名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年1月31日 Tevfik Tolga SAHIN氏の博士論文として提出された

New affinity molecules such as nucleic acid aptamers are in demand in the science and medical fields. Current aptamer selection technologies can generate unique aptamers with desired properties to targets of interest. My thesis describes a series of investigations on the protection of an oncolytic virus, the isolation of target cells from biological fluids, and aptamer-facilitated biomarker discovery. We tested individual aptamers and constructed a tetramer aptamer structure (quadramer) to increase virus infectivity. The quadramer protects vesicular stomatitis virus (VSV) during freeze–thaw cycles, shields the virus from neutralizing antibodies and increases viral active units. In addition to aptamers, we screened carbohydrate-based ice recrystallization inhibitors for the possible elimination of the cold chain of Vaccinia virus, VSV, and Herpes virus-1. N-octyl-gluconamide provides the longest shelf life for Vaccinia virus and Herpes virus-1 as tested according to the World Health Organization’s requirements for viral vaccines efficiency during transportation and distribution. We generated switchable aptamers capable of isolating cells expressing LIFR, NRP1, DLL4, uPAR, or PTCH1. These aptamers bind to the receptor positive cells in the presence of Mg2+ and Ca2+, and release the pure cells upon addition of EDTA. The aptamers were applied for a sequential positive immunomagnetic isolation of cells from mice bone marrow. We also utilized fluorescence-activated cell sorting (FACS) in our aptamer selections to develop switchable aptamers to positive isolation of monocytes from human blood. Moreover, we have selected non-switchable aptamers as an affinity probe to the cells expressing Axl receptor for immunofluorescent analysis and cell sorting. We determined aptamers to CD107a and applied them for biomarker discovery with mass spectrometry and found that CD107a was co-expressing with PD-1. Furthermore, we identified CD91 as binding partners to our aptamers in human monocytes using FACS and orbitrap mass spectrometry.

Tumour heterogeneity is a key hurdle for the effective treatment of cancer using oncolytic viruses (OVs). A better understanding of the pathways involved in delineating tumour cell resistance and hypersensitivity to OVs is critical in order to guide the development of new therapeutic strategies to enhance OVs. In this thesis, I performed a comparative genetic and epigenetic study of the murine OV-resistant colon cancer cell line CT26.WT and its hypersensitive subclone CT26.lacZ. This study led to the identification of retroviral insertion sites in AKAP7 and TP53RK genes, that are potentially involved in conveying sensitivity to infection by OVs and the dysregulation of the interferon antiviral response in the CT26.lacZ cell line. Gene overexpression and gene silencing experiments suggest a functional role of these proteins in controlling viral growth. Further investigation of these genes and their relationship to antiviral response pathways is warranted and may lead to novel strategies for improving the therapeutic activity of OVs.

Oncolytic viruses (OVs) are effective anti-cancer agents, however their abilities to induce anti-tumor immunity are not yet optimal. Mutanome epitopes are a novel source of tumor antigen formed as a result of mutations within the tumor genome. Within this project we attempted to combine B16F10 mutanome vaccination with OV therapy. We confirmed previous findings that significant immune responses to these epitopes can be generated. Furthermore, we designed and cloned a multi-epitope mutanome construct into MG1 Maraba virus and E1-/E3- deleted type 5 Adenovirus to use for heterologous prime-boost vaccination. While we demonstrated that these viruses induced T-cell responses to one mutanome epitope, we failed to detect responses to the other epitopes. Furthermore there was no effect seen on overall survival. This approach warrants further investigation because coupling mutanome vaccination with OV therapy has the potential to exploit the therapeutic effects of the OV while inducing anti-tumor immunity to tumor-unique antigens.

Small molecular Inhibitor of Apoptosis (IAP) antagonists, known as Smac mimetic compounds (SMCs), are a novel class of anti-cancer drugs currently undergoing clinical trials. SMCs were designed to mimic the function of the pro-apoptotic protein, Smac, which directly depletes cells of cIAP1 and cIAP2, and consequently renders tumour cells sensitive to death in the presence of proinflammatory ligands such as TNFα. The Korneluk lab recently reported that SMCs synergize with the attenuated oncolytic virus Vesicular stomatitis virus (VSVΔ51) by eliciting an enhanced immune response in mice, such that the combined therapy is vastly superior to stand-alone therapies. To improve on this SMC-mediated synergistic response, I generated variants of TNFα-armed VSVΔ51. Due to high ectopic expression of TNFα in infected cells, a five times lower viral dose of TNFα-armed VSVΔ51 combined with SMC treatment was sufficient to improve the survival rate as compared to SMC and VSVΔ51 co-therapy. This improved synergistic response is attributed to a bystander effect whereby the spread of TNFα from infected cells leads to the death of neighbouring, uninfected cells in the presence of a SMC. In addition, the double treatment induced vasculature collapse in solid tumours, revealing another mechanism by which cytokine-armed VSVΔ51 in combination with a SMC can induce cancer cell death. This approach demonstrates great potential for engineered oncolytic virus and SMCs as a new combination immunotherapy for cancer treatment.

Purpose: The objective of this thesis is to evaluate clinical studies that have used oncolytic viruses as treatment and to compare their treatment-outcomes on patients with malignant glioma. Method: This thesis is a systematic literature review where PubMed has been used as the database for data collection. Two searches were done using the search phrases oncolytic virus AND Glioma and oncolytic virus AND brain tumor. Several of the articles showed up multiple times in different searches. After having applied the inclusion criteria, ten of the seventeen articles were removed. Remaining were seven articles used for the thesis. Results: The study conducted by Forsythe et al., using reovirus showed the median overall survival (OS) to be 21 weeks and the median time to progression (TTP) was 4.3 weeks. The study conducted by Kicielinski et al., using REOLYSIN showed the median OS to be 140 days. Median TTP was 61 days. The study conducted by Geletneky et al., 2017 was the first dose-escalating clinical trial for the use of H-1 parvovirus. The median TTP was 111 days and the median OS was 464 days. The study conducted by Lang et al., DNX-2401 was used and in group A the median OS time was 9.5 months. In group B the median OS in the group was 13 months. In another example of an oncolytic adenovirus is ONYX-015, the median TTP after treatment for all patients was 46 days. The median OS for patients diagnosed with glioblastoma multiforme was 4.9 months and for patients with anaplastic astrocytoma and anaplastic oligodendroglioma was 11.3 months across. In a study conducted by Freeman et al. using newcastle disease virus, the OS ranged from 3-66 weeks from the start of treatment and TTP ranged from 2-53 weeks. The study conducted by Markert et al., the median OS from treatment with G207 was 7.5 months. The median TTP was around 2.5 months. Conclusion: Oncolytic viruses are promising agents for treatment against malignant gliomas. No definite outcomes of the treatment could be concluded, however, the median survival was extended in certain cases. The patients tolerated the oncolytic viruses well with no adverse effects correlated with the treatments. There are currently more virus vectors being tested as new developments are needed in this field.

OBJECTIVE: The overarching goal of this study was to examine the immunostimulatory potential of oHSV-1 rQNestin34.5v2 in syngeneic murine GBM models, perform in vitro screens for upregulation of immune checkpoint molecules in infected glioma cells, and evaluate the antitumor activity of the most promising combination immunovirotherapies. METHODS: The oncolytic activity of HSV-1 rQNestin34.5 was evaluated in CT-2A and GL261 syngeneic murine glioma models. Immunoassays were conducted to assess secretion of damage associated molecular patterns including ATP, HMGB1, Calreticulin, HSP70 and other proinflammatory mediators by infected glioma cells. In vitro screens for expression of inhibitory ligands by glioma cells following HSV-1 rQNestin34.5v2 infection at various doses were analyzed by flow cytometry. Intratumoral HSV-1 rQNestin34.5v2 administration and/or intraperitoneal anti-TIGIT (clone 1B4)/anti-NK1.1 treatments were performed in C57BL/6 mice bearing orthotopic CT-2A glioma to determine effect on overall survival. RESULTS: HSV-1 rQNestin34.5v2 exhibited greater capacity to infect CT-2A and minimal capacity to infect GL261 cells suggesting differences in permissiveness in HSV- 1 replication between the two GBM models. Infection stimulated immunogenic cell death as evidenced by surface expression of calreticulin and HSP70 and elevated extracellular release of ATP and HMGB1 in the GL261 model. CD155 and CD112 (both ligands of TIGIT) as well as PD-L1 were significantly highly expressed in glioma cells. TIGIT was found to be overexpressed in tumor infiltrating NK, CD4 and CD8 T cells suggesting systemic therapy with TIGIT blockade antibodies could have therapeutic utility in combination with HSV-1 rQNestin34.5v2 in GBM. Benefit in overall survival was not observed by anti-TIGIT monotherapy, and combination treatment with HSV-1 rQNestin34.5v2 exhibited modest therapeutic effect with a cure rate 25% in mice bearing intracranial CT-2A tumors. Depletion of NK cells prior to HSV-1 rQNestin34.5v2 administration attenuated brain edema and synergized with rQNestin34.5v2 virotherapy. CONCLUSION: Our findings show that the combination of HSV-1 rQNestin34.5v2 virotherapy with anti-TIGIT checkpoint blockade immunotherapy and/or NK cell inhibition represents a promising strategy to overcome primary resistance to immune checkpoint inhibitors in GBM. / 2025-02-03T00:00:00Z

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HPV-associated cancer and carcinoma of the prostate are responsible for significant worldwide morbidity and mortality. The viral transforming proteins E6 and E7 make human papilloma virus positive (HPV+) malignancies an attractive target for cancer immunotherapy however, therapeutic vaccination exerts limited efficacy in the setting of advanced disease. In prostatic carcinoma therapeutic vaccination shows some therapeutic activity but is infrequently curative. A strategy to induce substantial specific immune responses against multiple epitopes of E6 and E7 proteins based on an attenuated transgene from HPV serotypes 16 and 18, that is incorporated into MG1-Maraba virotherapy (MG1-E6E7), was designed. MG1-E6E7 is able to boost specific immunity following priming with either an adenoviral vector (Ad-E6E7) or customised synthetic peptide vaccines resulting in multifunctional CD8+ T cell responses of an enormous magnitude. MG1-E6E7 vaccination in the HPV+ murine model TC1 is curative against large tumours in a CD8+ dependent manner and results in durable immunologic memory. Using the same adenoviral prime and MG1 boosting strategy targeting the prostatic antigen, STEAP, immunity against multiple CD8+ STEAP epitopes was induced. In a murine prostate cancer model, STEAP specific oncolytic virotherapy significantly improved the survival of mice bearing advanced TRAMP-C2 tumours. One significant obstacle to therapeutic cancer vaccination is an immunosuppressive tumour microenvironment. MG1 Maraba is able to lethally infect HPV-associated and prostate cancer cells, increase the immunologic activity within the tumour microenvironment in vivo and exploit molecular hallmarks of HPV-positive cancer and prostatic carcinoma enabling infection of bulky tumours. Pre-clinical data generated within this thesis has been instrumental in securing funding for future clinical trials assessing the safety and activity of MG1 Maraba virotherapy for HPV-associated cancer and prostatic carcinoma. This promising approach has the potential to be directly translatable to human clinical oncology to tackle these two highly prevalent and frequently lethal groups of epithelial neoplasia. / Thesis / Doctor of Philosophy (PhD) / Carcinoma (epithelial cancer) is the most common form of human cancer and two frequently encountered types, namely HPV-associated and prostatic carcinoma are responsible for a substantial worldwide cancer burden. Current therapeutic options show limited clinical benefit and/ or significant long-term side effects for advanced carcinomas, therefore new treatments are urgently required. Oncolytic viruses represent an exciting new form of anti-tumour immunotherapy capable of infecting and killing cancerous cells; here we present a virus called MG1 Maraba that is able to exploit molecular characteristics of these cancers. When MG1 Maraba is engineered to target proteins from HPV-associated cancer and prostatic carcinoma, specific immune attack against these tumours occur in mouse cancer models. MG1 Maraba offers a novel, selective, safe and highly promising therapeutic approach against advanced carcinomas. Based on the information within this thesis human clinical trials assessing MG1 Maraba are due to take place for both HPV-associated and prostate cancer.

The field of immuno-oncology has made tremendous advances in the treatment of cancer. Adoptive cellular transfer (ACT) of tumor-specific T cells and oncolytic viruses (OVs) are powerful anti-tumor agents, but each modality faces significant challenges. Despite the promise of ACT against hematological malignancies, success has been limited in solid tumors. OVs preferentially lyse tumor cells, but have difficulty overcoming antiviral host factors when delivered systemically – therapeutic doses must therefore be quite high to achieve tumor delivery. One means of overcoming viral neutralization is by loading OV onto cellular carriers prior to treatment. Since engineered T cells and OVs both possess anticancer activity, and since viruses naturally associate with nearby circulating immune cells, employing T cells engineered with a T cell antigen coupler (TAC) receptor as viral carriers may offer an ideal combination. Our studies indicated that loading oncolytic maraba virus (MRB) onto T cells – engineered with a TAC receptor targeting HER2 – had no impact on the functionality or receptor expression of these T cells. OV loaded on the surface of these TAC-T cells enabled killing of a variety of tumor targets that may be otherwise resistant to TAC-T cell therapy. Efficacy remains to be elucidated in vivo using xenograft murine models due to the lack of a protective antiviral immune response, which ultimately resulted in encephalopathy. These observed toxicities were likely model-specific, as MRB has shown to be highly attenuated in healthy tissues of wild type models. While conceptually attractive, using TAC-T cells as viral carriers to deliver a multi-pronged, one-pot antitumor therapy directly to the site of the tumor requires further evaluation before considering human studies. / Thesis / Master of Science (MSc)