[Show abstract][Hide abstract] ABSTRACT: Previously, we showed that vesicular stomatitis virus (VSV) engineered to express a cDNA library from human melanoma cells (ASMEL, Altered Self Melanoma Epitope Library) was an effective systemic therapy to treat subcutaneous (s.c.) murine B16 melanomas. Here we show that intravenous treatment with the same ASMEL VSV-cDNA library was an effective treatment for established intra-cranial (i.c.) melanoma brain tumors. The optimal combination of antigens identified from the ASMEL which treated s.c. B16 tumors (VSV-N-RAS+VSV-CYTC-C+VSV-TYRP-1) was ineffective against i.c. B16 brain tumors. In contrast, combination of VSV-expressed antigens - VSV-HIF-2α+VSV-SOX-10+VSV-C-MYC+VSV-TYRP1 - from ASMEL which was highly effective against i.c. B16 brain tumors, had no efficacy against the same tumors growing subcutaneously. Correspondingly, i.c. B16 tumors expressed a HIF-2α(Hi), SOX-10(Hi), c-myc(Hi), TYRP1, N-RAS(lo) Cytc(lo) antigen profile, which differed significantly from the HIF-2α(lo), SOX-10(lo), c-myc(lo), TYRP1, N-RAS(Hi) Cytc(Hi) phenotype of s.c. B16 tumors, and was imposed upon the tumor cells by CD11b+ cells within the local brain tumor microenvironment. Combining T cell co-stimulation with systemic VSV-cDNA treatment, long term cures of mice with established i.c. tumors were achieved in about 75% of mice. Our data show that the anatomical location of a tumor profoundly affects the profile of antigens that it expresses.Molecular Therapy (2014); doi:10.1038/mt.2014.134.
[Show abstract][Hide abstract] ABSTRACT: Optimum clinical protocols require systemic delivery of oncolytic viruses in the presence of an intact immune system. We have shown that pre-conditioning with immune modulators, or loading virus onto carrier cells ex vivo, enhances virus-mediated anti-tumor activity. Our early trials of systemic reovirus delivery, showed that, following infusion, reovirus could be recovered from blood cells - but not from plasma - suggesting that rapid association with blood cells may protect virus from NAb. We therefore postulated that stimulation of potential carrier cells directly in vivo prior to i.v. viral delivery would enhance delivery of cell-associated virus to tumor. We show that mobilization of the CD11b+ cell compartment by GM-CSF immediately prior to i.v. reovirus, eliminated detectable tumor in mice with small B16 melanomas, and achieved highly significant therapy in mice bearing well-established tumors. Unexpectedly, cytokine conditioning therapy was most effective in the presence of pre-existing NAb. Consistent with this, reovirus bound by NAb effectively accessed monocytes/macrophages and was handed off to tumor cells. Thus, pre-conditioning with cytokine, stimulated recipient cells in vivo for enhanced viral delivery to tumors. Moreover, pre-existing NAb to an oncolytic virus may, therefore, even be exploited for systemic delivery to tumors, in the clinic.Molecular Therapy (2014); doi:10.1038/mt.2014.118.
[Show abstract][Hide abstract] ABSTRACT: Background
Oncolytic viruses are emerging as promising cancer therapies; a key challenge in their use is delivery of virus to tumour targets in the face of host immunity. In a recent translational clinical study we found that intravenously administered reovirus successfully reaches liver tumours, despite patients all having high concentrations of neutralising anti-reovirus antibodies. Functional virus was recoverable from several white-cell subsets, suggesting that systemically administered virus evades neutralisation by close cell-association (hitch-hiking). We hypothesised that this occurrence could be manipulated to enhance virotherapy by expanding the numbers of carrier cells in the circulation before viral administration.
We used immune competent mice (C57BL/6) that had been vaccinated with reovirus, or phosphate buffered saline (PBS) control, before establishing syngeneic melanoma (B16-Ova) tumours on their flank. Mice were then treated with one of three cytokines—interleukin 2, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF)—or PBS as control, for 3 days, either before or after virotherapy (reovirus) or PBS as control therapy. Unvaccinated mice were also tested with this regimen. Tumours were then measured daily or harvested for correlative analyses. Mice were humanely killed when tumours reached 1 cm in any dimension. Eight mice were used in each treatment group and experiments were repeated at least twice to ensure consistency of the findings.
Treatment with GM-CSF enhanced the number of circulating granulocytes, and enhanced macrophage infiltration into tumours. When reovirus-immune mice were treated with GM-CSF before virotherapy with reovirus, tumours regressed in all mice and were undetectable 60 days later; but reovirus-immune mice treated with either interleukin 2 or G-CSF before virotherapy, or treated with GM-CSF alone, all developed tumours that necessitated euthanasia. Moreover the combination of GM-CSF and reovirus was only effective if GM-CSF was administered before reovirus. The efficacy of GM-CSF plus reovirus was considerably diminished in unvaccinated reovirus-naive mice, but was restored if three cycles of GM-CSF plus reovirus were administered, suggesting that in this regimen anti-Rep antibodies contributed to the efficacy of therapy.
We found that oncolytic virotherapy with reovirus is enhanced by vaccinating mice against the virus and pretreatment with GM-CSF. Further in-vivo experiments, and associated in-vitro assays, are underway to characterise the mechanism involved. Pragmatically these data suggest a novel method for enhancing oncolytic virotherapy that can be readily implemented because both reovirus and GM-CSF are used clinically; accordingly a translational clinical trial protocol is being developed to explore this possibility further.
UK Medical Research Council, Royal Society of Medicine.
The Lancet 02/2014; 383:S42. · 39.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Injection of oncolytic vesicular stomatitis virus (VSV) into established B16ova melanomas results in tumor regression, in large part by inducing innate immune reactivity against the viral infection, mediated by MyD88- and type III interferon (IFN)-, but not TLR-4-, signaling. We show here that intratumoral (IT) treatment with lipopolysaccharide (LPS), a TLR-4 agonist, significantly enhanced the local therapy induced by VSV by combining activation of different innate immune pathways. Therapy was further enhanced by co-recruiting a potent antitumor, adaptive T-cell response by using a VSV engineered to express the ovalbumin tumor-associated antigen ova, in combination with LPS. However, the combination of IT LPS with systemically delivered VSV resulted in rapid morbidity and mortality in the majority of mice. Decreasing the intravenous (IV) dose of VSV to levels at which toxicity was ameliorated did not enhance therapy compared with IT LPS alone. Toxicity of the systemic VSV + IT LPS regimen was associated with rapidly elevated levels of serum tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, which neither systemic VSV, nor IT LPS, alone induced. These data show that therapy associated with direct IT injections of oncolytic viruses can be significantly enhanced by combination with agonists of innate immune activation pathways, which are not themselves activated by the virus alone. Importantly, they also highlight possible, unforeseen dangers of combination therapies in which an immunotherapy, even delivered locally at the tumor site, may systemically sensitize the patient to a cytokine shock-like response triggered by IV delivery of oncolytic virus.Molecular Therapy (2012); doi:10.1038/mt.2012.204.
[Show abstract][Hide abstract] ABSTRACT: Multiple intravenous injections of a cDNA library, derived from human melanoma cell lines and expressed using the highly immunogenic vector vesicular stomatitis virus (VSV), cured mice with established melanoma tumors. Successful tumor eradication was associated with the ability of mouse lymphoid cells to mount a tumor-specific CD4(+) interleukin (IL)-17 recall response in vitro. We used this characteristic IL-17 response to screen the VSV-cDNA library and identified three different VSV-cDNA virus clones that, when used in combination but not alone, achieved the same efficacy against tumors as the complete parental virus library. VSV-expressed cDNA libraries can therefore be used to identify tumor rejection antigens that can cooperate to induce anti-tumor responses. This technology should be applicable to antigen discovery for other cancers, as well as for other diseases in which immune reactivity against more than one target antigen contributes to disease pathology.
[Show abstract][Hide abstract] ABSTRACT: We have previously reported that a burst of vascular endothelial growth factor (VEGF) signaling to tumor-associated endothelium induces a proviral state, during which systemically delivered oncolytic reovirus can replicate in endothelium, thereby inducing immune-mediated vascular collapse and significant antitumor therapy. Using chimeric receptors, we show here that induction of the proviral state proceeds through VEGFR2, but not VEGFR1, signaling in endothelial cells. In contrast, innate immune activation by reovirus-exposed endothelial cells was predominantly through VEGFR1. By screening conventional chemotherapies for their ability to induce similar effects in combination with reovirus both in vitro and in vivo, we observed that the proviral state could also be induced in endothelial cells exposed to VEGF during rebound from paclitaxel-mediated inhibition of VEGF signaling. We translated these in vitro findings in vivo by careful scheduling of paclitaxel chemotherapy with systemic virotherapy, neither of which alone had therapeutic effects against B16 tumors. Systemic availability of reovirus during endothelial cell recovery from paclitaxel treatment allowed for endothelial replication of the virus, immune-mediated therapy, and tumor cures. Therefore, careful scheduling of combination viro- and chemotherapies, which preclinical testing suggests are individually ineffective against tumor cells, can lead to rational new clinical protocols for systemic treatments with oncolytic viruses.
[Show abstract][Hide abstract] ABSTRACT: Effective cancer immunotherapy requires the release of a broad spectrum of tumor antigens in the context of potent immune activation. We show here that a cDNA library of normal tissue, expressed from a highly immunogenic viral platform, cures established tumors of the same histological type from which the cDNA library was derived. Immune escape occurred with suboptimal vaccination, but tumor cells that escaped the immune pressure were readily treated by second-line virus-based immunotherapy. This approach has several major advantages. Use of the cDNA library leads to presentation of a broad repertoire of (undefined) tumor-associated antigens, which reduces emergence of treatment-resistant variants and also permits rational, combined-modality approaches in the clinic. Finally, the viral vectors can be delivered systemically, without the need for tumor targeting, and are amenable to clinical-grade production. Therefore, virus-expressed cDNA libraries represent a novel paradigm for cancer treatment addressing many of the key issues that have undermined the efficacy of immuno- and virotherapy to date.
Nature medicine 06/2011; 17(7):854-9. · 28.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DC) may be the most effective way of delivering oncolytic viruses to patients. Reovirus, a naturally occurring oncolytic virus, is currently undergoing early clinical trials; however, intravenous delivery of the virus is hampered by pre-existing antiviral immunity. Systemic delivery via cell carriage is a novel approach currently under investigation and initial studies have indicated its feasibility by using a variety of cell types and viruses. This study addressed the efficacy of human DC to transport virus in the presence of human neutralizing serum.
Following reovirus-loading, DC or T cells were cocultured with melanoma cells with or without neutralizing serum; the melanoma cells were then analyzed for cell death. Following reovirus loading, cells were examined by electron microscopy to identify mechanisms of delivery. The phagocytic function of reovirus-loaded DC was investigated by using labeled tumor cells and the ability of reovirus-loaded DC to prime T cells was also investigated.
In the presence of human neutralizing serum DC, but not T cells, were able to deliver reovirus for melanoma cell killing in vitro. Electron microscopy suggested that DC protected the virus by internalization, whereas with T cells it remained bound to the surface and hence accessible to neutralizing antibodies. Furthermore, DC loaded with reovirus were fully functional with regard to phagocytosis and priming of specific antitumor immune responses.
The delivery of reovirus via DC could be a promising new approach offering the possibility of combining systemic viral therapy for metastatic disease with induction of an antitumor immune response.
Clinical Cancer Research 03/2011; 17(9):2767-76. · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As well as inducing direct oncolysis, reovirus treatment of melanoma is associated with activation of innate and adaptive anti-tumour immune responses.
Here we characterise the effects of conditioned media from reovirus-infected, dying human melanoma cells (reoTCM), in the absence of live virus, to address the immune bystander potential of reovirus therapy. In addition to RANTES, IL-8, MIP-1α and MIP-1β, reovirus-infected melanoma cells secreted eotaxin, IP-10 and the type 1 interferon IFN-β. To address the mechanisms responsible for the inflammatory composition of reoTCM, we show that IL-8 and IFN-β secretion by reovirus-infected melanoma cells was associated with activation of NF-κB and decreased by pre-treatment with small molecule inhibitors of NF-κB and PKR; specific siRNA-mediated knockdown further confirmed a role for PKR. This pro-inflammatory milieu induced a chemotactic response in isolated natural killer (NK) cells, dendritic cells (DC) and anti-melanoma cytotoxic T cells (CTL). Following culture in reoTCM, NK cells upregulated CD69 expression and acquired greater lytic potential against tumour targets. Furthermore, melanoma cell-loaded DC cultured in reoTCM were more effective at priming adaptive anti-tumour immunity.
These data demonstrate that the PKR- and NF-κB-dependent induction of pro-inflammatory molecules that accompanies reovirus-mediated killing can recruit and activate innate and adaptive effector cells, thus potentially altering the tumour microenvironment to support bystander immune-mediated therapy as well as direct viral oncolysis.
Molecular Cancer 02/2011; 10:20. · 5.40 Impact Factor
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[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DC) are a clear choice for use in cancer immunotherapy, and much research has focused on generating DC for clinical use. Although DC therapy has been successful in inducing specific anti-tumour immune responses, these have rarely translated into clinical efficacy.
We examine some of the components of generating DC for therapy, including their culture, antigen loading and delivery, and discuss why DC therapy has not yet delivered substantial clinical benefit. We also examine more novel approaches, such as the potential for combination DC-based immunomodulatory strategies.
Given the highly immunosuppressive tumour environment, many of the approaches to DC vaccination are unlikely to result in effective therapy, as even successfully primed T cells may fail to infiltrate tumours or be anergized after entry. Broader approaches against multiple tumour-associated antigens in the context of overcoming tumour immune suppression are likely to prove more successful. The combination of oncolytic viral therapy with DC vaccines may promote an inflammatory tumour environment, inducing optimal DC activation, T cell priming and effective therapy.
Evolving DC-based therapeutic strategies addressing multiple components of tumour-immune system interactions may yield substantial benefits for patients.
[Show abstract][Hide abstract] ABSTRACT: JX-594 is a replication-competent Wyeth strain vaccinia virus that was genetically modified to inactive the endogenous thymidine kinase gene and to express human GM-CSF and LacZ genes. In development by Jennerex Inc and licensee Green Cross Corp, the modified virus is a novel therapy for treatment-refractive metastatic malignancies from various sites of origin. Targeted oncolytic virotherapy has demonstrated promise in preclinical studies, and more than ten viral species have subsequently entered clinical trials. JX-594 has been modified to augment the intrinsic targeting and oncolytic potential of the vaccinia virus and to enhance antitumor immunity by the expression of the GM-CSF transgene in situ. In vitro and in vivo animal studies have demonstrated the replication specificity of JX-594 for cancer cell lines and tumors, and the restriction of serum human GM-CSF expression to tumor-bearing animals, resulting in significantly reduced tumor burden and an increase in median survival. In phase I trials, JX-594 was well tolerated, with mild systemic toxicity reported. In a phase I trial in seven patients with melanoma, one partial response and one complete response after surgery were observed. In another phase I trial in patients with hepatic carcinoma, three out of ten evaluable patients had a partial response and six had stable disease; the MTD was also established. A phase II trial in patients (expected n = 30) with unresectable primary hepatocellular carcinoma was recruiting at the time of publication, with completion expected in March 2010, and a phase III trial in patients with hepatocellular carcinoma was planned for the second half of 2010. Further clinical investigations are needed to explore the potential of this agent as a single therapy and as part of multimodal treatment regimens.
Current opinion in investigational drugs (London, England: 2000) 12/2009; 10(12):1372-82. · 3.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oncolytic virotherapy may mediate antitumor effects via direct oncolysis or immune-mediated tumor regression. Although the ability of oncolytic viruses to generate adaptive antitumor immunity has been characterized, their interactions with the innate immune system are relatively unclear. Using a human in vitro system, this study investigates the innate immunological consequences of reovirus therapy and its potential to activate NK cell-mediated antitumor activity. Dendritic cells (DC) loaded with reovirus-infected human melanoma Mel888 cells (DC-MelReo), but not reovirus-infected tumor cells alone, induced IFN-gamma production within the NK cell population upon coculture with PBMC, in a cell-to-cell contact-dependent manner. DC-MelReo secreted the chemokines CCL2, 3, 4, 5, 7, 8, 11, and CXCL10; these culture supernatants induced NK cell chemotaxis. Coculture of DC-MelReo with purified NK cells induced reciprocal contact-dependent phenotypic DC maturation, while DC-MelReo elicited up-regulation of the activation marker CD69 on NK cells, in a partially contact and partially IL-12 dependent manner. Significantly, DC-MelReo induced NK cell cytotoxicity toward tumor cells by a type I IFN dependent mechanism. These data demonstrate that tumor infection by reovirus can act via DC to induce NK cell recruitment, activation, and cytotoxicity, along with reciprocal DC maturation. These findings suggest that reciprocal DC-NK cell interactions, following reovirus therapy, may play an important role in altering the immune milieu of the tumor microenvironment and mediating tumor regression.
The Journal of Immunology 10/2009; 183(7):4312-21. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reovirus is a naturally occurring oncolytic virus in clinical trials. Although tumor infection by reovirus can generate adaptive antitumor immunity, its therapeutic importance versus direct viral oncolysis is undefined. This study addresses the requirement for viral oncolysis and replication, and the relative importance of antitumor immunity and direct oncolysis in therapy.
Nonantigen specific T cells loaded with reovirus were delivered i.v. to C57BL/6 and severe combined immunodeficient mice bearing lymph node and splenic metastases from the murine melanoma, B16ova, with assessment of viral replication, metastatic clearance by tumor colony outgrowth, and immune priming. Human cytotoxic lymphocyte priming assays were done with dendritic cells loaded with Mel888 cells before the addition of reovirus.
B16ova was resistant to direct oncolysis in vitro, and failed to support reovirus replication in vitro or in vivo. Nevertheless, reovirus purged lymph node and splenic metastases in C57BL/6 mice and generated antitumor immunity. In contrast, reovirus failed to reduce tumor burden in severe combined immunodeficient mice bearing either B16ova or reovirus-sensitive B16tk metastases. In the human system, reovirus acted solely as an adjuvant when added to dendritic cells already loaded with Mel888, supporting priming of specific antitumor cytotoxic lymphocyte, in the absence of significant direct tumor oncolysis; UV-treated nonreplicating reovirus was similarly immunogenic.
The immune response is critical in mediating the efficacy of reovirus, and does not depend upon direct viral oncolysis or replication. The findings are of direct relevance to fulfilling the potential of this novel anticancer agent.
Clinical Cancer Research 07/2009; 15(13):4374-81. · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reovirus is a naturally occurring oncolytic virus currently in early clinical trials. However, the rapid induction of neutralizing antibodies represents a major obstacle to successful systemic delivery. This study addresses, for the first time, the ability of cellular carriers in the form of T cells and dendritic cells (DC) to protect reovirus from systemic neutralization. In addition, the ability of these cellular carriers to manipulate the subsequent balance of anti-viral versus anti-tumour immune response is explored. Reovirus, either neat or loaded onto DC or T cells, was delivered intravenously into reovirus-naive or reovirus-immune C57Bl/6 mice bearing lymph node B16tk melanoma metastases. Three and 10 days after treatment, reovirus delivery, carrier cell trafficking, metastatic clearance and priming of anti-tumour/anti-viral immunity were assessed. In naive mice, reovirus delivered either neat or through cell carriage was detectable in the tumour-draining lymph nodes 3 days after treatment, though complete clearance of metastases was only obtained when the virus was delivered on T cells or mature DC (mDC); neat reovirus or loaded immature DC (iDC) gave only partial early tumour clearance. Furthermore, only T cells carrying reovirus generated anti-tumour immune responses and long-term tumour clearance; reovirus-loaded DC, in contrast, generated only an anti-viral immune response. In reovirus-immune mice, however, the results were different. Neat reovirus was completely ineffective as a therapy, whereas mDC--though not iDC--as well as T cells, effectively delivered reovirus to melanoma in vivo for therapy and anti-tumour immune priming. Moreover, mDC were more effective than T cells over a range of viral loads. These data show that systemically administered neat reovirus is not optimal for therapy, and that DC may be an appropriate vehicle for carriage of significant levels of reovirus to tumours. The pre-existing immune status against the virus is critical in determining the balance between anti-viral and anti-tumour immunity elicited when reovirus is delivered by cell carriage, and the viral dose and mode of delivery, as well as the immune status of patients, may profoundly affect the success of any clinical anti-tumour viral therapy. These findings are therefore of direct translational relevance for the future design of clinical trials.
[Show abstract][Hide abstract] ABSTRACT: Early clinical trials are under way exploring the direct oncolytic potential of reovirus. This study addresses whether tumor infection by reovirus is also able to generate bystander, adaptive antitumor immunity.
Reovirus was delivered intravenously to C57BL/6 mice bearing lymph node metastases from the murine melanoma, B16-tk, with assessment of nodal metastatic clearance, priming of antitumor immunity against the tumor-associated antigen tyrosinase-related protein-2, and cytokine responses. In an in vitro human system, the effect of reovirus infection on the ability of Mel888 melanoma cells to activate and load dendritic cells for cytotoxic lymphocyte (CTL) priming was investigated.
In the murine model, a single intravenous dose of reovirus reduced metastatic lymph node burden and induced antitumor immunity (splenocyte response to tyrosinase-related protein-2 and interleukin-12 production in disaggregated lymph nodes). In vitro human assays revealed that uninfected Mel888 cells failed to induce dendritic cell maturation or support priming of an anti-Mel888 CTL response. In contrast, reovirus-infected Mel888 cells (reo-Mel) matured dendritic cells in a reovirus dose-dependent manner. When cultured with autologous peripheral blood lymphocytes, dendritic cells loaded with reo-Mel induced lymphocyte expansion, IFN-gamma production, specific anti-Mel888 cell cytotoxicity, and cross-primed CD8+ T cells specific against the human tumor-associated antigen MART-1.
Reovirus infection of tumor cells reduces metastatic disease burden and primes antitumor immunity. Future clinical trials should be designed to explore both direct cytotoxic and immunotherapeutic effects of reovirus.
Clinical Cancer Research 12/2008; 14(22):7358-66. · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ability of the immune system to effectively respond to human tumours is a matter of long-term controversy. There is an increasing body of recent evidence to support a role for the immune system in eliminating pre-clinical cancers, an old concept termed 'immunosurveillance'. 'Immunoediting' is an updated hypothesis, in which selection pressures applied by the immune response to tumours modulate tumour immunogenicity and growth. Tumour infiltration by immune cells has been shown to have powerful prognostic significance in a host of cancer types. Paradoxically, in some circumstances the immune system can promote tumour development. Cytotoxic therapies, including radiotherapy and chemotherapy, induce potentially immunogenic cell death, releasing tumour-associated antigens in the context of a 'danger' signal to the immune system. An understanding of the interaction between immune cells, tumour cells and treatment modalities will therefore guide the future combination of immunotherapy with conventional therapy to achieve optimal anti-tumour effects.