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Production and characterisation of therapeutical antibody Farletuzumab - Thiomab E154C S378C
Mochánová, Michaela ; Brynda, Jiří (advisor) ; Dračínská, Helena (referee)
5 Abstract The human folate receptor α (FRα) is a receptor that binds folic acid, which as one of the vitamins is required for basic cellular processes, cellular growth, and differentiation. FRα's expression is strictly regulated in healthy tissue but is highly overexpressed in some tumor types. A humanized monoclonal antibody against folate receptor α called Farletuzumab has been developed. Binding of Farletuzumab to FRα leads to the activation of the immune system via antibody-dependent cytotoxicity and complement-dependent cytotoxicity. To enhance the antitumor effect, selected amino acids in the Farletuzumab antibody molecule were mutated to cysteines, allowing the subsequent use of THIOMAB technology. These cysteines are used to specifically conjugate the antibody to an artificial effector molecule that triggers the cGAS-STING (cyclic GMP-AMP synthase - stimulator of interferon genes) signaling pathway. The combination of the antibody's own anti-tumor effect and the triggering of the immune response to the cGAS-STING signaling pathway maximizes the immune response against cancer cells. Therefore, the aim of my work was to express the antibody in a suitable eukaryotic cell system and then to do a basic characterization of the protein. The Farletuzumab Thiomab antibody was prepared by heterologous...
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In vitro saturation study of gallium-67 and zirconium-89 labelled monoclonal antibody ramucirumab on SKOV-3 cell line
Holodňáková, Nikola ; Bárta, Pavel (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Student: Nikola Holodňáková Supervisor: Mgr. Pavel Bárta, Ph.D. Name of the work: In vitro saturation study of gallium-67 and zirconium-89 labelled monoclonal antibody ramucirumab on SKOV-3 cell line. Targeted biological treatment becomes more and more important with the development of a new therapy in oncology. It stimulates immune system to eliminate cancer cells. Significant progress has been made since the introduction of monoclonal antibodies. They represent one of the newest possibility used in diagnosis and treatment of tumours. The ability of the monoclonal antibody ramucirumab is to recognize and bind specifically to tumour cell antigens such as the VEGF type 2 receptor (VEGFR-2) and thus to supress angiogenic process. Anti-angiogenic ramucirumab inhibits this receptor via blocking of VEGF binding sites, which prevents the growth of tumours. It is possible to increase antitumor effect of monoclonal antibodies by their combination with other molecules like radionuclides, toxins and cytostatics when forming the so called conjugates. Prepared immunoconjugates serve as diagnostic and therapeutic tools also in Nuclear Medicine. The aim of the experimental work in was the preparation of...
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Studies of properties of gene products of the Merkel cell carcinoma polyomavirus: Antibody preparation and expression vector construction.
Sauerová, Pavla ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Merkel cell polyomavirus (MCPyV) is a recently discovered human virus, having it's genome often integrated in a genome of Merkel carcinoma cells. Although this type of carcinoma is not so usual, it is very aggressive and it's incidence has been rising in last few years. It is not surprising that this virus is nowadays in the centre of scientific interest, as well as other pathogens and mechanisms affecting human life. Because the virus was discovered not so long ago, its research has been at the whole beginning. This diploma thesisaims to contribute to the study of this virus from the molecular-virology point of view. A neutralizing monoclonal antibody, type IgG2a, targeted against the main capsid protein of MCPyV, VP1, and recognizing its conformational epitote was prepared. This antibody was then used for a pilot study of VP1 VLPs MCPyV movement in mammalian cells. Results showed that the studied virus, at least particularly, utilizes caveolin-1-carrying vesicles for its movement in cells (colocalisation of VP1 VLPs and caveolin-1 was observedColocalisation with EEA1 marker of early endosomes, LamP2 marker of endolysosomal compartments or with BiP marker of endoplasmic reticulum was sporadic but significant. These preliminary results suggest that MCPyV might utilise an endocytic pathway leading...
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In vitro saturation study of gallium-67 and zirconium-89 labelled monoclonal antibody ramucirumab on SKOV-3 cell line
Holodňáková, Nikola ; Bárta, Pavel (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Student: Nikola Holodňáková Supervisor: Mgr. Pavel Bárta, Ph.D. Name of the work: In vitro saturation study of gallium-67 and zirconium-89 labelled monoclonal antibody ramucirumab on SKOV-3 cell line. Targeted biological treatment becomes more and more important with the development of a new therapy in oncology. It stimulates immune system to eliminate cancer cells. Significant progress has been made since the introduction of monoclonal antibodies. They represent one of the newest possibility used in diagnosis and treatment of tumours. The ability of the monoclonal antibody ramucirumab is to recognize and bind specifically to tumour cell antigens such as the VEGF type 2 receptor (VEGFR-2) and thus to supress angiogenic process. Anti-angiogenic ramucirumab inhibits this receptor via blocking of VEGF binding sites, which prevents the growth of tumours. It is possible to increase antitumor effect of monoclonal antibodies by their combination with other molecules like radionuclides, toxins and cytostatics when forming the so called conjugates. Prepared immunoconjugates serve as diagnostic and therapeutic tools also in Nuclear Medicine. The aim of the experimental work in was the preparation of...
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The preparation and the following in vitro saturation study of the radiopharmaceutical 99mTc-DTPA-ramucirumab on PC-3 cell line
Sabolová, Klaudia ; Bárta, Pavel (advisor) ; Janoušek, Jiří (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Student: Klaudia Sabolová Supervisor: Mgr. Pavel Bárta, Ph.D. Title of diploma thesis: The preparation and the following in vitro saturation study of the radiopharmaceutical 99m Tc-DTPA-ramucirumab on PC-3 cell line In cancer treatment, immunology is given prominence, which compared with chemotherapy and radiotherapy has a lower risk of side effects on healthy tissues. Immunotherapy includes application of monoclonal antibodies aimed at some tumour antigens using either non conjugated monoclonal antibodies or conjugated ones with an appropriate effector element, such as radionuclide. Angiogenesis plays the important role in pathogenesis of tumour diseases. Angiogenic process is regulated mostly by the interactions among vascular growth factors (VEGFs) and VEGF receptors (VEGFR). The main regulator of angiogenesis is VEGF-A. The blocking of the interaction among VEGF-A and its receptors VEGFR-1 and VEGFR-2 inhibits angiogenesis, and so does the growth of tumours. Ramucirumab is the monoclonal antibody with antiangiogenic effect, which blocks this interaction by its binding to the extracellular VEGFR-2 domain with high affinity. The presented study is focused on ramucirumab...
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In vitro saturation study of 99mTc-HYNIC-ramucirumab on SKOV3 cell line
Klimová, Juliána ; Bárta, Pavel (advisor) ; Maixnerová, Jana (referee)
v anglickom jazyku Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Student: Juliána Klimová Supervisor: Mgr. Pavel Bárta, Ph.D. Name of the work: In vitro saturation study of 99m Tc-HYNIC-ramucirumab on SKOV3 cell line. The passive immunotherapy is based on the use of already active immune system components (monoclonal antibodies), which play an important role in cancer cells elimination in the organism. The active immunotherapy tries to stimulate an active anticancer response via an appropriate form of an immunization. When monoclonal antibodies bind to cancer cells, those cells become a selected target for the following removal. The enhancement of the anti- cancer affect of monoclonal antibodies is possible due to the attachment of therapeutic agents like cytostatics, toxins and radionuclides. This presented master thesis is focused on the radiolabeling of the monoclonal antibody ramucirumab, which is directed against the vascular endothelial growth factor type 2 (VEGFR 2), which is often present in cells of some types of cancerous diseases. Within the experimental work, at first, there was a conjugation of chelating agent succinimidyl-6-hydrazino-nicotinamide (HYNIC) on the monoclonal antibody. After this step, radionuclide 99m...
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Preparation of plasmids for expression of interleukin 2-fusion monoclonal antibody
Slavata, Lukáš ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
Aim of the thesis was optimization of production of monoclonal antibody against interleu-kin 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. (The thesis is written in Czech.) Powered by TCPDF (www.tcpdf.org)
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Studies of properties of gene products of the Merkel cell carcinoma polyomavirus: Antibody preparation and expression vector construction.
Sauerová, Pavla ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Merkel cell polyomavirus (MCPyV) is a recently discovered human virus, having it's genome often integrated in a genome of Merkel carcinoma cells. Although this type of carcinoma is not so usual, it is very aggressive and it's incidence has been rising in last few years. It is not surprising that this virus is nowadays in the centre of scientific interest, as well as other pathogens and mechanisms affecting human life. Because the virus was discovered not so long ago, its research has been at the whole beginning. This diploma thesisaims to contribute to the study of this virus from the molecular-virology point of view. A neutralizing monoclonal antibody, type IgG2a, targeted against the main capsid protein of MCPyV, VP1, and recognizing its conformational epitote was prepared. This antibody was then used for a pilot study of VP1 VLPs MCPyV movement in mammalian cells. Results showed that the studied virus, at least particularly, utilizes caveolin-1-carrying vesicles for its movement in cells (colocalisation of VP1 VLPs and caveolin-1 was observedColocalisation with EEA1 marker of early endosomes, LamP2 marker of endolysosomal compartments or with BiP marker of endoplasmic reticulum was sporadic but significant. These preliminary results suggest that MCPyV might utilise an endocytic pathway leading...
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Preparation of affinity resin for the identification and preparation of glutamate carboxypeptidase II in biological material
Parolek, Jan ; Konvalinka, Jan (advisor) ; Bořek Dohalská, Lucie (referee)
For treatment of benign and malignant tumors it is desirable to find more specific and less burdening ways of therapy. The main objective of improving the treatment of inoperable tumors is as low as possible damage to healthy tissues during tumor tissue elimination. Using antibodies in research and therapy brought significant progress; antibodies are able not to only mark cells expressing certain molecules, but even to eliminate them. However, tumor cells are very similar to healthy cells and this similarity is one of the major problems in treatment of cancer; most of the substances toxic to tumor have also some adverse effect on the whole organism. For this reason, it is necessary to search new tumor-specific markers for treatment of tumor-based diseases. Monoclonal antibodies can be linked with a drug molecule (cytotoxic substance, radionuclide, etc.) and getting antibody- drug conjugate. These conjugates are very promising medicaments for carcinoma treatment because monoclonal antibody can find specific target and drug substance can be delivered locally with minimal harm to patient's organism. Glutamate carboxypeptidase II (GCPII) became one of the specific markers for the prostate cancer. GCPII is an integral membrane protein, which is highly expressed by epithelial cells of the prostate...
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Biochemical methods as tool for study of reproductive proteins
Postlerová, Pavla ; Zigo, Michal ; Pohlová, Alžběta ; Jonáková, Věra
Study of molecular mechanisms in reproduction is essential for the understanding of this outstanding process. Our lab studies proteins secreted by reproductive organs and sperm using various biochemical methods for a long time. We have expertise in protein extraction from spermatic cells using different approaches, and by kits for proteins from the sperm surface and distinct subcellular compartments. The proteins of reproductive organ fluids are separated by chromatographic methods, such as size exclusion chromatography, high-performance liquid chromatography with reverse phase (RP-HPLC) and affinity chromatography on matrices with various ligands. Proteins are subjected to SDS- or 2D-electrophoresis for their characterization and comparison of various extraction methods, different mammalian species, and sperm in different functional development. Electrophoretically separated proteins may be transferred onto nitrocellulose membrane (Western blot) for antibody detection or binding studies with lectin-labelled ligands (lectins, polysaccharides, zona pellucida glycoproteins). We use immunoprecipitation method with specific antibody for protein determination followed by the MALDI identification. Proteins are localized by immunofluorescent techniques on/in spermatic cells and tissue sections of reproductive organs. Isolation of proteins from reproductive tissues and fluids, and the antibody detection is crucial for the studying of reproductive protein origin.
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