Funcionalizando fragmentos de anticorpos através da bio-incorporação de aminoácidos não canônicos
Data
2023-09-12
Autores
Tognato, Rafael Cerioni 
Orientadores
Chagas, Jair Ribeiro
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O câncer de próstata é o segundo câncer mais recorrente em escala global e a quinta maior causa de morte derivada de câncer em indivíduos do sexo masculino. Duas enzimas têm ação essencial no câncer de próstata, tendo seus níveis elevados em indivíduos adoecidos: o antígeno prostático específico (PSA) e o antígeno prostático específico de membrana (PSMA). Fragmentos de anticorpos do tipo scFv são formados pela fusão dos domínios variáveis leve e pesado de imunoglobulinas convencionais, enquanto nanocorpos são constituídos pelo domínio variável pesado. A tecnologia de supressão do códon âmbar associada ao click chemistry permite a funcionalização desses fragmentos pela associação com diversas moléculas, incluindo fluoróforos e drogas através da incorporação de um aminoácido não-canônico nas proteínas. Objetivos: funcionalização de um scFv anti-PSA (LUP13A4) e um nanocorpo anti-PSMA (NB7) pela supressão do códon âmbar e click chemistry para associação de ambos com o fluoróforo Cy5 e do NB7 com o agente antineoplásico MMAE. Metodologia: os genes LUP13A4 e NB7 clonados no vetor modificado pGEX4T foram transformados em linhagens de E. coli DH5a e BL21(DE3) (cotransformadas com o vetor pEVOL). Os fragmentos foram expressos na ausência e presença do aminoácido não-canônico 4-azido-L-fenilalanina nas respectivas linhagens. A purificação ocorreu por cromatografia de afinidade via Strep-Tag II. A conjugação com o fluoróforo Cy5 ocorreu por reação de click chemistry com excesso molar de Cy5-DBCO. O LUP13A4-Cy5 foi testado via ELISA e por fluorescência, enquanto o NB7 foi testado em linhagens PC3 e LNCap. A conjugação com MMAE ocorreu de maneira análoga, e os conjugados foram aplicados em células PC3 e LNCap seguido de análise de viabilidade celular. Resultados e conclusão: as reações de click chemistry foram efetivas na formação dos fragmentos fluorescentes. O LUP13A4 permitiu a detecção de PSA purificado (500 a 100 ng) por imunoabsorção enzimática e fluorescência. O NB7-Cy5 marcou células PSMA positivas (LNCap) quando aplicados a 100, 20 e 5 μg, enquanto os conjugados NB7-MMAE diminuíram a viabilidade de celular de células LNCap nas concentrações de 0,05, 0,2 e 1 μM, com maior efetividade das construções contendo Val-Cit-PABA.
Prostate cancer is the second most recurrent cancer on a global scale and the fifth leading cause of cancer-related death in males. Two enzymes have an essential action in prostate cancer, with their levels being elevated in sick individuals: prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA). scFv fragments are formed by the fusion of the light and heavy variable domains of conventional immunoglobulins, while nanobodies are made up of the heavy variable domain. The amber codon suppression technology associated with click chemistry allows the functionalization of these fragments by association with various molecules, including fluorophores and drugs through the incorporation of a non-canonical amino acid into proteins. Objectives: functionalization of an anti-PSA scFv (LUP13A4) and an anti-PSMA nanobody (NB7) by suppression of the amber codon and click chemistry for association of both with the fluorophore Cy5 and NB7 with the antineoplastic agent MMAE. Methodology: the LUP13A4 and NB7 genes cloned into the modified pGEX4T vector were transformed into E. coli strains DH5a and BL21(DE3) (cotransformed with the pEVOL vector). The fragments were expressed in the absence and presence of the non-canonical amino acid 4-azido-L-phenylalanine in the respective lineages. Purification occurred by affinity chromatography via Strep-Tag II. Conjugation with the Cy5 fluorophore occurred via click chemistry reaction with a molar excess of Cy5-DBCO. LUP13A4-Cy5 was tested via ELISA and fluorescence, while NB7 was tested in PC3 and LNCap lines. Conjugation with MMAE occurred in an analogous manner, and the conjugates were applied to PC3 and LNCap cells followed by cell viability analysis. Results and conclusion: click chemistry reactions were effective in the formation of fluorescent fragments. LUP13A4 allowed the detection of purified PSA (500 to 100 ng) by enzyme-linked immunosorbent assay and fluorescence. NB7-Cy5 marked PSMA-positive cells (LNCap) when applied at 100, 20 and 5 μg, while NB7-MMAE conjugates decreased cell viability of LNCap cells at concentrations of 0.05, 0.2 and 1 μM, with greater effectiveness of constructs containing Val-Cit-PABA.
Prostate cancer is the second most recurrent cancer on a global scale and the fifth leading cause of cancer-related death in males. Two enzymes have an essential action in prostate cancer, with their levels being elevated in sick individuals: prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA). scFv fragments are formed by the fusion of the light and heavy variable domains of conventional immunoglobulins, while nanobodies are made up of the heavy variable domain. The amber codon suppression technology associated with click chemistry allows the functionalization of these fragments by association with various molecules, including fluorophores and drugs through the incorporation of a non-canonical amino acid into proteins. Objectives: functionalization of an anti-PSA scFv (LUP13A4) and an anti-PSMA nanobody (NB7) by suppression of the amber codon and click chemistry for association of both with the fluorophore Cy5 and NB7 with the antineoplastic agent MMAE. Methodology: the LUP13A4 and NB7 genes cloned into the modified pGEX4T vector were transformed into E. coli strains DH5a and BL21(DE3) (cotransformed with the pEVOL vector). The fragments were expressed in the absence and presence of the non-canonical amino acid 4-azido-L-phenylalanine in the respective lineages. Purification occurred by affinity chromatography via Strep-Tag II. Conjugation with the Cy5 fluorophore occurred via click chemistry reaction with a molar excess of Cy5-DBCO. LUP13A4-Cy5 was tested via ELISA and fluorescence, while NB7 was tested in PC3 and LNCap lines. Conjugation with MMAE occurred in an analogous manner, and the conjugates were applied to PC3 and LNCap cells followed by cell viability analysis. Results and conclusion: click chemistry reactions were effective in the formation of fluorescent fragments. LUP13A4 allowed the detection of purified PSA (500 to 100 ng) by enzyme-linked immunosorbent assay and fluorescence. NB7-Cy5 marked PSMA-positive cells (LNCap) when applied at 100, 20 and 5 μg, while NB7-MMAE conjugates decreased cell viability of LNCap cells at concentrations of 0.05, 0.2 and 1 μM, with greater effectiveness of constructs containing Val-Cit-PABA.