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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

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

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

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

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

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

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

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

Autologous tumor cell vaccines for post-operative active-specific immunotherapy of colorectal carcinoma: long-term patient survival and mechanism of function.
Jan. 2014 | Schirrmacher, Volker; Fournier, Philippe; Schlag, Peter
Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Surgery remains the primary curative treatment but nearly 50% of patients relapse as consequence of micrometastatic or minimal residual disease (MRD) at the time of surgery. Spontaneous T-cell-mediated immune responses to CRC tumor-associated antigens (TAAs) in tumor-draining lymph nodes and in the bone marrow (BM) lead to infiltration of the tumors by lymphocytes. Certain types of such tumor-infiltrating lymphocytes (TILs) have a positive and others a negative impact on the patients' prognosis. This review focuses on advances in CRC active-specific immunotherapy (ASI), in particular on results from randomized controlled clinical studies employing therapeutic autologous tumor cell vaccines. The observed improvement of long-term survival is explained by activation and mobilization of a pre-existing repertoire of tumor-reactive memory T cells which, according to recent discoveries, reside in distinct niches of patients' bone marrow in neighborhood with hematopoietic (HSC) and mesenchymal (MSC) stem cells. Interestingly, memory T cells also contain a subset of stem memory T cells (SMTs) in addition to effector (EMTs) and central memory T cells (CMTs). The mechanism of function of a therapeutic vaccine in a chronic disease is distinct from that of prophylactic vaccines which have to generate de novo protective immune responses. The advantage of autologous vaccines for mobilization of a broad and highly individual repertoire of memory T cells will be discussed. PMID 24219122

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

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

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

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

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

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

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

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

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

Treatment of advanced metastasized breast cancer with bone marrow-derived tumour-reactive memory T cells: a pilot clinical study.
Apr. 2009 | Schuetz, Florian; Ehlert, Katrin; Ge, Yingzi; Schneeweiss, Andreas; Rom, Joachim; Inzkirweli, Natalija; Sohn, Christoph; Schirrmacher, Volker; Beckhove, Philipp
Breast cancer patients frequently harbour tumour-reactive memory T cells in their bone marrow (BM) but not in the blood. After reactivation ex-vivo these cells rejected autologous breast tumours in xenotransplanted mice demonstrating therapeutic potential upon reactivation and mobilization into the blood. We conducted a clinical pilot study on metastasized breast cancer patients to investigate if ex-vivo reactivation of tumour-reactive BM memory T cells and their adoptive transfer is feasible and increases the frequencies of tumour-reactive T cells in the blood. PMID 18998129

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

Randomized clinical studies of anti-tumor vaccination: state of the art in 2008.
Dez. 2008 | Fournier, Philippe; Schirrmacher, Volker
This review elucidates state-of-the-art clinical studies on active specific immunotherapy with tumor vaccines. It refers solely to randomized studies and has a special focus on patient's survival, the most important parameter for any therapy. Of special interest, from a tumor immunological point of view, is a comparison between the results obtained with allogeneic tumor cell-derived vaccines and those obtained with autologous tumor cell-derived vaccines. Overall, autologous vaccines have given better results than allogeneic vaccines. Random mutations in cancer generate unique antigens in each individual case. The superiority of autologous vaccines suggests that unique tumor-associated antigens are particularly important in generating responsive T cells for a therapeutic effect. PMID 19093773

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

Detection and functional analysis of tumor infiltrating T-lymphocytes (TIL) in liver metastases from colorectal cancer.
Juli 2008 | Wagner, Philipp; Koch, Moritz; Nummer, Daniel; Palm, Sylvia; Galindo, Luis; Autenrieth, Daniel; Rahbari, Nuh; Schmitz-Winnenthal, Friedrich H; Schirrmacher, Volker; Büchler, Markus W; Beckhove, Philipp; Weitz, Jürgen
Tumor-infiltrating T lymphocytes (TIL) play an important role in primary colorectal cancer, but their activity in liver metastases has not yet been investigated. The aim of this study was to examine whether tumor-selective infiltration, activation, and cytotoxic activity of TIL can be demonstrated in situ in colorectal liver metastases. PMID 18521684

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

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

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

Tumor infiltrating T lymphocytes in colorectal cancer: Tumor-selective activation and cytotoxic activity in situ.
Nov. 2006 | Koch, Moritz; Beckhove, Philipp; Op den Winkel, Jan; Autenrieth, Daniel; Wagner, Philipp; Nummer, Daniel; Specht, Sebastian; Antolovic, Dalibor; Galindo, Luis; Schmitz-Winnenthal, Friedrich H; Schirrmacher, Volker; Büchler, Markus W; Weitz, Jürgen
To examine whether tumor-selective infiltration, activation, and cytotoxic activity of tumor infiltrating T lymphocytes (TIL) can be demonstrated in situ in colorectal cancer samples. PMID 17122624

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 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

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

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

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

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

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

Influence of adjuvant hormone therapy and chemotherapy on the immune system analysed in the bone marrow of patients with breast cancer.
Jan. 2003 | Solomayer, Erich-Franz; Feuerer, Markus; Bai, Lianhua; Umansky, Victor; Beckhove, Philipp; Meyberg, Gabriele C; Bastert, Gunther; Schirrmacher, Volker; Diel, Ingo Jakob
Bone marrow is a special compartment for antitumor immunological memory in patients with breastcancer. Until now, the influence of adjuvant systemic therapy on the immune system has only been investigated in peripheral blood and not in bone marrow. In this study, we analyzed the effect of hormone therapy and chemotherapy on the immune activation status in bone marrow. PMID 12538466

Cognate interactions between memory T cells and tumor antigen-presenting dendritic cells from bone marrow of breast cancer patients: bidirectional cell stimulation, survival and antitumor activity in vivo.
Nov. 2002 | Bai, Lianhua; Beckhove, Philipp; Feuerer, Markus; Umansky, Viktor; Choi, Carmen; Solomayer, Florian Schütz Erich-Franz; Diel, Ingo J; Schirrmacher, Volker
Dendritic cells (DC) and T cells were generated from Ficoll separated bone marrow (BM) mononuclear cells of primary operated breast cancer patients according to new cell culture protocols. BM-DC were capable of functioning as professional antigen-presenting cells (APCs) and of inducing autologous antigen-specific memory T-cell responses to either tetanus toxoid recall antigen or to breast cancer antigens. Treatment with lipopolysaccharide (LPS) resulted in phenotypic and functional maturation of BM-DC. When BM-DC, pulsed with breast cancer-associated tumor antigens, were cocultured with autologous patient-derived BM-T cells to allow for cognate breast cancer antigen recognition and stimulation, apoptosis of T cells-which occurred in noncognate coculture systems-was inhibited. Furthermore, in cocultures allowing for antigen-specific cognate interactions, the expression on BM-DC of CD83, MHC class II, CD40 and CD86 molecules was upregulated and the cytokines IL-12 and IFN-alpha were produced in significantly elevated amounts. Adoptive transfer of breast cancer-reactive memory T cells together with APCs into human breast cancer-bearing NOD/SCID mice caused a regression of the tumor and prolonged survival of the animals. This was not the case when such animals had been treated by transfer of reactivated BM T cells without BM-DCs. Our findings suggest that cognate interactions between cancer patient-derived memory BM-T cells and tumor antigen-presenting BM-DCs are important for reciprocal cell stimulation, survival and therapeutic activity. PMID 12455056

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

Generation of dendritic cells from human bone marrow mononuclear cells: advantages for clinical application in comparison to peripheral blood monocyte derived cells.
Jan. 2002 | Bai, L; Feuerer, M; Beckhove, P; Umansky, V; Schirrmacher, V
Dendritic cells (DCs) currently used for vaccination in clinical studies to induce immunity against malignant cells are normally generated from peripheral blood-derived monocytes. Here we studied conditions for the generation of DCs from unseparated human bone marrow (BM) mononuclear cells and compared them functionally with DCs from blood. The two types of DCs, from bone marrow (BM-DC) and peripheral blood (BL-DC), were generated in parallel from the same normal healthy donors by culturing in serum-free X-VIVO 20 medium containing GM-CSF and IL-4, and then the phenotypes and functions were compared. BM-DC generation occurred in 14 days and involved proliferative expansion from CD34 stem cells and differentiation while BL-DC generation occurred in 7 days from CD14 monocytes and involved only differentiation. A 7- to 25-fold higher number of DCs could be obtained from BM than from blood. BM-DC had similar phenotypes as BL-DC. The capacity to stimulate MLR reactivity in allogeneic T lymphocytes was higher with BM-DC than that with BL-DC. Also, the capacity to stimulate autologous memory T cell responses to tetanus toxoid (TT) or tuberculin (PPD) was higher with BM-DC than with BL-DC. These results suggest that BM-DC as produced here may be a very economic and useful source of professional antigen-presenting cells for anti-tumor immunotherapeutic protocols. PMID 11788884

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

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