Affinity-maturation engineering via phage display to optimize anti-idiotype antibodies for neutralizing antibody assays

AIM/BACKGROUND: Anti-idiotype antibodies (anti-IDs) targeting therapeutic antibodies play a crucial role in the development of pharmacokinetic (PK), antidrug antibody (ADA), and neutralizing antibody (NAb) assays during preclinical/clinical phases. However, the generation of fit-for-purpose anti-IDs poses significant challenges, especially for NAb assay development, which requires high-affinity anti-IDs to be effectively competed with the target for its binding to the drug. Our preliminary results from NAb screenings revealed that all 19 anti-IDs generated from a mouse immunization campaign in conjunction with a hybridoma platform failed to demonstrate the ability to effectively inhibit the binding of therapeutic tri-specific mAb1 to the target arm protein 1. METHOD/RESULTS: We subjected the selected parental clone to affinity maturation utilizing phage display technology aimed at enhancing binding affinity and mitigating matrix interference. Following four rounds of panning, our assessments via enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI) analysis demonstrated considerable enhancements in the binding affinity of 11 selected clones. Notably, three candidates further exhibited strong neutralizing capabilities to the drug target arm protein 1 within the matrix for NAb assays. CONCLUSION: Affinity-improved anti-IDs enabled the development of a robust and fit-for-purpose competitive ligand-binding NAb assay.