Targeting Glycosylated PD-1 Induces Potent Antitumor Immunity
Immunotherapies targeting programmed cell death protein 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) immune checkpoints represent a major breakthrough in cancer treatment. PD-1 is an inhibitory receptor expressed on the surface of activated T cells that dampens T-cell receptor (TCR)/CD28 signaling by engaging with its ligand PD-L1 expressed on cancer cells. Despite the clinical success of PD-1 blockade using mAbs, most patients do not respond to the treatment, and the underlying regulatory mechanisms of PD-1 remain incompletely defined. Here we show that PD-1 is extensively N-glycosylated in T cells and the intensities of its specific glycoforms are altered upon TCR activation. Glycosylation was critical for maintaining PD-1 protein stability and cell surface localization. Glycosylation of PD-1, especially at the N58 site, was essential for mediating its interaction with PD-L1. The mAb STM418 specifically targeted glycosylated PD-1, exhibiting higher binding affinity to PD-1 than FDA-approved PD-1 antibodies, potently inhibiting PD-L1/PD-1 binding, and enhancing antitumor immunity. Together, these findings provide novel insights into the functional significance of PD-1 glycosylation and offer a rationale for targeting glycosylated PD-1 as a potential strategy for immunotherapy.
T-cell activation is an intricately regulated process that involves peptide-MHC engagement of the T-cell receptor (TCR) and costimulatory signals (1). Upon T-cell activation, coinhibitory immune checkpoint proteins, such as programmed cell death protein 1 (PD-1), are induced and act as brakes on the activation (2). Cancer cells utilized the immune checkpoint proteins to avoid and suppress antitumor immune responses. The engagement of PD-1 with its ligand programmed death 1 ligand 1 (PD-L1), expressed on the surface of cancer cells, leads to T-cell exhaustion and immunosuppression (3). The inhibitory function of PD-l is primarily mediated by the tyrosine phosphatase SHP-2, which is recruited to the cytoplasmic domain of PD-1 and dephosphorylates the signaling molecules downstream of the TCR/CD28 (4, 5). PD-1 transcriptional regulation has been extensively studied, and several transcription factors, including nuclear factor of activated T cells 1 (NFATC1), forkhead box protein O1 (FOXO1), T-bet (also known as T-box transcription factor TBX21), and B lymphocyte–induced maturation protein 1 (BLIMP1) have been reported to regulate PD-1 expression (6). However, the posttranslational regulation of PD-1 (7, 8) remains less clear.
Glycosylation is an important posttranslational modification that covalently attaches carbohydrate moieties to proteins (9). N-linked glycosylation, in which N-acetylglucosamine is linked to the amide side chain, is regulated by the programmed remodeling of glycosyltransferases and glycosidases (10). The resulting abundant repertoire of glycans plays diverse essential roles in a large number of biological events. Recent studies on protein glycosylation (11, 12) revealed that glycans associated with the cell surface receptors substantially modify the structure and function of proteins through steric influences, and directly modulate protein turnover and intermolecular interactions. There is considerable evidence showing that T-cell glycosylation is crucial for immune regulation (13, 14). Although PD-1 is known to be a glycoprotein, the details of its glycan structure and functional significance of its glycosylation in regulating T-cell activation are not well understood.