Anti-drug antibody analysis
Plasma samples were analyzed for antibodies binding to inclacumab, and the titer of confirmed positive samples was reported. The detection and characterization of anti-inclacumab antibodies in participant plasma was performed using a multitiered approach with a validated bridging electrochemiluminescence immunoassay at PPD Bioanalytical Laboratory.In this assay, samples, positive controls, and a negative control were preincubated overnight with biotin-inclacumab. Any ADA present in the plasma formed a complex with the biotin-inclacumab; the complex was then bound to a streptavidin plate (Pierce). After a wash step to remove noncomplexed components, the ADAs were eluted using glycine and incubated with biotin-inclacumab and SULFO-TAG-inclacumab molecules to form a complex for detection. This complex was bound to a blocked streptavidin plate (Meso Scale Discovery) and detected by a chemiluminescent signal generated when voltage was applied. This resulting electrochemiluminescent signal was directly proportional to the amount of ADA present.Unknown samples were analyzed in a tiered approach. The first tier, a screening assay, identified samples as “potentially positive” when their results were equal to or above the plate-specific cut point. Samples with results below the plate-specific cut point were considered negative. The second tier, a confirmatory assay, identified “potentially positive” samples as “confirmed positive” when their results were at or above the confirmatory plate-specific cut point. Confirmed positive samples were then diluted (titered) until a negative response was obtained (third tier). The sample titer was reported as the interpolated titer result for each positive sample (inclusive of minimally required dilution).For the mAb-positive control, the sensitivity was 3.42 ng/mL for the screening assay and 10.3 ng/mL for the confirmatory assay. For the polyclonal antibody positive control, sensitivity was < 0.477 ng/mL for the screening assay and 0.900 ng/mL for the confirmatory assay. Drug tolerance was determined to be 400 μg/mL at 100, 500, and 2500 ng/mL of anti-inclacumab monoclonal and polyclonal antibodies in the screening and confirmatory assays.This method is applicable for the detection, confirmation, and titration of anti-inclacumab antibodies from a 20-μL participant plasma aliquot containing sodium citrate. Samples were frozen at approximately − 80 °C before analysis.