Rational Design of a Fibroblast Growth Factor 21-Based Clinical Candidate, LY2405319

Abstract

Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of plasma glucose coupled with a 10–30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.

FGF21-receptor agonists: an emerging therapeutic class for obesity-related diseases

“…The native mature form of FGF21 is a 19.4 kDa protein that is cleared rapidly from circulation most likely through the kidney, and has an estimated half-life of ∼1 h in rodents and 0.5-2 h in NHPs [31], [68]. In addition to the rapid renal clearance, human FGF21 protein undergoes rapid proteolysis upon injection into animals so that the 4 N-terminal amino acid residues [69] and the C-terminal 10 amino acid residues are cleaved off [68]. In vitro studies using recombinant truncated proteins showed that while the 4 N-terminal amino acid residues are not necessary for the signaling activity, the C-terminal 10 amino acid residues are essential for binding to KLB and signaling activity [43], [70].…”

“…In addition to the poor pharmacokinetics, native FGF21 is unstable and prone to aggregation in solution [68], [69]. Various engineering approaches have been undertaken to enable a stable liquid formulation at high concentration with a low viscosity.…”

“…LY2405319 is a FGF21 analog, designed to enable large-scale production in the yeast Pichia pastoris and to reduce aggregation in solution [69]. This modified protein lacks the N-terminal four amino acid residues, His-Pro-IlePro, which are functionally dispensable and prone to proteolysis in yeast as well as in human serum.…”

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

“…Truncation of N termini (NT) by six or more residues, or C termini (CT) by two or more residues, decreased in vitro potency more than 10-fold, suggesting that potent functional activity was derived from both termini of FGF21 (Micanovic et al, 2009;Yie et al, 2009). In diabetic animal models and patients with type 2 diabetes, recombinant fibroblast growth factor 21 (rFGF21) and its analogs demonstrated dose-dependent reductions in low-density lipoprotein cholesterol, apolipoproteins, fasting triglycerides, fasting insulin, and body weight, as well as dose-dependent elevations of high-density lipoprotein cholesterol and adiponectin (Adams et al, 2013;Gaich et al, 2013;Kharitonenkov et al, 2013;Smith et al, 2013). Although dose-dependent reductions in blood glucose have been observed in diabetic rodent and nonhuman primate models, thus far only a trend toward lower fasting blood glucose has been observed in human clinical studies (Gaich et al, 2013).…”

Circulating FGF21 levels are related to nutritional status and metabolic but not hormonal disturbances in polycystic ovary syndrome

“…The clinical studies do not permit clarification whether FGF21 predisposes to the development of carbohydrate disturbances or is a mechanism of ineffective compensation. Some authors suggest the use of anti-FGF receptor 1 (FGFR1) antibodies (R1MAbs) that mimic the metabolic effects of FGF21 on obesity and type 2 diabetes treatment (23,24,25,26,27).…”