A Glycoengineered Interferon-β Mutein (R27T) Generates Prolonged Signaling by an Altered Receptor-Binding Kinetics

Abstract

The glycoengineering approach is used to improve biophysical properties of protein-based drugs, but its direct impact on binding affinity and kinetic properties for the glycoengineered protein and its binding partner interaction is unclear. Type I interferon (IFN) receptors, composed of IFNAR1 and IFNAR2, have different binding strengths, and sequentially bind to IFN in the dominant direction, leading to activation of signals and induces a variety of biological effects. Here, we evaluated receptor-binding kinetics for each state of binary and ternary complex formation between recombinant human IFN-β-1a and the glycoengineered IFN-β mutein (R27T) using the heterodimeric Fc-fusion technology, and compared biological responses between them. Our results have provided evidence that the additional glycan of R27T, located at the binding interface of IFNAR2, destabilizes the interaction with IFNAR2 via steric hindrance, and simultaneously enhances the interaction with IFNAR1 by restricting the conformational freedom of R27T. Consequentially, altered receptor-binding kinetics of R27T in the ternary complex formation led to a substantial increase in strength and duration of biological responses such as prolonged signal activation and gene expression, contributing to enhanced anti-proliferative activity. In conclusion, our findings reveal N-glycan at residue 25 of R27T is a crucial regulator of receptor-binding kinetics that changes biological activities such as long-lasting activation. Thus, we believe that R27T may be clinically beneficial for patients with multiple sclerosis.

Emerging glyco‐based strategies to steer immune responses

“…Interesting in this respect are cytokines, whose glycosylation status regulates receptor binding, biological stability, and function [ 134 ]. Glycoengineered versions of interferon‐α and ‐β have been developed that show enhanced pharmacokinetic properties and prolonged signaling capacities [ 135 , 136 ]. Glycosylated forms of interferon‐β are currently used in the clinic for the treatment multiple sclerosis [ 137 ].…”

Antibody-Based Targeting of Interferon-Beta-1a Mutein in HER2-Positive Cancer Enhances Antitumor Effects Through Immune Responses and Direct Cell Killing

“…In our previous studies, we developed a glycoengineered variant of recombinant human IFN-β-1a, IFN-β-R27T, which has two N-glycosylation sites at the 80th (original site) and an additional one at the 25th amino acid residue due to a mutation of Thr to Arg at position 27 of IFN-β ( Song et al, 2014 ). IFN-β-R27T exhibited superior stability, solubility, productivity, and pharmacokinetic properties compared to wild-type IFN-β-1a ( Lee et al, 2019 ; Song et al, 2020 ). Herein, we constructed fusion proteins consisting of an anti-HER2 antibody (trastuzumab) with IFN-β mutein and investigated its antitumor effect on a HER2-positive model.…”

Glycosylation Heterogeneity of Hyperglycosylated Recombinant Human Interferon-β (rhIFN-β)

“…Surprisingly, rhIFN-β containing a larger proportion of higher antennary glycoforms showed more sustained bioactivity over time (Mastrangeli et al, 2015). Indeed, in our previous study, R27T exhibited more prolonged signaling than the mono-glycosylated rhIFN-β, with altered receptor-binding kinetics (Lee et al, 2018). A larger portion of higher antennary components in R27T may therefore influence cellular signaling effects.…”

“…To address these issues, in our previous study, an additional single-glycosylation site was introduced at amino acid 25 in rhIFN-β 1a, resulting in R27T in which Arg at position 27 is mutated to Thr (Song et al, 2014). The additional glycosylation site increases the half-life and in vitro biological activity, as well as thermostability, allowing less frequent dosing (Lee et al, 2018;Song et al, 2014).…”