Decoupling Drug Exposure, Binding ADA, and Functional Neutralization in Anti—PD-L1 Therapy Using Orthogonal ELISA Immunogenicity Assessment 2309558

Abstract Introduction Immune checkpoint inhibitors targeting PD-L1 can trigger anti-drug antibody (ADA) responses with varied biological effects. In anti-PD-L1 therapy, circulating drug levels and binding ADA detected via immunogenicity screening do not reliably predict loss of immune checkpoint blockade. Functional neutralizing antibodies (NAbs), which directly block PD-1/PD-L1 signaling, provide a distinct, biologically relevant immunogenicity measure. Accurate assessment thus demands separate evaluation of drug exposure, immunogenicity incidence, and functional neutralization. Methods We applied an ICH-aligned, tiered ELISA-based framework to to evaluate anti-PD-L1 therapy in human serum samples containing Pembrolizumab. Tier 1 used a sandwich therapeutic drug monitoring (TDM) ELISA to quantify total circulating drug. Tier 2 screened for binding ADA via drug-bridging ELISA. Tier 3 assessed NAb activity with a competitive receptor-blocking ELISA measuring PD-L1/PD-1 interaction inhibition. All assays were validated for sensitivity, specificity, precision, and drug tolerance. Results Binding ADA often appeared without loss of PD-L1 pathway inhibition, preserving pharmacological activity despite immunogenicity. Conversely, Tier 3 NAb-positive samples showed reduced PD-L1 blockade, even with detectable Pembrolizumab, confirming functional neutralization. These findings highlight drug exposure, binding ADA, and neutralizing activity as non-redundant variables in anti-PD-L1 therapy. Conclusion Overall, tiered, functionally informed immunogenicity assessment is essential for interpreting responses to anti-PD-L1 therapy. Integrating TDM, binding ADA detection, and NAb ELISAs distinguishes exposure, immunogenicity, and PD-1/PD-L1 blockade loss, enhancing mechanistic insights and risk assessment for checkpoint inhibitors. Funding Source n/a Topic Categories Translational and Interventional Immunology (TI)