An Interferon α2 Mutant Optimized by Phage Display for IFNAR1 Binding Confers Specifically Enhanced Antitumor Activities

Abstract

All ␣-interferons (IFN␣) bind the IFNAR1 receptor subunit with low affinity. Increasing the binding affinity was shown to specifically increase the antiproliferative potency of IFN␣2. Here, we constructed a phage display library by randomizing three positions on IFN␣2 previously shown to confer weak binding to IFNAR1. The tightest binding variant selected, comprised of mutations H57Y, E58N, and Q61S (YNS), was shown to bind IFNAR1 60-fold tighter compared with wild-type IFN␣2, and 3-fold tighter compared with IFN␤. Binding of YNS to IFNAR2 was comparable with wild-type IFN␣2. The YNS mutant conferred a 150-fold higher antiproliferative potency in WISH cells compared with wild-type IFN␣2, whereas its antiviral activity was increased by only 3.5-fold. The high antiproliferative activity was related to an induction of apoptosis, as demonstrated by annexin V binding assays, and to specific gene induction, particularly TRAIL. To determine the potency of the YNS mutant in a xenograft cancer model, we injected it twice a week to nude mice carrying transplanted MDA231 human breast cancer cells. After 5 weeks, no tumors remained in mice treated with YNS, whereas most mice treated with wild-type IFN␣2 showed visible tumors. Histological analysis of these tumors showed a significant antiangiogenic effect of YNS, compared with wild-type IFN␣2. This work demonstrates the application of detailed biophysical understanding in the process of protein engineering, yielding an interferon variant with highly increased biological potency. Type I interferons (IFNs)3 are multifunctional cytokines that orchestrate the antiviral innate immunity in vertebrates, acting in virtually every nucleated cell. They belong to the helical cytokine superfamily (1), and consist of at least 17 different members in humans: 13 different IFN␣ subtypes, IFN␤, IFN, and IFN, all of which share significant amino acid homology (2).All type I interferons induce their biological effects through a common receptor, composed of two distinct transmembrane proteins, namely IFNAR1 and IFNAR2 (3-5). Upon formation of the ternary complex, the interferon signal is transduced through receptor-associated Janus family kinases (JAK), which activate the signal transducers and activators of transcription (STAT) proteins. These, in turn, form homo-and heterodimers that translocate to the nucleus, where they directly regulate the transcription of specific interferon responsive genes (6).In addition to their important roles at the first line of defense against viral infections (7) and in modulation of the adaptive immune system (8), type I interferons are capable of inducing a significant antiproliferative activity (9). Consequently, interferons are widely used in the clinic for the treatment of multiple sclerosis and chronic hepatitis (10, 11), as well as for a number of malignancies (12). However, the efficiency of interferon treatment in the majority of cancers is modest, limited by side effects and varying resistance of malignant cells to this treatment (13,14).Despite their...

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