Characterization of abatacept mediated immunogenicity using domain-specific and neutralizing antibody assays: a biosimilar perspective

BACKGROUND: Abatacept, a CTLA-4-IgG1 Fc fusion protein, may induce anti-drug antibodies (ADAs) targeting distinct domains with varying functional and clinical implications. Antibodies directed against the CTLA-4 domain are of particular interest due to their similarity to endogenous CTLA-4, highlighting the importance of domain-specific immunogenicity assessment in biosimilar development. RESEARCH DESIGN AND METHODS: A domain-specific, multi-tier immunogenicity strategy was applied in a Phase 1, randomized, double-blind, parallel, single-dose study comparing DRL_Abatacept with US- and EU-licensed reference products in healthy subjects. ADA detection employed a drug-tolerant electrochemiluminescence bridging assay with acid dissociation, followed by confirmatory whole-molecule abatacept and CTLA-4 domain-specific assays, titer determination, and a cell-based neutralizing antibody (NAb) assay. RESULTS: ADA assays demonstrated high sensitivity (3.69-5.07 ng/mL), precision (≤12% CV), and drug tolerance up to 500 µg/mL, with no hook effect observed. The NAb assay showed sensitivity of 391.47 ng/mL and acceptable reproducibility (≤30% CV). ADA incidence at Day 85 was low and comparable across treatment arms, with predominantly low-titer responses and no clinically meaningful impact on safety. CONCLUSIONS: Only a subset of CTLA-4-specific binding ADAs exhibited neutralizing activity; within this Phase 1 study, domain-specific binding therefore did not consistently predict functional neutralization. A tiered, functionally anchored approach is essential for comprehensive immunogenicity assessment of complex fusion proteins. CLINICAL TRIAL REGISTRATION: EudraCT 2022-000926-94.