Rapid screening of paratope-targeting peptides for the detection of anti-PD-1 monoclonal antibodies

Abstract

Immune checkpoint blockade targeting PD-1 has emerged as a frontline therapy for diverse cancers, yet patient responses to anti-PD-1 monoclonal antibodies (mAbs) such as nivolumab and tislelizumab remain highly variable. These differences are closely linked to drug exposure, which affects both therapeutic benefit and adverse outcomes; however, the pharmacokinetic profiles of these antibodies are not well defined. This highlights the need for therapeutic drug monitoring (TDM) to guide precision immunotherapy. Yet, the development of rapid and reliable TDM methods is hindered by the lack of highly specific paratope-targeting biorecognition elements. In this study, we established a positive/negative F(ab')₂-based phage display screening strategy to efficiently identify paratope-targeting peptides for anti-PD-1 mAbs. Using this approach, we identified both cyclic and linear peptides, with two candidates showing good affinity and specificity toward anti-PD-1 mAbs while exhibiting minimal IgG binding. Cyclic peptides demonstrated higher specificity than their linear counterparts. Building on these peptides, we further developed a gold nanoparticle (AuNP)-based point-of-care detection platform that produced synchronized colorimetric signals, enabling simple, rapid, and accurate detection of anti-PD-1 mAbs. Application to clinical samples confirmed good accuracy compared to conventional ELISA. This strategy offers an effective tool for peptide screening and TDM of anti-PD-1 mAbs; it provides a broadly applicable framework for monitoring other therapeutic antibodies in clinical practice.