Doctoral theses
Natural killer (NK) cells are an essential part of immune system, providing self-surveillance of virally infected, stress transformed or cancerous cells. NKR-P1 receptors and their ligands from clec2 gene family represent an alternate missing-self recognition system of NK cells based on interaction of highly related C-type lectin-like receptors. Human NKR-P1 has been described more than twenty years ago but still remains the sole human orthologue of this receptor family, particularly numerous in rodents. On binding to its cognate ligand LLT1, NKR-P1 can relay inhibitory or co-stimulatory signals. Although being interesting targets for their potential role in tumor immune evasion and autoimmunity, nature of their interaction is still unclear. To elucidate the architecture of their interaction, we developed a generally applicable method for recombinant expression of human NKR-P1 and LLT1 and their homologues based on transfection of HEK293S GnTI- cells. Further, we described a stabilizing mutation His176Cys, that enables for expression of highly stable and soluble LLT1. Finally, we have crystallized LLT1 and human NKR-P1 in different glycosylation states both as individuals and in complex. While both structures of LLT1 and NKR-P1 follow the classical C-type lectin-like superfamily fold, contrary to LLT1, NKR-P1 forms a unique homodimer centered by its helix α1 that is similar to Dectin-1. Moreover, in the structure of their complex the α1/α2-centered dimers alternate in bivalent interaction of two distinct types. While the first is similar to manner of interaction of related complexes of human NKp65:KACL and mouse NKR-P1B:m12, the second one is unique.
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Natural killer cells (NK cells for short) are lymphocytes of the non-specific (innate) immune system. They excel in the ability to recognise and eliminate infected or cancerous cells rapidly. Although their role in immune surveillance of malignant transformation was confirmed years ago, ongoing research shows that this process is far from simple. NKp30 is one of the central activating receptors of NK cells with a potential for use in targeted immunotherapy. The oligomerisation of the extracellular ligand-recognition domain of NKp30 in solution depends on the presence of a C-terminal stalk region. However, the structure and role in signal transduction of these oligomers are still unclear. Moreover, the interaction of NKp30 with ligands is influenced by the presence of N-linked glycosylation.
In this work, we investigated whether and how the oligomerisation of NKp30 depends on its glycosylation. Our results show that NKp30 forms oligomers, regardless of whether glycosylation is complex or uniform (acquired by expression in the HEK293S GnTIcell line). In contrast, NKp30 was found to form only monomers when enzymatically deglycosylated. Furthermore, we characterised the interaction with the ligand B7-H6, again concerning oligomerisation and glycosylation. We solved the crystal structure of its complex with glycosylated NKp30, through which we revealed glycosylation-induced dimerisation of NKp30. This work provides new insight into the structural basis of NKp30 oligomerisation and complements our knowledge of how the glycosylation and the presence of the stalk region affect affinity for ligands. Unravelling of the actual mechanisms underlying NK cell activation, inhibition, and maturation, including the context of cooperation with other lymphocytes, remains a significant challenge. However, understanding these processes is a fundamental prerequisite for rational intervention in NK cell surveillance and its modulation by immunotherapeutics to eliminate tumours when our immunity fails.
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Natural killer cells (NK cells) are a family of lymphocytes with a natural ability to kill infected, harmed, or malignantly transformed cells. As these cells are part of the innate immunity, the cytotoxic mechanisms are activated upon recognizing specific patterns without prior antigen sensitization. This recognition is crucial for NK cell function in the maintenance of homeostasis and immunosurveillance. NK cells not only act directly towards malignant cells but also participate in the complex immune response by producing cytokines or crosstalk with other immune cells.
Cancer may be seen as a break of all immune defenses when malignant cells escape the immunity and invade surrounding tissues creating a microenvironment supporting tumor progression. This process may be reverted by interventions into immune response shaping with immunotherapy, which may lead to restoration of immune recognition. NK cells are also effector cells important for immunotherapy may be used for adoptive transfer, genetically modified with chimeric antigen receptors, or triggered with appropriate antibodies. NK cell receptors, responsible for target recognition and activation of cytotoxic machinery, may also be targeted in immunotherapy. However, this kind of immunoactive therapeutics may be designed only with the deep knowledge of NK cell receptor:ligand interaction.
This work describes the expression of soluble forms on NK cell receptors and their ligands and structural studies, providing detailed biophysical characteristics of NK cell immunocomplexes. Moreover, clustering of NKR-P1 receptor was described directly on the cell surface with superresolution microscopy, and the effect of receptor reorganization on signalization was verified by cytotoxic assay. Finally, NK cell-activating ligand B7-H6 was expressed with the coiled-coil sequence enabling its attachment on the polymeric carrier, together with tumor-targeting scFv. The biological activity of this ligand was verified in vitro, which suggests the utilization of these chimeric protein-polymer particles for targeted NK cell activation.
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Master theses
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Natural killer (NK) cells are an intensively studied part of immune system, possessing unique ability to recognize and induce death of tumor and virus-infected cells without prior antigen sensitization. Their function is regulated by a fine balance of signals induced by multiple activating and inhibitory cell surface receptors and their interaction with the ligands present on the target cell. Recent research in their C-type lectin-like receptors repertoire has shown that ligands of some of these previously orphan receptors lie within their own family, describing a lectin-lectin interaction. This is the case of human inhibitory receptor NKRP1 (gene KLRB1) and its ligand LLT1 (gene CLEC2D). Previous studies have shown that overproduction of LLT1 in cancer cells or lower production of NKRP1 in NK cells is connected to cancerous manifestations. This master’s thesis shows a successful production of the extracellular part of LLT1 utilizing a mammalian expression system based on transient transfection of modified human embryonic kidney (HEK) cell lines. It was found that the five cystein residues contained within the lectin domain of LLT1 tend to cause misfolding and formation of aggregates. Stabilization of the domain was achieved by restoration of the sixth cystein residue at the evolutionary conserved position utilizing a site directed mutagenesis approach. The reconstruction of disulfide bond was verified by mass spectrometry. The mutation of His 176 to Cys 176 led to significant improvement in yield and homogeneity of product that enabled successful crystallization in two different crystalline forms and solution of the structure at 2.0 Å.
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The Natural Killer (NK) cells have an important role in the nonspecific immunity of the organism. They have the ability to identify and to kill tumor cells and cells infected by a virus without preceding sensitization by antigen. Their function is directed by the amount of stimulation and inhibition receptors interacting with ligands on the tumor or infected cell. This thesis focuses on the preparation and the study of the complex of rat NK cellular inhibition receptor NKR-P1B („natural killer cell receptor – protein 1B“) and its ligand Clrb („C-type lectin-related ligand b“). The Clrb initiates the inhibition of NKR-P1B, meaning that if the cell express Clrb, it won’t be destroyed. If the cell gets infected by the rat cytomegalovirus, it loses Clrb from its surface and its destruction is therefore no longer prevented. Cells infected with this virus defend themselves from destruction by expression of the viral gene of C-type lectin RCTL, which is a homolog of Clrb. Transient transfection of human embryonic kidney 293 cell line with simple glycosylation (HEK293S GnTI−) was used for the recombinant preparation of the soluble form of these two receptors of the rat NK cells. The native forms of the receptors – disulfidic homodimers – were prepared as the fusion construct with IgG Fc (using vector pYD5), which after the cleavage using TEV protease provided purely dimeric form of the extracelular part of the receptors without any affinity tag. The complex of NKR-P1B with Clrb was characterized using fluorescence anisotropy and surface plasmon resonance. The ligand Clrb itself was used for the crystallization with the aim to determine its structure and the crystals of Clrb were obtained.
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Nitrilase well known for their unique property to effectively convert nitriles into corresponding carboxylic acids and ammonia. They can also form amides as by-products. In contrast to nitrile hydratases, they do not require cofactors or prosthetic groups. The research in this work is focused on nitrilase from filamentous fungus Arthroderma benhamiae and cyanide hydratase from Aspergillus niger K10. Genes of these enzymes were expressed using pET-30a(+) plasmid in the bacterium Escherichia coli strain BL21-Gold (DE3). The products obtained were purified by a series of ion exchange chromatography and gel filtration and subsequently characterized with respect to oligomeric state of the protein and its usability for protein crystallography. To obtain information regarding the structural arrangement of the individual proteins, electrophoretic separation in polyacrylamide gel, gel filtration, analytical ultracentrifugation, mass spectrometry, dynamic light scattering and drop coating deposition Raman spectroscopy were used.
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Transient transfection of mammalian cell lines is an effective approach for recombinant protein production, which can provide milligrams to grams of proteins in two weeks from cloning of the corresponding cDNA. Native glycosylated proteins prepared via this approach can be used for various purposes in molecular biology, immunology or pharmaceutical industry, i.e. initial phase of pre-clinical therapeutic protein research. One of the most used mammalian host cell lines is the human embryonic kidney cell line, that can be easily cultivated and chemically transfected. The amount of proteins produced by transiently transfected human embryonic kidney cells can be enhanced by a whole range of factors, i.e. co-expression or direct addition of acidic fibroblast growth factor to the culture medium, co-expression of cell cycle regulating proteins or anti-apoptotic proteins. Expression plasmid pTW5 was prepared and further modified by gene insertion of aFGF, cell cycle regulator p18, p21 or p27 (cyclin-dependent kinase inhibitors) or apoptosis inhibitor bcl-2 or bcl-x. These plasmids were then used for optimization of HEK293T cell line expression system. The impact of every single regulator and their combinations, including hitherto undescribed effect of combination of cell cycle regulator and anti-apoptotic protein, was monitored via model proteins – green fluorescent protein and secreted alkaline phosphatase. Furthermore, the general conditions of cell line cultivation during protein production were optimized.
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Natural killer or NK cells are immunocytes that mediate innate immunity against pathogens and tumors without pre-exposition to the antigen. They are holding rapid antiviral defense during the initial phase of immune response, before starting the production of antibodies and the development of specific cytotoxic T –lymphocytes. On the surface of NK cells is expressed wide range of inhibition and activation receptors. Important family of those receptors are C – type lectin like from which the family of NKR – P1 (“natural killer cell receptor – protein 1”) was discovered first. Diploma thesis deals with the preparation/study of mice NK cell activation receptor NKRP1C and searching for its binding partner. The soluble form of the protein NKR-P1C was prepared by recombinant expression using the transient transfection of HEK293 cell line (human embryonic kidney 293) with wild type or homogenous glycosylation as IgG – Fc fusion protein, from which was it possible to obtain pure dimer of NKR P1C, after process of affinity purification, TEV protease cleavage and HPLC chromatography. The fusion protein was bound to protein A labeled with a fluorescent probe DyLight 488. Mice tissues and cell lines were labeled by this complex for purpose of seeking ligand.
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Nitrilases are enzymes able to convert toxic nitriles to corresponding carboxylic acids or amides. Thus they might be used in the detoxification of dyes, herbicides and pharmaceutical intermediates and byproducts. They can be used also for enzymatic syntheses of carboxylic acids not available by standard procedures. The aim of this diploma thesis is a recombinant expression of nitrilases from Neurospora crassa and the optimization of their purification. Cells of E. coli (BL 21 Gold) were utilized as an expression system. The purification was performed by ion-exchange chromatography, chelation chromatography and gel filtration – all under reducing conditions. Purified enzymes were studied by sedimentation analysis in an analytical ultracentrifuge. They were also used for searching of optimal conditions for their crystallization.
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Natural killer cells, or NK cells are an integral component of innate immunity and fullfills the function of recognizing and killing tumor and virus-infected cells. Their function is regulated by signals produced by the interaction of inhibitory and stimulatory receptors on their surface with their specific ligands on the targer cell surface. NKp80 is an activating receptor of NK cells and forms specific complex with cell receptor AICL, both of which belong to the family of C-type lectin-like receptors. Overexpression of AICL receptor is preferably specific for tumor cells of myeloid character. This master´s thesis describes the production of AICL mutated form by expression in Escherichia coli BL21 Gold (DE3) followed by isolation and in vitro renaturation of the target protein. In a previous study it was found that an odd number of cysteines in the extracelular lectin domain of AICL causes wrong folding of the protein. Substituting an odd cystein for serine at position 87 lead to stable soluble form of AICL with an even number of cysteines in conserved positions, typical for CTLD receptors. Correctness of the formation of disulfide bonds between cysteines was verified by mass spectrometry. Significant amount of the protein gained allowed for setting up a wide variety of crystallization conditions.
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Auxin is one of the most important plant hormones, which provides development of a plant. PIN1 and PIN7 proteins belong to the PIN family of transporters which is among the most important auxin efflux carriers. This thesis deals with the of AtPIN1 and AtPIN7 auxin efflux carriers (from Arabidopsis thaliana) in human embryonic kidney 293 cell line. Biological activity of these proteins was tested by using radiolabeled auxins accumulation. Further inhibitors of auxin transport have been tested – NPA, CHPAA and BFA.
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NK (natural killer) cells play a key role in recognition and elimination of tumour, infected or altered cells. They do not possess any antigen-specific receptors on their surface, therefore they are classified as part of innate immunity. Recognition of target cell is based on other surface receptors. Inhibition receptors assure cell tolerance, by contrast activation receptors trigger cytotoxic mechanisms leading to apoptosis and thus to cell lysis. Thanks to this characteristics, NK cells are intensively studied in connection with tumour immunotherapy. One of the activating receptors is NKp80 recognising its ligand AICL. Both proteins belong into C-type lectin-like family. Complex is involved not only in direct lysis of malignant myeloid cells, but plays a crucial role in immunomodulation of inflammation. Objective of this master´s thesis is expression of receptor NKp80 and its ligand AICL. Receptor NKp80 was prepared in human embryonic kidney cell line (HEK 293S GnTI- ). Stable cell lines expressing NKp80 protein in constitutive or inducible way were prepared. Formation of disulfide bonds as well as occupation of sites predicted for N-glycosylation in NKp80 protein was verified by mass spectrometry. Furthermore, the preparation of AICL described by Mgr. Jiří Nový in E. coli expression system was optimised, which led to increased solubility of protein. Crystallization trials for NKp80, AICL and complex NKp80:AICL were set up. Crystals of NKp80 were obtained.
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NK cells are important part of immune system, recognizing and eliminating tumor cells and cells infected by viruses. For the target cell recognition, binding of ligands by activating receptors plays a crucial role. Activating receptor NKp30, protein of family of natural cytotoxicity receptors, is able to bind multiple ligands either present on tumor cell surface or being part of some viruses. B7-H6 is one of the ligands of NKp30 and its specific constitutive expression on some tumor cells and cell lines makes it an interesting biological target. Although the NKp30/B7-H6 complex structure has been solved, structural basis of some important features of their binding is not explained yet. Soluble form of NKp30 receptor binding domain creates oligomers, presence of which is dependent on C-terminus length of its domain and its N-glycosylation; however, structural insight into formation of the oligomers and their significance is not known. Furthermore, binding affinity of NKp30 to its ligands is dependent on presence of its glycosylation and glycosylation type. We have already found out that NKp30 oligomerization is dependent on its glycosylation. In my work, I attempted to gain detailed functional and structural information about oligomerization of NKp30 and its binding to B7-H6 by multimethodical approach including X-ray crystallography. Answering the questions described above would not only contribute to basic research of activating receptors of NK cells but possibly may have an impact on further research of cancer diagnostics and therapy.
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Natural killer (NK) cells are an intensively studied part of immune system possessing unique ability to recognize and induce death of tumor and virus-infected cells without prior antigen sensitization. Their function is regulated by a fine balance of signals induced by multiple activating and inhibitory cell surface receptors and their interaction with the ligands present on the target cell. This can be illustrated on the homodimeric rat inhibitory receptor NKR-P1B and its ligand Clrb which play, besides other things, crucial role in the immunological response of NK cells to the infection with rat cytomegalovirus (RCMV), one of the most studied NK cell function model in rat model organism. During RCMV infection the target cell downregulates cell surface expression of Clrb, thus decreasing inhibitory signal transmitted through the NKR-P1B receptor to the NK cell, which would ideally lead to NK cell activation and lysis of the infected cell. However, RCMV carries a gene for “decoy” surface receptor – RCTL that mimics Clrb and thus helps to escape the immunological response of NK cells. Moreover, while this escape strategy was demonstrated in the WAG rat strain, it has been shown that the NKR-P1B homologue from SD rat strain binds only Clrb and does not recognize RCTL. Thus the SD rat strain is less susceptible to the RCMV infection. This research aims to elucidate the molecular basis of the NKR-P1B:Clrb receptor-ligand recognition and is based on our previous successful results with homologous human NKRP1:LLT1 receptor-ligand pair. For protein crystallization of NK cell receptors and ligands, it was found out that the best recombinant expression system for production of soluble extracellular domains of these proteins is transiently or stably transfected HEK293S GnTIhuman cell line possessing homogeneous N-glycosylation profile. To increase the yield of recombinant proteins, we have optimized transposon-based doxycycline inducible mammalian cell expression system piggyBac within HEK293S GnTIcell line using Clrb soluble expression construct as the target protein.
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Natural killer (NK) cells are a subpopulation of effector lymphocytes with cytotoxic activity and cytokine-producing functions considered as an integral part of the innate immune response. Functions of NK cells include tumour elimination, engagement and regulation of antiviral immune responses and regulation of immune cells by production and secretion of chemokines and cytokines. CD69 is a C-type lectin-like transmembrane receptor expressed in NK cells. CD69 is an activating receptor and acts also as a very early marker of lymphocyte activation. Putative protein ligands have been described for CD69 in the last years: Galectin-1, S1P1, S100A8/S100A9 and Myl9/12. Galectin-1 is a prototypical lectin characterized by the presence of a common lectin structural fold and a carbohydrate recognition domain involved in carbohydrate binding. Galectin-1 was identified as a binding partner for CD69 based on biological and functional studies, but structural details about the complex are still missing. This thesis describes the successful establishment of an expression protocol for a tag-less cysteine-less mutant of galectin-1 and the study of the interaction between galectin-1 and NK cell receptors. The interaction was studied using microscale thermophoresis and confirmed as dependent on the presence of a N-glycan moiety on the NK cell receptor surface.
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The functions of the immune system include immunosurveillance of transformed cells, i.e., the ability to eliminate these cells before they become harmful to the organism. If the transformed cells succeed to escape the immune system surveillance, an oncological disease develops. The tumour immunotherapy aims to stimulate the immune system mechanisms to fight against the tumour. Lately, there´s an interest in using NK cells in the immunotherapy of tumours. These cells appertain to the innate immune system and participate in immunosurveillance. When an NK cell encounters a target cell, its activation depends on the integration of signals from the surface activating and inhibiting receptors which bind ligands on the surface of the target cell. Upon activation, NK cell exhibits a cytotoxic response against the target cell. The use of NK cells in immunotherapy includes, among others, the testing of bispecific fusion proteins which can bind a tumour surface antigen by one part and NK cell activating receptor by the other part. Thus, these fusion proteins mediate a contact between both cells and trigger the cytotoxic response. This work presents a preparation of bispecific fusion proteins which consist of an activating ligand MICA (for the receptor NKG2D) or B7H6 (for the receptor NKp30), and a nanobody targeting one of three tumour antigen HER2, CD20 or FGFR. Two proteins, MICA_antiHER2 and antiHER2_MICA, have been selected for further studies of their ability to bind the targeting molecules. The biological relevance of these results will be further evaluated in cytotoxicity experiments with NK cells.
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Natural killer cells (NK cells) are part of the immune system in human and other mammals. The task of these cells, which belong to the non-specific immunity, is to induce apoptosis in other cells of the body that may represent a threat for the body (i.e., tumour or virally infected cells). NK cells have a variety of surface receptors to recognize their target cells. A number of receptors are well-known today and they may be divided into groups based, e.g., on their structural similarities or on the type of signal which these receptors present to NK cells. Accordingly, we distinguish activation and inhibitory receptors. Inhibitory receptors inhibit NK cell response, while activation receptors elicit this response. During NK cell contact with another cell, the resulting NK cell behaviour is always the result of a certain balance of activation and inhibitory receptor responses. The NKp44 receptor is an immunoglobulin-like receptor. This receptor is very unique among other receptors in many respects, for example because it is associated with both activation and inhibitory motif. The ligand of this receptor is a proliferating cell nuclear antigen (PCNA). PCNA is a clamp protein important, inter alia, during DNA replication, in which it anchors other replisome proteins. This work is focused on recombinant preparation of NKp44 receptor (PCNA protein was already prepared earlier) and a study of interaction between both of these binding partners. NKp44 was prepared recombinantly in the HEK293S GnTI- expression system. The protein was prepared in several variants of the fusion constructs (some carrying mutation and differing in the orientation of the IgG Fc fragment). The most suitable candidate was then selected from the prepared constructs and used to analyze the binding between the two proteins. Biophysical methods of analytical ultracentrifugation and microscale thermophoresis were used for this purpose. Some interaction between proteins was observed by microscale thermophoresis, although the accuracy of the determination of the dissociation constant of the NKp44:PCNA complex was really very poor. On the other hand, analytical ultracentrifugation did not show any interaction.
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One of the key components of the innate immune system are natural killer (NK) cells. The task of these cells is to induce apoptosis in target cells (e.g., cancer or virally infected cells). The target cells are identified by their interaction with surface receptors of the NK cells. On the surface of the NK cells, there are activating and inhibiting receptors. One of the activating receptors is the natural cytotoxicity receptor NKp46. Several ligands of this receptor have been identified, one of them being the epithelial adhesin Epa1 of yeast Candida glabrata. The invasive candidiasis caused by this yeast is a feared complication for patients with haematological diseases. The use of the NK cells in immunotherapy includes bispecific fusion proteins which can bind to the NK receptor with one part and to tumour antigen with the other part. This work focuses on recombinant preparation of the NKp46 protein. To facilitate a study of the effects of O-glycosylation on the binding of the ligands, a mutation of the glycosylation site NKp46 T225A was prepared. A stably transfected HEK293S GnTI– and HEK293T cells had been prepared and these proteins were then extracellularly secreted. The Epa1 protein had been produced in E. coli bacterial expression system and purified. The binding ability of the Epa1 protein and lactose was verified by two measurements in solution (thermophoresis and isothermal titration calorimetry). Recombinant bispecific fusion proteins (that consist of the Epa1 protein and a nanobody-targeting tumour antigen HER2) were prepared to allow the study of the interactions of the NKp46 and the Epa1 proteins. The ability of the Epa1 and bispecific fusion proteins to bind to the surface of cells was verified by flow cytometry. The following lines were used in the process: the HEK293T transfected lines producing NKp46 and NKp46 T225A receptors, the NK92MI cell line, and the SK-BR3 cell line producing the HER2 receptor. Unfortunately, attempts to measure the specific binding of the Epa1 protein variants on the surface of the cells were not successful.
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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. IL2 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 IL2 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 reduction of systemic toxicity. The length of the peptide linker binding IL-2 and anti-IL-2 mAb plays an important role in this IC. By extending the linker between the anti-IL-2 mAb and IL-2 in the IC, higher biological activity of the protein could be achieved, allowing IL-2 to bind better to the mAb variable site. Thereby, there will be no risk of IL-2 diffusion far from the mAb and thus loss of biological activity and other benefits resulting from the association of IL2 and anti-IL-2 mAb.
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NK cells are an essential part of the immune system. They are the so and carry several receptors on their surface. Two types of called natural killers 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 respo nse 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 wi th potential use in targeted anticancer therapy. NKp30 has an activating ligand protein B7H6, 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 malig nantly transformed cell carries B7H6 on its surface, in most cases, it is recognized and eliminated almost at its formation. Not all tumour cells are B7H6 positive, so it is possible to visualize them to the immune system through surface decoration with H6. Moreover, such a ligand can be modified to amplify the immune response. This work is based on Pekar L. et al B7. (2020) publication, which described mutations that increase the affinity of the B7H6 binding site for NKp30. Based on their results, we pr five mutant forms of the entire extracellular domain of B7epared H6 in the human cell line HEK293T and measured binding affinity by isothermal titration calorimetry.
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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 antibody, and its extension could result in an increase in biological activity due to easier binding of IL-2 to the anti-IL-2 mAb paratope. In this work, a plasmid containing the gene for the mIL-2-S4B6 immunocytokine was prepared where murine IL-2 is linked to the S4B6 antibody heavy chain via a 35 amino acid glycineserine linker. Subsequently, the HEK293T cell line was stably transfected with this plasmid using the piggyBac system. Due to the high yields of recombinant protein provided by this method, the immunocytokine produced in this way will be further used for in vivo tests of its biological activity in a mouse model.
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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 assess which of the three available N-glycosylation sites of the NKp30 receptor is responsible for its homooligomerization. Another aim was the biophysical characterization of these homooligomers, including solving their structure.
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Malignant transformation of B-cells is manifested by a marked increase in the number of surface markers 20 (CD20, cluster of differentiation 20). In studying this trend, chimeric monoclonal antibodies targeting CD20 were introduced to induce apoptosis in B-lymphomas. Since the introduction of the first therapeutic monoclonal antibody rituximab, many others have been developed, with some still used to treat B-lymphomas today. Unfortunately, in many cases, resistance to these drugs is developing, and therefore the development of new types of therapeutics is still relevant. This work aims to develop a polymer-protein macromolecular conjugate capable of inducing apoptosis in CD20 positive leukemia cell lines. For this purpose, we work with biologically active vectors, so-called anti-CD20 nanobodies. This is a variable binding domain derived from the “heavy chain only” antibodies found in, e.g., llamas or camels. Compared to conventional antibodies, nanobodies are approximately ten times smaller, but their binding affinity for the antigen is not altered. For this reason, nanobodies are ideal candidates for attachment to a polymeric carrier, where poly-N-2-(hydroxypropyl)methacrylamide was chosen in this work. The transpeptidase reaction catalyzed by recombinant sortase A, which recognizes and transfers the C-terminal threonine in the LPXTG sequence to the N-terminal polyglycine, was selected for attachment of our engineered nanobodies. Therefore, for proper ligation, it was necessary to modify both the anti-CD20 nanobody and the polymeric carrier pHPMA with functional groups for the SrtA enzyme.
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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 modifications to the default construct, by extending the flexible linker with addition of IgG mutated hinge region or by replacing the nanobody with DARPin domain, were prepared. The ability of bispecific fusion protein to bind their binding partners was studied and the biological relevance of these proteins will be evaluated using cytotoxic experiments with NK cells in the future.
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Growth factors represent a group of significant substances in the metabolism of organisms. They are signaling molecules that control cell activity at the endocrine, paracrine, or autocrine levels. They act as key mediators binding to cell receptors, triggering a cascade of reactions leading to the regulation of genetic transcription in the cell nucleus and stimulation of cellular response. Growth factors influence various physiological functions such as cell proliferation, cell differentiation, and tissue healing. The utilization of growth factors is evident, for example, in regenerative medicine. For a similar purpose, research has been initiated to prepare the growth factor TGF-β3 with the possibility of attaching it to a polymeric carrier using a coiled-coil tag. This work focuses on the recombinant production of TGF-β3 – its analog with a latency-associated peptide (LAP) – and the application of techniques applicable to the intention of this work, specifically, attaching the protein to a polymeric carrier based on amino acids. Given the structural complexity with which growth factors are physiologically released from cells, the preparation of growth factors with a coiled-coil tag in vitro represents an unexplored challenge in the field of recombinant protein expression. In our HEK293T cell line expression system, it was possible to prepare the latent form of LAP-TGF-β3, whose biological activity was verified on rat mesenchymal stem cells. Several constructs of the growth factor with a coiled-coil tag theoretically enabling its controlled release from the polymeric carrier into the culture medium were designed and successfully cloned. As part of the experimental work, the potential of sortase A was also discovered as an alternative method for preparing the desired protein.
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Bachelor theses
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Mammalian cells have become the dominant system for recombinant expression of pharmaceutical proteins. This system is becoming suitable also for structural biology, with the advances in methodology of transient transfection of mammalian cells. This work dealt with optimization of recombinant expression in HEK293T and HEK293-6E cell lines in various media using easily quantifiable markers – secreted alkaline phosphatase (SEAP) and green fluorescent protein (GFP). Emphasis was placed on optimizing key factors behind the creation of transfection complex – the ratio of DNA to polyethyleneimine and the amount of DNA used. The positive influence of histone deacetylases inhibitor valproic acid and also of casein hydrolysate Tryptone N1 was also studied.
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HEK293 is a human cell line derived from embryonic kidney cells and is a frequently used system for the production of recombinant proteins. This work dealt with optimization of the composition of serum-free medium for HEK293S and HEK293T cell lines as a compensation for expensive commercial
media. The growth of culture and expression of reporter proteins SEAP and GFP was monitored as the
markers. I managed to create a new medium which contained, among other compounds, insulin (1 mg/l), transferrin (5 mg/l) and a mixture of trace elements. During the cultivation in a mixture of commercial medium EX CELL 293 with my new medium 293S cells grew faster than during the cultivation in commercial media (doubling time 20,47 ± 2,68 hours (srel = 13,1 %)). It seems that the new medium is suitable for transfection of HEK293 cell lines with a relatively high expression of recombinant proteins. Transfection ratio of DNA:PEI (w/w) for this medium is 1:2 to 1:3.
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T- and B- lymphocytes play a key role in immunity, because they provide specific immunity in organism. Receptor S1P1 regulates lymphocyte egress from lymphoid organs to blood, from which the lymphocytes travel to the site of infection. It was discovered that transmembrane helix 4 of S1P1 receptor interacts with transmembrane part of CD69 receptor and this interaction is responsible for retention of lymphocytes in lymphoid organs. The aim of this study is to prepare expression plasmids for production of these two receptors and their further research.
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Natural killer cells create an important part of innate immune system. Their importance lies in their ability to recognize and kill abnormal cells, especially tumour cells and virally infected ones, without previous activation. To recognize their targets, NK cells use a wide variety of surface receptors, both activating and inhibitory. If a ligand binds to an NK receptor, immune response is triggered. Examples of such ligand-receptor pairs are NKp80-AICL and NKRP1-LLT1 on human lymphocytes. Another ligand-receptor system of this kind is NKp65 and KACL, two recently discovered lectin receptors on human immunocytes. KACL is the last and most recently characterized member of CLEC2 receptor family in humans. Its expression is almost exclusively restricted to skin. NKp65, a close relative of NKp80, is a glycoprotein which stimulates NK92MI cell cytotoxicity upon KACL engagement. NKp65 has been shown to bind to KACL with a fairly high affinity by surface plasmon resonance measurement. This thesis aims at describing the cloning of expression vectors coding for NK cell receptors NKp65 and KACL, expression of these proteins in HEK293T cell line and their purification
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PIN1 protein belongs to a PIN family of transporters. This group of transporters is significantly involved in auxin transports outside of the cell. The auxin transport among plant cells gives an important role in plant growth, gravitropism, etc. The principle of auxin transport by this carrier protein is still not explored. This work deals with the production of PIN1 carrier protein by recombinant expression in human embryonic kidney 293 cell line. Production in these cells was checked by the accumulation of radioactive auxin and by the immunofluorescence.
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Aim of the thesis was optimization of production of monoclonal antibody against interleukin-2 (IL-2) as fusion construct with IL-2 by preparation of new expression vectors based on pTW5 plasmid and by replacing the native signal sequence with new one, originally from secreted alkaline phosphatase. This fusion antibody has interesting biological activity with therapeutic potential – prolonging the half-life of IL-2 in blood circulation.
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NK cells (natural killer cells) play a key role in innate immunity. Their function is to recognize and kill infected, stressed or malignantly transformed cells. A range of surface receptors promotes this recognition. Cytotoxic mechanisms, lead to induction of apoptosis in the target cell. Receptor NKp30 is one of cytotoxic reaction triggers. It belongs, with NKp46 and NKp44, to NCR (natural cytotoxicity receptors) family. This work describes preparation of NKp30 receptor with natural and simple glycosylation in expression system of human embryonic kidney cell line 293 (HEK293). It was found that glycosylated receptor NKp30 forms noncovalent oligomers. Equilibrium is formed in solution between oligomers and monomers. Oligomerization depends on glycosylation, deglycosylated protein doesn´t form oligomers. A recombinant endoglycosidase ENDO F1 was prepared for purposes of deglycosylation.
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NK cells, the cells of non-adaptive imune system, are able to recognise viraly infected or oncogenous cells by many inhibiting and activating receptors that are expressed on their surface and eliminate them consequently. For NKp30, activating receptor of NK cells included in NCR (Natural Killer Cell Receptors) family, lately there were identified several ligands, including membrane protein B7-H6 expressed on oncogenous cells surface and BAG6, cell core protein with wide spectrum of functions. Aim of this thesis was preparation of expression vectors coding for receptor NKp30 and his ligands B7-H6 and BAG-6, enabling expression of these proteins in HEK293 cell line.
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Besides recombinant protein expression in prokaryotic cell lines (E. coli), systems, that could quickly, reliably and stably produce recombinant proteins in human cell lines, come to the fore. These cell lines assure proper tertiary structure and posttranslational modification of the desired products. One of the ways to achieve production of recombinant proteins in human cell lines is the use of lentiviral vectors. This thesis describes the preparation of the lentiviral vector (plasmid) Daedalus, which contains a construct for recombinant expression of secreted alkaline phosphatase. For the preparation of the desired plasmid methods based on insertion of the secreted alkaline phosphatase gene using the restriction endonucleases and methods based on amplification by polymerase chain reaction (restriction-free cloning, transfer polymerase chain reaction and Gibson assembly) were used.
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The examined enzyme is C324S modified recombinant nitrilase from Arthroderma benhamiae CBS 112371 (GenBank: EFE30690). A recombinant form of this enzyme previously prepared by Mgr. John Blaha was analyzed using mass spectrometry with respect to disulfide disulfide bond pattern. The obtained data shows that on the C-terminus of the enzyme is an unpaired cysteine 324, which binds nonspecifically to other cysteines in the sequence of the protein leading to a heterogeneous mixture of disulfidic oligomers. Mutated form of the gene encoding the native form of this nitrilase was prepared by replacing cysteine 324 to serine and cloned into the plasmid pET-30a (+). The gene was expressed using the modified plasmid in Escherichia coli BL21-Gold (DE3) and the resulting product was subsequently purified using ion exchange chromatography and gel filtration. Oligomeric behaviour of the protein was examined using electrophoretic separation in polyacrylamide gels, gel filtration and analytical ultracentrifugation, both in non-reducing and reducing environment conditions.
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The Natural Killer (NK) cells play a vital role in the nonspecific immunity. They are capable of efficient immunity reaction without any antigen specific receptors on their surface. NK cells recognize non-self molecules and they also recognize their molecules serving as health markers, and absence of these molecules such as MHC glycoproteins on the target cell surface. The NK cells are able to recognize viral infection or tumor transformation in the organism. If natural killer cell is in contact with a cell carrying an abnormally low MHC class I glycoproteins, it will create a signal which informs the cell is infected with a virus. NK cells trigger apoptosis of the target cell without prior stimulation, proliferation and differentiation. They also promote inflammatory responses by the production of chemokines and cytokines. The response is always the interplay of activating and inhibitory signals that the cell receives from its surroundings. The latest research shows that the targeted modulation of NK cells leads to less complications in bone marrow transplantation. They can be potentially used in immunotherapy, e.g. in the treatment of autoimmune diseases. Therefore, NK cells are a highly-studied group of cells. This thesis is focused on a production of Clrb („Ctype-lectin-related protein b“). This protein is a ligand of a rat inhibitory NK cellular receptor NKRP1B. If the cell expresses Clrb on its surface, it won’t be destroyed. Vector for Clrb was prepared by recombinant cloning. We produced the protein in a human embryonic kidney 293 cells with simple glycosylation (HEK293S GnTI- ). Production of a monomeric form was verified by SDS-PAGE. Protein was also characterized by analytical ultracentrifugation and crystallization.
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Natural killer (NK) cells are cells of native immunity. Their task is to recognize and destroy tumor cells or, for example, viruses infected cells. NK cells use a number of surface receptors to recognize affected cells. These receptors may be activating, i.e., activate the cytotoxic response of NK cells leading to the induction of apoptosis in the target cell, or inhibitory ones, which, on the other hand, inhibit NK cell response. Whether a cytotoxic response occurs depends on both types of NK receptors. The NKp44 receptor belongs to the family of immunoglobulin receptors and it is one of the activating receptors. Its properties are very unique among other receptors. Other activating receptors of the immunoglobulin receptor family are NKp46 and NKp30. Recently, the interaction between the NKp44 receptor and the proliferating cell nuclear antigen (PCNA) has been described. PCNA is a clamp protein that plays a key role in DNA replication and repair as it anchors replication proteins. This work focuses on the production and optimization of a production of PCNA and on the preparation of expression vectors for the later production of NKp44 and NKp46. Interaction of NKp44 and PCNA will be investigated later. For the same reason, the expression vector for the NKp46 protein is also prepared in this work. PCNA protein was prepared recombinantly using expression systems of Escherichia coli BL21 (DE3) and human HEK293S GnTIcell line. The intracellular and secreted expression was performed in the HEK293S GnTIeukaryotic system and the protein was purified by affinity chelating chromatography followed by gel permeation chromatography. Purification of PCNA produced intracellularly was preceded by ion-exchange chromatography. In addition, vectors containing genes encoding NKp44 and NKp46 NK receptor expression constructs were prepared by introducing sections of their genes into plasmid pTW5sec for later secreted production in HEK293S GnTI- . The preparation of vectors with genes for NK receptors was successful, which was verified by DNA sequencing. PCNA was successfully prepared and analyzed by SDS electrophoresis and analytical ultracentrifugation.
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Natural killer cells (NK cells) are part of innate antitumor immunity. These cells have the capacity to prevent viral infection or malignant transformation without prior antigen sensitization. Activation of NK cells consist of different recognition strategies. Mechanism of activation is based on down-regulated expression of MHC gp I molecules on the cell surface. NK cells possess both activation and inhibitory receptors that transmit activation or inhibitory signals which determine if NK cells are activated or not, and thus whether the target cell will be lysed or spared. NKp30 is a type I transmembrane receptor which recognize the stress-induced cell surface ligand B7-H6. Interaction of these two proteins leads to the initiation of immune response. The main aim of this thesis is the preparation of cell ligand B7-H6 with coiled-coil peptide tag in human embryonic kidney cell lines HEK293 GnTI- and HEK293T. Successful preparation of B7-H6 with coiled-coil tag on its C-terminus was verified by mass spectrometry. Its interaction with natural cytotoxicity receptor NKp30 was also proven by sedimentation analysis.
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Natural killer cells (NK cells) are lymphocytes that possess cytotoxic activity against tumour or virally infected cells independent of preceding antigen sensitisation. To kill such cells, they utilise their activating and inhibitory surface receptors that interact with target cell surface molecules. The immune response carried by NK cells depends on the balance of both activating and inhibitory signals. Human NK cell surface receptor NKR-P1A belongs to the structural family of C-type lectin-like receptors. This receptor interacts with its ligand LLT1, which belongs to the same protein family, with low affinity and high specificity. The NKR-P1A:LLT1 complex formed between NK cell and its target cell inhibits NK cell cytotoxicity, and hence is a part of the regulation of immune response. This thesis studied the effect of S159A mutation on the stoichiometric state of soluble human NKR-P1A ectodomain in solution. Therefore, a mutant form of NKR-P1A G90-S225 S159A ectodomain was successfully produced in stably transfected human embryonic kidney cells 293 (HEK293S GnTI-). This construct was purified by affinity and size-exclusion chromatography, and analysed by SDS-PAGE and analytical ultracentrifugation. Our results show that the preclusion of N-linked glycosylation in the position 157 promotes the dimerization of the NKR-P1A ectodomain in solution.
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Natural killer cells (NK cells) are cells of innate immunity that play an essential role in the immune response of an organism. In contact with infected, stressed or tumour cells, the NK cells can trigger cytotoxic mechanisms. The initiation of mechanisms depends on the presence of activating or inhibitory ligands on the surface of the cells. On the surface of the NK cells, there are activating and inhibiting receptors that upon binding their respective ligands send a signal to the NK cell. One of the activating mechanisms is the decrease of expression in MHC gp I molecules on the surface of the infected cells. This molecule is a ligand of the inhibiting receptors. One of the activating receptors of NK cells is NKp46. This receptor belongs to the natural cytotoxicity receptor (NCR) family. NKp46 has many ligands, one of them being the adhesin Epa1 of yeast Candida glabrata. This thesis aims at preparing plasmids and producing extracellular domains of the NKp46 receptor and its ligand adhesin Epa1. The interaction of NKp46 and its ligand Epa1 remains to be the subject of future research. Plasmids containing NKp46 and Epa1 genes were successfully prepared and verified by DNA sequencing. The NKp46 protein was produced in eukaryotic expression system of HEK293S GnTI- cell line. A stably transfected HEK293S GnTI- cells had been prepared, and the NKp46 protein was then extracellularly secreted and isolated from cultivation medium using immobilized metal affinity chromatography and then purified using gel permeation chromatography. The Epa1 protein had been produced in the Escherichia coli bacterial expression system after which it was isolated from cell lysate similarly by the combination of immobilized metal affinity chromatography and gel permeation chromatography.
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Natural killer cells (NK cells) are an important part of innate immunity. On their surface they express a complex group of receptors that use different signalling motifs to activate or inhibit NK cell cytotoxic activity. NK cells are capable to kill aberrant cells (namely, viral, infected, and tumour cells) by using special cytotoxic mechanisms to trigger apoptosis. The activating receptors recognize tumour or stress-induced ligands, e.g., NKG2D receptor recognizes the MICA ligand and NKp30 recognizes the B7-H6 ligand. Therefore for human immune system it is only natural that cancer cells are destroyed by NK cells. The current therapeutic goals in the treatment of cancer are primarily focused on strengthening the body’s own natural ability to fight with cancer and one possible way is stimulation of NK cells to win this deadly fight. In addition to NK cells, antibodies are also widely used for the treatment of cancer, as well as other immune-related disorders. Most of them are monoclonal antibodies, but antibody fragments are getting attention and are being tested more and more in recent years. This work describes the preparation of three bifunctional fusion proteins: B7-H6-L-aHER2, MICA-L-aHER2, and aHER2-L-MICA, which contain immunoligands for the activating receptors of NK cell and VHH fragment of antiHER2 antibody, in the human embryonic kidney cell line HEK293T. The proteins were successfully prepared and their binding to the NK cell receptor was verified by analytical gel permeation chromatography.
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Natural killer cells (NK cells) are one of the basic elements of innate immunity. They play a key role in immune response against virus-infected, cancerous or otherwise stressed cells. NK cells express surface activating and inhibitory receptors. Activating receptors trigger cytotoxic mechanisms that lead to the target cell’s apoptosis. Inhibitory receptors provide cellular tolerance. The balance between these receptor signals determines the resultant NK cell response to the target cell. C-type lectin-like receptors include the activating receptor NKp80 and its ligand AICL. AICL is a myeloid-specific activating receptor expressed on tumor cells. The NKp80:AICL complex that assists in the cytolysis of malignant myeloid cells is being studied in the context of cancer immunotherapy. This bachelor thesis describes the preparation of vectors containing genes encoding AICL expression constructs, and the subsequent production of proteins in the human embryonic renal cell line (HEK293S GnTI- ). The expression constructs contain the extracellular domain of AICL, TEV protease site and Fc fragment. Two different constructs were prepared – one containing the native AICL sequence and the other carrying C87S mutation.
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Interleukin 2 is a growth factor of T cells as well as other lymphocytes, such as NK, NKT cells, dendritic and mast cells, which ensure its expression and secretion. IL-2 regulates immune cell homeostasis and is used to treat a variety of disorders including cancer and autoimmune diseases. In recent years, several cases of interleukin 2 complexed with anti-IL2 antibody have been shown to exhibit dramatically higher biological activity in vivo. These complexes have selective stimulatory activity for different IL2 receptors on target cell. This work follows up previous unsuccessful attempts to express and purify a sufficient amount of the murine IL2 immunocomplex with the S4B6 antibody linked by a 15 amino acid long glycine-serine linker. In this work, a plasmid containing the secreted fusion immunocomplex mIL2-S4B6 gene was prepared and stably transfected to the HEK293T cell line using piggyBac system. The protein was then isolated by chelation affinity chromatography and purified by gel permeation chromatography.
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Cancer is currently widespread disease and its successful treatment requires the elimination of all cancer cells in the body. One method of cancer treatment is immunotherapy, which seeks to elicit an immune response and activate the body’s anti-tumor defense mechanisms. Therapeutic antibodies are used to target tumor cells markers. One of such markers is the HER2 receptor which is overexpressed for example on the surface of breast cancer cells. Humanized monoclonal antibodies are often used as therapeutic antibodies, but other constructs such as bispecific particles, nanobodies or their analogs are also used. Nanobodies refer to recombinant antibody-derived variable domains that lack light chains in their structure. Such antibodies occur naturally, for example in camelid mammals or in certain cartilaginous fishes, such as sharks. This work describes the preparation of various glycoforms of the antiHER2 nanobody and verification of the effect of the glycosylation on the ability of nanobody to bind to the cell line that is overexpressing the HER2 receptor on its surface. A nanobody with complex natural glycosylation (produced in the HEK293T cell line) and a nanobody with uniform glycosylation (produced in the HEK293S GnTI- cell line) were prepared. The work also describes the cloning and production of antiHER2 nanobody with a mutated glycosylation site in the paratope. Glycosylation has been shown to negatively affect binding on HER2 receptor, an important finding for the future use of this nanobody in the preparation of potential immunoactive therapeutics.
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Every year, cancer tops the list of causes of death for people across all continents. Promising potential therapies, in addition to finding new drugs targeting more selectively the uncontrollably dividing cells, include so-called immunotherapy. The aim of this type of treatment is to increase the cytotoxicity of the effector cells, such as natural killer cells or T lymphocytes. This effect can be achieved by incubating effectors with selected cytokines or ligands that induce an increase in the number of activating receptors as well as to attract effectors to cancer cells. The main focus of this thesis was finding appropriate concentrations, combinations and ratios of natural killer cells activating ligands with cytokines to increase the percentage of activated NK cells. A plate surface coated with activation ligands was used to activate NK cells as a ment of an ideal tumour cell. The range of activation was measured by detecting the molecule CD107a, a surface marker of NK cell activation. This resulted in a simple synthetic system capable of verifying the biological activity of recombinantly prepared NK cell activation ligands. A possible future application of the results of this work is in further investigation of prepared bispecific fusion proteins that have therapeutic potential to activate NK cells.
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Natural killer cells belong to immune cells mediating non-specific immune response. On their surface, natural killer cells express many ligand-specific activation or inhibition receptors, among which belongs natural cytotoxicity activation receptor NKp30, which is able to trigger cytotoxic immune response upon interaction with its ligands. Natural cytotoxicity receptor NKp30 comprises of one immunoglobulin-like ligand-binding extracellular domain with short stalk domain, allowing the extracellular domain of NKp30 receptor to form oligomers, which leads to strengthening of ligand-receptor interaction. Furthermore, signalization leading to cell activation is affected by oligomerization of the extracellular domain of NKp30 receptor. This thesis focuses on preparation of constructs inspired by chimeric antigen receptor theory that carry extracellular domain of NKp30 receptor with stalk domain or only with ligandbinding domain and on validation of biological activity of prepared constructs after expression on the cell membrane. Two vectors for expression of the constructs inspired by chimeric antigen receptors carrying genes for extracellular domain of NKp30 receptor were prepared and to validate their biological activity, they were transfected into Jurkat cells.
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The subject of this study is a receptor NKp80, also known as killer cell lectin-like subfamily F, member 1 (KLRF1). It is an activating receptor which forms homodimers on the surface of natural killer (NK) cells. Receptor NKp80 binds to a ligand, AICL, which is naturally expressed on all myeloid cells. Upon a substantial increase in AICL expression, for example in cancer cell, the cell then becomes a target for an NK cells expressing the receptor NKp80. Ultimately, the complex NKp80:AICL is therefore a potential target for the immunotherapeutic treatment of myeloid leukaemia. The aim of the study was to the produce and purify a series of mutants of an extracellular domain of NKp80 by replacing cysteins by serines in a segment of extracellular domain called the stalk region. Here, by introducing the mutations, we studied their effect on homodimer formation. The proteins were prepared in HEK293S GnTI- cells using stable transfection. Altogether, we produced seven mutants with all possible combinations of mutations of the three cysteins in the stalk region. We then analysed the proteins using size exclusion chromatography and differential scanning fluorimetry. Lastly, we deglycosylated the proteins to verify that NKp80 is present in several glycoforms. Our results show that none of the variants of NKp80 form covalent dimers. However, regarding the elution volume of the proteins we may suggest that they form non-covalent dimers. In addition, we ascertained that there is no significant correlation between the position of a mutation or their combinations and the level of expression or their thermal stability. Finally, we confirmed the presence of several glycoforms in all prepared proteins.
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NK cells are an important part of the innate immune system. These are large granular lymphocytes that exhibit cytotoxic properties against cells infected with a virus, or protozoan, or have undergone malignant transformation. Due to its cytotoxic properties, NK cells also participate in regulation of other immune cells and their activity. NK cells express several activating and inhibiting receptors on their surface. Between signals from those receptors is a dynamic balance that determines the life and death of the target cell. If activation signals predominate, the target cell will be eliminated. If the inhibitory signals predominate, the cytotoxic response will not be triggered. The NKp30 receptor is one of the important activating receptors. Its involvement is in many cases crucial for the proper recognition and elimination of the target cell. Receptor NKp30 is activated by several ligands, including both viral, bacterial and parasitic ligands, as well as ligands produced by tumor cells. Galectin-3 is recently described ligand of NKp30, which inhibits its further activation upon formation of the ligand-receptor complex. Galectin-3 is expressed by a number of cells in the human body and it is also found in some cancer cells, where its expression is often increased above physiological levels. When galectin-3 enters the extracellular space, inhibition of the NKp30 receptor may contribute to tumor escape from the immune system. The NKp30 receptor can oligomerize in solution under certain conditions. The first requirement for its oligomerization is the presence of stalk domain. The second requirement is the presence of N-glycosylation. The aim of this thesis was to characterize the effect of presence of the carbohydrate recognition domain of galectin-3 (Gal-3-CRD) on the dissociation of oligomeric fraction of NKp30 by analytical gel permeation chromatography. Another aim was to characterize the binding of Gal-3-CRD to NKp30 depending on the type, or presence of N-glycosylation of NKp30.
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Immunotherapy is a promising tool to combat cancer, that has been attracting more and more attention at itself in the recent years. Its main objective is activating and boosting innate immune response towards malign cellular transformation and growth. This makes cytotoxic cells of the immune system the main targets of research, when studying and inventing new ways to combat cancer. Such cells of interest are, for instance, NK cells, which are cytotoxic cells of the innate immune response. Amongst their various functions is a process called „immune surveillance”. NK cells constantly scan other body cells, destroying those that are infected, defect or mutated. Upon finding a target cell, NK cell gets several activating and inhibiting signals from said target cell. Depending on which type of signal outweighs in its overall intensity, NK cell activates or leaves the target cell undamaged. When activated, NK cells have several ways to affect the target cell at their disposal: releasing effecter molecules that lead to lysis of the target cell and interacting and modulating other immune cells’ response towards cancerous formations via chemokines, cytokines and such. This makes NK cells quite useful in immunotherapy. One possibility how they can be engaged is via certain bispecific fusion proteins. Protein constructs tested nowadays are made up of two parts: first has an ability to recognize and bind to a specific tumor receptor whereas the other activates NK cells’ cytotoxic response. These molecules act as a bridge bringing a tumor cell and an NK cell close together, which triggers NK cell’s activation. This bachelor work attempted at preparing two fusion proteins and their subsequent use for the confirmation of transpeptidase activity of a bacterial enzyme sortase A. The first protein construct consisted of an activating ligand for NK cells (MICA) and a signal sequence of amino acids that is recognized by sortase A (sequence of amino acids LPETG). The second prepared protein is a bispecific fusion protein, that, besides including an activating ligand for NK cells (MICA) and a signal sequence of amino acids recognized by sortase A (sequence of amino acids LPETG) in its structure, also has at its beginning a nanobody against the tumor receptor CD20 (antiCD20). The signal sequence LPETG in the structure of both fusion proteins made it possible to use them as substrates for a ligation reaction catalyzed by sortase A. The possibility of sortase A usage as a tool when labeling proteins was confirmed and the efficiency of the reaction was determined.
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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 binding of the NKp30 receptor using gel permeation chromatography. It also deals with the preparation of galectin-3 with a C-terminal polyhistidine tag, which will be further used in the study of the interaction of galectin-3 with newly synthesized organic Gal-3 inhibitors using microscale thermophoresis. The exploitation of the results of this work may contribute to the unravelling of the structure of Gal-3 binding to the NKp30 receptor and the design of new effective drugs with therapeutic potential.
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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 SDS electrophoresis. After purification, 125 mg of pure protein was obtained.
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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 glycoprotein.
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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.
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