Biobetter Versions of Recombinant Human IFN-α2b for the Treatment of Viral Infections

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the infectious agent causing COVID-19 disease, whose pandemic has had far-reaching consequences on the global population. Since the detection of the first cases in late 2019, much has been learned about the mechanism of action of SARS-CoV-2 and the associated immune response to eradicate the infection. Recently, a clear correlation between disease severity and abnormal type I IFN response in patients has been established. Individuals with immune responses characterized by high concentrations of IFN-a2b and low blood levels of IL-6, TNF-alpha, and IL-1Ra were much less affected than those patients who exhibited an opposite scenario. Interestingly, recombinant human IFN-a2b (rhIFN-a2b) could mitigate the severity of symptoms, if given in the early stages of the disease, before reaching the inflammatory shock (cytokine storm) that characterizes the most severe cases. However, there are adverse effects associated with rhIFN-a2b-based therapy. Among them, the emergence of unwanted immune responses against the biologic can, in some cases, compromise the treatment’s safety and efficacy. In addition, rhIFN-a2b is a small cytokine, which results in rapid clearance from the bloodstream. This quick plasma clearance poses the need for frequent high doses to achieve the desired effect, which may, in turn, exacerbate unwanted effects associated with therapy.In this article we will address the most relevant strategies for the development of biobetters versions of rhIFN-a2b, as promising candidates for the treatment of COVID-19 and other human viral diseases.Fil: Mufarrege, Eduardo Federico. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Peña, Lucía Carolina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Echeverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentin

Analysis of expression mechanisms of genes encoding for cytochrome c oxidase plant subunits

En el presente trabajo realizamos un análisis en profundidad de las regiones promotoras de los genes que codifican las subunidades AtCox6a y AtCox6b de la cadena respiratoria mitocondrial, en Arabidopsis thaliana. A partir de los resultados obtenidos de las distintas experiencias realizadas resulta evidente el rol de los elementos site II en la expresión de los genes AtCOX6b. Una segunda parte de este trabajo consistió en estudiar el mecanismo mediante el cual ciertos intrones son capaces de participar en el aumento de la expresión génica. Por este motivo, comenzamos a estudiar algunas proteínas pertenecientes a un complejo conocido como el complejo de la unión entre exones (EJC, por sus siglas en inglés: Exon Junction Complex), e involucrado en forma directa con el aumento de la expresión génica mediada por intrones. En primer lugar, se intentó determinar de qué manera se produce la biogénesis del EJC, estudiando en profundidad tres de sus proteínas en Arabidopsis thaliana: AtMago, AtY14 y AtPym. De esta manera, se demostró la importancia de los elementos site II sugieriendo la existencia de un mecanismo coordinado de expresión génica que permitiría optimizar la biogénesis del complejo. Finalmente, se encontró que las modificaciones post-traduccionales (entre ellas la fosforilación) proveerían de una tercera vía de regulación de algunos componentes del EJC. Este hecho pone de manifiesto la importancia de contar con varias maneras de regular la estructura y función de este complejo, y consecuentemente la expresión de aquellos genes que dependen del mismo.In the present work we performed a detailed analysis of promoter regions of genes encoding the AtCox6b and AtCox6a subunits belonging to the mitochondrial respiratory chain in Arabidopsis thaliana. From the results of different experiments was evident the role of the site II elements in AtCOX6b gene expression. A second part of this work consisted in study the mechanism by which certain introns are able to participate in enhance gene expression. For this reason, we began to study some proteins belonging to a complex known as "Exon Junction Complex" (EJC), directly involved in gene expression enhancement mediated by introns. Firstly, we attempted to determine how EJC biogenesis occurs, studying in depth three of its proteins in Arabidopsis thaliana: AtMago, AtY14 and AtPym. Thus, we demonstrated the importance of site II elements suggesting the existence of a coordinated mechanism for gene expression that would optimize complex biogenesis. Finally, we found that post-translational modifications (including phosphorylation) would provide a third way of regulation of some components of the EJC. This fact highlights the importance of having several ways to regulate the structure and function of this complex, and consequently the expression of those genes that depend on it.Consejo Nacional de Investigaciones Científicas y Técnicas. Agencia Nacional de Promoción Científica y Tecnológica. Universidad Nacional del Litora

Common sets of promoter elements determine the expression characteristics of three arabidopsis genes encoding isoforms of mitochondrial cytochrome c oxidase subunit 6b

The promoters of the three Arabidopsis nuclear genes encoding mitochondrial cytochrome c oxidase subunit 6b (AtCOX6b) have similar expression patterns, with preferential expression in anthers and meristems, and are induced by sucrose and etiolation. Additionally, induction of AtCOX6b-1 by GA3 and AtCOX6b-3 by 6-benzylaminopurine was observed. Site II elements (TGGGCC/T) present in the three promoters bind common nuclear proteins and are important for basal and induced expression. Induction by sucrose requires, in addition, the integrity of elements with the sequence TACTAA. The results imply the participation of common regulatory factors in the expression of the three Arabidopsis COX6b genes.Fil: Mufarrege, Eduardo Federico. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Curi, Graciela Cristina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Gonzalez, Daniel Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Functional interconnections of Arabidopsis exon junction complex proteins and genes at multiple steps of gene expression

The exon junction complex (EJC) is deposited on mRNA after splicing and participates in several aspects of RNA metabolism, from intracellular transport to translation. In this work, the functional and molecular interactions of Arabidopsis homologues of Mago, Y14, and PYM, three EJC components that participate in intron-mediated enhancement of gene expression in animals, have been analysed. AtMago, AtY14, and AtPYM are encoded by single genes that show similar expression patterns and contain common regulatory elements, known as site II, that are required for expression. AtPYM and AtY14 are phosphorylated by plant extracts and this modification regulates complex formation between both proteins. In addition, overexpression of AtMago and AtY14 in plants produces an increase in AtPYM protein levels, while overexpression of AtPYM results in increased formation of a complex that contains the three proteins. The effect of AtMago and AtY14 on AtPYM expression is most likely to be due to intron-mediated enhacement of AtPYM expression, since the AtPYM gene contains a leader intron that is required for expression. Indeed, transient transformation asssays indicated that the three proteins are able to increase expression from reporter constructs that contain leader introns required for the expression of different genes. The results indicate that the plant homologues of Mago, Y14, and PYM are closely interconnected, not only through their function as EJC components but also at different steps of their own gene expression mechanisms, probably reflecting the importance of their interaction for the correct expression of plant genes.Fil: Mufarrege, Eduardo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Gonzalez, Daniel Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Curi, Graciela Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Specific sequence mutations in a long-lasting rhIFN-α2b version reduce in vitro and in vivo immunogenicity and increase in vitro protein stability

For decades, recombinant human interferon alpha (rhIFN-α2b) has been used to treat emerging and chronic viral diseases. However, rhIFN-α2b is immunogenic and has a short in vivo half-life. To solve these limitations, two long-lasting hyperglycosylated proteins with reduced immunogenicity were developed and designated as 4N-IFN(VAR1) and 4N-IFN(VAR3). Here, we continue to study the relevant characteristics of these therapeutic candidates. Thus, we demonstrated that both de-immunized IFN versions elicited significantly lower neutralizing antibody responses than the original molecule in HLA-DR1 transgenic mice, confirming our previous in vitro protein immunogenicity data. Also, we found that these biobetters exhibited remarkable stability when exposed to different physical factors that the protein product may encounter during its production process and storage, such as low pH, thermal stress, and repeated freezing/thawing cycles. Taking into consideration our previous and present results, 4N-IFN(VAR1) and 4N-IFN-4N(VAR3) appear to be valuable candidates for the treatment of human viral diseases.Fil: Mufarrege, Eduardo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: Peña, Lucía Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: Etcheverrigaray, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: De Groot, Anne S.. Epivax Inc.; Estados Unidos. University Of Rhode Island; Estados UnidosFil: Martin, William. Epivax Inc.; Estados Unido

Multiplexed Gene Expression as a Characterization of Bioactivity for Interferon Beta (IFN-β) Biosimilar Candidates: Impact of Innate Immune Response Modulating Impurities (IIRMIs)

Recombinant human interferon-β (rhIFN-β) therapy is the first-line treatment in relapsing-remitting forms of multiple sclerosis (MS). The mechanism of action underlying its therapeutic activity is only partially understood as IFN-βs induce the expression of over 1000 genes modifying multiple immune pathways. Currently, assessment of potency for IFN-β products is based on their antiviral effect, which is not linked to its therapeutic effect. Here, we explore the use of a multiplexed gene expression system to more broadly characterize IFN-β bioactivity. We find that MM6 cells stimulated with US-licensed rhIFN-βs induce a dose-dependent and reproducible pattern of gene expression. This pattern of gene expression was used to compare the bioactivity profile of biosimilar candidates with the corresponding US-licensed rhIFN-β products, Rebif and Betaseron. While the biosimilar candidate for Rebif matched the pattern of gene expression, there were differences in the expression of a subset of interferon-inducible genes including CXCL-10, CXCL-11, and GBP1 induced by the biosimilar candidate for Betaseron. Assessment of product impurities in both products suggested that the difference was rooted in the presence of innate immune response modulating impurities (IIRMIs) in the licensed product. These studies indicate that determining the expression levels for an array of reporter genes that monitor different pathways can be informative as part of the demonstration of biosimilarity or comparability for complex immunomodulatory products such as IFN-β, but the sensitivity of each gene to potential impurities in the product should be examined to fully understand the results.Fil: Mufarrege, Eduardo Federico. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: Haile, Lydia A.. Food And Drug Administration;Fil: Echeverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: Verthelyi, Daniela. Universidad Nacional del Litoral; Argentin

Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells: A new strategy for recombinant human FVIII (rhFVIII) production

Background: Recombinant protein overexpression in mammalian cells constitutes a real challenge in therapeutic protein production. Following the discovery of intron functionality in gene expression, various expression vectors that include them in their sequences have been developed. In this study, the main goal was to develop new lentiviral vectors (LVs) carrying different promoter and intron-containing 50UTR (50 untranslated region) combinations and the design of LVs for rhFVIII production in Chinese hamster ovary (CHO) cells. Results: By combining the human cytomegalovirus (CMV) or the elongation factor 1a (EF-1a) promoters along with different 50UTRs that included leader introns, between 2 and 12-fold increases were reached, when transient and stable expression of the enhanced green fluorescent protein (EGFP) and rhFVIII were analyzed. Also, new LVs provided with promoters and 50UTRs from high expression genes, according to a gene database, were designed. Three of them were shown to be superior to the EF-1a promoter in three widely used cell lines. Conclusion: In the present work, LVs containing different promoters and 50UTRs were designed. In transient and stable assays some of these constructs have shown higher activity compared with commercial promoters and, therefore, constitute promising candidates for therapeutic protein production.Fil: Mufarrege, Eduardo Federico. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Antuña, Sebastián. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: Marina Echeverrigaray. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Kratje, Ricardo Bertoldo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Prieto, Claudio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin

De-immunized and Functional Therapeutic (DeFT) versions of a long lasting recombinant alpha interferon for antiviral therapy

Interferon α (IFN-α) exerts potent antiviral, immunomodulatory, and antiproliferative activity and have proven clinical utility in chronic hepatitis B and C virus infections. However, repeated IFN-α administration induces neutralizing antibodies (NAb) against the therapeutic in a significant number of patients. Associations between IFN-α immunogenicity and loss of efficacy have been described. So as to improve the in vivo biological efficacy of IFN-α, a long lasting hyperglycosylated protein (4N-IFN) derived from IFN-α2b wild type (WT-IFN) was developed. However, in silico analysis performed using established in silico methods revealed that 4N-IFN had more T cell epitopes than WT-IFN. In order to develop a safer and more efficient IFN therapy, we applied the DeFT (De-immunization of Functional Therapeutics) approach to producing functional, de-immunized versions of 4N-IFN. Using the OptiMatrix in silico tool in ISPRI, the 4N-IFN sequence was modified to reduce HLA binding potential of specific T cell epitopes. Following verification of predictions by HLA binding assays, eight modifications were selected and integrated in three variants: 4N-IFN(VAR1), (VAR2) and (VAR3). Two of the three variants (VAR1 and VAR3) retained anti-viral function and demonstrated reduced T-cell immunogenicity in terms of T-cell proliferation and Th1 and Th2 cytokine levels, when compared to controls (commercial NG-IFN (non-glycosylated), PEG-IFN, WT-IFN and 4N-IFN). It was previously demonstrated that N-glycosylation improved IFN-α pharmacokinetic properties. Here, we further reduce immunogenicity as measured in vitro using T cell assays and cytokine profiling by modifying the T cell epitope content of a protein (de-immunizing). Taking into consideration the present results and previously reported immunogenicity data for commercial IFN-α2b variants, 4N-IFN(VAR1) and 4N-IFN-4N(VAR3) appear to be promising candidates for improved IFN-α therapy of HCV and HBV.Fil: Mufarrege, Eduardo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Giorgetti, Sofia Inés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Etcheverrigaray, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Terry, Frances. EpiVax Incorporated; Estados UnidosFil: Martin, William. EpiVax Incorporated; Estados UnidosFil: De Groot, Anne S.. EpiVax Incorporated; Estados Unidos. University of Rhode Island; Estados Unido

Development of IFNβ-1a versions with reduced immunogenicity and full in vitro biological activity for the treatment of multiple sclerosis

IFNβ (recombinant interferon Beta) has been widely used for the treatment of Multiple sclerosis for the last four decades. Despite the human origin of the IFNβ sequence, IFNβ is immunogenic, and unwanted immune responses in IFNβ-treated patients may compromise its efficacy and safety in the clinic. In this study, we applied the DeFT (De-immunization of Functional Therapeutics) approach to producing functional, de-immunized versions of IFNβ-1a. Two de-immunized versions of IFNβ-1a were produced in CHO cells and designated as IFNβ-1a(VAR1) and IFNβ-1a(VAR2). First, the secondary and tertiary protein structures were analyzed by circular dichroism spectroscopy. Then, the variants were also tested for functionality. While IFNβ-1a(VAR2) showed similar in vitro antiviral activity to the original protein, IFNβ-1a(VAR1) exhibited 40% more biological potency. Finally, in vivo assays using HLA-DR transgenic mice revealed that the de-immunized variants showed a markedly reduced immunogenicity when compared to the originator.Fil: Ricotti, Sonia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Garay, Alberto Sergio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Amadeo, Gabriel Ignacio. No especifíca;Fil: Anne De Groot. No especifíca;Fil: Martin, William. No especifíca;Fil: Mufarrege, Eduardo Federico. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin

Development of Magoh protein-overexpressing HEK cells for optimized therapeutic protein production

In the pharmaceutical industry, the need for high levels of protein expression in mammalian cells has prompted the search for new strategies, including technologies to obtain cells with improved mechanisms that enhance its transcriptional activity, folding, or protein secretion. Chinese Hamster Ovary (CHO) cells are by far the most used host cell for therapeutic protein expression. However, these cells produce specific glycans that are not present in human cells and therefore potentially immunogenic. As a result, there is an increased interest in the use of human-derived cells for therapeutic protein production. For many decades, human embryonic kidney (HEK) cells were exclusively used for research. However, two products for therapeutic indication were recently approved in the United States. It was previously shown that tethered Magoh, an Exon-junction complex core component, to specific mRNA sequences, have had significant positive effects on mRNA translational efficiency. In this study, a HEK Magoh-overexpressing cell line and clones, designated here as HEK-MAGO, were developed for the first time. These cells exhibited improved characteristics in protein expression, reaching —two- to threefold increases in rhEPO protein production in comparison with the wild-type cells. Moreover, this effect was promoter independent highlighting the versatility of this expression platform.Fil: Mufarrege, Eduardo Federico. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Benizio, Evangelina Leticia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Prieto, Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: Chiappini, Fabricio Alejandro. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Rodriguez, María Celeste. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Kratje, Ricardo Bertoldo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin