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Activating antitumor immune response using bispecific fusion proteins
Chytrá, Gabriela ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
Natural killer (NK) cells are lymphocytes of the innate immune system that recognize and eliminate transformed and potentially harmful cells in a mechanism termed immunosurveillance. Malignant cells strive to escape immunosurveillance, and if successful, oncological disease develops. To restore immune recognition, immunotherapy utilizing NK cell-directed therapeutic fusion proteins can be employed. Therapeutic fusion proteins target tumour markers expressed on the surface of malignant cells and, at the same time, stimulate immune response through binding to NK cell activating receptors, for example receptor NKG2D or NKp30. A relevant example of a tumour marker is the HER2 receptor, which is often overexpressed in several types of cancer, most notably breast carcinoma. This thesis describes the preparation of several bispecific fusion proteins with potential use in immunotherapy. Bispecific fusion proteins consist of an NK cell activating ligand (ligand MICA or B7-H6) and nanobody targeting selected tumour marker (receptor HER2), which are connected by flexible glycine-serine linker. The constructs of fusion proteins were prepared in two configurations - with nanobody located on the N-terminus and the ligand on the C-terminus and vice versa. In addition, bispecific fusion proteins introducing...
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Cancer Immunotherapy exploiting engineered antibody fragments against prostate-specific membrane antigen
Das, Gargi ; Bařinka, Cyril (advisor) ; Vaněk, Ondřej (referee) ; Ormsby, Tereza (referee)
Prostate cancer (PCa) remains a leading cause of male cancer-related mortality, necessitating thus the development of novel therapeutic approaches as conventional treatments have limited efficacy. Prostate-specific membrane antigen (PSMA) is an established biomarker for both imaging and therapy of PCa, as it is highly upregulated in neoplastic PCa tissues and metastatic castration- resistant prostate cancer. Consequently, immunological targeting of PSMA has gained significant attention as a therapeutic platform for the management of the disease. The thesis is focused on engineering of antibody fragments and fusion proteins derived from the high affinity anti-PSMA 5D3 monoclonal antibody that can be used as immune cell engagers to target and eliminate PSMA-positive cells. To this end, we engineered 5D3 single chain variable fragments (scFv) that were subsequently fused to anti-CD3 scFv and CP33 sequences, creating thus immune cell engagers targeting T-cells (BiTE) and monocytes (5D3-CP33), respectively. The engagers were expressed in insect cells, purified to homogeneity and their biophysical and functional characteristics evaluated using size exclusion chromatography, differential scanning fluorimetry, ELISA and flow cytometry. Ensuing cell-based assays revealed that both BiTE and 5D3-CP33 can...
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Pathofysiology of a tumour microenvironment of salivary glands cancer
Kuchař, Martin ; Skřivan, Jiří (advisor) ; Pokorný, Jaroslav (referee) ; Lohynská, Radka (referee)
Treatment options for salivary gland carcinomas (SGC), especially advanced ones, are limited. Immunotherapy, particularly therapy with immune checkpoint inhibitors (ICI), has brought significant progress and change in the treatment of malignant tumors. The effect and response to immunotherapy using ICI are largely driven by the characteristics of immune cells in the tumor tissue and, as it turns out, also in the peritumoral tissue. We conducted an immunohistochemical analysis of the expression of the immune checkpoint protein PD-1 and its ligand PD-L1 on the surface of tumor cells as well as tumor-infiltrating immune cells (TIIC) in samples of salivary carcinomas, separately in their centre and at their periphery. In addition to the above, an increasing amount of evidence suggests that resistance to ICI therapy is modulated by the interaction of the Fas receptor (CD95) and Fas ligand (FasL, CD178) between tumor cells and immune cells. We therefore decided to explore the expression and interaction of Fas-FasL between tumor cells and tumor-infiltrating immune cells in the centre of the tumor and in the peritumoral area of salivary carcinoma samples. Differential evaluation of the tumor centre and tumor periphery across various histological subtypes of SGC revealed the role of peripheral TIICs and...
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Immunological features of esophageal carcinoma in prognosis and therapy
Šnajdauf, Martin ; Lischke, Robert (advisor) ; Vrba, Radek (referee) ; Stříž, Ilja (referee)
Esophageal cancer belongs to the gastrointestinal malignancies with the worst prognosis. Current treatment options, including surgical resection, chemotherapy, radiotherapy, or a combination of these methods, have low efficacy and the five-year survival rate for patients with esophageal cancer is approximately 10 to 15 %. In the last decade, immunotherapy has become the leading treatment modality for metastatic tumors. However, the success of immunotherapeutic approaches does not only depend on the infiltration of the tumor microenvironment with immune cells but also on the phenotype of these infiltrating cells. The aim of this project was to evaluatethe immunological features of tumor-infiltrating lymphocytes obtained from different tissue compartments (tumor, peritumoral tissue, adjacent healthy tissue, and lymph node) of patients during surgical resection and to compare them with clinical and histopathological data of patients. We observed that the distribution of NK (natural killer) cells, CD8 and CD4 positive T cells was different in each tissue compartment. While the lymph nodes had the highest percentage of T cells, the opposite was seen in NK cells. The proportion of NK cells was the lowest in the lymph nodes. The expression of death receptors FasR and DR3 (death receptor 3) was the lowest...
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Novel methods of treatment of B cell malignancies based on immunotherapy with genetically modified T cells
Novotná, Natálie ; Otáhal, Pavel (advisor) ; Šmahel, Michal (referee)
CAR T cell therapy represents a promising method in treatment of hematological malignancies. Gene immunotherapy uses modified T cells that express a chimeric antigen receptor (CAR) on their surface. Modified T lymphocytes are able to recognize and destroy target cells based on specific surface markers. Although CAR T cell therapy is used in clinical practice, there is a number of limitations that reduce its effectiveness. The aim of this thesis is to explore new possibilities of making the entire therapy more efficient through endogenous secretion of interleukins (IL-7, IL-15, IL-21) under the control of inducible promoters, and thus to strengthen the persistence and expansion of CAR T cells in vivo. For this purpose, inducible expression systems containing the gene for CAR19 receptor specifically recognizing the CD19 molecule and the interleukin gene located under inducible NFAT or NR4A promoters, were constructed. The assembled vectors were electroporated into PBMC cells using the PiggyBac transposon system to achieve stable expression in T lymphocytes. After co-cultivation with RAMOS cell line, data were obtained by measurement on a flow cytometer and the ELISA method. Based on the results, it is evident that stimulated CAR T cells are able to generate higher concentrations of interleukins,...
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Small Molecules as Immune Modulators in Anticancer Therapy
Pavlovová, Anna ; Míšek, Jiří (advisor) ; Smrček, Stanislav (referee)
The aim of this bachelor thesis was to summarize knowledge about several selected therapeutic targets used for cancer immunotherapy and small molecules that can have an immunomodulatory effect on these targets. This is a relatively new and attractive topic in the field of biomedical sciences, which is constantly evolving. Some small molecules have already been approved for treatment of specific cancer diseases, and many more are currently undergoing various stages of clinical trials. This work should provide the reader with an overview of possible approaches to modulate the immune system using small molecules. Key words: small molecules, immunotherapy, cancer, checkpoint inhibitors, tumor microenvironment
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Inhibitory NK cell receptors and possibilities of manipulation of cytotoxic properties.
Švubová, Veronika ; Frič, Jan (advisor) ; Krulová, Magdaléna (referee)
Acute myeloid leukemia makes up for 18 % of all leukemias among pediatric and young adult patients. The complete remission rate (80-90 %) and the overall survival (70 %) of the patients is relatively high, nevertheless, the relapse rate is still almost at 50 % and the prognosis remains extremely bad. The relapse treatment is rather challenging because the persisting leukemic clones might in fact start to be refractory to chemotherapy. Lately, NK cells are being perceived as an attractive therapeutical tool for treatment of the relapses. NK cells are a subpopulation of innate lymphoid cells, possessing the ability to eliminate dysfunctional cells through cytotoxic activities and further perpetuate the immune response. One of the advantages of NK cells is their functional independency of specific antigens. In the light of growing evidence about the role of leukemic stem cells in context of acute myeloid leukemia, NK cells seem to offer a new perspective in therapeutical efforts to eliminate them via several cytotoxic mechanisms. Yet despite optimistic preliminary results, treating this disease has proved to be rather challenging and the NK cell-based immunotherapy is still facing several limitations. Transforming growth factor β is partially responsible for maintenance of leukemic stem cell...
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Production of a novel type of recombinant IL-2 immunocytokine
Bednaříková, Kristýna ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
Interleukin 2 is a glycoprotein that in humans consists of 133 amino acids and is produced by helper T cells to amplify immune responses. IL-2 has many immunostimulatory and immunoregulatory functions and has been shown to activate the cytotoxic function of natural killer cells, T lymphocytes and monocytes, and to promote cytotoxicity of these effector cells. In therapy, when given in higher doses, IL-2 stimulates effector lymphocytes and defends the body against pathogens and cancer cells. When IL-2 is administered in lower doses, it stimulates T regulatory cells that inhibit the immune response, thus maintaining autotolerance. While studies to date have shown that IL-2 is effective in the treatment of malignancies, considerable toxicity has also been observed. In recent years, studies have been published showing that when IL-2 is covalently bound through an oligopeptide linker to antiIL-2 monoclonal antibodies, there is an increase in its biological activity in vivo. Also, this antibody may sterically hinder binding to certain subunits of IL-2 receptor and thus contribute to the selective activation and expansion of natural killer cells and T effector cells, whereas regulatory T cells are not stimulated. The length of the peptide linker plays a key role in the association of the IL-2 with the anti-IL-2...
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Tumor microenvironment of soft tissue sarcomas and it's predictive significance in modern oncological treatment
Ozaniak, Andrej ; Ozaniak Střížová, Zuzana (advisor) ; Büchler, Tomáš (referee) ; Posová, Helena (referee)
Soft tissue sarcomas (STSs) are malignant tumors of mesenchymal origin, characterized by an extreme heterogeneity in histological composition, biological behavior, and clinical manifestation. Most STSs are chemo- and radiotherapy resistant. A key prognostic factor predicting the risk of distant metastases and affecting the overall survival is the tumor grade. However, grade has not been associated with the risk of local recurrence. Radical surgical procedure is in many cases the only possible treatment modality or at least plays a main role in the multimodal treatment. For patients with distant metastases, the treatment options are very limited. The chemosensitivity of STSs is generally very low, with the exception of several less common subtypes, and accounts for only 5-10% of the cases. In many cases, radiotherapy is a standard part of the treatment protocol. It is usually given in either neoadjuvant or adjuvant settings. However, radiotherapy administration in generalized patients does not improve the prognosis. Cancer immunotherapy is a therapeutic modality that utilizes the physiological cytotoxic antitumoral abilities of the immune cells. Therefore, it does not target the rapidly proliferating tumor cells but rather stimulates the immune cells. A wide variety of different strategies have been...
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Kombinace nádorové imunoterapie s blokací inhibitorů apoptózy
HAVLOVÁ, Aneta
The aim of this thesis was to study the possibility of combination of cancer immunotherapy with blockade of inhibitors of apoptosis. I described structure, functions, and the effect of these inhibitors on cancer development. Special attention was paid to the ways which can be used to block inhibitors of apoptosis. Finally I suggested the combination of blockade of these inhibitors with MBTA immunotherapy.
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