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

IFNß (recombinant interferon Beta) has been widely used for the treatment of Multiple sclerosis for the last four decades. Despite the human origin of the IFNß sequence, IFNß is immunogenic, and unwanted immune responses in IFNß-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 IFNß-1a. Two de-immunized versions of IFNß-1a were produced in CHO cells and designated as IFNß-1a(VAR1) and IFNß-1a(VAR2). First, the secondary and tertiary protein structures were analyzed by circular dichroism spectroscopy. Then, the variants were also tested for functionality. While IFNß-1a(VAR2) showed similar in vitro antiviral activity to the original protein, IFNß-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.

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.

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.

Development of highly stable and de-immunized versions of recombinant alpha interferon: Promising candidates for the treatment of chronic and emerging viral diseases.

Human interferon alpha (hIFN-α) administration constitutes the current FDA approved therapy for chronic Hepatitis B and C virus infections. Additionally, hIFN-α treatment efficacy was recently demonstrated in patients with COVID-19. Thus, hIFN-α constitutes a therapeutic alternative for those countries where vaccination is inaccessible and for people who did not respond effectively to vaccination. However, hIFN-α2b exhibits a short plasma half-life resulting in the occurrence of severe side effects. To optimize the cytokine's pharmacokinetic profile, we developed a hyperglycosylated IFN, referred to as GMOP-IFN. Given the significant number of reports showing neutralizing antibodies (NAb) formation after hIFN-α administration, here we applied the DeFT (De-immunization of Functional Therapeutics) approach to develop functional, de-immunized versions of GMOP-IFN. Two GMOP-IFN variants exhibited significantly reduced ex vivo immunogenicity and null antiproliferative activity, while preserving antiviral function. The results obtained in this work indicate that the new de-immunized GMOP-IFN variants constitute promising candidates for antiviral therapy.

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.

The glycosylation of anti-rhIFN-α2b recombinant antibodies influences the antigen-neutralizing activity.

OBJECTIVES: The influence of glycosylation on the antigen-neutralizing ability of two potential biotherapeutic anti-human IFN-α2b antibodies composed by murine and humanized single-chain Fv fused to human Fcγ1 (chimeric and humanized scFv-Fc, respectively) was studied. RESULTS: Chimeric antibodies produced in CHO-K1 and HEK293 mammalian cells showed no differences in the antigen-antibody affinity but demonstrated differences in the in vitro neutralization of IFN-α2b activity. On the other hand, the humanized antibodies produced in the same cell types showed differences in both the antigen-antibody affinity and the antigen-neutralizing ability. These differences are due to the scFv domain, as evidenced by its expression in CHO-K1 and HEK293 cells. In order to determine if the Fc glycosylation influences the antigen binding ability, both parameters were analyzed on chimeric and humanized deglycosylated scFv-Fc. Surprisingly, no differences in the antigen-antibody affinity were observed, but differences in the antigen-neutralizing ability of both chimeric and humanized antibodies, and their respectively deglycosylated glycoforms were found. CONCLUSIONS: Fc glycosylation influences the antigen neutralization ability of two anti-rhIFN-α2b recombinant antibodies. Although affinity is the widely accepted parameter to analyze antibody antigen binding, it does not appear to be sufficient to describe the behavior of recombinant antibodies in vitro. This work contributes with a high impact knowledge to develop therapeutic recombinant antibodies where glycosylation and producer cell lines must be taken into account for their influence on the antigen binding capacity and not only for their impact on the effector properties as it has been historically considered for antibodies.

Design and validation of an immuno-PCR assay for IFN-α2b quantification in human plasma.

Aim: Nowadays, IFN-α is considered a promising therapeutic target for systemic lupus erythematosus. An immuno-PCR (iPCR) was developed to quantify low amounts of IFN-α in human plasma followed by a deep analysis of the methodologic robustness throughout quality by design approach. Results: An accurate, sensitive, selective and versatile iPCR was validated. The critical iPCR procedural steps were identified, applying a Plackett-Burman design. Also, this assay demonstrated an outstanding LOD of 0.3 pg/ml. A significant aspect relies on its high versatility to detect and quantify other cytokines in human plasma as the appropriate biotinylated antibody is employed. Conclusion: This reliable iPCR assay can be clinically used as an alternative method for quantitating and detecting low IFN-α2b concentrations in human plasma samples.

Production of Therapeutic Enzymes by Lentivirus Transgenesis.

Since ERT for several LSDs treatment has emerged at the beginning of the 1980s with Orphan Drug approval, patients' expectancy and life quality have been improved. Most LSDs treatment are based on the replaced of mutated or deficient protein with the natural or recombinant protein.One of the main ERT drawback is the high drug prices. Therefore, different strategies trying to optimize the global ERT biotherapeutic production have been proposed. LVs, a gene delivery tool, can be proposed as an alternative method to generate stable cell lines in manufacturing of recombinant proteins. Since LVs have been used in human gene therapy, clinical trials, safety testing assays and procedures have been developed. Moreover, one of the main advantages of LVs strategy to obtain manufacturing cell line is the short period required as well as the high protein levels achieved.In this chapter, we will focus on LVs as a recombinant protein production platform and we will present a case study that employs LVs to express in a manufacturing cell line, alpha-Galactosidase A (rhαGAL), which is used as ERT for Fabry disease treatment.

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.

High yield process for the production of active human α-galactosidase a in CHO-K1 cells through lentivirus transgenesis.

Fabry disease is an X-linked recessive disorder caused by a deficiency in lysosomal α-Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high price. Selection of adequate high-expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α-Galactosidase A (rhαGAL) in Chinese hamster ovary (CHO-K1) cells. Herein, we describe for the first time the application of a strategy based on third-generation lentiviral particles (LP) transduction of suspension CHO-K1 cells to obtain high-producing rhαGAL clones (3.5 to 59.4 pg cell-1 d-1 ). After two purification steps, the active enzyme was recovered (2.4 × 106 U mg-1 ) with 98% purity and 60% overall yield. Michaelis-Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU-α-Gal at a comparable rate to Fabrazyme®, the current CHO-derived ERT available for Fabry disease. In addition, rhαGAL presented the same mannose-6-phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1334-1345, 2017.

An unusual cysteine VL87 affects the antibody fragment conformations without interfering with the disulfide bond formation.

The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: VH22 and VH92 in the variable heavy chain (VH) and VL23, VL87 and VL88 in the variable light chain (VL). The influence of two unusual contiguous Cys at positions VL87 and VL88 was studied by considering the wild type fragment and mutant variants: VL-C88S, VL-C87S, VL-C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that VL-C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the VL-C87 by a usual residue at this position like Tyr caused distant structural changes at the VH region that confers a higher mobility to the VH-CDR2 and VH-CDR3 loops improving the scFv binding to the antigen.

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.

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.

Development of Rabies Virus-Like Particles for Vaccine Applications: Production, Characterization, and Protection Studies.

Rabies is a viral infection of the central nervous system for which vaccination is the only treatment possible. Besides preexposure, vaccination is highly recommended for people living in endemic areas, veterinarians, and laboratory workers. Our group has developed rabies virus-like particles (RV-VLPs) with immunogenic features expressed in mammalian cells for vaccine applications. In this chapter the methods to obtain and characterize a stable HEK293 cell line expressing RV-VLPs are detailed. Further, analytical ultracentrifugation steps to purify the obtained VLPs are developed, as well as western blot, dynamic light scattering, and immunogold electron microscopy to analyze the size, distribution, shape, and antigenic conformation of the purified particles. Finally, immunization protocols are described to study the immunogenicity of RV-VLPs.

Immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate.

Rabies is one of the most lethal infectious diseases in the world, with a mortality approaching 100%. There are between 60,000 and 70,000 reported annual deaths, but this is probably an underestimation. Despite the fact that there are vaccines available for rabies, there is a real need of developing more efficacious and cheaper vaccines. This is particularly true for veterinary vaccines because dogs are still the main vector for rabies transmission to human beings. In a previous work, we described the development and characterization of rabies virus-like particles (RV-VLPs) expressed in HEK293 cells. We showed that RV-VLPs are able to induce a specific antibodies response. In this work, we show that VLPs are able to protect mice against virus challenge. Furthermore, we developed a VLPs expressing HEK-293 clone (sP2E5) that grows in serum free medium (SFM) reaching high cell densities. sP2E5 was cultured in perfusion mode in a 5 L bioreactor for 20 days, and the RV-VLPs produced were capable of triggering a protective immune response without the need of concentration or adjuvant addition. Further, these VLPs are able to induce the production of rabies virus neutralizing antibodies. These results demonstrate that RV-VLPs are a promising rabies vaccine candidate.

Target cells for antibodies detection in rabies vaccine control.

Rabies causes one of the most lethal zoonotic diseases, with more than 55,000 deaths reported annually. Prevention is based on pre-exposure vaccination of individuals at high risk of contracting rabies, and mass vaccination of dog, which are the main vector for transmission to humans. Post-exposure prophylaxis includes vaccination and rabies immunoglobulins treatment. The measurement of neutralizing antibodies in sera of vaccinated individuals is a primary concern to determine the efficacy of immunization schedules or the potency of new vaccines. Antibodies against rabies glycoprotein are considered an ideal indicator. In this work we showed the development of a VERO clone that is able to detect by fluorescence microscopy and flow cytometry, the presence of antibodies against the rabies glycoprotein, specifically in its native conformation anchored in the plasma membrane. These cells could trigger the development of a new rapid method for the detection of rabies virus neutralizing antibodies in vaccinated individuals.

High performance collection of cerebrospinal fluid in rats: evaluation of erythropoietin penetration after osmotic opening of the blood-brain barrier.

An important issue to be considered when studying a new drug for treatment of central nervous system (CNS) diseases is its ability to cross the blood-brain barrier (BBB) and distribute throughout the brain. As cerebrospinal fluid (CSF) has demonstrated to be an invaluable reservoir to study CNS availability of therapeutic proteins, we have developed an improved method for CSF sampling from the cisterna magna of rats. The technique enables the simple and rapid collection of adequate quantities (50-75 µl) of blood-free CSF, rendering a high percentage of animal survival (99%) without clinic or neurological consequences. Its success in avoiding blood contamination of CSF lays in the use of a mixture of lidocaine/ephinephrine topically injected in the rat's suboccipital area and neck. Another relevant feature of the methodology is its low cost, since the puncture device can be easily assembled with cheap and available materials and, more importantly, neither expensive stereotaxic equipment nor frame is required. The present method is demonstrated by studying the CSF pharmacokinetics of recombinant human erythropoietin (rhEPO), a well-studied therapeutic candidate for neurological diseases. Moreover, we applied this technique to evaluate a strategy of osmotic disruption of the BBB to achieve a faster delivery of rhEPO into the CNS.

A versatile ionic strength sensitive tag from a human GM-CSF-derived linear epitope.

A 7-mer hGM-CSF-derived linear epitope (APARSPS) is herein described as a novel and small tag taking into account its particular binding affinity in native conditions that could be easily modified by increasing or lowering the ionic strength. Thus, a 3.4 or 3.8-fold binding increment was observed in high NaCl concentration when the tag was fused to IFN-α2b or when the peptide was in its native environment, respectively. The high salt concentration increased the affinity of the binding interaction and improved the APARSPS epitope binding to the paratope allowing one to design an immunoaffinity chromatography purification step in which the high ionic strength was useful to adsorb the fusion protein to the immunoaffinity matrix and the low ionic strength at pH 9 was valuable to desorb it (a 470-fold purification with a 94%-purity was attained in only one step). Also, this short tag did not affect the functionality of the fusion protein and it was able to be detected both in the natural molecule (hGM-CSF) as in the tagged one with the same high detection limit: 273pg of each protein.