In vitro Characterization of Enhanced Human Immune Responses by GM-CSF Encoding HSV-1-Induced Melanoma Cells.

Purpose: We studied the innate and adaptive immune response against melanoma cells after JS-1 (wild-type herpes simplex virus 1, wt HSV-1) or Talimogene laherparepvec (T-VEC) infection and evaluated the antitumoral efficacy in human melanoma cells. We analyzed the putative synergistic biological and immunological effects of JS-1 or T-VEC combined with cytostatic drugs in human tumor and immune cells. T-VEC is a genetically modified strain of HSV-1. Genetic modifications (insertion of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene) were made to attenuate the virus and increase selectivity for cancer cells. In addition to the direct oncolytic effect, we investigated the immune stimulatory effects of T-VEC by comparing it with JS-1. JS-1 is identical T-VEC except for the inserted GM-CSF gene. Materials and Methods: We analyzed the effects of T-VEC and JS-1 with cytostatic drugs in human tumor-immune cell coculture experiments. After coculture, the surface markers CD80, CD83 and CD86 were measured by fluorescence-activated cell sorting and the cytokines, interleukin (IL)-2, IL-6, tumor necrosis factor (TNF)-α and GM-CSF, by enzyme-linked immunosorbent assays. Furthermore, we analyzed the potential of the viruses to induce T cell activation, measured on the basis of CD4, CD8 and CD69. Analysis of these markers and cytokines allows for conclusions to be drawn concerning the maturation of dendritic cells (DCs) and the immunostimulatory effects of the treatment. Results: We documented increased activation of human cytotoxic T lymphocytes after infection by both HSV-1 strains and treatment with cytostatic drugs without significant differences between T-VEC and JS-1. Conclusion: We demonstrated an immune response as a result of infection with both viruses, but T-VEC was in vitro not stronger than JS-1. The immunostimulatory effects of the viruses could be partially increased by chemotherapy, providing a rationale for future preclinical studies designed to explore T-VEC in combined regimens.

Virotherapy in Germany-Recent Activities in Virus Engineering, Preclinical Development, and Clinical Studies.

Virotherapy research involves the development, exploration, and application of oncolytic viruses that combine direct killing of cancer cells by viral infection, replication, and spread (oncolysis) with indirect killing by induction of anti-tumor immune responses. Oncolytic viruses can also be engineered to genetically deliver therapeutic proteins for direct or indirect cancer cell killing. In this review-as part of the special edition on "State-of-the-Art Viral Vector Gene Therapy in Germany"-the German community of virotherapists provides an overview of their recent research activities that cover endeavors from screening and engineering viruses as oncolytic cancer therapeutics to their clinical translation in investigator-initiated and sponsored multi-center trials. Preclinical research explores multiple viral platforms, including new isolates, serotypes, or fitness mutants, and pursues unique approaches to engineer them towards increased safety, shielded or targeted delivery, selective or enhanced replication, improved immune activation, delivery of therapeutic proteins or RNA, and redirecting antiviral immunity for cancer cell killing. Moreover, several oncolytic virus-based combination therapies are under investigation. Clinical trials in Germany explore the safety and potency of virotherapeutics based on parvo-, vaccinia, herpes, measles, reo-, adeno-, vesicular stomatitis, and coxsackie viruses, including viruses encoding therapeutic proteins or combinations with immune checkpoint inhibitors. These research advances represent exciting vantage points for future endeavors of the German virotherapy community collectively aimed at the implementation of effective virotherapeutics in clinical oncology.

Immunotherapy in gastrointestinal cancer: Recent results, current studies and future perspectives.

The new therapeutic approach of using immune checkpoint inhibitors as anticancer agents is a landmark innovation. Early studies suggest that immune checkpoint inhibition might also be effective in patients with gastrointestinal cancer. To improve the efficacy of immunotherapy, different strategies are currently under evaluation. This review summarises the discussion during the European Organisation for Research and Treatment of Cancer Gastrointestinal Tract Cancer Translational Research Meeting in Mainz in November 2014 and provides an update on the most recent results of immune therapy in gastrointestinal cancers. Knowledge of potential relationships between tumour cells and their microenvironment including the immune system will be essential in gastrointestinal malignancies. In this context, the density of T cell infiltration within colorectal cancer metastases has been associated with response to chemotherapy, and a high expression of programmed cell death ligand 1 (PD-L1) in advanced gastric cancer has been related with poor prognosis. Effective targets might include neo-antigens encoded from genes carrying tumour-specific somatic mutations. Tailored immunotherapy based on such mutations could enable the effective targeting of an individual patient's tumour with vaccines produced on demand. Other strategies considering checkpoint inhibitors have shown efficacy by targeting cytotoxic T-lymphocyte-associated protein 4 and PD-1 or PD-L1. DNA mismatch repair-deficient tumours appear to be potentially the best candidates for these therapies. Finally, the combination of oncolytic viruses with immunotherapy might boost antitumour activity as well. Further evaluation of these promising immunological therapeutic approaches will require large prospective clinical studies.

Oncolytic virotherapy as emerging immunotherapeutic modality: potential of parvovirus h-1.

Human tumors develop multiple strategies to evade recognition and efficient suppression by the immune system. Therefore, a variety of immunotherapeutic strategies have been developed to reactivate and reorganize the human immune system. The recent development of new antibodies against immune check points may help to overcome the immune silencing induced by human tumors. Some of these antibodies have already been approved for treatment of various solid tumor entities. Interestingly, targeting antibodies may be combined with standard chemotherapy or radiation protocols. Furthermore, recent evidence indicates that intratumoral or intravenous injections of replicative oncolytic viruses such as herpes simplex-, pox-, parvo-, or adenoviruses may also reactivate the human immune system. By generating tumor cell lysates in situ, oncolytic viruses overcome cellular tumor resistance mechanisms and induce immunogenic tumor cell death resulting in the recognition of newly released tumor antigens. This is in particular the case of the oncolytic parvovirus H-1 (H-1PV), which is able to kill human tumor cells and stimulate an anti-tumor immune response through increased presentation of tumor-associated antigens, maturation of dendritic cells, and release of pro-inflammatory cytokines. Current research and clinical studies aim to assess the potential of oncolytic virotherapy and its combination with immunotherapeutic agents or conventional treatments to further induce effective antitumoral immune responses.

Influence of the oncolytic parvovirus H-1, CTLA-4 antibody tremelimumab and cytostatic drugs on the human immune system in a human in vitro model of colorectal cancer cells.

INTRODUCTION: Tumor-directed and immune-system-stimulating therapies are of special interest in cancer treatment. Here, we demonstrate the potential of parvovirus H-1 (H-1PV) to efficiently kill colorectal cancer cells and induce immunogenicity of colorectal tumors by inducing maturation of dendritic cells (DCs) alone and also in combination with cytostatic drugs in vitro. Using our cell culture model, we have additionally investigated the effects of anti-CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) receptor antibody tremelimumab on this process. MATERIALS AND METHODS: Colon carcinoma cell lines were treated with different concentrations of cytostatic drugs or tremelimumab or were infected with H-1PV in different multiplicities of infection (MOIs), and viability was determined using MTT assays. Expression of CTLA-4 in colon carcinoma cell lines was measured by FACScan™. For the coculture model, we isolated monocytes using adherence, and differentiation into immature DCs (iDCs) was stimulated using interleukin-4 and granulocyte-macrophage colony-stimulating factor. Maturation of iDCs into mature DCs (mDCs) was induced by a cytokine cocktail. SW480 colon carcinoma cells were infected with H-1PV or treated with cytostatic drugs. Drug treated and H-1PV-infected SW480 colon carcinoma cells were cocultured with iDCs and expression of maturation markers was measured using FACScan™. Cytokine measurements were performed using enzyme-linked immunosorbent assay. RESULTS: Colon carcinoma cells SW480 were potently infected and killed by H-1PV. CTLA-4 expression in SW480 cells increased after infection with H-1PV and also after treatment with cytostatic drugs. Tremelimumab had no influence on viability of the colon carcinoma cell line. There was no maturation of iDCs after coculture with SW480; instead, H-1PV-infected or drug pretreated SW480 induced maturation. Cytokine production was higher for H-1PV-infected cells but was not significantly enhanced by tremelimumab treatment alone or in combination. Addition of tremelimumab did not interfere with the maturation process as measured by markers of maturation as well as by determination of cytokine levels. CONCLUSION: By enhancing both cell death and immunogenicity of tumors, H-1PV is of special interest for tumor-directed therapy. These features make it a promising candidate for clinical application in human colorectal cancer. As tremelimumab does not significantly interfere with this process, an interesting therapeutic combination of active enhancement of tumor immunogenicity and independent masking of the CTLA-4 silencing process on tumor cells is highlighted.