A Novel Neutralizing-antibody Assay for Detection of β-NGF Binding Domain-specific Antibodies Against a β-NGF/TNFR2 Bispecific Therapeutic Antibody.

Immunogenicity assessment for therapeutic bispecific antibodies often requires domain-specific characterization, including evaluation of neutralizing antibody (NAb) responses to individual domains. For our beta Nerve Growth Factor (β-NGF)-Tumor Necrosis Factor Receptor 2 (TNFR2) bispecific antibody, anti-drug antibody (ADA) mapping revealed a predominant immune response to the β-NGF binding domain, leading us to explore the correlation of β-NGF domain-specific NAbs with clinical impact. Because drug or target in the sample can interfere with NAb detection, the development of a NAb assay format with sufficient drug and target tolerance is required. To that end, we developed a domain-specific NAb assay, integrating a modified biotin-drug extraction and acid dissociation (BEAD) procedure that includes target blocking to eliminate false-positives. We termed this assay format the "BEAD with target blocking" (BEAD-TB) method. The workflow features sequential acid dissociation to liberate drug-bound NAbs, followed by selective enrichment of β-NGF domain-specific Nabs. We conducted a comprehensive assessment to ensure adequate sensitivity, selectivity, drug tolerance (up to 10 μg/mL), target tolerance (up to 10 ng/mL) and robustness for the assay to accurately detect β-NGF binding domian-specific NAbs in ADA-positive patients. Our domain specific NAb assay demonstrated a robust, innovative solution for detecting neutralizing responses in the context of complex bispecific biologics, leading to reliable immunogenicity evaluation in drug development.