Antitumor effects of Newcastle Disease Virus in vivo: local versus systemic effects.

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.

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.

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.

Immunization with virus-modified tumor cells.

Direct infection of tumor cells with viruses transfering protective or therapeutic genes-a frequently used procedure for production of tumor vaccines in human gene therapy-is often limited by the number of tumor cells that can reliably be infected, as well as by issues of selectivity and safety. In this review, we describe 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). Advantages of this virus are its good cell-binding properties, its selective replication in tumor cell cytoplasm, which is independent of cell proliferation, and its relative safety. Most important for its use as an adjuvant in human cancer vaccine are its ability to introduce T-cell costimulatory activity, to prevent anergy induction, and to induce locally chemokines (eg, RANTES, IP-10) and cytokines (eg, interferon alpha, beta [IFN-alpha, beta] and tumor necrosis factor-alpha [TNFalpha]) that affect T-cell recruitment and activation. A further development consists of attachment-via NDV-derived hemagluttinin-neuraminidase (HN) membrane-anchoring molecules-of universal defined bispecific reagents such as T-cell-activating anti-CD28 antibodies. Finally, we summarize the status of our clinical studies with the autologous virus modified live cell vaccine (ATV)-NDV.

Telomerase activity correlates with tumor aggressiveness and reflects therapy effect in breast cancer.

Telomerase appears to be an important factor for the control of cellular proliferation capacity and for tumorigenesis. Enzyme activity is highly increased in almost all human tumors and distinguishes them from benign lesions. Besides its diagnostic value, telomerase activity appears to be associated with tumor progression. The purpose of our study was to evaluate the significance of telomerase activity as a clinical marker in breast cancer. Twenty-five tumor samples from breast cancer patients were analyzed retrospectively for telomerase activity in chemotherapy-treated and untreated tumors. For each patient an identical number of cells was measured quantitatively for telomerase activity using the Telomerase-PCR-ELISA based on the TRAP (telomerase repeat amplification protocol) method. The findings were compared to clinical course, therapy and staging parameters. Telomerase activity was detected in all breast cancers. A significant correlation was found between enzyme activity and tumor size, lymph node status and stage: with ongoing tumor progression, telomerase activity appeared to increase in primary carcinomas. No correlation was seen between enzyme activity and the clinical course of patients. Without exception, telomerase activity was strongly decreased in all chemotherapy-treated tumors compared to untreated tumors. Our preliminary data indicate that telomerase activity is associated with aggressiveness of breast tumors and appears to mirror the anti-proliferative effects of chemotherapy.

Tumor-cell number and viability as quality and efficacy parameters of autologous virus-modified cancer vaccines in patients with breast or ovarian cancer.

PURPOSE: 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. MATERIALS AND METHODS: Included were 63 patients with primary breast cancer (a), 27 with metastatic pretreated breast cancer (b), and 31 with metastatic pretreated ovarian cancer (c). In addition to vaccine, cohorts b and c received nonspecific immunotherapy as supportive treatment. After cryoconservation and purification, the vaccines varied in applied numbers of viable cells and dead cell contaminations. We retrospectively hypothesized that an immunogenic vaccine should contain at least 1.5 x 10(6) viable tumor cells and viability should be at least 33%. Each cohort was thus divided into two groups; one that received vaccine type A (A), fulfilling both criteria; and the other type B (B), missing one or both criteria. RESULTS: Conventional prognostic factors were wall balanced between A and B in cohorts a and c. In cohort a, there was a benefit in survival (P = .026) and disease-free survival (P = .089) for A. In addition, in cohort a, the relative risk of dying in the group that received A as compared with B was 0.2 (univariate Cox model). There were also survival trends in favor of A versus B (P = .18 and P = .09, respectively) in cohorts b and c, with relative risks of 0.5 and 0.42, respectively. In cohort b, the survival benefit could not be ascribed to vaccine quality alone, because of prognostic imbalance in favor of A. CONCLUSION: In cohort c, like in cohort a, the survival benefit for A may be ascribed to the ATV-NDV vaccine quality, since prognostic factors were not biased. This could imply clinical effectivity in breast and ovarian cancer with ATV-NDV high-quality vaccine. Furthermore, the data provide clinically relevant information for standardization and quality control of autologous tumor-cell vaccines. A randomized study is urgently needed.

Newcastle disease virus-infected intact autologous tumor cell vaccine for adjuvant active specific immunotherapy of resected colorectal carcinoma.

An active specific immunization (ASI) procedure with two types of autologous tumor cell vaccines (ATVs) is tested for adjuvant immunotherapy of resected colorectal carcinoma to provide preliminary information on local immunological skin responses, side effects, and 2-year survival rates. For vaccine preparation, the tumor-derived freshly isolated and cryopreserved cells were thawed, purified by Percoll density centrifugation, and depleted of tumor-infiltrating lymphocytes by immunomagnetic beads. After inactivation by 200 Gy, the cells of this ATV were either infected by Newcastle disease virus (NDV) or they were admixed with Bacillus Calmette Guérin (BCG) organisms. Vaccination was performed in the arm beginning 6-8 weeks after operation, three times at 2-week intervals. Of 57 patients that received ASI, 48 were treated by virus-infected ATV (ATV-NDV) and 9 were treated with the BCG-admixed vaccine (ATV/BCG). The mean value of delayed hypersensitivity skin reactions from ATV-NDV-treated patients was 18 mm for the first vaccination and 26 and 29 mm for the succeeding ones. Although the application of ATV-NDV was associated with only mild side effects, the ATV/BCG vaccine led to long-lasting ulcers and to more serious side effects. The 2-year survival rate obtained with ATV-NDV was 97.9%, whereas the survival rate with ATV/BCG was 66.7%. The mean survival of 661 patients from a historical control was 73.8%. These data suggest that the type and quality of the tumor vaccine for ASI treatment is important. The findings with ATV-NDV necessitate corroboration in a prospective, randomized controlled study.

Isolation of a human melanoma adapted Newcastle disease virus mutant with highly selective replication patterns.

The apathogenic Newcastle disease virus (NDV) strain Ulster has been used successfully as an adjuvant component for active specific immunotherapy of malignant mouse lymphoma, and in nude mice it was shown to be able to lead to retardation of the s.c. growth of xenotransplanted human melanoma cells. In order to improve in vivo effectiveness of virotherapy of human tumors without significantly increasing the risk of unspecific viral replication in host cells, we adapted the virus for growth in a human melanoma line (MeWo M). For this purpose NDV Ulster was mutagenized and a variant was selected which could replicate and reinfect the tumor line. The mutant (NDV 1E 10) performed late lysis on the melanoma line. Replication was found to be at least 100 times more efficient in MeWo M than in 6 of 8 other human tumor cell lines of different tissue origin. In 10 of 11 murine cell lines, NDV 1E 10 did not replicate via multicycles. Chick embryonic fibroblasts were permissive for nonlytic replication. Neither the virulent wild-type NDV Italian nor the avirulent strain NDV Ulster shared these specific replication properties with the new variant. We also established MeWo melanoma sublines with different metastatic capacities and tested them as targets for NDV 1E 10 infection. The MeWo subpopulations exhibited comparatively small differences in permissivity for multicyclic replication, but the more metastatic MeWo Met, like allogeneic melanoma lines, was more resistant to lysis. NDV Italian, in contrast, showed no differences in replication and lysis on any of the tested melanoma lines. Trypsin-activation experiments suggested an incomplete cleavage of mutant envelope glycoprotein F by the permissive cell line and, thus, mechanisms of specific infection and replication not requiring fully activated envelope glycoproteins.

Human monoclonal antibodies directed against ovarian carcinoma.

In order to obtain human monoclonal antibodies for immunodetection or treatment of ovarian carcinoma, we generated hybridomas by fusing peripheral blood lymphocytes of patients with ovarian carcinoma and the mouse myeloma cell line X63.Ag8.653. The patients were immunized prior to collection of peripheral blood lymphocytes with autologous tumor cells admixed with New Castle Disease Virus. Immunocytologic studies of hybridoma supernatant with tumor cells fixed with methanol and air-dried tumor cells indicated that all 14 antibodies reactive with tumor cells were directed against cytoplasmic or nuclear antigens. One hybridoma designated as 1B3 was very stable and secreted a specific IgM antibody. This cell line expanded in nude mice and the monoclonal antibody 1B3 was effectively purified from ascites or supernatant fluid. In experiments with tissue sections partly derived from the patient whose peripheral blood lymphocytes were used for fusion, biotinylated 1B3 recognized ovarian tumor cells. There was no significant cross-reaction against normal tissue from stomach, small intestine, colon, lung, kidney, endometrium, placenta, or lymph node. Mammary carcinoma preparations were also stained by 1B3 while normal breast tissue was negative.

Prevention of metastatic spread by postoperative immunotherapy with virally modified autologous tumor cells. I. Parameters for optimal therapeutic effects.

Effective anti-metastatic therapy was achieved in a mouse tumor model by combining surgery with post-operative immunotherapy using virus-modified autologous tumor cells. No therapeutic effect was observed when using for immunotherapy the nonmodified autologous tumor ESb, which is only weakly immunogenic and highly metastatic. The viral modification was achieved by infecting the tumor with an avirulent strain of Newcastle disease virus (NDV), which led to expression of viral antigens and to an increase in the tumor cells' immunogenicity. Parameters which were of decisive influence for success or failure of therapy were the time of operation of the primary tumor and the dose of virus which was admixed to a standard dose of irradiated tumor cells. There was a low dose optimum of NDV at about 100 hemagglutinating units per 25 X 10(6) tumor cells. The therapeutic effect observed was less pronounced if the virus was given separately from the tumor cells. Post-operative immunotherapy with NDV-modified tumor cells had the following therapeutic effects: (1) disappearance of micrometastases from visceral organs as ascertained by a sensitive bioassay; (2) life prolongation in virtually all animals when compared to controls (operated only); (3) cures in about 50% of the treated animals. The possible mechanism of this therapeutic effect and its potential for clinical application are discussed.