Renato Mastrangeli

Biological Functions of Interferon β-1a Are Enhanced By Deamidation

Human type I Interferons (IFN-β, IFN-ɛ, IFN-κ, IFN-ω, and 12 subtypes of IFN-α) are a family of pleiotropic cytokines with antiviral, antiproliferative, and immunomodulatory activities. They signal through the same cell surface receptors, IFNAR1 and IFNAR2, yet evoking markedly differential potency. One differentiating factor of IFN-β from other type I interferons is the presence of a consensus sequence (NG) for deamidation. Comparing almost completely deamidated IFN-β-1a with untreated IFN-β-1a, this present study reports the increased activities in 3 in-vitro bioassays testing the antiviral, antiproliferative, and immunomodulatory properties, respectively, of the molecule. Deamidated IFN-β-1a has the potential to improve current therapies in multiple sclerosis, and its ability to potentiate the MHC-Class I expression suggests a clinical benefit in diseases where the downmodulation of the MHC-class I expression plays a role (eg, in immuno-oncology combination therapies or antiviral agents). The present study on IFN-β deamidation adds a new prospective on deamidation as part of a posttranslational modification code that allows the modulation of the biological properties of proteins. Moreover, it underlines the unique IFN-β-1a properties that differentiate this molecule from other members of the type I interferon family.

In vitro biological characterization of IFN-β-1a major glycoforms

Recombinant human interferon β-1a (IFN-β-1a) is extensively used as the first-line treatment of relapsing forms of multiple sclerosis. Its glycosylation is recognized as having a complex impact on a wide range of molecule characteristics and functions. The present study reports the enrichment of IFN-β-1a glycoforms and their physicochemical and biological characterization by means of electrospray ionization-mass spectrometry, sialic acid content, thermal denaturation and various in vitro bioassays (antiproliferative, antiviral, immunomodulatory and reporter gene assay). The glycoforms were fractionated by means of cation-exchange chromatography using recombinant IFN-β-1a derived from Chinese Hamster Ovary cell culture as starting material. The obtained fractions contained bi- and higher-antennarity glycans as described in the European Pharmacopoeia monograph (Nr. 1639E, Interferon beta 1a concentrated solution). The in vitro bioassay responses revealed a correlation mainly with the glycan antennarity. It is therefore suggested that all glycoforms have biological activity and play a role in modulating the overall IFN-β biological activity with higher-antennarity glycoforms being able to better sustain IFN-β-1a bioactivity over time. These data indicate the role of IFN-β-1a glycosylation in vivo and shed new light on the role of the glycosylation heterogeneity, in particular with regard to antennarity, on biological properties of glycoproteins.

A deamidated interferon-β variant binds to integrin αvβ3

HIGHLIGHTSInterferon‐beta contains a sequence motif prone to deamidation.Deamidation generates the DGR and iso‐DGR motifs.Deamidated interferon‐beta binds integrin &agr;v&bgr;3 with nanomolar affinity. ABSTRACT Human type I interferons are a family of pleiotropic cytokines with antiviral, anti‐proliferative and immunomodulatory activities. They signal through the same cell surface receptors IFNAR1 and IFNAR2 yet evoking markedly different physiological effects. One differentiating factor of interferon‐beta (IFN‐&bgr;) from other type I interferons is the presence of the Asn‐Gly‐Arg (NGR) sequence motif, which, upon deamidation, converts to Asp‐Gly‐Arg (DGR) and iso‐Asp‐Gly‐Arg (iso‐DGR) motifs. In other proteins, the NGR and iso‐DGR motifs are reported as CD13‐ and &agr;v&bgr;3, &agr;v&bgr;5, &agr;v&bgr;6, &agr;v&bgr;8 and &agr;5&bgr;1 integrin‐binding motifs, respectively. The scope of this study was to perform exploratory surface plasmon resonance (SPR) experiments to assess the binding properties of a deamidated IFN‐&bgr; variant to integrins. For this purpose, integrin &agr;v&bgr;3 was selected as a reference model within the iso‐DGR‐ integrin binding members. The obtained results show that deamidated IFN‐&bgr; binds integrin &agr;v&bgr;3 with nanomolar affinity and that the response was dependent on the deamidation extent. Based on these results, it can be expected that deamidated IFN‐&bgr; also binds to other integrin family members that are able to bind to the iso‐DGR binding motif. The novel binding properties could help elucidate specific IFN‐&bgr; attributes that under physiological conditions may be modulated by the deamidation.

In-vivo biological activity and glycosylation analysis of a biosimilar recombinant human follicle-stimulating hormone product (Bemfola) compared with its reference medicinal product (GONAL-f)

Recombinant human follicle-stimulating hormone (r-hFSH) is widely used in fertility treatment. Although biosimilar versions of r-hFSH (follitropin alfa) are currently on the market, given their structural complexity and manufacturing process, it is important to thoroughly evaluate them in comparison with the reference product. This evaluation should focus on how they differ (e.g., active component molecular characteristics, impurities and potency), as this could be associated with clinical outcome. This study compared the site-specific glycosylation profile and batch-to-batch variability of the in-vivo bioactivity of Bemfola, a biosimilar follitropin alfa, with its reference medicinal product GONAL-f. The focus of this analysis was the site-specific glycosylation at asparagine (Asn) 52 of the α-subunit of FSH, owing to the pivotal role of Asn52 glycosylation in FSH receptor (FSHR) activation/signalling. Overall, Bemfola had bulkier glycan structures and greater sialylation than GONAL-f. The nominal specific activity for both Bemfola and GONAL-f is 13,636 IU/mg. Taking into account both the determined potency and the nominal amount the average specific activity of Bemfola was 14,522 IU/mg (105.6% of the nominal value), which was greater than the average specific activity observed for GONAL-f (13,159 IU/mg; 97.3% of the nominal value; p = 0.0048), although this was within the range stated in the product label. A higher batch-to-batch variability was also observed for Bemfola versus GONAL-f (coefficient of variation: 8.3% vs 5.8%). A different glycan profile was observed at Asn52 in Bemfola compared with GONAL-f (a lower proportion of bi-antennary structures [~53% vs ~77%], and a higher proportion of tri-antennary [~41% vs ~23%] and tetra-antennary structures [~5% vs <1%]). These differences in the Asn52 glycan profile might potentially lead to differences in FSHR activation. This, together with the greater bioactivity and higher batch-to-batch variability of Bemfola, could partly explain the reported differences in clinical outcomes. The clinical relevance of the differences observed between GONAL-f and Bemfola should be further investigated.

Glycoengineered Antibodies: Towards the Next-Generation of Immunotherapeutics

Monoclonal antibodies (mAbs) are currently the largest and fastest growing class of biopharmaceuticals, and they address unmet medical needs, e.g., in oncology and in auto-immune diseases. Their clinical efficacy and safety is significantly affected by the structure and composition of their glycosylation profile which is commonly heterogeneous, heavily dependent on the manufacturing process, and thus susceptible to variations in the cell culture conditions. Glycosylation is therefore considered a critical quality attribute for mAbs. Commonly, in currently marketed therapeutic mAbs, the glycosylation profile is suboptimal in terms of biological properties such as antibody-dependent cell-mediated cytotoxicity or may give rise to safety concerns due to the presence of non-human glycans. This article will review recent innovative developments in chemo-enzymatic glycoengineering, which allow generating mAbs carrying single, well-defined, uniform Fc glycoforms, which confers the desired biological properties for the target application. This approach offers significant benefits such as enhanced Fc effector functions, improved safety profiles, higher batch-to-batch consistency, decreased risks related to immunogenicity and manufacturing process changes, and the possibility to manufacture mAbs, in an economical manner, in non-mammalian expression systems. Overall, this approach could facilitate and reduce mAb manufacturing costs which in turn would translate into tangible benefits for both patients and manufacturers. The first glycoengineered mAbs are about to enter clinical trials and it is expected that, once glycoengineering reagents are available at affordable costs, and in-line with regulatory requirements, that targeted remodeling of antibody Fc glycosylation will become an integral part in manufacturing the next-generation of immunotherapeutics.

How unique is interferon-β within the type I interferon family?

All type I interferons share structural homology and bind to a common heterodimeric receptor consisting of the IFNAR1 and IFNAR2 subunits, which are expressed on most cell types. Although binding to the same receptor pair, they evoke a broad range of activities within the cell affecting the expression of numerous genes and resulting in profound cellular changes. Differential activation results from multiple levels of cellular and molecular events including binding affinity, receptor density, cell type-specific variations, and post-translational modification of signaling molecules downstream. Within the type I interferon family the Asn-Gly-Arg (NGR) sequence motif is unique to interferon-β and, together with its deamidated variants Asp-Gly-Arg (DGR) and iso-Asp-Gly-Arg (iso-DGR), imparts additional binding specificities that go beyond that of the canonical IFNAR1/IFNAR2. These warrant further investigations and functional studies and may eventually shed new light on differential effects observed for this molecule in oncology and autoimmune diseases.