Purpose: Immunogenicity is a known risk factor for biotherapeutic drugs, which can have a direct impact on patient safety and drug efficacy. However, due to the complex nature of immunogenicity testing, developing a method that can unequivocally detect clinically relevant concentrations of anti-drug antibodies (ADA) in the presence of a circulating biotherapeutic drug or other interfering matrix components can be a challenge. The most common solution to this problem is the bridging assay, which uses the bivalent nature of ADA to form immunocomplexes between binding and detection components in a mixture of labeled biotherapeutics (master mix). Typically, the formation of these immune complexes is performed in solution and then the complexes are bound to a solid phase (e.g. ELISA, MSD) for detection. During development of an electrochemiluminescent bridging ADA assay for CDX-301, a recombinant FLT3 ligand, significant non-specific binding of master mix components (Biotin-CDX-301 & Ruthenium-CDX-301) was observed, leading to increased background and decreased sensitivity in both the screening assay and subsequent workflows. As FLT3 ligand exists in solution as a homodimer, it was hypothesized that dimers of Biotin-CDX-301 and Ruthenium-CDX-301 were exchanging during solution phase incubations and re-associating to include “heterodimers” of Biotin-CDX-301 and Ruthenium-CDX-301, contributing to false-positive assay signals.
Methods: Master mix concentrations and the minimum required serum dilution (MRD) were optimized to minimize non-specific binding and improve assay sensitivity and drug tolerance using Design of Experiment (DoE). These data showed that an equimolar solution of master mix reagents (Biotin-CDX-301 and Ruthenium-CDX-301) provided the best assay sensitivity and drug tolerance. An inverse relationship was observed when comparing MRD, such that increasing the MRD tended to improve drug tolerance, but decrease assay sensitivity. In addition, different blocking buffers, assay buffer additives (Magnesium Chloride, Triton X-100, Sodium Dodecyl Sulfate) incubation times, and solid phase capture formats were evaluated to decrease non-specific binding of master mix reagents.
Results: The evaluation showed that incubation times had the biggest impact on decreasing non-specific binding, lending support to the hypothesis that derivatized master mix monomers were exchanging during the solution phase incubations. An alternative sequential version of the bridging assay was evaluated to limit solution phase interaction of Biotin-CDX-301 and Ruthenium-CDX-301. Although this format dramatically decreased non-specific binding, assay sensitivity was negatively impacted and drug tolerance was inadequate to support in-study sampling at the designated collection points.
Conclusion: Based on the data obtained from these experiments, it was established that the most suitable assay format for the CDX-301 ADA assay was a master mix based bridging assay with an abbreviated solution phase incubation time.
Marley Kapsimalis– Senior Associate Scientist, Celldex Therapeutics, Inc., Nedham, Massachusetts
Jason DelCarpini– Senior Manager, Clinical Assays, Celldex Therapeutics, Needham, Massachusetts
Thomas Hawthorne– Director, Bioanalysis & Clinical Assays, Celldex Therapeutics, New Haven, Connecticut