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Preparation of a polymeric therapeutic targeting CD20 positive B-lymphomas and NK cells
Hejl, Maxmilián ; Vaněk, Ondřej (advisor) ; Bělonožníková, Kateřina (referee)
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] (pHPMA) was chosen in this work. The transpeptidase reaction catalyzed by recombinant sortase A, which recognizes and...
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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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