(T1430-06-36) Addressing Target Interference During the Development of a Neutralizing Anti-Drug Antibody Assay for the Clinical Support of a Bispecific Therapeutic Antibody

Purpose: This study aimed to develop and validate a competitive ligand binding assay to detect and characterize neutralizing anti-drug antibodies (NAbs) in human serum samples to support the clinical development of a bispecific therapeutic antibody. Developing NAb assays poses significant challenges, including drug tolerance and soluble target tolerance, which must be addressed to ensure reliable detection of neutralizing antibodies. Here, we demonstrate that availability of antibodies that compete for the same epitopes as the bispecific therapeutic antibody can be a powerful assay tool to overcome target interference in NAb assays.

Methods: The competitive ligand binding NAb assay was developed using electrochemiluminescence immunoassay (ECLIA). Assay optimization focused on the sample pretreatment to effectively address drug and soluble target interference, while ensuring that these optimizations did not compromise the assay's precision and sensitivity. For improved drug tolerance, SPEAD (Solid Phase Extraction with Acid Dissociation) pretreatment was optimized. Additionally, competing antibodies were used to improve the target tolerance of the assay.

Results: Several competing antibody clones were selected and tested for their ability to remove soluble target from target-spiked matrix samples. This revealed differences in assay target tolerance, highlighting that the selection of the appropriate competing antibody is crucial for effective removal of soluble target from matrix samples. We also observed that incorporating the selected competing antibody at various steps of the assay procedure, such as during the SPEAD incubation step or as part of the detection phase, impacted the assay's overall target tolerance. Specifically, incorporating the competing antibody during three steps of the assay proved optimal for the removal of soluble target.

Conclusion: Our findings indicate that both the selection of the competing antibody and its incorporation into the assay are critical for maximizing target tolerance while achieving reliable detection of neutralizing antibodies at the recommended assay sensitivity.