Publication: Diseño de variantes optimizadas de inmunotoxinas basadas en ribonucleasas fúngicas: efecto antitumoral in vitro e in vivo
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2021-06-10
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Universidad Complutense de Madrid
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Abstract
Las inmunotoxinas son biomoléculas quiméricas con aplicaciones terapéuticas que combinan la especifidad de un anticuerpo para reconocer y unirse a antígenos con la actividad citotóxica de una toxina. Su estructura consta de un dominio marcador, generalmente un anticuerpo o fragmento del mismo, que dirige la acción de un dominio tóxico o efector, que está compuesto por una toxina cuya actividad enzimática, una vez en el interior celular, provoca la muerte de la célula diana. Así, su mecanismo de acción implica el reconocimiento por parte del dominio marcador del antígeno tumoral presente en la superficie de la célula diana, la internación del complejo, y la posterior liberación del dominio tóxico en el interior celular que produce la muerte celular por su actividad tóxica. Por tanto, su elevada especifidad y potencia citotóxica, y la ausencia de mecanismos de resistencia específicos han convertido a las inmunotoxinas en potenciales agentes biológicos terapéuticos en el tratamiento del cáncer u otras enfermedades.
Desde la aparición de las primeras inmunotoxinas, las nuevas construcciones han evolucionado en cuanto a su diseño, producción, estabilidad y eficacia, minimizando las limitaciones y efectos secundarios observados en los primeros ensayos, como su elevada inmunogenicidad, gran tamaño molecular, corta vida media o la citotoxicidad inespecífica en células sanas. Gracias a ello, en los últimos años ha resurgido el interés en el estudio y desarrollo de nuevas inmunotoxinas para su aplicación clínica. Actualmente, tres de ellas han sido aprobadas por la FDA para el tratamiento de distintos cánceres hematológicos. Sin embargo, aún no ha sido aprobado ningún tratamiento clínico para tumores sólidos basado en inmunotoxinas. Con esta perspectiva, las investigaciones actuales se centran en la optimización de los diseños para mejorar sus propiedades farmacocinéticas, aumento en la capacidad de penetración en tumores sólidos y búsqueda de nuevas dianas terapéuticas, no solo para el tratamiento del cáncer, sino también para afecciones alérgicas, inflamatorias o parasitarias.
En la presente Tesis doctoral, se expone el trabajo realizado en el desarrollo y caracterización estructural y funcional de nuevos diseños optimizados de inmunotoxinas recombinantes basadas en la fusión de los dominios variables en cadena única del anticuerpo monoclonal A33 (scFvA33), que reconoce el antígeno GPA33, sobreexpresado en cáncer colorrectal, con dos RNasas fúngicas: la ribotoxina α-sarcina y la RNasa T1. Los objetivos planteados en esta Tesis se han dividido en dos bloques independientes: A) Profundización de los estudios sobre el efecto antitumoral in vivo de la IMTXA33αS, incluyendo la caracterización a nivel molecular de los tumores tratados, así como el efecto del tratamiento combinado de dos inmunotoxinas con distinta diana antigénica en la misma célula diana. B) Producción y caracterización de nuevos diseños optimizados de inmunotoxinas dirigidos a un incremento de su eficacia citotóxica. Para ello, se han seguido dos estrategias diferentes: B.1) Incorporación de un sitio de reconocimiento de la proteasa intracelular furina, para favorecer la liberación del dominio tóxico al citosol, obteniéndose las variantes IMTXA33furαS y scFvA33furT1. B.2) Obtención de una inmunotoxina con formato trimérico, IMTXTriA33αS, que debería mostrar un incremento en su avidez y carga tóxica...
Immunotoxins are chimeric molecules with high potential as therapeutic candidates, which combine antibody specificity to recognize and bind tumor-associated antigens, with the potency of the enzymatic activity of a toxin, in order to induce the death of target cells. They are composed of two domains: the target domain, usually a whole antibody, or a fragment, which specifically targets a tumor marker, and the toxic domain, responsible for their cytotoxicity. Their mechanism of action involves high affinity binding to the tumor antigen by the targeting domain, internalization of the complex by endocytosis, and subsequent release of the toxic domain that will cause target cell death. Due to these facts and the absence of resistance mechanisms have uncovered the immunotoxins as potential therapeutic agents for treatment of cancer and other diseases. The evolution of immunotoxin designs has focused on minimize their undesirable side effects, as high immunogenicity, large size, short half-life and unspecific cytotoxicity. Furthermore, multiple evidences have accumulated to hold up on pointing how the efficiency of immunotoxins depends on the following different aspects: the specificity and functional affinity of the antibody for the cell surface expressed tumor antigen, the complex internalization effectiveness, and the speed of the toxin release pathway and its own intrinsic potency and specificity for cell promotion. Thus, antibody engineering has driven to the development of different new formats that are being incorporated in immunotoxins constructs, leading to three new FDA-approved immunotoxins indicated for haematological cancers. However, no immunotoxins-based treatment against solid tumor have been approved. Within this perspective, current research is focused on the optimization of designs to improve their pharmacokinetic properties, increase the penetrability in solid tumors and search for new therapeutic targets, not only for cancer treatment, but also for allergic, inflammatory or parasitic affections. In the present doctoral thesis, it is presented the work carried out in the design, purification and both structural and functional characterization of new optimized designs of recombinant immunotoxins based on the fusion of single chain variable domains of the monoclonal antibody A33 (scFvA33), which recognizes the antigen GPA33, overexpressed in colorectal cancer, with two fungal RNases: the ribotoxin α-sarcin and RNase T1. The objectives proposed in this work have been divided into two independent blocks: A) In-depth studies on the anti-tumor effect of IMTXA33αS in vivo, including the molecular characterization of the treated tumors, as well as the effect of the combined treatment of two immunotoxins aimed to two different antigenic targeting the same tumoral cell. B) Production and characterization of new optimized designs of immunotoxins aimed at increasing their cytotoxic efficiency. For this purpose, two different approaches have been followed: B.1) Inclusion of a recognition and cleavage site for furin protease, to enhance the release of the toxic domain to the cytosol, resulting in the variants IMTXA33furαS and scFvA33furT1. B.2) Development of a trimeric immunotoxin based on α-sarcin, IMTXTriA33αS, which should exhibit an increase in its avidity and toxic load...
Immunotoxins are chimeric molecules with high potential as therapeutic candidates, which combine antibody specificity to recognize and bind tumor-associated antigens, with the potency of the enzymatic activity of a toxin, in order to induce the death of target cells. They are composed of two domains: the target domain, usually a whole antibody, or a fragment, which specifically targets a tumor marker, and the toxic domain, responsible for their cytotoxicity. Their mechanism of action involves high affinity binding to the tumor antigen by the targeting domain, internalization of the complex by endocytosis, and subsequent release of the toxic domain that will cause target cell death. Due to these facts and the absence of resistance mechanisms have uncovered the immunotoxins as potential therapeutic agents for treatment of cancer and other diseases. The evolution of immunotoxin designs has focused on minimize their undesirable side effects, as high immunogenicity, large size, short half-life and unspecific cytotoxicity. Furthermore, multiple evidences have accumulated to hold up on pointing how the efficiency of immunotoxins depends on the following different aspects: the specificity and functional affinity of the antibody for the cell surface expressed tumor antigen, the complex internalization effectiveness, and the speed of the toxin release pathway and its own intrinsic potency and specificity for cell promotion. Thus, antibody engineering has driven to the development of different new formats that are being incorporated in immunotoxins constructs, leading to three new FDA-approved immunotoxins indicated for haematological cancers. However, no immunotoxins-based treatment against solid tumor have been approved. Within this perspective, current research is focused on the optimization of designs to improve their pharmacokinetic properties, increase the penetrability in solid tumors and search for new therapeutic targets, not only for cancer treatment, but also for allergic, inflammatory or parasitic affections. In the present doctoral thesis, it is presented the work carried out in the design, purification and both structural and functional characterization of new optimized designs of recombinant immunotoxins based on the fusion of single chain variable domains of the monoclonal antibody A33 (scFvA33), which recognizes the antigen GPA33, overexpressed in colorectal cancer, with two fungal RNases: the ribotoxin α-sarcin and RNase T1. The objectives proposed in this work have been divided into two independent blocks: A) In-depth studies on the anti-tumor effect of IMTXA33αS in vivo, including the molecular characterization of the treated tumors, as well as the effect of the combined treatment of two immunotoxins aimed to two different antigenic targeting the same tumoral cell. B) Production and characterization of new optimized designs of immunotoxins aimed at increasing their cytotoxic efficiency. For this purpose, two different approaches have been followed: B.1) Inclusion of a recognition and cleavage site for furin protease, to enhance the release of the toxic domain to the cytosol, resulting in the variants IMTXA33furαS and scFvA33furT1. B.2) Development of a trimeric immunotoxin based on α-sarcin, IMTXTriA33αS, which should exhibit an increase in its avidity and toxic load...
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, leída el 27/01/2021