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.

Introduction of adhesive and costimulatory immune functions into tumor cells by infection with Newcastle Disease Virus.

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.

Virus potentiation of tumor vaccine T-cell stimulatory capacity requires cell surface binding but not infection.

This study elucidates a basically new mechanism of function of a virus-modified tumor cell vaccine which has been successful in mouse tumor models (metastatic ESb lymphoma and B16-F10 melanoma) in preventing or delaying metastatic spread and improving survival and which is being tested in clinical studies. Modification of tumor cells by a low dose of Newcastle disease virus (NDV), which caused this therapy effect, led to an augmentation of the tumor-specific cytotoxic CD8 T-cell (CTL) response and to increased CD4 T-helper activity in the absence of an antiviral T-cell response. When various noninfectious NDV preparations, which, according to newly established quantitative tests, had lost one or several of the viral functions, were tested, noninfectious virus particles with inactive fusion proteins and virus inactivated by UV light, which could fuse but could not replicate, were as active as infectious NDV in the tumor-specific CTL response. In contrast, NDV inactivated by heat treatment (NDV-HI) had no effect on the CTL response. NDV-HI, even when added to the cultures in excess, did not modulate the antitumor CTL response, which argues against a nonspecific adjuvant effect. There was no mitogenic effect of NDV. Because NDV-HI was not able to bind to the tumor cell surface and because hemagglutinin-neuraminidase c-DNA transfectants increased antigen-presenting function as virus-modified cells do, we propose that the NDV effect in the CTL response is caused by the introduction of functional viral hemagglutinin-neuraminidase molecules (1000 per virus particle) into the tumor cell surface, thereby facilitating cell-cell interactions through their cell-binding and neuraminidase activity.

Adhesive function of newcastle-disease virus hemagglutinin in tumor-host interaction.

Infection of metastatic lymphoma cells (ESbL) by a low dose of a non-lytic strain of Newcastle disease virus (NDV) leads to viral replication followed by strong cell surface expression of viral antigens, especially hemagglutinin neuraminidase (HN). The expressed HN was functional and facilitated cell-cell interactions and cell attachment. This was shown for NDV infected tumor cells, lymphocytes, fibroblasts and endothelial cells. The interactions could be strongly inhibited by antibodies against the viral HN protein. Increased binding was also seen with HN c-DNA transfectants expressing the HN as the only viral protein. Viral infection did not influence proliferation and lysability of the infected tumor cells. Following intravenous injection of tumor cells, the number of hepatic metastases was significantly reduced when the cells had been pre-infected with NDV. This reduction of metastases correlated with an increased survival time of the animals. As potential mechanisms of these NDV effects we propose augmentation of cell-eel interactions and immune functions and reduction of invasive capacity of NDV infected, as compared to non-infected tumor cells.

Viral hemagglutinin augments peptide-specific cytotoxic T cell responses.

In attempt to increase the induction of peptide-specific cytolytic T cells (CTL) we investigated the effect of the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) gene product on the activation of peptide-specific CTL. Spleen cells of CH3 mice immunized against the influenza nucleoprotein peptide 50-63 (NP 50-63) were restimulated in vitro (i) with peptide-pulsed syngeneic fibroblast cells (Ltk-) as antigen-presenting cells, which were in addition (ii) infected with NDV or (iii) stably transfected with the HN cDNA of NDV. A greater than sixfold increase in peptide-specific CTL responses was observed in cultures restimulated with peptide-pulsed Ltk- cells which co-expressed viral hemagglutinin due to either infection or transfection. A similar augmentation was seen in CTL responses against other types of antigen (major histocompatibility complex alloantigens, minor histocompatibility antigens or tumor antigens) when suboptimal cultures were stimulated with the respective antigen-presenting cells modified by NDV infection. These findings suggest that NDV or viral HN expressed on antigen-presenting cells or tumor cells can exert a T cell co-stimulatory function.

Specific membrane receptors for human interferon-gamma (IFN-gamma).

IFN-gamma is a T-cell derived lymphokine which possesses antitumoral activity for a variety of malignant cells by virtue of its direct effect on cell growth and by its immunomodulatory activity. All IFN-gamma actions are initiated by binding to high affinity cell surface receptors, which are constitutively expressed in virtually all cell lines from various tissues. Although the detailed structure of the IFN-gamma receptor is still elusive, the available data suggest that the high affinity IFN-gamma binding site is a heterodimeric molecule of 128 kDa comprised of two subunits of 53 and 75 kDa, which is invariantly expressed in distinct tumor cells, differing in their response to IFN-gamma. Thus, the capability and type of cellular response to IFN-gamma appears to be largely determined at a post-receptor level. Nevertheless, in sensitive cell lines, the magnitude of response is proportional to the quantity of receptor ligand interactions. This could be important for the definition of effective doses in clinical applications of IFN-gamma, as distinct tumor cells are heterogeneous with respect to quantity of IFN-gamma receptors, with greater 20-fold differences of the number of receptors per cell.

Quantitation and characterization of gamma-interferon receptors on human tumor cells.

The vast majority (71 of 77) of human tumor cells derived from various tissue origins were found to express specific membrane receptors for gamma-interferon (IFN-gamma). Six receptor-negative tumors were found among leukemic cells of lymphoid origin. Scatchard analysis with 125I-labeled human recombinant IFN-gamma revealed a similar binding affinity with a mean dissociation constant (Kd) of around 2 X 10(-11) M not only for various established cell lines, but also for leukemic and carcinoma cells derived from biopsy material. In contrast to similar KdS, large differences in the number of expressed IFN-gamma membrane receptors were found on distinct tumor cells of the same cell type ranging from a few hundred up to 2 X 10(4) for both carcinoma cells and leukemic cells. For comparison, the IFN-gamma receptor number on normal lymphocytes (mean, approximately 300/cell) and normal bone marrow cells (mean, approximately 1000/cell) was consistently found to be low. Cross-linking of membrane-bound 125I-IFN-gamma with disuccinimidyl suberate and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed, in both leukemia and carcinoma cells, three distinct complexes with molecular weights of approximately 70,000, 92,000, and 160,000, suggesting the existence of IFN-gamma receptor subunits. A dimeric structure of the functional IFN-gamma receptor with an estimated molecular weight of about 128,000 +/- 10,000 is proposed. Together with the Scatchard analysis, these data suggest the existence of a single class of high affinity IFN-gamma receptors in tumor cells of distinct tissue origin.