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Primary drug resistance and minimal residual disease are major challenges in the treatment of B cell neoplasms. Therefore, this study aimed to identify a novel treatment capable of eradicating malignant B cells and drug-resistant disease. Oncolytic viruses eradicate malignant cells by direct oncolysis and activation of anti-tumor immunity, have proven anti-cancer efficacy, and are safe and well tolerated in clinical use. Here, we demonstrate that the oncolytic virus coxsackievirus A21 can kill a range of B cell neoplasms, irrespective of an anti-viral interferon response. Moreover, CVA21 retained its capacity to kill drug-resistant B cell neoplasms, where drug resistance was induced by co-culture with tumor microenvironment support. In some cases, CVA21 efficacy was actually enhanced, in accordance with increased expression of the viral entry receptor ICAM-1. Importantly, the data confirmed preferential killing of malignant B cells and CVA21 dependence on oncogenic B cell signaling pathways. Significantly, CVA21 also activated natural killer (NK) cells to kill neoplastic B cells and drug-resistant B cells remained susceptible to NK cell-mediated lysis. Overall, these data reveal a dual mode of action of CVA21 against drug-resistant B cells and support the development of CVA21 for the treatment of B cell neoplasms.
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Oncolytic viruses (OVs) rapidly and specifically replicate in and kill tumor cells. OV-targeted infection of malignant cells has the potential to create an "inflammatory storm" that stimulates both innate and adaptive anti-tumor immune responses. The generation of anti-tumor immunity following OV treatment has been shown to be crucial for effective therapy. Therefore, establishing methodologies to measure the generation of anti-tumor T cell responses following OV infection in in vitro assays, which better mimic the complexity of the human tumor microenvironment (TME), will be critical to harness the full potential of OV therapy. Such experimental platforms will accelerate the development of next-generation OVs that are capable of overcoming immunosuppressive networks found within the tumor microenvironment. Here we describe a method that was designed to test the generation and quantification of human tumor-specific T cells following OV infection of 3D tumor spheroids cultured with or without fibroblasts.
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Antineoplásicos , Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos , Virosis , Humanos , Viroterapia Oncolítica/métodos , Neoplasias/terapia , Linfocitos T , Microambiente Tumoral , Virosis/terapiaRESUMEN
Improving the chances of curing patients with cancer who have had surgery to remove metastatic sites of disease is a priority area for cancer research. Pexa-Vec (Pexastimogene Devacirepvec; JX-594, TG6006) is a principally immunotherapeutic oncolytic virus that has reached late-phase clinical trials. We report the results of a single-center, nonrandomized biological end point study (trial registration: EudraCT number 2012-000704-15), which builds on the success of the presurgical intravenous delivery of oncolytic viruses to tumors. Nine patients with either colorectal cancer liver metastases or metastatic melanoma were treated with a single intravenous infusion of Pexa-Vec ahead of planned surgical resection of the metastases. Grade 3 and 4 Pexa-Vec-associated side effects were lymphopaenia and neutropaenia. Pexa-Vec was peripherally carried in plasma and was not associated with peripheral blood mononuclear cells. Upon surgical resection, Pexa-Vec was found in the majority of analyzed tumors. Pexa-Vec therapy associated with IFNα secretion, chemokine induction, and resulted in transient innate and long-lived adaptive anticancer immunity. In the 2 patients with significant and complete tumor necrosis, a reduction in the peripheral T-cell receptor diversity was observed at the time of surgery. These results support the development of presurgical oncolytic vaccinia virus-based therapies to stimulate anticancer immunity and increase the chances to cure patients with cancer.
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Neoplasias Hepáticas , Viroterapia Oncolítica , Virus Oncolíticos , Humanos , Leucocitos Mononucleares , Neoplasias Hepáticas/terapia , Terapia Neoadyuvante , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Virus Vaccinia/genéticaRESUMEN
Recent advances in cancer therapeutics clearly demonstrate the need for innovative multiplex therapies that attack the tumour on multiple fronts. Oncolytic or "cancer-killing" viruses (OVs) represent up-and-coming multi-mechanistic immunotherapeutic drugs for the treatment of cancer. In this study, we perform an in-vitro screen based on virus-encoded artificial microRNAs (amiRNAs) and find that a unique amiRNA, herein termed amiR-4, confers a replicative advantage to the VSVΔ51 OV platform. Target validation of amiR-4 reveals ARID1A, a protein involved in chromatin remodelling, as an important player in resistance to OV replication. Virus-directed targeting of ARID1A coupled with small-molecule inhibition of the methyltransferase EZH2 leads to the synthetic lethal killing of both infected and uninfected tumour cells. The bystander killing of uninfected cells is mediated by intercellular transfer of extracellular vesicles carrying amiR-4 cargo. Altogether, our findings establish that OVs can serve as replicating vehicles for amiRNA therapeutics with the potential for combination with small molecule and immune checkpoint inhibitor therapy.
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Vesículas Extracelulares , MicroARNs , Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos , Humanos , MicroARNs/genética , Neoplasias/terapia , Virus Oncolíticos/genéticaRESUMEN
In our clinical trials of oncolytic vesicular stomatitis virus expressing interferon beta (VSV-IFNß), several patients achieved initial responses followed by aggressive relapse. We show here that VSV-IFNß-escape tumors predictably express a point-mutated CSDE1P5S form of the RNA-binding Cold Shock Domain-containing E1 protein, which promotes escape as an inhibitor of VSV replication by disrupting viral transcription. Given time, VSV-IFNß evolves a compensatory mutation in the P/M Inter-Genic Region which rescues replication in CSDE1P5S cells. These data show that CSDE1 is a major cellular co-factor for VSV replication. However, CSDE1P5S also generates a neo-epitope recognized by non-tolerized T cells. We exploit this predictable neo-antigenesis to drive, and trap, tumors into an escape phenotype, which can be ambushed by vaccination against CSDE1P5S, preventing tumor escape. Combining frontline therapy with escape-targeting immunotherapy will be applicable across multiple therapies which drive tumor mutation/evolution and simultaneously generate novel, targetable immunopeptidomes associated with acquired treatment resistance.
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Proteínas de Unión al ADN/inmunología , Interferón beta/inmunología , Viroterapia Oncolítica/métodos , Virus Oncolíticos/inmunología , Proteínas de Unión al ARN/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Replicación Viral/inmunología , Animales , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Inmunoterapia/métodos , Interferón beta/metabolismo , Ratones Endogámicos C57BL , Mutación , Virus Oncolíticos/metabolismo , Virus Oncolíticos/fisiología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Virus de la Estomatitis Vesicular Indiana/metabolismo , Virus de la Estomatitis Vesicular Indiana/fisiologíaRESUMEN
Cancer somatic mutations have been identified as a source of antigens that can be targeted by cancer immunotherapy. In this work, expanding on previous studies, we analyze the HLA-presentation properties of mutations that are known to drive resistance to cancer targeted-therapies. We survey a large dataset of mutations that confer resistance to different drugs and occur in numerous genes and tumor types. We show that a significant number of them are predicted in silico to be potentially immunogenic across a large proportion of the human population. Further, by analyzing a cohort of patients carrying a small subset of these resistance mutations, we provide evidence that what is observed in the general population may be indicative of the mutations' immunogenic potential in resistant patients. Two of the mutations in our dataset had previously been experimentally validated by others and it was confirmed that some of their associated neopeptides elicit T-cell responses in vitro. The identification of potent cancer-specific antigens can be instrumental for developing more effective immunotherapies. In this work, we propose a novel list of drug-resistance mutations, several of which are recurrent, that could be of particular interest in the context of off-the-shelf precision immunotherapies such as therapeutic cancer vaccines.
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Simulación por Computador , Resistencia a Antineoplásicos/inmunología , Mutación , Neoplasias , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Humanos , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/terapia , Medicina de Precisión , Linfocitos T/inmunologíaRESUMEN
Vaccinia virus (VACV) possesses a great safety record as a smallpox vaccine and has been intensively used as an oncolytic virus against various types of cancer over the past decade. Different strategies were developed to make VACV safe and selective to cancer cells. Leading clinical candidates, such as Pexa-Vec, are attenuated through deletion of the viral thymidine kinase (TK) gene, which limits virus growth to replicate in cancer tissue. However, tumors are not the only tissues whose metabolic activity can overcome the lack of viral TK. In this study, we sought to further increase the tumor-specific replication and oncolytic potential of Copenhagen strain VACV ΔTK. We show that deletion of the anti-apoptosis viral gene F1L not only increases the safety of the Copenhagen ΔTK virus but also improves its oncolytic activity in an aggressive glioblastoma model. The additional loss of F1L does not affect VACV replication capacity, yet its ability to induce cancer cell death is significantly increased. Our results also indicate that cell death induced by the Copenhagen ΔTK/F1L mutant releases more immunogenic signals, as indicated by increased levels of IL-1ß production. A cytotoxicity screen in an NCI-60 panel shows that the ΔTK/F1L virus induces faster tumor cell death in different cancer types. Most importantly, we show that, compared to the TK-deleted virus, the ΔTK/F1L virus is attenuated in human normal cells and causes fewer pox lesions in murine models. Collectively, our findings describe a new oncolytic vaccinia deletion strain that improves safety and increases tumor cell killing.
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BACKGROUND: The oncolytic virus, coxsackievirus A21 (CVA21), has shown promise as a single agent in several clinical trials and is now being tested in combination with immune checkpoint blockade. Combination therapies offer the best chance of disease control; however, the design of successful combination strategies requires a deeper understanding of the mechanisms underpinning CVA21 efficacy, in particular, the role of CVA21 anti-tumor immunity. Therefore, this study aimed to examine the ability of CVA21 to induce human anti-tumor immunity, and identify the cellular mechanism responsible. METHODS: This study utilized peripheral blood mononuclear cells from i) healthy donors, ii) Acute Myeloid Leukemia (AML) patients, and iii) patients taking part in the STORM clinical trial, who received intravenous CVA21; patients receiving intravenous CVA21 were consented separately in accordance with local institutional ethics review and approval. Collectively, these blood samples were used to characterize the development of innate and adaptive anti-tumor immune responses following CVA21 treatment. RESULTS: An Initial characterization of peripheral blood mononuclear cells, collected from cancer patients following intravenous infusion of CVA21, confirmed that CVA21 activated immune effector cells in patients. Next, using hematological disease models which were sensitive (Multiple Myeloma; MM) or resistant (AML) to CVA21-direct oncolysis, we demonstrated that CVA21 stimulated potent anti-tumor immune responses, including: 1) cytokine-mediated bystander killing; 2) enhanced natural killer cell-mediated cellular cytotoxicity; and 3) priming of tumor-specific cytotoxic T lymphocytes, with specificity towards known tumor-associated antigens. Importantly, immune-mediated killing of both MM and AML, despite AML cells being resistant to CVA21-direct oncolysis, was observed. Upon further examination of the cellular mechanisms responsible for CVA21-induced anti-tumor immunity we have identified the importance of type I IFN for NK cell activation, and demonstrated that both ICAM-1 and plasmacytoid dendritic cells were key mediators of this response. CONCLUSION: This work supports the development of CVA21 as an immunotherapeutic agent for the treatment of both AML and MM. Additionally, the data presented provides an important insight into the mechanisms of CVA21-mediated immunotherapy to aid the development of clinical biomarkers to predict response and rationalize future drug combinations.
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Enterovirus , Leucemia Mieloide Aguda/terapia , Viroterapia Oncolítica , Virus Oncolíticos , Inmunidad Adaptativa , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Células Dendríticas/inmunología , Femenino , Humanos , Inmunidad Innata , Molécula 1 de Adhesión Intercelular/inmunología , Leucemia Mieloide Aguda/inmunología , Masculino , Persona de Mediana Edad , Linfocitos T Citotóxicos/inmunologíaRESUMEN
A clinical oncolytic herpes simplex virus (HSV) encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), talimogene laherparepvec, causes regression of injected and non-injected melanoma lesions in patients and is now licensed for clinical use in advanced melanoma. To date, limited data are available regarding the mechanisms of human anti-tumor immune priming, an improved understanding of which could inform the development of future combination strategies with improved efficacy. This study addressed direct oncolysis and innate and adaptive human immune-mediated effects of a closely related HSV encoding GM-CSF (HSVGM-CSF) alone and in combination with histone deacetylase inhibition. We found that HSVGM-CSF supported activation of anti-melanoma immunity via monocyte-mediated type I interferon production, which activates NK cells, and viral maturation of immature dendritic cells (iDCs) into potent antigen-presenting cells for cytotoxic T lymphocyte (CTL) priming. Addition of the histone deacetylase inhibitor valproic acid (VPA) to HSVGM-CSF treatment of tumor cells increased viral replication, viral GM-CSF production, and oncolysis and augmented the development of anti-tumor immunity. Mechanistically, VPA increased expression of activating ligands for NK cell recognition and induced expression of tumor-associated antigens, supporting innate NK cell killing and CTL priming. These data support the clinical combination of talimogene laherparepvec with histone deacetylase inhibition to enhance oncolysis and anti-tumor immunity.
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Inhibidores de Histona Desacetilasas/uso terapéutico , Melanoma/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/metabolismo , Simplexvirus/inmunología , Neoplasias Cutáneas/terapia , Ácido Valproico/uso terapéutico , Antígenos de Neoplasias/efectos de los fármacos , Antígenos de Neoplasias/metabolismo , Antineoplásicos Inmunológicos/uso terapéutico , Productos Biológicos/uso terapéutico , Supervivencia Celular/genética , Células Dendríticas/inmunología , Quimioterapia Combinada , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Herpesvirus Humano 1 , Humanos , Interferón Tipo I/metabolismo , Células Asesinas Naturales/inmunología , Células MCF-7 , Melanoma/patología , Virus Oncolíticos/genética , Simplexvirus/genética , Neoplasias Cutáneas/patología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The systemic delivery of therapeutic viruses, such as oncolytic viruses or vaccines, is limited by the generation of neutralizing antibodies. While pseudotyping of rhabdoviruses with the lymphocytic choriomeningitis virus glycoprotein has previously allowed for multiple rounds of delivery in mice, this strategy has not translated to other animal models. For the first time, we provide experimental evidence that antibodies generated against the lymphocytic choriomeningitis virus glycoprotein mediate robust complement-dependent viral neutralization via activation of the classical pathway. We show that this phenotype can be capitalized upon to deliver maraba virus pseudotyped with the lymphocytic choriomeningitis virus glycoprotein in a Fischer rat model in the face of neutralizing antibody through the use of complement modulators. This finding changes the understanding of the humoral immune response to arenaviruses, and also describes methodology to deliver viral vectors to their therapeutic sites of action without the interference of neutralizing antibody.
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Tumors are complex ecosystems composed of networks of interacting 'normal' and malignant cells. It is well recognized that cytokine-mediated cross-talk between normal stromal cells, including cancer-associated fibroblasts (CAFs), vascular endothelial cells, immune cells, and cancer cells, influences all aspects of tumor biology. Here we demonstrate that the cross-talk between CAFs and cancer cells leads to enhanced growth of oncolytic virus (OV)-based therapeutics. Transforming growth factor-ß (TGF-ß) produced by tumor cells reprogrammed CAFs, dampened their steady-state level of antiviral transcripts and rendered them sensitive to virus infection. In turn, CAFs produced high levels of fibroblast growth factor 2 (FGF2), initiating a signaling cascade in cancer cells that reduced retinoic acid-inducible gene I (RIG-I) expression and impeded the ability of malignant cells to detect and respond to virus. In xenografts derived from individuals with pancreatic cancer, the expression of FGF2 correlated with the susceptibility of the cancer cells to OV infection, and local application of FGF2 to resistant tumor samples sensitized them to virotherapy both in vitro and in vivo. An OV engineered to express FGF2 was safe in tumor-bearing mice, showed improved therapeutic efficacy compared to parental virus and merits consideration for clinical testing.
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Fibroblastos/metabolismo , Virus Oncolíticos/metabolismo , Microambiente Tumoral , Anciano , Animales , Antivirales/química , Línea Celular Tumoral , Chlorocebus aethiops , Técnicas de Cocultivo , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Microscopía Fluorescente , Persona de Mediana Edad , Trasplante de Neoplasias , Viroterapia Oncolítica/métodos , Neoplasias Ováricas/metabolismo , Transducción de Señal , Células del Estroma/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Células VeroRESUMEN
Oncolytic viruses, which preferentially lyse cancer cells and stimulate an antitumor immune response, represent a promising approach to the treatment of cancer. However, how they evade the antiviral immune response and their selective delivery to, and replication in, tumor over normal tissue has not been investigated in humans. Here, we treated patients with a single cycle of intravenous reovirus before planned surgery to resect colorectal cancer metastases in the liver. Tracking the viral genome in the circulation showed that reovirus could be detected in plasma and blood mononuclear, granulocyte, and platelet cell compartments after infusion. Despite the presence of neutralizing antibodies before viral infusion in all patients, replication-competent reovirus that retained cytotoxicity was recovered from blood cells but not plasma, suggesting that transport by cells could protect virus for potential delivery to tumors. Analysis of surgical specimens demonstrated greater, preferential expression of reovirus protein in malignant cells compared to either tumor stroma or surrounding normal liver tissue. There was evidence of viral factories within tumor, and recovery of replicating virus from tumor (but not normal liver) was achieved in all four patients from whom fresh tissue was available. Hence, reovirus could be protected from neutralizing antibodies after systemic administration by immune cell carriage, which delivered reovirus to tumor. These findings suggest new preclinical and clinical scheduling and treatment combination strategies to enhance in vivo immune evasion and effective intravenous delivery of oncolytic viruses to patients in vivo.