Abstract
Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.
Highlights
Fibroblast growth factor 21 (FGF21) is a novel class of drug candidates for the treatment of metabolic disorders, including type 2 diabetes and obesity
FGF21 belongs to the FGF superfamily, the function of FGF21 is largely limited in metabolism ([1] Long, 2011 #387)
To extend the circulating half-life, FGF21 was fused to the Fc fragment of human immunoglobulin G1 (IgG1) comprising the hinge region, CH2 and CH3 domains
Summary
Fibroblast growth factor 21 (FGF21) is a novel class of drug candidates for the treatment of metabolic disorders, including type 2 diabetes and obesity. FGF21 regulates glucose homeostasis through a combination of multiple mechanisms. It stimulates glucose uptake in adipose tissues, suppresses glucose output in liver and preserves bcell mass and islet function in pancreas [2,4,8]. Systemic administration of FGF21 improves insulin sensitivity, spares insulin secretion and ameliorates hyperinsulinemia [5,9]. The mechanism by which FGF21 lowers plasma triglycerides is partly through inhibiting hepatic fatty acid synthesis, while increasing fatty acid oxidation. FGF21 inhibits the nuclear maturation of sterol regulatory element binding protein 1, a master lipogenic transcription factor, and thereby decreasing hepatic fatty acid and triglyceride synthesis and secretion [5,10]. The weight-loss effect of FGF21 is largely accomplished by regulating energy metabolism. Peripheral or central administration of FGF21 increases basal metabolic rate and locomotor activity without reducing food intake [5,12,13]
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