Mitigation of pre-existing immunogenicity in ADA method development

BACKGROUND: Immunogenicity assessments are critical for evaluating biological therapeutics. Preexisting immune reactivity can affect clinical safety and efficacy and confound anti-drug antibody (ADA) assay cut point determination, complicating identification of treatment-emergent ADA. In the ALXN-X program, approximately 50% of naïve human serum samples showed preexisting reactivity, with some signals exceeding background by more than 100-fold, challenging ADA method development (cut points, sensitivity, drug tolerance). METHODS: Competitive inhibition experiments were conducted to map the binding site of the preexisting reactivity and depletion with protein A/G agarose was performed to further characterize the preexisting reactivity. A modified ADA assay with an engineered drug molecule was developed to mitigate its impact on assay performance. RESULTS: This preexisting reactivity binds to the C-terminal conserved framework sequence of the drug molecule. Characterization indicated that the preexisting reactivity is likely attributable to immunoglobulins. The modified ADA assay can effectively mitigate the impact of the preexisting reactivity on the assay performance, while maintaining the ability to detect ADA response specific to the unique epitopes of ALXN-X. CONCLUSIONS: Preexisting, immunoglobulin-mediated reactivity targeting ALXN-X's Cterminal framework sequence complicates ADA assay performance. A modified ADA assay mitigates this interference while preserving detection of ALXN-X-specific ADA, supporting more reliable immunogenicity assessment.