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Production of interleukin 2 in fusion with monoclonal antibody S4B6
Rožová, Dominika ; Vaněk, Ondřej (advisor) ; Kavan, Daniel (referee)
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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Preparation of reporter cell line for validation of recombinant protein biological activity
Kozojedová, Petra ; Vaněk, Ondřej (advisor) ; Moserová, Michaela (referee)
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 ligand- binding 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. Key words: NK cells, NKp30, reporter...
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Mutagenesis and expression of protein NKp80, the activation receptor of human lymphocytes
Pazderová, Kristýna ; Vaněk, Ondřej (advisor) ; Ječmen, Tomáš (referee)
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...
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Optimizing chimeric antigenic receptors (CARs) T-cells for immunotherapy of hematological malignancies
Mucha, Martin ; Otáhal, Pavel (advisor) ; Zadražil, Zdeněk (referee)
Immunotherapy based on chimeric antigen receptor (CAR)-expressing T lymphocytes has proven to be highly successful in the treatment of acute lymphoblastic leukemia (ALL), leading to development of CAR-based immunotherapies for other hematologic malignancies. Currently, efforts are underway to refine T cell modifications to make patient treatment more effective. Each time, this modification then needs to be empirically validated in in vitro experiments. We decided to study the effect of the cytokine IL-21 on the antitumor function of CD19-specific CAR T cells using in vitro assays. A construct that co-expressed IL-21 under the control of the inducible NFAT promoter together with CARs against CD19 was introduced into T cells. In a series of experiments, the properties of these cells were compared after coculture with tumor B cell lines and CLL cells obtained from patients. The results showed that CAR T cells that express IL-21 proliferate and activate better, even after repeated stimulation with leukemia cells. In addition to CARs specific against the CD19 molecule, we also investigated CARs specific against the CLL1 molecule, which has been described in the literature as one of the promising targets for the treatment of AML. We prepared CAR T cells against CLL1 producing IL-21. For this purpose, we...
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Interaction of a surface marker of immune cells with low-molecular weight ligands and their polymer conjugates
Šimonová, Lenka ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Millions of people worldwide die of cancer every year. In the last decade, im- munotherapy offered new treatment options achieving long-lasting remissions in a number of patients. Several new immunotherapy-based drugs have been ap- proved by Food and Drug Administration. However, majority of patients either do not respond or soon relapse. Combination of therapies as well as exploring new immune checkpoints seems promising. This thesis focuses on the new immunotherapeutic target CD73. CD73 is membrane ectonucleotidase, widely expressed on the regulatory leukocytes and on cancer cells. The enzymatically active CD73 contributes to the tumour mi- croenvironment by production of immunosuppressive adenosine. This novel im- mune checkpoint is being intensively studied. This thesis aims on development of new approaches for targeting and inhibition of CD73. Soluble recombinant CD73 (rhCD73) was prepared in mammalian expression system and transfectants stably expressing membrane-bound CD73 were prepared as well. Inhibitors necessary for both of my goals have been designed based on published inhibitor of CD73. Development and evaluation of novel antibody mimetic for CD73 characteri- sation was done. The so-called iBody, HPMA polymer conjugate decorated with CD73 inhibitor for targeting, fluorophore for...
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Influence of tumor microenvironment, cellular and humoral immunity on cancer pathogenesis.
Špaček, Jan ; Závadová, Eva (advisor) ; Petráková, Katarína (referee) ; Kopečková, Kateřina (referee)
Cancer is the second leading cause of death in the Czech Republic. Breast cancer and colorectal cancer have relatively high mortality rate. One of the areas of current clinical research in oncology is the study of prognostic biomarkers, which aims to optimize the decision-making process for a patient. Immune response and processes in the tumor microenvironment have been shown to influence to a large extent the biological nature of the tumor in terms of its aggressiveness and ability to metastasize in the host's body. There are certain tumors that could induce a strong immune response, while others do not. The ability to induce an anti-tumor cell response and to attract specific lymphocyte subpopulations directly into tumor tissue has been shown to be very closely related to the prognosis of cancer patients. There is evidence and correlation of the presence of so-called tumor infiltrating lymphocytes in tumor tissue and overall patient survival. Stratification of cancer patients based on immuno-predictors both in the plasma and directly in the tumor microenvironment makes it possible to identify suitable candidates for rediscovered modern anti-tumor immunotherapy, which can already be considered a standard therapeutic modality. In our projects, we focused on the identification of biomarkers that...
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Significance of Tim-3 protein expression on the surface of acute myeloid leukemia cells
Kořánová, Tereza ; Kuželová, Kateřina (advisor) ; Brdička, Tomáš (referee)
Acute myeloid leukemia (AML) is a cancer disorder of hematopoiesis, characterised by production of dysfunctional progenitor cells of myeloid cell lineage. Mutations provide malignant cells with the ability to proliferate independently of growth factors and to resist to cell death induction. In addition, transformed cells are often capable of escaping the immune system. T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) was originally discovered on the surface of immune cells, where it acts as an inhibitory receptor. Later, Tim- 3 was also found on leukemia cells, but its function on this cell type is much less clear. Upon ligation with galectin-9 (Gal-9), Tim-3 acts on AML blasts as an activating receptor. The Tim- 3/Gal-9 complex signalization stimulates NF-κB, β-catenin, HIF-1, PKC and mTOR pathways, which substitute to some extent the function of growth factors. They support continuous regeneration of leukemic cells, provide stimulatory signals, and further increase the production and secretion of Tim-3 and Gal-9 in a positive feedback loop. An important function of Tim-3 is the transport and localization of Gal-9, which blocks the activation of T lymphocytes by interacting with Tim-3 on their surface, inhibits formation of immunological synapse and execution of effector...
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Impact of pattern and functional properties of tumor-infiltrating immune cells for clinical outcome of head and neck cancer
Hladíková, Kamila ; Špíšek, Radek (advisor) ; Plzák, Jan (referee) ; Reiniš, Milan (referee)
Head and neck squamous cell carcinoma encompasses a complex and heterogeneous group of malignant diseases. Originally, this tumor type was associated with tobacco and alcohol consumption. However, a significantly expanding subset of tumors associated with oncogenic human papillomavirus infection arising in deep tonsillar crypts was identified within the last decades. Due to the essential role of the immune system in antiviral and anticancer immune response, the prognosis of patients is significantly influenced by the volume, composition and functional capacity of the immune infiltrate. The immunosuppressive landscape of head and neck cancer leads to unfavorable outcome of patients and decreased efficacy of immunotherapy. The response rate to standard treatment is high, however, standard therapy is accompanied by considerable toxicity influencing the quality of life. In 2016, the first immunotherapeutics for the treatment of patients with recurrent squamous cell carcinoma of the head and neck were approved - the anti-PD-1 immune checkpoint inhibitors nivolumab and pembrolizumab. This type of therapy, based on mitigation of immunosuppression, shows strong efficacy and less toxicity in combination with other therapies. Therefore, anti-PD-1 immunotherapy was recently approved in the first-line...
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Production of interleukin 2 in fusion with monoclonal antibody S4B6
Rožová, Dominika ; Vaněk, Ondřej (advisor) ; Kavan, Daniel (referee)
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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