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.
Highlights
Since the initial identification of FGF21 as a novel metabolic regulator [1], this protein has become the focus of intense research in the area of glucose and lipid homeostasis and an important target for drug discovery initiatives [2]
The strategic intent of our FGF21 biopharmaceutical re-engineering efforts were driven by the desire to create a stable, concentrated, and preserved solution preparation, while maintaining a favorable bioactivity profile that was amenable to a wide range of dosing demands in future clinical trials and potentially broader populations of patients
Adequate and effective treatment of the Type 2 diabetes (T2D) patient population with a protein therapy amenable to once-daily (QD) administration requires the development of a multi-use preserved formulation
Summary
Since the initial identification of FGF21 as a novel metabolic regulator [1], this protein has become the focus of intense research in the area of glucose and lipid homeostasis and an important target for drug discovery initiatives [2]. The potential metabolic benefits of FGF21 pharmacology in preclinical models are striking and provide a compelling rationale for ongoing life sciences research. Triglyceride lowering and a beneficial shift in plasma lipoprotein profiles suggest the potential of an FGF21-based therapy for amelioration of cardiovascular disease risk [2]. As the Type 2 diabetic patient population is exponentially growing [3], there was a need to select a cost-effective scalable expression platform and purification manufacturing process for an FGF21-based protein. The strategic intent of our FGF21 biopharmaceutical re-engineering efforts were driven by the desire to create a stable, concentrated, and preserved solution preparation, while maintaining a favorable bioactivity profile that was amenable to a wide range of dosing demands in future clinical trials and potentially broader populations of patients
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