High-Affinity Decoy PD-1 Mutant Screened from an Epitope-Specific Cell Library

Abstract

Immunotherapy with anti-programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) monoclonal antibodies has become routine in the treatment of many kinds of human cancers, such as lung cancer, intestinal cancer, and melanoma. The PD-1/PD-L1 pathway inhibits T cell activation in the micro-environment, making it an attractive target against cancer. Wild-type (WT) PD-1 ectodomain has been shown to have difficulty blocking PD-1/PD-L1 mixture formation due to its low affinity. The present work uses three-dimensional (3D) crystal complex structures to analyze the interaction by which PD-1 binds to PD-L1 or PD-L2. It also reports on a theoretical study of the binding mode between PD-1 and its clinical antibody Opdivo. Based on the theoretical binding analysis of PD-1 and its ligands (i.e., PD-L1 and PD-L2) or antibody (Opdivo), a small-content, epitope-oriented mammalian cell library was established for PD-1. After three rounds of cell sorting, the decoy PD-1 mutant 463, which presented a higher affinity than WT PD-1 to the PD-L1 (the affinity has increased by almost three orders of magnitude) was screened out. It exhibited an inhibitory effect against PD-1 to prevent it from forming mixtures with PD-L1, which was similar to the effect of the commercial anti-PD-L1 antibody atezolizumab (ATE). The median effective concentration (EC₅₀) value of the decoy mutant was 0.031 μg·mL⁻¹ in comparison with 0.063 μg·mL⁻¹ for ATE; both values were much lower than that of WT PD-1, at 2.571 μg·mL⁻¹. The 463 decoy mutant reversed the inhibitory function of PD-1 in T cell activation; furthermore, 10 mg·kg⁻¹ of 463 inhibited about 75% of tumor growth in vivo in a MC38 transgenic xenograft mice model, which was similar to the activity of ATE. More interestingly, an even lower dose of 463 (2 mg·kg⁻¹) showed a better effect than 10 mg·kg⁻¹ of WT PD-1. This work offers the decoy 463 with an improved curative effect, which holds potential to become a good option against PD-1/PD-L1-related cancers.

Keywords

Decoy PD-1 / PD-L1 / Mammalian cell library / Epitope-oriented

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