Programmed Cell Death Ligand 1 Protein Research Articles

Abstract 2180: The PD-L1 protein expression in Chinese patients with advanced esophageal cancers: a multi-center retrospective study

Abstract Introduction: Programmed cell death ligand-1 (PD-L1) is the ligand of programmed cell death-1 (PD-1), a member of the immunoglobulin gene superfamily that plays a role in programmed cell death. Accumulating evidence of anti-PD-1 therapy has showed improved survival benefit for esophageal cancer (EC) patients. PD-L1 has emerged as a crucial predictor for efficacy of immunotherapy. However, the understanding of PD-L1 as a biomarker is complicated by various immunohistochemistry platforms, PD-L1 antibodies, scoring systems, and cut-offs for immunotherapy. Although PD-L1 expression has been widely investigated, the real-world PD-L1 expression status in Chinese patients with advanced EC nationwide with unified testing platform, antibody, scoring system and cut-off is largely unrevealed. Methods: The present study was a nationwide multi-center retrospective analysis of data from six centers in China from August 9, 2021, to February 28, 2022. Patients with histologically or cytologically confirmed diagnoses of advanced EC were included. The primary outcome was the prevalence of high PD-L1 expression in patients with advanced EC. Immunohistochemical analysis of PD-L1 expression was performed by using the PD-L1 IHC 22C3 pharmDx assay in EC specimens. PD-L1 protein expression was assessed by using a combined positive score (CPS). The CPS≥ 10 was defined as a high PD-L1 high expression. The Chi-square test, Fisher’s exact test, or Wilcoxon rank sum test was used to compare the PD-L1 prevalence across demographic, clinicopathologic parameters, treatment status and other biomarkers. Results: Out of 488 enrolled patients with advanced EC, 482 patients were included in the final analysis. For all testing EC patients, 207 patients (42.9% [95% CI 38.48-47.50]) were PD-L1 high expression (CPS≥10). Between the PD-L1 high (CPS≥10) group and PD-L1 low (CPS<10) group, there were statistically significant differences in gender (P=0.046), alcohol consumption (P = 0.011), distant metastatic lesion number of patients with stage IV (P = 0.032), surgery (P = 0.012), systemic therapy (P = 0.004), and chemotherapy (P=0.004). Conclusion: In summary, the prevalence of high PD-L1 expression (CPS≥10) in Chinese patients with advanced EC in real-world setting using 22C3 assay is consistent with previous data in global clinical trials. Our findings of the related clinicopathological and treatment features to the PD-L1 expression can provide supplementary information for decision-making of immunotherapy and facilitate the better understanding of mechanism underlying PD-L1 and anti-tumor immunity. Citation Format: Liyan Xue, Jiaqi Wang, Dong Kuang, Jingping Yun, Yuan Li, Lili Jiang, Daoyuan Wu, Pei Duan, Shixun Lu, Yan Jin, Du He, Jing Qian, Wenmin Tang, Yan Wang, Jielin Li, Jianming Ying. The PD-L1 protein expression in Chinese patients with advanced esophageal cancers: a multi-center retrospective study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2180.

ANXA1-derived peptide for targeting PD-L1 degradation inhibits tumor immune evasion in multiple cancers

BackgroundImmune checkpoint inhibitors (ICIs) therapy targeting programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) shows promising clinical benefits. However, the relatively low response rate highlights the need to...

Expression and significance of immune checkpoint B7-homolog 4 in endometrial cancer

Objective: To investigate the expression of B7 homolog 4 (B7-H4) and its clinical significance in endometrial cancer. Methods: A total of 833 patients with endometrial cancer admitted to Peking Union Medical College Hospital, Chinese Academy of Medical Sciences from 2009 to 2019, were enrolled. The expression of B7-H4, mismatch repair (MMR), p53, programmed cell death ligand 1 (PD-L1) protein, and CD8+ T lymphocyte density in endometrial cancer tissues were detected by the EnVision two-step method of immunohistochemical staining. First-generation sequencing (Sanger method) was used to determine molecular subtyping of endometrial cancer. The χ2 test was used to compare the differences in positive expression rate of B7-H4 protein in endometrial cancer tissues with different clinicopathological features and molecular subtyping, PD-L1 protein expression, and CD8+ T lymphocyte density. Survival analyses [including recurrence-free survival (RFS) and disease-specific survival (DSS)] were performed for 664 patients with follow-up time≥3 months, with a median follow-up time of 31 months (range: 4-121 months), and the Cox proportional hazards regression model was used to analyze the relevant factors affecting the prognosis of patients with endometrial cancer. Results: (1) The median age of 833 patients was 58 years (range: 25-88 years); pathological type: 595 with endometrioid carcinoma, 238 with non-endometrioid carcinoma; surgical-pathological staging: 542 cases at stage Ⅰ, 38 cases at stage Ⅱ, 173 cases at stage Ⅲ, and 45 cases at stage Ⅳ. Molecular subtyping was performed in 590 patients, including 50 with POLE mutation, 163 with mismatch repair defect (MMR-d) type, 246 with nospecific molecular change (NSMP) type, and 131 with p53 mutation subtype. (2) B7-H4 protein was expressed with brownish-yellow stainind in the cell membrane and cytoplasm of endometrial carcinoma, and the positivity rate of B7-H4 protein was 71.5% (596/833). The positivity rates of B7-H4 protein among patients with different age, surgical-pathological stage, tumor grade, pathological type, depth of muscular invasion, presence or absence of lymphovascular space invasion, and molecular subtype were significantly different (all P<0.05). The positivity rates of B7-H4 protein among patients with different PD-L1 protein expression and CD8+ T lymphocyte density were not significantly different (P>0.05). The 5-year RFS (83.9%) and DSS (87.3%) of B7-H4 protein-positive patients had an increasing trend compared with the 5-year RFS (77.2%) and DSS (78.1%) of B7-H4 protein-negative patients, but there were not statistically significant differences (P=0.053, P=0.083). (3) Univariate analysis showed that the 5-year RFS and DSS of patients with different age, tumor grade, surgical-pathological stage, pathological type, depth of muscular invasion, lymphovascular space invasion, and molecular subtype were significantly different (all P<0.01). There were no significant differences in 5-year RFS (P=0.184, P=0.113) and DSS (P=0.549, P=0.247) among patients with different CD8+ T lymphocyte density and PD-L1 protein expression. Further analysis according to molecular subtype, the results of CD8+ T lymphocyte density and PD-L1 protein expression showed that the 5-year RFS and DSS of B7-H4 protein-positive patients were higher than those of B7-H4 protein-negative patients with NSMP subtype, low density of CD8+ T lymphocyte and PD-L1 protein-negative endometrial carcinoma (all P<0.05), however, there was no significant difference in 5-year DSS between B7-H4 protein-positive patients and B7-H4 protein-negative patients with PD-L1 protein-negative endometrial cancer (P=0.060). Multivariate analysis showed that positive expression of B7-H4 protein was an independent factor for 5-year RFS (HR=0.27, 95%CI: 0.09-0.78, P=0.016) and DSS (HR=0.16, 95%CI: 0.05-0.58, P=0.005) in patients with NSMP subtype endometrial carcinoma. In patients with low-density CD8+ T lymphocytes endometrial cancer, positive expression of B7-H4 protein was an independent factor for 5-year RFS (HR=0.45, 95%CI: 0.26-0.80, P=0.006), but it was not an independent factor for 5-year DSS. In patients with PD-L1 protein-negative endometrial cancer, B7-H4 protein was not an independent factor for 5-year RFS. Conclusion: B7-H4 protein expressed highly in endometrial carcinoma tissues, and its high expression is closely related to clinicopathological features, molecular subtype of p53 mutant and NSMP, and the favorable prognosis of patients with low density of CD8+ T lymphocyte immunophenotype endometrial carcinoma.

Novel phthalimides regulating PD-1/PD-L1 interaction as potential immunotherapy agents.

Programmed cell death 1(PD-1)/programmed cell death ligand 1(PD-L1) have emerged as one of the most promising immune checkpoint targets for cancer immunotherapy. Despite the inherent advantages of small-molecule inhibitors over antibodies, the discovery of small-molecule inhibitors has fallen behind that of antibody drugs. Based on docking studies between small molecule inhibitor and PD-L1 protein, changing the chemical linker of inhibitor from a flexible chain to an aromatic ring may improve its binding capacity to PD-L1 protein, which was not reported before. A series of novel phthalimide derivatives from structure-based rational design was synthesized. P39 was identified as the best inhibitor with promising activity, which not only inhibited PD-1/PD-L1 interaction (IC50=8.9nmol/L), but also enhanced killing efficacy of immune cells on cancer cells. Co-crystal data demonstrated that P39 induced the dimerization of PD-L1 proteins, thereby blocking the binding of PD-1/PD-L1. Moreover, P39 exhibited a favorable safety profile with a LD50>5000mg/kg and showed significant invivo antitumor activity through promoting CD8+ T cell activation. All these data suggest that P39 acts as a promising small chemical inhibitor against the PD-1/PD-L1 axis and has the potential to improve the immunotherapy efficacy of T-cells.

Butein suppresses PD-L1 expression via downregulating STAT1 in non-small cell lung cancer

PD-L1 (programmed cell death ligand 1) is frequently up-regulated in tumors and is critical in tumor immune escape. In addition to antibodies that block the interaction between PD-L1 and PD-1 (programmed cell death protein 1), small-molecule compounds that suppress PD-L1 expression also exhibit significant anti-tumor effects, emerging as a new strategy targeting PD-L1. By using a cell-based screening model, we found that butein, a natural chalcone compound, significantly reduced the cytoplasm and cell surface expression of PD-L1. This effect was further validated in various non-small cell lung cancer (NSCLC) cell lines and primary cells derived from clinical NSCLC tissues. Butein inhibited PD-L1 transcription, but not the half-life of PD-L1 protein. Butein reduced STAT1 level and butein-induced PD-L1 suppression was eliminated by the absence of STAT1. By co-culture system, butein improved tumor elimination by increasing the killing ability of CD8+ T cells. By in vivo study, we further confirmed that butein downregulated PD-L1 expression and improved infiltration of CD8+ T cells in tumor tissues. Taken together, our study suggested that butein could suppress the transcription of PD-L1 via downregulating STAT1, providing a theoretical basis for the application of butein in anti-tumor therapy.

Inhibition of MYC suppresses programmed cell death ligand-1 expression and enhances immunotherapy in triple-negative breast cancer.

Cancer immunotherapy has emerged as a promising strategy against triple-negative breast cancer (TNBC). One of the immunosuppressive pathways involves programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1), but many patients derived little benefit from PD-1/PD-L1 checkpoint blockades treatment. Prior research has shown that MYC, a master transcription amplifier highly expressed in TNBC cells, can regulate the tumor immune microenvironment and constrain the efficacy of immunotherapy. This study aims to investigate the regulatory relationship between MYC and PD-L1, and whether a cyclin-dependent kinase (CDK) inhibitor that inhibits MYC expression in combination with anti-PD-L1 antibodies can enhance the response to immunotherapy. Public databases and TNBC tissue microarrays were used to study the correlation between MYC and PD-L1. The expression of MYC and PD-L1 in TNBCs was examined by quantitative real-time polymerase chain reaction and Western blotting. A patient-derived tumor xenograft (PDTX) model was used to evaluate the influence of a CDK7 inhibitor THZ1 on PD-L1 expression. Cell proliferation and migration were detected by 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation and cell migration assays. Tumor xenograft models were established for in vivo verification. A high MYC expression level was associated with a poor prognosis and could alter the proportion of tumor-infiltrating immune cells (TIICs). The positive correlation between MYC and PD-L1 was confirmed by immunostaining samples from 165 TNBC patients. Suppression of MYC in TNBC caused a reduction in the levels of both PD-L1 messenger RNA and protein. In addition, antitumor immune response was enhanced in the TNBC cancer xenograft mouse model with suppression of MYC by CDK7 inhibitor THZ1. The combined therapy of CDK7 inhibitor THZ1 and anti-PD-L1 antibody appeared to have a synergistic effect, which might offer new insight for enhancing immunotherapy in TNBC.

Correlation Study on the Expression of INSR, IRS-1, and PD-L1 in Nonsmall Cell Lung Cancer.

Objective To study the expression and correlation of insulin receptor (INSR), insulin receptor substrate-1 (IRS-1), and programmed cell death ligand-1 (PD-L1) in nonsmall cell lung cancer (NSCLC). Methods 45 lung cancer tissues and 30 adjacent normal tissues of NSCLC patients diagnosed in the Second Affiliated Hospital of Shandong First Medical University from June 2019 to August 2020 were selected. The expressions of INSR, IRS-1, and PD-L1 proteins in tumor tissues and adjacent tissues of NSCLC were detected by immunohistochemical staining. Results The expression of INSR and IRS-1 in NSCLC was significantly higher than that in adjacent normal lung tissue (P < 0.05). INSR expression had statistical significance with the degree of pathological differentiation of nonsmall cell carcinoma (P = 0.031), but had no significant association with age, gender, pathological type, TNM stage, and lymph node metastasis status (P > 0.05). There was no significant correlation between IRS-1 positive expression and NSCLC patients' age, gender, pathological typing, degree of differentiation, TNM stage, and lymph node metastasis (P > 0.05). PD-L1 positive expression was correlated with lymph node metastasis of NSCLC (P = 0.028), while there was no significant correlation with gender, age, pathological type, TNM stage, and pathological differentiation degree of NSCLC patients (P > 0.05). Spearman correlation analysis showed that PD-L1 protein expression had a significant positive correlation with IRS-1 protein expression (r = 0.373), but was not correlated with the expression of INSR protein. Conclusion IRS-1 may be involved in the regulation of PD-L1 expression and mediate the occurrence of tumor immune escape, which is expected to become a new target for NSCLC immunotherapy and provide new clinical evidence for immunosuppressive therapy.

Research Advances of Immunotherapy of Exosome PD-L1 in Non-small Cell Lung Cancer

肿瘤免疫疗法在当前癌症治疗领域中愈来愈受瞩目，相关研究也层出不穷。对于非小细胞肺癌（non-small cell lung cancer, NSCLC）患者来说，近些年来，以程序性死亡受体1（programmed cell death 1, PD-1）/程序性死亡配体1（programmed cell death ligand 1, PD-L1）免疫抑制剂为代表的免疫检查点抑制剂（immune checkpoint inhibitors, ICIs）已经成为治疗恶性肿瘤的一种最具前景的治疗方案。免疫检查点阻断治疗包括抗细胞毒T淋巴细胞相关抗原4（cytotoxic T lymphocyte-associated antigen 4, CTLA-4）单抗、抗PD-1单抗和抗PD-L1单抗，其中最被人熟知的为PD-L1免疫疗法。目前ICIs在临床治疗中取得了很不错的治疗效果，但有效率较低，因此我们希望获得更高的治疗有效率。近几年外泌体PD-L1在NSCLC免疫治疗中发挥了重要作用。本文就肿瘤外泌体PD-L1蛋白对肿瘤微环境的影响、预测免疫治疗效果以及作为NSCLC免疫治疗的新型治疗策略作一综述。

Circulating monocytes associated with anti-PD-1 resistance in human biliary cancer induce Tcell paralysis.

Suppressive myeloid cells can contribute to immunotherapy resistance, but their role in response to checkpoint inhibition (CPI) in anti-PD-1 refractory cancers, such as biliary tract cancer (BTC), remains elusive. We use multiplexed single-cell transcriptomic and epitope sequencing to profile greater than 200,000 peripheral blood mononuclear cells from advanced BTC patients (n= 9) and matched healthy donors (n= 8). Following anti-PD-1 treatment, CD14+ monocytes expressing high levels of immunosuppressive cytokines and chemotactic molecules (CD14CTX) increase in the circulation of patients with BTC tumors that are CPI resistant. CD14CTX can directly suppress CD4+ Tcells and induce SOCS3 expression in CD4+ Tcells, rendering them functionally unresponsive. The CD14CTX gene signature associates with worse survival in patients with BTC as well as in other anti-PD-1 refractory cancers. These results demonstrate that monocytes arising after anti-PD-1 treatment can induce Tcell paralysis as a distinct mode of tumor-mediated immunosuppression leading to CPI resistance.

Cyclic adenosine monophosphate/phosphodiesterase 4 pathway associated with immune infiltration and PD-L1 expression in lung adenocarcinoma cells.

BackgroundThe cyclic adenosine monophosphate/phosphodiesterase 4 (cAMP/PDE4) pathway is involved in inflammation and immune regulation; however, the effect of cAMP/PDE4 on immune infiltration and immune evasion in lung adenocarcinoma (LUAD) remains unclear.MethodsCBioPortal, which is the The Cancer Genome Atlas (TCGA) online database, and the Kaplan Meier plotter were used to analyze the association between genes and the prognosis of TCGA-LUAD. Tumor Immune Estimation Resource (TIMER) was used to analyze the association between gene expression and immune infiltration. The Genecards database was used to identify the transcription factors of related genes. The lung adenocarcinoma cell line H1299 and A549 were treated with cAMP pathway drugs. Flow cytometry and qRT-PCR were used to detect the PD-L1 protein and gene expression, respectively. A one-way analysis of variance with Tukey’s post-hoc test or a Student’s t-test were used.ResultsIt was found that PDE4B and CREB1, which are downstream genes of the cAMP/PDE4 axis, were differentially expressed in LUAD and adjacent tissues and are correlated with the prognosis and immune infiltration of LUAD. In the CBioPortal database, cAMP pathway genes are closely related to programmed cell death-ligand 1 (PD-L1) expression in TCGA-LUAD. The protein-protein interaction revealed that there was a direct interaction between CREB1/CREBBP, which are the downstream molecules of the cAMP/PDE4 axis, and MYC; additionally, MYC was predicted to bind to the PD-L1 transcription site and regulate PD-L1 expression. CREB1 was also predicted to transcriptionally bind to both MYC and PD-L1. These results predicted the interaction network of cAMP/PDE4/CREB1/CREBP/MYC/PD-L1, and the core factor may be related to MYC. In the cell experiment, forskolin (an adenylate cyclase activator) and zardaverine (a PDE4 inhibitor) enhance the cAMP pathway and decrease PD-L1 expression, while SQ2253 (an adenylate cyclase inhibitor) inhibits the cAMP pathway and increases PD-L1 expression of the LUAD cell lines H1299 and A549, and MYC regulation by these drugs was positively correlated with PD-L1 regulation, which verified the regulation of the cAMP/PDE4 pathway on MYC and PD-L1.ConclusionsThis study showed that the cAMP/PDE4 pathway may play an important role in PD-L1 regulation and immune infiltration in LUAD.

USP7 Enables Immune Escape of Glioma Cells by Regulating PD-L1 Expression

ABSTRACT Background Glioma is the most common primary intracranial tumor. This study investigated the mechanism of ubiquitin-specific processing protease 7 (USP7) on the immune escape of glioma cells via the regulation of programmed cell death ligand-1 (PD-L1). Methods USP7 and PD-L1 expressions in glioma and normal brain tissues were detected using quantitative reverse transcriptase polymerase chain reaction and Western blot. The glioma cells U-251 MG were transfected with si-USP7 and pcDNA3.1-PD-L1, or treated with anti-PD-L1, after which the cell viability, colony-forming ability, and apoptosis rate were evaluated using cell counting kit-8, colony formation, and TdT-mediated dUTP nick-end labeling assays. Then, CD8+ T cells were purified, extracted, then co-cultured with U-251 MG cells. CD8+ T cell proliferation and the concentrations of interferon (IFN-γ), tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, interleukin (IL)-1β, and IL-10 were verified using carboxyfluorescein succinimidyl amino ester proliferation assay and enzyme-linked immunosorbent assay. Afterwards, the interaction between USP7 and PD-L1 and the ubiquitination level of PD-L1 were also assessed. Results USP7 was highly-expressed and PD-L1 mRNA levels did not change, while PD-L1 protein levels were up-regulated in the glioma cells. Silencing USP7 in U-251 MG cells limited the growth of the glioma cells, promoted glioma cell apoptosis, and facilitated the proliferation of CD8+ T cells, thus inhibiting immune escape. USP7 stabilized PD-L1 expression through deubiquitination. PD-L1 overexpression reversed the inhibitory effect of silencing USP7 on the immune escape of glioma cells. Conclusion USP7 stabilized PD-L1 through deubiquitination and accelerated the immune escape of glioma cells.

Method for detecting acetylated PD-L1 in cell lysates by immunoprecipitation and western blot analysis

Lysine acetylation is an important regulatory post-translational modification (PTM) that occurs sub-stoichiometrically, often representing less than 1% of the target protein. This makes studying endogenous protein acetylation extremely challenging. Recent reports suggest that several post-translational modifications (PTMs), including lysine acetylation, play a major role in the regulation the programmed cell death-ligand 1 (PD-L1), a clinically important protein target. An enrichment step is necessary to enable identification of the acetylated species by either antibody or mass spectrometry-based detection methods. This report describes a robust lab protocol for the enrichment and detection of endogenous acetylated PD-L1 protein. A recently developed acetyl lysine affinity matrix was utilized to enrich >90% of acetylated PD-L1 species, from a variety of cell lines, spanning a fourteen-fold range of target protein levels. Western blot analysis, using a highly sensitive PD-L1 antibody and optimized transfer times, was used to determine that the endogenous level of acetylated PD-L1 is in the range of 0.02–0.07% of total PD-L1. As validation, we demonstrate that acetylation levels increase to 0.11–0.17% of total PD-L1 after a 4h treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). The method described here is simple to perform in any lab equipped with tissue culture and western blot equipment.

Liposomal Co-delivery of PD-L1 siRNA/Anemoside B4 for Enhanced Combinational Immunotherapeutic Effect.

Combination therapy has gained a lot of attention thanks to its superior activity against cancer. In the present study, we report a cRGD-targeted liposomal preparation for co-delivery of programmed cell death ligand 1 (PD-L1) small interfering RNA (siRNA) and anemoside B4 (AB4)─AB4/siP-c-L─and evaluate its anticancer efficiency in mouse models of LLC and 4T1 tumors. AB4/siP-c-L showed a particle size of (180.7 ± 7.3) nm and a ζ-potential of (32.8 ± 1.5) mV, with high drug encapsulation, pH-sensitive release properties, and good stability in serum. AB4/siP-c-L demonstrated prolonged blood circulation and increased tumor accumulation. Elevated cellular uptake was dependent on the targeting ligand cRGD. This combination induced significant tumor inhibition in LLC xenograft tumor-bearing mice by downregulating PD-L1 protein expression and modulating the immunosuppressive microenvironment. Liposomes favored the antitumor T-cell response with long-term memory, without obvious toxicity. A similar tumor growth inhibition was also demonstrated in the 4T1 tumor model. In summary, our results indicate that cRGD-modified and AB4- and PD-L1 siRNA-coloaded liposomes have potential as an antitumor preparation, and this approach may lay a foundation for the development of a new targeted drug delivery system.

Abstract 5529: In vivo efficacy evaluation of ASC61, an oral PD-L1 inhibitor, in two tumor mouse models

Abstract Background: ASC61 is an oral inhibitor prodrug of programmed cell death-ligand 1 (PD-L1). ASC61 is converted to its pharmacologically active metabolite ASC61-A in the plasma. In vitro studies showed that ASC61-A could activate PD-L1 dimerization and membrane PD-L1 protein internalization, interfere PD-1/PD-L1 interaction and enhance T-cell activation. Here we report the in vivo efficacy of ASC61 in two tumor mouse models. ASC61 was found to have the best antitumor activity among all test drugs in the syngeneic tumor mouse model, BALB/c mice bearing CT-26-hPD-L1 tumors. Then a humanized mouse model, PD-1/PD-L1 dKI HuGEMM bearing hPD-L1 MC38 tumor, was used to further validate the therapeutic efficacy of ASC61. Methods: In the BALB/c mouse model, mice were inoculated subcutaneously with CT-26-hPD-L1 cells. When the average tumor volume reached approximately 69 mm3, mice were randomly assigned and treated twice a day (BID) with vehicle, reference drug (Atezolizumab), ASC61, GLC01-589 or GLC01-633 for 19 days. In the PD-1/PD-L1 dKI HuGEMM mouse model, MC38 hPD-L1 cells were subcutaneously inoculated in the right rear flank region of mice. Once the mean tumor size reached approximately 78.3 mm3, mice were randomized and treated BID for 16 days with vehicle, Atezolizumab, ASC61 (50 or 100 mg/kg). In both models, body weights and tumor volumes were measured regularly, and tumor growth inhibitions (TGI) of different groups were compared. Results: All mice showed a gradually increase in body weight during the study and no significant difference of body weight or body weight change was found among different groups in both mouse models. In the BALB/c mouse model, ASC61 50 mg/kg showed significantly inhibitory effects on the tumor growth with the best TGI value of 52.9% (p &amp;lt; 0.05) on Day 19, even better than that of the reference drug (40.77%). In the PD-1/PD-L1 dKI HuGEMM mouse model, Group 2 to 4 showed TGI values of 69.62%, 33.62% and 63.15% on Day 16, respectively. Atezolizumab 5 mg/kg BIW and ASC61 100 mg/kg BID produced similar significant antitumor efficacies (both p &amp;lt; 0.001). Survival analysis showed significant differences between Group 1 and Group 2 to 4 (p = 0.0002, 0.0338 and 0.0027, respectively). Conclusion: Results of this study show that ASC61 is well tolerated in both mouse models, and ASC61 has comparable antitumor efficacies as the FDA-approved PD-L1 antibody, Atezolizumab. ASC61 is an oral PD-L1 inhibitor, and thus will have better patient compliance than injective PD-1/PD-L1 antibody. ASC61 first in human clinical trial will begin soon. Citation Format: Jinzi J. Wu, Handan He. In vivo efficacy evaluation of ASC61, an oral PD-L1 inhibitor, in two tumor mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5529.

The worsening impact of programmed cell death ligand 1 in ovarian clear cell carcinomas.

To investigate the clinical significance of programmed cell death ligand 1 (PD-L1) expression in ovarian clear cell carcinoma (CCC). Patients with CCC who underwent primary surgery at our hospital between 1984 and 2014 were enrolled in this study. PD-L1 and mismatch repair (MMR) protein expression in tumor cells, tumor-infiltrating lymphocytes (TILs), including cluster of differentiation (CD) 8, CD4, forkhead box P3 (FOXP3), programmed cell death 1 (PD-1), and BAF250a, were evaluated using immunohistochemistry. The association between PD-L1 expression, clinicopathological features, prognosis, and expression of several proteins was investigated. Of the 125 patients with CCC, 17 had negative PD-L1 and 108 had positive PD-L1. Patients with positive PD-L1 expression showed a lower response to chemotherapy (p = 0.01). In addition, patients with positive PD-L1 showed worse progression-free survival (PFS, p = 0.01) and overall survival (OS, p = 0.01) than that in patients with negative PD-L1 expression. Multivariate analyses for PFS and OS showed that PD-L1 expression was an independent prognostic factor for PFS (hazard ratio [HR] 7.81, p < 0.01) and OS (HR 12.90, p < 0.01). PD-L1 expression was not associated with the expression of several TILs or proteins. The expression of PD-L1 was related to a lower response to chemotherapy and worse prognosis in CCC. These results may be useful for the development of new treatments.

Microsatellite Instability Analysis and Its Prognostic Value in Invasive Nonampullary Duodenal Adenocarcinoma

Purpose: Nonampullary duodenal adenocarcinoma (NADA) is a rare disease. Although several prognostic factors have been reported for this disease, they remain controversial due to their rarity. In this study, we retrospectively analyzed 54 cases of invasive NADA, focusing on the microsatellite instability (MSI) phenotype, programmed cell death-ligand 1 (PD-L1) expression, and prognostic factors. Methods: Expression of the PD-L1 protein and cell differentiation markers in tumors was detected by immunohistochemistry. Microsatellite markers (NR-21, NR-22, NR-24, BAT-25, and BAT-26) were amplified for MSI assessment by PCR. Results: The incidence of MSI in invasive NADA was 35.2%. No significant correlation between the MSI phenotype and clinicopathological factors was observed. Positive expression of PD-L1 by immune cells was common in advanced-stage disease (p = 0.054), and positive expression of PD-L1 in cancer cells correlated significantly with the histologically undifferentiated type (p = 0.016). Kaplan-Meier survival analysis demonstrated a significantly better overall survival (OS) in patients with MSI (p = 0.013) and at early-stage disease (p = 0.000) than in those with microsatellite-stable or at late tumor stages. Univariate and multivariate analyses showed that MSI (hazard ratio [HR]: 0.282, 95% confidence interval [CI]: 0.106–0.751, p = 0.011) and early tumor stage (stage I-II) (HR: 8.81, 95% CI: 2.545–30.500, p = 0.001) were independent better prognostic factors of OS. Conclusions: MSI and early tumor stage (stage I-II) were independent better prognostic factors of OS. A high proportion of MSI phenotypes and positive PD-L1 expression may be helpful for identifying immune checkpoint inhibitors as a novel therapeutic strategy.

Polysaccharide of Atractylodis Macrocephalae Rhizoma inhibits expression of immune checkpoint PD-L1 by targeting miR-34a in esophageal carcinoma cells

The immune checkpoint programmed cell death-ligand 1(PD-L1)-mediated immunosuppression is among the important features of tumor. PD-L1, an immunosuppressant, can induce T cell failure by binding to programmed cell death-1(PD-1). Thus, the key to restoring the function of T cells is inhibiting the expression of PD-L1. The Chinese medicinal Atractylodis Macrocephalae Rhizoma(AMR) has the anti-tumor, anti-inflammatory, antioxidant, and hypoglycemic activities, and the polysaccharide in AMR(PAMR) plays a crucial role in immunoregulation, but the influence on the immune checkpoints which are closely related to immunosuppression has not been reported. MicroRNA-34 a(miR-34 a) expression in esophageal carcinoma tissue is significantly lower than that in normal tissue. This study aims to investigate the inhibitory effect of PAMR on esophageal carcinoma cells, and the relationship between its inhibitory effect on PD-L1 expression and miR-34 a, which is expected to clarify the anti-tumor mechanism of PAMR. Firstly, different human esophageal carcinoma cell lines(EC9706, EC-1, TE-1, EC109 cells) were screend out, and expression of PD-L1 was determined. Then, EC109 cells, with high expression of PD-L1, were selected for further experiment. The result showed that PAMR suppressed EC109 cell growth. According to the real-time quantitative PCR(qPCR) and Western blot, it significantly suppressed the mRNA and protein expression of PD-L1, while promoting the expression of tumor suppressor miR-34 a. The confocal microscopy and luci-ferase assay proved that PAMR alleviated the inhibitory effect of PD-L1 while blocked miR-34 a. Additionally, the expression of PD-L1 was controlled by miR-34 a, and the combination of miR-34 a inhibitor with high-dose PAMR reversed the inhibitory effect of PAMR on PD-L1 protein expression. Thus, the PAMR may inhibit PD-L1 by increasing the expression of miR-34 a and regulating its downstream target genes. In conclusion, PAMR inhibits the expression of PD-L1 mainly by inducing miR-34 a.

Superinduction of immunosuppressive glioblastoma extracellular vesicles by IFN-γ through PD-L1 and IDO1.

BackgroundGlioblastoma (GBM), the most common primary brain tumor, has a median survival of 15–16 months. Immunotherapy is promising but GBM-mediated immunosuppression remains a barrier. GBMs express the interferon-gamma (IFN-γ)-responsive immunosuppressive molecules programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1). Extracellular vesicles (EVs) have also been implicated in GBM-mediated immunosuppression, in part through PD-L1. We therefore sought to determine if GBM IFN-γ exposure increased GBM EV-mediated immunosuppression and mechanisms underlying this.MethodsHuman GBM-derived cells were cultured in the presence/absence of IFN-γ. EVs were harvested. PD-L1, IDO1, and EV-associated protein expression was assessed. GBM EVs (+/−IFN-γ) were cultured with healthy donor monocytes. Immunosuppressive myeloid-derived suppressor cell (MDSC) and nonclassical monocyte (NCM) frequency was determined. Impact of GBM (+/−IFN-γ) EV-treated monocytes on CD3/CD28-mediated T cell proliferation was assessed. The impact of PD-L1 and IDO1 knockdown in GBM EVs in this system was evaluated.ResultsIFN-γ exposure increased PD-L1 and IDO1 expression in GBM cells and EVs without altering EV size or frequency. IFN-γ-exposed GBM EVs induced more MDSC and NCM differentiation in monocytes and these monocytes caused more T cell inhibition than IFN-γ-naive GBM EVs. PD-L1 and/or IDO1 knockdown in GBM cells abrogated the immunosuppressive effects of IFN-γ-exposed GBM EVs on monocytes.ConclusionsIFN-γ exposure such as might occur during an antitumor immune response results in superinduction of GBM EVs’ baseline immunosuppressive effects on monocytes. These effects are mediated by increased PD-L1 and IDO1 expression in GBM EVs. These data highlight mechanisms of GBM EV-mediated immunosuppression and identify therapeutic targets (PD-L1, IDO1) to reverse these effects.

Expression of programmed cell death ligand-1 protein in germinal center B-cell-like and non-germinal center B-cell-like subtypes of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma worldwide. However, there is still limited information on the expression of programmed cell death ligand-1 (PD-L1), a type 1 transmembrane protein of immunoglobulin B7/CD28 in the DLBCL subtypes. The present study aimed to identify the expression of PD-L1 in germinal center B-cell-like (GCB) subtype and non-germinal center B-cell-like (non-GCB) subtype of DLBCL. A total of 40 patient samples (formalin-fixed paraffin-embedded tissues) consisting of 20 cases of GCB subtype and 20 cases of non-GCB subtype of DLBCL were examined. The PD-L1 protein expressions were evaluated by using immunohistochemical staining in the tumor cells. The results showed a statistically significant difference (P=0.003) between the expression of PD-L1 in the GCB subtype and the non-GCB subtype of DLBCL. PD-L1 expression in the tumor cells were observed in 13 cases (65%) of non-GCB subtype and in three cases (15%) of the GCB subtype of DLBCL. In conclusion, it was found that the expression of PD-L1 protein in the tumor cells of the non-GCB subtype of DLBCL was significantly higher as compared with the tumor cells of the GCB subtype of DLBCL.

Molecular docking study of britannin binding to PD-L1 and related anticancer pseudoguaianolide sesquiterpene lactones

The pseudoguaianolide-type sesquiterpene lactone (SL) britannin (BRT), found in different Inula species, has been characterized as a potent anticancer agent acting via modulation of the transcription factor NFkB and the Nrf2-Keap1 signaling pathway. In addition, a BRT-induced down-regulation of the immune checkpoint PD-L1 (programmed cell death ligand 1) expressed on cancer cells has been evidenced. Here we have performed a docking analysis of the direct binding of BRT to the PD-L1 protein, both in its monomeric and dimeric state. BRT appears to form stable complexes with PD-L1, with a preference for the dimeric form, binding at the interface of the two monomers. The calculated empirical energy of interaction (ΔE) value reaches −63.1 kcal/mol for the BRT-PD-L1 dimer complex, not far from the value calculated with the reference PD-L1 ligand BMS-202 (ΔE = −73.4 kcal/mol) under identical conditions. We also studied the potential PD-L1 dimer binding of 15 pseudoguaianolide sesquiterpene lactones analogues to BRT, including helenalin, gaillardin, bigelovin, coronopilin, and others. The docking analysis predicted that the SL chamissonolide (CHM) can also form equally stable complexes with PD-L1 dimer (ΔE = −64.8 kcal/mol). Preliminary compound structure-PD-L1 binding relationships have been delineated. This computational study supports the proposed interaction of BRT with PD-L1 and provides a guidance to the design of novel PD-L1 binders incorporating a SL-like tricyclic core unit.