DEVELOPMENT AND QUALIFICATION OF BIOANALYTICAL METHODS FOR DEAMIDATED IFNβ-1a AND INVESTIGATION ABOUT THE MECHANISM OF ACTION

Interferon beta-1a (IFNβ-1a) is a recombinant IFNβ with the tradename Rebif involved in several biological activities. Recently, it has been reported that artificial deamidation of IFNb-1a increases its biological response. Given the therapeutical potential, an investigation on the deamidated variant has been carried out via different approaches to discover the mechanism underlying this biological effect. The antiviral and immunomodulatory activity of deamidated cytokine was assessed using two precise and accurate cell-based assays. As expected, deamidated IFNβ-1a showed an increase in the biological response and its canonical pathway and receptor binding affinity were thorough analysed. Deamidated interferon beta increases STAT1 phosphorylation and ISGs expression compared to its native form. A full in-depth analysis in receptor binding highlighted a change in receptor affinity in deamidated IFNβ-1a. In particular, deamidation destabilizes the interaction with IFNAR2 through a change in the H-bonds network, increasing the affinity to IFNAR1 and consequently to the whole receptor complex. The higher receptor binding and the consequent strong signaling and gene expression, explains the greater biological activity of the deamidated IFNβ-1a compared to the native fcorm. These results open new perspective on the therapeutic potential of this molecule which could have significant beneficial effects on patients with MS and beyond