Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana, United States of America.
PLoS One. 2013;8(3):e58575. doi: 10.1371/journal.pone.0058575. Epub 2013 Mar 11.
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.
成纤维细胞生长因子 21 是一种新型的激素调节剂，具有治疗多种代谢异常的潜力，如 2 型糖尿病、肥胖、肝脂肪变性和心血管疾病。人类重组野生型 FGF21（FGF21）已被证明可改善啮齿动物和非人类灵长类动物的代谢紊乱。然而，FGF21 的开发具有挑战性，需要对其氨基酸序列进行重新设计，以提高蛋白质表达和配方稳定性。在这里，我们报告了一种新型 FGF21 变体 LY2405319 的设计和特性。为了使具有每日一次给药方案的潜在药物产品得以开发，并在保存的、多用途的制剂中使用，通过 Leu118Cys 和 Ala134Cys 突变在 FGF21 中引入了一个额外的二硫键。通过删除四个 N 端氨基酸 His-Pro-Ile-Pro（HPIP）进一步优化了 FGF21，该氨基酸易受蛋白水解切割。此外，为了消除酵母中的一个 O-连接糖基化位点，引入了 Ser167Ala 突变，从而允许在毕赤酵母中进行大规模、均质的蛋白质生产。总之，FGF21 的重新设计使其生物制药特性得到了显著改善。在一系列体外和体内试验中评估了这些变化的影响，这些试验证实了 LY2405319 的生物学特性与 FGF21 基本相同。具体而言，在 7-14 天内，通过皮下给予 ob/ob 和饮食诱导肥胖（DIO）小鼠 LY2405319，可使血浆葡萄糖降低 25-50%，体重降低 10-30%。因此，LY2405319 表现出启动临床计划所需的所有生物制药和生物学特性，该计划旨在测试外源性 FGF21 给药是否会对人类相关代谢参数产生影响的假设。
