Programmed Cell Death Ligand 1 Antibody Research Articles

Novel Checkpoints and Cosignaling Molecules in Cancer Immunotherapy

The recent demonstration of the antitumor efficacy of checkpoint protein inhibition has resulted in the approval of blocking antibodies against the programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway in multiple different histologic findings. Therapeutic successes with PD-1/PD-L1 antibodies in melanoma and lung cancer have been followed by approvals in bladder, renal, and head and neck cancers and Hodgkin lymphoma, with others undoubtedly to come. However, PD-1 is only one of many checkpoints and agonistic regulatory molecules expressed on T cells by which maintenance of the balance between costimulatory and coinhibitory signaling pathways is perturbed in cancer. The manipulation of many of these molecules in cancer patients might be associated with clinical benefit. The majority of the T-cell cosignaling receptors belong to either the immunoglobulin superfamily or the tumor necrosis factor receptor superfamily. A total of 29 immunoglobulin superfamily and 26 tumor necrosis factor receptor superfamily cosignaling receptors have been identified that are expressed on T cells, providing fertile ground for development of inhibitory or agonistic antibodies and small molecules as cancer therapeutics. In the current work, we focus on some of the most promising new checkpoints and agonistic or cosignaling molecules that are in early clinical development as single agents or in combinations with PD-1/PD-L1, cytotoxic T-lymphocyte-associated protein 4 blockade, or chemotherapy with an emphasis on those that have reached the clinic and on important targets that are in late preclinical development.

P3.02c-097 Nivolumab in Elderly or Poor Performance Status Patients with Advanced Non-Small Cell Lung Cancer: Topic: IT Clinical

Nivolumab showed durable antitumor activity and survival benefit in previously treated patients with advanced non-small cell lung cancer (NSCLC). The aim of this study was to evaluate the efficacy and safety of nivolumab in elderly (≥75 year old) or poor performance status (PS; ≥2) patients with NSCLC, most of who have been excluded from clinical trials. This was a retrospective cohort study investigating the outcome of patients with advanced or post-operative recurrence NSCLC who received nivolumab from January to April 2016 in the National Cancer Center Hospital. Patient characteristics, the efficacy of nivolumab, survival, and adverse events were analyzed. Immunohistochemical (IHC) expression of programmed cell death ligand 1 (PD-L1) in pretreatment tissue (10 biopsy and 3 operation specimens) was assessed using the rabbit monoclonal PD-L1 antibody (clone 28-8; Abcam). Staining of ≥1% of tumor cells was considered as the cut-off value of positive PD-L1 expression. A total of 20 patients, including 10 elderly patients with non-squamous NSCLC and 10 poor PS patients (7 with non-squamous and 3 with squamous NSCLC), received nivolumab. Objective responses were observed in 6 patients: 4 (40%) elderly and 2 (20%) poor PS patients. The median progression-free survival was 2.8 months and 1.7 months in the elderly and poor PS groups, respectively. Of 13 cases with quantifiable IHC results, 10 cases were positive for PD-L1. PD-L1 expression was not predictive of a response, which occurred in 4 of 10 (40%) patients with PD-L1-postive tumors, 1 of 3 (33%) patients with PD-L1-negative tumors, and 1 of 7 (14%) patients with an unknown PD-L1 status (p = 0.52). Treatment-related adverse events led to discontinuation of nivolumab in 6 patients (1 elderly and 5 poor PS patients). Nivolumab had a clinically meaningful response for elderly or poor PS advanced NSCLC patients, but toxicity led to the discontinuation of nivolumab.

Validated programmed cell death ligand 1 immunohistochemistry assays (E1L3N and SP142) reveal similar immune cell staining patterns in melanoma when using the same sensitive detection system.

Tumour cell and/or immune cell programmed cell death ligand 1 (PD-L1) expression is considered as a potential biomarker for anti-PD1 and anti-PD-L1 immunotherapy. Currently, different PD-L1 assays are used. This study aims to compare the staining patterns of two PD-L1 antibody clones in melanoma metastases and correlate them with PD-L1 mRNA expression. The immunohistochemistry assays were optimized and validated independently on a Ventana Benchmark Ultra (Ventana Medical Systems Inc., Tucson, AZ, USA) (E1L3N) and XT (SP142), using the same detection system. In total, 46 melanoma metastases were stained with both validated immunohistochemistry assays. Stained slides were digitized for qualitative and semi-quantitative evaluation; done by pathologist and semi-automated software analysis. A subset of 21 melanoma metastases was selected for quantification of the PD-L1 mRNA expression. Accuracy and precision criteria were met for both assays. PD-L1 protein and mRNA expression showed remarkably good Spearman's coefficients of 0.90 (E1L3N) and 0.87 (SP142). Despite the remarkable correlation between both PD-L1 assays in expression patterns and quantification values (ρ > 0.90), E1L3N showed significantly more tumour cell staining than SP142. E1L3N and SP142 IHC assays were optimized and validated successfully and independently for sensitive and accurate PD-L1 detection. Concordance was best for immune cell scoring, while E1L3N tended to detect more tumour cells. Determination of the clinically relevant cut-off values for immune cell versus tumour cell detection requires further research.

Clinical and Histologic Features of Lichenoid Mucocutaneous Eruptions Due to Anti-Programmed Cell Death 1 and Anti-Programmed Cell Death Ligand 1 Immunotherapy.

Antagonist antibodies to programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) have shown remarkable activity in multiple tumor types. Recent US Food and Drug Administration approval of such agents for advanced melanoma, non-small cell lung cancer, and renal cell carcinoma has hastened the need to better characterize their unique toxicity profiles. To provide a clinical and pathologic description of the lichenoid mucocutaneous adverse effects seen in patients receiving anti-PD-1/PD-L1 treatment. Patients with advanced cancer who were referred to dermatology at Yale-New Haven Hospital, a tertiary care hospital, after developing cutaneous adverse effects while receiving an anti-PD-1 or PD-L1 antibody therapy either as monotherapy or in combination with another agent were identified. Medical records from 2010 to 2015 and available skin biopsy specimens were retrospectively reviewed. Patient demographic characteristics, concurrent medications, therapeutic regimen, type of disease, previous oncologic therapies, clinical morphology of cutaneous lesions, treatment of rash, peripheral blood eosinophil count, tumor response, and skin histologic characteristics if biopsies were available. Patients were 13 men and 7 women, with a mean (range) age of 64 (46-86) years. The majority of cases (16 [80%]) had a clinical morphology consisting of erythematous papules with scale in a variety of distributions. Biopsies were available from 17 patients; 16 (94%) showed features of lichenoid interface dermatitis. Eighteen patients were treated with topical corticosteroids, and only 1 patient required discontinuation of anti-PD-1/PD-L1 therapy. Only 4 of 20 patients (20%) developed peripheral eosinophilia. Sixteen patients (80%) were concurrently taking medications that have been previously reported to cause lichenoid drug eruptions. Papular and nodular eruptions with scale, as well as mucosal erosions, with lichenoid features on histologic analysis were a distinct finding seen with anti-PD-1/PD-L1 therapies and were generally manageable with topical steroids. Concurrent medications may play a role in the development of this cutaneous adverse effect.

Clinical and pathologic features of lung cancer expressing programmed cell death ligand 1 (PD-L1)

BackgroundProgrammed cell death 1 (PD-1) negatively regulates antigen receptor signaling upon binding by either of its ligands, programmed cell death ligand 1 or 2 (PD-L1/2). Blockade of this interaction with either PD-1 or PD-L1 antibodies has been successful in the treatment of human cancer, especially melanoma and non-small cell lung cancer. PD-L1 expression has been proposed as a predictor of tumor response. However, the relationships between PD-L1 expression and various clinicopathological characteristics remain unclear. Materials and methodsPD-L1 expression was examined in 220 non-small cell lung cancer specimens that were consecutively resected at our hospital after validating the E1L3N antibody immunohistochemical assay by comparing IHC and RT-PCR data for lung cancer cell lines. We evaluated the relationships between PD-L1 positivity, several clinical factors and the immunohistochemical expression of epithelial-mesenchymal transition (EMT), cancer stem cell and proliferative markers. ResultsPD-L1 was expressed in 22% of lung adenocarcinomas and 60% of squamous cell lung cancers. There was no significant association between PD-L1 expression and clinicopathological features in squamous cell lung cancer. However, in patients with lung adenocarcinoma, PD-L1 expression was significantly correlated with solid subtype histology, vimentin expression, increased Ki-67 labeling index and poor prognosis by multivariate analysis. ConclusionPD-L1 expression was associated with high proliferative activity and the EMT phenotype in adenocarcinoma but not in squamous cell carcinoma of the lung. PD-L1 expression was a significant poor prognostic factor in patients with lung adenocarcinoma.

Imaging, Biodistribution, and Dosimetry of Radionuclide-Labeled PD-L1 Antibody in an Immunocompetent Mouse Model of Breast Cancer.

The programmed cell death ligand 1 (PD-L1) participates in an immune checkpoint system involved in preventing autoimmunity. PD-L1 is expressed on tumor cells, tumor-associated macrophages, and other cells in the tumor microenvironment. Anti-PD-L1 antibodies are active against a variety of cancers, and combined anti-PD-L1 therapy with external beam radiotherapy has been shown to increase therapeutic efficacy. PD-L1 expression status is an important indicator of prognosis and therapy responsiveness, but methods to precisely capture the dynamics of PD-L1 expression in the tumor microenvironment are still limited. In this study, we developed a murine anti-PD-L1 antibody conjugated to the radionuclide Indium-111 ((111)In) for imaging and biodistribution studies in an immune-intact mouse model of breast cancer. The distribution of (111)In-DTPA-anti-PD-L1 in tumors as well as the spleen, liver, thymus, heart, and lungs peaked 72 hours after injection. Coinjection of labeled and 100-fold unlabeled antibody significantly reduced spleen uptake at 24 hours, indicating that an excess of unlabeled antibody effectively blocked PD-L1 sites in the spleen, thus shifting the concentration of (111)In-DTPA-anti-PD-L1 into the blood stream and potentially increasing tumor uptake. Clearance of (111)In-DTPA-anti-PD-L1 from all organs occurred at 144 hours. Moreover, dosimetry calculations revealed that radionuclide-labeled anti-PD-L1 antibody yielded tolerable projected marrow doses, further supporting its use for radiopharmaceutical therapy. Taken together, these studies demonstrate the feasibility of using anti-PD-L1 antibody for radionuclide imaging and radioimmunotherapy and highlight a new opportunity to optimize and monitor the efficacy of immune checkpoint inhibition therapy.

Programmed cell death ligand-1 (PD-L1) expression by immunohistochemistry: could it be predictive and/or prognostic in non-small cell lung cancer?

Blockade of immune checkpoints has recently emerged as a novel therapeutic strategy in various tumors. In particular, monoclonal antibodies targeting programmed cell death 1 (PD-1) or its ligand (PD-L1) have been most studied in lung cancer, and PD-1 inhibitors are now established agents in the management of non-small cell lung cancer (NSCLC). The reports on high-profile clinical trials have shown the association of PD-L1 expression by immunohistochemistry (IHC) with higher overall response rates to the PD-1/PD-L1 axis blockade suggesting that PD-L1 expression may serve as a predictive marker. Unfortunately, however, each PD-1 or PD-L1 inhibitor is coupled with a specific PD-L1 antibody, IHC protocol and scoring system for the biomarker assessment, making the head-to-head comparison of the studies difficult. Similarly, multiple clinical series that correlated PD-L1 expression with clinicopathologic and/or molecular variables and/or survival have reported conflicting results. The discrepancy could be explained by the differences in ethnicity and/or histologic types included in the studies, but it appears to be attributed in part to the differences in PD-L1 IHC methods. Thus, orchestrated efforts to standardize the PD-L1 IHC are warranted to establish the IHC as a predictive and/or prognostic biomarker in NSCLC.

Programmed cell death protein-1/programmed cell death ligand-1 pathway inhibition and predictive biomarkers: understanding transforming growth factor-beta role.

A deeper understanding of the key role of the immune system in regulating tumor growth and progression has led to the development of a number of immunotherapies, including cancer vaccines and immune checkpoint inhibitors. Immune checkpoint inhibitors target molecular pathways involved in immunosuppression, such as cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway, with the goal to enhance the host's own immune anticancer response. In phase I-III trials, anti-PD-1/PD-L1 antibodies have demonstrated to be effective treatment strategies by inducing significant durable tumor responses, with manageable toxicities, in patients with various malignancies, including those traditionally considered non-immunogenic, such as non-small cell lung cancer (NSCLC). Identification of predictive biomarkers to select patients for immune therapies is currently being investigated to improve their therapeutic efficacy. Transforming growth factor-β (TGF-β), a pleiotropic cytokine with immunosuppressive effects on multiple cell types of the innate and adaptive immune system, has emerged as one of the potential key factors modulating response to immune checkpoint inhibitors. However, due to the complexity of the anti-cancer immune response, the predictive value of many other factors related to cancer cells or tumor microenvironment needs to be further explored.

Abstract B98: Serial evaluation of PD-L1 expression on circulating tumor cells (CTCs)

Abstract BACKGROUND: Metastatic non-small cell lung cancer (NSCLC) tumors have adopted methods to evade immune detection and/or clearance. This can occur via overexpression of programmed cell death ligand 1 (PD-L1). Response rate, progression free survival and overall survival with PD-1 inhibitors are greater in tumors with high tumor PD-L1 expression (Garon et al, NEJM 2015, Paz-Ares et al, ASCO 2015). There has been interest in using PD-L1 tumor expression as a treatment selection criterion. Currently available methods of screening involve invasive tumor biopsy followed by histological grading of PD-L1 levels. Biopsies allow sampling from limited sections of the tumor, which may miss heterogeneity. CTC PD-L1 levels could aid in screening patients, and could supplement tissue PD-L1 biopsy results by evaluating a representative sampling from multiple tumor sites which shed cells into the blood. Additionally, following PD-L1 levels on CTCs serially over time may potentially yield information about modulation of tumor PD-L1 expression in the presence of PD-1 or PD-L1 antibodies or other anti-cancer agents. METHODS: We have developed a microfluidic device for rapid, size-based capture of CTCs from blood called Vortex HT chip. The Vortex HT chip utilizes inertial microfluidic flows to isolate CTCs with capture efficiency up to 40% and high purity (>80%). We used the Vortex HT device to capture CTCs from NSCLC patients undergoing PD-1 immunotherapies, both prior to initiating treatment and during treatment. PD-L1 expression was evaluated on CTCs using immunofluorescence staining. CTC number and PD-L1 expression were correlated with treatment response as evaluated by immune related response criteria (irRC) on serial CT scans. We also measured cell size and intensity levels of cytokeratin (CK) and CD45 on the collected cells. We developed a semi-automated algorithm to quantify fluorescence for these different markers on DAPI positive cells collected from each patient sample. We compared these results to PD-L1 expression on the initial tumor biopsy sections, as assayed by immunohistochemical staining and expression quantified with HALO software (Indica Labs). RESULTS: In patients receiving anti-PD-1 antibodies, PD-L1 expression on CTCs could be quantified and compared to a pre-treatment tumor biopsy, as well as to radiographic treatment response. Evaluating patient CTC count and PD-L1 expression at baseline and over time may lead to simple and non-invasive methods to predict response to immunotherapies. As an assay amenable to repeat testing, CTC PD-L1 levels may also be an important pharmacodynamics marker to assess the synergistic potential of combination immunotherapies. Further work is continuing to better understand this predictive biomarker. At the meeting, we will present correlation data between CTC and tumor biopsy PD-L1 expression assays and between the CTC assay and radiographic response to treatment. Citation Format: Jonathan W. Goldman, Manjima Dhar, James Che, Edward B. Garon, Siwen Hu-Lieskovan, Melissa Matsumoto, Brian R. Wolf, James M. Carroll, Matthew J. Crabtree, D. Andrew Tucker, Jennifer Strunck, Elodie Sollier, Rajan Kulkarni, Dino Di Carlo. Serial evaluation of PD-L1 expression on circulating tumor cells (CTCs). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B98.

Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging.

Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

PD-L1 is remarkably over-expressed in EBV-associated pulmonary lymphoepithelioma-like carcinoma and related to poor disease-free survival.

Programmed cell death-ligand 1 (PD-L1) and driver mutations are commonly seen in non-small-cell lung cancer (NSCLC). However, the prevelance of PD-L1 over-expression and its prognostic value in Epstein-Barr virus (EBV) associated pulmonary lymphoepithelioma-like carcinoma (LELC) remains poorly understood. A total of 214 NSCLC patients and 113 surgically treated pulmonary LELC patients were included. Paraffin-embedded tumor sections were stained with PD-L1 antibody. Correlations between PD-L1 expression and clinicopathological features as well as survival outcomes were analyzed. The frequency of PD-L1 over-expression in NSCLC was 51.4%. No significant association was observed between common driver mutations and PD-L1 over-expression. Remakably, the positive rate of PD-L1 in pulmonary LELC was 74.3%. High PD-L1 expression was associated with impaired disease-free survival (DFS) compared with low PD-L1 expression (p = 0.008). Multivariate analysis shows that PD-L1 expression level, N stage and M stage were independent prognostic factors for DFS. N stage and M stage but not PD-L1 expression level were significantly associated with overall survival (OS). PD-L1 over-expression was not related to common driver mutations in NSCLC. Pulmonary LELC have remarkably high incidence of PD-L1 expression. PD-L1 was a negative prognostic factor for DFS in surgically resected pulmonary LELC. These findings may provide a rationale for immunotarget therapy in this virus-associated lung cancer.

Positive expression of programmed death ligand-1 correlates with superior outcomes and might be a therapeutic target in primary pulmonary lymphoepithelioma-like carcinoma.

BackgroundPrimary pulmonary lymphoepithelioma-like carcinoma (LELC) is a rare subtype of non-small cell lung cancer (NSCLC), and no effective treatments have been defined for advanced disease. Programmed cell death-ligand 1 (PD-L1) is expressed in a group of cancers that may be suitable targets for specific immunotherapy.MethodsThis study investigated the expression and clinical value of PD-L1 in pulmonary LELC. Seventy-nine patients with pulmonary LELC were investigated. Paraffin-embedded tumor sections were stained with PD-L1 antibody. Correlations of PD-L1 expression with clinicopathologic parameters and outcomes were analyzed.ResultsFifty patients (63.3%) were PD-L1 positive. The 3-year and 5-year progression-free survival (PFS) rate was 76.0% and 68.0%, respectively, and the 3-year and 5-year overall survival (OS) rate was 88.0% and 79.0%, respectively. Kaplan–Meier analysis revealed that patients with positive PD-L1 expression had longer PFS and OS than those with negative PD-L1 expression (P=0.019 and P=0.042, respectively). In a multivariate Cox regression model including age, tumor size, stage, and PD-L1 expression status, the latter three factors were found to be independent predictors of PFS (P=0.023, P=0.000, and P=0.009, respectively), but only stage was found to be an independent factor for OS (P=0.007), and PD-L1 expression status showed a trend to be independently correlated with OS (P=0.080).ConclusionOur results showed that a large proportion of patients with pulmonary LELC had positive expression of PD-L1, supporting the potential use of anti-PD-1/PD-L1-targeted therapies in this distinct type of NSCLC.

The association between PD-L1 and EGFR status and the prognostic value of PD-L1 in advanced non-small cell lung cancer patients treated with EGFR-TKIs

Recent clinical trials have shown that immune-checkpoint blockade yields remarkable response in a subset of non-small cell lung cancer (NSCLC) patients. However, few studies directly focus on the association between epidermal growth factor receptor (EGFR) mutational status and programmed cell death-ligand 1 (PD-L1) expression. We examined whether PD-L1 is related to clinicopathologic factors and prognosis in patients with advanced NSCLC treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). One-hundred and seventy patients with advanced NSCLC were explored. Paraffin-embedded tumour sections were stained with PD-L1 antibody. EGFR mutation was examined by fluorescent quantitative polymerase chain reaction (PCR). The correlations between PD-L1 expression and EGFR status and survival parameters were analyzed. The overall frequency of PD-L1 over-expression was 65.9% (112/170). In lung adenocarcinoma, PD-L1 tended to be associated with mutant EGFR (PD-L1 overexpression in mutant and wild-type EGFR, 64/89 (71.9%) vs. 32/56 (57.1%), respectively; p=0.067). Subgroup analyses showed that high PD-L1 expression was associated with significantly shorter overall survival (OS) in EGFR wild-type patients (p=0.029) but not in EGFR mutant patients (p=0.932) treated with EGFR-TKIs. Even more, for EGFR mutant patients, higher expression of PD-L1 might only signal better outcome with TKIs. High PD-L1 expression was likely to be associated with the presence of EGFR mutation in advanced lung adenocarcinoma. For EGFR wild-type patients, the PD-L1 over expression can be considered as a poor prognostic indicator of OS.

PD-L1 is highly expressed in lung lymphoepithelioma-like carcinoma: A potential rationale for immunotherapy.

Programmed cell death-ligand 1 (PD-L1) and driver mutations are found in non-small cell lung cancers (NSCLCs) and may be suitable targets for specific therapies, but their roles in lymphoepithelioma-like carcinoma (LELC) of the lung are unclear. Sixty-six patients with pulmonary LELCs were investigated. Paraffin-embedded tumor sections were stained with PD-L1 antibody. Tumors with moderate-to-strong membrane staining in ≥5% of tumor cells were positive for PD-L1 overexpression. The presence of driver mutations in the genes for epidermal growth factor receptor (EGFR), KRAS, and BRAF were examined by direct sequencing. Anaplastic lymphoma kinase (ALK) and ROS1 levels were determined by immunohistochemistry. Correlations of PD-L1 expression and driver mutations with clinicopathologic parameters were analyzed. The overall frequency of PD-L1 overexpression and EGFR mutation was 75.8% and 12.1%, respectively. No KRAS, BRAF, ALK or ROS1 aberrations could be detected. PD-L1 expression was not associated with driver mutations. Multivariate analysis revealed that smoking and advanced stage were independent risk factors for poor overall survival, whereas PD-L1 positivity was not significantly associated with patient outcome. There are high PD-L1 expression and infrequent driver mutations in LELCs compared with conventional NSCLCs. The high expression of PD-L1 in EBV and inflammation associated LELC may provide a rationale for immunotherapy in this subtype of lung cancer.

A phase 1 study to evaluate the safety and tolerability of MEDI4736, an anti–PD-L1 antibody, in combination with tremelimumab in patients with advanced solid tumors.

TPS3120^ Background: MEDI4736 (M) is a human IgG1 monoclonal antibody that binds specifically to programmed cell death ligand 1 (PD-L1), expressed on immune cells and tumor cells, preventing bindin...