PD-L1 Expression Patterns Research Articles

Prognostic implications of PD-L1 expression in patients with soft tissue sarcoma.

BackgroundThe PD-1/PD-L1 axis plays a paramount role in the immune escape of tumor cells by negative regulation of T-cell functions. The aim of the present study was to characterize the PD-L1 expression pattern and its clinical implication in soft-tissue sarcomas (STS).MethodsWe analyzed PD-L1 expression in 82 STS patients with 5 subtypes: rhabdomyosarcoma, synovial sarcoma, Ewing sarcoma, epithelioid sarcoma, and mesenchymal chondrosarcoma.ResultsThe median age at diagnosis was 26 (range: 1–78) and the male to female ratio was 1.6. The majority (80 %) of patients showed locoregional disease rather than metastatic disease at diagnosis. Thirty-five cases (43 %) showed PD-L1 expression and the proportion of PD-L1 expression was significantly different according to histologic subtypes (P = 0.004); highest in epithelioid sarcoma (100 %, 7/7), followed by synovial sarcoma (53 %, 10/19), rhabdomyosarcoma (38 %, 12/32), and Ewing sarcoma (33 %, 6/18), while it was not expressed in mesenchymal chondrosarcoma (0 %, 0/6). STS patients with PD-L1 expression had worse overall survival compared with those without PD-L1 expression (5-year survival rate: 48 % vs. 68 %, P = 0.015). The Cox proportional hazard model adjusted for histologic subtype, initial metastasis, and PD-L1 expression showed that PD-L1 expression was significantly associated with shorter overall survival (P = 0.037, HR 2.57, 95 % CI 1.060–6.231).ConclusionWe have confirmed PD-L1 expression in various STS of young population and demonstrated its independent negative prognostic role, thereby suggesting the PD-1/PD-L1 axis as a potential therapeutic target for the treatment of young STS patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2451-6) contains supplementary material, which is available to authorized users.

Patterns of PD-L1 expression on circulating tumor cells in Japanese patients with advanced lung cancer.

e23036Background: Immune-checkpoint blockage with anti-PD-1 antibodies is effective against human malignancies including lung cancer. Although the expression of PD-L1 on tumor cells is expected as ...

Patterns of PD-1, PD-L1 and PD-L2 expression in pediatric solid tumors.

10561Background: Significant anti-tumor effects have been observed in a variety of malignancies via blockade of immune checkpoints, leading to T-cell-mediated killing of cancer cells. Interaction o...

Abstract P2-11-07: Mutually exclusive expression pattern of the immune co-inhibitory molecules B7-H4 and PD-L1 in triple negative breast cancer

Abstract B7-H4 (VTCN1, B7x, B7S1) is a transmembrane protein belonging to the B7 family of costimulatory proteins and has been shown to inhibit T cell proliferation, cytokine secretion, and cytotoxic lymphocyte (CTL) induction. B7-H4 expressed on tumor cells or macrophages has been associated with poor prognosis and impaired T cell function in renal cell and ovarian cancers. Here we show B7-H4 is abundantly expressed in human breast cancer with triple negative breast cancer (TNBC) having the highest overall B7-H4 mRNA expression. We developed a specific and sensitive immunohistochemistry (IHC) assay for evaluation of B7-H4 protein and quantified B7-H4 expression in 156 breast tumor samples. Approximately 70% of the breast tumor samples had detectable B7-H4 expression whereas none of the normal or benign breast tissues stained positive for B7-H4. Multiplex IHC and flow cytometry studies showed that the majority of B7-H4 expression was restricted to the tumor epithelial cells, the CD45+ immune cells were negative for B7-H4 expression. Interestingly none of the TNBC samples that were positive for B7-H4 showed detectable expression of PD-L1 suggesting that B7-H4 and PD-L1 checkpoint proteins may act in a mutually exclusive manner. To evaluate the role of B7-H4 on tumor immune evasion, we overexpressed murine or human B7-H4 on the mouse colon-26 (CT26) tumor cell line and injected these cells intravenously into Balb/c mice. By day 14 we observed significantly more tumors as well as larger percent tumor area in the lungs of mice given CT26 cells transduced with human or mouse B7-H4 as compared to vector control transduced cells. These data suggest B7-H4 expression in tumors can accelerate tumor growth in immune competent mice and that targeting B7-H4 may provide therapeutic benefit. Given the mutually exclusive expression patterns of B7-H4 and PD-L1 a B7-H4 targeting agent may provide particular benefit in those patients where current anti-PD-1/PD-L1 therapies are not effective. Citation Format: Shaffer DR, Nagashima K, Cortez-Retamozo V, Feldman I, Smith J, Zafari M, Larson R, Mabry R, Novorantseva T, Briskin M, Sathyanaryananan S. Mutually exclusive expression pattern of the immune co-inhibitory molecules B7-H4 and PD-L1 in triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-11-07.

Potential biomarker for checkpoint blockade immunotherapy and treatment strategy

Programmed cell death protein-1 (PD-1) and ligand (PD-L1) provide an important escape mechanism from immune attack, and blockade therapy of these proteins show promising clinical benefits in many types of cancer. PD-L1 can be induced by interferon-gamma (IFN-γ), hypoxia, or toll-like receptor (TLR)-mediated pathways that confer adaptive immune resistance, or upregulated by oncogenic signals leading to constitutive expression and resulting in intrinsic immune resistance. The PD-1/PD-L1 checkpoint blockade, which targets regulatory pathways in T cells to overcome immune resistance, is correlated to PD-L1 expression pattern and the presence of tumor-infiltrating lymphocytes (TILs). Meanwhile, immunogenic mutation loads show significant response to checkpoint blockade, which is probably due to PD-1/L1 status and TIL content. Finally, the clinical strategies to design effective checkpoint-targeting immunotherapiesare based on the classification of inducible/constitutive expression of PD-L1 and the presence of TILs.

Abstract B001: Development of a PD-L1 companion diagnostic IHC assay (SP142) for atezolizumab

Abstract Background: Understanding of immune tolerance mechanisms of cancer has prompted the development of cancer immunotherapies such as atezolizumab (anti-PD-L1, MPDL3280A). Robust, durable responses have been observed, leading to Breakthrough status designation by the FDA for atezolizumab for previously treated NSCLC and bladder cancer (UBC) patients. Roche/Ventana have developed a companion diagnostic (CDx) for atezoliziumab using a robust immunohistochemistry (IHC) assay and antibody clone (SP142), optimized to detect PD-L1 expression in both tumor cells (TC) and tumor-infiltrating immune cells (IC). Here we describe the characteristics of the SP142 assay, PD-L1 expression patterns observed by immunohistochemistry in TC and IC compartments, and their association with clinical efficacy for NSCLC and UBC patients. Methods: The anti-human PD-L1 rabbit monoclonal antibody SP142 was optimized for detection of both TC and IC expression of PD-L1 with the Ventana OptiView DAB IHC Detection Kit on the automated BenchMark ULTRA platform. The VENTANA PD-L1 (SP142) CDx assay was validated for use in formalin-fixed, paraffin-embedded samples of NSCLC and UBC in a series of studies addressing sensitivity, specificity, robustness, and precision. Formalin fixed, paraffin embedded tumor specimens were obtained from patients prescreened and/or enrolled into Genentech Ph I study PCD4989g, and PD-L1 expression was measured using the SP142 assay and PCR gene expression assays. Results: The SP142 assay met pre-defined acceptance criteria for TC and IC assessment of PD-L1 expression in NSCLC and UBC tumors, including >90% inter-reader concordance between pathologist readers. Rapid and durable responses were observed in the Ph I study (PCD4989g), and correlated with PD-L1 expression patterns observed by immunohistochemistry in TC and IC. In the same Ph I study (PCD4989g), PD-L1 expression as measured by PCR did not demonstrate the same predictive value as observed for IHC. Conclusions: The PD-L1 IHC (SP142) CDx is a robust assay to measure PD-L1 expression in both IC and TC. Further, the high reproducibility of results between pathologists shows that the scoring algorithms in UBC and NSCLC are precise, reproducible, and practical in the diagnostic setting. The results indicate that favorable atezolizumab efficacy for UBC is strongly associated with higher IC levels as assessed by the sensitive and specific PD-L1 IHC (SP142) CDx assay. In NSCLC, favorable atezolizumab efficacy is associated with tumors expressing either high TC or high IC levels by the PD-L1 IHC (SP142) CDx assay. These data support the predictive value of tumor PD-L1 levels by IHC for NSCLC and UBC patients receiving atezoliziumab. Citation Format: Zachary S. Boyd, Dustin Smith, Brian Baker, Bharathi Vennapusa, Hartmut Koeppen, Marcin Kowanetz, Sanjeev Mariathasan, Jean-Marie Bruey, Howard Mackey, Gregg Fine, Simonetta Mocci, Priti Hegde, J. Andrew Williams, Ian McCaffery. Development of a PD-L1 companion diagnostic IHC assay (SP142) for atezolizumab. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B001.

PD-L1 and PD1 Expression in Lymphomas

BackgroundBinding of PD-1 with ligands (e.g. PD-L1) expressed on tumor cells or native antigen-presenting cells results in down-regulation of effector T cell function and represents a potent mechanism of immune evasion across a number of solid tumors and lymphomas especially classical Hodgkin lymphoma (CHL) and aggressive virus-associated B-cell lymphomas. Several biomarkers (different antibodies, gene amplifications and mutations) are being investigated for identifying patients who may benefit from PD-1 checkpoint blockade therapies. It is well known that tumors which strongly express PD-L1 have a high clinical response rate to PD1 blockade. We aimed at identifying the expression of PD-L1 and PD1 in a diverse group of lymphomas. There are several antibodies used to detect PD-L1 expression, however the concordance between these antibodies is not known. We have previously tested concordance of 2 anti-PD-L1 antibodies in histiocytoses [1], but data in lymphomas are lacking. In the current study, we measured PD-L1 expression in lymphoma biopsy samples using 3 antibodies to identify if there was concordance in expression patterns of PD-L1 in various subtypes of lymphomas.MethodsFormalin fixed paraffin embedded tissues from 37 patients with lymphomas of B- and T-cell lineages (both EBV-positive and negative) were investigated for the expression of PD-1 (MRQ-22 antibody) and PD-L1 (MAB1561, SP142 and SP263) using automated immunohistochemical methods. Expression of PD-L1 on 5% or more lymphoma cells was considered positive.ResultsNeoplastic cells expression of PD-L1 was identified in 13/37 cases (35% of all cases). The strongest (3+/>50% cells) and consistent (4/4 cases) expression was observed in Hodgkin RS cells of CHL (2/2 nodular sclerosis and 2/2 lymphocyte depleted). Other B-cell lineage lymphomas positive for expression of PD-L1 included diffuse large B-cell lymphomas (5/9 DLBCL) and lymphomatoid granulomatosis (1/1 LYG). Small lymphocytic lymphoma (n=3), splenic marginal zone lymphoma (n=2) and follicular lymphomas (n=8) were all negative. One of four mantle cell lymphomas (MCL) was borderline (5% of cells) positive. One NK/T-cell lymphoma was strongly (>50% cells) positive; four peripheral T-cell lymphomas (PTCL) and one T cell lymphoblastic lymphoma were negative. PD-L1 expression was observed in both EBV- positive (2/4) and negative (2/3) malignancies. Concordance between any 2 anti-PD-L1 antibodies varied between 86 and 97% (Table 1) with all 3 Abs agreeing in 82%. PD-1 was expressed on malignant cells in 3 cases (2 PTCL and 1 DLBCL); reactive PD-1+ T-lymphocytes were absent in LD CHL and variably present in all other lymphomas.ConclusionsOver-expression of PD-L1was detected in classic HD, diffuse large B cell lymphoma, NK/T cell lymphoma and lymphomatoid granulomatosis. A high concordance in detection of PD-L1 between three antibodies indicates little need for companion diagnostic kits, but correlation between clinical responses and level of PDL1 and PD1 expression in lymphoma should be established in future clinical trials.Table 1Concordance between the PD-L1 antibodies.Antibodies (n)MAB1561SP142SP263MAB1561 (23)--20/23 (86%)SP142 (37)----34/35 (97%)SP263 (35)19/22 (86%)----

Interferon-Gamma Signaling in the Pathogenesis of Idiopathic Pneumonia Syndrome Following Allogeneic Bone Marrow Transplantation

Idiopathic pneumonia syndrome (IPS) is a life-threatening acute noninfectious diffuse lung injury occurring after allogeneic bone marrow transplantation (BMT). We have recently shown that absence of STAT1 signaling in recipient APC leads to accelerated Graft Versus Host Disease (GVHD) and Transplant-associated mortality and morbidity after lethal irradiation in fully MHC-mismatched allogeneic or in non-irradiated P→F1 BMT models. We now addressed the impact of recipient Type II interferon signaling on the development of lung injury after allogeneic BMT using a non-irradiated P→F1 model. 1x107 BMCs plus 2.5x107 SPCs from C57BL/6 (B6) mice were i.v. injected into IFNγR-deficient CB6F1 (F1.GRKO) or CB6F1 wild type (WT) mice. F1.GRKO recipients had significantly reduced survival (MST 32 days post-BMT vs. not reached p<0.001, Log-rank test). Pulmonary function tests were performed 3 weeks post-BMT on anaesthetized mice that were tracheotomized and mechanically ventilated using a computer-controlled animal ventilator (Flexivent, SCIREQ). Static compliance was derived from plateau pressure measurements between total lung capacity and functional residual capacity, and broadband forced oscillations were used to determine airway resistance at baseline and following increasing doses of methacholine (3.1, 12.5 and 50 mg/ml). Static compliance and inspiratory capacity were reduced in F1.GRKO recipients when compared with WT mice (p<0.01). In addition, airway resistance, both at baseline and in response to methacholine, was increased in the KO recipient mice (p<0.05). Histopathology was evaluated on H&E, Trichrome and PAS stained lung sections to assess for inflammation, fibrosis and mucus hyperplasia, respectively. Lungs from the KO mice showed increased interstitial inflammation involving bronchioles, vasculature and parenchyma (pathology index 10 vs 1; p<0.001), fibrosis (fibrosis score 2.8 vs 0.15; p<0.05) and a trend for mucus hyperplasia/metaplasia. To further evaluate the cellular mediators of the observed lung injury, we assessed the cellular composition of the bronchioalveolar lavage fluid (BAL) at day 30 post-BMT. We observed a 10-fold increase in the total cell number of the BAL in F1.GRKO recipient mice compared to WT mice (4x105 vs.4x104, p<0.01); the majority of the cells in the BAL from wild type mice were macrophages/monocytes, while neutrophils predominated in the F1.GRKO mice. Importantly, more than 50% of the CD45+ BAL cells were donor derived in the F1.GRKO recipients, compared to only 10% in the wild type recipients. Furthermore, the absolute cell number of donor CD4+ and CD8+ cells in the knockout mice significantly outnumbered that in their wild type counterparts (p<0.01). Thus, our results suggest that absence of recipient IFN-γR signaling promotes accumulation and/or trafficking of donor neutrophils and T cells to the lungs. To better understand the underlying mechanisms of enhanced infiltration of donor cells in the lungs of IFNγR-/- mice, the activation, proliferation, and differentiation status of donor lymphocytes were assessed by Flowcytometry after BMT. In line with the lung infiltration, there was significantly increased donor cell engraftment in PBMC, spleen, lymph nodes, and bone marrow in F1.GRKO recipients (all with p<0.01); Moreover, there were also significantly increased activation (measured as CD44high CD62Llow cells), Th1 and Tc1 differentiation of donor CD4 and CD8 cells (p<0.01). Furthermore, enhanced Th17 differentiation, but reduced Foxp3+ Treg cell differentiation was noticed. Interestigly, we observed significantly lower PD-L1 expression on recipient APC in the F1.GRKO mice compared to wild type mice, suggesting that absence of PD-L1 expression may contribute to reduced contraction of activated donor T cells. Interestingly, in line with these findings we also observed significantly reduced PD-L1 expression when single cell suspension from lung tissues were analyzed by flow. Reduced PD-L1 expression was observed on both recipient CD45+, and CD45- cells in F1.GRKO when tested on day 22 post-BMT, meanwhile there was no difference in PD-L1 expression pattern in the cells isolated from small intestine. These results suggest that IFN-γ induced PD-L1 expression on hematopoietic and non-hematopoietic cells in the lung may be of significance in protecting recipients against acute donor-cell mediated lung injury and IPS. DisclosuresLentzsch:Celgene: Membership on an entity's Board of Directors or advisory committees; Axiom: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

PD-L1 Antibodies to Its Cytoplasmic Domain Most Clearly Delineate Cell Membranes in Immunohistochemical Staining of Tumor Cells.

Blocking the programmed death-1 (PD-1) pathway has clinical benefit in metastatic cancer and has led to the approval of the mAbs pembrolizumab and nivolumab to treat melanoma and nivolumab for non-small cell lung cancer. Expression of PD-L1 on the cell surface of either tumor cells or infiltrating immune cells is associated with a higher likelihood of response to PD-1 blockade in multiple studies. Most mAbs to PD-L1 in use are directed to its extracellular domain and immunohistochemically stain tumor tissue with a mixture of cytoplasmic and membrane staining. Cytoplasmic staining obscures the interpretation of a positive reaction on the tumor cell membrane, and thus affects the accuracy of PD-L1 scoring systems. We developed a mAb to the cytoplasmic domain of PD-L1, 405.9A11 (9A11), which is both more selective for membranous PD-L1 and more sensitive in IHC and Western blotting, compared with previous mAbs specific for the PD-L1 extracellular domain. Here, we compare immunohistochemical staining patterns of PD-L1 expression in five types of tumors, using five PD-L1 mAbs: 9A11, 7G11, and three commercially available mAbs. We demonstrate that 9A11, as well as two other cytoplasmic domain-specific mAbs, E1L3N and SP142, can clearly delineate the membrane of PD-L1-positive cells in formalin-fixed paraffin-embedded tissue and facilitate interpretation of staining results.

Abstract A90: Tumor associated PDL1 expression pattern in microsopically tumor positive sentinel lymph nodes (SLN) in patients with melanoma

Abstract Background: Anti-PD1/PDL1 immunotherapy has demonstrated unprecedented clinical antitumor activity in patients with metastatic melanoma supporting planned testing as adjuvant therapy in patients with operable disease at high risk of recurrence and death. Characterization of PDL1 expression within clinically/radiologically negative but microscopically tumor positive sentinel lymph nodes is critical to our understanding of the relevance of this immune checkpoint pathway for adjuvant therapy. Methods: Cut sections (5 µm) of sentinel lymph node from 25 patients reported positive for melanoma micrometastasis were included. H&amp;E staining was performed, followed by PD-L1 immunohistochemical (IHC) staining using a preliminary IHC assay with anti-PD-L1 antibody clone 22C3. Slides from 2 patients were also stained using an anti-HMB45/MelA protocol to better ascertain the presence and/or localization of melanoma lesions in the tissue in order to facilitate interpretation of the PD-L1 staining in those samples. The slides were separately evaluated by 2 pathologists. Samples containing metastatic melanoma lesions were scored separately for PD-L1 expression in intratumoral (including along tumor periphery but with clear tumor cell labeling) and peritumoral (expression external to tumor nodule in immediately surrounding tissue) locations. For intratumoral signal, attempts were made to classify the expression as tumor cell associated (T), non-tumor cell associated (NT), or both (T/NT). Scores were assigned using a 0-5 semiquantitative scale assessing prevalence of positive cells where 0 = negative, 1 = minimal or rare, 2 = low, 3 = moderate, 4 = high, and 5 = very high. Samples where melanin content was too high to confidently assign a PD-L1 score were specifically noted. Results: Twenty patients where metastatic melanoma presence in regional sentinel nodes was confirmed by H&amp;E review of the cut sections were included in the final analysis of PDL1 expression in the target population. Table 1 summarizes the study results. Except for samples where melanin content was too high to confidently assign a PD-L1 score, all SLNs examined demonstrated evidence of intratumoral and/or peritumoral PD-L1 expression. Conclusions: PD-L1 expression is readily detectable intratumorally and/or peritumorally within melanoma micrometastases in the SLN. These results support the testing of a therapeutic role for PD1/PDL1 inhibition in the adjuvant therapy setting, targeting melanoma micrometastases. Patient with SLN+ Tumor Associated Expression PD-L1 Score / Tumor ---- PD-L1 Score / Peritumoral 1. 1 T, very weak ---- 1 2. 4, predominantly T, peripheral ---- 1 3. melanin confounds ---- 2 4. 0 ---- 2 (adjacent subcapsular sinus) 5. 4 T/NT---- 2 6. 2, predominantly NT ---- 3 7. 0 ---- 3 (adjacent subcapsular sinus) 8. 2.5, predominantly NT ---- 3 9. 4.5, predominantly T, weak ---- 2 (adjacent sinus) 10. melanin confounds ---- 0 11. 1 T ---- 2.5 12. 0* ---- 2 13. 1 T ---- 1 14. 2.5 NT ---- 2 15. 2 predominantly NT ---- 3 16. 1 T ---- 2 17. melanin confounds ---- 0 18. 1 ---- 1 19. 1 NT ---- 2.5 20. 2 T, melanin confounds ---- 2 *Very few tumor cells. In one region where a few tumor cells were present, a dense area of PD-L1 positivity was present directly adjacent but was impossible to say if all were non-tumor; this is interpreted as most likely. Citation Format: Ahmad A. Tarhini, Jennifer A. Yearley, Christopher Gibson, Uma N. M. Rao, Cindy Sander, John M. Kirkwood. Tumor associated PDL1 expression pattern in microsopically tumor positive sentinel lymph nodes (SLN) in patients with melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A90.

Tumor associated PD-L1 expression pattern in microscopically tumor positive sentinel lymph nodes in patients with melanoma.

BackgroundCharacterization of PD-L1 expression within clinically/radiologically negative but microscopically tumor positive sentinel lymph nodes (SLN) is important to our understanding of the relevance of this immune checkpoint pathway for adjuvant therapy.MethodsPatients included had primary cutaneous melanoma, Breslow thickness of 2.01–4.0 or >4 mm with or without tumor ulceration (T3a, T3b, T4a, T4b). All patients had microscopically tumor positive SLN. Hematoxylin and eosin (H&E) staining was performed, followed by PD-L1 immunohistochemical (IHC) staining using a preliminary IHC assay with anti-PD-L1 antibody clone 22C3. The slides were separately evaluated by two pathologists (JY and CG). Samples containing metastatic melanoma lesions were scored separately for PD-L1 expression in intratumoral and peritumoral locations, by utilizing two scoring methods.ResultsTwenty-four patients where metastatic melanoma presence in the SLN was confirmed by H&E review of the cut sections were included in the final analysis of PD-L1 expression. SLN tumor size ranged from 1 to 2 mm. For three patients, the melanin content was too high to confidently assign a PD-L1 score. For the remaining 21 patients, all had some evidence of either intratumoral or peritumoral PD-L1 expression. The frequency of intratumoral tumor-associated PD-L1 expression was: 0 % of tumor cells (3 pts, 14 %); <1 % (5 pts, 24 %); 1–10 % (6 pts, 29 %) and >10 % (7 pts, 33 %).ConclusionsTumor-associated PD-L1 expression is readily detectable within melanoma micrometastases in the SLN of the majority of patients. These results support the testing of a therapeutic role for PD1/PD-L1 inhibition in the adjuvant setting, targeting melanoma micrometastases.

Clinical pattern and implication of PD-L1 expression in soft-tissue sarcoma.

10565 Background: PD-1/PD-L1 axis plays a paramount role in tumor immune escape by negative regulation of T-cell functions. Recently, immune checkpoint inhibitors targeting this axis displayed prom...

Circulating and tumor-based biomarkers predict clinical activity in cancer patients treated with the engineered anti-PD-L1 antibody MPDL3280A

PD-L1 expressed in the tumor microenvironment regulates Th1 immune responses and mediates cancer immune evasion through interactions with PD-1 or B7.1 receptors on activated T cells. MPDL3280A, an engineered human monoclonal antibody, targets PD-L1 and inhibits its function. To identify immunologic predictive and pharmacodynamic biomarkers of MPDL3280A treatment, we performed a comprehensive analysis of tumors and blood samples collected at baseline and/or on treatment from ≈280 patients with locally advanced or metastatic solid tumors, including NSCLC, RCC, melanoma and bladder cancer. Regardless of tumor type, clinical responses were characterized by PD-L1 expression, the presence of markers of T cell activation (Th1 gene signature and CTLA4), and the absence of fractalkine at baseline in the tumor microenvironment. Elevated baseline expression of IFN-γ and IFN-γ-inducible genes (e.g., IDO1 and CXCL9) was associated with MPDL3280A response in melanoma but not NSCLC or RCC. On treatment, responding tumors showed increased infiltration of Th1-dominant immune infiltrate and evidence of adaptive PD-L1 up-regulation. In contrast, progressing tumors displayed the following patterns of tumor-infiltrating lymphocytes (TILs) and PD-L1 expression: (1) few/no TILs and absent PD-L1 expression (immunologic ignorance), (2) TILs present with minimal/no PD-L1 expression (non-functional immune responses), or (3) TILs residing solely around the tumor cell mass outer edge (excluded infiltrate), suggesting that resistance to MPDL3280A may be associated with impaired T cell trafficking and/or function. Profiling of ≈180 circulating biomarkers revealed that plasma concentrations of IL-18 and interferon-inducible T cell alpha chemoattractant (ITAC) increased in all patients following MPDL3280A treatment, representing a pharmacodynamic measurement of PD-L1 inhibition. In addition, analysis of PBMC showed an increase in T cell activation, as measured by IFN-γ and granzymes A and B gene expression in responders following MPDL3280A treatment, consistent with the observations in responding tumors. Baseline soluble PD-L1 was not associated with response. Some indication-specific biomarkers, such as plasma VEGF, decreased in responders with RCC but not with other indications. In NSCLC, a decrease in tumor burden markers, CA-125 and CEA, was associated with response. Similarly, IL-6 and IL-8 were differentially expressed on treatment in responders vs non-responders. Additionally, responders exhibited a decrease in circulating tumor DNA (ctDNA) in plasma, suggesting that ctDNA may be used to monitor MPDL3280A clinical activity in NSCLC. In conclusion, these data provide general and indication-specific mechanistic insights into immune checkpoint inhibition, potential mechanisms of response and resistance, as well as identification of potential predictive and pharmacodynamic biomarkers of anti-PD-L1/PD-1 clinical activity across multiple tumor types.

The role of splenic CD103+CD8α+cDCs in the induction of CTL responses during respiratory virus infection (168.11)

Abstract We have previously reported that large numbers of virus-specific CD8+ T cells were detected in the spleen following intranasal influenza virus infection. This finding does not seem simply due to the migration of cells from the draining mediastinal lymph nodes (mLN), but rather indicates that spleen can be a site for the induction of primary CTL responses. Although the integrin αEβ7 (CD103)+ migratory DCs have been known as a major DC subset which prime naïve influenza virus-specific CD8+ T cells in the mLN, what kind of DC subset is responsible for the induction of CTL responses in the spleen remains unclear. In this study, we found that increased numbers of CD103+DCs were detectable in the spleen following influenza virus infection, and the majority of CD103+DCs expressed CD8α. Although both CD103+CD11chi DCs (CD103+DCs) and CD103-CD11chi DCs (CD103-DCs) showed activated phenotype evidenced by the expression of co-stimulatory molecules at 9 days post infection, PDL-1 expression pattern was different between these two subsets. Immunohistochemical analysis showed that CD103+DCs localized in the T cell area (also in the B cell area) and purified CD103+DCs produced large amounts of IL-12p40 compared with CD103-DCs. These data suggest that splenic CD103+DCs may play a critical role in the development of CTL responses during influenza virus infection. We are currently investigating how efficiently splenic CD103+DCs act as antigen presenting cells.

Fine-Tuned Expression of Programmed Death 1 Ligands in Mature Dendritic Cells Stimulated by CD40 Ligand Is Critical for the Induction of an Efficient Tumor Specific Immune Response

During maturation, murine myeloid dendritic cells (DCs) upregulated the expressions of CD11c, CD25, CD40, CD80, CD86, MHC II and programmed death 1 ligands 1 and 2 (PD-L1 and PD-L2). Differential expression patterns of PD-L1 and PD-L2 were found when DCs were triggered by CD40 ligand and TNF-alpha. PD-L1 expression was repressed and PD-L2 expression remained unchanged in mature CD40-ligated DCs, whereas TNF-alpha stimulated DCs kept high expression of PD-L1 and significantly enhanced PD-L2 expression on DCs. Proliferations of T lymphocytes stimulated by immature DCs were enhanced by blockade of the PD-1 and PD-1 ligand interaction. But inhibitive effects were found in T lymphocytes stimulated by CD40-ligated DCs. With the fine-tuned expressions of PD-L1 and PD-L2, CD40-ligated DCs could sustain a longer activation period and elicit a more efficient T lymphocyte activation.

Regulation of PD-1, PD-L1, and PD-L2 expression during normal and autoimmune responses.

Newer members of the B7-CD28 superfamily include the receptor PD-1 and its two ligands, PD-L1 and PD-L2. Here, we characterize the expression of PD-1, PD-L1, and PD-L2 in tissues of naive miceand in target organs from two models of autoimmunity, the pancreas from non-obese diabetic (NOD) mice and brain from mice with experimental autoimmune encephalomyelitis (EAE). In naive mice, proteiexpression of PD-1, PD-L1, and PD-L2 was detected in the thymus, while PD-1 and PD-L1 were detected in the spleen. PD-L1, but not PD-L2, was also detected at low levels on cardiac endothelium, pancreatic islets, and syncyciotrophoblasts in the placenta. In pre-diabetic NOD mice, PD-1 and PD-L1 were expressed on infiltrating cells in the pancreatic islets. Furthermore, PD-L1 was markedly up-regulated on islet cells. In brains from mice with EAE, PD-1, PD-L1, and PD-L2 were expressed on infiltrating inflammatory cells, and PD-L1 was up-regulated on endothelium within EAE brain. The distinct expression patterns of PD-L1 and PD-L2 led us to compare their transcriptional regulation in STAT4(-/-), STAT6(-/-), or NF-kappaB p50(-/-)p65(+/-) dendritic cells (DC).PD-L2, but not PD-L1, expression was dramatically reduced in p50(-/-)p65(+/-) DC. Thus, PD-L1 and PD-L2 exhibit distinct expression patterns and are differentially regulated on the transcriptional level.

Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues

PD-1 is a member of the immunoglobulin superfamily expressed on immune cells, including T and B cells, and is involved in the delivery of inhibitory signal upon engagement of its ligands, PD-L1 and PD-L2. While the expression profile of PD-1 has been well documented, the analysis of PD-L1 and PD-L2 distributions on a protein basis has not been carried out because of the lack of available monoclonal antibodies specific for the molecules. In this study, we established two monoclonal antibodies, 1-111A and 122, specific for murine PD-L1 and PD-L2, respectively, and examined their expression profiles. Based on flow cytometric analyses, the expression of PD-L1 was detected in a variety of lymphohematopoietic cell types, including a minor proportion of T and B cells in the spleen, majority of pre-B cells and myeloid cells in bone marrow and subsets of thymocytes, while the expression of PD-L2 was not observed in the lymphohematopoietic cells at all. Notably, a significant proportion of the most immature lineage-marker-negative and c-Kit-positive bone marrow cells containing stem cells did express PD-L1. Following mitogenic stimulation, essentially all lymphocytes expressed PD-L1. Furthermore, a variety of leukemic lines also expressed PD-L1, while none of them did PD-L2. Thus, present results demonstrate the distinct expression patterns of PD-L1 and PD-L2 with the cells of lymphohematopoietic tissues exclusively expressing the former.