Construção, por desenho racional, e caracterização de variantes de afinidade da região variável do anticorpo monoclonal Rituximabe

• Ano de defesa: 2019
• Autor(a) principal: Pontes, Larissa Queiroz
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Palavras-chave em Português: Mutagênese sítio-dirigida; Engenharia de anticorpos; Rituximabe
• Link de acesso: http://www.repositorio.ufc.br/handle/riufc/40330

Cancer, also known as neoplasia, is a pathology characterized by uncontrolled proliferation of cells that can occur in different tissues. It is of major concern that drug development be capable of mitigate, reduce symptoms or prevent metastasis. Thereby the aforementioned, immunotherapy happens to be one of the most successful strategies on cancer treatment. Rituximab is a monoclonal antibody used in the treatment of lymphomas. Its mechanism of action is based on the interaction between it and the CD20 membrane protein expressed on B lymphocytes, guiding them to depletion. However, many patients are resistant to this treatment. In this study, we propose mutations on the variable region of Rituximab in order to increase its affinity to the CD20 epitope. Structural bioinformatics researchers of Fiocruz-CE in collaboration with our group suggested mutations based on structural and energetic features. These mutants were obtained in vitro by site-directed mutagenesis, specifically on its scFv format, and their sequences confirmed on Fiocruz-PE platform. The fragments were characterized by circular dichroism, had their capacity of binding confirmed by ELISA, and had their performance of protein denaturation and stability of antibody fragments evaluated by intrinsic tryptophan fluorescence. As results, our expression strategy led us to get all soluble fragments; it was possible to trace a secondary structure profile of β-sheet to the wt and to the mutants scFv; by ELISA, it was feasible to confirm the binding capacity of all fragments to peptide, which contains the epitope, where the quadruple mutant MUT3 was highlighted; and all the fragments presented a similar thermal denaturing profile by intrinsic tryptophan fluorescence. This work avenues future constructs on the scFv-Fc format, where it will be possible to chain the variants with highest affinities to an Fc domain. The resulting molecule will be more stable than the antibody fragment, will be capable of elicit immune effector functions, as well as will comprise higher affinity to the CD20 antigen. The perspective is the better is the affinity; the greater will be cytotoxic activity.