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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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Interactions of peptides and proteins with inorganic surfaces are important to both natural and artificial systems; however, a detailed understanding of such interactions is lacking. In this study, we applied new approaches to quantitatively measure the binding of amino acids and proteins to gold surfaces. Real-time surface plasmon resonance (SPR) measurements showed that TEM1-β-lactamase inhibitor protein (BLIP) interacts only weakly with Au nanoparticles (NPs). However, fusion of three histidine residues to BLIP (3H-BLIP) resulted in a significant increase in the binding to the Au NPs, which further increased when the histidine tail was extended to six histidines (6H-BLIP). Further increasing the number of His residues had no effect on the binding. A parallel study using continuous (111)-textured Au surfaces and single-crystalline, (111)-oriented, Au islands by ellipsometry, FTIR, and localized surface plasmon resonance (LSPR) spectroscopy further confirmed the results, validating the broad applicability of Au NPs as model surfaces. Evaluating the binding of all other natural amino acid homotripeptides fused to BLIP (except Cys and Pro) showed that aromatic and positively-charged residues bind preferentially to Au with respect to small aliphatic and negatively charged residues, and that the rate of association is related to the potency of binding. The binding of all fusions was irreversible. These findings were substantiated by SPR measurements of synthesized, free, soluble tripeptides using Au-NP-modified SPR chips. Here, however, the binding was reversible allowing for determination of binding affinities that correlate with the binding potencies of the related BLIP fusions. Competition assays performed between 3H-BLIP and the histidine tripeptide (3 His) suggest that Au binding residues promote the adsorption of proteins on the surface, and by this facilitate the irreversible interaction of the polypeptide chain with Au. The binding of amino acids to Au was simulated by using a continuum solvent model, showing agreement with the experimental values. These results, together with the observed binding potencies and kinetics of the BLIP fusions and free peptides, suggest a binding mechanism that is markedly different from biological protein-protein interactions.

We have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs) enabling the study of type I human interferons (Hu-IFN-Is) in mice. These “HyBNAR” (Hybrid IFNAR) mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2). HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase). Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFNα2A, IFNβ, and a human superagonist termed YNSα8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC) injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mice provides a convenient non-primate pre-clinical model to advance the study of human IFN-Is.

Background: IFN␤ constitutes an approved drug to treat multiple sclerosis (MS), but it has limited efficacy. Results: A modified human IFN variant, which exhibits both superagonist properties and 10-fold increased lifespan, outperforms IFN␤ in an animal MS model. Conclusion:This drug candidate has potential to supersede IFN␤ for the treatment of MS. Significance: Protein engineering allows development of more effective drugs to treat autoimmune diseases.

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