivity of 34.6 ng/mL with a suitably high degree of specificity, selectivity, and precision. To assess the neutralizing capacity of ADAs, we developed cell-based assays specific to IL-15 and TGF-β RII. These assays effectively discriminated the neutralizing activity of sera spiked with neutralizing antibody (NAb). The determination of positive NAb was based on absorbances corresponding to a threshold value of 30% inhibition of IL-15 or TGF-β RII activity. Each assay was validated using human sera from ongoing clinical trials. ADAs were detected in most sera tested with titers less than 10,000. None of the sera inhibited IL-15 and TGF-β RII activity by more than 30%. Interestingly, circulating TGF-β present in sera mimicked the action of NAb by binding to soluble TGF-β RII resulting in higher baseline neutralization activity. These methods proved to be suitable for detection of ADAs and NAbs in HCW9218 clinical samples or analogs sharing similar receptor/cytokine subunits and/or downstream signaling pathways.