Combined oncolytic and vaccination activities of parvovirus H-1 in a metastatic tumor model

Oncolytic viruses have emerged as a novel class of potent anticancer agents offering an improvement on chemo- and radiotherapy in terms of tumor targeting and reduction of side-effects. Among these agents, autonomous parvoviruses have attracted the attention of researchers for their ability to preferentially replicate in and kill transformed cells, and to suppress tumors in the absence of adverse reactions in various animal models. We have previously shown that lethally irradiated autologous tumor cells can support parvovirus H-1PV production and serve as carriers to deliver progeny H-1PV into the vicinity of lung metastases in a rat tumor model, resulting in H-1PV infection of and multiplication in metastatic cells. It is known that irradiated autologous (neoplastic) cells can also act as a therapeutic vaccine against the original tumor. Yet the ability of these cells to suppress metastases in the above model was found to be much increased as a result of their H-1PV infection. This prompted us to determine whether H-1PV boosted the tumor-suppressing capacity of the autologous vaccine by increasing its immunogenic potential and/or by making it a factory of oncolytic viruses able to reach and destroy the metastases. Both effects could be dissociated in the presence of neutralising antibodies which either prevent the progeny viruses from spreading to metastatic cells, or deplete the CD8 effector cells from the immune system. This strategy revealed that the H-1PV infection of tumor cells enhanced their ability to trigger an immune response for which uninfected tumor cells could be the targets, thereby amplifying and taking over from the direct viral oncolytic activity. This dual oncolytic/vaccinal effect of H-1PV holds out promises of clinical applications to cancer therapy.     

Immune cells participate in the oncosuppressive activity of parvovirus H-1PV and are activated as a result of their abortive infection with this agent

Treatment of cancers by means of viruses, that specifically replicate in (oncotropism) and kill (oncolysis) neoplastic cells, is increasingly gaining acceptance in the clinic. Among these agents, parvoviruses have been shown to possess not only direct oncolytic but also immunomodulating properties, serving as an adjuvant to prime the immune system to react against infected tumors. Here, we aimed to establish whether immunomodulating mechanisms participate in the recently reported therapeutic potential of parvoviruses against pancreatic carcinoma. Using adoptive transfer experiments we discovered that the transfer of splenocytes of donor rats harboring H-1PV-treated orthotopic PDAC tumors could significantly prolong the survival of na?ve tumor-bearing recipients, compared to those receiving cells from mock-treated donors. Closer investigation of immunological parameters in infected donor rats revealed that virus-induced interferon gamma production and cellular immune response played an important role in this effect. These data have also preclinical relevance since abortive H-1PV infection of human peripheral blood mononuclear cells or cocultivation of these cells with H-1PV-preinfected pancreatic cancer cells, resulted in enhancement of innate and adaptive immune reactivity. Taken together our data reveal that oncolytic H-1PV modulates the immune system into an anticancer state, and further support the concept of using parvoviruses in the fight against pancreatic cancer.     

Parvovirus H-1 infection of human glioma cells leads to complete viral replication and efficient cell killing

The extremely poor prognosis of malignant gliomas requires the investigation of other than standard therapies, i.e., the application of oncolytic viruses. In our study, we evaluated the effects of the oncosuppressive parvovirus H-1 on different established glioblastoma cell lines of rat and human origin and on short-term/low-passage cultures of human glioblastoma cells. We observed an efficient and dose-dependent killing of all glioma cell cultures at low multiplicities of infectious particles (MOI) per cell. Southern blot analysis of viral DNA amplification, RT-PCR analysis of viral RNA expression and Western blot analysis of the expression of viral structural (VP-1/VP-2) and nonstructural (NS-1) proteins demonstrated the biosynthesis of these viral macromolecular components in all of the cultures. Moreover, all the glioma cells were proficient for the production of infectious H-1 virus particles. The amount of virus production differed between a several fold increase of the input virus titer in most of the short-term/low-passage cultures up to 1,000-fold in one short-term glioma and in the rat cells. Glioma cells lines and, more importantly, short-term/low-passage cultures of human glioblastomas were found to be highly susceptible target cells for H-1 virus mediated cytotoxicity. The formation of fully infectious progeny particles in infected glioma cells offers the chance for the induction of secondary rounds of infection resulting in an advanced cytotoxic effect. These advantageous characteristics of H-1 virus infection of glioma cells, combined with the known low toxicity of H-1 virus in nontransformed cells, make parvovirus H-1 a promising candidate for oncolytic glioma therapy.     

Tumor cells as cellular vehicles to deliver gene therapies to metastatic tumors

A long-pursued goal in cancer treatment is to deliver a therapy specifically to metastases. As a result of the disseminated nature of the metastatic disease, carrying the therapeutic agent to the sites of tumor growth represents a major step for success. We hypothesized that tumor cells injected intravenously (i.v.) into an animal with metastases would respond to many of the factors driving the metastatic process, and would target metastases. Using a model of spontaneous metastases, we report here that i.v. injected tumor cells localized on metastatic lesions. Based on this fact, we used genetically transduced tumor cells for tumor targeting of anticancer agents such as a suicide gene or an oncolytic virus, with evident antitumoral effect and negligible systemic toxicity. Therefore, autologous tumor cells may be used as cellular vehicles for systemic delivery of anticancer therapies to metastatic tumors.     

Interferon γ improves the vaccination potential of oncolytic parvovirus H-1PV for the treatment of peritoneal carcinomatosis in pancreatic cancer

Oncolytic viruses with their capacity to specifically replicate in and kill tumor cells emerged as a novel class of cancer therapeutics. Rat oncolytic parvovirus (H-1PV) was used to treat different types of cancer in preclinical settings and was lately successfully combined with standard gemcitabine chemotherapy in treating pancreatic ductal adenocarcinoma (PDAC) in rats. Our previous work showed that the immune system and particularly the release of interferon-gamma (IFNγ) seem to mediate the anticancer effect of H-1PV in that model. Therefore, we reasoned that the therapeutic properties of H-1PV can be boosted with IFNγ for the treatment of late incurable stages of PDAC like peritoneal carcinomatosis. Rats bearing established orthotopic pancreatic carcinomas with peritoneal metastases were treated with a single intratumoral (i.t.) or intraperitoneal (i.p.) injection of 5x108 plaque forming units of H-1PV with or without concomitant IFNγ application. Intratumoral injection proved to be more effective than the intraperitoneal route in controlling the growth of both the primary pancreatic tumors and peritoneal carcinomatosis, accompanied by migration of virus from primary to metastatic deposits. Concomitant i.p. treatment of H-1PV with recIFNγ resulted in improved therapeutic effect yielding an extended animal survival, compared with i.p. treatment with H-1PV alone. IFNγ application enhanced the H-1PV-induced peritoneal macrophage and splenocyte responses against tumor cells while causing a significant reduction in the titers of H1-PV-neutralising antibodies in ascitic fluid. Thus, IFNγ co-application together with H-1PV might be considered as a novel therapeutic option to improve the survival of PDAC patients with peritoneal carcinomatosis.     

Potentiation of a recombinant oncolytic parvovirus by expression of Apoptin.

The oncotropic and oncolytic behaviors of certain autonomous rodent parvoviruses make them promising vectors for anticancer gene therapies. However, these parvoviruses are often not potent enough to kill all tumor cells equally well. With the aim of enhancing the intrinsic antitumor effect and the range of natural parvoviruses, a recombinant H1 parvovirus vector was constructed that produces the Apoptin protein, a tumor cell-specific, p53-independent, Bcl-2-insensitive apoptotic effector. We compared the apoptotic activity exerted by a recombinant hH1/Apoptin virus with that of a Green Fluorescent Protein (GFP)-transducing recombinant virus, hH1/GFP, in three human tumor cell lines differing in their susceptibility to wild-type parvovirus H1-induced killing. We found that in cells that were rather resistant to the basal cytotoxic effect of wild-type H1 or the GFP recombinant virus, a parvovirus that expressed Apoptin caused a pronounced, additional cytotoxic effect. In contrast to its enhanced cytotoxicity toward tumor cells, hH1/Apoptin virus was not more toxic to normal human fibroblasts than was the wild-type H1 virus. Taken together, these data indicate that enhancing the oncotropic behavior of wild-type H1 parvoviruses with the tumor-specific apoptotic potency of Apoptin should lead to an effective replicative parvoviral vector.     

Systemic therapy of spontaneous prostate cancer in transgenic mice with oncolytic herpes simplex viruses

Oncolytic viruses are an innovative therapeutic strategy for cancer, wherein viral replication and cytotoxicity are selective for tumor cells. Here we show the efficacy of systemically administered oncolytic viruses for the treatment of spontaneously arising tumors, specifically the use of oncolytic herpes simplex viruses (HSV) administered i.v. to treat spontaneously developing primary and metastatic prostate cancer in the transgenic TRAMP mouse, which recapitulates human prostate cancer progression. Four administrations of systemically delivered NV1023 virus, an HSV-1/HSV-2 oncolytic recombinant, to TRAMP mice at 12 or 18 weeks of age (presence of prostate adenocarcinoma or metastatic disease, respectively) inhibited primary tumor growth and metastases to lymph nodes. Expression of interleukin 12 (IL-12) from NV1042 virus, a derivative of NV1023, was additionally effective, significantly reducing the frequency of development of prostate cancer and lung metastases, even when the mice were treated after the onset of metastasis at 18 weeks of age. NV1042-infected cells, as detected by 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining for Lac Z expressed by the virus, were present in prostate tumors 1 week after the final virus injection and viral DNA was detected at 2 weeks after final virus injection by real-time PCR in primary and metastatic tumors but not in liver or blood. No toxicity was observed in any of the treated mice. The efficacy of the IL-12-expressing NV1042 virus in this aggressive prostate cancer model using a clinically relevant treatment paradigm merits its consideration for clinical studies.     

Cytokine-secreting herpes viral mutants effectively treat tumor in a murine metastatic colorectal liver model by oncolytic and T-cell-dependent mechanisms

In this model of hepatic micrometastases, the antitumor efficacy and role of the T-cell and natural killer (NK) cell populations were studied for oncolytic herpes simplex virus type-1 (HSV-1) viral mutants containing the granulocyte-monocyte colony stimulating factor (GM-CSF (NV1034)) or interluken-12 (IL-12 (NV1042)) cytokine genes. These were compared to saline and control virus (NV1023) in vitro and in vivo. HSV-1 mutants were assessed for cytotoxicity, replication and cytokine expression in CT-26 cells. A syngeneic micrometastatic liver model was then established in naive and immune cell-depleted animals to assess the antitumor efficacy of these viruses. In vitro cytotoxicity and viral replication were similar for each virus, resulting in greater than 80 and 98% cytotoxicity at multiplicity of infection of 1 and 10, respectively. Peak viral titers were 25- to 50-fold higher than initial titer and were not significantly different between viruses. In vivo, all three viruses reduced metastases relative to control, but cytokine-secreting viruses did so with greater efficacy compared to NV1023. This effect was abrogated by T-cell depletion, but not NK-cell depletion. Single-agent therapy with oncolytic viral agents containing GM-CSF or IL-12 is effective in a murine model of liver metastases and likely involves direct viral oncolysis and actions of specific immune effector cells.     

Carrier cell-based delivery of replication-competent HSV-1 mutants enhances antitumor effect for ovarian cancer

Oncolytic viruses capable of tumor-selective replication and cytolysis have shown early promise as cancer therapeutics. We have developed replication-competent attenuated herpes simplex virus type 1 (HSV-1) mutants, named HF10 and Hh101, which have been evaluated for their oncolytic activities. However, the host immune system remains a significant obstacle to effective intraperitoneal administration of these viruses in the clinical setting. In this study, we investigated the use of these HSV-1 mutants as oncolytic agents against ovarian cancer and the use of human peritoneal mesothelial cells (MCs) as carrier cells for intraperitoneal therapy. MCs were efficiently infected with HSV-1 mutants, and MCs loaded with HSV-1 mutants caused cell killing adequately when cocultured with cancer cells in the presence or absence of HSV antibodies. In a mouse xenograft model of ovarian cancer, the injection of infected carrier cells led to a significant reduction of tumor volume and prolonged survival in comparison with the injection of virus alone. Our results indicate that replication-competent attenuated HSV-1 exerts a potent oncolytic effect on ovarian cancer, which may be further enhanced by the utilization of a carrier cell delivery system, based on amplification of viral load and possibly on avoidance of neutralizing antibodies.     

Acute myeloid leukemia targeting by myxoma virus in vivo depends on cell binding but not permissiveness to infection in vitro

Some oncolytic viruses, such as myxoma virus (MYXV), can selectively target malignant hematopoietic cells, while sparing normal hematopoietic cells. This capacity for discrimination creates an opportunity to use oncolytic viruses as ex vivo purging agents of autologous hematopoietic cell grafts in patients with hematologic malignancies. However, the mechanisms by which oncolytic viruses select malignant hematopoietic cells are poorly understood. In this study, we investigated how MYXV specifically targets human AML cells. MYXV prevented chloroma formation and bone marrow engraftment of two human AML cell lines, KG-1 and THP-1. The reduction in human leukemia engraftment after ex vivo MYXV treatment was dose-dependent and required a minimum MOI of 3. Both AML cell lines demonstrated MYXV binding to leukemia cell membranes following co-incubation: however, evidence of productive MYXV infection was observed only in THP-1 cells. This observation, that KG-1 can be targeted in vivo even in the absence of in vitro permissive viral infection, contrasts with the current understanding of oncolytic virotherapy, which assumes that virus infection and productive replication is a requirement. Preventing MYXV binding to AML cells with heparin abrogated the purging capacity of MYXV, indicating that binding of infectious virus particles is a necessary step for effective viral oncolysis. Our results challenge the current dogma of oncolytic virotherapy and show that in vitro permissiveness to an oncolytic virus is not necessarily an accurate predictor of oncolytic potency in vivo.     

Systemic delivery of HER2-retargeted oncolytic-HSV by mesenchymal stromal cells protects from lung and brain metastases

Fully retargeted oncolytic herpes simplex viruses (o-HSVs) gain cancer-specificity from redirection of tropism to cancer-specific receptors, and are non-attenuated. To overcome the hurdles of systemic delivery, and enable oncolytic viruses (o-viruses) to reach metastatic sites, carrier cells are being exploited. Mesenchymal stromal cells (MSCs) were never tested as carriers of retargeted o-viruses, given their scarse-null expression of the cancer-specific receptors. We report that MSCs from different sources can be forcedly infected with a HER2-retargeted oncolytic HSV. Progeny virus spread from MSCs to cancer cells in vitro and in vivo. We evaluated the organ distribution and therapeutic efficacy in two murine models of metastatic cancers, following a single i.v. injection of infected MSCs. As expected, the highest concentration of carrier-cells and of viral genomes was in the lungs. Viral genomes persisted throughout the body for at least two days. The growth of ovarian cancer lung metastases in nude mice was strongly inhibited, and the majority of treated mice appeared metastasis-free. The treatment significantly inhibited also breast cancer metastases to the brain in NSG mice, and reduced by more than one-half the metastatic burden in the brain.     

Use of macrophages to target therapeutic adenovirus to human prostate tumors

New therapies are required to target hypoxic areas of tumors as these sites are highly resistant to conventional cancer therapies. Monocytes continuously extravasate from the bloodstream into tumors where they differentiate into macrophages and accumulate in hypoxic areas, thereby opening up the possibility of using these cells as vehicles to deliver gene therapy to these otherwise inaccessible sites. We describe a new cell-based method that selectively targets an oncolytic adenovirus to hypoxic areas of prostate tumors. In this approach, macrophages were cotransduced with a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, whose proliferation is restricted to prostate tumor cells using prostate-specific promoter elements from the TARP, PSA, and PMSA genes. When such cotransduced cells reach an area of extreme hypoxia, the E1A/B proteins are expressed, thereby activating replication of the adenovirus. The virus is subsequently released by the host macrophage and infects neighboring tumor cells. Following systemic injection into mice bearing subcutaneous or orthotopic prostate tumors, cotransduced macrophages migrated into hypoxic tumor areas, upregulated E1A protein, and released multiple copies of adenovirus. The virus then infected neighboring cells but only proliferated and was cytotoxic in prostate tumor cells, resulting in the marked inhibition of tumor growth and reduction of pulmonary metastases. This novel delivery system employs 3 levels of tumor specificity: the natural "homing" of macrophages to hypoxic tumor areas, hypoxia-induced proliferation of the therapeutic adenovirus in host macrophages, and targeted replication of oncolytic virus in prostate tumor cells.     

Sensitization of human keratinocytes to killing by parvovirus H-1 takes place during their malignant transformation but does not require them to be tumorigenic

To investigate the antineoplastic activity of parvoviruses, proliferating normal human epidermal cells and a series of established keratinocyte cell lines derived from squamous cell carcinomas or transformed in vitro, were compared for the outcome of H-1 virus infection. All established keratinocyte cell lines were more sensitive to killing by H-1 virus than normal epidermal cells, although to varying extents. Using a step-wise procedure for malignant transformation in vitro, we found that sensitization of transformed epidermal cells to H-1 virus can be dissociated from the acquisition of a tumorigenic phenotype. Thus, spontaneously- or SV40-immortalized human keratinocytes were moderately and highly sensitive to H-1 virus, respectively, and could be made tumorigenic by Harvey-ras oncogene transfection without a major change in their susceptibility to the virus. The capacity of human keratinocytes for replicating and expressing H-1 virus DNA appears to be a revealer of cellular alterations that take place in at least some pathways to malignant transformation but that may be insufficient to confer a tumorigenic potential.     

Potent systemic antitumor activity from an oncolytic herpes simplex virus of syncytial phenotype

Conditionally replicating (oncolytic) viruses, which selectively replicate in tumor cells but not in normal cells, show great promise as antitumor agents for cancer therapy. The principal antitumor activity of these viruses derives from their replication within tumor cells, which results in cell destruction and the production of progeny virions that can spread to adjacent tumor cells. However, one potential limitation of this approach is that viral gene deletions conferring tumor selectivity also result frequently in reduced potency of the virus in tumors. Therefore, strategies designed to enhance the potency of current oncolytic viruses will likely increase their chance of clinical success. Here we report the construction of an oncolytic herpes simplex virus (HSV) of which the infection also causes strong cell membrane fusion (syncytial formation). In vitro characterization on a variety of human tumor cells of different tissue origins showed that the plaques from this virus (Fu-10) are phenotypically unique and are significantly larger than those from the parental G207 virus, a well-characterized oncolytic HSV lacking fusogenic function. Furthermore, the syncytial formation caused by this virus depended on HSV replication, indicating that cell membrane fusion will only occur in dividing cells (such as tumor cells) where the virus can undergo a full infection cycle but not in normal cells where the viral replication is restricted. Systemic administration of Fu-10 into mice with established lung metastatic breast cancer resulted in a dramatic therapeutic effect. These studies demonstrate that incorporation of fusogenic function into an oncolytic virus can significantly increase the potency of viral oncolysis; this may lead to an enhanced clinical performance, especially in late-stage cancer patients.     

细胞荷载溶瘤病毒治疗肿瘤的研究进展

静脉注射的溶瘤病毒可被免疫系统完全排除而不能发挥其抗肿瘤作用。研究表明,溶瘤病毒用细胞包装后可以有效避免病毒被机体免疫系统清除,同时,病毒和载体细胞的生物学活性不受影响;另外采用具有肿瘤细胞、肿瘤基质或肿瘤解剖定位靶向性的细胞载体,可以将病毒准确地输送至肿瘤病灶,有效发挥溶瘤病毒的抗肿瘤效应。本文将对细胞荷载溶瘤病毒治疗肿瘤方面的研究进展做一综述。     

Combination of E2F-1 promoter-regulated oncolytic adenovirus and cytokine-induced killer cells enhances the antitumor effects in an orthotopic rectal cancer model

Due to the anatomical structure of the rectum, the treatment of rectal cancer remains challenging. Ad-E2F, an oncolytic adenovirus containing the E2F-1 promoter, can selectively replicate within and kill cancer cells derived from solid tumors. Thus, this virus provides a novel approach for the treatment of rectal cancer. Given the poor efficacy and possible adverse reactions that arise from the use of oncolytic virus alone and the results of our analysis of the efficacy of Ad-E2F in the treatment of rectal cancer, we investigated the use of oncolytic adenovirus in combination with adoptive immunotherapy using cytokine-induced killer (CIK) cells as a therapeutic treatment for rectal cancer. Our results illustrated that E2F-1 gene expression is higher in rectal cancer tissue than in normal tissue. Furthermore, the designed oncolytic adenovirus Ad-E2F is capable of selectively killing colorectal cell lines but has no significant effect on CIK cells. The results of in vitro and in vivo experiments demonstrated that combined therapy with Ad-E2F and CIK cells produce stronger antitumor effects than the administration of Ad-E2F or CIK cells alone. For low rectal cancers that are suitable for intratumoral injection, local injections of oncolytic viruses in combination with CIK cell-based adoptive immunotherapy may be suitable as a novel comprehensive therapeutic approach.     

Effect of a caspase inhibitor, zVADfmk, on the inhibition of breast cancer cells by herpes simplex virus type 1

The oncolytic effects of herpes simplex virus-1 (HSV-1) are limited, possibly because of premature death of infected cells by apoptosis, which limits the amount of progeny virus that is produced. It has been proposed that inhibition of apoptosis in infected tumor cells would allow increased viral persistence, replication and therapeutic effect. To test this hypothesis, we infected monocyte chemoattractant factor-7 (MCF-7) and MDA-MB-231 breast cancer cells with HSV-1 strain 17(+) and 17Δγ34.5 in the presence or absence of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVADfmk), a pan-caspase inhibitor. At low doses of HSV-1 strain 17(+) and 17Δγ34.5, the growth of MCF-7 cells was reduced to 37% or 42%, respectively, of uninfected cells. However, when cells were infected in the presence of zVADfmk, cell growth was further reduced to 24 and 33%. Similar results were seen in MDA-MB-231 cells. Cells treated with zVADfmk contained roughly 10 times more infectious viral particles than cells infected without zVADfmk, as shown by both plaque-forming and quantitative polymerase chain reaction assays. To model the situation within an infected tumor, supernatant fluids were collected from infected and non-infected cell cultures and then passed to non-infected cells. In the presence of zVADfmk, the cell growth inhibitory effect became stronger with repeated passages and was attributed to viral replication, because it could be prevented by anti-HSV antibody. These results suggest that caspases represent a novel target for drugs that increase the therapeutic efficacy of oncolytic herpes viruses against breast cancer.     

Effective infection, apoptotic cell killing and gene transfer of human hepatoma cells but not primary hepatocytes by parvovirus H1 and derived vectors

Autonomous parvoviruses preferentially replicate in and kill in vitro-transformed cells and reduce the incidence of spontaneous and implanted tumors in animals. Because of these natural oncotropic and oncolytic properties, parvoviruses deserve to be considered as potential antitumor vectors. Here, we assessed whether parvovirus H1 is able to kill human hepatoma cells by induction of apoptosis but spares primary human liver cells, and whether the former cells can efficiently be transduced by H1 virus-based vectors. Cell death, infectivity, and transgene transduction were investigated in Hep3B, HepG2, and Huh7 cells and in primary human hepatocytes with natural and recombinant H1 virus. All hepatoma cells were susceptible to H1 virus-induced cytolyis. Cell death correlated with H1 virus DNA replication, nonstructural protein expression, and with morphological features of apoptosis. H1 virus-induced apoptosis was more pronounced in p53-deleted Hep3B and p53-mutated Huh7 cells than in HepG2 cells which express wild-type p53. In Hep3B cells, apoptosis was partially inhibited by DEVD-CHO, a caspase-3 inhibitor. In contrast, H1 virus-infected primary hepatocytes were neither positive for nonstructural protein expression nor susceptible to H1 virus-induced killing. Infection with a recombinant parvovirus vector carrying the luciferase gene under control of parvovirus promoter P38 led to higher transgene activities in hepatoma cells than in the hepatocytes. Taken together, H1 virus kills human hepatoma cells at low virus multiplicity but not primary hepatocytes. Thus, recombinant H1 viruses carrying antitumor transgenes may be considered as potential therapeutic options for the treatment of hepatocellular carcinomas.