Objective While the majority of current diabetes treatments focus on reducing blood glucose levels, hypoglycemia represents a significant risk associated with insulin treatment. Glucagon plays a major regulatory role in controlling hypoglycemia in vivo, but its short half-life and hyperglycemic effects prevent its therapeutic use for non-acute applications. The goal of this study was to identify a modified form of glucagon suitable for prophylactic treatment of hypoglycemia without increasing baseline blood glucose levels. Methodology/Principal Findings Through application of the XTEN technology, we report the construction of a glucagon fusion protein with an extended exposure profile (Gcg-XTEN). The in vivo half-life of the construct was tuned to support nightly dosing through design and testing in cynomolgus monkeys. Efficacy of the construct was assessed in beagle dogs using an insulin challenge to induce hypoglycemia. Dose ranging of Gcg-XTEN in fasted beagle dogs demonstrated that the compound was biologically active with a pharmacodynamic profile consistent with the designed half-life. Prophylactic administration of 0.6 nmol/kg Gcg-XTEN to dogs conferred resistance to a hypoglycemic challenge at 6 hours post-dose without affecting baseline blood glucose levels. Consistent with the designed pharmacokinetic profile, hypoglycemia resistance was not observed at 12 hours post-dose. Importantly, the solubility and stability of the glucagon peptide were also significantly improved by fusion to XTEN. Conclusions/Significance The data show that Gcg-XTEN is effective in preventing hypoglycemia without the associated hyperglycemia expected for unmodified glucagon. While the plasma clearance of this Gcg-XTEN has been optimized for overnight dosing, specifically for the treatment of nocturnal hypoglycemia, constructs with significantly longer exposure profiles are feasible. Such constructs may have multiple applications such as allowing for more aggressive insulin treatment regimens, treating hypoglycemia due to insulin-secreting tumors, providing synergistic efficacy in combination therapies with long-acting GLP1 analogs, and as an appetite suppressant for treatment of obesity. The improved physical properties of the Gcg-XTEN molecule may also allow for novel delivery systems not currently possible with native glucagon.

Increasing the in vivo residence times of protein therapeutics could decrease their dosing frequencies. We show that genetic fusion of an unstructured recombinant polypeptide of 864 amino acids, called XTEN, to a peptide or protein provides an apparently generic approach to extend plasma half-life. Allometric scaling suggests that a fusion of XTEN to the exenatide peptide should increase exenatide half-life in humans from 2.4 h to a projected time of 139 h. We confirmed the biological activity of the exenatide-XTEN fusion in mice. As extended stability might exacerbate undesirable side effects in some cases, we show that truncating the XTEN sequence can regulate plasma half-life. XTEN lacks hydrophobic amino acid residues that often contribute to immunogenicity and complicate manufacture. Based on data on XTEN fusions to exenatide, glucagon, GFP and human growth hormone, we expect that XTEN will enable dosing of otherwise rapidly cleared protein drugs at up to monthly intervals in humans.

Abstract c-Met is a receptor tyrosine kinase (RTK) that is activated upon binding to its only known ligand, hepatocyte growth factor (HGF). Although essential in development, aberrant c-Met signaling can lead to oncogenesis by promoting proliferation, survival, metastasis, and angiogenesis. Deregulated expression/activation of cMet is found in many neoplasms, and the linkage to disease severity and poor prognosis make this RTK an attractive candidate for targeted anticancer therapy. Challenges of inhibiting c-Met/HGF signaling with a classic antibody therapeutic approach have included: (1) the inhibition of receptor activation required more than one antibody, and (2) bivalent antibodies caused receptor activation. Using a novel non-antibody protein technology platform, we have identified potent polypeptide inhibitors of HGF-mediated c-Met activation. This non-antibody class of proteins, termed Avimer (avidity multimer) polypeptides, are based on naturally occurring domain (e.g., A-domain) containing proteins and can be generated using a phage display library and expressed in E. coli. c-Met antagonist Avimer polypeptides potently inhibit HGF-mediated receptor activation, migration, and proliferation in in vitro assays. Improvement in pharmacokinetic profile gained through attaching polyethylene glycol molecules to avimers enabled in vivo studies. In multiple preclinical tumor models (U87MG glioblastoma, KP-4 pancreatic, U118 glioblastoma), Avimer-treated animals exhibited reduced cMet activation in xenografts and significantly decreased tumor growth. These data indicate that the c-Met antagonist Avimer polypeptides are potent c-Met inhibitors and offer an alternative to small molecules or antibodies in targeting cMet/HGF pathway for anticancer therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B233.

Nat. Biotechnol. 23, 1556–1561 (2005) In the print version of this article and the version originally published online, the second author's name was spelled incorrectly. The second author is Q Liu, not Q Lu as originally indicated. This correction is appended to the PDF version.

Nat. Biotechnol. 23, 1556–1561 (2005) In the print version of this article and the version originally published online, the second author's name was spelled incorrectly. The second author is Q Liu, not Q Lu as originally indicated. This correction is appended to the PDF version.