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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 kultivace zárodečných kmenových buněk jesetera
XIE, Xuan
The study in this thesis concerns a research area related to sturgeon germ cell biology including identification, purification and in vitro culture. The research objectives are: expounding fish spermatogonial stem cell characteristic and regulation of spermatogenesis; evaluation of the existent methods in fish germ cell purification and in vitro culture; investigation of in vitro amplification of sturgeon germ cells; development of an approach to purify germ cells in sturgeon; identification of type A spermatogonia (ASG) in sturgeon. Novelty and scientific originality. This work first time developed sturgeon germ cell growth in vitro and combined cell culture with transplantation. The application of monoclonal antibodies on recognition of sturgeon ASG represents a new, and currently only tool of germ cell identification. Important scientific problem solved in the field of research. This work of germ cell culture observed the effect of somatic cells and growth factors on the proliferation of sturgeon germ cells through evaluation of germ cell mitotic activity during culture, which contributed to the study on the regulation mechanism of germ cell self-renewal and differentiation. This thesis also developed a method using flow cytometry to purify sturgeon ASGs. A monoclonal antibody was also found that enable to identify ASGs of sturgeon specifically. Theoretical significance and applied value of the thesis. The thesis summarized the accumulated knowledge of spermatogonial stem cell and regulation of its self-renewal vs. differentiation in fish, compared available protocols and advances in enriching spermatogonia and reviewed development of in vitro germ cell culture conditions. The work of efficient enrichment of type A spermatogonia was achieved based on its physicochemical properties, which is applicable for commercial and endangered fishes without cell-labeling systems such as transgenes and cell antibodies. Monoclonal antibodies generated from Pacific blue fin tuna can also recognize antigens from sterlet ASGs, indicating some epitopes for ASGs are conserved between species. The research results present interest for characterization of ASGs in proteomics level. Implementation of scientific results. A serum-free culture condition for sturgeon germ cells was established. The germ cells under this in vitro condition can remain growth, survival and maintain their transplantability for more than 40 days. Therefore, this method is expected to provide a long term supply for germ cell transplantation and surrogate production. Moreover, based on the analysis of germ cell light scatter properties, a purified ASG population was isolated. No dye and transgenes are introduced during sorting, which is helpful for characterization of germ cell populations and downstream germ cell manipulation. The application of monoclonal antibody on sturgeon ASG can achieve the accuracy of the ASGs identification on in vitro culture.
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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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