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Newcastle disease virus mediates pancreatic tumor rejection via NK cell activation and prevents cancer relapse by prompting adaptive immunity.
Aug. 2017 | Schwaiger, Theresa; Knittler, Michael R; Grund, Christian; Roemer-Oberdoerfer, Angela; Kapp, Joachim-Friedrich; Lerch, Markus M; Mettenleiter, Thomas C; Mayerle, Julia; Blohm, Ulrike
Pancreatic cancer is the 8th most common cause of cancer-related deaths worldwide and the tumor with the poorest prognosis of all solid malignancies. In 1957, it was discovered that Newcastle disease virus (NDV) has oncolytic properties on tumor cells. To study the oncolytic properties of NDV in pancreatic cancer a single dose was administered intravenously in a syngeneic orthotopic tumor model using two different murine pancreatic adenocarcinoma cell lines (DT6606PDA, Panc02). Tumor growth was monitored and immune response was analyzed. A single treatment with NDV inhibited DT6606PDA tumor growth in mice and prevented recurrence for a period of three months. Tumor infiltration and systemic activation of NK cells, cytotoxic and helper T-cells was enhanced. NDV-induced melting of Panc02 tumors until d7 pi, but they recurred displaying unrestricted tumor growth, low immunogenicity and inhibition of tumor-specific immune response. Arrest of DT6606PDA tumor growth and rejection was mediated by activation of NK cells and a specific antitumor immune response via T-cells. Panc02 tumors rapidly decreased until d7 pi, but henceforth tumors characterized by the ability to perform immune-regulatory functions reappeared. Our results demonstrated that NDV-activated immune cells are able to reject tumors provided that an adaptive antitumor immune response can be initiated. However, activated NK cells that are abundant in Panc02 tumors lead to outgrowth of nonimmunogenic tumor cells with inhibitory properties. Our study emphasizes the importance of an adaptive immune response, which is initiated by NDV to mediate long-term tumor surveillance in addition to direct oncolysis. PMID 28857157

Newcastle disease virus establishes persistent infection in tumor cells in vitro: contribution of the cleavage site of fusion protein and second sialic acid binding site of hemagglutinin-neuraminidase.
Juni 2017 | Rangaswamy, Udaya S; Wang, Weijia; Cheng, Xing; McTamney, Patrick; Carroll, Danielle; Jin, Hong
Newcastle Disease Virus (NDV) is an oncolytic virus being developed for the treatment of cancer. Following infection of an ovarian human cancer cell line (OVCAR3) with a recombinant low pathogenic NDV, persistent infection (PI) was established in a subset of tumor cells. PI cells exhibited resistance to superinfection with NDV, and an anti-viral state as demonstrated by upregulation of interferon and interferon induced genes such as Myxoma resistance gene 1 (Mx1) and Retinoic acid-inducing gene-I (RIG-I). Viruses released from PI cells induced higher cell to cell fusion following infection compared to the parental virus in two tumor cell lines tested, HT1080 and HeLa, and remained to be attenuated in chickens. Two mutations, one in fusion (F) protein cleavage site, F117S (F117S), and another in hemagglutinin-neuraminidase (HN), G169R (HN169R) located in the second sialic acid binding region, were responsible for the hyperfusogenic phenotype. F117S improves F protein cleavage efficiency facilitating cell-to-cell fusion, while HN169R possesses a multi-faceted role in contributing to higher fusion, reduced receptor binding and lower neuraminidase activity, together resulting in increased fusion activity and reduced viral replication. Thus, establishment of PI in vitro involves viral genetic changes that facilitate efficient viral spread from cell to cell as a potential mechanism to escape host anti-viral responses. The results of our study also demonstrate a critical role of the second receptor binding region in the HN protein, which is conserved in several paramyxoviruses, in viral life cycle.IMPORTANCE Oncolytic Newcastle Disease Virus (NDV) could establish persistent infection in a tumor cell line resulting in a steady anti-viral state reflected by constitutively expressed interferon. Viruses isolated from persistently infected cells are highly fusogenic, and this phenotype has been mapped to two mutations each in the fusion (F) and Hemagglutinin-Neuraminidase (HN) proteins, respectively. The F117S mutation in the F protein cleavage site improved F protein cleavage efficiency while the HN169R mutation located at the second receptor binding site of HN protein contributed to a complex phenotype consisting of a modest increase in fusion and cell killing, lower neuraminidase activity and reduced viral growth. This study highlights the intricate nature of these two mutations in the glycoproteins of NDV in the establishment of persistent infection. The data also sheds light into the critical balance between the F and HN proteins required for efficient NDV infection and in avian pathogenicity. PMID 28592535

Evaluation of the oncolytic potential of R2B Mukteshwar vaccine strain of Newcastle disease virus (NDV) in a colon cancer cell line (SW-620).
Juni 2017 | Sharma, Kishan K; Kalyani, Irsadullakhan H; Mohapatra, Jogeswar; Patel, Satish D; Patel, Dharmesh R; Vihol, Priti D; Chatterjee, Abhijit; Patel, Dinesh R; Vyas, Bhavesh
Virotherapy is emerging as an alternative treatment of cancer. Among the candidate oncolytic viruses (OVs), Newcastle disease virus (NDV) has emerged as a promising non-engineered OV. In the present communication, we explored the oncolytic potential of R2B Mukteshwar strain of NDV using SW-620 colon cancer cells. SW-620 cells were xenografted in nude mice and after evaluation of the safety profile, 1 x 10(7) plaque forming units (PFU) of NDV were inoculated as virotherapeutic agent via the intratumoral (I/T) and intravenous (I/V) route. Tumor growth inhibition was compared with their respective control groups by gross volume and histopathological evaluation. Antibody titer and virus survival were measured by hemagglutination inhibition (HI)/serum neutralization test (SNT) and real-time PCR, respectively. During the safety trial, the test strain did not produce any abnormal symptoms nor weight loss in BALB/c mice. Significant tumor lytic activity was evident when viruses were injected via the I/T route. There was a 43 and 57% tumor growth inhibition on absolute and relative tumor volume basis, respectively, compared with mock control. On the same basis, the I/V route treatment resulted in 40 and 16% of inhibition, respectively. Histopathological examination revealed that the virus caused apoptosis, followed by necrosis, but immune cell infiltration was not remarkable. The virus survived in 2/2 mice until day 10 and in 3/6 mice by day 19, with both routes of administration. Anti-NDV antibodies were generated at moderate level and the titer reached a maximum of 1:32 and 1:64 via the I/T and I/V routes, respectively. In conclusion, the test NDV strain was found to be safe and showed oncolytic activity against the SW-620 cell line in mice. PMID 28578522

Immunobiology of Newcastle Disease Virus and Its Use for Prophylactic Vaccination in Poultry and as Adjuvant for Therapeutic Vaccination in Cancer Patients.
Mai 2017 | Schirrmacher, Volker
Newcastle disease (ND) is one of the most important diseases of poultry worldwide. In the last decades, molecular research has gained a lot of new information about its causative agent, newcastledisease virus (NDV). In poultry industry, certain strains of NDV have been used for preventive vaccination for more than 60 years. NDV has also been applied to cancer patients with beneficial effects for about 50 years, but this is less well known. The molecular basis for these differential effects of NDV in birds and man have been elucidated in the last decades and are explained in this review. The anti-neoplastic and immune-stimulatory properties in non-permissive hosts such as mouse and man have to do with the strong type I interferon responses induced in these foreign species. Additionally, NDV has the potential to break various types of tumor resistances and also to affect liver fibrosis. A main section is devoted to the benefits of clinical application of NDV and NDV-based vaccines to cancer patients. Reverse genetics technology allowed developing NDV into a vector suitable for gene therapy. Examples will be provided in which genetically engineered NDV is being used successfully as vector against new emerging viruses. PMID 28531117

Current status of clinical trials assessing oncolytic virus therapy for urological cancers.
März 2017 | Taguchi, Satoru; Fukuhara, Hiroshi; Homma, Yukio; Todo, Tomoki
Oncolytic virus therapy has recently been recognized as a promising new option for cancer treatment. Oncolytic viruses replicate selectively in cancer cells, thus killing them without harming normal cells. Notably, T-VEC (talimogene laherparepvec, formerly called OncoVEX(GM)(-)(CSF) ), an oncolytic herpes simplex virus type 1, was approved by the US Food and Drug Administration for the treatment of inoperable melanoma in October 2015, and was subsequently approved in Europe and Australia in 2016. The efficacies of many types of oncolytic viruses against urological cancers have been investigated in preclinical studies during the past decade, and some have already been tested in clinical trials. For example, a phase I trial of the third-generation oncolytic Herpes simplex virus type 1, G47Δ, in patients with prostate cancer was completed in 2016. We summarize the current status of clinical trials of oncolytic virus therapy in patients with the three major urological cancers: prostate, bladder and renal cell cancers. In addition to Herpes simplex virus type 1, adenoviruses, reoviruses, vaccinia virus, Sendai virus and Newcastle disease virus have also been used as parental viruses in these trials. We believe that oncolytic virus therapy is likely to become an important and major treatment option for urological cancers in the near future. PMID 28326624

Immune and viral therapies for malignant primary brain tumors.
März 2017 | Gardeck, Andrew M; Sheehan, Jordan; Low, Walter C
Glioblastoma multiforme (GBM) is a primary brain tumor with great lethality. Current standard of care with surgery, radiation therapy, and chemotherapy are ineffective in curing this disease. Recent advancements in biological therapies show promise in treating brain tumors. Areas covered: This article provides a review of: the peripheral activation of antigen presenting cells such as dendritic cells to stimulate T cells to recognize and destroy tumor cells within the brain; the ex vivo expansion and transfer of dendritic cells, T cells, and engineered T cells expressing chimeric antigen receptors to target cells bearing specific tumor antigens as well as monoclonal antibodies as immune check point inhibitors. Gene therapy approaches have also been utilized to employ viral vectors in transducing cells to express cytokines for activating immune responses to brain tumors. Finally, the article reviews engineering of viruses for oncolytic targeting and destruction of malignant tumors within the brain. Expert opinion: The ultimate goal of immune and viral approaches for treating malignant brain tumors is to cure this disease. Preclinical and clinical studies utilizing these biological therapeutic approaches for treating brain tumors have the potential to augment the current standard of care to provide potential curative therapies. PMID 28274139

Recombinant Newcastle disease virus expressing human TRAIL as a potential candidate for hepatoma therapy.
März 2017 | Wu, Yunzhou; He, Jinjiao; Geng, Jingshu; An, Ying; Ye, Xianlong; Yan, Shijun; Yu, Qingzhong; Yin, Jiechao; Zhang, Zhenyu; Li, Deshan
Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently proved for clinical trials. We have previously reported, for the first time, NDV Anhinga strain has an efficient cancer therapeutic efficacy in hepatoma. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. In this study, we have showed administration of a recombinant NDV Anhinga strain expressing soluble TRAIL (NDV/Anh-TRAIL) results in an efficient suppression of hepatocellular carcinoma without significant toxicity. The results show that recombinant NDV Anhinga strain expressing soluble TRAIL is a promising candidate for hepatoma therapy. PMID 28246027

Prostate cancer immunotherapy, particularly in combination with androgen deprivation or radiation treatment. Customized pharmacogenomic approaches to overcome immunotherapy cancer resistance.
Jan. 2017 | Alberti, C
Conventional therapeutic approaches for advanced prostate cancer - such as androgen deprivation, chemotherapy, radiation - come up often against lack of effectiveness because of possible arising of correlative cancer cell resistance and/or inadequate anti-tumor immune conditions. Whence the timeliness of resorting to immune-based treatment strategies including either therapeutic vaccination-based active immunotherapy or anti-tumor monoclonal antibody-mediated passive immunotherapy. Particularly attractive, as for research studies and clinical applications, results to be the cytotoxic T-lymphocyte check point blockade by the use of anti-CTLA-4 and PD-1 monoclonal antibodies, particularly when combined with androgen deprivation therapy or radiation. Unlike afore said immune check point inhibitors, both cell-based (by the use of prostate specific antigen carriers autologous dendritic cells or even whole cancer cells) and recombinant viral vector vaccines are able to induce immune-mediated focused killing of specific antigen-presenting prostate cancer cells. Such vaccines, either used alone or concurrently/sequentially combined with above-mentioned conventional therapies, led to generally reach, in the field of various clinical trials, reasonable results particularly as regards the patient's overall survival. Adoptive trasferred T-cells, as adoptive T-cell passive immunotherapy, and monoclonal antibodies against specific antigen-endowed prostate cancer cells can improve immune micro-environmental conditions. On the basis of a preliminary survey about various immunotherapy strategies, are here also outlined their effects when combined with androgen deprivation therapy or radiation. What's more, as regard the immune-based treatment effectiveness, it has to be pointed out that suitable personalized epigenetic/gene profile-achieved pharmacogenomic approaches to target identified gene aberrations, may lead to overcome - as well as for conventional therapies - possible prostate cancer resistance to immunotherapy. PMID 28098061

Mesenchymal stem cells enhance the oncolytic effect of Newcastle disease virus in glioma cells and glioma stem cells via the secretion of TRAIL.
Okt. 2016 | Kazimirsky, Gila; Jiang, Wei; Slavin, Shimon; Ziv-Av, Amotz; Brodie, Chaya
Newcastle disease virus (NDV) is an avian paramyxovirus, which selectively exerts oncolytic effects in cancer cells. Mesenchymal stem cells (MSCs) have been reported to affect tumor growth and deliver anti-tumor agents to experimental glioblastoma (GBM). Here, we explored the effects of NDV-infected MSCs derived from different sources, on glioma cells and glioma stem cells (GSCs) and the mechanisms involved in their effects. PMID 27724977

Recent advances in immuno-oncology and its application to urological cancers.
Sep. 2016 | Mataraza, Jennifer M; Gotwals, Philip
Recent advances in immuno-oncology have the potential to transform the practice of medical oncology. Antibodies directed against negative regulators of T-cell function (checkpoint inhibitors), engineered cell therapies and innate immune stimulators, such as oncolytic viruses, are effective in a wide range of cancers. Immune'based therapies have had a clinically meaningful impact on the treatment of advanced melanoma, and the lessons regarding use of single agents and combinations in melanoma may be applicable to the treatment of urological cancers. Checkpoint inhibitors, cytokine therapy and therapeutic vaccines are already showing promise in urothelial bladder cancer, renal cell carcinoma and prostate cancer. Critical areas of future immuno-oncology research include the prospective identification of patients who will respond to current immune-based cancer therapies and the identification of new therapeutic agents that promote immune priming in tumours, and increase the rate of durable clinical responses. PMID 27123757

Fifty Years of Clinical Application of Newcastle Disease Virus: Time to Celebrate!
Juli 2016 | Schirrmacher V,

This review provides an overview of 50 years of basic and clinical research on an oncolytic avian virus, Newcastle Disease Virus (NDV), which has particular anti-neoplastic and immune stimulatory properties. Of special interest is the fact that this biological agent induces immunogenic cell death and systemic anti-tumor immunity. Furthermore, localized oncolytic virotherapy with NDV was shown to overcome systemic tumor resistance to immune checkpoint blockade immunotherapy. Clinical experience attests to low side effects and a high safety profile. This is due among others to the strong virus-induced type I interferon response. Other viral characteristics are lack of interaction with host cell DNA, lack of genetic recombination and independence of virus replication from cell proliferation. In this millennium, new recombinant strains of viruses are being produced with improved therapeutic properties. Clinical applications include single case observations, case series studies and Phase I to III studies.

PMID ISSN 2227-9059

Genetic Modification of Oncolytic Newcastle Disease Virus for Cancer Therapy.
Mai 2016 | Cheng, Xing; Wang, Weijia; Xu, Qi; Harper, James; Carroll, Danielle; Galinski, Mark S; Suzich, JoAnn; Jin, Hong
Clinical development of a mesogenic strain of Newcastle disease virus (NDV) as an oncolytic agent for cancer therapy has been hampered by its select agent status due to its pathogenicity in avian species. Using reverse genetics, we have generated a lead candidate oncolytic NDV based on the mesogenic NDV-73T strain that is no longer classified as a select agent for clinical development. This recombinant NDV has a modification at the fusion protein (F) cleavage site to reduce the efficiency of F protein cleavage and an insertion of a 198-nucleotide sequence into the HN-L intergenic region, resulting in reduced viral gene expression and replication in avian cells but not in mammalian cells. In mammalian cells, except for viral polymerase (L) gene expression, viral gene expression is not negatively impacted or increased by the HN-L intergenic insertion. Furthermore, the virus can be engineered to express a foreign gene while still retaining the ability to grow to high titers in cell culture. The recombinant NDV selectively replicates in and kills tumor cells and is able to drive potent tumor growth inhibition following intratumoral or intravenous administration in a mouse tumor model. The candidate is well positioned for clinical development as an oncolytic virus. PMID 27009956

Recombinant Newcastle disease virus (NDV/Anh-IL-2) expressing human IL-2 as a potential candidate for suppresses growth of hepatoma therapy.
Mai 2016 | Wu, Yunzhou; He, Jinjiao; An, Ying; Wang, Xi; Liu, Yunye; Yan, Shijun; Ye, Xianlong; Qi, Jianying; Zhu, Shenglong; Yu, Qingzhong; Yin, Jiechao; Li, Deshan; Wang, Wenfei
Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical study and are currently approved for clinical trials. NDV Anhinga strain which is a mesogenic strain should be classified as lytic strain and has a therapeutic efficacy in hepatocellular cancer. In this study, we evaluated the capacity of NDV Anhinga strain to elicit immune reaction in vivo and the possibility for using as a vaccine vector for expressing tumor therapeutic factors. Interleukin-2 (IL-2) could boost the immune response against the tumor cells. Therefore, we use NDV Anhinga strain as backbone to construct a recombinant virus (NDV/Anh-IL-2) expressing IL-2. The virus growth curve showed that the production of recombinant NDV/Anh-IL-2 was slightly delayed compared to the wild type. The NDV/Anh-IL-2 strain could express soluble IL-2 and effectively inhibit the growth of hepatocellular carcinoma in vivo. 60 days post-treatment, mice which were completely cured by previous treatment were well protected when rechallenged with the same tumor cell. From the H&E-stained sections, intense infiltration of lymphocyte was observed in the NDV Anhinga strain treated group, especially in NDV/Anh-IL-2 group. The NDV Anhinga strain could not only kill the tumor directly, but could also elicit immune reaction and a potent immunological memory when killing tumor in vivo. In conclusion, the Anhinga strain could be an effective vector for tumor therapy; the recombinant NDV/Anh-IL-2 strain expressing soluble IL-2 is a promising candidate for hepatoma therapy. PMID 27174862

Newcastle disease virus chimeras expressing the Hemagglutinin- Neuraminidase protein of mesogenic strain exhibits an enhanced anti-hepatoma efficacy.
Mai 2016 | He, Jinjiao; Pan, Ziye; Tian, Guiyou; Liu, Xin; Liu, Yunye; Guo, Xiaochen; An, Ying; Song, Liying; Wu, Hongsong; Cao, Hongwei; Yu, Dan; Che, Ruixiang; Xu, Pengfei; Rasoul, Lubna M; Li, Deshan; Yin, Jiechao
Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, many researchers have reported that lentogenic NDV is a safe and effective agent for human cancer therapy. It had been demonstrated that the amino acid sequence of the fusion protein cleavage site is a major factor in the pathogenicity and anti-tumor efficacy of rNDV. However, the role of Hemagglutinin-Neuraminidase (HN) gene that contributes to virulence and anti-tumor efficacy remains undefined. To assess the role of HN gene in virus pathogenicity and anti-tumor efficacy, a reverse genetic system was developed using the lentogenic NDV Clone30 strain to provide backbone for gene exchange. Chimeras (rClone30-Anh(HN)) created by exchange of the HN gene of lentogenic strain Clone30 with HN gene of mesogenic strain produce no significant changes in virus pathogenicity as assessed by conducting the mean death time (MDT) and intracerebral pathogenicity index (ICPI) assays. In vitro, infection with chimeras could induce the formation of syncytium relative significantly in HepG2 cells. Furthermore, chimeras was shown to induce the cell apoptosis via MTT and Annexin V-PI assays, reduce mitochondrial membrane potential and increase the mRNA transcription level of caspase 3. In vivo, ICR mice carrying tumor of hepatoma H22 cells were treated via intratumoral injection of chimeric virus. The treatment of chimera shows an obvious suppression in tumor volume. These results suggest that it could be an ideal approach to enhance the antitumor ability of Newcastle disease virus and highlighted the potential therapeutic application of rClone30-Anh(HN) as a viral vector to deliver foreign genes for treatment of cancers. PMID 27164362

Oncolytic newcastle disease virus triggers cell death of lung cancer spheroids and is enhanced by pharmacological inhibition of autophagy.
Feb. 2016 | Hu, Lulu; Sun, Sulan; Wang, Tianpeng; Li, Yingchun; Jiang, Ke; Lin, Guibin; Ma, Yan; Barr, Martin P; Song, Fei; Zhang, Guirong; Meng, Songshu
Lung cancer stem cells (CSCs) have recently been isolated from lung cancer patient samples and have been reported to be responsible for tumor initiation, treatment resistance and tumor recurrence. We have previously shown that oncolytic Newcastle disease virus (NDV), strain FMW (NDV/FMW) induces apoptosis in drug-resistant lung cancer cells. However, how NDV exerts its oncolytic effect on lung CSCs remains to be investigated. Here we show that NDV/FMW replicates in, and lyses CSC-enriched lung cancer spheroids and inhibits the 3D growth potential of lung cancer spheroid and agar colonies. We demonstrate that NDV/FMW triggers caspase-dependent apoptosis in lung cancer spheroids as shown by increased caspase-3 processing and Poly (ADP-ribose) polymerase (PARP) cleavage. Notably, NDV/FMW infection results in the degradation of microtubule-associated protein 1 light chain 3 (LC3) II and P62, two hallmarks of autophagy maturation, indicating that NDV/FMW promotes autophagy flux in lung cancer cell spheroids. This was further confirmed by the appearance of an increased number of double-membrane vesicles as detected by transmission electron microscopy. We also show that NDV/FMW promotes autophagy degradation in lung cancer spheroids via inhibition of the AKT/mTOR pathway. In addition, treatment of spheroids with the autophagy inhibitor, chloroquine increases NDV/FMW-induced cytotoxicity. Collectively, our data show that oncolytic NDV/FMW may be a potential strategy in targeting lung CSCs. PMID 26885450

Viro-immune therapy: A new strategy for treatment of pancreatic cancer.
Jan. 2016 | Ibrahim, Andrea Marie; Wang, Yao-He
Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly lethal disease with less than 5% survival at five years. This is largely due to metastatic disease, which is already present in the majority of patients when diagnosed. Even when the primary cancer can be removed by radical surgery, local recurrence occurs within one year in 50%-80% of cases. Therefore, it is imperative to develop new approaches for the treatment of advanced cancer and the prevention of recurrence after surgery. Tumour-targeted oncolytic viruses (TOVs) have become an attractive therapeutic agent as TOVs can kill cancer cells through multiple mechanisms of action, especially via virus-induced engagement of the immune response specifically against tumour cells. To attack tumour cells effectively, tumour-specific T cells need to overcome negative regulatory signals that suppress their activation or that induce tolerance programmes such as anergy or exhaustion in the tumour microenvironment. In this regard, the recent breakthrough in immunotherapy achieved with immune checkpoint blockade agents, such as anti-cytotoxic T-lymphocyte-associate protein 4, programmed death 1 (PD-1) or PD-L1 antibodies, has demonstrated the possibility of relieving immune suppression in PDAC. Therefore, the combination of oncolytic virotherapy and immune checkpoint blockade agents may synergistically function to enhance the antitumour response, lending the opportunity to be the future for treatment of pancreatic cancer. PMID 26811622

Overcoming Barriers in Oncolytic Virotherapy with HDAC Inhibitors and Immune Checkpoint Blockade.
Jan. 2016 | Marchini, Antonio; Scott, Eleanor M; Rommelaere, Jean
Oncolytic viruses (OVs) target and destroy cancer cells while sparing their normal counterparts. These viruses have been evaluated in numerous studies at both pre-clinical and clinical levels and the recent Food and Drug Administration (FDA) approval of an oncolytic herpesvirus-based treatment raises optimism that OVs will become a therapeutic option for cancer patients. However, to improve clinical outcome, there is a need to increase OV efficacy. In addition to killing cancer cells directly through lysis, OVs can stimulate the induction of anti-tumour immune responses. The host immune system thus represents a "double-edged sword" for oncolytic virotherapy: on the one hand, a robust anti-viral response will limit OV replication and spread; on the other hand, the immune-mediated component of OV therapy may be its most important anti-cancer mechanism. Although the relative contribution of direct viral oncolysis and indirect, immune-mediated oncosuppression to overall OV efficacy is unclear, it is likely that an initial period of vigorous OV multiplication and lytic activity will most optimally set the stage for subsequent adaptive anti-tumour immunity. In this review, we consider the use of histone deacetylase (HDAC) inhibitors as a means of boosting virus replication and lessening the negative impact of innate immunity on the direct oncolytic effect. We also discuss an alternative approach, aimed at potentiating OV-elicited anti-tumour immunity through the blockade of immune checkpoints. We conclude by proposing a two-phase combinatorial strategy in which initial OV replication and spread is maximised through transient HDAC inhibition, with anti-tumour immune responses subsequently enhanced by immune checkpoint blockade. PMID 26751469

Newcastle Disease Virus: Potential Therapeutic Application for Human and Canine Lymphoma.
Dez. 2015 | Sánchez, Diana; Pelayo, Rosana; Medina, Luis Alberto; Vadillo, Eduardo; Sánchez, Rogelio; Núñez, Luis; Cesarman-Maus, Gabriela; Sarmiento-Silva, Rosa Elena
Research on oncolytic viruses has mostly been directed towards the treatment of solid tumors, which has yielded limited information regarding their activity in hematological cancer. It has also been directed towards the treatment of humans, yet veterinary medicine may also benefit. Several strains of the Newcastle disease virus (NDV) have been used as oncolytics in vitro and in a number of in vivo experiments. We studied the cytolytic effect of NDV-MLS, a low virulence attenuated lentogenic strain, on a human large B-cell lymphoma cell line (SU-DHL-4), as well as on primary canine-derived B-cell lymphoma cells, and compared them to healthy peripheral blood mononuclear cells (PBMC) from both humans and dogs. NDV-MLS reduced cell survival in both human (42% ± 5%) and dog (34% ± 12%) lymphoma cells as compared to untreated controls. No significant effect on PBMC was seen. Cell death involved apoptosis as documented by flow-cytometry. NDV-MLS infections of malignant lymphoma tumors in vivo in dogs were confirmed by electron microscopy. Early (24 h) biodistribution of intravenous injection of 1 × 10(12) TCID50 (tissue culture infective dose) in a dog with T-cell lymphoma showed viral localization only in the kidney, the salivary gland, the lung and the stomach by immunohistochemistry and/or endpoint PCR. We conclude that NDV-MLS may be a promising agent for the treatment of lymphomas. Future research is needed to elucidate the optimal therapeutic regimen and establish appropriate biosafety measures. PMID 26703717

Oncolytic Newcastle disease virus expressing chimeric antibody enhanced anti-tumor efficacy in orthotopic hepatoma-bearing mice.
Dez. 2015 | Wei, Ding; Li, Qian; Wang, Xi-Long; Wang, Yuan; Xu, Jing; Feng, Fei; Nan, Gang; Wang, Bin; Li, Can; Guo, Ting; Chen, Zhi-Nan; Bian, Huijie
Oncolytic virus which arms the therapeutic gene to enhance anti-tumor activity is a prevalent strategy to improve oncovirotherapy of cancer. Newcastle disease virus (NDV) is a naturally oncolytic virus used for cancer therapy. Previously, we generated a mouse-human chimeric HAb18 antibody (cHAb18) against tumor-associated antigen CD147 and demonstrated the inhibition of invasion and migration of hepatocellular carcinoma (HCC) cells. Here, we constructed a recombinant NDV carrying intact cHAb18 gene (rNDV-18HL) based on Italien strain using a reverse genetics system. PMID 26689432

Enhanced therapeutic effect using sequential administration of antigenically distinct oncolytic viruses expressing oncostatin M in a Syrian hamster orthotopic pancreatic cancer model.
Dez. 2015 | Nistal-Villan, Estanislao; Bunuales, Maria; Poutou, Joanna; Gonzalez-Aparicio, Manuela; Bravo-Perez, Carlos; Quetglas, Jose I; Carte, Beatriz; Gonzalez-Aseguinolaza, Gloria; Prieto, Jesus; Larrea, Esther; Hernandez-Alcoceba, Ruben
The limited efficacy of current treatments against pancreatic cancer has prompted the search of new alternatives such as virotherapy. Activation of the immune response against cancer cells is emerging as one of the main mechanisms of action of oncolytic viruses (OV). Direct oncolysis releases tumor antigens, and viral replication within the tumor microenvironment is a potent danger signal. Arming OV with immunostimulatory transgenes further enhances their therapeutic effect. However, standard virotherapy protocols do not take full advantage of OV as cancer vaccines because repeated viral administrations may polarize immune responses against strong viral antigens, and the rapid onset of neutralizing antibodies limits the efficacy of redosing. An alternative paradigm based on sequential combination of antigenically distinct OV has been recently proposed. PMID 26671477

Oncolysis by paramyxoviruses: preclinical and clinical studies.
Dez. 2015 | Matveeva, Olga V; Guo, Zong S; Senin, Vyacheslav M; Senina, Anna V; Shabalina, Svetlana A; Chumakov, Peter M
Preclinical studies demonstrate that a broad spectrum of human malignant cells can be killed by oncolytic paramyxoviruses, which include cells of ecto-, endo-, and mesodermal origin. In clinical trials, significant reduction in size or even complete elimination of primary tumors and established metastases are reported. Different routes of viral administration (intratumoral, intravenous, intradermal, intraperitoneal, or intrapleural), and single- versus multiple-dose administration schemes have been explored. The reported side effects are grade 1 and 2, with the most common among them being mild fever. Some advantages in using para-myxoviruses as oncolytic agents versus representatives of other viral families exist. The cytoplasmic replication results in a lack of host genome integration and recombination, which makes paramyxoviruses safer and more attractive candidates for widely used therapeutic oncolysis in comparison with retroviruses or some DNA viruses. The list of oncolytic paramyxovirus representatives includes attenuated measles virus (MV), mumps virus (MuV), low pathogenic Newcastle disease (NDV), and Sendai (SeV) viruses. Metastatic cancer cells frequently overexpress on their surface some molecules that can serve as receptors for MV, MuV, NDV, and SeV. This promotes specific viral attachment to the malignant cell, which is frequently followed by specific viral replication. The paramyxoviruses are capable of inducing efficient syncytium-mediated lyses of cancer cells and elicit strong immunomodulatory effects that dramatically enforce anticancer immune surveillance. In general, preclinical studies and phase 1-3 clinical trials yield very encouraging results and warrant continued research of oncolytic paramyxoviruses as a particularly valuable addition to the existing panel of cancer-fighting approaches. PMID 26640815

Viral Infection of Tumors Overcomes Resistance to PD-1-immunotherapy by Broadening Neoantigenome-directed T-cell Responses.
Okt. 2015 | Woller, Norman; Gürlevik, Engin; Fleischmann-Mundt, Bettina; Schumacher, Anja; Knocke, Sarah; Kloos, Arnold M; Saborowski, Michael; Geffers, Robert; Manns, Michael P; Wirth, Thomas C; Kubicka, Stefan; Kühnel, Florian
There is evidence that viral oncolysis is synergistic with immune checkpoint inhibition in cancer therapy but the underlying mechanisms are unclear. Here, we investigated whether local viral infection of malignant tumors is capable of overcoming systemic resistance to PD-1-immunotherapy by modulating the spectrum of tumor-directed CD8 T-cells. To focus on neoantigen-specific CD8 T-cell responses, we performed transcriptomic sequencing of PD-1-resistant CMT64 lung adenocarcinoma cells followed by algorithm-based neoepitope prediction. Investigations on neoepitope-specific T-cell responses in tumor-bearing mice demonstrated that PD-1 immunotherapy was insufficient whereas viral oncolysis elicited cytotoxic T-cell responses to a conserved panel of neoepitopes. After combined treatment, we observed that PD-1-blockade did not affect the magnitude of oncolysis-mediated antitumoral immune responses but a broader spectrum of T-cell responses including additional neoepitopes was observed. Oncolysis of the primary tumor significantly abrogated systemic resistance to PD-1-immunotherapy leading to improved elimination of disseminated lung tumors. Our observations were confirmed in a transgenic murine model of liver cancer where viral oncolysis strongly induced PD-L1 expression in primary liver tumors and lung metastasis. Furthermore, we demonstrated that combined treatment completely inhibited dissemination in a CD8 T-cell-dependent manner. Therefore, our results strongly recommend further evaluation of virotherapy and concomitant PD-1 immunotherapy in clinical studies. PMID 26112079

Oncolytic Newcastle disease virus as a prospective anti-cancer therapy. A biologic agent with potential to break therapy resistance.
Okt. 2015 | Schirrmacher, Volker
Oncolytic viruses (OVs) selectively replicate in tumor cells and cause cancer cell death. Most OVs in clinical studies are genetically engineered. In contrast, the avian Newcastle disease virus (NDV) is a naturally oncolytic RNA virus. While anti-viral immunity is considered a major problem in achieving maximal tumor cell killing by OVs, this review discusses the importance of NDV immunogenic cell death (ICD) and how anti-viral immune responses can be integrated to induce maximal post-oncolytic T-cell-mediated anti-tumor immunity. Since replication of NDV is independent of host cell DNA replication (which is the target of many cytostatic drugs and radiotherapy) and because of other findings, oncolytic NDV is a candidate agent to break therapy resistance of tumor cells. Areas covered: Properties of this avian paramyxovirus are summarized with special emphasis to its anti-neoplastic and immune-stimulatory properties. The review then discusses prospective anti-cancer therapies, including treatments with NDV alone, and combinations with an autologous NDV-modified tumor cell vaccine or with a viral oncolysate pulsed dendritic cell vaccine. Various combinatorial approaches between these and with other modalities are also reviewed. Expert opinion: Post-oncolytic anti-tumor immunity based on ICD is in the expert's opinion of greater importance for long-term therapeutic effects than maximal tumor cell killing. Of the various combinatorial approaches discussed, the most promising and feasible for clinical practice appears to be the combination of systemic NDV pre-treatment with anti-tumor vaccination. PMID 26436571

HN Protein of Newcastle Disease Virus Induces Apoptosis Through SAPK/JNK Pathway.
Sep. 2015 | Rajmani, R S; Gandham, Ravi Kumar; Gupta, Shishir Kumar; Sahoo, A P; Singh, Prafull Kumar; Kumar, Rajiv; Saxena, Shikha; Chaturvedi, Uttara; Tiwari, Ashok K
Many viral proteins are responsible for causing induction of apoptosis in the target cells. Hemagglutinin neuraminidase (HN), a multifunctional protein of Newcastle disease virus (NDV), is one of such proteins. The present study was undertaken to determine the apoptotic potential of the HN gene in cultured human cervical cancer cell line (HeLa cell) and to elucidate the molecular mechanisms involved. The results of the study indicate that HN protein causes apoptosis in HeLa cells, as observed by the translocation of Phosphatidylserine, activation of caspases, cleavage of poly (ADP-ribose) polymerase (PARP), and DNA fragmentation. Further, we report that expression of HN protein upregulates the SAPK/JNK pathway leading to transactivation of c-Jun which in turn activates apoptosis signaling. The results of our study provide an insight into the mechanism through which HN induces apoptosis. PMID 26306526

Sensitivity of Human Malignant Melanoma Cell Lines to Newcastle Disease Virus.
Sep. 2015 | Pap, Marianna; Bátor, Judit; Szeberényi, József
Virotherapy may be a promising alternative to chemotherapy of malignant melanoma. In clinical trials using strains of Newcastle disease virus (NDV), only a fraction of patients with cancer responded to virotherapy. In the present study, we tried to find a correlation between the susceptibility of human melanoma cell lines to NDV and growth factor signaling pathways. PMID 26408702

Long-term survival of a breast cancer patient with extensive liver metastases upon immune and virotherapy: a case report.
Sep. 2015 | Schirrmacher, Volker; Stücker, Wilfried; Lulei, Maria; Bihari, Akos-Sigmund; Sprenger, Tobias
Liver metastases in breast cancer are associated with a poor prognosis. We report long-term survival of a patient with breast cancer and liver metastases. After operation the patient declined further standard therapy. Instead, she was treated with local hyperthermia, Newcastle disease virus and dendritic cell vaccination at the Immunological and Oncological Center Cologne (IOZK), Germany. A continuous high quality of life was reported and the patient survived more than 66 months after initial diagnosis. No recurrence or further metastases developed under treatment. Following treatment, a long-lasting tumor-reactive memory T-cell responsiveness could be documented. This possibly explains the favorable course of disease. Since this combination of therapies is not restricted to a particular tumor type, further exploration is warranted. PMID 26020523

Recombinant Newcastle Disease virus Expressing IL15 Demonstrates Promising Antitumor Efficiency in Melanoma Model.
Sep. 2015 | Niu, Zeshan; Bai, Fuliang; Sun, Tian; Tian, Hui; Yu, Dan; Yin, Jiechao; Li, Siming; Li, Tianhe; Cao, Hongwei; Yu, Qingzhong; Wu, Yunzhou; Ren, Guiping; Li, Deshan
Recombinant Newcastle Disease Virus (rNDV) has shown oncolytic therapeutic effect in preclinical studies. Previous data indicate that rNDV carrying IL2 has shown promise in cancer therapy. Due to the significant side effects of IL2, IL15 has been introduced into cancer therapy. A number of studies have suggested that IL15 efficiently enhances the activities of CTL and NK cells and inhibits the tumor recurrence and metastasis. Furthermore, IL15 is less toxic than IL2. Therefore, we hypothesize that a recombinant NDV expressing IL15 would be a promising agent for the treatment of malignant tumors. The human IL15 gene or IL2 gene was incorporated into the genome of lentogenic LaSota strain at the position between the HN and L genes (namely rNDV-IL15 or rNDV-IL2). The two viruses efficiently infected tumor cells and expressed IL15 or IL2 protein. Melanoma tumor-bearing mice were treated by intra-tumoral (i.t.) injection of rNDV-IL15 or rNDV-IL2. Both rNDV-IL15 and rNDV-IL2 effectively suppressed tumor growth compared with rNDV. The 120-day survival rate of rNDV-IL15- treated group was 12.5% higher than that of rNDV-IL2 group, although the difference was not statistically significant, both recombinant viruses had strong abilities to induce CD41 T cell and CTL cell responses. However, rNDV-IL15 significantly induced more IFN-γ release and stimulated more CD81 T cells infiltration in the tumor sites compared with rNDV-IL2. In the tumor re-challenged experiment, the survival rates of rNDV-IL15 group and rNDV-IL2 group were statistically higher than that of PBS group. The survival rate of rNDV-IL15 group was 26.67% higher than that of rNDV-IL2 group although the difference was not statistically significant. In conclusion, rNDV-IL15 is a promising antitumor agent against melanoma. PMID 24645750

Recombinant Newcastle Disease Virus Encoding IL-12 and/or IL-2 as Potential Candidate for Hepatoma Carcinoma Therapy.
Aug. 2015 | Ren, Guiping; Tian, Guiyou; Liu, Yunye; He, Jinjiao; Gao, Xinyu; Yu, Yinhang; Liu, Xin; Zhang, Xu; Sun, Tian; Liu, Shuangqing; Yin, Jiechao; Li, Deshan
Interleukins as immunomodulators are promising therapeutic agents for cancer therapy. Previous studies showed that there was an improved antitumor immunity in tumor-bearing mice using recombinant Newcastle disease virus carrying for interleukin-2. Interleukin-12 is a promising antitumor cytokine too. So we investigated and compared the antitumor effect of genetically engineered Newcastle disease virus strains expressing both interleukin-12 and/or interleukin-2 (rClone30-interleukin-2, rClone30-interleukin-12, and rClone30-interleukin-12-interleukin-2). In vitro studies showed that rClone30s could efficiently infect tumor cells and express interleukin-12 and/or interleukin-2. 3-(4,5-Dimethylthiazol-2-y)-2,5-diphenyl-tetrazolium bromide results showed rClone30s possessed strong cytotoxic activities against multiple tumor cell lines (U251, HepG2, A549, and Hela). Animal studies showed that rClone30-interleukin-12-interleukin-2 was more effective in inhibition of murine hepatoma carcinoma tumors, with the mean tumor volume (day 14) of 141.70 mm(3) comparing 165.67 mm(3) of rClone30-interleukin-12 group, 210.47 mm(3) of rClone30-interleukin-2 group, 574.70 mm(3) of rClone30 group, and 1206.83 mm(3) of phosphate-buffered saline group. Moreover, the rClone30-interleukin-12-interleukin-2 treated mice secreted more interferon γ (333.518 pg/mL) and its downstream cytokine interferon-γ induced protein 10 (16.006 pg/mL) in tumor than the rClone30-interleukin-12 group (interferon γ: 257.548 pg/mL; interferon-γ induced protein 10: 13.601 pg/mL), rClone30-interleukin2 group (interferon γ: 124.601 pg/mL; interferon-γ induced protein 10: 9.779 pg/mL), or rClone30 group (interferon γ: 48.630 pg/mL; interferon-γ induced protein 10:1.650 pg/mL). For the survival study, rClone30-interleukin12-interleukin2 increased the survival rate (12 of 16) of the tumor-bearing mice versus 11 of 16 in rClone30-interleukin-12 group, 10 of 16 in rClone30-interleukin-2 group, 7 of 16 in Clone30 group, and 0/16 in phosphate-buffered saline group, respectively. To determine whether the mice treated with recombinant virus developed protective immune response, the mice were rechallenged with the same tumor cells. The results showed that viral-treated mice were significantly protected from rechallenge. These results suggest that expressing both interleukin-2 and/or interleukin-12 could be ideal approaches to enhance the antitumor ability of Newcastle disease virus, and rClone30-interleukin-12-interleukin-2 is slightly superior over rClone30-interleukin-12 and rClone30-interleukin-2 alone. PMID 26303327

The therapeutic effect of death: Newcastle disease virus and its antitumor potential.
Aug. 2015 | Cuadrado-Castano, Sara; Sanchez-Aparicio, Maria T; García-Sastre, Adolfo; Villar, Enrique
Programmed cell death is essential to survival of multicellular organisms. Previously restricted to apoptosis, the concept of programmed cell death is now extended to other mechanisms, as programmed necrosis or necroptosis, autophagic cell death, pyroptosis and parthanatos, among others. Viruses have evolved to manipulate and take control over the programmed cell death response, and the infected cell attempts to neutralize viral infections displaying different stress signals and defensive pathways before taking the critical decision of self-destruction. Learning from viruses and their interplay with the host may help us to better understand the complexity of the self-defense death response that when altered might cause disorders as important as cancer. In addition, as the fields of immunotherapy and oncolytic viruses advance as promising novel cancer therapies, the programmed cell death response reemerges as a key point for the success of both therapeutic approaches. In this review we summarize the research of the multimodal cell death response induced by Newcastle disease viruses (NDV), considered nowadays a promising viral oncolytic therapeutic, and how the manipulation of the host programmed cell death response can enhance the NDV antitumor capacity. PMID 26221764

Human renal carcinoma cells respond to Newcastle disease virus infection through activation of the p38 MAPK/NF-κB/IκBα pathway.
Aug. 2015 | Ch'ng, Wei-Choong; Abd-Aziz, Noraini; Ong, Meng-Hua; Stanbridge, Eric J; Shafee, Norazizah
Newcastle disease virus (NDV) is an oncolytic virus that is known to have a higher preference to cancer cells than to normal cells. It has been proposed that this higher preference may be due to defects in the interferon (IFN) responses of cancer cells. The exact mechanism underlying this process, however, remains to be resolved. In the present study, we examined the antiviral response towards NDV infection of clear cell renal cell carcinoma (ccRCC) cells. ccRCC is associated with mutations of the von Hippel-Lindau tumor suppressor gene VHL, whose protein product is important for eliciting cellular responses to changes in oxygen levels. The most common first line treatment strategy of ccRCC includes IFN. Unfortunately, most ccRCC cases are diagnosed at a late stage and often are resistant to IFN-based therapies. Alternative treatment approaches, including virotherapy using oncolytic viruses, are currently being investigated. The present study was designed to investigate the mechanistic pathways underlying the response of ccRCC cells to oncolytic NDV infection. PMID 25930675

Antineoplastic activity of Newcastle disease virus strain D90 in oral squamous cell carcinoma.
Juli 2015 | Zhang, Chun-Xiao; Ye, Long-Wei; Liu, Ying; Xu, Xiao-Ya; Li, Dan-Rui; Yang, Yan-Qing; Sun, Lu-Lu; Yuan, Jie
Newcastle disease virus (NDV), an avian paramyxovirus, possesses the ability to kill tumor cells. Here, we report the effects of NDV strain D90, which was isolated in China, against oral squamous cell carcinoma (OSCC) cells. In this study, we showed that the cell death induced by D90 was apoptotic. Furthermore, the apoptosis induced by D90 was dependent on the mitochondrial pathway, and the death receptor pathway may be not involved. Bax and Bcl-2 also played a role in the apoptosis induced by D90. Lymph node metastasis is a serious problem for oral cancer; we therefore evaluated the impact of D90 on the migration and invasion of OSCC cells. NDV D90 affected microtubules and microfilaments to inhibit the motility of OSCC prior to apoptosis. The effects of D90 on the migration and invasion rates of OSCC cells were evaluated by migration and invasion assays. Subsequently, the changes in sp1, RECK, MMP-2, and MMP-9 induced by a low concentration of D90 were detected by western blot and gelatin zymography. D90 significantly inhibited the invasion and metastasis of OSCC cells by decreasing the expression of sp1 and increasing the expression of RECK to suppress the expression and activity of MMP-2 and MMP-9. PMID 25877754

Recombinant Immunomodulating Lentogenic or Mesogenic Oncolytic Newcastle Disease Virus for Treatment of Pancreatic Adenocarcinoma.
Juni 2015 | Buijs, Pascal; van Nieuwkoop, Stefan; Vaes, Vincent; Fouchier, Ron; van Eijck, Casper; van den Hoogen, Bernadette
Oncolytic Newcastle disease virus (NDV) might be a promising new therapeutic agent for the treatment of pancreatic cancer. We evaluated recombinant NDVs (rNDVs) expressing interferon (rNDV-hIFNβ-F₀) or an IFN antagonistic protein (rNDV-NS1-F₀), as well as rNDV with increased virulence (rNDV-F₃aa) for oncolytic efficacy in human pancreatic adenocarcinoma cells. Expression of additional proteins did not hamper virus replication or cytotoxic effects on itself. However, expression of interferon, but not NS1, resulted in loss of multicycle replication. Conversely, increasing the virulence (rNDV-F₃aa) resulted in enhanced replication of the virus. Type I interferon was produced in high amounts by all tumor cells inoculated with rNDV-hIFNβ -F₀, while inoculation with rNDV-NS1-F₀ resulted in a complete block of interferon production in most cells. Inoculation of human pancreatic adenocarcinoma cells with rNDV-F₃aa caused markedly more cytotoxicity compared to rNDV-F₀, while inoculation with rNDVβ-hIFN -F₀ and rNDV-NS1-F₀ induced cytotoxic effects comparable to those induced by the parental rNDV-F₀. Evaluation in vivo using mice bearing subcutaneous pancreatic cancer xenografts revealed that only intratumoral injection with rNDV-F₃aa resulted in regression of tumors. We conclude that although lentogenic rNDVs harboring proteins that modulate the type I interferon pathway proteins do have an oncolytic effect, a more virulent mesogenic rNDV might be needed to improve oncolytic efficacy. PMID 26110582

[Expressing foreign genes by Newcastle disease virus for cancer therapy].
Juni 2015 | Bai, F L; Tian, H; Yu, Q Z; Renl, G P; Li, D S
An interesting aspect of Newcastle disease virus (NDV) is the ability to selectively replicate in tumor cells. Recently, using reverse genetics technology to enhance the oncolytic properties and therapeutic potential of NDV for tumor therapy has become popular in immunocompetent carcinoma tumor models. Expressing foreign genes by recombinant NDV (rNDV-FG) has been shown to be more effective in cancer therapy in preclinical studies. This paper provides an overview of the current studies on the cytotoxic and anti-cancer effects of rNDV-FG via direct oncolysis and immune stimulation. Safety of rNDV-FG as a therapeutic agent for cancer immunotherapy and virotherapy is also discussed. PMID 26065249

IPS-1 differentially induces TRAIL, BCL2, BIRC3 and PRKCE in type I interferons-dependent and -independent anticancer activity.
Mai 2015 | Kumar, S; Ingle, H; Mishra, S; Mahla, R S; Kumar, A; Kawai, T; Akira, S; Takaoka, A; Raut, A A; Kumar, H
RIG-I-like receptors are the key cytosolic sensors for RNA viruses and induce the production of type I interferons (IFN) and pro-inflammatory cytokines through a sole adaptor IFN-β promoter stimulator-1 (IPS-1) (also known as Cardif, MAVS and VISA) in antiviral innate immunity. These sensors also have a pivotal role in anticancer activity through induction of apoptosis. However, the mechanism for their anticancer activity is poorly understood. Here, we show that anticancer vaccine adjuvant, PolyIC (primarily sensed by MDA5) and the oncolytic virus, Newcastle disease virus (NDV) (sensed by RIG-I), induce anticancer activity. The ectopic expression of IPS-1 into type I IFN-responsive and non-responsive cancer cells induces anticancer activity. PolyIC transfection and NDV infection upregulate pro-apoptotic gene TRAIL and downregulate the anti-apoptotic genes BCL2, BIRC3 and PRKCE. Furthermore, stable knockdown of IPS-1, IRF3 or IRF7 in IFN-non-responsive cancer cells show reduced anticancer activity by suppressing apoptosis via TRAIL and anti-apoptotic genes. Collectively, our study shows that IPS-1 induces anticancer activity through upregulation of pro-apoptotic gene TRAIL and downregulation of the anti-apoptotic genes BCL2, BIRC3 and PRKCE via IRF3 and IRF7 in type I IFN-dependent and -independent manners. PMID 25950488

Transplanted tumor growth and the incidence of T-lymphocyte populations in the spleen of newcastle virus-treated mice.
Mai 2015 | Martić, Ana Jurin; Ivanković, Siniša; Antica, Mariastefania; Hiršl, Nevenka; Jukić, Tomislav; Jurin, Mislav
C3Hf/HZgr mice were transplanted with SCCVII carcinoma cells and treated with Newcastle disease virus (NDV). The treatment slows down the growth of transplanted tumor. Furthermore, by using specific monoclonal antibodies, the frequencies of CD4+, CD8+, and CD4+CD25+ T lymphocytes were determined in the spleen of tumorous mice at particular times following tumor transplantation and/or NDV application. The incidence of lymphocytes CD4+ and CD8+ decreased and of CD4+CD25+ increased in the spleen of mice during the time following tumor transplantation. However, the frequency of regulatory CD4+CD25+ T lymphocytes in the spleen is very low, while CD4+ and CD8+ increased to normal level following intraperitoneal (i.p.) NDV injection in tumor-bearing mice. Thus, besides directly destroying transplanted tumor, NDV seems to be involved against growing tumor by reducing the frequency of regulatory T lymphocytes maintaining the frequency of CD4+ and CD8+ T lymphocytes within the control values pointing to its role in immunomodulation. PMID 25763836

Enhancement of the proapoptotic properties of newcastle disease virus promotes tumor remission in syngeneic murine cancer models.
Mai 2015 | Cuadrado-Castano, Sara; Ayllon, Juan; Mansour, Mena; de la Iglesia-Vicente, Janis; Jordan, Stefan; Tripathi, Shashank; García-Sastre, Adolfo; Villar, Enrique
Newcastle disease virus (NDV) is considered a promising agent for cancer therapy due to its oncolytic properties. These include preferential replication in transformed cells, induction of innate and adaptive immune responses within tumors, and cytopathic effects in infected tumor cells due to the activation of apoptosis. To enhance the latter and thus possibly enhance the overall oncolytic activity of NDV, we generated a recombinant NDV encoding the human TNF receptor Fas (rNDV-B1/Fas). rNDV-B1/Fas replicates to similar titers as its wild-type (rNDV-B1) counterpart; however, overexpression of Fas in infected cells leads to higher levels of cytotoxicity correlated with faster and increased apoptosis responses, in which both the intrinsic and extrinsic pathways are activated earlier. Furthermore, in vivo studies in syngeneic murine melanoma models show an enhancement of the oncolytic properties of rNDV-B1/Fas, with major improvements in survival and tumor remission. Altogether, our data suggest that upregulation of the proapoptotic function of NDV is a viable approach to enhance its antitumor properties and adds to the currently known, rationally based strategies to design optimized therapeutic viral vectors for the treatment of cancer. PMID 25761895

Progress in oncolytic virotherapy for the treatment of thyroid malignant neoplasm.
Apr. 2015 | Guan, Mingxu; Romano, Gaetano; Coroniti, Roberta; Henderson, Earl E
Thyroid malignant neoplasm develops from follicular or parafollicular thyroid cells. A higher proportion of anaplastic thyroid cancer has an adverse prognosis. New drugs are being used in clinical treatment. However, for advanced thyroid malignant neoplasm such as anaplastic thyroid carcinoma, the major impediment to successful control of the disease is the absence of effective therapies. Oncolytic virotherapy has significantly progressed as therapeutics in recent years. The advance is that oncolytic viruses can be designed with biological specificity to infect, replicate and lyse tumor cells. Significant advances in virotherapy have being achieved to improve the accessibility, safety and efficacy of the treatment. Therefore, it is necessary to summarize and bring together the main areas covered by these investigations for the virotherapy of thyroid malignant neoplasm. We provide an overview of the progress in virotherapy research and clinical trials, which employ virotherapy for thyroid malignant neoplasm as well as the future prospect for virotherapy of thyroid malignant neoplasms. PMID 25366264

Inhibitory and apoptosis-inducing effects of Newcastle disease virus strain AF2240 on mammary carcinoma cell line.
März 2015 | Ahmad, Umar; Ahmed, Ismaila; Keong, Yong Yoke; Abd Manan, Nizar; Othman, Fauziah
Breast cancer is the malignant tumour that developed from cells of the breast and is the first leading cause of cancer death among women worldwide. Surgery, radiotherapy, and chemotherapy are the available treatments for breast cancer, but these were reported to have side effects. Newcastle disease virus (NDV) known as Avian paramyxovirus type-1 (APMV1) belongs to the genus Avulavirus in a family Paramyxoviridae. NDV is shown to be a promising anticancer agent, killing tumour cells while sparing normal cells unharmed. In this study, the oncolytic and cytotoxic activities of NDV AF2240 strain were evaluated on MDA-MB-231, human mammary carcinoma cell line, using MTT assay, and its inhibitory effects were further studied using proliferation and migration assays. Morphological and apoptotic-inducing effects of NDV on MD-MB-231 cells were observed using phase contrast and fluorescence microscopes. Detection of DNA fragmentation was done following terminal deoxyribonucleotide transferase-mediated Br-dUTP nick end labeling staining (TUNEL) assay, which confirmed that the mode of death was through apoptosis and was quantified by flow cytometry. Furthermore, analysis of cellular DNA content demonstrated that the virus caused an increase in the sub-G1 phase (apoptotic peak) of the cell cycle. It appears that NDV AF2240 strain is a potent anticancer agent that induced apoptosis in time-dependent manner. PMID 25821783

Immune Suppression during Oncolytic Virotherapy for High-Grade Glioma; Yes or No?
Feb. 2015 | Koks, Carolien A E; De Vleeschouwer, Steven; Graf, Norbert; Van Gool, Stefaan W
Oncolytic viruses have been seriously considered for glioma therapy over the last 20 years. The oncolytic activity of several oncolytic strains has been demonstrated against human glioma cell lines and in in vivo xenotransplant models. So far, four of these stains have additionally completed the first phase I/II trials in relapsed glioma patients. Though safety and feasibility have been demonstrated, therapeutic efficacy in these initial trials, when described, was only minor. The role of the immune system in oncolytic virotherapy for glioma remained much less studied until recent years. When investigated, the immune system, adept at controlling viral infections, is often hypothesized to be a strong hurdle to successful oncolytic virotherapy. Several preclinical studies have therefore aimed to improve oncolytic virotherapy efficacy by combining it with immune suppression or evasion strategies. More recently however, a new paradigm has developed in the oncolytic virotherapy field stating that oncolytic virus-mediated tumor cell death can be accompanied by elicitation of potent activation of innate and adaptive anti-tumor immunity that greatly improves the efficacy of certain oncolytic strains. Therefore, it seems the three-way interaction between oncolytic virus, tumor and immune system is critical to the outcome of antitumor therapy. In this review we discuss the studies which have investigated how the immune system and oncolytic viruses interact in models of glioma. The novel insights generated here hold important implications for future research and should be incorporated into the design of novel clinical trials. PMID 25663937

Newcastle disease virotherapy induces long-term survival and tumor-specific immune memory in orthotopic glioma through the induction of immunogenic cell death.
Dez. 2014 | Koks, Carolien A; Garg, Abhishek D; Ehrhardt, Michael; Riva, Matteo; Vandenberk, Lien; Boon, Louis; De Vleeschouwer, Steven; Agostinis, Patrizia; Graf, Norbert; Van Gool, Stefaan W
The oncolytic features of several naturally oncolytic viruses have been shown on Glioblastoma Multiforme cell lines and in xenotransplant models. However, orthotopic glioma studies in immunocompetent animals are lacking. Here we investigated Newcastle disease virus (NDV) in the orthotopic, syngeneic murine GL261 model. Seven days after tumor induction, mice received NDV intratumorally. Treatment significantly prolonged median survival and 50% of animals showed long-term survival. We demonstrated immunogenic cell death (ICD) induction in GL261 cells after NDV infection, comprising calreticulin surface exposure, release of HMGB1 and increased PMEL17 cancer antigen expression. Uniquely, we found absence of secreted ATP. NDV-induced ICD occurred independently of caspase signaling and was blocked by Necrostatin-1, suggesting the contribution of necroptosis. Autophagy induction following NDV infection of GL261 cells was demonstrated as well. In vivo, elevated infiltration of IFN-γ(+) T cells was observed in NDV-treated tumors, along with reduced accumulation of myeloid derived suppressor cells. The importance of a functional adaptive immune system in this paradigm was demonstrated in immunodeficient Rag2(-/-) mice and in CD8(+) T cell depleted animals, where NDV slightly prolonged survival, but failed to induce long-term cure. Secondary tumor induction with GL261 cells or LLC cells in mice surviving long-term after NDV treatment, demonstrated the induction of a long-term, tumor-specific immunological memory response by ND virotherapy. For the first time, we describe the therapeutic activity of NDV against GL261 tumors, evidenced in an orthotopic mouse model. The therapeutic effect relies on the induction of ICD in the tumor cells, which primes adaptive antitumor immunity. PMID 25208916

Intravenously injected Newcastle disease virus in non-human primates is safe to use for oncolytic virotherapy.
Nov. 2014 | Buijs, P R A; van Amerongen, G; van Nieuwkoop, S; Bestebroer, T M; van Run, P R W A; Kuiken, T; Fouchier, R A M; van Eijck, C H J; van den Hoogen, B G
Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic potential. Detailed preclinical information regarding the safety of oncolytic NDV is scarce. In this study, we evaluated the toxicity, biodistribution and shedding of intravenously injected oncolytic NDVs in non-human primates (Macaca fascicularis). Two animals were injected with escalating doses of a non-recombinant vaccine strain, a recombinant lentogenic strain or a recombinant mesogenic strain. To study transmission, naive animals were co-housed with the injected animals. Injection with NDV did not lead to severe illness in the animals or abnormalities in hematologic or biochemistry measurements. Injected animals shed low amounts of virus, but this did not lead to seroconversion of the contact animals. Postmortem evaluation demonstrated no pathological changes or evidence of virus replication. This study demonstrates that NDV generated in embryonated chicken eggs is safe for intravenous administration to non-human primates. In addition, our study confirmed results from a previous report that naïve primate and human sera are able to neutralize egg-generated NDV. We discuss the implications of these results for our study and the use of NDV for virotherapy. PMID 25257305

Strong T‑cell costimulation can reactivate tumor antigen‑specific T cells in late‑stage metastasized colorectal carcinoma patients: results from a phase Ⅰ clinical study.
Nov. 2014 | Schirrmacher, Volker; Schlude, Christoph; Weitz, Jürgen; Beckhove, Philipp
T‑cell costimulation is necessary to induce a response of naïve T cells. Whether T‑cell costimulation can also cause reactivation of unreactive, possibly anergized memory T cells (MTCs) from late‑stage cancer patients is unknown. To investigate this question, we developed a bispecific anti‑CD28 fusion protein (bsHN‑CD28) which can easily be attached to the vaccine ATV‑NDV. This virus‑modified autologous tumor cell vaccine has already shown effectivity in colon cancer patients following resection of liver metastases. In this phase Ⅰ clinical study, 14 colorectal carcinoma (CRC) patients with late‑stage disease which could not be operated anymore with curative intent were treated with the vaccine ATV‑NDV to which bsHN‑CD28 was attached. No severe adverse events were recorded. All patients showed an immunological response of tumor‑reactive T cells, at least once during the course of five vaccinations. Also, we demonstrate a dose‑response relationship with the costimulatory molecule added to the vaccine. A partial response of metastases was documented in four patients. The study suggests that the three‑component vaccine is safe and can reactivate possibly anergized T cells from a chronic disease like advanced‑stage cancer. PMID 25354198

Apoptotic induction of lung adenocarcinoma A549 cells infected by recombinant RVG Newcastle disease virus (rL-RVG) in vitro.
Nov. 2014 | Yan, Yulan; Liang, Bing; Zhang, Jin; Liu, Yang; Bu, Xuefeng
Newcastle disease virus (NDV) is a member of the genus Avulavirus in the Paramyxoviridae family and its antitumor properties depend on its ability to kill malignant cells while not affecting normal cells. The present study investigated a recombinant avirulent NDV LaSota strain (wild-type NDV strain) expressing the rabies virus glycoprotein (rL-RVG), examined its oncolytic effect on the lung adenocarcinoma A549 cell line and evaluated its potential to serve as a vaccine against lung cancer. A549 cells were infected with the rL-RVG virus and analyzed by MTT, western blot, polymerase chain reaction (PCR), immunofluorescence, terminal deoxynucleotidyl transferase dUTP nick end labeling and flow-cytometric analyses. PCR, western blot and immunofluorescence showed that the RVG gene and protein were stably expressed in A549 cells following infection with rL-RVG. The growth of A549 cells in the rL-RVG group was inhibited more effectively compared to those infected with the wild-type NDV strain. MTT results showed that cell growth inhibition rates in the rL-RVG group were significantly higher than those in the NDV group (P<0.05). Early apoptosis in the rL-RVG group was also more evident, with the apoptotic index being increased in rL-RVG group. The expression of the pro-apoptotic proteins caspase-3, -8 and -9 increased. The expression of caspase-3 decreased following application of the broad-specificity caspase inhibitor Z-VAD-FMK. However, the expression of the inhibitory apoptosis protein B-cell lymphoma 2 (bcl-2) did not change, but bcl-2-associated X/bcl-2 ratio was higher in the rL-RVG group than that in the NDV group. The rL-RVG strain was able to suppress lung cancer cell growth and promote lung cancer cell apoptosis to a greater extent than the wild-type NDV strain. Therefore, the rL-RVG strain is a potent antitumor agent. PMID 25322856

Emerging treatment strategies for glioblastoma multiforme.
Nov. 2014 | Carlsson, Steven K; Brothers, Shaun P; Wahlestedt, Claes
Glioblastoma multiforme (GBM) is the deadliest form of brain tumor with a more than 90% 5-year mortality. GBM has a paltry median survival of 12.6 months attributed to the unique treatment limitations such as the high average age of onset, tumor location, and poor current understandings of the tumor pathophysiology. The resection techniques, chemotherapic strategies, and radiation therapy currently used to treat GBM have slowly evolved, but the improvements have not translated to marked increases in patient survival. Here, we will discuss the recent progress in our understanding of GBM pathophysiology, and the diagnostic techniques and treatment options. The discussion will include biomarkers, tumor imaging, novel therapies such as monoclonal antibodies and small-molecule inhibitors, and the heterogeneity resulting from the GBM cancer stem cell population. PMID 25312641

Long-term remission of prostate cancer with extensive bone metastases upon immuno- and virotherapy: A case report.
Nov. 2014 | Schirrmacher, Volker; Bihari, Akos-Sigmund; Stücker, Wilfried; Sprenger, Tobias
The present study reports the case of a patient with hormone-refractory metastatic prostate cancer who had failed standard therapy, but then achieved complete remission following combined treatment with local hyperthermia (LHT), Newcastle disease virus and dendritic cell (DC) vaccination, which was an unusual combination. In August 2005, the patient underwent a radical prostatectomy. Despite standard treatment, the patient developed progressive bone metastases and stopped conventional therapy in June 2007. Starting in October 2007, the patient was treated with LHT, oncolytic virotherapy and DC vaccination. Prostate-specific antigen (PSA)-levels, with the highest level of 233.8 ng/ml in January 2008, decreased to 0.8 ng/ml in late February 2008. In March 2008, a reduction in bone metastases could be detected by positron emission tomography/computed tomography. Since then, the PSA levels have remained low and the patient is doing well. The treatment induced a long-lasting antitumor memory T-cell response. This possibly explains the long-term effectiveness of this novel experimental combined treatment approach. PMID 25364402

Effect of recombinant Newcastle disease virus transfection on lung adenocarcinoma A549 cells in vivo.
Nov. 2014 | Yan, Yulan; Jia, Lijuan; Zhang, Jin; Liu, Yang; Bu, Xuefeng
Newcastle disease virus (NDV) has been reported to selectively duplicate in and then destroy tumor cells, whilst sparing normal cells. However, the effect of NDV on lung cancer has yet to be elucidated. In the present study, recombinant NDV (rl-RVG) was applied to lung adenocarcinoma A549 cell tumor-bearing mice to explore its effect on the proliferation of the cells and the immune response of the mice. Following rl-RVG transfection, RVG and NDV gene expression, decreased tumor growth, subcutaneous tumor necrosis, tumor apoptosis and an increased number of cluster of differentiation (CD)3(-)/CD49(+) natural killer cells were more evident in the rl-RVG group. The present study demonstrated that rl-RVG transfection effectively restrained lung adenocarcinoma A549 cell growth in vivo, which may have been accomplish by inducing tumor cell apoptosis and regulating the cell immune response. PMID 25364430

Immunomodulatory effects of Newcastle disease virus AF2240 strain on human peripheral blood mononuclear cells.
Okt. 2014 | Lam, Han Yuen; Yusoff, Khatijah; Yeap, Swee Keong; Subramani, Tamilselvan; Abd-Aziz, Suraini; Omar, Abdul Rahman; Alitheen, Noorjahan Banu
Immunotherapy has raised the attention of many scientists because it hold promise to be an attractive therapeutic strategy to treat a number of disorders. In this study, the immunomodulatory effects of low titers of Newcastle disease virus (NDV) AF2240 on human peripheral blood mononuclear cells (PBMC) were analyzed. We evaluated cytokine secretion and PBMC activation by cell proliferation assay, immunophenotyping and enzyme linked immunosorbent assay. The proliferation of the human PBMC was measured to be 28.5% and 36.5% upon treatment with 8 hemaglutinin unit (HAU) and 2 HAU of NDV respectively. Interestingly, the percentage of cells with activating markers CD16 and CD56 were increased significantly. Furthermore, the intracellular perforin and granzyme levels were also increased upon virus infection. Human PBMC treated with NDV titer 8 HAU was found to stimulate the highest level of cytokine production including interferon-γ, interleukin-2 and interleukin-12. The release of these proteins contributes to the antitumor effect of PBMC against MCF-7 breast cancer cells. Based on the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, activated human PBMC showed high cytolytic efficiency towards human breast tumor cells. In summary, NDV was able to stimulate PBMC proliferation, cytokine secretion and cytolytic activity. PMID 25317070

Multimodal cancer therapy involving oncolytic newcastle disease virus, autologous immune cells, and bi-specific antibodies.
Okt. 2014 | Schirrmacher, Volker; Fournier, Philippe
This paper focuses on oncolytic Newcastle disease virus (NDV). This paper summarizes (i) the peculiarities of this virus as an anti-cancer and immune stimulatory agent and (ii) the approaches to further harness this virus as a vector to combat cancer. Special emphasis is given on combining virus therapy with cell therapy and on improving tumor targeting. The review will include some of the authors work on NDV, bi-specific antibodies, and cell therapy as building blocks for a new perspective of multimodal cancer therapy. The broad anti-tumor immune reactivation includes innate and adaptive, tumor antigen (TA) specific and TA independent activities. PMID 25309868

Newcastle disease virus interaction in targeted therapy against proliferation and invasion pathways of glioblastoma multiforme.
Sep. 2014 | Abdullah, Jafri Malin; Mustafa, Zulkifli; Ideris, Aini
Glioblastoma multiforme (GBM), or grade IV glioma, is one of the most lethal forms of human brain cancer. Current bioscience has begun to depict more clearly the signalling pathways that are responsible for high-grade glioma initiation, migration, and invasion, opening the door for molecular-based targeted therapy. As such, the application of viruses such as Newcastle disease virus (NDV) as a novel biological bullet to specifically target aberrant signalling in GBM has brought new hope. The abnormal proliferation and aggressive invasion behaviour of GBM is reported to be associated with aberrant Rac1 protein signalling. NDV interacts with Rac1 upon viral entry, syncytium induction, and actin reorganization of the infected cell as part of the replication process. Ultimately, intracellular stress leads the infected glioma cell to undergo cell death. In this review, we describe the characteristics of malignant glioma and the aberrant genetics that drive its aggressive phenotype, and we focus on the use of oncolytic NDV in GBM-targeted therapy and the interaction of NDV in GBM signalling that leads to inhibition of GBM proliferation and invasion, and subsequently, cell death. PMID 25243137

Attacking Postoperative Metastases using Perioperative Oncolytic Viruses and Viral Vaccines.
Aug. 2014 | Tai, Lee-Hwa; Auer, Rebecca
Surgical resection of solid primary malignancies is a mainstay of therapy for cancer patients. Despite being the most effective treatment for these tumors, cancer surgery has been associated with impaired metastatic clearance due to immunosuppression. In preclinical surgery models and human cancer patients, we and others have demonstrated a profound suppression of both natural killer (NK) and T cell function in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Oncolytic viruses (OV) were originally designed to selectively infect and replicate in tumors, with the primary objective of directly lysing cancer cells. It is becoming increasingly clear, however, that OV infection results in a profound inflammatory reaction within the tumor, initiating innate and adaptive immune responses against it that is critical for its therapeutic benefit. This anti-tumor immunity appears to be mediated predominantly by NK and cytotoxic T cells. In preclinical models, we found that preoperative OV prevents postoperative NK cell dysfunction and attenuates tumor dissemination. Due to theoretical safety concerns of administering live virus prior to surgery in cancer patients, we characterized safe, attenuated versions of OV, and viral vaccines that could stimulate NK cells and reduce metastases when administered in the perioperative period. In cancer patients, we observed that in vivo infusion with oncolytic vaccinia virus and ex vivo stimulation with viral vaccines promote NK cell activation. These preclinical studies provide a novel and clinically relevant setting for OV therapy. Our challenge is to identify safe and promising OV therapies that will activate NK and T cells in the perioperative period preventing the establishment of micrometastatic disease in cancer patients. PMID 25161958

Mitophagy promotes replication of oncolytic Newcastle disease virus by blocking intrinsic apoptosis in lung cancer cells.
Aug. 2014 | Meng, Gang; Xia, Mao; Wang, Diancheng; Chen, Aiping; Wang, Yongshan; Wang, Hongwei; Yu, Decai; Wei, Jiwu
Apoptosis contributes to antitumor effect of Newcastle disease virus (NDV). Autophagy is a protective response under cellular stress including viral infection. How autophagy interferes with oncolysis of NDV remains unclear. In this study, we found that NDV La Sota strain induced autophagy and preserved autophagic flux in non-small cell lung cancer cells. NDV-induced autophagy promoted viral replication by blocking cancer cells from caspase-dependent apoptosis. Moreover, we found that NDV recruited SQSTM1-mediated mitophagy to control cytochrome c release, and thus blocked intrinsic pro-apoptotic signaling. Finally, we observed an enhanced oncolysis in NSCLC cells treated with NDV in the presence of an autophagy inhibitor 3-methyladenine (3-MA). Interestingly, a more profound antitumor effect could be achieved when administration of 3-MA was postponed to 24 h after NDV infection. Our findings unveil a novel way that NDV subverts mitophagy to favor its replication by blocking apoptosis, and provide rationale for systemic therapeutic cohort combining NDV with autophagy inhibitors in cancer therapy. PMID 25051374

Pharmacological modulation of autophagy enhances Newcastle disease virus-mediated oncolysis in drug-resistant lung cancer cells.
Aug. 2014 | Jiang, Ke; Li, Yingchun; Zhu, Qiumin; Xu, Jiansheng; Wang, Yupeng; Deng, Wuguo; Liu, Quentin; Zhang, Guirong; Meng, Songshu
Oncolytic viruses represent a promising therapy against cancers with acquired drug resistance. However, low efficacy limits its clinical application. The objective of this study is to investigate whether pharmacologically modulating autophagy could enhance oncolytic Newcastle disease virus (NDV) strain NDV/FMW virotherapy of drug-resistant lung cancer cells. PMID 25078870

ISG12a mediates cell response to Newcastle disease viral infection.
Aug. 2014 | Liu, Nianli; Long, Ying; Liu, Bin; Yang, Darong; Li, Chen; Chen, Tianran; Wang, Xiaohong; Liu, Chen; Zhu, Haizhen
Newcastle disease virus (NDV) oncolysis is believed to be facilitated by a defective Type I interferon (IFN) response. We compared hepatocellular carcinoma (HCC)-derived cell lines and found that TRAIL-resistant cells were more susceptible to NDV oncolysis than were TRAIL-sensitive cells. In examining the IFN response, we found that basal expression of the IFN-stimulated gene (ISG)-12a was low in TRAIL-resistant but high in TRAIL-sensitive cells, and ISG12a over-expression or silencing enhanced or reduced their TRAIL sensitivities, respectively. Moreover, ISG12a over-expression in TRAIL-resistant cells decreased NDV replication but surprisingly increased oncolysis while ISG12a silencing had the opposite effect on TRAIL-sensitive cells. Finally, RIG-I and Noxa appear to also contribute to NDV oncolysis. Together, these results suggest that high basal ISG12a may inhibit NDV replication and oncolysis, while low basal ISG12a may allow sufficient NDV replication for induction of ISG12a, and other factors required for NDV oncolysis, with implications for future therapeutics. PMID 24999841

Going viral with cancer immunotherapy.
Juli 2014 | Lichty, Brian D; Breitbach, Caroline J; Stojdl, David F; Bell, John C
Recent clinical data have emphatically shown the capacity of our immune systems to eradicate even advanced cancers. Although oncolytic viruses (OVs) were originally designed to function as tumour-lysing therapeutics, they have now been clinically shown to initiate systemic antitumour immune responses. Cell signalling pathways that are activated and promote the growth of tumour cells also favour the growth and replication of viruses within the cancer. The ability to engineer OVs that express immune-stimulating 'cargo', the induction of immunogenic tumour cell death by OVs and the selective targeting of OVs to tumour beds suggests that they are the ideal reagents to enhance antitumour immune responses. Coupling of OV therapy with tumour antigen vaccination, immune checkpoint inhibitors and adoptive cell therapy seems to be ready to converge towards a new generation of multimodal therapeutics to improve outcomes for cancer patients. PMID 24990523

Autologous tumor vaccine modified with recombinant new castle disease virus expressing IL-7 promotes antitumor immune response.
Juli 2014 | Zhao, Lixiang; Mei, Yu; Sun, Qing; Guo, Linghua; Wu, Yan; Yu, Xiao; Hu, Bo; Liu, Xiufan; Liu, Haiyan
Autologous tumor vaccine modified with nonlytic Newcastle disease virus (ATV-NDV) is a promising vaccine for cancer immunotherapy. IL-7 plays a critical role in lymphocyte development and homeostasis. To improve the efficacy of ATV-NDV, we inserted the murine IL-7 gene into the genome of nonlytic NDV strain LX using reverse genetic system. The insertion of the IL-7 gene neither affected the main features of NDV replication nor its tumor selectivity. The gene product was biologically active and stable. Then we tested the antitumor effects of the autologous tumor vaccine modified with LX/(IL-7) in the murine tumor models. We showed that tumor cells modified with LX/IL-7 induced a strong antitumor activity both in prophylaxis and therapeutic models. The IFN-γ production and the cytotoxicity of tumor-specific CD8(+) T cells were significantly enhanced after immunization with tumor cells modified with LX/(IL-7) in both models. Although the tumor-infiltrating CD4(+) T cells and CD8(+) T cells were both increased and their IFN-γ productions also were upregulated, the antitumor activity of the tumor vaccine modified with LX/(IL-7) was dependent on CD8(+) T cells. Our results demonstrated that the autologous tumor vaccine modified with NDV strain LX/(IL-7) could promote the antitumor immune responses mediated by CD8(+) T cells and significantly improve the efficacy of the ATV-NDV. PMID 24943214

Chemotherapy and Oncolytic Virotherapy: Advanced Tactics in the War against Cancer.
Juni 2014 | Nguyen, Andrew; Ho, Louisa; Wan, Yonghong
Cancer is a traitorous archenemy that threatens our survival. Its ability to evade detection and adapt to various cancer therapies means that it is a moving target that becomes increasingly difficult to attack. Through technological advancements, we have developed sophisticated weapons to fight off tumor growth and invasion. However, if we are to stand a chance in this war against cancer, advanced tactics will be required to maximize the use of our available resources. Oncolytic viruses (OVs) are multi-functional cancer-fighters that can be engineered to suit many different strategies; in particular, their retooling can facilitate increased capacity for direct tumor killing (oncolytic virotherapy) and elicit adaptive antitumor immune responses (oncolytic immunotherapy). However, administration of these modified OVs alone, rarely induces successful regression of established tumors. This may be attributed to host antiviral immunity that acts to eliminate viral particles, as well as the capacity for tumors to adapt to therapeutic selective pressure. It has been shown that various chemotherapeutic drugs with distinct functional properties can potentiate the antitumor efficacy of OVs. In this review, we summarize the chemotherapeutic combinatorial strategies used to optimize virally induced destruction of tumors. With a particular focus on pharmaceutical immunomodulators, we discuss how specific therapeutic contexts may alter the effects of these synergistic combinations and their implications for future clinical use. PMID 24967214

Oncolytic therapy of a recombinant Newcastle disease virus D90 strain for lung cancer.
Juni 2014 | Chai, Zheng; Zhang, Peiyi; Fu, Fang; Zhang, Xueyun; Liu, Ying; Hu, Lihua; Li, Xi
Lung cancer is one of the leading causes of deaths from cancer worldwide. Tumor virotherapy using naturally oncolytic Newcastle disease virus (NDV) has been shown to be safe and effective in preclinical studies and clinical trials. Previously, we have reported the NDV D90 strain that was isolated from natural source has an antiproliferative effect in human lung cancer cell line A549. PMID 24885546

Oncolytic Newcastle Disease Virus as Cutting Edge between Tumor and Host.
Mai 2014 | Fournier, Philippe; Schirrmacher, Volker
Oncolytic viruses (OVs) replicate selectively in tumor cells and exert anti-tumor cytotoxic activity. Among them, Newcastle Disease Virus (NDV), a bird RNA virus of the paramyxovirus family, appears outstanding. Its anti-tumor effect is based on: (i) oncolytic activity and (ii) immunostimulation. Together these activities facilitate the induction of post-oncolytic adaptive immunity. We will present milestones during the last 60 years of clinical evaluation of this virus. Two main strategies of clinical application were followed using the virus (i) as a virotherapeutic agent, which is applied systemically or (ii) as an immunostimulatory agent combined with tumor cells for vaccination of cancer patients. More recently, a third strategy evolved. It combines the strategies (i) and (ii) and includes also dendritic cells (DCs). The first step involves systemic application of NDV to condition the patient. The second step involves intradermal application of a special DC vaccine pulsed with viral oncolysate. This strategy, called NDV/DC, combines anti-cancer activity (oncolytic virotherapy) and immune-stimulatory properties (oncolytic immunotherapy) with the high potential of DCs (DC therapy) to prime naive T cells. The aim of such treatment is to first prepare the cancer-bearing host for immunocompetence and then to instruct the patient's immune system with information about tumor-associated antigens (TAAs) of its own tumor together with danger signals derived from virus infection. This multimodal concept should optimize the generation of strong polyclonal T cell reactivity targeted against the patient's TAAs and lead to the establishment of a long-lasting memory T cell repertoire. PMID 24833054

Two is better than one: Complementing oncolytic virotherapy with gemcitabine to potentiate antitumor immune responses.
Mai 2014 | Gujar, Shashi A; Clements, Derek; Lee, Patrick Wk
Oncolytic viruses (OVs) preferentially infect and kill cancer cells. Additionally, OV-induced immune responses subvert cancer-associated immunosuppression and promote antitumor immunity. We have recently demonstrated that the complementation of oncolytic virotherapy with gemcitabine accentuates its immunostimulatory effects, hence exerting superior antineoplastic activity. PMID 24804161

Oncolytic viruses and their application to cancer immunotherapy.
Apr. 2014 | Chiocca, E Antonio; Rabkin, Samuel D
Oncolytic viruses (OV) selectively replicate and kill cancer cells and spread within the tumor, while not harming normal tissue. In addition to this direct oncolytic activity, OVs are also very effective at inducing immune responses to themselves and to the infected tumor cells. OVs encompass a broad diversity of DNA and RNA viruses that are naturally cancer selective or can be genetically engineered. OVs provide a diverse platform for immunotherapy; they act as in situ vaccines and can be armed with immunomodulatory transgenes or combined with other immunotherapies. However, the interactions of OVs with the immune system may affect therapeutic outcomes in opposing fashions: negatively by limiting virus replication and/or spread, or positively by inducing antitumor immune responses. Many aspects of the OV-tumor/host interaction are important in delineating the effectiveness of therapy: (i) innate immune responses and the degree of inflammation induced; (ii) types of virus-induced cell death; (iii) inherent tumor physiology, such as infiltrating and resident immune cells, vascularity/hypoxia, lymphatics, and stromal architecture; and (iv) tumor cell phenotype, including alterations in IFN signaling, oncogenic pathways, cell surface immune markers [MHC, costimulatory, and natural killer (NK) receptors], and the expression of immunosuppressive factors. Recent clinical trials with a variety of OVs, especially those expressing granulocyte macrophage colony-stimulating factor (GM-CSF), have demonstrated efficacy and induction of antitumor immune responses in the absence of significant toxicity. Manipulating the balance between antivirus and antitumor responses, often involving overlapping immune pathways, will be critical to the clinical success of OVs. PMID 24764576

Gene expression profiling in PC12 cells infected with an oncolytic Newcastle disease virus strain.
Apr. 2014 | Balogh, András; Bátor, Judit; Markó, Lajos; Németh, Mária; Pap, Marianna; Sétáló, György; Müller, Dominik N; Csatary, Laszlo K; Szeberényi, József
Although the oncolytic potential of natural, non-engineered Newcastle disease virus (NDV) isolates are well-known, cellular mechanisms determining NDV sensitivity of tumor cells are poorly understood. The aim of the present study was to look for gene expression changes in PC12 pheochromocytoma cells infected with an attenuated NDV strain that may be related to NDV susceptibility. PC12 cells were infected with the NDV strain MTH-68/H for 12h at a titer corresponding to the IC₅₀ value. Total cytoplasmic RNA samples isolated from control and MTH-68/H-infected cells were analyzed using a rat specific Affymetrix exon chip. Genes with at least 2-fold increase or decrease in their expression were identified. MTH-68/H-induced gene expression changes of 9 genes were validated using quantitative reverse transcriptase PCR. A total of 729 genes were up- and 612 genes were down-regulated in PC12 cells infected with MTH-68/H. Using the DAVID functional annotation clustering tool, the up- and down-regulated genes can be categorized into 176 and 146 overlapping functional gene clusters, respectively. Gene expression changes affecting the most important signaling mechanisms (Toll-like receptor signaling, RIG-I-like receptor signaling, interferon signaling, interferon effector pathways, apoptosis pathways, endoplasmic reticulum stress pathways, cell cycle regulation) are analyzed and discussed in detail in this paper. NDV-induced gene expression changes described in this paper affect several regulatory mechanisms and dozens of putative key proteins that may determine the NDV susceptibility of various tumors. Further characterization of these proteins may identify susceptibility markers to predict the chances of virotherapeutic treatment of human tumors. PMID 24637408

Genetically engineered Newcastle disease virus expressing interleukin 2 is a potential drug candidate for cancer immunotherapy.
Apr. 2014 | Bai, Fuliang; Niu, Zeshan; Tian, Hui; Li, Siming; Lv, Zheng; Zhang, Tianyuan; Ren, Guiping; Li, Deshan
Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, several clinical trials have reported that mesogenic NDV is a safe and effective agent for human cancer therapy. Interleukin 2 (IL2) is a cytokine that stimulates T cell propagation to trigger innate and adaptive immunity. IL2 has been used for cancer therapy and has achieved curative effects. In this study, a recombinant NDV LaSota strain expressing human interleukin 2 (rLaSota/IL2) was generated. The ability of rLaSota/IL2 to express human IL2 was detected in the infected tumor cells. In addition, the activity of IL2 was analyzed. The antitumor potential of rLaSota/IL2 was studied by xenograph mice carrying H22 and B16-F10 cells. Tumor-specific CD4(+) and CD8(+) T cells and MHC II were also analyzed in the two tumor-bearing models. Our study showed that rLaSota/IL2 significantly stimulated tumor-specific cytotoxic T-lymphocyte (CTL) responses and increased regulatory CD4(+) and cytotoxic CD8(+) T cells proliferation. The treatment with rLaSota/IL2 led to tumor regression in tumor-bearing mice and prolonged the survival of tumor-bearing mice. Furthermore, tumor challenging experiments demonstrated that rLaSota/IL2 invoked mice a unique capacity to remember a pathogen through the generation of memory T cells, which protect the host in the event of reinfection and form adaptive immune system. The result indicates that tumor-infiltrating CD4(+) T regulatory cells may denote the effective regression of tumors. Taken together, rLaSota/IL2 has potential for immunotherapy and oncolytic therapy of cancers and may be an ideal candidate for clinical application in future cancer therapy. PMID 24613899

Recombinant Newcastle disease virus Anhinga strain (NDV/Anh-EGFP) for hepatoma therapy.
März 2014 | Wu, Yunzhou; Yan, Shijun; Lv, Zheng; Chen, Lin; Geng, Jingshu; He, Jinjiao; Yu, Qingzhong; Yin, Jiechao; Ren, Guiping; Li, Deshan
Hepatocellular carcinoma remains one of the most common malignant tumors in the world. Newcastle disease virus (NDV) has been proved to be an efficient oncolytic agent. NDV tumor killing efficacy is not only dependening on the NDV strain but the type of tumor targeted. It is significant to discover more effective and safe oncolytic strains. We investigated the effectiveness of genetically engineered NDV Anhinga strain in hepatoma treatment. The modified virus containing an insertion of enhanced green fluorescent protein (EGFP), named NDV/Anh-EGFP. The antitumor efficacy of the recombinant virus on hepatoma was examined both in vivo and in vitro. NDV Anhinga strain, which could be classified as a lytic strain, is an effective oncolytic agent on hepatoma. There was no significant difference in the TCID50 and growth capability between the recombinant NDV and the parental. NDV/Anh-EGFP can obviously inhibit hepatocarcinoma development in vitro and in vivo. We demonstrate Anhinga strain could become a potent candidate for clinical carcinoma therapy especially for hepatocarcinoma. PMID 23819497

Localized oncolytic virotherapy overcomes systemic tumor resistance to immune checkpoint blockade immunotherapy.
März 2014 | Zamarin, Dmitriy; Holmgaard, Rikke B; Subudhi, Sumit K; Park, Joon Seok; Mansour, Mena; Palese, Peter; Merghoub, Taha; Wolchok, Jedd D; Allison, James P
Preexisting lymphocytic infiltration of tumors is associated with superior prognostic outcomes in a variety of cancers. Recent studies also suggest that lymphocytic responses may identify patients more likely to benefit from therapies targeting immune checkpoints, suggesting that therapeutic efficacy of immune checkpoint blockade can be enhanced through strategies that induce tumor inflammation. To achieve this effect, we explored the immunotherapeutic potential of oncolytic Newcastle disease virus (NDV). We find that localized intratumoral therapy of B16 melanoma with NDV induces inflammatory responses, leading to lymphocytic infiltrates and antitumor effect in distant (nonvirally injected) tumors without distant virus spread. The inflammatory effect coincided with distant tumor infiltration with tumor-specific CD4(+) and CD8(+) T cells, which was dependent on the identity of the virus-injected tumor. Combination therapy with localized NDV and systemic CTLA-4 blockade led to rejection of preestablished distant tumors and protection from tumor rechallenge in poorly immunogenic tumor models, irrespective of tumor cell line sensitivity to NDV-mediated lysis. Therapeutic effect was associated with marked distant tumor infiltration with activated CD8(+) and CD4(+) effector but not regulatory T cells, and was dependent on CD8(+) cells, natural killer cells, and type I interferon. Our findings demonstrate that localized therapy with oncolytic NDV induces inflammatory immune infiltrates in distant tumors, making them susceptible to systemic therapy with immunomodulatory antibodies, which provides a strong rationale for investigation of such combination therapies in the clinic. PMID 24598590

Different responses of human pancreatic adenocarcinoma cell lines to oncolytic Newcastle disease virus infection.
Feb. 2014 | Buijs, P R A; van Eijck, C H J; Hofland, L J; Fouchier, R A M; van den Hoogen, B G
Newcastle disease virus (NDV) is a naturally occurring oncolytic virus with clinically proven efficacy against several human tumor types. Selective replication in and killing of tumor cells by NDV is thought to occur because of differences in innate immune responses between normal and tumor cells. In our effort to develop oncolytic virotherapy with NDV for patients with pancreatic cancer, we evaluated the responses to NDV infection and interferon (IFN) treatment of 11 different established human pancreatic adenocarcinoma cell lines (HPACs). Here we show that all HPACs were susceptible to NDV. However, this NDV infection resulted in different replication kinetics and cytotoxic effects. Better replication resulted in more cytotoxicity. No correlation was observed between defects in the IFN pathways and NDV replication or NDV-induced cytotoxicity. IFN production by HPACs after NDV infection differed substantially. Pretreatment of HPACs with IFN resulted in diminished NDV replication and decreased the cytotoxic effects in most HPACs. These findings suggest that not all HPACs have functional defects in the innate immune pathways, possibly resulting in resistance to oncolytic virus treatment. These data support the rationale for designing recombinant oncolytic NDVs with optimized virulence that should likely contain an antagonist of the IFN pathways. PMID 24384773

Oncolytic viruses as therapeutic cancer vaccines.
Nov. 2013 | Bartlett, David L; Liu, Zuqiang; Sathaiah, Magesh; Ravindranathan, Roshni; Guo, Zongbi; He, Yukai; Guo, Zong Sheng
Oncolytic viruses (OVs) are tumor-selective, multi-mechanistic antitumor agents. They kill infected cancer and associated endothelial cells via direct oncolysis, and uninfected cells via tumor vasculature targeting and bystander effect. Multimodal immunogenic cell death (ICD) together with autophagy often induced by OVs not only presents potent danger signals to dendritic cells but also efficiently cross-present tumor-associated antigens from cancer cells to dendritic cells to T cells to induce adaptive antitumor immunity. With this favorable immune backdrop, genetic engineering of OVs and rational combinations further potentiate OVs as cancer vaccines. OVs armed with GM-CSF (such as T-VEC and Pexa-Vec) or other immunostimulatory genes, induce potent anti-tumor immunity in both animal models and human patients. Combination with other immunotherapy regimens improve overall therapeutic efficacy. Coadministration with a HDAC inhibitor inhibits innate immunity transiently to promote infection and spread of OVs, and significantly enhances anti-tumor immunity and improves the therapeutic index. Local administration or OV mediated-expression of ligands for Toll-like receptors can rescue the function of tumor-infiltrating CD8+ T cells inhibited by the immunosuppressive tumor microenvironment and thus enhances the antitumor effect. Combination with cyclophosphamide further induces ICD, depletes Treg, and thus potentiates antitumor immunity. In summary, OVs properly armed or in rational combinations are potent therapeutic cancer vaccines. PMID 24020520

TRAIL is involved in the tumoricidal activity of mouse natural killer cells stimulated by Newcastle disease virus in vitro.
Sep. 2013 | Song, De-Zhi; Liang, Ying; Xiao, Qing; Yin, Jun; Gong, Jin-Ling; Lai, Zhen-Ping; Zhang, Zeng-Feng; Gao, Ling-Xi; Fan, Xiao-Hui
Newcastle disease virus (NDV) is a potential antitumor agent, and its antitumor effect has been evaluated in preclinical tests. However, the mechanisms of NDV-based antitumor therapy are still not completely clear. In the present study we found that NDV-stimulation enhanced the killing ability of mouse spleen natural killer (NK) cells towards mouse hepatoma cell lines, and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) plays an important role in this tumoricidal activity. NDV stimulation induced up-regulation of TRAIL both at the mRNA and protein levels in NK cells. Blocking TRAIL by antibody (Ab) almost completely eliminated the killing effect of NK cells on hepatoma cell lines. Furthermore, neutralizing interferon (IFN)-γ by Ab could inhibit TRAIL expression and tumoricidal activity of NDV-stimulated NK cells. These results indicated a substantial role of TRAIL as an effector molecule in NDV-induced NK cells mediated tumoricidal activity. The NDV stimulation triggered TRAIL expression in mouse spleen NK cells could be mediated by IFN-γ induction. PMID 23959779

The oncolytic activity of Newcastle disease virus in clear cell renal carcinoma cells in normoxic and hypoxic conditions: the interplay between von Hippel-Lindau and interferon-β signaling.
Juli 2013 | Ch'ng, Wei-Choong; Stanbridge, Eric J; Yusoff, Khatijah; Shafee, Norazizah
Viral-mediated oncolysis is a promising cancer therapeutic approach offering an increased efficacy with less toxicity than the current therapies. The complexity of solid tumor microenvironments includes regions of hypoxia. In these regions, the transcription factor, hypoxia inducible factor (HIF), is active and regulates expression of many genes that contribute to aggressive malignancy, radio-, and chemo-resistance. To investigate the oncolytic efficacy of a highly virulent (velogenic) Newcastle disease virus (NDV) in the presence or absence of HIF-2α, renal cell carcinoma (RCC) cell lines with defective or reconstituted wild-type (wt) von Hippel-Lindau (VHL) activity were used. We show that these RCC cells responded to NDV by producing only interferon (IFN)-β, but not IFN-α, and are associated with increased STAT1 phosphorylation. Restoration of wt VHL expression enhanced NDV-induced IFN-β production, leading to prolonged STAT1 phosphorylation and increased cell death. Hypoxia augmented NDV oncolytic activity regardless of the cells' HIF-2α levels. These results highlight the potential of oncolytic NDV as a potent therapeutic agent in the killing of hypoxic cancer cells. PMID 23506478

Oncolytic virus therapy for cancer: the first wave of translational clinical trials.
März 2013 | Patel, Manish R; Kratzke, Robert A
The field of oncolytic virus therapy, the use of live, replicating viruses for the treatment of cancer, has expanded rapidly over the past decade. Preclinical models have clearly demonstrated anticancer activity against a number of different cancer types. Several agents have entered clinical trials and promising results have led to late stage clinical development for some viruses. The early clinical trials have demonstrated that oncolytic viruses by themselves have potential to result in tumor regression. Engineering of viruses to express novel genes have also led to the use of these vectors as a novel form of gene therapy. As a result, interest in oncolytic virus therapy has gained traction. The following review will focus on the first wave of clinical translation of oncolytic virus therapy, what has been learned so far, and potential challenges ahead for advancing the field. PMID 23313629

Prostate-specific antigen-retargeted recombinant newcastle disease virus for prostate cancer virotherapy.
März 2013 | Shobana, Raghunath; Samal, Siba K; Elankumaran, Subbiah
Oncolytic virus (OV) therapies of cancer are based on the use of replication-competent, tumor-selective viruses with limited toxicity. Newcastle disease virus (NDV), an avian paramyxovirus, is a promising OV and is inherently tumor selective and cytotoxic only to tumor cells. Replication is restricted in normal cells. Despite encouraging phase I/II clinical trials with NDV, further refinements for tumor-specific targeting are needed to enhance its therapeutic index. Systemically delivered NDV fails to reach solid tumors in therapeutic concentrations and also spreads poorly within the tumors due to barriers including complement, innate immunity, and the extracellular matrix. Overcoming these hurdles is paramount to realizing the exceptional oncolytic efficacy of NDV. We engineered the F protein of NDV and generated a recombinant NDV (rNDV) whose F protein is cleavable exclusively by prostate-specific antigen (PSA). The rNDV replicated efficiently and specifically in prostate cancer (CaP) cells and 3-dimensional prostaspheres but failed to replicate in the absence of PSA. Induction of intracellular PSA production by a synthetic androgen analog (R1881) enhanced fusogenicity in androgen-responsive CaP cells. Further, PSA-cleavable rNDV caused specific lysis of androgen-independent and androgen-responsive/nonresponsive CaP cells and prostaspheres, with a half-maximal effective concentration (EC50) ranging from a multiplicity of infection of 0.01 to 0.1. PSA-retargeted NDV efficiently lysed prostasphere tumor mimics, suggesting efficacy in vivo. Also, PSA-cleavable NDV failed to replicate in chicken embryos, indicating no pathogenicity for chickens. Prostate-specific antigen targeting is likely to enhance the therapeutic index of rNDV owing to tumor-restricted replication and enhanced fusogenicity. PMID 23345509

Virotherapy of neuroendocrine tumors.
Feb. 2013 | Essand, Magnus
Most patients with small intestinal neuroendocrine tumors (SI-NETs), also referred to as midgut carcinoids, present with systemic disease at the time of diagnosis with metastases primarily found in regional lymph nodes and the liver. Curative treatment is not available for these patients and there is a need for novel and specific therapies. Engineered oncolytic viruses may meet the need and play an important role in the future management of SI-NET liver metastases. This review focuses on adenovirus as the oncolytic anti-cancer agent and its potential curative role for SI-NET liver metastases, but it also summarizes the use of oncolytic viruses for NETs in general. It discusses how specific features of neuroendocrine cell biology can be used to engineer viruses to become selective for infection of NET cells and/or replication within NET cells. In addition, it points out the advantages and shortcomings of using replicating viruses in the treatment of cancer and addresses research fields that can increase the efficacy of virus-based therapy. PMID 22378183

Bispecific antibodies and trispecific immunocytokines for targeting the immune system against cancer: preparing for the future.
Feb. 2013 | Fournier, Philippe; Schirrmacher, Volker
Monoclonal anti-tumor antibodies (mAbs) that are clinically effective usually recruit, via their constant fragment (Fc) domain, Fc receptor (FcR)-positive accessory cells of the immune system and engage these additionally against the tumor. Since T cells are FcR negative, these important cells are not getting involved. In contrast to mAbs, bispecific antibodies (bsAbs) can be designed in such a way that they involve T cells. bsAbs are artificially designed molecules that bind simultaneously to two different antigens, one on the tumor cell, the other one on an immune effector cell such as CD3 on T cells. Such dual antibody constructs can cross-link tumor cells and T cells. Many such bsAb molecules at the surface of tumor cells can thus build a bridge to T cells and aggregate their CD3 molecules, thereby activating them for cytotoxic activity. BsAbs can also contain a third binding site, for instance a Fc domain or a cytokine that would bind to its respective cytokine receptor. The present review discusses the pros and cons for the use of the Fc fragment during the development of bsAbs using either cell-fusion or recombinant DNA technologies. The recombinant antibody technology allows the generation of very efficient bsAbs containing no Fc domain such as the bi-specific T-cell engager (BiTE). The strong antitumor activity of these molecules makes them very interesting new cancer therapeutics. Over the last decade, we have developed another concept, namely to combine bsAbs and multivalent immunocytokines with a tumor cell vaccine. The latter are patient-derived tumor cells modified by infection with a virus. The virus-Newcastle Disease Virus (NDV)-introduces, at the surface of the tumor cells, viral molecules that can serve as general anchors for the bsAbs. Our strategy aims at redirecting, in an Fc-independent fashion, activities of T cells and accessory cells against autologous tumor antigens. It creates very promising perspectives for a new generation of efficient and safe cancer therapeutics that should confer long-lasting anti-tumor immunity. PMID 23329400

The use of oncolytic viruses to overcome lung cancer drug resistance.
Okt. 2012 | Beljanski, Vladimir; Hiscott, John
Intrinsic and acquired drug resistance remains a fundamental obstacle to successful applications of anticancer therapies for lung cancer. Combining conventional therapies with immunotherapeutic approaches is a promising strategy to circumvent lung cancer drug resistance. Genetically modified oncolytic viruses (OVs) kill tumor cells via completely unique mechanisms compared to small molecule chemotherapeutics typically used in lung cancer treatment and can also be used to deliver specific toxic, therapeutic or immunomodulatory genes to tumor cells. Recent pre-clinical and clinical studies with oncolytic vaccine approaches have revealed promising combination strategies that enhance oncolysis of tumor cells and circumvent tumor resistance mechanisms. As clinical trials with oncolytic vaccines progress, and as the knowledge acquired from these studies builds a foundation demonstrating OVs safety and efficacy, novel combination approaches could soon have a major impact on the clinical management of patients diagnosed with lung cancer. PMID 22910124

Two avirulent, lentogenic strains of Newcastle disease virus are cytotoxic for some human pancreatic tumor lines in vitro.
Sep. 2012 | Walter, Robert J; Attar, Bashar M; Rafiq, Asad; Delimata, Megan; Tejaswi, Sooraj
Pancreatic cancer is the fourth leading cause of cancer death in the U.S. Highly infectious Newcastle disease virus (NDV) strains are known to be very cytotoxic for an array of human tumor cell types in vitro and in vivo but the effects of these and avirulent NDV strains on pancreatic neoplasms are little known. PMID 22964957

Velogenic newcastle disease virus as an oncolytic virotherapeutics: in vitro characterization.
Aug. 2012 | Kumar, Rajiv; Tiwari, Ashok K; Chaturvedi, Uttara; Kumar, G Ravi; Sahoo, Aditya P; Rajmani, R S; Saxena, Lovleen; Saxena, Shikha; Tiwari, Sangeeta; Kumar, Sudesh
Cancer is one of the killer diseases in humans and needs alternate curative measures despite recent improvement in modern treatment modalities. Oncolytic virotherapy seems to be a promising nonconventional way to treat cancers. Newcastle disease virus (NDV), a poultry virus, is nonpathogenic to human and domestic animals and has a long history of being used in oncotherapy research in several preclinical studies. The ability of NDV to successfully infect and destroy cancer cells is dependent on the strain and the pathotype of the virus. Adaptation of viruses to heterologous hosts without losing its replicative and oncolytic potential is prerequisite for use as cancer virotherapeutics. In the present study, velogenic NDV was adapted for replication in HeLa cells, and its cytotoxic potential was evaluated by observing morphological, biochemical, and nuclear landmarks of apoptosis. Our results indicated that the NDV-induced apoptosis in HeLa cells was dependent on upregulation of TNF-related apoptosis-inducing ligand (TRAIL) and caspases activation. Different determinants of apoptosis evaluated in the present study indicated that this strain could be a promising candidate for cancer therapy in future. PMID 22644640

Cell-type-specific innate immune response to oncolytic Newcastle disease virus.
Aug. 2012 | Biswas, Moanaro; Kumar, Sandeep R P; Allen, Adria; Yong, Wang; Nimmanapalli, Ramadevi; Samal, Siba K; Elankumaran, Subbiah
Virotherapy of cancer exploits the potential of naturally occurring and engineered oncolytic viruses to selectively replicate in and cause cytotoxicity to tumor cells without affecting healthy normal cells. The tumor selectivity of Newcastle disease virus (NDV), a member of the family Paramyxoviridae, depends on the differential type I interferon (IFN) response. Further understanding of the key mechanisms and immune effector molecules involved will aid in augmenting the oncolytic properties of NDV. Here we report on the infection kinetics and innate immune responses to a recombinant LaSota strain of NDV (rLaSota eGFP) in human tumor and normal cells. We observed varying replicative fit and cytotoxicity of rLaSota eGFP depending on the tumor cell type, with severely restricted replication in normal cells. The absence of retinoic acid-inducible gene I (RIG-I), a cytosolic RNA sensor, determined sensitivity to NDV. Productive NDV infection with a moderate IFN-α induction in human multiple myeloma cells suggested a role for IFN-independent mechanisms or lack of type I IFN reinforcement by RIG-I. Proinflammatory cytokines and chemokines were altered differentially in infected normal and tumor cells. Our results suggest that tumor selectivity is dependent on variations in the cellular antiviral response to infection with NDV and RIG-I expression. PMID 22808996

Recent clinical experience with oncolytic viruses.
Aug. 2012 | Donnelly, O G; Errington-Mais, F; Prestwich, R; Harrington, K; Pandha, H; Vile, R; Melcher, A A
There has been interest in using viruses to treat cancer for over a century. Recent clinical efforts, driven on by significant preclinical advances, have focussed on the safety of using replication-competent viruses. Recently published clinical trials of six oncolytic viruses (adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus and vaccinia) have added to the accumulating data that endorse oncolytic viruses as a safe and well tolerated treatment approach. Conclusive evidence of efficacy remains to be demonstrated, but randomised clinical trials are now underway. PMID 21740364

Newcastle disease virus: a promising agent for tumour immunotherapy.
Juli 2012 | Zhao, Lixiang; Liu, Haiyan
Malignant tumours are a major cause of mortality in humans. Currently used therapeutic regimens have not improved survival rates of patients suffering from malignant tumours much because of their limited efficacy and side-effects. A therapeutic approach that uses Newcastle disease virus (NDV) represents an attractive new tool for tumour immunotherapy. The present review highlights the mechanisms and advances that are likely to have considerable impact on NDV virotherapy. Significant evidence exists regarding the oncolytic effects of NDV, suggesting its potential use in the treatment of various tumours. Furthermore, clinical trials have suggested that several NDV strains have the potential for cancer virotherapy with few side-effects compared with traditional treatments. Many studies have been performed to investigate the oncolytic mechanisms of NDV. Apoptosis following NDV infection may contribute to the observed oncolytic effects; however, NDV could also stimulate both innate and adaptive antitumour immune responses. For many years, different approaches have been investigated (or are in the process of being developed) regarding the use of NDV for the treatment of malignancies. Recent advances using reverse genetics have provided a means of generating recombinant NDV strains with improved oncolytic and immune regulatory properties. PMID 22211810

Construction of recombinant Newcastle disease virus Italien strain for oncolytic virotherapy of tumors.
Juli 2012 | Wei, Ding; Sun, Na; Nan, Gang; Wang, Yuan; Liu, Hong-Qi; Peeters, Ben; Chen, Zhi-Nan; Bian, Huijie
Newcastle disease virus (NDV) is a naturally oncolytic virus that has been shown to be safe and effective for cancer therapy. Tumor virotherapy using NDV emerged in the 1950s and has advanced more recently by the increased availability of reverse genetics technology. In this study, we constructed a reverse genetics system based on the virulent and oncolytic NDV Italien strain, and generated two recombinant NDVs carrying a gene encoding either enhanced green fluorescent protein or firefly luciferase. We evaluated the replication and antitumor characteristics of these viruses in vitro and in vivo. Our data showed that the insertion of exogenous reporter genes did not affect NDV replication and sensitivity to type I interferon. The recombinant NDVs kept the property of tumor-selective replication both in vitro and in vivo and strongly induced syncytium formation leading to cell death. Moreover, the recombinant NDVs significantly prolonged the survival of tumor-bearing athymic mice (p=0.017) and suppressed the loss of body weight after intratumoral injection. Taken together, our study provides a novel platform to develop recombinant oncolytic viruses based on the NDV Italien strain and shows the efficiency of recombinant NDV Italien for oncolytic virotherapy of tumors. PMID 22372930

Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication.
Mai 2012 | Meng, Chunchun; Zhou, Zhizhi; Jiang, Ke; Yu, Shengqing; Jia, Lijun; Wu, Yantao; Liu, Yanqing; Meng, Songshu; Ding, Chan
Newcastle disease virus (NDV) can replicate in tumor cells and induce apoptosis in late stages of infection. However, the interaction between NDV and cells in early stages of infection is not well understood. Here, we report that, shortly after infection, NDV triggers the formation of autophagosomes in U251 glioma cells, as demonstrated by an increased number of double-membrane vesicles, GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) a dot formations, and elevated production of LC3II. Moreover, modulation of NDV-induced autophagy by rapamycin, chloroquine or small interfering RNAs targeting the genes critical for autophagosome formation (Atg5 and Beclin-1) affects virus production, indicating that autophagy may be utilized by NDV to facilitate its own production. Furthermore, the class III phosphatidylinositol 3-kinase (PI3K)/Beclin-1 pathway plays a role in NDV-induced autophagy and virus production. Collectively, our data provide a unique example of a paramyxovirus that uses autophagy to enhance its production. PMID 22398914

Apoptin enhances the oncolytic properties of Newcastle disease virus.
Mai 2012 | Wu, Yantao; Zhang, Xiaorong; Wang, Xiaobo; Wang, Li; Hu, Shunlin; Liu, Xiufan; Meng, Songshu
Naturally occurring strains of Newcastle disease virus (NDV) have demonstrated the potential to kill cancer cells in both preclinical and clinical studies. Previous studies showed that apoptin, the VP3 protein of chicken infectious anemia virus, is a p53-independent, Bcl-2-insensitive apoptotic protein with the ability to specifically induce apoptosis in transformed cells. In this study, we tested the hypothesis that apoptin enhances NDV-mediated tumor cell death. PMID 21865658

Anti-leukemic activity of Newcastle disease virus strains AF2240 and V4-UPM in murine myelomonocytic leukemia in vivo.
März 2012 | Alabsi, Aied M; Ali, Rola; Ideris, Aini; Omar, Abdul Rahman; Bejo, Mohd Hair; Yusoff, Khatijah; Ali, Abdul Manaf
Newcastle disease virus (NDV) is a member of the Paramyxoviridae that has caused severe economic losses in poultry industry worldwide. Several strains of NDV were reported to induce cytolysis to cancerous cell lines. It has prompted much interest as anticancer agent because it can replicate up to 10,000 times better in human cancer cells than in most normal cells. In this study, two NDV strains, viserotropic-velogenic strain AF2240 and lentogenic strain V4-UPM, showed cytolytic activity and apoptosis induction against Mouse myelomoncytic leukemia (WEHI 3B). The cytolytic effects of NDV strains were determined using microtetrazolium (MTT) assay. The cytolytic dose - fifty percent (CD(50)) were 2 and 8HAU for AF2240 and V4-UPM strains, respectively. Cells treated with NDV strains showed apoptotic features compared to the untreated cells under fluorescence microscope. NDV induced activation of caspase-3 and DNA laddering in agarose gel electrophoresis which confirmed the apoptosis. The anti-leukemic activity of both strains was evaluated on myelomoncytic leukemia BALB/c mice. The results indicated that both NDV strains significantly decreased liver and spleen weights. It also decreased blasts cell percentage in blood, bone marrow and spleen smears of treated mice (p<0.05). Histopathological studies for spleen and liver confirmed the hematological results of blood and bone marrow. From the results obtained, the exposure to both NDV stains AF2240 and V4-UPM showed similar results for Ara-c. In conclusion NDV strains AF2240 and V4-UPM can affect WEHI 3B leukemia cells in vitro and in vivo. PMID 22133641

Targeting pediatric cancer stem cells with oncolytic virotherapy.
März 2012 | Friedman, Gregory K; Cassady, Kevin A; Beierle, Elizabeth A; Markert, James M; Gillespie, G Yancey
Cancer stem cells (CSCs), also termed "cancer-initiating cells" or "cancer progenitor cells," which have the ability to self-renew, proliferate, and maintain the neoplastic clone, have recently been discovered in a wide variety of pediatric tumors. These CSCs are thought to be responsible for tumorigenesis and tumor maintenance, aggressiveness, and recurrence due to inherent resistance to current treatment modalities such as chemotherapy and radiation. Oncolytic virotherapy offers a novel, targeted approach for eradicating pediatric CSCs using mechanisms of cell killing that differ from conventional therapies. Moreover, oncolytic viruses have the ability to target specific features of CSCs such as cell-surface proteins, transcription factors, and the CSC microenvironment. Through genetic engineering, a wide variety of foreign genes may be expressed by oncolytic viruses to augment the oncolytic effect. We review the current data regarding the ability of several types of oncolytic viruses (herpes simplex virus-1, adenovirus, reovirus, Seneca Valley virus, vaccinia virus, Newcastle disease virus, myxoma virus, vesicular stomatitis virus) to target and kill both CSCs and tumor cells in pediatric tumors. We highlight advantages and limitations of each virus and potential ways in which next-generation engineered viruses may target resilient CSCs. PMID 22430386

Oncolytic Newcastle disease virus for cancer therapy: old challenges and new directions.
März 2012 | Zamarin, Dmitriy; Palese, Peter
Newcastle disease virus (NDV) is an avian paramyxovirus, which has been demonstrated to possess significant oncolytic activity against mammalian cancers. This review summarizes the research leading to the elucidation of the mechanisms of NDV-mediated oncolysis, as well as the development of novel oncolytic agents through the use of genetic engineering. Clinical trials utilizing NDV strains and NDV-based autologous tumor cell vaccines will expand our knowledge of these novel anticancer strategies and will ultimately result in the successful use of the virus in the clinical setting. PMID 22393889

Newcastle disease virus induces apoptosis in cisplatin-resistant human lung adenocarcinoma A549 cells in vitro and in vivo.
Jan. 2012 | Meng, Songshu; Zhou, Zhizhi; Chen, Fei; Kong, Xiangang; Liu, Huairan; Jiang, Ke; Liu, Wenbo; Hu, Maozhi; Zhang, Xiaorong; Ding, Chan; Wu, Yantao
Cisplatin (DDP) is widely used in lung cancer chemotherapy. However, cisplatin resistance represents a major obstacle in effective clinical treatment. This study aims to investigate whether Newcastle disease virus (NDV) exhibits an oncolytic effect on cisplatin-resistant A549 lung cancer cells. We found that NDV induced A549/DDP cell apoptosis via the caspase pathway, particularly involving caspase-9, while the mitogen-activated protein kinase (MAPK) and Akt pathways also contributed to apoptotic induction. Furthermore, NDV displayed oncolytic effects in a mouse A549/DDP lung cancer model. Collectively, our data indicate that NDV could overcome the cisplatin resistance in lung cancer cells in vitro and in vivo. PMID 22095029

Newcastle disease virus LaSota strain kills human pancreatic cancer cells in vitro with high selectivity.
Jan. 2012 | Walter, Robert J; Attar, Bashar M; Rafiq, Asad; Tejaswi, Sooraj; Delimata, Megan
Pancreatic cancer is highly resistant to treatment. Previously, we showed that Newcastle disease virus (NDV) strain 73-T was highly cytotoxic to a range of tumor types in vitro and in vivo but the effects of NDV on pancreatic tumors are unknown. We determined the cytotoxicity of the lentogenic LaSota strain of NDV (NDV-LS) toward 7 different human pancreatic tumor cell lines and 4 normal human cell lines (keratinocytes, fibroblasts, pancreatic ductal cells, and vascular endothelial cells). PMID 22233946

Newcastle disease virus induces pro-inflammatory conditions and type I interferon for counter-acting Treg activity.
Jan. 2012 | Fournier, Philippe; Arnold, Annette; Wilden, Holger; Schirrmacher, Volker
Newcastle disease virus (NDV) is a negative sense RNA paramyxovirus of birds which in human tumor cells, in contrast to human non-tumor cells, has shown replication competence leading to tumor cell death (i.e., tumor selectivity and viral oncolysis). Our study demonstrates that this virus induces high levels of pro-inflammatory cytokines in the bronchial lavage fluid of mice after nasal application and also in vitro in human dendritic cells (DCs). NDV is known as a very efficient inductor of type I interferon (IFN). The presented data show the key role played by the cell surface receptor to type I IFN (IFNAR) but not by the interferon transcription factors IRF-3 and IRF-7 in the induction of the important pro-inflammatory cytokine IL-12 upon transcription of NDV genes in DCs. We show that NDV activates in infected cells the helicase RIG-I. In Tregs, the activation of RIG-I was shown in other studies to inhibit the suppressive function of these cells. We thus conclude that NDV in tumor therapy may help to stimulate T effector cells but also to block Treg cells, thereby alleviating a brake to antitumor activity. PMID 22102168

Safety and clinical usage of newcastle disease virus in cancer therapy.
Dez. 2011 | Lam, Han Yuen; Yeap, Swee Keong; Rasoli, Mehdi; Omar, Abdul Rahman; Yusoff, Khatijah; Suraini, Abd Aziz; Alitheen, Noorjahan Banu
Newcastle disease virus (NDV) is an avian virus that causes deadly infection to over 250 species of birds, including domestic and wild-type, thus resulting in substantial losses to the poultry industry worldwide. Many reports have demonstrated the oncolytic effect of NDV towards human tumor cells. The interesting aspect of NDV is its ability to selectively replicate in cancer cells. Some of the studies have undergone human clinical trials, and favorable results were obtained. Therefore, NDV strains can be the potential therapeutic agent in cancer therapy. However, investigation on the therapeutic perspectives of NDV, especially human immunological effects, is still ongoing. This paper provides an overview of the current studies on the cytotoxic and anticancer effect of NDV via direct oncolysis effects or immune stimulation. Safety of NDV strains applied for cancer immunotherapy is also discussed in this paper. PMID 22131816

Oncolytic viruses for the treatment of neuroendocrine tumors.
Nov. 2011 | Essand, M; Leja, J; Giandomenico, V; Oberg, K E
Oncolytic viruses are emerging as anticancer agents, and they have also shown great promise for use against neuroendocrine tumors. Many viruses have a natural tropism for replication in tumor cells. Others can be genetically engineered to selectively kill tumor cells. Viruses have some advantages as therapeutic agents over current cytotoxic drugs and small molecules. They replicate in tumor cells and thereby increase in number over time leading to increased dosage. They are immunogenic and can alter the immunosuppressive tumor microenvironment and activate immune effector cells. They have also been shown to be able to kill drug-resistant cancer stem cells. This article reviews the recent literature on oncolytic viruses used so far for neuroendocrine tumors and indicates important issues to focus on in the future. PMID 21626470

Importance of retinoic acid-inducible gene I and of receptor for type I interferon for cellular resistance to infection by Newcastle disease virus.
Okt. 2011 | Fournier, Philippe; Wilden, Holger; Schirrmacher, Volker
Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic properties which shows promising effects in the treatment of cancer. Anti-cancer effects are due to the virus ability: i) to replicate in and kill tumor cells, leading finally to their selective elimination; and ii) to induce the stimulation of antitumor activities in immune cells. NDV does not harm normal cells and has a high safety profile. In this study, we first report a direct correlation between the degree of cell resistance to NDV infection and the cellular expression of the retinoic acid-inducible gene I (RIG-I) which is a cytosolic viral RNA receptor. RIG-I plays an important role in the recognition of and response to infection by RNA viruses. We also demonstrate that impairment of the interferon (IFN) pathway through deletion of the receptor for type I IFN (IFNR1) in primary macrophages leads to NDV replication. In tumor cells, addition of exogenous IFN-α4 is shown to lead to tumor growth reduction and inhibition of viral replication. Finally, increase of the RIG-I concentration of tumor cells via plasmid transfection is shown to be associated with a stronger resistance to NDV infection. These findings shed new light on the crucial role played by the cytosolic receptor RIG-I and the plasma membrane receptor IFNR1 as key molecules to protect cells against infection by NDV. PMID 21971670

Analysis of three properties of Newcastle disease virus for fighting cancer: tumor-selective replication, antitumor cytotoxicity, and immunostimulation.
Sep. 2011 | Fournier, Philippe; Bian, Huijie; Szeberényi, József; Schirrmacher, Volker
Newcastle disease virus (NDV), a bird paramyxovirus, is an antitumor agent which has shown benefits to cancer patients. Its antineoplastic efficacy appears to be associated with three properties of the virus: 1. Selective replication in tumor cells. This feature can be studied at the RNA level, for example by RT-PCR, and at the protein level by immunochemistry. 2. Oncolytic properties (of some strains). The use of cultures of tumor cell lines represents a selective model to study direct viral oncolysis at the cellular level. The capacity of NDV to lyse tumor cells can be analyzed in vitro using cytotoxic assays based on the WST1 chemical reagent. The endoplasmic reticulum stress, which is induced by infection with the oncolytic NDV strain MTH-68/H and which plays an important role in the viral oncolytic effects, can be analyzed by Western blotting using specific monoclonal antibodies. Such stress appears as a key component of NDV cytotoxicity. 3. Immunostimulatory capacity. We describe an in vitro test called "Tumor Neutralisation Assay" which allows the analysis of bystander antitumor immune effects induced in human peripheral blood mononuclear cells by NDV. There are two variants, one for oncolytic NDV strains and the other one for nonlytic NDV strains. NDV may use several mechanisms to exert its tumor-killing action: direct cytotoxicity against cancer cells but also nonspecific as well as active-specific antitumor immune responses from the host organism. All the methods described here allow to evaluate the different oncolytic and immunostimulatory capacities of various strains of NDV. They are crucial to harness optimal antitumor activity by appropriate combinations of virus strains and application regimens. PMID 21948477

Therapeutic effects of a fusogenic newcastle disease virus in treating head and neck cancer.
Sep. 2011 | Li, Pingdong; Chen, Chun-Hao; Li, Sen; Givi, Babak; Yu, Zhenkun; Zamarin, Dmitriy; Palese, Peter; Fong, Yuman; Wong, Richard J
Newcastle disease virus (NDV) is a paramyxovirus that is pathogenic in birds but causes only mild flulike symptoms in human beings. NDV(F3aa)-GFP is a genetically modified, fusogenic NDV. We assessed the utility of NDV(F3aa)-GFP in treating head and neck squamous cell carcinoma. PMID 21928411

Improvement of dendritic cell therapy in glioblastoma multiforme WHO 4 by Newcastle disease virus
Sep. 2011 | Nesselhut, J

2011 ASCO Annual Meeting; Oral Abstract Session, Developmental Therapeutics - Clinical Pharmacology and Immunotherapy

Background: Glioblastoma multiforme (GBM, WHO grade 4 glioma) is an aggressive disease with an unfavorable prognosis. The current first line treatment comprises radical operation and radiotherapy combined with temozolomide chemotherapy. The reported median overall survival time after primary diagnosis is less than 15 months. After failure of first-line therapy there is currently no effective therapy, and side effects from further treatments may potentially impair quality of life. We report that immunotherapy with monocyte-derived dendritic cells (MoDC) combined with oncolytic Newcastle Disease Virus (NDV) can improve the efficacy of dendritic cell based immune therapy for relapsed GBM. Methods: After isolating monocytes from peripheral blood of n=37 patients with stage IV GBM who failed first-line radio-chemotherapy (PFI: 8 months), MoDC were generated using standard protocols. In 19 patients, NDV was added to the MoDC on day 5. These patients were also pretreated with an infusion with NDV one day before vaccination. The MoDC were harvested on day 7 of culture and administered to the patients intradermally. Results: Improvement of the clinical response was observed in patients, who received combination of NDV with MoDC vs. MoDC alone (47% vs. 11%). The median survival after onset of DC-therapy was 3 months with MoDC alone and 10 months (23 months after primary diagnosis) in combination with NDV. The 1- year-survival rates after onset of DC therapy were 6% and 32%, respectively. The therapy was well tolerated without any major side effects. IFN-gamma Elispot analyses from patients who received NDV injections and NDV-primed MoDC show that MoDC primed with NDV can induce a specific CD4 and CD8 T-cell response against NDV whereas healthy donors show no specific T-cell response. Conclusions: The efficacy of dendritic-cell based therapy for GBM can be improved by combination with NDV and may prolong overall survival of patients after failure of first-line therapy. For a first time we demonstrate that NDV primed MoDC induces a NDV specific T-cell response in humans, which may lead to in-vivo lysis of NDV-infected tumor cells.

Caspase- and p38-MAPK-dependent induction of apoptosis in A549 lung cancer cells by Newcastle disease virus.
Juli 2011 | Bian, Jianchun; Wang, Kai; Kong, Xiangang; Liu, Huairan; Chen, Fei; Hu, Maozhi; Zhang, Xiaorong; Jiao, Xinan; Ge, Baoxue; Wu, Yantao; Meng, Songshu
Newcastle disease virus (NDV) has a potential oncolytic effect due to its ability to induce apoptosis in tumor cells. However, previous studies have indicated discrepancies regarding the apoptosis signaling pathways induced by NDV in tumor cells. Here, we show that NDV infection induces simultaneous activation of intrinsic and extrinsic death pathways in A549 human lung cancer cells. In contrast, endoplasmic reticulum (ER) stress is not activated in NDV-induced apoptosis. We demonstrate for the first time that mitogen-activated protein kinase (MAPK) pathways are activated in NDV-infected A549 cells, and p38 MAPK is involved in NDV-induced cell death. Together, our findings provide novel insights into the underlying mechanisms by which NDV induces apoptosis in tumor cells. PMID 21625975

Important role of interferon regulatory factor (IRF)-3 in the interferon response of mouse macrophages upon infection by Newcastle disease virus.
Juni 2011 | Wilden, Holger; Schirrmacher, Volker; Fournier, Philippe
Newcastle disease virus (NDV) is an interesting agent for activating innate immune activity in macrophages including secretion of TNF-α and IFN-α, upregulation of TRAIL and activation of NF-κB and iNOS. However, the molecular mechanism of such cellular activities remains largely unknown. Tumor selectivity of replication of NDV has been described to be linked to deviations in tumor cells of the type I interferon response. We therefore focused on the interferon response to NDV of macrophages as part of innate anti-viral and anti-tumor activity. In particular, we investigated the functional significance of the interferon regulatory factor genes (IRF)-3 and IRF-7. Deletion of the IRF-3 or IRF-7 gene was found to increase susceptibility of mouse macrophages to virus infection. Surprisingly, NDV replicated better in IRF-3 KO than in IRF-7 KO macrophages. Further analysis showed that IRF-3 KO macrophages have a lower basal and NDV-induced RIG-I expression in comparison to IRF-7 KO macrophages. This might explain why, in IRF-3 KO macrophages, the secretion of type I interferons after NDV infection is delayed, when compared to IRF-7 KO and wild-type macrophages. In addition, IRF-3 KO cells showed reduced NDV-induced levels of IRF-7. This effect could be prevented by priming the cells first by interferon-α. Further results indicated that an early production of type I interferon rather than high maximal levels at later time points are important for resistance to infection by NDV. In conclusion, these results demonstrate an important role of IRF-3 for the innate anti-viral response to NDV of mouse macrophages. PMID 21567079

Oncolytic specificity of Newcastle disease virus is mediated by selectivity for apoptosis-resistant cells.
Mai 2011 | Mansour, Mena; Palese, Peter; Zamarin, Dmitriy
Newcastle disease virus (NDV) is a negative-sense RNA virus that has been shown to possess oncolytic activity. NDV's selective replication in tumor cells has been previously suggested to be due to the lack of a proper antiviral response in these cells. Here we demonstrate that NDV possesses oncolytic activity in tumor cells capable of a robust type I interferon (IFN) response, suggesting that another mechanism underlies NDV's tumor specificity. We show that the oncolytic selectivity of NDV for tumor cells is dependent upon tumor cell resistance to apoptosis. Utilizing the human non-small-cell lung cancer cell line A549 overexpressing the antiapoptotic protein Bcl-xL, we show significant enhancement of oncolytic activity and NDV replication. Interestingly, while the Bcl-xL-overexpressing cells were resistant to apoptotic stimuli induced by chemotherapeutic agents and early viral replication, during the subsequent viral cycles, we observed a paradoxical increase in apoptosis in response to NDV. The increased oncolytic activity seen was secondary to enhanced viral replication and syncytium formation. The induction of a type I IFN response was enhanced in Bcl-xL cells. Overall, these findings propose a new mechanism for cancer cell specificity for NDV, making it an attractive anticancer agent for chemoresistant tumors with enhanced antiapoptotic activity. PMID 21471241

Targeting of IL-2 and GM-CSF immunocytokines to a tumor vaccine leads to increased anti-tumor activity.
Apr. 2011 | Fournier, Philippe; Aigner, Maximilian; Schirrmacher, Volker
Fusion proteins combining antibodies with cytokines such as IL-2 and GM-CSF appear to be promising reagents for tumor therapy. In this study, we combined such immunocytokines with the tumor vaccine ATV-NDV consisting of irradiated tumor cells infected with Newcastle disease virus (NDV). The two fusion proteins bsF-GMCSF and tsHN-IL2-GM-CSF, binding, respectively, to the viral fusion protein (F) or to hemagglutinin-neuraminidase (HN) expressed on the surface of the vaccine cells and containing GM-CSF or GM-CSF and IL-2-activities were produced by recombinant antibody technology. The purified molecules showed the expected binding specificity and biological activity inherent to the respective cytokine. Using a newly established in vitro tumor neutralisation assay (TNA), we showed improved antitumoral effect through tumor growth inhibition when human peripheral blood mononuclear cells from healthy donors were stimulated with immunocytokine modified versus non-modified tumor vaccine cells. These effects induced by the fusion proteins, in the presence of a suboptimal T cell activation signal 1 provided by bsHN-CD3, occured only when these were bound to the tumor vaccine. Furthermore, it was shown that CD14+ monocytes could be activated by the GM-CSF cytokine fused within the recombinant proteins and that they contributed essentially to the antitumor effect in the TNA. The data presented here suggest an easy way for a broad clinical development and application of tumor-targeted IL-2- and GM-CSF-based immunocytokines based on the associated increase of anti-tumor activity mediated by T cells and monocytes. PMID 21424118

IL-15 and type I interferon are required for activation of tumoricidal NK cells by virus-infected dendritic cells.
Apr. 2011 | Boudreau, Jeanette E; Stephenson, Kyle B; Wang, Fuan; Ashkar, Ali A; Mossman, Karen L; Lenz, Laurel L; Rosenthal, Kenneth L; Bramson, Jonathan L; Lichty, Brian D; Wan, Yonghong
There is increasing evidence that natural killer (NK) cells play an important role in antitumor immunity following dendritic cell (DC) vaccination. Little is known, however, about the optimal stimulation of DCs that favors NK activation in tumor-bearing hosts. In this study, we demonstrate that treatment with toll-like receptor (TLR) ligands and infection with a mutant vesicular stomatitis virus (VSV-ΔM51) both induced DC maturation. Further, inoculation of these DCs led to robust NK-mediated protection against tumor challenge. Strikingly, only VSV-ΔM51-infected DCs were capable of suppressing the growth of established tumors, suggesting that additional signals provided by viral infection may be required to activate tumoricidal NK cells in tumor-bearing hosts. VSV-ΔM51 infection of DCs induced greater type I interferon (IFN I) production than TLR ligand treatment, and disruption of the IFN I pathway in DCs eliminated their ability to induce NK activation and tumor protection. However, further studies indicated that IFN I alone was not sufficient to activate NK cells, especially in the presence of a tumor, and DC-derived IL-15 was additionally required for tumoricidal NK activation. These results suggest that induction of IFN I by VSV-ΔM51 allows DCs to overcome tumor-associated immunosuppression and facilitate IL-15-mediated priming of tumoricidal NK cells. Thus, the mode of DC maturation should be carefully considered when designing DC-based cancer immunotherapies. PMID 21307131

[Roles of Newcastle disease virus in human acute monocytic leukemia in vitro and in vivo].
Feb. 2011 | Wang, Ya-Jun; Song, Chun; Li, Xiao-Hui; Zhang, Jian-Bai
Some research has shown that Newcastle disease virus (NDV) is effective in the treatment of various tumors, including transferred melanoma and well differentiated renal cell carcinoma. This study aimed to evaluate the effect of NDV against human acute monocytic leukemia SHI-1 cells in vitro and in vivo. PMID 21342628

Antitumor vaccination by Newcastle Disease Virus Hemagglutinin-Neuraminidase plasmid DNA application: changes in tumor microenvironment and activation of innate anti-tumor immunity.
Jan. 2011 | Ni, Jing; Galani, Ioanna E; Cerwenka, Adelheid; Schirrmacher, Volker; Fournier, Philippe
A plasmid encoding the Hemagglutinin-Neuraminidase (HN) protein of Newcastle Disease Virus (pHN) was tested for its capacity to stimulate innate anti-tumor activity in tumor-bearing mice. We observed that application of the pHN plasmid at the ear pinna site (i.e.) of mice induces higher levels of systemic interferon-α and reduced tumor growth in the prophylactic mammary carcinoma DA3 tumor model in comparison to application of a control plasmid not encoding the HN protein. Analysis of the tumor microenvironment revealed a significant increase in NK cell infiltration and decrease in infiltration of CD11b(+)Gr-1(high) myeloid cells bearing the myeloid-derived suppressor cell (MDSC) phenotype after vaccination with the pHN DNA compared to a control DNA. Finally, innate immunity and partially type I IFN responses were proved important for the reduction of s.c. RMA-S tumor growth after pHN vaccination, as shown with the use of RAG2(-/-) and RAG2(-/-)IFNAR1(-/-) mice. These data demonstrate that triggering innate immunity by pHN application at the ear pinna of mice modulates the immune cell compartment in the tumor microenvironment and reduces tumor growth. This highlights thus the potential adjuvant activity of the HN gene in tumor therapy. PMID 21172381

[Progress in using Newcastle disease virus for tumor therapy: a review].
Nov. 2010 | Wu, Yunzhou; Hao, Jingbo; Li, Deshan
Naturally existing Newcastle disease virus (NDV) can specifically execute oncolytic ability in clinical studies. Reports from clinical trials using NDV as oncolytic agents showed promise and warrant results in cancer therapy. In recent years, reverse genetics technology has been used widely in the studies of NDV virology. More recently, the technology was applied to optimize the oncolytic efficacy of NDV, for instance, modification of the F gene, and expression of GM-CSF, IFN-gamma, IL-2 or TNF-alpha. NDV is widely investigated in cancer therapy and will definitely offer a prosperous future for clinical cancer therapeutics. We reviewed the developments of cancer therapy by recombinant NDV using reverse genetics technology, as well as our own experience in this domain. PMID 21090105

Use of attenuated paramyxoviruses for cancer therapy.
Nov. 2010 | Lech, Patrycja J; Russell, Stephen J
Paramyxoviruses, measles virus (MV), mumps virus (MuV) and Newcastle disease virus (NDV), are well known for causing measles and mumps in humans and Newcastle disease in birds. These viruses have been tamed (attenuated) and successfully used as vaccines to immunize their hosts. Remarkably, pathogenic MuV and vaccine strains of MuV, MV and NDV efficiently infect and kill cancer cells and are consequently being investigated as novel cancer therapies (oncolytic virotherapy). Phase I/II clinical trials have shown promise but treatment efficacy needs to be enhanced. Technologies being developed to increase treatment efficacy include: virotherapy in combination with immunosuppressive drugs (cyclophosphamide); retargeting of viruses to specific tumor types or tumor vasculature; using infected cell carriers to protect and deliver the virus to tumors; and genetic manipulation of the virus to increase viral spread and/or express transgenes during viral replication. Transgenes have enabled noninvasive imaging or tracking of viral gene expression and enhancement of tumor destruction. PMID 21087107

Transcriptome analysis and cytokine profiling of naive T cells stimulated by a tumor vaccine via CD3 and CD25.
Nov. 2010 | Fournier, Philippe; Aigner, Maximilian; Schirrmacher, Volker
T-cell receptor engagement by peptide/MHC complexes constitutes the main signal for the activation of naive T cells, but for a productive generation and maintenance of effector cells, full activation requires additional signals driven by costimulatory molecules present on activated antigen-presenting cells. Herein we describe T cell costimulation via CD25, the interleukin (IL)-2 receptor, during priming of naive T cells with a tumor vaccine. To this end, we produced, purified and characterized the fusion protein bsHN-IL2 which contains the IL-2 cytokine and an antibody scFv fragment directed towards the Hemagglutinin-Neuraminidase (HN) protein of Newcastle Disease Virus (NDV). Tumor vaccine cells were modified by infection with this virus which allows the attachment of the immunocytokine bsHN-IL2. In the presence of CD3-mediated signal 1, the vaccine/bsHN-IL2 provided via CD25 a strong bystander antitumor effect in vitro leading to tumor growth inhibition, even stronger than the vaccine/bsHN-CD28 which provides costimulation via CD28. Transcriptome analysis of naive T cells which were stimulated with the vaccine/bsHN-IL2 showed, similarly to the vaccine/bsHN-CD28, upregulation of 71 genes belonging to different signalling pathways, including PLC-γ1, Grb-2, Vav-1 and PDE-4A. Analysis of the supernatants of activated T cells with ligand-bound tumor vaccine showed that the vaccine/bsHN-IL2, in contrast to the vaccine/bsHN-CD28, did not lead to the production of additional IL-2. We report here the first transcriptome analysis of IL-2 receptor mediated costimulatory signals. The findings provide new insights into mechanisms of function of IL-2 during T cell priming. PMID 21042712

Genetically engineered oncolytic Newcastle disease virus effectively induces sustained remission of malignant pleural mesothelioma.
Okt. 2010 | Silberhumer, Gerd R; Brader, Peter; Wong, Joyce; Serganova, Inna S; Gönen, Mithat; Gonzalez, Segundo Jaime; Blasberg, Ronald; Zamarin, Dmitriy; Fong, Yuman
Malignant pleural mesothelioma is a highly aggressive tumor. Alternative treatment strategies such as oncolytic viral therapy may offer promising treatment options in the future. In this study, the oncolytic efficacy and induction of tumor remission by a genetically engineered Newcastle disease virus [NDV; NDV(F3aa)-GFP; GFP, green fluorescent protein] in malignant pleural mesothelioma is tested and monitored by bioluminescent tumor imaging. The efficacy of NDV(F3aa)-GFP was tested against several mesothelioma cell lines in vitro. Firefly luciferase-transduced MSTO-211H* orthotopic pleural mesothelioma tumor-bearing animals were treated with either single or multiple doses of NDV(F3aa)-GFP at different time points (days 1 and 10) after tumor implantation. Tumor burden was assessed by bioluminescence imaging. Mesothelioma cell lines exhibited dose-dependent susceptibility to NDV lysis in the following order of sensitivity: MSTO-211H > MSTO-211H* > H-2452 > VAMT > JMN. In vivo studies with MSTO-211H* cells showed complete response to viral therapy in 65% of the animals within 14 days after treatment initiation. Long-term survival in all of these animals was >50 days after tumor installation (control animals, <23 d). Multiple treatment compared with single treatment showed a significantly better response (P = 0.005). NDV seems to be an efficient viral oncolytic agent in the therapy of malignant pleural mesothelioma in an orthotopic pleural mesothelioma tumor model. PMID 20858727

Optimization studies for the coupling of bispecific antibodies to viral anchor molecules of a tumor vaccine.
Sep. 2010 | Fournier, Philippe; Aigner, Maximilian; Schirrmacher, Volker
Tumor vaccines have to provide several signals for T cell activation. Among them, signal 1 (through TCR/CD3) and signal 2 (through CD28) are the most important. We herein describe a procedure to introduce anti-CD3 and anti-CD28 signals into any tumor cell which is susceptible to infection by Newcastle disease virus (NDV). We developed the ATV-NDV tumor vaccine which consists of patient-derived tumor cells (ATV) modified through infection by NDV. We tested for further improvement of vaccine efficiency the addition of two bispecific single-chain antibodies. They bind with one arm to the viral hemagglutinin-neuraminidase (HN) or fusion (F) protein of NDV expressed at the surface of the vaccine cells while the second arm is directed either against CD3 or CD28 of T cells. The aim of this study was to optimize the coupling of these new reagents to the tumor vaccine. When anti-CD3 and anti-CD28 molecules bind to the same anchoring viral molecule (e.g. HN), competition for binding could occur under certain conditions. This was not the case when the bispecific reagents bound to separate viral molecules (HN or F, respectively). When using transfectants expressing HN and F either separately or on the same cell, we show that T cell activation works best when anti-CD3 and anti-CD28 are attached to the same stimulator cell. The clinical application of such a combined therapy with ATV-NDV vaccine cells and bi-specific antibodies allows to modify the strength of signal 1 and 2 in a quantitative and predictable way according to the immune status of the T cells and the requirements of the patients' immune system. PMID 20878068

The hemagglutinin-neuraminidase gene of Newcastle Disease Virus: a powerful molecular adjuvant for DNA anti-tumor vaccination.
Sep. 2010 | Ni, Jing; Schirrmacher, Volker; Fournier, Philippe
Plasmid-encoded DNA vaccine is a novel and potentially powerful tool for cancer therapy. Since the strength of immune responses induced by DNA vaccine is usually rather low, a major goal in DNA vaccine development is to enhance vaccine-induced immunity. In this study, we investigated an approach based on the use of a viral surface protein with pleiotropic function as a potential immune enhancer. To this end, we prepared bicistronic DNA plasmids encoding the hemagglutinin-neuraminidase (HN) protein of Newcastle Disease Virus in addition to a tumor target antigen. We demonstrate a higher tumor antigen-specific T cell-mediated immune response and a lower humoral response upon vaccination with a bicistronic DNA plasmid with incorporated HN gene. In a prophylactic immunization tumor model with the surrogate tumor antigen beta-galactosidase (β-gal) and in a therapeutic immunization tumor model with the xenogeneic tumor antigen human Epithelial Cell Adhesion Molecule (hEpCAM), HN gene incorporation into the DNA vaccine led to better survival and tumor regression in mice. There was also cross protection in the therapeutic tumor model against a second challenge by the parental mouse mammary carcinoma cells in mice vaccinated with the bicistronic plasmids. This is the first report describing the HN protein as an immunomodulator for enhanced antigen-specific T cell responses via DNA plasmids. The results show that co-expression of HN with a tumor target antigen through bicistronic vectors ensures precise temporal and spatial co-delivery to direct anti-tumor immune responses preferentially towards Th1. PMID 20709006

[Anti-tumor immunity of Newcastle disease virus HN protein is influenced by differential subcellular targeting].
Aug. 2010 | Wang, Kaibing; Sui, Hong; Li, Lejing; Li, Xi; Wang, Lei
Hemagglutinin-neuraminidase (HN) protein of newcastle disease virus is an important immunogen for oncolysis. We designed three different expression plasmids encoding the HN protein targeted to different subcellular compartments: cytoplasmic (Cy-HN), secreted (Sc-HN) and membrane-anchored (M-HN). On the basis of antitumor effect in vitro, the aim of this study is to investigate the anti-tumor immunity effect of HN protein in vivo. PMID 20704816

New perspectives in cancer virotherapy: bringing the immune system into play.
Juli 2010 | Boisgerault, Nicolas; Tangy, Frédéric; Gregoire, Marc
Despite constant advances in medically orientated cancer studies, conventional treatments by surgery, chemotherapy or radiotherapy remain partly ineffective against numerous cancers. Oncolytic virotherapy - the use of replication-competent viruses that specifically target tumor cells - has opened up new perspectives for improved treatment of these pathologies. Certain viruses demonstrate a natural, preferential tropism for tumor cells, while others can be genetically modified to show such an effect. Several of these viruses have already been used in preclinical and clinical trials in different tumor models; these studies have provided encouraging results and, thus, confirm the growing interest presented by this therapeutic strategy. The role of the immune system in the efficacy of cancer virotherapy has been poorly documented for a long time; however, several recent reports have presented evidence of synergistic effects between both direct viral oncolysis and the activation of specific, anti-tumor immune responses. These findings offer an exciting outlook for the future of cancer virotherapy. PMID 20635927

Hyperthermia potentiates oncolytic herpes viral killing of pancreatic cancer through a heat shock protein pathway.
Juli 2010 | Eisenberg, David P; Carpenter, Susanne G; Adusumilli, Prasad S; Chan, Mei-Ki; Hendershott, Karen J; Yu, Zhenkun; Fong, Yuman
Oncolytic herpes simplex virus-1 (HSV-1) is designed to specifically infect, replicate in, and lyse cancer cells. This study investigates a novel therapeutic regimen, combining the effects of NV1066 (a recombinant HSV-1) and hyperthermia in the treatment of pancreatic cancer. PMID 20633729

Antitumor efficacy of viral therapy using genetically engineered Newcastle disease virus [NDV(F3aa)-GFP] for peritoneally disseminated gastric cancer.
Mai 2010 | Song, Kyo Young; Wong, Joyce; Gonzalez, Lorena; Sheng, Gang; Zamarin, Dmitriy; Fong, Yuman
Peritoneal dissemination is a common and fatal clinical manifestation of gastric cancer with few effective therapies available. Natural Newcastle disease virus (NDV) has been shown to be an effective oncolytic agent, and recent advances now allow genetic manipulation of this virus to improve cancer killing and safety. This study was designed to investigate the effectiveness of a genetically engineered NDV in the treatment of peritoneally disseminated gastric carcinoma. NDV mutant virus containing a modified F cleavage site and insertion of enhanced green fluorescent protein (GFP), NDV(F3aa)-GFP, was tested in vitro against human gastric cancer cells by standard cytotoxicity at different multiplicities of infection. To test NDV(F3aa)-GFP in vivo in a peritoneal carcinomatosis gastric tumor model, MKN-74 human gastric cancer cells were injected intraperitoneally (IP) in severe combined immunodeficient mice. Mice were treated with NDV(F3aa)-GFP either once or multiple times after tumor challenge. Effective killing of MKN-74 cells by NDV(F3aa)-GFP was found in vitro. This cancer killing was dose-related and correlated with viral replication. GFP expression was a good marker of infection. The virus was also effective as an antitumor therapy in a peritoneal cancer model that simulates clinical disease. Half the animals treated with virus had no evidence of disease. Genetically engineered NDV [NDV(F3aa)-GFP] administered IP is an effective antitumor therapy against peritoneal carcinomatosis from human gastric cancer in a xenograft model, without significant toxicity. These data provide further rationale for clinical trials involving NDV for peritoneal carcinomatosis from gastric cancer. PMID 20393691

The anti-tumor effect of Newcastle disease virus HN protein is influenced by differential subcellular targeting.
Apr. 2010 | Sui, Hong; Bai, Yuxian; Wang, Kaibing; Li, Xi; Song, Chun; Fu, Fang; Zhang, Yongxin; Li, Lejing
Immunotherapy is emerging as a major player in the current standard of care for aggressive cancers such as non-small cell lung cancer (NSCLC). The Newcastle disease virus with its tumor-specific replicative and oncolytic abilities is a promising immunotherapeutic candidate. A DNA vaccine expressing the major immunogenic hemagglutinin-neuraminidase (HN) protein of this virus has shown promising results as an immunotherapeutic agent. PMID 20130861

Rac1 is required for oncolytic NDV replication in human cancer cells and establishes a link between tumorigenesis and sensitivity to oncolytic virus.
Apr. 2010 | Puhlmann, J; Puehler, F; Mumberg, D; Boukamp, P; Beier, R
Oncolytic Newcastle disease virus (NDV) replicates selectively in most human tumor cells but not in normal cells. The relationship between tumorigenesis and the selective susceptibility of most tumor cells to oncolytic NDV replication is poorly understood. A multistage skin carcinogenesis model derived from non-tumorigenic HaCaT cells was used to systematically investigate the molecular mechanisms involved in the oncolytic NDV-sensitivity associated with tumorigenic transformation. No significant differences in interferon signaling were observed between the virus-sensitive tumor cells and the virus-resistant non-tumorigenic parental cells. Oncogenic H-Ras, which had been used for tumorigenic transformation, was shown to be necessary for virus replication but was not sufficient to render cells susceptible to NDV replication. By using an siRNA screening approach to search for virus-sensitizing genes in the tumorigenic cells, we could identify the small GTPase Rac1 as an oncogenic protein that is essential for NDV replication and anchorage-independent growth in tumorigenic cells. Furthermore, Rac1 expression was sufficient to render non-tumorigenic cells susceptible to NDV replication and to oncolytic cytotoxicity. This study establishes Rac1 as a link between tumorigenesis and oncolytic virus sensitivity in the HaCaT multistage skin carcinogenesis model. PMID 20101224

Oncolytic virus therapy for prostate cancer.
Apr. 2010 | Fukuhara, Hiroshi; Homma, Yukio; Todo, Tomoki
The use of replication-competent viruses that can selectively replicate in and destroy neoplastic cells is an attractive strategy for treating cancer. Various oncolytic viruses have been taken to clinical trials since a recombinant virus was first applied to cancer patients a decade ago. The concept of the therapy is simple: infectious virus kills the host cancer cells in the course of viral replication. It is important, however, that the virus does not harm the surrounding normal tissue. Oncolytic viruses can be classified largely into two groups: DNA viruses genetically engineered to achieve cancer specificity (e.g. adenovirus, herpes simplex virus and vaccinia) and RNA viruses of which human is not the natural host (e.g. Newcastle disease virus and reovirus). Prostate cancer has always been one of the major targets of oncolytic virus therapy development. The result of six clinical trials for prostate cancer has been published and several trials are now going on. Forty-eight of 83 (58%) patients evaluated in the phase I studies demonstrated a >25% decrease in serum prostate-specific antigen level without evidence of severe toxicities. The result shows the oncolytic virus therapy is promising toward clinical application. Here, we review the recent advances in the field and summarize the results from clinical trials. PMID 19832925

Engineered newcastle disease virus as an improved oncolytic agent against hepatocellular carcinoma.
Feb. 2010 | Altomonte, Jennifer; Marozin, Sabrina; Schmid, Roland M; Ebert, Oliver
Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, which is currently under investigation as a clinical oncolytic agent. Several clinical trials have reported NDV to be a safe and effective agent for cancer therapy; however, there remains a clear need for improvement in therapeutic outcome. The endogenous NDV fusion (F) protein directs membrane fusion, which is required for virus entry and cell-cell fusion. Here, we report a novel NDV vector harboring an L289A mutation within the F gene, which resulted in enhanced fusion and cytotoxicity of hepatocellular carcinoma (HCC) cells in vitro, as compared with the rNDV/F3aa control virus. In vivo administration of the recombinant vector, termed rNDV/F3aa(L289A), via hepatic arterial infusion in immune-competent Buffalo rats bearing multifocal, orthotopic liver tumors resulted in tumor-specific syncytia formation and necrosis, with no evidence of toxicity to the neighboring hepatic parenchyma. Furthermore, the improved oncolysis conferred by the L289A mutation translated to significantly prolonged survival compared with control NDV. Taken together, rNDV/F(L289A) represents a safe, yet more effective vector than wild-type NDV for the treatment of HCC, making it an ideal candidate for clinical application in HCC patients. PMID 19809404

The oncolytic activity of Newcastle disease virus NDV-HUJ on chemoresistant primary melanoma cells is dependent on the proapoptotic activity of the inhibitor of apoptosis protein Livin.
Dez. 2009 | Lazar, Itay; Yaacov, Barak; Shiloach, Tamar; Eliahoo, Elad; Kadouri, Luna; Lotem, Michal; Perlman, Riki; Zakay-Rones, Zichria; Panet, Amos; Ben-Yehuda, Dina
Patients with advanced melanoma usually do not benefit from conventional chemotherapy treatment. There is therefore a true need for a new kind of therapy for melanoma. One factor responsible for the poor prognosis of melanoma is the inhibitor of apoptosis protein (IAP) family member Livin. In this study, we applied a novel approach for the treatment of melanoma, using a unique strain of the oncolytic Newcastle disease virus (NDV-HUJ). We found that, unlike chemotherapeutic drugs, NDV-HUJ, a one-cycle replicating virus, overcomes the resistance to apoptosis of melanoma primary cultures that over express the Livin protein. In contrast, melanoma tumor cells that do not express Livin are relatively resistant to NDV-HUJ treatment. Furthermore, we show that NDV-HUJ-induced oncolysis is attributed to the dual function of Livin: although Livin inhibits apoptosis through the inhibition of caspases, under the robust apoptotic stimulation of NDV-HUJ, caspases can cleave Livin to create a truncated protein with a paradoxical proapoptotic activity. Thus, NDV-HUJ is a potent inducer of apoptosis that can overcome the antiapoptotic effect of Livin and allow cleavage of Livin into the proapoptotic tLivin protein. Moreover, the results indicate that the interferon system, which is functional in melanoma, is not involved in NDV-induced oncolysis. Taken together, our data offer the possibility of a new viral oncolytic treatment for chemoresistant melanoma. PMID 19864394

Polarization of human monocyte-derived dendritic cells to DC1 by in vitro stimulation with Newcastle Disease Virus.
Sep. 2009 | Fournier, P; Arnold, A; Schirrmacher, V
Dendritic cell (DC)-based tumor vaccines have been tested extensively to treat cancer patients. However, the results of several DC-based clinical trials have been disappointing. Amelioration of such a modality for cancer treatment seems warranted, i.e. by improving DC immunogenicity and polarization. The goal of our study was to evaluate the potential for immune activation of human DCs by incubating them in vitro with the Newcastle Disease Virus (NDV), a paramyxovirus with strong immunostimulatory properties. PMID 19785053

Activation of natural killer cells by newcastle disease virus hemagglutinin-neuraminidase.
Juli 2009 | Jarahian, Mostafa; Watzl, Carsten; Fournier, Philippe; Arnold, Annette; Djandji, Dominik; Zahedi, Sarah; Cerwenka, Adelheid; Paschen, Annette; Schirrmacher, Volker; Momburg, Frank
The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3zeta lacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV. PMID 19515783

Newcastle disease virus: a promising vector for viral therapy, immune therapy, and gene therapy of cancer.
Juli 2009 | Schirrmacher, Volker; Fournier, Philippe
This review deals with the avian paramyxovirus Newcastle disease virus (NDV) and describes properties that explain its oncolytic activity, its tumor-selective replication behavior, and its immune-stimulatory capacity with human cells. The strong interferon response of normal cells upon contact with NDV appears to be the basis for the good tolerability of the virus in cancer patients and for its immune stimulatory properties, whereas the weak interferon response of tumor cells explains the tumor selectivity of replication and oncolysis. Various concepts for the use of this virus for cancer treatment are pointed out and results from clinical studies are summarized. Reverse genetics technology has made it possible recently to clone the genome and to introduce new foreign genes thus generating new recombinant viruses. These can, in the future, be used to transfer new therapeutic genes into tumors and also to immunize against new emerging pathogens. The modular nature of gene transcription, the undetectable rate of recombination, and the lack of a DNA phase in the replication cycle make NDV a suitable candidate for the rational design of a safe and stable vaccine and gene therapy vector. PMID 19565923

Genetically engineered Newcastle disease virus for malignant melanoma therapy.
Juni 2009 | Zamarin, D; Vigil, A; Kelly, K; García-Sastre, A; Fong, Y
Despite the advances in cancer therapies in the past century, malignant melanoma continues to present a significant clinical challenge due to lack of chemotherapeutic response. Systemic therapy with immunostimulatory agents such as interferon and interleukin-2 (IL-2) has shown some promise, though each is associated with significant side effects. Over the past 50 years, oncolytic Newcastle disease virus (NDV) has emerged as an alternative candidate for cancer therapy. The establishment of reverse-genetics systems for the virus has allowed us to further manipulate the virus to enhance its oncolytic activity. Introduction of immunomodulatory molecules, especially IL-2, into the NDV genome was shown to enhance the oncolytic potential of the virus in a murine syngeneic colon carcinoma model. We hypothesize that a recombinant NDV expressing IL-2 would be an effective agent for therapy of malignant melanoma. We show that recombinant NDV possesses a strong cytolytic activity against multiple melanoma cell lines, and is effective in clearing established syngeneic melanoma tumors in mice. Moreover, introduction of murine IL-2 into NDV significantly enhanced its activity against syngeneic melanomas, resulting in increased overall animal survival and generation of antitumor immunity. These findings warrant further investigations of IL-2-expressing NDV as an antimelanoma agent in humans. PMID 19242529

The extrinsic RNA-sensing pathway for adjuvant immunotherapy of cancer.
Mai 2009 | Seya, Tsukasa; Matsumoto, Misako
Infection with RNA viruses presents a typical pattern of virus products, double-stranded RNA (dsRNA), and induces the maturation of antigen-presenting dendritic cell (mDC). There are several dsRNA sensors that are differentially distributed on the cell membrane and in the cytoplasm and are variably expressed depending on the cell type. Among these sensors, TLR3 links to the adaptor TICAM-1 (TRIF), which is characterized by its unique multipronged signaling cascades for cytokine/chemokine production, apoptosis and autophagy in both immune and tumor cells. In the context of mDC maturation, various cellular events are further induced in response to dsRNA; these include cross-priming followed by CD8+ CTL induction, NK activation and proliferation of CD4+ T cells including Th1, Th2, Treg and Th17 cells. In this review, we focus on the potential role of dsRNA in modulating the inflammatory milieu around mDCs and tumor-associated antigens to drive specific cellular effectors against the tumor. PMID 19184005

Enhancement of oncolytic properties of recombinant newcastle disease virus through antagonism of cellular innate immune responses.
Apr. 2009 | Zamarin, Dmitriy; Martínez-Sobrido, Luis; Kelly, Kaitlyn; Mansour, Mena; Sheng, Gang; Vigil, Adam; García-Sastre, Adolfo; Palese, Peter; Fong, Yuman
Newcastle disease virus (NDV) has been previously shown to possess oncolytic activity, causing specific lysis of cancerous but not normal cells. Here we show that despite these findings, the oncolytic efficiency of naturally occurring NDV strains can still be relatively low, as many tumors exhibit strong innate immune responses that suppress viral replication and spread. To overcome this problem, we generated a recombinant fusogenic NDV expressing influenza NS1 protein, a protein exhibiting interferon (IFN)-antagonist and antiapoptotic functions in human and mouse cells. Interestingly, the resultant virus was dramatically enhanced in its ability to form syncytia, lyse a variety of human and mouse tumor cell lines, and suppressed the induction of the cellular IFN responses. Using the aggressive syngeneic murine melanoma model, we show that the NDV-NS1 virus is more effective than virus not expressing NS1 in clearing the established footpad tumors and results in higher overall long-term animal survival. In addition, mice treated with NDV-NS1 exhibited no signs of toxicity to the virus and developed tumor-specific cytotoxic T lymphocyte (CTL) responses. These findings demonstrate that modulation of innate immune responses by NDV results in enhancement of its oncolytic properties and warrant further investigation of this strategy in design of oncolytic NDV vectors against human tumors. PMID 19209145

Newcastle disease virus represses the activation of human hepatic stellate cells and reverses the development of hepatic fibrosis in mice.
März 2009 | Li, Ya-Lin; Wu, Jiao; Wei, Ding; Zhang, Da-Wei; Feng, Hao; Chen, Zhi-Nan; Bian, Huijie
Activated hepatic stellate cells (HSCs) are the crucial factor responsible for liver fibrosis and involved in development of hepatocellular carcinoma (HCC) by interaction with tumour cells. Newcastle disease virus (NDV) has the oncolytic characteristics of intrinsically selective replication in neoplasia cells and transformed cells. But, NDV replication in HSCs and effects on hepatic fibrosis have not been reported. PMID 19192169

Cutting Edge: TLR-Dependent viral recognition along with type I IFN positive feedback signaling masks the requirement of viral replication for IFN-{alpha} production in plasmacytoid dendritic cells.
März 2009 | Kumagai, Yutaro; Kumar, Himanshu; Koyama, Shohei; Kawai, Taro; Takeuchi, Osamu; Akira, Shizuo
Plasmacytoid dendritic cells (pDCs) recognize RNA virus infection via TLRs and consequently produce vast amounts of type I IFN. Because nucleic acid-sensing TLRs reside in the intracellular membrane compartment, it is presumable that pDCs do not require cytoplasmic viral replication to recognize the infection. By checking Newcastle disease virus (NDV) RNA abundance in GFP(+) and GFP(-) pDCs from Ifna6gfp mice, we found that NDV replication was not detected in IFN-producing pDCs. GFP(+) pDC was induced in response to replication-incompetent NDV. In contrast, the replication-incompetent NDV failed to induce IFN-producing pDCs in type I IFNR-deficient mice. The lack of IFNR signaling led to the replication of NDV and the subsequent RIG-I-like helicase-dependent IFN-alpha production in pDCs. These results showed that detection of viruses via TLRs together with a type I IFN feedback system circumvents the requirement for viral replication-dependent recognition in pDCs. PMID 19299691

Expression of RIG-I, IRF3, IFN-beta and IRF7 determines resistance or susceptibility of cells to infection by Newcastle Disease Virus.
März 2009 | Wilden, Holger; Fournier, Philippe; Zawatzky, Rainer; Schirrmacher, Volker
Newcastle Disease Virus (NDV) is an avian paramyxovirus with anti-neoplastic and immune-stimulatory properties which has raised considerable interest for cancer therapy. To better understand the molecular nature of the tumor selective replication of NDV, we investigated the cellular responses of murine normal and tumor cells after infection by NDV. To this end, we compared the basal expression of different antiviral proteins as well as the expression induced by the addition of NDV to the cells in vitro and in vivo. Primary macrophages were found to be resistant to NDV infection and exhibited a high basal and induced expression of various antiviral genes. In contrast, macrophage-derived RAW tumor cells were highly susceptible to NDV infection and displayed a low expression of several antiviral genes. Macrophage-derived J774 tumor cells were intermediate with regard to NDV replication and antiviral gene expression. The responsiveness to exogenously added IFN-alpha was found highest in normal macrophages, lowest in the RAW cells, and intermediate in the J774 cells. We also analysed dendritic cells as well as additional normal and tumor cell types. A strong inverse correlation was obeserved between the susceptibility to infection and the basal expression of the antiviral genes RIG-I, IRF3, IRF7 and IFN-beta. A strong expression of these genes can explain the resistance of normal cells to NDV infection and a weak antiviral gene expression the broad susceptibility of tumor cells. PMID 19287954

Cross-infection of tumor cells by contact with T lymphocytes loaded with Newcastle disease virus.
März 2009 | Pfirschke, Christina; Schirrmacher, Volker
Oncolytic virotherapy, a new type of cancer therapy involving viruses with oncolytic and immunostimulatory potential, is based on tumor selective viral replication, resulting in a specific lysis of tumor cells. Effective tumor targeting of oncolytic viruses remains a major problem because only a fraction of systemically applied viruses can reach the tumor tissue. We describe for the first time in an in vitro co-culture system that T lymphocytes can be loaded with Newcastle disease virus (NDV) in such a way that the virus load will be transferred to the tumor target cells upon contact of the T cells with tumor cells. The effectiveness of this NDV 'hitchhiking' on T cells can be influenced by the amount of virus, the ratio of T cells to tumor cells, the activation status of the T cells and by the virulence of the virus as shown by flow cytometry, quantitative real-time PCR and fluorescence microscopy. In a tumor neutralization assay in vitro, monolayers of human tumor cells could be completely and effectively destroyed by the addition of polyclonally activated human T cells loaded with oncolytic NDV. This process involves the formation of large T cell clusters as revealed by phase-contrast microscopy. Loading of oncolytic NDV onto activated T cells and adoptive transfer into a tumor-bearing host might enhance the efficacy of adoptive T cell therapy of tumors as well as tumor targeting of oncolytic viruses. PMID 19287952

Newcastle disease virus-induced cytopathic effect in infected cells is caused by apoptosis.
März 2009 | Ravindra, P V; Tiwari, Ashok K; Ratta, Barkha; Chaturvedi, Uttara; Palia, Sudesh Kumar; Chauhan, R S
The velogenic Newcastle disease virus (NDV) causes highly infectious and economically significant Newcastle disease (ND) in birds of various species. In cell culture NDV induces cytopathic effect (CPE) characterized by rounding, vacuolation, syncytia formation and cell death. Aside from cell to cell fusion caused by the F and HN glycoprotein of the virus molecular events leading to cell death are not known. In the current study, NDV-infected Vero cells, at 48 h p.i., showed nuclear condensation, cytoplasm blebbing, DNA fragmentation, and phosphatidylserine translocation to the cell surface. In addition, virus-infected cells demonstrated decreased DNA content and an increased Bax to Bcl-2 ratio, p53 level and caspase 3, 8, 9 expression compared to mock-infected cells. Based on these results, it was concluded that CPE in NDV-infected cells was caused by to the induction of apoptosis with the involvement of p53 and the Bax, dependent apoptotic pathways. PMID 19152817

Newcastle diseases virus strain V4UPM displayed oncolytic ability against experimental human malignant glioma.
Feb. 2009 | Zulkifli, M M; Ibrahim, R; Ali, A M; Aini, I; Jaafar, H; Hilda, S S; Alitheen, N B; Abdullah, J M
Newcastle disease virus (NDV) is a virus of paramyxovirus family and lately has been studied for the treatment of cancer in human. In this study, we successfully determined the oncolysis potential of NDV vaccine, V4UPM tested on the human glioblastoma multiform cell line (DBTRG.05MG) and human glioblastoma astrocytoma cell line (U-87MG) in vitro and in vivo. The V4UPM strain is a modified V4 strain developed as thermostable feed pellet vaccine for poultry. PMID 18937888

Antitumoral immune response by recruitment and expansion of dendritic cells in tumors infected with telomerase-dependent oncolytic viruses.
Feb. 2009 | Edukulla, Ramakrishna; Ramakrishna, Edukulla; Woller, Norman; Mundt, Bettina; Knocke, Sarah; Gürlevik, Engin; Saborowski, Michael; Malek, Nisar; Manns, Michael P; Wirth, Thomas; Kühnel, Florian; Kubicka, Stefan
Virotherapy can potentially be used to induce tumor-specific immune responses and to overcome tumor-mediated tolerance mechanisms because apoptotic tumor cells are exposed together with viral danger signals during oncolysis. However, insufficient numbers of dendritic cells (DC) present at the site of oncolysis can limit a tumor-specific immune response and the resulting therapeutic benefit. We investigated MHC class I peptide-specific immune responses against model antigens ovalbumin (OVA) and hemagglutinin (HA) in mouse tumor models that support efficient replication of the oncolytic adenovirus hTert-Ad. Virotherapy resulted in peptide-specific cytotoxic T-cell responses against intracellular tumor antigens. Triggering of DC and T-cell infiltration to the oncolytic tumors by macrophage inflammatory protein 1alpha (MIP-1alpha, CCL3) and Fms-like tyrosine kinase-3 ligand (Flt3L) enhanced both antitumoral and antiviral immune responses. Although immune-mediated clearance of the virus can restrict therapeutic efficacy of virotherapy, MIP-1alpha/FLT3L-augmented hTert-Ad virotherapy inhibited local tumor growth more effectively than virotherapy alone. In agreement with the hypothesis that immune-mediated mechanisms account for improved outcome in MIP-1alpha/FLT3L virotherapy, we observed systemic antitumoral effects by MIP-1alpha/FLT3L virotherapy on uninfected lung metastasis in immunocompetent mice but not in nude mice. Furthermore, MIP-1alpha/FLT3L virotherapy of primary tumors was strongly synergistic with tumor DC vaccination in inhibition of established lung metastasis. Combined viroimmunotherapy resulted in long-term survival of 50% of treated animals. In summary, improvement of cross-presentation of tumor antigens by triggering of DC and T-cell infiltration during virotherapy enhances antitumoral immune response that facilitates an effective viroimmunotherapy of primary tumors and established metastases. PMID 19190348

Selective oncolytic effect of an attenuated Newcastle disease virus (NDV-HUJ) in lung tumors.
Nov. 2008 | Yaacov, B; Eliahoo, E; Elihaoo, E; Lazar, I; Ben-Shlomo, M; Greenbaum, I; Panet, A; Zakay-Rones, Z
Newcastle disease virus (NDV), an avian paramyxovirus, has a potential oncolytic effect that may be of significance in the treatment of a variety of cancer diseases. An attenuated lentogenic isolate of NDV (HUJ) demonstrated a selective cytopathic effect upon a panel of human and mouse lung tumor cells, as compared to human nontumorigenic lung cells. The virus-selective oncolytic effect is apoptosis dependent, and related to higher levels of viral transcription, translation and progeny virus formation. Furthermore, NDV-HUJ oncolytic activity is directed in-cis and not through induction of cytokines, that may act in-trans on neighboring cells. Development of primary lung tumors and of the consequent metastasis in mice inoculated with mouse lung tumor cells 3LL-D122 was decreased following treatment with NDV-HUJ. The preferential killing of the tumor cells is not due to a deficiency in the interferon (IFN) system, as expression of the IFN-beta gene, in the infected cells, is properly induced. Moreover, pretreatment with IFN effectively protected the tumor cells from the virus oncolytic effect. We conclude therefore, that NDV-HUJ should have a significant benefit in the treatment of lung cancer as well as other malignancies. PMID 18535620

Recombinant Newcastle disease virus as a vaccine vector for cancer therapy.
Okt. 2008 | Vigil, Adam; Martinez, Osvaldo; Chua, Mark A; García-Sastre, Adolfo
Naturally occurring strains of Newcastle disease virus (NDV) are currently being investigated in multiple clinical trials for oncolytic cancer therapy in the United States and abroad. We have previously reported, for the first time, the development of recombinant NDVs designed for enhanced cancer therapeutic efficacy. Specifically, we have shown that NDV engineered to express interleukin-2 (IL-2) generates a robust therapeutic response associated with increased tumor-specific T-cell infiltration after intratumoral administration in mice. We have now demonstrated that this therapeutic response is dependent on T cells and we have investigated the potential to focus the NDV-induced immune response toward a tumor-associated antigen (TAA) to enhance the inherent therapeutic efficacy of NDV further. We found that intratumoral treatments of tumor-bearing mice with recombinant NDV expressing a model TAA elicited an enhanced tumor-specific response, resulting in a significant increase in the number of complete tumor regressions compared with control NDV. Additionally, coadministration of NDV expressing a model TAA with NDV expressing IL-2 enhanced the TAA-directed response and led to more complete tumor regressions. Our results show that TAA-directed immunotherapy by oncolytic recombinant NDV alone or in combination with IL-2 results in an enhanced therapeutic efficacy and warrant consideration in the development of cancer therapies based on the use of oncolytic NDV. PMID 18714310

Efficiency of adjuvant active specific immunization with Newcastle disease virus modified tumor cells in colorectal cancer patients following resection of liver metastases: results of a prospective randomized trial.
Okt. 2008 | Schulze, T; Kemmner, W; Weitz, J; Wernecke, K-D; Schirrmacher, V; Schlag, P M
Metastatic disease is a major cause of mortality in colorectal cancer patients. Even after complete resection of isolated liver metastases, recurrence develops in the majority of patients. Therefore, development of strategies to prevent recurrent liver metastases is of major clinical importance. The present prospectively randomised phase III trial investigates the efficiency of active specific immunotherapy (ASI) after liver resection for hepatic metastases of colorectal cancer. PMID 18488223

Activation of human T cells by a tumor vaccine infected with recombinant Newcastle disease virus producing IL-2.
Sep. 2008 | Janke, Markus; Peeters, Ben; Zhao, Heng; de Leeuw, Olav; Moorman, Rob; Arnold, Annette; Ziouta, Yvonne; Fournier, Philippe; Schirrmacher, Volker
A new recombinant (rec) Newcastle disease virus (NDV) with incorporated human interleukin 2 (IL-2) as foreign therapeutic gene [rec(IL-2)] will be described. The foreign gene in rec(IL-2) did not affect the main features of NDV replication nor its tumor selectivity. Biologically active IL-2 was produced in high amounts by tumor cells infected with rec(IL-2). Tumor vaccine cells infected by rec(IL-2) stimulated human T cells to exert anti-tumor activity in vitro in a tumor neutralization assay. These effects were significantly increased when compared to vaccine infected by rec(-) virus without IL-2 gene. After incubation with rec(IL-2) infected tumor cells, T cells showed increased expression of the activation marker CD69 and produced increased amounts of IFNgamma when compared to T cells co-incubated with rec(-) infected tumor cells. CD8 T cells incubated with rec(IL-2) infected tumor cells showed upregulation of perforin, cell surface exposure of the degranulation marker CD107a and increased anti-tumor cytotoxic activity. Purified T cells from lymph nodes of head and neck squamous cell carcinoma (HNSCC) patients could be stimulated to secrete IFNgamma in an ELISPOT assay upon 40 h of stimulation with rec(IL-2) infected autologous tumor cells [ATV-rec(IL-2)] but not upon stimulation with rec(IL-2) infected allogeneic U937 tumor cells. This suggests direct activation of patient derived tumor antigen-specific memory T cells by ATV-rec(IL-2). In conclusion, the already inherent immunostimulatory properties of NDV could be further augmented by the introduction of the therapeutic gene IL-2. Active specific immunization of patients with ATV-rec(IL-2) should provide the microenvironment at the vaccination site with IL-2 and avoid side effects as seen after systemic IL-2 application. PMID 18813797

Oncolytic vaccinia virus: from bedside to benchtop and back.
Aug. 2008 | Thorne, Steve H
The field of oncolytic viral therapy has undergone a major shift in focus in the last few years. Less research has been directed at making incremental improvements in original vectors based mainly on strains of adenovirus and HSV; instead a variety of different viral strains have been suggested as potential backbones for future oncolytic viruses (including Newcastle disease virus, reovirus, vesicular stomatitis virus, polio virus, retrovirus, Sindbis virus, picornavirus, mumps and measles virus), with many of these progressing to clinical trials. Of these, vaccinia virus represents a particularly promising candidate. It possesses a variety of intrinsic molecular properties suitable for an oncolytic virus (such as rapid life cycle and lysis of infected cells, and an ability to infect various cell types), in addition to undergoing extensive study both in the laboratory and in the clinic. Although not a natural human pathogen, there are extensive data on the effects of vaccinia infection in humans. Preclinical models incorporating new oncolytic vaccinia strains, as well as data from the first clinical trials that have utilized the next-generation oncolytic vaccinia strains for the potential treatment of cancer have been described. PMID 18683104

Virus combinations and chemotherapy for the treatment of human cancers.
Aug. 2008 | Kumar, Shantanu; Gao, Lu; Yeagy, Brian; Reid, Tony
Oncolytic viruses possess an inherent trophism for tumor cells or have been engineered in a variety of ways to selectively replicate in and destroy cancer cells. Because of the unique mode of tumor destruction, oncolytic virotherapy has the potential to augment the antineoplastic activity of chemotherapy and radiation therapy. Many oncolytic viruses, such as adenovirus, HSV, vaccinia, measles, reovirus, Newcastle disease virus and coxsackie virus, have entered into clinical trials and their efficacy and safety have been demonstrated with few, minor, side effects. Data obtained from several clinical trials of the oncolytic adenovirus, ONYX-015, in patients with cancer have been described in detail. Some preclinical studies of oncolytic viruses have demonstrated promising results, mainly when administered in combination with chemotherapeutic drugs. In this review, the clinical use of oncolytic viruses in combination with chemotherapy and radiation therapy, and future directions to enhance the efficacy of oncolytic virotherapy, are discussed. PMID 18683102

Recombinant Newcastle disease virus expressing human interleukin-2 serves as a potential candidate for tumor therapy.
Juli 2008 | Zhao, Heng; Janke, Markus; Fournier, Philippe; Schirrmacher, Volker
A recombinant Newcastle disease virus (NDV) containing human interleukin-2 (IL-2) gene was generated by applying reverse genetics technique and further evaluated for its suitability to express and deliver IL-2 for cancer therapy. We have further analyzed the ability of rNDV/IL2 to express IL-2 in several human tumor cell lines, including the human breast carcinoma cell line MCF-7, the human colon-adenocarcinoma cell line HT29, and human Jurkat cell line. IL-2 expressed by tumor cells infected with rNDV/IL-2 was stable up till 16 days, at body temperature, and with biological activity. Expression kinetics indicated that the expression level of IL-2 was already high at 24 h after infection and reached the highest level at 48 h after infection. As NDV was proposed as a very promising oncolytic agent in a new age of therapeutic viruses, our data strongly support the application of recombinant NDV/IL-2 virus as an anti-tumor agent. PMID 18538434

Oncolytic virotherapy as a personalized cancer vaccine.
Juni 2008 | Li, Qi-Xiang; Liu, Guohong; Wong-Staal, Flossie
Oncolytic virotherapy has demonstrated multimodal antitumor mechanisms in both preclinical and clinical settings for cancer treatment, including antitumor immunity. Compared with conventional immunotherapy, oncolytic viruses have the advantages of simultaneous cytoreduction and conferring personalized anticancer immunity, but without the need of personalized manufacture. Additionally, oncolytic viruses can be further engineered to delete immunosuppressive viral components and to insert transgenes that enhance antitumor immunity. Finally, combination with new immunomodulating agents (e.g., cyclophosphamide) or cell therapy approaches will likely further augment specific antitumor immunity of virotherapy. Virotherapy could become a new paradigm for potent, safe and practical therapeutic vaccines for cancer. PMID 18500742

Oncolytic virotherapy: molecular targets in tumor-selective replication and carrier cell-mediated delivery of oncolytic viruses.
Apr. 2008 | Guo, Z Sheng; Thorne, Stephen H; Bartlett, David L
Tremendous advances have been made in developing oncolytic viruses (OVs) in the last few years. By taking advantage of current knowledge in cancer biology and virology, specific OVs have been genetically engineered to target specific molecules or signal transduction pathways in cancer cells in order to achieve efficient and selective replication. The viral infection and amplification eventually induce cancer cells into cell death pathways and elicit host antitumor immune responses to further help eliminate cancer cells. Specifically targeted molecules or signaling pathways (such as RB/E2F/p16, p53, IFN, PKR, EGFR, Ras, Wnt, anti-apoptosis or hypoxia) in cancer cells or tumor microenvironment have been studied and dissected with a variety of OVs such as adenovirus, herpes simplex virus, poxvirus, vesicular stomatitis virus, measles virus, Newcastle disease virus, influenza virus and reovirus, setting the molecular basis for further improvements in the near future. Another exciting new area of research has been the harnessing of naturally tumor-homing cells as carrier cells (or cellular vehicles) to deliver OVs to tumors. The trafficking of these tumor-homing cells (stem cells, immune cells and cancer cells), which support proliferation of the viruses, is mediated by specific chemokines and cell adhesion molecules and we are just beginning to understand the roles of these molecules. Finally, we will highlight some avenues deserving further study in order to achieve the ultimate goals of utilizing various OVs for effective cancer treatment. PMID 18328829

Brain tumor therapy by combined vaccination and antisense oligonucleotide delivery with nanoparticles.
Apr. 2008 | Schneider, Thomas; Becker, Andreas; Ringe, Kerstin; Reinhold, Annegret; Firsching, Raimund; Sabel, Bernhard A
We examined a "double-punch" approach to overcome the escape of glioblastoma cells to the immune surveillance: increasing the immune systems activation by an active specific immunization (ASI) with Newcastle-Disease-Virus infected tumor cells and blocking the TGF-beta production by delivery of TGF-beta antisense oligonucleotides using polybutyl cyanoacrylate nanoparticles (NPs). Gene delivery was first evaluated using the CMV-beta-gal plasmid as a reporter gene. Fischer rats received implantation of glioblastoma cells into the brain and were then treated with combined ASI/NP-anti-TGF-beta formulation. Massive staining of tumor cells was seen after NP delivery of the plasmid beta-galactosidase, indicating gene transfer by nanoparticles to tumor cells. When treated with NP-anti-TGF-beta after having been immunized, the rats survived longer than untreated controls, had reduced TGF-beta-levels and showed increased rates of activated CD25+ T cells. In summary, nanoparticles are useful to deliver plasmids and antisense oligonucleotides to brain tumors. A combined immunization/gene delivery of TGF-beta antisense oligonucleotides may be a promising approach for brain tumor therapy. PMID 18304655

An effective tumor vaccine optimized for costimulation via bispecific and trispecific fusion proteins.
März 2008 | Aigner, Maximilian; Janke, Markus; Lulei, Maria; Beckhove, Philipp; Fournier, Philippe; Schirrmacher, Volker
T cell costimulation has great therapeutic potential if it can be optimized and controlled. To achieve this, we engineered T cell-activating fusion proteins and immunocytokines that specifically attach to viral antigens of a virus-infected tumor vaccine. We employed the avian Newcastle Disease Virus because this agent is highly efficient for human tumor cell infection, and leads to introduction of viral hemagglutinin-neuraminidase (HN) molecules at the tumor cell surface. Here, we demonstrated the strong potentiation of the T cell stimulatory activity of such a vaccine upon attachment of bispecific or trispecific fusion proteins which bind with one arm to viral HN molecules of the vaccine, and with the other arm either to CD3 (signal 1), to CD28 (costimulatory signal 2a), or to interleukin-2 receptor (costimulatory signal 2b) on T cells. A vaccine with a combination of all three signals triggered the strongest activation of naïve human T cells, thereby inducing the most durable bystander antitumor activity in vitro. Adoptive transfer of such polyclonally activated cells into immunodeficient mice bearing human breast carcinoma caused tumor regression. Furthermore, tumor-reactive memory T cells from draining lymph nodes of carcinoma patients could be efficiently reactivated in a short-term ELISpot assay using an autologous tumor vaccine with optimized signals 1 and 2, but not with a similarly modified vaccine from an unrelated tumor cell line. Our data describe new bioactive molecules which in combination with an established virus-modified tumor vaccine greatly augments the antitumor activity of T cells from healthy donors and cancer patients. PMID 18360705

Generation of a recombinant oncolytic Newcastle disease virus and expression of a full IgG antibody from two transgenes.
Feb. 2008 | Pühler, F; Willuda, J; Puhlmann, J; Mumberg, D; Römer-Oberdörfer, A; Beier, R
The most advanced oncolytic Newcastle disease virus (NDV) strains that are used in clinical trials for the treatment of cancer are wild-type mesogenic strains. These virus strains have an inherent, nongenetically engineered, oncolytic activity and selectively replicate in tumor cells but not in normal human cells. To date no investigations have been performed with genetically engineered mesogenic NDV regarding the oncolytic activity. We describe here the generation of recombinant viruses of the mesogenic naturally oncolytic NDV strain MTH68. We show that not only one, but also two additional transgenes coding for amino-acid chains with a molecular weight of 25 and 50 kDa can be inserted into the viral genome without affecting viral growth, oncolytic potency or tumor-selective replication of the virus. Transgenic expression of the heavy and light chains of a monoclonal antibody, as separate additional transcriptional cassettes, leads to the expression of full immunoglobulin G (IgG) monoclonal antibody by recombinant NDV. Infection of tumor cells with antibody-transgenic viruses results in the efficient production and secretion of a functional full size IgG antibody by the tumor cells, that specifically binds to its target-antigen in tumor tissue. This approach will allow to combine the advantages of oncolytic RNA viruses and monoclonal antibodies in a single powerful anticancer agent with improved or even new therapeutic properties. PMID 18200068

Recombinant Newcastle disease virus (NDV) with inserted gene coding for GM-CSF as a new vector for cancer immunogene therapy.
Nov. 2007 | Janke, M; Peeters, B; de Leeuw, O; Moorman, R; Arnold, A; Fournier, P; Schirrmacher, V
This is the first report describing recombinant (rec) Newcastle disease virus (NDV) as vector for gene therapy of cancer. The gene encoding granulocyte/macrophage colony-stimulating factor (GM-CSF) was inserted as an additional transcription unit at two different positions into the NDV genome. The rec virus with the strongest production of the gene product (rec(GM-CSF)) was selected for our study. The insertion of the new foreign gene did neither affect the main features of NDV replication nor its tumor selectivity. The gene product was biologically active and stable. Tumor vaccine cells infected by rec(GM-CSF) stimulated human peripheral blood mononuclear cells (PBMC) to exert antitumor bystander effects in vitro in a tumor neutralization assay. These effects were significantly increased when compared to vaccine infected by rec(-) virus. Furthermore, rec(GM-CSF) led to a much higher interferon-alpha (IFN-alpha) production than rec(-) when added as virus or as virus-modified vaccine to PBMC. Two distinct cell types, monocytes and plasmacytoid dendritic cells were shown to contribute to the augmented IFN-alpha response of PBMC. In conclusion, the already inherent anti-neoplastic and immunostimulatory properties of NDV could be further augmented by the introduction of a therapeutic gene whose product initiates a broad cascade of immunological effects in the microenvironment of the vaccine. PMID 17914407

Host mediated anti-tumor effect of oncolytic Newcastle disease virus after locoregional application.
Okt. 2007 | Apostolidis, Leonidas; Schirrmacher, Volker; Fournier, Philippe
Several strains of the Newcastle disease virus (NDV) have raised considerable interest in recent years for clinical application because of their oncolytic properties. In this study we characterized virological, immunological and anti-tumor properties of some NDV strains. The oncolytic strain MTH-68/H was the most potent interferon-alpha inducer and, after UV light inactivation, it was the only tested NDV strain which induced in human PBMC anti-tumor activity in vitro. Upon systemic application to mice bearing a virus susceptible intradermal tumor, no significant anti-tumor effects were observed with the two oncolytic strains Italian and MTH-68/H while the treatment had significant side effects as seen by loss of body weight. In contrast, when using a locoregional application model for treatment of liver metastases of luciferase transfected CT26 colon carcinoma cells, MTH-68/H showed a significant delay in tumor growth, as well as prolonged survival but no effects on body weight. Surprisingly, this CT26 murine tumor cell transfectant was resistant in vitro to virus infection and oncolysis. These results suggest: i) that locoregional application of oncolytic NDV is more effective than systemic i.v. application; and ii) that oncolytic NDV can mediate effects even against a virus-resistant tumor line. The involvement of host anti-tumor immune responses as an important mechanism in therapies based on oncolytic NDV will be discussed. PMID 17912426

Use of reverse genetics to enhance the oncolytic properties of Newcastle disease virus.
Sep. 2007 | Vigil, Adam; Park, Man-Seong; Martinez, Osvaldo; Chua, Mark A; Xiao, Sa; Cros, Jerome F; Martínez-Sobrido, Luis; Woo, Savio L C; García-Sastre, Adolfo
Naturally occurring strains of Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently in clinical trials. Here, we have evaluated the possibility to enhance the cancer therapeutic potential of NDV by means of reverse genetics. Mice bearing s.c. implanted CT26 tumors were treated with intratumoral (i.t.) injections of a recombinant NDV modified to contain a highly fusogenic F protein. These treated mice exhibited significant reduction in tumor development compared with mice treated with the unmodified virus. Furthermore, mice in a CT26 metastatic tumor model treated with an i.v. injection of the genetically engineered NDV exhibited prolonged survival compared with wild-type control virus. In addition, we examined whether the oncolytic properties of NDV could be improved by expression of immunostimulatory molecules. In this regard, we engineered several NDVs to express granulocyte macrophage colony-stimulating factor, IFN-gamma, interleukin 2 (IL-2), or tumor necrosis factor alpha, and evaluated their therapeutic potential in an immunocompetent colon carcinoma tumor model. Mice bearing s.c. CT26 tumors treated with i.t. injections of recombinant NDV expressing IL-2 showed dramatic reductions in tumor growth, with a majority of the mice undergoing complete and long-lasting remission. Our data show the use of reverse genetics to develop enhanced recombinant NDV vectors as effective therapeutic agents for cancer treatment. PMID 17804743

In vitro induction of apoptosis in tumor cells by inactivated NDV and IAV.
Juni 2007 | Yang, ShuYan; Liu, WeiQuan; Cui, HuanXian; Sun, ShaoGuang; Wang, JiGui
We examined how Newcastle disease virus (NDV) and influenza A virus (IAV) inactivated by 5% formaldehyde, used either alone or in combination, can induce apoptosis in both HeLa and SP2/0 cells. Inactive NDV and IAV demonstrated enhanced rates of lysis in apoptotic tumor cells and greater antitumor effects when combined. Our study supports the argument that viral replication does not cause virally induced apoptosis. PMID 17600467

Phase 1 clinical experience using intravenous administration of PV701, an oncolytic Newcastle disease virus.
März 2007 | Lorence, Robert M; Roberts, M Scot; O'Neil, James D; Groene, William S; Miller, Jeffrey A; Mueller, Stephen N; Bamat, Michael K
PV701 is a naturally-attenuated, non-recombinant, oncolytic strain of Newcastle disease virus that displays preclinical intravenous (IV) efficacy. PV701 is selective at killing human cancer cells versus normal human cells based on tumor specific defects in the interferon (IFN)-mediated antiviral response. This oncolytic virus displays a broad spectrum of antitumor activity in vitro and in vivo. Preclinical models successfully predicted key clinical parameters including the mechanism of toxicity, two complementary strategies (desensitization and slow infusion) to reduce toxicity, and the starting dose for phase 1 trials. In three phase 1 trials of 114 patients using IV administration of PV701, Wellstat Biologics Corporation has evaluated the effects of dose, schedule, and infusion rate for PV701. Three general classes of side effects were seen: flu-like symptoms; tumor-site-specific adverse events (AEs); and infusion reactions. The first PV701 dose desensitized the patient to the side effects of further doses, allowing a marked increase in the maximum tolerated dose for subsequent doses compared to the first dose. Tumor responses were first noted at the higher doses achieved using desensitization. In the most recent phase 1 trial of 19 patients at Hamilton, Ontario, that employed desensitization, high repeat doses, and a slower infusion rate (Hamilton Regimen), there were six responses (4 major; 2 minor) and a total of six patients with survival for at least 2 years. In addition, patient tolerability improved using the Hamilton Regimen compared to IV bolus dosing used previously. Phase 2 studies of this novel biologic agent are about to begin. PMID 17346107

Oncolytic virus therapy--foreword.
März 2007 | Kasuya, H; Takeda, S; Shimoyama, S; Shikano, T; Nomura, N; Kanazumi, N; Nomoto, S; Sugimoto, H; Nakao, A
We are very pleased and proud to be able to publish this special issue of Current Cancer Drug Targets devoted to oncolytic virus therapy covering basic and clinical research on adenovirus, vaccinia virus, herpes virus, and Newcastle disease virus. In these papers, we welcome the world's top authorities in the field who have generously contributed their latest review articles for exclusive publication in this special issue. Moreover, this issue also includes a range of opinion from government drug organizations. Here we simply wish to bring together the newest knowledge and experience in the field of cutting-edge oncolytic virus therapy for researchers and every kind of cancer therapist. The Foreword presents a historical perspective on the development of oncolytic virus together with the encouraging results of recent clinical trials (e.g., H101 has been tested in clinical trial of nearly 250 patients and approved for human use by the Chinese FDA, while PV701 has been tried in over 110 patients, as described in our special issue). PMID 17346102

p53-independent endoplasmic reticulum stress-mediated cytotoxicity of a Newcastle disease virus strain in tumor cell lines.
Feb. 2007 | Fábián, Zsolt; Csatary, Christine M; Szeberényi, József; Csatary, Laszlo K
While Newcastle disease virus (NDV) causes serious infections in birds, it is apparently nonpathogenic in mammalian species, including humans. Previous observations and small-scale clinical trials indicated that NDV exerts oncolytic effects. Isolates of NDV were found to have selective affinity to transformed cells. We previously showed that the attenuated NDV strain MTH-68/H causes apoptotic cell death in cultures of PC12 rat pheochromocytoma cells. The aim of the present study was to extend MTH-68/H cytotoxicity testing with human tumor cell lines and to analyze certain biochemical aspects of its oncolytic effect. MTH-68/H was found to be able to kill a wide range of transformed cells by apoptosis. While caspase-8 and caspase-9 are not involved in MTH-68/H-induced apoptosis, activation of caspase-3 and caspase-12 was detected in virus-infected PC12 cells. A human glioblastoma cell line with repressible expression of the p53 protein did not show any difference in MTH-68/H sensitivity in its p53-expressing and p53-depleted states, indicating that the apoptotic process induced by MTH-68/H does not depend on p53. Apoptosis was accompanied by virus replication in two tumor cell lines tested (PC12 cells and HeLa human cervical cells), and signs of endoplasmic reticulum stress (phosphorylation of protein kinase R-like endoplasmic reticulum kinase and eIF2alpha) were also detected in transformed cells. In contrast, proliferation of nontransformed mouse and rat fibroblast cell lines and human primary fibroblasts was not affected by MTH-68/H treatment. MTH-68/H thus selectively kills tumor cell cultures by inducing endoplasmic reticulum stress leading to p53-independent apoptotic cell death. PMID 17215292

Systemic efficacy with oncolytic virus therapeutics: clinical proof-of-concept and future directions.
Jan. 2007 | Liu, Ta-Chiang; Kirn, David
Oncolytic viruses that can destroy cancer cells have been engineered in a variety of ways with the aim of improving their selectivity and efficacy. Here, we review data from clinical investigations of these virotherapeutic agents, specifically those that have shown systemic efficacy: vaccinia, measles, mumps, viruses, Newcastle disease virus, and reovirus. Further directions for optimizing i.v. delivery and efficacy are discussed. PMID 17234747

Newcastle disease virus exerts oncolysis by both intrinsic and extrinsic caspase-dependent pathways of cell death.
Juli 2006 | Elankumaran, Subbiah; Rockemann, Daniel; Samal, Siba K
Newcastle disease virus (NDV), an avian paramyxovirus, is tumor selective and intrinsically oncolytic. Here, we present evidence that genetically modified, recombinant NDV strains are cytotoxic to human tumor cell lines of ecto-, endo-, and mesodermal origin. We show that cytotoxicity against tumor cells is due to multiple caspase-dependent pathways of apoptosis independent of interferon signaling competence. The signaling pathways of NDV-induced, cancer cell-selective apoptosis are not well understood. We demonstrate that NDV triggers apoptosis by activating the mitochondrial/intrinsic pathway and that it acts independently of the death receptor/extrinsic pathway. Caspase-8-methylated SH-SY5Y neuroblastoma cells are as sensitive to NDV as other caspase-8-competent cells. This demonstrates that NDV is likely to act primarily through the mitochondrial death pathway. NDV infection results in the loss of mitochondrial membrane potential and the subsequent release of the mitochondrial protein cytochrome c, but the second mitochondrion-derived activator of caspase (Smac/DIABLO) is not released. In addition, we describe early activation of caspase-9 and caspase-3. In contrast, cleavage of caspase-8, which is predominantly activated by the death receptor pathway, is a TNF-related, apoptosis-inducing ligand (TRAIL)-induced late event in NDV-mediated apoptosis of tumor cells. Our data, therefore, indicate that the death signal(s) generated by NDV in tumor cells ultimately converges at the mitochondria and that it acts independently of the death receptor pathway. Our cytotoxicity studies demonstrate that recombinant NDV could be developed as a cancer virotherapy agent, either alone or in combination with therapeutic transgenes. We have also shown that trackable oncolytic NDV could be developed without any reduction in oncolytic efficacy. PMID 16840332

Tumor selective replication of Newcastle disease virus: association with defects of tumor cells in antiviral defence.
Juni 2006 | Fiola, Christoph; Peeters, Ben; Fournier, Philippe; Arnold, Annette; Bucur, Mariana; Schirrmacher, Volker
To investigate tumor-selective viral replication, we compared several tumorigenic human cell lines to nontumorigenic human cells from the blood for the sensitivity to become infected by a recombinant lentogenic strain of Newcastle Disease Virus (NDV) with incorporated transgene EGFP (NDFL-EGFP). Although fluorescence signals in nontumorigenic cells were only weak or missing completely, a massive and long-lasting transgene-expression was observed in all tumor cell lines. The majority of tumor cells (50-95%) could be infected, and viral replication was associated with an increase in the cell surface density of viral antigens. To clarify the underlying mechanism of the observed difference in virus susceptibility we examined the kinetics of interferon-induced antiviral enzymes because NDV is a strong type-I interferon inducer. This analysis revealed several defects of tumor cells in their antiviral defence responses: They showed no response to UV-inactivated NDV, whereas nontumorigenic cells reacted with induction of high-levels of the antiviral enzymes PKR and MxA. Upon coincubation with live NDV, tumor cells showed a delayed response in the increased expression of the antiviral enzymes in comparison with PBMC. In nontumorigenic cells the replication cycle of NDV stopped after the production of positive-strand RNA, while tumor cells continued in the replication cycle and copied viral genomes 10-50 hr after infection. These results can explain the tumor selective replication behavior of this interesting antineoplastic virus. PMID 16470838

A phase 1 clinical study of intravenous administration of PV701, an oncolytic virus, using two-step desensitization.
Apr. 2006 | Laurie, Scott A; Bell, John C; Atkins, Harold L; Roach, Joanne; Bamat, Michael K; O'Neil, James D; Roberts, M Scot; Groene, William S; Lorence, Robert M
In a previous phase 1 study, adverse events, especially flu-like symptoms, were observed mainly following the first i.v. bolus dose of PV701, an oncolytic Newcastle disease virus. Desensitization to adverse events of subsequent doses occurred, allowing a 10-fold increase in the maximum tolerated dose for these doses. Although one-step desensitization (a single desensitizing dose with higher subsequent doses) addressed the tolerability of high repeat doses, additional testing was required to further improve tolerability of the initial dose. This study tested the hypothesis that two-step desensitization, using two dose increments before high repeat doses, would be well tolerated. PMID 16638865

Combined action of virus injection and local tumor irradiation on tumor growth in mice.
Apr. 2006 | Cović, Dinko; Ivanković, Sinisa; Hirsl, Nevenka; Rupcić, Boris; Samija, Mirko; Jurin, Mislav
The dynamics of SCCVII transplantable tumor growth in C3H/H mice was determined after local tumor irradiation and/or virus (NDV LaSota) i.p. injection. The virus applied alone significantly suppressed tumor growth, particularly until the 19th day after tumor transplantation. Local irradiation with 30 Gy resulted in tumor disappearance followed with its regrowth about 15 days later. However, if the virus was injected after the irradiation, there was no tumor growth until the end of the 31 day observation period. It should be noted that virus application prior to local irradiation did not have any additional influence on tumor growth. Thus, the pronounced efficacy of virus applied after tumor irradiation deserves attention. It is possible that the virus injected after irradiation induced a chain of cytokine production joining the action of tumor destruction induced by irradiation. This should be further studied in clarifying the approaches to combined tumor therapy with possible cell-free vaccine production. PMID 16617596

Phase I/II trial of intravenous NDV-HUJ oncolytic virus in recurrent glioblastoma multiforme.
Dez. 2005 | Freeman, Arnold I; Zakay-Rones, Zichria; Gomori, John M; Linetsky, Eduard; Rasooly, Linda; Greenbaum, Evgeniya; Rozenman-Yair, Shira; Panet, Amos; Libson, Eugene; Irving, Charles S; Galun, Eithan; Siegal, Tali
We undertook a Phase I/II trial in patients with apparent recurrent glioblastoma multiforme (GBM) based on imaging studies to determine the safety and tumor response of repetitive intravenous administration of NDV-HUJ, the oncolytic HUJ strain of Newcastle disease virus. The first part of the study utilized an accelerated intrapatient dose-escalation protocol with one-cycle dosage steps of 0.1, 0.32, 0.93, 5.9, and 11 billion infectious units (BIU) of NDV-HUJ (1 BIU = 1 x 10(9) EID(50) 50% egg infectious dose) followed by three cycles of 55 BIU. Virus was administered by intravenous infusion over 15 min. In the second part, patients received three cycles of 11 BIU. All patients without progressive disease were maintained with two doses of 11 BIU iv weekly. Eleven of the 14 enrolled patients (11-58 years, Karnofsky performance scale 50-90%) received treatment. Toxicity was minimal with Grade I/II constitutional fever being seen in 5 patients. Maximum tolerated dose was not achieved. Anti-NDV hemagglutinin antibodies appeared within 5-29 days. NDV-HUJ was recovered from blood, saliva, and urine samples and one tumor biopsy. One patient achieved a complete response. Intravenous NDV-HUJ is well tolerated. The findings of good tolerability and encouraging responses warrant the continued evaluation of NDV-HUJ in GBM, as well as other cancers. PMID 16257582

A tumor vaccine containing anti-CD3 and anti-CD28 bispecific antibodies triggers strong and durable antitumor activity in human lymphocytes.
Dez. 2005 | Haas, Claudia; Lulei, Maria; Fournier, Philippe; Arnold, Annette; Schirrmacher, Volker
We recently reported on newly designed virus-targeted bispecific CD3- and CD28-binding molecules for human T-cell activation. When bound via one arm to a human virus-modified tumor cell vaccine, these reagents caused a polyclonal T-cell response and overcame the potential various T-cell evasion mechanisms of tumor cells. In our current study, we demonstrated the induction of strong antitumor activity in human lymphocytes upon coincubation with a virus-modified tumor vaccine containing anti-CD3 and anti-CD28 bispecific antibodies. Blood mononuclear cells or purified T cells that were coincubated with such a tumor vaccine for 3 days were able to destroy monolayers of human breast carcinoma and other carcinoma cells. Serial transfer to new tumor cell monolayers revealed antitumor cytotoxic activity in such effector cells that lasted for about 10 days. Nontumor target cells appeared to be much less sensitive to the activated effector cells. Although the bispecific molecules alone did not activate effector cells, their binding to virus-infected tumor cells was important and more effective than their binding to free virus. Antitumor activity of the activated effector cells was mediated through soluble factors as well as through direct cell contact of effector cells with the nontargeted bystander tumor cells. Since the virus-modified tumor vaccine is well tolerated and already exhibits a certain effectiveness in cancer patients, the combination with new bispecific molecules has the potential to introduce additional antitumor effects. The reagents can also be combined with Newcastle Disease Virus (NDV)-based oncolytic virotherapy. PMID 16108015

Virus-based therapies for colon cancer.
Dez. 2005 | Morse, Michael A
Viral vectors are under development for anticancer therapy. As they can infect tumours and activate the immune system, viral vectors may directly destroy cancers (oncolysis), deliver genes with antitumour activity directly to the cancer cells, or act as cancer vaccines. Better insights into the biology of the various vectors in use (e.g., poxvectors, adenovirus, adeno-associated virus, reovirus, Newcastle disease virus) are making it possible to engineer viruses that are more tumour-specific, efficient at tumour infection, and which have enhanced safety due to incorporation of safeguards should dissemination occur. As considerable research has focused on therapy of colon cancer with viral vectors, this review will illustrate the major concepts of viral therapy of cancers with examples from studies targeting colorectal carcinoma. PMID 16318426

Tumor-targeted gene transfer in vivo via recombinant Newcastle disease virus modified by a bispecific fusion protein.
Juli 2005 | Bian, Huijie; Fournier, Philippe; Peeters, Ben; Schirrmacher, Volker
Previously we have demonstrated that a recombinant Newcastle disease virus (NDV) carrying the transgene EGFP can be retargeted to IL-2 receptor positive tumor cells by a bispecific fusion protein alphaHN-IL-2 in vitro. The purpose of the present study was to investigate the specificity and efficiency of gene delivery to tumor cells in vivo via this modified RNA virus. Prior ex vivo infection of murine lymphoma cells by the modified virus resulted in selective EGFP expression in IL-2R+ target tumor cells in vivo. Direct fluorescence microscopy and immunohistology showed viral replication in target positive tumor tissue resulting in much more EGFP expression than in target negative tumor tissue, 24 h after intratumoral injection of the alphaHN-IL-2 modified NDV. A quantitative real-time RT-PCR for EGFP mRNA. confirmed the selective gene expression in IL-2R+ tumor cells. Biodistribution studies showed that EGFP transgene delivery was reduced by 35-100% in liver, spleen, kidney, lung and thymus by the modified virus, while 98% of the transgene was delivered to IL-2R+ tumors. In conclusion, the modification of NDV by the bispecific protein does not compromise severely the efficiency of gene delivery into IL-2R-positive tumors, but greatly reduces viral gene expression in IL-2R-negative tumors and in normal tissues. PMID 16010418

Targeting vaccinia to solid tumors with local hyperthermia.
Mai 2005 | Chang, Eugene; Chalikonda, Sricharan; Friedl, Josef; Xu, Hui; Phan, Giao Q; Marincola, Francesco M; Alexander, H Richard; Bartlett, David L
We have previously demonstrated that mutant vaccinia viruses target tumors in vivo after systemic delivery, and they have potential as vectors for tumor-directed gene therapy. We hypothesized that hyperthermia may augment vaccinia delivery to tumors after systemic injection, as hyperthermia increases the permeability of the endothelial vasculature to nanoparticles. In our in vitro experiments, we have shown that hyperthermia does not alter tumor cells' susceptibility to the intrinsic cytopathogenicity of the vaccinia virus compared with normothermic controls. Hyperthermia also does not change the viral infectivity or the level of viral marker gene expression when compared with normothermia. In an in vitro model of endothelial cell monolayer permeability, we have demonstrated that hyperthermia increases the permeability of the monolayer to vaccinia virus and that this phenomenon is completely reversible. In vivo we have demonstrated that the tumors that were treated with systemic vaccinia under conditions of hyperthermia (41.5 degrees C for 30 min) had significantly higher levels of vaccinia marker gene activity (>100-fold) than those treated under normothermic conditions (p < 0.05) and that this effect was specific to tumor. We also demonstrated that mice with 1 cm subcutaneous tumors treated with a systemically delivered, conditionally replicating vaccinia under conditions of hyperthermia had complete tumor regression in 50% and significantly improved antitumor response, compared with normothermic viral-treated controls (mean tumor volume of 110 mm(3) vs 3169 mm(3), 13 days after treatment) and compared with hyperthermic, nonvirally treated control animals (p < 0.0001). Regional hyperthermia improves vaccinia targeting to tumors, and thereby enhances the antitumor response. PMID 15871675

Clinical trials of antitumor vaccination with an autologous tumor cell vaccine modified by virus infection: improvement of patient survival based on improved antitumor immune memory.
Apr. 2005 | Schirrmacher, Volker
For active specific immunotherapy of cancer patients, we designed the autologous virus-modified tumor cell vaccine ATV-NDV. The rationale of this vaccine is to link multiple tumor-associated antigens (TAAs) from individual patient-derived tumor cells with multiple danger signals (DS) derived from virus infection (dsRNA, HN, IFN-alpha). This allows activation of multiple innate immune responses (monocytes, dendritic cells, and NK cells) as well as adaptive immune responses (CD4 and CD8 memory T cells). Preexisting antitumor memory T cells from cancer patients could be activated by antitumor vaccination with ATV-NDV as seen by augmentation of antitumor memory delayed-type hypersensitivity (DTH) responses. In a variety of phase II vaccination studies, an optimal formulation of this vaccine could improve long-term survival beyond what is seen in conventional standard therapies. A new concept is presented which proposes that a certain threshold of antitumor immune memory plays an important role (1) in the control of residual tumor cells which remain after most therapies and (2) for long-term survival of treated cancer patients. This immune memory is T-cell based and most likely maintained by persisting TAAs from residual dormant tumor cells. Such immune memory was prominent in the bone marrow in animal tumor models as well as in cancer patients. Immunization with a tumor vaccine in which individual TAAs are combined with DS from virus infection appears to have a positive effect on antitumor immune memory and on patient survival. PMID 15838708

T-cell triggering by CD3- and CD28-binding molecules linked to a human virus-modified tumor cell vaccine.
März 2005 | Haas, Claudia; Lulei, Maria; Fournier, Philippe; Arnold, Annette; Schirrmacher, Volker
The aim was to develop T cell costimulatory molecules that are broadly applicable to augment anti-tumor immune responses upon application of a virus-modified tumor vaccine to cancer patients. We generated recombinant bispecific single-chain antibodies with one specificity directed against the CD3 or the CD28 antigen on human T cells and the other against the viral target molecule hemagglutinin-neuraminidase (HN) of Newcastle Disease Virus (NDV). By re-directing unstimulated primary human T cells against HN-expressing NDV-infected tumor cells, the bispecific molecule bsHN-CD3 cross-linked effector and target cells and rapidly induced cytotoxicity at nanomolar concentrations. The bsHN-CD28 molecule exerted T cell co-stimulatory function. Maximal T cell activation was achieved with tumor cells infected by NDV and modified with both new stimulatory molecules. This was revealed by T cell proliferation, upregulation of CD69 and CD25 and by release of cytokines, interferons and chemokines. The new molecules combine high-effectivity with specificity and safety. PMID 15752830

Selective gene transfer in vitro to tumor cells via recombinant Newcastle disease virus.
Feb. 2005 | Bian, Huijie; Fournier, Philippe; Moormann, Rob; Peeters, Ben; Schirrmacher, Volker
We developed a novel strategy to target recombinant Newcastle disease virus (NDV) to tumor cells for gene therapy. Modifying the virus with a bispecific fusion protein allowed virus receptor-independent tumor cell binding and gene transfer. The targeting molecule (alpha)HN-IL-2 contains an scFv antibody cloned from a neutralizing hemagglutinin-neuraminidase (HN)-specific hybridoma linked to the human cytokine IL-2. A recombinant NDV expressing the enhanced green fluorescent protein (NDFL-EGFP) was applied to show the expression of foreign genes in virus-infected tumor cells. At 24 hours after infection with the modified virus (NDFL-EGFP/(alpha)HN-IL-2), FACS analysis and fluorescence microscopy revealed neutralization of natural infection in IL-2 receptor-negative Jurkat leukemia cells, but targeted expression of EGFP in IL-2 receptor-positive human leukemia-derived MT-2 cells. The targeted gene delivery of NDFL-EGFP/(alpha)HN-IL-2 in MT-2 cells was blocked by the target ligand human IL-2. Selective virus entry to IL-2 receptor bearing tumor cells was also observed in a mixture of Jurkat and MT-2 cell lines. These results demonstrate that a recombinant NDV carrying a foreign gene can be successfully targeted to a specific tumor through a bispecific protein, which thereby increases the selectivity of gene transfer. PMID 15605075

Selective gene transfer to tumor cells by recombinant Newcastle Disease Virus via a bispecific fusion protein.
Jan. 2005 | Bian, Huijie; Fournier, Philippe; Moormann, Rob; Peeters, Ben; Schirrmacher, Volker
Much interest exists presently in development of vectors for gene therapy of tumors based on RNA viruses because these viruses replicate in the cytoplasm and do not integrate into DNA. The negative stranded paramyxovirus, Newcastle Disease Virus (NDV) from chicken has the additional advantages of preferential replication in tumor cells and of oncolytic and immunostimulatory properties. We here describe the bispecific fusion protein alphaHN-IL-2 which binds to NDV, inhibits its normal cell binding property and introduces a new binding specificity for the interleukin-2 receptor (IL-2R). We demonstrate selective gene transfer to tumor cells expressing IL-2R via the bispecific fusion protein when using recombinant NDV carrying as marker gene the enhanced green fluorescence protein (NDFL-EGFP). Hemadsorption (HA) and neuraminidase activities (NA) of the HN protein of NDV were shown to be blocked by alphaHN-IL-2 simultaneously and the absence of HA-activity of modified NDV was confirmed in vivo. Retargeted virus-binding to IL-2R positive tumor cells was not sufficient for the process of cellular infection. It required in addition membrane fusion via the viral F-protein. By modification of recombinant NDV with a bispecific molecule, our results demonstrate a novel and safe strategy for selective gene transfer to targeted tumor cells. PMID 15645128

Effects of febrile temperature on adenoviral infection and replication: implications for viral therapy of cancer.
Dez. 2004 | Thorne, Stephen H; Brooks, Gabriel; Lee, Yeun-Ling; Au, Tina; Eng, Lawrence F; Reid, Tony
We previously conducted a phase I/II study using arterial infusions of ONYX-015 (dl1520), a replication-selective adenoviral vector, with E1b deleted, for patients with metastatic colorectal cancer. No dose-limiting toxicities occurred, but >90% of the patients experienced fever. The effects of temperature on the replication of dl1520 in normal and transformed cells had not been studied. Therefore, replication and cell viability assays were performed with a panel of nontransformed and transformed cell lines cultured at 37 and 39.5 degrees C and treated with adenovirus type 5 (Ad5) or dl1520. Ad5-mediated cytolytic effects were inhibited and production of infectious particles decreased by >1,000-fold in the nontransformed cells at 39.5 degrees C. Seven of nine of the tumor cell lines retained significant cell-killing effects when treated with Ad5 at 39.5 degrees C. When dl1520 was used, no cytolytic effects were observed at 39.5 degrees C in the nontransformed cell lines; however, cytolytic effects occurred in six of nine tumor cell lines at 39.5 degrees C. Notably, a subset of the tumor cell lines demonstrated increased dl1520-mediated cytolytic effect and replication at 39.5 degrees C. Suppression of Ad5 and dl1520 replication at 39.5 degrees C was not related to p53 status or HSP70 expression. Also, at 39.5 degrees C, E1a expression was inhibited in nontransformed cells but was still abundant in the transformed cells, indicating that a novel early block in viral replication occurred in the nontransformed cells. Fever may therefore augment the therapeutic index of oncolytic viruses by inhibiting replication in normal cells while permitting or enhancing viral replication in some tumor cells. PMID 15596850

Antitumor vaccination in patients with head and neck squamous cell carcinomas with autologous virus-modified tumor cells.
Nov. 2004 | Karcher, Jochen; Dyckhoff, Gerhard; Beckhove, Philipp; Reisser, Christoph; Brysch, Michael; Ziouta, Yvonne; Helmke, Burkhard H; Weidauer, Hagen; Schirrmacher, Volker; Herold-Mende, Christel
Prognosis of patients with advanced head and neck squamous cell carcinomas (HNSCC) is still poor. Therefore, we analyzed whether antitumor vaccination with a virus-modified autologous tumor cell vaccine is feasible and safe in HNSCC patients. Furthermore, we determined the influence on disease-free survival and overall survival and the vaccination-induced antitumor reactivity. In a nonrandomized pilot study, 20 patients were vaccinated postoperatively. Vaccine was prepared from the tumor cell cultures of patients by infection of the cells with Newcastle Disease Virus, followed by gamma-irradiation, and vaccine was applied up to five times. Antitumor immune reactivity was determined in the skin by delayed type hypersensitivity skin reaction and in the blood by enzyme-linked immunospot assay. Establishment of tumor cell cultures was successful in about 80% of the cases. After vaccination, we observed no severe side effects. Percentages of survival of vaccinated patients with stage III and stage IV tumors (n = 18) were 61% at 5 years. Immune monitoring revealed significant increases of antitumor delayed type hypersensitivity reactivity especially in disease-free patients, and in a significant proportion of vaccinated patients the presence of tumor-reactive T-cells in the peripheral blood even 5 to 7 years after the last vaccination. Postoperative vaccination with virus-modified autologous tumor cells seems to be feasible and safe and may improve the prognosis of HNSCC patients with advanced tumors. This could be supported by antitumor immune responses that we observed especially in long-term surviving patients. PMID 15520216

Antitumor vaccination of patients with glioblastoma multiforme: a pilot study to assess feasibility, safety, and clinical benefit.
Okt. 2004 | Steiner, Hans Herbert; Bonsanto, Matteo Mario; Beckhove, Philipp; Brysch, Michael; Geletneky, Karsten; Ahmadi, Rezvan; Schuele-Freyer, Rebecca; Kremer, Paul; Ranaie, Golamreza; Matejic, Dejana; Bauer, Harald; Kiessling, Marika; Kunze, Stefan; Schirrmacher, Volker; Herold-Mende, Christel
Prognosis of patients with glioblastoma is poor. Therefore, in glioblastoma patients, we analyzed whether antitumor vaccination with a virus-modified autologous tumor cell vaccine is feasible and safe. Also, we determined the influence on progression-free survival and overall survival and on vaccination-induced antitumor reactivity. PMID 15452186

High cell surface expression of Newcastle disease virus proteins via replicon vectors demonstrates syncytia forming activity of F and fusion promotion activity of HN molecules.
Juli 2004 | Zeng, Jinyang; Fournier, Philippe; Schirrmacher, Volker
For functional studies, the hemagglutinin-neuraminidase (HN) and the fusion protein (F) of Newcastle disease virus (NDV) were expressed in BHK cells using two vectors which are based on the Semliki Forest virus (SFV) replicon. The first system of high protein expression works by transfection of RNA which before has been in vitro transcribed from a vector containing the gene for the SFV self-amplifying replicase (REP) and a foreign gene using the SP6 promoter. A high level of protein (HN or F) expression was detected 18-20 h after transfection. To study the host range of this expression system, a panel of different cell lines were compared for transfections with SFV RNA. A wide range of expression efficiency was observed, the highest being BHK cells. The second system is based on a DNA plasmid in which the SFV-REP and a foreign gene are expressed in cells under the transcriptional and translational control of the cytomegalovirus immediate-early enhancer T7 promoter. DNA-electroporated BHK cells expressed also high levels of the recombinant proteins but at a delayed time point (40-48 h) as compared with the corresponding RNA. Co-expression of the two NDV proteins, HN and F, via this DNA vector in the same cells led to syncytium formation in the cell monolayer, showing that both proteins expressed in this way, were functionally active. F alone, expressed via this vector, displayed residual fusion activity suggesting its proteolytic cleavage and its functional independence from HN. PMID 15254725

MTH-68/H oncolytic viral treatment in human high-grade gliomas.
Apr. 2004 | Csatary, L K; Gosztonyi, G; Szeberenyi, J; Fabian, Z; Liszka, V; Bodey, B; Csatary, C M
Application of virus therapy to treat human neoplasms has over a three decade history. MTH-68/H, a live attenuated oncolytic viral strain of the Newcastle disease virus, is one of the viruses used in the treatment of different malignancies. Here we report on the administration of MTH-68/H to patients with glioblastoma multiforme, the most common and most aggressive neuroectodermal neoplasm with a poor prognosis, averaging six months to a year. Four cases of advanced high-grade glioma were treated with MTH-68/H after the conventional modalities of anti-neoplastic therapies had failed. This treatment resulted in survival rates of 5-9 years, with each patient still living today. Against all odds, each patient resumed a lifestyle that resembles their previous daily routines and enjoys a good quality of life, Each of these patients has regularly received MTH-68/H as their sole form of onco-therapy for a number of years now without interruption. PMID 15072452

Importance of serine 200 for functional activities of the hemagglutinin-neuraminidase protein of Newcastle Disease Virus.
Feb. 2004 | Fournier, Philippe; Zeng, Jinyang; Von Der Lieth, Claus-W; Washburn, Birgit; Ahlert, Thorsten; Schirrmacher, Volker
Newcastle disease virus (NDV) is an avian paramyxovirus with replication competence in human tumor cells and interesting anti-neoplastic and immune stimulatory properties. In order to increase tumor selectivity of replication, we prepared mutants from the avirulent strain Ulster with monocyclic replication cycle and adapted them for multicyclic replication in human melanoma cells. Two mutants (M1 and M2) showed interesting functional differences: while M2 showed T cell co-stimulatory effects in a tumor-specific cytotoxic T lymphocyte (CTL) assay, M1 did not. A distinct difference of these 2 virus mutants appeared also when testing their capacity to induce interferon-alpha and -beta as well as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) molecules in human monocytes. Sequence analysis of the hemagglutinin-neuraminidase (HN) molecules of the 2 virus mutants showed 7 non-silent mutational differences. Upon cloning of the HN mutant genes into an expression vector and transfection of cells, only HN derived from M2 (HN-M2) was detected at the cell surface by immunostaining with specific antibodies and showed hemadsorption and neuraminidase activity. In order to define which amino acid was responsible for the loss of functional activity of HN derived from M1 (HN-M1), distinct HN mutants were generated via site-directed mutagenesis and tested. Substitution of serine 200 by a proline abrogated HN expression and its hemadsorption and neuraminidase activities. Molecular modeling revealed that proline 200 in HN influences flexibility of a loop near the entrance to the neuraminidase active site, a function that may be crucial for the functions of this viral protein. PMID 14767547

Overview of phase I studies of intravenous administration of PV701, an oncolytic virus.
Feb. 2004 | Lorence, Robert M; Pecora, Andrew L; Major, Pierre P; Hotte, Sebastien J; Laurie, Scott A; Roberts, M Scot; Groene, William S; Bamat, Michael K
PV701 is an attenuated, non-recombinant, oncolytic strain of Newcastle disease virus that displays preclinical intravenous (i.v.) efficacy. PV701 selectively lyses tumor cells versus normal cells based on tumor-specific defects in the interferon-mediated antiviral response. In three phase I trials in 113 patients, the effects of dose, schedule and i.v. infusion rate were evaluated. Three types of adverse events were seen: flu-like, tumor-site-specific and those occurring during infusion. The first PV701 dose desensitized the patient to the side effects of further doses, allowing a 5- to 10-fold increase in the maximum-tolerated dose for subsequent doses compared with the first dose. Tumor responses were first noted at the higher doses achieved using desensitization. In 95 evaluable patients, there were ten responses (six major and four minor), with five of these responses occurring in the most recent trial of 18 patients that employed desensitization, high repeat doses and a slower infusion rate. Phase II studies are planned. PMID 14755888

Recombinant Newcastle disease virus as a vaccine vector.
Juni 2003 | Huang, Z; Elankumaran, S; Panda, A; Samal, S K
Veterinary vaccines remained conventional for more than fifty years. Recent advances in the recombinant genetic engineering techniques brought forward a leap in designing vaccines for veterinary use. A novel approach of delivering protective immunogens of many different pathogens in a single virus vector was made possible with the introduction of a "reverse genetics" system for nonsegmented negative-sense RNA viruses. Newcastle disease virus (NDV), a nonsegmented negative-sense virus, is one of the major viruses of economic importance in the poultry industry throughout the world. Despite the availability of live virus vaccines of good potency, the intrinsic ability of attenuated strains to revert in virulence makes control of this disease by vaccination difficult. Armed with the knowledge of virulence factors of this virus, it is now possible to produce genetically stable vaccines and to engineer mutations that enhance immunogenicity. The modular nature of the genome of this virus facilitates engineering additional genes from several different pathogens or tumor-specific antigens to design contemporary vaccines for animals and humans. This review will summarize the developments in using NDV as a vaccine vector and the potential of this approach in designing next generation vaccines for veterinary use. PMID 12817444

TNF-related apoptosis-inducing ligand mediates tumoricidal activity of human monocytes stimulated by Newcastle disease virus.
Feb. 2003 | Washburn, Birgit; Weigand, Markus A; Grosse-Wilde, Anne; Janke, Markus; Stahl, Heiko; Rieser, Eva; Sprick, Martin R; Schirrmacher, Volker; Walczak, Henning
The Newcastle disease virus (NDV) has antineoplastic and immunostimulatory properties, and it is currently clinically tested in anticancer therapy. However, the tumoricidal mechanisms of NDV tumor therapy are not fully understood. The results presented here demonstrate that NDV-stimulated human monocytes (Mphi) kill various human tumor cell lines and that this tumoricidal activity is mediated by TRAIL. In contrast to soluble TRAIL-R2-Fc, soluble CD95-Fc and TNF-R2-Fc showed only minimal blocking of the antitumor effect. TRAIL expression is induced on human Mphi after stimulation with NDV and UV-inactivated NDV. These results show that TRAIL induction on human Mphi after NDV stimulation is independent from viral replication and that TRAIL mediates the tumoricidal activity of NDV-stimulated human Mphi. PMID 12574346

Postirradiation malignant fibrous histiocytoma of the larynx: a case report.
Sep. 2002 | Guney, Ercihan; Yigitbasi, Orhan Gazi; Balkanli, Sülegman; Canoz, Ozlem M
A 63-year-old man presented with malignant fibrous histiocytoma of the larynx occurring 16 years after radiation treatment for squamous cell carcinoma of the larynx. Postirradiation sarcoma of the larynx is an unusual tumor. The location, the histopathologic and immunohistochemical appearance of the tumor, and the time elapsed since the initial treatment make it probable that this tumor is associated with prior radiation treatment. The possibility of postirradiation sarcomas after radiation therapy should not be a major factor influencing treatment decisions in the patients with head and neck cancer. Wide surgical resection of the tumor seems to be an efficient means in the management of this tumor. PMID 12239696

Human tumor cell infection by Newcastle Disease Virus leads to upregulation of HLA and cell adhesion molecules and to induction of interferons, chemokines and finally apoptosis.
Juni 2002 | Washburn, B; Schirrmacher, V
In order to analyse immune-stimulatory effects of infection of human tumor cells with Newcastle Disease Virus (NDV), gamma-irradiated human breast carcinoma, colon-carcinoma or glioblastoma cells from defined cell lines were modified either by true infection with live virus or by cell surface adsorption of UV-inactivated replication deficient virus. Modification with live but not inactive NDV induced in all human tumor cells IFN-beta and the chemokines RANTES and IFN-gamma-inducible protein-10 (IP-10). In addition, infection by live NDV induced upregulation of HLA-ABC-molecules in all tumor lines tested and HLA-DR molecules in breast carcinoma lines. Two cell adhesion molecules, ICAM-I (CD54) and LFA-3 (CD58), were also upregulated on human tumor cells after infection with live NDV. When infection of MCF-7 breast carcinoma cells by NDV was performed in the presence of neutralizing anti-IFN-beta antibodies no upregulation of HLA molecules was observed suggesting an important role of IFN-beta in this process. Forty-eight to 72 hours after infection of the irradiated tumor cells with live NDV, many tumor cells were dead or in early or late stages of apoptosis. These results provide explanations for the function of the virus-modified autologous tumor vaccine ATV-NDV with which promising clinical results have already been obtained. PMID 12063554

Phase I trial of intravenous administration of PV701, an oncolytic virus, in patients with advanced solid cancers.
Apr. 2002 | Pecora, Andrew L; Rizvi, Naiyer; Cohen, Gary I; Meropol, Neal J; Sterman, Daniel; Marshall, John L; Goldberg, Stuart; Gross, Peter; O'Neil, James D; Groene, William S; Roberts, M Scot; Rabin, Harvey; Bamat, Michael K; Lorence, Robert M
PV701, a replication-competent strain of Newcastle disease virus, causes regression of tumor xenografts after intravenous administration. This phase I study was designed to define the maximum-tolerated dose (MTD) and safety of single and multiple intravenous doses of PV701 as a single agent in patients with cancer. PMID 11980996

Heat shock and heat shock protein 70i enhance the oncolytic effect of replicative adenovirus.
Dez. 2001 | Haviv, Y S; Blackwell, J L; Li, H; Wang, M; Lei, X; Curiel, D T
Replication-competent viruses are currently being evaluated for their cancer cell-killing properties. These vectors are designed to induce tumor regression after selective viral propagation within the tumor. However, replication-competent viruses have not resulted heretofore in complete tumor eradication in the clinical setting. Recently, heat shock has been reported to partially alleviate replication restriction on an avian adenovirus (Ad) in a human lung cancer cell line. Therefore, we hypothesized that heat shock and overexpression of heat shock protein (hsp) would support the oncolytic effect of a replication-competent human Ad. To this end, we tested the oncolytic and burst kinetics of a replication-competent Ad after exposure to heat shock or to inducible hsp 70 overexpression by a replication-deficient Ad (Adhsp 70i). Heat-shock resulted in augmentation of Ad burst and oncolysis while decreasing total intracellular Ad DNA. Overexpression of hsp 70i also enhanced Ad-mediated oncolysis but did not decrease intracellular Ad DNA levels. We conclude that heat shock and Adhsp 70i enhance the Ad cell-killing potential via distinct mechanisms. A potential therapeutic implication would be the use of local hyperthermia to augment oncolysis by increasing the burst of replication-competent Ad. The role of hsp in Ad-mediated oncolysis should be additionally explored. PMID 11731408

Preliminary results of active specific immunization with modified tumor cell vaccine in glioblastoma multiforme.
Okt. 2001 | Schneider, T; Gerhards, R; Kirches, E; Firsching, R
Treatment for glioblastoma multiforme has failed to show any progress for decades. While specific immunization with tumor cells modified with Newcastle-Disease-Virus (NDV) has been reported successful in some extracerebral tumors, its effect on glioblastoma is unknown. We report on 11 patients, in whom this approach was analyzed. PMID 11678429

Newcastle disease virus therapy of human tumor xenografts: antitumor effects of local or systemic administration.
Okt. 2001 | Phuangsab, A; Lorence, R M; Reichard, K W; Peeples, M E; Walter, R J
Previously we showed that a single local injection of the avian paramyxovirus Newcastle disease virus (NDV) strain 73-T caused long-lasting, complete tumor regression of human neuroblastoma and fibrosarcoma xenografts in athymic mice. Here we report the antitumor effects of NDV administered by either the intratumoral (IT) route to treat a variety of human carcinoma xenografts or by the systemic (intraperitoneal, IP) route to treat neuroblastoma xenografts (6.5-12 mm in diameter). For IT treatments, mice were randomized into treatment groups and given a single IT injection of NDV 73-T, vehicle (phosphate buffered saline, PBS), or UV-inactivated NDV. For systemic therapy, mice (n=18) with subcutaneous IMR-32 human neuroblastoma xenografts received IP injections of NDV (5 x 10(9) PFU). Significant tumor growth inhibition (77-96%) was seen for epidermoid (KB8-5-11), colon (SW620 and HT29), large cell lung (NCIH460), breast (SKBR3), prostate (PC3), and low passage colon (MM17387) carcinoma xenografts treated IT with NDV. In all cases, tumors treated IT with PBS or replication-incompetent, UV-inactivated NDV displayed rapid tumor growth. After a single IP injection of NDV, complete regression of IMR-32 neuroblastomas was observed in 9 of 12 mice without recurrence for the 3-9 month follow-up period. Six mice with recurrent neuroblastomas after one IP injection received one to three additional IP treatments with NDV. Three of these six mice showed complete regression without recurrence. These data show that: (1) NDV administered either IT or IP is an effective antitumor therapy in this system, (2) replication competency is necessary for maximal effect, and (3) multiple NDV doses can be more effective than a single dose. These studies provide further rationale for the preclinical study of NDV as an oncolytic agent. PMID 11595126

Antitumor effects of Newcastle Disease Virus in vivo: local versus systemic effects.
Apr. 2001 | Schirrmacher, V; Griesbach, A; Ahlert, T
Newcastle Disease Virus (NDV) has interesting anti-neoplastic and pleiotropic immune stimulatory properties. The virus preferentially replicates in and kills tumor cells and appears to be safe and to varying degrees effective in phase II-clinical studies in the US and in Europe. Here we have compared various lytic and non-lytic strains of NDV with regard to their antitumor effects after local or systemic application. As tumor models we used human metastatic melanoma xenotransplants in nude mice and murine metastatic colon carcinoma (CT26), renal carcinoma (Renca) and lymphoma (ESb) cell lines. Intra or peri-tumoral application of NDV or NDV infected tumor cells showed more pronounced antitumor activity than systemic application even when in the latter case much higher dose ranges were used. In the CT26 colon carcinoma model the non-lytic strain Ulster showed stronger antitumor activity than the lytic strain 73T. In the human MeWo melanoma xentransplant model strong antitumor bystander effects were observed by 20% admixture of melanoma cells pre-infected in vitro with NDV (either strain Ulster or Italien). Virus therapy of pre-established human melanomas by intra-tumoral injection of NDV was effective with the lytic strain Italien but not with the non-lytic strain Ulster. Systemic anti-metastatic effects were never observed with NDV alone in contrast to previous results obtained with NDV modified tumor vaccines. PMID 11295039

Newcastle disease virus (NDV): brief history of its oncolytic strains.
März 2000 | Sinkovics, J G; Horvath, J C
While genetically engineered viruses are now being tested for the virus therapy of human cancers, some naturally occurring viruses display unmatched oncolytic activity. Newcastle disease virus (NDV) excels as an oncolytic agent. PMID 10680736

Human tumor cell modification by virus infection: an efficient and safe way to produce cancer vaccine with pleiotropic immune stimulatory properties when using Newcastle disease virus.
Juni 1999 | Schirrmacher, V; Haas, C; Bonifer, R; Ahlert, T; Gerhards, R; Ertel, C
Direct infection of tumor cells with viruses transferring protective or therapeutic genes, a frequently used procedure for production of tumor vaccines in human gene therapy, is an approach which is often limited by the number of tumor cells that can reliably be infected as well as by issues of selectivity and safety. We report an efficient, selective and safe way of infecting human tumor cells with a natural virus with interesting pleiotropic immune stimulatory properties, the avian paramyxovirus Newcastle disease virus (NDV). Two of the six viral genes (HN and F) modify the tumor cell surface by introduction of new adhesion molecules for lymphocyte interactions and other viral genes stimulate host cell genes and local production of cytokines and chemokines which can recruit a broad antitumor response in vivo. A large variety of human tumor cells is shown to be efficiently infected by NDV with viral replication being independent of tumor cell proliferation. Such properties make NDV a suitable agent for modification of noncultured freshly isolated and gamma-irradiated patient-derived tumor cells. For the apathogenic non-lytic strain NDV-Ulster which is used in our clinical vaccine trials, we demonstrate selective replication in tumor cells as compared with corresponding normal cells. Furthermore, we present evidence that new virions produced by infected tumor cells are non-infectious using three different quantitative test methods. Our results demonstrate feasibility and broad applicability of this strategy of human tumor vaccine modification. Post-operative vaccination with the autologous virus-modified vaccine ATV-NDV thus provides a reasonable potential for pleiotropic modifications of the immune response of cancer patients against their own tumor. PMID 10341877

Tumor stimulator cell modification by infection with Newcastle Disease Virus: analysis of effects and mechanism in MLTC-CML cultures.
Feb. 1999 | Schirrmacher, V; Jurianz, K; Roth, C; Griesbach, A; Bonifer, R; Zawatzky, R
Effects of tumor stimulator cell modification by infection with Newcastle Disease Virus (NDV) are described as analysed in vitro in mixed lymphocyte tumor cell cultures (MLTC). Direct antitumor effects were seen with human melanoma or colon-carcinoma cells in a dose- and time-dependent manner when using live but not UV inactivated virus. When T cell stimulation was measured by [3H]-thymidine uptake, NDV infected tumor stimulator cells did not show an augmentation but rather an inhibitory effect in comparison to non-infected stimulator cells. Virus infected tumor stimulator cells were, however, capable of augmenting the induction of tumor specific cytotoxic T cells in MLTC-CML assays when using murine ESb lymphoma immune cells and syngeneic NDV modified ESb cells as stimulators. A CML stimulatory effect was also shown for NDV modified third party cells and thereof derived conditioned medium. These effects are most likely explained by interferon- which is induced in tumor cells by NDV infection and by interferon-á which is induced in responder cells when stimulated with NDV infected stimulator cells. PMID 9917494

Introduction of adhesive and costimulatory immune functions into tumor cells by infection with Newcastle Disease Virus.
Jan. 1999 | Haas, C; Ertel, C; Gerhards, R; Schirrmacher, V
We demonstrate in this study that infection of tumor cells by Newcastle Disease Virus (NDV) leads to changes in tumor cell surface adhesiveness and tumor immune costimulatory function. While adsorbtion of virions to the cell surface occurs after short-term (10 min) incubation and leads to cells expressing viral antigens at low antigen density (LAD), viral replication in the cytoplasm occurs within 5-24 h leading to tumor cells expressing viral antigens at high antigen density (HAD) as shown by quantitative FACS flow cytometry. Virus infected tumor cells showed an increased adhesiveness for erythrocytes and lymphocytes. When IL-2 preactivated human lymphocytes with cytotoxic potential were coincubated with 51Cr-labeled NDV-infected or non-infected human colon carcinoma cells increased lysis of the virus infected targets was observed. The virus mediated cell adhesion could be inhibited by monoclonal antibody (mAb) against the hemagglutinin-neuraminidase (HN) molecule but not by antibody against the fusion protein. HN cDNA transfectants also mediated increased lymphocyte adhesion in comparison to wild-type or neo-vector transfected control cells. Further experiments demonstrated that not only the adhesion domain of HN but also the neuraminidase plays a role in cell-cell interactions. A comparison of an NDV neuraminidase mutant of the strain Australian Victoria (AV-L1) with the parental AV strain revealed pronounced differences in their capacity to mediate lymphocyte binding and costimulatory activity. The mutant with highly decreased neuraminidase activity was very similar to NDV Ulster in adhesive and costimulatory activity while the parental line with high neuraminidase activity was negative for both functions. Costimulatory effects of NDV Ulster and AV-L1 were revealed when virions and suboptimal concentrations of anti-CD3 mAbs were coated to microtiter plates for induction of murine CD4 T cell proliferation. In human autologous mixed lymphocyte-tumor cell cultures up-regulation of T cell activation markers CD69 and CD25 was seen with NDV modified but not with non-modified tumor cells. PMID 9824618

Tumor-cell number and viability as quality and efficacy parameters of autologous virus-modified cancer vaccines in patients with breast or ovarian cancer.
Juli 1997 | Ahlert, T; Sauerbrei, W; Bastert, G; Ruhland, S; Bartik, B; Simiantonaki, N; Schumacher, J; Häcker, B; Schumacher, M; Schirrmacher, V
We investigated quality and efficacy criteria of an autologous, physically and immunologically purified, Newcastle disease virus (NDV)-modified, irradiated tumor-cell vaccine (ATV-NDV) by analyzing three independent cohorts (a through c) of patients vaccinated between 1991 and 1995. PMID 9193327

Complete regression of human fibrosarcoma xenografts after local Newcastle disease virus therapy.
Dez. 1994 | Lorence, R M; Katubig, B B; Reichard, K W; Reyes, H M; Phuangsab, A; Sassetti, M D; Walter, R J; Peeples, M E
We have recently demonstrated that a single local injection of the avian pathogen Newcastle disease virus (NDV; strain 73-T) causes complete regression of human neuroblastoma xenografts in athymic mice (R. M. Lorence, K. W. Reichard, B. B. Katubig, H. M. Reyes, A. Phuangsab, B. R. Mitchell, C. J. Cascino, R. J. Walter, and M. E. Peeples. J. Natl. Cancer Inst., 86: 1228-1233, 1994). In this report, we tried to determine if this in vivo antineoplastic effect of NDV extends to human sarcomas. Athymic mice with s.c. HT1080 fibrosarcoma xenografts (7-14 mm) were randomly divided into two groups and treated i.t. with a single injection of either 10(7) plaque-forming units of NDV or phosphate-buffered saline. Complete tumor regression occurred in 8 of 10 mice treated with NDV while unabated tumor growth occurred in all 9 mice treated with phosphate-buffered saline (P < 0.001). To determine if complete tumor regression was long lasting, the 8 mice were monitored for 1 year, during which time no tumor recurred. To test the antitumor effects of NDV on tumors derived from a fresh human sarcoma, a similar experiment was performed in athymic mice using TH15145 synovial sarcoma xenografts at their first and second passages. Of 9 mice with TH15145 xenografts, a single i.t. injection of NDV (10(7) plaque-forming units) caused complete regression of 3 tumors and > 80% regression in 3 more tumors. In contrast, tumors in all 5 mice treated with phosphate-buffered saline exhibited unabated growth (P < 0.03 for > 80% tumor regression). Since HT1080 fibrosarcoma cells express the N-ras oncogene, we explored the effects that transfection of this oncogene has on the sensitivity to NDV. Cultured human fibroblasts that were made tumorigenic following N-ras-transfection were found to be 1000-fold more sensitive to NDV than normal fibroblasts in a cytotoxicity assay. Oncogene expression by the HT1080 fibrosarcoma may therefore contribute to the long-lasting complete regression of this sarcoma following a single local injection of NDV. PMID 7954437

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