New approach enhances PD-L1 detection to aid in immunotherapy selection

Just as a driver’s night vision improves by simply cleaning the headlights, scientists at The University of Texas MD Anderson Cancer Center have discovered that removing an unwanted element from a protein known to suppress the immune system may eventually help clinicians better observe which cancer patients will benefit by treatment with immune checkpoint blockades.

Heng-Huan Lee, Ph.D., a postdoctoral fellow and Ying-Nai Wang, Ph.D., an instructor in the Department of Molecular and Cellular Oncology, demonstrated how to enhance detection of the programmed death ligand 1 (PD-L1) and potentially better predict how some patients will respond to anti-PD1/PD-L1 immunotherapy.

Immunotherapy drugs that block the PD-L1/PD-1 connection free T cells to attack cancer by releasing the PD-1 brake on the T cell. The presence of PD-L1 in the tumor indicates potential vulnerability to checkpoint blockade, so an accurate assessment is important.

Results from the study, which reported on removal of certain sugar-based molecules called glycans and the resultant impact on PD-L1, were published in the July 18 online issue of Cancer Cell. Glycans are commonly linked to tumor formation.

“Reactivation of T-cell immunity by PD-1/PD-L1 immune checkpoint blockade has been shown to be a promising cancer therapeutic strategy,” said Lee. “However heavy glycosylation of PD-L1 hinders its detection by the PD-L1 antibodies and can lead to inaccurate readouts from a variety of bioassays.”

Such inaccurate readouts of PD-L1’s presence are a clinical challenge to stratifying patients.  The research team developed a method to resolve this by removing glycan groups from cell surface antigens via enzymatic digestion, a process called sample deglycosylation.

Removal of the glycan structures significantly improved PD-L1 signal intensity and binding affinity. This allowed for more accurate measurement of PD-L1 levels, which may provide a timely approach to reducing false-negative patient stratification for guiding anti-PD-1/PD-L1 therapy.

Zeroing in on N-Linked glycosylation

Specifically, the scientists focused on N-linked glycosylation, so-called for the glycans attachment to nitrogen atoms. Changes in N-linked glycosylation have been linked to several diseases including cancer.

N-linked glycosylation of cell surface PD-L1 accounts for more than half of the molecular weight of PD-L1 polypeptides. Glycosylation of PD-L1 could render its polypeptide region inaccessible to PD-L1 antibody bindings, making PD-L1 hard to detect and leading to inaccurate immunohistochemical results in some patient samples. The potential end result is conflicting therapeutic outcomes.

The team, using human lung and basal-like breast cancer cell lines, removed the entire N-linked glycosylation through treatment with recombinant glycosidase, resulting in a homogenous pattern of PD-L1.  Subsequent experiments supported the notion that glycans on the PD-L1 antigen region hinder its interaction with, and subsequent detection by PD-L1 antibodies.

“The removal of PD-L1 N-linked glycosylation by enzymatic digestion of tissue samples can be used to increase homogeneity of target proteins and quantitatively facilitate antibody based detection,” said Lee. “Since cell surface proteins are frequently N-linked glycosylated at different levels, this deglycosylation method can be used as a general approach to decrease antigen heterogeneity and eliminate structural hindrance prior to antibody detection.”

Explaining how deglycosylation enhances anti-PD-L1 signaling

The research team further explored deglycosylation by the immunohistochemical (IHC) staining of 200 patient samples from a multi-organ carcinoma tissue microarray, which included breast, lung, colon, prostate and pancreatic cancers.

 “These results revealed that the number of patients with positive IHC staining for PD-L1 increased significantly by more than two folds after deglycosylation, indicating that N-linked glycosylation of PD-L1 critically affects its recognition by the PD-L1 antibody,” said Lee.

Together with other disease parameters such as tumor mutational burden and immune cell infiltration, Lee believes that deglycosylation of PD-L1 is an effective method to improve the predictive power of PD-L1 as a biomarker for immune checkpoint therapy.

Mien-Chie Hung, Ph.D., formerly of the Department of Molecular & Cellular Oncology, was senior author on the paper.