GLYCOSYLATION OF PD-L1 MODULATES PD-L1 EXPRESSION LEVEL AND ITS IMMUNOSUPPRESSION ACTIVITY AND DEGLYCOSYLATION OF PATIENT SAMPLES IS BENEFICIAL TO THERAPEUTIC SELECTION (ADT05)
Mien-Chie Hung, PhD (University of Texas, MD Anderson Cancer Center, Houston, TX, USA; China Medical University, Taichung, Taiwan) described the modulation of PD-L1 expression level by N-glycosylation which prevents proteasome-dependent degradation of PD-L1, leading to immunosuppression activity. In endoplasmic reticulum of mammalian cells, PD-L1 is glycosylated at multiple sites that contain an “NXT” consensus motif. Glycosylation of PD-L1 by epidermal growth factor receptor (EGFR) pathway protects PD-L1 from its degradation, which, in turn, suppresses T cell effector activity and causes immunosuppression. In contrast, phosphorylation of PD-L1 by 5' AMP-activated protein kinase (AMPK) results in abnormal glycosylation and leads to PD-L1 degradation, resulting in the decreased PD-L1 localization level on the cell membrane. Based on this pathway, an AMPK activator, metformin, decreases the PD-L1 expression on the cell surface and enhances cytotoxic T lymphocytes activity. In mice, when metformin was combined with anti-CTLA-4 antibody, a synergistic anti-tumor effect was observed. Dr. Hung’s group then developed a monoclonal antibody specifically recognizes glycosylated PD-L1 and tried to target glycosylated PD-L1 with an anti-PD-L1-ADC, an antibody drug conjugate that was conjugated with monomethyl auristatin E (MMAE, synthetic antimitotic and antineoplastic agent) which exhibited enhanced anti-tumor immunity. They further investigated to answer another clinically important question “How do the certain PD-L1 negative patients respond to PD-L1 inhibitors?” One hypothesis was the N-linked glycan structure of PD-L1 might hinder antibody-based detection against the PD-L1 antigen, classified as PD-L1 negative. To examine this hypothesis, PD-L1 was deglycosylated with PNGase F treatment in basal-like carcinoma (BLBC) and lung cancer cells and was able to detect enhanced PD-L1 signal with immunofluorescence assay. Moreover, anti-PD-L1 signal was enhanced after N-glycosylation removal in lung cancer TMA (thrombotic microangiopathy; n = 149) and rectal cancer TMA (n = 92). PD-L1 expression detected by this deglycosylation method accompanied with immunohistochemistry analysis predicted clinical response better than the conventional method (n = 82). In addition, deglycosylation of patient samples is beneficial to therapeutic selection to prevent false-negative detection of PD-L1 in lung cancer cohort (n = 44 out of 95). Together, AMPK activation enhanced cytotoxic T lymphocytes activity, and the administration of metformin in combination with anti-CTLA-4 therapy resulted in a synergistic anti-tumor effect. In a diagnostic side, the deglycosylation may decrease false-negative detection of PD-L1 and better predict response to PD-L1 axis immunotherapies.
OPPOSING FUNCTIONS OF INTERFERON SIGNALING IN HOMEOSTASIS AND REGULATION OF ANTI-CANCER IMMUNITY (SY09)
Andy J. Minn, MD, PhD (Abramson Family Cancer Research Inst., Philadelphia, PA, USA) discussed about dichotomous functions of interferon (IFN) signaling in immunostimulation and immunosuppression as well as a resistance mechanism to cancer therapies. First, the mechanisms of the viral infection and anti-viral responses was described. The chronic INF signaling that causes high expression of INF-stimulated genes (ISGs), immunosuppression (PD-L1, IL-10), and limited immune-mediated pathology, all relates to anti-viral effects and immunomodulatory effects. Magnitude and duration of these IFN-mediated anti-viral signals can dictate outcome in infection and cancer. For example, inflammatory gene expression via DNA damage responses and anti-viral activity were observed after genotoxic cancer therapy such as radiation, chemotherapy, or epigenetic drugs. Radiation also improved the efficacy of immune checkpoint blockade (ICB) in mice. In patients with advanced and metastatic melanoma and NSCLC, pembrolizumab with hypofractionated radiotherapy reinvigorated a systemic response despite previous progression on anti-PD-1 therapy (RADVAX: phase I trial, NCT02303990). Further analysis in mice identified that relapse after radiation therapy + anti-CTLA-4 was “paradoxically” associated with ISGs expression with both active and persistent signatures. Epigenomic and transcriptomic features of relapsed and resistant tumors demonstrated elevated ISGs and resistance genes and expression of ISGs in cancer cells were found to be an unfavorable prognostic factor in contrast to ones in immune cells (unpublished). Interestingly, immune:cancer ISG ratio was better predictive for response than tumor mutational burden (TMB) (unpublished). Studies of IFN pathway in preclinical model demonstrated that inhibition of tumor IFN signaling improved ICB response as well as tumor interferon-gamma receptor (IFNGR) knockout restored response despite poor MHC-I and neoantigens (unpublished). Similarly, disrupting tumor IFN-gamma signaling increased intratumoral CD8 cells while disruption of tumor IFNG signaling enhanced IFNG signaling in immune cells (unpublished). These opposing roles of IFN signaling tightly controlled adaptive and innate anti-tumor immunity with increased cancer cell ISGs and inhibitory pathways (e.g. PD-L1), decreased cytotoxic effectors (TRAILR2), T cell dysfunction, as well as decreased IFN-gamma and cytotoxic T cell function (unpublished). Also, potential pathogenic mutations in IFN pathway predicted clinical ICB response in patients with NSCLC treated with anti-PD-1 + anti-CTLA-4 (unpublished). These data suggest that IFN pathway is an immunomodulatory target for anti-tumor activity and that an immune:cancer ISG ratio and mutations in IFN pathway are proposed to be predictive markers for response to the treatment.
Glossary
ADC = an antibody drug conjugate
AMPK = 5' AMP-activated protein kinase
EGFR = epidermal growth factor receptor
ERAD = endoplasmic-reticulum-associated protein degradation
ICB = immune check blockade
IFN = interferon
IFNGR = interferon-gamma receptor
IHC = immunohistochemistry
ISG = interferon-stimulated genes
MMAE = monomethyl auristatin E
NSCLC = non-small cell lung cancer
PD-1 = programmed cell death protein-1
PD-L1 = Programmed death-ligand 1
TMA = thrombotic microangiopathy
TMB = tumor mutational burden