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abstract: Immunotherapy has been revitalized with the advent of immune checkpoint blockade treatments, and neo-antigens are the targets of immune system in cancer patients who respond to the treatments. The cancer vaccine field is focused on using neo-antigens from unique point mutations of genomic sequence in the cancer patient for making personalized cancer vaccines. However, we choose a different path to find frameshift neo-antigens at the mRNA level and develop broadly effective cancer vaccines based on frameshift antigens. In this dissertation, I have summarized and characterized all the potential frameshift antigens from microsatellite regions in human, dog and mouse. A list of frameshift antigens was validated by PCR in tumor samples and the mutation rate was calculated for one candidate – SEC62. I develop a method to screen the antibody response against frameshift antigens in human and dog cancer patients by using frameshift peptide arrays. Frameshift antigens selected by positive antibody response in cancer patients or by MHC predictions show protection in different mouse tumor models. A dog version of the cancer vaccine based on frameshift antigens was developed and tested in a small safety trial. The results demonstrate that the vaccine is safe and it can induce strong B and T cell immune responses. Further, I built the human exon junction frameshift database which includes all possible frameshift antigens from mis-splicing events in exon junctions, and I develop a method to find potential frameshift antigens from large cancer immunosignature dataset with these databases. In addition, I test the idea of ‘early cancer diagnosis, early treatment’ in a transgenic mouse cancer model. The results show that ii early treatment gives significantly better protection than late treatment and the correct time point for treatment is crucial to give the best clinical benefit. A model for early treatment is developed with these results. Frameshift neo-antigens from microsatellite regions and mis-splicing events are abundant at mRNA level and they are better antigens than neo-antigens from point mutations in the genomic sequences of cancer patients in terms of high immunogenicity, low probability to cause autoimmune diseases and low cost to develop a broadly effective vaccine. This dissertation demonstrates the feasibility of using frameshift antigens for cancer vaccine development. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2018

Induction of an endogenous antibody response by therapeutic vaccination could provide an alternative to cost-intensive monoclonal antibody-based treatments for cancer. Since the target of a cancer vaccine will most likely be a self-antigen, self-tolerance of the immune system must be circumvented. Using fusion proteins consisting of the self-antigen to be targeted and a part derived from a foreign antigen, it is possible to break tolerance against the self-antigen. Furthermore, a potent adjuvant is required to support an immune response against a self-molecule. Currently no adjuvant suitable for this purpose is approved for use in humans. This thesis describes the development of a therapeutic vaccine targeting the vasculature of tumors. As tumor cells have developed strategies to escape immune surveillance, targeting of molecules associated with the tumor stroma is an interesting alternative. The alternatively spliced extra domain-A and B (ED-A and ED-B) of fibronectin and the glycan-binding protein galectin-1 are selectively expressed during events of tumor angiogenesis. We have designed recombinant proteins to target ED-B, ED-A and galectin-1, containing bacterial thioredoxin (TRX) as a non-self part, resulting in TRX-EDB, TRX-EDA and TRX-Gal-1. Vaccination against ED-B induced anti-ED-B antibodies and inhibited growth of subcutaneous fibrosarcoma. Immunization against ED-A decreased tumor burden and reduced the number of lung metastases in the MMTV-PyMT model for metastatic mammary carcinoma in a therapeutic setting. Analysis of the tumor tissue from ED-B and ED-A-immunized mice indicated an attack of the tumor vasculature by the immune system. Finally, we show that galectin-1 immunization reduced tumor burden and increased leukocyte numbers in the tumor tissue. Galectin-1 is pro-angiogenic and immunosuppressive, and therefore allows simultaneous targeting of fundamental characteristics of tumorigenesis. We furthermore show that the biodegradable squalene-based Montanide ISA 720 combined with CpG oligo 1826 (M720/CpG) is at least as potent as Freund’s adjuvant with respect to breaking self-tolerance, when comparing several immunological parameters. Freund’s is a potent but toxic adjuvant used in the majority of preclinical studies. The work presented in this thesis shows that therapeutic cancer vaccines targeting the tumor vasculature are a feasible and promising approach for cancer therapy.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22469号 / 工博第4730号 / 新制||工||1739(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 秋吉 一成, 教授 近藤 輝幸, 教授 梅田 眞郷 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Cancer is a group of diseases caused by aberrant continuously proliferating cells capable of metastasis. Despite significant advances in preventive, diagnostic and treatment measures, cancer is one of the major causes of death in the United States, second only to heart diseases. Main treatment approaches are surgery, radiotherapy, chemotherapy, and the recently expanding immunotherapeutic approaches. The main challenge in treating cancer is the ability of cancer cells to mutate and develop resistance to drug treatments therefore lowering the efficacy of chemotherapy in preventing metastatic tumors. Cancer vaccines are a treatment modality that employs the potential of the immune system to recognize and eliminate tumor cells by unmasking tumor cell antigens and generating an effective anti-tumor immune response with an immune memory capable of preventing metastases formation. This dissertation describes and evaluates an innovative cell-particle hybrid cancer vaccine construct involving irradiated tumor cell surface-engineered with polymeric particles using streptavidin-biotin cross-linking. The tumor cells were biotinylated indirectly using biotin-linked antibodies targeting a surface integrin and the particles were loaded with an immune adjuvant and coated with streptavidin. The tumor cells served as the source of tumor antigens and the anchored particles served to confine loaded immune adjuvant to the tumor cells. The vaccine construct was designed to co-deliver tumor antigens and the immune adjuvant to the same antigen presenting cell, a criteria that has been suggested recently to be important for optimal cancer vaccine potency. The first report on this cell-particle construct was published in my master’s thesis defended in May 2013. In that report, the feasibility of assembling the cell-particle hybrid was demonstrated. However, loading of the immune adjuvant, CpG ODN (cytosine phosphate guanine oligonucleotide), into streptavidin-coated particles was not optimal. In the current studies, this problem was addressed and the cancer vaccine potential of the cell-particle construct was assessed. We first evaluated a new TLR4 (toll like receptor 4) agonist, PET lipid A (pentaeryhtritol lipid A), for its potential use in cancer vaccines with the intention to incorporate it in the cell-particle hybrid. PET lipid A is a fully synthetic lipid A analog that has been demonstrated to have immunostimulatory properties. We evaluated the potential use of PET lipid A in cancer vaccine applications and the effect of particulate formulations on its adjuvant properties. Results showed improved in vitro immunostimulatory properties for particle based formulations. Upon testing the immunostimulatory properties of PET lipid A in vivo, moderate enhancement in antigen specific cytotoxic T cells stimulation was observed when PET lipid A was delivered in particles, which then translated into a corresponding trend toward increased survival in a prophylactic tumor study. PET lipid A was concluded to be a weak potential cancer vaccine adjuvant and was not chosen as the immune adjuvant to use in the cell-particle hybrid assembly. Instead, CpG ODN (TLR9 agonist) was chosen due to its strong record of efficacy as a cancer vaccine adjuvant. The second part of this research project aimed at addressing the challenges we encountered previously in achieving acceptable CpG ODN loading of the final streptavidin-coated PLGA (Polylactic-co-glycolic acid) particles. The approach taken was to modify the method used earlier to make the particles in order to circumvent CpG ODN loss. In the modified method the number of steps required to make streptavidin-coated CpG ODN-loaded PLGA particles was reduced and the fabrication media was altered to allow simultaneous particle fabrication and activation of surface carboxyl groups. The modified method resulted in 5-fold higher loading in the final streptavidin-coated particles compared to the original method. Subsequent to establishing the feasibility of constructing the cell-particle hybrid and characterizing the assembled hybrid in vitro, the in vivo cancer vaccine potential of the designed construct was examined. Two independent murine tumor models were chosen for this purpose, namely prostate cancer and melanoma. The proposed cell-particle hybrid vaccine construct had significant therapeutic outcomes in the prostate cancer tumor model where mice vaccinated with cell-particle hybrids were the only group to show significant improvement in survival compared to untreated controls whereas no other vaccine formulation had such an effect. Unfortunately, no prophylactic benefit was observed from any of the vaccine formulations used in the melanoma tumor model involving irradiated GM-CSF (granulocyte macrophage colony stimulating factor)-secreting B16.F10 cells. In vitro examination of the immunostimulatory properties of all cell lines used in these studies revealed that transfected and parent B16.F10 cells (representing murine melanoma) were possibly immunoinhibitory whereas RM11 (representing murine prostate cancer) cells lacked such immunosuppressive effect in vitro. Our objective was to design and evaluate a new cancer vaccine construct that improved the immunostimulatory properties of irradiated tumor cell based vaccines. The approach taken was to surface engineer tumor cells with immune adjuvant loaded polymeric particles. We reported a simple method for fabricating streptavidin-coated PLGA particles and a versatile method of tumor cell surface engineering. We found that the efficacy of tumor cell-based vaccines can be inconsistent across tumor models and the in vitro immunosuppressive effect of tumor cells might be a contributing factor.

Vaccines have become an important approach for the treatment of cancer. Cancer vaccines help the immune system to detect and eradicate tumor cells. Also, cancer vaccines are designed to stimulate an effective immune response that can create long-term immune memory to prevent tumor recurrence. This treatment approach involves the administration of a vaccine comprising or encoding an antigen and can often be combined with an adjuvant to further promote the immune response. The goal of this research was to study the effect of the polyanhydride composition of prophylactic cancer vaccine formulations on the tumor-specific immune response. To achieve this goal, three different amphiphilic polyanhydride copolymers were generated comprising different ratios of 1,6-bis-(p-carboxyphenoxy)-hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) or sebacic anhydride (SA) monomers. These copolymers were used to fabricate particles encapsulating a model antigen, ovalbumin (OVA), using a double emulsion solvent evaporation technique. The ability of the three different compositions of amphiphilic polyanhydride copolymers (50:50 CPTEG:CPH, 20:80 CPTEG:CPH, and 20:80 CPH:SA) encapsulating OVA to elicit immune responses was investigated. Further, the impact of soluble unmethylated oligodeoxynucleotides containing deoxycytidyl-deoxyguanosine dinucleotides (CpG ODN), an immunologic adjuvant, on the immune response to the three formulations was also studied. The immune response to cancer vaccines was measured after treatment of C57BL/6J mice with two subcutaneous injections, seven days apart, of 50 μg OVA encapsulated in particles composed of different polyanhydride copolymers with or without 25 μg CpG ODN. In vivo studies showed that 20:80 CPTEG:CPH particles encapsulating OVA significantly stimulated the highest level of CD8+ T lymphocytes, generated the highest serum titers of OVA-specific IgG antibodies, and produced longer survival in comparison to formulations involving the other polyanhydride copolymers. The results also revealed that supplementing the vaccine formulations with CpG ODN did not enhance the immunogenicity of OVA. These results accentuate the crucial role of the copolymer composition of polyanhydrides in stimulating the immune response and improving cancer vaccine efficacy.