An interferon α2 mutant optimized by phage display for IFNAR1 binding confers specifically enhanced antitumor activities

All alpha-interferons ( IFN alpha) bind the IFNAR1 receptor subunit with low affinity. Increasing the binding affinity was shown to specifically increase the antiproliferative potency of IFN alpha 2. Here, we constructed a phage display library by randomizing three positions on IFN alpha 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 alpha 2, and 3-fold tighter compared with IFN beta. Binding of YNS to IFNAR2 was comparable with wild-type IFN alpha 2. The YNS mutant conferred a 150-fold higher antiproliferative potency in WISH cells compared with wild-type IFN alpha 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 alpha 2 showed visible tumors. Histological analysis of these tumors showed a significant anti-angiogenic effect of YNS, compared with wild-type IFN alpha 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.