Asn25 Deamidation as an Allosteric Tool to Increase IFNß-1a Biological Activity.

Interferon beta (IFNß) is a well-known cytokine, belonging to the type I family, that exerts antiviral, immunomodulatory, and antiproliferative activity. It has been reported that the artificially deamidated form of recombinant IFNß-1a at Asn25 position shows an increased biological activity. As a deepening of the previous study, the molecular mechanism underlying this biological effect was investigated in this work by combining experimental and computational techniques. Specifically, the binding to IFNAR1 and IFNAR2 receptors and the canonical pathway of artificially deamidated IFNß-1a molecule were analyzed in comparison to the native form. As a result, a change in receptor affinity of deamidated IFNß-1a with respect to the native form was observed, and to better explore this molecular interaction, molecular dynamics simulations were carried out. Results confirmed, as previously hypothesized, that the N25D mutation can locally change the interaction network of the mutated residue but also that this effect can be propagated throughout the molecule. In fact, many residues not involved in the interaction with IFNAR1 in the native form participate to the recognition in the deamidated molecule, enhancing the binding to IFNAR1 receptor and consequently an increase of signaling cascade activation. In particular, a higher STAT1 phosphorylation and interferon-stimulated gene expression was observed under deamidated IFNß-1a cell treatment. In conclusion, this study increases the scientific knowledge of deamidated IFNß-1a, deciphering its molecular mechanism, and opens new perspectives to novel therapeutic strategies.

A deamidated interferon-ß variant binds to integrin αvß3.

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-ß) from other type I interferons is the presence of theAsn-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 αvß3, αvß5, αvß6, αvß8 and α5ß1 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-ß variant to integrins. For this purpose, integrin αvß3 was selected as a reference model within the iso-DGR- integrin binding members. The obtained results show that deamidated IFN-ß binds integrin αvß3 with nanomolar affinity and that the response was dependent on the deamidation extent. Based on these results, it can be expected that deamidated IFN-ß 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-ß attributes that under physiological conditions may be modulated by the deamidation.

Demonstration of physicochemical and functional similarity between the proposed biosimilar adalimumab MSB11022 and Humira®.

Biosimilars are biological products that are highly similar to existing products approved by health authorities. Demonstration of similarity starts with the comprehensive analysis of the reference product and its proposed biosimilar at the physicochemical and functional levels. Here, we report the results of a comparative analysis of a proposed biosimilar adalimumab MSB11022 and its reference product, Humira®. Three batches of MSB11022 and up to 23 batches of Humira® were analyzed by a set of state-of-the-art orthogonal methods. Primary and higher order structure analysis included N/C-terminal modifications, molecular weight of heavy and light chains, C-terminal lysine truncation, disulfide bridges, secondary and tertiary structures, and thermal stability. Purity ranged from 98.4%-98.8% for MSB11022 batches (N = 3) and from 98.4%-99.6% for Humira® batches (N = 19). Isoform analysis showed 5 isoform clusters within the pI range of 7.94-9.14 and 100% glycan site occupancy for both MSB11022 and Humira®. Functional analysis included Fab-dependent inhibition of tumor necrosis factor (TNF)-induced cytotoxicity in L929-A9 cell line and affinity to soluble and transmembrane forms of TNF, as well as Fc-dependent binding to Fcγ and neonatal Fc receptors and C1q complement proteins. All tested physicochemical and functional parameters demonstrated high similarity of MSB11022 and Humira®, with lower variability between MSB11022 and Humira® batches compared with variability within individual batches of Humira®. Based on these results, MSB11022 is anticipated to have safety and efficacy comparable to those of Humira®.