US20140094406A1 - Chimeric fibroblast growth factor 21 proteins and methods of use - Google Patents

Chimeric fibroblast growth factor 21 proteins and methods of use Download PDF

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US20140094406A1
US20140094406A1 US14/097,116 US201314097116A US2014094406A1 US 20140094406 A1 US20140094406 A1 US 20140094406A1 US 201314097116 A US201314097116 A US 201314097116A US 2014094406 A1 US2014094406 A1 US 2014094406A1
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Regina GOETZ
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Abstract

The present invention relates to a chimeric protein that includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine fibroblast growth factor (“FGF”) and the C-terminus includes a C-terminal portion of an FGF21 molecule. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. The present invention also relates to pharmaceutical compositions including chimeric proteins according to the present invention, methods for treating a subject suffering from diabetes, obesity, or metabolic syndrome, and methods of screening for compounds with enhanced binding affinity for the βKlotho-FGF receptor complex involving the use of chimeric proteins of the present invention.

Description

  • This application claims priority benefit of U.S. Provisional Patent Application No. 61/656,778, filed Jun. 7, 2012, and U.S. Provisional Patent Application No. 61/664,081, filed Jun. 25, 2012, each of which is hereby incorporated by reference in its entirety.
  • This invention was made with government support under grant numbers DE13686, DK077276, AG019712, DK091392, and DK067158 awarded by the U.S. National Institutes of Health. The government has certain rights in this invention.
  • FIELD OF THE INVENTION
  • The present invention relates to chimeric fibroblast growth factor (“FGF”) proteins and uses thereof.
  • BACKGROUND OF THE INVENTION
  • Type 2 diabetes is a chronic progressive disorder, which results from end-organ resistance to the action of insulin in combination with insufficient insulin secretion from the pancreas. The metabolic abnormalities associated with insulin resistance and secretory defects, in particular the hyperglycemia, lead over the course of years to extensive irreversible damage to multiple organs including heart, blood vessels, kidney, and eye. Currently, nearly 200 million or 2.9% of the world population have type 2 diabetes (World Health Organization, Diabetes Fact Sheet No 312, January 2011; Wild et al., “Global Prevalence of Diabetes: Estimates for the Year 2000 and Projections for 2030,” Diabetes Care 27(5):1047-1053 (2004)), and its prevalence is rising at an alarmingly fast pace in parallel with the rise in the prevalence of overweight and obesity (World Health Organization, Obesity and Overweight Fact Sheet No 311, January 2011). Until the end of the 20th century, type 2 diabetes was observed only in adults but what was once known as “adult-onset diabetes” is now also diagnosed in children and adolescents, and this growing incidence can be related to the increase in overweight and obesity among children and adolescents. The prevalence of pre-diabetes, an intermediate metabolic stage between normal glucose homeostasis and diabetes, is even greater than that of type 2 diabetes. Currently, nearly 80 million or 26% of the population in the United States alone have pre-diabetes (Center for Disease Control and Prevention, National Diabetes Fact Sheet 2011), and as such are at high risk for progressing to type 2 diabetes. Type 2 diabetes ranks among the ten leading causes of death worldwide, and the World Health Organization projects that mortality from diabetes (90% of which is type 2) will more than double within the next decade (World Health Organization, Diabetes Fact Sheet No 312, January 2011). Type 2 diabetes also is a major cause of disability. As a consequence of diabetic retinopathy, about 10% of all patients with diabetes in the world develop severe visual impairment and 2% become blind 15 years into the disease (World Health Organization, Diabetes Fact Sheet N° 312, January 2011). Diabetic neuropathy, which affects up to half of all patients with diabetes worldwide (World Health Organization, Diabetes Fact Sheet No 312, January 2011), accounts for the majority of nontraumatic lower-limb amputations. Indeed, in its recently published first worldwide report on non-infectious diseases, the World Health Organization considers diabetes, together with other chronic non-infectious diseases like cancer and heart disease, a global economic and social burden, which exceeds that imposed by infectious diseases such as HIV/AIDS.
  • The current drug therapy for type 2 diabetes is focused on correcting the hyperglycemia in the patients. Although a number of small molecules and biologics with different mechanisms of anti-hyperglycemic action are available for use as mono-therapy or combination therapy, most, if not all of these have limited efficacy, limited tolerability, and significant adverse effects (Moller, “New Drug Targets for Type 2 Diabetes and the Metabolic Syndrome,” Nature 414(6865):821-827 (2001)). For example, treatment with sulfonylureas, glinides, thiazolidinediones, or insulin has been associated with weight gain, which is an undesired effect since overweight is considered a driving force in the pathogenesis of type 2 diabetes. Some of these treatments have also been associated with increased risk of hypoglycemia. A limitation specific to the thiazolidinediones is the potential for adverse cardiovascular effects (DeSouza et al., “Therapeutic Targets to Reduce Cardiovascular Disease in Type 2 Diabetes,” Nat Rev Drug Discov 8(5):361-367 (2009)). A meta-analysis of clinical data on the thiazolidinedione rosiglitazone (Avandia®), which was widely used for the treatment of type 2 diabetes, found that the drug increased the risk of myocardial infarction in patients with type 2 diabetes (Nissen et al., “Effect of Rosiglitazone on the Risk of Myocardial Infarction and Death from Cardiovascular Causes,” N Engl J Med 356(24):2457-2471 (2007)). Of all diabetic complications, cardiovascular disease is the main cause of morbidity and mortality in patients with diabetes (World Health Organization, Diabetes Fact Sheet No 312, January 2011; Center for Disease Control and Prevention, National Diabetes Fact Sheet 2011), and hence an aggravation of cardiovascular risk by drug treatment is absolutely unacceptable. In the wake of the debate about the cardiovascular safety of thiazolidinediones, the FDA issued a guidance on evaluating cardiovascular risk in new anti-diabetic therapies to treat type 2 diabetes (Opar A, “Diabetes Drugs Pass Cardiovascular Risk Check,” Nat Rev Drug Discov 8(5):343-344 (2009)). Meanwhile, thiazolidinediones lost their popularity. Even for glucagon-like peptide-1 agonists, one of the latest class of drugs introduced for the treatment of type 2 diabetes, concerns about safety have been raised, namely the potential for carcinogenicity (Opar A, “Diabetes Drugs Pass Cardiovascular Risk Check,” Nat Rev Drug Discov 8(5):343-344 (2009)). Therefore, novel therapies that are more effective and safer than existing drugs are needed. Since the currently available drugs do not directly target complications of advanced diabetic disease, especially cardiovascular disease, therapies that are not only effective in lowering blood glucose but also reduce cardiovascular risk factors such as dyslipidemia are particularly desired.
  • A search conducted by Eli Lilly & Co. for potential novel biotherapeutics to treat type 2 diabetes led to the discovery of fibroblast growth factor (FGF) 21 as a protein that stimulates glucose uptake into adipocytes in an insulin-independent fashion (Kharitonenkov et al., “FGF-21 as a Novel Metabolic Regulator,” J Clin Invest 115(6):1627-1635 (2005)). FGF21 has since emerged as a key endocrine regulator not only of glucose metabolism but also of lipid metabolism, and has become one of the most promising drug candidates for the treatment of type 2 diabetes, obesity, and metabolic syndrome. In mouse models of diabetes and obesity, pharmacologic doses of FGF21 lower plasma glucose and increase insulin sensitivity (Kharitonenkov et al., “FGF-21 as a Novel Metabolic Regulator,” J Clin Invest 115(6):1627-1635 (2005); Coskun et al., “Fibroblast growth factor 21 corrects obesity in mice,” Endocrinology 149(12):6018-6027 (2008)). Concurrently, FGF21 lowers plasma triglyceride and cholesterol, enhances lipolysis and suppresses lipogenesis, and accelerates energy expenditure (Kharitonenkov et al., “FGF-21 as a Novel Metabolic Regulator,” J Clin Invest 115(6):1627-1635 (2005); Coskun et al., “Fibroblast growth factor 21 corrects obesity in mice,” Endocrinology 149(12):6018-6027 (2008)). In obese mice, FGF21 causes weight loss, in lean mice, it is weight neutral (Kharitonenkov et al., “FGF-21 as a Novel Metabolic Regulator,” J Clin Invest 115(6):1627-1635 (2005); Coskun et al., “Fibroblast growth factor 21 corrects obesity in mice,” Endocrinology 149(12):6018-6027 (2008)). Thus, FGF21 has some of the most desired characteristics of a drug for the treatment of type 2 diabetes; not only does it improve glycemic control, but also directly affects cardiovascular risk factors, such as hypertriglyceridemia, and reduces obesity, which is considered the single most important promoter of type 2 diabetes. Importantly, FGF21 does not induce hypoglycemia (Kharitonenkov et al., “FGF-21 as a Novel Metabolic Regulator,” J Clin Invest 115(6):1627-1635 (2005)), a side effect that can occur with several of the current anti-diabetic therapies, including insulin. Moreover, FGF21 does not exhibit any mitogenic activity in mice (Kharitonenkov et al., “FGF-21 as a Novel Metabolic Regulator,” J Clin Invest 115(6):1627-1635 (2005)), ruling out the possibility of a carcinogenic risk. The findings on FGF21 therapy in mouse models of diabetes have been reproduced in diabetic rhesus monkeys (Kharitonenkov et al., “The Metabolic State of Diabetic Monkeys is Regulated by Fibroblast Growth Factor-21,” Endocrinology 148(2):774-781 (2007)), and are currently followed up with clinical trials in humans (Kharitonenkov et al., “FGF21 Reloaded: Challenges of a Rapidly Growing Field,” Trends Endocrinol Metab 22(3):81-86 (2011)). However, there is a need for more effective FGF21 therapeutics.
  • The present invention overcomes these and other deficiencies in the art.
  • SUMMARY OF THE INVENTION
  • One aspect of the present invention relates to a chimeric protein. The chimeric protein includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine fibroblast growth factor (“FGF”) and the C-terminus includes a C-terminal portion of an FGF21 molecule. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification.
  • Another aspect of the present invention relates to a method for treating a subject suffering from a disorder. This method involves selecting a subject suffering from the disorder and providing a chimeric FGF protein, where the chimeric FGF protein includes an N-terminus coupled to a C-terminus. The N-terminus includes a portion of a paracrine FGF and the C-terminus includes a C-terminal portion of FGF21. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. This method also involves administering a therapeutically effective amount of the chimeric FGF protein to the selected subject under conditions effective to treat the disorder.
  • Another aspect of the present invention relates to a method of making a chimeric FGF protein possessing enhanced endocrine activity. This method involves introducing one or more modifications to an FGF protein, where the modification decreases the affinity of the FGF protein for heparin and/or heparan sulfate and coupling a Klotho co-receptor binding domain to the modified FGF protein's C-terminus, whereby a chimeric FGF protein possessing enhanced endocrine activity is made.
  • Yet another aspect of the present invention relates to a method of facilitating fibroblast growth factor receptor (“FGFR”)-βKlotho co-receptor complex formation. This method involves providing a cell that includes a βKlotho co-receptor and an FGFR and providing a chimeric FGF protein. The chimeric FGF protein includes a C-terminal portion of FGF21 and a portion of a paracrine FGF, where the portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. This method also involves contacting the cell and the chimeric FGF protein under conditions effective to cause FGFR-βKlotho co-receptor complex formation.
  • Yet a further aspect of the present invention relates to a method of screening for agents capable of facilitating FGFR-βKlotho complex formation in the treatment of a disorder. This method involves providing a chimeric FGF that includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine FGF and the C-terminus includes a C-terminal portion of FGF21. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. This method also involves providing a binary βKlotho-FGFR complex and providing one or more candidate agents. This method further involves combining the chimeric FGF, the binary βKlotho-FGFR complex, and the one or more candidate agents under conditions permitting the formation of a ternary complex between the chimeric FGF and the binary βKlotho-FGFR complex in the absence of the one or more candidate agents. This method also involves identifying the one or more candidate agents that decrease ternary complex formation between the chimeric FGF and the binary βKlotho-FGFR complex compared to the ternary complex formation in the absence of the one or more candidate agents as suitable for treating the disorder.
  • Fibroblast growth factors (FGFs) 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. To decipher the role of HS in endocrine FGF signaling, we generated FGF19 and FGF23 mutant ligands devoid of HS binding and compared their signaling capacity with that of wild-type ligands. The data presented herein show that the mutated ligands retain full metabolic activity demonstrating that HS does not participate in the formation of the endocrine FGF signaling complex. Here it is shown that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. A paracrine FGF is converted into an endocrine ligand by diminishing heparan sulfate binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor binding site in order to home the ligand into the target tissue. The ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGS. 1A-1D are schematic diagrams showing side-by-side comparison of the HS-binding site of FGF2, FGF19, and FGF23, and two working models for the endocrine FGF signaling complex. FIG. 1A shows interactions of FGF2 (schematic representation) with a heparin hexasaccharide (shown as sticks) as observed in the crystal structure of the 2:2 FGF2-FGFR1c dimer (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety)). The heparin hexasaccharide consists of three disaccharide units of 1→4 linked N-sulfated-6-O-sulfated D-glucosamine and 2-O-sulfated L-iduronic acid. Note that the heparin hexasaccharide interacts with both side chain and backbone atoms of residues in the HS-binding site of FGF2. Dashed lines denote hydrogen bonds. K128, R129, and K134, which make the majority of hydrogen bonds with the heparin hexasaccharide, are boxed. The β-strand nomenclature follows the original FGF1 and FGF2 crystal structures (Ago et al., J. Biochem. 110:360-363 (1991); Eriksson et al., Proc. Nat'l. Acad. Sci. U.S.A. 88:3441-3445 (1991); Zhang et al., Proc. Nat'l. Acad. Sci. U.S.A. 88:3446-3450 (1991); Zhu et al., Science 251:90-93 (1991), which are hereby incorporated by reference in their entirety). Please note that compared to the prototypical β-trefoil fold seen in soybean trypsin inhibitor (PDB ID: 1TIE; (Onesti et al., J. Mol. Biol. 217:153-176 (1991), which is hereby incorporated by reference in its entirety)) and interleukin 1β (PDB ID: 1I1B; (Finzel et al., J. Mol. Biol. 209:779-791 (1989), which is hereby incorporated by reference in its entirety)), the β10-β11 strand pairing in FGF2 and other paracrine FGFs is less well defined. FIGS. 1B and 1C show cartoon representation of the crystal structures of FGF19 (PDB ID: 2P23; (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety)) (FIG. 1B) and FGF23 (PDB ID: 2P39; (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety)) (FIG. 1C) shown in the same orientation as the FGF2 structure in FIG. 1A. Side chains of residues that map to the corresponding HS-binding sites of these ligands are shown as sticks. Residues selected for mutagenesis to knock out residual HS binding in FGF19 and FGF23 are boxed. NT and CT indicate N- and C-termini of the FGFs. FIG. 1D is a schematic of two working models for the endocrine FGF-FGFR-Klotho coreceptor signal transduction unit. A recent study on the ternary complex formation between FGF21, FGFR1c, and βKlotho supports the 1:2:1 model rather than the 2:2:2 model (Ming et al., J. Biol. Chem. 287:19997-20006 (2012), which is hereby incorporated by reference in its entirety). For comparison, a schematic of the paracrine FGF-FGFR-HS signaling unit is shown, which was made based on the crystal structure of the 2:2:2 FGF2-FGFR1c-HS complex (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety)). HS engages both paracrine FGF and receptor to enhance binding of FGF to its primary and secondary receptors thus promoting receptor dimerization. A question mark denotes whether or not HS is also a component of the endocrine FGF signaling complex.
  • FIG. 2 shows a sequence alignment of the endocrine FGFs, FGF1 and FGF2. The amino acid sequences of the mature human FGF19, FGF21, and FGF23 ligands are aligned. Also included in the alignment are the human sequences of FGF1 and FGF2, prototypical paracrine FGFs, which were used in the experiments described herein, in which FGF1 and FGF2 were converted into endocrine FGF ligands. Residue numbers corresponding to the human sequence of FGF1 (SEQ ID NO:1) (GenBank Accession No. AAH32697, which is hereby incorporated by reference in its entirety), FGF2 (SEQ ID NO: 121) (GenBank Accession No. EAX05222, which is hereby incorporated by reference in its entirety), FGF19 (SEQ ID NO:338) (GenBank Accession No. NP005108, which is hereby incorporated by reference in its entirety), FGF21 (SEQ ID NO: 233) (GenBank Accession No. NP061986, which is hereby incorporated by reference in its entirety), and FGF23 (SEQ ID NO:351) (GenBank accession no. AAG09917, which is hereby incorporated by reference in its entirety) are in parenthesis to the left of the alignment. Secondary structure elements are labeled, and residues containing these elements for known secondary structures are boxed. Gaps (dashes) were introduced to optimize the sequence alignment. The β-trefoil core domain for known FGF crystal structures is shaded gray. Blue bars on top of the alignment indicate the location of the HS-binding regions. HS-binding residues selected for mutagenesis are shaded blue.
  • FIGS. 3A-3G show Surface plasmon resonance (“SPR”) results relating to knockout of residual heparin binding in FGF19 and FGF23 by site-directed mutagenesis. FIG. 3A shows an overlay of SPR sensorgrams illustrating heparin binding of FGF2, FGF19, FGF21, and FGF23 (left panel) and an exploded view of the binding responses for FGF19-, FGF21-, and FGF23-heparin interactions (right panel). Heparin was immobilized on a biosensor chip, and 400 nM of FGF2, FGF19, FGF21, or FGF23 were passed over the chip. Note that FGF19, FGF21, and FGF23 exhibit measurable, residual heparin binding and that differences in heparin binding exist between these three endocrine FGFs. FIGS. 3B-3D show overlays of SPR sensorgrams illustrating binding of FGF19 to heparin (FIG. 3B) and lack of interaction between the FGF19K149A mutant and heparin (FIG. 3C) and between the FGF19K149A, R157A mutant and heparin (FIG. 3D). Heparin was immobilized on a biosensor chip, and increasing concentrations of FGF19 were passed over the chip. Thereafter, FGF19K149A or FGF19K149A, R157A was injected over the heparin chip at the highest concentration tested for the wild-type ligand. FIGS. 3E-3G show overlays of SPR sensorgrams illustrating binding of FGF23 to heparin (FIG. 3E), poor interaction between the FGF23R48A, N49A mutant and heparin (FIG. 3F), and lack of interaction between the FGF23R140A, R143A mutant and heparin (FIG. 3G). Heparin was immobilized on a biosensor chip, and increasing concentrations of FGF23 were passed over the chip. FGF23R48A, N49A FGF23R140A, R143A was then injected over the heparin chip at the highest concentration tested for the wild-type ligand.
  • FIGS. 4A-4D show results demonstrating that HS is dispensable for the metabolic activity of FGF19 and FGF23. FIG. 4A shows results of an immunoblot analysis of phosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) in H4IIE hepatoma cells following stimulation with the FGF19K149A mutant, the FGF19K149A, R157A mutant, or wild-type FGF19. Numbers above the lanes give the amounts of protein added in ng ml−1. Total 44/42 MAPK protein expression was used as a loading control. FIG. 4B shows results of an immunoblot analysis of phosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) in a HEK293-αKlotho cell line following stimulation with the FGF23R48A, N49A mutant, the FGF23R140A, R143A mutant, or wild-type FGF23. Numbers above the lanes give the amounts of protein added in ng ml−1. Total 44/42 MAPK and αKlotho protein expression were used as loading controls. FIG. 4C shows graphical results of a quantitative analysis of CYP7A1 and CYP8B1 mRNA expression in liver tissue from mice treated with FGF19K149A, FGF19K149A, R157A, FGF19, or vehicle. 1 mg of protein per kg of body weight was given. Data are presented as mean±SEM; ***, P<0.001 by Student's t test. FIG. 4D shows graphical results of analysis of serum phosphate concentrations (serum Pi) in mice before and 8 h after intraperitoneal injection of FGF23R48A, N49A, FGF23R140A, R143A, FGF23, or vehicle. Wild-type mice were given a single dose of protein (0.29 mg kg body weight−1), whereas Fgf23 knockout mice received two doses of 0.71 mg kg body weight−1 each. Data are presented as mean±SEM; *, P<0.05, and **, P<0.01 by ANOVA.
  • FIGS. 5A-5G show design and results relating to the conversion of FGF2 into an endocrine ligand. FIG. 5A is a schematic of human FGF2, FGF19, FGF21, FGF23, and engineered FGF2-FGF19, FGF2-FGF21, and FGF2-FGF23 chimeras. Amino acid boundaries of each ligand and of each component of the chimeras are labeled with residue letter and number. The β-trefoil core domain for the known ligand crystal structures is shaded gray. HS-binding residues mutated in the FGF2 portion of chimeras are labeled with residue letter and number. Also labeled are the arginine residues of the proteolytic cleavage site in the C-terminal region of FGF23 that were mutated to glutamine in both FGF23 and the FGF2-FGF23 chimeras. FIGS. 5B and 5C show overlays of SPR sensorgrams illustrating binding of FGF2WTcore-FGF21C-tail (FIG. 5B) and FGF2ΔHBScore-FGF21C-tail (FIG. 5C) to heparin, and fitted saturation binding curves. Heparin was immobilized on a biosensor chip, and increasing concentrations of FGF2WTcore-FGF21C-tail or FGF2ΔHBScore-FGF21C-tail were passed over the chip. Dissociation constants (KDs) were derived from the saturation binding curves. FIGS. 5D and 5E show overlays of SPR sensorgrams illustrating binding of FGF2WTcore-FGF23C-tail (FIG. 5D) and FGF2ΔHBScore-FGF23C-tail (FIG. SE) to heparin. Increasing concentrations of FGF2WTcore-FGF23C-tail or FGF2ΔHBScore-FGF23C-tail were passed over a chip containing immobilized heparin. FIGS. 5F and 5G show results of immunoblot analysis for Egr1 expression in HEK293 cells following stimulation with chimeras or native FGFs as denoted. Numbers above the lanes give the amounts of protein added in nanomolar. GAPDH protein expression was used as a loading control.
  • FIG. 6 is a schematic illustrating the conversion of FGF1 into an endocrine ligand. Shown are schematic drawings of human FGF1, FGF19, FGF21, FGF23, and exemplary FGF1-FGF19, FGF1-FGF21, and FGF1-FGF23 chimeras according to the present invention. Amino acid boundaries of each ligand and of each component of the chimeras are labeled with residue letter and number. The β-trefoil core domain for the known ligand crystal structures is shaded gray. HS-binding residues mutated in the FGF1 portion of chimeras are labeled with residue letter and number. Also labeled are the arginine residues of the proteolytic cleavage site in the C-terminal region of FGF23 that were mutated to glutamine in both FGF23 and the FGF1-FGF23 chimeras.
  • FIGS. 7A-7G show results demonstrating that the FGF2ΔHBScore-FGF23C-tail chimera exhibits FGF23-like activity. FIGS. 7A and 7B show overlays of SPR sensorgrams illustrating inhibition by FGF2ΔHBScore-FGF23C-tail (FIG. 7A) or FGF23 (FIG. 7B) of αKlotho-FGFR1c binding to FGF23 immobilized on a biosensor chip. Increasing concentrations of FGF2ΔHBScore-FGF23C-tail FGF23 were mixed with a fixed concentration of αKlotho-FGFR1c complex, and the mixtures were passed over a FGF23 chip. FIG. 7C shows an overlay of SPR sensorgrams illustrating failure of FGF2 to inhibit αKlotho-FGFR1c binding to FGF23. FGF2 and αKlotho-FGFR1c complex were mixed at a molar ratio of 15:1, and the mixture was passed over a biosensor chip containing immobilized FGF23. FIGS. 7D and 7E show overlays of SPR sensorgrams illustrating no inhibition by FGF2ΔHBScore-FGF23C-tail (FIG. 7D) or FGF23 (FIG. 7E) of βKlotho-FGFR1c binding to FGF21. FGF2ΔHBScore-FGF23C-tail or FGF23 were mixed with βKlotho-FGFR1c complex at a molar ratio of 10:1, and the mixtures were passed over a biosensor chip containing immobilized FGF21. FIG. 7F shows analysis of serum phosphate concentrations (serum Pi) in mice before and 8 h after intraperitoneal injection of FGF2ΔHBScore-FGF23C-tail, FGF2WTcore-FGF23C-tail, FGF23, or vehicle. Wild-type mice and αKlotho knockout mice were given 0.21 mg and 0.51 mg of protein, respectively, per kg of body weight. Data are presented as mean±SEM; **, P<0.01; ***, P<0.001 by ANOVA. FIG. 7G shows quantitative analysis of CYP27B1 mRNA expression in renal tissue from mice injected with FGF2ΔHBScore-FGF23C-tail, FGF2WTcore-FGF23C-tail, FGF23, or vehicle. 0.21 mg of protein per kg of body weight were injected. Data are presented as mean±SEM; ***, P<0.001 by ANOVA.
  • FIGS. 8A-8G show results demonstrating that the FGF2ΔHBScore-FGF21C-tail chimera exhibits FGF21-like activity. FIGS. 8A-8B show overlays of SPR sensorgrams illustrating inhibition by FGF2ΔHBScore-FGF21C-tail (FIG. 8A) or FGF21 (FIG. 8B) of βKlotho-FGFR1c binding to FGF21 immobilized on a biosensor chip. Increasing concentrations of FGF2ΔHBScore-FGF21C-tail or FGF21 were mixed with a fixed concentration of βKlotho-FGFR1c complex, and the mixtures were passed over a FGF21 chip. FIG. 8C shows an overlay of SPR sensorgrams illustrating failure of FGF2 to inhibit βKlotho-FGFR1c binding to FGF21. FGF2 and βKlotho-FGFR1c complex were mixed at a molar ratio of 15:1, and the mixture was passed over a biosensor chip containing immobilized FGF21. FIGS. 8D-8E show overlays of SPR sensorgrams illustrating no inhibition by FGF2ΔHBScore-FGF21C-tail (FIG. 8D) or FGF21 (FIG. 8E) of αKlotho-FGFR1c binding to FGF23. FGF2ΔHBScore-FGF21C-tail or FGF21 were mixed with αKlotho-FGFR1c complex at a molar ratio of 10:1, and the mixtures were passed over a biosensor chip containing immobilized FGF23. FIG. 8F shows results of immunoblot analysis for Egr1 expression in HEK293-βKlotho cells stimulated with FGF2ΔHBScore-FGF21C-tail or FGF21. Numbers above the lanes give the amounts of protein added in ng ml−1. GAPDH protein expression was used as a loading control. Note that the FGF2ΔHBScore-FGF21C-tail chimera is more potent than native FGF21 at inducing Egr1 expression suggesting that the chimera has agonistic property. This is expected since the core domain of FGF2 has inherently greater binding affinity for FGFR than the core domain of FGF21 (see FIGS. 10A and 10C). FIG. 8G shows graphical results of analysis of blood glucose concentrations in mice before and at the indicated time points after intraperitoneal injection of insulin alone, insulin plus FGF2ΔHBScore-FGF21C-tail chimera, insulin plus FGF21, or vehicle alone. 0.5 units of insulin per kg of body weight and 0.3 mg of FGF21 ligand per kg of body weight were injected. Blood glucose concentrations are expressed as percent of pre-injection values. Data are presented as mean±SEM.
  • FIGS. 9A-9C show the glucose-lowering effects in ob/ob mice of FGF1 variants according to the present invention. FIG. 9A shows graphical results of analysis of blood glucose concentrations in ob/ob mice before and at the indicated time points after subcutaneous injection of FGF 1 or FGF21. FIG. 9B shows graphical results of analysis of blood glucose concentrations in ob/ob mice before and at the indicated time points after subcutaneous injection of FGF1, FGF1ΔNT, or FGF1ΔHBS. FIG. 9C shows graphical results of analysis of blood glucose concentrations in ob/ob mice before and at the indicated time points after subcutaneous injection of FGF1 or FGF1ΔHBScore-FGF21C-tail chimera. For the experiments shown in FIGS. 9A-9C, ob/ob mice were injected with a bolus of 0.5 mg of FGF protein per kg of body weight. Data are presented as mean±SD.
  • FIGS. 10A-10F show results demonstrating that endocrine FGFs have low binding affinity for FGFR1c compared to FGF2. FIGS. 10A-10D show overlays of SPR sensorgrams illustrating binding of FGFR1c to FGF2 (FIG. 10A), FGF19 (FIG. 10B), FGF21 (FIG. 10C), and FGF23 (FIG. 10D), and fitted saturation binding curves. Increasing concentrations of FGFR1c ligand-binding domain were passed over a biosensor chip containing immobilized FGF2, FGF19, FGF21, or FGF23. FIG. 10E shows an overlay of SPR sensorgrams illustrating binding of αKlotho-FGFR1c complex to FGF23. Increasing concentrations of αKlotho-FGFR1c complex were passed over a biosensor chip containing immobilized FGF23. FIG. 10F shows an overlay of SPR sensorgrams showing lack of interaction between the C-terminal tail peptide of FGF23 and FGFR1c. FGF23C-tail was immobilized on a biosensor chip and increasing concentrations of FGFR1c ligand-binding domain were passed over the chip. Dissociation constants (KDs) given in FIGS. 10A-10E were derived from the saturation binding curves.
  • FIG. 11 shows an alignment of the C-terminal tail sequences of human FGF19 (SEQ ID NO:338) (GenBank Accession No. NP005108, which is hereby incorporated by reference in its entirety), FGF21 (SEQ ID NO:233) (GenBank Accession No. NP061986, which is hereby incorporated by reference in its entirety), and FGF23 (SEQ ID NO:351) (GenBank accession no. AAG09917, which is hereby incorporated by reference in its entirety). Residue numbers are in parenthesis to the left of the alignment. Gaps (dashes) were introduced to optimize the alignment. Residues that are identical between FGF19 and FGF21 are shaded gray. Note that 40% of these residues map to the most C-terminal sequence.
  • FIG. 12 shows an alignment of the C-terminal tail sequences of human FGF21 (SEQ ID NO:233) (GenBank Accession No. NP061986, which is hereby incorporated by reference in its entirety), FGF19 (SEQ ID NO:338) (GenBank Accession No. NP005108, which is hereby incorporated by reference in its entirety), and variants of FGF21 harboring a single amino acid substitution or insertion for a residue unique to FGF19. Residue numbers for the sequences of native FGF21 and FGF19 are in parenthesis to the left of the alignment. Gaps (dashes) were introduced to optimize the alignment. In the sequence of native FGF19, residues unique to FGF19 are bold and boxed, and in the sequences of the variants of the FGF21 C-terminal tail, introduced FGF19 residues are highlighted in the same manner.
  • FIG. 13 shows an alignment of the C-terminal tail sequences of human FGF21 (SEQ ID NO:233) (GenBank Accession No. NP061986, which is hereby incorporated by reference in its entirety), FGF19 (SEQ ID NO:338) (GenBank Accession No. NP005108, which is hereby incorporated by reference in its entirety), and variants of FGF21 in which residues unique to FGF19 progressively replace the corresponding residues of FGF21 or are inserted into the FGF21 sequence. Residue numbers for the sequences of native FGF21 and FGF19 are in parenthesis to the left of the alignment. Gaps (dashes) were introduced to optimize the alignment. In the sequence of native FGF19, residues unique to FGF19 are bold and boxed, and in the sequences of variants of the FGF21 C-terminal tail, introduced FGF19 residues are highlighted in the same manner.
  • DETAILED DESCRIPTION OF THE INVENTION
  • One aspect of the present invention relates to a chimeric protein. The chimeric protein includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine fibroblast growth factor (“FGF”) and the C-terminus includes a C-terminal portion of an FGF21 molecule. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification.
  • As described by Goetz et al. (Goetz et al., “Molecular Insights into the Klotho-Dependent, Endocrine Mode of Action of Fibroblast Growth Factor 19 Subfamily Members,” Mol Cell Biol 3417-3428 (2007), which is hereby incorporated by reference in its entirety), the mammalian fibroblast growth factor (FGF) family comprises 18 polypeptides (FGF1 to FGF10 and FGF16 to FGF23), which participate in a myriad of biological processes during embryogenesis, including but not limited to gastrulation, body plan formation, somitogenesis, and morphogenesis of essentially every tissue/organ such as limb, lung, brain, and kidney (Bottcher et al., “Fibroblast Growth Factor Signaling During Early Vertebrate Development,” Endocr Rev 26:63-77 (2005), and Thisse et al., “Functions and Regulations of Fibroblast Growth Factor Signaling During Embryonic Development,” Dev Biol 287:390-402 (2005), which are hereby incorporated by reference in their entirety).
  • FGFs execute their biological actions by binding to, dimerizing, and activating FGFR tyrosine kinases, which are encoded by four distinct genes (Fgfr1 to Fgfr4). Prototypical FGFRs consist of an extracellular domain composed of three immunoglobulin-like domains, a single-pass transmembrane domain, and an intracellular domain responsible for the tyrosine kinase activity (Mohammadi et al., “Structural Basis for Fibroblast Growth Factor Receptor Activation,” Cytokine Growth Factor Rev 16:107-137 (2005), which is hereby incorporated by reference in its entirety).
  • The number of principal FGFRs is increased from four to seven due to a major tissue-specific alternative splicing event in the second half of the immunoglobulin-like domain 3 of FGFR1 to FGFR3, which creates epithelial lineage-specific “b” and mesenchymal lineage-specific “c” isoforms (Mohammadi et al., “Structural Basis for Fibroblast Growth Factor Receptor Activation,” Cytokine Growth Factor Rev 16:107-137 (2005) and Ornitz et al., “Fibroblast Growth Factors,” Genome Biol 2(3): reviews 3005.1-reviews 3005.12 (2001), which are hereby incorporated by reference in their entirety). Generally, the receptor-binding specificity of FGFs is divided along this major alternative splicing of receptors whereby FGFRb-interacting FGFs are produced by epithelial cells and FGFRc-interacting FGFs are produced by mesenchymal cells (Ornitz et al., “Fibroblast Growth Factors,” Genome Biol 2(3): reviews 3005.1-reviews 3005.12 (2001), which is hereby incorporated by reference in its entirety). These reciprocal expression patterns of FGFs and FGFRs result in the establishment of specific paracrine FGF signaling loops between the epithelium and the mesenchyme, which is essential for proper organogenesis and patterning during embryonic development as well as tissue homeostasis in the adult organism.
  • Based on sequence homology and phylogenetic and structural considerations, the eighteen mammalian FGFs are grouped into six subfamilies (Itoh et al., “Fibroblast growth factors: from molecular evolution to roles in development, metabolism, and disease,” J Biochem 149:121-130 (2011); Mohammadi et al., “Structural basis for fibroblast growth factor receptor activation,” Cytokine Growth Factor Rev 16:107-137 (2005), which are hereby incorporated by reference in its entirety). The FGF core homology domain (approximately 120 amino acids long) is flanked by N- and C-terminal sequences that are highly variable in both length and primary sequence, particularly among different FGF subfamilies. The core region of FGF19 shares the highest sequence identity with FGF21 (38%) and FGF23 (36%), and therefore, these ligands are considered to form a subfamily.
  • Based on mode of action, the eighteen mammalian FGFs are grouped into paracrine-acting ligands (five FGF subfamilies) and endocrine-acting ligands (one FGF subfamily) comprising FGF19, FGF21 and FGF23 (Itoh and Ornitz, “Fibroblast Growth Factors: From Molecular Evolution to Roles in Development, Metabolism and Disease,” J. Biochem. 149:121-130 (2011); Mohammadi et al., “Structural Basis for Fibroblast Growth Factor Receptor Activation,” Cytokine Growth Factor Rev. 16:107-137 (2005), which are hereby incorporated by reference in their entirety).
  • Paracrine FGFs direct multiple processes during embryogenesis, including gastrulation, somitogenesis, organogenesis, and tissue patterning (Itoh and Ornitz, “Fibroblast Growth Factors: From Molecular Evolution to Roles in Development, Metabolism and Disease,” J. Biochem. 149:121-130 (2011); Bottcher and Niehrs, “Fibroblast Growth Factor Signaling During Early Vertebrate Development,” Endocr. Rev. 26:63-77 (2005); Thisse et al., “Functions and Regulations of Fibroblast Growth Factor Signaling During Embryonic Development,” Dev. Biol. 287:390-402 (2005), which are hereby incorporated by reference in their entirety), and also regulate tissue homeostasis in the adult (Hart et al., “Attenuation of FGF Signalling in Mouse Beta-cells Leads to Diabetes,” Nature 408:864-868 (2000); Jonker et al., “A PPARγ-FGF1 Axis is Required for Adaptive Adipose Remodelling and Metabolic Homeostasis,” Nature 485:391-394 (2012), which is hereby incorporated by reference in its entirety).
  • Endocrine FGFs control major metabolic processes such as bile acid homeostasis (Inagaki et al., “Fibroblast Growth Factor 15 Functions as an Enterohepatic Signal to Regulate Bile Acid Homeostasis,” Cell Metab. 2:217-225 (2005), which is hereby incorporated by reference in its entirety), and hepatic glucose and protein metabolism (Kir et al., “FGF19 as a Postprandial, Insulin-Independent Activator of Hepatic Protein and Glycogen Synthesis,” Science 331:1621-1624 (2011); Potthoff et al., “FGF15/19 Regulates Hepatic Glucose Metabolism by Inhibiting the CREB-PGC-1α Pathway,” Cell Metab. 13:729-738 (2011), which are hereby incorporated by reference in their entirety) (FGF19), glucose and lipid metabolism (Badman et al., “Hepatic Fibroblast Growth Factor 21 Is Regulated by PPARα and Is a Key Mediator of Hepatic Lipid Metabolism in Ketotic States,” Cell Metab. 5:426-437 (2007); Inagaki et al., “Endocrine Regulation of the Fasting Response by PPARalpha-mediated Induction of Fibroblast Growth Factor 21,” Cell Metab. 5:415-425 (2007); Kharitonenkov et al., “FGF-21 as a Novel Metabolic Regulator,” J. Clin. Invest. 115:1627-1635 (2005); Potthoff et al., “FGF21 Induces PGC-1alpha and Regulates Carbohydrate and Fatty Acid Metabolism During the Adaptive Starvation Response,” Proc. Nat'l. Acad. Sci. U.S.A. 106:10853-10858 (2009), which are hereby incorporated by reference in their entirety) (FGF21), and phosphate and vitamin D homeostasis (White et al., “Autosomal Dominant Hypophosphataemic Rickets is Associated with Mutations in FGF23,” Nat. Genet. 26:345-348 (2000); Shimada et al., “Targeted Ablation of Fgf23 Demonstrates an Essential Physiological Role of FGF23 in Phosphate and Vitamin D Metabolism,” J. Clin. Invest. 113:561-568 (2004), which are hereby incorporated by reference in their entirety) (FGF23). Thus, these ligands have attracted much attention as potential drugs for the treatment of various inherited or acquired metabolic disorders (Beenken and Mohammadi, “The FGF Family: Biology, Pathophysiology and Therapy,” Nat. Rev. Drug Discov. 8:235-253 (2009); Beenken and Mohammadi, “The Structural Biology of the FGF19 Subfamily,” in Endocrine FGFs and Klothos (Kuro-o, M. ed.), Landes Bioscience. pp 1-24 (2012), which are hereby incorporated by reference in their entirety).
  • FGFs share a core homology region of about one hundred and twenty amino acids that fold into a β-trefoil (Ago et al., J. Biochem. 110:360-363 (1991); Eriksson et al., Proc. Nat'l. Acad. Sci. U.S.A. 88:3441-3445 (1991); Zhang et al., Proc. Nat'l. Acad. Sci. U.S.A. 88:3446-3450 (1991); Zhu et al., Science 251:90-93 (1991), which are hereby incorporated by reference in their entirety) consisting of twelve β strands in paracrine FGFs (β1-β12) and eleven β strands in endocrine FGFs (β1-β10 and β12) (Mohammadi et al., “Structural Basis for Fibroblast Growth Factor Receptor Activation,” Cytokine Growth Factor Rev. 16:107-137 (2005); Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which are hereby incorporated by reference in their entirety). The conserved core region is flanked by divergent N- and C-termini, which play a critical role in conferring distinct biological activity on FGFs (Mohammadi et al., “Structural Basis for Fibroblast Growth Factor Receptor Activation,” Cytokine Growth Factor Rev. 16:107-137 (2005); Olsen et al., Genes Dev. 20:185-198 (2006), which are hereby incorporated by reference in their entirety).
  • All FGFs interact with pericellular heparan sulfate (HS) glycosaminoglycans albeit with different affinities (Asada et al., Biochim. Biophys. Acta. 1790:40-48 (2009), which is hereby incorporated by reference in its entirety). The HS-binding site of FGFs is comprised of the β1-β2 loop and the region between β10 and β12 strands (Mohammadi et al., “Structural Basis for Fibroblast Growth Factor Receptor Activation,” Cytokine Growth Factor Rev. 16:107-137 (2005), which is hereby incorporated by reference in its entirety). HS interacts with both side chain and main chain atoms of the HS-binding site in paracrine FGFs (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety). The HS-binding site of endocrine FGFs deviates from the common conformation adopted by paracrine FGFs such that interaction of HS with backbone atoms of the HS-binding site is precluded (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety). As a result, compared to paracrine FGFs, endocrine FGFs exhibit poor affinity for HS (Beenken and Mohammadi, “The FGF Family: Biology, Pathophysiology and Therapy,” Nat. Rev. Drug Discov. 8:235-253 (2009); Asada et al., Biochim. Biophys. Acta. 1790:40-48 (2009), which are hereby incorporated by reference in their entirety). The poor HS affinity enables these ligands to diffuse freely away from the site of their secretion and enter the blood circulation to reach their distant target organs (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007); Asada et al., Biochim. Biophys. Acta. 1790:40-48 (2009), which are hereby incorporated by reference in their entirety).
  • By contrast, owing to their high HS affinity (Asada et al., Biochim. Biophys. Acta. 1790:40-48 (2009), which is hereby incorporated by reference in its entirety), paracrine FGFs are mostly immobilized in the vicinity of the cells secreting these ligands, and hence can only act within the same organ. There is emerging evidence that differences in HS-binding affinity among paracrine FGFs translate into the formation of ligand-specific gradients in the pericellular matrix (Kalinina et al., Mol. Cell Biol. 29:4663-4678 (2009); Makarenkova et al., Sci. Signal 2:ra55 (2009), which are hereby incorporated by reference in their entirety), which contribute to the distinct functions of these ligands (Beenken and Mohammadi, “The FGF Family: Biology, Pathophysiology and Therapy,” Nat. Rev. Drug Discov. 8:235-253 (2009); Itoh and Ornitz, “Fibroblast Growth Factors: From Molecular Evolution to Roles in Development, Metabolism and Disease,” J. Biochem. 149:121-130 (2011), which are hereby incorporated by reference in their entirety).
  • Besides controlling ligand diffusion in the extracellular space, HS promotes the formation of the 2:2 paracrine FGF-FGFR signal transduction unit (Schlessinger et al., Mol. Cell 6:743-750 (2000); Mohammadi et al., Curr. Opin. Struct. Biol. 15:506-516 (2005), which are hereby incorporated by reference in their entirety). HS engages both ligand and receptor to enhance the binding affinity of FGF for receptor and promote dimerization of ligand-bound receptors. Owing to their poor HS-binding affinity, endocrine FGFs rely on Klotho co-receptors to bind their cognate FGFR (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007); Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006); Ogawa et al., Proc. Nat'l. Acad. Sci. U.S.A. 104:7432-7437 (2007); Urakawa et al., Nature 444:770-774 (2006), which are hereby incorporated by reference in their entirety). Klotho co-receptors are single-pass transmembrane proteins with an extracellular domain composed of two type I β-glycosidase domains (Ito et al., Mech. Dev. 98:115-119 (2000); Kuro-o et al., Nature 390:45-51 (1997), which are hereby incorporated by reference in their entirety). Klotho co-receptors constitutively associate with FGFRs to enhance the binding affinity of endocrine FGFs for their cognate FGFRs in target tissues (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007); Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006); Ogawa et al., Proc. Nat'l. Acad. Sci. U.S.A. 104:7432-7437 (2007); Urakawa et al., Nature 444:770-774 (2006), which are hereby incorporated by reference in their entirety). αKlotho is the co-receptor for FGF23 (Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006); Urakawa et al., Nature 444:770-774 (2006), which are hereby incorporated by reference in their entirety), and βKlotho is the co-receptor for both FGF19 and FGF21 (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007); Ogawa et al., Proc. Nat'l. Acad. Sci. U.S.A. 104:7432-7437 (2007), which are hereby incorporated by reference in their entirety). The C-terminal region of endocrine FGFs mediates binding of these ligands to the FGFR-α/βKlotho co-receptor complex (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007); Goetz et al., Proc. Nat'l. Acad. Sci. U.S.A 107:407-412 (2010); Micanovic et al., J. Cell Physiol. 219:227-234 (2009); Wu et al., J. Biol. Chem. 283:33304-33309 (2008); Yie et al., FEBS Lett, 583:19-24 (2009); Goetz et al., Mol. Cell Biol. 32:1944-1954 (2012), which are hereby incorporated by reference in their entirety).
  • βKlotho promotes binding of FGF21 to its cognate FGFR by engaging ligand and receptor simultaneously through two distinct binding sites (Goetz et al., “Klotho Coreceptors Inhibit Signaling by Paracrine Fibroblast Growth Factor 8 Subfamily Ligands,” Mol Cell Biol 32:1944-1954 (2012), which is hereby incorporated by reference in its entirety). βKlotho plays the same role in promoting binding of FGF19 to its cognate FGFR (Goetz et al., “Klotho Coreceptors Inhibit Signaling by Paracrine Fibroblast Growth Factor 8 Subfamily Ligands,” Mol Cell Biol 32:1944-1954 (2012), which is hereby incorporated by reference in its entirety). The binding site for βKlotho was mapped on FGF21 and FGF19 to the C-terminal region of each ligand that follows the β-trefoil core domain (Goetz et al., “Klotho Coreceptors Inhibit Signaling by Paracrine Fibroblast Growth Factor 8 Subfamily Ligands,” Mol Cell Biol 32:1944-1954 (2012), which is hereby incorporated by reference in its entirety). In the course of these studies, it was found that the C-terminal tail peptides of FGF21 and FGF19 share a common binding site on βKlotho, and that the C-terminal tail of FGF19 binds tighter than the C-terminal tail of FGF21 to this site (Goetz et al., “Klotho Coreceptors Inhibit Signaling by Paracrine Fibroblast Growth Factor 8 Subfamily Ligands,” Mol Cell Biol 32:1944-1954 (2012), which is hereby incorporated by reference in its entirety).
  • Endocrine FGFs still possess residual HS-binding affinity, and moreover, there are differences in this residual binding affinity among the endocrine FGFs (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety). These observations raise the possibility that HS may play a role in endocrine FGF signaling. Indeed, there are several reports showing that HS can promote endocrine FGF signaling in the presence as well as in the absence of Klotho co-receptor. It has been shown that HS augments the mitogenic signal elicited by endocrine FGFs in BaF3 cells over-expressing FGFR and Klotho co-receptor by at least two-fold (Suzuki et al., Mol. Endocrinol. 22:1006-1014 (2008), which is hereby incorporated by reference in its entirety). In addition, even in the absence of Klotho co-receptor, HS enables endocrine FGFs to induce proliferation of BaF3 cells over-expressing FGFR (Yu et al., Endocrinology 146:4647-4656 (2005); Zhang et al., J. Biol. Chem. 281:15694-15700 (2006), which are hereby incorporated by reference in their entirety). Compared to paracrine FGFs, however, significantly higher concentrations of both ligand and HS are needed, and the proliferative response of cells to endocrine FGFs still lags behind that of paracrine FGFs by about one order of magnitude (Zhang et al., J. Biol. Chem. 281:15694-15700 (2006), which is hereby incorporated by reference in its entirety).
  • As used herein, the terms “chimeric polypeptide” and “chimeric protein” encompass a polypeptide having a sequence that includes at least a portion of a full-length sequence of first polypeptide sequence and at least a portion of a full-length sequence of a second polypeptide sequence, where the first and second polypeptides are different polypeptides. A chimeric polypeptide also encompasses polypeptides that include two or more non-contiguous portions derived from the same polypeptide. A chimeric polypeptide or protein also encompasses polypeptides having at least one substitution, wherein the chimeric polypeptide includes a first polypeptide sequence in which a portion of the first polypeptide sequence has been substituted by a portion of a second polypeptide sequence.
  • As used herein, the term “N-terminal portion” of a given polypeptide sequence is a contiguous stretch of amino acids of the given polypeptide sequence that begins at or near the N-terminal residue of the given polypeptide sequence. An N-terminal portion of the given polypeptide can be defined by a contiguous stretch of amino acids (e.g., a number of amino acid residues). Similarly, the term “C-terminal portion” of a given polypeptide sequence is a contiguous length of the given polypeptide sequence that ends at or near the C-terminal residue of the given polypeptide sequence. A C-terminal portion of the given polypeptide can be defined by a contiguous stretch of amino acids (e.g., a number of amino acid residues).
  • The term “portion,” when used herein with respect to a given polypeptide sequence, refers to a contiguous stretch of amino acids of the given polypeptide's sequence that is shorter than the given polypeptide's full-length sequence. A portion of a given polypeptide may be defined by its first position and its final position, in which the first and final positions each correspond to a position in the sequence of the given full-length polypeptide. The sequence position corresponding to the first position is situated N-terminal to the sequence position corresponding to the final position. The sequence of the portion is the contiguous amino acid sequence or stretch of amino acids in the given polypeptide that begins at the sequence position corresponding to the first position and ending at the sequence position corresponding to the final position. A portion may also be defined by reference to a position in the given polypeptide sequence and a length of residues relative to the referenced position, whereby the sequence of the portion is a contiguous amino acid sequence in the given full-length polypeptide that has the defined length and that is located in the given polypeptide in reference to the defined position.
  • As noted above, a chimeric protein according to the present invention may include an N-terminus coupled to a C-terminus. N-terminus and C-terminus are used herein to refer to the N-terminal region or portion and the C-terminal region or portion, respectively, of the chimeric protein of the present invention. In some embodiments of the present invention, the C-terminal portion and the N-terminal portion of the chimeric protein of the present invention are contiguously joined. In alternative embodiments, the C-terminal portion and the N-terminal portion of the chimeric protein of the present invention are coupled by an intervening spacer. In one embodiment, the spacer may be a polypeptide sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues. In some embodiments, the C-terminal portion and/or the N-terminal portion of the chimeric protein of the present invention may include additional portion(s) coupled to the C-terminal residue and/or the N-terminal residue of the chimeric protein of the present invention, respectively. In some embodiments, the additional portion(s) may be a polypeptide sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues. In some embodiments, the N-terminal portion and/or the C-terminal portion having such additional portion(s) will maintain the activity of the corresponding naturally occurring N-terminal portion and/or C-terminal portion, respectively. In some embodiments, the N-terminal portion and/or the C-terminal portion having such additional portion(s) will have enhanced and/or prolonged activity compared to the corresponding naturally occurring N-terminal portion and/or C-terminal portion, respectively. In other embodiments, the C-terminal portion and/or the N-terminal portion of the chimeric protein of the present invention do not include any additional portion(s) coupled to the C-terminal residue and/or the N-terminal residue of the chimeric protein of the present invention, respectively.
  • The portion of the paracrine FGF may be derived from any suitable paracrine FGF. Suitable paracrine FGFs in accordance with the present invention include FGF1, FGF2, and ligands of the FGF4 and FGF9 subfamilies. Certain embodiments of the present invention may include a full-length amino acid sequence of a paracrine FGF, rather than a portion of a paracrine FGF.
  • In one embodiment, the portion of the paracrine FGF is derived from a mammalian FGF. In one embodiment, the portion of the paracrine FGF is derived from a vertebrate FGF. In one embodiment, the portion of the paracrine FGF is derived from a human FGF. In one embodiment, the paracrine FGF is derived from a non-human mammalian FGF. In one embodiment, the portion of the paracrine FGF is derived from a non-human vertebrate FGF. In one embodiment, the paracrine FGF is derived from an ortholog of human FGF, or a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species.
  • In one embodiment according to the present invention, the portion of the paracrine FGF of the chimeric protein includes an N-terminal portion of the paracrine FGF.
  • In one embodiment, the paracrine FGF is FGF1. In one embodiment, the portion of the FGF1 is from human FGF1 having the following amino acid sequence (GenBank Accession No. AAH32697, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 1):
  • 1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI
    LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF
    LERLEENHYN TYISKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
  • In one embodiment, the portion of the paracrine FGF includes an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 150 to 155 of SEQ ID NO: 1 (human FGF1). In one embodiment, the portion of the paracrine FGF includes amino acid residues 1-150, 1-151, 1-152, 1-153, 1-154, 1-155, 2-150, 2-151, 2-152, 2-153, 2-154, 2-155, 3-150, 3-151, 3-152, 3-153, 3-154, 3-155, 4-150, 4-151, 4-152, 4-153, 4-154, 4-155, 5-150, 5-151, 5-152, 5-153, 5-154, 5-155, 6-150, 6-151, 6-152, 6-153, 6-154, 6-155, 7-150, 7-151, 7-152, 7-153, 7-154, 7-155, 8-150, 8-151, 8-152, 8-153, 8-154, 8-155, 9-150, 9-151, 9-152, 9-153, 9-154, 9-155, 10-150, 10-151, 10-152, 10-153, 10-154, 10-155, 11-150, 11-151, 11-152, 11-153, 11-154, 11-155, 12-150, 12-151, 12-152, 12-153, 12-154, 12-155, 13-150, 13-151, 13-152, 13-153, 13-154, 13-155, 14-150, 14-151, 14-152, 14-153, 14-154, 14-155, 15-150, 15-151, 15-152, 15-153, 15-154, 15-155, 16-150, 16-151, 16-152, 16-153, 16-154, 16-155, 17-150, 17-151, 17-152, 17-153, 17-154, 17-155, 18-150, 18-151, 18-152, 18-153, 18-154, 18-155, 19-150, 19-151, 19-152, 19-153, 19-154, 19-155, 20-150, 20-151, 20-152, 20-153, 20-154, 20-155, 21-150, 21-151, 21-152, 21-153, 21-154, 21-155, 22-150, 22-151, 22-152, 22-153, 22-154, 22-155, 23-150, 23-151, 23-152, 23-153, 23-154, 23-155, 24-150, 24-151, 24-152, 24-153, 24-154, 24-155, 25-150, 25-151, 25-152, 25-153, 25-154, or 25-155 of FGF1 (SEQ ID NO: 1). In one embodiment, the portion of the paracrine FGF includes amino acid residues 1-150 or 25-150 of SEQ ID NO: 1.
  • In one embodiment, the portion of the paracrine FGF includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 150 to 155 of SEQ ID NO: 1 (human FGF1). In one embodiment, the portion of the paracrine FGF includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence homology to an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 150 to 155 of SEQ ID NO: 1 (human FGF1).
  • Percent (%) amino acid sequence identity with respect to a given polypeptide sequence identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent (%) amino acid sequence homology with respect to a given polypeptide sequence identified herein is the percentage of amino acid residues in a candidate sequence that are identical to or strongly similar to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence homology. Strongly similar amino acid residues may include, for example, conservative amino acid substitutions known in the art. Alignment for purposes of determining percent amino acid sequence identity and/or homology can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
  • In one embodiment of the present invention, the portion of the paracrine FGF of the chimeric protein is derived from an ortholog of human FGF1. In one embodiment, the portion of FGF1 is derived from Papio Anubis, Pongo abelii, Callithrix jacchus, Equus caballus, Pan troglodytes, Loxodonta Africana, Canis lupus familiaris, Ailuropoda melanoleuca, Saimiri boliviensis boliviensis, Sus scrofa, Otolemur garnettii, Rhinolophus ferrumequinum, Sorex araneus, Oryctolagus cuniculus, Cricetulus griseus, Sarcophilus harrisii, Mus musculus, Cavia porcellus, Monodelphis domestica, Desmodus rotundus, Bos taurus, Ornithorhynchus anatinus, Taeniopygia guttata, Dasypus novemcinctus, Xenopus Silurana tropicalis, Heterocephalus glaber, Pteropus alecto, Tupaia chinensis, Columba livia, Ovis aries, Gallus gallus, Vicugna pacos, Anolis carolinensis, Otolemur garnettii, Felis catus, Pelodiscus sinensis, Latimeria chalumnae, Tursiops truncates, Mustela putorius furo, Nomascus leucogenys, Gorilla gorilla, Erinaceus europaeus, Procavia capensis, Dipodomys ordii, Petromyzon marinus, Echinops telfairi, Macaca mulatta, Pteropus vampyrus, Myotis lucifugus, Microcebus murinus, Ochotona princeps, Rattus norvegicus, Choloepus hoffmanni, Ictidomys tridecemlineatus, Tarsius syrichta, Tupaia belangeri, Meleagris gallopavo, Macropus eugenii, or Danio rerio. The portions of an ortholog of human paracrine FGF1 include portions corresponding to the above-identified amino acid sequences of human FGF1. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.
  • In one embodiment, the portion of the FGF1 of the chimeric protein of the present invention is derived from an ortholog of human FGF1 having the amino acid sequence shown in Table 1.
  • TABLE 1
    Amino acid sequence of human FGF1 (SEQ ID NO: 1)(GenBank accession no.
    AAH32697, which is hereby incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Papio anubis (olive baboon) FGF1(SEQ ID NO: 2)
    (GenBank accession no. NP_001162557, which is hereby incorporated by
    reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP ANYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Pongo abelii (Sumatran orangutan) FGF1(SEQ ID
    NO: 3) (GenBank accession no. NP_001127073, which is hereby
    incorporated by reference in its entirety)
    60                                                                 M
    61 AEGEITTFTA LTEKFNLPPG NYKKPKLLYC SNGGHFLRIL PDGTVDGTRD RSDQHIQLQL
    121 SAESVGEVYI KSTETGQYLA MDTDGLLYGS QTPNEECLFL ERLEENHYNT YISKKHAEKN
    181 WFVGLKKNGS CKRGPRTHYG QKAILFLPLP VSSD
    Amino acid sequence of Callithrix jacchus (white-tufted-ear marmoset)
    FGF1(SEQ ID NO: 4) (GenBank accession no. XP_002744341, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFDLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Equus caballus (horse) FGF1(SEQ ID NO: 5)
    (GenBank accession no. NP_001157358, which is hereby incorporated by
    reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Pan troglodytes (chimpanzee) FGF1(SEQ ID NO: 6)
    (GenBank accession no. JAA29511, which is hereby incorporated by
    reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPS GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Loxodonta africana (elephant) FGF1(SEQ ID NO: 7)
    (GenBank accession no. XP_003404621, which is hereby incorporated by
    reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKGTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Canis lupus familiaris (dog) FGF1(SEQ ID NO: 8)
    (GenBank accession no. XP_849274, which is hereby incorporated by
    reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYMKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Ailuropoda melanoleuca (giant panda) FGF1(SEQ
    ID NO: 9) (GenBank accession no. XP_002912581, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPA GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Saimiri boliviensis boliviensis (Bolivian
    squirrel monkey) FGF1(SEQ ID NO: 10) (GenBank accession no.
    XP_003920596, which is hereby incorporated by reference in its
    entirety):
    1 MAEGEITTFT ALTEKFDLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDLHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Sus scrofa (pig) FGF1(SEQ ID NO: 11) (GenBank
    accession no. XP_003124058, which is hereby incorporated by reference
    in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTSGLLYG SQTPSEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Otolemur garnettii (small-eared galago)
    FGF1(SEQ ID NO: 12) (GenBank accession no. XP_003782135, which is
    hereby incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTQ DRSDQHIQLQ
    61 LSAESVGEVY IKSTQTGQYL AMDSDGLLYG SQTPNEECLF LERLEENHYN TYVSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Rhinolophus ferrumequinum (greater horseshoe
    bat) FGF1(SEQ ID NO: 13) (GenBank accession no. ACC62496, which is
    hereby incorporated by reference in its entirety):
    1 MAEGEVTTFT ALTEKFNLPT GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DKSDQHIQLQ
    61 LSAESVGEVY IKSTESGQYL AMDSDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Sorex araneus (European shrew) FGF1(SEQ ID
    NO: 14) (GenBank accession no. ACE75805, which is hereby incorporated
    by reference in its entirety):
    1 MAEGEITTFG ALMEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGHYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Oryctolagus cuniculus (rabbit) FGF1(SEQ ID
    NO: 15) (GenBank accession no. NP_001164959, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEVTTFT ALTEKFNLPA GNYKLPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPSEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Cricetulus griseus (Chinese hamster) FGF1(SEQ
    ID NO: 16) (GenBank accession no. XP_003502469, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFS ALTERFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESAGEVY IKGTETGQYR NMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Sarcophilus harrisii (Tasmanian devil) FGF1(SEQ
    ID NO: 17) (GenBank accession no. XP_003756738, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRNDQHIQLQ
    61 LSAESVGEVY IKSTESGQYL AMDTDGLLYG SQTPTEECLF LERLEENHYN TYISKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSE
    Amino acid sequence of Mus musculus (house mouse) FGF1(SEQ ID NO: 18)
    (GenBank accession no. NP_034327, which is hereby incorporated by
    reference in its entirety):
    1 MAEGEITTFA ALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESAGEVY IKGTETGQYL AMDTEGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Cavia porcellus (domestic guinea pig) FGF1(SEQ
    ID NO: 19) (GenBank accession no. XP_003477242, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFA ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAEGVGEVY IQSTETGQYL AMDTDGLLYG SQTPSEECLF LERLEENHYN TYTSKKHVEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSD
    Amino acid sequence of Monodelphis domestica (gray short-tailed
    opossum) FGF1(SEQ ID NO: 20) (GenBank accession no. XP_001368921, which
    is hereby incorporated by reference in its entirety):
    1 MAEGEITTFT ALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRNDQHIQLQ
    61 LSTESVGEVY IKSTESGQYL AMDTDGLLYG SQTPSEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKKGPRTHY GQKAILFLPL PVSSE
    Amino acid sequence of Desmodus rotundus (common vampire bat) FGF1(SEQ
    ID NO: 21) (GenBank accession no. JAA45191, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEVTTFT ALTEKFNLPL ESYKKPKLLY CSNGGHFLRI LPDGTVDGTR DKSDQHIQLQ
    61 LSAESVGEVY IKSTGSGQYL AMDSAGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVNSD
    Amino acid sequence of Bos taurus (cattle) FGF1(SEQ ID NO: 22) (GenBank
    accession no. NP_776480, which is hereby incorporated by reference in
    its entirety):
    1 MAEGETTTFT ALTEKFNLPL GNYKKPKLLY CSNGGYFLRI LPDGTVDGTK DRSDQHIQLQ
    61 LCAESIGEVY IKSTETGQFL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK
    121 HWFVGLKKNG RSKLGPRTHF GQKAILFLPL PVSSD
    Amino acid sequence of Ornithorhynchus anatinus (platypus) FGF1(SEQ ID
    NO: 23) (GenBank accession no. XP_001514861, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALMEKFDLPL GNYKKPRLLY CSNGGYFLRI QPDGKVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTESGHYL AMDTEGLLYG SQAPSEDCLF LERLEENHYN TYVSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVASD
    Amino acid sequence of Taeniopygia guttata (zebra finch) FGF1(SEQ ID
    NO: 24) (GenBank accession no. XP_002193287, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFS ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGVVH IQSTQSGQYL AMDTNGLLYG SQLPPGECLF LERLEENHYN TYVSKMHADK
    121 NWFVGLKKNG TSKLGPRTHY GQKAILFLPL PVAAD
    Amino acid sequence of Dasypus novemcinctus (nine-banded armadillo)
    FGF1(SEQ ID NO: 25) (GenBank accession no. ACO06224, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFM ALMEKFNLPL ENYKHPRLLY CRNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSAETGQYL AMDTDGLLYG SETPSEECLF MEKLEENNYN TYISKKHAEK
    121 KWFVGLKKDG SSKRGPQTHY GQKAILFLPL PVSSD
    Amino acid sequence of Xenopus Silurana tropicalis (western clawed
    frog) FGF1(SEQ ID NO: 26) (GenBank accession no. ACJ50585, which is
    hereby incorporated by reference in its entirety):
    1 MAEGDITTFN PIAESFSLPI GNYKKPKLLY CNNGGYFLRI LPDGVVDGTR DRDDLYITLK
    61 LSAQSQGEVH IKSTETGSYL AMDSSGQLYG TLTPNEESLF LETLEENHYN TYKSKKYAEN
    121 NWFVGIKKNG ASKKGSRTHY GQKAILFLPL PASPD
    Amino acid sequence of Heterocephalus glaber (naked mole-rat) FGF1(SEQ
    ID NO: 27) (GenBank accession no. EHA99379, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRSDQHIQLQ
    61 LSAEGVGEVY IKSTETGQYL AMDTDGLLYG SQTASEECLF LERLEENHYN TYISKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Pteropus alecto (black flying fox) FGF1(SEQ ID
    NO: 28) (GenBank accession no. ELK02961, which is hereby incorporated
    by reference in its entirety):
    1 MAEGEVTTFT ALTERFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DKSDQHIQLQ
    61 LSAESVGEVY IKSTESGQYL AMDSDGLLYG SQTPDEDCLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Tupaia chinensis (Chinese tree shrew) FGF1(SEQ
    ID NO: 29) (GenBank accession no. ELW69091, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFA ALTEKFDLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LTAENVGEVY IKSTETGQYL AMDADGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK
    121 NWFVALKKNG SCKLGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Columba livia (rock pigeon) FGF1(SEQ ID NO: 30)
    (GenBank accession no. EMC79997, which is hereby incorporated by
    reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTQSGQYL AMDPTGLLYG SQLLGEECLF LERIEENHYN TYVSKKHADK
    121 NWFVGLKKNG NSKLGPRTHY GQKAILFLPL PVSAD
    Amino acid sequence of Ovis aries (sheep) FGF1(SEQ ID NO: 31) (GenBank
    accession no. XP_004008958, which is hereby incorporated by reference
    in its entirety):
    1 MAEGETTTFR ALTEKFNLPL GNYKKPKLLY CSNGGYFLRI LPDGRVDGTK DRSDQHIQLQ
    61 LYAESIGEVY IKSTETGQFL AMDTNGLLYG SQTPSEECLF LERLEENHYN TYISKKHAEK
    121 NWFIGLKKNG SSKLGPRTHF GQKAILFLPL PVSSD
    Amino acid sequence of Gallus gallus (chicken) FGF1(SEQ ID NO: 32)
    (GenBank accession no. NP_990511, which is hereby incorporated by
    reference in its entirety):
    1 MAEGEITTFT ALTERFGLPL GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRSDQHIQLQ
    61 LSAEDVGEVY IKSTASGQYL AMDTNGLLYG SQLPGEECLF LERLEENHYN TYISKKHADK
    121 NWFVGLKKNG NSKLGPRTHY GQKAILFLPL PVSAD
    Amino acid sequence of Vicugna pacos (alpaca) FGF1(SEQ ID NO: 33)
    (Ensembl accession no. ENSVPAP00000007810; partial sequence
    corresponding to human FGF1 residues 58 to 155, which is hereby
    incorporated by reference in its entirety):
    1 QLQLSAESVG EVYIKSTETG QYLAMDTDGL LHGSQTPNEE CLFLERLEEN HYNTYTSKKH
    61 AEKNWFVGLK KNGSCKRGPR THYGQKAILF LPLPVSSD
    Amino acid sequence of Anolis carolinensis (anole lizard) FGF1(SEQ ID
    NO: 34) (Ensembl accession no. ENSACAP00000013203, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTERFALPM ENYKKPKLLY CSNGGHFLRI LPDGKVDGTM DRNDSYIQLL
    61 LTAEDVGVVY IKGTETGQYL AMDANGHLYG SQLPTEECLF VETLEENHYN TYTSKMHGDK
    121 KWYVGLKKNG KGKLGPRTHR GQKAILFLPL PVSPD
    Amino acid sequence of Otolemur garnettii (bushbaby) FGF1(SEQ ID
    NO: 35) (Ensembl accession no. ENSOGAP00000004540, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTQ DRSDQHIQLQ
    61 LSAESVGEVY IKSTQTGQYL AMDSDGLLYG SQTPNEECLF LERLEENHYN TYVSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Felis catus (cat) FGF1(SEQ ID NO: 36) (Ensembl
    accession no. ENSFCAP00000008457, which is hereby incorporated by
    reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Pelodiscus sinensis (Chinese softshell turtle)
    FGF1(SEQ ID NO: 37) (Ensembl accession no. ENSPSIP00000016356, which is
    hereby incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPL GNYKNPKLLY CSNGGYFLRI HPDGKVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTESGQFL AMDANGLLYG SLSPSEECLF LERMEENHYN TYISKKHADK
    121 NWFVGLKKNG SCKLGPRTHY GQKAVLFLPL PVSAD
    Amino acid sequence of Latimeria chalumnae (coelacanth) FGF1(SEQ ID
    NO: 38) (Ensembl accession no. ENSLACP00000015106, which is hereby
    incorporated by reference in its entirety):
    1 MAEDKITTLK ALAEKFNLPM GNYKKAKLLY CSNGGYFLRI PPDGKVEGIR ERSDKYIQLQ
    61 MNAESLGMVS IKGVEAGQYL AMNTNGLLYG SQSLTEECLF MEKMEENHYN TYRSKTHADK
    121 NWYVGIRKNG SIKPGPRTHI GQKAVLFLPL PASSD
    Amino acid sequence of Tursiops truncatus (dolphin) FGF1(SEQ ID NO: 39)
    (Ensembl accession no. ENSTTRP00000004470, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYASKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Mustela putorius furo (ferret) FGF1(SEQ ID
    NO: 40) (Ensembl accession no. ENSMPUP00000007888, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALMEKFNLPA GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Nomascus leucogenys (gibbon) FGF1(SEQ ID NO: 41)
    (Ensembl accession no. ENSNLEP00000011873, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Gorilla gorilla (gorilla) FGF1(SEQ ID NO: 42)
    (Ensembl accession no. ENSGGOP00000017663, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Erinaceus europaeus (hedgehog) FGF1(SEQ ID
    NO: 43) (Ensembl accession no. ENSEEUP00000005318, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Procavia capensis (hyrax) FGF1(SEQ ID NO: 44)
    (Ensembl accession no. ENSPCAP00000010969, which is hereby
    incorporated by reference in its entirety)(partial sequence
    corresponding to human FGF1 residues 1 to 91):
    1 MAEGEITTFT ALTEKFNLPL ENYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKGTETGQYL AMDTDGLLYG S
    Amino acid sequence of Dipodomys ordii (kangaroo rat) FGF1(SEQ ID
    NO: 45) (Ensembl accession no. ENSDORP00000006889, which is hereby
    incorporated by reference in its entirety) (partial sequence
    corresponding to human FGF1 residues 1 to 16 and 58 to 155):
    1 MAEGEITTFT ALTERF---- ---------- ---------- ---------- -------QLQ
    61 LSAESVGEVY IKSTETGQYL AMDADGLLYG SQTPDEECLF LERLEENHYN TYIAKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Petromyzon marinus (lamprey) FGF1(SEQ ID NO: 46)
    (Ensembl accession no. ENSPMAP00000010683, which is hereby
    incorporated by reference in its entirety)(partial sequence
    corresponding to human FGF1 residues 1 to 93):
    1 MEVGHIGTLP VVPAGPVFPG SFKEPRRLYC RSAGHHLQIL GDGTVSGTQD ENEPHAVLQL
    61 QAVRRGVVTI RGLCAERFLA MSTEGHLYGA VR
    Amino acid sequence of Echinops telfairi (lesser hedgehog tenrec)
    FGF1(SEQ ID NO: 47) (Ensembl accession no. ENSETEP00000014504, which is
    hereby incorporated by reference in its entirety)(partial sequence
    corresponding to human FGF1 residues 58 to 155)
    1 QLKLVAESVG VVYIKSIKTG QYLAMNPDGL LYGSETPEEE CLFLETLEEN HYTTFKSKKH
    61 VEKNWFVGLR KNGRVKIGPR THQGQKAILF LPLPVSSD
    Amino acid sequence of Macaca mulatta (rhesus monkey) FGF1(SEQ ID
    NO: 48) (Ensembl accession no. ENSMMUP00000030943, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Pteropus vampyrus (megabat) FGF1(SEQ ID NO: 49)
    (Ensembl accession no. ENSPVAP00000004349, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEVTTFT ALTERFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DKSDQHIQLQ
    61 LSAESVGEVY IKSTESGQYL AMDSDGLLYG SQTPDEDCLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Myotis lucifugus (microbat) FGF1(SEQ ID NO: 50)
    (Ensembl accession no. ENSMLUP00000006481, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEVTTFT ALTERFNLPL ENYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTESGQYL AMDSDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Microcebus murinus (mouse lemur) FGF1(SEQ ID
    NO: 51) (Ensembl accession no. ENSMICP00000008602, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESAGEVY IKSTQTGRYL AMDADGLLYG SQTPNEECLF LERLEENHYN TYVSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Ochotona princeps (pika) FGF1(SEQ ID NO: 52)
    (Ensembl accession no. ENSOPRP00000011739, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEVTTFS ALTEKFNLPG GNYKLPKLLY CSNGGHFLRI LPDGTVDGTR DRSDLH----
    61 -------EVF IKSTETGQYL AMDTDGLLYG SQTPSEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGIKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Rattus norvegicus (rat) FGF1(SEQ ID NO: 53)
    (Ensembl accession no. ENSRNOP00000018577, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFA ALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESAGEVY IKGTETGQYL AMDTEGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Choloepus hoffmanni (sloth) FGF1(SEQ ID NO: 54)
    (Ensembl accession no. ENSCHOP00000010964, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALMEKFNLPP GNYMKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDLHIQLQ
    61 LSAESVGEVY IKSAETGQYL AMDTGGLLYG SQTPSEECLF LERLEENHYN TYVSKKHAEK
    121 NWFVGLKKNG SSKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Ictidomys tridecemlineatus (squirrel) FGF1(SEQ
    ID NO: 55) (Ensembl accession no. ENSSTOP00000021782, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Tarsius syrichta (tarsier) FGF1(SEQ ID NO: 56)
    (Ensembl accession no. ENSTSYP00000006804, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYVSKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Tupaia belangeri (tree shrew) FGF1(SEQ ID
    NO: 57) (Ensembl accession no. ENSTBEP00000010264, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFA ALTEKFDLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    61 LTAENVGEVY IKSTETGQYL AMDADGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK
    121 NWFVALKKNG SCKLGPRTHY GQKAILFLPL PVSSD
    Amino acid sequence of Meleagris gallopavo (turkey) FGF1(SEQ ID NO: 58)
    (Ensembl accession no. ENSMGAP00000016398; partial sequence
    corresponding to human FGF1 residues 1 to 56, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTERFGLPL GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRSDQH
    Amino acid sequence of Macropus eugenii (wallaby) FGF1(SEQ ID NO: 59)
    (Ensembl accession no. ENSMEUP00000015084, which is hereby
    incorporated by reference in its entirety):
    1 MAEGEITTFT ALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRNDQHIQLQ
    61 LSAESVGEVY IKSTESGQYL AMDTNGLLYG SQTPSEECLF LERLEENHYN TYISKKHAEK
    121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSE
    Amino acid sequence of Danio rerio (zebrafish) FGF1(SEQ ID NO: 60)
    (Ensembl accession no. ENSDARP00000008825, which is hereby
    incorporated by reference in its entirety):
    1 MTEADIAVKS SPRDYKKLTR LYCMNGGFHL QILADGTVAG AADENTYSIL RIKATSPGVV
    61 VIEGSETGLY LSMNEHGKLY ASSLVTDESY FLEKMEENHY NTYQSQKHGE NWYVGIKKNG
    121 KMKRGPRTHI GQKAIFFLPR QVEQEED
  • As noted above, the portion of the paracrine FGF may be modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. In one embodiment, the modified portion of the paracrine FGF includes one or more substitutions, additions, or deletions.
  • In one embodiment, the one or more substitutions are located at one or more amino acid residues of SEQ ID NO: 1 selected from N33, K127, K128, N129, K133, R134, R137, Q142, K143, and combinations thereof. In one embodiment, the one or more substitutions are selected from N33T, K127D, K128Q, N129T, K133V, R134L, R137H, Q142M, K143T/L/I, and combinations thereof. In one embodiment, the modification is one or more substitutions which are located at one or more amino acid residues corresponding to residues of SEQ ID NO: 1 selected from N33, K127, K128, N129, K133, R134, R137, Q142, K143, and combinations thereof. In one embodiment, the modification is one or more substitutions which are located at one or more amino acid residues corresponding to residues of SEQ ID NO: 1 selected from N33, K127, K128, N129, K133, R134, R137, Q142, K143, and combinations thereof. Amino acid residues corresponding to those of SEQ ID NO:1 may be determined by, for example, sequence analysis and structural analysis.
  • Also encompassed within the present invention are portions of paracrine FGFs other than FGF1 (e.g., FGF2, FGF4, FGF5, FGF6, FGF9, FGF16, and FGF20). The portions derived from paracrine FGFs other than FGF1 include portions corresponding to the above-identified amino acid sequences of FGF1. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.
  • It will be understood that the portion of the paracrine FGF according to the present invention may be derived from a nucleotide sequence that encodes a paracrine FGF protein. For example, in one embodiment, the nucleotide sequence is the nucleotide sequence that encodes human FGF1 (GenBank Accession No. BC032697, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 61), as follows:
  •  91                                  ATGGCTGAAG GGGAAATCAC CACCTTCACA
    121 GCCCTGACCG AGAAGTTTAA TCTGCCTCCA GGGAATTACA AGAAGCCCAA ACTCCTCTAC
    181 TGTAGCAACG GGGGCCACTT CCTGAGGATC CTTCCGGATG GCACAGTGGA TGGGACAAGG
    241 GACAGGAGCG ACCAGCACAT TCAGCTGCAG CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT
    301 ATAAAGAGTA CCGAGACTGG CCAGTACTTG GCCATGGACA CCGACGGGCT TTTATACGGC
    361 TCACAGACAC CAAATGAGGA ATGTTTGTTC CTGGAAAGGC TGGAGGAGAA CCATTACAAC
    421 ACCTATATAT CCAAGAAGCA TGCAGAGAAG AATTGGTTTG TTGGCCTCAA GAAGAATGGG
    481 AGCTGCAAAC GCGGTCCTCG GACTCACTAT GGCCAGAAAG CAATCTTGTT TCTCCCCCTG
    541 CCAGTCTCTT CTGATTAA
  • In another embodiment of the present invention, the portion of the paracrine FGF of the chimeric protein may be derived from a nucleotide sequence that encodes an ortholog of human FGF1. Nucleotide sequences that encode FGF1 orthologs are shown in Table 2.
  • TABLE 2
    Olive Baboon FGF1 gene coding sequence (1-155) (SEQ ID NO: 62)
    (GenBank accession no. NM_001169086, which is hereby
    incorporated by reference in its entirety):
    1 ATGGCTGAAG GGGAAATCAC CACGTTCACA GCCCTGACCG AGAAGTTTAA TCTGCCTCCA
    61 GCGAATTACA AGAAGCCCAA ACTGCTCTAC TGTAGCAACG GGGGACACTT CTTGAGGATC
    121 CTTCCGGATG GCACAGTGGA TGGGACAAGG GACAGGAGCG ACCAGCACAT TCAGCTGCAG
    181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CCGAGACTGG CCAGTACTTG
    241 GCCATGGACA CCGACGGGCT TTTATACGGC TCACAGACAC CAAATGAGGA ATGTTTGTTC
    301 CTGGAAAGGC TGGAGGAGAA CCATTACAAC ACCTACATAT CCAAGAAGCA CGCAGAGAAG
    361 AATTGGTTTG TTGGCCTCAA GAAGAATGGA AGCTGCAAAC GTGGTCCTCG GACTCACTAT
    421 GGCCAGAAAG CAATCTTGTT TCTTCCCCTG CCAGTCTCTT CTGATTAA
    Sumatran orangutan FGF1 gene coding sequence (60-214) (SEQ ID NO: 63)
    (GenBank accession no. NM_001133601, which is hereby incorporated by
    reference in its entirety):
    211                                  ATGGCTGAAG GGGAAATCAC CACCTTCACA
    241 GCCCTGACCG AGAAGTTTAA TCTGCCTCCA GGGAATTACA AGAAGCCCAA ACTCCTCTAC
    301 TGTAGCAACG GGGGCCACTT CTTGAGGATC CTTCCGGATG GCACAGTGGA TGGGACAAGG
    361 GACAGGAGCG ACCAGCACAT TCAGCTGCAG CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT
    421 ATAAAGAGTA CCGAGACTGG CCAGTACTTG GCCATGGACA CCGACGGGCT TTTATACGGC
    481 TCACAGACAC CAAATGAGGA ATGTTTGTTC CTGGAAAGGC TGGAGGAGAA CCATTACAAC
    541 ACCTATATAT CCAAGAAGCA TGCAGAGAAG AATTGGTTTG TTGGCCTCAA GAAGAATGGA
    601 AGCTGCAAAC GCGGTCCTCG GACTCACTAT GGCCAGAAAG CAATCTTGTT TCTCCCCCTG
    661 CCAGTCTCTT CCGATTAA
    White-tufted-ear marmoset FGF1 gene coding sequence (1-155) (SEQ ID
    NO: 64) (GenBank accession no. XM_002744295, which is
    hereby incorporated by reference in its entirety):
    130          A TGGCTGAAGG GGAAATCACC ACCTTCACAG CCCTGACCGA GAAGTTTGAT
    181 CTGCCTCCAG GGAATTACAA GAAGCCCAAA CTCCTCTACT GTAGCAATGG GGGCCACTTC
    241 TTGAGGATCC TTCCGGATGG CACAGTGGAT GGGACAAGGG ACAGGAGCGA CCAGCACATT
    301 CAGCTGCAGC TCAGTGCGGA AAGCGTGGGG GAGGTGTATA TAAAGAGTAC CGAGACTGGC
    361 CAGTACTTGG CCATGGACAC CGACGGGCTT TTATACGGCT CACAGACACC AAATGAGGAA
    421 TGTTTGTTCC TGGAGAGGCT GGAGGAGAAC CATTACAACA CCTATATATC CAAGAAACAT
    481 GCAGAGAAGA ATTGGTTTGT CGGCCTCAAG AAGAATGGAA GCTGTAAACG TGGTCCTCGG
    541 ACTCACTATG GTCAGAAAGC GATCTTGTTT CTCCCCCTGC CAGTTTCTTC TGATTAA
    Horse FGF1 gene coding sequence (1-155) (SEQ ID NO: 65)
    (GenBank accession no. NM_001163886, which is
    hereby incorporated by reference in its entirety):
    34                                     ATGGCTG AAGGAGAAAT CACAACCTTC
    61 ACGGCCCTGA CCGAGAAGTT TAATCTGCCT CCAGGGAATT ACAAGAAGCC CAAACTCCTC
    121 TACTGTAGCA ATGGGGGCCA CTTCCTGAGG ATCCTTCCAG ATGGCACAGT GGATGGGACA
    181 AGGGACAGGA GCGACCAGCA CATTCAGCTG CAGCTCAGTG CGGAAAGCGT GGGGGAGGTG
    241 TATATAAAGA GTACCGAGAC TGGCCAGTAC TTGGCCATGG ACACCGACGG GCTGTTGTAC
    301 GGCTCACAGA CACCAAACGA GGAATGTTTG TTCCTGGAAA GGCTGGAGGA AAACCATTAC
    361 AACACCTACA CATCCAAGAA GCATGCAGAG AAGAACTGGT TCGTTGGTCT CAAGAAGAAT
    421 GGGAGCTGCA AACGCGGTCC TCGGACTCAC TATGGGCAGA AAGCAATCTT GTTTCTTCCC
    481 CTGCCCGTCT CCTCTGACTA A
    Chimpanzee FGF1 gene coding sequence (1-155) (SEQ ID NO: 66)
    (GenBank accession no. GABD01003589, which is hereby
    incorporated by reference in its entirety):
    80                     A TGGCTGAAGG GGAAATCACC ACCTTCACAG CCCTGACCGA
    121 GAAGTTTAAT CTGCCTTCAG GGAATTACAA GAAGCCCAAA CTCCTCTACT GTAGCAACGG
    181 GGGCCACTTC CTGAGGATCC TTCCGGATGG CACAGTGGAT GGGACAAGGG ACAGGAGCGA
    241 CCAGCACATT CAGCTGCAGC TCAGTGCGGA AAGCGTGGGG GAGGTGTATA TAAAGAGTAC
    301 CGAGACTGGC CAGTACTTGG CCATGGACAC CGACGGGCTT TTATACGGCT CACAGACACC
    361 AAATGAGGAA TGTTTGTTCC TGGAACGGCT GGAGGAGAAC CATTACAACA CCTATATATC
    421 CAAGAAGCAT GCAGAGAAGA ATTGGTTTGT TGGCCTCAAG AAGAATGGAA GCTGCAAACG
    481 CGGTCCTCGG ACTCACTATG GCCAGAAAGC AATCTTGTTT CTCCCCCTGC CAGTCTCTTC
    541 CGATTAA
    Elephant FGF1 gene coding sequence (1-155) (SEQ ID NO: 67) (GenBank
    accession no. XM_003404573, which is hereby incorporated
    by reference in its entirety):
    1 ATGGCCGAAG GGGAAATCAC AACTTTCACA GCCCTGACAG AGAAGTTCAA CCTGCCTCCA
    61 GGGAATTACA AGAAGCCCAA ACTCCTCTAC TGTAGCAATG GAGGTCACTT CTTAAGGATC
    121 CTTCCAGATG GCACAGTGGA TGGCACCAGG GACAGGAGTG ACCAGCACAT TCAGCTGCAG
    181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGGGCA CCGAGACTGG CCAGTACTTG
    241 GCCATGGACA CCGACGGGCT TTTATACGGC TCACAGACAC CAAATGAGGA ATGTTTGTTC
    301 CTGGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACACAT CCAAGAAGCA CGCAGAGAAG
    361 AATTGGTTCG TTGGTCTCAA GAAGAATGGA AGCTGCAAAC GCGGTCCTCG GACTCACTAT
    421 GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTCTCCT CTGATTAA
    Dog FGF1 gene coding sequence (1-155) (SEQ ID NO: 68)
    (GenBank accession no. XM_844181, which is hereby
    incorporated by reference in its entirety):
    164                                                ATGGCTG AAGGGGAAAT
    181 CACAACCTTC ACTGCCCTGA CGGAGAAGTT TAATCTGCCT CCGGGGAATT ACATGAAGCC
    241 CAAACTCCTC TACTGTAGCA ACGGGGGCCA CTTCCTGAGG ATCCTTCCAG ATGGCACAGT
    301 GGATGGGACA AGGGACAGGA GCGACCAGCA CATTCAGCTG CAGCTCAGCG CGGAAAGCGT
    361 GGGGGAGGTG TATATAAAGA GCACCGAGAC TGGCCAGTAC TTGGCCATGG ACACCGATGG
    421 GCTTCTGTAC GGCTCACAGA CACCGAATGA GGAATGTTTG TTCCTGGAAA GGCTGGAGGA
    481 AAACCATTAC AACACCTACA CATCCAAGAA GCATGCAGAA AAAAATTGGT TTGTTGGTCT
    541 CAAGAAGAAT GGAAGCTGCA AACGCGGTCC TCGGACTCAC TATGGTCAAA AAGCAATTTT
    601 GTTTCTCCCC CTGCCAGTGT CCTCTGATTA A
    Giant panda FGF1 gene coding sequence (1-155) (SEQ ID NO: 69) (GenBank
    accession no. XM_002912535, which is hereby incorporated
    by reference in its entirety):
    146                            ATGGC TGAAGGGGAG ATCACAACCT TCACCGCCCT
    181 GACGGAGAAG TTTAATCTGC CTGCGGGGAA TTACAAGAAG CCCAAACTCC TCTACTGTAG
    241 CAACGGGGGC CACTTCCTGA GGATCCTTCC AGATGGCACA GTGGACGGGA CGAGGGACAG
    301 GAGCGACCAG CACATTCAAC TGCAGCTCAG CGCGGAAAGC GTAGGGGAGG TGTACATAAA
    361 GAGCACCGAG ACCGGCCAGT ACTTGGCCAT GGACACCGAT GGGCTTCTGT ACGGCTCACA
    421 GACACCAAAT GAGGAATGTT TGTTCCTGGA AAGGCTGGAG GAAAACCATT ACAACACCTA
    481 CACATCCAAG AAGCACGCGG AGAAGAATTG GTTTGTTGGT CTCAAGAAGA ATGGAAGCTG
    541 CAAACGTGGT CCTCGGACTC ACTATGGCCA GAAAGCAATT CTGTTTCTCC CCCTGCCAGT
    601 CTCCTCTGAT TAA
    Bolivian squirrel monkey FGF1 gene coding sequence (1-155)
    (SEQ ID NO: 70) (GenBank accession no. XM_003920547,
    which is hereby incorporated by reference in its entirety):
    130          A TGGCTGAAGG GGAAATCACC ACCTTTACAG CCCTGACCGA GAAGTTTGAT
    181 CTGCCTCCAG GGAATTACAA GAAGCCCAAA CTCCTCTACT GTAGCAACGG GGGCCACTTC
    241 TTGAGGATCC TTCCGGATGG CACAGTGGAT GGGACCAGGG ACAGGAGCGA TCTTCACATT
    301 CAGCTGCAGC TCAGTGCGGA AAGCGTGGGG GAGGTGTATA TAAAGAGTAC CGAGACTGGC
    361 CAGTACTTGG CCATGGACAC CGACGGGCTT TTATACGGCT CACAGACACC AAATGAGGAA
    421 TGTTTGTTCC TGGAAAGGCT GGAGGAGAAC CATTACAACA CCTATATATC CAAGAAACAC
    481 GCAGAGAAGA ATTGGTTTGT TGGCCTCAAG AAGAATGGAA GCTGCAAGCG CGGTCCTCGG
    541 ACTCACTATG GCCAGAAAGC AATCTTGTTT CTCCCCCTGC CAGTCTCTTC TGATTAA
    Pig FGF1 gene coding sequence (1-155) (SEQ ID NO: 71)
    (GenBank accession no. XM_003124010, which
    is hereby incorporated by reference in its entirety):
    35                                      ATGGCT GAAGGCGAAA TCACAACCTT
    61 CACGGCCCTG ACCGAGAAGT TTAATCTGCC TCCAGGAAAT TACAAGAAGC CCAAGCTCCT
    121 CTACTGCAGC AACGGGGGCC ATTTCCTCAG GATCCTTCCA GATGGCACAG TGGATGGGAC
    181 CAGGGACAGG AGCGACCAGC ACATTCAGCT GCAGCTCAGT GCGGAAAGCG TGGGGGAGGT
    241 GTATATAAAG AGTACGGAGA CTGGCCAGTA CTTGGCCATG GACACCAGCG GGCTTTTGTA
    301 CGGCTCACAG ACACCCAGTG AGGAGTGTTT GTTCCTGGAG AGGCTGGAGG AAAACCATTA
    361 CAATACCTAC ACATCCAAGA AGCACGCAGA GAAGAACTGG TTCGTTGGCC TCAAGAAGAA
    421 TGGAAGCTGC AAACGCGGTC CTCGGACTCA CTATGGCCAG AAAGCCATCC TGTTTCTCCC
    481 CCTGCCAGTA TCCTCGGATT AA
    Small-eared galago FGF1 gene coding sequence (1-155) (SEQ ID NO: 72)
    (GenBank accession no. XM_003782087, which is hereby
    incorporated by reference in its entirety):
    28                              ATG GCTGAAGGGG AAATCACAAC CTTCACAGCC
    61 CTCACAGAGA AGTTTAATCT GCCTCTAGGA AATTACAAGA AGCCCAAGCT CCTCTACTGT
    121 AGCAACGGGG GTCACTTTCT GAGGATCCTG CCGGATGGCA CCGTGGATGG GACACAAGAC
    181 AGGAGCGACC AGCACATTCA GCTGCAGCTC AGTGCGGAAA GCGTGGGGGA GGTGTATATA
    241 AAGAGTACCC AGACTGGCCA GTACTTGGCC ATGGACTCCG ACGGGCTTTT ATACGGCTCA
    301 CAAACACCAA ATGAGGAATG CCTGTTCCTG GAACGGCTGG AGGAAAACCA TTACAACACC
    361 TATGTGTCCA AGAAGCACGC CGAGAAGAAT TGGTTTGTCG GTCTCAAGAA GAACGGAAGT
    421 TGCAAACGTG GTCCTCGGAC TCACTACGGC CAGAAAGCAA TCTTGTTTCT CCCCCTGCCA
    481 GTCTCCTCTG ATTAA
    Greater horseshoe bat FGF1 gene coding sequence (1-155) (SEQ ID NO: 73)
    (GenBank accession no. DP000705, which is hereby incorporated
    by reference in its entirety):
    190120                                           T TAATCAGAGG AGACTGGCAG
    190141 GGGGAGAAAC AGGATTGCTT TCTGGCCATA GTGAGTCCGA GGACCGCGCT TGCAGCTTCC
    190201 ATTCTTCTTG AGCCCAACGA ACCAATTCTT TTCTGCGTGC TTCTTGGACG TGTAGGTGTT
    190261 GTAATGGTTT TCCTCCAGCC TTTCCAGGAA CAGACATTCC TCATTTGGTG TCTG
    194466      TGAGC CGTACAAAAG CCCGTCGGAG TCCATGGCCA AGTACTGGCC ACTCTCGGTG
    194521 CTCTTTATAT ACACCTCCCC CACGCTTTCC GCACTGAGCT GCAGCTGAA
    208114                                     TGTGCTG GTCACTCTTG TCCCTTGTCC
    208141 CATCCACTGT GCCATCTGGA AGGATCCTCA GGAAGTGGCC CCCGTTGCTG CAGTAGAGAA
    208201 GTTTGGGTTT CTTGTAATTC CCTGTAGGCA GATTAAACTT CTCAGTAAGG GCTGTGAACG
    208261 TGGTGACTTC CCCTTCGGCC AT
    European shrew FGF1 gene coding sequence (1-155) (SEQ ID NO: 74)
    (GenBank accession no. DP000767, which is hereby incorporated
    by reference in its entirety):
    138344                                                CTAGTCG GAGGAGACGG
    138361 GCAGGGGGAG AAACAAGATC GCTTTCTGGC CGTAGTGAGT CCGGGGACCA CGCTTGCAGC
    138421 TTCCGTTCTT CTTCAGACCA ACAAACCAAT TCTTCTCGGC ATGCTTCTTG GAGGTATAGG
    138481 TGTTGTAATG GTTTTCCTCC AGCCTTTCCA GAAACAGACA TTCCTCATTC GGTGTTTG
    143512                                                         TGAGCCGTA
    143521 TAAAAGCCCG TCGGTGTCCA TGGCCAAGTA ATGGCCAGTC TCCGTGCTCT TTATATACAC
    143581 CTCCCCCACG CTTTCCGCAC TGAGCTGCAG CTGAA
    157009                                                     TG TGCTGGTCGC
    157021 TGCGGTCCCT GGTCCCATCC ACTGTGCCGT CCGGGAGGAT GCGCAGGAAG TGGCCCCCGT
    157081 TGCTGCAGTA CAGGAGTTTG GGCTTCTTGT AGTTCCCTGG TGGCAGGTTA AACTTCTCCA
    157141 TGAGGGCCCC AAAGGTGGTG ATCTCCCCCT CGGCCAT
    Rabbit FGF1 gene coding sequence (1-155) (SEQ ID NO: 75) (GenBank
    accession no. NM_001171488, which is hereby incorporated
    by reference in its entirety):
    1 ATGGCTGAGG GGGAGGTCAC CACCTTCACA GCCCTGACCG AGAAGTTCAA CCTGCCTGCA
    61 GGGAACTACA AGTTGCCCAA ACTCCTCTAC TGCAGCAACG GGGGCCACTT CCTGAGGATC
    121 CTGCCGGACG GCACTGTGGA CGGCACAAGG GACAGGAGCG ACCAGCACAT TCAGCTGCAG
    181 CTGAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CGGAGACCGG CCAGTACTTG
    241 GCCATGGACA CCGACGGCCT TTTATACGGC TCGCAAACGC CCAGTGAGGA GTGTTTGTTC
    301 CTGGAACGGC TGGAGGAGAA CCACTACAAC ACCTACACGT CCAAGAAGCA CGCCGAGAAG
    361 AACTGGTTCG TGGGGCTGAA GAAAAACGGG AGCTGCAAGC GCGGTCCTCG GACTCACTAC
    421 GGCCAGAAAG CCATCTTGTT CCTCCCCCTG CCGGTCTCCT CCGACTAA
    Chinese hamster FGF1 gene coding sequence (1-155) (SEQ ID NO: 76)
    (GenBank accession no. XM_003502421, which is hereby
    incorporated by reference in its entirety):
    1 ATGGCTGAAG GAGAAATCAC CACCTTCTCA GCCCTGACAG AGAGATTTAA TCTGCCTCCA
    61 GGAAACTACA AGAAGCCCAA ACTGCTCTAC TGCAGCAACG GGGGCCACTT CTTGAGGATC
    121 CTTCCAGATG GCACAGTGGA TGGGACAAGG GACAGGAGTG ACCAGCACAT TCAGCTGCAG
    181 CTGAGTGCGG AAAGCGCGGG CGAAGTGTAT ATAAAGGGTA CAGAGACAGG CCAGTACAGG
    241 AACATGGACA CGGATGGCCT TTTATACGGC TCACAGACAC CAAATGAAGA ATGCCTGTTC
    301 CTGGAAAGGC TGGAAGAAAA CCATTACAAC ACTTATACAT CCAAGAAGCA CGCAGAGAAG
    361 AACTGGTTTG TGGGCCTCAA GAAAAACGGG AGCTGCAAGC GTGGTCCTCG GACTCACTAT
    421 GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCTGTATCTT CTGACTAG
    Tasmanian devil FGF1 gene coding sequence (1-155) (SEQ ID NO: 77)
    (GenBank accession no. XM_003756690, which is hereby
    incorporated by reference in its entirety):
    24                          ATGGCCG AAGGGGAGAT CACAACCTTC ACAGCCCTGA
    61 CCGAAAGATT TAATCTGCCA CTGGGGAATT ACAAGAAGCC CAAGCTTCTC TACTGTAGCA
    121 ATGGGGGCCA CTTTTTGAGG ATTCTTCCTG ATGGTAAAGT GGATGGGACA AGGGACAGAA
    181 ATGATCAACA CATTCAACTG CAACTAAGCG CGGAAAGCGT GGGTGAGGTG TATATAAAGA
    241 GCACTGAGTC TGGCCAGTAT TTGGCTATGG ACACCGATGG ACTTTTATAC GGCTCACAGA
    301 CACCCACTGA AGAATGCTTG TTCCTGGAGA GATTGGAGGA GAATCATTAC AACACCTACA
    361 TATCAAAGAA GCATGCGGAG AAAAATTGGT TTGTGGGCCT CAAGAAAAAT GGAAGCTGCA
    421 AAAGAGGTCC CAGGACTCAC TATGGCCAGA AAGCCATCCT CTTCCTTCCC CTCCCTGTGT
    481 CCTCTGAGTA A
    House mouse FGF1 gene coding sequence (1-155) (SEQ ID NO: 78)
    (GenBank accession no. NM_010197, which is hereby
    incorporated by reference in its entirety):
    188        ATG GCTGAAGGGG AGATCACAAC CTTCGCAGCC CTGACCGAGA GGTTCAACCT
    241 GCCTCTAGGA AACTACAAAA AGCCCAAACT GCTCTACTGC AGCAACGGGG GCCACTTCTT
    301 GAGGATCCTT CCTGATGGCA CCGTGGATGG GACAAGGGAC AGGAGCGACC AGCACATTCA
    361 GCTGCAGCTC AGTGCGGAAA GTGCGGGCGA AGTGTATATA AAGGGTACGG AGACCGGCCA
    421 GTACTTGGCC ATGGACACCG AAGGGCTTTT ATACGGCTCG CAGACACCAA ATGAGGAATG
    481 TCTGTTCCTG GAAAGGCTGG AAGAAAACCA TTATAACACT TACACCTCCA AGAAGCATGC
    541 GGAGAAGAAC TGGTTTGTGG GCCTCAAGAA GAACGGGAGC TGTAAGCGCG GTCCTCGGAC
    601 TCACTATGGC CAGAAAGCCA TCTTGTTTCT GCCCCTCCCG GTGTCTTCTG ACTAG
    Domestic guinea pig FGF1 gene coding sequence (1-154) (SEQ ID NO: 79)
    (GenBank accession no. XM_003477194, which is hereby
    incorporated by reference in its entirety):
    1 ATGGCTGAAG GAGAAATCAC AACTTTTGCA GCCCTGACTG AGAAGTTTAA TCTGCCTCCA
    61 GGGAATTATA AGAAGCCCAA ACTGCTCTAC TGCAGCAATG GGGGCCACTT CCTGAGGATC
    121 CTTCCAGACG GCACAGTGGA CGGCACAAGA GACAGGAGCG ACCAGCACAT TCAGCTGCAG
    181 CTCAGTGCGG AAGGCGTGGG GGAGGTGTAT ATACAGAGCA CCGAGACCGG CCAGTACTTG
    241 GCCATGGACA CCGACGGGCT TTTATACGGC TCACAGACAC CAAGTGAGGA ATGCTTGTTC
    301 CTGGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACACAT CCAAGAAGCA TGTGGAGAAG
    361 AATTGGTTTG TTGGCCTCAA GAAGAACGGA AGCTGCAAGC GTGGTCCTCG GACTCACTAT
    421 GGCCAGAAAG CAATCTTGTT CCTCCCCTTG CCAGTCTCTG ATTAG
    Gray short-tailed opossum FGF1 gene coding sequence (1-155) (SEQ ID
    NO: 80) (GenBank accession no. XM_001368884,
    which is hereby incorporated by reference in its entirety):
    1 ATGGCCGAAG GGGAGATCAC AACCTTCACA GCCCTGACTG AAAGATTTAA CCTGCCACTG
    61 GGGAATTACA AGAAACCCAA GCTTCTCTAC TGTAGCAATG GGGGCCATTT CTTGAGGATC
    121 CTTCCTGATG GCAAAGTGGA TGGGACACGG GACAGAAATG ATCAACACAT TCAACTGCAG
    181 CTGAGCACGG AAAGTGTGGG TGAGGTGTAT ATAAAGAGCA CTGAGTCTGG CCAGTATTTG
    241 GCTATGGACA CCGATGGACT TTTATATGGC TCACAGACAC CCAGTGAAGA ATGCTTGTTT
    301 CTGGAGAGGT TGGAGGAGAA TCATTACAAC ACCTACACAT CGAAGAAGCA TGCAGAGAAA
    361 AATTGGTTTG TTGGTCTCAA GAAGAATGGA AGCTGCAAAA AGGGTCCCAG GACTCACTAC
    421 GGCCAGAAAG CCATCCTGTT CCTTCCCCTC CCTGTGTCCT CTGAGTAA
    Common vampire bat FGF1 gene coding sequence (1-155) (SEQ ID NO: 81)
    (GenBank accession no. GABZ01008334, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCTGAAG GGGAAGTCAC CACGTTCACA GCTCTGACTG AGAAGTTTAA TCTGCCTCTG
    61 GAGAGTTACA AGAAGCCCAA ACTTCTCTAC TGCAGCAACG GTGGCCACTT CCTGAGGATC
    121 CTTCCAGATG GTACAGTGGA TGGGACAAGG GACAAGAGCG ACCAGCACAT TCAGCTGCAG
    181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTAC ATAAAGAGCA CCGGGAGTGG CCAGTACTTG
    241 GCCATGGACT CCGCCGGGCT TTTGTATGGC TCACAGACAC CAAATGAGGA ATGTTTGTTC
    301 CTGGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACACAT CCAAGAAGCA TGCAGAAAAG
    361 AATTGGTTCG TGGGGCTCAA GAAGAATGGA AGCTGCAAGC GTGGCCCCCG GACTCATTAT
    421 GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTCAACT CTGATTAA
    Cattle FGF1 gene coding sequence (1-155) (SEQ ID NO: 82) (GenBank
    accession no. NM_174055, which is hereby incorporated
    by reference in its entirety):
    918                   ATG GCTGAAGGAG AAACCACGAC CTTCACGGCC CTGACTGAGA
    961 AGTTTAACCT GCCTCTAGGC AATTACAAGA AGCCCAAGCT CCTCTACTGC AGCAACGGGG
    1021 GCTACTTCCT GAGAATCCTC CCAGATGGCA CAGTGGATGG GACGAAGGAC AGGAGCGACC
    1081 AGCACATTCA GCTGCAGCTC TGTGCGGAAA GCATAGGGGA GGTGTATATT AAGAGTACGG
    1141 AGACTGGCCA GTTCTTGGCC ATGGACACCG ACGGGCTTTT GTACGGCTCA CAGACACCCA
    1201 ATGAGGAATG TTTGTTCCTG GAAAGGTTGG AGGAAAACCA TTACAACACC TACATATCCA
    1261 AGAAGCATGC AGAGAAGCAT TGGTTCGTTG GTCTCAAGAA GAACGGAAGG TCTAAACTCG
    1321 GTCCTCGGAC TCACTTCGGC CAGAAAGCCA TCTTGTTTCT CCCCCTGCCA GTCTCCTCTG
    1381 ATTAA
    Platypus FGF1 gene coding sequence (1-155) (SEQ ID NO: 83) (GenBank
    accession no. XM_001514811, which is hereby incorporated
    by reference in its entirety):
    1 ATGGCGGAGG GTGAAATCAC CACGTTCACA GCCCTGATGG AGAAGTTCGA CCTACCCCTG
    61 GGCAACTACA AAAAGCCTAG GCTGCTCTAC TGCAGCAATG GCGGCTACTT CCTGCGCATC
    121 CAGCCAGACG GTAAAGTGGA CGGGACCAGG GATCGGAGCG ATCAGCACAT TCAACTGCAG
    181 CTAAGCGCGG AAAGCGTGGG CGAGGTGTAT ATAAAGAGCA CCGAGTCTGG CCACTATTTG
    241 GCTATGGACA CCGAAGGACT TTTATATGGC TCACAGGCAC CCAGTGAAGA CTGCTTGTTC
    301 CTGGAGCGGC TGGAGGAGAA CCACTATAAC ACGTACGTGT CCAAGAAGCA CGCTGAGAAG
    361 AATTGGTTTG TCGGTCTCAA GAAGAACGGG AGCTGCAAAC GAGGTCCCCG GACTCACTAC
    421 GGCCAGAAAG CCATCCTCTT CCTCCCGCTC CCCGTGGCAT CCGACTAG
    Zebra finch FGF1 gene coding sequence (1-155) (SEQ ID NO: 84) (GenBank
    accession no. XM_002193251, which is hereby incorporated
    by reference in its entirety):
    1 ATGGCCGAGG GGGAGATCAC CACCTTCAGC GCCCTGACGG AGAAGTTCAA CCTGCCCCCG
    61 GGGAACTACA AGAAGCCCAA ACTGCTGTAC TGCAGCAACG GGGGGCATTT CCTGCGCATC
    121 CTCCCGGACG GCACCGTGGA TGGCACCAGG GACCGCAGCG ACCAGCACAT TCAGCTCCAG
    181 CTGAGTGCAG AGAGCGTGGG GGTGGTGCAC ATCCAGAGCA CCCAGTCGGG GCAGTACCTG
    241 GCCATGGACA CCAACGGGCT GCTCTACGGC TCGCAGCTGC CACCCGGTGA GTGTCTGTTC
    301 CTGGAAAGGC TGGAGGAGAA CCATTACAAC ACCTACGTCT CCAAAATGCA CGCGGACAAG
    361 AACTGGTTTG TGGGGCTGAA GAAGAACGGG ACAAGCAAGC TGGGCCCGCG GACTCACTAC
    421 GGCCAGAAGG CGATCCTGTT CCTGCCGCTG CCCGTGGCGG CCGACTGA
    Nine-banded armadillo FGF1 gene coding sequence (1-155) (SEQ ID NO: 85)
    (GenBank accession no. DP001080, which is hereby incorporated by
    reference in its entirety):
    178389         TT AATCAGAGGA GACTGGCAGG GGAAGAAACA AGATAGCTTT CTGGCCATAG
    178441 TGAGTCTGAG GACCACGTTT GCTGCTTCCG TCCTTCTTGA GACCAACAAA CCATTTCTTC
    178501 TCTGCATGCT TCTTGGATAT GTAGGTGTTG TAATTGTTTT CTTCCAGCTT TTCCATGAAC
    178561 AAGCATTCCT CACTTGGTGT CTC
    182873                                                          TGAGCCAT
    182881 ATAAAAGCCC GTCGGTGTCC ATGGCTAAGT ACTGGCCGGT CTCTGCACTC TTTATATACA
    182941 CCTCCCCCAC GCTTTCCGCA CTGAGCTGCA GCTGAA
    197786                            TGTGT TGGTCGCTCC TGTCCCTTGT CCCATCCACC
    197821 GTGCCATCTG GAAGGATCCT CAAGAAGTGG CCCCCGTTTC TGCAGTAGAG GAGTCTGGGG
    197881 TGCTTGTAAT TTTCTAGGGG CAGGTTGAAC TTCTCCATCA GGGCCATGAA GGTTGTGATC
    197941 TCCCCTTCAG CCAT
    Xenopus Silurana tropicalis FGF1 gene coding sequence (1-155) (SEQ ID
    NO: 86) (GenBank accession no. FJ428265, which is hereby incorporated
    by reference in its entirety):
    1 ATGGCAGAGG GAGACATCAC AACATTCAAC CCCATTGCAG AGTCCTTCAG TCTTCCAATT
    61 GGCAACTACA AGAAACCAAA ACTTCTGTAC TGTAATAATG GAGGGTATTT TTTGCGCATC
    121 CTCCCAGATG GGGTTGTGGA TGGAACAAGA GACAGAGATG ACCTTTACAT TACACTGAAG
    181 TTAAGCGCAC AAAGCCAAGG GGAGGTGCAT ATCAAAAGCA CAGAGACAGG GAGTTACTTA
    241 GCCATGGACT CCAGTGGACA GTTGTATGGA ACTCTCACAC CAAATGAAGA AAGCCTGTTT
    301 CTGGAGACAT TAGAAGAGAA TCACTATAAC ACATACAAGT CAAAGAAGTA TGCAGAAAAT
    361 AACTGGTTTG TGGGGATAAA GAAGAACGGG GCAAGCAAAA AGGGATCAAG GACTCACTAT
    421 GGACAAAAAG CCATCCTTTT TCTGCCGCTG CCAGCATCAC CTGACTAG
    Heterocephalus glaber FGF1 gene coding sequence (1-155) (SEQ ID NO: 87)
    (generated using SMS Reverse Translate tool on the ExPASy Bioinformatics
    Resource website (www.expasy.org):
    1 ATGGCGGAAG GCGAAATTAC CACCTTTACC GCGCTGACCG AAAAATTTAA CCTGCCGCCG
    61 GGCAACTATA AAAAACCGAA ACTGCTGTAT TGCAGCAACG GCGGCCATTT TCTGCGCATT
    121 CTGCCGGATG GCAAAGTGGA TGGCACCCGC GATCGCAGCG ATCAGCATAT TCAGCTGCAG
    181 CTGAGCGCGG AAGGCGTGGG CGAAGTGTAT ATTAAAAGCA CCGAAACCGG CCAGTATCTG
    241 GCGATGGATA CCGATGGCCT GCTGTATGGC AGCCAGACCG CGAGCGAAGA ATGCCTGTTT
    301 CTGGAACGCC TGGAAGAAAA CCATTATAAC ACCTATATTA GCAAAAAACA TGCGGAAAAA
    361 AACTGGTTTG TGGGCCTGAA AAAAAACGGC AGCTGCAAAC GCGGCCCGCG CACCCATTAT
    421 GGCCAGAAAG CGATTCTGTT TCTGCCGCTG CCGGTGAGCA GCGAT
    Black flying fox FGF1 gene coding sequence (1-155) (SEQ ID NO: 88)
    (generated using SMS Reverse Translate tool on the ExPASy Bioinformatics
    Resource website (www.expasy.org):
    1 ATGGCGGAAG GCGAAGTGAC CACCTTTACC GCGCTGACCG AACGCTTTAA CCTGCCGCCG
    61 GGCAACTATA AAAAACCGAA ACTGCTGTAT TGCAGCAACG GCGGCCATTT TCTGCGCATT
    121 CTGCCGGATG GCACCGTGGA TGGCACCCGC GATAAAAGCG ATCAGCATAT TCAGCTGCAG
    181 CTGAGCGCGG AAAGCGTGGG CGAAGTGTAT ATTAAAAGCA CCGAAAGCGG CCAGTATCTG
    241 GCGATGGATA GCGATGGCCT GCTGTATGGC AGCCAGACCC CGGATGAAGA TTGCCTGTTT
    301 CTGGAACGCC TGGAAGAAAA CCATTATAAC ACCTATACCA GCAAAAAACA TGCGGAAAAA
    361 AACTGGTTTG TGGGCCTGAA AAAAAACGGC AGCTGCAAAC GCGGCCCGCG CACCCATTAT
    421 GGCCAGAAAG CGATTCTGTT TCTGCCGCTG CCGGTGAGCA GCGAT
    Chinese tree shrew FGF1 gene coding sequence (1-155) (SEQ ID NO: 89)
    (generated using SMS Reverse Translate tool on the ExPASy Bioinformatics
    Resource website (www.expasy.org):
    1 ATGGCGGAAG GCGAAATTAC CACCTTTGCG GCGCTGACCG AAAAATTTGA TCTGCCGCCG
    61 GGCAACTATA AAAAACCGAA ACTGCTGTAT TGCAGCAACG GCGGCCATTT TCTGCGCATT
    121 CTGCCGGATG GCACCGTGGA TGGCACCCGC GATCGCAGCG ATCAGCATAT TCAGCTGCAG
    181 CTGACCGCGG AAAACGTGGG CGAAGTGTAT ATTAAAAGCA CCGAAACCGG CCAGTATCTG
    241 GCGATGGATG CGGATGGCCT GCTGTATGGC AGCCAGACCC CGAACGAAGA ATGCCTGTTT
    301 CTGGAACGCC TGGAAGAAAA CCATTATAAC ACCTATATTA GCAAAAAACA TGCGGAAAAA
    361 AACTGGTTTG TGGCGCTGAA AAAAAACGGC AGCTGCAAAC TGGGCCCGCG CACCCATTAT
    421 GGCCAGAAAG CGATTCTGTT TCTGCCGCTG CCGGTGAGCA GCGAT
    Rock pigeon FGF1 gene coding sequence (1-155) (SEQ ID NO: 90) (generated
    using SMS Reverse Translate tool on the ExPASy Bioinformatics Resource
    website (www.expasy.org):
    1 ATGGCGGAAG GCGAAATTAC CACCTTTACC GCGCTGACCG AAAAATTTAA CCTGCCGCCG
    61 GGCAACTATA AAAAACCGAA ACTGCTGTAT TGCAGCAACG GCGGCCATTT TCTGCGCATT
    121 CTGCCGGATG GCAAAGTGGA TGGCACCCGC GATCGCAGCG ATCAGCATAT TCAGCTGCAG
    181 CTGAGCGCGG AAAGCGTGGG CGAAGTGTAT ATTAAAAGCA CCCAGAGCGG CCAGTATCTG
    241 GCGATGGATC CGACCGGCCT GCTGTATGGC AGCCAGCTGC TGGGCGAAGA ATGCCTGTTT
    301 CTGGAACGCA TTGAAGAAAA CCATTATAAC ACCTATGTGA GCAAAAAACA TGCGGATAAA
    361 AACTGGTTTG TGGGCCTGAA AAAAAACGGC AACAGCAAAC TGGGCCCGCG CACCCATTAT
    421 GGCCAGAAAG CGATTCTGTT TCTGCCGCTG CCGGTGAGCG CGGAT
    Sheep FGF1 gene coding sequence (1-155) (SEQ ID NO: 91)
    (GenBank accession no. XM_004008909, which is
    hereby incorporated by reference in its entirety):
    361 ATGGCTGAAG GAGAAACCAC AACCTTCAGG GCCCTGACTG AGAAGTTTAA CCTGCCTCTA
    421 GGCAATTACA AGAAGCCCAA GCTCCTCTAT TGCAGCAACG GGGGCTACTT CCTGAGAATC
    481 CTCCCAGATG GCAGAGTGGA TGGGACGAAG GACAGGAGCG ACCAGCACAT TCAGCTGCAG
    541 CTCTATGCGG AAAGCATAGG GGAGGTGTAT ATTAAGAGTA CGGAGACTGG CCAGTTCTTG
    601 GCCATGGACA CCAACGGGCT TTTGTACGGC TCACAAACAC CCAGTGAGGA ATGTTTGTTC
    661 CTGGAAAGGC TGGAGGAAAA CCATTATAAC ACCTACATAT CCAAGAAGCA TGCAGAGAAG
    721 AATTGGTTCA TTGGTCTCAA GAAGAACGGA AGCTCCAAAC TCGGTCCTCG GACTCACTTC
    781 GGCCAGAAAG CCATCTTGTT TCTCCCCCTG CCAGTTTCCT CTGATTAA
    Chicken FGF1 gene coding sequence (1-155) (SEQ ID NO: 92) (GenBank
    accession no. NM_205180, which is hereby incorporated by
    reference in its entirety):
    52                                                         ATGGCCGAG
    61 GGGGAGATAA CCACCTTCAC CGCCCTGACC GAGCGCTTCG GCCTGCCGCT GGGCAACTAC
    121 AAGAAGCCCA AACTCCTGTA CTGCAGCAAC GGGGGCCACT TCCTACGGAT CCTGCCGGAC
    181 GGCAAGGTGG ACGGGACGCG GGACCGGAGT GACCAGCACA TTCAGCTGCA GCTCAGCGCG
    241 GAAGATGTGG GCGAGGTCTA TATAAAGAGC ACAGCGTCGG GGCAGTACCT GGCAATGGAC
    301 ACCAACGGGC TCCTGTATGG CTCGCAGCTA CCAGGCGAGG AGTGCTTGTT CCTTGAGAGG
    361 CTCGAGGAGA ACCATTACAA CACATACATC TCCAAAAAGC ACGCAGACAA GAACTGGTTC
    421 GTCGGGCTGA AGAAAAACGG GAACAGCAAG CTGGGGCCGC GGACTCACTA TGGGCAAAAG
    481 GCGATCCTCT TCCTCCCATT GCCGGTGTCG GCTGACTGA
    Alpaca FGF1 gene coding sequence (1-155, excluding 1-57) (SEQ ID NO: 93)
    (Ensembl accession no. ENSVPAT00000008395, which is hereby incorporated
    by reference in its entirety):
    1 CAGCTGCAGC TCAGTGCGGA AAGCGTGGGG GAGGTGTATA TAAAGAGTAC CGAGACTGGC
    61 CAGTACTTGG CCATGGACAC CGACGGGCTT TTGCACGGCT CACAGACACC AAATGAGGAA
    121 TGTTTGTTCC TGGAAAGGCT GGAGGAGAAC CATTACAACA CCTACACGTC CAAGAAGCAC
    181 GCCGAAAAGA ATTGGTTTGT TGGTCTCAAG AAGAATGGAA GCTGCAAACG CGGTCCTCGG
    241 ACTCACTACG GCCAGAAGGC GATCTTGTTT CTCCCCTTGC CAGTCTCCTC TGATTAA
    Anole lizard FGF1 gene coding sequence (1-155) (SEQ ID NO: 94) (Ensembl
    accession no. ENSACAT00000013467, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCTGAAG GTGAAATAAC AACATTCACA GCCTTGACCG AGAGGTTTGC TCTCCCAATG
    61 GAGAATTACA AGAAGCCCAA ACTCCTGTAT TGCAGCAATG GAGGCCACTT CCTGAGGATC
    121 CTTCCAGATG GAAAAGTGGA TGGCACCATG GACCGGAATG ACAGCTATAT TCAGTTGCTG
    181 TTAACAGCAG AAGATGTGGG TGTGGTATAT ATAAAAGGCA CTGAGACCGG GCAGTACTTG
    241 GCCATGGATG CCAATGGACA TTTATATGGC TCGCAGTTGC CAACAGAAGA GTGTTTATTT
    301 GTGGAAACGC TGGAAGAAAA CCATTACAAT ACATATACCT CAAAGATGCA TGGCGATAAG
    361 AAGTGGTATG TTGGCTTGAA AAAGAATGGG AAAGGCAAAC TGGGGCCACG GACTCATCGC
    421 GGCCAAAAGG CAATACTTTT CCTTCCACTG CCAGTATCAC CTGATTAG
    Bushbaby FGF1 gene coding sequence (1-155) (SEQ ID NO: 95) (Ensembl
    accession no. ENSOGAT00000005081, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCTGAAG GGGAAATCAC AACCTTCACA GCCCTCACAG AGAAGTTTAA TCTGCCTCTA
    61 GGAAATTACA AGAAGCCCAA GCTCCTCTAC TGTAGCAACG GGGGTCACTT TCTGAGGATC
    121 CTGCCGGATG GCACCGTGGA TGGGACACAA GACAGGAGCG ACCAGCACAT TCAGCTGCAG
    181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CCCAGACTGG CCAGTACTTG
    241 GCCATGGACT CCGACGGGCT TTTATACGGC TCACAAACAC CAAATGAGGA ATGCCTGTTC
    301 CTGGAACGGC TGGAGGAAAA CCATTACAAC ACCTATGTGT CCAAGAAGCA CGCCGAGAAG
    361 AATTGGTTTG TCGGTCTCAA GAAGAACGGA AGTTGCAAAC GTGGTCCTCG GACTCACTAC
    421 GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTCTCCT CTGATTAA
    Cat FGF1 gene coding sequence (1-155) (SEQ ID NO: 96) (Ensembl accession
    no. ENSFCAT00000009123, which is hereby incorporated
    by reference in its entirety):
    1 ATGGCTGAAG GGGAAATCAC AACCTTCACG GCCCTGACGG AGAAGTTCAA TCTGCCTCCA
    61 GGGAATTACA AGAAACCCAA ACTCCTCTAC TGTAGCAACG GGGGCCACTT CCTGAGGATC
    121 CTTCCAGATG GCACAGTGGA TGGGACGAGG GACAGGAGCG ACCAGCACAT TCAGCTGCAG
    181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CCGAGACTGG CCAGTACTTG
    241 GCCATGGACA CCGACGGGCT TTTGTACGGC TCACAGACAC CAAATGAGGA ATGCTTGTTC
    301 CTGGAAAGGC TGGAAGAAAA CCATTACAAC ACCTACACAT CCAAGAAGCA CGCAGAAAAG
    361 AATTGGTTTG TGGGTCTCAA GAAGAATGGA AGCTGCAAAC GCGGTCCCCG GACTCACTAT
    421 GGCCAGAAGG CAATTTTGTT TCTCCCCCTG CCAGTCTCCT CTGATTAA
    Chinese softshell turtle FGF1 gene coding sequence (1-155)
    (SEQ ID NO: 97) (Ensembl accession no. ENSPSIT00000016432,
    which is hereby incorporated by reference in its entirety):
    131            ATGGCTGAAG GGGAAATAAC AACGTTCACC GCCCTGACCG AAAAATTCAA
    181 CCTTCCCCTG GGGAATTACA AGAATCCCAA ACTCTTATAT TGCAGCAATG GAGGCTACTT
    241 CTTGAGGATA CATCCAGATG GCAAAGTAGA TGGGACAAGG GACCGAAGTG ACCAACACAT
    301 TCAGCTGCAG CTAAGTGCGG AAAGCGTGGG TGAGGTATAT ATAAAGAGCA CTGAGTCTGG
    361 ACAGTTTTTG GCTATGGACG CCAATGGACT TTTATATGGA TCACTGTCAC CGAGTGAGGA
    291 ATGCTTATTC TTGGAAAGAA TGGAAGAAAA TCATTATAAC ACCTACATCT CCAAGAAGCA
    351 TGCAGACAAG AACTGGTTCG TTGGCTTAAA GAAGAATGGA AGCTGCAAAC TGGGACCGCG
    411 GACGCACTAC GGCCAAAAGG CCGTCCTTTT CCTTCCACTG CCAGTGTCAG CTGATTAA
    Coelacanth FGF1 gene coding sequence (1-155) (SEQ ID NO: 98) (Ensembl
    accession no. ENSLACT00000015212, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCTGAAG ACAAAATAAC AACACTGAAG GCCTTGGCTG AAAAATTTAA CCTTCCTATG
    61 GGAAATTACA AGAAAGCAAA ACTCCTCTAC TGCAGCAACG GAGGGTATTT CCTGCGAATA
    121 CCCCCAGACG GGAAAGTGGA AGGAATTAGA GAACGAAGCG ACAAGTACAT TCAGCTGCAA
    181 ATGAATGCAG AAAGTTTAGG CATGGTGTCT ATAAAGGGTG TGGAGGCAGG GCAATACCTA
    241 GCTATGAATA CAAATGGACT CCTGTATGGA TCTCAGTCTC TAACTGAAGA ATGCCTTTTC
    301 ATGGAAAAGA TGGAAGAAAA CCACTACAAC ACATACAGGT CTAAGACACA TGCAGATAAA
    361 AACTGGTATG TTGGCATTAG AAAGAACGGT AGCATCAAAC CAGGACCAAG GACTCACATT
    421 GGCCAAAAGG CTGTTCTTTT TCTCCCTCTG CCTGCCTCGA GTGATTAG
    Dolphin FGF1 gene coding sequence (1-155) (SEQ ID NO: 99) (Ensembl
    accession no. ENSTTRT00000004742, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCTGAAG GGGAAATCAC AACCTTCACA GCCCTGACCG AGAAGTTTAA TCTGCCTCCA
    61 GGGAATTACA AGAAGCCCAA ACTCCTCTAC TGTAGCAACG GGGGCCACTT CCTGAGGATC
    121 CTTCCAGATG GCACAGTGGA TGGGACAAGG GACAGGAGTG ACCAGCACAT TCAGCTGCAG
    181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CGGAGACTGG CCAGTACTTG
    241 GCCATGGACA CCGACGGGCT TTTGTACGGC TCACAGACAC CCAATGAGGA ATGTTTGTTC
    301 CTGGAAAGGT TGGAGGAAAA CCATTACAAC ACCTACGCAT CCAAGAAGCA TGCAGAAAAG
    361 AATTGGTTCG TTGGTCTCAA GAAGAACGGA AGCTGCAAAC GCGGTCCTCG GACTCACTAC
    421 GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTCTCCT CCGATTAA
    Ferret FGF1 gene coding sequence (1-155) (SEQ ID NO: 100) (Ensembl
    accession no. ENSMPUT00000008013, which is hereby incorporated by
    reference in its entirety):
    1                                      ATGGCT GAAGGGGAAA TCACAACCTT
    61 CACAGCCCTG ATGGAGAAGT TTAATCTGCC TGCGGGGAAT TACAAGAAGC CCAAACTCCT
    121 CTACTGTAGC AATGGGGGCC ACTTCCTGAG GATCCTTCCA GATGGCACAG TGGACGGCAC
    181 AAGGGACAGG AGCGACCAGC ACATTCAGCT GCAGCTCAGT GCGGAAAGCG TGGGGGAGGT
    241 GTACATAAAG AGTACCGAGA CTGGCCAGTA CTTGGCCATG GACACCGATG GGCTTTTGTA
    301 CGGCTCACAA ACACCAAATG AGGAATGTCT GTTCCTGGAA AGGCTGGAGG AAAACCATTA
    361 CAACACCTAC ACATCCAAGA AGCACGCTGA GAAGAATTGG TTTGTAGGTC TCAAGAAGAA
    421 CGGAAGCTGC AAACGCGGTC CTCGGACTCA CTATGGCCAG AAAGCAATTC TGTTTCTCCC
    481 CCTGCCAGTC TCCTCTGATT AA
    Gibbon FGF1 gene coding sequence (1-155) (SEQ ID NO: 101) (Ensembl
    accession no. ENSNLET00000012455, which is hereby incorporated by
    reference in its entirety):
    241                                                   ATGG CCGAAGGGGA
    301 AATCACCACC TTCACAGCCC TGACCGAGAA GTTTAATCTG CCTCCAGGGA ATTACAAGAA
    361 GCCCAAACTC CTCTACTGTA GCAACGGGGG CCACTTCTTG AGGATCCTTC CGGATGGCAC
    421 AGTGGATGGG ACAAGGGACA GGAGCGACCA GCACATTCAG CTGCAGCTCA GTGCGGAAAG
    481 CGTGGGGGAG GTGTATATAA AGAGTACCGA GACTGGCCAG TACTTGGCCA TGGACACCGA
    541 CGGGCTTTTA TACGGCTCAC AGACACCAAA TGAGGAATGT TTGTTCCTGG AAAGGCTGGA
    601 GGAGAACCAT TACAACACCT ATATATCCAA GAAGCATGCA GAGAAGAATT GGTTTGTTGG
    661 CCTCAAGAAG AATGGAAGCT GCAAACGCGG TCCTCGGACT CACTATGGCC AGAAAGCAAT
    721 CTTGTTTCTC CCCCTGCCAG TCTCTTCTGA TTAA
    Gorilla FGF1 gene coding sequence (1-155) (SEQ ID NO: 102) (Ensembl
    accession no. ENSGGOT00000025344, which is hereby incorporated by
    reference in its entirety):
    121                                                   ATGG CTGAAGGGGA
    181 AATCACCACC TTCACAGCCC TGACCGAGAA GTTTAATCTG CCTCCAGGGA ATTACAAGAA
    241 GCCCAAACTC CTCTACTGTA GCAATGGGGG CCACTTCTTG AGGATCCTTC CGGATGGCAC
    301 AGTGGATGGG ACAAGGGACA GGAGCGACCA GCACATTCAG CTGCAGCTCA GTGCGGAAAG
    361 CGTGGGGGAG GTGTATATAA AGAGTACCGA GACTGGCCAG TACTTGGCCA TGGACACCGA
    421 CGGGCTTTTA TACGGCTCAC AGACACCAAA TGAGGAATGT TTGTTCCTGG AAAGGCTGGA
    481 GGAGAACCAT TACAACACCT ATATATCCAA GAAGCATGCA GAGAAGAATT GGTTTGTTGG
    541 CCTCAAGAAG AATGGAAGCT GCAAACGCGG TCCTCGGACT CACTATGGCC AGAAAGCAAT
    601 CTTGTTTCTC CCCCTGCCAG TCTCTTCCGA TTAA
    Hedgehog FGF1 gene coding sequence (1-155) (SEQ ID NO: 103) (Ensembl
    accession no. ENSEEUT00000005832, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCTGAAG GAGAAATCAC CACCTTCACG GCCCTGACTG AGAAGTTTAA TCTGCCACTA
    61 GGGAATTACA AGAAGCCCAA GCTCCTCTAC TGTAGCAACG GGGGCCACTT CCTGAGGATC
    121 CTTCCAGATG GCACCGTGGA TGGGACAAGG GACAGGAGCG ACCAGCATAT TCAGCTGCAG
    181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CGGAGACTGG CCAGTACTTG
    241 GCCATGGACA CCGACGGGCT TTTATACGGC TCACAAACAC CAAATGAGGA ATGTCTGTTC
    301 CTTGAAAGGC TGGAAGAGAA CCATTACAAT ACCTACACAT CCAAGAAGCA TGCCGAGAAG
    361 AACTGGTTTG TTGGCCTCAA GAAGAATGGA AGCTGCAAGC GTGGTCCTCG GACTCATTAT
    421 GGCCAGAAAG CTATTTTGTT TCTCCCCCTG CCAGTTTCCT CTGATTAA
    Hyrax FGF1 gene coding sequence (1-155, excluding 1-90) (SEQ ID NO: 104)
    (Ensembl accession no. ENSPCAT00000011746, which is hereby incorporated
    by reference in its entirety):
    1 ATGGCTGAAG GCGAAATCAC AACCTTCACA GCCCTGACTG AGAAGTTTAA CCTGCCACTA
    61 GAGAATTACA AGAAGCCCAA ACTCCTCTAC TGTAGCAACG GAGGCCACTT CCTGAGGATC
    121 CTTCCGGACG GCACAGTGGA TGGCACCAGG GACAGGAGTG ACCAGCACAT TCAGCTGCAG
    181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGGGCA CCGAGACTGG CCAGTACTTG
    241 GCCATGGACA CCGACGGGCT TTTATATGGC TCA
    Kangaroo rat FGF1 gene coding sequence (1-155, excluding 1-16 and
    58-155) (SEQ ID NO: 105) (Ensembl accession no. ENSDORT00000007345,
    which is hereby incorporated by reference in its entirety):
    1 ATGGCTGAAG GGGAAATCAC AACCTTCACA GCCCTGACGG AAAGGTTTAA ----------
    ---------- ---------- ---------- ---------- ---------- ----------
    51 ---------- ---------- ---------- ---------- ---------T TCAGCTGCAA
    62 CTGAGTGCGG AAAGCGTGGG GGAGGTCTAT ATAAAGAGCA CCGAGACTGG CCAATACTTG
    122 GCCATGGATG CCGACGGGCT TTTATACGGC TCACAGACAC CTGATGAAGA ATGCTTGTTC
    182 CTGGAGAGGC TGGAAGAAAA TCATTATAAC ACCTACATAG CCAAGAAACA TGCTGAAAAG
    242 AATTGGTTTG TCGGCCTCAA AAAGAATGGA AGCTGCAAGC GTGGTCCTCG GACTCACTAT
    302 GGCCAGAAAG CAATCCTGTT CCTCCCCTTG CCTGTCTCCT CTGATTAG
    Lamprey FGF1 gene coding sequence (1-155, excluding 94-155) (SEQ ID
    NO: 106) (Ensembl accession no. ENSPMAT00000010729, which is hereby
    incorporated by reference in its entirety):
    1 ATGGAGGTGG GCCACATCGG CACGCTGCCC GTGGTCCCCG CGGGGCCCGT GTTCCCCGGC
    61 AGTTTCAAGG AGCCACGGCG CCTCTACTGC CGCAGCGCGG GCCACCACCT CCAGATCCTG
    121 GGGGACGGCA CCGTGAGTGG CACCCAGGAC GAGAACGAGC CCCACGCCGT TCTGCAGCTG
    181 CAGGCGGTGC GCCGCGGGGT GGTGACGATC CGTGGGCTCT GCGCCGAGAG GTTCCTCGCC
    241 ATGAGCACGG AGGGACACCT GTACGGGGCG GTGAGG
    Lesser hedgehog tenrec FGF1 gene coding sequence (1-155, excluding 1-57)
    (SEQ ID NO: 107) (Ensembl accession no. ENSETET00000017851, which is
    hereby incorporated by reference in its entirety):
    1 CAGCTGAAGC TCGTTGCCGA AAGCGTGGGG GTGGTGTATA TAAAGAGCAT CAAGACCGGC
    61 CAGTACTTGG CCATGAACCC CGACGGGCTT TTATACGGCT CCGAGACCCC AGAGGAAGAA
    121 TGCTTGTTCC TGGAAACGCT GGAGGAAAAC CACTACACCA CCTTCAAATC TAAGAAGCAC
    181 GTAGAGAAGA ATTGGTTCGT TGGTCTCCGG AAGAATGGAA GGGTCAAGAT CGGGCCTCGG
    241 ACTCACCAAG GCCAGAAAGC AATCTTGTTC CTGCCCCTCC CGGTGTCCTC TGATTAA
    Rhesus monkey FGF1 gene coding sequence (1-155) (SEQ ID NO: 108)
    (Ensembl accession no. ENSMMUT00000033070, which is
    hereby incorporated by reference in its entirety):
    36                                       ATGGC TGAAGGGGAA ATCACCACGT
    61 TCACAGCCCT GACCGAGAAG TTTAATCTGC CTCCAGGGAA TTACAAGAAG CCCAAACTGC
    121 TCTACTGTAG CAATGGGGGC CACTTCTTGA GGATCCTTCC GGATGGCACA GTGGATGGGA
    181 CAAGGGACAG GAGCGACCAG CACATTCAGC TGCAGCTCAG TGCGGAAAGC GTGGGGGAGG
    241 TGTATATAAA GAGTACCGAG ACTGGCCAGT ACTTGGCCAT GGACACCGAC GGGCTTTTAT
    301 ACGGCTCACA GACACCAAAT GAGGAATGTT TGTTCCTGGA AAGGCTGGAG GAGAACCATT
    361 ACAACACCTA TACATCCAAG AAGCACGCAG AGAAGAATTG GTTTGTTGGC CTCAAGAAGA
    421 ATGGAAGCTG CAAACGTGGT CCTCGGACTC ACTATGGCCA GAAAGCAATC TTGTTTCTTC
    481 CCCTGCCAGT CTCTTCTGAT TAA
    Megabat FGF1 gene coding sequence (1-155) (SEQ ID NO: 109) (Ensembl
    accession no. ENSPVAT00000004596, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCCGAGG GGGAAGTCAC GACGTTCACG GCCCTGACCG AGAGGTTTAA CCTGCCTCCA
    61 GGGAATTACA AGAAGCCCAA ACTTCTCTAC TGCAGCAACG GGGGCCACTT CCTGAGGATC
    121 CTCCCAGATG GCACAGTGGA TGGGACAAGG GACAAGAGCG ACCAGCACAT TCAGCTGCAG
    181 CTCAGTGCGG AAAGTGTGGG GGAGGTGTAT ATAAAGAGCA CCGAGAGTGG CCAGTACTTG
    241 GCCATGGACT CCGACGGGCT TTTGTACGGC TCACAGACAC CAGATGAGGA CTGTTTGTTC
    301 CTGGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACACAT CCAAGAAGCA CGCAGAGAAG
    361 AATTGGTTTG TTGGGCTCAA GAAGAATGGA AGCTGCAAGC GCGGTCCCCG GACTCACTAC
    421 GGCCAGAAAG CGATCCTGTT TCTCCCCCTG CCAGTCTCCT CTGATTAG
    Microbat FGF1 gene coding sequence (1-155) (SEQ ID NO: 110) (Ensembl
    accession no. ENSMLUT00000007098, which is hereby incorporated by
    reference in its entirety):
    66      ATGGC TGAGGGGGAA GTCACCACAT TCACGGCCCT GACCGAGAGG TTCAATCTGC
    121 CTCTGGAGAA CTACAAGAAG CCCAAGCTTC TCTACTGCAG CAACGGGGGC CACTTCCTGC
    181 GGATCCTCCC AGACGGCACC GTGGACGGGA CGAGGGACAG GAGCGACCAG CACATTCAGC
    241 TGCAGCTCAG TGCGGAAAGC GTGGGGGAGG TGTATATAAA GAGCACCGAG AGTGGCCAGT
    301 ACTTGGCCAT GGACTCCGAC GGGCTTTTGT ACGGCTCACA AACACCCAAT GAGGAATGTT
    361 TGTTCCTGGA AAGGCTGGAG GAGAACCACT ACAACACCTA CACGTCCAAG AAGCACGCAG
    421 AAAAGAATTG GTTCGTTGGG CTCAAGAAGA ACGGAAGCTG CAAGCGTGGT CCTCGGACGC
    481 ATTATGGCCA GAAAGCAATC TTGTTTCTCC CCCTGCCAGT CTCCTCCGAT TAA
    Mouse lemur FGF1 gene coding sequence (1-155) (SEQ ID NO: 111) (Ensembl
    accession no. ENSMICT00000009454, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCCGAAG GGGAGATCAC AACCTTCACG GCCCTCACCG AGAAGTTTAA CCTGCCTCCG
    61 GGGAACTACA AGAAGCCCAA GCTCCTCTAC TGCAGCAACG GCGGCCACTT CCTGCGCATC
    121 CTTCCCGACG GCACCGTGGA TGGCACGAGA GACAGGAGCG ACCAGCACAT TCAGCTGCAG
    181 CTCAGTGCGG AAAGCGCGGG GGAGGTGTAT ATAAAGAGCA CCCAGACTGG CCGGTACTTG
    241 GCCATGGACG CCGACGGGCT TTTATACGGC TCACAAACAC CAAATGAGGA ATGTTTGTTC
    301 CTGGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACGTAT CCAAGAAGCA CGCAGAGAAG
    361 AATTGGTTTG TTGGCCTCAA GAAGAATGGA AGTTGCAAAC GCGGCCCCCG GACTCACTAT
    421 GGCCAGAAAG CAATCTTGTT TCTGCCCCTG CCAGTCTCCT CTGATTAA
    Pika FGF1 gene coding sequence (1-155, excluding 57-67) (SEQ ID NO: 112)
    (Ensembl accession no. ENSOPRT00000012854, which is hereby incorporated
    by reference in its entirety):
    1 ATGGCCGAGG GAGAAGTCAC CACCTTCTCA GCCCTGACGG AGAAGTTCAA TCTGCCTGGA
    61 GGAAACTACA AGTTGCCCAA GCTCCTTTAC TGTAGCAACG GAGGCCACTT CCTGAGGATC
    121 CTTCCAGATG GCACAGTGGA TGGGACCAGG GACAGGAGCG ACCTGCACA- ----------
    170 ---------- ---------- -GAGGTGTTT ATAAAGAGTA CGGAGACTGG CCAGTACTTG
    209 GCTATGGACA CCGATGGCCT TTTATATGGC TCGCAGACAC CCAGTGAGGA GTGTTTGTTC
    269 CTGGAGCGGC TGGAGGAGAA CCACTACAAC ACCTACACAT CCAAGAAGCA TGCCGAGAAG
    329 AACTGGTTTG TGGGCATCAA GAAGAATGGA AGCTGCAAGC GTGGTCCTCG GACTCACTAC
    389 GGCCAGAAAG CCATCTTGTT TCTCCCTCTG CCAGTCTCTT CTGACTAA
    Rat FGF1 gene coding sequence (1-155) (SEQ ID NO: 113)
    (Ensembl accession no. ENSRNOT00000018577, which is hereby
    incorporated by reference in its entirety):
    268                              ATG GCCGAAGGGG AGATCACAAC CTTTGCAGCC
    301 CTGACCGAGA GGTTCAATCT GCCTCTAGGG AACTACAAAA AACCCAAACT GCTCTACTGC
    361 AGCAACGGGG GCCACTTCTT GAGGATTCTT CCCGATGGCA CCGTGGATGG GACCAGGGAC
    421 AGGAGCGACC AGCACATTCA GCTGCAGCTC AGTGCGGAAA GCGCGGGCGA AGTGTATATA
    481 AAGGGTACAG AGACTGGCCA GTACTTGGCC ATGGACACCG AAGGGCTTTT ATACGGCTCG
    541 CAGACACCAA ATGAAGAATG CCTATTCCTG GAAAGGCTAG AAGAAAACCA TTATAACACT
    601 TACACATCCA AGAAGCACGC GGAGAAGAAC TGGTTTGTGG GCCTCAAGAA GAACGGGAGT
    661 TGTAAGCGCG GTCCTCGGAC TCACTACGGC CAGAAAGCCA TCTTGTTTCT CCCCCTCCCG
    721 GTATCTTCTG ACTAA
    Sloth FGF1 gene coding sequence (1-155) (SEQ ID NO: 114) (Ensembl
    accession no. ENSCHOT00000012416, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCTGAAG GGGAAATCAC AACCTTCACA GCTCTGATGG AGAAGTTTAA CCTGCCACCA
    61 GGGAATTACA TGAAGCCCAA ACTCCTCTAC TGTAGCAACG GGGGCCACTT CTTGAGGATC
    121 CTTCCAGACG GCACAGTGGA TGGGACAAGG GACAGGAGCG ACCTGCACAT TCAGCTGCAG
    181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTG CGGAGACCGG CCAGTACTTA
    241 GCCATGGACA CCGGCGGGCT TTTATACGGC TCACAGACAC CAAGTGAGGA ATGCCTGTTC
    301 CTAGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACGTAT CCAAGAAGCA TGCGGAGAAG
    361 AACTGGTTCG TTGGCCTAAA GAAGAATGGA AGCAGCAAAC GCGGCCCCCG GACTCACTAT
    421 GGCCAGAAAG CCATCTTGTT TCTTCCCCTG CCAGTCTCCT CTGATTAA
    Squirrel FGF1 gene coding sequence (1-155) (SEQ ID NO: 115) (Ensembl
    accession no. ENSSTOT00000029249, which is hereby incorporated by
    reference in its entirety):
    1                                                              ATGG
    5 CTGAAGGGGA AATCACAACC TTCACAGCCC TGACCGAGAA GTTCAATCTG CCTCCAGGGA
    65 ACTACAAGAA GCCCAAACTG CTCTACTGTA GCAACGGAGG CCACTTCTTG AGGATCCTTC
    125 CTGATGGCAC AGTGGATGGG ACAAGAGACA GGAGCGACCA ACACATTCAG CTGCAGCTCA
    185 GTGCGGAAAG CGTGGGGGAG GTGTATATAA AGAGTACCGA GACCGGCCAG TACTTGGCCA
    245 TGGACACCGA CGGGCTTTTA TATGGCTCAC AGACCCCAAA TGAGGAATGC TTATTCCTGG
    305 AAAGGCTGGA GGAAAACCAT TACAACACGT ACACATCCAA GAAGCATGCA GAGAAGAATT
    365 GGTTTGTTGG CCTCAAGAAG AACGGAAGCT GCAAGCGCGG TCCCCGGACT CACTATGGCC
    425 AGAAAGCGAT CTTGTTTCTC CCACTGCCTG TCTCCTCTGA TTAG
    Tarsier FGF1 gene coding sequence (1-155) (SEQ ID NO: 116) (Ensembl
    accession no. ENSTSYT00000007425, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCCGAAG GGGAAATCAC AACCTTCACA GCCCTGACCG AGAAGTTCAA CCTGCCCCCG
    61 GGGAATTACA AGAAGCCCAA ACTCCTCTAC TGCAGCAACG GGGGCCACTT CTTGAGGATC
    121 CTTCCGGATG GCACTGTGGA TGGAACGAGG GACAGGAGCG ACCAGCACAT TCAGCTGCAG
    181 CTCAGCGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CCGAGACCGG CCAGTACTTG
    241 GCCATGGACA CCGACGGGCT TTTGTACGGC TCACAGACAC CAAATGAGGA GTGTCTGTTC
    301 CTGGAAAGGC TGGAAGAGAA TCATTACAAT ACCTACGTGT CCAAGAAGCA TGCGGAGAAG
    361 AATTGGTTTG TCGGCCTCAA GAAGAATGGA AGCTGCAAAC GCGGTCCTCG GACTCACTAT
    421 GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTTTCCT CTGATTAA
    Tree shrew FGF1 gene coding sequence (1-155) (SEQ ID NO: 117) (Ensembl
    accession no. ENSTBET00000011861, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCTGAAG GGGAAATCAC GACCTTCGCA GCCCTGACCG AGAAGTTTGA TCTGCCTCCA
    61 GGGAATTACA AGAAGCCCAA ACTTCTCTAC TGTAGCAACG GGGGCCATTT CTTGAGGATT
    121 CTTCCAGATG GCACCGTGGA TGGGACAAGA GACAGGAGCG ACCAGCACAT TCAGCTGCAG
    181 CTCACTGCGG AAAACGTGGG GGAGGTGTAC ATAAAGAGTA CGGAGACTGG CCAGTACTTG
    241 GCCATGGACG CCGACGGGCT TTTATATGGC TCACAGACAC CAAACGAGGA ATGTTTGTTC
    301 CTGGAAAGGC TGGAGGAGAA CCATTACAAC ACCTACATAT CCAAGAAGCA CGCAGAGAAG
    361 AATTGGTTTG TTGCCCTCAA GAAGAACGGA AGCTGCAAAC TCGGTCCTCG GACTCACTAT
    421 GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTCTCCT CTGATTAA
    Turkey FGF1 gene coding sequence (1-155, excluding 57-155) (SEQ ID
    NO: 118) (Ensembl accession no. ENSMGAT00000017372, which is hereby
    incorporated by reference in its entirety):
    1 ATGGCCGAGG GGGAGATAAC CACCTTCACA GCCCTGACCG AGCGCTTCGG CCTGCCGCTG
    61 GGCAACTACA AGAAGCCCAA ACTCCTGTAC TGCAGCAACG GGGGCCACTT CCTACGGATC
    121 CTGCCGGACG GCAAGGTGGA CGGGACGCGG GACCGGAGCG ACCAGCAC
    Wallaby FGF1 gene coding sequence (1-155) (SEQ ID NO: 119) (Ensembl
    accession no. ENSMEUT00000016544, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCCGAAG GGGAGATCAC AACCTTCACA GCCCTGACCG AAAGATTTAA CCTGCCACTG
    61 GGGAATTACA AGAAGCCCAA GCTTCTCTAC TGTAGCAATG GGGGCCACTT TTTGAGGATC
    121 CTTCCTGATG GCAAAGTGGA TGGGACAAGG GACAGAAATG ATCAACACAT TCAACTGCAA
    181 CTAAGCGCGG AAAGCGTGGG TGAGGTGTAT ATAAAGAGCA CTGAGTCTGG GCAGTATTTG
    241 GCCATGGACA CCAATGGACT TTTATATGGC TCACAGACCC CCAGCGAAGA ATGCTTATTC
    301 CTGGAGAGGT TGGAGGAGAA TCATTACAAC ACCTACATAT CAAAGAAGCA TGCGGAGAAA
    361 AATTGGTTTG TTGGCCTCAA GAAGAACGGA AGTTGCAAAA GAGGTCCCAG GACTCACTAT
    421 GGCCAGAAAG CCATCCTATT CCTTCCCCTC CCTGTGTCCT CTGAGTAA
    Zebrafish FGF1 gene coding sequence (1-147) (SEQ ID NO: 120) (Ensembl
    accession no. ENSDART00000005842, which is hereby incorporated by
    reference in its entirety):
    178                                                               ATG
    181 ACCGAGGCCG ATATTGCGGT AAAGTCCAGC CCGCGCGACT ATAAAAAACT GACGCGGCTG
    241 TACTGTATGA ATGGAGGATT TCACCTTCAG ATCCTGGCGG ACGGGACAGT GGCTGGAGCA
    124 GCAGACGAAA ACACATACAG CATACTGCGC ATAAAAGCAA CAAGTCCAGG AGTGGTGGTG
    184 ATCGAAGGAT CAGAAACAGG TCTTTACCTC TCGATGAATG AACATGGCAA GCTGTACGCT
    244 TCATCATTAG TGACGGATGA AAGTTATTTC CTGGAGAAGA TGGAGGAAAA CCACTACAAC
    304 ACATATCAGT CTCAAAAGCA CGGTGAAAAC TGGTACGTCG GAATAAAAAA GAACGGGAAA
    364 ATGAAACGGG GCCCAAGAAC TCACATCGGA CAAAAGGCCA TTTTCTTTCT TCCACGACAG
    424 GTGGAGCAGG AAGAGGACTG A
  • As noted above, also encompassed within the present invention are portions of paracrine FGFs other than FGF1 (e.g., FGF2, FGF4, FGF5, FGF6, FGF9, FGF16, and FGF20). The portions derived from paracrine FGF2 include portions corresponding to the above-identified amino acid sequences of FGF1. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.
  • In one embodiment, the paracrine FGF is FGF2. In one embodiment, the portion of the FGF2 is derived from human FGF2 having the amino acid sequence of SEQ ID NO: 121 (GenBank Accession No. EAX05222, which is hereby incorporated by reference in its entirety), as follows:
  • 1 MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI
    61 KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY
    121 TSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS
  • In one embodiment, the portion of the paracrine FGF includes an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 151 to 155 of SEQ ID NO: 121. In one embodiment, the portion of the paracrine FGF includes amino acid residues 1-151, 1-152, 1-153, 1-154, 1-155, 2-151, 2-152, 2-153, 2-154, 2-155, 3-151, 3-152, 3-153, 3-154, 3-155, 4-151, 4-152, 4-153, 4-154, 4-155, 5-151, 5-152, 5-153, 5-154, 5-155, 6-151, 6-152, 6-153, 6-154, 6-155, 7-151, 7-152, 7-153, 7-154, 7-155, 8-151, 8-152, 8-153, 8-154, 8-155, 9-151, 9-152, 9-153, 9-154, 9-155, 10-151, 10-152, 10-153, 10-154, 10-155, 11-151, 11-152, 11-153, 11-154, 11-155, 12-151, 12-152, 12-153, 12-154, 12-155, 13-151, 13-152, 13-153, 13-154, 13-155, 14-151, 14-152, 14-153, 14-154, 14-155, 15-151, 15-152, 15-153, 15-154, 15-155, 16-151, 16-152, 16-153, 16-154, 16-155, 17-151, 17-152, 17-153, 17-154, 17-155, 18-151, 18-152, 18-153, 18-154, 18-155, 19-151, 19-152, 19-153, 19-154, 19-155, 20-151, 20-152, 20-153, 20-154, 21-155, 21-151, 21-152, 21-153, 21-154, 21-155, 22-151, 22-152, 22-153, 22-154, 22-155, 23-151, 23-152, 23-153, 23-154, 23-155, 24-151, 24-152, 24-153, 24-154, 24-155, 25-151, 25-152, 25-153, 25-154, or 25-155 of FGF2 (SEQ ID NO: 121). In one embodiment, the portion of the paracrine FGF includes amino acid residues 1-151 or 1-152 of SEQ ID NO: 121.
  • In one embodiment, the portion of the paracrine FGF of the chimeric protein includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the corresponding amino acid sequence of native paracrine FGF (e.g., SEQ ID NO: 121). In one embodiment, the portion of the paracrine FGF includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 151 to 155 of SEQ ID NO: 121. In one embodiment, the portion of the paracrine FGF includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence homology to the corresponding amino acid sequence of native paracrine FGF (e.g., SEQ ID NO: 121). In one embodiment, the portion of the paracrine FGF includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence homology to an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 151 to 155 of SEQ ID NO: 121.
  • Also encompassed within the present invention are portions of paracrine FGFs other than FGF2 (e.g., FGF1, FGF4, FGF5, FGF6, FGF9, FGF16, and FGF20). The portions derived from paracrine FGFs other than FGF2 include portions corresponding to the above-identified amino acid sequences of FGF2. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.
  • In one embodiment of the present invention, the portion of the paracrine FGF is derived from an ortholog of a human paracrine FGF. In one embodiment of the present invention, the portion of the paracrine FGF of the chimeric protein is derived from an ortholog of human FGF2. In one embodiment, the portion of the FGF2 is derived from Gorilla gorilla, Pongo abelii, Macaca mulatta, Pan troglodytes, Pan paniscus, Saimiri boliviensis boliviensis, Nomascus leucogenys, Equus caballus, Bos taurus, Papio Anubis, Vicugna pacos, Ovis aries, Capreolus capreolus, Loxodonta Africana, Sus scrofa, Ailuropoda melanoleuca, Choloepus hoffmanni, Bubalus bubalis, Canis lupus familiaris, Rattus norvegicus, Heterocephalus glaber, Otolemur garnettii, Mus musculus, Ictidomys tridecemlineatus, Felis catus, Cavia porcellus, Sarcophilus harrisii, Monodelphis domestica, Oryctolagus cuniculus, Meleagris gallopavo, Gallus gallus, Taeniopygia guttata, Cynops pyrrhogaster, Xenopus laevis, Didelphis albiventris, Myotis lucifugus, Anolis carolinensis, Dasypus novemcinctus, Tupaia belangeri, Xenopus silurana tropicalis, Latimeria chalumnae, Tetraodon nigroviridis, Gasterosteus aculeatus, Takifugu rubripes, Oncorhynchus mykiss, Salmo salar, Danio rerio, Oreochromis niloticus, or Oryzias latipes. The portions of an ortholog of human paracrine FGF include portions corresponding to the above-identified amino acid sequences of FGF2. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.
  • In one embodiment, the portion of the FGF2 of the chimeric protein of the present invention is derived from an ortholog of human FGF2 having the amino acid sequence shown in Table 3.
  • TABLE 3
    Amino acid sequence of Gorilla gorilla (gorilla)
    FGF2 (SEQ ID NO: 122) (Ensembl accession no.
    ENSGGOP00000004720, which is hereby incorporated
    by reference in its entirety):
    104                                                 MAAGSI TTLPALPEDG
    120 GSGAFPPGHF KDPKRLYCKN GGFFLRIHPD GRVDGVREKS DPHIKLQLQA EERGVVSIKG
    180 VCANRYLAMK EDGRLLASKC VTDECFFFER LESNNYNTYR SRKYTSWYVA LKRTGQYKLG
    240 SKTGPGQKAI LFLPMSAKS
    Amino acid sequence of Pongo abelii (sumatran orangutan)
    FGF2(SEQ ID NO: 123) (GenBank accession no.
    XP_002815172, which is hereby incorporated by
    reference in its entirety):
    168                                                    MAA GSITTLPALP
    181 EDGGSGAFPP GHFKDPKRLY CKNGGFFLRI HPDGRVDGVR EKSDPHIKLQ LQAEERGVVS
    241 IKGVCANRYL AMKEDGRLLA SKCVTDECFF FERLESNNYN TYRSRKYTSW YVALKRTGQY
    301 KLGSKTGPGQ KAILFLPMSA KS
    Amino acid sequence of Macaca mulatta (rhesus monkey)
    FGF2 (SEQ ID NO: 124) (GenBank accession no.
    XP_001099284, which is hereby incorporated by
    reference in its entirety):
    83                         MAAGSITT LPALPEDGGS GAFPPGHFKD PKRLYCKNGG
    121 FFLRIHPDGR VDGVREKSDP HIKLQLQAEE RGVVSIKGVC ANRYLAMKED GRLLASKCVT
    181 DECFFFERLE SNNYNTYRSR KYTSWYVALK RTGQYKLGSK TGPGQKAILF LPMSAKS
    Amino acid sequence of Pan troglodytes (chimpanzee)
    FGF2 (SEQ ID NO: 125) (GenBank accession no.
    NP_001103711, which is hereby incorporated by
    reference in its entirety):
    134               MAAGSIT TLPALPEDGG SGAFPPGHFK DPKRLYCKNG GFFLRIHPDG
    181 RVDGVREKSD PHIKLQLQAE ERGVVSIKGV CANRYLAMKE DGRLLASKCV TDECFFFERL
    241 ESNNYNTYRS RKYTSWYVAL KRTGQYKLGS KTGPGQKAIL FLPMSAKS
    Amino acid sequence of Pan paniscus (Pygmy chimpanzee)
    FGF2 (SEQ ID NO: 126) (GenBank accession no.
    XP_003816481, which is hereby incorporated by
    reference in its entirety):
    112                                                         MAAGSITTL
    121 PALPEDGGSG AFPPGHFKDP KRLYCKNGGF FLRIHPDGRV DGVREKSDPH IKLQLQAEER
    181 GVVSIKGVCA NRYLAMKEDG RLLASKCVTD ECFFFERLES NNYNTYRSRK YTSWYVALKR
    241 TGQYKLGSKT GPGQKAILFL PMSAKS
    Amino acid sequence of Saimiri boliviensis boliviensis
    (Bolivian squirrel monkey) FGF2 (SEQ ID NO: 127)
    (GenBank accession no. XP_003936290, which is
    hereby incorporated by reference in its entirety):
    1 MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI
    61 KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY
    121 TSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS
    Amino acid sequence of Nomascus leucogenys
    (Northern white-cheeked gibbon) FGF2 (SEQ ID NO: 128)
    (GenBank accession no. XP_003271404, which is
    hereby incorporated by reference in its entirety):
    1 MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI
    61 KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY
    121 TSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS
    Amino acid sequence of Equus caballus (horse) FGF2 (SEQ ID NO: 129)
    (GenBank accession no. NP_001182150, which is hereby
    incorporated by reference in its entirety):
    1 MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI
    61 KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY
    121 SSWYVALKRT GQYKLGPKTG PGQKAILFLP MSAKS
    Amino acid sequence of Bos taurus (cattle) FGF2
    (SEQ ID NO: 130) (GenBank accession no. NP_776481,
    which is hereby incorporated by reference in its entirety):
    1 MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI
    61 KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY
    121 SSWYVALKRT GQYKLGPKTG PGQKAILFLP MASKS
    Amino acid sequence of Papio anubis (Olive baboon) FGF2
    (SEQ ID NO: 131) (GenBank accession no. XP_003899210,
    which is hereby incorporated by reference in its entirety):
    1 MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI
    61 KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY
    121 TSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS
    Amino acid sequence of Vicugna pacos (alpaca) FGF2
    (SEQ ID NO: 132) (Ensembl accession no. ENSVPAP00000009804,
    which is hereby incorporated by reference in its entirety):
    111                                                        MAAGSITTLP
    121 ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI KLQLQAEERG
    181 VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY SSWYVALKRT
    241 GQYKLGPKTG PGQKAILFLP MSAKS
    Amino acid sequence of Ovis aries (sheep) FGF2 (SEQ ID NO: 133)
    (GenBank accession no. NP_001009769, which is hereby
    incorporated by reference in its entirety):
    1 MAAGSITTLP ALPEDGGSSA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI
    61 KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY
    121 SSWYVALKRT GQYKLGPKTG PGQKAILFLP MSAKS
    Amino acid sequence of Capreolus capreolus (Western roe deer)
    FGF2 (partial amino acid sequence corresponding to human FGF2
    residues
    42 to 149) (SEQ ID NO: 134) (GenBank accession no.
    AAF73226, which is hereby incorporated by reference
    in its entirety):
    1 RIHPDGRVDG VREKSDPHIK LQLQAEERGV VSIKGVCANR YLAMKEDGRL LASKCVTDEC
    61 FFFERLESNN YNTYRSRKYS SWYVALKRTG QYKLGPKTGP GQKAILFL
    Amino acid sequence of Loxodonta africana (elephant) FGF2
    (partial amino acid sequence corresponding to human FGF2
    residues
    60 to 155) (SEQ ID NO: 135) (Ensembl accession no.
    ENSLAFP00000008249, which is hereby incorporated by reference
    in its entirety):
    1 VKLQLQAEER GVVSIKGVCA NRYLAMKEDG RLLASRCVTD ECFFFERLES NNYNTYRSRK
    61 YTSWYVALKR TGQYKLGSKT GPGQKAILFL PMSAKS
    Amino acid sequence of Sus scrofa (pig) FGF2 (partial
    amino acid sequence corresponding to human FGF2 residues
    36 to 155) (SEQ ID NO: 136) (GenBank accession no. CAE11791
    and Ensembl accession no. ENSSSCP00000009695, which is
    hereby incorporated by reference in its entirety):
    1 NGGFFLRIHP DGRVDGVREK SDPHIKLQLQ AEERGVVSIK GVCANRYLAM KEDGRLLASK
    61 CVTDECFFFE RLESNNYNTY RSRKYSSWYV ALKRTGQYKL GPKTGPGQKA ILFLPMSAKS
    Amino acid sequence of Ailuropoda melanoleuca (panda)
    FGF2 (partial amino acid sequence corresponding to human FGF2
    residues
    60 to 155) (SEQ ID NO: 137) (Ensembl accession no.
    ENSAMEP00000018489, which is hereby incorporated by
    reference in its entirety):
    1 VKLQLQAEER GVVSIKGVCA NRYLAMKEDG RLLASKCVTD ECFFFERLES NNYNTYRSRK
    61 YSSWYVALKR TGQYKLGPKT GPGQKAILFL PMSAKS
    Amino acid sequence of Choloepus hoffmanni (sloth) FGF2
    (SEQ ID NO: 138) (Ensembl accession no. ENSCHOP00000010051,
    which is hereby incorporated by reference in its entirety):
    14                                                           MAAGSIT
    21 TLPALPEDGG SGALPPGHFK DPKRLYCKNG GFFLRIHPDG RVDGVREKSD PHIKLQLQAE
    81 ERGVVSIKGV CANRYLAMKE DGRLQASKCV TDECFFFERL ESNNYNTYRS RKYSSWYVAL
    141 KRTGQYKLGP KTGPGQKAIL FLPMSAKS
    Amino acid sequence of Bubalus bubalis (water buffalo) FGF2
    (SEQ ID NO: 139) (GenBank accession no. AFH66795, which is
    hereby incorporated by reference in its entirety):
    1 MAAGSITTLP PLPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI
    61 KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESS NYNTYRSRKY
    121 SSWYVALKRT GQYKLGPKTG PGQKAILFLP MSAKS
    Amino acid sequence of Canis lupus familiaris (dog) FGF2
    (SEQ ID NO: 140) (GenBank accession no. XP_003432529,
    which is hereby incorporated by reference in its entirety):
    40                                           M AAGSITTLPA LPEDGGSGAF
    61 PPGHFKDPKR LYCKKGGFFL RIHPDGRVDG VREKSDPHVK LQLQAEERGV VSIKGVCANR
    121 YLAMKEDGRL LASKCVTDEC FFFERLESNN YNTYRSRKYS SWYVALKRTG QYKLGPKTGP
    181 GQKAILFLPM SAKS
    Amino acid sequence of Rattus norvegicus (Norway rat) FGF2
    (SEQ ID NO: 141) (GenBank accession no. NP_062178,
    which is hereby incorporated by reference in its entirety):
    1 MAAGSITSLP ALPEDGGGAF PPGHFKDPKR LYCKNGGFFL RIHPDGRVDG VREKSDPHVK
    61 LQLQAEERGV VSIKGVCANR YLAMKEDGRL LASKCVTEEC FFFERLESNN YNTYRSRKYS
    121 SWYVALKRTG QYKLGSKTGP GQKAILFLPM SAKS
    Amino acid sequence of Heterocephalus glaber (naked mole-rat)
    FGF2 (partial amino acid sequence corresponding to human FGF2
    residues 22 to 155) (SEQ ID NO: 142) (GenBank accession no.
    EHB17407, which is hereby incorporated by reference
    in its entirety):
    1 ppghfkdpkr lycknggffl rihpdgrvdg vreksdphvk lqlqaeergv vsikgvcanr
    61 ylamkedgrl laskcvtdec ffferlesnn yntyrsrkys swyvalkrtg qyklgsktgp
    121 gqkailflpm saks
    Amino acid sequence of Otolemur garnettii (bushbaby) FGF2
    (SEQ ID NO: 143) (Ensembl accession no. ENSOGAP00000021960,
    which is hereby incorporated by reference in its entirety):
    52                                                         MAAGSITTL
    61 PSLPEDGGSD AFPPGHFKDP KRLYCKNGGF FLRIHPDGRV DGVREKSDPY IKLQLQAEER
    121 GVVSIKGVCA NRYLAMKEDG RLLASKLITD ECFFFERLES NNYNTYRSRK YSSWYVALKR
    181 TGQYKLGSKT GPGQKAILFL PMSAKS
    Amino acid sequence of Mus musculus (house mouse) FGF2
    (SEQ ID NO: 144) (GenBank accession no. NP_032032,
    which is hereby incorporated by reference in its entirety):
    1 MAASGITSLP ALPEDGGAAF PPGHFKDPKR LYCKNGGFFL RIHPDGRVDG VREKSDPHVK
    61 LQLQAEERGV VSIKGVCANR YLAMKEDGRL LASKCVTEEC FFFERLESNN YNTYRSRKYS
    121 SWYVALKRTG QYKLGSKTGP GQKAILFLPM SAKS
    Amino acid sequence of Ictidomys tridecemlineatus (squirrel)
    FGF2 (partial amino acid sequence corresponding to human FGF2
    residues
    12 to 155) (SEQ ID NO: 145) (Ensembl accession no.
    ENSSTOP00000015653, which is hereby incorporated by reference
    in its entirety):
    1 LPEDGGGGAF PPGHFKDPKR LYCKNGGFFL RIHPDGRVDG VREKSDPHIK LQLQAEDRGV
    61 VSIKGVCANR YLAMKEDGRL LASKCVTDEC FFFERLESNN YNTYRSRKYS SWYVALKRTG
    121 QYKLGSKTGP GQKAILFLPM SAKS
    Amino acid sequence of Felis catus (domestic cat) FGF2
    (partial amino acid sequence corresponding to human FGF2
    residues
    25 to 130) (SEQ ID NO: 146) (GenBank accession no.
    ABY47638, which is hereby incorporated by reference
    in its entirety):
    1 HFKDPKRLYC KNGGFFLRIH PDGRVDGVRE KSDPHIKLQL QAEERGVVSI KGVCANRYLA
    61 MKEDGRLLAS KCVTDECFFF ERLESNNYNT YRSRKYSSWY VALKRT
    Amino acid sequence of Cavia porcellus (guinea pig) FGF2
    (partial amino acid sequence corresponding to human FGF2
    residues
    60 to 155) (SEQ ID NO: 147) (Ensembl accession no.
    ENSCPOP00000004847, which is hereby incorporated by
    reference in its entirety):
    1 VKLQLQAEDR GVVSIKGVCA NRYLAMKEDG RLLASKCVTD ECFFFERLES NNYNTYRSRK
    61 YSSWYVALKR TGQYKLGSKT GPGQKAILFL PMSAKS
    Amino acid sequence of Sarcophilus harrisii (Tasmanian devil)
    FGF2 (SEQ ID NO: 148) (Ensembl accession no.
    ENSSHAP00000012215, which is hereby incorporated by
    reference in its entirety):
    48                                                    MAA GSITTLPALA
    61 GDGASGGAFP PGHFQDPKRL YCKNGGFFLR IHPDGHVDGI REKSDPHIKL QLQAEERGVV
    121 SIKGVCANRY LAMKEDGRLL ALKCVTEECF FFERLESNNY NTYRSRKYSN WYVALKRTGQ
    181 YKLGSKTGPG QKAILFLPMS AKS
    Amino acid sequence of Monodelphis domestica (gray short-tailed
    opossum) FGF2 (SEQ ID NO: 149) (GenBank accession no.
    NP_001029148, which is hereby incorporated by reference
    in its entirety):
    1 MAAGSITTLP ALSGDGGGGG AFPPGHFKDP KRLYCKNGGF FLRIHPDGRV DGIREKSDPN
    61 IKLQLQAEER GVVSIKGVCA NRYLAMKEDG RLLALKYVTE ECFFFERLES NNYNTYRSRK
    121 YSNWYVALKR TGQYKLGSKT GPGQKAILFL PMSAKS
    Amino acid sequence of Oryctolagus cuniculus (rabbit) FGF2
    (SEQ ID NO: 150) (GenBank accession no. XP_002717284,
    which is hereby incorporated by reference in its entirety):
    1 MAAESITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI
    61 KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY
    121 SSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS
    Amino acid sequence of Meleagris gallopavo (turkey) FGF2
    (partial amino acid sequence corresponding to human FGF2
    residues 31 to 155) (SEQ ID NO: 151) (Ensembl accession no.
    ENSMGAP00000010977, which is hereby incorporated by
    reference in its entirety):
    1 RLYCKNGGFF LRINPDGRVD GVREKSDPHI KLQLQAEERG VVSIKGVSAN RFLAMKEDGR
    61 LLALKCATEE CFFFERLESN NYNTYRSRKY SDWYVALKRT GQYKPGPKTG PGQKAILFLP
    121 MSAKS
    Amino acid sequence of Gallus gallus (chicken) FGF2
    (SEQ ID NO: 152) (GenBank accession no. NP_990764
    1 maagaagsit tlpalpddgg ggafppghfk dpkrlyckng gfflrinpdg rvdgvreksd
    61 PHIKLQLQAE ERGVVSIKGV SANRFLAMKE DGRLLALKCA TEECFFFERL ESNNYNTYRS
    121 RKYSDWYVAL KRTGQYKPGP KTGPGQKAIL FLPMSAKS
    Amino acid sequence of Taeniopygia guttata (zebra finch)
    FGF2 (SEQ ID NO: 153) (GenBank accession no. XP_002188397,
    which is hereby incorporated by reference in its entirety):
    1 MAAAGGIATL PDDGGSGAFP PGHFKDPKRL YCKNGGFFLR INPDGKVDGV REKSDPHIKL
    61 QLQAEERGVV SIKGVSANRF LAMKEDGRLL ALKYATEECF FFERLESNNY NTYRSRKYSD
    121 WYVALKRTGQ YKPGPKTGPG QKAILFLPMS AKS
    Amino acid sequence of Cynops pyrrhogaster (Japanese
    firebelly newt) FGF2 (SEQ ID NO: 154) (GenBank accession no.
    BAB63249, which is hereby incorporated by reference
    in its entirety):
    1 MAAGSITSLP ALPEDGNGGT FTPGGFKEPK RLYCKNGGFF LRINSDGKVD GAREKSDSYI
    61 KLQLQAEERG VVSIKGVCAN RYLAMKDDGR LMALKWITDE CFFFERLESN NYNTYRSRKY
    121 SDWYVALKRT GQYKNGSKTG AGQKAILFLP MSAKS
    Amino acid sequence of Xenopus laevis (African clawed frog)
    FGF2 (SEQ ID NO: 155) (GenBank accession no. NP_001093341,
    which is hereby incorporated by reference in its entirety):
    1 MAAGSITTLP TESEDGGNTP FSPGSFKDPK RLYCKNGGFF LRINSDGRVD GSRDKSDSHI
    61 KLQLQAVERG VVSIKGITAN RYLAMKEDGR LTSLRCITDE CFFFERLEAN NYNTYRSRKY
    121 SSWYVALKRT GQYKNGSSTG PGQKAILFLP MSAKS
    Amino acid sequence of Didelphis albiventris (white-eared
    opossum) FGF2 (SEQ ID NO: 156) (GenBank accession no.
    ABL77404, which is hereby incorporated by reference
    in its entirety):
    1 MAAGSITTLP ALSGDGGGGG AFPPGHFKDP KRLYCKNGGF FLRIHPDGRV DGIREKSDPN
    61 IKLQLQAEER GVVSIKGVCA NRYLAMKEDG RLLALKYVTE ECFFFERLES NNYNTYRSRK
    121 YSNWYVALKR TGQYKLGSKT GPGQKAILFS PCLLRC
    Amino acid sequence of Myotis lucifugus (microbat) FGF2
    (partial amino acid sequence corresponding to human FGF2
    residues
    60 to 155) (SEQ ID NO: 157) (Ensembl accession no.
    ENSMLUP00000017859, which is hereby incorporated by
    reference in its entirety):
    1 VKLQLQAEER GVVSIKGVCA NRYLAMKEDG RLQASKCVTD ECFFFERLES NNYNTYRSRK
    61 YSSWYVALKR NGQYKLGPKT GPGQKAILFL PMSAKS
    Amino acid sequence of Anolis carolinensis (anole lizard) FGF2
    (partial amino acid sequence corresponding to human FGF2
    residues 16 to 155) (SEQ ID NO: 158) (Ensembl accession no.
    ENSACAP00000011657, which is hereby incorporated by reference
    in its entirety):
    1 AAAASFPPGP FKDPKRLYCK NGGFFLRINP DGGVDGVREK SDPNIKLLLQ AEERGVVSIK
    61 GVCANRFLAM NEDGRLLALK YVTDECFFFE RLESNNYNTY RSRKYRDWYI ALKRTGQYKL
    121 GPKTGRGQKA ILFLPMSAKS
    Amino acid sequence of Dasypus novemcinctus (armadillo)
    FGF2 (partial amino acid sequence corresponding to human
    FGF2 residues
    1 to 94) (SEQ ID NO: 159) (Ensembl accession no.
    ENSDNOP00000011351, which is hereby incorporated by
    reference in its entirety):
    124    MAAGSIT TLPALPEDGG SGAFPPGHFK DPKRLYCKNG GFFLRIHPDG RVDGVREKSD
    181 PNIKLQLQAE ERGVVSIKGV CANRYLAMRE DGRLQAS
    Amino acid sequence of Tupaia belangeri (tree shrew) FGF2
    (SEQ ID NO: 160) (Ensembl accession no. ENSTBEP00000000985,
    which is hereby incorporated by reference in its entirety):
    1 AGVRAEREEA PGSGDSRGTD PAARSLIRRP DAAAREALLG ARSRVQGSST SWPASSRTGI
    61 KLPDDSGQGM GGYPLDRPSR STGRGLGGAP DPAVKLQLQA EERGVVSIKG VCANRYLAMK
    121 EDGRLLASKC VTDECFFFER LESNNYNTYR SRKYSSWYVA LKRTGQYKLG SKTGPGQKAI
    181 LFLPMSAKS
    Amino acid sequence of Xenopus silurana tropicalis (western
    clawed frog) FGF2 (SEQ ID NO: 161) (GenBank accession no.
    NP_001017333, which is hereby incorporated by
    reference in its entirety):
    1 MAAGSITTLP TESEDGNTPF PPGNFKDPKR LYCKNGGYFL RINSDGRVDG SRDKSDLHIK
    61 LQLQAVERGV VSIKGITANR YLAMKEDGRL TSLKCITDEC FFYERLEANN YNTYRSRKNN
    121 SWYVALKRTG QYKNGSTTGP GQKAILFLPM SAKS
    Amino acid sequence of Latimeria chalumnae (coelacanth)
    FGF2 (SEQ ID NO: 162) (Ensembl accession no.
    ENSLACP00000019200, which is hereby incorporated by
    reference in its entirety):
    1 MAAGGITTLP AVPEDGGSST FPPGNFKEPK RLYCKNGGYF LRINPDGRVD GTREKNDPYI
    61 KLQLQAESIG VVSIKGVCSN RYLAMNEDCR LFGLKYPTDE CFFHERLESN NYNTYRSKKY
    121 SDWYVALKRT GQYKPGPKTG LGQKAILFLP MSAKS
    Amino acid sequence of Tetraodon nigroviridis (spotted green
    pufferfish) FGF2 (SEQ ID NO: 163) (GenBank accession no.
    CAG04681, which is hereby incorporated by reference
    in its entirety):
    34                                     MATGGIT TLPSTPEDGG SSGFPPGSFK
    61 DPKRLYCKNG GFFLRIKSDG VVDGIREKSD PHIKLQLQAT SVGEVVIKGV CANRYLAMNR
    121 DGRLFGTKRA TDECHFLERL ESNNYNTYRS RKYPTMFVGL TRTGQYKSGS KTGPGQKAIL
    181 FLPMSAKC
    Amino acid sequence of Gasterosteus aculeatus (stickleback)
    FGF2 (SEQ ID NO: 164) (Ensembl accession no.
    ENSGACP00000022078, which is hereby incorporated by
    reference in its entirety):
    1 MATAGFATLP STPEDGGSGG FTPGGFKDPK RLYCKNGGFF LRIRSDGGVD GIREKSDAHI
    61 KLQIQATSVG EVVIKGVCAN RYLAMNRDGR LFGVRRATDE CYFLERLESN NYNTYRSRKY
    121 PGMYVALKRT GQYKSGSKTG PGQKAILFLP MSAKC
    Amino acid sequence of Takifugu rubripes (fugu rubripes)
    FGF2 (SEQ ID NO: 165) (GenBank accession no. CAD19830,
    which is hereby incorporated by reference in its entirety):
    1 MATGGITTLP STPEDGGSGG FPPGSFKDPK RLYCKNGGFF LRIRSDGAVD GTREKTDPHI
    61 KLQLQATSVG EVVIKGVCAN RYLAMNRDGR LFGMKRATDE CHFLERLESN NYNTYRSRKY
    121 PNMFVGLTRT GNYKSGTKTG PCQKAILFLP MSAKY
    Amino acid sequence of Oncorhynchus mykiss (rainbow trout)
    FGF2 (SEQ ID NO: 166) (GenBank accession no. NP_001118008,
    which is hereby incorporated by reference in its entirety):
    1 MATGEITTLP ATPEDGGSGG FLPGNFKEPK RLYCKNGGYF LRINSNGSVD GIRDKNDPHN
    61 KLQLQATSVG EVVIKGVSAN RYLAMNADGR LFGPRRTTDE CYFMERLESN NYNTYRSRKY
    121 PEMYVALKRT GQYKSGSKTG PGQKAILFLP MSARR
    Amino acid sequence of Salmo salar (salmon) FGF2
    (SEQ ID NO: 167) (GenBank accession no. ACJ02099,
    which is hereby incorporated by reference in its entirety):
    1 MATGEITTLP ATPEDGGSGG FPPGNFKDPK RLYCKNGGYF LRINSNGSVD GIREKNDPHK
    61 QPQFVRAWTL QGVKRSTGML AHVDSNASHN CVKVAGCSLG EFGSMSNRPH NRRPRVATPA
    121 QDLHIRLLHL RDRLKPATRT ADKTEEYFCL
    Amino acid sequence of Danio rerio (zebrafish) FGF2
    (SEQ ID NO: 168) (GenBank accession no. AAP32155,
    which is hereby incorporated by reference in its entirety):
    1 MATGGITTLP AAPDAENSSF PAGSFRDPKR LYCKNGGFFL RINADGRVDG ARDKSDPHIR
    61 LQLQATAVGE VLIKGICTNR FLAMNADGRL FGTKRTTDEC YFLERLESNN YNTYRSRKYP
    121 DWYVALKRTG QYKSGSKTSP GQKAILFLPM SAKC
    Amino acid sequence of Oreochromis niloticus (Nile tilapia)
    FGF2 (SEQ ID NO: 169) (GenBank accession no.
    XP_003443412, which is hereby incorporated by
    reference in its entirety):
    1 MATGGITTLP ATPEDGGSSG FPPGNFKDPK RLYCKNGGFF LRIKSDGGVD GIREKNDPHI
    61 KLQLQATSVG EVVIKGICAN RYLAMNRDGR LFGARRATDE CYFLERLESN NYNTYRSRKY
    121 PNMYVALKRT GQYKSGSKTG PGQKAILFLP MSAKC
    Amino acid sequence of Oryzias latipes (medaka) FGF2
    (SEQ ID NO: 170) (Ensembl accession no.
    ENSORLP00000025834, which is hereby incorporated by
    reference in its entirety):
    1 MATGEITTLP SPAENSRSDG FPPGNYKDPK RLYCKNGGLF LRIKPDGGVD GIREKKDPHV
    61 KLRLQATSAG EVVIKGVCSN RYLAMHGDGR LFGVRQATEE CYFLERLESN NYNTYRSKKY
    121 PNMYVALKRT GQYKPGNKTG PGQKAILFLP MSAKY
  • As noted above, the portion of the paracrine FGF may be modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. In one embodiment, the modification of the paracrine FGF includes one or more substitutions, additions, or deletions.
  • In one embodiment, the modification is one or more substitutions located at one or more amino acid residues of SEQ ID NO: 121 selected from N36, K128, R129, K134, K138, Q143, K144, C78, C96, and combinations thereof. In one embodiment, the one or more substitutions are selected from N36T, K128D, R129Q, K134V, K138H, Q143M, K144T/L/I, C78S, C96S, and combinations thereof. In one embodiment, the modification is one or more substitutions which are located at one or more amino acid residues corresponding to residues of SEQ ID NO: 121 selected from N36, K128, R129, K134, K138, Q143, K144, C78, C96, and combinations thereof. In one embodiment, the modification is one or more substitutions which are located at one or more amino acid residues corresponding to residues of SEQ ID NO: 121 selected from N36, K128, R129, K134, K138, Q143, K144, C78, C96, and combinations thereof. Amino acid residues corresponding to those of SEQ ID NO: 121 may be determined by, for example, sequence analysis and structural analysis.
  • It will be understood that the portion of the paracrine FGF according to the present invention may be derived from a nucleotide sequence that encodes a paracrine FGF protein. For example, in one embodiment, nucleotide sequence is the nucleotide sequence that encodes human FGF2 (GenBank Accession No. NM002006, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 171), as follows:
  • 468                                                    ATG GCAGCCGGGA
    481 GCATCACCAC GCTGCCCGCC TTGCCCGAGG ATGGCGGCAG CGGCGCCTTC CCGCCCGGCC
    541 ACTTCAAGGA CCCCAAGCGG CTGTACTGCA AAAACGGGGG CTTCTTCCTG CGCATCCACC
    601 CCGACGGCCG AGTTGACGGG GTCCGGGAGA AGAGCGACCC TCACATCAAG CTACAACTTC
    661 AAGCAGAAGA GAGAGGAGTT GTGTCTATCA AAGGAGTGTG TGCTAACCGT TACCTGGCTA
    721 TGAAGGAAGA TGGAAGATTA CTGGCTTCTA AATGTGTTAC GGATGAGTGT TTCTTTTTTG
    781 AACGATTGGA ATCTAATAAC TACAATACTT ACCGGTCAAG GAAATACACC AGTTGGTATG
    841 TGGCACTGAA ACGAACTGGG CAGTATAAAC TTGGATCCAA AACAGGACCT GGGCAGAAAG
    901 CTATACTTTT TCTTCCAATG TCTGCTAAGA GCTGA
  • In another embodiment of the present invention, the portion of the paracrine FGF of the chimeric protein may be derived from a nucleotide sequence that encodes an ortholog of human FGF2. Nucleotide sequences that encode FGF2 orthologs are shown in Table 4.
  • TABLE 4
    Gorilla FGF2 gene coding sequence (amino acids (“aa”) 104-258) (SEQ ID
    NO: 172) (Ensembl accession no. ENSGGOT00000004842, which is hereby
    incorporated by reference in its entirety):
    310             ATGGCAGCC GGGAGCATCA CCACGCTGCC CGCCTTGCCC GAGGATGGCG
    359 GCAGCGGCGC CTTCCCGCCC GGCCACTTCA AGGACCCCAA GCGGCTGTAC TGCAAAAACG
    419 GGGGCTTCTT CCTGCGCATC CACCCCGACG GCCGAGTTGA CGGGGTCCGG GAGAAGAGCG
    479 ACCCTCACAT CAAGCTACAA CTTCAAGCAG AAGAGAGAGG AGTTGTGTCT ATCAAAGGAG
    539 TGTGTGCTAA CCGTTACCTT GCTATGAAGG AAGATGGAAG ATTACTGGCT TCTAAATGTG
    599 TTACGGATGA GTGTTTCTTT TTTGAACGAT TGGAATCTAA TAACTACAAT ACTTACCGGT
    659 CAAGGAAATA CACCAGTTGG TATGTGGCAC TGAAACGAAC TGGGCAGTAT AAACTTGGAT
    719 CCAAAACAGG ACCTGGGCAG AAAGCTATAC TTTTTCTTCC AATGTCTGCT AAGAGCTGA
    Sumatran orangutan FGF2 gene coding sequence (aa 168-322) (SEQ ID
    NO: 173) (GenBank accession no. XM_002815126, which is hereby
    incorporated by reference in its entirety):
    504                          ATGGCAG CCGGGAGCAT CACCACGCTG CCCGCCTTGC
    541 CCGAGGATGG CGGCAGCGGC GCCTTCCCGC CGGGCCACTT CAAGGACCCC AAGCGGCTGT
    601 ACTGCAAAAA CGGGGGCTTC TTCCTGCGCA TCCACCCCGA CGGCCGAGTT GACGGGGTCC
    661 GAGAGAAGAG CGACCCTCAC ATCAAACTAC AACTTCAAGC AGAAGAAAGA GGAGTTGTGT
    721 CTATCAAAGG AGTGTGTGCT AACCGCTACC TTGCTATGAA GGAAGATGGA AGATTACTGG
    781 CTTCTAAATG TGTTACGGAT GAGTGTTTCT TTTTTGAACG ATTGGAATCT AATAACTACA
    841 ATACTTACCG GTCAAGGAAA TACACCAGTT GGTATGTGGC ACTGAAACGA ACTGGGCAGT
    901 ATAAACTTGG ATCCAAAACA GGACCTGGGC AGAAAGCTAT ACTTTTTCTT CCAATGTCTG
    961 CTAAGAGCTG A
    Rhesus monkey FGF2 gene coding sequence (aa 83-237) (SEQ ID NO: 174)
    (GenBank accession no. XM_001099284, which is hereby incorporated by
    reference in its entirety):
    247       ATGG CAGCCGGGAG CATCACCACG CTGCCCGCCT TGCCCGAGGA TGGCGGCAGC
    301 GGCGCCTTCC CGCCTGGCCA CTTCAAGGAC CCCAAGCGGC TGTACTGCAA AAACGGGGGC
    361 TTCTTCCTGC GCATTCACCC CGACGGCCGA GTTGACGGGG TCCGGGAGAA GAGCGACCCT
    421 CACATCAAAT TACAACTTCA AGCAGAAGAG AGAGGAGTTG TGTCTATCAA AGGAGTGTGT
    481 GCTAACCGTT ACCTTGCTAT GAAGGAAGAT GGAAGATTAC TGGCTTCTAA ATGTGTTACA
    541 GATGAGTGTT TCTTTTTTGA ACGATTGGAA TCTAATAACT ACAATACTTA CCGGTCAAGG
    601 AAATACACCA GTTGGTATGT GGCACTGAAA CGAACTGGGC AATATAAACT TGGATCCAAA
    661 ACAGGACCTG GGCAGAAAGC TATACTTTTT CTTCCAATGT CTGCTAAGAG CTGA
    Chimpanzee FGF2 gene coding sequence (aa 134-288) (SEQ ID NO: 175)
    (GenBank accession no. NM_001110241, which is hereby incorporated by
    reference in its entirety):
    400                                           A TGGCAGCCGG GAGCATCACC
    421 ACGCTGCCCG CCTTGCCCGA GGATGGCGGC AGCGGCGCCT TCCCGCCCGG CCACTTCAAG
    481 GACCCCAAGC GGCTGTACTG CAAAAACGGG GGCTTCTTCC TGCGCATCCA CCCCGACGGC
    541 CGAGTTGACG GGGTCCGGGA GAAGAGCGAC CCTCACATCA AGCTACAACT TCAAGCAGAA
    601 GAGAGAGGAG TTGTGTCTAT CAAAGGAGTG TGTGCTAACC GTTACCTTGC TATGAAGGAA
    661 GATGGAAGAT TACTGGCTTC TAAATGTGTT ACGGATGAGT GTTTCTTTTT TGAACGATTG
    721 GAATCTAATA ACTACAATAC TTACCGGTCA AGGAAATACA CCAGTTGGTA TGTGGCACTG
    781 AAACGAACTG GGCAGTATAA ACTTGGATCC AAAACAGGAC CTGGGCAGAA AGCTATACTT
    841 TTTCTTCCAA TGTCTGCTAA GAGCTGA
    Pygmy chimpanzee FGF2 gene coding sequence (112-266) (SEQ ID NO: 176)
    (GenBank accession no. XM_003816433, which is hereby incorporated by
    reference in its entirety):
    334                                     ATGGCAG CCGGGAGCAT CACCACGCTG
    361 CCCGCCTTGC CCGAGGATGG CGGCAGCGGC GCCTTCCCGC CCGGCCACTT CAAGGACCCC
    421 AAGCGGCTGT ACTGCAAAAA CGGGGGCTTC TTCCTGCGCA TCCACCCCGA CGGCCGAGTT
    481 GACGGGGTCC GGGAGAAGAG CGACCCTCAC ATCAAGCTAC AACTTCAAGC AGAAGAGAGA
    541 GGAGTTGTGT CTATCAAAGG AGTGTGTGCT AACCGTTACC TTGCTATGAA GGAAGATGGA
    601 AGATTACTGG CTTCTAAATG TGTTACGGAT GAGTGTTTCT TTTTTGAACG ATTGGAATCT
    661 AATAACTACA ATACTTACCG GTCAAGGAAA TACACCAGTT GGTATGTGGC ACTGAAACGA
    721 ACTGGGCAGT ATAAACTTGG ATCCAAAACA GGACCTGGGC AGAAAGCTAT ACTTTTTCTT
    781 CCAATGTCTG CTAAGAGCTG A
    Bolivian squirrel monkey FGF2 gene coding sequence (1-155) (SEQ ID
    NO: 177) (GenBank accession no. XM_003936241, which is hereby
    incorporated by reference in its entirety):
    23                         ATGGCAGC CGGGAGCATC ACCACGCTGC CCGCCCTGCC
    61 CGAAGACGGC GGCAGCGGCG CCTTCCCGCC CGGCCACTTC AAAGACCCCA AGCGGCTGTA
    121 CTGCAAAAAC GGGGGCTTCT TCCTGCGAAT CCACCCCGAC GGCCGAGTGG ACGGGGTCCG
    181 GGAGAAGAGC GACCCTCACA TCAAACTACA ACTTCAAGCA GAAGAGAGAG GAGTTGTATC
    241 TATCAAAGGA GTGTGTGCTA ACCGTTACCT TGCTATGAAG GAAGATGGAA GATTACTGGC
    301 TTCTAAATGT GTTACGGACG AGTGTTTCTT TTTTGAACGA TTGGAATCTA ATAACTACAA
    361 TACTTACCGA TCAAGGAAAT ACACCAGTTG GTATGTGGCA CTGAAACGAA CTGGGCAGTA
    421 TAAACTTGGA TCCAAAACAG GACCTGGGCA GAAAGCTATA CTTTTTCTTC CAATGTCTGC
    481 TAAGAGCTGA
    Northern white-cheeked gibbon FGF2 gene coding sequence (aa 1-155) (SEQ
    ID NO: 178) (GenBank accession no. XM_003271356, which is hereby
    incorporated by reference in its entirety):
    435                                                    ATG GCAGCCGGGA
    481 GCATCACCAC GCTGCCCGCC TTGCCGGAGG ATGGCGGCAG CGGCGCCTTC CCGCCCGGCC
    541 ACTTCAAGGA CCCCAAGCGG CTGTACTGCA AAAACGGGGG TTTCTTCCTG CGCATCCACC
    601 CCGACGGTCG AGTTGACGGG GTCCGGGAGA AGAGCGACCC TCACATCAAA CTACAACTTC
    661 AAGCAGAAGA GAGAGGAGTT GTGTCTATCA AAGGAGTGTG TGCTAACCGT TACCTTGCTA
    721 TGAAGGAAGA TGGAAGATTA CTGGCTTCTA AATGTGTTAC GGATGAGTGT TTCTTTTTTG
    781 AACGATTGGA ATCTAATAAC TACAATACTT ACCGGTCAAG GAAATACACC AGTTGGTATG
    841 TGGCACTGAA ACGAACTGGG CAGTATAAAC TTGGATCCAA AACAGGACCT GGGCAGAAAG
    901 CTATACTTTT TCTTCCAATG TCTGCTAAGA GCTGA
    Horse FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 179) (GenBank
    accession no. NM_001195221, which is hereby incorporated by reference in
    its entirety):
    54                                                           ATGGCAG
    61 CCGGGAGCAT CACCACGCTG CCCGCCCTGC CCGAGGACGG CGGCAGCGGC GCCTTCCCGC
    121 CCGGCCACTT CAAGGACCCC AAGCGGCTCT ACTGCAAAAA CGGGGGCTTC TTCCTGCGCA
    181 TCCACCCCGA CGGCCGAGTG GACGGGGTCC GGGAGAAGAG CGACCCTCAC ATCAAACTAC
    241 AACTTCAAGC AGAAGAGAGA GGGGTTGTGT CTATCAAAGG AGTGTGTGCG AACCGTTATC
    301 TTGCTATGAA GGAAGATGGA AGGTTACTGG CTTCTAAATG TGTTACGGAC GAGTGTTTCT
    361 TTTTTGAACG ATTGGAATCT AATAACTACA ATACTTACCG GTCAAGGAAA TACTCCAGTT
    421 GGTATGTGGC CCTGAAACGA ACGGGGCAGT ATAAACTTGG ACCCAAAACA GGACCTGGAC
    481 AGAAAGCTAT ACTTTTTCTT CCAATGTCTG CTAAGAGCTG A
    Cattle FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 180) (GenBank
    accession no. NM_174056, which is hereby incorporated by reference
    in its entirety):
    104                                                ATGGCCG CCGGGAGCAT
    121 CACCACGCTG CCAGCCCTGC CGGAGGACGG CGGCAGCGGC GCTTTCCCGC CGGGCCACTT
    181 CAAGGACCCC AAGCGGCTGT ACTGCAAGAA CGGGGGCTTC TTCCTGCGCA TCCACCCCGA
    241 CGGCCGAGTG GACGGGGTCC GCGAGAAGAG CGACCCACAC ATCAAACTAC AACTTCAAGC
    301 AGAAGAGAGA GGGGTTGTGT CTATCAAAGG AGTGTGTGCA AACCGTTACC TTGCTATGAA
    361 AGAAGATGGA AGATTACTAG CTTCTAAATG TGTTACAGAC GAGTGTTTCT TTTTTGAACG
    421 ATTGGAGTCT AATAACTACA ATACTTACCG GTCAAGGAAA TACTCCAGTT GGTATGTGGC
    481 ACTGAAACGA ACTGGGCAGT ATAAACTTGG ACCCAAAACA GGACCTGGGC AGAAAGCTAT
    541 ACTTTTTCTT CCAATGTCTG CTAAGAGCTG A
    Olive baboon FGF2 gene coding sequence (1-155) (SEQ ID NO: 181) (GenBank
    accession no. XM_003899161, which is hereby incorporated by reference in
    its entirety):
    467                                                   ATGG CAGCCGGGAG
    481 CATCACCACG CTGCCCGCCT TGCCCGAGGA TGGCGGCAGC GGCGCCTTCC CGCCCGGCCA
    541 CTTCAAGGAC CCCAAGCGGC TGTACTGCAA AAACGGGGGC TTCTTCCTGC GCATTCACCC
    601 CGACGGCCGA GTTGACGGGG TCCGGGAGAA GAGCGACCCT CACATCAAAT TACAACTTCA
    661 AGCAGAAGAG AGAGGAGTTG TGTCTATCAA AGGAGTGTGT GCTAACCGTT ACCTTGCTAT
    721 GAAGGAAGAT GGAAGATTAC TGGCTTCTAA ATGTGTTACG GATGAGTGTT TCTTTTTTGA
    781 ACGATTGGAA TCTAATAACT ACAATACTTA CCGGTCAAGG AAATACACCA GTTGGTATGT
    841 GGCACTGAAA CGAACTGGGC AGTATAAACT TGGATCCAAA ACAGGACCTG GGCAGAAAGC
    901 TATACTTTTT CTTCCAATGT CTGCTAAGAG CTGA
    Alpaca FGF2 gene coding sequence (aa 111-265) (SEQ ID NO: 182) (Ensembl
    accession no. ENSVPAT00000010536, which is hereby incorporated by
    reference in its entirety):
    341                                  ATGGCAGCTG GGAGCATCAC CACGCTGCCC
    361 GCCCTGCCGG AGGACGGCGG CAGCGGCGCC TTCCCGCCCG GCCACTTCAA GGACCCCAAG
    421 CGGTTGTACT GCAAAAACGG GGGCTTCTTC CTGCGCATCC ACCCCGACGG CCGAGTGGAC
    481 GGGGTCCGGG AGAAGAGCGA CCCTCACATC AAACTACAAC TTCAAGCAGA AGAGAGAGGG
    541 GTCGTGTCTA TCAAAGGAGT GTGTGCAAAC CGTTACCTTG CTATGAAGGA AGATGGAAGA
    601 TTACTGGCTT CTAAATGTGT CACAGACGAG TGTTTCTTTT TTGAACGATT GGAATCTAAT
    661 AACTACAATA CTTACCGGTC AAGGAAATAC TCCAGTTGGT ATGTGGCACT GAAACGAACT
    721 GGGCAGTACA AACTTGGACC CAAAACAGGA CCTGGGCAGA AAGCTATACT TTTCCTTCCA
    781 ATGTCTGCTA AGAGCTGA
    Sheep FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 183) (GenBank
    accession no. NM_001009769, which is hereby incorporated by reference in
    its entirety):
    1 ATGGCCGCCG GGAGCATCAC CACGCTGCCA GCCCTGCCGG AGGACGGCGG CAGCAGCGCT
    61 TTCCCGCCCG GCCACTTTAA GGACCCCAAG CGGCTGTACT GCAAGAACGG GGGCTTCTTC
    121 CTGCGCATCC ACCCCGACGG CCGAGTGGAC GGGGTCCGCG AGAAGAGCGA CCCTCACATC
    181 AAACTACAAC TTCAAGCAGA AGAGAGAGGG GTTGTGTCTA TCAAAGGAGT GTGTGCAAAC
    241 CGTTACCTTG CTATGAAAGA AGATGGAAGA TTACTAGCTT CTAAATGTGT TACAGACGAG
    301 TGTTTCTTTT TTGAACGATT GGAGTCTAAT AACTACAATA CTTACCGGTC AAGGAAATAC
    361 TCCAGTTGGT ATGTGGCACT GAAACGAACT GGGCAGTATA AACTTGGACC CAAAACAGGA
    421 CCTGGGCAGA AAGCTATACT TTTTCTTCCA ATGTCTGCTA AGAGCTGA
    Western roe deer FGF2 gene coding sequence (1-108; partial amino acid
    sequence corresponding to human FGF2 residues 42 to 149) (SEQ ID
    NO: 184) (GenBank accession no. AF152587, which is hereby
    incorporated by reference in its entirety):
    1 GCGCATCCAC CCCGACGGCC GAGTGGACGG GGTCCGCGAG AAGAGTGACC CTCACATCAA
    61 ACTACAACTT CAAGCAGAAG AGAGAGGGGT TGTGTCTATC AAAGGAGTGT GTGCGAACCG
    121 TTATCTTGCT ATGAAAGAAG ACGGAAGATT ATTGGCTTCA AAATGTGTTA CAGACGAATG
    181 TTTCTTTTTT GAACGATTGG AGTCTAATAA CTACAATACT TACCGGTCAA GGAAATACTC
    241 CAGTTGGTAT GTGGCACTGA AACGAACTGG GCAGTATAAA CTTGGACCCA AAACAGGACC
    301 TGGGCAGAAA GCTATACTTT TTCTT
    Elephant FGF2 gene coding sequence (1-96; partial amino acid sequence
    corresponding to human FGF2 residues 60 to 155) (SEQ ID NO: 185)
    (Ensembl accession no. ENSLAFT00000008249, which is hereby
    incorporated by reference in its entirety):
    1 GTTAAACTAC AGCTTCAAGC AGAAGAGAGA GGTGTTGTGT CTATCAAAGG AGTGTGTGCC
    61 AACCGTTATC TGGCTATGAA GGAAGATGGA AGATTGCTGG CTTCTAGATG TGTGACAGAT
    121 GAATGTTTCT TCTTTGAACG ACTGGAATCT AATAACTACA ATACTTACCG GTCAAGGAAA
    181 TACACCAGTT GGTATGTGGC ACTGAAACGA ACGGGGCAGT ATAAACTTGG ATCCAAAACA
    241 GGACCTGGAC AGAAAGCTAT ACTTTTTCTT CCCATGTCTG CTAAGAGC
    Pig FGF2 gene coding sequence (1-120; partial amino acid sequence
    corresponding to human FGF2 residues 36 to 155) (SEQ ID NO: 186)
    (GenBank accession no. AJ577089 and Ensembl accession
    no. ENSSSCT00000009952, which is hereby incorporated by
    reference in its entirety):
    1 GAACGGGGGC TTCTTCCTGC GCATCCACCC CGACGGCCGA GTGGATGGGG TCCGGGAGAA
    61 GAGCGACCCT CACATCAAAC TACAACTTCA AGCAGAAGAG AGAGGGGTTG TGTCTATCAA
    121 AGGAGTGTGT GCAAACCGTT ATCTTGCTAT GAAGGAAGAT GGAAGATTAC TGGCTTCTAA
    181 ATGTGTTACA GACGAGTGTT TCTTTTTTGA ACGACTGGAA TCTAATAACT ACAATACTTA
    241 CCGGTCGAGG AAATACTCCA GTTGGTATGT GGCACTGAAA CGAACTGGGC AGTATAAACT
    301 TGGACCCAAA ACAGGACCTG GGCAGAAAGC TATACTTTTT CTTCCAATGT CTGCTAAGAG
    361 C
    Panda FGF2 gene coding sequence (1-96; partial amino acid sequence
    corresponding to human FGF2 residues 60 to 155) (SEQ ID NO: 187)
    (Ensembl accession no. ENSAMET00000019232, which is hereby
    incorporated by reference in its entirety):
    1 GTCAAACTGC AACTTCAAGC GGAAGAGAGA GGGGTTGTAT CCATCAAAGG AGTATGTGCA
    61 AATCGCTATC TTGCCATGAA GGAAGATGGA AGATTACTGG CTTCTAAATG TGTTACCGAT
    121 GAGTGTTTCT TTTTTGAGCG ACTGGAATCT AATAACTACA ATACTTACCG GTCAAGGAAA
    181 TACTCCAGTT GGTATGTGGC ACTGAAACGA ACTGGGCAGT ATAAACTTGG ACCCAAAACA
    241 GGACCTGGGC AGAAAGCTAT ACTTTTTCTT CCAATGTCTG CTAAGAGC
    Sloth FGF2 gene coding sequence (aa 14-168) (SEQ ID NO: 188) (Ensembl
    accession no. ENSCHOT00000011394, which is hereby incorporated by
    reference in its entirety):
    40                                           A TGGCAGCCGG GAGCATCACC
    61 ACGCTGCCCG CCCTGCCCGA GGACGGAGGC AGCGGCGCCT TACCGCCCGG CCACTTCAAA
    121 GATCCCAAGC GGCTCTACTG CAAAAACGGG GGCTTCTTCC TGCGTATCCA TCCCGACGGC
    181 AGAGTGGACG GGGTCCGGGA GAAGAGCGAC CCCCACATCA AACTACAACT TCAAGCAGAA
    241 GAGAGAGGGG TTGTGTCTAT CAAAGGTGTG TGTGCAAACC GATATCTTGC TATGAAGGAA
    301 GATGGAAGAT TACAGGCTTC TAAATGTGTA ACGGACGAGT GTTTCTTTTT TGAACGATTG
    361 GAATCTAATA ACTACAATAC GTACCGATCA AGGAAATACT CCAGTTGGTA TGTGGCACTG
    421 AAACGAACTG GGCAATATAA ACTTGGACCC AAAACAGGAC CTGGGCAGAA AGCCATACTT
    481 TTTCTTCCAA TGTCTGCTAA GAGCTGA
    Water buffalo FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 189)
    (GenBank accession no. JQ326277, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCCGCCG GGAGCATCAC CACGCTGCCA CCCCTGCCGG AGGACGGCGG CAGCGGCGCT
    61 TTCCCGCCCG GCCACTTCAA GGACCCCAAG CGGCTGTACT GCAAGAACGG GGGCTTCTTC
    121 CTGCGCATCC ACCCCGACGG CCGAGTGGAC GGGGTCCGCG AGAAGAGCGA CCCACACATC
    181 AAACTACAAC TTCAAGCAGA AGAGAGAGGG GTTGTGTCTA TCAAAGGAGT GTGTGCAAAC
    241 CGTTACCTTG CTATGAAAGA AGATGGAAGA TTACTAGCTT CCAAATGTGT TACAGACGAG
    301 TGTTTCTTTT TTGAACGATT GGAGTCTAGT AACTACAATA CTTACCGGTC AAGGAAATAC
    361 TCCAGTTGGT ATGTGGCACT GAAACGAACT GGGCAGTATA AACTTGGACC CAAAACAGGA
    421 CCTGGGCAGA AAGCTATACT TTTTCTTCCA ATGTCTGCTA AGAGCTGA
    Dog FGF2 gene coding sequence (aa 40-194) (SEQ ID NO: 190) (GenBank
    accession no. XM_003432481, which is hereby incorporated by reference in
    its entirety):
    118                                                               ATG
    121 GCAGCCGGGA GCATCACCAC GCTGCCCGCC CTGCCGGAGG ACGGCGGCAG CGGCGCCTTC
    181 CCGCCCGGCC ACTTCAAGGA CCCCAAGAGG CTGTACTGCA AAAAAGGGGG CTTCTTCCTG
    241 CGGATCCACC CCGACGGCCG GGTGGACGGG GTCCGGGAGA AGAGCGATCC CCACGTCAAA
    301 TTGCAACTTC AAGCAGAAGA GAGAGGCGTT GTGTCCATCA AAGGAGTATG TGCAAATCGC
    361 TATCTTGCTA TGAAGGAAGA TGGAAGATTA CTGGCTTCTA AATGTGTTAC TGACGAGTGC
    421 TTCTTTTTTG AACGATTGGA ATCTAATAAC TACAATACTT ACCGGTCAAG GAAATACTCC
    481 AGTTGGTATG TGGCACTGAA ACGAACTGGG CAGTATAAAC TTGGACCAAA AACAGGACCT
    541 GGGCAGAAAG CTATACTTTT TCTTCCAATG TCTGCTAAGA GCTGA
    Norway rat FGF2 gene coding sequence (aa 1-154) (SEQ ID NO: 191)
    (GenBank accession no. NM_019305, which is hereby
    incorporated by reference in its entirety):
    533                                                          ATGGCTGC
    541 CGGCAGCATC ACTTCGCTTC CCGCACTGCC GGAGGACGGC GGCGGCGCCT TCCCACCCGG
    601 CCACTTCAAG GATCCCAAGC GGCTCTACTG CAAGAACGGC GGCTTCTTCC TGCGCATCCA
    661 TCCAGACGGC CGCGTGGACG GCGTCCGGGA GAAGAGCGAC CCACACGTCA AACTACAGCT
    721 CCAAGCAGAA GAGAGAGGAG TTGTGTCCAT CAAGGGAGTG TGTGCGAACC GGTACCTGGC
    781 TATGAAGGAA GATGGACGGC TGCTGGCTTC TAAGTGTGTT ACAGAAGAGT GTTTCTTCTT
    841 TGAACGCCTG GAGTCCAATA ACTACAACAC TTACCGGTCA CGGAAATACT CCAGTTGGTA
    901 TGTGGCACTG AAACGAACTG GGCAGTATAA ACTCGGATCC AAAACGGGGC CTGGACAGAA
    961 GGCCATACTG TTTCTTCCAA TGTCTGCTAA GAGCTGA
    Naked mole-rat FGF2 gene coding sequence (1-134; partial amino acid
    sequence corresponding to human FGF2 residues 22 to 155)
    (SEQ ID NO: 192) (GenBank accession no. JH173674, which is hereby
    incorporated by reference in its entirety):
    378500                     C CACCCGGCCA CTTCAAGGAC CCAAAGCGGC
    378531 TGTACTGCAA AAACGGGGGC TTCTTCCTGC GCATCCACCC CGACGGCCGC
    378581 GTGGACGGGG TCCGGGAGAA GAGCGACCCT CACG
    418784    TCAAACT ACAACTTCAA GCAGAAGAGA GAGGAGTTGT GTCTATTAAG
    418831 GGAGTGTGTG CGAACCGTTA CCTTGCTATG AAGGAAGATG GAAGATTACT
    418881 GGCTTCT
    433983   AAATGTGT TACAGATGAG TGTTTCTTTT TTGAACGATT GGAATCTAAT
    434031 AACTACAATA CTTATCGGTC AAGGAAATAC TCCAGTTGGT ATGTGGCACT
    434081 GAAACGAACT GGACAATATA AACTTGGATC CAAAACAGGA CCGGGGCAGA
    434131 AAGCTATACT TTTTCTTCCA ATGTCTGCTA AGAGCTGA
    Bushbaby FGF2 gene coding sequence (aa 52-206) (SEQ ID NO: 193) (Ensembl
    accession no. ENSOGAT00000025228, which is hereby incorporated by
    reference in its entirety):
    154                                     ATGGCAG CCGGGAGCAT CACCACGCTG
    181 CCCTCCCTGC CCGAGGACGG CGGCAGCGAC GCCTTTCCGC CCGGCCACTT CAAGGACCCC
    241 AAGCGACTGT ACTGCAAAAA CGGGGGCTTC TTCCTGCGCA TCCACCCCGA CGGCCGAGTG
    301 GACGGGGTCC GGGAGAAGAG CGACCCTTAC ATCAAACTAC AACTTCAAGC AGAAGAGAGA
    361 GGAGTTGTGT CTATCAAAGG AGTGTGTGCG AACCGTTACC TTGCTATGAA GGAAGACGGA
    421 AGATTGCTGG CTTCTAAATT GATTACAGAC GAGTGCTTCT TTTTTGAACG ACTGGAATCT
    481 AATAACTACA ATACTTACCG GTCAAGAAAA TACTCCAGTT GGTATGTGGC ACTGAAACGA
    541 ACTGGACAGT ATAAACTTGG ATCCAAAACA GGACCTGGGC AGAAAGCTAT ACTTTTTCTT
    601 CCAATGTCTG CTAAGAGCTG A
    House mouse FGF2 gene coding sequence (aa 1-154) (SEQ ID NO: 194)
    (GenBank accession no. NM_008006, which is hereby
    incorporated by reference in its entirety):
    198                   ATG GCTGCCAGCG GCATCACCTC GCTTCCCGCA CTGCCGGAGG
    241 ACGGCGGCGC CGCCTTCCCA CCAGGCCACT TCAAGGACCC CAAGCGGCTC TACTGCAAGA
    301 ACGGCGGCTT CTTCCTGCGC ATCCATCCCG ACGGCCGCGT GGATGGCGTC CGCGAGAAGA
    361 GCGACCCACA CGTCAAACTA CAACTCCAAG CAGAAGAGAG AGGAGTTGTG TCTATCAAGG
    421 GAGTGTGTGC CAACCGGTAC CTTGCTATGA AGGAAGATGG ACGGCTGCTG GCTTCTAAGT
    481 GTGTTACAGA AGAGTGTTTC TTCTTTGAAC GACTGGAATC TAATAACTAC AATACTTACC
    541 GGTCACGGAA ATACTCCAGT TGGTATGTGG CACTGAAACG AACTGGGCAG TATAAACTCG
    601 GATCCAAAAC GGGACCTGGA CAGAAGGCCA TACTGTTTCT TCCAATGTCT GCTAAGAGCT
    661 GA
    Squirrel FGF2 gene coding sequence (1-144; partial amino acid sequence
    corresponding to human FGF2 residues 12 to 155) (SEQ ID NO: 195)
    (Ensembl accession no. ENSSTOT00000022105, which is hereby
    incorporated by reference in its entirety):
    1 CTGCCCGAGG ACGGCGGCGG CGGCGCCTTC CCGCCCGGCC ACTTTAAGGA CCCCAAGCGG
    61 CTCTACTGCA AAAACGGAGG CTTCTTCCTG CGCATCCACC CCGACGGCCG AGTGGACGGG
    121 GTCCGGGAGA AGAGCGACCC CCACATCAAG CTCCAGCTTC AAGCCGAAGA CCGAGGGGTT
    181 GTGTCCATCA AGGGAGTGTG TGCAAACCGA TACCTGGCCA TGAAGGAGGA CGGGAGGCTC
    241 CTGGCTTCTA AATGTGTTAC GGACGAGTGT TTCTTTTTTG AACGACTGGA ATCAAATAAC
    301 TACAATACTT ACCGGTCAAG GAAATACTCC AGTTGGTATG TGGCCCTGAA ACGAACAGGG
    361 CAGTATAAAC TTGGATCCAA AACAGGACCT GGGCAGAAAG CTATACTTTT TCTTCCAATG
    421 TCTGCTAAGA GC
    Domestic cat FGF2 gene coding sequence (1-106; partial amino acid
    sequence corresponding to human FGF2 residues 25 to 130) (SEQ ID NO:
    196) (GenBank accession no. EU314952, which is hereby incorporated by
    reference in its entirety):
    1 CCACTTCAAG GACCCCAAGC GTCTGTACTG CAAAAACGGG GGCTTCTTCC TGCGCATCCA
    61 CCCCGACGGC CGAGTGGATG GGGTCCGGGA GAAGAGCGAC CCTCACATCA AACTGCAACT
    121 TCAGGCAGAA GAGAGAGGGG TTGTGTCCAT CAAAGGAGTC TGTGCAAACC GCTATCTTGC
    181 CATGAAGGAA GATGGAAGAT TACTGGCTTC TAAATGTGTT ACGGACGAGT GTTTCTTTTT
    241 TGAACGATTG GAATCTAATA ACTACAATAC TTATCGGTCA AGGAAATACT CCAGCTGGTA
    301 TGTGGCACTG AAACGAAC
    Guinea pig FGF2 gene coding sequence (1-96; partial amino acid sequence
    corresponding to human FGF2 residues 60 to 155) (SEQ ID NO: 197)
    (Ensembl accession no. ENSCPOT00000005443, which is hereby
    incorporated by reference in its entirety):
    1 GTTAAACTAC AACTTCAAGC CGAAGACAGA GGAGTTGTGT CTATCAAGGG AGTCTGTGCG
    61 AACCGTTACC TTGCTATGAA GGAAGACGGA AGATTATTGG CTTCCAAATG TGTTACAGAT
    121 GAATGTTTCT TTTTTGAACG ACTGGAATCT AATAACTACA ACACTTACCG GTCAAGGAAA
    181 TACTCCAGTT GGTATGTGGC ACTGAAACGA ACTGGACAAT ATAAACTTGG GTCCAAAACA
    241 GGACCAGGGC AGAAAGCCAT ACTTTTTCTT CCAATGTCTG CGAAGAGC
    Tasmanian devil FGF2 gene coding sequence (aa 48-203) (SEQ ID NO: 198)
    (Ensembl accession no. ENSSHAP00000012215, which is hereby incorporated
    by reference in its entirety):
    142                        ATGGCCGCG GGCAGCATCA CCACGTTGCC GGCCCTGGCC
    181 GGGGATGGAG CCAGCGGGGG CGCCTTTCCC CCGGGCCACT TCCAGGACCC CAAGCGGCTG
    241 TACTGCAAGA ACGGAGGCTT CTTCTTGCGC ATCCATCCCG ACGGTCACGT GGACGGCATC
    301 CGCGAGAAGA GCGATCCGCA CATTAAACTT CAGCTTCAGG CAGAAGAGAG AGGAGTAGTG
    361 TCTATTAAAG GAGTTTGTGC CAACCGCTAT CTTGCCATGA AAGAGGATGG CAGATTACTG
    421 GCTCTGAAAT GTGTGACTGA AGAGTGTTTC TTCTTTGAAC GTCTAGAGTC CAACAATTAC
    481 AACACTTATC GCTCAAGGAA ATACTCCAAT TGGTATGTGG CATTGAAACG CACAGGCCAG
    541 TATAAGCTTG GATCCAAGAC TGGACCAGGG CAGAAAGCCA TCCTTTTCCT TCCCATGTCT
    601 GCTAAGAGCT GA
    Gray short-tailed opossum FGF2 gene coding sequence (aa 1-155) (SEQ ID
    NO: 199) (GenBank accession no. NM_001033976, which is hereby
    incorporated by reference in its entirety):
    29                               AT GGCCGCAGGC AGCATCACCA CGCTGCCAGC
    61 CCTGTCCGGG GACGGAGGCG GCGGGGGCGC CTTTCCCCCG GGCCACTTCA AGGACCCCAA
    121 GCGGCTGTAC TGCAAGAACG GAGGCTTCTT CCTGCGCATC CACCCCGACG GCCGTGTGGA
    181 CGGCATCCGC GAGAAGAGCG ACCCGAACAT TAAACTACAA CTTCAGGCAG AAGAGAGAGG
    241 AGTGGTGTCT ATTAAAGGAG TATGTGCCAA TCGCTATCTT GCCATGAAGG AAGATGGAAG
    301 ATTATTGGCT TTGAAATATG TGACCGAAGA GTGTTTCTTT TTCGAACGCT TGGAGTCCAA
    361 CAACTACAAC ACTTATCGCT CGAGGAAATA TTCCAATTGG TACGTGGCAC TGAAACGAAC
    421 GGGGCAGTAC AAGCTTGGAT CCAAGACTGG CCCGGGGCAG AAAGCCATCC TTTTCCTCCC
    481 CATGTCTGCT AAGAGCTGA
    Rabbit FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 200) (GenBank
    accession no. XM_002717238, which is hereby incorporated by reference in
    its entirety):
    1 ATGGCAGCCG AGAGCATCAC CACGCTGCCC GCCCTGCCGG AGGATGGAGG CAGCGGCGCC
    61 TTCCCGCCCG GCCACTTCAA GGACCCCAAG CGGCTGTACT GCAAAAACGG GGGTTTCTTC
    121 CTGCGTATCC ACCCCGACGG CCGCGTGGAC GGGGTCCGGG AGAAGAGCGA CCCACACATC
    181 AAATTACAAC TTCAAGCAGA AGAGAGAGGA GTTGTATCCA TCAAAGGTGT GTGTGCAAAC
    241 CGTTACCTTG CTATGAAGGA AGATGGAAGA CTGCTGGCTT CTAAATGTGT TACAGACGAG
    301 TGCTTCTTTT TTGAACGACT GGAGTCTAAT AACTACAATA CTTACCGGTC AAGGAAATAT
    361 TCCAGCTGGT ATGTGGCACT GAAACGAACT GGGCAGTATA AACTTGGATC CAAAACAGGA
    421 CCTGGGCAGA AGGCTATACT TTTTCTTCCA ATGTCTGCTA AGAGCTGA
    Turkey FGF2 gene coding sequence (1-125; partial amino acid sequence
    corresponding to human FGF2 residues 31 to 155) (SEQ ID NO: 201)
    (Ensembl accession no. ENSMGAT00000011845, which is hereby
    incorporated by reference in its entirety):
    1 CGGCTCTACT GTAAGAACGG CGGCTTCTTC CTGCGCATCA ATCCCGACGG CAGAGTGGAC
    61 GGCGTCCGCG AGAAGAGCGA TCCGCACATC AAACTGCAGC TTCAGGCAGA AGAAAGAGGA
    121 GTGGTATCAA TCAAAGGTGT AAGTGCAAAC CGCTTTCTGG CTATGAAGGA GGATGGCAGA
    181 TTGCTGGCAC TGAAATGTGC AACAGAAGAA TGTTTCTTTT TTGAGCGTTT GGAATCTAAT
    241 AATTATAACA CTTACCGGTC ACGGAAGTAC TCTGATTGGT ATGTGGCACT GAAAAGAACT
    301 GGACAGTACA AGCCCGGACC AAAAACTGGA CCTGGACAGA AAGCTATCCT TTTTCTTCCA
    361 ATGTCTGCTA AAAGC
    Gallus gallus FGF2 gene coding sequence (aa 1-158) (SEQ ID NO: 202)
    (GenBank accession no. NM_205433, which is hereby incorporated by
    reference in its entirety):
    98                                         ATG GCGGCGGGGG CGGCGGGGAG
    121 CATCACCACG CTGCCGGCGC TGCCCGACGA CGGGGGCGGC GGCGCTTTTC CCCCCGGGCA
    181 CTTCAAGGAC CCCAAGCGGC TCTACTGCAA GAACGGCGGC TTCTTCCTGC GCATCAACCC
    241 CGACGGCAGG GTGGACGGCG TCCGCGAGAA GAGCGATCCG CACATCAAAC TGCAGCTTCA
    301 AGCAGAAGAA AGAGGAGTAG TATCAATCAA AGGCGTAAGT GCAAACCGCT TTCTGGCTAT
    361 GAAGGAGGAT GGCAGATTGC TGGCACTGAA ATGTGCAACA GAGGAATGTT TCTTTTTCGA
    421 GCGCTTGGAA TCTAATAACT ATAACACTTA CCGGTCACGG AAGTACTCTG ATTGGTATGT
    481 GGCACTGAAA AGGACTGGAC AGTACAAGCC CGGACCAAAA ACTGGACCTG GACAGAAAGC
    541 TATCCTTTTT CTTCCAATGT CTGCTAAAAG CTGA
    Zebra finch FGF2 gene coding sequence (aa 1-153) (SEQ ID NO: 203)
    (GenBank accession no. XM_002188361, which is hereby
    incorporated by reference in its entirety):
    1 ATGGCGGCGG CGGGGGGCAT CGCTACGCTG CCCGACGACG GCGGCAGCGG CGCCTTTCCC
    61 CCGGGGCACT TCAAGGACCC CAAGCGCCTG TACTGCAAGA ACGGCGGCTT CTTCCTGCGC
    121 ATCAACCCCG ACGGGAAGGT GGACGGCGTC CGCGAGAAGA GCGACCCGCA CATCAAGCTG
    181 CAGCTTCAGG CGGAGGAACG AGGAGTGGTG TCCATCAAAG GTGTCAGTGC CAATCGCTTC
    241 CTGGCCATGA AAGAGGATGG CAGATTGCTG GCCTTGAAAT ATGCAACAGA AGAATGTTTC
    301 TTTTTTGAAC GTTTGGAATC CAATAACTAT AACACTTACC GGTCACGGAA ATACTCGGAT
    361 TGGTATGTGG CACTGAAAAG AACTGGACAG TACAAACCTG GACCAAAAAC TGGACCTGGA
    421 CAGAAAGCTA TCCTTTTCCT TCCTATGTCT GCTAAAAGCT GA
    Japanese firebelly newt FGF2 gene coding sequence (aa 1-155) (SEQ ID
    NO: 204) (GenBank accession no. AB064664, which is hereby incorporated
    by reference in its entirety):
    384                          ATGGCTG CTGGGAGCAT CACCAGTCTC CCTGCCCTAC
    421 CCGAGGACGG GAATGGCGGC ACCTTCACAC CCGGCGGATT CAAAGAGCCG AAGAGGCTGT
    481 ACTGCAAGAA CGGGGGCTTC TTTCTCCGGA TCAACTCCGA CGGCAAGGTG GACGGAGCCC
    541 GGGAGAAGAG CGACTCCTAC ATTAAACTGC AGCTTCAAGC AGAAGAGCGC GGTGTGGTGT
    601 CCATCAAGGG AGTATGTGCA AACCGCTATC TCGCTATGAA GGATGATGGC AGGCTGATGG
    661 CGCTGAAATG GATAACCGAT GAATGCTTCT TTTTCGAGCG ACTGGAGTCC AACAACTATA
    721 ACACGTATCG ATCACGGAAA TATTCCGATT GGTATGTGGC GCTGAAAAGA ACTGGGCAAT
    781 ACAAAAATGG ATCAAAAACC GGAGCAGGAC AGAAAGCAAT CCTTTTTCTA CCCATGTCGG
    841 CCAAGAGTTG A
    African clawed frog FGF2 gene coding sequence (aa 1-155) (SEQ ID
    NO: 205) (GenBank accession no. NM_001099871, which is
    hereby incorporated by reference in its entirety):
    335                                      ATGGCG GCAGGGAGCA TCACAACTCT
    361 GCCAACTGAA TCCGAGGATG GGGGAAACAC TCCTTTTTCA CCAGGGAGTT TTAAAGACCC
    421 CAAGAGGCTC TACTGCAAGA ACGGGGGCTT CTTCCTCAGG ATAAACTCAG ACGGGAGAGT
    481 GGACGGGTCA AGGGACAAAA GTGACTCGCA CATAAAATTA CAGCTACAAG CTGTAGAGCG
    541 GGGAGTGGTA TCAATAAAGG GAATCACTGC AAATCGCTAC CTTGCCATGA AGGAAGATGG
    601 GAGATTAACA TCGCTGAGGT GTATAACAGA TGAATGCTTC TTTTTTGAAC GACTGGAAGC
    661 TAATAACTAC AACACTTACC GGTCTCGGAA ATACAGCAGC TGGTATGTGG CACTAAAGCG
    721 AACCGGGCAG TACAAAAATG GATCGAGCAC TGGACCGGGA CAAAAAGCTA TTTTATTTCT
    781 CCCAATGTCC GCAAAGAGCT GA
    White-eared opossum FGF2 gene coding sequence (aa 1-156) (SEQ ID
    NO: 206) (GenBank accession no. EF057322, which is hereby
    incorporated by reference in its entirety):
    1 ATGGCAGCAG GCAGCATCAC CACATTGCCG GCCCTGTCCG GGGACGGAGG CGGCGGGGGA
    61 GCCTTTCCTC CAGGCCACTT CAAGGACCCC AAGCGGCTGT ACTGCAAGAA CGGAGGCTTC
    121 TTCCTGCGCA TCCACCCCGA CGGCCGCGTG GACGGCATCC GCGAGAAGAG CGACCCGAAC
    181 ATTAAACTAC AACTTCAGGC AGAAGAGAGA GGAGTAGTGT CTATTAAAGG AGTATGTGCC
    241 AACCGATATC TTGCCATGAA GGAGGATGGC AGATTATTGG CTTTGAAATA TGTGACCGAA
    301 GAGTGTTTCT TTTTTGAACG TTTGGAGTCC AACAACTACA ACACTTATCG CTCAAGAAAA
    361 TATTCCAATT GGTATGTGGC ACTGAAACGA ACGGGGCAGT ATAAGCTTGG ATCCAAGACT
    421 GGCCCGGGGC AGAAAGCCAT CCTTTTCTCC CCATGTCTGC TAAGATGCTG A
    Microbat FGF2 gene coding sequence (1-96; partial amino acid sequence
    corresponding to human FGF2 residues 60 to 155) (SEQ ID NO: 207)
    (Ensembl accession no. ENSMLUT00000027717, which is hereby
    incorporated by reference in its entirety):
    1 GTCAAACTCC AACTTCAAGC AGAAGAGAGA GGGGTCGTGT CTATCAAAGG AGTGTGTGCC
    61 AACCGCTATC TCGCTATGAA GGAGGACGGC CGGTTACAGG CTTCTAAATG TGTTACGGAT
    121 GAGTGTTTCT TTTTTGAACG GTTGGAATCC AATAACTACA ACACTTACCG GTCAAGAAAG
    181 TACTCCAGTT GGTATGTGGC ATTGAAGCGG AATGGGCAGT ATAAACTTGG ACCCAAAACA
    241 GGACCTGGCC AGAAAGCCAT ACTTTTTCTT CCCATGTCTG CTAAGAGC
    Anole lizard FGF2 gene coding sequence (1-140; partial amino acid
    sequence corresponding to human FGF2 residues 16 to 155) (SEQ ID
    NO: 208) (Ensembl accession no. ENSACAT00000011897, which
    is hereby incorporated by reference in its entirety):
    1 GCGGCGGCGG CCTCTTTCCC CCCGGGCCCC TTCAAGGACC CCAAGCGCCT CTACTGCAAG
    61 AACGGGGGCT TCTTCCTGCG GATCAACCCC GACGGCGGCG TGGACGGCGT CCGAGAGAAG
    121 AGCGACCCCA ACATCAAATT GCTGCTCCAG GCAGAGGAGA GAGGTGTAGT GTCCATCAAA
    181 GGTGTATGCG CAAACCGTTT CCTGGCTATG AATGAAGACG GTCGATTGTT AGCACTGAAA
    241 TACGTAACAG ATGAATGCTT CTTTTTTGAA CGCTTGGAAT CTAATAATTA CAATACTTAT
    301 CGGTCTCGTA AATACCGTGA TTGGTACATT GCACTGAAAC GAACTGGTCA GTACAAACTT
    361 GGACCAAAAA CTGGACGAGG CCAGAAAGCT ATCCTTTTCC TTCCAATGTC TGCCAAAAGT
    Armadillo FGF2 gene coding sequence (124-217; partial amino acid
    sequence corresponding to human FGF2 residues 1 to 94) (SEQ
    ID NO: 209) (Ensembl accession no. ENSDNOT00000014647, which
    is hereby incorporated by reference in its entirety):
    361          A TGGCAGCCGG GAGCATCACC ACGCTGCCCG CTCTGCCCGA GGACGGCGGC
    421 AGCGGCGCCT TCCCGCCGGG CCACTTCAAG GACCCCAAGC GGCTGTACTG CAAAAACGGG
    481 GGCTTCTTCC TGCGCATCCA TCCCGACGGC CGAGTGGACG GGGTCCGGGA GAAGAGCGAC
    541 CCTAACATCA AACTACAACT TCAAGCAGAA GAGAGAGGGG TCGTGTCTAT CAAAGGCGTG
    601 TGTGCGAACC GTTACCTTGC TATGCGGGAA GACGGAAGAC TCCAGGCGTC T
    Tree shrew FGF2 gene coding sequence (1-189) (SEQ ID NO: 210) (Ensembl
    accession no. ENSTBET00000001143, which is hereby incorporated by
    reference in its entirety):
    1 GCGGGGGTTA GAGCTGAGAG GGAGGAGGCA CCGGGGAGCG GTGACAGCCG GGGGACCGAT
    61 CCCGCCGCGC GTTCGCTCAT CAGGAGGCCG GATGCTGCAG CGCGAGAGGC GCTTCTTGGA
    121 GCCAGGAGCC GGGTTCAGGG CAGCTCCACC TCCTGGCCAG CCTCGTCACG AACCGGGATC
    181 AAGTTGCCGG ACGACTCAGG TCAAGGAATG GGCGGCTATC CTCTGGACCG CCCGAGCCGG
    241 AGCACAGGGC GAGGGCTGGG CGGTGCCCCG GACCCTGCCG TAAAACTACA GCTTCAAGCG
    301 GAAGAGAGAG GGGTCGTGTC TATCAAAGGA GTGTGTGCAA ACCGTTACCT GGCCATGAAG
    361 GAGGATGGGC GACTGCTGGC TTCTAAATGT GTTACAGATG AGTGTTTCTT TTTTGAACGA
    421 CTGGAATCTA ATAACTACAA TACTTACCGG TCCCGAAAGT ACTCCAGCTG GTATGTGGCA
    481 CTGAAACGAA CTGGGCAGTA TAAACTTGGA TCCAAAACAG GACCTGGGCA GAAAGCTATA
    541 CTTTTTCTTC CAATGTCTGC TAAAAGC
    Western clawed frog FGF2 gene coding sequence (aa 1-154) (SEQ ID
    NO: 211) (GenBank accession no. NM_001017333, which is
    hereby incorporated by reference in its entirety):
    171                                                        ATGGCAGCAG
    181 GAAGCATCAC AACCCTACCA ACCGAATCTG AGGATGGAAA CACTCCTTTC CCACCGGGGA
    241 ACTTTAAGGA CCCCAAGAGG CTCTACTGCA AGAATGGGGG CTACTTCCTC AGGATTAACT
    301 CAGACGGGAG AGTGGACGGA TCAAGGGATA AAAGTGACTT ACACATAAAA TTACAGCTAC
    361 AAGCAGTAGA GCGGGGAGTG GTATCAATAA AGGGAATCAC TGCAAATCGC TACCTTGCCA
    421 TGAAGGAAGA TGGGAGATTA ACATCGCTGA AGTGTATAAC AGATGAATGC TTCTTTTATG
    481 AACGATTGGA AGCTAATAAC TACAACACTT ACCGGTCTCG GAAAAACAAC AGCTGGTATG
    541 TGGCACTAAA GCGAACTGGG CAGTATAAAA ATGGATCGAC CACTGGACCA GGACAAAAAG
    601 CTATTTTGTT TCTCCCAATG TCAGCAAAAA GCTGA
    Coelacanth FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 212)
    (Ensembl accession no. ENSLACT00000019333, which is hereby
    incorporated by reference in its entirety):
    1                       ATGGCTGCGG GAGGAATCAC TACCCTGCCG GCGGTACCTG
    41 AGGATGGAGG CAGCAGCACC TTCCCTCCAG GAAACTTCAA GGAGCCCAAG AGACTTTACT
    101 GTAAGAATGG AGGCTATTTC TTAAGGATAA ACCCCGATGG AAGAGTGGAT GGAACAAGGG
    161 AGAAAAATGA TCCTTATATA AAATTACAAC TGCAAGCTGA ATCTATAGGA GTGGTGTCGA
    221 TAAAGGGAGT TTGTTCAAAC CGTTACCTAG CGATGAATGA AGACTGTAGA CTTTTTGGAT
    281 TGAAATATCC AACGGATGAA TGTTTCTTCC ATGAGAGGCT GGAGTCCAAC AACTACAATA
    341 CTTATCGTTC AAAGAAGTAT TCGGATTGGT ATGTGGCGCT GAAACGGACT GGTCAGTACA
    401 AACCTGGGCC AAAAACTGGA CTGGGACAAA AAGCAATCCT TTTCCTTCCG ATGTCTGCCA
    461 AGAGTTGA
    Spotted green pufferfish FGF2 gene coding sequence (aa 34-188) (SEQ ID
    NO: 213) (Ensembl accession no. ENSTNIT00000016254, which is hereby
    incorporated by reference in its entirety):
    1 ATGGCCACGG GAGGGATCAC GACGCTTCCA TCCACACCTG AAGACGGCGG CAGCAGCGGC
    61 TTTCCTCCCG GCAGCTTCAA GGATCCCAAA AGGCTCTACT GTAAAAACGG AGGTTTCTTC
    121 CTGAGGATCA AGTCCGACGG GGTCGTGGAC GGAATCCGGG AGAAGAGTGA CCCCCACATA
    181 AAGCTTCAGC TCCAGGCGAC CTCTGTGGGG GAGGTGGTCA TCAAGGGGGT GTGCGCTAAC
    241 CGCTATCTGG CCATGAACAG AGATGGACGG CTGTTCGGAA CGAAACGAGC CACGGACGAA
    301 TGCCATTTCT TAGAGCGGCT TGAGAGCAAC AACTACAACA CTTACCGCTC CAGGAAGTAC
    361 CCAACCATGT TTGTGGGACT GACGCGGACG GGCCAGTACA AGTCTGGGAG CAAAACTGGA
    421 CCGGGCCAAA AGGCCATCCT TTTTCTTCCG ATGTCCGCCA AATGCTAA
    Stickleback FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 214)
    (Ensembl accession no. ENSGACT00000022120, which is hereby
    incorporated by reference in its entirety):
    1                    AT GGCCACGGCA GGCTTCGCGA CGCTTCCCTC CACGCCCGAA
    43 GACGGCGGCA GCGGCGGCTT CACCCCCGGG GGATTCAAGG ATCCCAAGAG GCTGTACTGC
    103 AAAAACGGGG GCTTCTTCTT GAGGATCAGG TCCGACGGAG GTGTAGATGG AATCAGGGAG
    163 AAGAGCGACG CCCACATAAA GCTCCAAATC CAGGCGACGT CGGTGGGGGA GGTGGTCATC
    223 AAAGGAGTCT GTGCCAACCG CTATCTGGCC ATGAACAGAG ACGGCCGGCT GTTCGGAGTG
    283 AGACGGGCGA CGGACGAATG CTACTTCCTG GAGCGGCTGG AGAGTAACAA CTACAACACC
    343 TACCGCTCCA GGAAGTACCC CGGCATGTAC GTGGCTCTGA AGCGGACCGG CCAGTACAAG
    403 TCCGGGAGCA AAACCGGACC CGGTCAAAAG GCCATTCTGT TCCTCCCCAT GTCGGCTAAG
    463 TGCTAA
    Fugu rubripes FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 215)
    (Ensembl accession no. ENSTRUT00000022363, which is hereby incorporated
    by reference in its entirety):
    127       ATGG CCACGGGAGG GATCACAACA CTTCCATCCA CACCTGAAGA CGGCGGCAGC
    181 GGCGGTTTTC CTCCCGGGAG CTTCAAGGAT CCCAAAAGGC TGTACTGTAA AAACGGCGGC
    241 TTCTTCCTGA GGATCAGGTC CGACGGGGCC GTGGACGGAA CCCGGGAGAA GACTGACCCC
    301 CACATAAAGC TTCAGCTCCA GGCGACCTCT GTGGGGGAGG TGGTCATCAA GGGGGTTTGT
    361 GCTAATCGTT ATCTGGCCAT GAACAGAGAT GGACGACTGT TTGGAATGAA ACGAGCGACG
    421 GATGAATGCC ACTTCTTAGA GCGGCTCGAG AGCAACAACT ACAACACCTA CCGCTCCAGG
    481 AAGTACCCCA ACATGTTTGT GGGACTGACG CGAACTGGCA ACTACAAGTC TGGGACTAAA
    541 ACTGGACCGG GCCAAAAGGC CATCCTCTTT CTTCCGATGT CGGCCAAATA CTAA
    Rainbow trout FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 216)
    (GenBank accession no. NM_001124536, which is hereby incorporated by
    reference in its entirety):
    390                                A TGGCCACAGG AGAAATCACC ACTCTACCCG
    421 CCACACCTGA AGATGGAGGC AGTGGCGGCT TCCTTCCAGG AAACTTTAAG GAGCCCAAGA
    481 GGTTGTACTG TAAAAATGGA GGCTACTTCT TGAGGATAAA CTCTAACGGA AGCGTGGACG
    541 GGATCAGAGA TAAGAACGAC CCCCACAATA AGCTTCAACT CCAGGCGACC TCAGTGGGGG
    601 AAGTAGTAAT CAAAGGGGTC TCAGCCAACC GCTATCTGGC CATGAATGCA GATGGAAGAC
    661 TGTTTGGACC GAGACGGACA ACAGATGAAT GCTACTTCAT GGAGAGGCTG GAGAGTAACA
    721 ACTACAACAC CTACCGCTCT CGAAAGTACC CTGAAATGTA TGTGGCACTG AAAAGGACTG
    781 GCCAGTACAA GTCAGGATCC AAAACTGGAC CCGGCCAAAA AGCCATCCTC TTCCTCCCCA
    841 TGTCAGCCAG ACGCTGA
    Salmon FGF2 gene coding sequence (1-150) (SEQ ID NO: 217) (GenBank
    accession no. EU816603, which is hereby incorporated by reference in its
    entirety):
    99402                                              ATGGCCACA GGAGAAATCA
    99421 CCACTCTACC CGCCACACCT GAAGATGGAG GCAGTGGCGG CTTCCCTCCA GGAAACTTTA
    99481 AGGATCCCAA GAGGCTGTAC TGTAAAAACG GGGGCTACTT CTTGAGAATA AACTCTAATG
    99541 GAAGCGTGGA CGGGATCCGA GAGAAGAACG ACCCCCACA
    100968                                                    AAC AGCCTCAATT
    100981 TGTCAGGGCA TGGACTCTTC AAGGTGTCAA ACGTTCCACA GGGATGCTGG CCCATGTTGA
    101041 CTCCAACGCT TCCCACAATT GTGTCAAGGT GGCTGGATGT TCTTTGGGAG
    101845                           AATTTG GCAGTATGTC CAACCGGCCT CATAACCGCA
    101881 GACCACGTGT AGCTACACCA GCCCAGGACC TCCACATCCG GCTTCTTCAT CTACGGGATC
    101941 GTCTGAAACC AGCCACCCGA ACAGCTGATA AAACTGAGGA GTATTTCTGT CTGTAA
    Zebrafish FGF2 gene coding sequence (aa 1-154) (SEQ ID NO: 218) (GenBank
    accession no. AY269790, which is hereby incorporated by reference in its
    entirety):
    43                                               ATGGCCAC CGGAGGGATC
    61 ACCACACTCC CGGCCGCTCC GGACGCCGAA AACAGCAGCT TTCCCGCGGG CAGCTTCAGG
    121 GATCCCAAGC GCCTGTACTG CAAAAACGGA GGATTCTTCC TGCGGATCAA CGCGGACGGC
    181 CGAGTGGACG GAGCCCGAGA CAAGAGCGAC CCGCACATTC GTCTGCAGCT GCAGGCGACG
    241 GCAGTGGGTG AAGTACTCAT TAAAGGCATC TGTACCAACC GTTTCCTTGC CATGAACGCA
    301 GACGGACGAC TGTTCGGGAC GAAAAGGACC ACAGATGAAT GTTATTTCCT GGAGCGCCTG
    361 GAGTCCAACA ACTACAACAC ATACAGATCC CGCAAGTATC CCGACTGGTA CGTGGCTCTG
    421 AAGAGAACCG GCCAGTATAA AAGCGGCTCT AAAACCAGCC CGGGACAGAA GGCCATCCTG
    481 TTTCTGCCCA TGTCGGCCAA ATGCTGA
    Nile tilapia FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 219)
    (GenBank accession no. XM_003443364, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCCACGG GAGGAATCAC AACACTTCCC GCTACACCTG AAGACGGCGG CAGCAGCGGC
    61 TTTCCTCCTG GGAACTTCAA GGACCCTAAA AGGCTGTACT GTAAAAATGG TGGCTTCTTC
    121 TTGAGGATAA AATCTGATGG AGGAGTGGAT GGAATACGAG AGAAAAACGA CCCCCACATA
    181 AAGCTTCAAC TCCAGGCGAC CTCAGTGGGA GAAGTGGTCA TCAAAGGGAT TTGTGCAAAC
    241 CGATATCTGG CAATGAACAG AGATGGACGA CTGTTTGGAG CGAGAAGAGC AACAGATGAG
    301 TGCTACTTCT TAGAGCGGCT CGAGAGCAAC AACTACAACA CCTACCGCTC CAGGAAGTAC
    361 CCAAACATGT ACGTGGCGCT GAAGCGGACT GGCCAGTACA AGTCTGGAAG CAAAACTGGA
    421 CCGGGTCAAA AGGCAATTCT CTTTCTCCCA ATGTCTGCTA AATGCTAA
    Medaka FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 220) (Ensembl
    accession no. ENSORLT00000025835, which is hereby incorporated by
    reference in its entirety):
    1 ATGGCTACGG GAGAAATCAC AACACTTCCC TCCCCAGCTG AAAACAGCAG AAGCGATGGC
    61 TTTCCTCCAG GGAACTACAA GGATCCTAAG AGGCTCTACT GTAAAAATGG AGGTTTGTTT
    121 TTGAGGATTA AACCTGATGG AGGAGTGGAT GGAATCCGGG AAAAAAAAGA TCCCCACGTT
    181 AAGCTTCGCC TTCAGGCTAC CTCAGCGGGA GAGGTGGTGA TCAAAGGAGT TTGTTCAAAC
    241 AGATATCTGG CGATGCATGG AGATGGACGT CTATTTGGAG TGAGACAAGC AACAGAGGAA
    301 TGCTACTTCT TGGAGCGACT AGAGAGCAAC AACTATAACA CCTATCGCTC TAAAAAGTAC
    361 CCAAACATGT ACGTGGCACT GAAGCGGACA GGCCAGTACA AACCTGGAAA CAAAACTGGA
    421 CCAGGTCAAA AGGCCATTCT CTTTCTGCCT ATGTCTGCCA AGTACTAA
  • As noted above, also encompassed within the present invention are portions of paracrine FGFs other than FGF1 and/or FGF2 (e.g., FGF4, FGF5, FGF6, FGF9, FGF16, and FGF20). The portion of the paracrine FGF may be from human FGF4, FGF5, FGF6, FGF9, FGF16, and/or FGF20 having the amino acid sequences shown in Table 5, or orthologs thereof.
  • TABLE 5
    Amino acid sequence of human FGF4(SEQ ID NO: 221) (GenBank accession
    no. NP_001998, which is hereby incorporated by reference in its
    entirety):
    1 MSGPGTAAVA LLPAVLLALL APWAGRGGAA APTAPNGTLE AELERRWESL VALSLARLPV
    61 AAQPKEAAVQ SGAGDYLLGI KRLRRLYCNV GIGFHLQALP DGRIGGAHAD TRDSLLELSP
    121 VERGVVSIFG VASRFFVAMS SKGKLYGSPF FTDECTFKEI LLPNNYNAYE SYKYPGMFIA
    181 LSKNGKTKKG NRVSPTMKVT HFLPRL
    Amino acid sequence of human FGF5(SEQ ID NO: 222) (GenBank Accession
    No. NP_004455, which is hereby incorporated by reference in its
    entirety):
    1 MSLSFLLLLF FSHLILSAWA HGEKRLAPKG QPGPAATDRN PRGSSSRQSS SSAMSSSSAS
    61 SSPAASLGSQ GSGLEQSSFQ WSPSGRRTGS LYCRVGIGFH LQIYPDGKVN GSHEANMLSV
    121 LEIFAVSQGI VGIRGVFSNK FLAMSKKGKL HASAKFTDDC KFRERFQENS YNTYASAIHR
    181 TEKTGREWYV ALNKRGKAKR GCSPRVKPQH ISTHFLPRFK QSEQPELSFT VTVPEKKKPP
    241 SPIKPKIPLS APRKNTNSVK YRLKFRFG
    Amino acid sequence of human FGF6(SEQ ID NO: 223) (NP_066276, which
    is hereby incorporated by reference in its entirety):
    1 MALGQKLFIT MSRGAGRLQG TLWALVFLGI LVGMVVPSPA GTRANNTLLD SRGWGTLLSR
    61 SRAGLAGEIA GVNWESGYLV GIKRQRRLYC NVGIGFHLQV LPDGRISGTH EENPYSLLEI
    121 STVERGVVSL FGVRSALFVA MNSKGRLYAT PSFQEECKFR ETLLPNNYNA YESDLYQGTY
    181 IALSKYGRVK RGSKVSPIMT VTHFLPRI
    Amino acid sequence of human FGF9(SEQ ID NO: 224) (GenBank accession
    no. NP_002001, which is hereby incorporated by reference in its
    entirety):
    1 MAPLGEVGNY FGVQDAVPFG NVPVLPVDSP VLLSDHLGQS EAGGLPRGPA VTDLDHLKGI
    61 LRRRQLYCRT GFHLEIFPNG TIQGTRKDHS RFGILEFISI AVGLVSIRGV DSGLYLGMNE
    121 KGELYGSEKL TQECVFREQF EENWYNTYSS NLYKHVDTGR RYYVALNKDG TPREGTRTKR
    181 HQKFTHFLPR PVDPDKVPEL YKDILSQS
    Amino acid sequence of human FGF16(SEQ ID NO: 225) (GenBank
    accession no. NP_003859, which is hereby incorporated by
    reference in its entirety):
    1 MAEVGGVFAS LDWDLHGFSS SLGNVPLADS PGFLNERLGQ IEGKLQRGSP TDFAHLKGIL
    61 RRRQLYCRTG FHLEIFPNGT VHGTRHDHSR FGILEFISLA VGLISIRGVD SGLYLGMNER
    121 GELYGSKKLT RECVFREQFE ENWYNTYAST LYKHSDSERQ YYVALNKDGS PREGYRTKRH
    181 QKFTHFLPRP VDPSKLPSMS RDLFHYR
    Amino acid sequence of human FGF20(SEQ ID NO: 226) (GenBank
    accession no. NP_062825, which is hereby incorporated by
    reference in its entirety):
    1 MAPLAEVGGF LGGLEGLGQQ VGSHFLLPPA GERPPLLGER RSAAERSARG GPGAAQLAHL
    61 HGILRRRQLY CRTGFHLQIL PDGSVQGTRQ DHSLFGILEF ISVAVGLVSI RGVDSGLYLG
    121 MNDKGELYGS EKLTSECIFR EQFEENWYNT YSSNIYKHGD TGRRYFVALN KDGTPRDGAR
    181 SKRHQKFTHF LPRPVDPERV PELYKDLLMY T
  • It will be understood that the portion of the paracrine FGF according to the present invention may be derived from a nucleotide sequence that encodes human FGF4, FGF5, FGF6, FGF9, FGF16, and/or FGF20 having the nucleotide sequences shown in Table 6, or orthologs thereof.
  • TABLE 6
    Human FGF4 gene coding sequence (1-206) (SEQ ID NO: 227) (GenBank
    accession no. NM_002007, which is hereby incorporated by reference in
    its entirety):
    320                     A TGTCGGGGCC CGGGACGGCC GCGGTAGCGC TGCTCCCGGC
    361 GGTCCTGCTG GCCTTGCTGG CGCCCTGGGC GGGCCGAGGG GGCGCCGCCG CACCCACTGC
    421 ACCCAACGGC ACGCTGGAGG CCGAGCTGGA GCGCCGCTGG GAGAGCCTGG TGGCGCTCTC
    481 GTTGGCGCGC CTGCCGGTGG CAGCGCAGCC CAAGGAGGCG GCCGTCCAGA GCGGCGCCGG
    541 CGACTACCTG CTGGGCATCA AGCGGCTGCG GCGGCTCTAC TGCAACGTGG GCATCGGCTT
    601 CCACCTCCAG GCGCTCCCCG ACGGCCGCAT CGGCGGCGCG CACGCGGACA CCCGCGACAG
    661 CCTGCTGGAG CTCTCGCCCG TGGAGCGGGG CGTGGTGAGC ATCTTCGGCG TGGCCAGCCG
    721 GTTCTTCGTG GCCATGAGCA GCAAGGGCAA GCTCTATGGC TCGCCCTTCT TCACCGATGA
    781 GTGCACGTTC AAGGAGATTC TCCTTCCCAA CAACTACAAC GCCTACGAGT CCTACAAGTA
    841 CCCCGGCATG TTCATCGCCC TGAGCAAGAA TGGGAAGACC AAGAAGGGGA ACCGAGTGTC
    901 GCCCACCATG AAGGTCACCC ACTTCCTCCC CAGGCTGTGA
    Human FGF5 gene coding sequence (1-268) (SEQ ID NO: 228) (GenBank
    Accession No. NM_004464, which is hereby incorporated by reference in
    its entirety):
    238                                                               ATG
    241 AGCTTGTCCT TCCTCCTCCT CCTCTTCTTC AGCCACCTGA TCCTCAGCGC CTGGGCTCAC
    301 GGGGAGAAGC GTCTCGCCCC CAAAGGGCAA CCCGGACCCG CTGCCACTGA TAGGAACCCT
    361 AGAGGCTCCA GCAGCAGACA GAGCAGCAGT AGCGCTATGT CTTCCTCTTC TGCCTCCTCC
    421 TCCCCCGCAG CTTCTCTGGG CAGCCAAGGA AGTGGCTTGG AGCAGAGCAG TTTCCAGTGG
    481 AGCCCCTCGG GGCGCCGGAC CGGCAGCCTC TACTGCAGAG TGGGCATCGG TTTCCATCTG
    541 CAGATCTACC CGGATGGCAA AGTCAATGGA TCCCACGAAG CCAATATGTT AAGTGTTTTG
    601 GAAATATTTG CTGTGTCTCA GGGGATTGTA GGAATACGAG GAGTTTTCAG CAACAAATTT
    661 TTAGCGATGT CAAAAAAAGG AAAACTCCAT GCAAGTGCCA AGTTCACAGA TGACTGCAAG
    721 TTCAGGGAGC GTTTTCAAGA AAATAGCTAT AATACCTATG CCTCAGCAAT ACATAGAACT
    781 GAAAAAACAG GGCGGGAGTG GTATGTGGCC CTGAATAAAA GAGGAAAAGC CAAACGAGGG
    841 TGCAGCCCCC GGGTTAAACC CCAGCATATC TCTACCCATT TTCTGCCAAG ATTCAAGCAG
    901 TCGGAGCAGC CAGAACTTTC TTTCACGGTT ACTGTTCCTG AAAAGAAAAA GCCACCTAGC
    961 CCTATCAAGC CAAAGATTCC CCTTTCTGCA CCTCGGAAAA ATACCAACTC AGTGAAATAC
    1021 AGACTCAAGT TTCGCTTTGG ATAA
    Human FGF6 gene coding sequence (1-208) (SEQ ID NO: 229) (NM_020996,
    which is hereby incorporated by reference in its entirety):
    45                                                 ATGGCC CTGGGACAGA
    61 AACTGTTCAT CACTATGTCC CGGGGAGCAG GACGTCTGCA GGGCACGCTG TGGGCTCTCG
    121 TCTTCCTAGG CATCCTAGTG GGCATGGTGG TGCCCTCGCC TGCAGGCACC CGTGCCAACA
    181 ACACGCTGCT GGACTCGAGG GGCTGGGGCA CCCTGCTGTC CAGGTCTCGC GCGGGGCTAG
    241 CTGGAGAGAT TGCCGGGGTG AACTGGGAAA GTGGCTATTT GGTGGGGATC AAGCGGCAGC
    301 GGAGGCTCTA CTGCAACGTG GGCATCGGCT TTCACCTCCA GGTGCTCCCC GACGGCCGGA
    361 TCAGCGGGAC CCACGAGGAG AACCCCTACA GCCTGCTGGA AATTTCCACT GTGGAGCGAG
    421 GCGTGGTGAG TCTCTTTGGA GTGAGAAGTG CCCTCTTCGT TGCCATGAAC AGTAAAGGAA
    481 GATTGTACGC AACGCCCAGC TTCCAAGAAG AATGCAAGTT CAGAGAAACC CTCCTGCCCA
    541 ACAATTACAA TGCCTACGAG TCAGACTTGT ACCAAGGGAC CTACATTGCC CTGAGCAAAT
    601 ACGGACGGGT AAAGCGGGGC AGCAAGGTGT CCCCGATCAT GACTGTCACT CATTTCCTTC
    661 CCAGGATCTA A
    Human FGF9 gene coding sequence (1-208) (SEQ ID NO: 230) (GenBank
    accession no. NM_002010, which is hereby incorporated by reference in
    its entirety):
    838                                                               ATG
    841 GCTCCCTTAG GTGAAGTTGG GAACTATTTC GGTGTGCAGG ATGCGGTACC GTTTGGGAAT
    901 GTGCCCGTGT TGCCGGTGGA CAGCCCGGTT TTGTTAAGTG ACCACCTGGG TCAGTCCGAA
    961 GCAGGGGGGC TCCCCAGGGG ACCCGCAGTC ACGGACTTGG ATCATTTAAA GGGGATTCTC
    1021 AGGCGGAGGC AGCTATACTG CAGGACTGGA TTTCACTTAG AAATCTTCCC CAATGGTACT
    1081 ATCCAGGGAA CCAGGAAAGA CCACAGCCGA TTTGGCATTC TGGAATTTAT CAGTATAGCA
    1141 GTGGGCCTGG TCAGCATTCG AGGCGTGGAC AGTGGACTCT ACCTCGGGAT GAATGAGAAG
    1201 GGGGAGCTGT ATGGATCAGA AAAACTAACC CAAGAGTGTG TATTCAGAGA ACAGTTCGAA
    1261 GAAAACTGGT ATAATACGTA CTCATCAAAC CTATATAAGC ACGTGGACAC TGGAAGGCGA
    1321 TACTATGTTG CATTAAATAA AGATGGGACC CCGAGAGAAG GGACTAGGAC TAAACGGCAC
    1381 CAGAAATTCA CACATTTTTT ACCTAGACCA GTGGACCCCG ACAAAGTACC TGAACTGTAT
    1441 AAGGATATTC TAAGCCAAAG TTGA
    Human FGF16 gene coding sequence (1-207) (SEQ ID NO: 231) (GenBank
    accession no. NM_003868, which is hereby incorporated by reference in
    its entirety):
    1 ATGGCAGAGG TGGGGGGCGT CTTCGCCTCC TTGGACTGGG ATCTACACGG CTTCTCCTCG
    61 TCTCTGGGGA ACGTGCCCTT AGCTGACTCC CCAGGTTTCC TGAACGAGCG CCTGGGCCAA
    121 ATCGAGGGGA AGCTGCAGCG TGGCTCACCC ACAGACTTCG CCCACCTGAA GGGGATCCTG
    181 CGGCGCCGCC AGCTCTACTG CCGCACCGGC TTCCACCTGG AGATCTTCCC CAACGGCACG
    241 GTGCACGGGA CCCGCCACGA CCACAGCCGC TTCGGAATCC TGGAGTTTAT CAGCCTGGCT
    301 GTGGGGCTGA TCAGCATCCG GGGAGTGGAC TCTGGCCTGT ACCTAGGAAT GAATGAGCGA
    361 GGAGAACTCT ATGGGTCGAA GAAACTCACA CGTGAATGTG TTTTCCGGGA ACAGTTTGAA
    421 GAAAACTGGT ACAACACCTA TGCCTCAACC TTGTACAAAC ATTCGGACTC AGAGAGACAG
    481 TATTACGTGG CCCTGAACAA AGATGGCTCA CCCCGGGAGG GATACAGGAC TAAACGACAC
    541 CAGAAATTCA CTCACTTTTT ACCCAGGCCT GTAGATCCTT CTAAGTTGCC CTCCATGTCC
    601 AGAGACCTCT TTCACTATAG GTAA
    Human FGF20 gene coding sequence (1-211) (SEQ ID NO: 232) (GenBank
    accession no. NM_019851, which is hereby incorporated by reference in
    its entirety):
    134               ATGGCTC CCTTAGCCGA AGTCGGGGGC TTTCTGGGCG GCCTGGAGGG
    181 CTTGGGCCAG CAGGTGGGTT CGCATTTCCT GTTGCCTCCT GCCGGGGAGC GGCCGCCGCT
    241 GCTGGGCGAG CGCAGGAGCG CGGCGGAGCG GAGCGCGCGC GGCGGGCCGG GGGCTGCGCA
    301 GCTGGCGCAC CTGCACGGCA TCCTGCGCCG CCGGCAGCTC TATTGCCGCA CCGGCTTCCA
    361 CCTGCAGATC CTGCCCGACG GCAGCGTGCA GGGCACCCGG CAGGACCACA GCCTCTTCGG
    421 TATCTTGGAA TTCATCAGTG TGGCAGTGGG ACTGGTCAGT ATTAGAGGTG TGGACAGTGG
    481 TCTCTATCTT GGAATGAATG ACAAAGGAGA ACTCTATGGA TCAGAGAAAC TTACTTCCGA
    541 ATGCATCTTT AGGGAGCAGT TTGAAGAGAA CTGGTATAAC ACCTATTCAT CTAACATATA
    601 TAAACATGGA GACACTGGCC GCAGGTATTT TGTGGCACTT AACAAAGACG GAACTCCAAG
    661 AGATGGCGCC AGGTCCAAGA GGCATCAGAA ATTTACACAT TTCTTACCTA GACCAGTGGA
    721 TCCAGAAAGA GTTCCAGAAT TGTACAAGGA CCTACTGATG TACACTTGA
  • As noted above, the chimeric protein includes a portion of a paracrine FGF coupled to a C-terminal region derived from an FGF21 molecule. FGF21 is an endocrine FGF expressed primarily by the pancreas (Fon Tacer et al., “Research Resource: Comprehensive Expression Atlas of the Fibroblast Growth Factor System in Adult Mouse,” Mol Endocrinol 24(10):2050-2063 (2010), which is hereby incorporated by reference in its entirety) and has metabolic effects similar to that of FGF19, such as increased energy metabolism, weight loss, lowered blood glucose levels, and resistance to obesity and diabetes (Kharitonenkov et al., “FGF-21 as a Novel Metabolic Regulator,” J Clin Invest 115(6), 1627-1635 (2005); Coskun et al., “Fibroblast growth factor 21 corrects obesity in mice,” Endocrinology 149(12):6018-6027 (2008), which are hereby incorporated by reference in their entirety). Transgenic mice overexpressing FGF21 are also resistant to diet-induced obesity (Kharitonenkov et al., “FGF-21 as a Novel Metabolic Regulator,” J Clin Invest 115(6), 1627-1635 (2005), which is hereby incorporated by reference in its entirety). Moreover, in diabetic rodent models, FGF21 administration lowers blood glucose and triglyceride levels (Kharitonenkov et al., “FGF-21 as a Novel Metabolic Regulator,” J Clin Invest 115(6), 1627-1635 (2005), which is hereby incorporated by reference in its entirety).
  • In one embodiment, the C-terminal portion of FGF21 of the chimeric protein of the present invention is from human FGF21 having the amino acid sequence of SEQ ID NO: 233 (GenBank Accession No. NP061986, which is hereby incorporated by reference in its entirety), as follows:
  • 1 MDSDETGFEH SGLWVSVLAG LLLGACQAHP IPDSSPLLQF
    GGQVRQRYLY TDDAQQTEAH
    61 LEIREDGTVG GAADQSPESL LQLKALKPGV IQILGVKTSR
    FLCQRPDGAL YGSLHFDPEA
    121 CSFRELLLED GYNVYQSEAH GLPLHLPGNK SPHRDPAPRG
    PARFLPLPGL PPALPEPPGI
    181 LAPQPPDVGS SDPLSMVGPS QGRSPSYAS.
  • In one embodiment, the C-terminal portion of FGF21 of the chimeric protein of the present invention includes a β-Klotho co-receptor binding domain.
  • In one embodiment, the C-terminal portion of FGF21 of the chimeric protein of the present invention includes amino acid residues 168-209 of SEQ ID NO: 233.
  • In one embodiment, the C-terminal portion of FGF21 of the chimeric protein further includes one or more substitutions, deletions, or additions. In one embodiment, the C-terminal portion of FGF21 of the chimeric protein further includes one or more substitutions, deletions, or additions while retaining the ability to bind β-Klotho. In one embodiment, the C-terminal portion of FGF21 of the chimeric protein further includes one or more substitutions, deletions, or additions while retaining the ability to selectively bind β-Klotho. In one embodiment, the C-terminal portion of FGF21 of the chimeric protein further includes one or more substitutions, additions, or deletions to enhance binding affinity for β-Klotho.
  • In one embodiment of the present invention, the C-terminal portion of the chimeric protein according to the present invention is or is derived from a mammalian FGF21. In one embodiment of the present invention, the C-terminal portion of the chimeric protein according to the present invention is or is derived from a vertebrate FGF21. In one embodiment, the C-terminal portion of the chimeric protein according to the present invention is derived from a non-human vertebrate FGF21. It will be understood that this includes orthologs of human FGF21, or a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. In one embodiment of the present invention, the C-terminal portion of FGF21 of the chimeric protein according to the present invention is derived from human, pongo abelii, pan troglodytes, canis lupus familiaris, bos taurus, equus caballus, ailuropoda melanoleuca, oryctolagus cuniculus, gorilla gorilla, nomascus leucogenys, procavia capensis, cavia porcellus, tupaia belangeri, sorex araneus, ictidomys tridecemlineatus, loxodonta africana, sus scrofa, felis catus, otolemur garnettii, rattus norvegicus, mus musculus, vicugna pacos, anolis carolinensis, gadus morhua, latimeria chalumnae, tursiops truncatus, mustela putorius furo, takifugu rubripes, dipodomys ordii, echinops telfairi, macaca mulatta, microcebus murinus, ochotona princeps, xiphophorus maculatus, gasterosteus aculeatus, sarcophilus harrisii, macropus eugenii, xenopus tropicalis, danio rerio, bos grunniens mutus, saimiri boliviensis boliviensis, callithrix jacchus, tupaia chinensis, papio anubis, pteropus alecto, heterocephalus glaber, cricetulus griseus, ovies aries, pan paniscus, macaca fascicularis, mesocricetus auratus, or oreochromis niloticus.
  • In one embodiment of the present invention, the portion of FGF21 of the chimeric protein of the present invention is from an ortholog of human FGF21 having an amino acid sequence as shown in Table 7. The portions of an ortholog of human FGF21 of a chimeric protein according to the present invention include portions corresponding to the above-identified amino acid sequences of human FGF21. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.
  • TABLE 7
    Pongo abelii (Sumatran orangutan) FGF21 (GenBank Accession No.
    XP_002829565, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 234)
    1 MDSDETGFEH SGLWVPVLAG LLLGACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQQTEAH
    61 LEIREDGTVG GAADQSPESL LQLKALKPGV IQILGVKTSR FLCQRPDGAL YGSLHFDPEA
    121 CSFRELLLED GYNVYQSEAH GLPLHLPGNK SPHRDPAPRG PARFLPLPGL PPAPPEPPGI
    181 LAPQPPDVGS SDPLSMVGPS QGRSPSYAS
    Pan troglodytes (chimpanzee) FGF21 (GenBank Accession No. XP_524333,
    which is hereby incorporated by reference in its entirety)
    (SEQ ID NO: 235)
    1 MDSDETGFEH SGLWVSVLAG LLLGACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQQTEAH
    61 LEIREDGTVG GAADQSPESL LQLKALKPGV IQILGVKTSR FLCQRPDGAL YGSLHFDPEA
    121 CSFRELLLED GYNVYQSEAH GLPLHLPGNK SPHRDPAPRG PARFLPLPGL PPAPPEPPGI
    181 LAPQPPDVGS SDPLSMVGPS QGRSPSYTS
    Canis lupus familiaris (dog) FGF21 (GenBank Accession No. XP_541510,
    which is hereby incorporated by reference in its entirety)
    (SEQ ID NO: 236)
    1 MGWAEAGFEH LGLWVPVLAV LLLEACRAHP IPDSSPLLQF GGQVRQRYLY TDDAQETEAH
    61 LEIRADGTVV GAARQSPESL LELKALKPGV IQILGVKTSR FLCQGPDGTL YGSLHFDPVA
    121 CSFRELLLED GYNIYHSETL GLPLRLRPHN SAYRDLAPRG PARFLPLPGL LPAPPEPPGI
    181 LAPEPPDVGS SDPLSMVGPS QGRSPSYAS
    Bos taurus (bovine) FGF21 (GenBank Accession No. XP_001789639, which
    is hereby incorporated by reference in its entirety)
    (SEQ ID NO: 237)
    1 MGWDEAKFKH LGLWVPVLAV LLLGTCRAHP IPDSSPLLQF GGQVRQRYLY TDDAQETEAH
    61 LEIRADGTVV GAARQSPESL LELKALKPGV IQILGVKTSR FLCQGPDGKL YGSLHFDPKA
    121 CSFRELLLED GYNVYQSETL GLPLRLPPQR SSNRDPAPRG PARFLPLPGL PAAPPDPPGI
    181 LAPEPPDVGS SDPLSMVGPS YGRSPSYTS
    Equus caballus (horse) FGF21 (GenBank Accession No. XP_001489202,
    which is hereby incorporated by reference in its entirety)
    (SEQ ID NO: 238)
    1 MDWDKTGFKY QGLWVPVLAV LLLGACQSHP IPDSSPLLQF GGQVRQRHLY TDDAQETEAH
    61 LEIRADGTVA GAVHRSPESL LELKALKPGV IQILGVKTSR FLCQGPDGTL YGSLHFDPVA
    121 CSFRELLLED GYNVYQSETL GLPLRLPHHS SPYQDPAPRA PARFLPLPGF PPAPPEPPGI
    181 PAPEPPDVGS SDPLSMVGPS RSRSPSYTS
    Ailuropoda melanoleuca (giant panda) FGF21 (GenBank Accession No.
    XP_002917910, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 239)
    1 MGWDEARSEQ LGLWVPVLAV LLLEACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQETEAH
    61 LAIRADGTVV GAASRSPESL LELKALKPGV IQILGVKTSR FLCQGPDGTL YGSVRFDPVA
    121 CSFRELLLED GYNIYHSETL GLPLRLPAHN SPYRDSAPRG PARFLPLPGL LPVPPDPPGI
    181 LGPEPPDVGS SDPLSMVGPS QGRSPSYAS
    Oryctolagus cuniculus (rabbit) FGF21 (GenBank Accession No.
    XP_002723745, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 240)
    1 MDWGKAKCRP PGLWVPALAA LLLGACQAHP IPDSSPLLQF GDQVRQQHLY TDDAQETEAH
    61 LEIRADGTVV GAARRSPESL LQMKALQPGI IQILGVQTSR FLCQRPDGTL YGSLHFDREA
    121 CSFRELLRED GYNVYLSEAL GLPLRLSPGS SPRRAPAPRG PARFLPLPGL PPDLPEPPGL
    181 LAAAPPDVDS PDPLSMVQPA LDQSPSYTS
    Gorilla gorilla (gorilla) FGF21 (Ensembl Accession No.
    ENSGGOP00000001229, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 241)
    1 MDSDETGFEH SGLWVSVLAG LLLGACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQQTEAH
    61 LEIREDGTVG GAADQSPESL LQLKALKPGV IQILGVKTSR FLCQRPDGAL YGSLHFDPEA
    121 CSFRELLLED GYNVYQSEAH GLPLHLPGNK SPHRDPAPRG PARFLPLPGL PPAPPEPPGI
    181 LAPQPPDVGS SDPLSMVGPS QGRSPSYAS
    Nomascus leucogenys (Northern white-cheeked gibbon) FGF21 (Ensembl
    Accession No. ENSNLEP00000005639, which is hereby incorporated by
    reference in its entirety) (SEQ ID NO: 242)
    1 MDSDETGFEH SGLWVPVLAG LLLGACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQQTEAH
    61 LEIREDGTVG GAADQSPESL LQLKALKPGV IQILGVKTSR FLCQRPDGAL YGSLHFDPEA
    121 CSFRELLLED GYNVYQSEAH GLPLHLPGNK SPHRDPAPRG PARFLPLPGL PPAPPEPPGI
    181 LAPQPPDVGS SDPLSMVGPS QGRSPSYAS
    Procavia capensis (hyrax) FGF21 (Ensembl Accession No.
    ENSOGAG00000001210, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 243)
    1 MDWAKFGIEH PGLWVPVMAV LLLGACQGYP IPDSSPLLQF GGQVRQRYLY TDDAQETEAH
    61 LEIRADGTVV GAAHRSPESL LELKALKPGI IQILGVKTSR FLCQGPDGVL YGSLRFDPVA
    121 CSFRELLLED GYNVYQSEAH GLPLRLPSHN SPQRDLASRV PARFLPLPGR LTVLPEPSGV
    181 LGPEPPDVDS SDPLSMVGPS QGRSPSYAS
    Cavia porcellus (guinea pig) FGF21 (Ensembl Accession No.
    ENSCPOP00000000237, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 244)
    1 MDWARTECER PRLWVSMLAI LLVGACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQDTEVH
    61 LEIRADGSVR GIAHRSPESL LELKALKPGV IQILGIRTSR FLCQRPDGSL YGSLHFDPEA
    121 CSFRELLLAD GYNVYKSEAH GLPLHLLRGD SLSQEPAPPG PARFLPLPGL PATPPEPPRM
    181 LPPGPPDVGS SDPLSMVGPL WDRSPSYTS
    Tupaia belangeri (tree shrew) FGF21 (Ensembl Accession No.
    ENSTBEP00000013946, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 245)
    1 MGWDKARFEH LGAWAPVLAV LLLGACQAYP IPDSSPLLQF GGQVRQRYLY TDDTQDTEAH
    61 LEIRADGTVV GAAHQSPESL LELKALKPGV IQILGVKTSR FLCQRPDGAL YGSLHFDPEA
    121 CSFRELLLED GYNIYQSEAR GLPLRLPPHD SPHRDRTPRG PARFLPLPGL PLVPPELPGV
    181 LALEPPDVGS SDPLSMMGPS QGQSPSYAS
    Sorex araneus (shrew) FGF21 (Ensembl Accession No.
    ENSSARP00000002784, which is hereby incorporated by
    reference in its entirety) (SEQ ID NO: 246)
    1 MVWDKARGQQ LGLWAPMLLG LLLGACQAHP LPDSSPLLQF GGQVRLRFLY TDDAQRTGAH
    61 LEIRADGTVQ GAAHRTPECL LELKALKPGV IQILGVSTSR FLCQRPDGVL YGSLRFDPEA
    121 CSFRELLLQD GYNVYQSEAL GLPLYLHPPS APVSQEPASR GAVRFLPLPG LPPASLEPPR
    181 PPAPVPPDVG SSDPLSMVGP PERHSPSYTS
    Ictidomys tridecemlineatus (squirrel) FGF21 (SEQ ID NO: 247)
    1 MDWVKAKLEP LGLWVLVLAA LVLGACQAYP IPDSSPLLQF GGQVRQRYLY TDDAQETEAH
    61 LEIRADGTVV GAAHQSPESL LELKALKPGV IQILGVKTSR FLCQRPDGVL YGSLHFDPEA
    121 CSFREQLLED GYNVYQSESH GLPVRLPPNS PYRDPAPPGP ARFLPLPGLP PAALEPPGIL
    181 GPEPPDVGSS DPLSMVGPLQ GRSPSYAS
    Loxodonta africana (elephant) FGF21 (Ensembl Accession No.
    ENSLAFP00000016854, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 248)
    1 MDWAKFGLE HPGLWVPVMA VLLLGACQGH PIPDSSPLLQ FGGQVRQRYL YTDDQETEAH
    60 LEIRADGTVA GAAHRSSESL LELKALKPGI IQILGVKTSR FLCQGPDGVL YGSLHFDPAA
    120 CSFRELLLED GYNVYWSEAH GLPIRLPSHN SPYRDPASRV PARFLPLPGL LPMLQEPPGV
    180 LAPEPPDVDS SDPLSMVGPS QGRSPSYAS
    Sus scrofa (pig) FGF21 (GenBank Accession No. NP_001156882, which is
    hereby incorporated by reference in its entirety) (SEQ ID NO: 249)
    1 MGWAEAKFER LGLWVPVLAV LLGACQARPI PDSSPLLQFG GQVRQRYLYT DDAQETEAHL
    61 EIRADGTVAG VARQSPESLL ELKALKPGVI QILGVQTSRF LCQGPDGRLY GSLHFDPEAC
    121 SFRELLLEDG YNVYQSEALG LPLRLPPHRS SNRDLAPRGP ARFLPLPGLP PAPPEPPGIL
    181 APEPPDVGSS DPLSMVGPSH GRSPSYTS
    Felis catus (cat) FGF21 (Ensembl Accession No. ENSFCAP00000006832,
    which is hereby incorporated by reference in its entirety) (SEQ ID
    NO: 250)
    1 MDWDEAGSQ RLGLWVVLGV LLPEACQAHP IPDSSPLLQF GGQVRQRFLY TDDAQETEVH
    60 LEIKADGTVV GTARRSPESL LELKALKPGV IQILGVKTSR FLCQGPDGTL YGSLRFDPAA
    120 CSFRELLLED GYNIYHSETL GLPLRLPPHN SPYRDLAPRA PARFLPLPGL LPAPPEPPGI
    180 LAPEPPDVGS SDPLSMVGPS QGRSPSYAS
    Otolemur garnettii (bushbaby) FGF21 (Ensembl Accession No.
    ENSOGAG00000003581, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 251)
    1 DKARTGFKH PGPWFPLLAV LLLGACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQETEAH
    60 LEIREDGTVV GAAQQSPESL LELKALKPGV IQILGVKTSR FLCQRPDGGL YGSLYFDPKA
    120 CSFRELLLED GYNVYWSETY GLPLHLPPAN SPYWGPSLRS PARFLPLPGP PAASPELPGI
    180 LALEPPDVGS SDPLSMVGPS QGRSPSYAS
    Rattus norvegicus (Norway rat) FGF21 (GenBank Accession No.
    NP_570108, which is hereby incorporated by reference
    in its entirety) (SEQ ID NO: 252)
    1 MDWMKSRVGA PGLWVCLLLP VFLLGVCEAY PISDSSPLLQ FGGQVRQRYL YTDDDQDTEA
    61 HLEIREDGTV VGTAHRSPES LLELKALKPG VIQILGVKAS RFLCQQPDGT LYGSPHFDPE
    121 ACSFRELLLK DGYNVYQSEA HGLPLRLPQK DSQDPATRGP VRFLPMPGLP HEPQEQPGVL
    181 PPEPPDVGSS DPLSMVEPLQ GRSPSYAS
    Mus musculus (house mouse) FGF21 (GenBank Accession No. NP_064397,
    which is hereby incorporated by reference in its entirety) (SEQ ID
    NO: 253)
    1 MEWMRSRVGT LGLWVRLLLA VFLLGVYQAY PIPDSSPLLQ FGGQVRQRYL YTDDDQDTEA
    61 HLEIREDGTV VGAAHRSPES LLELKALKPG VIQILGVKAS RFLCQQPDGA LYGSPHFDPE
    121 ACSFRELLLE DGYNVYQSEA HGLPLRLPQK DSPNQDATSW GPVRFLPMPG LLHEPQDQAG
    181 FLPPEPPDVG SSDPLSMVEP LQGRSPSYAS
    Vicugna pacos (alpaca) FGF21 (Ensembl Accession No.
    ENSVPAP00000005562, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 254); partial sequence corresponding to human
    FGF21 residues
    1 to 78, 169 to 171, and 183 to 209
    1 MDWDEAKFEH RGLWVPVLTV LLLGACQARP IPDSSPLLQF GGQVRQRYLY TDDAQETEAH
    61 LEIRADGTVV GVARQPE--- ---------- ---------- ---------- ----------
    121 ---------- ---------- ---------- ---------- --------GI P---------
    181 --PEPPDVGS SDPLSMVGPS YSRSPSYTS
    Anolis carolinensis (anole lizard) FGF21 (Ensembl Accession No.
    ENSACAP00000016895, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 255)
    1 CKSKGGGKGG ERMWVDLVFW AALLRTAPAL PLRNSNPIYQ FDGQVRLRHL YTADEQTHLH
    61 LEILPDGTVG GSRFQNPFSL MEIKAVKPGV IRMQAKKTSR FLCMKPNGRL YGSLFYSEEA
    121 CNFHEKVLSD GYNLYYSENY NIPVSLSSAG NLGQSRQLPP FSQFLPLVNK IPLEPVLEDF
    181 DFYGHQLDVE SADPLSILGQ NPGFMSPSYV F
    Gadus morhua (cod) FGF21 (Ensembl Accession No. ENSGMOP00000013789,
    which is hereby incorporated by reference in its entirety) (SEQ ID
    NO: 256)
    1 LLLATLLHIG LSFYVPDSGP LLWLGDQVRE RHLYTAESHR RGLFLEMSPD GQVTGSAAQT
    61 PLSVLELRSV RAGDTVIRAR LSSLYLCVDR AGHLTGQRQY TESDCTFREV ILEDGYTHFL
    121 SVHHGLPISL APRHSPGRQG LRFSRFLPLR SSLSEDRVAE PPDSPLNLDS EDPLGMGLGS
    181 LLSPAFSM
    Latimeria chalumnae (coelacanth) FGF21 (Ensembl Accession No.
    ENSLACP00000003781, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 257)
    1 MLCQSFVILS QKFIFGLFLT GLGLTGLAWT RPFQDSNPIL QYSDSIRLRH LYTASESRHL
    61 HLQINSDGQV GGTTKQSPYS LLEMKAVKTG FVVIRGKKSA RYLCMERSGR LYGSLQYTEK
    121 DCTFKEVVLA DGYNLYVSEE HQATVTLSPM RARIAQGKKI PPFSHFLPMV NKVPVEDVAA
    181 EMEFVQVLRE MTADVDSPDP FGMTWEESVH SPSFFA
    Tursiops truncatus (dolphin) FGF21 (Ensembl Accession No.
    ENSTTRP00000013808, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 258)
    1 MGWDKTKLEH LGLWVPVLAV LLGPCQAHPI PDSSPLLQFG GQVRQRYLYT DDAQETEAHL
    61 EIRADGTVVG TARRSPEGVK TSRFLCQGPE GRLYGSLHFN PQACSFRELL LEDGYNVYQS
    121 EALGIPLRLP PHRSSNWDLA PRGPARFLPL PGFLPPPLEP PGILAPEPPN VGSSDPLSMV
    181 GPSHGRSPSY TS
    Mustela putorius furo (ferret) FGF21 (Ensembl Accession No.
    ENSMPUP00000003687, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 259)
    1 MGWEEARSEH LGLWVPVLAV LLLGACQAYP IPDSSPLLQF GGQVRQRYLY TDDAQETEAH
    61 LEIRADGTVV GAARRSPESL LELKALKPGV IQILGVKTSR FLCQGPNGTL YGSFHFDPVA
    121 CSFREVLLED GYNIYHSETL GLPLRLPPHN SPHRDLAPRG PARFLPLPGL LPATPESRGI
    181 PAPEPPNVGS SDPLSMVGPL QGQSPSYTS
    Takifugu rubripes (fugu) FGF21 (Ensembl Accession No.
    ENSTRUP00000033950, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 260)
    1 FIYLFIQTAL FSPSKWFNFY LPDSNPLLSF DSHGRGIHLY TDNQRRGMYL QMSTDGSVSG
    61 SDVQTANSVL ELKSVRNGHV VIRGKSSSLF LCMDSRGRLW GQRHPTEADC TFREVLLADG
    121 YTRFLSLHNG TPVSLAPKQS PDQHTVPFTR FLPLRNTLAE ESMSEPPSNQ QRYFNIDSDD
    181 LLGMDLNAMV SPQFSGDK
    Dipodomys ordii (kangaroo rat) FGF21 (Ensembl Accession No.
    ENSDORP00000001155, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 261)
    1 MDQAKTRVGA RGLGGLVLAV IILGACKARP IPDSSPLLQF GGQVRLRHLY TDDTQETEAH
    61 LEIRADGTVV GTAHRSPESL LELKALKPGV IQILGIKTSR FLCQRPDGTL YGSLHFDPEV
    121 CSFQELLLED GYNIYRSEAL GLPLRLSPDP APWGPARFLP LPGVPPAPPE PPGILAPEPP
    181 DVGSSDPLSM VGLLQGRSPS YAS
    Echinops telfairi (lesser hedgehog tenrec) FGF21 (Ensembl Accession
    No. ENSETEP00000008707, which is hereby incorporated by reference in
    its entirety) (SEQ ID NO: 262)
    1 MGCTKSGWKS PGLWVPVLAS LLLGGCGAHP IPDSSPLLQF GGQVRQRYLY TDDAQTTEAH
    61 LEIRADGTVG GVAHQSPEKF LSQWREKPLR SLHFDPAACS FREKLLEDGY NLYHSETHGL
    121 PLRLPPRGGD PSSQPGARFP PLPGQLPQLQ ETPGVLAPEP PDVGSSDPLS MVGPWRGQSP
    181 SYAS
    Macaca mulatta (rhesus monkey) FGF21 (Ensembl Accession No.
    ENSMMUP00000031540, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 263)
    1 MDSDETGFEH SGLWVPVLAG LLLGACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQQTEAH
    61 LEIREDGTVG GAAHQSPESE CGPEPGSEGG GAVGGAEGPG LLGLREAGLG PGSWLHFDPE
    121 ACSFRELLLE NGYNVYQSEA HGLPLHLPGN KSPHRDPASQ GPARFLPLPG LPPAPPEPPG
    181 ILAPQPPDVG SSDPLSMVGP SQARSPSYAS
    Microcebus murinus (mouse lemur) FGF21 (Ensembl Accession No.
    ENSMICP00000012089, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 264)
    1 MGWDEAGAGF EHPGLWFPML GVLLLGACQA YPIPDSSPLL QFGGQVRQRH LYTDDIQETE
    61 AHLEIRADGT VVGAARQSPE LELKALKPGV IQILGVKTSR FLCQRPDGAL YGSLHFDPEC
    121 SFRELLLEDG YNVYCPYLPL HLSPRIELAG SRSALPLPPA PERRILAPEP PDGSSDPLSM
    181 VGPSQGRSPS YAS
    Ochotona princeps (pika) FGF21 (Ensembl Accession No.
    ENSOPRP00000006754, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 265)
    1 KDMDGLQPPG LRVPVLAALL LGVGQARPIP DSSPLLQFGG QVRQRHLYTD DAQESEVHLE
    61 IRADGTVAGT ARRSPESLLE MKALKPGVIQ ILGVHTSRFL CQRPDGTLYG SLHFDHKACS
    121 FREQLLEDGY NVYHSETHGL PLRLSPDRAP RGPARFLPLP GPPPDLLVPP LPPDVLAPEP
    181 PDVDSPDPLS MVGPLQGQSP SYTS
    Xiphophorus maculatus (platyfish) FGF21 (Ensembl Accession No.
    ENSXMAP00000001576, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 266)
    1 CPFPFLFLIL SLPFFSSSFY IPESNPIFAF RNQLREVHLY TENHRRGLYV EIHLDGRVTG
    61 SDAQSPYSVL QIKSVKPGHV VIKGQTSSLF LCMDDSGNLR GQTTYDEADC SFRELLLADG
    121 YTRFLNSQHG VPLSLASRNS PDRHSVPFTR FLPLRNTLTV SEESTKTQRD FNLDSDDLLG
    181 MG
    Gasterosteus aculeatus (stickleback) FGF21 (Ensembl Accession No.
    ENSGACP00000010703, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 267)
    1 SLLLMVPLPF CSSFYLTDSS PLLPFNNQVK EVHLYTAENH RRAMYLQIAL DGSVSGSDAR
    61 STYSVLQLKS IQPGHVVIRG KASSMFLCVD SGGRLRGQGP YSEADCSFRE LLLGDGYTRF
    121 LSSQHGSPLS LASRPSPDPN SVPFTRFLPI RTAPEAESVI EEPPSNQRYV NVDSEDLLGM
    181 GLNTVVSPQF SA
    Sarcophilus harrisii (tasmanian devil) FGF21 (Ensembl Accession No.
    ENSSHAP00000005963, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 268); partial sequence corresponding to human
    FGF21 residues 3 to 172
    1 VSAMGLRERA PRYLAPLLSL LLACRASGHP LPDSSPMLLF GGQVRLRHLY TDVGQEAEAH
    61 VELASDGTVR AAARRSPNSL LELKAVKPGI VRILAVHSSR FLCMRPNGEL YGAIHYDPSA
    121 CNFRERLLGD GYNVYESEAH GRTLRLPPKA APGPAGPSRF LPLPG
    Macropus eugenii (wallaby) FGF21 (Ensembl Accession No.
    ENSMEUP00000013936, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 269)
    1 TEEPSTGSRH LGQWAPGLPG PLLSLLLAYR GWGSPIPDSS PMLLFGGQVR LRHLYTDDGQ
    61 DTEAHVELGP DGVVRAVAER SPNSLLELKA VKPGVIRILA VQSSRFLCMR PNGELYGAVH
    121 YDPSACNFRE HLLGDGYNVY ESETHRRTLR LSPSLGQAGP SRFLPLPGDW LPGPDPPWAQ
    181 GPEPPDVGSA DPLSMVGAVQ GLSPSYSS
    Xenopus tropicalis (Western clawed frog) FGF21 (Ensembl Accession
    No. ENSXETP00000009917, which is hereby incorporated by reference
    in its entirety) (SEQ ID NO: 270); partial sequence corresponding to
    human FGF21 residues 1 to 169
    1 RGGRTKKKTL LRKWLCLLAI MLSRSRFSLA NPIQNSNPIL SNDNQVRTQY LYTDNNNMHL
    61 YLQITHNGVV TGTEEKNDYG VLEIKAVKAG VVVIKGIRSN LYLCMDSRHQ LYASAYDKDD
    121 CHFHEKITPD NYNMYSSEKH SEYVSLAPLK GSQMARFLPI
    Danio rerio (zebrafish) FGF21 (Ensembl Accession No.
    ENSDARP00000094287, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 271)
    1 MLLACFFIFF ALFPHLRWCM YVPAQNVLLQ FGTQVRERLL YTDGLFLEMN PDGSVKGSPE
    61 KNLNCVLELR SVKAGETVIQ SAATSLYLCV DDQDKLKGQH HYSALDCTFQ ELLLDGYSFF
    121 LSPHTNLPVS LLSKRQKHGN PLSRFLPVSR AEDSRTQEVK QYIQDINLDS DDPLGMGHRS
    181 HLQTVFSPSL HTKK
    Bos grunniens mutus (yak) FGF21 (GenBank Accession No. ELR56628,
    which is hereby incorporated by reference in its entirety)
    (SEQ ID NO: 272)
    1 MGWDEAKFKH LGLWVPVLAV LLLGTCRAHP IPDSSPLLQF GGQVRQRYLY TDDAQETEAH
    61 LEIRADGTVV GAARQSPESL LELKALKPGV IQILGVKTSR FLCQGPDGKL YGSLHFDPKA
    121 CSFRELLLED GYNVYQSETL GLPLRLPPQR SSNRDPAPRG PARFLPLPGL PAEPPDPPGI
    181 LAPEPPDVGS SDPLSMVGPS YGRSPSYTS
    Saimiri boliviensis boliviensis (Bolivian squirrel monkey) FGF21
    (GenBank Accession No. XP_003940375, which is hereby incorporated by
    reference in its entirety) (SEQ ID NO: 273)
    1 MGSEEVALER PALWVSVLAG LLLGTCQAYP IPDSSPLLQF GGQVRQRYLY TDDAQQTEAH
    61 LEIREDGTVA GAAHQSPESL LQLKALKPGV IQILGVKTSR FLCQRPDGAL YGSLYFDPEA
    121 CSFRELLLED GYNVYQSVAH SLPLHLPGGR SPPWDPAPRG PARFLPLPGL PPEPPEAPGI
    181 LAPEPPDVGS SDPLSMVGPS QGQSPSYTS
    Callithrix jacchus (white-tufted-ear marmoset) FGF21 (GenBank
    Accession No. XP_003735669, which is hereby incorporated by
    reference in its entirety) (SEQ ID NO: 274)
    1 MGSEEVGLEH PALWVSVLAG LLLGTCQAHP IPDSSPLLQF GGQVRQRYLY TDDAQQKEAH
    61 LEIXEDGTVA GAATKVPKVS LLQLKALKPG VIQILGVKTS RFLCQRPDGA LYGSLHFDPE
    121 ACSFRELLLE DGYNVYQSVA HGLPLHLPES RSPPRDPAPR GPARFLPLPG LPPEPPEPPG
    181 ILAPEPPDVG SSDPLSMVGP SQGQSPSYAS
    Tupaia chinensis (Chinese tree shrew) FGF21 (GenBank Accession No.
    ELW47159, which is hereby incorporated by reference in its entirety)
    (SEQ ID NO: 275)
    1 MGWDKARFEH LGAWAPVLAV LLLGACQAYP IPDSSPLLQF GGQVRQRYLY TDDTQDTEAH
    61 LEIRADGTVV GAAHQSPESL LELKALKPGV IQILGVKTSR FLCQRPDGAL YGSLHFDPEA
    121 CSFRELLLED GYNIYQSEAR GLPLRLPPHD SPHRDRTPQG PARFLPLPGL PLVPPELPGV
    181 LALEPPDVGS SDPLSMMGPS QGQSPSYAS
    Papio anubis (olive baboon) FGF21 (GenBank Accession No.
    XP_003915900, which is hereby incorporated by
    reference in its entirety) (SEQ ID NO: 276)
    1 MDSDETGFEH SGLWVPVLAG LLLGACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQQTEAH
    61 LEIREDGTVG GAAHQSPESK CGPEPGSEGG GALHFDPEAC SFRELLLENG YNVYQSEAHG
    121 LPLHLPGNKS PHRDPASRGP ARFLPLPGLP PAPPEPPGIL APQPPDVGSS DPLSMVGPSQ
    181 ARSPSYAS
    Pteropus alecto (black flying fox) FGF21 (GenBank Accession No.
    ELK18566, which is hereby incorporated by reference in its entirety)
    (SEQ ID NO: 277)
    1 MGWGKARLQH PGLWGPVLAV LLGACQAHPI LDSSPLFQFG SQVRRRYLYT DDAQDTEAHL
    61 EIRADGTVAG AARRSPESLL ELKALKPGVI QVLGVKTSRF LCQRPDGTLY GSLHFDPAAC
    121 SFRELLLKDG YNVYQSEALA RPLRLPPYSS PSSDPARRGP ARFLPLPGPP PEPPQPPGRL
    181 APEPPDVGSS DPLSMVWPSR GRSPSYTS
    Heterocephalus glaber (naked mole-rat) FGF21 (GenBank Accession No.
    EHB06286, which is hereby incorporated by reference in its entirety)
    (SEQ ID NO: 278)
    1 MDWARAESER PGLWVPAVLA VLLLGACQAH PIPDSSPLLQ FGGQVRQRHL YTDDAQDTEV
    61 HLEIRADGSV GGAAHRSPES LLELKALKPG VIQILGVRTS RFLCQRPDGT LYGSLHFDPE
    121 ACSFRELLLA DGYNIYQSEA YGLPLRMLPS DSASRDPVPP GPARFLPLPG LHPPPLEPPG
    181 MLPPEPPDVG SSDPLSMVGP LQGRSPSYAF
    Cricetulus griseus (Chinese hamster) FGF21 (GenBank Accession No.
    XP_003508726, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 279)
    1 MDWMKSGVGV PGLWVPLLPI FLLGVSQAHP IPDSSPLLQF GGQVRHRHLY TDDNQETEVH
    61 LEIRQDGTVI GTTHRSPESL LELKALKPEV IPVLGVKASR FLCQQPDGTL YGSPHFDPEA
    121 CSFRELLLED GYNVYQSEVH GLPLRLPQRD SPNQAPASWG PVPPLPVPGL LHQPQELPGF
    181 LAPEPPDVGS SDPLSMVGPL QGRSPSYAS
    Ovis aries (sheep) FGF21 (GenBank Accession No. XP_004015845, which
    is hereby incorporated by reference in its entirety)
    (SEQ ID NO: 280)
    1 MGWDEAKFKH LGLWVPVLAV LLLGTCRAHP IPDSSPLLQF GGQVRQRYLY TDDAQETEAH
    61 LEIRADGTVV GAARQSPESL LELKALKPGV IQIFGVKTSR FLCQGPDGKL YGSLHFDPKA
    121 CSFRELLLED GYNVYQSETL GLPLRLPPQR SSNRDPAPRG PPKPQLHFLK TSAVQYWPRY
    181 EKVPAFLHPF PG
    Pan paniscus (pygmy chimpanzee) FGF21 (GenBank Accession No.
    XP_003814163, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 281); partial sequence corresponding to human
    FGF21 residues
    1 to 116 and 195 to 201
    1 MDSDETGFEH SGLWVSVLAG LLLGACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQQTEAH
    61 LEIREDGTVG GAADQSPESL LQLKALKPGV IQILGVKTSR FLCQRPDGAL YGSVSF----
    121 ---------- ---------- ---------- ----Q----- ---------- -----DPP--
    181 --HHPP---C S---SYMSPS Q---PG---
    Macaca fascicularis (crab-eating macaque) FGF21 (GenBank Accession
    No. EHH59757, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 282); partial sequence corresponding to human
    FGF21 residues
    1 to 116
    1 MDSDETGFEH SGLWVPVLAG LLLGACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQQTEAH
    61 LEIREDGTVG GAAHQSPESL LQLKALKPGV IQILGVKTSR FLCQKPDGAL YGSVSF
    Mesocricetus auratus (golden hamster) FGF21 (GenBank Accession No.
    ACB30542, which is hereby incorporated by reference in its entirety)
    (SEQ ID NO: 283); partial sequence corresponding to human FGF21
    residues 90 to 193
    1 VIQILGVKAA RFPCQQPDGS LYGSPHFDPE ACSFRELLLE DGYNVYQSEA HGLPLRLPQR
    61 DAPSQPPASW GPVRFLPVPG LFQPPHDLPG RPAPEPPDVG SSDP
    Oreochromis niloticus (Nile tilapia) FGF21 (GenBank Accession No.
    XP_003438516, which is hereby incorporated by reference in its
    entirety) (SEQ ID NO: 284); partial sequence corresponding to human
    FGF21 residues 59 to 209
    1 MYLQMNMDGR VTGSDAQTPY SLMQLKSVKP GHVIIKGPSS SLFLCVDSEG NLRGQSHYSE
    61 TSCTFREMLL ADGYTRFISS QYGFPMSLAS RHSPDRHALP FTRFLPLRNN LKTDSVSEQL
    121 PNNQRLFNVD SDDLLGMGLN SMGSPQFSMD K
  • In certain embodiments according to the present invention, the C-terminal portion of FGF21 of the chimeric protein of the present invention includes a polypeptide sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to amino acid residues 168-209 of SEQ ID NO: 233. In certain embodiments according to the present invention, the C-terminal portion of FGF21 of the chimeric protein of the present invention includes a polypeptide sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence homology to amino acid residues 168-209 of SEQ ID NO: 233.
  • It will be understood that the portion of FGF21 of the chimeric protein of the present invention may be derived from a nucleotide sequence that encodes a vertebrate or a non-vertebrate FGF21 protein. In one embodiment, the portion of FGF21 of the chimeric protein of the present invention may be derived from a nucleotide sequence that encodes a mammalian FGF21 protein. Nucleotide sequences encoding a vertebrate FGF21 protein according to the present invention may include, but are not limited to, those shown in Table 8. The portion of FGF21 of the chimeric protein of the present invention derived from an ortholog of human FGF21 include portions corresponding to the above-identified amino acid sequences of FGF21. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.
  • TABLE 8
    Human FGF21 gene coding sequence (SEQ ID NO: 285) (GenBank Accession
    No. NM_019113, which is hereby incorporated by reference in its
    entirety)
    151 ATGGACTCGG ACGAGACCGG GTTCGAGCAC TCAGGACTGT GGGTTTCTGT GCTGGCTGGT
    211 CTTCTGCTGG GAGCCTGCCA GGCACACCCC ATCCCTGACT CCAGTCCTCT CCTGCAATTC
    271 GGGGGCCAAG TCCGGCAGCG GTACCTCTAC ACAGATGATG CCCAGCAGAC AGAAGCCCAC
    331 CTGGAGATCA GGGAGGATGG GACGGTGGGG GGCGCTGCTG ACCAGAGCCC CGAAAGTCTC
    391 CTGCAGCTGA AAGCCTTGAA GCCGGGAGTT ATTCAAATCT TGGGAGTCAA GACATCCAGG
    451 TTCCTGTGCC AGCGGCCAGA TGGGGCCCTG TATGGATCGC TCCACTTTGA CCCTGAGGCC
    511 TGCAGCTTCC GGGAGCTGCT TCTTGAGGAC GGATACAATG TTTACCAGTC CGAAGCCCAC
    571 GGCCTCCCGC TGCACCTGCC AGGGAACAAG TCCCCACACC GGGACCCTGC ACCCCGAGGA
    631 CCAGCTCGCT TCCTGCCACT ACCAGGCCTG CCCCCCGCAC TCCCGGAGCC ACCCGGAATC
    691 CTGGCCCCCC AGCCCCCCGA TGTGGGCTCC TCGGACCCTC TGAGCATGGT GGGACCTTCC
    751 CAGGGCCGAA GCCCCAGCTA CGCTTCCTGA
    Pongo abelii (Sumatran orangutan) FGF21 gene coding sequence (SEQ ID
    NO: 286) (GenBank Accession No. XM_002829519, which is hereby
    incorporated by reference in its entirety)
    165     ATGGAC TCGGACGAGA CCGGGTTCGA GCACTCAGGA CTGTGGGTTC CTGTGCTGGC
    221 TGGTCTTCTG CTGGGAGCCT GCCAGGCACA CCCCATCCCT GACTCCAGTC CTCTCCTGCA
    281 ATTCGGGGGC CAAGTCCGGC AGCGGTACCT CTACACAGAT GATGCCCAGC AGACAGAAGC
    341 CCACCTGGAG ATCAGGGAGG ATGGGACGGT GGGGGGCGCT GCTGACCAGA GCCCCGAAAG
    401 TCTCCTGCAG CTGAAAGCCT TGAAGCCGGG AGTTATTCAA ATCTTGGGAG TCAAGACATC
    461 CAGGTTCCTG TGCCAGAGGC CAGATGGGGC CCTGTATGGA TCGCTCCACT TTGACCCTGA
    521 GGCCTGCAGC TTCCGGGAGC TGCTTCTTGA GGACGGATAC AATGTTTATC AGTCCGAGGC
    581 CCATGGCCTC CCGCTGCACC TGCCGGGAAA CAAGTCCCCA CACCGGGACC CTGCACCCCG
    641 AGGACCAGCT CGCTTCCTGC CACTACCAGG CCTGCCCCCC GCACCCCCAG AGCCGCCCGG
    701 AATCCTGGCC CCCCAGCCCC CCGATGTGGG CTCCTCGGAC CCTCTGAGCA TGGTGGGACC
    761 TTCCCAGGGC CGAAGCCCCA GCTATGCTTC CTGA
    Pan troglodytes (chimpanzee) FGF21 gene coding sequence (SEQ ID
    NO: 287) (GenBank Accession No. XM_524333, which is hereby
    incorporated by reference in its entirety)
    573   ATGGACTC GGACGAGACC GGGTTCGAGC ACTCAGGACT GTGGGTTTCT GTGCTGGCTG
    631 GTCTTCTGCT AGGAGCCTGC CAGGCACACC CCATCCCTGA CTCCAGTCCT CTCCTGCAAT
    691 TCGGGGGCCA AGTCCGGCAG CGGTACCTCT ACACAGATGA TGCCCAGCAG ACAGAAGCCC
    751 ACCTGGAGAT CAGGGAGGAT GGGACGGTGG GGGGCGCTGC TGACCAGAGC CCCGAAAGTC
    811 TCCTGCAGCT GAAAGCCTTG AAGCCGGGAG TTATTCAAAT CTTGGGAGTC AAGACATCCA
    871 GGTTCCTGTG CCAGAGGCCA GATGGGGCCC TGTATGGATC GCTCCACTTT GACCCTGAGG
    931 CCTGCAGCTT CCGGGAGCTG CTTCTTGAGG ACGGATACAA TGTTTACCAG TCCGAGGCCC
    991 ACGGCCTCCC GCTGCACCTG CCGGGGAACA AGTCCCCACA CCGGGACCCT GCACCCCGAG
    1051 GACCAGCTCG CTTCCTGCCA CTACCAGGCC TGCCCCCCGC ACCCCCGGAG CCACCCGGAA
    1111 TCCTGGCCCC CCAGCCCCCC GATGTGGGCT CCTCAGACCC TCTGAGCATG GTGGGACCTT
    1171 CCCAGGGCCG AAGCCCCAGC TACACTTCCT GA
    Canis lupus familiaris (dog) FGF21 gene coding sequence (SEQ ID
    NO: 288) (GenBank Accession No. XM_541510, which is hereby
    incorporated by reference in its entirety)
    1 ATGGGCTGGG CCGAGGCCGG GTTCGAGCAC CTGGGACTGT GGGTCCCTGT GCTGGCTGTG
    61 CTTTTGCTGG AAGCCTGCCG GGCACATCCG ATCCCTGACT CCAGCCCCCT CCTACAATTT
    121 GGAGGTCAAG TTCGACAGCG GTACCTCTAC ACCGACGATG CCCAGGAGAC AGAGGCCCAC
    181 CTAGAGATCA GGGCCGATGG CACAGTGGTG GGGGCTGCCC GCCAGAGCCC TGAAAGTCTC
    241 CTGGAGCTGA AAGCCCTAAA GCCAGGGGTC ATTCAAATCT TGGGAGTCAA AACATCCAGG
    301 TTCCTGTGCC AGGGCCCAGA TGGGACACTA TATGGCTCGC TCCATTTCGA CCCTGTGGCC
    361 TGCAGTTTCC GAGAACTGCT TCTTGAGGAT GGGTACAACA TCTACCACTC CGAGACCCTT
    421 GGTCTCCCGC TTCGCCTGCG CCCCCACAAC TCCGCATACC GGGACTTGGC ACCCCGCGGG
    481 CCTGCCCGCT TCCTGCCACT GCCAGGCCTG CTTCCAGCAC CCCCAGAGCC TCCAGGGATC
    541 CTGGCCCCGG AGCCTCCTGA CGTGGGCTCC TCGGACCCTC TGAGCATGGT GGGGCCTTCA
    601 CAGGGCCGGA GTCCCAGCTA TGCTTCCTAA
    Bos taurus (bovine) FGF21 gene coding sequence (SEQ ID NO: 289)
    (GenBank Accession No. XP_001789587, which is hereby
    incorporated by reference in its entirety)
    1 ATGGGCTGGG ACGAGGCCAA GTTCAAGCAC TTGGGACTGT GGGTCCCTGT GCTGGCTGTC
    61 CTCCTGCTAG GAACCTGCCG GGCGCATCCC ATTCCAGACT CCAGCCCCCT CCTCCAGTTT
    121 GGGGGCCAAG TCCGCCAGCG GTACCTCTAC ACGGATGATG CCCAGGAGAC AGAGGCCCAC
    181 CTGGAGATCA GGGCCGATGG CACAGTGGTG GGGGCAGCCC GCCAGAGCCC CGAAAGTCTC
    241 TTGGAGCTGA AAGCCCTGAA GCCAGGCGTC ATTCAGATCT TGGGAGTTAA AACATCCAGG
    301 TTTCTCTGCC AGGGGCCAGA TGGGAAGCTG TACGGATCGC TGCACTTTGA CCCCAAAGCC
    361 TGCAGCTTTC GGGAGCTGCT TCTTGAAGAT GGATACAACG TCTACCAGTC GGAGACCCTG
    421 GGCCTTCCAC TCCGCCTGCC CCCCCAGCGC TCGTCCAACC GGGACCCGGC CCCGCGGGGA
    481 CCTGCTCGCT TCCTTCCACT GCCGGGCCTG CCCGCGGCGC CCCCGGATCC TCCAGGGATC
    541 TTGGCCCCCG AGCCTCCCGA CGTGGGCTCC TCGGATCCCC TGAGTATGGT GGGACCCTCG
    601 TATGGCCGAA GCCCCAGCTA CACTTCTTGA
    Equus caballus (horse) FGF21 gene coding sequence (SEQ ID NO: 290)
    (GenBank Accession No. XM_001489152, which is hereby incorporated by
    reference in its entirety)
    1 ATGGACTGGG ACAAGACGGG GTTCAAGTAC CAGGGACTGT GGGTCCCTGT GCTGGCTGTC
    61 CTTCTGCTGG GAGCCTGCCA GTCACACCCC ATCCCTGACT CCAGTCCCCT CCTCCAATTC
    121 GGGGGCCAAG TCAGGCAGCG CCACCTCTAC ACAGATGATG CCCAGGAGAC AGAGGCGCAC
    181 CTGGAGATCA GGGCTGACGG CACTGTGGCA GGGGCTGTCC ACCGGAGCCC AGAAAGTCTC
    241 TTGGAGCTGA AAGCCCTGAA GCCAGGGGTA ATTCAAATCT TGGGAGTCAA GACATCCAGG
    301 TTTCTGTGCC AGGGGCCAGA CGGGACGCTG TACGGATCGC TCCACTTCGA CCCCGTGGCC
    361 TGCAGCTTCC GGGAGCTGCT TCTCGAAGAC GGCTACAACG TTTACCAGTC TGAGACCCTT
    421 GGCCTCCCAC TCCGCCTGCC CCACCACAGC TCCCCATACC AGGATCCGGC CCCTCGGGCA
    481 CCCGCCCGCT TCCTGCCGCT GCCAGGCTTT CCCCCAGCAC CCCCGGAGCC TCCAGGGATC
    541 CCGGCCCCCG AGCCCCCGGA CGTGGGCTCC TCGGACCCCC TGAGCATGGT GGGGCCTTCA
    601 CGCAGCCGGA GCCCCAGCTA CACTTCCTGA
    Ailuropoda melanoleuca (giant panda) FGF21 gene coding sequence (SEQ
    ID NO: 291) (GenBank Accession No. XM_002917864, which is hereby
    incorporated by reference in its entirety)
    1 ATGGGCTGGG ACGAGGCCAG GTCCGAGCAG CTGGGGCTGT GGGTCCCTGT GCTGGCTGTC
    61 CTTTTGCTGG AAGCTTGCCA GGCACACCCT ATCCCTGACT CCAGCCCCCT CCTCCAATTC
    121 GGAGGCCAAG TTCGACAGCG GTACCTCTAC ACGGACGATG CCCAGGAGAC AGAGGCCCAC
    181 CTAGCGATCA GGGCTGATGG CACAGTGGTG GGGGCTGCCA GCCGGAGCCC AGAAAGTCTC
    241 TTGGAGCTGA AAGCCCTGAA ACCGGGGGTC ATTCAAATCC TGGGAGTGAA AACATCTAGG
    301 TTCCTGTGCC AGGGCCCAGA TGGGACACTG TACGGATCGG TCCGCTTCGA CCCCGTAGCC
    361 TGCAGCTTCC GGGAACTGCT CCTGGAGGAT GGGTACAACA TCTACCACTC TGAGACCCTC
    421 GGCCTCCCAC TTCGCCTGCC CGCCCACAAC TCTCCATACC GGGACTCGGC GCCCCGGGGG
    481 CCTGCCCGCT TCCTGCCCCT GCCAGGCCTG CTTCCGGTCC CCCCGGACCC CCCAGGGATC
    541 CTGGGCCCCG AGCCTCCCGA CGTGGGCTCC TCGGACCCCC TGAGCATGGT GGGGCCTTCA
    601 CAGGGCCGAA GTCCCAGCTA CGCTTCCTGA
    Oryctolagus cuniculus (rabbit) FGF21 gene coding sequence (SEQ
    ID NO: 292) (GenBank Accession No. XM_002723699, which is
    hereby incorporated by reference in its entirety)
    1 ATGGACTGGG GCAAGGCCAA GTGCCGGCCC CCGGGGCTGT GGGTCCCCGC GCTCGCTGCC
    61 CTGCTGCTGG GGGCCTGCCA GGCACACCCC ATCCCCGACT CCAGCCCCCT CCTCCAGTTT
    121 GGGGACCAAG TGCGGCAGCA GCACCTGTAC ACGGACGATG CGCAGGAAAC AGAAGCCCAC
    181 CTGGAGATCA GGGCGGATGG CACGGTGGTG GGGGCTGCCC GGAGGAGCCC AGAAAGTCTC
    241 TTGCAGATGA AAGCCTTACA ACCGGGGATC ATTCAGATCT TGGGGGTCCA GACGTCCAGG
    301 TTCCTCTGCC AGAGGCCGGA TGGCACGCTC TACGGCTCGC TCCACTTCGA CCGCGAGGCC
    361 TGCAGCTTCC GGGAGCTGCT GCGTGAGGAT GGGTACAACG TTTACCTCTC GGAGGCCCTG
    421 GGCCTGCCCC TGCGCCTGTC CCCCGGCAGC TCCCCACGCA GGGCGCCGGC CCCCCGGGGA
    481 CCAGCCCGCT TCCTGCCGCT GCCCGGCCTG CCGCCAGACC TTCCGGAACC GCCAGGCCTC
    541 CTGGCCGCCG CGCCCCCCGA TGTCGACTCC CCGGACCCCC TGAGCATGGT GCAGCCTGCG
    601 CTGGACCAGA GCCCCAGCTA CACCTCCTGA
    Gorilla gorilla (gorilla) FGF21 gene coding sequence (SEQ ID
    NO: 293) (Ensembl Accession No. ENSGGOT00000001253, which is hereby
    incorporated by reference in its entirety)
    151 ATGGACTCGG ACGAGACCGG GTTCGAGCAC TCAGGACTGT GGGTTTCTGT GCTGGCTGGT
    211 CTTCTGCTGG GAGCCTGCCA GGCACACCCC ATCCCTGACT CCAGTCCTCT CCTGCAATTC
    271 GGGGGCCAAG TCCGGCAGCG GTACCTCTAC ACAGATGATG CCCAGCAGAC AGAAGCCCAC
    331 CTGGAGATCA GGGAGGATGG GACGGTGGGG GGTGCTGCTG ACCAGAGCCC TGAAAGTCTC
    391 CTGCAGCTGA AAGCCTTGAA GCCGGGAGTT ATTCAAATCT TGGGAGTCAA GACATCCAGG
    451 TTCCTGTGCC AGAGGCCAGA TGGGGCCCTG TATGGATCGC TCCACTTTGA CCCTGAGGCC
    511 TGCAGCTTCC GGGAGCTGCT TCTTGAGGAC GGATACAATG TTTACCAGTC CGAGGCCCAC
    571 GGCCTCCCGC TGCACCTGCC GGGGAACAAG TCCCCACACC GGGACCCTGC ACCCCGAGGA
    631 CCAGCTCGCT TCCTGCCACT ACCAGGCCTG CCCCCCGCAC CCCCGGAGCC ACCCGGAATC
    691 CTGGCCCCCC AGCCCCCCGA TGTGGGCTCC TCGGACCCTC TGAGCATGGT GGGACCTTCC
    751 CAGGGCCGAA GCCCCAGCTA CGCTTCCTGA
    Nomascus leucogenys (Northern white-cheeked gibbon) FGF21 gene
    coding sequence (SEQ ID NO: 294) (Ensembl Accession No.
    ENSNLET00000005931, which is hereby incorporated by reference
    in its entirety)
    587       ATGG ACTCGGACGA GACCGGGTTC GAGCACTCAG GACTGTGGGT TCCTGTGCTG
    647 GCTGGTCTTC TGCTGGGAGC CTGCCAGGCA CACCCCATCC CTGACTCCAG TCCTCTCCTG
    707 CAATTCGGGG GCCAAGTCCG GCAGCGGTAC CTCTACACAG ATGATGCCCA GCAGACAGAA
    767 GCCCACCTGG AGATCAGGGA GGATGGGACG GTGGGGGGCG CTGCTGACCA GAGCCCTGAA
    831 AGTCTCCTGC AGCTGAAAGC CTTGAAGCCG GGAGTTATTC AAATCTTGGG AGTCAAGACA
    891 TCCAGGTTCC TATGCCAGAG GCCAGATGGG GCCCTGTATG GATCGCTCCA CTTTGACCCT
    951 GAGGCCTGCA GCTTCCGGGA GCTGCTTCTT GAGGACGGAT ACAATGTTTA CCAGTCCGAG
    1011 GCCCATGGCC TCCCGCTGCA CCTGCCGGGG AACAAGTCCC CACACCGGGA CCCTGCACCC
    1071 CGAGGACCAG CTCGCTTCCT GCCACTACCA GGCCTGCCCC CTGCACCCCC AGAGCCGCCC
    1131 GGAATCCTGG CCCCCCAGCC CCCCGATGTG GGCTCCTCGG ACCCTCTGAG CATGGTGGGA
    1191 CCTTCCCAGG GCCGAAGCCC CAGCTACGCT TCCTGA
    Procavia capensis (hyrax) FGF21 gene coding sequence (SEQ ID
    NO: 295) (Ensembl Accession No. ENSPCAT00000001288, which is hereby
    incorporated by reference in its entirety)
    1 ATGGACTGGG CCAAGTTTGG GATCGAGCAC CCGGGACTGT GGGTCCCGGT GATGGCAGTA
    61 CTTCTGCTGG GAGCCTGCCA AGGATACCCT ATTCCTGACT CCAGCCCCCT TCTCCAATTC
    121 GGAGGCCAGG TCCGGCAACG TTACCTCTAC ACAGATGACG CGCAGGAGAC CGAGGCCCAC
    181 CTGGAGATCC GAGCAGACGG CACGGTGGTG GGGGCTGCCC ACCGGAGCCC CGAGAGTCTC
    241 TTGGAGCTGA AAGCTTTGAA GCCCGGCATA ATTCAGATCT TGGGAGTCAA GACATCCAGA
    301 TTCCTCTGCC AGGGTCCTGA TGGGGTGCTG TATGGATCGC TCCGTTTTGA CCCAGTGGCC
    361 TGCAGCTTCC GGGAGCTGCT TCTTGAAGAT GGATACAATG TTTACCAGTC TGAGGCCCAC
    421 GGCCTCCCGC TTCGCCTACC ATCCCACAAT TCCCCACAGA GGGACCTGGC GTCCCGGGTG
    481 CCAGCCCGCT TCCTGCCACT GCCAGGCCGG CTCACGGTGC TCCCAGAACC TTCGGGGGTC
    541 CTGGGCCCTG AGCCCCCCGA TGTGGACTCC TCAGACCCCC TGAGCATGGT GGGGCCTTCG
    601 CAGGGCCGAA GCCCCAGTTA CGCCTCCTGA
    Cavia porcellus (guinea pig) FGF21 gene coding sequence (SEQ ID
    NO: 296) (Ensembl Accession No. ENSCPOT00000000273, which is hereby
    incorporated by reference in its entirety)
    1 ATGGACTGGG CCCGGACTGA GTGTGAGCGC CCAAGGCTGT GGGTCTCCAT GCTGGCCATC
    61 CTTCTGGTGG GAGCCTGCCA GGCACACCCT ATCCCTGACT CCAGCCCCCT CCTCCAGTTT
    121 GGGGGCCAGG TCCGGCAGCG GTACCTCTAC ACAGATGATG CTCAGGACAC TGAAGTGCAC
    181 CTGGAGATCA GGGCCGATGG CTCAGTACGG GGCATTGCCC ACAGGAGCCC TGAAAGTCTC
    241 CTGGAGCTGA AAGCCTTGAA GCCAGGAGTC ATTCAGATCT TGGGAATCAG GACTTCCAGG
    301 TTCCTGTGCC AGAGGCCCGA TGGGAGTCTG TATGGATCAC TCCACTTTGA TCCTGAGGCC
    361 TGCAGCTTCC GGGAGCTGCT GCTTGCTGAT GGCTACAATG TCTACAAGTC TGAAGCCCAC
    421 GGCCTCCCTC TGCACCTGCT GCGCGGTGAC TCTCTATCGC AGGAACCAGC ACCCCCAGGA
    481 CCAGCCCGAT TTCTGCCACT ACCAGGCCTG CCCGCAACAC CCCCGGAGCC ACCCAGGATG
    541 CTGCCCCCAG GGCCCCCAGA TGTGGGCTCC TCGGACCCTT TGAGCATGGT GGGGCCTTTA
    601 TGGGACCGAA GCCCCAGCTA TACTTCCTGA
    Tupaia belangeri (tree shrew) FGF21 gene coding sequence (SEQ ID
    NO: 297) (Ensembl Accession No. ENSTBET00000016056, which is hereby
    incorporated by reference in its entirety)
    1 ATGGGCTGGG ACAAGGCCCG GTTCGAGCAC CTGGGAGCGT GGGCTCCTGT GCTGGCTGTC
    61 CTCCTCCTGG GAGCCTGCCA GGCATACCCC ATCCCTGACT CCAGCCCCCT CCTACAATTC
    121 GGGGGCCAGG TCCGGCAGCG GTACCTCTAC ACGGACGACA CGCAGGACAC AGAAGCCCAC
    181 CTTGAGATCA GGGCCGACGG CACCGTGGTG GGGGCCGCCC ACCAAAGCCC GGAAAGTCTC
    241 CTGGAGCTGA AAGCCTTGAA GCCGGGGGTC ATTCAAATCC TGGGAGTCAA GACCTCCAGG
    301 TTCCTGTGCC AGAGGCCAGA CGGGGCCCTG TACGGGTCGC TTCACTTCGA CCCCGAGGCC
    361 TGCAGCTTCC GGGAGCTGCT TCTCGAGGAT GGATACAACA TTTACCAGTC TGAGGCTCGT
    421 GGCCTCCCCC TGCGCCTGCC GCCCCACGAC TCCCCACATC GGGACCGGAC CCCTCGGGGA
    481 CCAGCTCGTT TCCTGCCGCT GCCTGGCCTG CCCCTGGTTC CTCCAGAGCT GCCAGGGGTC
    541 CTGGCCCTTG AGCCCCCCGA CGTGGGCTCC TCAGACCCGC TGA
    Sorex araneus (shrew) FGF21 gene coding sequence (SEQ ID NO: 298)
    (Ensembl Accession No. ENSSART00000003074, which is hereby
    incorporated by reference in its entirety)
    1 ATGGTCTGGG ACAAGGCCAG GGGGCAGCAG TTGGGACTGT GGGCCCCCAT GCTGCTGGGC
    61 TTGCTGCTGG GTGCCTGCCA GGCACACCCC CTCCCTGACT CCAGCCCCCT CCTCCAATTT
    121 GGGGGCCAAG TCCGACTGAG GTTCCTGTAC ACCGACGATG CCCAGAGGAC AGGGGCGCAC
    181 CTGGAGATCA GGGCCGACGG CACAGTGCAG GGTGCGGCCC ACAGGACCCC AGAATGTCTC
    241 CTGGAGCTGA AAGCCTTGAA GCCAGGCGTA ATTCAAATCC TTGGGGTCAG CACATCCAGA
    301 TTCCTGTGCC AGCGGCCCGA TGGGGTCCTG TATGGATCGC TTCGCTTTGA CCCAGAGGCC
    361 TGCAGTTTCC GGGAACTTCT TCTCCAGGAT GGATATAACG TTTACCAGTC TGAGGCCCTG
    421 GGTCTCCCGC TCTACCTACA CCCGCCCAGT GCCCCAGTGT CCCAGGAACC AGCCTCACGG
    481 GGCGCCGTCC GCTTCCTGCC ACTGCCAGGA CTGCCACCTG CCTCCCTGGA GCCCCCCAGG
    541 CCCCCCGCCC CGGTGCCTCC AGACGTGGGT TCCTCAGACC CCCTGA
    Ictidomys tridecemlineatus (squirrel) FGF21 gene coding sequence
    (SEQ ID NO: 299)
    1 ATGTACCCCA TCCCTGACTC AAGCCCCCTC CTCCAATTTG GGGGCCAAGT CCGGCAGCGG
    61 TACCTGTACA CAGATGATGC CCAGGAGACT GAGGCCCACC TGGAGATCAG GGCTGATGGC
    121 ACCGTGGTGG GGGCTGCCCA TCAAAGCCCG GAAAGTCTCT TGGAACTGAA AGCCTTGAAG
    181 CCTGGGGTCA TTCAAATCTT GGGGGTCAAA ACATCCAGGT TCCTGTGCCA GAGGCCAGAT
    241 GGAGTGCTGT ATGGATCGCT CCACTTTGAC CCTGAGGCCT GCAGCTTCCG GGAGCAGCTT
    301 CTGGAGGACG GGTACAACGT TTACCAGTCA GAATCCCACG GCCTCCCCGT GCGCCTGCCC
    361 CCTAACTCAC CATACCGGGA CCCAGCGCCG CCAGGACCAG CCCGCTTCCT TCCACTGCCA
    421 GGCCTGCCCC CAGCAGCCCT GGAGCCGCCA GGGATCCTGG GCCCTGAGCC CCCTGATGTG
    481 GGCTCCTCCG ACCCACTCAG CATGGTGGGG CCTTTGCAGG GCCGAAGCCC CAGTTACGCT
    541 TCCTGA
    Loxodonta africana (elephant) FGF21 gene coding sequence (SEQ ID
    NO: 300) (Ensembl Accession No. ENSLAFT00000022429, which is hereby
    incorporated by reference in its entirety)
    1 ATGGACTGGG CCAAGTTTGG GTTGGAGCAC CCAGGACTGT GGGTCCCTGT GATGGCTGTC
    61 CTTCTGCTGG GAGCCTGCCA GGGACACCCC ATCCCTGACT CCAGCCCCCT CCTCCAATTC
    121 GGGGGCCAGG TCCGGCAACG TTACCTCTAC ACAGATGATC AGGAGACCGA GGCCCACCTG
    181 GAGATCAGAG CAGATGGCAC AGTGGCGGGA GCCGCTCACC GGAGCTCTGA GAGTCTCTTG
    241 GAGCTGAAAG CTTTGAAGCC TGGAATAATT CAGATCTTGG GGGTCAAGAC ATCCCGGTTC
    301 CTGTGCCAGG GGCCTGATGG GGTGCTGTAC GGATCGCTCC ATTTCGACCC AGCCGCCTGC
    361 AGCTTCCGGG AGCTGCTTCT TGAAGATGGA TACAATGTTT ACTGGTCCGA GGCCCATGGA
    421 CTCCCAATCC GCCTGCCCTC CCACAACTCC CCATATAGGG ACCCAGCATC CCGGGTACCA
    481 GCCCGCTTCC TGCCACTGCC AGGCCTGCTC CCAATGCTCC AAGAACCTCC AGGGGTCCTG
    541 GCCCCTGAGC CCCCTGATGT GGACTCCTCA GACCCCCTGA GCATGGTGGG GCCTTCACAG
    601 GGCCGAAGCC CCAGCTATGC CTCCTGA
    Sus scrofa (pig) FGF21 gene coding sequence (SEQ ID NO: 301)
    (GenBank Accession No. NM_001163410, which is hereby
    incorporated by reference in its entirety
    131 ATGGGCTGGG CCGAGGCCAA GTTCGAGCGC TTGGGACTGT GGGTCCCTGT GCTGGCTGTC
    191 CTGCTGGGAG CCTGCCAGGC ACGTCCCATT CCTGACTCCA GCCCCCTCCT CCAATTTGGG
    251 GGCCAAGTGC GCCAACGATA CCTCTACACG GATGATGCCC AGGAAACTGA AGCCCACCTG
    311 GAGATCAGAG CTGATGGCAC CGTGGCAGGG GTAGCCCGCC AGAGCCCTGA AAGTCTCTTG
    371 GAGCTGAAAG CCCTGAAGCC AGGGGTCATT CAAATTTTGG GAGTCCAGAC ATCCCGGTTC
    431 CTGTGCCAGG GGCCAGACGG GAGACTGTAC GGATCGCTCC ACTTCGACCC TGAGGCCTGC
    491 AGCTTCCGGG AGCTGCTTCT TGAGGATGGC TACAACGTTT ACCAGTCTGA GGCCCTTGGC
    551 CTCCCACTCC GGCTGCCTCC GCACCGCTCC TCCAACCGGG ACCTGGCCCC CCGGGGACCT
    611 GCTCGCTTCC TGCCACTGCC AGGCCTGCCC CCGGCACCCC CGGAGCCGCC AGGGATCTTG
    671 GCCCCTGAAC CTCCCGACGT GGGCTCCTCG GACCCCCTGA GCATGGTGGG GCCTTCACAC
    731 GGCCGGAGCC CCAGCTACAC TTCTTGA
    Felis catus (cat) FGF21 gene coding sequence (SEQ ID NO: 302)
    (Ensembl Accession No. ENSFCAT00000007367, which is hereby
    incorporated by reference in its entirety)
    1 ATGGGCTGGG ACGAGGCCGG GTCCCAGCGC CTGGGACTGT GGGTCGTGCT GGGGGTCCTT
    61 TTGCCGGAAG CCTGCCAGGC ACACCCTATC CCTGACTCCA GCCCCCTCCT CCAATTCGGG
    121 GGCCAAGTTC GACAGCGGTT CCTCTACACG GACGACGCCC AGGAGACAGA GGTCCACCTC
    181 GAGATCAAGG CTGATGGCAC AGTGGTGGGG ACCGCTCGCC GGAGCCCTGA GAGTCTCTTG
    241 GAGCTAAAAG CCCTGAAGCC GGGGGTAATT CAAATCTTGG GGGTCAAAAC GTCCAGGTTC
    301 CTGTGCCAGG GCCCAGATGG GACACTGTAT GGATCGCTCC GCTTTGACCC CGCAGCCTGC
    361 AGCTTCCGGG AACTGCTCCT GGAGGACGGA TACAACATCT ACCACTCGGA GACCCTCGGG
    421 CTCCCACTCC GCCTGCCCCC CCACAACTCC CCATACCGGG ACTTGGCCCC CCGGGCACCT
    481 GCCCGCTTCC TGCCGCTGCC AGGCCTGCTT CCGGCACCCC CGGAGCCTCC AGGGATCCTG
    541 GCCCCCGAGC CCCCGGACGT GGGCTCCTCG GACCCTCTGA GCATGGTGGG GCCTTCCCAG
    601 GGCCGAAGTC CCAGCTACGC TTCCTGA
    Otolemur garnettii (bushbaby) FGF21 gene coding sequence (SEQ ID NO:
    303) (Ensembl Accession No. ENSOGAT00000003585, which is hereby
    incorporated by reference in its entirety)
    1 GACAAGGCCA GGACTGGGTT CAAGCACCCA GGACCATGGT TTCCCCTGCT GGCTGTACTT
    61 TTGTTGGGAG CCTGCCAGGC ACACCCTATC CCTGACTCCA GCCCCCTACT CCAGTTTGGT
    121 GGCCAAGTCC GGCAGCGGTA CCTCTACACA GATGATGCCC AGGAGACAGA AGCCCACCTG
    181 GAGATCAGGG AAGATGGCAC AGTGGTGGGG GCTGCACAAC AGAGCCCTGA AAGTCTCTTG
    241 GAGCTGAAAG CTTTAAAGCC AGGGGTCATT CAAATCTTGG GAGTCAAGAC ATCCAGGTTC
    301 CTGTGCCAGA GGCCAGATGG GGGCCTATAT GGATCGCTCT ACTTTGACCC CAAGGCCTGC
    361 AGTTTCCGGG AGCTGCTTCT TGAGGATGGA TACAACGTTT ACTGGTCTGA GACCTATGGC
    421 CTCCCACTGC ACCTGCCTCC TGCCAATTCC CCATACTGGG GCCCATCCCT TCGGAGCCCA
    481 GCCCGCTTCC TGCCACTGCC AGGCCCTCCT GCAGCATCCC CAGAGCTGCC GGGGATCTTG
    541 GCCCTGGAAC CCCCCGATGT GGGCTCCTCG GACCCTCTGA GCATGGTGGG GCCTTCGCAG
    601 GGCCGAAGCC CCAGCTATGC TTCCTGA
    Rattus norvegicus (Norway rat) FGF21 gene coding sequence (SEQ
    ID NO: 304) (GenBank Accession No. NM_130752, which is
    hereby incorporated by reference in its entirety)
    1 ATGGACTGGA TGAAATCTAG AGTTGGGGCC CCGGGACTGT GGGTCTGTCT CCTGCTGCCT
    61 GTCTTCCTGC TGGGGGTGTG CGAGGCATAC CCCATCTCTG ACTCCAGCCC CCTCCTCCAG
    121 TTTGGGGGTC AAGTCCGACA GAGGTATCTC TACACAGATG ACGACCAGGA CACCGAAGCC
    181 CACCTGGAGA TCAGGGAGGA CGGAACAGTG GTGGGCACAG CACACCGCAG TCCAGAAAGT
    241 CTCCTGGAGC TCAAAGCCTT GAAGCCAGGG GTCATTCAAA TCCTGGGTGT CAAAGCCTCT
    301 AGGTTTCTTT GCCAACAACC AGATGGAACT CTCTATGGAT CGCCTCACTT TGATCCTGAG
    361 GCCTGCAGTT TCAGAGAGCT GCTGCTTAAG GACGGATACA ATGTGTACCA GTCTGAGGCC
    421 CATGGCCTGC CCCTGCGTCT GCCCCAGAAG GACTCCCAGG ATCCAGCAAC CCGGGGACCT
    481 GTGCGCTTCC TGCCCATGCC AGGCCTGCCC CACGAGCCCC AAGAGCAACC AGGAGTCCTT
    541 CCCCCAGAGC CCCCAGATGT GGGTTCCTCC GACCCCCTGA GCATGGTAGA GCCTTTGCAA
    601 GGCCGAAGCC CCAGCTATGC ATCTTGA
    Mus musculus (house mouse) FGF21 gene coding sequence (SEQ ID
    NO: 305) (GenBank Accession No. NM_020013, which is
    hereby incorporated by reference in its entirety)
    185     ATGGAA TGGATGAGAT CTAGAGTTGG GACCCTGGGA CTGTGGGTCC GACTGCTGCT
    241 GGCTGTCTTC CTGCTGGGGG TCTACCAAGC ATACCCCATC CCTGACTCCA GCCCCCTCCT
    301 CCAGTTTGGG GGTCAAGTCC GGCAGAGGTA CCTCTACACA GATGACGACC AAGACACTGA
    361 AGCCCACCTG GAGATCAGGG AGGATGGAAC AGTGGTAGGC GCAGCACACC GCAGTCCAGA
    421 AAGTCTCCTG GAGCTCAAAG CCTTGAAGCC AGGGGTCATT CAAATCCTGG GTGTCAAAGC
    481 CTCTAGGTTT CTTTGCCAAC AGCCAGATGG AGCTCTCTAT GGATCGCCTC ACTTTGATCC
    541 TGAGGCCTGC AGCTTCAGAG AACTGCTGCT GGAGGACGGT TACAATGTGT ACCAGTCTGA
    601 AGCCCATGGC CTGCCCCTGC GTCTGCCTCA GAAGGACTCC CCAAACCAGG ATGCAACATC
    661 CTGGGGACCT GTGCGCTTCC TGCCCATGCC AGGCCTGCTC CACGAGCCCC AAGACCAAGC
    721 AGGATTCCTG CCCCCAGAGC CCCCAGATGT GGGCTCCTCT GACCCCCTGA GCATGGTAGA
    781 GCCTTTACAG GGCCGAAGCC CCAGCTATGC GTCCTGA
    Vicugna pacos (alpaca) FGF21 gene coding sequence (SEQ ID NO: 306)
    (Ensembl accession no. ENSVPAT00000005993, which is hereby
    incorporated by reference in its entirety) (1-209, excluding 79-168
    and 172-182)
    1 ATGGACTGGG ACGAGGCCAA GTTCGAGCAT CGGGGACTGT GGGTCCCAGT GCTCACTGTC
    61 CTTCTGCTGG GAGCCTGCCA GGCACGCCCC ATTCCTGACT CCAGCCCCCT CCTCCAATTC
    121 GGGGGCCAAG TCCGGCAGCG GTACCTCTAC ACGGATGACG CCCAGGAGAC AGAAGCCCAC
    181 CTGGAGATCA GGGCTGATGG CACAGTGGTG GGGGTGGCCC GCCAG---CC CGAA------
    241 ---------- ---------- ---------- ---------- ---------- ----------
    301 ---------- ---------- ---------- ---------- ---------- ----------
    361 ---------- ---------- ---------- ---------- ---------- ----------
    421 ---------- ---------- ---------- ---------- ---------- ----------
    481 ---------- ---------- ----GGAATT CCT------- ---------- ----------
    541 ------CCCG AGCCTCCTGA CGTGGGCTCC TCAGACCCCC TGAGCATGGT GGGGCCTTCA
    601 TACAGCAGAA GCCCCAGCTA CACTTCCTGA
    Anolis carolinensis (anole lizard) FGF21 gene coding sequence
    (SEQ ID NO: 307) (Ensembl accession no. ENSACAT00000017230,
    which is hereby incorporated by reference in its entirety)
    1 TGTAAAAGCA AGGGAGGAGG GAAGGGGGGA GAGAGGATGT GGGTAGACCT AGTTTTCTGG
    61 GCTGCCTTGC TCCGCACAGC TCCTGCTCTT CCCTTGCGGA ATTCCAACCC CATCTACCAA
    121 TTTGATGGGC AGGTCCGGCT TCGGCACCTC TACACAGCAG ATGAACAGAC GCACCTCCAC
    181 TTGGAGATCT TGCCAGACGG TACCGTGGGT GGATCCAGGT TTCAGAATCC CTTCAGTTTG
    241 ATGGAGATCA AAGCTGTGAA GCCAGGAGTC ATTCGCATGC AGGCCAAGAA GACCTCTAGA
    301 TTTCTCTGTA TGAAACCCAA TGGACGACTG TATGGCTCGC TGTTCTACTC TGAGGAGGCA
    361 TGCAACTTCC ATGAGAAGGT TCTCAGCGAT GGCTACAACC TCTACTATTC TGAAAACTAC
    421 AACATACCTG TCAGCCTCAG CTCGGCAGGG AACCTGGGTC AGAGCCGTCA GTTGCCTCCC
    481 TTCTCCCAAT TCCTGCCGTT AGTCAACAAA ATTCCTCTTG AGCCTGTGCT TGAAGACTTT
    541 GACTTCTATG GACATCAATT GGATGTTGAA TCAGCTGATC CTTTGAGCAT TTTAGGACAA
    601 AACCCTGGTT TCATGAGTCC GAGCTATGTC TTC
    Gadus morhua (cod) FGF21 gene coding sequence (SEQ ID NO: 308)
    (Ensembl accession no. ENSGMOT00000014151, which is hereby
    incorporated by reference in its entirety)
    1 CTCCTCCTCG CCACCCTCCT CCACATCGGC CTCTCCTTCT ACGTCCCCGA CTCCGGCCCC
    61 CTGCTGTGGC TGGGCGACCA GGTCAGGGAG AGACACCTCT ACACAGCAGA GAGCCACCGG
    121 AGGGGGCTGT TCCTGGAGAT GAGCCCGGAC GGTCAGGTGA CAGGAAGTGC TGCTCAGACG
    181 CCGCTCAGTG TTCTGGAGCT GAGGTCGGTC AGAGCAGGAG ATACGGTCAT CAGAGCGCGC
    241 CTCTCCTCTC TCTACCTGTG TGTGGACAGG GCAGGTCACC TGACAGGACA GAGACAGTAC
    301 ACAGAGTCCG ACTGCACCTT CAGAGAGGTC ATCCTTGAGG ACGGCTACAC CCACTTCCTG
    361 TCCGTGCACC ACGGACTTCC TATTTCGCTG GCGCCGAGAC ACTCCCCAGG GAGACAGGGG
    421 CTGCGCTTCA GCAGGTTCCT CCCGCTGAGG AGCAGTCTGT CAGAGGATAG GGTCGCCGAG
    481 CCCCCAGACA GCCCACTGAA CCTGGACTCT GAAGACCCCC TGGGGATGGG TCTGGGTTCG
    541 CTCCTCAGCC CGGCCTTCTC CATG
    Latimeria chalumnae (coelacanth) FGF21 gene coding sequence (SEQ ID
    NO: 309) (Ensembl accession no. ENSLACT00000003815, which is hereby
    incorporated by reference in its entirety)
    1 ATGTTATGCC AGAGTTTTGT GATATTAAGT CAGAAATTCA TTTTTGGGCT CTTTTTGACT
    61 GGATTGGGGC TAACAGGATT GGCTTGGACA AGGCCCTTCC AGGATTCCAA TCCCATCCTG
    121 CAGTATTCCG ATTCCATCCG GCTCCGACAT CTGTACACTG CCAGTGAGAG TCGGCACCTT
    181 CACCTACAAA TCAACTCGGA TGGACAGGTG GGAGGGACAA CCAAGCAAAG CCCTTACAGT
    241 CTGTTGGAGA TGAAGGCGGT GAAGACAGGT TTTGTGGTCA TCAGGGGCAA GAAAAGCGCC
    301 CGTTACCTCT GTATGGAACG TAGTGGACGG CTCTATGGAT CGCTGCAGTA TACAGAAAAA
    361 GACTGCACCT TCAAAGAGGT TGTGTTGGCA GATGGATACA ACCTGTATGT CTCAGAGGAA
    421 CACCAGGCCA CAGTGACGCT GAGCCCCATG AGGGCGAGGA TAGCGCAAGG GAAAAAGATC
    481 CCACCCTTTT CCCATTTCCT TCCAATGGTG AACAAGGTGC CTGTGGAGGA TGTTGCCGCT
    541 GAGATGGAGT TTGTCCAGGT GCTGCGGGAA ATGACGGCCG ACGTGGACTC TCCGGATCCC
    601 TTTGGAATGA CCTGGGAAGA ATCGGTTCAC AGTCCGAGCT TTTTTGCC
    Tursiops truncatus (dolphin) FGF21 gene coding sequence (SEQ ID
    NO: 310) (Ensembl accession no. ENSTTRT00000014561, which is hereby
    incorporated by reference in its entirety)
    1 ATGGGCTGGG ACAAGACCAA ACTCGAGCAC CTGGGACTGT GGGTCCCTGT GCTAGCTGTC
    61 CTGCTGGGAC CCTGCCAGGC ACATCCCATT CCTGACTCCA GCCCCCTCCT CCAATTTGGG
    121 GGCCAAGTCC GCCAGCGATA CCTCTACACG GATGACGCCC AGGAGACGGA GGCCCACCTG
    181 GAGATCAGGG CTGATGGCAC AGTGGTGGGG ACGGCCCGCC GGAGCCCCGA AGGAGTTAAA
    241 ACATCCAGGT TCCTGTGCCA GGGGCCAGAG GGGAGGCTGT ATGGATCGCT CCACTTCAAC
    301 CCCCAGGCCT GCAGCTTCCG GGAGCTGCTT CTTGAGGATG GATACAACGT TTACCAGTCT
    361 GAGGCTCTTG GCATTCCCCT CCGCCTGCCC CCGCACCGCT CCTCCAACTG GGACCTGGCC
    421 CCCCGGGGAC CTGCTCGCTT CCTGCCGCTG CCAGGCTTCC TCCCGCCACC CCTGGAGCCT
    481 CCAGGGATCT TGGCCCCCGA GCCTCCCAAC GTAGGTTCCT CGGACCCCTT GAGCATGGTG
    541 GGACCTTCAC ATGGCCGAAG CCCCAGCTAC ACTTCCTGA
    Mustela putorius furo (ferret) FGF21 gene coding sequence (SEQ ID
    NO: 311) (Ensembl accession no. ENSMPUT00000003755, which is hereby
    incorporated by reference in its entirety)
    188        ATG GGCTGGGAAG AGGCCAGGTC CGAGCACCTG GGGCTGTGGG TCCCTGTGCT
    241 GGCGGTCCTT TTGCTGGGAG CCTGCCAGGC ATACCCTATT CCTGACTCCA GCCCCCTCCT
    301 CCAATTTGGA GGCCAAGTTC GACAGCGGTA CCTCTACACA GACGACGCTC AGGAGACGGA
    361 GGCCCACCTA GAGATCAGGG CTGATGGCAC GGTGGTGGGG GCTGCCCGCC GGAGCCCCGA
    421 AAGTCTCTTG GAGCTGAAAG CCCTGAAGCC AGGGGTCATT CAGATCTTGG GAGTGAAAAC
    481 ATCCAGGTTC CTGTGCCAGG GCCCGAATGG GACACTGTAC GGATCGTTCC ACTTCGACCC
    541 CGTAGCCTGC AGCTTCCGGG AAGTGCTTCT GGAAGATGGA TACAACATCT ACCACTCTGA
    601 GACCCTGGGC CTCCCACTGC GCCTGCCCCC CCACAACTCC CCACACAGGG ACCTGGCGCC
    661 CCGGGGGCCT GCCCGCTTCC TGCCCCTGCC AGGCCTGCTT CCGGCCACCC CGGAGTCCCG
    721 GGGGATCCCA GCCCCCGAGC CTCCCAACGT GGGCTCCTCA GACCCCCTGA GCATGGTGGG
    781 GCCTTTGCAG GGTCAAAGTC CCAGCTACAC TTCCTGA
    Takifugu rubripes (fugu) FGF21 gene coding sequence (SEQ ID NO: 312)
    (Ensembl accession no. ENSTRUT00000034076, which is hereby
    incorporated by reference in its entirety)
    1 TTTATTTATT TATTTATTCA AACTGCACTT TTTTCCCCTT CCAAATGGTT CAACTTTTAT
    61 CTCCCTGACT CCAACCCGCT CTTATCCTTT GACAGTCATG GCAGAGGCAT CCACCTCTAC
    121 ACAGATAATC AAAGGCGAGG GATGTATCTG CAGATGAGCA CAGATGGAAG CGTTTCCGGG
    181 AGTGATGTCC AGACGGCGAA CAGTGTGCTG GAACTGAAGT CAGTCAGAAA CGGCCACGTC
    241 GTCATCCGAG GAAAATCGTC TTCTCTGTTT CTCTGTATGG ACAGCAGAGG CCGTTTATGG
    301 GGGCAGAGGC ACCCCACTGA GGCCGACTGC ACTTTCAGGG AAGTGTTGCT GGCAGATGGA
    361 TACACTCGCT TCCTGTCCCT GCACAACGGA ACTCCTGTGT CTCTGGCACC TAAACAATCT
    421 CCAGACCAGC ACACAGTCCC CTTCACTCGT TTCCTGCCGC TCAGGAATAC ACTGGCAGAG
    481 GAGAGCATGT CTGAACCACC ATCAAACCAA CAGAGATATT TTAACATTGA CTCTGATGAT
    541 CTTCTTGGAA TGGATTTAAA TGCGATGGTC AGTCCTCAGT TTTCAGGGGA CAAGTGA
    Dipodomys ordii (kangaroo rat) FGF21 gene coding sequence (SEQ
    ID NO: 313) (Ensembl accession no. ENSDORT00000001234, which
    is hereby incorporated by reference in its entirety)
    1 ATGGACCAGG CAAAGACCAG GGTTGGGGCC CGGGGGCTGG GGGGCCTTGT GCTGGCTGTC
    61 ATAATTCTGG GAGCATGCAA GGCACGGCCT ATCCCTGACT CCAGCCCCCT CCTCCAATTT
    121 GGGGGTCAAG TTCGGCTTCG GCACCTCTAC ACAGATGACA CTCAGGAGAC GGAAGCCCAT
    181 CTGGAGATCA GGGCAGATGG CACGGTAGTG GGGACTGCCC ACCGGAGCCC TGAAAGTCTC
    241 TTGGAGCTGA AAGCCTTGAA GCCAGGAGTC ATTCAAATCT TAGGGATCAA GACATCCAGA
    301 TTCTTATGCC AGAGACCAGA CGGGACACTG TATGGATCAC TCCACTTTGA CCCTGAGGTT
    361 TGCAGCTTCC AGGAGCTGCT TCTGGAAGAT GGATACAACA TTTACCGTTC TGAAGCCCTG
    421 GGTCTCCCCC TGCGCCTGTC CCCAGATCCA GCACCCTGGG GGCCAGCCCG CTTCCTGCCC
    481 CTGCCTGGTG TGCCCCCCGC ACCGCCGGAG CCCCCCGGGA TCCTGGCTCC CGAACCCCCT
    541 GATGTCGGCT CCTCCGACCC TCTGAGTATG GTGGGACTGT TGCAGGGCCG AAGCCCCAGC
    601 TATGCATCCT GA
    Echinops telfairi (lesser hedgehog tenrec) FGF21 gene coding
    sequence (SEQ ID NO: 314) (Ensembl accession no.
    ENSETET00000010721, which is hereby incorporated by reference
    in its entirety)
    1 ATGGGTTGCA CCAAATCTGG GTGGAAGTCC CCGGGACTGT GGGTCCCTGT GCTGGCCAGC
    61 CTTCTGCTGG GAGGCTGCGG AGCACACCCC ATCCCTGACT CCAGCCCCCT CCTCCAATTC
    121 GGGGGCCAAG TCCGGCAGCG ATACCTCTAT ACGGATGACG CCCAGACCAC CGAGGCCCAC
    181 CTGGAGATCA GAGCGGATGG CACAGTGGGG GGCGTCGCCC ACCAGAGCCC AGAGAAGTTC
    241 CTGAGTCAAT GGCGTGAAAA GCCCCTGAGA TCACTCCATT TCGACCCAGC CGCCTGCAGC
    301 TTCCGGGAGA AGCTTCTAGA AGACGGATAC AACTTGTACC ACTCTGAGAC CCACGGCCTC
    361 CCCCTCCGCC TCCCACCCCG TGGGGGCGAC CCCTCTTCTC AGCCTGGGGC CCGCTTCCCA
    421 CCGCTGCCGG GCCAGCTCCC ACAACTCCAA GAGACGCCAG GGGTCCTCGC CCCCGAACCC
    481 CCCGACGTGG GCTCTTCAGA CCCCCTGAGC ATGGTGGGGC CTTGGCGAGG GCAAAGTCCC
    541 AGTTATGCCT CCTGA
    Macaca mulatta (rhesus monkey) FGF21 gene coding sequence (SEQ ID
    NO: 315) (Ensembl accession no. ENSMMUT00000038440, which is hereby
    incorporated by reference in its entirety)
    1 ATGGACTCGG ACGAGACCGG GTTCGAGCAC TCAGGACTGT GGGTTCCTGT GCTGGCTGGT
    61 CTTCTGCTGG GAGCCTGCCA GGCACACCCC ATCCCTGACT CCAGTCCTCT CCTGCAATTC
    121 GGGGGCCAAG TCCGGCAACG GTACCTCTAC ACAGATGATG CCCAGCAGAC AGAAGCCCAC
    181 CTGGAGATCA GGGAGGATGG GACAGTGGGG GGCGCTGCTC ACCAGAGCCC CGAAAGTGAG
    241 TGTGGGCCAG AGCCTGGGTC TGAGGGAGGA GGGGCTGTGG GAGGTGCTGA GGGACCTGGA
    301 CTCCTGGGTC TGAGGGAGGC AGGGCTGGGG CCTGGATCCT GGCTCCACTT TGACCCTGAG
    361 GCCTGCAGCT TCCGGGAGCT GCTTCTTGAG AACGGATACA ATGTTTACCA GTCCGAGGCC
    421 CACGGCCTCC CACTGCACCT GCCGGGAAAC AAGTCCCCAC ACCGGGACCC TGCATCCCAA
    481 GGACCAGCTC GCTTCCTGCC ACTACCAGGC CTGCCCCCCG CACCCCCGGA GCCGCCAGGA
    541 ATCCTCGCCC CCCAGCCCCC CGATGTGGGC TCCTCGGACC CTCTGAGCAT GGTGGGACCT
    601 TCCCAGGCCC GAAGCCCCAG CTATGCTTCC TGA
    Microcebus murinus (mouse lemur) FGF21 gene coding sequence (SEQ ID
    NO: 316) (Ensembl accession no. ENSMICT00000013258, which is hereby
    incorporated by reference in its entirety)
    1 ATGGGCTGGG ACGAGGCCGG CGCCGGGTTC GAGCACCCAG GACTGTGGTT TCCCATGCTG
    61 GGTGTCCTGC TGCTGGGAGC CTGCCAGGCG TACCCCATCC CTGACTCCAG CCCCCTCCTC
    121 CAATTTGGCG GCCAAGTCCG GCAGCGGCAC CTCTACACAG ACGATATCCA GGAGACAGAA
    181 GCCCACCTGG AGATCAGGGC GGACGGCACA GTGGTGGGGG CCGCCCGACA GAGCCCTGAG
    241 TTGGAGCTGA AAGCCTTAAA GCCAGGGGTC ATTCAAATCT TGGGAGTCAA GACCTCCAGG
    301 TTCCTGTGCC AGAGGCCAGA CGGGGCCCTG TACGGATCGC TCCACTTTGA CCCCGAGTGC
    361 AGCTTCCGGG AGCTGCTTCT TGAGGATGGA TACAACGTCT ACTGTCCCTA CCTCCCGCTG
    421 CACCTGTCCC CACGCATCGA ACTGGCCGGA TCACGCTCTG CGCTGCCACT GCCCCCAGCA
    481 CCTGAACGCA GGATTTTGGC CCCGGAGCCC CCGGATGGCT CCTCGGACCC TCTGAGCATG
    541 GTGGGGCCTT CGCAGGGCCG AAGTCCCAGC TATGCTTCCT GA
    Ochotona princeps (pika) FGF21 gene coding sequence (SEQ ID NO: 317)
    (Ensembl accession no. ENSOPRT00000007373, which is hereby
    incorporated by reference in its entirety)
    1 AAAGACATGG ACGGGCTCCA GCCTCCGGGG CTGCGGGTTC CTGTGCTGGC TGCCCTGCTT
    61 TTGGGAGTTG GCCAGGCACG CCCCATCCCT GATTCTAGCC CTCTCCTCCA ATTCGGGGGC
    121 CAGGTCCGGC AGAGGCACCT CTACACGGAT GACGCCCAGG AATCGGAAGT ACACCTGGAG
    181 ATCCGGGCAG ACGGCACCGT GGCAGGGACT GCCCGCCGGA GCCCTGAAAG TCTCTTAGAA
    241 ATGAAAGCGT TGAAGCCAGG CGTCATTCAG ATCCTGGGGG TCCACACATC CAGGTTCCTG
    301 TGCCAGAGAC CAGACGGGAC GCTGTACGGC TCGCTCCACT TCGACCACAA GGCCTGCAGC
    361 TTCCGGGAGC AGCTGCTGGA GGATGGGTAC AACGTGTACC ACTCAGAGAC ACACGGCCTC
    421 CCGCTGCGCC TGTCTCCAGA CCGAGCCCCC CGGGGCCCAG CCCGCTTCCT GCCACTGCCA
    481 GGCCCTCCTC CTGACCTCCT GGTGCCACCC CTGCCACCGG ACGTCCTAGC CCCTGAGCCC
    541 CCCGACGTGG ACTCCCCAGA CCCCCTGAGC ATGGTGGGGC CCTTGCAGGG CCAAAGCCCC
    601 AGCTACACTT CCTGA
    Xiphophorus maculatus (platyfish) FGF21 gene coding sequence (SEQ ID
    NO: 318) (Ensembl accession no. ENSXMAT00000001579, which is hereby
    incorporated by reference in its entirety)
    1 TGCCCGTTCC CCTTCCTTTT CTTAATCCTC TCTCTTCCCT TTTTCTCTTC CTCGTTTTAC
    61 ATCCCAGAAT CCAACCCAAT CTTTGCCTTC AGGAATCAGC TCAGAGAGGT GCATCTCTAC
    121 ACAGAAAATC ACAGACGGGG TTTGTATGTG GAGATACATC TGGATGGGAG AGTGACTGGA
    181 AGTGATGCTC AGAGTCCTTA TAGTGTGTTG CAGATAAAGT CTGTTAAACC GGGTCATGTG
    241 GTCATAAAGG GACAGACATC GTCCCTGTTC CTCTGCATGG ACGACTCCGG GAATCTAAGA
    301 GGACAGACAA CCTATGACGA GGCTGACTGC TCCTTCAGGG AACTGCTGCT GGCCGATGGC
    361 TACACCCGTT TCCTGAACTC ACAACATGGC GTTCCTTTAT CACTGGCATC CAGAAACTCT
    421 CCAGATCGAC ACTCCGTTCC TTTCACAAGA TTTTTACCTC TCAGGAATAC TTTAACGGTT
    481 TCAGAAGAAT CAACAAAAAC TCAGAGGGAC TTCAACCTGG ACTCGGACGA CCTTCTCGGG
    541 ATGGGA
    Gasterosteus aculeatus (stickleback) FGF21 gene coding sequence (SEQ
    ID NO: 319) (Ensembl accession no. ENSGACT00000010725, which
    is hereby incorporated by reference in its entirety)
    1 TCTCTCCTCC TCATGGTCCC ACTTCCTTTC TGTTCATCCT TTTATCTCAC TGACTCCAGC
    61 CCACTTCTAC CCTTCAATAA TCAAGTCAAA GAGGTGCACC TCTACACAGC AGAGAATCAC
    121 AGAAGAGCGA TGTACCTGCA GATCGCTCTG GACGGGAGCG TGTCGGGAAG CGACGCTCGG
    181 TCCACTTACA GTGTGCTGCA GCTGAAATCT ATCCAGCCGG GCCACGTGGT CATCAGAGGG
    241 AAGGCCTCCT CCATGTTCCT CTGCGTGGAC AGCGGGGGCC GTTTGAGAGG ACAGGGGCCG
    301 TACTCAGAGG CCGACTGCAG CTTCAGGGAG CTGCTGCTGG GGGATGGCTA CACCCGGTTC
    361 CTGTCCTCGC AGCACGGGTC CCCGCTGTCT CTGGCGTCGA GGCCTTCCCC GGATCCCAAC
    421 TCGGTGCCCT TCACTCGATT CCTACCCATC CGGACCGCCC CCGAGGCTGA GAGCGTGATC
    481 GAAGAGCCAC CGAGCAATCA GAGATACGTC AACGTGGACT CCGAGGATCT TCTTGGAATG
    541 GGCCTGAACA CTGTGGTCAG TCCTCAGTTC TCGGCG
    Sarcophilus harrisii (Tasmanian devil) FGF21 gene coding sequence
    (SEQ ID NO: 320) (Ensembl accession no. ENSSHAT00000006017,
    which is hereby incorporated by reference in its entirety) (1-209,
    excluding 1-2 and 173-209)
    132            GTGTCTGCC ATGGGCCTGA GGGAGCGAGC TCCCAGGTAC CTGGCCCCGC
    181 TGCTGTCCTT GCTCTTGGCC TGCAGGGCCT CGGGTCACCC CCTCCCGGAT TCCAGCCCCA
    241 TGCTCCTGTT TGGGGGGCAG GTCCGCCTCC GGCACCTCTA CACGGATGTG GGCCAGGAGG
    301 CCGAGGCCCA CGTGGAACTG GCGTCCGACG GCACAGTCCG GGCGGCAGCG CGGAGGAGTC
    361 CCAACAGTCT CCTGGAGCTG AAGGCTGTGA AGCCGGGCAT CGTCCGAATC CTGGCCGTCC
    421 ACAGCTCTCG GTTTCTGTGT ATGAGGCCCA ACGGGGAGCT GTACGGAGCG ATACACTACG
    481 ACCCTTCCGC CTGCAACTTT CGGGAGCGCC TGCTGGGGGA CGGCTACAAC GTGTACGAGT
    541 CCGAGGCTCA CGGGAGGACC CTCCGCCTGC CCCCCAAGGC CGCACCGGGA CCCGCCGGAC
    601 CTTCTCGCTT CCTGCCGCTC CCCGGC
    Macropus eugenii (wallaby) FGF21 gene coding sequence (SEQ ID
    NO: 321) (Ensembl accession no. ENSMEUT00000015309, which is
    hereby incorporated by reference in its entirety)
    1 ACAGAGGAGC CTTCTACTGG GTCCAGGCAC CTGGGACAAT GGGCTCCCGG GCTGCCTGGT
    61 CCTCTGCTGT CCTTGCTCCT GGCCTACAGG GGCTGGGGCT CCCCCATCCC TGATTCCAGC
    121 CCCATGCTCC TGTTTGGTGG CCAGGTCCGC CTCCGACACC TGTACACAGA TGATGGCCAG
    181 GACACGGAGG CCCATGTGGA GCTGGGGCCA GATGGAGTGG TTCGAGCTGT GGCTGAGAGG
    241 AGCCCCAACA GTCTTCTGGA ACTGAAGGCG GTGAAGCCTG GAGTCATCCG AATCCTCGCT
    301 GTCCAGAGCT CTCGGTTTCT GTGTATGAGG CCCAACGGGG AACTGTATGG AGCGGTACAC
    361 TATGACCCTT CTGCCTGCAA CTTTCGGGAA CATCTGCTGG GGGATGGTTA TAATGTGTAT
    421 GAATCAGAGA CTCACAGAAG GACCCTCCGT CTGTCCCCAT CCCTGGGTCA GGCTGGCCCC
    481 TCTCGCTTCC TGCCACTTCC AGGCGACTGG CTGCCCGGCC CTGATCCACC TTGGGCACAG
    541 GGCCCTGAGC CCCCAGACGT GGGCTCTGCA GACCCCCTGA GCATGGTGGG GGCCGTGCAG
    601 GGCCTCAGCC CCAGCTACTC CTCCTGA
    Xenopus tropicalis (Western clawed frog) FGF21 gene coding sequence
    (SEQ ID NO: 322) (Ensembl accession no. ENSXETT00000009917, which is
    hereby incorporated by reference in its entirety) (1-209, excluding
    170-209)
    1 AGAGGGGGTA GGACCAAAAA AAAGACGTTA CTCAGGAAAT GGCTTTGCCT TTTAGCCATT
    61 ATGTTGAGTA GGTCAAGGTT TTCTTTAGCA AATCCTATCC AGAATTCGAA CCCAATCTTA
    121 TCCAACGACA ACCAAGTACG GACTCAGTAT TTATACACAG ATAACAATAA CATGCACCTG
    181 TATCTTCAGA TCACCCACAA TGGAGTAGTA ACTGGTACCG AAGAAAAGAA TGACTATGGT
    241 GTGCTGGAAA TAAAGGCAGT AAAAGCTGGG GTTGTAGTTA TAAAAGGAAT TCGAAGCAAT
    301 CTCTACCTAT GCATGGATTC TAGACACCAA TTGTATGCGT CGGCATATGA TAAAGATGAC
    361 TGCCATTTCC ATGAAAAGAT CACACCAGAT AATTACAACA TGTATAGCTC AGAGAAGCAT
    421 TCAGAATACG TGTCCTTAGC TCCATTAAAA GGAAGCCAGA TGGCTCGTTT TCTACCTATA
    Danio rerio (zebrafish) FGF21 gene coding sequence (SEQ ID NO: 323)
    (Ensembl accession no. ENSDART00000103511, which is hereby
    incorporated by reference in its entirety)
    30                                A TGCTTCTTGC CTGCTTTTTT ATATTTTTTG
    61 CTCTTTTTCC TCATCTTCGG TGGTGTATGT ATGTTCCTGC ACAGAACGTG CTTCTGCAGT
    121 TTGGCACACA AGTCAGGGAA CGCCTGCTTT ACACAGATGG GTTGTTTCTT GAAATGAATC
    181 CAGATGGCTC CGTCAAAGGC TCTCCTGAAA AGAATCTAAA TTGTGTGCTG GAGCTGCGTT
    241 CAGTCAAAGC GGGTGAAACC GTCATCCAGA GTGCAGCTAC ATCTCTCTAC CTCTGCGTCG
    301 ATGATCAAGA CAAGCTGAAA GGACAGCATC ATTACTCTGC ACTAGACTGC ACCTTTCAGG
    361 AATTGCTACT GGATGGATAT TCGTTTTTCC TTTCTCCACA CACTAATCTT CCCGTATCGC
    421 TCCTCTCGAA ACGTCAGAAA CACGGCAATC CTCTTTCTCG CTTCCTCCCT GTTAGCAGAG
    481 CAGAGGACAG CCGGACACAG GAGGTGAAAC AGTATATTCA GGATATAAAC CTGGACTCTG
    541 ACGACCCACT AGGAATGGGA CATCGGTCAC ACTTACAGAC CGTCTTCAGT CCCAGTCTGC
    572 ATACTAAAAA ATGA
    Bos grunniens mutus (yak) FGF21 gene coding sequence (SEQ ID
    NO: 324) (generated using SMS Reverse Translate tool on the
    ExPASy Bioinformatics Resource website (www.expasy.org))
    1 ATGGGCTGGG ATGAAGCGAA ATTTAAACAT CTGGGCCTGT GGGTGCCGGT GCTGGCGGTG
    61 CTGCTGCTGG GCACCTGCCG CGCGCATCCG ATTCCGGATA GCAGCCCGCT GCTGCAGTTT
    121 GGCGGCCAGG TGCGCCAGCG CTATCTGTAT ACCGATGATG CGCAGGAAAC CGAAGCGCAT
    181 CTGGAAATTC GCGCGGATGG CACCGTGGTG GGCGCGGCGC GCCAGAGCCC GGAAAGCCTG
    241 CTGGAACTGA AAGCGCTGAA ACCGGGCGTG ATTCAGATTC TGGGCGTGAA AACCAGCCGC
    301 TTTCTGTGCC AGGGCCCGGA TGGCAAACTG TATGGCAGCC TGCATTTTGA TCCGAAAGCG
    361 TGCAGCTTTC GCGAACTGCT GCTGGAAGAT GGCTATAACG TGTATCAGAG CGAAACCCTG
    421 GGCCTGCCGC TGCGCCTGCC GCCGCAGCGC AGCAGCAACC GCGATCCGGC GCCGCGCGGC
    481 CCGGCGCGCT TTCTGCCGCT GCCGGGCCTG CCGGCGGAAC CGCCGGATCC GCCGGGCATT
    541 CTGGCGCCGG AACCGCCGGA TGTGGGCAGC AGCGATCCGC TGAGCATGGT GGGCCCGAGC
    601 TATGGCCGCA GCCCGAGCTA TACCAGCTAA
    Saimiri boliviensis boliviensis (Bolivian squirrel monkey) FGF21
    gene coding sequence (SEQ ID NO: 325) (GenBank accession no.
    XM_003940326, which is hereby incorporated by reference
    in its entirety)
    163                                               atgggctc ggaggaggtc
    181 GCGTTGGAGC GCCCTGCACT GTGGGTCTCT GTGTTGGCTG GTCTCCTGCT GGGAACCTGC
    241 CAGGCATACC CCATCCCTGA CTCTAGTCCC CTCCTGCAAT TTGGAGGCCA AGTCCGGCAG
    301 CGGTACCTCT ACACAGATGA CGCTCAGCAG ACAGAAGCCC ACCTGGAGAT CAGGGAAGAT
    361 GGCACGGTGG CGGGGGCTGC CCACCAGAGC CCCGAAAGTC TCTTGCAGCT GAAAGCCTTA
    421 AAGCCAGGGG TTATTCAAAT CTTGGGAGTC AAGACCTCCA GGTTCCTGTG CCAGAGGCCG
    481 GACGGGGCCC TGTACGGATC GCTCTACTTT GACCCCGAGG CCTGCAGCTT CCGGGAGCTG
    541 CTTCTTGAGG ACGGATACAA TGTGTACCAG TCCGTGGCCC ACAGCCTCCC GCTGCACCTG
    601 CCAGGGGGCA GGTCCCCACC CTGGGACCCT GCACCTCGAG GACCAGCTCG CTTCCTGCCG
    661 CTACCAGGCC TGCCCCCCGA ACCCCCCGAG GCGCCAGGAA TCCTGGCCCC CGAGCCCCCC
    721 GATGTGGGCT CCTCAGACCC TCTGAGCATG GTGGGGCCTT CCCAAGGCCA AAGCCCCAGC
    781 TACACTTCCT GA
    Callithrix jacchus (white-tufted-ear marmoset) FGF21 gene coding
    sequence (SEQ ID NO: 326) (GenBank accession no. XM_003735621,
    which is hereby incorporated by reference in its entirety)
    1 ATGGGCTCGG AGGAGGTCGG GTTGGAGCAC CCTGCACTGT GGGTTTCTGT GCTGGCTGGT
    61 CTCCTGCTGG GAACCTGCCA GGCGCACCCC ATCCCTGACT CCAGTCCCCT CCTGCAATTT
    121 GGAGGCCAAG TCCGGCAGCG GTACCTCTAC ACAGATGACG CCCAGCAGAA AGAAGCCCAC
    181 CTGGAGATCN AGGAAGATGG CACAGTGGCC GGGGCTGCCA CCAAAGTCCC GAAAGTGAGT
    241 CTCTTGCAGC TGAAAGCCTT AAAGCCAGGG GTTATTCAAA TCTTGGGAGT CAAGACATCC
    301 AGGTTCCTGT GCCAGAGGCC AGACGGGGCG CTGTATGGAT CGCTCCACTT TGACCCCGAG
    361 GCCTGCAGCT TCCGGGAGCT GCTTCTTGAG GACGGATACA ATGTGTACCA GTCTGTGGCC
    421 CACGGCCTCC CGCTGCACCT GCCAGAGAGC AGGTCACCAC CCCGGGACCC TGCACCCCGA
    481 GGACCAGCTC GCTTCCTGCC ACTACCAGGC CTGCCCCCTG AACCCCCAGA GCCGCCAGGA
    541 ATCCTGGCCC CTGAGCCCCC CGACGTGGGC TCCTCAGACC CTCTGAGCAT GGTGGGGCCT
    601 TCCCAAGGCC AAAGCCCCAG CTACGCTTCC TGA
    Tupaia chinensis (Chinese tree shrew) FGF21 gene coding sequence
    (SEQ ID NO: 327)(generated using SMS Reverse Translate tool on the
    ExPASy Bioinformatics Resource website (www.expasy.org))
    1 ATGGGCTGGG ATAAAGCGCG CTTTGAACAT CTGGGCGCGT GGGCGCCGGT GCTGGCGGTG
    61 CTGCTGCTGG GCGCGTGCCA GGCGTATCCG ATTCCGGATA GCAGCCCGCT GCTGCAGTTT
    121 GGCGGCCAGG TGCGCCAGCG CTATCTGTAT ACCGATGATA CCCAGGATAC CGAAGCGCAT
    181 CTGGAAATTC GCGCGGATGG CACCGTGGTG GGCGCGGCGC ATCAGAGCCC GGAAAGCCTG
    241 CTGGAACTGA AAGCGCTGAA ACCGGGCGTG ATTCAGATTC TGGGCGTGAA AACCAGCCGC
    301 TTTCTGTGCC AGCGCCCGGA TGGCGCGCTG TATGGCAGCC TGCATTTTGA TCCGGAAGCG
    361 TGCAGCTTTC GCGAACTGCT GCTGGAAGAT GGCTATAACA TTTATCAGAG CGAAGCGCGC
    421 GGCCTGCCGC TGCGCCTGCC GCCGCATGAT AGCCCGCATC GCGATCGCAC CCCGCAGGGC
    481 CCGGCGCGCT TTCTGCCGCT GCCGGGCCTG CCGCTGGTGC CGCCGGAACT GCCGGGCGTG
    541 CTGGCGCTGG AACCGCCGGA TGTGGGCAGC AGCGATCCGC TGAGCATGAT GGGCCCGAGC
    601 CAGGGCCAGA GCCCGAGCTA TGCGAGCTAA
    Papio anubis (olive baboon) FGF21 gene coding sequence (SEQ ID
    NO: 328) (GenBank accession no. XM_003915851, which is
    hereby incorporated by reference in its entirety)
    1 ATGGACTCGG ACGAGACCGG GTTCGAGCAC TCAGGACTGT GGGTTCCTGT GCTGGCTGGT
    61 CTTCTGCTGG GAGCCTGCCA GGCACACCCC ATCCCTGACT CCAGTCCTCT CCTGCAATTC
    121 GGGGGCCAAG TCCGGCAACG GTACCTCTAC ACAGATGATG CCCAGCAGAC AGAAGCCCAC
    181 CTGGAGATCA GGGAGGATGG GACAGTGGGG GGCGCTGCTC ACCAGAGCCC CGAAAGTAAG
    241 TGTGGGCCAG AGCCTGGGTC TGAGGGAGGA GGGGCTCTCC ACTTTGACCC TGAGGCCTGC
    301 AGCTTCCGCG AGCTGCTTCT TGAGAACGGA TACAATGTTT ACCAGTCCGA GGCCCACGGC
    361 CTCCCACTGC ACCTGCCGGG AAACAAGTCC CCACACCGGG ACCCTGCATC CCGAGGACCA
    421 GCTCGCTTCC TGCCACTACC AGGCCTGCCC CCCGCACCCC CAGAGCCACC AGGAATCCTC
    481 GCCCCCCAGC CCCCCGATGT GGGCTCCTCG GACCCTCTGA GCATGGTGGG ACCTTCCCAG
    541 GCCCGAAGCC CTAGCTACGC TTCCTGA
    Pteropus alecto (black flying fox) FGF21 gene coding sequence
    (SEQ ID NO: 329) (generated using SMS Reverse Translate tool on the
    ExPASy Bioinformatics Resource website (www.expasy.org))
    1 ATGGGCTGGG GCAAAGCGCG CCTGCAGCAT CCGGGCCTGT GGGGCCCGGT GCTGGCGGTG
    61 CTGCTGGGCG CGTGCCAGGC GCATCCGATT CTGGATAGCA GCCCGCTGTT TCAGTTTGGC
    121 AGCCAGGTGC GCCGCCGCTA TCTGTATACC GATGATGCGC AGGATACCGA AGCGCATCTG
    181 GAAATTCGCG CGGATGGCAC CGTGGCGGGC GCGGCGCGCC GCAGCCCGGA AAGCCTGCTG
    241 GAACTGAAAG CGCTGAAACC GGGCGTGATT CAGGTGCTGG GCGTGAAAAC CAGCCGCTTT
    301 CTGTGCCAGC GCCCGGATGG CACCCTGTAT GGCAGCCTGC ATTTTGATCC GGCGGCGTGC
    361 AGCTTTCGCG AACTGCTGCT GAAAGATGGC TATAACGTGT ATCAGAGCGA AGCGCTGGCG
    421 CGCCCGCTGC GCCTGCCGCC GTATAGCAGC CCGAGCAGCG ATCCGGCGCG CCGCGGCCCG
    481 GCGCGCTTTC TGCCGCTGCC GGGCCCGCCG CCGGAACCGC CGCAGCCGCC GGGCCGCCTG
    541 GCGCCGGAAC CGCCGGATGT GGGCAGCAGC GATCCGCTGA GCATGGTGTG GCCGAGCCGC
    601 GGCCGCAGCC CGAGCTATAC CAGCTAA
    Heterocephalus glaber (naked mole-rat) FGF21 gene coding sequence
    (SEQ ID NO: 330) (generated using SMS Reverse Translate tool on the
    ExPASy Bioinformatics Resource website (www.expasy.org))
    1 ATGGATTGGG CGCGCGCGGA AAGCGAACGC CCGGGCCTGT GGGTGCCGGC GGTGCTGGCG
    61 GTGCTGCTGC TGGGCGCGTG CCAGGCGCAT CCGATTCCGG ATAGCAGCCC GCTGCTGCAG
    121 TTTGGCGGCC AGGTGCGCCA GCGCCATCTG TATACCGATG ATGCGCAGGA TACCGAAGTG
    181 CATCTGGAAA TTCGCGCGGA TGGCAGCGTG GGCGGCGCGG CGCATCGCAG CCCGGAAAGC
    241 CTGCTGGAAC TGAAAGCGCT GAAACCGGGC GTGATTCAGA TTCTGGGCGT GCGCACCAGC
    301 CGCTTTCTGT GCCAGCGCCC GGATGGCACC CTGTATGGCA GCCTGCATTT TGATCCGGAA
    361 GCGTGCAGCT TTCGCGAACT GCTGCTGGCG GATGGCTATA ACATTTATCA GAGCGAAGCG
    421 TATGGCCTGC CGCTGCGCAT GCTGCCGAGC GATAGCGCGA GCCGCGATCC GGTGCCGCCG
    481 GGCCCGGCGC GCTTTCTGCC GCTGCCGGGC CTGCATCCGC CGCCGCTGGA ACCGCCGGGC
    541 ATGCTGCCGC CGGAACCGCC GGATGTGGGC AGCAGCGATC CGCTGAGCAT GGTGGGCCCG
    601 CTGCAGGGCC GCAGCCCGAG CTATGCGTTT TAA
    Cricetulus griseus (Chinese hamster) FGF21 gene coding sequence (SEQ
    ID NO: 331) (GenBank accession no. XM_003508678, which is hereby
    incorporated by reference in its entirety)
    1 ATGGACTGGA TGAAATCTGG AGTTGGGGTC CCGGGACTGT GGGTCCCTCT GCTGCCTATC
    61 TTCCTGCTGG GGGTCTCCCA GGCACACCCC ATCCCTGACT CCAGCCCCCT CCTCCAGTTT
    121 GGGGGTCAAG TCCGGCACAG GCACCTCTAC ACAGATGACA ACCAGGAAAC TGAAGTCCAC
    181 CTGGAGATTA GGCAGGATGG CACGGTGATA GGGACCACAC ACCGCAGCCC AGAAAGTCTC
    241 CTGGAGCTCA AAGCCTTGAA GCCAGAGGTC ATCCCAGTGC TGGGTGTCAA GGCCTCCAGG
    301 TTTCTTTGCC AACAACCAGA CGGAACCCTG TATGGATCGC CTCACTTTGA TCCTGAGGCC
    361 TGCAGTTTCA GGGAGCTCTT GCTTGAGGAT GGATACAATG TGTACCAATC TGAAGTCCAT
    421 GGCCTGCCCC TGCGCCTGCC CCAGAGGGAC TCTCCAAACC AGGCCCCAGC ATCCTGGGGA
    481 CCTGTGCCCC CCCTGCCAGT GCCAGGACTG CTCCACCAGC CCCAGGAGCT ACCAGGGTTC
    541 CTGGCCCCAG AACCTCCAGA TGTGGGCTCC TCTGACCCAC TGAGCATGGT GGGACCTTTG
    601 CAGGGCCGAA GCCCCAGCTA TGCTTCCTGA
    Ovis aries (sheep) FGF21 gene coding sequence (SEQ ID NO: 332)
    (GenBank accession no. XM_004015796, which is hereby
    incorporated by reference in its entirety)
    1 ATGGGCTGGG ACGAGGCCAA GTTCAAGCAC TTGGGACTGT GGGTCCCTGT GCTGGCTGTC
    61 CTCCTGCTAG GAACCTGCCG GGCGCATCCA ATTCCAGACT CCAGCCCCCT CCTCCAGTTT
    121 GGGGGCCAAG TCCGCCAGCG GTACCTCTAC ACGGATGATG CCCAGGAGAC AGAGGCCCAC
    181 CTGGAGATCA GGGCCGATGG CACAGTGGTG GGGGCGGCCC GCCAGAGTCC CGAAAGTCTC
    241 TTGGAGCTGA AAGCCCTGAA GCCAGGAGTC ATTCAGATCT TTGGAGTTAA AACATCCAGG
    301 TTCCTGTGCC AGGGGCCAGA TGGGAAGCTG TATGGATCGC TGCACTTTGA CCCCAAAGCC
    361 TGCAGCTTCC GGGAGCTGCT TCTTGAAGAT GGGTACAATG TCTACCAGTC GGAGACCCTG
    421 GGCCTTCCAC TCCGCCTGCC GCCGCAGCGC TCATCCAACC GGGACCCGGC CCCGCGGGGA
    481 CCTCCGAAGC CCCAGCTACA CTTCTTGAAG ACGTCCGCTG TGCAGTACTG GCCACGTTAT
    541 GAGAAGGTCC CAGCTTTTCT GCACCCCTTC CCCGGCTGA
    Pan paniscus (pygmy chimpanzee) FGF21 gene coding sequence (SEQ
    ID NO: 333) (GenBank accession no. XM_003814115, which is
    hereby incorporated by reference in its entirety) (1-209, excluding
    117-194 and 202-209)
    573                                    ATGGACTC GGACGAGACC GGGTTCGAGC
    601 ACTCAGGACT GTGGGTTTCT GTGCTGGCTG GTCTTCTGCT GGGAGCCTGC CAGGCACACC
    661 CCATCCCTGA CTCCAGTCCT CTCCTGCAAT TCGGGGGCCA AGTCCGGCAG CGGTACCTCT
    721 ACACAGATGA TGCCCAGCAG ACAGAAGCCC ACCTGGAGAT CAGGGAGGAT GGGACGGTGG
    781 GGGGCGCTGC TGACCAGAGC CCCGAAAGTC TCCTGCAGCT GAAAGCCTTG AAGCCGGGAG
    841 TTATTCAAAT CTTGGGAGTC AAGACATCCA GGTTCCTGTG CCAGAGGCCA GATGGGGCCC
    901 TGTATGGATC GGTGAGTTTC ---------- ---------- ---------- ----------
    ---------- ---------- ---------- ---------- ---------- ----------
    921 ---------- ----CAG--- ---------- ---------- ---------- ----------
    924 ---------- -------GAC CCTCCT---- --------CA CCACCCACCA ---------T
    946 GCTCC----- ----TCCTAT ATGTCGCCCTCACAG------ ---CCTGGG
    Macaca fascicularis (crab-eating macaque) FGF21 gene coding sequence
    (SEQ ID NO: 334) (generated using SMS Reverse Translate tool on the
    ExPASy Bioinformatics Resource website (www.expasy.org)) (1-209,
    excluding 117-209)
    1 ATGGATAGCG ATGAAACCGG CTTTGAACAT AGCGGCCTGT GGGTGCCGGT GCTGGCGGGC
    61 CTGCTGCTGG GCGCGTGCCA GGCGCATCCG ATTCCGGATA GCAGCCCGCT GCTGCAGTTT
    121 GGCGGCCAGG TGCGCCAGCG CTATCTGTAT ACCGATGATG CGCAGCAGAC CGAAGCGCAT
    181 CTGGAAATTC GCGAAGATGG CACCGTGGGC GGCGCGGCGC ATCAGAGCCC GGAAAGCCTG
    241 CTGCAGCTGA AAGCGCTGAA ACCGGGCGTG ATTCAGATTC TGGGCGTGAA AACCAGCCGC
    301 TTTCTGTGCC AGAAACCGGA TGGCGCGCTG TATGGCAGCG TGAGCTTTTA A
    Mesocricetus auratus (golden hamster) FGF21 gene coding sequence
    (SEQ ID NO: 335) (GenBank accession no. EU497769, which
    is hereby incorporated by reference in its entirety) (1-209,
    excluding 1-89 and 194-209)
    1 GGTCATCCAA ATCCTGGGTG TCAAGGCTGC TAGGTTTCCT TGCCAGCAAC CAGACGGAAG
    61 CCTGTACGGA TCGCCTCACT TCGATCCCGA GGCCTGCAGT TTCCGGGAGC TCCTGCTTGA
    121 GGATGGATAC AATGTGTACC AGTCGGAAGC CCACGGCCTG CCCCTGCGCC TGCCCCAGAG
    181 GGACGCTCCG AGCCAGCCCC CAGCATCCTG GGGACCGGTG CGCTTCCTGC CAGTGCCCGG
    241 ACTGTTCCAG CCGCCCCACG ACCTCCCAGG GCGCCCGGCC CCAGAGCCTC CGGACGTGGG
    301 CTCCTCCGAC CCAC
    Nile tilapia FGF21 gene coding sequence (SEQ ID NO: 336) (GenBank
    accession no. XM_003438468, which is hereby incorporated by
    reference in its entirety) (1-209, excluding 1-58)
    1 ATGTATTTGC AGATGAACAT GGATGGGAGA GTCACAGGAA GTGATGCTCA GACACCTTAC
    61 AGTTTGATGC AGCTGAAATC AGTTAAACCA GGCCATGTAA TCATTAAAGG ACCATCATCA
    121 TCTCTTTTTC TCTGTGTGGA CAGCGAAGGC AATCTGAGAG GGCAGAGTCA CTACTCAGAA
    181 ACCAGCTGCA CCTTCAGAGA AATGCTGCTG GCTGACGGAT ACACCCGTTT CATTTCCTCA
    241 CAATATGGAT TTCCCATGTC ACTGGCATCA AGACATTCCC CAGATCGACA CGCGCTTCCC
    301 TTTACGCGGT TCCTACCACT GAGGAATAAC TTGAAAACGG ATAGCGTATC AGAGCAGCTG
    361 CCAAACAATC AGAGACTCTT CAACGTGGAC TCTGATGACC TTCTTGGAAT GGGTCTAAAT
    421 TCTATGGGCA GTCCTCAGTT TTCTATGGAC AAATAA
  • In one embodiment of the present invention, the chimeric protein may include one or more substitutions for or additions of amino acids from another FGF. In one embodiment, the C-terminal portion from FGF21 includes a modification that includes a substitution for or addition of amino acid residues from an FGF19 (including a human FGF19 and orthologs of human FGF19). In one embodiment the FGF19 is a human FGF19 protein having an amino acid sequence of SEQ ID NO: 337 (GenBank Accession No. NP005108, which is hereby incorporated by reference in its entirety) or a portion or ortholog thereof, as follows:
  • 1 MRSGCVVVHV WILAGLWLAV AGRPLAFSDA GPHVHYGWGD PIRLRHLYTS GPHGLSSCFL
    61 RIRADGVVDC ARGQSAHSLL EIKAVALRTV AIKGVHSVRY LCMGADGKMQ GLLQYSEEDC
    121 AFEEEIRPDG YNVYRSEKHR LPVSLSSAKQ RQLYKNRGFL PLSHFLPMLP MVPEEPEDLR
    181 GHLESDMFSS PLETDSMDPF GLVTGLEAVR SPSFEK

    Exemplary substitutions and additions of such residues are shown in FIGS. 12 and 13.
  • In one embodiment, the C-terminal portion from FGF21 includes a modification that includes a substitution of amino acid residues from an FGF19 molecule. In one embodiment, the modification includes a substitution for or addition of amino acid residues 169 to 216 of SEQ ID NO: 337 (FGF19). In one embodiment, the modification is a substitution of amino acid residues from SEQ ID NO: 337 (FGF19) for corresponding amino acid residues of SEQ ID NO: 233 (FGF21). The corresponding residues of FGFs may be identified by sequence analysis and/or structural analysis. See FIGS. 2, 11, 12, and 13. In one embodiment, the modification includes a substitution of a contiguous stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acid residues 169 to 216 of SEQ ID NO: 337 (FGF19) for the corresponding contiguous stretch of amino acid residues of SEQ ID NO: 233 (FGF21). In one embodiment, amino acid residues 168 to 209, 191 to 209, or 198 to 209 of SEQ ID NO: 233 (FGF21) are substituted with the corresponding amino acid residues selected from the sequence including amino acid residues 169 to 216 of SEQ ID NO: 337 (FGF19).
  • In one embodiment, the modification includes a substitution of one or more individual amino acid residues from residues 169 to 216 of SEQ ID NO: 337 (FGF19) for the corresponding amino acid residues of SEQ ID NO: 233 (FGF21). In one embodiment, the C-terminal portion includes substitutions of one or more of amino acid residues 168, 169, 170, 171, 173, 174, 177, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 189, 191, 194, 195, 196, 199, 200, 201, 202, 207, 208, or 209 of SEQ ID NO: 233 (FGF21) for the corresponding amino acid residues of SEQ ID NO:337 (FGF19).
  • In one embodiment of the present invention, the C-terminal portion from FGF21 includes a modification that includes an addition of amino acid residues that are present in the corresponding C-terminal portion from FGF19. As shown in FIGS. 11, 12, and 13, FGF19 residues that are absent in the corresponding C-terminal portion of FGF21 may be identified by sequence analysis and/or structural analysis. In one embodiment, the modification includes an addition of amino acid residues selected from residues 204 to 216, 197 to 216, 174 to 216, or 169 to 216 of SEQ ID NO: 337 (FGF19). In one embodiment, the modification includes an addition of amino acid residue 204 of SEQ ID NO: 337 (FGF19). In one embodiment, the modification includes an addition of amino acid residues 178, 179, 180, 181, and/or 182 of SEQ ID NO: 337 (FGF19) individually or in combination.
  • It will be understood that the C-terminal portion from FGF21 that includes a substitution of amino acid residues from an FGF19 molecule may be derived using a nucleotide sequence that encodes a human FGF19 protein having a nucleotide sequence of SEQ ID NO: 338 (Human FGF19 gene coding sequence (1-216); GenBank Accession No. NM005117, which is hereby incorporated by reference in its entirety) or a portion or ortholog thereof, as follows:
  • 464    ATGCGGA GCGGGTGTGT GGTGGTCCAC GTATGGATCC TGGCCGGCCT CTGGCTGGCC
    521 GTGGCCGGGC GCCCCCTCGC CTTCTCGGAC GCGGGGCCCC ACGTGCACTA CGGCTGGGGC
    581 GACCCCATCC GCCTGCGGCA CCTGTACACC TCCGGCCCCC ACGGGCTCTC CAGCTGCTTC
    641 CTGCGCATCC GTGCCGACGG CGTCGTGGAC TGCGCGCGGG GCCAGAGCGC GCACAGTTTG
    701 CTGGAGATCA AGGCAGTCGC TCTGCGGACC GTGGCCATCA AGGGCGTGCA CAGCGTGCGG
    761 TACCTCTGCA TGGGCGCCGA CGGCAAGATG CAGGGGCTGC TTCAGTACTC GGAGGAAGAC
    821 TGTGCTTTCG AGGAGGAGAT CCGCCCAGAT GGCTACAATG TGTACCGATC CGAGAAGCAC
    881 CGCCTCCCGG TCTCCCTGAG CAGTGCCAAA CAGCGGCAGC TGTACAAGAA CAGAGGCTTT
    941 CTTCCACTCT CTCATTTCCT GCCCATGCTG CCCATGGTCC CAGAGGAGCC TGAGGACCTC
    1001 AGGGGCCACT TGGAATCTGA CATGTTCTCT TCGCCCCTGG AGACCGACAG CATGGACCCA
    1061 TTTGGGCTTG TCACCGGACT GGAGGCCGTG AGGAGTCCCA GCTTTGAGAA GTAA
  • In one embodiment, the chimeric protein of the present invention includes the amino acid sequence of SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, or SEQ ID NO: 342, as shown in Table 9.
  • TABLE 9
    Description of Chimeric Protein Sequence
    Amino acid sequence of a SEQ ID NO: 339
    FGF1/FGF21 chimera composed MAEGEITTFT ALTEKFNLPP GNYKKPKLLY
    of residues M1 to L150 of human CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    FGF1 harboring LSAESVGEVY IKSTETGQYL AMDTDGLLYG
    K127D/K128Q/K133V triple SQTPNEECLF LERLEENHYN TYISKKHAEK
    mutation (bold) and residues P168 NWFVGLDQNG SCVRGPRTHY GQKAILFLPL
    to S209 of human FGF21 (bold) PGLPPALPEP PGILAPQPPD VGSSDPLSMV
    GPSQGRSPSY AS
    Amino acid sequence of a SEQ ID NO: 340
    FGF1/FGF21 chimera composed                           KPKLLY
    of residues K25 to L150 of human CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ
    FGF1 harboring LSAESVGEVY IKSTETGQYL AMDTDGLLYG
    K127D/K128Q/K133V triple SQTPNEECLF LERLEENHYN TYISKKHAEK
    mutation (bold) and residues P168 NWFVGLDQNG SCVRGPRTHY GQKAILFLPL
    to S209 of human FGF21 (bold) PGLPPALPEP PGILAPQPPD VGSSDPLSMV
    GPSQGRSPSY AS
    Amino acid sequence of a SEQ ID NO: 341
    FGF2/FGF21 chimera composed MAAGSITTLP ALPEDGGSGA FPPGHFKDPK
    of residues M1 to M151 of human RLYCKNGGFF LRIHPDGRVD GVREKSDPHI
    FGF2 harboring KLQLQAEERG VVSIKGVCAN RYLAMKEDGR
    K128D/R129Q/K134V triple LLASKCVTDE CFFFERLESN NYNTYRSRKY
    mutation (bold) and residues P168 TSWYVALDQT GQYVLGSKTG PGQKAILFLP
    to S209 of human FGF21 (bold) MPGLPPALPEPPGILAPQPP DVGSSDPLSM
    VGPSQGRSPS YAS
    Amino acid sequence of a SEQ ID NO: 342
    FGF2/FGF21 chimera composed                           HFKDPK
    of residues H25 to M151 of RLYCKNGGFF LRIHPDGRVD GVREKSDPHI
    human FGF2 harboring KLQLQAEERG VVSIKGVCAN RYLAMKEDGR
    K128D/R129Q/K134V triple LLASKCVTDE CFFFERLESN NYNTYRSRKY
    mutation (bold) and residues P168 TSWYVALDQT GQYVLGSKTG PGQKAILFLP
    to S209 of human FGF21 (bold) MPGLPPALPE PPGILAPQPP DVGSSDPLSM
    VGPSQGRSPS YAS
  • Chimeric proteins according to the present invention may be isolated proteins or polypeptides. The isolated chimeric proteins of the present invention may be prepared for use in accordance with the present invention using standard methods of synthesis known in the art, including solid phase peptide synthesis (Fmoc or Boc strategies) or solution phase peptide synthesis. Alternatively, peptides of the present invention may be prepared using recombinant expression systems.
  • Accordingly, another aspect of the present invention relates to an isolated nucleic acid molecule encoding a chimeric protein according to the present invention. In one embodiment, the nucleic acid molecule includes the nucleotide sequence of SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, or SEQ ID NO: 346 (as shown in Table 10).
  • TABLE 10
    Description of Chimeric Protein Sequence
    Nucleotide sequence of a SEQ ID NO: 343
    FGF1/FGF21 chimera composed ATGGCTGAAG GGGAAATCAC CACCTTCACA
    of residues M1 to L150 of human GCCCTGACCG AGAAGTTTAA TCTGCCTCCA
    FGF1 harboring GGGAATTACA AGAAGCCCAA ACTCCTCTAC
    K127D/K128Q/K133V triple TGTAGCAACG GGGGCCACTT CCTGAGGATC
    mutation (bold) and residues P168 CTTCCGGATG GCACAGTGGA TGGGACAAGG
    to S209 of human FGF21 (bold) GACAGGAGCG ACCAGCACAT TCAGCTGCAG
    CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT
    ATAAAGAGTA CCGAGACTGG CCAGTACTTG
    GCCATGGACA CCGACGGGCT TTTATACGGC
    TCACAGACAC CAAATGAGGA ATGTTTGTTC
    CTGGAAAGGC TGGAGGAGAA CCATTACAAC
    ACCTATATAT CCAAGAAGCA TGCAGAGAAG
    AATTGGTTTG TTGGCCTCGATCAGAATGGG
    AGCTGCGTTC GCGGTCCTCG GACTCACTAT
    GGCCAGAAAG CAATCTTGTT TCTCCCCCTG
    CCAGGCCTGC CCCCCGCACT CCCGGAGCCA
    CCCGGAATCC TGGCCCCCCA GCCCCCCGAT
    GTGGGCTCCT CGGACCCTCT GAGCATGGTG
    GGACCTTCCC AGGGCCGAAG CCCCAGCTAC
    GCTTCC
    Nucleotide sequence of a SEQ ID NO: 344
    FGF1/FGF21 chimera composed              AAGCCCAA ACTCCTCTAC
    of residues K25 to L150 of human TGTAGCAACG GGGGCCACTT CCTGAGGATC
    FGF1 harboring CTTCCGGATG GCACAGTGGA TGGGACAAGG
    K127D/K128Q/K133V triple GACAGGAGCG ACCAGCACAT TCAGCTGCAG
    mutation (bold) and residues P168 CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT
    to S209 of human FGF21 (bold) ATAAAGAGTA CCGAGACTGG CCAGTACTTG
    GCCATGGACA CCGACGGGCT TTTATACGGC
    TCACAGACAC CAAATGAGGA ATGTTTGTTC
    CTGGAAAGGC TGGAGGAGAA CCATTACAAC
    ACCTATATAT CCAAGAAGCA TGCAGAGAAG
    AATTGGTTTG TTGGCCTCGATCAGAATGGG
    AGCTGCGTTC GCGGTCCTCG GACTCACTAT
    GGCCAGAAAG CAATCTTGTT TCTCCCCCTG
    CCAGGCCTGC CCCCCGCACT CCCGGAGCCA
    CCCGGAATCC TGGCCCCCCA GCCCCCCGAT
    GTGGGCTCCT CGGACCCTCT GAGCATGGTG
    GGACCTTCCC AGGGCCGAAG CCCCAGCTAC
    GCTTCC
    Nucleotide sequence of a SEQ ID NO: 345
    FGF2/FGF21 chimera composed ATG GCACAGTGGA
    of residues M1 to M151 of human GCATCACCAC GCTGCCCGCC TTGCCCGAGG
    FGF2 harboring ATGGCGGCAG CGGCGCCTTC CCGCCCGGCC
    K128D/R129Q/K134V triple ACTTCAAGGA CCCCAAGCGG CTGTACTGCA
    mutation (bold) and residues P168 AAAACGGGGG CTTCTTCCTG CGCATCCACC
    to S209 of human FGF21 (bold) CCGACGGCCG AGTTGACGGG GTCCGGGAGA
    AGAGCGACCC TCACATCAAG CTACAACTTC
    AAGCAGAAGA GAGAGGAGTT GTGTCTATCA
    AAGGAGTGTG TGCTAACCGT TACCTGGCTA
    TGAAGGAAGA TGGAAGATTA CTGGCTTCTA
    AATGTGTTAC GGATGAGTGT TTCTTTTTTG
    AACGATTGGA ATCTAATAAC TACAATACTT
    ACCGGTCAAG GAAATACACC AGTTGGTATG
    TGGCACTGGATCAGACTGGG CAGTATGTTC
    TTGGATCCAA AACAGGACCT GGGCAGAAAG
    CTATACTTTT TCTTCCAATG CCAGGCCTGC
    CCCCCGCACT CCCGGAGCCA CCCGGAATCC
    TGGCCCCCCA GCCCCCCGAT GTGGGCTCCT
    CGGACCCTCT GAGCATGGTG GGACCTTCCC
    AGGGCCGAAG CCCCAGCTAC GCTTCC
    Nucleotide sequence of a SEQ ID NO: 346
    FGF2/FGF21 chimera composed                                C
    of residues H25 to M151 of ACTTCAAGGA CCCCAAGCGG CTGTACTGCA
    human FGF2 harboring AAAACGGGGG CTTCTTCCTG CGCATCCACC
    K128D/R129Q/K134V triple CCGACGGCCG AGTTGACGGG GTCCGGGAGA
    mutation (bold) and residues P168 AGAGCGACCC TCACATCAAG CTACAACTTC
    to S209 of human FGF21 (bold) AAGCAGAAGA GAGAGGAGTT GTGTCTATCA
    AAGGAGTGTG TGCTAACCGT TACCTGGCTA
    TGAAGGAAGA TGGAAGATTA CTGGCTTCTA
    AATGTGTTAC GGATGAGTGT TTCTTTTTTG
    AACGATTGGA ATCTAATAAC TACAATACTT
    ACCGGTCAAG GAAATACACC AGTTGGTATG
    TGGCACTGGATCAGACTGGG CAGTATGTTC
    TTGGATCCAA AACAGGACCT GGGCAGAAAG
    CTATACTTTT TCTTCCAATG CCAGGCCTGC
    CCCCCGCACT CCCGGAGCCA CCCGGAATCC
    TGGCCCCCCA GCCCCCCGAT GTGGGCTCCT
    CGGACCCTCT GAGCATGGTG GGACCTTCCC
    AGGGCCGAAG CCCCAGCTAC GCTTCC
  • Another aspect of the present invention relates to a nucleic acid construct including a nucleic acid molecule encoding a chimeric protein according to the present invention, a 5′ DNA promoter sequence, and a 3′ terminator sequence. The nucleic acid molecule, the promoter, and the terminator are operatively coupled to permit transcription of the nucleic acid molecule.
  • Also encompassed are vectors or expression vectors including such nucleic acid molecules and host cells including such nucleic acid molecules. Nucleic acid molecules according to the present invention can be expressed in a host cell, and the encoded polynucleotides isolated, according to techniques that are known in the art.
  • Generally, the use of recombinant expression systems involves inserting the nucleic acid molecule encoding the amino acid sequence of the desired peptide into an expression system to which the molecule is heterologous (i.e., not normally present). One or more desired nucleic acid molecules encoding a peptide of the invention may be inserted into the vector. When multiple nucleic acid molecules are inserted, the multiple nucleic acid molecules may encode the same or different peptides. The heterologous nucleic acid molecule is inserted into the expression system or vector in proper sense (5′→3′) orientation relative to the promoter and any other 5′ regulatory molecules, and correct reading frame.
  • The preparation of the nucleic acid constructs can be carried out using standard cloning procedures well known in the art as described by Joseph Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Harbor 1989). U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in a suitable host cell.
  • A variety of genetic signals and processing events that control many levels of gene expression (e.g., DNA transcription and messenger RNA (“mRNA”) translation) can be incorporated into the nucleic acid construct to maximize protein production. For the purposes of expressing a cloned nucleic acid sequence encoding a desired protein, it is advantageous to use strong promoters to obtain a high level of transcription. Depending upon the host system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV 5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene. Common promoters suitable for directing expression in mammalian cells include, without limitation, SV40, MMTV, metallothionein-1, adenovirus Ela, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV-LTR.
  • There are other specific initiation signals required for efficient gene transcription and translation in prokaryotic cells that can be included in the nucleic acid construct to maximize protein production. Depending on the vector system and host utilized, any number of suitable transcription and/or translation elements, including constitutive, inducible, and repressible promoters, as well as minimal 5′ promoter elements, enhancers or leader sequences may be used. For a review on maximizing gene expression see Roberts and Lauer, “Maximizing Gene Expression On a Plasmid Using Recombination In Vitro,” Methods in Enzymology 68:473-82 (1979), which is hereby incorporated by reference in its entirety.
  • A nucleic acid molecule encoding an isolated protein of the present invention, a promoter molecule of choice, including, without limitation, enhancers, and leader sequences; a suitable 3′ regulatory region to allow transcription in the host, and any additional desired components, such as reporter or marker genes, are cloned into the vector of choice using standard cloning procedures in the art, such as described in Joseph Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Harbor 1989); Frederick M. Ausubel, SHORT PROTOCOLS IN MOLECULAR BIOLOGY (Wiley 1999); and U.S. Pat. No. 4,237,224 to Cohen and Boyer, which are hereby incorporated by reference in their entirety.
  • Once the nucleic acid molecule encoding the protein has been cloned into an expression vector, it is ready to be incorporated into a host. Recombinant molecules can be introduced into cells, without limitation, via transfection (if the host is a eukaryote), transduction, conjugation, mobilization, or electroporation, lipofection, protoplast fusion, mobilization, or particle bombardment, using standard cloning procedures known in the art, as described by JOSEPH SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Harbor 1989), which is hereby incorporated by reference in its entirety.
  • A variety of suitable host-vector systems may be utilized to express the recombinant protein or polypeptide. Primarily, the vector system must be compatible with the host used. Host-vector systems include, without limitation, the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria.
  • Purified proteins may be obtained by several methods readily known in the art, including ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, gel filtration, and reverse phase chromatography. The protein is preferably produced in purified form (preferably at least about 80% or 85% pure, more preferably at least about 90% or 95% pure) by conventional techniques. Depending on whether the recombinant host cell is made to secrete the protein into growth medium (see U.S. Pat. No. 6,596,509 to Bauer et al., which is hereby incorporated by reference in its entirety), the protein can be isolated and purified by centrifugation (to separate cellular components from supernatant containing the secreted protein) followed by sequential ammonium sulfate precipitation of the supernatant. The fraction containing the protein is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the protein of interest from other proteins. If necessary, the protein fraction may be further purified by HPLC.
  • Another aspect of the present invention relates to a pharmaceutical composition that includes a chimeric protein according to the present invention and a pharmaceutically acceptable carrier.
  • “Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.
  • The term “pharmaceutically acceptable” means it is, within the scope of sound medical judgment, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and is commensurate with a reasonable benefit/risk ratio.
  • In one embodiment, the pharmaceutical composition includes an organotropic targeting agent. In one embodiment, the targeting agent is covalently linked to the chimeric protein via a linker that is cleaved under physiological conditions.
  • Chimeric and/or modified proteins according to the present invention may also be modified using one or more additional or alternative strategies for prolonging the in vivo half-life of the protein. One such strategy involves the generation of D-peptide chimeric proteins, which consist of unnatural amino acids that are not cleaved by endogenous proteases. Alternatively, the chimeric and/or modified proteins may be fused to a protein partner that confers a longer half-life to the protein upon in vivo administration. Suitable fusion partners include, without limitation, immunoglobulins (e.g., the Fc portion of an IgG), human serum albumin (HAS) (linked directly or by addition of the albumin binding domain of streptococcal protein G), fetuin, or a fragment of any of these. The chimeric and/or modified proteins may also be fused to a macromolecule other than protein that confers a longer half-life to the protein upon in vivo administration. Suitable macromolecules include, without limitation, polyethylene glycols (PEGs). Methods of conjugating proteins or peptides to polymers to enhance stability for therapeutic administration are described in U.S. Pat. No. 5,681,811 to Ekwuribe, which is hereby incorporated by reference in its entirety. Nucleic acid conjugates are described in U.S. Pat. No. 6,528,631 to Cook et al., U.S. Pat. No. 6,335,434 to Guzaev et al., U.S. Pat. No. 6,235,886 to Manoharan et al., U.S. Pat. No. 6,153,737 to Manoharan et al., U.S. Pat. No. 5,214,136 to Lin et al., or U.S. Pat. No. 5,138,045 to Cook et al., which are hereby incorporated by reference in their entirety.
  • The pharmaceutical composition according to the present invention can be formulated for administration orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • Another aspect of the present invention relates to a method for treating a subject suffering from a disorder. This method involves selecting a subject suffering from the disorder and administering the pharmaceutical composition according to the present invention to the selected subject under conditions effective to treat the disorder. In one embodiment the disorder is diabetes, obesity, or metabolic syndrome.
  • Another aspect of the present invention relates to a method for treating a subject suffering from a disorder. This method involves selecting a subject suffering from the disorder and providing a chimeric FGF protein, where the chimeric FGF protein includes an N-terminus coupled to a C-terminus. The N-terminus includes a portion of a paracrine FGF and the C-terminus includes a C-terminal portion of FGF21. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. This method also involves administering a therapeutically effective amount of the chimeric FGF protein to the selected subject under conditions effective to treat the disorder.
  • Suitable chimeric proteins for use in accordance with this aspect of the present invention are described above and throughout the present application.
  • In one embodiment, the selected subject is a mammal. In one embodiment, the selected subject is a human. In another embodiment, the selected subject is a rodent.
  • In one embodiment, the selected subject is in need of increased FGF21-βKlotho-FGF receptor (“FGFR”) complex formation.
  • In one embodiment, the disorder is a selected from diabetes, obesity, and metabolic syndrome. As used herein, diabetes includes type I diabetes, type II diabetes, gestational diabetes, and drug-induced diabetes. In yet another embodiment, the subject has obesity. In yet another embodiment, the subject has metabolic syndrome.
  • The chimeric protein of the present invention or pharmaceutical composition thereof can be used to treat a number of conditions. In one embodiment, the condition is one which the therapeutic outcome includes a decrease in blood glucose, a decrease in blood fructosamine, an increase in energy expenditure, an increase in fat utilization, a decrease in body weight, a decrease in body fat, a decrease in triglycerides, a decrease in free fatty acids, an increase in fat excretion, an improvement, or even a preservation, of pancreatic β-cell function and mass, a decrease in total blood cholesterol, a decrease in blood low-density lipoprotein cholesterol, an increase in blood high-density lipoprotein cholesterol, an increase in blood adiponectin, an increase in insulin sensitivity, an increase in leptin sensitivity, a decrease in blood insulin, a decrease in blood leptin, a decrease in blood glucagon, an increase in glucose uptake by adipocytes, a decrease in fat accumulation in hepatocytes, and/or an increase in fat oxidation in hepatocytes. Each of these parameters can be measured by standard methods, for example, by measuring oxygen consumption to determine metabolic rate, using scales to determine weight, and measuring lean body mass composition or mass to determine fat. Moreover, the presence and amount of triglycerides, free fatty acids, glucose and leptin can be determined by standard methods (e.g., blood test).
  • Additional conditions that are treatable in accordance with the present invention include one or more of type 1 diabetes, type 2 diabetes, gestational diabetes, drug-induced diabetes, high blood glucose, metabolic syndrome, lipodystrophy syndrome, dyslipidemia, insulin resistance, leptin resistance, atherosclerosis, vascular disease, inflammatory disease, fibrotic disease, hypercholesterolemia, hypertriglyceridemia, non-alcoholic fatty liver disease, overweight, and obesity.
  • In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof is administered with a pharmaceutically-acceptable carrier.
  • The chimeric protein according to the present invention or pharmaceutical composition thereof can be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes. The most suitable route may depend on the condition and disorder of the recipient. Formulations including chimeric proteins according to the present invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Those skilled in the art can readily optimize pharmaceutically effective dosages and administration regimens for therapeutic compositions including the chimeric protein according to the present invention, as determined by good medical practice and the clinical condition of the individual patient.
  • When in vivo administration of a chimeric protein of the present invention or is employed, normal dosage amounts may vary from, for example, about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day. In one embodiment, the dosage may be from about 1 μg/kg/day to 10 mg/kg/day, depending upon the route of administration. In one embodiment, the chimeric protein according to the present invention is administered at a dose of about 0.1 to 10 mg/kg once or twice daily. In one embodiment, the chimeric protein according to the present invention is administered at a dose of about 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 mg/kg. In one embodiment, the dosage is the same as that of a native FGF21 therapeutic. In one embodiment, the dosage is less than that of a native FGF21 therapeutic, but has the same effect as a higher dosage of a native FGF21 therapeutic. Guidance as to particular dosages and methods of delivery of proteins is provided in the literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212, which are hereby incorporated by reference in their entirety. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue.
  • Where sustained-release administration of a chimeric protein of the present invention is desired in a formulation with release characteristics suitable for the treatment of any disease or disorder requiring administration of the chimeric protein of the present invention, microencapsulation is contemplated. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., “Preparation and Characterization of Poly(D,L-lactide-co-glycolide) Microspheres for Controlled Release of Human Growth Hormone,” Nat. Med. 2:795-799 (1996); Yasuda, “Sustained Release Formulation of Interferon,” Biomed. Ther. 27:1221-1223 (1993); Hora et al., “Controlled Release of Interleukin-2 from Biodegradable Microspheres,” Nat. Biotechnol. 8:755-758 (1990); Cleland, “Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems,” in VACCINE DESIGN: THE SUBUNIT AND ADJUVANT APPROACH 439-462 (Powell and Newman, eds. 1995); WO 97/03692; WO 96/40072; WO 96/07399; and U.S. Pat. No. 5,654,010, which are hereby incorporated by reference in their entirety. The sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids, can be cleared quickly within the human body. Moreover, the degradability of this polymer can be adjusted from months to years depending on its molecular weight and composition. Lewis, “Controlled release of bioactive agents from lactide/glycolide polymer,” in: BIODEGRADABLE POLYMERS AS DRUG DELIVERY SYSTEMS 1-41 (M. Chasin and R. Langer eds. 1990), which is hereby incorporated by reference in its entirety.
  • The chimeric protein of the present invention or pharmaceutical composition thereof may be administered as frequently as necessary in order to obtain the desired therapeutic effect. Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. For other patients, it may be necessary to have long-term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each particular patient. For other patients, it will be necessary to prescribe not more than one or two doses per day.
  • In some embodiments, the chimeric protein of the present invention or a pharmaceutical composition thereof is administered in a therapeutically effective amount in combination with a therapeutically effective amount of a second agent. In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof is administered in conjunction with the second agent, i.e., the respective periods of administration are part of a single administrative regimen. In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof and the second agent are administered concurrently, i.e., the respective periods of administration overlap each other. In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof and the second agent are administered non-concurrently, i.e., the respective periods of administration do not overlap each other. In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof and the second agent are administered sequentially, i.e., the chimeric protein of the present invention or pharmaceutical composition thereof is administered prior to and/or after the administration of the second agent. In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof and the second agent are administered simultaneously as separate compositions. In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof and the second agent are administered simultaneously as part of the same compositions.
  • In one embodiment, the second agent is an anti-inflammatory agent, an antifibrotic agent, an antihypertensive agent, an anti-diabetic agent, a triglyceride-lowering agent, and/or cholesterol-lowering drug such as a drug of the “statin” class. In one embodiment, the second agent is insulin. In one embodiment, the insulin is rapid acting, short acting, regular acting, intermediate acting, or long acting insulin. In one embodiment, the insulin is and/or comprises Humalog®, Lispro, Novolog®, Apidra®, Humulin®, Aspart, regular insulin, NPH, Lente, Ultralente, Lantus®, Glargine, Levemir®, or Detemir. In one embodiment, the second agent is a statin. In one embodiment, the statin is and/or comprises Atorvastatin (e.g., Lipitor® or Torvast®), Cerivastatin (e.g., Lipobay® or Baycol®), Fluvastatin (e.g., Lescol® or LescolXL®), Lovastatin (e.g., Mevacor®, Altocor®, or Altoprev®) Mevastatin, Pitavastatin (e.g., Livalo® or Pitava®), Pravastatin (e.g., Pravachol®, Selektine, or Lipostat®) Rosuvastatin (e.g., Crestor®), Simvastatin (e.g., Zocor® or Lipex®), Vytorin®, Advicor®, Besylate Caduet® or Simcor®.
  • In one embodiment of the present invention, the chimeric protein according to the present invention or the pharmaceutical composition thereof is administered with an anti-inflammatory agent, an antifibrotic agent, an antihypertensive agent, an antidiabetic agent, a triglyceride-lowering agent, and/or a cholesterol-lowering agent.
  • Another aspect of the present invention relates to a method of making a chimeric FGF protein possessing enhanced endocrine activity. This method involves introducing one or more modifications to an FGF protein, where the modification decreases the affinity of the FGF protein for heparin and/or heparan sulfate and coupling a Klotho co-receptor binding domain to the modified FGF protein's C-terminus, whereby a chimeric FGF protein possessing enhanced endocrine activity is made.
  • In one embodiment, the method includes selecting a Klotho co-receptor binding domain, where the Klotho co-receptor binding domain is selected to target an endocrine FGF target tissue. In one embodiment, the Klotho co-receptor binding domain is selected to home the chimeric FGF protein into a target tissue of endocrine FGF. In one embodiment, the Klotho co-receptor binding domain is selected to target white adipose tissue, brown adipose tissue, skeletal muscle, pancreas, and/or liver.
  • In one embodiment, the Klotho co-receptor binding domain includes a β-Klotho co-receptor binding domain. In one embodiment, the β-Klotho co-receptor binding domain includes a C-terminal portion from FGF21. In one embodiment, the C-terminal portion from the FGF21 includes amino acid residues 168-209 of SEQ ID NO: 233. In one embodiment, the C-terminal portion derived from FGF21 further includes one or more substitutions while retaining the ability to bind β-Klotho. In one embodiment, the C-terminal portion derived from FGF21 further includes one or more substitutions to enhance its binding affinity for β-Klotho. In one embodiment, the C-terminal portion from FGF21 is derived from a mammalian FGF21. In one embodiment, the C-terminal portion derived from FGF21 is from a vertebrate FGF21. Suitable FGF21 molecules, C-terminal portions thereof, and modifications thereto, are described above.
  • In one embodiment, the chimeric FGF protein has greater binding affinity for FGFR than native endocrine FGF ligand having the Klotho co-receptor binding domain. In one embodiment, the chimeric FGF protein possesses enhanced endocrine activity compared to the chimeric FGF protein in the absence of the modification or the Klotho co-receptor binding domain. In one embodiment, the native endocrine FGF ligand having the Klotho co-receptor binding domain is native FGF21. In one embodiment, the FGFR is FGFR1c, FGFR2c, or FGFR4.
  • In one embodiment, the chimeric FGF protein has greater stability than a native endocrine FGF ligand possessing the Klotho co-receptor binding domain. In one embodiment, increasing the stability includes an increase in thermal stability of the protein as compared to either wild type protein or native endocrine FGF ligand. In one embodiment, increasing the stability includes increasing the half-life of the protein in the blood circulation as compared to wild type protein or native endocrine FGF ligand.
  • In one embodiment, the method involves introducing one or more modifications to the FGF protein, where the modification alters the receptor-binding specificity of the FGF protein. In one embodiment, the method involves introducing one or more modifications to the FGF protein, where the modification alters the receptor-binding affinity of the FGF protein.
  • In one embodiment, the FGF is derived from a mammalian FGF. In one embodiment, the FGF is derived from a vertebrate FGF. In one embodiment, the FGF protein is a paracrine FGF molecule. In one embodiment the FGF molecule is FGF1 or FGF2. In one embodiment, the FGF protein is an FGF protein that possesses intrinsically greater binding affinity for FGF receptor than a native endocrine FGF ligand. In one embodiment, the FGF protein is an FGF protein that possesses intrinsically greater thermal stability than a native endocrine FGF ligand. In one embodiment, the method involves introducing one or more modifications to the FGF protein, where the modification alters receptor-binding specificity and/or receptor-binding affinity of the FGF protein. In one embodiment, the method involves introducing one or more modifications to the FGF protein, where the modification alters the stability of the FGF protein. For example, receptor-binding specificity of FGF1, which by nature binds to all the seven principal FGFRs, may be altered to, for example, reduce any risk for adverse effects (e.g., mitogenicity). Paracrine FGFs, portions of paracrine FGFs, and modifications thereto are described above.
  • In one embodiment, the chimeric FGF protein is effective to treat diabetes, obesity, and/or metabolic syndrome.
  • Suitable methods of generating chimeric proteins according to the present invention include standard methods of synthesis known in the art, as described above.
  • Yet another aspect of the present invention relates to a method of facilitating fibroblast growth factor receptor (“FGFR”)-βKlotho co-receptor complex formation. This method involves providing a cell that includes a βKlotho co-receptor and an FGFR and providing a chimeric FGF protein. The chimeric FGF protein includes a C-terminal portion of FGF21 and a portion of a paracrine FGF, where the portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. This method also involves contacting the cell and the chimeric FGF protein under conditions effective to cause FGFR-βKlotho co-receptor complex formation.
  • The portion of the paracrine FGF may also be modified to alter receptor-binding specificity and/or receptor-binding affinity of the FGF, as noted above. Suitable portions of the paracrine FGFs for use in accordance with the present invention, as well as modifications to alter receptor-binding specificity and/or receptor-binding affinity of the FGF, are described above. Suitable modifications to the paracrine FGFs for use in accordance with the present invention are also described above. Suitable C-terminal portions from FGF21 are described above and throughout the present application.
  • In one embodiment according to the present invention, βKlotho is mammalian βKlotho. In one embodiment, βKlotho is human or mouse βKlotho. In one particular embodiment of the present invention, βKlotho is human or mouse βKlotho having the amino acid sequence of SEQ ID NO: 347 (i.e., GenBank Accession No. NP783864, which is hereby incorporated by reference in its entirety) or SEQ ID NO: 348 (i.e., GenBank Accession No. NP112457, which is hereby incorporated by reference in its entirety), respectively, as follows:
  • SEQ ID NO: 347:
    1 MKPGCAAGSP GNEWIFFSTD EITTRYRNTM SNGGLQRSVI LSALILLRAV TGFSGDGRAI
    61 WSKNPNFTPV NESQLFLYDT FPKNFFWGIG TGALQVEGSW KKDGKGPSIW DHFIHTHLKN
    121 VSSTNGSSDS YIFLEKDLSA LDFIGVSFYQ FSISWPRLFP DGIVTVANAK GLQYYSTLLD
    181 ALVLRNIEPI VTLYHWDLPL ALQEKYGGWK NDTIIDIFND YATYCFQMFG DRVKYWITIH
    241 NPYLVAWHGY GTGMHAPGEK GNLAAVYTVG HNLIKAHSKV WHNYNTHFRP HQKGWLSITL
    301 GSHWIEPNRS ENTMDIFKCQ QSMVSVLGWF ANPIHGDGDY PEGMRKKLFS VLPIFSEAEK
    361 HEMRGTADFF AFSFGPNNFK PLNTMAKMGQ NVSLNLREAL NWIKLEYNNP RILIAENGWF
    421 TDSRVKTEDT TAIYMMKNFL SQVLQAIRLD EIRVFGYTAW SLLDGFEWQD AYTIRRGLFY
    481 VDFNSKQKER KPKSSAHYYK QIIRENGFSL KESTPDVQGQ FPCDFSWGVT ESVLKPESVA
    541 SSPQFSDPHL YVWNATGNRL LHRVEGVRLK TRPAQCTDFV NIKKQLEMLA RMKVTHYRFA
    601 LDWASVLPTG NLSAVNRQAL RYYRCVVSEG LKLGISAMVT LYYPTHAHLG LPEPLLHADG
    661 WLNPSTAEAF QAYAGLCFQE LGDLVKLWIT INEPNRLSDI YNRSGNDTYG AAHNLLVAHA
    721 LAWRLYDRQF RPSQRGAVSL SLHADWAEPA NPYADSHWRA AERFLQFEIA WFAEPLFKTG
    781 DYPAAMREYI ASKHRRGLSS SALPRLTEAE RRLLKGTVDF CALNHFTTRF VMHEQLAGSR
    841 YDSDRDIQFL QDITRLSSPT RLAVIPWGVR KLLRWVRRNY GDMDIYITAS GIDDQALEDD
    901 RLRKYYLGKY LQEVLKAYLI DKVRIKGYYA FKLAEEKSKP RFGFFTSDFK AKSSIQFYNK
    961 VISSRGFPFE NSSSRCSQTQ ENTECTVCLF LVQKKPLIFL GCCFFSTLVL LLSIAIFQRQ
    1021 KRRKFWKAKN LQHIPLKKGK RVVS
    SEQ ID NO: 348:
    1 MKTGCAAGSP GNEWIFFSSD ERNTRSRKTM SNRALQRSAV LSAFVLLRAV TGFSGDGKAI
    61 WDKKQYVSPV NPSQLFLYDT FPKNFSWGVG TGAFQVEGSW KTDGRGPSIW DRYVYSHLRG
    121 VNGTDRSTDS YIFLEKDLLA LDFLGVSFYQ FSISWPRLFP NGTVAAVNAQ GLRYYRALLD
    181 SLVLRNIEPI VTLYHWDLPL TLQEEYGGWK NATMIDLFND YATYCFQTFG DRVKYWITIH
    241 NPYLVAWHGF GTGMHAPGEK GNLTAVYTVG HNLIKAHSKV WHNYDKNFRP HQKGWLSITL
    301 GSHWIEPNRT DNMEDVINCQ HSMSSVLGWF ANPIHGDGDY PEFMKTGAMI PEFSEAEKEE
    361 VRGTADFFAF SFGPNNFRPS NTVVKMGQNV SLNLRQVLNW IKLEYDDPQI LISENGWFTD
    421 SYIKTEDTTA IYMMKNFLNQ VLQAIKFDEI RVFGYTAWTL LDGFEWQDAY TTRRGLFYVD
    481 FNSEQKERKP KSSAHYYKQI IQDNGFPLKE STPDMKGRFP CDFSWGVTES VLKPEFTVSS
    541 PQFTDPHLYV WNVTGNRLLY RVEGVRLKTR PSQCTDYVSI KKRVEMLAKM KVTHYQFALD
    601 WTSILPTGNL SKVNRQVLRY YRCVVSEGLK LGVFPMVTLY HPTHSHLGLP LPLLSSGGWL
    661 NMNTAKAFQD YAELCFRELG DLVKLWITIN EPNRLSDMYN RTSNDTYRAA HNLMIAHAQV
    721 WHLYDRQYRP VQHGAVSLSL HCDWAEPANP FVDSHWKAAE RFLQFEIAWF ADPLFKTGDY
    781 PSVMKEYIAS KNQRGLSSSV LPRFTAKESR LVKGTVDFYA LNHFTTRFVI HKQLNTNRSV
    841 ADRDVQFLQD ITRLSSPSRL AVTPWGVRKL LAWIRRNYRD RDIYITANGI DDLALEDDQI
    901 RKYYLEKYVQ EALKAYLIDK VKIKGYYAFK LTEEKSKPRF GFFTSDFRAK SSVQFYSKLI
    961 SSSGLPAENR SPACGQPAED TDCTICSFLV EKKPLIFFGC CFISTLAVLL SITVFHHQKR
    1021 RKFQKARNLQ NIPLKKGHSR VFS
  • In one particular embodiment of the present invention, βKlotho is human or mouse βKlotho encoded by a nucleotide sequence having the nucleotide sequences of SEQ ID NO: 349 (GenBank Accession No. NM175737, which is hereby incorporated by reference in its entirety) and SEQ ID NO: 350 (GenBank Accession No. NM031180, which is hereby incorporated by reference in its entirety), as follows:
  • SEQ ID NO: 349 (Human βKlotho gene coding sequence):
    98        ATG AAGCCAGGCT GTGCGGCAGG ATCTCCAGGG AATGAATGGA TTTTCTTCAG
    151 CACTGATGAA ATAACCACAC GCTATAGGAA TACAATGTCC AACGGGGGAT TGCAAAGATC
    211 TGTCATCCTG TCAGCACTTA TTCTGCTACG AGCTGTTACT GGATTCTCTG GAGATGGAAG
    271 AGCTATATGG TCTAAAAATC CTAATTTTAC TCCGGTAAAT GAAAGTCAGC TGTTTCTCTA
    331 TGACACTTTC CCTAAAAACT TTTTCTGGGG TATTGGGACT GGAGCATTGC AAGTGGAAGG
    391 GAGTTGGAAG AAGGATGGAA AAGGACCTTC TATATGGGAT CATTTCATCC ACACACACCT
    451 TAAAAATGTC AGCAGCACGA ATGGTTCCAG TGACAGTTAT ATTTTTCTGG AAAAAGACTT
    511 ATCAGCCCTG GATTTTATAG GAGTTTCTTT TTATCAATTT TCAATTTCCT GGCCAAGGCT
    571 TTTCCCCGAT GGAATAGTAA CAGTTGCCAA CGCAAAAGGT CTGCAGTACT ACAGTACTCT
    631 TCTGGACGCT CTAGTGCTTA GAAACATTGA ACCTATAGTT ACTTTATACC ACTGGGATTT
    691 GCCTTTGGCA CTACAAGAAA AATATGGGGG GTGGAAAAAT GATACCATAA TAGATATCTT
    751 CAATGACTAT GCCACATACT GTTTCCAGAT GTTTGGGGAC CGTGTCAAAT ATTGGATTAC
    811 AATTCACAAC CCATATCTAG TGGCTTGGCA TGGGTATGGG ACAGGTATGC ATGCCCCTGG
    871 AGAGAAGGGA AATTTAGCAG CTGTCTACAC TGTGGGACAC AACTTGATCA AGGCTCACTC
    931 GAAAGTTTGG CATAACTACA ACACACATTT CCGCCCACAT CAGAAGGGTT GGTTATCGAT
    991 CACGTTGGGA TCTCATTGGA TCGAGCCAAA CCGGTCGGAA AACACGATGG ATATATTCAA
    1051 ATGTCAACAA TCCATGGTTT CTGTGCTTGG ATGGTTTGCC AACCCTATCC ATGGGGATGG
    1111 CGACTATCCA GAGGGGATGA GAAAGAAGTT GTTCTCCGTT CTACCCATTT TCTCTGAAGC
    1171 AGAGAAGCAT GAGATGAGAG GCACAGCTGA TTTCTTTGCC TTTTCTTTTG GACCCAACAA
    1231 CTTCAAGCCC CTAAACACCA TGGCTAAAAT GGGACAAAAT GTTTCACTTA ATTTAAGAGA
    1291 AGCGCTGAAC TGGATTAAAC TGGAATACAA CAACCCTCGA ATCTTGATTG CTGAGAATGG
    1351 CTGGTTCACA GACAGTCGTG TGAAAACAGA AGACACCACG GCCATCTACA TGATGAAGAA
    1411 TTTCCTCAGC CAGGTGCTTC AAGCAATAAG GTTAGATGAA ATACGAGTGT TTGGTTATAC
    1471 TGCCTGGTCT CTCCTGGATG GCTTTGAATG GCAGGATGCT TACACCATCC GCCGAGGATT
    1531 ATTTTATGTG GATTTTAACA GTAAACAGAA AGAGCGGAAA CCTAAGTCTT CAGCACACTA
    1591 CTACAAACAG ATCATACGAG AAAATGGTTT TTCTTTAAAA GAGTCCACGC CAGATGTGCA
    1651 GGGCCAGTTT CCCTGTGACT TCTCCTGGGG TGTCACTGAA TCTGTTCTTA AGCCCGAGTC
    1711 TGTGGCTTCG TCCCCACAGT TCAGCGATCC TCATCTGTAC GTGTGGAACG CCACTGGCAA
    1771 CAGACTGTTG CACCGAGTGG AAGGGGTGAG GCTGAAAACA CGACCCGCTC AATGCACAGA
    1831 TTTTGTAAAC ATCAAAAAAC AACTTGAGAT GTTGGCAAGA ATGAAAGTCA CCCACTACCG
    1891 GTTTGCTCTG GATTGGGCCT CGGTCCTTCC CACTGGCAAC CTGTCCGCGG TGAACCGACA
    1951 GGCCCTGAGG TACTACAGGT GCGTGGTCAG TGAGGGGCTG AAGCTTGGCA TCTCCGCGAT
    2011 GGTCACCCTG TATTATCCGA CCCACGCCCA CCTAGGCCTC CCCGAGCCTC TGTTGCATGC
    2071 CGACGGGTGG CTGAACCCAT CGACGGCCGA GGCCTTCCAG GCCTACGCTG GGCTGTGCTT
    2131 CCAGGAGCTG GGGGACCTGG TGAAGCTCTG GATCACCATC AACGAGCCTA ACCGGCTAAG
    2191 TGACATCTAC AACCGCTCTG GCAACGACAC CTACGGGGCG GCGCACAACC TGCTGGTGGC
    2251 CCACGCCCTG GCCTGGCGCC TCTACGACCG GCAGTTCAGG CCCTCACAGC GCGGGGCCGT
    2311 GTCGCTGTCG CTGCACGCGG ACTGGGCGGA ACCCGCCAAC CCCTATGCTG ACTCGCACTG
    2371 GAGGGCGGCC GAGCGCTTCC TGCAGTTCGA GATCGCCTGG TTCGCCGAGC CGCTCTTCAA
    2431 GACCGGGGAC TACCCCGCGG CCATGAGGGA ATACATTGCC TCCAAGCACC GACGGGGGCT
    2491 TTCCAGCTCG GCCCTGCCGC GCCTCACCGA GGCCGAAAGG AGGCTGCTCA AGGGCACGGT
    2551 CGACTTCTGC GCGCTCAACC ACTTCACCAC TAGGTTCGTG ATGCACGAGC AGCTGGCCGG
    2611 CAGCCGCTAC GACTCGGACA GGGACATCCA GTTTCTGCAG GACATCACCC GCCTGAGCTC
    2671 CCCCACGCGC CTGGCTGTGA TTCCCTGGGG GGTGCGCAAG CTGCTGCGGT GGGTCCGGAG
    2731 GAACTACGGC GACATGGACA TTTACATCAC CGCCAGTGGC ATCGACGACC AGGCTCTGGA
    2791 GGATGACCGG CTCCGGAAGT ACTACCTAGG GAAGTACCTT CAGGAGGTGC TGAAAGCATA
    2851 CCTGATTGAT AAAGTCAGAA TCAAAGGCTA TTATGCATTC AAACTGGCTG AAGAGAAATC
    2911 TAAACCCAGA TTTGGATTCT TCACATCTGA TTTTAAAGCT AAATCCTCAA TACAATTTTA
    2971 CAACAAAGTG ATCAGCAGCA GGGGCTTCCC TTTTGAGAAC AGTAGTTCTA GATGCAGTCA
    3031 GACCCAAGAA AATACAGAGT GCACTGTCTG CTTATTCCTT GTGCAGAAGA AACCACTGAT
    3091 ATTCCTGGGT TGTTGCTTCT TCTCCACCCT GGTTCTACTC TTATCAATTG CCATTTTTCA
    3151 AAGGCAGAAG AGAAGAAAGT TTTGGAAAGC AAAAAACTTA CAACACATAC CATTAAAGAA
    3211 AGGCAAGAGA GTTGTTAGCT AA
    SEQ ID NO: 350 (House mouse(βKlotho gene coding sequence):
    12  ATGAAGACA GGCTGTGCAG CAGGGTCTCC GGGGAATGAA TGGATTTTCT TCAGCTCTGA
    61 TGAAAGAAAC ACACGCTCTA GGAAAACAAT GTCCAACAGG GCACTGCAAA GATCTGCCGT
    121 GCTGTCTGCG TTTGTTCTGC TGCGAGCTGT TACCGGCTTC TCCGGAGACG GGAAAGCAAT
    181 ATGGGATAAA AAACAGTACG TGAGTCCGGT AAACCCAAGT CAGCTGTTCC TCTATGACAC
    241 TTTCCCTAAA AACTTTTCCT GGGGCGTTGG GACCGGAGCA TTTCAAGTGG AAGGGAGTTG
    301 GAAGACAGAT GGAAGAGGAC CCTCGATCTG GGATCGGTAC GTCTACTCAC ACCTGAGAGG
    361 TGTCAACGGC ACAGACAGAT CCACTGACAG TTACATCTTT CTGGAAAAAG ACTTGTTGGC
    421 TCTGGATTTT TTAGGAGTTT CTTTTTATCA GTTCTCAATC TCCTGGCCAC GGTTGTTTCC
    481 CAATGGAACA GTAGCAGCAG TGAATGCGCA AGGTCTCCGG TACTACCGTG CACTTCTGGA
    541 CTCGCTGGTA CTTAGGAATA TCGAGCCCAT TGTTACCTTG TACCATTGGG ATTTGCCTCT
    601 GACGCTCCAG GAAGAATATG GGGGCTGGAA AAATGCAACT ATGATAGATC TCTTCAACGA
    661 CTATGCCACA TACTGCTTCC AGACCTTTGG AGACCGTGTC AAATATTGGA TTACAATTCA
    721 CAACCCTTAC CTTGTTGCTT GGCATGGGTT TGGCACAGGT ATGCATGCAC CAGGAGAGAA
    781 GGGAAATTTA ACAGCTGTCT ACACTGTGGG ACACAACCTG ATCAAGGCAC ATTCGAAAGT
    841 GTGGCATAAC TACGACAAAA ACTTCCGCCC TCATCAGAAG GGTTGGCTCT CCATCACCTT
    901 GGGGTCCCAT TGGATAGAGC CAAACAGAAC AGACAACATG GAGGACGTGA TCAACTGCCA
    961 GCACTCCATG TCCTCTGTGC TTGGATGGTT CGCCAACCCC ATCCACGGGG ACGGCGACTA
    1021 CCCTGAGTTC ATGAAGACGG GCGCCATGAT CCCCGAGTTC TCTGAGGCAG AGAAGGAGGA
    1081 GGTGAGGGGC ACGGCTGATT TCTTTGCCTT TTCCTTCGGG CCCAACAACT TCAGGCCCTC
    1141 AAACACCGTG GTGAAAATGG GACAAAATGT ATCACTCAAC TTAAGGCAGG TGCTGAACTG
    1201 GATTAAACTG GAATACGATG ACCCTCAAAT CTTGATTTCG GAGAACGGCT GGTTCACAGA
    1261 TAGCTATATA AAGACAGAGG ACACCACGGC CATCTACATG ATGAAGAATT TCCTAAACCA
    1321 GGTTCTTCAA GCAATAAAAT TTGATGAAAT CCGCGTGTTT GGTTATACGG CCTGGACTCT
    1381 CCTGGATGGC TTTGAGTGGC AGGATGCCTA TACGACCCGA CGAGGGCTGT TTTATGTGGA
    1441 CTTTAACAGT GAGCAGAAAG AGAGGAAACC CAAGTCCTCG GCTCATTACT ACAAGCAGAT
    1501 CATACAAGAC AACGGCTTCC CTTTGAAAGA GTCCACGCCA GACATGAAGG GTCGGTTCCC
    1561 CTGTGATTTC TCTTGGGGAG TCACTGAGTC TGTTCTTAAG CCCGAGTTTA CGGTCTCCTC
    1621 CCCGCAGTTT ACCGATCCTC ACCTGTATGT GTGGAATGTC ACTGGCAACA GATTGCTCTA
    1681 CCGAGTGGAA GGGGTAAGGC TGAAAACAAG ACCATCCCAG TGCACAGATT ATGTGAGCAT
    1741 CAAAAAACGA GTTGAAATGT TGGCAAAAAT GAAAGTCACC CACTACCAGT TTGCTCTGGA
    1801 CTGGACCTCT ATCCTTCCCA CTGGCAATCT GTCCAAAGTT AACAGACAAG TGTTAAGGTA
    1861 CTATAGGTGT GTGGTGAGCG AAGGACTGAA GCTGGGCGTC TTCCCCATGG TGACGTTGTA
    1921 CCACCCAACC CACTCCCATC TCGGCCTCCC CCTGCCACTT CTGAGCAGTG GGGGGTGGCT
    1981 AAACATGAAC ACAGCCAAGG CCTTCCAGGA CTACGCTGAG CTGTGCTTCC GGGAGTTGGG
    2041 GGACTTGGTG AAGCTCTGGA TCACCATCAA TGAGCCTAAC AGGCTGAGTG ACATGTACAA
    2101 CCGCACGAGT AATGACACCT ACCGTGCAGC CCACAACCTG ATGATCGCCC ATGCCCAGGT
    2161 CTGGCACCTC TATGATAGGC AGTATAGGCC GGTCCAGCAT GGGGCTGTGT CGCTGTCCTT
    2221 ACATTGCGAC TGGGCAGAAC CTGCCAACCC CTTTGTGGAT TCACACTGGA AGGCAGCCGA
    2281 GCGCTTCCTC CAGTTTGAGA TCGCCTGGTT TGCAGATCCG CTCTTCAAGA CTGGCGACTA
    2341 TCCATCGGTT ATGAAGGAAT ACATCGCCTC CAAGAACCAG CGAGGGCTGT CTAGCTCAGT
    2401 CCTGCCGCGC TTCACCGCGA AGGAGAGCAG GCTGGTGAAG GGTACCGTCG ACTTCTACGC
    2461 ACTGAACCAC TTCACTACGA GGTTCGTGAT ACACAAGCAG CTGAACACCA ACCGCTCAGT
    2521 TGCAGACAGG GACGTCCAGT TCCTGCAGGA CATCACCCGC CTAAGCTCGC CCAGCCGCCT
    2581 GGCTGTAACA CCCTGGGGAG TGCGCAAGCT CCTTGCGTGG ATCCGGAGGA ACTACAGAGA
    2641 CAGGGATATC TACATCACAG CCAATGGCAT CGATGACCTG GCTCTAGAGG ATGATCAGAT
    2701 CCGAAAGTAC TACTTGGAGA AGTATGTCCA GGAGGCTCTG AAAGCATATC TCATTGACAA
    2761 GGTCAAAATC AAAGGCTACT ATGCATTCAA ACTGACTGAA GAGAAATCTA AGCCTAGATT
    2821 TGGATTTTTC ACCTCTGACT TCAGAGCTAA GTCCTCTGTC CAGTTTTACA GCAAGCTGAT
    2881 CAGCAGCAGT GGCCTCCCCG CTGAGAACAG AAGTCCTGCG TGTGGTCAGC CTGCGGAAGA
    2941 CACAGACTGC ACCATTTGCT CATTTCTCGT GGAGAAGAAA CCACTCATCT TCTTCGGTTG
    3001 CTGCTTCATC TCCACTCTGG CTGTACTGCT ATCCATCACC GTTTTTCATC ATCAAAAGAG
    3061 AAGAAAATTC CAGAAAGCAA GGAACTTACA AAATATACCA TTGAAGAAAG GCCACAGCAG
    3121 AGTTTTCAGC TAA
  • In one embodiment, the FGFR is FGFR1c, FGFR2c, or FGFR4. In one embodiment of the present invention, the FGF receptor is FGFR1c receptor. In one particular embodiment, the FGFR1c receptor is the human FGFR1c receptor (GenBank Accession No. NP075598, which is hereby incorporated by reference in its entirety). In another embodiment, the FGF receptor is FGFR2c receptor. In one particular embodiment, the FGFR2c receptor is the human FGFR2c receptor (GenBank Accession No. NP000132, which is hereby incorporated by reference in its entirety). In another embodiment, the FGF receptor is FGFR4 receptor. In one particular embodiment, the FGFR4 receptor is the human FGFR4 receptor (GenBank Accession No. NP002002, which is hereby incorporated by reference in its entirety).
  • In one embodiment, the method of facilitating FGFR-βKlotho co-receptor complex formation is carried out in vitro. In one embodiment, the method is carried out in an adipocyte. In another embodiment, the method is carried out in a skeletal muscle cell, a pancreatic β cell, or a hepatocyte.
  • In one embodiment, the method of facilitating FGFR-Klotho co-receptor complex formation is carried out in vivo. In one embodiment, the method is carried out in a mammal. In one particular embodiment, the mammal is a mouse.
  • Yet a further aspect of the present invention relates to a method of screening for agents capable of facilitating FGFR-βKlotho complex formation in the treatment of a disorder. This method involves providing a chimeric FGF that includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine FGF and the C-terminus includes a C-terminal portion of FGF21. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. This method also involves providing binary βKlotho-FGFR complex and providing one or more candidate agents. This method further involves combining the chimeric FGF, the binary βKlotho-FGFR complex, and the one or more candidate agents under conditions permitting the formation of a ternary complex between the chimeric FGF and the binary βKlotho-FGFR complex in the absence of the one or more candidate agents. This method also involves identifying the one or more candidate agents that decrease ternary complex formation between the chimeric FGF and the binary βKlotho-FGFR complex compared to the ternary complex formation in the absence of the one or more candidate agents as suitable for treating the disorder.
  • The portion of the paracrine FGF may also be modified to alter receptor-binding specificity and/or reduce receptor-binding affinity compared to the portion without the modification.
  • Suitable chimeric proteins for use in accordance with this aspect of the present invention are described above and throughout the present application. Suitable paracrine FGFs, as well as suitable modifications to decrease binding affinity for heparin and/or heparan sulfate, to alter receptor-binding specificity and/or to reduce receptor-binding affinity compared to the portion without the modification, are also described above.
  • In one embodiment, the modulation is a competitive interaction between the chimeric FGF molecule and the one or more candidate agents for binding to the binary βKlotho-FGFR complex.
  • In one embodiment, the FGFR is FGFR1c, FGFR2c, or FGFR4.
  • In one embodiment, the disorder is a selected from diabetes, obesity, and metabolic syndrome. In one embodiment, the disorder is diabetes selected from type II diabetes, gestational diabetes, or drug-induced diabetes. In one embodiment, the disorder is type I diabetes. In one embodiment, the disorder is obesity. In one embodiment, the disorder is metabolic syndrome.
  • In one embodiment of the screening aspects of the present invention, a plurality of compounds or agents is tested. Candidate agents may include small molecule compounds or larger molecules (e.g., proteins or fragments thereof). In one embodiment, the candidate compounds are biomolecules. In one embodiment, the biomolecules are proteins. In one embodiment, the biomolecules are peptides. In one embodiment, the candidates are peptides or peptide mimetics having similar structural features to native FGF ligand. In one embodiment, the candidate agent is a second chimeric FGF molecule. In one particular embodiment, the peptides are synthetic peptides. In one embodiment, the compounds are small organic molecules.
  • In one embodiment of the screening aspects of the present invention, the method is carried out using a cell-based assay. In one embodiment, the identifying is carried out using a cell-based assay.
  • In one embodiment of the screening aspects of the present invention, the method is carried out using a binding assay. In one embodiment, the binding assay is a direct binding assay. In one embodiment, the binding assay is a competition-binding assay. In one embodiment, the modulation stabilizes the ternary complex between the chimeric FGF molecule and the binary βKlotho-FGFR complex. In one embodiment, the stabilization is compared to the native ternary complex.
  • In one embodiment, the modulation is an allosteric or kinetic modulation. In one embodiment, the allosteric or kinetic modulation is compared to the native ternary complex. Such stabilization or allosteric or kinetic modulation can be measured using methods known in the art (e.g., by use of surface plasmon resonance (SPR) spectroscopy experiments as described in the Examples infra).
  • In one embodiment, the binding assay is carried out using surface plasmon resonance spectroscopy. In one embodiment, the identifying is carried out using a binding assay. In one embodiment, the identifying is carried out using surface plasmon resonance spectroscopy.
  • In one embodiment of the screening aspects of the present invention, the cell-based assay is carried out with adipocytes. In one embodiment, the cell-based assay is carried out with skeletal muscle cells. In one embodiment, the cell-based assay is carried out with pancreatic β cells. In one embodiment, the cell-based assay is carried out with hepatocytes. In one embodiment, stimulation of glucose uptake is the assay readout. In one embodiment, induction of glucose transporter 1 gene expression is the assay readout. In one embodiment, a dose-response curve is generated for the stimulation of glucose uptake by a candidate compound to determine potency and efficacy of the candidate compound. In one embodiment, a dose-response curve is generated for the induction of glucose transporter 1 gene expression by a candidate compound to determine potency and efficacy of the candidate compound. For example, if the dose-response curve is shifted to the left compared to that obtained for the chimeric FGF protein, the candidate compound has greater potency than the chimeric FGF protein and/or native FGF21. In one embodiment, an IC50 value is derived from the dose-response curve of a candidate compound to determine potency of the candidate compound. An IC50 value smaller than that obtained for the chimeric FGF protein identifies a candidate compound as more potent than the chimeric FGF protein and/or native FGF21.
  • In one embodiment of the screening aspects of the present invention, the cell-based assay is carried out with mammalian cells ectopically expressing βKlotho. In one particular embodiment, the cells are HEK293 cells. In one embodiment, activation of FGF receptor is the assay readout. In one embodiment, tyrosine phosphorylation of an FGF receptor substrate is used as readout for FGF receptor activation. In one particular embodiment, the FGF receptor substrate is FGF receptor substrate 2α. In one embodiment, activation of downstream mediators of FGF signaling is used as readout for (or an indicator of) FGF receptor activation. In one particular embodiment, the downstream mediator of FGF signaling is 44/42 mitogen-activated protein kinase. In one embodiment, the downstream mediator of FGF signaling is a transcription factor. In one particular embodiment, the transcription factor is early growth response 1. In one embodiment, a dose-response curve is generated for βKlotho-dependent activation of FGF receptor by a candidate compound to determine potency and efficacy of the candidate compound. For example, if the dose-response curve is shifted to the left compared to that obtained for the chimeric FGF protein, the candidate compound is more potent than the chimeric FGF protein and/or native FGF21. In one embodiment, an IC50 value is derived from the dose-response curve of a candidate compound to determine potency of the candidate compound. An IC50 value smaller than that obtained for the chimeric FGF protein identifies a candidate compound as more potent than the chimeric FGF protein and/or native FGF21.
  • In one embodiment of the screening aspects of the present invention, the surface plasmon resonance spectroscopy-based assay is carried out using the chimeric FGF protein as ligand coupled to a biosensor chip. In one embodiment, mixtures of βKlotho ectodomain with increasing concentrations of a candidate compound are passed over a biosensor chip containing chimeric FGF protein. In one embodiment, mixtures of the binary complex of FGFR ligand-binding domain and βKlotho ectodomain with increasing concentrations of a candidate compound are passed over a biosensor chip containing chimeric FGF protein. In one particular embodiment, the FGFR ligand-binding domain is the FGFR1c ligand-binding domain. In one embodiment, an inhibition-binding curve is plotted for a candidate compound to determine potency of the candidate compound. For example, if the inhibition-binding curve is shifted to the left compared to that obtained for the chimeric FGF protein, the candidate compound has greater potency than the chimeric FGF protein and/or native FGF21. In one embodiment, an IC50 value is derived from the inhibition-binding curve of a candidate compound to determine potency of the candidate compound. An IC50 value smaller than that obtained for containing chimeric FGF protein identifies a candidate compound as more potent than the chimeric FGF protein and/or native FGF21. In one embodiment, the inhibition constant Ki is determined for a candidate compound to determine potency of the candidate compound. A Ki value smaller than that obtained for native FGF21 identifies a candidate compound as more potent than the chimeric FGF protein and/or native FGF21.
  • In one embodiment of the screening aspects of the present invention, the method is carried out in vivo. In one embodiment, the method is carried out in a mammal. In one particular embodiment, the mammal is a mouse. In one embodiment, the mammal has obesity, diabetes, or a related metabolic disorder. In one embodiment, the ability of a candidate compound to potentiate the hypoglycemic effect of insulin is used as readout for FGF21-like metabolic activity. This involves fasting the mammal for a period of time prior to insulin injection and measuring fasting blood glucose levels. The mammal is then injected with insulin alone or co-injected with insulin plus a candidate compound. Blood glucose levels are measured at several time points after the injection. If a candidate compound potentiates the hypoglycemic effect of insulin to a greater degree than the chimeric FGF protein and/or native FGF21 does, the candidate compound exhibits enhanced efficacy. Likewise, if a candidate compound potentiates the hypoglycemic effect of insulin to a similar degree than the chimeric FGF protein and/or native FGF21 does but at a lower dose compared to that of the chimeric FGF protein and/or native FGF21 and/or for a longer period of time compared to the chimeric FGF protein and/or native FGF21, the candidate compound has enhanced agonistic properties. In one embodiment, the ability of a candidate compound to elicit a hypoglycemic effect in a mammal with diabetes, obesity, or a related metabolic disorder is used as readout for FGF21-like metabolic activity. This involves injecting a mammal suffering from diabetes, obesity, or a related metabolic disorder with the candidate compound. Blood glucose levels are measured before the injection and at several time points thereafter. If a candidate compound has a greater hypoglycemic effect than the chimeric FGF protein and/or native FGF21 does, the candidate compound exhibits enhanced efficacy. Likewise, if a candidate compound shows a similar hypoglycemic effect than the chimeric FGF protein and/or native FGF21 does but at a lower dose compared to that of the chimeric FGF protein and/or native FGF21 and/or for a longer period of time compared to the chimeric FGF protein and/or native FGF21, the candidate compound has enhanced agonistic properties.
  • EXAMPLES Example 1 Purification of FGF, FGFR, and Klotho Proteins
  • The N-terminally hexahistidine-tagged, mature form of human FGF19 (SEQ ID NO: 338) (R23 to K216), human FGF21 (SEQ ID NO:233) (H29 to S209; FIG. 5A), and human FGF23 (SEQ ID NO: 351) (Y25 to I251; FIG. 5A) was refolded in vitro from bacterial inclusion bodies, and purified by published protocols (Ibrahimi et al., Hum. Mol. Genet. 13:2313-2324 (2004); Plotnikov et al., Cell 101:413-424 (2000), which is hereby incorporated by reference in its entirety). The amino acid sequence of human FGF23 (SEQ ID NO:351) (GenBank accession no. AAG09917, which is hereby incorporated by reference in its entirety) is as follows:
  • 1 MLGARLRLWV CALCSVCSMS VLRAYPNASP LLGSSWGGLI HLYTATARNS YHLQIHKNGH
    61 VDGAPHQTIY SALMIRSEDA GFVVITGVMS RRYLCMDFRG NIFGSHYFDP ENCRFQHQTL
    121 ENGYDVYHSP QYHFLVSLGR AKRAFLPGMN PPPYSQFLSR RNEIPLIHFN TPIPRRHTRS
    181 AEDDSERDPL NVLKPRARMT PAPASCSQEL PSAEDNSPMA SDPLGVVRGG RVNTHAGGTG
    241 PEGCRPFAKF I
  • HS-binding site mutants of FGF19 (K149A) and FGF23 (R140A/R143A) were purified from bacterial inclusion bodies by similar protocols as the wild-type proteins. In order to minimize proteolysis of FGF23 wild-type and mutant proteins, arginine residues 176 and 179 of the proteolytic cleavage site 176RXXR179 were replaced with glutamine as it occurs in the phosphate wasting disorder “autosomal dominant hypophosphatemic rickets” (ADHR) (White et al., Nat. Genet. 26:345-348 (2000); White et al., Kidney Int. 60:2079-2086 (2001), which are hereby incorporated by reference in their entirety). Human FGF1 (SEQ ID NO:1) (M1 to D155; FIG. 6), N-terminally truncated human FGF1 (K25 to D155, termed FGF1ΔNT; FIG. 6), human FGF2 (SEQ ID NO: 121) (M1 to 5155; FIG. 5A), and human FGF homologous factor 1B (FHF1B; M1 to T181) were purified by published protocols (Plotnikov et al., Cell 101:413-424 (2000); Olsen et al., J. Biol. Chem. 278:34226-34236 (2003), which are hereby incorporated by reference in their entirety).
  • Chimeras composed of the core domain of FGF2 (M1 to M151) and the C-terminal region of either FGF21 (P168 to S209) or FGF23 (R161 to I251) (termed FGF2WTcore-FGF21C-tail and FGF2WTcore-FGF23C-tail, respectively; FIG. 5A) were purified by the same protocol as that for native FGF2 (Plotnikov et al., Cell 101:413-424 (2000), which is hereby incorporated by reference in its entirety). Analogous chimeras containing three mutations in the HS-binding site of the FGF2 core (K128D/R129Q/K134V) (termed FGF2ΔHBScore-FGF21C-tail and FGF2ΔHBScore-FGF23C-tail, respectively, FIG. 5A) were purified from the soluble bacterial cell lysate fraction by ion-exchange and size-exclusion chromatographies. In order to minimize proteolysis of the chimeras containing the C-terminal sequence from R161 to I251 of FGF23, arginine residues 176 and 179 of the proteolytic cleavage site 176RXXR179 located within this sequence were replaced with glutamine as it occurs in ADHR (White et al., Nat. Genet. 26:345-348 (2000); White et al., Kidney Int. 60:2079-2086 (2001), which are hereby incorporated by reference in their entirety). In addition, in order to prevent disulfide-mediated dimerization of FGF2 and chimeric FGF2 proteins, cysteine residues 78 and 96 were mutated to serine. An HS-binding site mutant of FGF1 (K127D/K128Q/K133V) (termed FGF1ΔHBScore; FIG. 6) and chimeras composed of the core domain of the HS-binding site mutant of FGF1 (M1 to L150, K127D/K128Q/K133V) and the C-terminal region of either FGF19 (L169 to K216) or FGF21 (P168 to S209) (termed FGF1ΔHBScore-FGF19C-tail and FGF1ΔHBScore-FGF21C-tail, respectively; FIG. 6) were purified from the soluble bacterial cell lysate fraction by ion-exchange and size-exclusion chromatographies. The N-terminally hexahistidine-tagged C-terminal tail peptide of FGF23 (S180 to I251, termed FGF23C-tail) was purified by a published protocol (Goetz et al., Proc. Nat'l. Acad. Sci. U.S.A 107:407-412 (2010), which is hereby incorporated by reference in its entirety). The ligand-binding domain of human FGFR1c (D142 to R365) was refolded in vitro from bacterial inclusion bodies, and purified by published protocols (Ibrahimi et al., Hum. Mol. Genet. 13:2313-2324 (2004); Plotnikov et al., Cell 101:413-424 (2000), which are hereby incorporated by reference in their entirety). The ectodomain of murine αKlotho (A35 to K982) and the ectodomain of murine βKlotho (F53 to L995) were expressed in HEK293 cells as fusion proteins with a C-terminal FLAG tag (Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006); Kurosu et al., Science 309:1829-1833 (2005), which are hereby incorporated by reference in their entirety). The binary complex of FGFR1c ligand-binding domain with αKlotho ectodomain (referred to as αKlotho-FGFR1c complex) was prepared by a published protocol (Goetz et al., Proc. Nat'l. Acad. Sci. U.S.A 107:407-412 (2010), which is hereby incorporated by reference in its entirety). The binary complex of FGFR1c ligand-binding domain with βKlotho ectodomain (referred to as βKlotho-FGFR1c complex) was prepared in the same fashion as the αKlotho-FGFR1c complex.
  • Example 2 Analysis of FGF-Heparin and FGF-FGFR-α/βKlotho Interactions by Surface Plasmon Resonance Spectroscopy
  • Surface plasmon resonance (SPR) experiments were performed on a Biacore 2000 instrument (Biacore AB), and the interactions were studied at 25° C. in HBS-EP buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) polysorbate 20). To study endocrine FGF-heparin interactions, a heparin chip was prepared by immobilizing biotinylated heparin (Sigma-Aldrich) on flow channels of a research-grade streptavidin chip (Biacore AB). The coupling density was ˜5 fmol mm−2 of flow channel. To measure binding of chimeric FGF2 proteins to heparin, biotinylated heparin was coupled to a streptavidin chip at an approximately 4-fold lower density as judged based on the binding responses obtained for FGF1. To study FGF-FGFR-α/βKlotho interactions, FGF chips were prepared by covalent coupling of FGF proteins through their free amino groups on flow channels of research grade CM5 chips (Biacore AB). Proteins were injected over a chip at a flow rate of 50 μl min−1, and at the end of each protein injection (180 and 300 s, respectively), HBS-EP buffer (50 μl min−1) was flowed over the chip to monitor dissociation for 180 or 240 s. The heparin chip surface was regenerated by injecting 50 μl of 2.0 M NaCl in 10 mM sodium acetate, pH 4.5. For FGF chips, regeneration was achieved by injecting 2.0 M NaCl in 10 mM sodium/potassium phosphate, pH 6.5. To control for nonspecific binding in experiments where an FGF ligand was immobilized on the chip, FHF1B, which shares structural similarity with FGFs but does not exhibit any FGFR binding (Olsen et al., J. Biol. Chem. 278:34226-34236 (2003), which is hereby incorporated by reference in its entirety), was coupled to the control flow channel of the chip (˜15-30 fmol mm−2). In experiments where heparin was immobilized on the chip, the control flow channel was left blank. The data were processed with BiaEvaluation software (Biacore AB). For each protein injection over the heparin chip, the nonspecific responses from the control flow channel were subtracted from the responses recorded for the heparin flow channel. Similarly, for each protein injection over a FGF chip, the nonspecific responses from the FHF1B control flow channel were subtracted from the responses recorded for the FGF flow channel. Where possible, equilibrium dissociation constants (KDs) were calculated from fitted saturation binding curves. Fitted binding curves were judged to be accurate based on the distribution of the residuals (even and near zero) and χ2 (<10% of Rmax).
  • To examine whether the K149A mutation abrogates residual heparin binding of FGF19, increasing concentrations of wild-type FGF19 were passed over a heparin chip. Thereafter, the FGF19K149A mutant was injected over the heparin chip at the highest concentration tested for the wild-type ligand. The effect of the R140A/R143A double mutation in the HS-binding site of FGF23 on residual heparin binding of FGF23 was examined in the same fashion as was the effect of the HS-binding site mutation in FGF19.
  • To verify that the K128D/R129Q/K134V triple mutation in the HS-binding site of the FGF2 core domain diminishes heparin-binding affinity of the FGF2 core, increasing concentrations of FGF2ΔHBScore-FGF21C-tail and FGF2ΔHBScore-FGF23C-tail were passed over a heparin chip. As a control, binding of FGF2WTcore-FGF21C-tail and FGF2WTcore-FGF23C-tail to heparin was studied.
  • To examine whether the FGF2ΔHBScore-FGF23C-tail chimera can compete with FGF23 for binding to the αKlotho-FGFR1c complex, FGF23 was immobilized on a chip (˜16 fmol mm−2 of flow channel). Increasing concentrations of FGF2ΔHBScore-FGF23C-tail were mixed with a fixed concentration of αKlotho-FGFR1c complex in HBS-EP buffer, and the mixtures were injected over the FGF23 chip. As controls, the binding competition was carried out with FGF23 or FGF2 as the competitor in solution. As an additional specificity control, competition of the FGF2ΔHBScore-FGF23C-tail chimera with FGF21 for binding to the αKlotho-FGFR1c complex was studied. αKlotho-FGFR1c complex was mixed with FGF2ΔHBScore-FGF23C-tail or FGF23 at a molar ratio of 1:10, and the mixture was injected over a chip containing immobilized FGF21 (˜12 fmol mm−2 of flow channel).
  • To test whether the FGF2ΔHBScore-FGF21C-tail chimera can compete with FGF21 for binding to the βKlotho-FGFR1c complex, increasing concentrations of FGF2ΔHBScore-FGF21C-tail were mixed with a fixed concentration of βKlotho-FGFR1c complex in HBS-EP buffer, and the mixtures were passed over a chip containing immobilized FGF21 (˜19 fmol mm−2 of flow channel). As controls, the binding competition was carried out with FGF21 or FGF2 as the competitor in solution. As an additional specificity control, competition of the FGF2ΔHBScore-FGF21C-tail chimera with FGF23 for binding to the αKlotho-FGFR1c complex was studied. αKlotho-FGFR1c complex was mixed with FGF2ΔHBScore-FGF21C-tail or FGF21 at a molar ratio of 1:10, and the mixture was injected over a chip containing immobilized FGF23 (˜12 fmol mm−2 of flow channel).
  • To measure binding of FGFR1c to each of the three endocrine FGFs, increasing concentrations of FGFR1c ligand-binding domain were injected over a chip containing immobilized FGF19, FGF21, and FGF23 (˜30 fmol mm−2 of flow channel). As a control, binding of FGFR1c to FGF2 immobilized on a chip was studied. As additional controls, binding of the αKlotho-FGFR1c complex to FGF23 and binding of FGFR1c to the C-terminal tail peptide of FGF23 was measured.
  • Example 3 Analysis of Phosphorylation of FRS2α and 44/42 MAP Kinase in Hepatoma and Epithelial Cell Lines
  • To examine whether the FGF19K149A and FGF23R140A/R143A mutants can activate FGFR in a α/βKlotho-dependent fashion, induction of tyrosine phosphorylation of FGFR substrate 2α (FRS2α) and downstream activation of MAP kinase cascade was used as readout for FGFR activation. Subconfluent cells of the H4IIE rat hepatoma cell line, which endogenously expresses βKlotho (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007), which is hereby incorporated by reference in its entirety), were serum starved for 16 h and then stimulated for 10 min with the FGF19K149A mutant or wild-type FGF19 (0.2 ng ml−1 to 2.0 μg ml−1). Similarly, subconfluent cells of a HEK293 cell line ectopically expressing the transmembrane isoform of murine αKlotho (Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006), which is hereby incorporated by reference in its entirety) were treated with the FGF23R140A/R143A mutant or wild-type FGF23 (0.1 to 100 ng ml−1). After stimulation, the cells were lysed (Kurosu et al., Science 309:1829-1833 (2005), which is hereby incorporated by reference in its entirety), and cellular proteins were resolved on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The protein blots were probed with antibodies to phosphorylated FRS2α, phosphorylated 44/42 MAP kinase, total (phosphorylated and nonphosphorylated) 44/42 MAP kinase, and αKlotho. Except for the anti-αKlotho antibody (KM2119) (Kato et al., Biochem. Biophys. Res. Commun. 267:597-602 (2000), which is hereby incorporated by reference in its entirety), all antibodies were from Cell Signaling Technology.
  • Example 4 Analysis of Egr1 Protein Expression in an Epithelial Cell Line
  • To examine whether the FGF2ΔHBScore-FGF21C-tail and FGF2ΔHBScore-FGF23C-tail chimeras can activate FGFR in a HS-dependent fashion, induction of protein expression of the transcription factor early growth response 1 (Egr1), a known downstream mediator of FGF signaling, was used as readout for FGFR activation. HEK293 cells were serum starved overnight and then stimulated for 90 min with FGF2ΔHBScore-FGF21C-tail or FGF2ΔHBScore-FGF23C-tail (0.1 and 0.3 nM). Cell stimulation with FGF2WTcore-FGF21C-tail, FGF2WTcore-FGF23C-tail, FGF21, and FGF23 served as controls. To test whether the FGF2ΔHBScore-FGF21C-tail chimera can activate FGFR in a βKlotho-dependent fashion, HEK293 cells transfected with murine βKlotho were serum starved overnight and then stimulated for 90 min with FGF2ΔHBScore-FGF21C-tail or FGF21 (3 to 300 ng ml−1). After stimulation, the cells were lysed (Kurosu et al., Science 309:1829-1833 (2005), which is hereby incorporated by reference in its entirety), and cellular proteins were resolved on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The protein blots were probed with antibodies to Egr1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The anti-Egr1 antibody was from Cell Signaling Technology and the anti-GAPDH antibody was from Abcam.
  • Example 5 Analysis of CYP7A1 and CYP8B1 mRNA Expression in Murine Liver Tissue
  • To examine the metabolic activity of the FGF19K149A mutant in vivo, 6- to 8-week old C57BL/6 mice were fasted overnight and then given intraperitoneally a single dose (1 mg kg body weight−1) of FGF19K149A or FGF19 as a control. 6 h after the injection, the mice were sacrificed, and liver tissue was excised and frozen. Total RNA was isolated from liver tissue, and mRNA levels of cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) were measured using quantitative real time RT-PCR as described previously (Inagaki et al., Cell Metab. 2:217-225 (2005); Kim et al., J. Lipid Res. 48:2664-2672 (2007), which are hereby incorporated by reference in their entirety). The Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center at Dallas had approved the experiments.
  • Example 6 Measurement of Serum Phosphate in Mice
  • The metabolic activity of the FGF23R140A/R143A mutant was examined both in normal mice and in Fgf23 knockout mice. 4- to 5-week old C57BL/6 mice were given intraperitoneally a single dose (0.29 mg kg body weight−1) of FGF23R140A/R143A or FGF23 as a control. Before the injection and 8 h after the injection, blood was drawn from the cheek pouch and spun at 3,000×g for 10 min to obtain serum. Phosphate concentration in serum was measured using the Phosphorus Liqui-UV Test (Stanbio Laboratory). 6- to 8-week old Fgf23 knockout mice (Sitara et al., Matrix Biol. 23:421-432 (2004), which is hereby incorporated by reference in its entirety) (56) were given two injections of FGF23R140A/R143A or FGF23 at 8 h intervals (0.71 mg kg body weight−1 each), and blood samples were collected for phosphate analysis before the first injection and 8 h after the second injection.
  • To test whether the FGF2ΔHBScore-FGF23C-tail chimera exhibits FGF23-like metabolic activity, 5- to 6-week old C57BL/6 mice were given a single injection of FGF2ΔHBScore-FGF23C-tail (0.21 mg kg body weight−1). As controls, mice were injected with FGF2WTcore-FGF23C-tail or FGF23. Before the injection and 8 h after the injection, blood samples were collected for measurement of serum phosphate. To confirm that αKlotho is required for the metabolic activity of the FGF2ΔHBScore-FGF23C-tail chimera, 7- to 8-week old αKlotho knockout mice (Lexicon Genetics) were injected once with FGF2ΔHBScore-FGF23C-tail or FGF23 as a control (0.51 mg kg body weight−1). Before the injection and 8 h after the injection, blood samples were collected for phosphate analysis. The Harvard University Animal Care and Research committee board had approved all the experiments.
  • Example 7 Analysis of CYP27B1 mRNA Expression in Murine Renal Tissue
  • The ability of the FGF2ΔHBScore-FGF23C-tail chimera to reduce renal expression of 25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1) was used as another readout for FGF23-like metabolic activity. C57BL/6 mice injected with FGF2ΔHBScore-FGF23C-tail, FGF2WTcore-FGF23C-tail, or FGF23 were sacrificed 8 h after the protein injection, and renal tissue was excised and frozen. CYP27B1 mRNA levels in total renal tissue RNA were measured using real time quantitative PCR as described previously (Nakatani et al., FASEB J. 23:3702-3711 (2009); Ohnishi et al., Kidney Int. 75:1166-1172 (2009), which are hereby incorporated by reference in their entirety). The Harvard University Animal Care and Research committee board had approved the experiments.
  • Example 8 Insulin Tolerance Test in Mice
  • The ability of the FGF2ΔHBScore-FGF21C-tail chimera to potentiate the hypoglycemic effect of insulin was used as readout for FGF21-like metabolic activity (Ohnishi et al., FASEB J. 25:2031-2039 (2011), which is hereby incorporated by reference in its entirety). 8- to 12-week old C57BL/6 mice were kept on normal chow. On the day of the insulin tolerance test, mice were fasted for 4 h and then bled from the cheek pouch for measuring fasting blood glucose levels. Thereafter, mice were administered intraperitoneally insulin (0.5 units kg body weight−1) alone or insulin (0.5 units·kg body weight−1) plus FGF2ΔHBScore-FGF21C-tail chimera (0.3 mg kg body weight−1). As a control, mice were co-injected with insulin plus FGF21. At the indicated time points after the injection (FIG. 7G), blood was drawn from the tail vein. Glucose concentrations in the blood samples were determined using Bayer Contour® blood glucose test strips (Bayer Corp.). The Harvard University Animal Care and Research committee board had approved the experiments.
  • Example 9 Analysis of Blood Glucose in Ob/Ob Mice
  • ob/ob mice were injected subcutaneously with FGF1ΔNT, FGF1ΔHBS, or FGF1ΔHBScore-FGF21C-tail chimera. Injection of native FGF1 or native FGF21 served as controls. A single bolus of 0.5 mg of protein per kg of body weight was injected. This dose was chosen on the basis that maximal efficacy of the hypoglycemic effect of native FGF1 is seen at this dose. Before the protein injection and at the indicated time points after the injection (FIGS. 9A-9C), blood glucose concentrations were measured using an OneTouch Ultra glucometer (Lifescan). The Institutional Animal Care and Use Committee at the Salk Institute for Biological Sciences at La Jolla had approved the experiments.
  • Example 10 Statistical Analysis
  • Data are expressed as mean±SEM. A Student's t test or analysis of variance (ANOVA) was used as appropriate to make statistical comparisons. A value of P<0.05 was considered significant.
  • Example 11 HS is Dispensable for the Metabolic Activity of FGF19 and FGF23
  • In order to engineer endocrine FGFs devoid of HS binding, the FGF19 crystal structure (PDB ID: 2P23; (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety) was compared with that of FGF2 bound to a heparin hexasaccharide (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety)). This analysis shows that solvent-exposed residues K149, Q150, Q152, and R157 of FGF19 lie at the corresponding HS-binding site of this ligand, and hence could account for the residual HS binding of FGF19 (FIGS. 1A, 1B, and 2). Likewise, comparative analysis of the FGF23 crystal structure (PDB ID: 2P39; (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety) (29)) with that of heparin-bound FGF2 (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety)) points to R48, N49, R140, and R143 as candidates mediating the residual HS binding of this ligand (FIGS. 1A, 1C, and 2). In agreement with the structural predictions, replacement of K149 alone in FGF19 with alanine and combined substitution of R140 and R143 in FGF23 for alanine were sufficient to abolish residual HS binding of these ligands (FIGS. 3B-3G).
  • To test the impact of knocking out residual HS binding of FGF19 on the signaling by this ligand, H4IIE hepatoma cells were stimulated with the FGF19K149A mutant or wild-type FGF19. H4IIE cells endogenously express FGFR4 and βKlotho (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007), which is hereby incorporated by reference in its entirety), the cognate receptor and co-receptor, respectively, for FGF19. The FGF19K149A mutant was as effective as wild-type FGF19 in inducing tyrosine phosphorylation of FRS2α and downstream activation of MAP kinase cascade (FIG. 4A). These data show that elimination of residual HS binding has no impact on the ability of FGF19 to signal in cultured cells. To test whether the same holds true for FGF23 signaling, HEK293 cells, which naturally express two of the three cognate receptors of FGF23, namely FGFR1c and FGFR3c (Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006), which is hereby incorporated by reference in its entirety) were transfected with the transmembrane isoform of αKlotho, the co-receptor of FGF23. These cells were treated with the FGF23R140A/R143A double mutant or wild-type FGF23. The FGF23R140A/R143A mutant had the same capacity as wild-type FGF23 in inducing phosphorylation of FRS2α and downstream activation of MAP kinase cascade (FIG. 4B). These data show that similar to FGF19, FGF23 does not need to bind HS in order to activate FGFR in cultured cells.
  • To substantiate the findings in cells, the metabolic activity of wild-type and mutated ligands in vivo were compared. Mice were injected with the FGF19K149A mutant or wild-type FGF19 and liver gene expression of CYP7A1 and CYP8B1, which are key enzymes in the major bile acid biosynthetic pathway (Russell, D. W., Annu. Rev. Biochem. 72:137-174 (2003), which is hereby incorporated by reference in its entirety), was analyzed. Like wild-type FGF19, the FGF19K149A mutant markedly decreased CYP7A1 and CYP8B1 mRNA levels (FIG. 4C), demonstrating that knockout of residual HS binding does not affect the metabolic activity of FGF19. To examine whether residual HS binding is also dispensable for the metabolic activity of FGF23, mice were injected with the FGF23R140A/R143A mutant or wild-type FGF23 and serum phosphate concentrations were measured. The FGF23R140A/R143A mutant reduced serum phosphate as effectively as wild-type FGF23 (FIG. 4D). Moreover, when injected into Fgf23 knockout mice, the FGF23R140A/R143A mutant exhibited as much of phosphate-lowering activity as wild-type FGF23 (FIG. 4D). These data show that, as in the case of FGF19, abolishment of residual HS binding does not impact the metabolic activity of FGF23 leading to the conclusion that HS is not a component of the endocrine FGF signal transduction unit (FIG. 1D).
  • Example 12 Conversion of a Paracrine FGF into an Endocrine Ligand Confirms that HS is Dispensable for the Metabolic Activity of Endocrine FGFs
  • If HS is dispensable for the metabolic activity of endocrine FGFs, then it should be feasible to convert a paracrine FGF into an endocrine FGF by eliminating HS-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor binding site. Reducing HS-binding affinity will allow the ligand to freely diffuse and enter the blood circulation while attaching the C-terminal tail of an endocrine FGF will home the ligand into its target tissues. FGF2, a prototypical paracrine FGF, was chosen for conversion into FGF23-like and FGF21-like ligands, respectively. FGF2 was selected as paracrine ligand for this protein engineering exercise because it preferentially binds to the “c” isoform of FGFR1, the principal receptor mediating the metabolic activity of FGF23 (Gattineni et al., Am. J. Physiol. Renal Physiol. 297:F282-291 (2009); Liu et al., J. Am. Soc. Nephrol. 19:2342-2350 (2008), which are hereby incorporated by reference in their entirety) and FGF21 (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007), which is hereby incorporated by reference in its entirety), respectively. In the crystal structure of heparin-bound FGF2 (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety)), K128, R129, and K134 mediate the majority of hydrogen bonds with heparin and hence mutation of these residues was predicted to cause a major reduction in HS-binding affinity of FGF2 (FIGS. 1A, 2, and 5A). Accordingly, these three residues were mutated and then the short C-terminal tail of the mutated FGF2 was replaced with the C-terminal tail of FGF23 (R161 to I251) or the C-terminal tail of FGF21 (P168 to S209) (FIG. 5A). The resulting chimeras were termed FGF2ΔHBScore-FGF23C-tail and FGF2ΔHBScore-FGF21C-tail (FIG. 5A). To demonstrate that reduction in HS-binding affinity is required for converting FGF2 into an endocrine ligand, two control chimeras were made in which the HS-binding site of the FGF2 core was left intact (FGF2WTcore-FGF23C-tail and FGF2WTcore-FGF21C-all; FIG. 5A).
  • Consistent with the structural prediction, FGF2ΔHBScore-FGF23C-tail and FGF2ΔHBScore-FGF21C-tail exhibited poor binding affinity for HS compared to the corresponding control chimeras with intact HS-binding site (FIGS. 5B-5E). Since HS is an obligatory cofactor in paracrine FGF signaling, the FGF2ΔHBScore-FGF23C-tail and FGF2ΔHBScore-FGF21C-tail chimeras were predicted to lose the ability to activate FGFR1c in an HS-dependent fashion. To test this, HEK293 cells, which endogenously express FGFR1c, were stimulated with FGF2ΔHBScore-FGF23C-tail or FGF2WTcore-FGF23C-tail. Induction of protein expression of the transcription factor Egr1, a known downstream mediator of FGF signaling, was used as readout for FGFR activation. As shown in FIG. 5G, the FGF2HBScore-FGF23C-tail chimera, like native FGF23, was ineffective in inducing Egr1 expression at concentrations at which the FGF2WTcore-FGF23C-tail chimera elicited a near maximal effect. The same observations were made for the FGF2ΔHBScore-FGF21C-tail chimera (FIG. 5F). These data show that, similar to native FGF23 and FGF21, the FGF2ΔHBScore-FGF23C-tail and FGF2ΔHBScore-FGF21C-tail chimeras lost the ability to activate FGFR in an HS-dependent, paracrine fashion.
  • To determine whether the FGF2ΔHBScore-FGF23C-tail and FGF2ΔHBScore-FGF21C-tail chimeras gained the ability to signal in a Klotho co-receptor-dependent, endocrine fashion, it was first analyzed whether these chimeras can form ternary complexes with FGFR1c and Klotho co-receptor. To this end, a SPR-based binding competition assay was employed. FGF23 was immobilized onto a SPR biosensor chip, and mixtures of a fixed concentration of binary αKlotho-FGFR1c complex with increasing concentrations of FGF2ΔHBScore-FGF23C-tail chimera were passed over the chip. FGF2ΔHBScore-FGF23C-tail competed, in a dose-dependent fashion, with immobilized FGF23 for binding to the αKlotho-FGFR1c complex (FIG. 7A), demonstrating that the chimera, like native FGF23 (FIG. 7B), is able to form a ternary complex with FGFR1c and αKlotho. To test whether the FGF2ΔHBScore-FGF21C-tail chimera can likewise form a ternary complex with FGFR1c and βKlotho, FGF21 was coupled to a SPR biosensor chip, and mixtures of the binary βKlotho-FGFR1c complex with FGF2ΔHBScore-FGF21C-tail were passed over the chip. FGF2ΔHBScore-FGF21C-tail effectively competed with immobilized FGF21 for binding to the βKlotho-FGFR1c complex (FIG. 8A), demonstrating that the chimera, like native FGF21 (FIG. 8B), is capable of binding to the binary complex of FGFR1c and βKlotho. Notably, native FGF2 failed to compete with FGF23 for binding to the αKlotho-FGFR1c complex (FIG. 7C), and with FGF21 for binding to the βKlotho-FGFR1c complex (FIG. 8C) since it lacks the Klotho co-receptor binding domain. To further confirm the binding specificity of the FGF2ΔHBScore-FGF23C-tail chimera for the αKlotho-FGFR1c complex, FGF2ΔHBScore-FGF23C-tail and βKlotho-FGFR1c complex were mixed at a molar ratio of 10:1, and the mixture was injected over a chip containing immobilized FGF21. FGF2ΔHBScore-FGF23C-tail, like native FGF23, failed to compete with FGF21 for binding to the βKlotho-FGFR1c complex (FIGS. 7D and 7E). Similarly, the FGF2ΔHBScore-FGF21C-tail chimera, like native FGF21, failed to compete with FGF23 for binding to the αKlotho-FGFR1c complex (FIGS. 8D and 8E). For the FGF2ΔHBScore-FGF21C-tail chimera, we investigated whether it is able to activate FGFR1c in a βKlotho-dependent fashion in cells. HEK293 cells were transfected with βKlotho and then stimulated with FGF2ΔHBScore—FGF21C-tail or FGF21. Similar to native FGF21, the FGF2ΔHBScore-FGF21C-tail chimera induced Egr1 protein expression in HEK293-βKlotho cells (FIG. 8F), indicating that the chimera is capable of activating FGFR1c in the presence of βKlotho.
  • To provide definite proof for the ligand conversion, the metabolic activity of the chimeras in vivo was tested. Specifically, the ability of the FGF2ΔHBScore-FGF23C-tail chimera to lower serum phosphate and to reduce renal gene expression of CYP27B1, which catalyzes the conversion of vitamin D into its bioactive form, was examined. Mice were injected with FGF2ΔHBScore-FGF23C-tail or as controls, FGF23 or FGF2WTcore-FGF23C-tail, and serum phosphate concentrations and renal CYP27B1 mRNA levels were measured. Similar to native FGF23, the FGF2ΔHBScore-FGF23C-tail chimera caused a decrease in serum phosphate in wild-type mice (FIG. 7F). The chimera also induced a marked decrease in CYP27B1 mRNA levels, just like the native FGF23 ligand (FIG. 7G). These data show that the FGF2ΔHBScore-FGF23C-tail chimera acts as an FGF23-like hormone. Importantly, the FGF2WTcore-FGF23C-tail chimera failed to decrease serum phosphate or CYP27B1 mRNA levels (FIGS. 7F and 7G). This is expected because, owing to its high affinity for HS, this chimera should be trapped in the vicinity of the injection site and hence not be able to enter the blood circulation. Moreover, these data show that adding the Klotho co-receptor binding site is not sufficient to convert a paracrine FGF into an endocrine ligand. To confirm that the metabolic activity of the FGF2ΔHBScore-FGF23C-tail chimera is dependent on αKlotho, αKlotho knockout mice were injected with FGF2ΔHBScore-FGF23C-tail or FGF23 as a control, and serum concentrations of phosphate were measured. As shown in FIG. 7F, FGF2ΔHBScore-FGF23C-tail failed to lower serum phosphate, demonstrating that the chimera, like native FGF23 (FIG. 7F), requires αKlotho for metabolic activity.
  • To determine whether the FGF2ΔHBScore-FGF21C-tail chimera exhibits FGF21-like metabolic activity, its ability to potentiate the hypoglycemic effect of insulin was examined (Ohnishi et al., FASEB J. 25:2031-2039 (2011), which is hereby incorporated by reference in its entirety). Mice were injected with insulin plus FGF2ΔHBScore-FGF21C-tail, insulin plus FGF21, or insulin alone, and blood glucose concentrations were monitored for up to one hour after the injection. Similar to FGF21, the FGF2ΔHBScore-FGF21C-tail chimera enhanced the hypoglycemic effect of insulin (FIG. 8G), demonstrating that the chimera acts as an FGF21-like hormone.
  • To substantiate further the concept of FGF ligand conversion, another FGF21-like ligand was engineered using FGF1 as paracrine FGF, and the metabolic activity of the engineered protein was tested in vivo in a mouse model of diabetes and obesity. Besides serving as an additional proof-of-concept, the use of FGF1 for this particular ligand conversion was appealing because FGF1 on its own plays an essential role in glucose metabolism (Jonker et al., “A PPARγ-FGF1 Axis is Required for Adaptive Adipose Remodelling and Metabolic Homeostasis,” Nature 485:391-394 (2012), which is hereby incorporated by reference in its entirety). Notably, similar to FGF21, FGF1 is induced postprandially in gonadal white adipose tissue by the nuclear hormone receptor PPARγ (peroxisome proliferator activated receptor-γ) (Jonker et al., “A PPARγ-FGF1 Axis is Required for Adaptive Adipose Remodelling and Metabolic Homeostasis,” Nature 485:391-394 (2012); Dutchak et al., “Fibroblast Growth Factor-21 Regulates PPARγ Activity and the Antidiabetic Actions of Thiazolidinediones,” Cell 148:556-567 (2012), which are hereby incorporated by reference in their entirety). FGF1 is required for the remodeling of adipose tissue to adjust to fluctuations in nutrient availability (Jonker et al., “A PPARγ-FGF1 Axis is Required for Adaptive Adipose Remodelling and Metabolic Homeostasis,” Nature 485:391-394 (2012), which is hereby incorporated by reference in its entirety), and this process is influenced by FGF21 (Hotta et al., “Fibroblast Growth Factor 21 Regulates Lipolysis in White Adipose Tissue But is Not Required for Ketogenesis and Triglyceride Clearance in Liver,” Endocrinology 150:4625-4633 (2009); Dutchak et al., “Fibroblast Growth Factor-21 Regulates PPARγ Activity and the Antidiabetic Actions of Thiazolidinediones,” Cell 148:556-567 (2012), which are hereby incorporated by reference in their entirety). As part of a positive feedback loop, FGF21 stimulates PPARγ activity in adipocytes (Dutchak et al., “Fibroblast Growth Factor-21 Regulates PPARγ Activity and the Antidiabetic Actions of Thiazolidinediones,” Cell 148:556-567 (2012), which is hereby incorporated by reference in its entirety), raising the intriguing possibility that FGF21 regulates FGF1 signaling in adipose tissue through PPARγ. An FGF1ΔHBScore-FGF21C-tail chimera was generated in the same manner as the FGF2ΔHBScore-FGF21C-tail chimera (FIGS. 5 and 6). Specifically, K127, K128, and K133 of FGF1, which correspond to the key HS-binding residues identified in the crystal structure of heparin-bound FGF2 (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety)), were mutated and then the short C-terminal tail of the mutated FGF1 was replaced with the C-terminal tail of FGF21 (P168 to S209) (FIG. 6). A full-length FGF1 protein harboring the HS-binding site mutations was used as a control (FIG. 6). Consistent with the structural prediction, this protein exhibited poor binding affinity for HS compared to wild-type FGF1 as evidenced by the fact that, unlike the wild-type ligand, the mutant protein did not bind to a Heparin sepharose column. A subcutaneous bolus injection of the FGF1ΔHBScore-FGF21C-tail chimera elicited a hypoglycemic effect in ob/ob mice (FIG. 9C), demonstrating that the chimera has metabolic activity. The effect was of similar magnitude as that observed for native FGF1 (FIG. 9C), which itself has a much greater hypoglycemic effect in ob/ob mice than native FGF21 (FIG. 9A). The HS-binding site mutant of FGF1, which was included as a control in these experiments, showed a similar hypoglycemic effect as the wild-type ligand (FIG. 9B), indicating that the loss in HS-binding affinity had no impact on the metabolic activity of FGF1. To alter the receptor-binding specificity of FGF1 such that FGF1 selectively binds to the “c” splice isoform of FGFR1, the principal receptor mediating the metabolic activity of FGF21, an N-terminally truncated FGF1 protein was made (FIG. 6). The truncated FGF1 ligand lacked twenty four residues from the N-terminus including the nine residues that are critical for the promiscuous binding of FGF1 to both splice isoforms of FGFR1-3 (Beenken et al., “Plasticity in Interactions of Fibroblast Growth Factor 1 (FGF1) N Terminus with FGF Receptors Underlies Promiscuity of FGF1,” J Biol Chem 287(5):3067-3078 (2012), which is hereby incorporated by reference in its entirety). Based on the crystal structures of FGF1-FGFR complexes, the truncation was also predicted to reduce the receptor-binding affinity of FGF1, and hence the ligand's mitogenicity. The truncated FGF1 protein induced a similar hypoglycemic effect in ob/ob mice as native FGF1 did, indicating that the metabolic activity of FGF1 is mediated through the “c” splice isoform of FGFR. Together, these findings provide a starting point for engineering FGF1 ligands that have no mitogenicity but the same or enhanced metabolic activity compared to native FGF1.
  • The demonstrated ability to convert a paracrine FGF into an endocrine ligand by means of reducing HS-binding affinity of the paracrine FGF and adding the Klotho co-receptor binding site substantiates that HS does not participate in the formation of the endocrine FGF signal transduction unit. The dispensability of HS for the metabolic activity of endocrine FGFs has an intriguing implication as to how these FGFs have evolved to become hormones. It appears that these ligands have lost the requirement to bind HS in order to signal, while acquiring the ability to bind Klotho co-receptors, which is necessary to direct these ligands to their target organs.
  • In the target tissue, Klotho co-receptors constitutively associate with cognate receptors of endocrine FGFs to offset the inherently low receptor-binding affinity of endocrine FGFs (FIGS. 10B-10D; Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007); Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006); Ogawa et al., Proc. Nat'l. Acad. Sci. U.S.A. 104:7432-7437 (2007); Urakawa et al., Nature 444:770-774 (2006), which are hereby incorporated by reference in their entirety). This low binding affinity is due to the fact that key receptor-binding residues in the β-trefoil core of endocrine FGFs are replaced by residues that are suboptimal for receptor binding (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety). To measure the degree to which Klotho co-receptors enhance the receptor-binding affinity of endocrine FGFs, SPR experiments were conducted using FGF23 and FGFR1c and αKlotho co-receptor as an example (see FIGS. 10A-10F). The SPR data show that αKlotho enhances the affinity of FGF23 for FGFR1c by over 20-fold (FIGS. 10D and 10E). The affinity of FGF23 for FGFR1c in the presence of αKlotho is comparable to that of FGF2 for FGFR1c in the absence of its HS cofactor (FIGS. 10A and 10E). It should be noted, however, that HS further increases the binding affinity of FGF2 for FGFR1c by at least an order of magnitude (Pantoliano et al., Biochemistry 33:10229-10248 (1994); Roghani et al., J. Biol. Chem. 269:3976-3984 (1994), which are hereby incorporated by reference in their entirety). Hence, the receptor-binding affinity of FGF23 in the presence of αKlotho co-receptor still is lower than that of FGF2 in the presence of HS cofactor. These observations imply that the signaling capacity of the endocrine FGF signal transduction unit should be weaker than that of the paracrine FGF signaling unit. Indeed, cell-based studies show that even in the presence of their Klotho co-receptor, endocrine FGFs are inferior to paracrine FGFs at activating FGFR-induced intracellular signaling pathways (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007); Urakawa et al., Nature 444:770-774 (2006), which are hereby incorporated by reference in their entirety).
  • The finding that endocrine FGFs do not need to rely on HS for signaling has another important implication in regard to the role of Klotho co-receptors. Since FGFR dimerization is a prerequisite for FGF signaling in general, it is proposed that Klotho co-receptors not only enhance the binding affinity of endocrine ligand for receptor but also promote receptor dimerization upon ligand binding. In other words, Klotho co-receptors must fulfill the same dual role that HS plays in signaling by paracrine FGFs (FIG. 1D). The ligand conversion also provides the framework for the rational design of endocrine FGF-like molecules for the treatment of metabolic disorders. An FGF23-like molecule, for example, will be useful for the treatment of inherited or acquired hyperphosphatemia, and an FGF21-like molecule, for example, for the treatment of type 2 diabetes, obesity, and related metabolic disorders.
  • Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims (47)

1. A chimeric protein comprising:
an N-terminus coupled to a C-terminus, wherein the N-terminus comprises a portion of a paracrine fibroblast growth factor (“FGF”) and the C-terminus comprises a C-terminal portion of an FGF21 molecule and wherein the portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification.
2. The chimeric protein according to claim 1, wherein the paracrine FGF is FGF 1 or FGF2.
3. The chimeric protein according to claim 1, wherein the portion of the paracrine FGF comprises an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 150 to 155 of SEQ ID NO: 1.
4. The chimeric protein according to claim 1, wherein the portion of the paracrine FGF comprises an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 151 to 155 of SEQ ID NO: 121.
5. The chimeric protein according to claim 3, wherein the portion of the paracrine FGF comprises amino acid residues 1-150, 1-151, 1-152, 1-153, 1-154, 1-155, 2-150, 2-151, 2-152, 2-153, 2-154, 2-155, 3-150, 3-151, 3-152, 3-153, 3-154, 3-155, 4-150, 4-151, 4-152, 4-153, 4-154, 4-155, 5-150, 5-151, 5-152, 5-153, 5-154, 5-155, 6-150, 6-151, 6-152, 6-153, 6-154, 6-155, 7-150, 7-151, 7-152, 7-153, 7-154, 7-155, 8-150, 8-151, 8-152, 8-153, 8-154, 8-155, 9-150, 9-151, 9-152, 9-153, 9-154, 9-155, 10-150, 10-151, 10-152, 10-153, 10-154, 10-155, 11-150, 11-151, 11-152, 11-153, 11-154, 11-155, 12-150, 12-151, 12-152, 12-153, 12-154, 12-155, 13-150, 13-151, 13-152, 13-153, 13-154, 13-155, 14-150, 14-151, 14-152, 14-153, 14-154, 14-155, 15-150, 15-151, 15-152, 15-153, 15-154, 15-155, 16-150, 16-151, 16-152, 16-153, 16-154, 16-155, 17-150, 17-151, 17-152, 17-153, 17-154, 17-155, 18-150, 18-151, 18-152, 18-153, 18-154, 18-155, 19-150, 19-151, 19-152, 19-153, 19-154, 19-155, 20-150, 20-151, 20-152, 20-153, 20-154, 20-155, 21-150, 21-151, 21-152, 21-153, 21-154, 21-155, 22-150, 22-151, 22-152, 22-153, 22-154, 22-155, 23-150, 23-151, 23-152, 23-153, 23-154, 23-155, 24-150, 24-151, 24-152, 24-153, 24-154, 24-155, 25-150, 25-151, 25-152, 25-153, 25-154, or 25-155 of SEQ ID NO: 1.
6. The chimeric protein according to claim 4, wherein the portion of the paracrine FGF comprises amino acid residues 1-151, 1-152, 1-153, 1-154, 1-155, 2-151, 2-152, 2-153, 2-154, 2-155, 3-151, 3-152, 3-153, 3-154, 3-155, 4-151, 4-152, 4-153, 4-154, 4-155, 5-151, 5-152, 5-153, 5-154, 5-155, 6-151, 6-152, 6-153, 6-154, 6-155, 7-151, 7-152, 7-153, 7-154, 7-155, 8-151, 8-152, 8-153, 8-154, 8-155, 9-151, 9-152, 9-153, 9-154, 9-155, 10-151, 10-152, 10-153, 10-154, 10-155, 11-151, 11-152, 11-153, 11-154, 11-155, 12-151, 12-152, 12-153, 12-154, 12-155, 13-151, 13-152, 13-153, 13-154, 13-155, 14-151, 14-152, 14-153, 14-154, 14-155, 15-151, 15-152, 15-153, 15-154, 15-155, 16-151, 16-152, 16-153, 16-154, 16-155, 17-151, 17-152, 17-153, 17-154, 17-155, 18-151, 18-152, 18-153, 18-154, 18-155, 19-151, 19-152, 19-153, 19-154, 19-155, 20-151, 20-152, 20-153, 20-154, 20-155, 21-151, 21-152, 21-153, 21-154, 21-155, 22-151, 22-152, 22-153, 22-154, 22-155, 23-151, 23-152, 23-153, 23-154, 23-155, 24-151, 24-152, 24-153, 24-154, 24-155, 25-151, 25-152, 25-153, 25-154, or 25-155 of SEQ ID NO: 121.
7. The chimeric protein according to claim 3, wherein the modification comprises one or more substitutions located at one or more amino acid residues of SEQ ID NO: 1 selected from the group consisting of N33, K127, K128, N129, K133, R134, R137, Q142, K143, and combinations thereof.
8. The chimeric protein according to claim 7, wherein the one or more substitutions are selected from the group consisting of N33T, K127D, K128Q, N129T, K133V, R134L, R137H, Q142M, K143T/L/I, and combinations thereof.
9. The chimeric protein according to claim 4, wherein the modification comprises one or more substitutions located at one or more amino acid residues of SEQ ID NO: 121 selected from the group consisting of N36, K128, R129, K134, K138, Q143, K144, C78, C96, and combinations thereof.
10. The chimeric protein according to claim 9, wherein the one or more substitutions are selected from the group consisting of N36T, K128D, R129Q, K134V, K138H, Q143M, K144T/L/I, C78S, C96S, and combinations thereof.
11. The chimeric protein according to claim 1, wherein the C-terminal portion comprises a β-Klotho co-receptor binding domain.
12. The chimeric protein according to claim 1, wherein the C-terminal portion comprises amino acid residues 168-209 of SEQ ID NO: 233.
13. The chimeric protein according to claim 12, wherein the C-terminal portion further comprises one or more deletions or substitutions while retaining the ability to bind β-Klotho.
14. The chimeric protein according to claim 12, wherein the C-terminal portion of FGF21 further comprises one or more substitutions, additions, or deletions to enhance binding affinity for β-Klotho compared to the C-terminal portion without the modification.
15. A pharmaceutical composition comprising the chimeric protein of claim 1 and a pharmaceutically-acceptable carrier.
16. The pharmaceutical composition according to claim 15 further comprising:
one or more agents selected from the group consisting of an anti-inflammatory agent, an antifibrotic agent, an antihypertensive agent, an antidiabetic agent, a triglyceride-lowering agent, and a cholesterol-lowering agent.
17. The pharmaceutical composition according to claim 15 further comprising an organotropic targeting agent.
18. A method for treating a subject suffering from a disorder, the method comprising:
selecting a subject suffering from the disorder;
providing a chimeric fibroblast growth factor (“FGF”) protein, wherein the chimeric FGF protein comprises an N-terminus coupled to a C-terminus, wherein the N-terminus comprises a portion of a paracrine FGF and the C-terminus comprises a C-terminal portion of FGF21, and wherein the portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification; and
administering a therapeutically effective amount of the chimeric FGF protein to the selected subject under conditions effective to treat the disorder.
19. The method according to claim 18, wherein the paracrine FGF is FGF1 or FGF2.
20. The method according to claim 18, wherein the portion of the paracrine FGF comprises an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 150 to 155 of SEQ ID NO: 1.
21. The method according to claim 18, wherein the portion of the paracrine FGF comprises an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 151 to 155 of SEQ ID NO: 121.
22. The method according to claim 20, wherein the portion of the paracrine FGF comprises amino acid residues 1-150, 1-151, 1-152, 1-153, 1-154, 1-155, 2-150, 2-151, 2-152, 2-153, 2-154, 2-155, 3-150, 3-151, 3-152, 3-153, 3-154, 3-155, 4-150, 4-151, 4-152, 4-153, 4-154, 4-155, 5-150, 5-151, 5-152, 5-153, 5-154, 5-155, 6-150, 6-151, 6-152, 6-153, 6-154, 6-155, 7-150, 7-151, 7-152, 7-153, 7-154, 7-155, 8-150, 8-151, 8-152, 8-153, 8-154, 8-155, 9-150, 9-151, 9-152, 9-153, 9-154, 9-155, 10-150, 10-151, 10-152, 10-153, 10-154, 10-155, 11-150, 11-151, 11-152, 11-153, 11-154, 11-155, 12-150, 12-151, 12-152, 12-153, 12-154, 12-155, 13-150, 13-151, 13-152, 13-153, 13-154, 13-155, 14-150, 14-151, 14-152, 14-153, 14-154, 14-155, 15-150, 15-151, 15-152, 15-153, 15-154, 15-155, 16-150, 16-151, 16-152, 16-153, 16-154, 16-155, 17-150, 17-151, 17-152, 17-153, 17-154, 17-155, 18-150, 18-151, 18-152, 18-153, 18-154, 18-155, 19-150, 19-151, 19-152, 19-153, 19-154, 19-155, 20-150, 20-151, 20-152, 20-153, 20-154, 20-155, 21-150, 21-151, 21-152, 21-153, 21-154, 21-155, 22-150, 22-151, 22-152, 22-153, 22-154, 22-155, 23-150, 23-151, 23-152, 23-153, 23-154, 23-155, 24-150, 24-151, 24-152, 24-153, 24-154, 24-155, 25-150, 25-151, 25-152, 25-153, 25-154, or 25-155 of SEQ ID NO: 1.
23. The method according to claim 21, wherein the portion of the paracrine FGF comprises amino acid residues 1-151, 1-152, 1-153, 1-154, 1-155, 2-151, 2-152, 2-153, 2-154, 2-155, 3-151, 3-152, 3-153, 3-154, 3-155, 4-151, 4-152, 4-153, 4-154, 4-155, 5-151, 5-152, 5-153, 5-154, 5-155, 6-151, 6-152, 6-153, 6-154, 6-155, 7-151, 7-152, 7-153, 7-154, 7-155, 8-151, 8-152, 8-153, 8-154, 8-155, 9-151, 9-152, 9-153, 9-154, 9-155, 10-151, 10-152, 10-153, 10-154, 10-155, 11-151, 11-152, 11-153, 11-154, 11-155, 12-151, 12-152, 12-153, 12-154, 12-155, 13-151, 13-152, 13-153, 13-154, 13-155, 14-151, 14-152, 14-153, 14-154, 14-155, 15-151, 15-152, 15-153, 15-154, 15-155, 16-151, 16-152, 16-153, 16-154, 16-155, 17-151, 17-152, 17-153, 17-154, 17-155, 18-151, 18-152, 18-153, 18-154, 18-155, 19-151, 19-152, 19-153, 19-154, 19-155, 20-151, 20-152, 20-153, 20-154, 20-155, 21-151, 21-152, 21-153, 21-154, 21-155, 22-151, 22-152, 22-153, 22-154, 22-155, 23-151, 23-152, 23-153, 23-154, 23-155, 24-151, 24-152, 24-153, 24-154, 24-155, 25-151, 25-152, 25-153, 25-154, or 25-155 of SEQ ID NO: 121.
24. The method according to claim 20, wherein the modification comprises one or more substitutions located at one or more amino acid residues of SEQ ID NO: 1 selected from the group consisting of N33, K127, K128, N129, K133, R134, R137, Q142, K143, and combinations thereof.
25. The method according to claim 24, wherein the one or more substitutions are selected from the group consisting of N33T, K127D, K128Q, N129T, K133V, R134L, R137H, Q142M, K143T/L/I, and combinations thereof.
26. The method according to claim 21, wherein the modification comprises one or more substitutions located at one or more amino acid residues of SEQ ID NO: 121 selected from the group consisting of N36, K128, R129, K134, K138, Q143, K144, C78, C96, and combinations thereof.
27. The method according to claim 26, wherein the one or more substitutions are selected from the group consisting of N36T, K128D, R129Q, K134V, K138H, Q143M, K144T/L/I, C78S, C96S, and combinations thereof.
28. The method according to claim 18, wherein the C-terminal portion comprises a β-Klotho co-receptor binding domain.
29. The method according to claim 18, wherein the C-terminal portion comprises amino acid residues 168-209 of SEQ ID NO: 233.
30. The method according to claim 29, wherein the C-terminal portion of FGF21 further comprises one or more substitutions, additions, or deletions while retaining the ability to bind β-Klotho.
31. The method according to claim 29, wherein the C-terminal portion of FGF21 further comprises one or more substitutions, additions, or deletions to enhance binding affinity for β-Klotho compared to the C-terminal portion without the modification.
32. The method according to claim 18, wherein the disorder is a selected from the group consisting of diabetes, obesity, and metabolic syndrome.
33. The method according to claim 18, wherein the disorder is type II diabetes, gestational diabetes, or drug-induced diabetes.
34. The method according to claim 18, wherein the disorder is type I diabetes.
35. The method according to claim 18, wherein the disorder is obesity.
36. The method according to claim 18, wherein the disorder is metabolic syndrome.
37. The method according to claim 18, wherein said administering is carried out parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes.
38. The method according to claim 18, wherein the chimeric FGF protein is administered with a pharmaceutically-acceptable carrier.
39. The method according to claim 18, wherein the selected subject is a mammal.
40. The method according to claim 18, wherein the selected subject is a human.
41. The method according to claim 18, wherein the chimeric FGF is co-administered with one or more agents selected from the group consisting of an anti-inflammatory agent, an antifibrotic agent, an antihypertensive agent, an antidiabetic agent, a triglyceride-lowering agent, and a cholesterol-lowering agent.
42. A method of making a chimeric fibroblast growth factor (“FGF”) protein possessing enhanced endocrine activity, the method comprising:
introducing one or more modifications to a FGF protein, wherein the modification decreases the affinity of the FGF protein for heparin and/or heparan sulfate; and
coupling a Klotho co-receptor binding domain to the modified FGF protein's C-terminus, whereby a chimeric FGF protein possessing enhanced endocrine activity is made.
43-59. (canceled)
60. A method of facilitating fibroblast growth factor receptor (“FGFR”)-βKlotho co-receptor complex formation, the method comprising:
providing a cell comprising a βKlotho co-receptor and an FGFR;
providing a chimeric fibroblast growth factor (“FGF”) protein comprising a C-terminal portion of FGF21 and a portion of a paracrine FGF, wherein the portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification; and
contacting the cell with the chimeric FGF protein under conditions effective to cause FGFR-βKlotho co-receptor complex formation.
61-75. (canceled)
76. A method of screening for agents capable of facilitating fibroblast growth factor receptor (“FGFR”)-βKlotho complex formation in the treatment of a disorder, the method comprising:
providing a chimeric fibroblast growth factor (“FGF”) comprising an N-terminus coupled to a C-terminus, wherein the N-terminus comprises a portion of a paracrine FGF and the C-terminus comprises a C-terminal portion of FGF21, and wherein the portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification;
providing binary βKlotho-FGFR complex;
providing one or more candidate agents;
combining the chimeric FGF, the binary βKlotho-FGFR complex, and the one or more candidate agents under conditions permitting the formation of a ternary complex between the chimeric FGF and the binary βKlotho-FGFR complex in the absence of the one or more candidate agents; and
identifying the one or more candidate agents that decrease ternary complex formation between the chimeric FGF and the binary βKlotho-FGFR complex compared to the ternary complex formation in the absence of the one or more candidate agents as suitable for treating the disorder.
77-84. (canceled)
US14/097,116 2012-06-07 2013-12-04 Chimeric fibroblast growth factor 21 proteins and methods of use Abandoned US20140094406A1 (en)

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