Cancer immunotherapy has become a leading treatment modality in metastatic diseases. Although this novel therapy has changed the therapeutic algorithms and patients' outcomes in multiple malignancies, certain proportions of patients still fail to respond to these approaches. In our studies, we aimed to address the main mechanisms of tumor resistance to cancer immunotherapy. We have systematically defined the main challenges in adoptive cell transfer. We have focused on two key mechanisms of the tumor resistance to immunotherapy: poor trafficking of adoptively transferred immune cells into tumors, and the death receptor-induced apoptosis of the tumor-infiltrating immune cells. In our work, we have gone beyond the tumor tissue and searched for the immune cell populations and novel targets that would help to challenge the two mechanisms of resistance. Our data uncovered the therapeutic potential of the paratumoral tissue compartments and, thus, provided new avenues on how to challenge solid tumors by immunotherapy.

The general idea of this study was to develop candidate specific IgY antibodies for an oral therapy targeting the ESBL-producing gram negative bacteria. As the family of ESBLs constantly grows and there is lack of their clear classification in the literature, the specific aim was to build a proof of concept study based on the parental enzyme ß-lactamase TEM-1 to investigate different specific IgYs strategies to inhibit the growth of TEM-1 producing E.coli. This research included a bioinformatic analysis of the TEM-1 structure in the context of TEM-derivative ESBLs. Then, two IgY strategies were designed to target the ß-lactamase TEM-1-producing E.coli (BW25113 ΔbamBΔtolC) with IgYs: as a complement to antibiotics (IgYs against the enzyme TEM-1 used in combination with ampicillin) and as an alternative to antibiotics (IgYs against the bacteria TEM-1-producing E.coli without the addition of ampicillin). A good inhibitory effect of (a)TIgY, (a)p2IgY (in the presence of ampicillin) and eIgY, hIgY (without the ampicillin) on TEM-1-producing E.coli was observed in vitro. Moreover, they had the typical configuration of avian antibodies and were highly specific to their antigens. This study presents a model system to develop specific IgYs against a therapeutic target of interest. The activity of these IgYs complementary or alternatively to antibiotics should be further investigated in vivo in an animal infectious model. IgYs developed in this study might also be good candidates for further investigation as a broad-spectrum treatment against a variety of ESBL-producing E.coli. The aTIgY which was developed against the whole TEM-1 might also target its derivatives, as they have similar 3D structure with single amino acids mutations in the sequence. The ap2IgY was generated against catalytic and conservative residues, characteristic for the whole class A of ß-lactamases, thus it might target also the active site of ESBL-s from this class. The strategy used to generate eIgY and hIgY was efficient and IgYs could be generated directly against ESBL-producing bacteria.

Major capside protein of Polyomaviridae family viruses is able to selfassemble into virus-like particle (VLP) even without the presence of minor proteins, bind exogenous DNA non-specifically and recognise the receptor on the cellular surface. These characteristics determine its use as vector in gene therapy or immunotherapy. It was discovered before that MPyV VLPs significantly stimulate immune system and have strong adjuvant effect. Chimeric VLP derived from mouse polyomavirus carrying exogenous antigene or epitop is supposed to elicit specifically targeted immune response after immunisation. The main obstacle is choice of immunogene that is strong enough to cause adequate immune response. The goal of this thesis was to construct chimeric particles carrying epitop of malignant melanoma, one of the most immunogenic tumours, on their surface, using methods of genetic engineering. For future research of particle's immunogenic properties three types of particles were developed - particles with human and mouse melanoma epitopes, respectively and control particles with ovalbumine epitop. For the purpose of production of chimeric protein was used baculovirus expression system. It was verified then, with the use of electron microscopy, that introduction of tumour antigen into one of surface loops of VP1...

Genetically engineered oncolytic viruses (OVs) have been proven to be effective anti-cancer agents. However, the heterogeneity of tumours and obligate attenuation of OVs to achieve safety can limit their efficacy. Our lab has previously shown that diverse small molecules, which we have termed “Viral Sensitizers”, used in combination with OVs can potentiate the infection of cancer cells by OVs over 1000-fold in some cases, resulting in cancer-specific killing in both in vitro and in vivo tumour models. We observed that a subset of viral sensitizer compounds ultimately acts by reducing the expression of IFNb, thereby inhibiting antiviral signaling. Here, we aimed to further refine the mechanism of action of this class of compounds. Our results suggest that VSe1 and more stable analogs such as VSe1-28 inhibit nuclear accumulation of NF-kB p65 and expression of various antiviral cytokines including, TNFa, IL-6, IFITM1, and MX2 in multiple oncolytic VSV-resistant cancer cell lines but not in normal cells. This was also observed in vivo in CT26wt immune-competent mouse tumour models, where our group has already demonstrated the therapeutic benefit of combining VSe1-28 with oncolytic VSV. Using various biochemical methods, we have determined that VSe1 and its analog VSe1-28 lead to these effects at least in part through covalent modification of NF-kB p65. In sum, this study provides a new understanding of how these novel viral sensitizers work at the molecular level. This new understanding will not only aid in the discovery and development of improved molecules but also their clinical translation in combination with oncolytic viruses.

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There is a rise in prevalence of antibiotic resistance in Streptococcus pneumoniae, and its FDA-approved vaccines often do not mount effective immune responses in children, the elderly, or the immunocompromised. One reason these vaccines are generally less effective is because they do not utilize T-cell help. T-cell help can be accessed when di-, tri-, or tetra-saccharides positioned inside major histocompatibility complex (MHC) II are presented to T-cell receptors as a target antigen. Pairing MHC II-antigen complexes with T-cell receptors enables development of B and T lymphocytes that are highly specific to these antigens, granting an increase in antibody affinity and cell memory. One problem with today's vaccines against S. pneumoniae, in contrast, is that extracted, polymeric sugars cannot be presented to T-cells by MHC because they do not fit inside the MHC II complex due to their large molecular size. Thus, FDA-approved vaccines generate antibodies which have inadequate affinities and are largely nonspecific in their targets. This thesis covers the synthesis of a functionalized S. pneumoniae serotype 6A disaccharide and trisaccharide, which are core components of the repeating unit of natural capsular polysaccharides, and can be used to obtain necessary T-cell help in working vaccines and good monoclonal antibodies.

Immunotherapy is a promising tool in the fight against cancer and aims to recruit patients own immune systems to seek out and destroy malignant cells. Options such as oncolytic viruses (OVs), autologous tumour vaccines and chimeric antigen receptors have shown clinical success to date, yet there remain significant hurdles to overcome. Here, we demonstrate a novel vaccine combining irrCell priming and infected cell boosting dramatically improves the tumour-specific CTL response against CT26 tumours and can be further enhanced using additional immunogenic factors (armed OVs, adjuvants). We also developed a novel fluorescence-based high-throughput screening platform to identify compounds that sensitize resistant solid tumours to killing by CAR-T cells, which ultimately revealed cardiac glycosides as putative tumour sensitizers. Overall, this thesis identifies several novel enhancers of the anticancer immune response, including a heterologous irr:ICV vaccine regimen and the potential ability to identify molecules to overcome resistance to CAR-T therapy.

Glioblastoma multiforme (GBM) is the most common malignant brain cancer that invades normal brain tissue and impedes surgical eradication, resulting in early local recurrence and high mortality. In addition, most therapeutic agents lack permeability across the blood brain barrier (BBB), further reducing the efficacy of chemotherapy. Thus, effective treatment against GBM requires tumor specific targets and efficient intracranial drug delivery. With the most recent advances in immunotherapy, genetically engineered T cells with chimeric antigen receptors (CARs) are becoming a promising approach for treating cancer. By transducing T lymphocytes with CAR constructs containing a tumor-associated antigen (TAA) recognition domain linked to the constant regions of a signaling T cell receptor, CAR T cells may recognize a predefined TAA with high specificity in a non-MHC restricted manner, and is independent of antigen processing. Active T cells can travel across the BBB, providing additional advantage for drug delivery and tumor targeting. Here we review the CAR design and technical innovations, the major targets that are in pre-clinical and clinical development with a focus on GBM, and multiple strategies developed to improve CAR T cell efficacy.

In the aftermath of cancer surgery, Natural killer (NK) cells are severely suppressed. NK cells are critical for anti-tumour surveillance and their postoperative dysfunction creates an opportunity for metastases. I hypothesized that NK cell suppression is mediated by multiple suppressive mechanisms of surgery-induced Myeloid Derived Suppressor Cells (Sx-MDSCs). In this thesis, I first show that NK cell dysfunction is far worse than previously described. In a cohort of colorectal cancer (CRC) surgery patients (n=42), the ability of NK cells to secrete IFN-gamma in response to stimulation was suppressed for up to 2 months after surgery. Secondly, since Sx-MDSCs have been poorly characterized in humans, I thoroughly phenotyped Sx-MDSCs from cancer surgery patients using flow cytometry (n=32 patient samples) and single-cell RNA sequencing (n=6 patient samples). Additionally, upon screening a library of 150 compounds, I showed that Sx-MDSC rely on PI3K signaling for their suppression of NK cells in ex vivo NK cell suppression assays. The third part of this thesis explores the contribution of Sx-MDSCs to the rapid reduction in postoperative arginine, the perioperative importance of arginine for NK cells, and the therapeutic effects of a perioperative arginine enriched supplement (AES) on metastases in murine models of surgical stress. Here, I showed that perioperative AES attenuates postoperative metastases by accelerating NK cell recovery after surgery. These promising preclinical data combined with evidence from the scientific literature led us to initiate a Phase II randomized-controlled clinical trial assessing the ability of perioperative AES to improve NK cell function after surgery in CRC patients (n=12/arm). In the last part of this thesis, I present the results from our clinical trial, which showed only a transient and, at best, modest improvement in NK cell function. Importantly, this may have been heavily influenced by poor postoperative patient compliance in taking the AES. In conclusion, this body of work describes the multifactorial role that Sx-MDSCs play in mediating postoperative NK cell suppression, and that safe, effective, and targeted perioperative interventions should be further investigated as a strategy to attenuate metastatic disease recurrence after surgery.

Ovarian cancer (OVCA) is the most lethal gynecological cancer with a 5-year survival rate less than 50%. Despite new therapeutic strategies, such as targeted therapies and immune checkpoint blockers (ICBs), tumor recurrence and drug resistance remain key obstacles in achieving long term therapeutic success. Therefore, there is an urgent need to understand the cellular and molecular mechanisms of immune dysregulation in chemoresistant ovarian cancer in order to harness the host’s immune system to improve cancer survival. Early diagnosis and residual disease are key determinants of favorable survival in OVCA; however, CA125 which is the conventional marker is not reliable and has modest diagnostic accuracy. There is therefore an urgent need to discover reliable biomarkers to optimize individualized treatment and diagnostic recommendations. Plasma gelsolin (pGSN; an actin binding protein) is the secreted isoform of the gelsolin (GSN) gene implicated in inflammatory disorders, colon cancer and prostate cancer. Increased expression of total GSN is associated with poor survival of patients with gynecological cancers. As to whether this is due to pGSN is yet to be investigated. Increased expression of pGSN is significantly associated with the down-regulation of immune cell markers; however, the exact mechanism has not been explored. If and how pGSN is involved in the cellular and molecular mechanisms of OVCA remains to be determined. In our current research, we have demonstrated that pGSN is involved in the regulation of immune cells, early diagnosis, tumor recurrence and chemoresistance in OVCA, using standard in vitro techniques and human clinical samples (North America, Asia and public datasets). We have shown that pGSN is highly expressed and secreted in chemoresistant OVCA cells than their chemosensitive counterparts. pGSN, secreted and transported via exosomes, upregulated HIF1α–mediated pGSN expression in chemoresistant OVCA cells in an autocrine manner as well as conferred cisplatin resistance in otherwise chemosensitive OVCA cells. pGSN also induced the OVCA expression of the antioxidant and tumor growth promoter, glutathione (GSH), by activating Nuclear factor erythroid 2-related factor 2 (NRF2), a response that attenuated cisplatin (CDDP)-induced apoptosis. In human tumor tissues, increased pGSN mRNA and protein expressions were significantly associated with advanced tumor stage, suboptimal residual disease, tumor recurrence, chemoresistance and poor survival regardless of patients’ ethnic background and histologic subtypes. Increased Infiltration of CD8+ T cells was significantly associated with favorable patient survival; however, increased pGSN hindered the survival impact of these infiltrated CD8+ T cells. Further investigation revealed that pGSN induced CD8+ T cell death via caspase-3 activation, an action that resulted in decreased IFNγ levels. Increased epithelial pGSN expression was significantly associated with reduced survival benefits of infiltrated M1 macrophages, through caspase-3-dependent apoptosis as well as reduced production of TNFα and iNOS. The clinical application of circulatory pGSN as a biomarker for early detection and patients’ survival was investigated. Pre-operative circulating pGSN presented as a favorable and independent biomarker for early disease detection and residual disease prediction compared with CA125. The test accuracy of pGSN was significantly enhanced when combined with CA125 in multianalyte index assay. The findings suggest that pGSN is a potential target for chemoresistant OVCA and presents as a diagnostic marker for early stage disease and surgical outcomes, interventions that could maximize the therapeutic success of immunotherapies.