Construção de scFv anti-FGF2 a partir do hibridoma secretor do MAb 3F12E7
Data
2015-07-31
Autores
Santos, Deny Anderson dos 
Orientadores
Moraes, Jane Zveiter de
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Já foi demonstrado o potencial da terapia antiangiogênica, tanto que sua utilização foi aprovada para o tratamento de alguns tumores, quando associada a terapias convencionais citotóxicas. Dentre as drogas antiangiogênicas, encontram-se os anticorpos monoclonais contra fatores angiogênicos. O anticorpo monoclonal (MAb) anti-hFGF2 3F12E7, produzido por nosso grupo, por meio de imunização com o gene FGF2, mostrou atividade inibidora de crescimento de tumor, bem como de metástases, em estudos pré-clínicos. Além disso, o MAb 3F12E7 parece ter utilidade em métodos de diagnóstico. Com o objetivo de melhorar a capacidade deste anticorpo, propusemos a construção de um fragmento variável de cadeia única (scFv) de 3F12E7. Os anticorpos na forma de scFv, uma vez que são moléculas de proteína pequenas, podem infiltrar-se mais facilmente em massas tumorais sólidas e podem ter seu potencial terapêutico e diagnóstico aumentado. Além desta vantagem sobre o uso do anticorpo completo, a scFv, quando injetada em seres humanos, não provoca resposta imune contra marcadores iso e alotípicos presentes na porção constante das cadeias de imunoglobulina. Assim, RNA total foi isolado de células de hibridoma 3F12E7. O cDNA dos fragmentos variáveis da cadeia pesada (FvH) e leve (FvL) foram obtidos com a utilização de oligonucleotídeos específicos. Os fragmentos de DNA que codificam para os domínios FvH e FvL de 3F12E7 foram então amplificados por PCR. Fragmentos de DNA de 325 pb e 340 pb, que correspondem a FvL e FvH, respectivamente, foram purificados a partir do gel de agarose após eletroforese e ligados ao vetor pGEM-T Easy®. Bactérias competentes foram transformadas 85 e clonadas e as colônias resistentes à ampicilina foram selecionadas, expandidas e submetidas a minipreparações para isolamento do plasmídeo. Os plasmídeos foram selecionados, usando enzimas de restrição, sequenciados e analisados quanto à homologia com outras imunoglobulinas. Após confirmação de homologia, foi possível desenhar um fragmento variável de cadeia única, que contém um ?linker? que codifica para uma sequência de 15 aminoácidos (Gly4-Ser)3, e que une os fragmentos FvL e FvH. Além disso, foi incluído um peptídeo sinal, para orientar a expressão da scFv em superfície celular. O vetor pcDNA3.1, contendo a construção, foi usado para transfectar células de mamífero. Assim, por ELISA e imunofluorescência, foi possível avaliar a capacidade da scFv 3F12E7 em reconhecer FGF-2. A scFv 3F12E7, obtida a partir do hibridoma 3F12E7, reconhece FGF-2.
TRACT Anti-angiogenic therapy has already shown its potential and has been approved for the treatment of some tumors when associated with conventional cytotoxic therapies. Among the anti-angiogenic drugs, there are monoclonal antibodies against angiogenic factors. The anti-hFGF-2 MAb 3F12E7 produced by our group using gene immunization has shown tumor as well as metastasis growth inhibiting activities in pre-clinical studies. In addition, the MAb 3F12E7 has appeared to have utility in diagnostic approaches. Aiming to better assess the capabilities of this antibody, we proposed the construction of a single chain variable fragment (scFv) of 3F12E7. Antibodies in the form of scFv, since they are small protein molecules, can easily infiltrate into solid tumor masses and may have their therapeutic and diagnosis potential increased. Besides this advantage over the use of the complete antibody, scFv, when injected into humans, does not provoke immune response against the iso and allotypic markers present in the constant portion of immunoglobulin chains. In this way, the total RNA was isolated from 3F12E7 hybridoma cells. The cDNA of heavy (FvH) and light (FvL) variable fragments were synthesized with specific oligonucleotides. The DNA fragments encoding for the FvH and FvL domains of 3F12E7 were then amplified by PCR. DNA fragments of 325 bp and 340 bp, which correspond to FVL and FVH, respectively, were purified from the agarose gel upon electrophoresis and bound to the pGEM-T Easy® vector. Competent bacteria were transformed, cloned and the ampicillin-resistant colonies were selected, expanded and subjected to miniprep for plasmid isolation. Plasmids were screened using restriction enzymes, sequenced and 87 analyzed for homology with other immunoglobulins. After homology confirmation, it was possible to draw a single chain variable fragment, containing a ?linker? coding for 15-aminoacid residue peptide (Gly4-Ser)3, that joined the FvL and FvH fragments, and a signal peptide, for cell surface expression. The pcDNA3.1 vector, containing the construction, was used to transfect mammalian cells. By ELISA and immunofluorescence, it was possible to evaluate the ability of scFv 3F12E7 to recognize FGF-2. The scFv 3F12E7, obtained from the hybridoma 3F12E7 recognizes FGF-2.
TRACT Anti-angiogenic therapy has already shown its potential and has been approved for the treatment of some tumors when associated with conventional cytotoxic therapies. Among the anti-angiogenic drugs, there are monoclonal antibodies against angiogenic factors. The anti-hFGF-2 MAb 3F12E7 produced by our group using gene immunization has shown tumor as well as metastasis growth inhibiting activities in pre-clinical studies. In addition, the MAb 3F12E7 has appeared to have utility in diagnostic approaches. Aiming to better assess the capabilities of this antibody, we proposed the construction of a single chain variable fragment (scFv) of 3F12E7. Antibodies in the form of scFv, since they are small protein molecules, can easily infiltrate into solid tumor masses and may have their therapeutic and diagnosis potential increased. Besides this advantage over the use of the complete antibody, scFv, when injected into humans, does not provoke immune response against the iso and allotypic markers present in the constant portion of immunoglobulin chains. In this way, the total RNA was isolated from 3F12E7 hybridoma cells. The cDNA of heavy (FvH) and light (FvL) variable fragments were synthesized with specific oligonucleotides. The DNA fragments encoding for the FvH and FvL domains of 3F12E7 were then amplified by PCR. DNA fragments of 325 bp and 340 bp, which correspond to FVL and FVH, respectively, were purified from the agarose gel upon electrophoresis and bound to the pGEM-T Easy® vector. Competent bacteria were transformed, cloned and the ampicillin-resistant colonies were selected, expanded and subjected to miniprep for plasmid isolation. Plasmids were screened using restriction enzymes, sequenced and 87 analyzed for homology with other immunoglobulins. After homology confirmation, it was possible to draw a single chain variable fragment, containing a ?linker? coding for 15-aminoacid residue peptide (Gly4-Ser)3, that joined the FvL and FvH fragments, and a signal peptide, for cell surface expression. The pcDNA3.1 vector, containing the construction, was used to transfect mammalian cells. By ELISA and immunofluorescence, it was possible to evaluate the ability of scFv 3F12E7 to recognize FGF-2. The scFv 3F12E7, obtained from the hybridoma 3F12E7 recognizes FGF-2.
Descrição
Citação
SANTOS, Deny Anderson dos. Construção de scFv anti-FGF2 a partir do hibridoma secretor do MAb 3F12E7. 2015. 106 f. Dissertação (Mestrado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.