Generation and functional evaluation of novel monoclonal antibodies targeting glycosylated human stem cell factor.

Human stem cell factor (hSCF) is an early-acting growth factor that promotes proliferation, differentiation, migration, and survival in several tissues. It plays a crucial role in hematopoiesis, gametogenesis, melanogenesis, intestinal motility, and in development and recovery of nervous and cardiovascular systems. Potential therapeutic applications comprise anemia treatment, mobilization of hematopoietic stem/progenitor cells to peripheral blood, and increasing gene transduction efficiency for gene therapy. Developing new tools to characterize recombinant hSCF in most native-like form as possible is crucial to understand the complexity of its in vivo functions and for improving its biotechnological applications. The soluble domain of hSCF was expressed in HEK293 cells. Highly purified rhSCF showed great molecular mass variability due to the presence of N- and O-linked carbohydrates, and it presented a 2.5-fold increase on proliferative activity compared to bacteria-derived hSCF. Three hybridoma clones producing monoclonal antibodies (mAbs) with high specificity for the glycoprotein were obtained. 1C4 and 2D3 mAbs were able to detect bacteria-derived and glycosylated rhSCF and demonstrated to be excellent candidates to develop a sandwich ELISA assay for rhSCF quantification, with detection limits of 0.18 and 0.07 ng/ml, respectively. Interestingly, 1A10 mAb only recognized glycosylated rhSCF, suggesting that sugar moieties might be involved in epitope recognition. 1A10 mAb showed the highest binding affinity, and it constituted the best candidate for immunodetection of the entire set rhSCF glycoforms in western blot assays, and for intracellular cytokine staining. Our work shows that combining glycosylated rhSCF expression with hybridoma technology is a powerful strategy to obtain specific suitable immunochemical assays and thus improve glycoprotein-producing bioprocesses. KEY POINTS: • Soluble glycosylated human SCF exerted improved proliferative activity on UT-7 cells. • Three mAbs with high specificity targeting glycosylated human SCF were obtained. • mAbs applications comprise sandwich ELISA, western blot, and immunofluorescence assays.

Screening of CHO-K1 endogenous promoters for expressing recombinant proteins in mammalian cell cultures.

For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, new endogenous promoters were discovered based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico-derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.

Development, Production, and Characterization of Hepatitis B Subviral Envelope Particles as a Third-Generation Vaccine.

Vaccination still represents the most efficient and inexpensive strategy in the control of hepatitis B virus (HBV) infection. However, about 10% of the population vaccinated with the current S yeast-derived vaccine fail to induce an adequate immune response. Our group has developed a new-generation hepatitis B vaccine candidate composed by the three surface proteins of the HBV. Here we describe the methods to develop and characterize a stable CHO-K1 recombinant cell line able to produce and secrete hepatitis B subviral envelope particles (HBV-SVPs) containing L and M glycoproteins in addition to S glycoprotein. In addition, Western blot and immunogold electron microscopy techniques to evaluate the size, morphology, and composition of the particles are explained. Finally, immunization protocols are described in order to study the immunogenicity of HBV-SVPs and the ability of the antibodies triggered by these particles to recognize the binding site of HBV with the hepatocyte.

Rational design of novel fusion rabies glycoproteins displaying a major antigenic site of foot-and-mouth disease virus for vaccine applications.

Chimeric virus-like particles are self-assembling structures composed of viral proteins that had been modified to incorporate sequences from different organisms, being able to trigger immune responses against the heterologous sequence. However, the identification of suitable sites for that purpose in the carrier protein is not an easy task. In this work, we describe the generation of rabies chimeric VLPs that expose a major antigenic site of foot-and-mouth disease virus (FMDV) by identifying suitable regions in rabies glycoprotein (RVG), as a proof of concept of a novel heterologous display platform for vaccine applications. To identify adequate sites for insertion of heterologous sequences without altering the correct folding of RVG, we identified regions that were evolutionally non-conserved in Lyssavirus glycoproteins and performed a structural analysis of those regions using a 3D model of RVG trimer that we generated. The heterologous sequence was inserted in three different sites within RVG sequence. In every case, it did not affect the correct folding of the protein and was surface exposed, being recognized by anti-FMDV antibodies in expressing cells as well as in the surface of VLPs. This work sets the base for the development of a heterologous antigen display platform based on rabies VLPs. KEY POINTS: • Adequate regions for foreign epitope display in RVG were found. • G-H loop of FMDV was inserted in three regions of RVG. • The foreign epitope was detected by specific antibodies on fusion proteins. • G-H loop was detected on the surface of chimeric VLPs.

Chimeric VLPs Based on HIV-1 Gag and a Fusion Rabies Glycoprotein Induce Specific Antibodies against Rabies and Foot-and-Mouth Disease Virus.

Foot and mouth disease is a livestock acute disease, causing economic losses in affected areas. Currently, control of this disease is performed by mandatory vaccination campaigns using inactivated viral vaccines. In this work, we describe the development of a chimeric VLP-based vaccine candidate for foot-and-mouth disease virus (FMDV), based on the co-expression of the HIV-1 Gag protein and a novel fusion rabies glycoprotein (RVG), which carries in its N-term the FMDV main antigen: the G-H loop. It is demonstrated by confocal microscopy that both Gag-GFP polyprotein and the G-H loop colocalize at the cell membrane and, that the Gag polyprotein of the HIV virus acts as a scaffold for enveloped VLPs that during the budding process acquires the proteins that are being expressed in the cell membrane. The obtained VLPs were spherical particles of 130 ± 40 nm in diameter (analyzed by TEM, Cryo-TEM and NTA) carrying an envelope membrane that efficiently display the GH-RVG on its surface (analyzed by gold immunolabeling). Immunostainings with a FMDV hyperimmune serum showed that the heterologous antigenic site, genetically fused to RVG, is recognized by specific G-H loop antibodies. Additionally, the cVLPs produced expose the G-H loop to the liquid surrounding (analyzed by specific ELISA). Finally, we confirmed that these FMD cVLPs are able to induce a specific humoral immune response, based on antibodies directed to the G-H loop in experimental animals.

Novel erythropoietin-based therapeutic candidates with extra N-glycan sites that block hematopoiesis but preserve neuroplasticity.

Neurological disorders affect millions of people causing behavior-cognitive disabilities. Nowadays they have no effective treatment. Human erythropoietin (hEPO) has been clinically used because of its neurotrophic and cytoprotective properties. However, the erythropoietic activity (EA) should be considered as a side effect. Some analogs like non-sialylated EPO, carbamylated EPO, or EPO peptides have been developed showing different weaknesses: erythropoiesis preservation, low stability, potential immunogenicity, or fast clearance. Herein, we used a novel strategy that blocks the EA but preserves hEPO neurobiological actions. N-glycoengineering was accomplished to add a new glycosylation site within the hEPO sequence responsible for its EA. hEPO-derivatives were produced by CHO.K1 cells, affinity-purified and functionally analyzed studying their in vitro and in vivo EA, their in vitro neuronal plasticity in hippocampal neurons and their neuroprotective action by rescuing hippocampal neurons from apoptosis. Muteins Mut 45_47 (K45 > N45 + N47 > T47), Mut 104 (S104 > N104), and Mut 151_153 (G151 > N151 + K153 > T153) lost their EA but preserved their neuroprotection activity and enhanced neuroplasticity more efficiently than hEPO. Interestingly, Mut 45_47 resulted in a promising candidate to explore as neurotherapeutic considering not only its biopotency but also its pharmacokinetic potential due to the hyperglycosylation.

Bifunctional GM-CSF-derived peptides as tools for O-glycoengineering and protein tagging.

Rapid development of effective biotherapeutics has been a concern during the last couple decades. In our work we designed two novel peptide tags, GMOP and mGMOP, derived from the N-terminal region of human granulocyte and macrophage colony stimulating factor (hGM-CSF), which contain four and six potential O-glycosylation sites, respectively. These peptide tags were fused to the N-terminus of human interferon-α2b (hIFN-α2b), a therapeutic antiviral and antiproliferative protein rapidly cleared from circulation. Two new molecules were obtained which, consistently with the presence of O-glycans, showed higher molecular masses, more negatively charged isoforms, and higher sialic acid content compared to wild-type IFN. In vitro bioactivity of purified chimeras revealed a similar antiviral specific biological activity (SBA) compared to unmodified IFN. A reduction of antiproliferative SBA was only observed for mGMOP-IFN. Pharmacokinetic studies in rats showed a notable improvement in terminal half-life (t1/2elim) (3.3 and 2.8 times-longer) and a marked reduction of the apparent clearance (CLapp, 3.7 and 4.1-fold lower for GMOP-IFN and mGMOP-IFN in comparison with native IFN, respectively). Furthermore, the in vitro thermal and plasma stability of both proteins was improved. Finally, a monoclonal antibody (mAb) that recognizes an N-terminal GM-CSF epitope was able to bind both chimeras in western blots and ELISAs. This demonstrates the potential of both peptides to behave as bifunctional tags to create novel long-acting biotherapeutics and to facilitate detection and purification.

Pharmacokinetics Versus In Vitro Antiproliferative Potency to Design a Novel Hyperglycosylated hIFN-α2 Biobetter.

PURPOSE: IFN4N is a glycoengineered version of recombinant human interferon alpha 2 (rhIFN-α2) that was modified to exhibit four N-glycosylation sites. It shows reduced in vitro specific biological activity (SBA) mainly due to R23 mutation by N23. However, it has improved pharmacokinetics and led to a high in vivo antitumor activity in mice. In order to prepare a new IFN-based biobetter, this work compares the influence of glycosylation (affecting pharmacokinetics) with the in vitro antiproliferative SBA on the in vivo efficacy. METHODS: Based on IFN4N, three groups of muteins were designed, produced, and characterized. Group A: variants with the same glycosylation degree (4N) but higher in vitro antiproliferative SBA (R23 restored); group B: muteins with higher glycosylation degree (5N) but similar in vitro antiproliferative activity; and group C: variants with improved glycosylation (5N and 6N) and in vitro antiproliferative bioactivity. RESULTS: Glycoengineering was successful for improving pharmacokinetics, and R23 restoration considerably increased in vitro antiproliferative activity of new muteins compared to IFN4N. Hyperglycosylation was able to improve the in vivo efficacy similarly to or even better than R23 restoration. Additionally, the highest glycosylated mutein exhibited the lowest immunogenicity. CONCLUSIONS: Hyperglycosylation constitutes a successful strategy to prepare a novel IFN biobetter.

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.

Characterization of hepatitis B virus surface antigen particles expressed in stably transformed mammalian cell lines containing the large, middle and small surface protein.

Vaccination still represents the most efficient and inexpensive strategy in the control of hepatitis B virus (HBV) infection. However, about 10% of the population vaccinated with the current yeast derived vaccine, consisting of the non-glycosylated form of the small envelope protein (S) of the HBV, fail to display an adequate immune response. Therefore, there is a need for the development of new vaccines with enhanced immunogenicity. On this regard, new generation vaccines containing L and preS2-containing HBV surface proteins in addition to S, have proven to be able to bypass the lack of response of the standard vaccine. In this work, we describe the development of stable recombinant CHO-K1 and HEK293 cell lines able to produce and secrete hepatitis B subviral envelope particles (HBV-SVPs) composed by the three surface proteins of the HBV. In turn, we demonstrated that these particles induced a specific humoral immune response in experimental animals and triggered the production of antibodies with the ability to recognize the binding site of HBV with the hepatocyte. Thus, these HBV-SVPs represent a promising candidate as a new generation vaccine in order to enhance the immunogenicity of the conventional yeast derived HBV vaccine.

Rabies VLPs adjuvanted with saponin-based liposomes induce enhanced immunogenicity mediated by neutralizing antibodies in cattle, dogs and cats.

We carried out an investigation on rabies virus-like particles (RV-VLPs) expressed in HEK293 cells using serum free medium. These RV-VLPs were formulated with two different adjuvants in order to analyse the enhancement of the triggered immune response and its stability. In experiments in mice, RV-VLPs showed an enhanced humoral immune response when injected with adjuvant, in contrast to the obtained for the RV-VLPs without adjuvant addition. Besides, higher titers of neutralizing antibodies were induced when RV-VLPs were formulated with LipoSap® in comparison with the obtained with Alhydrogel®. At the same time, the positive effect of this adjuvant in vaccine's potency and stability was demonstrated, showing that LipoSap® significantly increases the value obtained in NIH efficiency test for rabies vaccine, and proving that this value is maintained after 15 months storage at 4 °C. Further, we showed that RV-VLPs induces an immune response based on neutralizing antibodies when cat, dogs and bovines were vaccinated with only one dose of RV-VLPs. These results demonstrated that this vaccine candidate could be applied for the prevention of rabies in pets as well as for the control of paralytic rabies in cattle.

Identification and characterization of human interferon alpha inhibitors through a WISH cell line-based reporter gene assay.

Interferons (IFNs) are important glycoproteins which can stimulate or inhibit up to three hundred different genes encoding proteins involved in antiviral defense mechanisms, inflammation, adaptive immunity, angiogenesis and among other processes. Nevertheless, different genetic alterations may lead to interferon alpha (IFN-α) overproduction in human autoimmune diseases like systemic lupus erythematosus. As a consequence, IFN-α is a central molecule whose activity must be regulated to block their harmful effect on those disorders where the endogenous cytokine production constitutes the etiology of the illnesses. In this work, we evaluate the biological activity of eighty-eight compounds, from our own chemo-library, to find potential IFN-α inhibitors by using a reporter gene assay (RGA) WISH-Mx2/EGFP. We identified some compounds able to modulate negatively the IFN-α activity. The most active IFN-α inhibitors were further studied achieving promising results. In addition, some combinations of the most active compounds were analyzed accomplishing a stronger effect to decrease the IFN-α activity than each compound alone. Furthermore, the complete inhibition of the cytokine activity was reached with some combinations of compounds.

A simplified roller bottle platform for the production of a new generation VLPs rabies vaccine for veterinary applications.

Rabies is a neglected disease with an estimated annual mortality of 55,000 human deaths, affecting mainly low-income countries. Over 95% of these cases result from virus transmission through the bite of infected dogs and for this reason there is a real need for a cheap and effective rabies veterinary vaccine to be used in mass vaccination campaigns. In this work, we describe the establishment of a simple platform for the production of a virus-like particles based rabies vaccine using mammalian cells and roller bottles as culture system. Adherent cells were cultured during more than 15 days and VLPs were continuously produced and secreted to the culture supernatant. Immunogenicity and protective efficacy of VLPs were tested through rabies virus neutralizing antibody test and NIH potency test. These viral particles induced high titer of long lasting neutralizing antibodies and protected mice against active virus challenge. Therefore, this development represents a promising platform for the production of a new generation and virus-free rabies vaccine candidate for veterinary applications.

Effect of ANITVNITV peptide fusion on the bioactivity and pharmacokinetics of human IFN-α2b and a hyper-N-glycosylated variant.

Different strategies have been developed and successfully applied to biotherapeutics in order to improve their in vivo efficacy. The genetic fusion to natural or synthetic glycosylated peptides constitutes a promising strategy since it conserves the protein sequence and results in the improvement of the pharmacokinetic properties. The ANITVNITV peptide described by Perlmann and coworkers presents 9 amino acids and 2 potential N-glycosylation sites. Its fusion to FSH resulted in the increase of the molecular mass and negative charge of the protein. Consequently, the pharmacokinetics was considerably improved. The aim of the present study was to compare the influence of ANITVNITV peptide fusion on the physicochemical, biological and pharmacokinetic properties of native hIFN-α2b (IFNwt), which contains a single O-glycosylation site, and a hyperglycosylated variant (IFN4N), that bears, in addition, 4 N-linked glycans. The resulting molecules, IFNwtNter and IFN4NNter, evidenced a higher molecular mass and negative charge compared to IFNwt and IFN4N, respectively. Therefore, the pharmacokinetic properties of the new molecules were significantly improved. The molecules obtained by the synthetic peptide fusion strategy evidenced a decrease in their in vitro antiviral specific biological activities (SBA). However, in vitro antiproliferative SBA was differentially modified for IFNwtNter and IFN4NNter in comparison with the parental molecules. For IFNwtNter, a reduction in the antiproliferative SBA was also observed. Remarkably, the addition of the ANITVNITV peptide to the N-terminus of IFN4N had a positive impact on its growth-inhibitory activity. This feature together with its improved pharmacokinetics encourages the development of IFN4NNter as an IFN-α based biobetter.

Characterization of cellular states of CHO-K1 suspension cell culture through cell cycle and RNA-sequencing profiling.

Chinese hamster ovary (CHO) derived cell lines are the preferred host system for the production of therapeutic proteins. The aim of this work was to explore the regulation of suspension-adapted CHO-K1 host cell line bioprocesses, especially under a temperature gradient from 37 °C to 31 °C. We analyzed cell cycle behavior through flow cytometry of propidium iodide stained cells and high throughput transcriptome dynamics by RNA sequencing. We found a cell culture state characterized by G0/G1 synchronization, mainly during the late exponential growth phase and towards the last days of the stationary phase. We successfully identified key genes and pathways connected with the particular culture states, such as response to low temperature, modulation of the cell cycle, regulation of DNA replication and repair, apoptosis, among others. The most important gene expression changes occurred throughout the stationary phase when gene up-regulation markedly prevailed. Our RNA-seq data analysis enabled the identification of target genes for mechanism-based cell line engineering and bioprocess modification, an essential step to translate gene expression data from CHO-K1 host cells into bioprocess-related knowledge. Further efforts aim at increasing desirable phenotypes of CHO cells, and promoting efficient production of high quality therapeutic proteins can highly benefit from this type of studies.

Strategies to Develop Therapeutic N- and O-Hyperglycosylated Proteins.

Glycoengineering by N- and/or O-hyperglycosylation represents a procedure to introduce potential sites for adding N- and/or O-glycosyl structures to proteins with the aim of producing biotherapeutics with improved pharmacodynamic and pharmacokinetic properties. In this chapter, a detailed description of the steps routinely performed to generate new proteins having high content of N- and/or O-glycosyl moieties is carried out. The rational strategy involves the initial stage of designing N- and/or O-hyperglycosylated muteins to be expressed by mammalian cells and includes the upstream and downstream processing stages necessary to develop hyperglycosylated versions of the proteins of interest with the purpose of beginning the long road toward producing biobetters.

Strategy for erythroid differentiation in ex vivo cultures: Lentiviral genetic modification of human hematopoietic stem cells with erythropoietin gene.

If cultured in appropriate conditions, such as supplementing culture media with costly cytokines and growth factors, hematopoietic stem/progenitor cells (HSPCs) from different origins have shown to be an adequate source of erythroid cells. This requirement turns erythroid cells production into a complicated process to be scaled-up for future applications. The aim of our work was to genetically modify HSPCs with human erythropoietin (hEPO) sequence by lentiviral transgenesis in order for cells to secrete the hormone into the culture medium. Initially, we evaluated erythroid differentiation in colony forming units (CFU) assays and further analyzed cell expansion and erythroid differentiation throughout time in suspension cultures by flow cytometry and May-Grünwald-Giemsa staining. Additionally, we studied hEPO production and its isoforms profile. The different assessment approaches demonstrated erythroid differentiation, which was attributed to the hEPO secreted by the HSPCs. Our data demonstrate that it is possible to develop culture systems in which recombinant HSPCs are self-suppliers of hEPO. This feature makes our strategy attractive to be applied in biotechnological production processes of erythroid cells that are currently under development.

A highly efficient modified human serum albumin signal peptide to secrete proteins in cells derived from different mammalian species.

Signal peptides (SPs) are key elements in the production of recombinant proteins; however, little information is available concerning different SP in mammalian cells other than CHO. In order to study the efficiency of different SPs to direct the traffic along the secretory pathway of the green fluorescence protein (GFP) and a scFv-Fc fusion protein; CHO-K1, HEK293 and NS0 cell lines were transfected in a transient and stable way. SP of human azurocidin (AZ), modified human albumin (mSA), modified Cricetulus griseus Ig kappa chain V III region MOPC 63 like (mIgκ C) and modified human Ig kappa chain V III region VG (mIgκ H) were evaluated. The efficiency of SPs to translocate a propeptide across the ER membrane was evaluated by fluorescence microscopy and flow cytometry for the GFP inside the secretory pathway, and by antigen-specific indirect ELISA for the scFv-Fc outside the cell. The mSA SP was successful in directing the secretion of the active proteins in these different types of mammalian cells, regardless of the transgene copy number. The goal of this work was to demonstrate that a modified version of SA SP might be used in different mammalian cells employing the same expression vector.

Glycosylation and antiproliferative activity of hyperglycosylated IFN-α2 potentiate HEK293 cells as biofactories.

Both CHO and HEK cells are interesting hosts for the production of biotherapeutics due to their ability to introduce post-translational modifications such as glycosylation. Even though oligosaccharide structures attached to proteins are conserved among eukaryotes, many differences have been found between therapeutic glycoproteins expressed in hamster and human derived cells. In this work, a hyperglycosylated IFN-α2b mutein (IFN4N) was produced in CHO and HEK cell lines and an extensive characterization of their properties was performed. IFN4NCHO exhibited a higher average molecular mass and more acidic isoforms compared to IFN4NHEK. In agreement with these results, a 2-times higher sialic acid content was found for IFN4NCHO in comparison with the HEK-derived protein. This result was in agreement with monosaccharide quantification and glycan's analysis using WAX chromatography and HILIC coupled to mass spectrometry; all methods supported the existence of highly sialylated and also branched structures for IFN4NCHO glycans, in contrast with smaller and truncated structures among IFN4NHEK glycans. Unexpectedly, those remarkable differences in the glycosylation pattern had not a considerable impact on the clearance rate of both molecules in rats. In fact, although IFN4NHEK reached maximum plasma concentration 3-times faster than IFN4NCHO, their elimination profile did not differ significantly. Also, despite the in vitro antiviral specific biological activity of both proteins was the same, IFN4NHEK was more efficient as an antiproliferative agent in different tumor-derived cell lines. Accordingly, IFN4NHEK showed a higher in vivo antitumor activity in animal models. Our results show the importance of an appropriate host selection to set up a bioprocess and potentiate the use of HEK293 cells for the production of a new hyperglycosylated protein-based pharmaceutical.

Screening and characterization of molecules that modulate the biological activity of IFNs-I.

Type I Interferons (IFNs-I) are species-specific glycoproteins which play an important role as primary defence against viral infections and that can also modulate the adaptive immune system. In some autoimmune diseases, interferons (IFNs) are over-produced. IFNs are widely used as biopharmaceuticals for a variety of cancer indications, chronic viral diseases, and for their immuno-modulatory action in patients with multiple sclerosis; therefore, increasing their therapeutic efficiency and decreasing their side effects is of high clinical value. In this sense, it is interesting to find molecules that can modulate the activity of IFNs. In order to achieve that, it was necessary to establish a simple, fast and robust assay to analyze numerous compounds simultaneously. We developed four reporter gene assays (RGAs) to identify IFN activity modulator compounds by using WISH-Mx2/EGFP, HeLa-Mx2/EGFP, A549-Mx2/EGFP, and HEp2-Mx2/EGFP reporter cell lines (RCLs). All of them present a Z' factor higher than 0.7. By using these RGAs, natural and synthetic compounds were analyzed simultaneously. A total of 442 compounds were studied by the Low Throughput Screening (LTS) assay using the four RCLs to discriminate between their inhibitory or enhancing effects on IFN activity. Some of them were characterized and 15 leads were identified. Finally, one promising candidate with enhancing effect on IFN-α/-ß activity and five compounds with inhibitory effect were described.