title: An interferon alpha2 mutant optimized by phage display for IFNAR1 binding confers specifically enhanced antitumor activities.
abstract: All alpha-interferons (IFNalpha) bind the IFNAR1 receptor subunit with low affinity. Increasing the binding affinity was shown to specifically increase the antiproliferative potency of IFNalpha2. Here, we constructed a phage display library by randomizing three positions on IFNalpha2 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 IFNalpha2, and 3-fold tighter compared with IFNbeta. Binding of YNS to IFNAR2 was comparable with wild-type IFNalpha2. The YNS mutant conferred a 150-fold higher antiproliferative potency in WISH cells compared with wild-type IFNalpha2, 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 IFNalpha2 showed visible tumors. Histological analysis of these tumors showed a significant anti-angiogenic effect of YNS, compared with wild-type IFNalpha2. This work demonstrates the application of detailed biophysical understanding in the process of protein engineering, yielding an interferon variant with highly increased biological potency.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel.
authors.forename: Eyal
authors.initials: E
authors.lastname: Kalie
authors.forename: Diego A
authors.initials: DA
authors.lastname: Jaitin
authors.forename: Renne
authors.initials: R
authors.lastname: Abramovich
authors.forename: Gideon
authors.initials: G
authors.lastname: Schreiber
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000627:therapeutic use*; D016899:Interferon-beta / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008819:Mice, Nude; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D019151:Peptide Library; D010766:Phosphorylation; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D053633:Receptor, Interferon alpha-beta / Q000378:metabolism*; D050791:STAT Transcription Factors / Q000378:metabolism; D015398:Signal Transduction; D015533:Transcriptional Activation; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D016898:Interferon-alpha; D019151:Peptide Library; D021122:Protein Subunits; D050791:STAT Transcription Factors; D053633:Receptor, Interferon alpha-beta; D016899:Interferon-beta