Rational design of a fibroblast growth factor 21-based clinical candidate, LY2405319.
Rational design of a fibroblast growth factor 21-based clinical candidate, LY2405319.
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DOI：
10.1371/journal.pone.0058575
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Moller DE
中科院分区：
文献类型：
--
作者：
Kharitonenkov A;Beals JM;Micanovic R;Strifler BA;Rathnachalam R;Wroblewski VJ;Li S;Koester A;Ford AM;Coskun T;Dunbar JD;Cheng CC;Frye CC;Bumol TF;Moller DE
成纤维细胞生长因子21是一种新的激素调节因子，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。人重组野生型FGF21 （FGF21）已被证明可以改善啮齿动物和非人灵长类动物的代谢紊乱。然而，FGF21作为药物的开发具有挑战性，需要对其氨基酸序列进行重组，以提高蛋白质表达和配方稳定性。在这里，我们报告了一种新的FGF21变体LY2405319的设计和表征。为了开发一种每日一次给药的潜在药物，在保存的多用途配方中，通过Leu118Cys和Ala134Cys突变在FGF21中引入了一个额外的二硫键。进一步优化FGF21，删除4个n端氨基酸His-Pro-Ile-Pro (HPIP)，进行蛋白水解裂解。此外，为了消除酵母中o链糖基化位点，引入了Ser167Ala突变，从而允许在毕赤酵母中大规模、均匀地生产蛋白质。总之，重组FGF21导致其生物制药性能的显著改善。这些变化的影响通过一组体外和体内试验进行了评估，证实LY2405319的生物学特性与FGF21基本相同。具体来说，在ob/ob和饮食诱导肥胖（DIO）小鼠中皮下给予LY2405319 7-14天，血糖降低25-50%，体重减轻10-30%。因此，LY2405319显示了启动临床项目所需的所有生物制药和生物学特性，该临床项目旨在验证外源性FGF21的施用会对人类疾病相关代谢参数产生影响的假设。
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.
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DOI：
10.1073/pnas.0701600104
发表时间：
2007-05-01
影响因子：
11.1
作者：
Ogawa, Yasushi;Kurosu, Hiroshi;Kuro-o, Makoto
通讯作者：
Kuro-o, Makoto
影响因子：
3.7
作者：
Fransson, J;Hallen, D;FlorinRobertsson, E
通讯作者：
FlorinRobertsson, E
影响因子：
5.6
作者：
Micanovic, Radmila;Raches, David W.;Kharitonenkov, Alexei
通讯作者：
Kharitonenkov, Alexei
影响因子：
5.6
作者：
Kharitonenkov, Alexei;Dunbar, James D.;Shanafelt, Armen B.
通讯作者：
Shanafelt, Armen B.
影响因子：
3.6
作者：
Itoh, Nobuyuki
通讯作者：
Itoh, Nobuyuki