Purpose: Therapeutic targets such as homodimeric or trimeric cytokines can induce false positive signals which is a common problem in bridging methods used to detect anti-drug antibodies (ADA). For example, a recent report described the development of an ADA assay where drug bound target was removed from the assay to eliminate the target-dependent false positive signal. The presence of two soluble targets further complicated development of the ADA assay used to support development of our bi-specific antibody. Using the U-PLEX™ Assay Platform, we have developed a novel method that is able to simultaneously measure ADA dependent responses and cytokines target responses.
Methods: The method employs five different specialized linker molecules with specific binding for biotinylated proteins. The following antibodies were biotinylated; bi-specific Ab, bi-specific arm A Ab, bi-specific arm B Ab, homodimeric cytokine A Ab, trimeric cytokine B Ab. The bi-specific Ab was also conjugated with ruthenium. The biotinylated antibodies, linkers and ruthenylated drug were used to create a master mix able to detect and differentiate between ADA and target signal. After an acid treatment, serum samples were neutralized and incubated with master mix. Following that incubation, samples were placed on the U-PLEX plate and incubated. Then the plate was washed, read buffer was added and the electrochemiluminescent signal was detected on an MSD plate reader.
Results: The selectivity of the method and the absence of cross reactivity between ADA and target spots was demonstrated. Screening of 40 individuals was done to determine spot specific cut points. The method sensitivity for the bi-specific arm A ADA and bi-specific arm B ADA was 2.0 ng/mL for both. The homodimeric cytokine A and trimeric cytokine B were detected at concentrations greater than 500ng/mL and 1.6ng/mL, respectively. The drug tolerance of the method to interference from unlabeled drug was measured at 100ng/mL of arm A and B domain ADAs. Detection of both ADAs tolerated 1: 500 molar excess of drug. The ADA and target specific responses were reproducible and assay precision was acceptable.
Conclusion: We have successfully developed a novel multiple spots U-PLEX ADA assay suitable for detection of two domain specific ADAs and two soluble targets in a single well measurement. The method significantly reduced analysis time and sample volume requirements. With good assay sensitivity, specificity, drug tolerance, and reproducibility, it holds great promise for supporting potential therapeutic bi-specific Ab development and other potential immunogenicity applications.