title: 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.
authors.affiliation: Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana, United States of America. a.kharch@lilly.com
authors.forename: Alexei
authors.initials: A
authors.lastname: Kharitonenkov
authors.forename: John M
authors.initials: JM
authors.lastname: Beals
authors.forename: Radmila
authors.initials: R
authors.lastname: Micanovic
authors.forename: Beth A
authors.initials: BA
authors.lastname: Strifler
authors.forename: Radhakrishnan
authors.initials: R
authors.lastname: Rathnachalam
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Wroblewski
authors.forename: Shun
authors.initials: S
authors.lastname: Li
authors.forename: Anja
authors.initials: A
authors.lastname: Koester
authors.forename: Amy M
authors.initials: AM
authors.lastname: Ford
authors.forename: Tamer
authors.initials: T
authors.lastname: Coskun
authors.forename: James D
authors.initials: JD
authors.lastname: Dunbar
authors.forename: Christine C
authors.initials: CC
authors.lastname: Cheng
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Frye
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Bumol
authors.forename: David E
authors.initials: DE
authors.lastname: Moller
mesh_terms: D016475:3T3 Cells; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D015195:Drug Design; D005346:Fibroblast Growth Factors / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D015870:Gene Expression; D014644:Genetic Variation; D056945:Hep G2 Cells; D006801:Humans; D000090265:Klotho Proteins; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins; D013696:Temperature
chemical_list: C418622:Klb protein, mouse; C585266:LY2405319; D008565:Membrane Proteins; D011994:Recombinant Proteins; C414620:fibroblast growth factor 21; D005346:Fibroblast Growth Factors; D000090265:Klotho Proteins
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