Brucellosis is the most common zoonotic disease worldwide. The intracellular localization of Brucella hinders the action of drugs that poorly cross cell membrane barriers. Additionally, when the immune response fails to clear the infection, chronic brucellosis ensues that becomes more challenging to treat with antibiotics. Therefore, two approaches, intracellular drug delivery and immunostimulation, have been explored in this dissertation, with an aim to develop a better therapeutic against Brucella infection in mice. First, to overcome the cell membrane barriers, drug loaded nanoparticles were tested to treat B. melitensis infection in mice. Gentamicin loaded block-ionomer complexes (BICs) and magnetite block-ionomer complexes (MBICs) were tested in vitro and along with clusters of MBICs (MBIClusters) were tested in vivo as tools to deliver gentamicin intracellularly. While these complexes showed very high efficacy compared to free gentamicin against Brucella in macrophage cell culture, they failed to show similar efficacies in mice. Histopathological examination of kidneys from mice treated with MBICs or MBIClusters showed deposition of brown pigment-laden macrophages in peri-renal adipose tissue and the pigment was confirmed as MBICs or MBIClusters based on special staining for iron. Additionally, it was found that doxycycline-gentamicin (DG) treatment results in better clearance of Brucella from infected mice compared to doxycycline alone. Secondly, two vaccine candidates, irradiated B. neotomae (IBN) and outer membrane vesicles (OMVs), were tested as immunostimulants to treat chronic B. melitensis infection in mice in combination with antibiotics. The non-ionic block co-polymer Pluronic P85, when mixed with OMVs as an adjuvant showed significantly higher protection against B. melitensis challenge in vaccinated mice compared to those vaccinated with OMVs alone. When tested as immunostimulants, there was no additive effect of vaccines and antibiotics on Brucella clearance from mice. However, IBN enhanced the production of IFN-γ while OMVs were associated with enhanced antibody production. This enhancement in the immune system resulted in the control of Brucella growth after the end of treatment. When given without antibiotics, vaccine alone failed to clear any Brucella from infected mice. The use of these vaccine candidates in combination with antibiotics shows a potential to prevent relapses in cases of brucellosis. / Ph. D.

Recombinant oncolytic virus (OV) vaccines that encode tumour-associated antigens are potent boosting agents for adoptive transfer of tumor-specific T cells (adoptive T cell therapy or ACT). Current strategies to exploit boosting vaccines for ACT rely on a priori knowledge of targetable tumour epitopes and isolation of matched epitope-specific T cells. Therefore, booster vaccines must be developed on a patient-by-patient basis, which severely limits clinical feasibility. To overcome the requirement for individualized pairing of vaccines and T cells, we propose a “universal” strategy for boosting tumor-specific T cells where the boost is provided through a synthetic receptor that can be engineered into any T cell and a matched vaccine. To this end, we are employing chimeric antigen receptors (CARs), which confer MHC-independent antigen specificity to engineered T cells, and a paired OV vaccine that encodes the CAR target. As proof-of-concept, we have developed and evaluated a model where murine TCR transgenic T cells are engineered with boosting CARs against a surrogate antigen for studies in immunocompetent hosts. In chapter 3, I optimized a murine CAR-T cell manufacturing protocol that allows for generation of highly-transduced T cells that maintain a predominantly central memory (Tcm) phenotype. This protocol leads to generation of highly functional CAR-T cell products that can be cryopreserved at the end of ex vivo culture for future use in adoptive transfer and vaccination studies. In chapter 4, I evaluated the in vivo boosting potential of our dual-specific CAR-T cells with paired OV vaccines. Adoptive transfer of these CAR-engineered tumor-specific T cells followed by vaccination with paired oncolytic vesicular stomatitis virus (VSV) vaccine leads to robust, but variable and transient, CAR-mediated expansion of tumour-specific CD8+ T-cells, resulting in delayed tumour progression in aggressive syngeneic tumour models. In chapter 5, I investigated the role of OV-induced type I interferon (IFN-I) responses on CAR-T cell boosting. I found that CAR-T cell expansion and anti-tumour function following OV vaccination is limited by the IFN-I response and can be further enhanced by blocking interferon alpha and beta receptor subunit 1 (IFNAR1). This IFN-I-mediated T cell suppression was found to be T cell-extrinsic and related to premature termination of OV infection and antigen expression in vivo. In chapter 6, I investigated the role of CD4+ T cell help in vaccine-mediated T cell boosting and evaluated different genetic engineering strategies to integrate pro-survival STAT5 signaling into the CAR-T cell product in an effort to improve persistence and long-term anti-tumour efficacy. The work presented herein describes a novel and clinically feasible approach to enhancing adoptive T cell therapies and contributes to the basic understanding of T cell biology in the context of CAR-engineering and cancer vaccination. / Thesis / Doctor of Philosophy (PhD) / Despite recent advances in cancer prevention, detection, and treatment, 2 in 5 Canadians are expected to be diagnosed with cancer in their lifetime and approximately 1 in 4 will succumb to their disease. New, more specific therapies are needed to improve responses to treatment and reduce therapy-related side effects. Cell therapy is a new way to treat cancer that uses the patient’s own immune cells as a living drug. The immune cells are taken from a patient’s blood or tumour, trained to attack cancer in the laboratory, and infused back into the patient where they will find and kill cancer cells. A major challenge with this strategy is that the trained immune cells do not always survive in the patient for long enough to get rid of the tumour. To “boost” the immune cells, we are developing a new strategy where the immune cells are genetically modified and combined with a vaccine to enhance their anti-tumor activity. Just like a vaccine against a bacteria or virus, this vaccine will tell the modified immune cells to turn on, make more of themselves, and to find and kill the cancer cells. By delivering this “go” signal through a vaccine, we think that the immune cells will be better able to survive and generate a stronger, longer-lasting immune response against the cancer. This thesis tests this approach in relevant mouse models of cancer and aims to understand how we can best design the immune cells and vaccine to work together in their tumour-killing activities.

Yes / Phosphoinositide 3-kinase δ (PI3Kδ) has a key role in lymphocytes, and inhibitors that target this PI3K have been approved for treatment of B cell malignancies1–3. Although studies in mouse models of solid tumours have demonstrated that PI3Kδ inhibitors (PI3Kδi) can induce anti-tumour immunity4,5, its effect on solid tumours in humans remains unclear. Here we assessed the effects of the PI3Kδi AMG319 in human patients with head and neck cancer in a neoadjuvant, double-blind, placebo-controlled randomized phase II trial (EudraCT no. 2014-004388-20). PI3Kδ inhibition decreased the number of tumour-infiltrating regulatory T (Treg) cells and enhanced the cytotoxic potential of tumour-infiltrating T cells. At the tested doses of AMG319, immune-related adverse events (irAEs) required treatment to be discontinued in 12 out of 21 of patients treated with AMG319, suggestive of systemic effects on Treg cells. Accordingly, in mouse models, PI3Kδi decreased the number of Treg cells systemically and caused colitis. Single-cell RNA-sequencing analysis revealed a PI3Kδi-driven loss of tissue-resident colonic ST2 Treg cells, accompanied by expansion of pathogenic T helper 17 (TH17) and type 17 CD8+ T (TC17) cells, which probably contributed to toxicity; this points towards a specific mode of action for the emergence of irAEs. A modified treatment regimen with intermittent dosing of PI3Kδi in mouse models led to a significant decrease in tumour growth without inducing pathogenic T cells in colonic tissue, indicating that alternative dosing regimens might limit toxicity. / Research Development Fund Publication Prize Award winner, May 2022.

Cancer immunotherapy has long been proposed as a powerful approach to curing tumors, based on the natural function of the immune system in protecting its host with specificity, thus holding the potential for developing long-term memory that prevents tumor recurrence. However, the immunosuppressive feature of the tumor microenvironment prevents the patients' own immune system from functioning normally in the fight against cancer. As one of the most potent cancer immunotherapies, immunostimulatory cytokines have been shown to elicit anti-tumor immune responses in preclinical studies, but their clinical application is limited by severe immune-related adverse events upon systemic administration. None of the current delivery strategies can fully address issues of toxicities and sustainably supply cytokines over the course of a few days without compromising cytokines' structural integrity. Herein, we have developed a novel formulation to anchor potent cytokine molecules to the surface of large-sized particles (1 µm) for local cancer treatment. The cytokines are confined in tumors and have minimal systemic exposure over a few days following intratumoral injection, thereby eliciting anti-tumor immunity while avoiding the systemic toxicities caused by the circulating cytokines. Such particle-anchored cytokines can be synergistic with other immunotherapies, including immune checkpoint blockade antibodies and tumor antigens, to safely promote tumor regressions in various syngeneic tumor models and genetically engineered murine tumor models. / Doctor of Philosophy / Cancer immunotherapy is a promising method to treat cancer by harnessing the power of the body's immune system, which naturally fights off diseases and can remember and prevent diseases from returning. Unfortunately, cancers create a hostile environment that weakens the immune system's ability to combat the disease effectively. Among the treatments explored, immunostimulatory cytokines (unique proteins that boost the immune system) have shown great promise in laboratory studies for their ability to fight cancer. However, when these proteins are administered to patients, they can cause severe side effects due to their systemic dissemination throughout the body. Herein, by attaching the potent cytokines to large-sized particles (1 µm), and injecting them directly into the tumor, their cancer-fighting abilities are focused precisely where they are most needed. This targeted delivery minimizes the cytokines' presence in the rest of the body, dramatically reducing the risk of side effects associated with their systemic dissemination. This method not only shows promise on its own but also enhances the effectiveness of other cancer treatments. Our findings suggest a new, safer way to encourage the body's defense system to fight cancer more effectively.

Yes / Big data and machine learning result in agile and robust healthcare by expanding raw data into useful patterns for data-enhanced decision support. The available datasets are mostly small and unbalanced, resulting in non-optimal classification when the algorithms are implemented. In this study, five novel machine learning experiments are conducted to address the challenges of small datasets by expanding these into big data and then utilising Random Forests. The experiments are based on personalised adaptable strategies for both balanced and unbalanced datasets. Multiple datasets from cryotherapy and immunotherapy are considered, however, hereby only immunotherapy is used. In the first experiment, artificially generated data is presented by increasing the observations of the dataset, each new data is four-time larger than the previous one, resulting in better classification. In the second experiment, the effect of volume on classification is considered based on the number of attributes. The attributes of each new dataset are built based on conditional probabilities. It did not make any difference, in obtained classification, when the number of attributes is increased to more than 879. In the third simulation experiment, classes of data are classified manually by dividing the data into a two-dimensional plane. This experiment is first performed on small data and then on expanded big data: by increasing observations, an accuracy of 73.68% is attained. In the fourth experiment, the visualisation of the enlarged data did not provide better insights. In the fifth experiment, the impact of correlations among datasets’ attributes on classification is observed, however, no improvements in performance are achieved. The experiments generally improved performance by comparing the classification results using the original and artificial data.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24998号 / 医博第5032号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 佐藤 俊哉, 教授 山本 洋介, 教授 永井 洋士 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Immunotherapy has significantly improved cancer treatment. However, many tumors are resistant to current immunotherapy due to the highly immunosuppressive tumor microenvironment (TME). Tumor cells can evade immune activation by externalizing phosphatidylserine (PS) on cell surface to trigger anti-inflammatory signals and induce immune tolerance. Recent studies show that PS is upregulated in TME and further increased after chemotherapy. For effective immunotherapy of tumors, the exposed PS needs to be blocked to relieve immunosuppressive TME and sensitize tumors to immune stimulants. </p> <p><br></p> <p>In this study, PS exposure level increased after the chemotherapy Doxil treatment on B16F10 melanoma cells, and the PS exposure reduced the response of antigen-presenting cells (APCs) to immune stimulants such as lipopolysaccharide. Dipicolylamine-Zn (DPA-Zn) shielded the PS exposure resulting from doxorubicin (DOX) treatment and reduced immunosuppressive interaction between tumors and APCs. The PS blockade by DPA-Zn improves the tumor response rate immune stimulants such as GM-CSF, STING agonist cyclic dinucleotides (CDN), anti-PD-L1 antibody. Among the combination at the tested doses, Doxil + DPA-Zn + CDN was the optimal combination that enhanced anti-tumor effect most significantly and prolonged the survival time in immune-cold B16F10 melanoma model. However, the anti-tumor efficacy was limited, which is attributed to poor tumor retention of CDN and DPA-Zn. </p> <p><br></p> <p>To prolong the intratumoral release of DPA-Zn and CDN and maximize the anti-tumor immunity, CDN was formulated as liposomes (CDN@lip), which significantly delayed the release of CDN in vitro and improved anti-tumor efficacy compared with free CDN formulation. Alginate hydrogel showed the potential to sustain release of DPA-Zn. DPA-Zn was loaded in the alginate hydrogel via electrostatic interaction, and the release rate was controlled by additional zinc gluconate. However, zinc caused detrimental effects on skin and can cause mice death at a high dose. To avoid the side effect of subcutaneously administered Zn, the dose of DPA-Zn in alginate hydrogel was readjusted based on the maximum tolerated dose study, and zinc gluconate was replaced with CaSO4.</p>

Brain tumors are among the most devastating types of solid tumors to treat. Standard of care for glioblastoma (GBMs), the most aggressive form of primary brain tumors, has failed to improve the current survival rates in the past decades. Despite many other solid tumors, recent advances in cancer immunotherapies have also shown disappointing outcomes in GBMs. The heterogenous nature of GBMs, the immunosuppressive tumor microenvironment and the restrictive role of blood brain barrier (BBB) are some of the main challenges faced for treating GBMs. Electroporation-based treatments have demonstrated promising results, treating preclinical models of GBMs. It has been shown that low and high frequency irreversible electroporation treatments shift the immunosuppressive tumor microenvironment and reversibly open large areas of blood brain barrier (BBB). In this dissertation, in vitro cell culture models are utilized to study electroporation-based treatments for achieving a more optimized treatment for glioblastoma. We are proposing to utilize the immunomodulatory effects of electroporation treatments to improve the outcomes of immunotherapies in the brain. / Doctor of Philosophy / Despite the current advancements in treating solid tumors, brain tumors remain among the most difficult cancers to treat. The special structure of the brain as an organ as well as tumor complexity can lead to treatment failure. It is also known that infiltration of the immune cells within the tumor mass is limited due to the tumor's immunosuppressive nature. Hence, the use of newly advancing immunotherapy techniques is limited in the brain. Local treatments have become one of the most promising tools against brain tumors. Such treatments include methods that use excessive heating of the tissue to kill the tumors. Relying on heat for tissue destruction could damage the critical structures near the tumor and will reduce the favorable immune response after the treatment. A new treatment modality known as electroporation has been introduced for non-thermal treatment of brain tumors. Due to its non-thermal nature, electroporation treatments will allow for sparing of critical structures and can lead to a more robust immune response comparing to thermal treatment modalities. In this dissertation, we utilize electroporation-based treatments to try to overcome some of the challenges associated with treating brain tumors such as tumor heterogeneity and immune suppression.

Tesis por compendio / [ES] La inmunoterapia es un tratamiento prometedor para el cáncer de mama triple negativo (TNBC), pero los pacientes recaen, lo que destaca la necesidad de comprender los mecanismos de resistencia. En esta tesis doctoral hemos descubierto que, en el tumor primario de cáncer de mama, las células tumorales que resisten el ataque de los linfocitos T son quiescentes. Las células cancerosas quiescentes (QCC) forman nichos con baja infiltración inmune. Estas células QCC exhiben mayor capacidad de regenerar tumores 2 y tienen un perfil de expresión génica relacionado con resistencia a quimioterapia y pluripotencia. Adaptamos la secuenciación de ARN unicelular para obtener también una resolución espacial precisa que nos permitiese analizar los infiltrados dentro y fuera del nicho de QCC. Este análisis transcriptómico reveló la inducción de programas relacionados con la hipoxia e identificó células T más agotadas, fibroblastos supresores y células dendríticas disfuncionales dentro de las áreas de QCC. Esto pone de manifiesto los fenotipos diferenciales en las células infiltrantes según su ubicación intratumoral. Fuimos capaces además de identificar la activación HIF1a específicamente en las QCC como el responsable del fenotipo de exclusión y disfuncionalidad inmune. La activación forzada de HIF1a en células tumorales era suficiente para recapitular el fenotipo observado en las áreas con QCC. Por todo esto, hemos demostrado que las QCC constituyen reservorios resistentes a la inmunoterapia al orquestar un medio inmunosupresor hipóxico localizado que bloquea la función de las células dendríticas y por tanto de los linfocitos T. La eliminación de las QCC es la clave que promete contrarrestar la resistencia a la inmunoterapia y prevenir la recurrencia de la enfermedad en el TNBC. / [CA] La immunoteràpia és un tractament prometedor per al càncer de mama triple negatiu (TNBC), però els pacients recauen, fent destacar la necessitat de comprendre els mecanismes de resistència. En aquesta tesi doctoral hem descobert que al tumor primari de càncer de mama, les cèl·lules tumorals que resisteixen l'atac dels limfòcits T són quiescents. Les cèl·lules canceroses quiescents (QCC) formen nínxols amb baixa infiltració immune. Aquestes cèl·lules QCC exhibeixen més capacitat de regenerar tumors i tenen un perfil d'expressió gènica relacionat amb resistència a quimioteràpia i pluripotència. Hem adaptat la sequ¿enciació d'ARN unicel·lular per obtenir també una resolució espacial precisa que ens permetés analitzar els infiltrats dins i fora del nínxol de QCC. Aquesta anàlisi transcriptòmica va revelar la inducció de programes relacionats 3 amb la hipòxia i va identificar cèl·lules T més esgotades, fibroblasts supressors i cèl·lules dendrítiques disfuncionals dins de les àrees de QCC. Això posa de manifest els fenotips diferencials a les cèl·lules infiltrants segons la seva ubicació intratumoral. Vam ser capaços a més d'identificar l'activació de HIF1a específicament a les QCC com a responsable del fenotip d'exclusió i disfuncionalitat immune. L'activació forçada de HIF1a en cèl·lules tumorals era suficient per recapitular el fenotip observat a les àrees amb QCC. Per tot això, hem demostrat que les QCC constitueixen reservoris resistents a la immunoteràpia en orquestrar un micro-ambient immunosupressor hipòxic localitzat que bloqueja la funció de les cèl·lules dendrítiques i per tant dels limfòcits T. L'eliminació de les QCC és la clau que promet contrarestar la resistència a la immunoteràpia i prevenir la recurrència de la malaltia al TNBC. / [EN] Immunotherapy is a promising treatment for Triple-Negative Breast Cancer (TNBC), but many patients relapse or do not respond, highlighting the need to understand mechanisms of resistance. In this doctoral thesis we discovered that in primary breast cancer, tumor cells that resist T cell attack are quiescent. These Quiescent Cancer Cells (QCCs) form clusters with reduced immune infiltration. They also display superior tumorigenic capacity and higher expression of chemotherapy resistance and stemness genes. We adapted single-cell-RNA-sequencing with precise spatial resolution to profile infiltrating cells (stromal and immune cells) inside and outside the QCC niche. This transcriptomic analysis revealed hypoxia-induced programs and identified the presence of more abundant exhausted T-cells, tumor-protective fibroblasts, and dysfunctional dendritic cells inside clusters of QCCs. This uncovered differential phenotypes in infiltrating cells based on their intra-tumor location with respect to QCCs. We were also able to identify HIF1a expression in QCC as the driver of immune exclusion and dysfunction. Forced activation of a HIF1a program in cancer cells recapitulated the immune phenotype observed in the QCCs' niche. Thus, QCCs constitute immunotherapyresistant reservoirs by orchestrating a local immune-suppressive milieu that blocks DC activation impairing T-cell function. Eliminating QCCs holds the promise to counteract immunotherapy resistance and prevent disease recurrence in TNBC. / Baldominos Flores, P. (2024). Study of immune resistant mechanisms in mouse models of breast cancer [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203657 / Compendio

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25161号 / 医博第5047号 / 新制||医||1070(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 小川 誠司, 教授 伊藤 能永 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM