Biologic based therapeutics are used for treatment of many diseases and represent the most advanced therapies available. These drugs often generate immune responses in patients that may impact drug efficacy and adversely affect patient safety. Regulatory agencies responsible for drug approval recommend an assessment of anti-drug immune responses including a screening assay to detect the presence of anti-drug antibodies (ADA), titer, assessment of drug neutralizing activity, and classification of ADA responses with respect to antibody class and subclass. The industry uses singleplex assays to detect and characterize ADA responses in a tiered sequential manner. This presentation will introduce the concepts of Immunogenicity and share a novel multiplexed based approach to detect and characterize ADA responses.

We selected Humira, a human IgG1 antibody that binds to TNFa as the biologic drug for this study which has been shown to induce ADA responses. Biotinylated Humira was used to capture and isolate ADA from clinical samples using streptavidin coated plates. This also removed non-specific antibodies and other matrix components from the purified ADA preparation. The ADA were subsequently mixed with Luminex bead sets each coupled to an Fc specific anti-human Ig class or subclass to delineate the different classes and subclasses from each other. These included human IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE. The beads were washed and incubated with biotinylated Humira to detect drug specific ADA captured on each bead set. Streptavidin-phycoerythrin was added followed by analysis of the bead sets in a Luminex Flexmap-3D instrument. The median fluorescence intensity (MFI) of >50 beads per set was calculated and used in the final data analysis.

The data show that each isotype specific bead set had good specificity for the respective isotype. The detection of ADA in samples was based on a statistically derived cut point based on a set of 50 drug-naïve samples analyzed in the assay 6 times across several days. This cut point was set so that 95% of drug naïve samples fall below the cut point and samples with drug specific antibodies present would generate a signal above the cut point. The cut point was set to yield a 5% false positive rate to ensure all samples with ADA would be detected. A separate cut point was calculated for each specific antibody class or subclass. Clinical samples from psoriasis patients and other autoimmune disease patients were analyzed for the presence of ADA. The results show the presence of ADA and a mixture of different isotypes present. The sensitivity of the assay was determined to be less than 50 ng/ml using a human anti-Humira antibody as a positive control. The presentation will also show data from clinical samples with respect to the presence of ADA and the respective isotypes present.

The multiplexed approach described here demonstrate an advantage over single assay approaches with respect to minimizing clinical sample freeze thaws, cost and labor, while providing a robust and reliable method for ADA evaluation. This method yielded results that were comparable to the FDA guidelines for assessment of Immunogenicity.