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Just as a driver¡¯s night vision improves by simply cleaning the headlights, scientists at Â鶹ӳ»­ 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 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.