Efficient Method for High-Throughput Screening of Compound Libraries Targeting Human PD-L1

Abstract

The PD-1/PD-L1 immune checkpoint is a pivotal target for cancer immunotherapy. Monoclonal antibodies (mAbs) targeting the PD-1/PD-L1 interaction have achieved clinical success but face limitations, including high production costs, suboptimal tumor penetration, and potential immunogenicity. To address these challenges, we present the DNA-linked Inhibitor Antibody Assay (DIANA)-a robust, high-throughput screening platform optimized for identifying and characterizing low-molecular-weight inhibitors of human PD-L1. DIANA integrates competitive binding with qPCR detection, enabling single-well determination of dissociation constants (K d) and rapid screening of thousands of compounds. The assay was validated using three FDA-approved mAbs (atezolizumab, avelumab, and durvalumab), the PD-L1-binding macrocyclic peptide WL12, and the native PD-1 receptor, yielding K d values consistent with the literature. DIANA demonstrated a broad dynamic range spanning more than 4 orders of magnitude, excellent robustness (Z '-factor = 0.94), and high tolerance to DMSO (up to 10%). We applied DIANA to screen two libraries: a 5,280-compound in-house library (pooled format) and a 1,298-compound commercial peptidomimetic library (individual format). While very weak initial hits were detected, none were confirmed in follow-up manual (non-HTS) experiments or in an orthogonal cell-based assay. Nonetheless, DIANA's sensitivity, scalability, and minimal sample requirements establish it as a powerful tool for accelerating the discovery of next-generation PD-1/PD-L1 inhibitors and overcoming key limitations of conventional screening methods.