Protein engineering strategies for sustained glucagon-like peptide-1 receptor-dependent control of glucose homeostasis

OBJECTIVE—We have developed a novel platform for display and delivery of bioactive peptides that links the biological properties of the peptide to the pharmacokinetic properties of an antibody. Peptides engineered in the MIMETIBODY platform have improved biochemical and biophysical properties that are quite distinct from those of Fc-fusion proteins. CNTO736 is a glucagon-like peptide 1 (GLP-1) receptor agonist engineered in our MIMETIBODY platform. It retains many activities of native GLP-1 yet has a significantly enhanced pharmacokinetic profile. Our goal was to develop a long-acting GLP-1 receptor agonist with sustained efficacy.RESEARCH DESIGN AND METHODS—In vitro and in vivo activity of CNTO736 was evaluated using a variety of rodent cell lines and diabetic animal models.RESULTS—Acute pharmacodynamic studies in diabetic rodents demonstrate that CNTO736 reduces fasting and postprandial glucose, decreases gastric emptying, and inhibits food intake in a GLP-1 receptor–specific manner. Reduction of food intake following CNTO736 dosing is coincident with detection of the molecule in the circumventricular organs of the brain and activation of c-fos in regions protected by the blood-brain barrier. Diabetic rodents dosed chronically with CNTO736 have lower fasting and postprandial glucose and reduced body weight.CONCLUSIONS—Taken together, our data demonstrate that CNTO736 produces a spectrum of GLP-1 receptor–dependent actions while exhibiting significantly improved pharmacokinetics relative to the native GLP-1 peptide.Drug development strategies for therapeutic peptides continue to be challenging despite advances in technologies such as pegylation and lipidation (1–4). Although important biological processes are regulated by peptides, successful development of peptide drugs has been limited and transformation of a metabolically labile peptide into a drug remains challenging. In contrast, considerable advances have been made in the development of antibody therapeutics (5,6). A technology that could link the activity of a target peptide with the pharmacokinetic characteristics of an antibody would be a valuable addition to tools available for drug discovery. To address this need, we developed the MIMETIBODY platform as a novel technology for the display and delivery of bioactive peptides. Using protein design tools, we linked an antibody Fc domain to a bioactive glucagon-like peptide 1 (GLP-1) analog and engineered the construct for optimal biochemical and biophysical properties.GLP-1 is a 30–amino acid peptide secreted from L-cells of the intestine following nutrient ingestion (7–10). GLP-1 is rapidly degraded in vivo with a half-life of <2 min and cleared via the kidney (11,12). When circulating glucose concentrations are elevated, GLP-1 increases insulin and decreases glucagon secretion from the pancreas and slows gastric emptying, thereby reducing glucose appearance in the circulation and enhancing glucose clearance from the circulation (13–15). In rodent models, GLP-1 expands β-cell mass via induction of β-cell proliferation and neogenesis and reduction of β-cell apoptosis (16–20). The cytoprotective actions of GLP-1 also promote survival of human islets (21,22). Furthermore, GLP-1 reduces food intake, and therapy with GLP-1 receptor agonists has been associated with weight loss in clinical studies (23,24). Thus, GLP-1 receptor agonists are attractive therapeutic candidates for the treatment of type 2 diabetes.CNTO736 is a GLP-1 receptor agonist engineered in our MIMETIBODY platform that incorporates a GLP-1 peptide analogue genetically fused to a domain that includes the Fc portion of an antibody (25,26). In addition to an amino acid substitution in the peptide rendering it resistant to dipeptidyl peptidase IV (27,28), the increased molecular weight and pharmacokinetic properties of an Fc were expected to enable sustained delivery of a GLP-1 receptor agonist. We demonstrate that CNTO736 dose-dependently increases cAMP and insulin secretion from islets in a glucose-dependent manner. In rodent models of type 2 diabetes, acute dosing with CNTO736 lowers fasting and postprandial blood glucose with a significantly longer duration of action than native GLP-1, and chronic dosing with CNTO736 decreases body weight. Although CNTO736 is a large molecule that is not likely to efficiently cross the blood-brain barrier, it can be detected in the circumventricular organs of the brain following peripheral dosing, and c-fos expression is detected in regions that are protected by the blood-brain barrier. Food intake is reduced in mice and rats following peripheral dosing with CNTO736, correlating with the appearance of the molecule in the hypothalamus. Hence, the generation of stable bioactive peptide therapeutics with optimized pharmacokinetic properties may provide a new option for the treatment of metabolic disorders.