Caracterização do anticorpo monocional anti-anti-idiotípico 5.G8 ligante de GD3
Nenhuma Miniatura disponível
Data
2015-10-28
Autores
Silva, Tabata de Almeida da [UNIFESP]
Orientadores
Moraes, Jane Zveiter de Moraes [UNIFESP]
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O melanoma é uma doença potencialmente fatal, geralmente derivada da pele, para a qual as terapias convencionais são pouco eficientes. Estudos em nível molecular mostraram que, durante o processo de transformação maligna do melanócito, ocorrem alterações no padrão de expressão de gangliosídeos. O gangliosídeo GD3 está entre os que passam a ser hiperexpressos e tem sido considerado um marcador importante do melanoma humano e um potencial alvo para imunoterapias. Um anticorpo monoclonal (mAb) anti-GD3 é disponível comercialmente, o mAb R24. Apesar de promover regressão de melanoma metastático em pacientes, é um anticorpo tóxico e, por isso, tem aplicação terapêutica limitada. Na tentativa de obter uma alternativa para esse anticorpo, foram produzidos mAbs anti-idiotipo (Id), e anti-anti-Id de R24. O mAb anti-anti-Id 5.G8, in vitro, reconhece linhagens de células de melanoma, promove citotoxidade dependente de anticorpo, fixação de complemento e se liga especificamente a frações enriquecidas de lipídios, extraídas de células de melanoma humano. In vivo, mostrou ser capaz de inibir o crescimento tumoral. O presente trabalho buscou analisar a ligação do mAb 5.G8 à linhagem celular de melanoma humano SKMel-28 e comparar as sequências das porções variáveis das cadeias leves e pesadas, FVL FVH, respectivamente, dos mAbs 5.G8 e R24. Em ensaios de citometria de fluxo, foi possível observar que o mAb 5.G8, além de se ligar a GD3, reconhece molécula de natureza proteica presente em células SKMel-28 e em linhagem de melanócitos murinos 83 transformados para expressar GD3, ST8D4. Por western blotting, utilizando extrato de células SKMel-28, foi verificado que o mAb 5.G8 se liga à molécula de peso molecular entre 12 e 17 kDa. A análise das sequências nucleotídicas e peptídicas das regiões variáveis dos mAbs R24 e 5.G8 mostrou 100% de homologia entre FvLs e, nas FvHs, foram identificados quatro nucleotídeos diferentes, resultando em dois aminoácidos diferentes. Um deles, o aminoácido polar neutro, glutamina, encontrado no mAb R24, foi substituído por um aminoácido polar básico, lisina, no mAb 5.G8 e está localizado na framework1 (FR1), posição 2, que faz parte da zona de Vernier. Este achado pode explicar as diferentes propriedades dos dois anticorpos.
Melanoma is a potentially fatal disease, usually derived from the skin, for which conventional therapies are not efficient. Studies showed that changes occur in the expression pattern of gangliosides during the process of malignant transformation of melanocytes. The hyper expressed ganglioside GD3 has been considered an important marker of human melanoma and a potential target for immunotherapy. An anti-GD3 monoclonal antibody (mAb) is commercially available, the mAb R24. Although it has been demonstrated that mAb R24 causes regression of metastatic melanoma in patients, it is a toxic antibody and its therapeutic application is limited. In an attempt to obtain an alternative to R24, we produced anti-R24 idiotype (Id) and anti-anti-R24 Id mAbs. In vitro, the anti-anti-Id mAb 5.G8 recognizes melanoma cell lines, promotes antibodydependent cytotoxicity, complement fixation and specifically binds to lipidenriched fractions extracted from human melanoma cells. In vivo experiments, it was shown to inhibit tumor growth. The present study analyzed the binding of mAb 5.G8 to the human melanoma cell line SKMel-28 and compared the sequences of the variable portions of the light and heavy chains, FvL and FvH, respectively, of mAbs 5.G8 and R24. By flow cytometry assays, it was observed that, besides to bind to GD3, mAb 5.G8 recognizes a protein molecule on SKMel-28 cells and on murine melanocytes transformed to express GD3 sintase, ST8D4. By western blotting, using SKMel-28 cell extract, mAb 5.G8 bound to a molecule of molecular weight between 12-17-kDa. 85 The analysis of the nucleotide and peptide sequences of variable regions of R24 and 5.G8 mAbs showed 100% homology between FvLs and four different nucleotides were identified in the FvHs, resulting in two different amino acids. One of them, the neutral polar amino acid glutamine, found in mAb R24, was replaced by the basic polar amino acid lysine in 5.G8 mAb and is located in the framework1 (FR1) at position 2, part of the Vernier zone. This finding may explain the different proprieties of the two antibodies.
Melanoma is a potentially fatal disease, usually derived from the skin, for which conventional therapies are not efficient. Studies showed that changes occur in the expression pattern of gangliosides during the process of malignant transformation of melanocytes. The hyper expressed ganglioside GD3 has been considered an important marker of human melanoma and a potential target for immunotherapy. An anti-GD3 monoclonal antibody (mAb) is commercially available, the mAb R24. Although it has been demonstrated that mAb R24 causes regression of metastatic melanoma in patients, it is a toxic antibody and its therapeutic application is limited. In an attempt to obtain an alternative to R24, we produced anti-R24 idiotype (Id) and anti-anti-R24 Id mAbs. In vitro, the anti-anti-Id mAb 5.G8 recognizes melanoma cell lines, promotes antibodydependent cytotoxicity, complement fixation and specifically binds to lipidenriched fractions extracted from human melanoma cells. In vivo experiments, it was shown to inhibit tumor growth. The present study analyzed the binding of mAb 5.G8 to the human melanoma cell line SKMel-28 and compared the sequences of the variable portions of the light and heavy chains, FvL and FvH, respectively, of mAbs 5.G8 and R24. By flow cytometry assays, it was observed that, besides to bind to GD3, mAb 5.G8 recognizes a protein molecule on SKMel-28 cells and on murine melanocytes transformed to express GD3 sintase, ST8D4. By western blotting, using SKMel-28 cell extract, mAb 5.G8 bound to a molecule of molecular weight between 12-17-kDa. 85 The analysis of the nucleotide and peptide sequences of variable regions of R24 and 5.G8 mAbs showed 100% homology between FvLs and four different nucleotides were identified in the FvHs, resulting in two different amino acids. One of them, the neutral polar amino acid glutamine, found in mAb R24, was replaced by the basic polar amino acid lysine in 5.G8 mAb and is located in the framework1 (FR1) at position 2, part of the Vernier zone. This finding may explain the different proprieties of the two antibodies.
Descrição
Citação
SILVA, Tabata de Almeida da. Caracterização do anticorpo monoclonal anti-antiidiotípico 5.G8 ligante de gd3. 2015. 93 f. Dissertação (Mestrado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.