Chimeric antigen receptors (CARs) are artificial molecules composed of an antibody derived antigen recognition domain which is fused with the signal transduction domain derived from the physiological TCR. CAR technology used to transduce patients T-cells and endow them with the specificity to a certain surface antigen, has been a major breakthrough in cancer immunotherapy in the last decade. This strategy has been most successful for treating hematologic malignancies. Various CAR approaches and applications are currently tested mainly in the United States where many clinical trials have been launched. In contrast, in the Czech Republic, there are only a few teams focused on this topic with no clinical trials going on. During my work on this diploma thesis and in close collaboration with MUDr. Pavel Otáhal, PhD., who is working on implementation of CAR technology into the Czech clinics for the treatment of B-cell malignancies, individual functional CARs were prepared and tested. CAR expressing Jurkat T-cell lines were generated using a lentiviral vector transduction system. CAR functionality was determined by two different assays. We have shown that individual CARs are able to recognize the B-cell lineage specific antigens CD19 and CD20 and significantly up-regulate the activation molecule CD69 upon...

The aim of this thesis was study of replacement of LPS in innate immunity based cancer immunotherapy. Application of poly (I:C) with mixture of mannan-SMCC + R-848.HCl leads to synergy, higher reduction of tumor growth and significant prolongation of survival. The significance and mechanism of action of neutrophils have also been studied within this therapy.

The goal of this thesis was to find agonists of Toll-like recptors that would interact with ligands of phagocytic receptors synergistically, would cause a strong suppresion of tumour growth, and could be safely used in human medicine.

This study examines the role of innate and adaptive immunity in the immunotherapy based on the combination of the ligands stimulating phagocytosis anchored in the tumour cells membrane and the mixture of TLR agonists. This immunotherapy is primarily focused on the innate immunity activation and induces strong inflammatory infiltration, which neutrophils and NK cells are part of. Therefore, the next aim of this study was to evaluate the anti-tumour activity of neutrophils and NK cells. For examination malignant melanoma and pancreatic adenocarcinoma mouse tumour models were used.

The aim of this thesis was to find optimal therapy based on combination of membrane-anchored phagocytic ligands (mannose-(G)5-(K)10-STE, mannan-BAM, mannan-SMCC) with LPS (ligand of signal receptor)for treatment of murine melanoma B16-F10. Mixture of mannan-BAM with LPS applied in pulse regime proved to be the most effective, resulting in heigh reduction of tumor growth and significant prolongation of survival.

The aim of this thesis is to improve the therapeutic effect of the immunotherapy based on the synergy of TLR agonists with phagocytosis stimulating ligands. Furthermore, this thesis is focused on the information transfer to the specific immunity, as well as it pursues the study of the specific immunity relevance during cancer immunotherapy.

The aim of this thesis was to study how to increase effectiveness of cancer immunotherapy based on synergy of compounds stimulating phagocytosis and TLR agonist. Tumor microenvironment was modified by enzymes, which catalised conversion of lactate to pyruvate or acetate. It was monitored effect of enzymes on tumor size, survival of experimental mice and cytotoxicity on tumor cells.

Cancer is the second most common cause of death in the world, and the number of people with the disease increases each year. The therapy of the disease currently stands on four pillars; surgery, chemotherapy, radiotherapy, and immunotherapy. Through the past few years, immunotherapy has become the fastest developing treatment modality. However, despite its unprecedented efficacy in some patients, the majority of patients still does not respond to the therapy. Therefore, there is a need to investigate the mechanisms that make immunotherapy inefficient. Cell-based cancer immunotherapy is the treatment modality which uses live ex vivo-produced tumor-targeting immune cells to treat cancer. One of the mechanisms that may compromise its therapeutic efficacy is the expression of inhibitory molecules on the surface of the produced immune cells. Tim-3 is the inhibitory molecule which attracts attention in recent years. Tim-3 expression in the tumor cells and the tumor-infiltrating immune cells is often associated with worse prognosis and more aggressive forms of the disease. However, its role in the in vitro or ex vivo-produced immune cells is difficult to predict. In this work, an in vitro study model which is based on in vitro-produced antigen-specific CD8+ T cells with high expression of Tim-3 has been...

V ANGLICKÉM JAZYCE Immunotherapy of ovarian carcinoma with dendritic cells Anticancer immunotherapy is a therapeutical strategy aimed at elicitation and maintenance of immune responses against cancer cells. In this study we have focused on immunotherapy of ovarian cancer, because it is one of the most common gynaecological tumors with poor prognosis and high mortality. Our immunotherapy protocol involves preparing dendritic cells (DC) from monocytes isolated from patient's peripheral blood, which are subsequently pulsed with irradiated cells of established ovarian cancer cell line. These immature pulsed DC are maturated and subsequently co-cultivated with autologous T lymphocytes. The aim of this study was to demonstrate, that DC are able to elicit specific immune response after addition of suitable mature agens in combination with apoptotic ovarian tumor cells. Our observations indicate that 24 hours are sufficient for induction of tumor cells apoptosis. Additionally, we have shown that DC successfully ingested most of the apoptotic tumor cells after 4 hours of co-incubation. Furthermore, we have found out that ingestion of apoptotic cells by dendritic cells, which are stimulated with polyI:C, inhibits maturation of DC and consequently also production of cytokines IL-12p70, IL-6 and TNF-α. Whereas...

1 Abstract This doctoral thesis is focused on the synthesis and characterization of novel hybrid polysaccharide-based polymers applicable for biomedicine, specifically for a conceptually new bimodal cancer treatment - immunoradiotherapy. For this purpose, polysaccharides β-glucan from Auricularia auricula-judae and κ-carrageenan from Kappaphycus alvarezii, exhibiting immunostimulatory and anticancer activities, were chosen to be grafted with thermoresponsive poly(2-isopropyl-2-oxazoline-co-2-butyl-2-oxazoline)s (POXs) (with different graft lengths and grafting densities) that induced a lower critical solution temperature of the final polymers. The thermoresponsive behavior of resulting polymers was studied with temperature-dependent light scattering methods, fluorescence measurements and also nuclear magnetic spectroscopy to select a polymer material with the most suitable properties for the intended application, aiming at a polymer depot formation after the injection of a polymer solution into the body. The chosen polymer, β-glucan-graft-POX with graft length of 2500 Da, was then modified to bear 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid and a fluorescent dye Dyomics-615 at the graft ends and tested first in vitro to investigate its immunostimulatory properties and also the cellular uptake....