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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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Studies on structure and biological functions of NKR-P1 receptors
Rozbeský, Daniel ; Novák, Petr (advisor) ; Konvalinka, Jan (referee) ; Drbal, Karel (referee)
Natural killer (NK) cells play a significant role in the detection and destruction of virally infected and tumor cells. The NKR-P1 receptors regulate NK cell function by an alternative missing-self recognition system. Although the NKR-P1 receptors were among the first surface NK receptors identified on rodent NK cells more than 20 years ago, there is still very little known about their biological function and their physiological ligands. Furthermore, no three-dimensional structure of any of the NKR-P1 family receptors has been published so far. To understand the functional architecture of mouse NKR-P1 receptors, we developed a simple and efficient protocol providing large amounts of pure soluble NKR-P1 proteins. The crystal structure of mouse NKR-P1A, determined at 1.7 A resolution, is the first structure of a representative of the NKR-P1 family. Crystal structure is formed by a compact C-type lectin-like domain and an extended loop that participates in domain swapping. A potential role of the swapped loop has been suggested in natural ligand binding by in silico studies. However, chemical cross-linking and H/D exchange in combination with high resolution mass spectrometry revealed this loop in close proximity to the compact core in solution. The discrepancy between the crystal and solution structure...
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Spontaneous Regression of Melanoma in Pigs of the MeLiM Strain
Plánská, Daniela
Melanoma is a skin tumour arising from melanocytes - skin cells bearing pigment melanin. Melanoma belongs among immunogenic tumours, which is probably associated with a relatively high incidence of partial spontaneous regression (SR). Melanoma-bearing Libechov Minipigs (MeLiM) represent a specially bred animal model that is mostly affected by nodular melanomas, which fully regressed in about 2/3 of the affected animals. Our interest was to examine immune response (associated with melanoma cell destruction) and the role of proteins related to the extracellular matrix (reflecting tissue remodeling) during SR of MeLiM melanoma. We performed an extensive time-lapse study of skin melanomas taken from individuals of the MeLiM strain at 3, 4, 6, 8, 10, 12, 20 and 32 weeks (5-10 samples in each age category) in which we immunohistochemically detected the expression of collagen IV, laminin, fibronectin, tenascin C, as well as MMP-2 and we monitored the proportion of basic immune subpopulations in blood and tumour by flow cytometry. The higher expression of collagen IV, laminin and MMP-2 positively correlated with the appearance of melanoma cells. The expression of collagen IV and laminin indicates a possible survival of tumour cells due to the interaction with these proteins, the presence of MMP-2 in these...
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Spontaneous Regression of Melanoma in Pigs of the MeLiM Strain
Plánská, Daniela ; Horák, Vratislav (advisor) ; Smetana, Karel (referee) ; Bartůňková, Jiřina (referee)
Melanoma is a skin tumour arising from melanocytes - skin cells bearing pigment melanin. Melanoma belongs among immunogenic tumours, which is probably associated with a relatively high incidence of partial spontaneous regression (SR). Melanoma-bearing Libechov Minipigs (MeLiM) represent a specially bred animal model that is mostly affected by nodular melanomas, which fully regressed in about 2/3 of the affected animals. Our interest was to examine immune response (associated with melanoma cell destruction) and the role of proteins related to the extracellular matrix (reflecting tissue remodeling) during SR of MeLiM melanoma. We performed an extensive time-lapse study of skin melanomas taken from individuals of the MeLiM strain at 3, 4, 6, 8, 10, 12, 20 and 32 weeks (5-10 samples in each age category) in which we immunohistochemically detected the expression of collagen IV, laminin, fibronectin, tenascin C, as well as MMP-2 and we monitored the proportion of basic immune subpopulations in blood and tumour by flow cytometry. The higher expression of collagen IV, laminin and MMP-2 positively correlated with the appearance of melanoma cells. The expression of collagen IV and laminin indicates a possible survival of tumour cells due to the interaction with these proteins, the presence of MMP-2 in these...
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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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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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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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