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Preparation of the high-affinity binding domain of protein B7-H6
Jeníček, Jakub ; Vaněk, Ondřej (advisor) ; Čermáková, Michaela (referee)
Natural killer cells are part of innate immunity and play a key role in defending the organism. Their role in the defense against tumors and anti-tumor therapy has been the subject of multiple research projects because tumors are among the most frequent causes of death worldwide. Tumor therapy is often complicated and invasive; therefore, finding new therapeutic approaches that target naturally occurring defense mechanisms is advantageous. One of the key mechanisms used by NK cells to recognize tumor cells and eliminate them is signaling via their receptor NKp30. The binding of an activation ligand to this receptor can induce the activation of a cytotoxic response, leading to the elimination of the tumor cell. One of the activating ligands that can bind to NKp30 is B7-H6, a cell surface protein found on certain types of tumors. However, the interaction between B7-H6 and NKp30 has not been wholly described yet. This thesis focuses on different methods, which can be used for obtaining the B7-H6 domain bearing a fluorescent label, that could be used to visualize NKp30 on the cell surface, thus allowing for further description of the interaction between these molecules. KEY WORDS NK cells, NKp30, B7-H6, sortase A, HEK293
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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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The role of N-glycosylation in homooligomerization of natural cytotoxicity receptor NKp30
Tulpa, Matouš ; Vaněk, Ondřej (advisor) ; Bělonožníková, Kateřina (referee)
NK cells play a key role in the defence against cells that have been infected by a virus, a protozoan or have undergone malignant transformation. In addition, they also regulate the activity and quantity of other cells of the immune system. Target cells are recognized using their activating and inhibitory receptors, from which they receive activating and inhibitory signals, on which the cytotoxic response of NK cells depends. There is a dynamic balance between the signals that determines the life and death of the target cell. If activation signals prevail, the target cell will be eliminated. If inhibitory signals prevail, then a cytotoxic response will not be triggered. The NKp30 receptor, which belongs to the immunoglobulin-like receptor superfamily, is an important activating receptor that recognizes a number of ligands, including hemagglutinin of vaccinia and ectromelia virus, human cytomegalovirus pp65 protein, B7-H6, BAG-6, and galectin-3. The extracellular domain of the NKp30 receptor is capable of homooligomerization in solution under certain conditions. The first requirement is the presence of N-glycosylation, the second requirement is the presence of a 15 amino acid long "stalk" domain that connects the ligand binding domain with the transmembrane α-helix. The aim of this thesis was to...
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Recombinant expression of rat NK cell receptor Clr-b in the presence of fluorinated analogues of monosaccharides
Urbanová, Zuzana ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
NK cells are part of innate immunity that, besides eliminating damaged cells, also produce chemokines and cytokines, which affect the cells of adaptive immunity. NK cells express activating and inhibitory receptors on their surface. The balance between them keeps the NK cells inactive. When the balance is disrupted, the cytotoxic mechanisms of the cell are activated. Receptors NKR-P1B and NKR-P1D are two rat NK cell inhibitory receptors whose ligand is protein Clr-b, a receptor belonging to the C-type lectin-like receptor family. This work aimed to recombinantly produce Clr-b in the presence of seven fluorinated analogues of monosaccharides as potential inhibitors of N-glycosylation. The protein was successfully expressed in the HEK293T cell line as a construct containing the extracellular part of Clr-b, the Fc fragment of human IgG, and a histidine tag multiple times, each time in the presence of one of the compounds. As glycosylation plays a major role in the functionality of many proteins, inhibition of glycosylation appears to be a promising way of treatment of different diseases such as cancer or multiple sclerosis. The aim here was to assess the effect of fluorinated analogues of monosaccharides on the native N-glycosylation of the HEK293T cell line using the Clr-b construct as a model...
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Production and characterization of galectin-3 mutant
Dubanych, Yurii ; Vaněk, Ondřej (advisor) ; Ječmen, Tomáš (referee)
Natural killer cells are large granular lymphocytes of innate immunity that are characterized by the ability to kill cancer and virus-damaged cells without prior activation. Cytotoxic functions of NK cells are regulated on the one hand through surface receptors recognizing MHC-I molecules, on the other hand by the presence of a set of activating and inhibitory receptors that are under normal conditions in balance with each other. Therefore, the fate of the target cell depends not only on the expression of MHC-I, but also on the expression of ligands that activate NK cell receptors. One of the activating receptors of NK cells is NKp30. Three specific cellular ligands have been discovered for NKp30: human BCL-2-associated athanogen 6 (BAG-6, also known as BAT3), tumour antigen B7-H6, and the newly discovered ligand galectin-3. All these ligands are often expressed by cancer cells, where BAG-6 and Gal-3 inhibit NK cell functions, which may be a mechanism for tumour escape from the immune system. Therefore, Gal-3 is a new potential drug target that, by inhibiting Gal-3, can help the immune system defend itself against malignantly transformed cells. This bachelor's thesis includes the verification of the effect of the Cys173 - Ser173 mutation in the carbohydrate recognition domain of galectin-3 on the...
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Preparation of mutated forms of protein B7-H6
Malá, Viktorie ; Vaněk, Ondřej (advisor) ; Bělonožníková, Kateřina (referee)
NK cells are an essential part of the immune system. They are the so-called natural killers and carry several receptors on their surface. Two types of receptors are crucial for immune surveillance, according to their function, called inhibitory and activating. Inhibitory receptors provide auto-tolerance. The second type is activating receptors, whose activation signals toward initiating a cytotoxic response or involving other immune system components (e.g., macrophages). The activating receptor NKp30 (also known as NCR3 or CD337) is a protein of the Natural Cytotoxicity Receptors (NCR) family. It is one of the primary human NK cell activating receptors with potential use in targeted anticancer therapy. NKp30 has an activating ligand protein B7-H6, which is not found on the surface of healthy cells. It is exclusively found on highly stressed cells, e.g., infected or malignantly transformed cells. If a malignantly transformed cell carries B7-H6 on its surface, in most cases, it is recognized and eliminated almost at its formation. Not all tumour cells are B7-H6 positive, so it is possible to visualize them to the immune system through surface decoration with B7- H6. Moreover, such a ligand can be modified to amplify the immune response. This work is based on Pekar L. et al. (2020) publication, which...
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Recombinant expression of the mouse NK cell receptor NKR-P1C
Rudenko, Anastassiya ; Vaněk, Ondřej (advisor) ; Čermáková, Michaela (referee)
NK cells are at the forefront of the body's defense against various threats, such as viruses or cancer cells. NK cells can recognize and destroy harmful cells in several ways that are very different from the defense mechanisms of, for example, T- or B-lymphocytes. NK cells carry a wide range of activating and inhibitory receptors on their surface that distinguish healthy cells from harmful or infected cells. NKR-P1 (natural killer receptor-protein 1) is the first discovered family of NK cell receptors similar to C-type lectins, which includes both activating and inhibitory receptors. NKR-P1C is an activating receptor capable of forming homodimers on the surface of NK cells. The major ligands of the NKR-P1 receptor family are the immunoenvasins of murine cytomegalovirus and other molecules structurally distinct from MHC class I glycoproteins. This work focuses on the recombinant expression of the NKR-P1C receptor protein. Using the pUT7 plasmid, we prepared a stably transfected HEK293T cell line carrying the gene for expression of the extracellular domain of NKR-P1C fused to the Fc fragment of IgG using the PiggiyBac system, and between them, they carry the TEV protease cleavage site. The protein was purified by affinity and gel permeation chromatography, and its purity was subsequently verified by...
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Production of IL-2 fusion antibodies and determination of their biological activity
Frantová, Eliška ; Vaněk, Ondřej (advisor) ; Kubíčková, Božena (referee)
Interleukin 2 is a small cytokine with many important immune functions. It is used mainly as a T cell growth factor, but it also acts on other immune cells, especially NK and NK-T cells. IL- 2 at higher doses induces the differentiation and proliferation of the cell population of effector and memory T cells, which are characterized by cytotoxicity and are able to effectively defend the organism against pathogens and / or tumor cells. In contrast, low-dose IL-2 stimulates the Treg population, which suppresses immune responses and helps prevent autoimmune diseases. However, in cancer therapy, stimulation of this cell population is undesirable. Because free IL- 2 is toxic to the body at high doses, strategies have previously been proposed to potentiate the biological effect of IL-2. One of the most promising appears to be the single-stranded recombinant fusion construct, where IL-2 is covalently linked via an oligopeptide linker to an anti-IL-2 monoclonal antibody (mAb). Based on the findings of the studies of IL-2 / anti-IL-2 mAb immunocomplexes, this immunocytokine (IC) could provide significant therapeutic benefits in vivo, as compared to free IL-2, especially very robust strengthening of biological activity, selective stimulation of specific cell populations according to the selected antibody and...
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