Journal Articles

Experimental Immunotherapy

Preclinical and clinical cancer immunotherapy approaches

B-cell receptor-mediated NFATc1 activation induces IL-10/STAT3/PD-L1 signaling in diffuse large B-cell lymphoma

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Knowledge of PD-L1 expression and its regulation in B-cell lymphoma cells is limited. Investigating mechanisms that control PD-L1 expression in B-cell lymphoma cells might identify biomarkers that predict the efficacy of immunotherapy with anti-PD-1/PD-L1 antibodies. In addition, identification of mechanisms that regulate PD-L1 may also identify molecules that can be targeted to improve the clinical efficacy of immune checkpoint inhibitors. In this study, we used proteomic approaches and patient-derived B-cell lymphoma cell lines to investigate mechanisms that regulate PD-L1 expression. We found that PD-L1 expression, particularly in non-germinal center B cell-derived diffuse large B-cell lymphoma (DLBCL), is controlled and regulated by several interactive signaling pathways, including the B-cell receptor (BCR) and JAK2/STAT3 signaling pathways. We found in PD-L1-positive B-cell lymphoma cells that BCR-mediated NFATc1 activation up-regulates IL-10 chemokine expression. Released IL-10 activates the JAK2/STAT3 pathway, leading to STAT3-induced PD-L1 expression. IL-10 antagonist antibody abrogates IL-10/STAT3 signaling and PD-L1 protein expression. We also found that BCR pathway inhibition by BTK inhibitors (ibrutinib, acalabrutinib, and BGB-3111) blocks both NFATc1 and STAT3 activation, thereby inhibiting IL-10 and PD-L1 expression. Finally, we validated the PD-L1 signaling network in two primary DLBCL cohorts, consisting of 428 and 350 cases and showed significant correlations between IL-10, STAT3, and PD-L1. Thus, our findings reveal a complex signaling network regulating PD-L1 expression in B-cell lymphoma cells and suggest that PD-L1 expression can be modulated by small molecule inhibitors to potentiate immunotherapies.

Author Info: (1) Department of Hematology, The Second Hospital of Dalian Medical University, Dalian, China. (2) Department of Hematopathology, The University of Texas MD Anderson Cancer Center

Author Info: (1) Department of Hematology, The Second Hospital of Dalian Medical University, Dalian, China. (2) Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States. (3) Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States. (4) Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States. (5) Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China. (6) Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States. (7) Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States. (8) Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States. (9) Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States. (10) Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX, United States. (11) Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States. (12) Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States; lvpham@mdanderson.org.

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Antibody-Neutralized Reovirus Is Effective in Oncolytic Virotherapy

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Immunotherapy is showing promise for otherwise incurable cancers. Oncolytic viruses (OVs), developed as direct cytotoxic agents, mediate their antitumor effects via activation of the immune system. However, OVs also stimulate antiviral immune responses, including the induction of OV-neutralizing antibodies. Current dogma suggests that the presence of preexisting antiviral neutralizing antibodies in patients, or their development during viral therapy, is a barrier to systemic OV delivery, rendering repeat systemic treatments ineffective. However, we have found that human monocytes loaded with preformed reovirus-antibody complexes, in which the reovirus is fully neutralized, deliver functional replicative reovirus to tumor cells, resulting in tumor cell infection and lysis. This delivery mechanism is mediated, at least in part, by antibody receptors (in particular FcgammaRIII) that mediate uptake and internalization of the reovirus/antibody complexes by the monocytes. This finding has implications for oncolytic virotherapy and for the design of clinical OV treatment strategies. Cancer Immunol Res; 1-13. (c)2018 AACR.

Author Info: (1) Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom. (2) Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United

Author Info: (1) Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom. (2) Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom. (3) Leiden University Medical Centre, Department of Molecular Cell Biology, Leiden, the Netherlands. (4) Leiden University Medical Centre, Department of Molecular Cell Biology, Leiden, the Netherlands. (5) Department of Immunology, Mayo Clinic, Rochester, Minnesota. (6) Department of Immunology, Mayo Clinic, Rochester, Minnesota. (7) Oncolytics Biotech Incorporated, Calgary, Alberta, Canada. (8) Leiden University Medical Centre, Department of Molecular Cell Biology, Leiden, the Netherlands. (9) Department of Immunology, Mayo Clinic, Rochester, Minnesota. (10) Institute of Cancer Research, London, United Kingdom. (11) Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom. e.ilett@leeds.ac.uk.

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CAR-T Cells Based on Novel BCMA Monoclonal Antibody Block Multiple Myeloma Cell Growth

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The cell-surface protein B cell maturation antigen (BCMA, CD269) has emerged as a promising target for CAR-T cell therapy for multiple myeloma. In order to create a novel BCMA CAR, we generated a new BCMA monoclonal antibody, clone 4C8A. This antibody exhibited strong and selective binding to human BCMA. BCMA CAR-T cells containing the 4C8A scFv were readily detected with recombinant BCMA protein by flow cytometry. The cells were cytolytic for RPMI8226, H929, and MM1S multiple myeloma cells and secreted high levels of IFN-gamma in vitro. BCMA-dependent cytotoxicity and IFN-gamma secretion were also observed in response to CHO (Chinese Hamster Ovary)-BCMA cells but not to parental CHO cells. In a mouse subcutaneous tumor model, BCMA CAR-T cells significantly blocked RPMI8226 tumor formation. When BCMA CAR-T cells were given to mice with established RPMI8226 tumors, the tumors experienced significant shrinkage due to CAR-T cell activity and tumor cell apoptosis. The same effect was observed with 3 humanized BCMA-CAR-T cells in vivo. These data indicate that novel CAR-T cells utilizing the BCMA 4C8A scFv are effective against multiple myeloma and warrant future clinical development.

Author Info: (1) ProMab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, USA. robert.berahovich@promab.com. (2) ProMab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, USA. huazhou369@gmail.com. (3) ProMab Biotechnologies

Author Info: (1) ProMab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, USA. robert.berahovich@promab.com. (2) ProMab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, USA. huazhou369@gmail.com. (3) ProMab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, USA. shirley.xu@promab.com. (4) ProMab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, USA. yuehua.wei@promab.com. (5) ProMab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, USA. jasper.guan@promab.com. (6) ProMab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, USA. jian.guan@promab.com. (7) ProMab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, USA. hizkia.harto@promab.com. (8) Forevertek Biotechnology Co., Ltd., Building M0, Oversea Graduate Park National High-Tech Industrial Zone, Changsha 410003, China. promab8807@126.com. (9) Forevertek Biotechnology Co., Ltd., Building M0, Oversea Graduate Park National High-Tech Industrial Zone, Changsha 410003, China. yangkaihuai520@126.com. (10) Forevertek Biotechnology Co., Ltd., Building M0, Oversea Graduate Park National High-Tech Industrial Zone, Changsha 410003, China. shuying256@163.com. (11) ProMab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, USA. simon.li@promab.com. Forevertek Biotechnology Co., Ltd., Building M0, Oversea Graduate Park National High-Tech Industrial Zone, Changsha 410003, China. simon.li@promab.com. (12) ProMab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, USA. john@promab.com. (13) ProMab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, USA. vita.gol@promab.com.

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Natural killer cells and other innate lymphoid cells in cancer

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Immuno-oncology is an emerging field that has revolutionized cancer treatment. Most immunomodulatory strategies focus on enhancing T cell responses, but there has been a recent surge of interest in harnessing the relatively underexplored natural killer (NK) cell compartment for therapeutic interventions. NK cells show cytotoxic activity against diverse tumour cell types, and some of the clinical approaches originally developed to increase T cell cytotoxicity may also activate NK cells. Moreover, increasing numbers of studies have identified novel methods for increasing NK cell antitumour immunity and expanding NK cell populations ex vivo, thereby paving the way for a new generation of anticancer immunotherapies. The role of other innate lymphoid cells (group 1 innate lymphoid cell (ILC1), ILC2 and ILC3 subsets) in tumours is also being actively explored. This Review provides an overview of the field and summarizes current immunotherapeutic approaches for solid tumours and haematological malignancies.

Author Info: (1) Innate Pharma Research Labs, Innate Pharma, Marseille, France. Aix Marseille University, CNRS, INSERM, CIML, Marseille, France. (2) Aix Marseille University, CNRS, INSERM, CIML, Marseille

Author Info: (1) Innate Pharma Research Labs, Innate Pharma, Marseille, France. Aix Marseille University, CNRS, INSERM, CIML, Marseille, France. (2) Aix Marseille University, CNRS, INSERM, CIML, Marseille, France. CHU Bordeaux, Service d'Hematologie Clinique et Therapie Cellulaire, F-33000, Bordeaux, France. (3) Aix Marseille University, CNRS, INSERM, CIML, Marseille, France. (4) Innate Pharma Research Labs, Innate Pharma, Marseille, France. vivier@ciml.univ-mrs.fr. Aix Marseille University, CNRS, INSERM, CIML, Marseille, France. vivier@ciml.univ-mrs.fr. Service d'Immunologie, Marseille Immunopole, Hopital de la Timone, Assistance Publique-Hopitaux de Marseille, Marseille, France. vivier@ciml.univ-mrs.fr.

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Chemotherapy Combines Effectively with Anti-PD-L1 Treatment and Can Augment Antitumor Responses

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Immunotherapy with checkpoint inhibitors has proved to be highly effective, with durable responses in a subset of patients. Given their encouraging clinical activity, checkpoint inhibitors are increasingly being tested in clinical trials in combination with chemotherapy. In many instances, there is little understanding of how chemotherapy might influence the quality of the immune response generated by checkpoint inhibitors. In this study, we evaluated the impact of chemotherapy alone or in combination with anti-PD-L1 in a responsive syngeneic tumor model. Although multiple classes of chemotherapy treatment reduced immune cell numbers and activity in peripheral tissues, chemotherapy did not antagonize but in many cases augmented the antitumor activity mediated by anti-PD-L1. This dichotomy between the detrimental effects in peripheral tissues and enhanced antitumor activity was largely explained by the reduced dependence on incoming cells for antitumor efficacy in already established tumors. The effects of the various chemotherapies were also agent specific, and synergy with anti-PD-L1 was achieved by different mechanisms that ultimately helped establish a new threshold for response. These results rationalize the combination of chemotherapy with immunotherapy and suggest that, despite the negative systemic effects of chemotherapy, effective combinations can be obtained through distinct mechanisms acting within the tumor.

Author Info: (1) Genentech, South San Francisco, CA 94080; and cubasr@gene.com. (2) Genentech, South San Francisco, CA 94080; and. (3) Genentech, South San Francisco, CA 94080; and

Author Info: (1) Genentech, South San Francisco, CA 94080; and cubasr@gene.com. (2) Genentech, South San Francisco, CA 94080; and. (3) Genentech, South San Francisco, CA 94080; and. (4) Genentech, South San Francisco, CA 94080; and. (5) Genentech, South San Francisco, CA 94080; and. (6) Genentech, South San Francisco, CA 94080; and. (7) Roche Pharmaceutical Research and Early Development, Roche Innovation Center Munich, 82377 Penzberg, Germany. (8) Roche Pharmaceutical Research and Early Development, Roche Innovation Center Munich, 82377 Penzberg, Germany. (9) Genentech, South San Francisco, CA 94080; and. (10) Genentech, South San Francisco, CA 94080; and.

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Combined analysis of antigen presentation and T cell recognition reveals restricted immune responses in melanoma

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The quest for tumor-associated-antigens (TAAs) and neo-antigens is a major focus of cancer immunotherapy. Here we combine a neo-antigen prediction-pipeline and human-leukocyte-antigen (HLA)-peptidomics to identify TAAs and neo-antigens in 16 tumors derived from seven melanoma patients, and characterize their interactions with their TILs. Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neo-antigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell co-signatures, allowing us to discover remarkable antigenic and TIL similarities between metastases from the same patient. Furthermore, we reveal that two neo-antigen-specific clonotypes killed 90% of autologous melanoma cells, both in vitro and in vivo, showing that a limited set of neo-antigen-specific T-cells may play a central role in melanoma tumor rejection. Our findings indicate that combining HLA-peptidomics with neo-antigen predictions allows robust identification of targetable neo-antigens, which could successfully guide personalized cancer-immunotherapies.

Author Info: (1) Department of Molecular Cell Biology, Weizmann Institute of Science Yardena.Samuels@weizmann.ac.il. (2) Department of Molecular Cell Biology, Weizmann Institute of Science. (3) Department of Molecular

Author Info: (1) Department of Molecular Cell Biology, Weizmann Institute of Science Yardena.Samuels@weizmann.ac.il. (2) Department of Molecular Cell Biology, Weizmann Institute of Science. (3) Department of Molecular Cell Biology, Weizmann Institute of Science. (4) Department of Immunology, Weizmann Institute of Science. (5) Biology, Technion - Israel Intitute of Technology. (6) Department of Immunology, Weizmann Institute of Science. (7) Department of Immunology, Weizmann Institute of Science. (8) Department of Molecular Cell Biology, Weizmann Institute of Science. (9) Surgical Oncology and Genomic Medicine, University of Texas MD Anderson Cancer Center. (10) Center for Bioinformatics and Computational Biology, University of Maryland, College Park. (11) Department of Molecular Cell Biology, Weizmann Institute of Science. (12) TCR Discovery, BioNTech Cell & Gene Therapies GmbH. (13) TCR Discovery, BioNTech Cell & Gene Therapies GmbH. (14) Molecular Cell Biology, The Weizmann Institute of Science. (15) Department of Life Sciences Core Facilities, Weizmann Institute of Science. (16) Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center. (17) Genomic Medicine, University of Texas MD Anderson Cancer Center. (18) Genomic Medicine, University of Texas MD Anderson Cancer Center. (19) Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center. (20) Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center. (21) Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center. (22) Department of Systems Biology, University of Texas MD Anderson Cancer Center. (23) Department of Molecular Cell Biology, Weizmann Institute of Science. (24) Department of Diagnostic Radiology, The University of Texas MD Anderson Cancer Center. (25) Translational Medicine, MedImmune. (26) Surgery Branch, National Cancer Institute. (27) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital. (28) Immunotherapy, TRON gGmbH. (29) Department of Immunology, weizmann INstitute. (30) CCR, NCI, NIH. (31) Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center. (32) Department of Immunology, Weizmann Institute of Science. (33) Biology, Technion - Israel Intitute of Technology.

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Stromal Fibroblasts Mediate Anti-PD-1 Resistance via MMP-9 and Dictate TGF-beta Inhibitor Sequencing in Melanoma

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Although anti-PD-1 therapy has improved clinical outcomes for select patients with advanced cancer, many patients exhibit either primary or adaptive resistance to checkpoint inhibitor immunotherapy. The role of the tumor stroma in the development of these mechanisms of resistance to checkpoint inhibitors remains unclear. We demonstrated that pharmacological inhibition of the TGF-beta signaling pathway synergistically enhanced the efficacy of anti-CTLA-4 immunotherapy but failed to augment anti-PD-1/PD-L1 responses in an autochthonous model of BRAF(V600E) melanoma. Additional mechanistic studies revealed that TGF-beta pathway inhibition promoted the proliferative expansion of stromal fibroblasts, thereby, facilitating MMP-9-dependent cleavage of PD-L1 surface expression, leading to anti-PD-1 resistance in this model. Further work demonstrated that melanomas escaping anti-PD-1 therapy exhibited a mesenchymal phenotype associated with enhanced TGF-beta signaling activity. Delayed TGF-beta inhibitor therapy, following anti-PD-1 escape, better served to control further disease progression and was superior to a continuous combination of anti-PD-1 and TGF-beta inhibition. This work illustrates that formulating immunotherapy combination regimens to enhance the efficacy of checkpoint blockade requires an in-depth understanding of the impact of these agents on the tumor microenvironment. These data indicated that stromal fibroblast MMP-9 may desensitize tumors to anti-PD-1 and suggests that TGF-beta inhibition may generate greater immunologic efficacy when administered following the development of acquired anti-PD-1 resistance.

Author Info: (1) NIEHS/IIDL, NIH. (2) Internal Medicine/Medical Oncology, Duke University Medical Center. (3) Internal Medicine/Medical Oncology, Duke University Medical Center. (4) Medicine, Duke University Medical Center

Author Info: (1) NIEHS/IIDL, NIH. (2) Internal Medicine/Medical Oncology, Duke University Medical Center. (3) Internal Medicine/Medical Oncology, Duke University Medical Center. (4) Medicine, Duke University Medical Center. (5) Internal Medicine/Medical Oncology, Duke University Medical Center. (6) Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill. (7) Internal Medicine III, University Hospital Regensburg. (8) Pharmacology & Cancer Biology, Duke University Medical Center. (9) Internal Medicine/Medical Oncology and Pharmacology/Cancer Biology, Duke University Medical Center hanks004@mc.duke.edu.

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Successful treatment with intralesional talimogene laherparepvec in two patients with immune checkpoint inhibitors refractory advanced melanoma

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Monoclonal antibodies that block the programmed death-1 (anti-PD-1) or cytotoxic T-lymphocyte antigen-4 (CTLA-4) immune checkpoint receptors (pembrolizumab, nivolumab, ipilimumab, or the combination of nivolumab with ipilimumab) are approved treatment option for patients with advanced melanoma. Over half of all patients are refractory to these immunotherapies and are in need of alternative or complementary treatment options. Talimogene laherparepvec (T-VEC) is a first-in-class intralesionally delivered oncolytic immunotherapy, which has proven efficacy in the treatment of advanced melanoma. A proportion of patients treated with T-VEC will benefit from an abscopal response of noninjected metastases indicative of a systemic antitumor immune response elicited by the intratumoral injections. At present it remains unknown whether the systemic antitumor responses elicited by T-VEC are nonredundant with immune-checkpoint blockade. Recent data on potential synergy between T-VEC and both PD-1 and CTLA-4 blockade suggest that the mechanism of action may be complementary. We report on the successful treatment with intralesional T-VEC of two female patients with locoregionally advanced BRAF V600 wild-type melanoma who previously progressed on anti-PD-1 and anti-CTLA-4 inhibitors.

Author Info: (1) Department of Medical Oncology. Department of Dermatology. (2) Department of Medical Oncology. (3) Department of Medical Oncology. (4) Department of Medical Oncology. (5) Department

Author Info: (1) Department of Medical Oncology. Department of Dermatology. (2) Department of Medical Oncology. (3) Department of Medical Oncology. (4) Department of Medical Oncology. (5) Department of Medical Oncology. (6) Department of Radiotherapy, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB). (7) Department of Nuclear Medicine, Hopital Erasme, Universite Libre de Bruxelles (ULB), Brussels, Belgium. (8) Department of Dermatology. (9) Department of Dermatology. (10) Department of Medical Oncology.

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Co-inhibition of TIGIT, PD1, and Tim3 reverses dysfunction of Wilms tumor protein-1 (WT1)-specific CD8+ T lymphocytes after dendritic cell vaccination in gastric cancer

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Dendritic cell (DC) vaccines have been shown to stimulate tumor antigen-specific CD8+ T cells; however, this strategy has demonstrated variable clinical efficacy likely due to immune escape mechanisms that can induce tumor-specific CD8+ T cell dysfunction. Herein, we evaluated the functional characteristics of DC vaccine-induced CD8+ T cells with regard to immune checkpoint inhibitors in gastric cancer patients who were administered Wilms tumor protein-1 (WT1)-targeted DC vaccine. We observed the upregulation of the inhibitory molecule, TIGIT and the inhibitory T cell co-receptors PD1 and Tim3 in limiting WT1-specific CD8+ T cell growth and function in GC patients. TIGIT-expressing PD1+Tim3- CD8+ T cells were the largest subset, while TIGIT+PD1+Tim3+ was the most dysfunctional subset of WT1-specific CD8+ T cells in gastric cancer patients. Importantly, the co-inhibition of TIGIT, PD1, and Tim3 pathways enhanced the growth, proliferation, and cytokine production of WT1-specific CD8+ T cells. In conclusion, our data suggests that targeting TIGIT, PD1, and Tim3 pathways may be important in reversing immune escape in patients with advanced gastric cancer.

Author Info: (1) Department of Oncology, Beijing Biohealthcare Biotechnology Co., Ltd China. (2) Department of Oncology, Beijing Biohealthcare Biotechnology Co., Ltd China. (3) Department of Gastroenterology, Beijing

Author Info: (1) Department of Oncology, Beijing Biohealthcare Biotechnology Co., Ltd China. (2) Department of Oncology, Beijing Biohealthcare Biotechnology Co., Ltd China. (3) Department of Gastroenterology, Beijing Tiantan Hospital, Capital Medical University China. (4) Key Laboratory of Digestive System Tumors, Second Hospital of Lanzhou University China. (5) Department of Oncology, Beijing Anzhen Hospital Affiliated to The Capital Medical University, Beijing Institute of Heart Lung and Blood Vessel Diseases China. (6) Department of Biotherapy Center, Gansu Provincial Hospital China. (7) Department of Hemotology, Gansu Provincial Hospital China. (8) Department of Biochemistry and Molecular Biology, Hainan Medical College China. (9) Department of Oncology, Beijing Biohealthcare Biotechnology Co., Ltd China. (10) Department of Oncology, Beijing Biohealthcare Biotechnology Co., Ltd China. (11) Department of Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College Beijing, China.

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Oncolytic Newcastle disease virus induces autophagy-dependent immunogenic cell death in lung cancer cells

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In addition to direct oncolysis, oncolytic viruses trigger immunogenic cell death (ICD) and primes antitumor immunity. We have previously shown that oncolytic Newcastle disease virus (NDV), strain FMW (NDV/FMW), induces apoptosis and/or autophagy in cancer cells. In this study, we investigated whether oncolytic NDV can induce ICD in lung cancer cells and whether apoptosis or autophagy plays a role in NDV-triggered ICD. To this end, we examined cell surface expression of calreticulin (CRT) on NDV-infected lung cancer cells and measured ICD determinants, high mobility group box 1 (HMGB1), heat shock protein 70/90 (HSP70/90) and ATP in supernatants following viral infection. Flow cytometric analysis using anti-CRT antibody and PI staining of NDV-infected lung cancer cells showed an increase in the number of viable (propidium iodide-negative) cells, suggesting the induction of CRT exposure upon NDV infection. In addition, confocal and immunoblot analysis using anti-CRT antibody showed that an enhanced accumulation of CRT on the cell surface of NDV-infected cells, indicating the translocation of CRT to the cell membrane upon NDV infection. We further demonstrated that NDV infection induced the release of secreted HMGB1 and HSP70/90 by examining the concentrated supernatants of NDV-infected cells. Furthermore, pre-treatment with either the pan-caspase inhibitor z-VAD-FMK or the necrosis inhibitor Necrostain-1, had no impact on NDV-induced release of ICD determinants in lung cancer cells. Rather, depletion of autophagy-related genes in lung cancer cells significantly inhibited the induction of ICD determinants by NDV. Of translational importance, in a lung cancer xenograft model, treatment of mice with supernatants from NDV-infected cells significantly inhibited tumour growth. Together, these results indicate that oncolytic NDV is a potent ICD-inducer and that autophagy contributes to NDV-mediated induction of ICD in lung cancer cells.

Author Info: (1) Department of Neurosurgery, Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute Shenyang, China. Central Laboratory, Cancer Hospital of China Medical University

Author Info: (1) Department of Neurosurgery, Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute Shenyang, China. Central Laboratory, Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute Shenyang, China. Institute of Cancer Stem Cell, Dalian Medical University Dalian, China. (2) Institute of Cancer Stem Cell, Dalian Medical University Dalian, China. (3) Department of Neurosurgery, Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute Shenyang, China. (4) Thoracic Oncology Research Group, Trinity Translational Medicine Institute, Trinity Centre for Health Sciences St. James's Hospital & Trinity College Dublin Dublin, Ireland. (5) Department of Oncology, Shanghai Tenth People's Hospital, Tongji University Shanghai, China. Tongji University Cancer Center Shanghai, China. Department of Oncology, Dermatology Hospital, Tongji University Shanghai, China. (6) Central Laboratory, Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute Shenyang, China. (7) Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science Shanghai, China. (8) Institute of Cancer Stem Cell, Dalian Medical University Dalian, China. (9) Department of Neurosurgery, Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute Shenyang, China.

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