We describe a new class of potent PD-L1/PD-1 inhibitors based on a terphenyl scaffold that is derived from the rigidified biphenyl-inspired structure. Using in silico docking, we designed and then experimentally demonstrated the effectiveness of the terphenyl-based scaffolds in inhibiting PD-1/PD-L1 complex formation using various biophysical and biochemical techniques. We also present a high-resolution structure of the complex of PD-L1 with one of our most potent inhibitors to identify key PD-L1/inhibitor interactions at the molecular level. In addition, we show the efficacy of our most potent inhibitors in activating the antitumor response using primary human immune cells from healthy donors.

The clinical successes seen with anti-programmed cell death protein 1/programmed death-ligand 1 (anti-PD-[L]1) agents have galvanized the field of immuno-oncology. We evaluated the landscape and trends in immunotherapy trials involving the PD-(L)1 axis in intrathoracic tumors.

Purpose: To investigate the prognostic significance of programmed cell death ligand-1 (PD-L1) and phosphorylated ERK (p-ERK) and their interactions in T-cell lymphoma (TCL). Methods: The mRNA levels of PD-L1 and ERK in TCL samples were analyzed. Formalin-fixed paraffin-embedded tissues from 69 TCL patients were collected to detect the expression of PD-L1 and p-ERK by multiplexed immunofluorescence staining. The total PD-L1 and p-ERK was measured by western blotting, and membrane PD-L1 was determined using flow cytometry. Results: PD-L1 and ERK mRNA levels were significantly upregulated in TCL. The expression rates of PD-L1 and p-ERK were 52.2% and 27.5%, respectively. PD-L1 expression correlated with stage (R=0.304, P=0.011) and IPI score (R=0.313, P=0.009), and p-ERK expression correlated with stage (R=0.330, P=0.006) and IPI score (R=0.376, P=0.002). PD-L1 expression positively correlated with p-ERK expression (R=0.355, P=0.003). Patients with co-expression of PD-L1 and p-ERK had the worst overall survival (P=0.007). In three TCL cell lines with PD-L1 expression, we demonstrated that the expression of p-ERK was upregulated after stimulation with PD-1, suggesting that ERK signaling was activated. Conclusions: The PD-1/PD-L1 axis activates intracellular ERK signaling in tumor cells and that PD-L1, p-ERK or their combination are potential biomarkers for predicting the prognosis in TCL patients.

Objective: To investigate the relationship between programmed death ligand 1 (PD-L1) expression using 5%, 25%, 50% cutoffs in tumor cells (TC) and postsurgical survival in non-small-cell lung cancer (NSCLC) patients. For samples with tumor infiltrating lymphocytes (TIL), correlation between PD-L1 expression in TIL using 1% cutoff and postsurgical survival was also evaluated. Methods: Primary NSCLC tumor surgical samples staging I to IIIA of 126 patients who underwent surgical procedures from September 2009 to August 2012 in Shanghai Chest Hospital, Shanghai Jiao Tong University were retrospectively included. PD-L1 protein expression was detected by immunohistochemistry (IHC) assays. A rabbit anti-human PD-L1 (E1L3N) monoclonal antibody (1:300, CST#13684, Cell Signaling Technology) was used for PD-L1 IHC staining. PD-L1 expression was evaluated both on TC and TIL. Univariate and multivariate analyses for postsurgical survival were done using Kaplan-Meier and Cox regression model, respectively. Results: The median postsurgical survival for all patients was 44.1 months [95% confidence interval (CI): 33.9-70.0 months). The median postsurgical survival for PD-L1 expression percentage 0, 1-50% and ≥50% were 51.9 months (95%CI: 33.9-70.0 months), 33.2 months (95%CI: 20.8-45.6 months) and 14.7 months (95%CI: 1.9-27.6 months), respectively (P = 0.002). Clinical stage and PD-L1 expression in TC (25% cutoff or 50% cutoff values) were found to be independent predictors for longer postsurgical survival in all cohort. Ninety (71.4%) of the 126 samples were identified to concurrent TIL. The median postsurgical survival time was 39.6 months (95% CI: 31.8-47.4 months) in patients with TIL. PD-L1 expression in TC (25% cutoff or 50% cutoff values) was found to be the independent predictor for longer postsurgical survival time in patients with TIL. Conclusion: PD-L1 negative expression in TC at 25% or 50% cutoff values was the independent predictor for longer postsurgical survival time in both NSCLC samples and NSCLC samples with TIL. For patients with PD-L1 high expression at 25% or 50% cutoff values, PD-L1 blocking may be considered.

New biphenyl-based chimeric compounds containing pomalidomide were developed and evaluated for their activity to inhibit and degrade the programmed cell death-1/programmed cell death- ligand 1 (PD-1/PD-L1) complex. Most of the compounds displayed excellent inhibitory activity against PD-1/PD-L1, as assessed by the homogenous time-resolved fluorescence (HTRF) binding assay. Among them, compound 3 is one of the best with an IC50 value of 60 nM. Using an ex vivo PD-1/PD-L1 blockade cell line bioassay that expresses human PD-1 and PD-L1, we show that compounds 4 and 5 significantly restore the repressed immunity in this co-culture model. Western blot data, however, demonstrated that these anti-PD-L1/pomalidomide chimeras could not reduce the protein levels of PD-L1.

Due to recent advantages in cancer therapy, immune checkpoint inhibitors (ICIs) are new classes of drugs targeting programmed cell death protein 1 (PD-1) or its ligand programmed cell death protein 1-ligand 1 (PD-L1) used in many cancer therapies. Acute interstitial nephritis (AIN) is a potential and deleterious immune-related adverse events (irAE) in the kidney observed in patients receiving ICIs and the most common biopsy-proven diagnosis in patients who develop acute kidney injury (AKI). Based on previous reports, AIN in patients receiving ICIs is associated with tubular positivity for PD-L1, implicating that PD-L1 positivity reflects susceptibility to develop renal complications with these agents. It remains unclear if PD-L1 positivity is acquired specifically during ICI therapy or expressed independently in the kidney.

Programmed cell death protein 1 (PD-1) and its ligand Programmed death ligand 1 (PD-L1) have gained massive attention in cancer research due to recent availability and their targeted antitumor effects. Their role in prostate cancer is still undetermined. We constructed tissue microarrays from prostatectomy specimens from 535 prostate cancer patients. Following validation of antibodies, immunohistochemistry was used to evaluate the expression of PD-1 in lymphocytes and PD-L1 in epithelial and stromal cells of primary tumors. PD-L1 expression was commonly seen in tumor epithelial cells (92% of cases). Univariate survival analysis revealed a positive association between a high density of PD-1+ lymphocytes and worse clinical failure-free survival, limited to a trend (p = 0.084). In subgroups known to indicate unfavorable prostate cancer prognosis (Gleason grade 9, age < 65, preoperative PSA > 10, pT3) patients with high density of PD-1+ lymphocytes had a significantly higher risk of clinical failure (p = < 0.001, p = 0.025, p = 0.039 and p = 0.011, respectively). In the multivariate analysis, high density of PD-1+ lymphocytes was a significant negative independent prognostic factor for clinical failure-free survival (HR = 2.48, CI 95% 1.12-5.48, p = 0.025).

Extracellular interaction between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) leads to tumour-associated immune escape. Here we show that the immunosuppression activity of PD-L1 is stringently modulated by ubiquitination and N-glycosylation. We show that glycogen synthase kinase 3β (GSK3β) interacts with PD-L1 and induces phosphorylation-dependent proteasome degradation of PD-L1 by β-TrCP. In-depth analysis of PD-L1 N192, N200 and N219 glycosylation suggests that glycosylation antagonizes GSK3β binding. In this regard, only non-glycosylated PD-L1 forms a complex with GSK3β and β-TrCP. We also demonstrate that epidermal growth factor (EGF) stabilizes PD-L1 via GSK3β inactivation in basal-like breast cancer. Inhibition of EGF signalling by gefitinib destabilizes PD-L1, enhances antitumour T-cell immunity and therapeutic efficacy of PD-1 blockade in syngeneic mouse models. Together, our results link ubiquitination and glycosylation pathways to the stringent regulation of PD-L1, which could lead to potential therapeutic strategies to enhance cancer immune therapy efficacy.

The trans-membrane proteins of the B7 family programmed cell death ligand-1 (PD-L1) and programmed death-1 (PD-1) play important roles in inhibiting immune responses and enhancing self-tolerance via T-cell modulation. Several therapeutic antibodies are used to promote T-cell proliferation by preventing interactions between PD-1/PD-L1. Recombinant technology appears to be quite useful in the production of such potent antibodies. In this study, we constructed recombinant molecules by cloning variable regions of the PD-L1 molecule into pMH3 vectors and transferring them into mammalian cell lines for expression. G418 supplementation was used to screen the recombinant clones, which were then maintained on serum-free medium. The full-length antibody was isolated and purified from the medium supernatant at a concentration of 0.5-0.8 mg/ml. Antibody binding affinity was investigated using ELISA and immunofluorescence methods. The protein-protein interactions (PPI) were determined using a docking approach. The SWISS model was utilized for homology modeling, while ZDOCK, Chimera, and PyMOL were used to validate 3D models. The Ramachandran plots were constructed using the SWISS model, which revealed that high-quality structures had a value of more than 90%. Current technologies allow for the accurate determination of antigen-antibody interactions.

The expression of immune checkpoint proteins such as programmed cell death protein 1 (PD-1) and its ligand (PD-L1) has been shown to correlate with patient prognosis in many malignant cancers. The expression of PD-L1 is controlled by c-Myc; however, further upstream regulation of PD-L1 expression is largely unknown. We have previously shown that atypical protein kinase C lambda/iota (aPKCλ) phosphorylates the Forkhead box protein O1 (FoxO1) transcription factor at Ser218 to suppress its DNA-binding ability, thereby regulating c-Myc expression and controlling physiologic and pathologic endothelial proliferation. The presence of phosphorylation of FoxO1 at Ser218 (pSer218 FoxO1) in cutaneous angiosarcoma (CAS) strongly correlates with poor patient prognosis. Here, we reported that patients with PD-L1+ cells in CAS lesions showed significantly worse prognosis compared to those that were PD-L1- . Expression of PD-L1 correlated with that of aPKCλ or the presence of pSer218FoxO1. Moreover, suppression of aPKCλ expression or inhibition of its activity in HUVECs or AS-M, an established human angiosarcoma cell line, resulted in decreased PD-L1 expression. Our results suggest that combined treatment with immune checkpoint inhibitors and aPKCλ inhibitors could be a novel treatment strategy for CAS patients.

The addition of programmed cell death protein-1 (PD-1) or programmed death-ligand 1 (PD-L1) inhibitors to first-line chemotherapy (CT) improved the outcomes of advanced NSCLC. Nonetheless, no direct comparison exists between these combination treatments.

Lymphatic endothelial cells (LECs) comprise lymphatic capillaries and vessels that guide immune cells to lymph nodes (LNs) and form the subcapsular sinus and cortical and medullary lymphatic structures of the LN. During an active immune response, the lymphatics remodel to accommodate the influx of immune cells from the tissue, but factors involved in remodeling are unclear. Here, we determined that a TSS motif within the cytoplasmic domain of programmed death ligand 1 (PD-L1), expressed by LECs in the LN, participates in lymphatic remodeling. Mutation of the TSS motif to AAA does not affect surface expression of PD-L1, but instead causes defects in LN cortical and medullary lymphatic organization following immunostimulant, Poly I:C, administration in vivo. Supporting this observation, in vitro treatment of the LEC cell line, SVEC4-10, with cytokines TNFα and IFNα significantly impeded SVEC4-10 movement in the presence of the TSS-AAA cytoplasmic mutation. The cellular movement defects coincided with reduced F-actin polymerization, consistent with differences previously found in dendritic cells. Here, in addition to loss of actin polymerization, we define STAT3 and Paxillin as important PD-L1 binding partners. STAT3 and Paxillin were previously demonstrated to be important at focal adhesions for cellular motility. We further demonstrate the PD-L1 TSS-AAA motif mutation reduced the amount of pSTAT3 and Paxillin bound to PD-L1 both before and after exposure to TNFα and IFNα. Together, these findings highlight PD-L1 as an important component of a membrane complex that is involved in cellular motility, which leads to defects in lymphatic organization.

PD-L1 and HER-2 are routine biomarkers for gastric cancer (GC). However, little research has been done to investigate the correlation among PD-L1, HER-2, immune microenvironment, and clinical features in GC.

The programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) checkpoint blockade is central to Immuno-Oncology based therapies, and alternatives to antibody blockers of this interaction are an active area of research due to antibody related toxicities. Recently, small molecule compounds that induce PD-L1 dimerization and occlusion of PD-1 binding site have been identified and developed for clinical trials. This mechanism invokes an oligomeric state of PD-L1 not observed in cells previously, as PD-L1 is generally believed to function as a monomer. Therefore, understanding the cellular lifecycle of the induced PD-L1 dimer is of keen interest. Our report describes a moderate but consistent increase in the PD-L1 rate of degradation observed upon protein dimerization as compared to the monomer counterpart. This subtle change, while not resolved by measuring total PD-L1 cellular levels by western blotting, triggered investigations of the overall protein distribution across various cellular compartments. We show that PD-L1 dimerization does not lead to rapid internalization of neither transfected nor endogenously expressed protein forms. Instead, evidence is presented that dimerization results in retention of PD-L1 intracellularly, which concomitantly correlates with its reduction on the cell surface. Therefore, the obtained data for the first time points to the ability of small molecules to induce dimerization of the newly synthesized PD-L1 in addition to the protein already present on the plasma membrane. Overall, this work serves to improve our understanding of this important target on a molecular level in order to guide advances in drug development.

The high prevalence of oral squamous cell carcinoma (OSCC) in South Asia is associated with habitual areca nut chewing. Arecoline, a primary active carcinogen within areca nut extract, is known to promote OSCC pathological development. Dysregulation of N6-methyladenosine (m6A) modification has begun to emerge as a significant contributor to cancer development and progression. However, the biological effects and molecular mechanisms of m6A modification in arecoline-promoted OSCC malignance remain elusive. We reveal that chronic arecoline exposure substantially induces upregulation of fat mass and obesity-associated protein (FTO), MYC, and programmed cell death-ligand 1 (PD-L1) in OSCC cells. Moreover, upregulation of PD-L1 is observed in OSCC cell lines and tissues and is associated with areca nut chewing in OSCC patients. We also demonstrate that arecoline-induced FTO promotes the stability and expression levels of PD-L1 transcripts through mediating m6A modification and MYC activity, respectively. PD-L1 upregulation confers superior cell proliferation, migration, and resistance to T-cell killing to OSCC cells. Blockage of PD-L1 by administration of anti-PD-L1 antibody shrinks tumor size and improves mouse survival by elevating T-cell-mediated tumor cell killing. Therefore, targeting PD-L1 might be a potential therapeutic strategy for treating PD-L1-positive OSCC patients, especially those with habitual areca nut chewing.

Programmed cell death ligand-1 (PD-L1) detection assays have not been standardized for patients with colorectal cancer, and the prognostic value of PD-L1 expression is unclear. We compared the PD-L1 expression patterns in colorectal cancer samples using various immunohistochemical assays using 3 primary PD-L1 antibodies (assay 1, MIH1; assay 2, E1L3; and assay 3, 22C3) and investigated the prognostic implication of PD-L1 expression using each. Additionally, PD-L1 gene amplification was evaluated using FISH. The percentage scorings and positivity rates of the 3 assays differed; the degrees of correlation and concordance between assays 2 and 3 were relatively high, whereas assay 1 was an outlier. Multivariate analyses indicated that PD-L1 positivity in tumor cells and its negativity in tumor-infiltrating lymphocytes were independent predictors of poorer overall and disease-free survival in patients with colorectal cancer. PD-L1 gene amplification was found in 2 patients (PD-L1/CEP ratio, 5.60 and 5.84, respectively); both had strong PD-L1 expression according to immunohistochemistry. Overall, our study showed that PD-L1 expression status in tumor and immune cells is an independent prognostic factor in patients with colorectal cancer. Standardizations of both PD-L1 detection using immunohistochemistry and the cut-off for positivity are necessary. Finally, PD-L1 gene amplification was found in a small fraction of samples, suggesting the possibility of an ancillary test for PD-L1 evaluation.

The relationship between immune checkpoint status and disease outcome is a major focus of research in cutaneous T-cell lymphoma (CTCL), a disfiguring neoplastic dermatological disorder. Mycosis fungoides (MF) and Sézary syndrome (SS) are the two most common types of CTCL.

Bladder cancer (BC) is the second most common urologic malignancy and the ninth most common malignancy worldwide. Surgical resection is the mainstay of treatment for patients with early-stage disease, whereas therapeutic options are limited for patients with advanced-stage or residual BC. Programmed cell death ligand-1 (PD-L1) is an important target for immunotherapy. It is known that PD-L1 is overexpressed in BC; a clinical trial involving PD-L1 immune checkpoint inhibitors in advanced BC is ongoing. In the present study, we used Western blot and quantitative real-time PCR (qPCR) to define the expression level of PD-L1 after cisplatin treatment in BC-derived cell lines. The signal activation was also evaluated by Western blot in BC-derived cell lines. We found that chemotherapeutic drug cisplatin can induce PD-L1 but not PD-L2 expression in BC-derived cell lines. Furthermore, the expression level of PD-L1 was increased in a dose- and time-dependent manner after cisplatin treatment. The cisplatin-induced PD-L1 expression is mainly mediated by ERK1/2 but not Akt/mTOR signal pathway. Moreover, we found that cisplatin activates transcription factor activator protein-1 (AP-1) to regulate PD-L1 expression. The chemotherapy drug such as cisplatin may trigger resistance of BC through PD-L1 up-regulation. The present study suggests that PD-L1 antibody should be used concomitantly with chemotherapy in the setting of advanced and metastatic BC.

Pathways in the B7:CD28 family of costimulatory molecules regulate T cell activation and tolerance. B7-dependent responses in Cd28(-/-)Ctla4(-/-) T cells together with reports of stimulatory and inhibitory functions for Programmed Death-1 Ligand 1 or 2 molecules (PD-L1 or PD-L2) have suggested additional receptors for these B7 family members. We show that B7-1 and PD-L1 interacted with affinity intermediate to that of B7-1:CD28 and B7-1:CTLA-4. The PD-L1:B7-1 interface overlapped with the B7-1:CTLA-4 and PD-L1:PD-1 (Programmed Death-1) interfaces. T cell activation and cytokine production were inhibited by the interaction of B7-1 with PD-L1. The responses of PD-1-deficient versus PD-1,B7-1 double-deficient T cells to PD-L1 and of CD28,CTLA-4 double-deficient versus CD28,CTLA-4,PD-L1 triple-deficient T cells to B7-1 demonstrated that PD-L1 and B7-1 interact specifically to inhibit T cell activation. Our findings point to a substantial bidirectional inhibitory interaction between B7-1 and PD-L1 and add an additional dimension to immunoregulatory functions of the B7:CD28 family.

Programmed death ligand 2 (PD-L2) is the second ligand of programmed death 1 (PD-1) protein. In autoimmune myocarditis, the protective roles of PD-1 and its first ligand programmed death ligand 1 (PD-L1) have been well documented; however, the role of PD-L2 remains unknown. In this study, we report that PD-L2 deficiency exacerbates myocardial inflammation in mice with experimental autoimmune myocarditis (EAM). EAM was established in wild-type (WT) and PD-L2-deficient mice by immunization with murine cardiac myosin peptide. We found that PD-L2-deficient mice had more serious inflammatory infiltration in the heart and a significantly higher myocarditis severity score than WT mice. PD-L2-deficient dendritic cells (DCs) enhanced CD4+ T cell proliferation in the presence of T cell receptor and CD28 signaling. These data suggest that PD-L2 on DCs protects against autoreactive CD4+ T cell expansion and severe inflammation in mice with EAM.