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Using viruses to treat cancer is a novel approach to an age-old disease. Oncolytic viruses are native or recombinant viruses that have the innate or enhanced capability to infect tumour cells, replicate within the tumour microenvironment and subsequently lyse those cells. One representative, the vaccinia virus (VACV), belongs to the orthopoxvirus genus of the Poxviridae family. GLV-1h68, a recombinant and attenuated vaccinia virus devel- oped by the Genelux Corporation, is a member of this family currently being tested in various phase I/II clinical trials under the name GL-ONC1. It has been shown to specif- ically replicate in tumour cells while sparing healthy tissue and to metabolise prodrug at or transport immunological payloads to the site of affliction. Since imaging modalities offer little insight into viral replication deep within the body, and because oncolytic virotherapy is dependent on replication within the target tissue, the need for a monitoring system is evident. Pharmacokinetic analysis of this oncolytic agent was to give insight into the dynamics present in tumours during treatment. This, in turn, would give clinicians the opportunity to monitor the efficacy as early as possible after the onset of treatment, to observe treatment progression and possibly to gauge prognosis, without resorting to invasive procedures, e.g. biopsies. A criteria for viable biomarkers was that it had to be directly dependent on viral replica- tion. Ideally, a marker for treatment efficacy would be specific to the treatment modality, not necessarily the treatment type. Such a marker would be highly detectable (high sen- sitivity), specific for the treatment (high specificity), and present in an easily obtained specimen (blood). Taking this into consideration, the biomarkers were chosen for their potential to be indicators of viral replication. Thus, the biomarkers analysed in this thesis are: the native proteins expressed by the viral genes A27L and B5R, the virally encoded recombinant proteins β-galactosidase, β-glucuronidase, green fluorescent protein (GFP), carboxypeptidase G2 (CPG2) and carcinoembryonic antigen (CEA). Each marker is under the control of one of five different promoters present. All recombinant viruses used in this thesis express A27L, B5R, GFP and β-glucuronidase and all are derived from the parental virus GLV-1h68. In addition to these markers, GLV-1h68 expresses β-galactosidase; GLV-1h181 expresses CPG2. [...]
Oncolytic viral therapies have shown great promise pre-clinically and in human clinical trials for the treatment of various cancers. Oncolytic viruses selectively infect and replicate in cancer cells, destroying tumor tissue via cell lysis, while leaving noncancerous tissues unharmed. Vaccinia virus (VACV) is arguably one of the safest viruses, which has been intensively studied in molecular biology and pathogenesis as a vaccine for the eradication of smallpox in more than 200 million people. It has fast and efficient replication, and cytoplasmic replication of the virus lessens the chance of recombination or integration of viral DNA into the genome of host cells. Anti-tumor therapeutic efficacy of VACV has been demonstrated for human cancers in xenograft models with a variety of tumor types. In addition recombinant oncolytic VACVs carrying imaging genes represent an advance in treatment strategy that combines tumor-specific therapeutics as well as diagnostics.
As for other targeted therapies, a number of challenges remain for the clinical translation of oncolytic virotherapy. These challenges include the potential safety risk of replication of oncolytic virus in non-tumor tissue, the relatively poor virus spread throughout solid tumor tissue and the disadvantageous ratio between anti-viral and anti-tumoral immunity. However, manipulation of components of the tumor microenvironment may help oncolytic virus infection in killing the tumor tissue and thereby increasing the anti-tumor efficacy. Furthermore, dogs with natural cancer are considered as one of the best animal models to develop new drugs for cancer therapy. Traditionally, rodent cancer models have been used for development of cancer therapeutics. However, they do not adequately represent several features that define cancer in humans, including biology of initiation of tumor, the complexity of cancer recurrence and metastasis and outcomes to novel therapies. However, the tumor microenvironment, histopathology, molecular and genomics data from dog tumors has significant similarities with corresponding human tumors. These advantages of pet dog cancers provide a unique opportunity to integrate canine cancer patients in the studies designed for the development of new cancer drugs targeted against both human and canine cancers. This dissertation centers on the use of VACV strains in canine cancer xenografts with the aim of understanding the effects of modulation of tumor microenvironment on VACV-mediated tumor therapy.
In the first studies, wild-type VACV strain LIVP6.1.1 was tested for its oncolytic efficiency in canine soft tissue sarcoma (STSA-1) and canine prostate carcinoma (DT08/40) cells in culture and xenografts models. LIVP6.1.1 infected, replicated within, and killed both STSA-1 and DT08/40 cells in cell culture. The replication of virus was more efficient in STSA-1 cells compared to DT08/40 cells. In xenograft mouse models, LIVP6.1.1 was safe and effective in regressing both STSA-1 and DT08/40 xenografts. However, tumor regression was faster in STSA-1 xenografts compared to DT08/40 xenografts presumably due to more efficient replication of virus in STSA-1 cells. Biodistribution profiles revealed persistence of virus in tumors 5 and 7 weeks post virus injection in STSA-1 and DT08/40 xenografts, respectively, with the virus mainly cleared from all other major organs. Immunofluorescence staining detected successful colonization of VACV in the tumor. Consequently, LIVP6.1.1 colonization in the tumor showed infiltration of innate immune cells mainly granulocytes and macrophages in STSA-1 tumor xenografts. These findings suggest that virotherapy-mediated anti-tumor mechanism in xenografts could be a combination of direct viral oncolysis of tumor cells and virus-dependent infiltration of tumor-associated host immune cells.
In further studies, the effects of modulation of tumor angiogenesis of VACV therapy were analyzed in canine cancer xenografts. GLV-1h109 VACV strain (derived from prototype virus GLV-1h68) encoding the anti-VEGF single chain antibody GLAF-1 was characterized for its oncolytic efficacy in STSA-1 and DT08/40 cancer cells in culture and tumor xenografts. Concomitantly, the effects of locally expressed GLAF- 1 in tumors on virus replication, host immune infiltration, tumor vascularization and tumor growth were also evaluated.
GLV-1h109 was shown to be similar to the parental virus GLV-1h68 in expression of the two marker genes that both virus strains have in common (Ruc-GFP and gusA) in cell cultures. Additionally, the anti-VEGF single-chain antibody GLAF-1 was expressed by GLV-1h109 in both cell cultures and tumor xenografts. The insertion of GLAF-1 did not significantly affect the replication and cytotoxicity of GLV-1h109 in the STSA-1 and DT08/40 cell lines, although at early time points (24-48 hpi), the replication of GLV-1h109 was higher in STSA-1 cells compared to DT08/40 cells. In addition, STSA-1 cells were more susceptible to lysis with GLV-1h109 than DT08/40 cells. GLV-1h109 achieved a significant inhibition of tumor growth in both STSA-1 and DT08/40 canine xenografts models. Consequently, the significant regression of tumor growth was initiated earlier in STSA-1 tumor xenografts compared to regression in DT08/40 xenografts. The reason for the higher efficacy of GLV-1h109 in STSA-1 xenografts than DT08/40 xenografts was attributed to more efficient replication of virus in STSA-1 cells. In addition, tumor-specific virus infection led to a continued presence of GLAF-1 in peripheral blood, which could be useful as a pharmacokinetic marker to monitor virus colonization and persistence in GLV-1h109- injected xenograft mice. GLAF-1 is a single-chain antibody targeting human and murine VEGF. It was demonstrated that GLAF-1 was functional and recognized both canine and human VEGF with equal efficiency.
Histological analysis of tumor sections 7 days after GLV-1h109 injection confirmed that colonization of VACV and intratumoral expression of GLAF-1 translated into a significant decrease in blood vessel number compared to GLV-1h68 or PBS-treated control tumors. Subsequently, reduction in blood vessel density significantly improved the spread and replication of VACV as observed by FACS analysis and standard plaque assay, respectively. Inhibition of tumor angiogenesis and increased replication of virus further improved the infiltration of innate immune cells mainly granulocytes and macrophages in STSA-1 tumor xenografts. Both the results, i.e. improved virus spread and increased infiltration of innate immune cells in tumor, were explained by a phenomenon called “vascular normalization”, where anti-VEGF therapy normalizes the heterogeneous tumor vasculature thereby improving delivery and spread of VACV. In summary, the effects of inhibition of tumor angiogenesis on virus spread and replication were demonstrated using a vaccinia virus caring an anti- angiogenic payload targeting vascular endothelial growth factor (VEGF) in canine cancer xenografts.
In the final studies, the effects of VACV therapy on modulation of the immune system were analyzed in canine cancer patients enrolled in a phase I clinical trial. V-VET1 (clinical grade LIVP6.1.1 VACV) injection significantly increased the percentages of CD3+CD8+ T lymphocytes at 21 days after initiation of treatment. CD3+CD8+ T lymphocytes are mainly cytotoxic T lymphocytes that have potential to lyse cancer cells. Subsequently, the frequency of immune suppressor cells, mainly MDSCs and Treg was also analyzed in peripheral blood of canine cancer patients. Increase in the MDSC population and decreased CD8/Treg ratio is known to have inhibitory effects on the functions of cytotoxic T cells. We demonstrated that injection of V-VET1 in canine cancer patients significantly reduced the percentages of MDSCs at 21 days post initiation of treatment. Additionally, CD8/Treg ratio was increased 21 days after initiation of V-VET1 treatment. We also showed that changes in the frequency of immune cells neither depends on dose of virus nor depends on tumor type according to the data observed from this clinical trial with eleven analyzed patients.
This preclinical and clinical data have important clinical implications of how VACV therapy can be used for the treatment of canine cancers. Moreover, dogs with natural cancers can be used as an ideal animal model to improve the oncolytic virotherapy for human cancers. Furthermore, modulation of tumor microenvironment mainly tumor angiogenesis and tumor immunity has significant impact on the success of oncolytic virotherapy.
In initial experiments, the well characterized VACV strain GLV-1h68 and three wild-type LIVP isolates were utilized to analyze gene expression in a pair of autologous human melanoma cell lines (888-MEL and 1936 MEL) after infection. Microarray analyses, followed by sequential statistical approaches, characterized human genes whose transcription is affected specifically by VACV infection. In accordance with the literature, those genes were involved in broad cellular functions, such as cell death, protein synthesis and folding, as well as DNA replication, recombination, and repair. In parallel to host gene expression, viral gene expression was evaluated with help of customized VACV array platforms to get better insight over the interplay between VACV and its host. Our main focus was to compare host and viral early events, since virus genome replication occurs early after infection. We observed that viral transcripts segregated in a characteristic time-specific pattern, consistent with the three temporal expression classes of VACV genes, including a group of genes which could be classified as early-stage genes. In this work, comparison of VACV early replication and respective early gene transcription led to the identification of seven viral genes whose expression correlated strictly with replication. We considered the early expression of those seven genes to be representative for VACV replication and we therefore referred to them as viral replication indicators (VRIs). To explore the relationship between host cell transcription and viral replication, we correlated viral (VRI) and human early gene expression. Correlation analysis revealed a subset of 114 human transcripts whose early expression tightly correlated with early VRI expression and thus early viral replication. These 114 human molecules represented an involvement in broad cellular functions. We found at least six out of 114 correlates to be involved in protein ubiquitination or proteasomal function. Another molecule of interest was the serine-threonine protein kinase WNK lysine-deficient protein kinase 1 (WNK1). We discovered that WNK1 features differences on several molecular biological levels associated with permissiveness to VACV infection. In addition to that, a set of human genes was identified with possible predictive value for viral replication in an independent dataset. A further objective of this work was to explore baseline molecular biological variances associated with permissiveness which could help identifying cellular components that contribute to the formation of a permissive phenotype. Therefore, in a subsequent approach, we screened a set of 15 melanoma cell lines (15-MEL) regarding their permissiveness to GLV-1h68, evaluated by GFP expression levels, and classified the top four and lowest four cell lines into high and low permissive group, respectively. Baseline gene transcriptional data, comparing low and highly permissive group, suggest that differences between the two groups are at least in part due to variances in global cellular functions, such as cell cycle, cell growth and proliferation, as well as cell death and survival. We also observed differences in the ubiquitination pathway, which is consistent with our previous results and underlines the importance of this pathway in VACV replication and permissiveness. Moreover, baseline microRNA (miRNA) expression between low and highly permissive group was considered to provide valuable information regarding virus-host co-existence. In our data set, we identified six miRNAs that featured varying baseline expression between low and highly permissive group. Finally, copy number variations (CNVs) between low and highly permissive group were evaluated. In this study, when investigating differences in the chromosomal aberration patterns between low and highly permissive group, we observed frequent segmental amplifications within the low permissive group, whereas the same regions were mostly unchanged in the high group. Taken together, our results highlight a probable correlation between viral replication, early gene expression, and the respective host response and thus a possible involvement of human host factors in viral early replication. Furthermore, we revealed the importance of cellular baseline composition for permissiveness to VACV infection on different molecular biological levels, including mRNA expression, miRNA expression, as well as copy number variations. The characterization of human target genes that influence viral replication could help answering the question of host cell response to oncolytic virotherapy and provide important information for the development of novel recombinant vaccinia viruses with improved features to enhance replication rate and hence trigger therapeutic outcome.
Aim of this thesis was to study the contribution of the hosts immune system during tumor regression. A wild-type rejection model was studied in which tumor regression is mediated through an adaptive, T cell host response (Research article 1). Additionally, the relationship between VACV infection and cancer rejection was assessed by applying organism-specific microarray platforms to infected and non-infected xenografts. It could be shown that tumor rejection in this nude mouse model was orchestrated solely by the hosts innate immune system without help of the adaptive immunity. In a third study the inflammatory baseline status of 75 human cancer cell lines was tested in vitro which was correlated with the susceptibility to VACV and Adenovirus 5 (Ad5) replication of the respective cell line (Manuscript for Research article 3). Although xenografts by themselves lack the ability to signal danger and do not provide sufficient proinflammatory signals to induce acute inflammation, the presence of viral replication in the oncolytic xenograft model provides the "tissue-specific trigger" that activates the immune response and in concordance with the hypothesis, the ICR is activated when chronic inflammation is switched into an acute one. Thus, in conditions in which a switch from a chronic to an acute inflammatory process can be induced by other factors like the immune-stimulation induced by the presence of a virus in the target tissue, adaptive immune responses may not be necessary and immune-mediated rejection can occur without the assistance of T or B cells. However, in the regression study using neu expressing MMC in absence of a stimulus such as a virus and infected cancer cells thereafter, adaptive immunity is needed to provoke the switch into an acute inflammation and initiate tissue rejection. Taken together, this work is supportive of the hypothesis that the mechanisms prompting TSD differ among immune pathologies but the effect phase converges and central molecules can be detected over and over every time TSD occurs. It could be shown that in presence of a trigger such as infection with VACV and functional danger signaling pathways of the infected tumor cells, innate immunity is sufficient to orchestrate rejection of manifested tumors.