Inclusion of a Furin Cleavage Site Enhances Antitumor Efficacy against Colorectal Cancer Cells of Ribotoxin α-Sarcin- or RNase T1-Based Immunotoxins

Immunotoxins are chimeric molecules that combine the specificity of an antibody to recognize and bind tumor antigens with the potency of the enzymatic activity of a toxin, thus, promoting the death of target cells. Among them, RNases-based immunotoxins have arisen as promising antitumor therapeutic agents. In this work, we describe the production and purification of two new immunoconjugates, based on RNase T1 and the fungal ribotoxin α-sarcin, with optimized properties for tumor treatment due to the inclusion of a furin cleavage site. Circular dichroism spectroscopy, ribonucleolytic activity studies, flow cytometry, fluorescence microscopy, and cell viability assays were carried out for structural and in vitro functional characterization. Our results confirm the enhanced antitumor efficiency showed by these furin-immunotoxin variants as a result of an improved release of their toxic domain to the cytosol, favoring the accessibility of both ribonucleases to their substrates. Overall, these results represent a step forward in the design of immunotoxins with optimized properties for potential therapeutic application in vivo

Fungal Ribotoxins

Fungal ribotoxins constitute a family of extracellular ribonucleases with exquisite specificity against rRNA (ribonucleic acid). They induce apoptotic death of cells after inhibiting protein translation. Ribosomes become functionally incompetent because ribotoxins cleave one single phosphodiester bond, located at a unique and universally conserved loop, needed for elongation factors function. As secreted proteins, ribotoxins need to cross the membrane of their target cells in order to exert their catalytic activity, and they do it without receptor mediation. Using lipid model systems, it has been shown that they are able to enter cells with membranes enriched in acidic phospholipids. Both membrane-interacting and ribosomal-recognition activities are characterised by distinct structural features. Even though the natural function of ribotoxins is not known yet, their production by entomopathogenic fungi has suggested their insecticidal role. After decades of detailed study, the biotechnological potential of ribotoxins in pest control and as antitumour agents is becoming evident

Inmunotoxinas antitumorales basadas en ribonucleasas extracelulares: tras los pasos de Paul Ehrlich

Han pasado más de 100 años desde que el laureado premio Nobel, Paul Ehrlich, introdujese el concepto de “bala mágica”, refiriéndose a una molécula con la capacidad de actuar específicamente sobre un determinado tipo de células. Este concepto supuso una revolución en la terapia y tratamiento de enfermedades, pues implicaba actuar solamente sobre las células dañadas, sin afectar al resto del organismo. Décadas después, su teoría de la actividad específica se hizo realidad revolucionando la terapia convencional, con la aparición del anticuerpo monoclonal y la aplicación de su exquisita especificidad en la inmunoterapia. Las inmunotoxinas suponen un paso más allá, combinando la especificidad de un anticuerpo por un antígeno tumoral con una actividad enzimática responsable de la muerte de la célula diana. Así, el dominio marcador dirige la acción del dominio tóxico. Este diseño ha evolucionado hacia la obtención de construcciones de menor tamaño más efectivas frente a tumores sólidos, siendo su máximo exponente las scFvinmunotoxinas recombinantes. La utilización de anticuerpos que reconocen distintos antígenos específicos de tumor, como dominio tóxico; y de diferentes tipos de toxinas o RNasas, como dominio tóxico, han permitido crear una gran diversidad de construcciones, potencialmente interesantes en el tratamiento contra el cáncer. Nuestro grupo de investigación lleva décadas ahondando en las relaciones estructura-función de ribonucleasas extracelulares microbianas, habiendo estudiado en profundidad los mecanismos de actuación de las ribotoxinas α-sarcina e hirsutelina A, dos ribonucleasas citotóxicas, capaces de inactivar el ribosoma. En la presente Tesis se han utilizado estas ribotoxinas, o variantes de ellas, como constituyentes del dominio toxico de inmunotoxinas; así como la RNasaT1, otra ribonucleasa microbiana, pero de carácter no tóxico. Las inmunotoxinas cuya caracterización se presentan en esta Tesis Doctoral tienen como dominio marcador los dominios variables del anticuerpo monoclonal A33 (scFvA33), que reconocen específicamente al antígeno GPA33, antígeno específico de tumor, que se presenta en el 95% de los tumores de colon, estando ausente en tejidos normales. En la presente tesis doctoral se aborda la caracterización estructural y funcional de estas inmunotoxinas basadas en RNasas extracelulares. Los resultados indican una elevada especificidad y eficacia citotóxica de las inmunotoxinas purificadas. Los resultados se discuten en términos de las diferencias encontradas en cuanto a dicha eficacia y especificidad y su capacidad o no para interaccionar con membranas celulares; y en sus diferencias como ribonucleasas

A New Optimized Version of a Colorectal Cancer-Targeted Immunotoxin Based on a Non-Immunogenic Variant of the Ribotoxin α-Sarcin

15 Pág.Due to its incidence and mortality, cancer remains one of the main risks to human health and lifespans. In order to overcome this worldwide disease, immunotherapy and the therapeutic use of immunotoxins have arisen as promising approaches. However, the immunogenicity of foreign proteins limits the dose of immunotoxins administered, thereby leading to a decrease in its therapeutic benefit. In this study, we designed two different variants of non-immunogenic immunotoxins (IMTXA33αSDI and IMTXA33furαSDI) based on a deimmunized variant of the ribotoxin α-sarcin. The inclusion of a furin cleavage site in IMTXA33furαSDI would allow a more efficient release of the toxic domain to the cytosol. Both immunotoxins were produced and purified in the yeast Pichia pastoris and later functionally characterized (both in vitro and in vivo), and immunogenicity assays were carried out. The results showed that both immunotoxins were functionally active and less immunogenic than the wild-type immunotoxin. In addition, IMTXA33furαSDI showed a more efficient antitumor effect (both in vitro and in vivo) due to the inclusion of the furin linker. These results constituted a step forward in the optimization of immunotoxins with low immunogenicity and enhanced antitumor activity, which can lead to potential better outcomes in cancer treatment.This research was funded by the Complutense University of Madrid (grant numbers PR75/18-21563 and PR87/19-22627) and the PID2020-116692RB-I00 grant from the I+D+I Retos Investigación of the Spanish Ministry of Science and Innovation. J.N. was supported by a predoctoral fellowship from the Spanish Ministry of Science and Innovation (FPU15/04121). J.T.-A. was granted funding by the Community of Madrid through the FOODAL project (FOODAL-CM; S2018/BAA-4574) co-funded by the ESF and ERDF R&D projects of Tecnologías 2018.Peer reviewe

Preparation of an engineered safer immunotoxin against colon carcinoma based on the ribotoxin hirsutellin A

Immunotoxins are chimeric proteins composed of an antibody domain that specifically directs the action of the toxic domain, resulting in the death of the targeted cells. Over recent years, immunotoxins have been widely studied and the number of different constructions has increased exponentially. Protein engineering has allowed the design of optimized versions of immunotoxins with an improved tumor binding affinity, stability or cytotoxic efficacy, although sometimes this has compromised the safety of the patient in terms of undesirable adverse secondary reactions. A triple mutant at three Trp residues (HtA3DW) of the ribotoxin hirsutellin A retains its specific ribonucleolytic activity, although cell internalization capacity is lacking.This toxin variant has been fused to the single chain variable fragment A33 (scFvA33). This immunoconjugate (IMTXA33HtA3DW) was produced in the methylotrophic yeast Pichia pastoris and purified using nickelnitrilotriacetic acid affinity chromatography. Both target and toxic domains were characterized. The immunotoxin showed an exquisite specific binding against GPA33-positive culture cells, which results in the death of the targeted cells because of specific ribonucleolytic activity against ribosomes of the engineered hirsutellin A variant. IMTXA33HtA3DW represents a promising structure in the search for an improved immunotoxin without compromising the safety of patients

Efficient in vivo antitumor effect of an immunotoxin based on ribotoxin α-sarcin in nude mice bearing human colorectal cancer xenografts

Tagging of RNases, such as the ribotoxin α-sarcin, with the variable domains of antibodies directed to surface antigens that are selectively expressed on tumor cells endows cellular specificity to their cytotoxic action. A recombinant single-chain immunotoxin based on the ribotoxin α-sarcin (IMTXA33αS), produced in the generally regarded as safe (GRAS) yeast Pichia pastoris, has been recently described as a promising candidate for the treatment of colorectal cancer cells expressing the glycoprotein A33 (GPA33) antigen, due to its high specific and effective cytotoxic effect on in vitro assays against targeted cells. Here we report the in vivo antitumor effectiveness of this immunotoxin on nude mice bearing GPA33-positive human colon cancer xenografts. Two sets of independent assays were performed, including three experimental groups: control (PBS) and treatment with two different doses of immunotoxin (50 or 100 μg/ injection) (n = 8). Intraperitoneal administration of IMTXA33αS resulted in significant dose-dependent tumor growth inhibition. In addition, the remaining tumors excised from immunotoxin-treated mice showed absence of the GPA33 antigen and a clear inhibition of angiogenesis and proliferative capacity. No signs of immunotoxin-induced pathological changes were observed from specimens tissues.Overall these results show efficient and selective cytotoxic action on tumor xenografts, combined with the lack of severe side effects, suggesting that IMTXA33αS is a potential therapeutic agent against colorectal cancer

A multifunctionalized potyvirus-derived nanoparticle that targets and internalizes into cancer cells

Plant viral nanoparticles (VNPs) are attractive to nanomedicine researchers because of their safety, ease of production, resistance, and straightforward functionalization. In this paper, we developed and successfully purified a VNP derived from turnip mosaic virus (TuMV), a well-known plant pathogen, that exhibits a high affinity for immunoglobulins G (IgG) thanks to its functionalization with the Z domain of staphylococcal Protein A via gene fusion. We selected cetuximab as a model IgG to demonstrate the versatility of this novel TuMV VNP by developing a fluorescent nanoplatform to mark tumoral cells from the Cal33 line of a tongue squamous cell carcinoma. Using confocal microscopy, we observed that fluorescent VNP–cetuximab bound selectively to Cal33 and was internalized, revealing the potential of this nanotool in cancer research.Ministerio de Universidades (España)Ministerio de Ciencia e Innovación (España)Comunidad de MadridInstituto Nacional de Investigación y Tecnología Agraria y Alimentaria (España)ERANetUniversidad Complutense de MadridDepto. de Genética, Fisiología y MicrobiologíaDepto. de Biología CelularFac. de Ciencias BiológicasFac. de MedicinaTRUEpu

α-sarcin and RNase T1 based immunoconjugates: the role of intracellular trafficking in cytotoxic efficiency

Toxins have been thoroughly studied for their use as therapeutic agents in search of an improvement in toxic efficiency together with a minimization of their undesired side effects. Different studies have shown how toxins can follow different intracellular pathways which are connected with their cytotoxic action inside the cells. The work herein presented describes the different pathways followed by the ribotoxin a-sarcin and the fungal RNase T1,as toxic domains of immunoconjugates with identical binding domain, the single chain variable fragment of a monoclonal antibody raised against the glycoprotein A33. According to the results obtained both immunoconjugates enter the cells via early endosomes and, while a-sarcin can translocate directly into the cytosol to exert its deathly action, RNase T1 follows a pathway that involves lysosomes and the Golgi apparatus. These facts contribute to explaining the different cytotoxicity observed against their targeted cells, and reveal how the innate properties of the toxic domain, apart from its catalytic features, can be a key factor to be considered for immunotoxin optimization