FGF21 Analogs of Sustained Action Enabled by Orthogonal Biosynthesis Demonstrate Enhanced Antidiabetic Pharmacology in Rodents

Abstract

Fibroblast growth factor 21 (FGF21) mitigates many of the pathogenic features of type 2 diabetes, despite a short circulating half-life. PEGylation is a proven approach to prolonging the duration of action while enhancing biophysical solubility and stability. However, in the absence of a specific protein PEGylation site, chemical conjugation is inherently heterogeneous and commonly leads to dramatic loss in bioactivity. This work illustrates a novel means of specific PEGylation, producing FGF21 analogs with high specific activity and salutary biological activities. Using homology modeling and structure-based design, specific sites were chosen in human FGF21 for site-specific PEGylation to ensure that receptor binding regions were preserved. The in vitro activity of the PEGylated FGF21 ana-logs corresponded with the site of PEG placement within the binding model. Site-specific PEGylated analogs demonstrated dramatically increased circulating half-life and enhanced efficacy in db/db mice. Twice-weekly dosing of an optimal FGF21 analog reduced blood glucose, plasma lipids, liver triglycerides, and plasma glucagon and enhanced pancreatic insulin content, islet number, and glucose-dependent insulin secretion. Restoration of insulin sensitivity was demonstrated by the enhanced ability of insulin to induce Akt/protein kinase B phosphorylation in liver, muscle, and adipose tissues. PEGylation of human FGF21 at a specific and preferred site confers superior metabolic pharmacology.

Development of a Novel Long-Acting Antidiabetic FGF21 Mimetic by Targeted Conjugation to a Scaffold Antibody

“…Together, the data suggest that the durable effects of CVX-343 may partially rely on sustained alterations at the transcriptional level after ligand binding and receptor signaling. In mice treated with CVX-343, insulin staining and proliferative state of b cells were increased, consistent with previous reports Mu et al, 2012), suggesting that the antidiabetic effects of FGF21 mimetics in vivo may be effected not only through liver and adipose but also by enhancing b-cell secretory capacity. FGF21 was recently reported to regulate bone homeostasis and to mediate PPARg actions in mice Wei et al, 2012).…”

“…A number of midprotein sitespecific PEGylated FGF21 molecules have also been characterized, all of which had reduced in vitro potency compared with the native protein (Mu et al, 2012). As with application of the CovX technology, these PEGylated FGF21 variants had improved half-lives ranging ∼15-34 hours.…”

“…In the case of FGF21, however, N or C terminal genetic fusion may impair the bioactivity of the protein, as the termini have been shown to be important for biologic function (Micanovic et al, 2009;Yie et al, 2009). Polyethylene glycol (PEG)ylation has also been used to improve the pharmacokinetic property of proteins and peptides, although PEGylated FGF21 exhibits lower intrinsic potency compared with the wild-type (WT) protein (Mu et al, 2012). We aimed to create a long-acting FGF21 molecule with comparable in vitro potency and full physiologic efficacy of WT FGF21 protein using CovX conjugation technology.…”

Mechanistic Investigation of the Preclinical Pharmacokinetics and Interspecies Scaling of PF-05231023, a Fibroblast Growth Factor 21–Antibody Protein Conjugate

“…Site-specific conjugation of 30-kDa polyethylene glycol to FGF21 demonstrated 10-to 50-fold improvements in CL/F compared with rFGF21, albeit at the expense of 5-to 10-fold reductions in in vitro potency (Mu et al, 2012). Fusion of NT (Fc-FGF21) or CT of FGF21 (FGF21-Fc) to the Fc fragment of hIgG 1 right-shifted in vitro potency 2-to 4-fold and 1000-fold, respectively (Hecht et al, 2012).…”

Therapeutic opportunities for alcoholic steatohepatitis and nonalcoholic steatohepatitis: exploiting similarities and differences in pathogenesis

“…Furthermore, FGF21 leads to a decrease in free fatty acids by inhibiting lipolysis in adipose tissue (167). FGF21 has been reported to attenuate hepatic steatosis in animal models and therefore may serve as a novel therapeutic approach (168)(169)(170)(171)(172); pegylated FGF21 (BMS-986036) is currently under investigation in NASH patients (NCT02413372).…”