Programmed Death Ligand-1 Immune Checkpoint Research Articles

Immunohistochemistry of immune checkpoint markers PD-1 and PD-L1 in prostate cancer

Recent availability of immune checkpoint inhibitors has facilitated research involving programmed cell death protein 1 (PD-1) and its ligand, programmed death-ligand 1 (PD-L1). However, the incidence and clinical implication of PD-1 and PD-L1 expression in prostate cancer remain poorly understood. The current study aimed to determine the status of PD-1/PD-L1 expression in prostate cancer specimens and its prognostic significance.We immunohistochemically stained for PD-1 and PD-L1 in our tissue microarray (TMA) consisting of radical prostatectomy specimens. The expression of PD-1/PD-L1 was designated as positive when moderate to strong staining or weak staining was seen in at least 1% or 10%, respectively, of tumor cells and/or associated immune cells. We then evaluated the relationship between the expression of each protein and clinicopathological features available for our patient cohort.PD-1 and PD-L1 were positive in 3 (1.5%) and 1 (0.5%) of 201 non-neoplastic prostate tissues, and also in 17 (7.7%) and 29 (13.2%) of 220 prostate cancers, respectively. PD-1 and PD-L1 were also expressed in tumor-infiltrating lymphocytes/macrophages in 172 (78.2%) and 33 (15.0%) cases, respectively. PD-L1 expression in tumor cells was more often seen in high pT stage (pT2: 10.8% vs pT3/4: 20.4%; P = .072; pT2/3a: 11.4% vs pT3b/4: 31.6%; P = .013) or lymph node-positive (pN0: 10.1% vs pN1: 27.3%; P = .086) cases, whereas PD-1 expression in tumor cells was not significantly associated with pT/pN stage. In addition, there were no statistically significant associations between PD-1/PD-L1 expression in tumor cells or tumor-infiltrating lymphocytes/macrophages versus patient age, preoperative prostate-specific antigen level, or Gleason score. Kaplan–Meier analysis coupled with log-rank test further revealed no significant associations between PD-1/PD-L1 expression in tumor cells (P = .619/P = .315), tumor-infiltrating lymphocytes/macrophages (P = .954/P = .155), or either or both of them (P = .964/P = .767) versus disease recurrence after radical prostatectomy.PD-1/PD-L1 expression was detected in a subset of prostate cancers. In particular, PD-L1 expression was considerably up-regulated in nonorgan-confined tumors. However, PD-1/PD-L1 expression in our TMA was found to be not very helpful in predicting tumor recurrence in prostate cancer patients who underwent radical prostatectomy.

Epidermal Growth Factor Receptor (EGFR) Pathway, Yes-Associated Protein (YAP) and the Regulation of Programmed Death-Ligand 1 (PD-L1) in Non-Small Cell Lung Cancer (NSCLC).

The epidermal growth factor receptor (EGFR) pathway is a well-studied oncogenic pathway in human non-small cell lung cancer (NSCLC). A subset of advanced NSCLC patients (15–55%) have EGFR-driven mutations and benefit from treatment with EGFR-tyrosine kinase inhibitors (TKIs). Immune checkpoint inhibitors (ICIs) targeting the PD-1/PDL-1 axis are a new anti-cancer therapy for metastatic NSCLC. The anti-PD-1/PDL-1 ICIs showed promising efficacy (~30% response rate) and improved the survival of patients with metastatic NSCLC, but the role of anti-PD-1/PDL-1 ICIs for EGFR mutant NSCLC is not clear. YAP (yes-associated protein) is the main mediator of the Hippo pathway and has been identified as promoting cancer progression, drug resistance, and metastasis in NSCLC. Here, we review recent studies that examined the correlation between the EGFR, YAP pathways, and PD-L1 and demonstrate the mechanism by which EGFR and YAP regulate PD-L1 expression in human NSCLC. About 50% of EGFR mutant NSCLC patients acquire resistance to EGFR-TKIs without known targetable secondary mutations. Targeting YAP therapy is suggested as a potential treatment for NSCLC with acquired resistance to EGFR-TKIs. Future work should focus on the efficacy of YAP inhibitors in combination with immune checkpoint PD-L1/PD-1 blockade in EGFR mutant NSCLC without targetable resistant mutations.

IMMUNE CHECKPOINTS INDOLEAMINE 2,3-DIOXYGENASE 1 AND PROGRAMMED DEATH-LIGAND 1 IN ORAL MUCOSAL DYSPLASIA

Objectives Oral mucosal dysplasia is a potentially malignant disorder that is associated with risk of transformation to carcinoma. During malignant transformation, dysplastic cells escape from immune-mediated destruction. We hypothesized that adaptive immunity is inhibited by activation of distinct immune checkpoint molecules, such as indoleamine 2,3-dioxygenase 1 (IDO1) and programmed death-ligand 1 (PD-L1). We collected 64 oral dysplasia samples from 47 patients. Nine biopsies from alveolar mucosa during wisdom teeth extractions were used as healthy controls. Tissue samples were stained and scored for IDO1 and PD-L1. Additionally, dysplasia grades and inflammatory cell infiltration were evaluated. Nine patients were followed up to 36 months to evaluate dysplasia progression, inflammation, and immune checkpoint molecule expression. Findings Dysplastic epithelium had significantly lower IDO1 expression than that of healthy controls. Cells positive for PD-L1 in the lamina propria were mainly in dysplastic samples and seldom in healthy controls. Dysplasia grade associated negatively with epithelium IDO1 and positively with IDO1 and PD-L1 expression in the lamina propria. There was a positive association between dysplasia grade and level of inflammatory cell infiltration. During follow-up, dysplasia grade, inflammatory cell infiltration, and the immune checkpoint expression fluctuated over time. Conclusions The immune checkpoint molecules IDO1 and PD-L1 are modulated during oral epithelial dysplastic changes and their expression is associated with inflammatory cell infiltration in the lamina propria. As immune checkpoint molecule expression fluctuates over time, these molecules are not useful as biomarkers for oral mucosal dysplasia progression.

Combined Inhibition of TGF-β Signaling and the PD-L1 Immune Checkpoint Is Differentially Effective in Tumor Models.

Antibodies blocking the programmed death-ligand 1 (PD-L1) have shown impressive and durable responses in clinical studies. However, this type of immunotherapy is only effective in a subset of patients and not sufficient for rejection of all tumor types. In this study, we explored in two mouse tumor models whether the antitumor effect could be enhanced by the combined blockade of PD-L1 and transforming growth factor-β (TGF-β), a potent immunosuppressive cytokine. The effect of anti-PD-L1 mouse monoclonal (mAb) and a TGF-β type I receptor small molecule kinase inhibitor (LY364947) was evaluated in the highly immunogenic mouse MC38 colon adenocarcinoma and the poorly immunogenic mouse KPC1 pancreatic tumor model. In the MC38 tumor model, LY364947 monotherapy did not show any antitumor effect, whereas treatment with anti-PD-L1 mAb significantly delayed tumor outgrowth. However, combination therapy showed the strongest therapeutic efficacy, resulting in improved long-term survival compared with anti-PD-L1 mAb monotherapy. This improved survival was associated with an increased influx of CD8+ T cells in the tumor microenvironment. In the KPC1 tumor model, LY364947 did not enhance the antitumor effect of anti-PD-L1 mAb. Despite this, delayed KPC1 tumor outgrowth was observed in the LY364947-treated group and this treatment led to a significant reduction of CD4+ T cells in the tumor microenvironment. Together, our data indicate that an additive anti-tumor response of dual targeting PD-L1 and TGF-β is dependent on the tumor model used, highlighting the importance of selecting appropriate cancer types, using in-depth analysis of the tumor microenvironment, which can benefit from combinatorial immunotherapy regimens.

Abstract B027: Antitumor effects of the programmed death receptor-1 and the programmed death-ligand 1 blockade in human colorectal carcinoma in a humanized orthotopic mouse model

Abstract Background: The immune system plays an important role in tumor immune surveillance and progression. Immune checkpoint blockade is a new approach for cancer immunotherapy. The programmed death receptor-1 (PD-1) and the programmed death-ligand 1 (PD-L1) are immune checkpoint molecules and their expression results in negative regulation of T-cells primarily within the tumor microenvironment by preventing the killing of cancer cells by cytotoxic T-lymphocytes. Antibody blocking of the PD-1/PD-L1 signal exhibits promising therapeutic effects in non-small cell lung cancer and melanoma in patients. Due to its early success, more trials have been conducted to evaluate their efficacy for different tumors. We have developed a patient-derived orthotopic xenograft mouse model for colorectal carcinoma (CRC). Here, we investigate the potential efficacy of PD-1/PD-L1 blockade in our humanized orthotopic mouse models for human CRC. Methods: All studies were conducted under approved guidelines of the Institutional Animal Care and Use Committee and the Investigative Review Board of Ochsner Clinic Foundation. Humanized mice were established by intraperitoneal injection of donor human peripheral blood mononuclear cells (PBMCs) into recombinase activating gene 2 (Rag2) and common cytokine receptor gamma chain gene (IL2Rγ) double knockout (Rag2-/-/IL2Rγ-/-) Rag2 mice. Luciferase-tagged CRC cells were injected intra-rectally into Rag2 mice. One group of mice received a combination of anti-PD-1 and anti-PD-L1 antibodies (nivolumab, 200 µg/mouse and atezolizumab, 200 µg/mouse, intravenous injection) once a week for 3 weeks. Tumor growth was measured weekly by bioluminescent imaging (BLI). At necropsy, the CRC tumor weights were measured. Human CD45+ hematopoietic cells, CD4+ and CD8+ T-cells, and CD20+ B cells were detected in peritoneal lavage, blood, and tumor by flow cytometry. The presence of human immune cells (humanization) and tumor-infiltrating human lymphocytes was further confirmed by immunohistochemistry staining on paraffin-embedded tissue slides of mouse spleen and tumor, respectively. Results: Blood from mice receiving human PBMCs contained on average 31.4% CD45+ human cells (n=9) when tested by FACS analysis. Smaller tumor growth was observed in mice given PBMCs. This may be due to alloreactivity based on the recognition of MHC alloantigens in the transplanted tumor cells. However, mice further treated with the combination therapy of anti-PD-1 and anti-PD-L1 antibodies exhibited better antitumor response as well as less development of lung metastases compared to untreated controls. On average, tumor weight was reduced by 36% and lung metastases measured by ex-vivo BLI was reduced by 82% (n=5). In addition, circulating CD326+ tumor cells were reduced by 21% and CD3, CD4, CD8 and CD20 positive human lymphocytes were also reduced in the combination treatment group. Conclusion: Our study provides preclinical evidence of establishment of humanized Rag2 mice to be used as an orthotopic xenograft model for CRC; and treatment benefit for CRC through targeting immune checkpoint molecules PD-1 and PD-L1. The effect of immune checkpoint blockade in combination with conventional chemotherapy using CRC patient-derived specimens and PBMCs from MHC matched donors or autologous PBMCs will be further investigated. Citation Format: Li Li, Xin Zhang, Grace Maresh, Linh Hellmers, Avi Patel, Ravan Moret, Sarah Cohen, David Margolin. Antitumor effects of the programmed death receptor-1 and the programmed death-ligand 1 blockade in human colorectal carcinoma in a humanized orthotopic mouse model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B027.

Prognostic value of immune checkpoint molecules in head and neck cancer: a meta-analysis.

Immune checkpoint molecules are important targets in cancer immunotherapy, but their association with prognosis in patients with head and neck cancer is controversial. In this meta-analysis, we searched for 12 immune checkpoint molecules in the PubMed, Embase and Cochrane Library databases and retrieved 52 studies with 7127 participants. Among the molecules included in the search, indoleamine 2, 3-dioxygenase (IDO), programmed death ligand 1 (PD-L1), and programmed death 1 (PD-1) met the inclusion criteria for further analysis. Higher expression of IDO was associated with poorer overall survival in head and neck cancer patients (P = 0.011), but higher expression of PD-L1 correlated with better overall survival specifically in nasopharyngeal carcinoma patients (P = 0.01). In a sensitivity analysis, higher PD-L1 expression correlated with better progression-free survival (P = 0.043), and was associated with better overall survival in Caucasian subjects (P = 0.02), nasopharyngeal carcinoma patients (P = 0.015), and studies with small sample sizes (P = 0.001). PD-1 had no prognostic significance. There was no publication bias affecting the results. Thus, among the immune checkpoint molecules, IDO and PD-L1 are potential prognostic predictors in head and neck cancer.

The Role of Yes-Associated Protein (YAP) in Regulating Programmed Death-Ligand 1 (PD-L1) in Thoracic Cancer.

The programmed death-ligand 1(PD-L1)/PD-1 pathway is an immunological checkpoint in cancer cells. The binding of PD-L1 and PD-1 promotes T-cell tolerance and helps tumor cells escape from host immunity. Immunotherapy targeting the PD-L1/PD-1 axis has been developed as an anti-cancer therapy and used in treating advanced human non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM). Yes-associated protein (YAP) is a key mediator of the Hippo/YAP signaling pathway, and plays important roles in promoting cancer development, drug resistance and metastasis in human NSCLC and MPM. YAP has been suggested as a new therapeutic target in NSCLC and MPM. The role of YAP in regulating tumor immunity such as PD-L1 expression has just begun to be explored, and the correlation between YAP-induced tumorigenesis and host anti-tumor immune responses is not well known. Here, we review recent studies investigating the correlation between YAP and PD-L1 and demonstrating the mechanism by which YAP regulates PD-L1 expression in human NSCLC and MPM. Future work should focus on the interactions between Hippo/YAP signaling pathways and the immune checkpoint PD-L1/PD-1 pathway. The development of new synergistic drugs for immune checkpoint PD-L1/PD-1 blockade in NSCLC and MPM is warranted.

CRISPR/Cas9-Mediated Disruption of PD-L1 Reduces the T Cell Suppressive Effect of Wharton's Jelly Mesenchymal Stromal Cells and Their Extracellular Vesicles

BackgroundMesenchymal stem/stromal cells (MSCs) are a promising treatment for acute graft-versus-host disease and other inflammatory diseases. MSCs express programmed death-ligand 1 (PD-L1) that has been reported to contribute to MSC-mediated immune suppression through T-cell receptor (TCR)-mediated inhibitory signaling. MSCs actively secrete extracellular vesicles (EVs) that have immune modulatory properties resembling their cell of origin. Although the release of EVs by MSCs has been extensively reported, the presence and functional impact of immune inhibitory checkpoint molecules in MSC-derived EVs is still being determined. We sought to determine if PD-L1 and other immune inhibitory checkpoint molecules are enriched in MSC-derived EVs. In this study we report that PD-L1 is enriched in MSC-EVs, and that PD-L1(+) EVs and PD-L1(-) EVs differ in their T cell immune suppressive capacity.MethodsUmbilical cords and healthy donor blood samples were collected on separate IRB-approved protocols after written informed consent (HSC#1546/HSC#5929). Umbilical cord Wharton's jelly-MSCs (WJMSC) were isolated from umbilical cords under GMP conditions and characterized by morphology, multiparameter flow cytometry (MPFC), and differentiation capacity. Three clinical-grade WJMSC cell lines were cultured at low passage (2-5) in EV-depleted media. EVs were isolated from the media by differential ultracentrifugation and the 100K pellets enriched with exosomes were characterized by Western blot (WB), nanoparticle tracking analysis (NTA), and electron microscopy (EM). To further identify EV-enriched immune proteins, the WJMSC and EV proteomes were characterized by Tandem Mass Tag (TMT) labeling and LC MS/MS, using isobaric labels to compare protein quantities between samples. Gene disruption of PD-L1 was performed through site-specific CRISPR/Cas9 introducing a single cysteine nucleotide deletion in exon 3; the genetic mutation was confirmed by Sanger sequencing. The absence of PD-L1 transcript was confirmed by RT-PCR, and the absence of PD-L1 protein was confirmed by WB, immunofluorescence, MPFC, and IFNγ-response. PBMC were isolated from healthy donor blood samples; T cells were activated by CD3/CD28 beads and induced CD154 was measured on CD4(+) T cells by MPFC. Alternatively, CMV-experienced T cells were activated with CMV-pp65 peptide and induced IFNγ was measured on CD8(+) T cells by MPFC. Activated T cells were treated with PD-L1(+) and PD-L1(-) WJMSCs or their derived EVs. Phospho-Zap-70 (Tyr319)/Syk (Tyr352) MPFC was used to analyze T-cell receptor (TCR)-mediated signaling.ResultsWJMSCs were CD73(+), CD90(+), CD105(+), and CD34(-), CD45(-), CD11b(-), CD19(-) and HLA-DR(-); and inhibited proliferation in a mixed lymphocyte reaction. WJMSCs-derived EVs were cup-shaped 100-200nm nanoparticles by EM and were CD9(+), CD81(+), CD73(+), CD90(+), and CD105(+) by WB. Quantitative proteomic profiling revealed a total of 1,634 proteins across WJMSC-derived EV preparations. Proteins that were highly enriched in MSC-derived EVs and that were classified under “negative regulation of lymphocyte activation” (Gene Ontology Biological Processes; GOBP) included PDL1, CD276, DLG1, TNR6, ERBB2, TGFβ1, LYN, and HMGB3. Both WJMSCs and EVs suppressed CD4(+) T cell activation by CD3/CD28 beads and CD8(+) T cell activation by CMV peptide. CRISPR/Cas9 edited WJMSC exhibited normal morphology and differentiation characteristics. PD-L1 gene disruption efficiently reduced PD-L1 protein expression on WJMSC and their derived EVs, and reduced the ability of both to suppress T cell activation. Similarly, blocking PD-L1 with a specific internalization antibody decreased WJMSC and EV-mediated T cell activation.ConclusionProteomic analyses reveal that PD-L1 is enriched in WJMSC-derived EVs. CRISPR/Cas9-mediated disruption of PD-L1 in WJMSCs alone did not affect the release of EVs in vitro. Gene-edited WJMSCs stably generate PD-L1(-) EVs. The PD-L1(-) EVs generated from PD-L1(-) WJMSC did not suppress T cell activation in co-culture. These findings indicate that PD-L1 is enriched in WJMSC EVs and is biologically active. WJMSC-EVs can deliver membrane-bound PD-L1 to T cells, resulting in attenuated TCR signaling. DisclosuresMcGuirk:Fresenius Biotech: Research Funding; Bellicum Pharmaceuticals: Research Funding; Astellas Pharma: Research Funding; Pluristem Ltd: Research Funding; Gamida Cell: Research Funding; Kite Pharma: Honoraria, Other: travel accommodations, expenses, speaker ; Novartis Pharmaceuticals Corporation: Honoraria, Other: speaker, Research Funding.

Immune checkpoints indoleamine 2,3-dioxygenase 1 and programmed death-ligand 1 in oral mucosal dysplasia.

Oral mucosal dysplasia is a histologic feature of potentially malignant disorders that is associated with the risk of transformation to carcinoma. Dysplastic cells use many strategies during their transformation to cancer, including escape from the immune mediated destruction. We hypothesized that adaptive immunity is inhibited by activation of distinct immune checkpoint molecules, such as indoleamine 2,3-dioxygenase 1 (IDO1) and programmed death-ligand 1 (PD-L1). We collected 63 oral dysplasia samples from 47 patients. Nine biopsies from alveolar mucosa were taken during wisdom teeth extractions were used as healthy controls. Tissue samples were stained and scored for IDO1 and PD-L1. Additionally, dysplasia grades and inflammatory cell infiltration were evaluated. Eight patients were followed up to 36months to evaluate dysplasia progression, inflammation, and immune checkpoint molecules expression. Dysplastic epithelium had significantly lower IDO1 expression than that of healthy controls. PD-L1 positive cells in the lamina propria were mainly in dysplastic samples and seldom in healthy controls. Dysplasia grade was associated negatively with epithelium IDO1 and positively with IDO1 and PD-L1 expression in the lamina propria. There was a positive association between dysplasia grade and level of inflammatory cell infiltration. During follow-up, dysplasia grade, inflammatory cell infiltration, and the immune checkpoint expression fluctuated over time. Immune checkpoint molecules IDO1 and PD-L1 are modulated during oral epithelial dysplastic changes, and their expression is associated with inflammatory cell infiltration in the lamina propria. As immune checkpoint molecules expression fluctuates over time, these molecules are not useful as biomarkers for oral mucosal dysplasia progression.

The Roles of microRNAs in Regulating the Expression of PD-1/PD-L1 Immune Checkpoint.

Engagement of programmed death-ligand 1 (PD-L1) with its receptor programmed death 1 (PD-1) on T cells has been speculated to play a major role in suppressing the immune system, which helps tumor cells evade anti-tumor immunity. With the development of whole genome sequencing technologies, microRNAs have gained more attention as an important new layer of molecular regulation. Recent studies have revealed that altered expression of microRNAs play a pivotal role in immune checkpoint and various cellular processes in cancer. In this review, we focused on the latest progress about microRNAs research which involves the regulation of PD-1/PD-L1 immune checkpoint.

Abstract 2645: Development of a first in class APRIL fully blocking antibody BION-1301 for the treatment of multiple myeloma

Abstract APRIL, or tumor necrosis factor super family member 13 (TNFSF13), is a ligand for the receptors B-cell maturation antigen (BCMA) and transmembrane activator calcium modulator and cyclophilin ligand (CAML) interactor (TACI). APRIL serum levels are enhanced in patients diagnosed with multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and colorectal carcinoma and are correlated with poor prognosis. As a paracrine factor produced by osteoclasts, macrophages and other cells in the bone marrow niche, APRIL binds to BCMA to drive proliferation and survival of human Multiple Myeloma (MM) cells and induces resistance to several standard of care agents. Using a mouse anti-human APRIL blocking antibody 01A1 originally discovered using Aduro’s B-Select platform, we demonstrated that osteoclast-induced MM cell in vitro proliferation and survival is dependent on APRIL. In these co-cultures, cytolytic activity of lenalidomide and bortezomib is significantly enhanced by 01A in a dose-dependent fashion2. Importantly, APRIL drives and 01A inhibits expression of an immunosuppressive gene set including the immune checkpoint programmed death ligand-1 (PD-L1), interleukin-10, vascular endothelial growth factor, and transforming growth factor beta. The human APRIL antagonist antibody 01A was humanized and designated BION-1301. The antibody binds an epitope overlapping the BCMA and TACI binding sites potently and fully blocks BCMA and TACI binding (IC50 <1 nM). Biophysical and functional experiments indicated that BION-1301 recapitulated all characteristics of 01A. In vivo, BION-1301 was shown to suppress T cell-independent B cell responses to NP-Ficoll. Furthermore, APRIL blockade demonstrated single agent anti-multiple myeloma activity in a humanized SCID model2 confirming its activity in vivo, and potentially indicating that BION-1301 is active targeting multiple myeloma cells in a tumor-protective bone marrow microenvironment. To our knowledge, BION-1301 is a first-in-class humanized APRIL antagonist demonstrated in to inhibit multiple myeloma survival, drug resistance and an immune suppressive phenotype preclinical. These data suggest a rationale to develop BION-1301 as a single agent, and in combination with lenalidomide, bortezomib, or possibly checkpoint inhibitors such as anti-PD-1. BION-1301 is expected to enter Phase 1 in 2017. 1) Guadagnoli at al. Blood. 2011 Jun 23;117(25):6856-65 2) Yu-Tzu et al. Blood. 2016 Jun 23;127(25):3225-36 Citation Format: John Dulos, Lilian Driessen, Marc Snippert, Marco Guadagnoli, Astrid Bertens, David Lutje Hulsik, Tai Yu Tzu, Kenneth Anderson, Jan Paul Medema, Kate Cameron, Hans Eenennaam, Andrea Elsas. Development of a first in class APRIL fully blocking antibody BION-1301 for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2645. doi:10.1158/1538-7445.AM2017-2645

Abstract 570: Transparent tumor tomography (T3): 3D spatial immunoanalysis for PD-L1 immune checkpoint blockade therapy

Abstract Untangling the complexity of programmed death-ligand 1 (PD-L1) expression within a heterogeneous tumor microenvironment is an urgent challenge in PD-1/PD-L1 immune checkpoint blockade therapy. Here, we address this challenge with a method, termed transparent tumor tomography (T3), facilitating three-dimensional (3D) visualization and spatial analysis of distributions of multiple biomarkers regarding to cancer cells, vasculature, and immune cells in context in the tumor microenvironment. With T3 analysis of transgenic mouse mammary tumors immunostained against Her2, CD45, Ki-67, CD31, and PD-L1, we reveal that PD-L1 expression within the tumor microenvironment is highly adaptable for efficiently preventing immune cell infiltration into the tumor. Stronger correlation of Her2 and PD-L1 expression in the tumor periphery where has a high CD45+ immune infiltrate density is determined by tumor-wide analysis. Also, tomographic analysis shows blood vessels expressing PD-L1 in the tumor core, where PD-L1 expression is lower. Furthermore, high-resolution T3 image localizes PD-L1 expression to a region between the endothelium and the surrounding smooth muscle cells in blood vessels. We investigate spatial pharmacokinetics of anti-PD-L1 antibody in the whole mouse mammary tumor in the context of hypoxia, CD31+ blood vessels, and target PD-L1+ cells. The 3D anti-PD-L1 antibody distribution is fit to a two-compartment pharmacokinetic model, yielding estimated distribution half-life of 4.7 min and terminal half-life of 2.5 days. We also evaluate anti-tumor immune responses after PD-L1 blockade therapy using T3. We observe broad distribution of tumor infiltrating CD3+CD8+ cytotoxic T cells in 3D tumor section following combination therapy of radiation and anti-PD-L1 antibody compared to PBS, anti-PD-L1 antibody alone, or radiation alone treatment group. Moreover, we apply T3 for immunoanalysis of whole core needle biopsy. We spatially map PD-L1 expression and granzymeB-producing CD3+CD8+ cytotoxic T cells in pre-treatment and in-treatment core needle biopsies at cellular resolution and in three dimensions. Meanwhile, T3 analysis is nondestructive, allowing secondary analysis by IHC and/or IF. We anticipate that T3 can be applied broadly to facilitate preclinical studies of immunotherapy and also find use in spatial, multiparameter analysis of patient biopsies, particularly to improve predictive testing and analysis of immune responses to tumor immunotherapy. Citation Format: Steve Seung-Young Lee, Vytautas P. Bindokas, Stephen J. Kron. Transparent tumor tomography (T3): 3D spatial immunoanalysis for PD-L1 immune checkpoint blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 570. doi:10.1158/1538-7445.AM2017-570

Targeting the PD-1/PD-L1 Immune Checkpoint in EGFR-Mutated or ALK-Translocated Non-Small-Cell Lung Cancer.

Immune checkpoint inhibitors, notably antibodies targeting programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), have modified the management of patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC). Several PD-1/PD-L1 inhibitors have been approved by health authorities for this indication and others are in clinical development. However, only a subset of patients truly benefits from these agents. For patients with mutated EGFR or translocated ALK NSCLC, for whom an immune checkpoint inhibitor can be prescribed after progression on tyrosine kinase inhibitors and chemotherapy, information is scarce and sometimes contradictory. Phase III randomized clinical trials have evaluated different immune checkpoint inhibitors (nivolumab, pembrolizumab, atezolizumab) vs. chemotherapy as second- or subsequent-line therapy in NSCLC, but included very few patients with EGFR/ALK-positive disease. Subgroup analyses found that these patients did not benefit from immune checkpoint inhibitors. Retrospective data show progression-free survival lasting only 1.2-2.1months. Preclinical data suggested a lower expression of PD-L1 in EGFR/ALK-positive patients compared to EGFR/ALK-negative patients. Our objective herein is to provide an up-to-date review of available data from the various publications on the impact of immune checkpoint inhibitors in patients with EGFR/ALK-positive NSCLC.

PD-L1 protein expression assessed by immunohistochemistry is neither prognostic nor predictive of benefit from adjuvant chemotherapy in resected non-small cell lung cancer

The expression of programmed death (PD) ligand 1 (PD-L1) protein expression assessed by immunohistochemistry (IHC) has been correlated with response and survival benefit from anti-PD-1/PD-L1 immune checkpoint inhibitor therapies in advanced non-small cell lung carcinoma (NSCLC). The efficacy of several agents appears correlated with PD-L1 expression. It remains controversial whether PD-L1 is prognostic in NSCLC. We assessed the prognostic value of PD-L1 IHC and its predictive role for adjuvant chemotherapy in early stage NSCLC. Tumor sections from three pivotal adjuvant chemotherapy trials (IALT, JBR.10, CALGB 9633) using the E1L3N antibody were studied in this pooled analysis. PD-L1 staining intensity and percentage in both tumor cells (TCs) and immune cells (ICs) were scored by two pathologists. The average or consensus PD-L1 expression levels across intensities and/or percent cells stained were correlated with clinicopathological and molecular features, patient survivals and potential benefit of adjuvant chemotherapy. Results from 982 patients were available for analysis. Considering staining at any intensities for overall PD-L1 expression, 314 (32.0%), 204 (20.8%) and 141 (14.3%) tumor samples were positive for PD-L1 staining on TCs using cut-offs at ≥1%, ≥10% and ≥25%, respectively. For PD-L1 expressing ICs, 380 (38.7%), 308 (31.4%) and 148 (15.1%) were positive at ≥ 1%, ≥10% and 25% cut-offs, respectively. Positive PD-L1 was correlated with squamous histology, intense lymphocytic infiltrate, and KRAS but not with TP53 mutation. EGFR mutated tumors showed statistically non-significant lower PD-L1 expression. PD-L1 expression was neither prognostic with these cut-offs nor other exploratory cut-offs, nor were predictive for survival benefit from adjuvant chemotherapy. PD-L1 IHC is not a prognostic factor in early stage NSCLC patients. It is also not predictive for adjuvant chemotherapy benefit in these patients.

Immunoinhibitory checkpoint deficiency in medium and large vessel vasculitis

Giant cell arteritis (GCA) causes autoimmune inflammation of the aorta and its large branches, resulting in aortic arch syndrome, blindness, and stroke. CD4+ T cells and macrophages form organized granulomatous lesions in the walls of affected arteries, destroy the tunica media, and induce ischemic organ damage through rapid intimal hyperplasia and luminal occlusion. Pathogenic mechanisms remain insufficiently understood; specifically, it is unknown whether the unopposed activation of the immune system is because of deficiency of immunoinhibitory checkpoints. Transcriptome analysis of GCA-affected temporal arteries revealed low expression of the coinhibitory ligand programmed death ligand-1 (PD-L1) concurrent with enrichment of the programmed death-1 (PD-1) receptor. Tissue-residing and ex vivo-generated dendritic cells (DC) from GCA patients were PD-L1lo, whereas the majority of vasculitic T cells expressed PD-1, suggesting inefficiency of the immunoprotective PD-1/PD-L1 immune checkpoint. DC-PD-L1 expression correlated inversely with clinical disease activity. In human artery-SCID chimeras, PD-1 blockade exacerbated vascular inflammation, enriched for PD-1+ effector T cells, and amplified tissue production of multiple T-cell effector cytokines, including IFN-γ, IL-17, and IL-21. Arteries infiltrated by PD-1+ effector T cells developed microvascular neoangiogenesis as well as hyperplasia of the intimal layer, implicating T cells in the maladaptive behavior of vessel wall endogenous cells. Thus, in GCA, a breakdown of the tissue-protective PD1/PD-L1 checkpoint unleashes vasculitic immunity and regulates the pathogenic remodeling of the inflamed arterial wall.

Abstract B084: Transparent tumor tomography (T3): Spatial analysis for PD-L1 checkpoint blockade immunotherapy

Abstract Untangling the complexity of programmed death-ligand 1 (PD-L1) expression within a heterogeneous tumor microenvironment is an urgent challenge in PD-1/PD-L1 immune checkpoint blockade therapy. Here, we address this challenge with a method, termed transparent tumor tomography (T3), facilitating three-dimensional (3D) visualization and spatial analysis of distributions of multiple biomarkers including Her2, CD45, Ki-67, CD31, and PD-L1 throughout transgenic mouse mammary tumors. Based on our T3 imaging, we reveal that PD-L1 expression within the tumor microenvironment is highly adaptable to tumor status for efficiently preventing immune cell infiltration into the tumor. We also determine spatial pharmacokinetics of anti-PD-L1 antibody in the whole mouse mammary tumor in the context of hypoxia, CD31+ blood vessels, and target PD-L1+ cells. Finally, we demonstrate broad distribution of tumor infiltrating CD3+CD8+ cytotoxic T cells following radiation and anti-PD-L1 antibody therapy. These results provide unique spatial insights into PD-L1 cloaking and PD-L1 blockade therapy. Citation Format: Steve Seung-Young Lee. Transparent tumor tomography (T3): Spatial analysis for PD-L1 checkpoint blockade immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B084.

A Mini-Review for Cancer Immunotherapy: Molecular Understanding of PD-1/PD-L1 Pathway & Translational Blockade of Immune Checkpoints.

Interference of the binding of programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) has become a new inspiring immunotherapy for resisting cancers. To date, the FDA has approved two PD-1 monoclonal antibody drugs against cancer as well as a monoclonal antibody for PD-L1. More PD-1 and PD-L1 monoclonal antibody drugs are on their way in clinical trials. In this review, we focused on the mechanism of the PD-1/PD-L1 signaling pathway and the monoclonal antibodies (mAbs) against PD-1 and PD-L1, which were approved by the FDA or are still in clinical trials. And also presented is the prospect of the PD-1/PD-L1 immune checkpoint blockade in the next generation of immunotherapy.

MYC regulates the antitumor immune response through CD47 and PD-L1.

The MYC oncogene codes for a transcription factor that is overexpressed in many human cancers. Here we show that MYC regulates the expression of two immune checkpoint proteins on the tumor cell surface: the innate immune regulator CD47 (cluster of differentiation 47) and the adaptive immune checkpoint PD-L1 (programmed death-ligand 1). Suppression of MYC in mouse tumors and human tumor cells caused a reduction in the levels of CD47 and PD-L1 messenger RNA and protein. MYC was found to bind directly to the promoters of the Cd47 and Pd-l1 genes. MYC inactivation in mouse tumors down-regulated CD47 and PD-L1 expression and enhanced the antitumor immune response. In contrast, when MYC was inactivated in tumors with enforced expression of CD47 or PD-L1, the immune response was suppressed, and tumors continued to grow. Thus, MYC appears to initiate and maintain tumorigenesis, in part, through the modulation of immune regulatory molecules.

Activation of the PD-1/PD-L1 immune checkpoint confers tumor cell chemoresistance associated with increased metastasis.

The ability of tumor cells to avoid immune destruction (immune escape) as well as their acquired resistance to anti-cancer drugs constitute important barriers to the successful management of cancer. Interaction between the Programmed Death Ligand 1 (PD-L1) on the surface of tumor cells with the Programmed Death-1 (PD-1) receptor on cytotoxic T lymphocytes leads to inactivation of these immune effectors and, consequently, immune escape. Here we show that the PD-1/PD-L1 axis also leads to tumor cell resistance to conventional chemotherapeutic agents. Using a panel of PD-L1-expressing human and mouse breast and prostate cancer cell lines, we found that incubation of breast and prostate cancer cells in the presence of purified recombinant PD-1 resulted in resistance to doxorubicin and docetaxel as determined using clonogenic survival assays. Co-culture with PD-1-expressing Jurkat T cells also promoted chemoresistance and this was prevented by antibody blockade of either PD-L1 or PD-1 or by silencing of the PD-L1 gene. Moreover, inhibition of the PD-1/PD-L1 axis using anti-PD-1 antibody enhanced doxorubicin chemotherapy to inhibit metastasis in a syngeneic mammary orthotopic mouse model of metastatic breast cancer. To further investigate the mechanism of tumor cell survival advantage upon PD-L1 ligation, we show that exposure to rPD-1 promoted ERK and mTOR growth and survival pathways leading to increased cell proliferation. Overall, the findings of this study indicate that combinations of chemotherapy and immune checkpoint blockade may limit chemoresistance and progression to metastatic disease.

Advances of programmed death ligand 1 and its inhibitor in non-small cell lung cancer

Programmed death-ligand 1 (PD-L1), which is highly expressed in NSCLC, can be divided into two categories: membrane PD-L1 and soluble PD-L1. PD-L1 participates in tumor immune escape through combining with its receptor. PD-L1 immune checkpoint inhibitors have entered the phase Ⅰ studies and showed a good application prospect. It is also found that the sensitivity of PD-L1 immune checkpoint inhibitors is strongly associated with the expression of PD-L1 in tumor. Therefore, PD-L1 can be used as a biomarker to predict its curative effect. Key words: Carcinoma, non-small cell lung; Immunosuppressive agents; Programmed death ligand 1