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Patent: Fusion Proteins for Treating Metabolic Disorders

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Patent: Fusion Proteins for Treating Metabolic Disorders University of Dayton eCommons Chemistry Faculty Publications Department of Chemistry 3-28-2013 Patent: Fusion Proteins for Treating Metabolic Disorders Brian R. Boettcher Shari L. Caplan Douglas S. Daniels Norio Hamamatsu Stuart Licht See next page for additional authors Follow this and additional works at: https://ecommons.udayton.edu/chm_fac_pub Part of the Other Chemistry Commons, and the Physical Chemistry Commons Author(s) Brian R. Boettcher, Shari L. Caplan, Douglas S. Daniels, Norio Hamamatsu, Stuart Licht, and Stephen Craig Weldon US 2013 0079500A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/007.9500 A1 Boettcher et al. (43) Pub. Date: Mar. 28, 2013 (54) FUSION PROTEINS FORTREATING (22) Filed: Sep. 25, 2012 METABOLIC DSORDERS Related U.S. Application Data (71) Applicants: Brian R. Boettcher, Winchester, MA (US); Shari L. Caplan, Lunenburg, MA (60) gyal application No. 61/539,280, filed on Sep. (US); Douglas S. Daniels, Arlington, s MA (US); Norio Hamamatsu, Belmont, Publication Classificati MA (US): Stuart Licht, Cambridge, MA DCOSSO (US); Stephen Craig Weldon, (51) Int. Cl. Leominster, MA (US) C07K 9/00 (2006.01) (72) Inventors: Brian R. Boettcher, Winchester, MA (52) t l. 530/387.3 (US); Shari L. Caplan, Lunenburg, MA ' ' '''''''''''''''''''" (US); Douglas S. Daniels, Arlington, (57) ABSTRACT MA (US); Norio Hamamatsu, Belmont, MA (US): Stuart Licht, Cambridge, MA The invention relates to the identification of fusion proteins (US); Stephen Craig Weldon, comprising polypeptide and protein variants of fibroblast Leominster, MA (US) growth factor 21 (FGF21) with improved pharmaceutical properties. Also disclosed are methods for treating FGF21 (21) Appl. No.: 13/626,194 associated disorders, including metabolic conditions. Patent Application Publication Mar. 28, 2013 Sheet 1 of 9 US 2013/007.9500 A1 Fed plasma glucose st st 38t 3t sis s : a : Fed plasma insulin p<3.35 %&. “elicie, Cre-saw &NOW& 3. O FIG 1B Patent Application Publication Mar. 28, 2013 Sheet 2 of 9 US 2013/007.9500 A1 Body weight * is 3 vs. weiicle, 38-ya kiss, FIG C Liver lipid content * 1: 15 w8. weigii, pre-way & iOvá. is x 5 w8. 335, one-way Aki. A 3. 1. FIG D Patent Application Publication Mar. 28, 2013 Sheet 3 of 9 US 2013/007.9500 A1 Fed plasma glucose g Fed plasma insulin * psi), Sws. Wehle, one-ky3y Al-WA FIG 2B Patent Application Publication Mar. 28, 2013 Sheet 4 of 9 US 2013/0079500 A1 Body weight 38,085 vs. Vehicle, pre-way A-C. A ig33.85 ws, J:395. of e-way A$3. Rik FIG 2C Liver lipid content SO O FIG 2D Patent Application Publication Mar. 28, 2013 Sheet 5 of 9 US 2013/007.9500 A1 Fed plasma glucose 3S 30 35 30 25 O Fed plasma insulin * 3:. 35 was, weiicie, 3rg-ysay Akiva, FIG 3B Patent Application Publication Mar. 28, 2013 Sheet 6 of 9 US 2013/007.9500 A1 Body weight ps: (3 y& Welcie, one-way s-wa FIG 3C Liver lipid content * -8.5 wi: . Whitia, ini-sway & ECA it 3.5 was it 385, in-stay &NO, & FIG 3D Patent Application Publication Mar. 28, 2013 Sheet 7 of 9 US 2013/007.9500 A1 O(3. - " 3:00 s N"m N X o 100- y 3E. w w N t f2, eifty x86iwww. * Q- t12eff -9.4hr al5 - {- 3 : 48 $85 OB 33 88 8: 8 - Tire at -KXr re-is-F82; 38R, P78, A3338 ... --5-82; 38, 716 FIG 4A Patent Application Publication Mar. 28, 2013 Sheet 8 of 9 US 2013/007.9500 A1 Single dose mouse PKIV 1 mg/kg Patent Application Publication Mar. 28, 2013 Sheet 9 of 9 US 2013/007.9500 A1 Anita 3 20 hours .W.Anima 3 Ainal 6 15 days i.W. 5 days S.C. ; 28. 3.} : & 33 3: 833 33 33 33 Temperature (C) FG4D US 2013/007.9500 A1 Mar. 28, 2013 FUSION PROTEINS FORTREATING Metabolism 8,169) (Lundasen et al. (2007) Biochemical and METABOLIC DISORDERS Biophysical Research Communications 360, 437). 0006 FGF21 regulates adipocyte homeostasis through FIELD OF THE INVENTION activation of an AMPK/SIRT1/PGC1C. pathway to inhibit PPARY expression and increase mitochondrial function. 0001. The present invention relates to new fusion proteins (Chau et al. (2010) PNAS 107, 12553) FGF21 also increases comprising fibroblast growth factor 21 (FGF21) known to glucose uptake by skeletal muscle as measured in cultured improve metabolic profiles in subjects to whom they are human myotubes and isolated mouse tissue. FGF21 treatment administered. of rodent islet cells leads to improved function and survival through activation of ERK1/2 and Akt pathways. (Wente et al. BACKGROUND OF THE INVENTION (2006) Diabetes 55, 2470) FGF21 treatment also results in altered gene expression for lipogenesis and fatty acid oxida 0002. The fibroblast growth factor (FGF) family is char tion enzymes in rodent livers, likely through HNF4C. and acterized by 22 genetically distinct, homologous ligands, Foxa2 signaling. which are grouped into seven subfamilies. FGF-21 is most 0007. A difficulty associated with using FGF-21 directly closely related to, and forms a subfamily with, FGF-19 and as a biotherapeutic is that its half-life is very short. (Khari FGF-23. This FGF subfamily regulates diverse physiological tonenkov, A. et al. (2005) Journal of Clinical Investigation processes uncommon to classical FGFs, namely energy and 115:1627-1635) In mice, the half-life of human FGF21 is 0.5 bile acid homeostasis, glucose and lipid metabolism, and to 1 hours, and in cynomolgus monkeys, the half-life is 2 to 3 phosphate as well as Vitamin D homeostasis. Moreover, hours. FGF21 may be utilized as a multi-use, sterile pharma unlike other FGFs, this subfamily acts in an endocrine fash ceutical formulation. However, it has been determined that ion. (Moore, D. D. (2007) Science 316, 1436-8) (Beenken et preservatives, i.e., m-cresol, have an adverse affect on its al. (2009) Nature Reviews Drug Discovery 8, 235). stability under these conditions. 0003 FGF21 is a 209 amino acid polypeptide containing a 0008. In developing an FGF21 protein for use as a thera 28 amino acid leader sequence (SEQ ID NO:5). Human peutic in the treatment of type 1 and type 2 diabetes mellitus FGF21 has about 79% amino acid identity to mouse FGF21 and other metabolic conditions, an increase in half-life and and about 80% amino acid identity to rat FGF21. Fibroblast stability would be desirable. FGF21 proteins having growth factor 21 enhanced half-life and stability would allow for less frequent 0004 (FGF21) has been described as a treatment for dosing of patients being administered the protein. Clearly, ischemic vascular disease, wound healing, and diseases asso there is a need to develop a stable aqueous protein formula ciated with loss of pulmonary, bronchia or alveolar cell func tion for the therapeutic protein FGF21. tion. (Nishimura et al. (2000) Biochimica et Biophysica Acta, 0009 Furthermore, significant challenge in the develop 1492:203-206: patent publication WO01/36640; and patent ment of FGF21 as a protein pharmaceuticals, is to cope with publication WO01/18172) Although FGF-21 activates FGF its physical and chemical instabilities. The compositional receptors and downstream signaling molecules, including variety and characteristics of proteins define specific behav FRS2a and ERK, direct interaction of FGFRs and FGF-21 has iors such as folding, conformational stability, and unfolding/ not been detected. Studies have identified B-klotho, which is denaturation. Such characteristics should be addressed when highly expressed in liver, adipocytes and pancreas, as a deter aiming to stabilize proteins in the course of developing phar minant of the cellular response to FGF-21 and a cofactor maceutical formulation conditions utilizing aqueous protein which mediates FGF-21 signaling through FGFRs (Kurosu, solutions (Wang, W., Int. J. of Pharmaceutics, 18, (1999)). A H. et al. (2007) J Biol Chem 282, 26687-95). FGF21 is a desired effect of stabilizing therapeutic proteins of interest, potentagonist of the FGFR1 (IIIc), FGFR2(IIIc) and FGFR3 e.g., the proteins of the present invention, is increasing resis (IIIc) B-klotho signaling complexes. tance to proteolysis and enzymatic degradation, thereby improving protein stability and reducing protein aggregation. 0005 FGF-21 has been shown to induce insulin-indepen dent glucose uptake. FGF-21 has also been shown to amelio rate hyperglycemia in a range of diabetic rodent models. In SUMMARY OF THE INVENTION addition, transgenic mice over-expressing FGF-21 were 0010. The invention relates to the identification of new found to be resistant to diet-induced metabolic abnormalities, fusion proteins which comprise fibroblast growth factor 21 and demonstrated decreased body weight and fat mass, and (FGF21) and which have improved pharmaceutical proper enhancements in insulin sensitivity (Badman, M. K. et al. ties over the wild-type FGF21 and variants thereof under (2007) Cell Metab 5, 426-37). Administration of FGF-21 to pharmaceutical formulation conditions, e.g., are more stable, diabetic non-human primates caused a decline in fasting possess the ability to improve metabolic parameters for Sub plasma glucose, triglycerides, insulin and glucagon levels, jects to whom they are administered, are less Susceptible to and led to significant improvements in lipoprotein profiles proteolysis and enzymatic degradation, and are less likely to including a nearly 80% increase in HDL cholesterol (Khari aggregate and form complexes. The fusion proteins of the tonenkov, A. et al. (2007) Endocrinology 148, 774-81). invention comprise truncations, mutations, and variants of Recent studies investigating the molecular mechanisms of FGF21. FGF21 action have identified FGF21 as an important endo 0011. Also disclosed are methods for treating FGF21-as crine hormone that helps to control adaptation to the fasting Sociated disorders, as well as other metabolic, endocrine, and state.
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