Glycan-Controlled Human PD-1 Variants Displaying Broad-Spectrum High Binding to PD-1 Ligands Potentiate T Cell

Although therapeutic immunoglobulin G (IgG) antibodies that regulate the activity of immune checkpoints bring innovation to the field of immuno-oncology, they are still limited in their efficiency to infiltrate the tumor microenvironment due to their large molecular size (150 kDa) and the necessity of additional engineering works to ablate effector functions for antibodies targeting immune cells. To address these issues, the human PD-1 (hPD-1) ectodomain, a small protein moiety of 14–17 kDa, has been considered as a therapeutic agent. Here, we used bacterial display-based high-throughput directed evolution to successfully isolate glycan-controlled (aglycosylated or only single- N -linked glycosylated) human PD-1 variants exhibiting over 1000-fold increased hPD-L1 binding affinity compared to that of wild-type hPD-1. The resulting hPD-1 variants, aglycosylated JYQ12 and JYQ12-2 with a sin…