PD-L1 glycosylation and its impact on binding to clinical antibodies

Abstract

Immune checkpoint inhibitors, including PD-L1/PD-1, are key regulators of immune response and promising targets in cancer immunotherapy. N-glycosylation of PD-L1 affects its interaction with PD-1 but little is known about the distribution of glycoforms at its four NXS/T sequons. We optimized LC-MS/MS methods using collision energy modulation for the site-specific resolution of specific glycan motifs. Using these methods, we demonstrate that PD-L1 expressed on the surface of breast cancer cells carries mostly complex glycans with high proportion of polyLacNAc structures at the N219 sequon. PD-L1 from whole cell lysate contained, in addition, large proportion of high mannose glycans at all sites. Contrary to the full-length protein, the secreted form of PD-L1 expressed in breast cancer or HEK293 cells demonstrated minimum N219 occupancy and low contribution of the polyLacNAc structures. Molecular modeling of PD-L1/PD-1 interaction with N-glycans suggests that glycans at the N219 site of PD-L1 and N74 and N116 of PD-1 are involved in glycan-glycan interactions, but the impact of this potential interaction on the protein function remains at this point unknown. In addition, the interaction of PD-L1 with clinical antibodies is also affected by glycosylation. In conclusion, our study demonstrates that PD-L1 expressed in the MDA-MB-231 breast cancer cells carries polyLacNAc glycans mostly at the N219 sequon which displays the highest variability in occupancy and is most likely to directly influence the interaction with PD-1.Competing Interest StatementThe authors have declared no competing interest.View Full Text