Desarrollo de un anticuerpo de afinidad mejorada contra el antígeno HspX con potencial diagnóstico para tuberculosis.
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Date
2025
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Universidad de Concepción
Abstract
La tuberculosis es la segunda causa de muerte debido a enfermedades infecciosas a nivel mundial. Según datos de la Organización Mundial de la Salud (OMS), en promedio cada año se reportan 10 millones de nuevos casos y 1.3 millones de decesos. La creciente carga sanitaria mundial de la tuberculosis se ve agravada por el alarmante incremento del número de personas infectadas con VIH y de cepas multirresistentes a antibióticos. El mejor pronóstico de la tuberculosis se obtiene con el diagnóstico temprano de la infección, no obstante, los métodos diagnósticos habitualmente empleados (microscópicos, microbiológicos y moleculares) presentan importantes limitaciones: sensibilidad y especificidad variables, diagnóstico subóptimo y, un gran consumo de tiempo y recursos.
En el presente trabajo se mejoró la afinidad de un anticuerpo dirigido contra el antígeno HspX derivado de MTB mediante mutagénesis parsimoniosa. Para ello, se seleccionaron posiciones candidatas a mutagénesis de las regiones CDR basándose en el modelado estructural del anticuerpo. Se introdujeron mutaciones en dieciocho posiciones dentro de cada CDR objetivo utilizando la estrategia de mutagénesis parsimoniosa. Posteriormente, se generaron bibliotecas de variantes scFv mediante el método de Kunkel. Tras varias rondas de selección, se analizaron las potenciales variantes con afinidad mejorada, evaluando sus secuencias de CDR y sustituciones de aminoácidos. Este enfoque permitió identificar ocho variantes que aumentaron la afinidad del anticuerpo. La variante más prometedora exhibió un valor IC50 de 38 nM y tenía dos sustituciones: Lys50 y Glu50, dentro del CDR-H2, que reflejaron un aumento de 28,14 veces en la afinidad de unión con respecto al anticuerpo original.
Estos hallazgos brindan información valiosa sobre la ingeniería de anticuerpos y podrían contribuir al desarrollo de ensayos diagnósticos más sensibles y confiables para la tuberculosis.
Tuberculosis is the second leading cause of death due to infectious diseases worldwide. According to the World Health Organization (WHO), an average of 10 million new cases and 1.3 million deaths are reported each year. The global health burden of TB is further exacerbated by the rising number of HIV-infected individuals and the increasing prevalence of multidrug-resistant (M. tuberculosis) strains. Early diagnosis is crucial for improving TB prognosis; however, conventional diagnostic methods—including microscopic, microbiological, and molecular techniques—have significant limitations, such as variable sensitivity and specificity, suboptimal diagnostic accuracy, and high time and resource demands. In the present work, the affinity of an antibody targeting Mycobacterium tuberculosis-derived HspX antigen was improved by parsimonious mutagenesis. Candidate positions for mutagenesis within the complementarity-determining regions (CDRs) were identified based on structural modeling of the antibody. Mutations were introduced at eighteen positions within each target CDR using the parsimonious mutagenesis approach, followed by the generation of scFv variant libraries via the Kunkel method. After multiple rounds of selection, potential variants with improved affinity were analyzed by evaluating their CDR sequences and amino acid substitutions. This approach allowed the identification of eight variants with increased binding affinity. The most promising variant exhibited an IC50 value of 38 nM and contained two key substitutions: Lys50 and Glu50, within CDR-H2, resulting in a 28.14-fold increase in binding affinity compared to the original antibody. These findings provide valuable insights into antibody engineering and may contribute to the development of more sensitive and reliable diagnostic assays for tuberculosis.
Tuberculosis is the second leading cause of death due to infectious diseases worldwide. According to the World Health Organization (WHO), an average of 10 million new cases and 1.3 million deaths are reported each year. The global health burden of TB is further exacerbated by the rising number of HIV-infected individuals and the increasing prevalence of multidrug-resistant (M. tuberculosis) strains. Early diagnosis is crucial for improving TB prognosis; however, conventional diagnostic methods—including microscopic, microbiological, and molecular techniques—have significant limitations, such as variable sensitivity and specificity, suboptimal diagnostic accuracy, and high time and resource demands. In the present work, the affinity of an antibody targeting Mycobacterium tuberculosis-derived HspX antigen was improved by parsimonious mutagenesis. Candidate positions for mutagenesis within the complementarity-determining regions (CDRs) were identified based on structural modeling of the antibody. Mutations were introduced at eighteen positions within each target CDR using the parsimonious mutagenesis approach, followed by the generation of scFv variant libraries via the Kunkel method. After multiple rounds of selection, potential variants with improved affinity were analyzed by evaluating their CDR sequences and amino acid substitutions. This approach allowed the identification of eight variants with increased binding affinity. The most promising variant exhibited an IC50 value of 38 nM and contained two key substitutions: Lys50 and Glu50, within CDR-H2, resulting in a 28.14-fold increase in binding affinity compared to the original antibody. These findings provide valuable insights into antibody engineering and may contribute to the development of more sensitive and reliable diagnostic assays for tuberculosis.
Description
Tesis presentada para optar al grado de Doctor en Biotecnología Molecular
Keywords
Tuberculosis, Mycobacterium tuberculosis, Anticuerpos bacterianos