Constructs for modulating FGF21 / FGFR / beta-klotho signaling pathway and treating diseases and disorders, and methods thereof

Abstract

Described herein is a construct including an FGF21 polypeptide, which includes residues 186-198 and residues 200-209 of FGF21. The FGF21 polypeptide includes one or more mutations that confers FGF21 polypeptide stronger affinity with β-Klotho as compared with a corresponding wild-type FGF21 polypeptide. Also described herein is a composition including the construct, as well as methods of modulating the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway and methods of treating, ameliorating and / or preventing diseases or disorders associated with mis-regulation (such as up- or down-regulation) of the signaling pathways.

Description

[0001]Attorney Docket No.047162-7483WO1 CONSTRUCTS FOR MODULATING FGF21 / FGFR / BETA-KLOTHO SIGNALING PATHWAY AND TREATING DISEASES AND DISORDERS, AND METHODS THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No.63 / 581,860, filed September 11, 2023, which is incorporated herein by reference in its entirety. SEQUENCE LISTING The XML file named " 047162-7483WO1_Seq Listing.xml " created on August 31, 2024, comprising 81,737 bytes, is hereby incorporated by reference in its entirety. BACKGROUND FGF19 and FGF21 (as well FGF23) stand out from the canonical FGFs by exhibiting hallmarks of circulating hormones, and are thus termed endocrine FGFs. FGF19 and FGF21 both function as hormones that bind specifically to receptors located in liver, fat tissue, and the hypothalamus, regulating metabolic functions (such as bile acid synthesis and lipogenesis) and also stimulating insulin sensitivity, energy expenditure, and weight loss. Unlike canonical FGFs that require heparan sulfate proteoglycans (HSPGs) to activate FGFRs, endocrine FGFs do not have this requirement, but instead are specifically dependent on Klotho co-receptors for FGFR activation. There are two Klothos, encoded by different genes. α-Klotho is required for FGF23- dependent signaling, and β-Klotho is essential for FGF19- or FGF21-dependent signaling in specific tissues and organs. Although different FGFRs are expressed throughout the body, expression of Klotho proteins is limited to specific tissues — α-Klotho expression is confined to the kidney and parathyroid, whereas β-Klotho expression is limited to adipose tissue, liver, pancreas and hypothalamus. FGF19 or FGF23 bind to FGFRs (such as FGFR1c) via the FGF-core region, and bind to β-Klotho via the C-terminus tail, forming the FGF / FGFR / β-Klotho ternary complex and resulting in stimulation of tyrosine kinase activity, which activate downstream signaling events (see e.g., WO 2019 / 010314 A1, which is incorporated herein in its entirety by 1 52997671.2 Attorney Docket No.047162-7483WO1 reference). Mis-regulations of the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway are implicated in various diseases or disorders including obesity, diabetes, pancreatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), and acute alcohol toxicity. In addition, certain cancers, such as a subtype of liver cancer, are driven by aberrant activation of FGF19 / FGFR4 / β- Klotho pathway. There is a need for novel compounds and / or constructs that modulate these signaling pathways, as well as compositions, constructs, and / or methods that can treat, ameliorate, and / or prevent diseases or disorders associated with these signaling pathways. The present invention addresses this need. SUMMARY Non-natural construct In some aspects, the present invention is directed to a non-natural construct, such as a non-natural soluble construct. In some embodiments, the construct comprises an FGF21 polypeptide comprising residues 186-198 and residues 200-209 of FGF21. In some embodiments, the FGF21 polypeptide comprises at least one mutation selected from the group consisting of L126, L194, V197, P199, Q201, R203, Y207, A208, and S209. In some embodiments, the FGF21 polypeptide comprises at least one mutation selected from the group consisting of L126R, L194F, V197I, V197L, P199G, Q201L, Q201I, Q201P, Q201R, Q201W, R203W, Y207W, A208E, A208W, and S209G. In some embodiments, the FGF21 polypeptide comprises residues 186-209 of FGF21. In some embodiments, the FGF21 polypeptide lacks all or some of residues 1-185 of FGF21. In some embodiments, the FGF21 polypeptide lacks all or some of residues 29-185 of FGF21. In some embodiments, the FGF21 polypeptide lacks all or some of residues 169-185 of FGF21. In some embodiments, the FGF21 polypeptide comprises residues 169-209 of FGF21. In some embodiments, the FGF21 polypeptide lacks all or some of residues 1-168 of FGF21. In some embodiments, the FGF21 polypeptide lacks all or some of residues 29-168 of FGF21. In some embodiments, the FGF21 polypeptide comprises residues 29-209 of FGF21. 2 52997671.2 Attorney Docket No.047162-7483WO1 In some embodiments, the FGF21 polypeptide lacks all or some of residues 1-28 of FGF21. In some embodiments, the FGF21 polypeptide further comprises at least one sequence selected from the group consisting of RKK, RRS, and RKS conjugated to the C-terminus of the residues 200-209 of FGF21. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, V197I, Q201L, R203W, Y207W, A208W and S209G, and the sequence RKK conjugated to the C-terminus of the residues 200-209 of FGF21. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, V197I, Q201R, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, V197I, Q201R, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, V197L, Q201R, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, V197L, Q201R, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, Q201I, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, Q201L, R203W, Y207W, A208W and S209G, and the sequence RKS conjugated to the C- terminus of the residues 200-209 of FGF21. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, Q201P, R203W, Y207W, A208W and S209G, and the sequence RRS conjugated to the C- terminus of the residues 200-209 of FGF21. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, Q201R, R203W, Y207W, A208W and S209G, and the sequence RRS conjugated to the C- terminus of the residues 200-209 of FGF21. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, Q201R, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, Q201W, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide comprises the mutations R203W, Y207W and A208W. 3 52997671.2 Attorney Docket No.047162-7483WO1 In some embodiments, the FGF21 is a human FGF21 having a protein sequence set forth in SEQ ID NO:1. In certain embodiments, the FGF21 polypeptide comprises residues 29-209 of SEQ ID NO: 1 with one or more mutations as described herein, wherein the FGF21 polypeptide lacks residues 1-28 of SEQ ID NO: 1. In another example, the FGF21 polypeptide comprises residues 29-209 with one or more mutations as described in more detail herein and at least one sequence selected from the group consisting of RKK, RRS, and RKS conjugated to the C-terminus (e.g., the C-terminus of the residues 200-209 of FGF21), and wherein the FGF21 polypeptide lacks residues 1-28 of SEQ ID NO: 1. In some embodiments, the construct further comprises a stability enhancing domain. In some embodiments, the stability enhancing domain is attached to an N-terminus of the FGF21 polypeptide, or the stability enhancing domain is attached to a C-terminus of the FGF21 polypeptide. In some embodiments, the stability enhancing domain is at least one selected from the group consisting of albumin, an Fc region of an antibody, a glutathione S-transferase, a small ubiquitin-like modifier (SUMO), an O-glycosylation region of a Drosophila Spitz protein, and a thioredoxin. In some embodiments, the FGF21 polypeptide and the stability enhancing domain are linked through a linker polypeptide comprising about 1-18 amino acids. In some embodiments, the dissociation constant (Kd) of the FGF21 polypeptide with β-Klotho is about 20% or less of the Kdof a corresponding wild-type FGF21 polypeptide. In some embodiments, the Koff of the FGF21 polypeptide with β-Klotho is about 20% or less of the Koffof a corresponding wild-type FGF21 polypeptide. In some embodiments, the construct binds to β-Klotho and sequester β-Klotho from pathways downstream of a fibroblast growth factor receptor (FGFR) and β-Klotho. In some embodiments, the construct binds to both an FGFR and β-Klotho and activate pathways downstream of the FGFR and β-Klotho. In some embodiments, the FGFR is FGFR1c. Composition In some aspects, the present invention is directed to a composition. In some embodiments, the composition comprises the construct herein and a pharmaceutically acceptable carrier. 4 52997671.2 Attorney Docket No.047162-7483WO1 Method of modulating FGF21 / FGFR / β-Klotho signaling pathway or FGF19 / FGFR / β-Klotho signaling pathway In some aspects, the present invention is directed to a method of modulating FGF21 / FGFR / β-Klotho signaling pathway or FGF19 / FGFR / β-Klotho signaling pathway in a cell. In some embodiments, the method comprises contacting the surface of the cell with the construct herein. In some embodiments, the construct binds to β-Klotho but not FGFR, thereby sequestering β-Klotho from the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway and down-regulating the signaling pathway. In some embodiments, the construct binds to both β-Klotho and FGFR, thereby upregulating the FGF21 / FGFR / β-Klotho signaling pathway. In some embodiments, the cell is from a cell line, a primary cell, or a cell in the body of a subject. In some embodiments, the cell is a cell in or derived from adipose tissue, liver, pancreas, or hypothalamus. Method of treating, ameliorating and / or preventing a disease or disorder In some aspects, the present invention is directed to a method of treating, ameliorating and / or preventing a disease or disorder in a subject in need thereof. In some embodiments, the method comprises administering to the subject an effective amount of the construct herein, or the composition herein. In some embodiments, the disease or disorder is at least one selected from obesity, diabetes, pancreatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), acute alcohol toxicity, and cancers driven by an aberrant activation of FGF19 / FGFR4 / β-Klotho pathway. In some embodiments, the construct or the composition is administered via at least one route selected from the group consisting of inhalational, oral, rectal, vaginal, parenteral, intracranial, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, and intravenous. In some embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of exemplary embodiments will be better 5 52997671.2 Attorney Docket No.047162-7483WO1 understood when read in conjunction with the appended drawings. For the purpose of illustrating, non-limiting embodiments are shown in the drawings. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. Figs.1A-1C are schematic representations of FGF21 C-terminus tail variants (CV) according to some embodiments. The locations of the amino acid changes from the wild-type FGF21 are indicated as arrows with corresponding mutations. Figs.2A-2B are schematic representations of FGF21 protein variants with N-terminal fusion partner proteins, in accordance with some embodiments. The fusion partner proteins include histidine-tag, SUMO, mouse Serum Albumin (mSA), Spitz, and Fc region from human IgG1. Sizes of regions in all schemes are proportional to the length of the amino acids except regions of Fc and mSA, which have molecular weights of 32 kDa and 65.9 kDa, respectively. Figs.3A-3N illustrates the results of some exemplary sensorgrams from the binding kinetics measurements between Fc-FGF21 variants and sKLB using the bio-layer interferometry (BLI). Fc-FGF21 variants were immobilized on the biosensors coated with anti-Fc antibodies (AHC) which were dipped into wells containing a series of concentrations of sKLB. Reference-subtracted sensorgrams (blue lines) are fitted with 1:1 Langmuir binding model (red lines) to obtain kinetic rate constants, konand koff, and the dissociation constant, KD, which are listed in Fig.3N. Fig.4A illustrates the results of some exemplary BLI sensorgrams from the binding kinetics measurements between His-FGF21 wt or His-FGF21 CV16 and sKLB. His-FGF21 variants were immobilized onto the Amine Reactive 2nd Generation (AR2G) biosensors using standard amine coupling method utilizing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Reference-subtracted sensorgrams (blue lines) are fitted with 1:1 Langmuir binding model (red lines) to obtain kinetic rate constants, konand koff, and the dissociation constant, KD, which are listed in Fig. 4B. Fig.5A shows the results of some exemplary immunoblotting analyses demonstrating the cellular activities of His-FGF21 wt and His-FGF21 CV16 in L6 cells co-expressing β- Klotho and FGFR1c, in accordance with some embodiments. Cellular activities were revealed by the levels of phosphorylated MAPK by immunoblotting using anti-MAPK and anti-pMAPK antibodies. Fig.5B illustrates the results of glucose uptake assays for His- FGF21 wt (red dots) and His-FGF21 CV16 (blue squares), in accordance with some 6 52997671.2 Attorney Docket No.047162-7483WO1 embodiments.3T3-L1 fibroblasts were chemically differentiated into adipocytes and treated with either His-FGF21 wt or His-FGF21 CV16. The cellular glucose amount in each sample were measured as luminescence levels. The data represent the mean of normalized luminescence ± SD of four independent experiments. EC50values for His-FGF21 wt and His- FGF21 CV16 are 1.84 ± 0.346 nM and 0.29 ± 0.071 nM (in value ± SEM), respectively. Figs.6A-6E: Calcium release and MAPK activation in pancreatic acinar (AR42J) cells stimulated with His-FGF21 wt and His-FGF21 CV16, in accordance with some embodiments. Figs.6A-6D: Time course of Ca2+release in AR42J cells (expressing endogenous FGFR1c and ^-Klotho). For Ca2+release studies, cells were loaded with a fluorescent Ca2+indicator (Calbryte 520 AM) and then stimulated with indicated concentrations of His-FGF21 wt or His-FGF21 CV16. Total amount of Ca2+release was analyzed as a change in fluorescence. Fig.6E: Analysis of MAPK response. AR42J cells were left unstimulated or stimulated with indicated concentrations of His-FGF21 wt or His- FGF21 CV16 (10 minutes, 37 ^C), and the cellular activities were monitored by the levels of phosphorylated MAPK using anti-pMAPK antibodies. Fig.7A illustrates the results of some exemplary BLI sensorgrams from the binding kinetics measurements between SUMO-FGF21 variants and sKLB, in accordance with some embodiments. SUMO-FGF21 variants were immobilized onto the Amine Reactive 2nd Generation (AR2G) biosensors using standard amine coupling method utilizing 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Reference-substracted sensorgrams (blue lines) are fitted with 1:1 Langmuir binding model (red lines) to obtain kinetic rate constants, konand koffand the dissociation constant, KD, which are listed in Fig.7B. Fig.7C shows some exemplary western blots demonstrating the cellular activities of SUMO-FGF21 wt and SUMO-FGF21 CV16 in L6 cells co-expressing β- Klotho and FGFR1c, in accordance with some embodiments. Cellular activities were monitored by the levels of phosphorylated MAPK by immunoblotting using anti-MAPK and anti-pMAPK antibodies. Fig.8A illustrates the results of some exemplary BLI sensorgrams from the binding kinetics measurements between mSA-FGF21 variants and sKLB. mSA-FGF21 variants were immobilized onto the Amine Reactive 2nd Generation (AR2G) biosensors using standard amine coupling method utilizing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Reference-substracted sensorgrams (blue lines) are fitted with 1:1 Langmuir binding model (red lines) to obtain kinetic rate constants, kon and 7 52997671.2 Attorney Docket No.047162-7483WO1 koff, and the dissociation constant, KD, which are listed in Fig.8B. Fig.8C shows the results of some exemplary western blots demonstrating the cellular activities of mSA-FGF21 wt and mSA-FGF21 CV16 in L6 cells co-expressing β-Klotho and FGFR1c, in accordance with some embodiments. Cellular activities were monitored by the levels of phosphorylated MAPK by immunoblotting using anti-MAPK and anti-pMAPK antibodies. Fig.9A illustrates the results of some exemplary BLI sensorgrams from the binding kinetics measurements between Spitz-FGF21 variants and sKLB, in accordance with some embodiments. Spitz-FGF21 variants were immobilized onto the Amine Reactive 2nd Generation (AR2G) biosensors using standard amine coupling method utilizing 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Reference-substracted sensorgrams (blue lines) are fitted with 1:1 Langmuir binding model (red lines) to obtain kinetic rate constants, konand koff, and the dissociation constant, KD, which are listed in Fig.8B. Fig.9C shows the results of some exemplary western blots demonstrating the cellular activities of mSA-FGF21 wt and mSA-FGF21 CV16 in L6 cells co-expressing β-Klotho and FGFR1c, in accordance with some embodiments. Cellular activities were monitored by the levels of phosphorylated MAPK by immunoblotting using anti-MAPK and anti-pMAPK antibodies. Figs.10A-10E illustrate the Gaussian accelerated molecular dynamics simulation results of FGF21 wt SIR1 (Figs.10A-10B) and FGF21 wt SIR2 peptides (Figs.10C-10E), in accordance with some embodiments. In the folding free energy landscape (Fig.10A) and conformational population density (Fig.10B) of FGF21 wt SIR1 peptide, the X- and Y-axes represent the backbone RMSD of FGF21 wt D192-V197 and D187-S190 carboxylate side chain to backbone amide distance, respectively. The dotted lines indicate the cutoff between the folded and unfolded states. In the folding free energy landscape (Fig.10C) and conformational population density (Fig.10D) of FGF21 wt SIR2 peptide, backbone RMSD of S204-A208 and S204-Y207 backbone amide distance are used as measurement metrics. FGF21 wt SIR2 peptide has three populations: folded, intermediate, and unfolded states. An activation free energy ( ^G^) between the folded and unfolded states, estimated from the global minimum of the folding free energy landscape, is ~ 2.5 kcal / mol. Conformational population density plots indicate the folded state of SIR1 peptide was rarely sampled, whereas SIR2 has a slight propensity to sample the folded state. Fig.10E demonstrate that conformational population densities of FGF21 CV16 SIR2 variants observed in Gaussian accelerated molecular dynamics simulations. Conformational state is measured by backbone 8 52997671.2 Attorney Docket No.047162-7483WO1 RMSD of S204-W208 and S204-W207 backbone amide distance. FGF21 CV16 SIR2 has markedly increased population in the folded state (39%), suggesting FGF21 CV16 SIR2 has higher propensity than FGF21 wt SIR2 to form the folded conformation similar to the observed crystallographic conformation (PDB: 5VAQ). Fig.11A: The crystal structure of sKLB bound to CT16, in accordance with some embodiments. sKLB is shown as a green surface representation, and CT16 is shown as an orange ball-and-stick representation. A nanobody used as a crystallization chaperone is not shown for clarity. Figs.11B-11E: Comparison of overall conformations of CT16 and CTWT when bound to sKLB, in accordance with some embodiments. Site 1-interacting regions (Figs.11B-11C) for sKLB:CT16 (Fig.11B) and sKLB:CTWT (Fig.11C) structure. Site 2- interacting regions (Figs.11D-11E) for sKLB:CT16 (Fig.11D) and sKLB:CTWT (Fig.11E) structure. sKLB molecules are shown as cartoon representations in green for sKLB:CT16 structure and in grey for sKLB:CTWT structure. Amino acid residues in CT16 that are different from CTWT are highlighted with red dashed circles. Color schemes in this figure are used for all the figures for structures except when otherwise noted. Fig.12A: Comparison of structures between sKLB bound to CT16 and sKLB bound to CTWT (PDB ID: 5VAQ), in accordance with some embodiments. sKLB is shown as Cα trace representation, and the backbone atoms of CT16 and CTWT are shown as sticks. Cα atoms of sKLB are overlaid with RMSD of 0.29 Å for KL1, 0.46 Å for KL2, and 2.12 Å for both domains. Figs.12B-12C: Side chain atoms of site 1 (Fig.12B) and site 2 (Fig.12C) residues that are making contacts with CT16 are highlighted as sticks. sKLB molecules are shown as cartoon representation whereas CT16 and CTWT are shown as Cα trace representation. Figs.13A-13B demonstrate that the interactions on site 2 that are responsible for the superior binding affinity to β-Klotho and higher cellular activities of FGF21 CV16 compared to FGF21 wt. Figs.13A-13B: Inter-molecular contacts that contribute to the overall binding affinities between CT16 (orange) and sKLB (green) (Fig.13A), and between CTWT (purple) and sKLB (grey) (Fig.13B) are labeled. Fig.14 is a schematic diagram describing a proposed mechanism for the high-affinity binding of FGF21 CV16 to β-Klotho, in accordance with some embodiments. Fig.14A: FGF21 CV16 is in equilibrium with linear and folded conformation. A helical segment in SIR2 can be formed owing to the π-π interaction between W203 and W208. Fig.14B: Helical conformation in SIR2 facilitates the interaction towards site 2 of β-Klotho. Fig.14C: Through multiple intra- and inter-molecular interactions in SIR1 of CV16 and site 1, the binding 9 52997671.2 Attorney Docket No.047162-7483WO1 process is complete, rendering the dissociation process less favorable. Figs.15A-15D illustrate the results of the calcium release activation in Chinese Hamster Ovary cell line (CHO) stimulated with FGF21 WT or its FGF21 C-terminus tail variant 16 (FGF21 CV16), in accordance with some embodiments. Figs.15A-15D: Time course of Ca2+release in CHO cells (expressing endogenous FGFR1c and β-Klotho) were loaded with a fluorescent Ca2+indicator (Calbryte 520 AM) and then stimulated with increasing concentrations of the (Figs.15A-15B) N-terminal His-tagged, and (Figs.15C- 15D) Human Fc fused, WT FGF21 or its CV16 variant as indicated, and Ca2+release was analyzed as a change in fluorescence. Figs.16A-16C demonstrate that some FGF21 constructs according to some embodiments herein are more potent in activating the FGF21 / FGFR / β-Klotho signaling than wild-type FGF21. Specifically, cellular activity of various FGF21 proteins measured in L6 cells co-expressing β-Klotho and FGFR1 through detection of phosphorylated MAPK. Immunoblots with β-tubulin antibody (loading control) and phosphorylated MAPK antibody for (Fig.16A) His-FGF21 wt (top) and His-FGF21 CV30 (bottom), (Fig.16B) Fc-FGF21 wt (top) and Fc-FGF21 CV16 (bottom), and (Fig.16C) Fc-FGF21 wt (top) and Fc-FGF21 CV30 (bottom). Figs.17A-17C demonstrate that some FGF21 constructs according to some embodiments herein are more potent in activating the FGF21 / FGFR / β-Klotho signaling than wild-type FGF21. Specifically, cellular activity of various FGF21 proteins measured in L6 cells co-expressing β-Klotho and FGFR1 through detection of phosphorylated MAPK. Graphs from signal in AlphaLISA for phosphorylated MAPK normalized to total MAPK for (Fig.17A) Fc-FGF21 wt (black circles) and Fc-FGF21 CV25 (white circles), (Fig.17B) FGF21 wt (black circles) and FGF21 CV16 (white circles), and (Fig.18C) FGF21 wt (black circles) and FGF21 CV25 (white circles). DETAILED DESCRIPTION The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact and may also include embodiments in which additional features may be formed between the first 10 52997671.2 Attorney Docket No.047162-7483WO1 and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed. FGF21 is an important regulator of metabolic homeostasis. While most circulating FGF21 is normally produced in liver, FGF21 is also produced in adipose tissue and in the pancreas. Moreover, a variety of nutrient stresses (e.g., alcohol, sugar) as well as fasting and refeeding induce FGF21 expression in liver. When pharmacologically administered to diabetic or obese mice, FGF21 causes body weight loss, improves insulin sensitivity, and lowers blood glucose and triglyceride levels; similar metabolic effects were also observed in FGF21 treated non-human primates. There is a significant interest in using a FGF21 analog as a therapeutic agent to treat patients with metabolic diseases such as type 2 diabetes and non-alcoholic fatty liver disease (NASH), pancreatitis, acute alcohol toxicity among other indications. FGF21 belongs to the family of the endocrine FGF that also includes FGF19 and FGF23, which act as circulating hormones and bind specifically to the Klotho family of surface receptors resulting in formation of a ternary complexes with and selective activation of the c-isoforms of FGFRs. It was shown that FGF19 and FGF21 interact with and bind specifically to β-Klotho whereas FGF23 binds specifically to α-Klotho. It has been shown that endocrine FGFs exert their physiological functions only in tissues or cells which express Klotho proteins. Importantly, endocrine FGFs contain unique C-terminus regions that specifically and strongly binds to their respective Klotho receptors, to provide a ‘zip-code’ or a targeting signal for FGFR activation by endocrine FGFs. Moreover, elucidations of the crystal structures of FGF19, FGF21, and FGF23 in complex with the extracellular region of α- and β-Klotho protein revealed detailed atomic interactions that determine the specificity of endocrine FGFs towards Klotho proteins, and demonstrated how FGFRs are activated in a Klotho-dependent manner. The structural information that provides detailed molecular interactions between FGF21 and β-Klotho is the key to enhance the interactions and to develop FGF21 variants with improved potencies. Here, the present study presents new FGF21 variants with extremely high affinities to β-Klotho that were identified using the structural information. These FGF21 variants exhibit much improved potencies in various cell-based assays. Furthermore, various fusion proteins with potent FGF21 variants were developed to improve biochemical and pharmacological properties of the new variants. 11 52997671.2 Attorney Docket No.047162-7483WO1 In the study described herein (“the present study”), certain polypeptide constructs were designed by introducing mutations in wild-type fibroblast growth factor 21 (FGF21) fragments. The polypeptide constructs of the present study were confirmed to have stronger (or at least comparable) binding affinity toward β-Klotho as compared to the corresponding wild-type FGF21 polypeptides as determined by dissociation constant Kd determinations. The polypeptide constructs of the present study were also confirmed to have stronger or enhanced binding activity to β-Klotho binding as compared to the corresponding wild-type FGF21 polypeptides as determined by increased Koffdeterminations. Depending on the configuration of the FGF21-derived polypeptide (such as the presence or absence of the FGFR-binding core region), the constructs herein can either down- regulate the FGF / FGFR / β-Klotho signaling by sequestering β-Klotho from endogenous FGF21 and FGF19, or up-regulate FGF21 / FGFR / β-Klotho signaling by acting as FGF21 analog having stronger receptor / co-receptor binding. As such, the construct herein can be used to treat, ameliorate and / or prevent various diseases or disorders caused by and / or involving mis-regulation of the FGF / FGFR / β-Klotho signaling pathways. Accordingly, in some aspects, the present invention is directed to a non-natural construct comprising an FGF21 polypeptide. FGF / FGFR / β-Klotho signaling pathways are implicated in various diseases and disorders. For example, expression of FGF21 in liver (where β-Klotho is also expressed) is upregulated upon various nutrient stresses such as starvation, alcohol, and sugar. Studies have shown that FGF21 is an important regulator of metabolic homeostasis. When pharmacologically administered to diabetic or obese animals, FGF21 causes body weight loss, improves insulin sensitivity, and lowers blood glucose and triglyceride levels. Therefore, the constructs herein are expected to be useful as pharmaceutical compositions and for treating various diseases and disorders caused by and / or involving mis-regulation of the FGF / FGFR / β-Klotho signaling pathways. In some aspects, the present invention is directed to a composition, such as a pharmaceutical composition, including the non-natural construct of the disclosure. In some aspects, the present invention is directed to a method of modulating the FGF / FGFR / β-Klotho signaling pathways. In some aspects, the present invention is directed to a method of treating, ameliorating and / or preventing a disease or disorder caused by and / or involving mis-regulation of the FGF / FGFR / β-Klotho signaling pathways. 12 52997671.2 Attorney Docket No.047162-7483WO1 Definitions As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, peptide chemistry, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components. In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process. In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.” “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. The term “antibody,” as used herein, refers to an immunoglobulin molecule that specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources, and can be immunoreactive portions of intact 13 52997671.2 Attorney Docket No.047162-7483WO1 immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, scFv antibodies, single-domain antibodies such as sdAb (either VL or VH), such as camelid antibodies (Riechmann, 1999, J. Immunol. Meth.231:25-38), camelid VHH domains, composed of either a VL or a VH domain that exhibit sufficient affinity for the target, and multispecific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide brudge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger & Hudson, 2005, Nature Biotech.23:1126-1136). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)( U.S. Patent No.: 6,703,199, which describes fibronectin polypeptide minibodies). The antibody fragment also includes a human antibody or a humanized antibody or a portion of a human antibody or a humanized antibody. By the term “applicator,” as the term is used herein, is meant any device including, but not limited to, a hypodermic syringe, a pipette, and the like, for administering the compounds and compositions of the disclosure. As used herein, a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. As used herein, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health. As used herein, the terms “effective amount” or “therapeutically effective amount” or 14 52997671.2 Attorney Docket No.047162-7483WO1 “pharmaceutically effective amount” of a compound are used interchangeably to refer to the amount of the compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system. As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system. The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter. The term “FGF21 polypeptide” as used herein encompasses naturally-occurring FGF21 (e.g., human FGF21), fragments of naturally-occurring FGF21 (e.g., fragments of human FGF21), as well as variants of any of thereof. “Homologous” as used herein, refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous. By way of example, the DNA sequences 5’-ATTGCC-3’ and 5’-TATGGC-3’ share 50% homology. As used herein, the term “immunoglobulin” or “Ig” is defined as a class of proteins that function as antibodies. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitor-urinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most mammals. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen. The terms “inhibit” and “antagonize”, as used herein, mean to reduce a molecule, a 15 52997671.2 Attorney Docket No.047162-7483WO1 reaction, an interaction, a gene, an mRNA, and / or a protein’s expression, stability, function or activity by a measurable amount or to prevent entirely. Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., antagonists. “Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition and / or compound of the disclosure in a kit. The instructional material of the kit may, for example, be affixed to a container that contains the compound and / or composition of the disclosure or be shipped together with a container which contains the compound and / or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website. “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the co-existing materials of its natural state is “isolated.” An isolated nucleic acid or protein may exist in substantially purified form, or may exist in a non-native environment such as, for example, a host cell. An “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence. 16 52997671.2 Attorney Docket No.047162-7483WO1 As used herein, the term “modulate” is meant to refer to any change in biological state, i.e. increasing, decreasing, and the like. For example, the term “modulate” may be construed to refer to the ability to regulate positively or negatively the expression, stability or activity of a target protein, including but not limited to transcription of a target protein mRNA, stability of a target protein mRNA, translation of a target protein mRNA, target protein stability, target protein post-translational modifications, target protein activity, or any combination thereof. Further, the term modulate may be used to refer to an increase, decrease, masking, altering, overriding or restoring of activity, including but not limited to, target protein activity. “Naturally-occurring” as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man is a naturally-occurring sequence. The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame. “Parenteral” administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques. As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound useful within the disclosure with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to: intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, intracranial and topical administration. As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the composition, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious 17 52997671.2 Attorney Docket No.047162-7483WO1 manner with any of the components of the composition in which it is contained. “Pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent; sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances employed in pharmaceutical formulations, or any combination thereof. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof. “Polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term “protein” typically refers to large polypeptides. The term 18 52997671.2 Attorney Docket No.047162-7483WO1 “peptide” typically refers to short polypeptides. Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus. As used herein, a “peptidomimetic” is a compound containing non-peptidic structural elements that is capable of mimicking the biological action of a parent peptide. A peptidomimetic may or may not comprise peptide bonds. As used herein, the term “prevent” or “prevention” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease. Disease and disorder are used interchangeably herein. By the term “specifically bind” or “specifically binds,” as used herein, is meant that a first molecule (e.g., an antibody) preferentially binds to a second molecule (e.g., a particular antigenic epitope), but does not necessarily bind only to that second molecule. As used herein, a “subject” refers to a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human. As used herein, the term “treatment” or “treating” is defined as the application or administration of a therapeutic agent, i.e., a composition useful within the disclosure (alone or in combination with another pharmaceutical agent), to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject (e.g., for diagnosis or ex vivo applications), who has a disease or disorder, a symptom of a disease or disorder or the potential to develop a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder or the potential to develop the disease or disorder. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains an essential property of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. In some examples, changes in the sequence of peptide 19 52997671.2 Attorney Docket No.047162-7483WO1 variants are limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. A variant of a nucleic acid or peptide may be a naturally occurring such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis. The alteration of one or more amino acids in the reference sequence may be a modification selected from the group consisting of substitution, addition, deletion, and modification of at least one amino acid in the reference polypeptide. An exemplary variant is a polypeptide that includes an alteration at one or more amino acids compared to human FGF21 (SEQ ID NO: 1). Another example of a variant is a polypeptide that includes an alteration at one or more amino acids compared to a fragment of human FGF21 (SEQ ID NO: 1). The terms “identical” or “percent identity,” in the context of two or more nucleic acids, or two or more polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides, or amino acids, that are the same. For example, about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the algorithms can account for gaps and the like. As used herein, the term “include” means “comprising, consisting of, or consisting essentially of.” For example, the phrase A includes B and C means: in some embodiments, A comprises B and C (and other elements can also be part of A); in some embodiments, A consists of B and C (and A has no further elements apart from B and C); in some embodiments, A consists essentially of B and C (and C can only have elements other than B and C if the additional element does not materially affect the basic and novel characteristics of A). Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for 20 52997671.2 Attorney Docket No.047162-7483WO1 convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Non-Natural Construct In some aspects, the present invention is directed to a non-natural soluble construct. In some embodiments, the construct includes residues 186-198 and residues 200-209 of fibroblast growth factor 21 (FGF21). In some embodiments, the residue numbers of FGF21 used herein refer to those for the protein sequence set forth in SEQ ID NO:1 below: Fibroblast growth factor 21 precursor [Homo sapiens], NP_061986.1 MDSDETGFEHSGLWVSVLAGLLLGAC AHPIPDSSPLL FGG VR RYLYTDDAQ L The signal sequence of human FGF21 is residues 1-28 of SEQ ID NO: 1. The mature human FGF21, which lacks the signal sequence (residues 1-28 of SEQ ID NO: 1), corresponds to residues 29-209 of SEQ ID NO: 1. In certain embodiments, the FGF21 polypeptide described herein has an amino acid sequence that has at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, or has an amino acid sequence that has at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of residues 29-209 of SEQ ID NO: 1 (the mature human FGF21 protein). In yet additional aspects, the FGF21 polypeptide described herein has an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, or has an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 21 52997671.2 Attorney Docket No.047162-7483WO1 or 99% sequence identity to the amino acid sequence of residues 29-209 of SEQ ID NO: 1 (the mature human FGF21 protein). In yet additional aspects, the FGF21 polypeptide has an amino acid sequence that has at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a specific polypeptide described herein. In some embodiments, FGF21 polypeptides including a P199 mutation (such as the P199G mutation) and / or a A208 mutation (such as the A208E mutation) are contemplated herein. Without wishing to be limited by any theory, in certain embodiments the P199 mutation, such as the P199G mutation, can reduce the in vivo cleavage of the FGF21 polypeptide. Without wishing to be limited by any theory, in certain embodiments the A208 mutation, such as the A208E mutation, can reduce the in vivo cleavage of the FGF21 polypeptide. In some embodiments, FGF21 polypeptides including the L126 mutation (such as an L126R mutation) are contemplated herein. Without wishing to be limited by any theory, in certain embodiments the L126 mutation, such as the L126R mutation, can reduce aggregation of the FGF21 polypeptide. It is worth noting that, although the numbering of FGF21 residues used herein are made in reference to SEQ ID NO:1, the constructs described herein is not limited to the human FGF21 having the protein sequence set forth in SEQ ID NO:1. For example, one of ordinary skill in the art would understand that orthologs of FGF21 (i.e., FGF21 in other species) may have different sequences and different length, but the residues corresponding to residues 186-198 and residues 200-209 of SEQ ID NO:1, as well as residues that should be mutated (described elsewhere herein) can be readily determined by conventional tools such as sequence alignment. In some embodiments, the FGF21 polypeptide includes at least one mutation selected from the group consisting of L126, L194, V197, P199, Q201, R203, Y207, A208, and S209. In some embodiments, the FGF21 polypeptide includes the mutation L126. In some embodiments, the FGF21 polypeptide includes the mutation L194. In some embodiments, the FGF21 polypeptide includes the mutation V197. In some embodiments, the FGF21 polypeptide includes the mutation P199. In some embodiments, the FGF21 polypeptide includes the mutation Q201. In some embodiments, the FGF21 polypeptide includes the mutation R203. In some embodiments, the FGF21 polypeptide includes the mutation Y207. In some embodiments, the FGF21 polypeptide includes the mutation A208. In some 22 52997671.2 Attorney Docket No.047162-7483WO1 embodiments, the FGF21 polypeptide includes the mutation S209. In some embodiments, the FGF21 polypeptide includes at least one mutation selected from the group consisting of L126R, L194F, V197I, V197L, P199G, Q201L, Q201I, Q201P, Q201R, Q201W, R203W, Y207W, A208E, A208W, and S209G. In some embodiments, the FGF21 polypeptide includes the mutation L126R. In some embodiments, the FGF21 polypeptide includes the mutation L194F. In some embodiments, the FGF21 polypeptide includes the mutation V197I. In some embodiments, the FGF21 polypeptide includes the mutation V197L. In some embodiments, the FGF21 polypeptide includes the mutation P199G. In some embodiments, the FGF21 polypeptide includes the mutation Q201L. In some embodiments, the FGF21 polypeptide includes the mutation Q201I. In some embodiments, the FGF21 polypeptide includes the mutation Q201P. In some embodiments, the FGF21 polypeptide includes the mutation Q201R. In some embodiments, the FGF21 polypeptide includes the mutation Q201W. In some embodiments, the FGF21 polypeptide includes the mutation R203W. In some embodiments, the FGF21 polypeptide includes the mutation selected from the group consisting of Y207W. In some embodiments, the FGF21 polypeptide includes the mutation A208E. In some embodiments, the FGF21 polypeptide includes the mutation A208W. In some embodiments, the FGF21 polypeptide includes the mutation S209G. In some embodiments, in the FGF21 polypeptide, the segment corresponding to residues 186-198 and the segment corresponding to residues 200-209 of SEQ ID NO:1 are part of a continuous polypeptide chain. In some embodiments, the segment corresponding to residues 186-198 are conjugated to the N-terminus of the segment corresponding to residues 200-209 of SEQ ID NO:1 FGF21. In some embodiments, the FGF21 polypeptide includes residues 186-209 of FGF21, which, according to some embodiments, include both the segment corresponding to residues 186-198 and the segment corresponding to residues 200-209 of FGF21. In some embodiments, the FGF21 polypeptide includes residues 169-209 of FGF21, which, according to some embodiments, include both the segment corresponding to residues 186-198 and the segment corresponding to residues 200-209 of FGF21. In some embodiments, the FGF21 polypeptide includes residues 29-209 of FGF21, which, according to some embodiments, include both the segment corresponding to residues 186-198 and the segments corresponding to residues 200-209 of FGF21. In some embodiments, the FGF21 polypeptide includes residues 186-209 of FGF21 or residues 169-209 of FGF21, which include the C-terminus tail of FGF21, which binds 23 52997671.2 Attorney Docket No.047162-7483WO1 specifically to β-klotho. In some embodiments, the polypeptide does not include residues 29-169 of FGF21 (the “core region” responsible for binding FGF receptors), or portions thereof, which renders the FGF21 polypeptide unable to specifically bind FGF receptors, such as FGFR1c. In some embodiments, the FGF21 polypeptide lacking the core region is able to bind to and sequester β-klotho from the FGF / FGFR / β-klotho signaling pathways, thereby down-regulating this pathway. In some embodiments, the FGF21 polypeptide includes residues 29-209 of FGF21. In some embodiments, the FGF21 polypeptide including residues 29-209 of FGF21, which includes both the core region responsible for binding FGF receptors and the C-terminus tail region responsible for binding to β-klotho. In some embodiments, the FGF21 polypeptide including both the core region and the tail region is able to activate the FGF21 / FGFR / β- klotho signaling pathways with potency higher than wild-type FGF21. In some embodiments, the FGF21 polypeptide further includes 2-5 basic and / or hydrophilic amino acid residues at the C-terminus region of the residues 200-209 of FGF21. One of ordinary skill in the art would understand basic amino acid residues include e.g., arginine (R) and lysine (K), and hydrophilic residues include serine (S), threonine (T), and some other amino acid residues. In some embodiments, the amino acid sequence RKK, RRS or RKS is conjugated to the C-terminus of the residues 200-209 of FGF21. In some embodiments, the FGF21 polypeptide includes the mutations L194F, V197I, Q201L, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, V197I, Q201L, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L194F, V197I, Q201L, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the corresponding wild-type FGF21. In yet additional embodiments, the FGF21 polypeptide includes the mutations L194F, V197I, Q201L, R203W, Y207W, A208W and S209G but otherwise has the same sequence as the mature human FGF21 (which excludes the signal sequence). In yet additional embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, V197I, Q201L, R203W, Y207W, A208W and S209G but otherwise has the same sequence as the mature human FGF21. In some embodiments, the FGF21 polypeptide further includes the sequence RKK conjugated to the C-terminus of the residues 200-209 of FGF21 (CV3). In some embodiments, the FGF21 polypeptide includes the mutations L194F, V197I, Q201R, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 24 52997671.2 Attorney Docket No.047162-7483WO1 polypeptide includes the mutations L126R, P199G, L194F, V197I, Q201R, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L194F, V197I, Q201R, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV4). In additional embodiments, the FGF21 polypeptide includes the mutations L194F, V197I, Q201R, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the mature human FGF21. In yet additional embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, V197I, Q201R, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the mature human FGF21. In some embodiments, the FGF21 polypeptide includes the mutations L194F, V197I, Q201R, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, V197I, Q201R, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide includes the mutations L194F, V197I, Q201R, R203W, Y207W and A208W, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV6). In additional embodiments, the FGF21 polypeptide includes the mutations L194F, V197I, Q201R, R203W, Y207W and A208W, but otherwise has the same sequence as the mature human FGF21. In yet additional embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, V197I, Q201R, R203W, Y207W and A208W, but otherwise has the same sequence as the mature human FGF21. In some embodiments, the FGF21 polypeptide includes the mutations L194F, V197L, Q201R, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, V197L, Q201R, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L194F, V197L, Q201R, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV11). In additional embodiments, the FGF21 polypeptide includes the mutations L194F, V197L, Q201R, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the mature human FGF21. In yet additional embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, V197L, Q201R, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the mature human FGF21. In some embodiments, the FGF21 polypeptide includes the mutations L194F, V197L, Q201R, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, V197L, Q201R, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide includes the mutations L194F, 25 52997671.2 Attorney Docket No.047162-7483WO1 V197L, Q201R, R203W, Y207W and A208W, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV12). In additional embodiments, the FGF21 polypeptide includes the mutations L194F, V197L, Q201R, R203W, Y207W and A208W, but otherwise has the same sequence as the mature human FGF21. In yet additional embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, V197L, Q201R, R203W, Y207W and A208W, but otherwise has the same sequence as the mature human FGF21. In some embodiments, the FGF21 polypeptide includes the mutations L194F, Q201I, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, Q201I, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide includes the mutations L194F, Q201I, R203W, Y207W and A208W, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV16). In additional embodiments, the FGF21 polypeptide includes the mutations L194F, Q201I, R203W, Y207W and A208W, but otherwise has the same sequence as the mature human FGF21. In yet additional embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, Q201I, R203W, Y207W and A208W, but otherwise has the same sequence as the mature human FGF21. In some embodiments, the FGF21 polypeptide includes the mutations L194F, Q201L, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, Q201L, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L194F, Q201L, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the corresponding wild-type FGF21. In additional embodiments, the FGF21 polypeptide includes the mutations L194F, Q201L, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the mature human FGF21. In yet additional embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, Q201L, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the corresponding wild-type FGF21. In some embodiments, the FGF21 polypeptide further includes the sequence RKS conjugated to the C-terminus of the residues 200-209 of FGF21 (CV18). In some embodiments, the FGF21 polypeptide includes the mutations L194F, Q201P, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, Q201P, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L194F, Q201P, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the corresponding wild-type FGF21. In additional embodiments, the FGF21 polypeptide includes 26 52997671.2 Attorney Docket No.047162-7483WO1 the mutations L194F, Q201P, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the mature human FGF21. In yet additional embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, Q201P, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the mature human FGF21. In some embodiments, the FGF21 polypeptide further includes the sequence RRS conjugated to the C-terminus of the residues 200-209 of FGF21 (CV21). In some embodiments, the FGF21 polypeptide includes the mutations L194F, Q201R, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, Q201R, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L194F, Q201R, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the corresponding wild-type FGF21. In additional embodiments, the FGF21 polypeptide includes the mutations L194F, Q201R, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the mature human FGF21. In yet additional embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, Q201R, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the mature human FGF21. In some embodiments, the FGF21 polypeptide further includes the sequence RRS conjugated to the C-terminus of the residues 200-209 of FGF21 (CV24). In some embodiments, the FGF21 polypeptide includes the mutations L194F, Q201R, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, Q201R, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide includes the mutations L194F, Q201R, R203W, Y207W and A208W, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV25). In additional embodiments, the FGF21 polypeptide includes the mutations L194F, Q201R, R203W, Y207W and A208W, but otherwise has the same sequence as the mature human FGF21. In yet additional embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, Q201R, R203W, Y207W and A208W, but otherwise has the same sequence as the mature human FGF21. In some embodiments, the FGF21 polypeptide includes the mutations L194F, Q201W, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, Q201W, R203W, Y207W, A208W and S209G. In some embodiments, the FGF21 polypeptide includes the mutations L194F, Q201W, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV28). In additional embodiments, the FGF21 27 52997671.2 Attorney Docket No.047162-7483WO1 polypeptide includes the mutations L194F, Q201W, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the mature human FGF21. In yet additional embodiments, the FGF21 polypeptide includes the mutations L126R, P199G, L194F, Q201W, R203W, Y207W, A208W and S209G, but otherwise has the same sequence as the mature human FGF21. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide comprises the mutations L126R, P199G, L194F, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide comprises the mutations L194F, R203W, Y207W and A208W, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV30). In additional embodiments, the FGF21 polypeptide comprises the mutations L194F, R203W, Y207W and A208W, but otherwise has the same sequence as the mature human FGF21. In yet additional embodiments, the FGF21 polypeptide comprises the mutations L126R, P199G, L194F, R203W, Y207W and A208W, but otherwise has the same sequence as the mature human FGF21. In some embodiments, the FGF21 polypeptide comprises the mutations R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide comprises the mutations L126R, P199G, R203W, Y207W and A208W. In some embodiments, the FGF21 polypeptide comprises the mutations R203W, Y207W and A208W, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV30). In some embodiments, the FGF21 polypeptide comprises the mutations R203W, Y207W and A208W, but otherwise has the same sequence as the mature human FGF21. In additional embodiments, the FGF21 polypeptide comprises the mutations L126R, P199G, R203W, Y207W and A208W, but otherwise has the same sequence as the mature human FGF21. In some embodiments, the construct further includes a stability enhancing domain. In some embodiments, the stability enhancing domain is attached to an N-terminus of the FGF21 polypeptide. In some embodiments, the stability enhancing domain is attached to a C- terminus of the FGF21 polypeptide. Non-limiting examples of stability enhancing domain includes albumin, an Fc region of an antibody, a glutathione S-transferase, a small ubiquitin- like modifier (SUMO), an O-glycosylation region of a Drosophila Spitz protein, a thioredoxin, and the like. In some embodiments, the FGF21 polypeptide and the stability enhancing domain are linked through a linker polypeptide. In some embodiments, the linker peptide ranges from about 1 amino acid to about 18 amino acids. In some embodiments, the linker peptide is a 28 52997671.2 Attorney Docket No.047162-7483WO1 flexible linker, such as a peptide rich in glycine (G) and / or serine (S), or a peptide cleavable by an enzyme. In some embodiments, the FGF21 polypeptide herein binds to β-klotho tighter than a corresponding wild-type FGF21 polypeptide. In some embodiments, the dissociation constant (Kd) of the FGF21 polypeptide with β-Klotho is about 20% or less, such as 10% or less or 5% or less, of a Kdwith a corresponding wild-type FGF21 polypeptide. In some embodiments, the FGF21 polypeptide herein binds to β-klotho for longer than a corresponding wild-type FGF21 polypeptide. In some embodiments, the Koffof the FGF21 polypeptide with β-Klotho is about 20% or less, such as 10% or less or 5% or less, of the Koffof a corresponding wild-type FGF21 polypeptide. In some embodiments, the construct binds to β-Klotho and sequesters β-Klotho from pathways downstream of a fibroblast growth factor receptor (FGFR) and β-Klotho. In some embodiments, the construct binds to both an FGFR and β-Klotho and activates pathways downstream of the FGFR and β-Klotho. In some embodiments, the FGFR is FGFR1c. Compositions In some aspects, the present invention is directed to a composition, such as a pharmaceutical composition including the non-natural construct herein. Details of the formulations of the composition can be found in the “Pharmaceutical Compositions and Formulations” section. Method of Modulating FGF / FGFR / β-Klotho Signaling Pathways In some aspects, the present study is directed to a method of modulating FGF / FGFR / β-Klotho signaling pathways, such as the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway. In some embodiments, the method includes contacting the surface of the cell, which has an FGF / FGFR / β-Klotho signaling pathway, with the construct herein. In some embodiments, the construct is the same as or similar to those as described in the “Non- Natural Construct” section. As described elsewhere herein, the construct herein can be configured as a positive regulator or a negative regulator of the pathways, such as by adding or removing the FGF core sequences from the FGF21 polypeptide. In some embodiments, the construct binds to β-Klotho but not FGFR, thereby 29 52997671.2 Attorney Docket No.047162-7483WO1 sequestering β-Klotho from the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway and down-regulating the signaling pathway. In some embodiments, the construct binds to both β-Klotho and FGFR, thereby up- regulating the FGF21 / FGFR / β-Klotho signaling pathway. In some embodiments, the cell is from a cell line, a primary cell, or a cell in the body of a subject (such as a mammalian subject or a human subject). In some embodiments, the cell is a cell in or derived from an adipose tissue, a liver, a pancreas or a hypothalamus. Method of Treating, Ameliorating and / or Preventing a Disease or Disorder In some aspects, the present study is directed to a method of treating, ameliorating and / or preventing a disease or disorder in a subject in need thereof. In some embodiments, the disease or disorder is caused by and / or involves a misregulation of an FGF / FGFR / β-Klotho signaling pathway (such as the FGF21 / FGFR / β- Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway). In some embodiments, the disease or disorder is caused by or involves an up-regulated FGF / FGFR / β- Klotho signaling pathway or a down-regulated FGF / FGFR / β-Klotho signaling pathway. In some embodiments, the disease or disorder is at least one selected from obesity, diabetes, pancreatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), acute alcohol toxicity, and liver cancer and other cancers that are driven by aberrant activation of FGF19 / FGFR4 / β-Klotho pathway in tumor cells. In some embodiments, the method includes administering to the subject an effective amount of the construct herein or the composition (such as the pharmaceutical composition) herein. In some embodiments, the construct or the composition is administered via at least one route selected from the group consisting of inhalational, oral, rectal, vaginal, parenteral, intracranial, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, and intravenous. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. Pharmaceutical Compositions and Formulations The disclosure also encompasses the use of pharmaceutical compositions of the disclosure to practice the methods of the disclosure. 30 52997671.2 Attorney Docket No.047162-7483WO1 Such pharmaceutical compositions may be provided in a form suitable for administration to a subject, and may comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The compositions of the disclosure may comprise a physiologically acceptable salt, such as a compound contemplated within the disclosure in combination with a physiologically acceptable cation or anion, as is well known in the art. In certain embodiments, the pharmaceutical compositions useful for practicing the method of the disclosure may be administered to deliver a dose of between 1 ng / kg / day and 100 mg / kg / day. In other embodiments, the pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of between 1 ng / kg / day and 500 mg / kg / day. The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient. Pharmaceutical compositions that are useful in the methods of the disclosure may be suitably developed for inhalational, oral, rectal, vaginal, parenteral, topical, intracranial, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit. As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- 31 52997671.2 Attorney Docket No.047162-7483WO1 third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the disclosure is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs. In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of at least one compound of the disclosure and a pharmaceutically acceptable carrier. Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents. As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other 32 52997671.2 Attorney Docket No.047162-7483WO1 “additional ingredients” that may be included in the pharmaceutical compositions of the disclosure are known in the art and described, for example in Genaro, ed. (1985, Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference. Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl para- hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol. Powdered and granular formulations of a pharmaceutical preparation of the disclosure may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations. A pharmaceutical composition of the disclosure may also be prepared, packaged, or 33 52997671.2 Attorney Docket No.047162-7483WO1 sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally- occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents. Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Administration / Dosing The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the manifestation of symptoms associated with the disease or condition. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. Administration of the compositions of the present invention to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or condition in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided 34 52997671.2 Attorney Docket No.047162-7483WO1 doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the disclosure is from about 0.01 and 50 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation. The compound can be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be 35 52997671.2 Attorney Docket No.047162-7483WO1 achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of cancer in a patient. In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physical taking all other factors about the patient into account. Compounds of the disclosure for administration may be in the range of from about 1 ^g to about 7,500 mg, about 20 ^g to about 7,000 mg, about 40 ^g to about 6,500 mg, about 80 ^g to about 6,000 mg, about 100 ^g to about 5,500 mg, about 200 ^g to about 5,000 mg, about 400 ^g to about 4,000 mg, about 800 ^g to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments therebetween. In some embodiments, the dose of a compound of the disclosure is from about 0.5 ^g and about 5,000 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or 36 52997671.2 Attorney Docket No.047162-7483WO1 partial increments thereof. In certain embodiments, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient. The term “container” includes any receptacle for holding the pharmaceutical composition. For example, in certain embodiments, the container is the packaging that contains the pharmaceutical composition. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound’s ability to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient. Routes of Administration Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, intracranial, and topical administration. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein. 37 52997671.2 Attorney Docket No.047162-7483WO1 Oral Administration For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, a paste, a gel, toothpaste, a mouthwash, a coating, an oral rinse, or an emulsion. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients which are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Patents Nos.4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation. Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin. Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil. For oral administration, the compounds of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents; fillers; lubricants; disintegrates; or wetting agents. If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and 38 52997671.2 Attorney Docket No.047162-7483WO1 OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non- aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl para-hydroxy benzoates or sorbic acid). Liquid formulations of a pharmaceutical composition of the disclosure which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use. A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycollate. Known surface-active agents include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc. Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a “granulation.” For example, solvent-using “wet” granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of 39 52997671.2 Attorney Docket No.047162-7483WO1 a wet granulated mass from which the solvent must then be evaporated. Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e. drug) by forming a solid dispersion or solid solution. U.S. Patent No.5,169,645 discloses directly compressible wax-containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) will melt. The present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds useful within the methods of the disclosure, and a further layer providing for the immediate release of one or more compounds useful within the methods of the disclosure. Using a wax / pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release. Parenteral Administration As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques. Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as 40 52997671.2 Attorney Docket No.047162-7483WO1 sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non- toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. Additional Administration Forms Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos.6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos.20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03 / 35041, WO 03 / 35040, WO 03 / 35029, WO 03 / 35177, WO 03 / 35039, WO 02 / 96404, WO 02 / 32416, WO 01 / 97783, WO 01 / 56544, WO 01 / 32217, WO 98 / 55107, WO 98 / 11879, WO 97 / 47285, WO 93 / 18755, and WO 90 / 11757. 41 52997671.2 Attorney Docket No.047162-7483WO1 Controlled Release Formulations and Drug Delivery Systems Controlled- or sustained-release formulations of a pharmaceutical composition of the disclosure may be made using conventional technology. In some cases, the dosage forms to be used can be provided as slow or controlled-release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the disclosure. Thus, single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps, and caplets, which are adapted for controlled-release are encompassed by the present invention. Most controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood level of the drug, and thus can affect the occurrence of side effects. Most controlled-release formulations are designed to initially release an amount of drug that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds. The term “controlled-release component” in the context of the present invention is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres or a combination thereof that facilitates the controlled-release of the active ingredient. In certain embodiments, the formulations of the present invention may be, but are not 42 52997671.2 Attorney Docket No.047162-7483WO1 limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations. The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form. For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation. In a preferred embodiment of the disclosure, the compounds of the disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation. The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours. The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration. The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration. As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration. As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the 43 52997671.2 Attorney Docket No.047162-7483WO1 scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction and assaying conditions with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application. It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein. EXAMPLES The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Example 1: Identification and characterization of new FGF21 variants In order to identify new FGF21 variants exhibiting enhanced binding affinity to β- Klotho, a variety of mutations were generated in the C-terminus tail (CT) of FGF21; the region responsible for specific binding of FGF21 to β-Klotho (Figs.1A-1C). Additional variants were obtained by generating constructs that direct the synthesis of fusion proteins connected to the N terminal region of FGF21. The fusion proteins include Sumo protein, murine Albumin, Spitz and human Fc domain of human IgG1 (Figs.2A-2B). The new FGF21 variants were generated in order to enhance binding affinity to β -Klotho, increase expression level, improve protein stability resulting in superior pharmacological properties and activities of the new FGF21 variants. A scheme describing 14 new FGF21 variants with mutations in the C-terminus tail regions is illustrated in Figs.1A-1C. The constructs containing the FGF21 variants were generated in the form of genetically engineered fusion proteins containing an Fc region of 44 52997671.2 Attorney Docket No.047162-7483WO1 human IgG1 connected to their N-termini with a flexible linker (Figs.2A-2B). This expression system enabled efficient production of significant quantities of the FGF21 variants with high purity and homogeneity for further molecular characterizations. The binding affinity measurements of these Fc-FGF21 variants to soluble extracellular domain of β- Klotho (sKLB) were performed using biolayer interferometry (BLI). The reference subtracted BLI responses are fitted to the 1:1 binding model to obtain kinetic parameters and dissociation constants (Figs.3A-3N). The present study compared the kinetics parameters obtained from BLI measurements to monitor whether the Fc-fusion on the N-termini of FGF21 variants may alter their binding to sKLB. Comparison of the binding kinetics parameters between Fc-FGF21 wt (Fig.3N) and His-FGF21 wt (Fig.4B) revealed minor and insignificant differences in the overall dissociation constants (KD), strongly indicating that the N-terminal Fc-fusion on FGF21 variants does not influence the binding affinity towards sKLB. The binding kinetics parameters from the BLI measurements (Figs.3A-3N) indicate that most of Fc-FGF21 variants showed improved binding affinities to sKLB in comparison to Fc-FGF21 wt. Moreover, because of their distinct features (Figs.3A-3M), the characteristics of BLI sensorgrams themselves can be used to identify high-affinity variants as changes in the koff rates reflected the improvements in the overall binding affinities. This can be readily seen from the kinetic parameter values listed in Fig.3M; the differences between KD values among Fc-FGF21 variants almost exclusively originate from the differences in koffvalues. To further explore the properties of FGF21 CV16, the present study used an additional approach to establish and compare the binding affinities of FGF21 wt and FGF21 CV16 to sKLB (Figs.4A-4B). In these experiments His-FGF21 wt or His-FGF21 CV16 were immobilized on amine-reactive biosensors AR2G using standard amine coupling reaction. The sensors were subsequently dipped into wells containing solutions with a series of concentrations of sKLB. Note that the absence of Lys residues in the CT regions of all three FGF21 proteins prevents the CT regions from being covalently linked to the sensors and enables proper binding of sKLB after the amine coupling reactions. The binding kinetics parameters from the BLI measurements (Figs.4A-4B) confirmed that FGF21 CV16 exhibits around 40-fold higher binding affinity to β-Klotho compared to FGF21 wt and that the difference in the overall KD values between FGF21 wt and FGF21 CV16 to sKLB originates almost exclusively from the difference in the koffvalues. 45 52997671.2 Attorney Docket No.047162-7483WO1 Example 2: Enhanced cellular activities of new FGF21 variants To examine the abilities of His-FGF21 wt and His-FGF21 CV16 to stimulate cellular signaling, L6 myoblast cell line co-expressing FGFR1c and β-Klotho were stimulated with increasing concentrations of either His-FGF21 wt or His-FGF21 CV16, and the levels of phosphorylated MAPK (pMAPK) are monitored using immunoblotting analyses, 5 nM of FGF1 was used as a positive control. The results shown in Fig.5A demonstrate that both His- FGF21 wt and His-FGF21 CV16 activate cellular signaling in L6 myoblast cell line co- expressing FGFR1c and β-Klotho in a dose-dependent manner as monitored by pMAPK levels, with His-FGF21 CV16 exhibiting higher potency as compared to His-FGF21 wt. The rat L6 cell is a desirable host cell line for the functional assays regarding the signaling activities by FGFRs because it is known to have minimal expression of FGFRs. FGFR1c is chosen because FGF21 is known to activate signaling by c-isoforms of, but not b-isoforms of FGFR1, and to lesser extent, FGFR2 and FGFR3. It was also reported that signaling by FGFR4 is not detected upon stimulation by FGF21. All signaling by FGF21 requires the presence of β-Klotho. To gain further insights into the cellular activity of FGF21 CV16 the present study analyzed its capacity to stimulate glucose uptake by differentiated 3T3-L1 cells derived adipocytes (Fig.5B). When 3T3-L1 fibroblasts are chemically induced to differentiate into adipocytes, expression level of β-Klotho is significantly increased and FGFR signaling can be activated upon addition of human FGF21. FGF21 induced signaling then induce the expression of GLUT4 which is the main transporter responsible for the cellular glucose uptake. Therefore, it was anticipated that the level of cellular glucose uptake in 3T3 adipocytes can be used to determine the potencies of FGF21 variants in a well-controlled system. The present study treated, in quadruplicates, the cells with series of concentrations of His-FGF21 wt and His-FGF21 CV16 after 3T3-L1 fibroblasts were differentiated into adipocytes and compared the level of cellular uptake of glucose. As shown in Fig.5B, His- FGF21 wt and His-FGF21 wt CV16 induced glucose uptake in 3T3-L1 adipocytes in dose- dependent manners, with the EC50 values of 1.84 nM and 0.29 nM, respectively. This also confirms that the binding affinities of FGF21 variants to sKLB measured by BLI and the cellular activities as monitored with the level of phosphorylated MAPK in L6 cells co- expressing β-Klotho and FGFR1c can directly be translated into other cellular activities such as the level of glucose uptake in adipocytes. The present study next compared the level of Ca2+release in pancreatic acinar (AR42J) cells stimulated with either His-FGF21 wt or His-FGF21 CV16. Time course of 46 52997671.2 Attorney Docket No.047162-7483WO1 Ca2+release in AR42J cells (expressing endogenous FGFR1c and ^-Klotho) were quantitively measured. For Ca2+release experiments, cells were loaded with a fluorescent Ca2+indicator and then stimulated with increasing concentrations of His-FGF21 wt or His- FGF21 CV16. The level of Ca2+release was then analyzed as a change in the fluorescence emission. Results from the experiments presented in Fig.6A indicate that Ca2+release was not detected upon 0.5 nM stimulation with His-FGF21 wt while stimulation of AR42J cells with 0.5 nM of His-FGF21 CV16 induced strong Ca2+release in these cells. Also, robust Ca2+release was detected upon stimulation with 2 nM of His-FGF21 CV16 while stimulation with 2 nM of His-FGF21 wt gave small but detectable level of Ca2+release. In addition, stimulation with 10 nM of His-FGF21 CV16 induced a robust Ca2+release signal similar to Ca2+release induced by saturating concentrations of His-FGF21 wt or by His-FGF21 CV16 (50 nM). The cellular activities of AR42J cells stimulated by either His-FGF21 wt or His- FGF21 CV16 were monitored by the level of phosphorylated MAPK in the immunoblotting analyses. Results from the experiments presented in Fig.6B clearly demonstrate that the potency of His-FGF21 CV16 to activate MAPK response in AR42J cells is higher as compared to the potency of His-FGF21 wt, and the degree of improvement is similar to the enhancements in the activities in other stimulated cells such as L6 cells co-expressing FGFR1c and β-Klotho, as shown in Fig.5A. As mentioned, additional variants were obtained by generating constructs that direct the synthesis of fusion proteins connected to the N terminal region of FGF21 including the small Ubiquitin-like Modifier (SUMO), murine Serum Albumin (mSA), Spitz, and human Fc region of human IgG1. The sequences of these fusion partners are listed below: Small Ubiquitin-like Modifier (SUMO) E A P T S L 47 52997671.2 Attorney Docket No.047162-7483WO1 QTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGT FLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPK L A N P S ese us on construct o var ants were generate to mprove express on evel, biochemical properties, and / or superior pharmacological activities of the new FGF21 variants. Other modifications that can be applied to improve the pharmacological properties include but not limited to: chemical modification such as PEGylation, lipidation, and glycosylation, and other N-terminal fusion constructs such as Fc regions from other IgG subtypes, human serum albumin, and albumin-binding domain. The binding kinetic parameters from BLI measurements (Figs.3A-3N, 4A-4B, 7A- 7C, 8A-8C and 9A-9C) indicate that all fusion partner proteins on the N-termini of FGF21 variants did not alter the binding affinities of FGF21 variants to sKLB. Importantly, all fusion proteins with FGF21 CV16 retain high affinity binding to sKLB, similar to His-FGF21 CV16. Moreover, all fusion proteins exhibit similar cellular activities to His-FGF21 CV16 in L6 cells co-expressing FGFR1c and β-Klotho (Figs.7C, 8C and 9C). This confirms that the enhancement of binding affinity to β-Klotho directly impacts the cellular potency of FGF21, and that FGF21 CV16 exhibits significantly enhanced potency than FGF21 wt regardless of the fusion partner on the N-terminus. MAPK activation as assayed by immunoblotting: To examine the abilities of other FGF21 variants to stimulate cellular signaling, L6 myoblast cell line co-expressing FGFR1c and β-Klotho were stimulated with increasing concentrations of FGF21 variants, and the 48 52997671.2 Attorney Docket No.047162-7483WO1 levels of phosphorylated MAPK (pMAPK) were monitored with immunoblot analysis. The results shown in Fig.16A demonstrate that both His-FGF21 wt and His-FGF21 CV30 activate cellular signaling in L6 myoblast cell line co-expressing FGFR1c and β-Klotho in a dose-dependent manner as monitored by pMAPK levels, with His-FGF21 CV30 exhibiting higher potency as compared to His-FGF21 wt. The results shown in Fig.16B demonstrate that both Fc-FGF21 wt and Fc-FGF21 CV16 activate cellular signaling in L6 myoblast cell line co-expressing FGFR1c and β-Klotho in a dose-dependent manner as monitored by pMAPK levels, with Fc-FGF21 CV16 exhibiting higher potency as compared to His-FGF21 wt. The results shown in Fig.16C demonstrate that both Fc-FGF21 wt and Fc-FGF21 CV30 activate cellular signaling in L6 myoblast cell line co-expressing FGFR1c and β-Klotho in a dose-dependent manner as monitored by pMAPK levels, with Fc-FGF21 CV30 exhibiting higher potency as compared to His-FGF21 wt. This confirms that the enhancement of binding affinity to β-Klotho directly impacts the cellular potency of FGF21, and that FGF21 CV30 exhibits significantly enhanced potency than FGF21 wt regardless of the fusion partner on the N-terminus. This confirms that Fc-FGF21 CV16 exhibits significantly enhanced potency over Fc-FGF21 wt. MAPK activation as assayed by AlphaLISA: To examine the abilities of other FGF21 variants to stimulate cellular signaling, L6 myoblast cell line co-expressing FGFR1c and β-Klotho were stimulated with increasing concentrations of FGF21 variants, and the levels of phosphorylated MAPK (pMAPK) were monitored with AlphaLISA (Revvity). The results shown in Fig.17A demonstrate that both Fc-FGF21 wt and Fc-FGF21 CV25 activate cellular signaling in L6 myoblast cell line co-expressing FGFR1c and β-Klotho in a dose- dependent manner as monitored by pMAPK levels, with Fc-FGF21 CV25 exhibiting higher potency as compared to Fc-FGF21 wt. The results shown in Fig.17B demonstrate that both FGF21 wt (no tag) and FGF21 CV16 activate cellular signaling in L6 myoblast cell line co- expressing FGFR1c and β-Klotho in a dose-dependent manner as monitored by pMAPK levels, with FGF21 CV16 exhibiting higher potency as compared to FGF21 wt. The results shown in Fig.17C demonstrate that both FGF21 wt (no tag) and FGF21 CV25 activate cellular signaling in L6 myoblast cell line co-expressing FGFR1c and β-Klotho in a dose- dependent manner as monitored by pMAPK levels, with FGF21 CV25 exhibiting higher potency as compared to FGF21 wt. This confirms that the enhancement of binding affinity to β-Klotho directly impacts the cellular potency of FGF21, and that FGF21 CV25 exhibits significantly enhanced potency than FGF21 wt regardless of the fusion partner on the N- terminus or the lack of fusion partner. This confirms that FGF21 CV16 exhibits significantly 49 52997671.2 Attorney Docket No.047162-7483WO1 enhanced potency over FGF21 wt demonstrating that a fusion partner is not required. Example 3: MD simulation results of C-tail of FGF21 CV16 provide insights on the mechanism of high affinity binding to β-Klotho The present study applied Gaussian accelerated molecular dynamics (GAMD) simulation, an efficient unconstrained enhanced sampling method that lowers the energetic barrier for conformational transition of the simulated system, to explore conformational space for C-terminus tail (CT) of FGF21 wt (CTWT) and FGF21 CV16 (CT16). The peptide simulations were limited to 10-14 amino acids length since peptides longer than 20 amino acids often collapse to form metastable partially folded state that reduce sampling efficacy. The first stretch of FGF21, site 1-interacting region (SIR1), is composed of residues 186-198, and the second stretch, site 2-interacting region (SIR2), is composed of residues 200-209. GAMD can be used to derive the potential free energy of the simulated system. The folding free energy can be visualized in the potential of mean force (PMF) energy landscape to indicate the energetic wells of the states. This landscape was constructed by correlating the energy of the simulated system to the degree of SIR1 peptide folding, i.e., backbone RMSD of residues D192-V197, and the distance between carboxylate side chain of D187 and backbone amide of S190, which form the hydrogen-bond that maintains the turn structure in SIR1. Free energy landscape for SIR1 of wild-type FGF21 shows a wide coverage of the conformational space with multiple shallow metastable energetic wells (Fig.10A). Strikingly, the folded state of both FGF21 CTs was rarely sampled by GAMD even after 1.2 μs total simulation time, suggesting that the SIR1 region of FGF21 is intrinsically disordered and does not form the turn conformation observed in the atomic structures until interacting with β-Klotho. This was also observed in the conformational population landscape which provides the information on the frequency of sampling of the folded, intermediate, and unfolded states during the GAMD simulations. SIR1 of wild-type FGF21 sampled mostly (67.3%) unfolded state, less frequently (30%) the intermediate state, and hardly (1%) folded state (Fig.10B). SIR2 of FGF21 wt forms a single α-helical turn when bound to β-Klotho as shown in the crystal structure. The folding energetics of SIR2 could be examined by the folding free energy difference between the folded and unfolded conformational states. Three distances, backbone amide nitrogen atoms of R203-A208, S204-A208, and S204-Y207 that form backbone amide hydrogen-bonds to maintain the α-helical turn structure of the SPS motif, can be used as structural measurement metrics. Using the S204-Y207 backbone amide distance as an example (Figs.10C-10D), the folding free energy landscape of wild-type 50 52997671.2 Attorney Docket No.047162-7483WO1 FGF21 SIR2 region shows two well-defined energy wells that are separated by an activation free energy barrier (ΔG ^) of roughly 2.5 kcal / mol between the folded and unfolded conformational states. On the other hand, an intermediate state (RMSD between 1.0 Å and 3.0 Å) is energetically similar to the folded state (RMSD < 1.0 Å). In other words, the folded and intermediate states are distinct from the unfolded states even when FGF21-CT is in the unbound form, while the folded and intermediate states would frequently exchange since there is no significant energy barrier between the two. The conformational population landscape of WT FGF21 SIR2 suggests the unfolded states (58%) is more frequently sampled than the sum of the folded (15%) and intermediate state (27%). This is in contrast to SIR1 where multiple metastable unfolded and intermediate states are found and the folded state is rarely sampled (Figs.8A-8B). SIR2 have few metastable states and the folded state is sampled frequently, indicating that α-helical turn of SPS motif is intrinsically stable and accessible even in the unbound form. Analysis of a computational model of CT16 built based on the conformation of sKLB-bound CT regions of WT FGF21 (CTWT) suggests that two tryptophan substitutions in CT16, R203W and A208W, may interact with each other via edge-to-face π-π stacking between their indole rings and hydrogen-bonding between A208W indole NH to S204 carbonyl oxygen. It was hypothesized that these two intramolecular interactions may bring R203W and A208W in close proximity and retain them in such configuration for longer period of time, which would then promote the propensity of the SPS motif residues between them to adopt the α-helical turn, thus shifting the equilibrium to favor the folded conformational states for β-Klotho binding. To test this hypothesis, the present study examined the population of folded and unfolded conformational states sampled during the GAMD simulation of SIR2 of FGF21CT16. Analyses of the population ratio for the GAMD simulation indicate that SIR2 of CT16 has higher population fraction in the folded state (39%) and lower fraction in the combined intermediate and unfolded states (61%) than SIR2 of FGF21CTWT shown in Figure 8D. This suggests the amino acid substitutions in CT16 shift the folding equilibrium toward the folded state. Therefore, R203W / A208W substitutions in FGF21 CV16 would promote the propensity of SPS motif folding with the specific intramolecular interactions, thereby reducing the entropic cost during the folding event, as compared to the WT. This increase in the folded FGF21 SPS motif in FGF21 CV16 might have strong implication in the binding to β-Klotho, in which the folded SPS motif will be more likely to bind to the target than the unfolded SPS motif. Furthermore, the mutations may also reduce the rate the folded 51 52997671.2 Attorney Docket No.047162-7483WO1 SPS motif from unfolding, hence retaining the folded state population for longer duration. These retention changes may have implication on the dissociation constant of the complex of FGF21-CT and β-Klotho, in which the intrinsic unfolding of the bound CT may lead to its eventual dissociation. Reducing the rate of unfolding of the bound CT may therefore reduce the off-rate of overall interactions between FGF21 and β-Klotho. Example 4: Crystal structure of FGF21 CV16 peptide in complex with sKLB To gain insights on how FGF21 CV16 binds to sKLB with high affinity, the present study determined the crystal structure of sKLB in complex with a C-terminus tail peptide from FGF21 CV16 (CT16). Diffraction-quality crystals were obtained from multiple crystallization conditions when sKLB containing two mutations, N308Q and N611Q, in complex with CT16 and the nanobody Nb30 was screened for crystallization. After the optimization of each crystallization conditions, large plate-like and pyramid-shaped crystals could be obtained by hanging drop vapor diffusion method using the solution containing 13% polyethylene glycol 3,350 and 10 mM citric acid / bis-tris (1:1) buffer at pH 5.0. The X-ray diffraction data from the crystals were processed and scaled to a resolution cutoff of 3.0 Å. The structure was determined with molecular replacement using an existing model of sKLB in complex with Nb30 followed by iterative refinement steps to build the model of CT16. The final model (Rwork / Rfree=22.8 / 25.7 %) exhibits good overall geometry and atomic parameters. sKLB contains two glycoside hydrolase-like domains, KL1 and KL2 (Fig.11A), each of which has its catalytic Glu residue replaced into either Asn or Ala, which renders them to be inactive glycoside hydrolases, and the two domains are connected by a linker region. The two domains are intrinsically dynamic and the inter-domain orientations vary depending on the crystallization packing condition and / or the presence of different nanobodies. Just like C- terminus tail peptide from wild type FGF21 (CTWT), the C-terminus tail peptide from FGF21 CV16 (CT16) binds to sKLB spanning KL1 and KL2 domains (Fig.11A). Comparison of crystal structures of sKLB in complex with CT16 and with CTWT (PDB ID: 5VAQ) reveals that, when bound to sKLB, overall conformation of CT16 is very similar to the conformation of CTWT. Figs.11B-11C show site 1-interacting region (SIR1) for CT16 and CTWT, respectively, whereas Figs.11D-11E show site 2-interacting region (SIR2) for CT16 and CTWT, respectively. SIR1 of both CT16 and CTWT adopt unique conformation comprised of multiple turns which make rather large interface that interact with the surface of sKLB in KL1 domain and KL1-KL2 interface. An amino acid substitution in CT16, L194F, within SIR1 did not induce any conformational changes on CT16 as compared to CTWT. 52 52997671.2 Attorney Docket No.047162-7483WO1 SIR2 of both CT16 and CTWT adopt a small helical structure to bind to the pocket of sKLB in KL2. There are four amino acid substitutions in CT16, Q201I, R203W, Y207W, and E208W, within SIR2, and they did not induce significant conformational changes on CT16 as compared to CTWT. The linker regions (residues 197-200) in CT16 and CTWT are the regions whose structures are most different from one another when overall structures are overlaid, and this might be due to the intrinsic flexibilities of the linker regions in the peptides and / or the flexibilities in the interdomain orientation of β-Klotho. Overall structure of CT16-bound sKLB is similar to the structure of WT-bound sKLB (Fig.12A); both structures contain two glycoside hydrolase-like domains, KL1 and KL2, which are similarly occupied by the peptides. Superposition of Cα atoms from the entire structures results in the root-mean-square deviation (RMSD) of 2.12 Å, whereas superposition of individual domains yields Cα RMSD of 0.29 Å and 0.46 Å for KL1 (residues 53-507) and KL2 (residues 515-969), respectively. Results shown in Fig.12B (site 1) and Fig.12C (site 2) indicate that each site for CT16-bound sKLB is comprised of residues that are almost identical as residues in site 1 and site 2 for CTWT-bound sKLB, and that the structural fold of each site is not altered by binding of different peptides. The intrinsic flexibilities in inter-domain orientation allow sKLB to be crystallized in various conformations depending on the crystallization packing interface created by molecular interactions facilitated by the nanobody utilized, and these inter-domain movements in β-Klotho could be necessary to form adequate inter-molecular interactions for the formation of signaling complexes. Comparison of structures between CTWT-bound sKLB and CT16-bound sKLB shown in Figs.12A-12C suggests that the inter-domain flexibilities might have caused the structural differences which originate from the molecular interactions during the crystallization process. Indeed, the analysis of these two structures using the DynDom (www dot cmp dot uea dot ac dot uk / dyndom) results in the inter-domain angle changes of 13.2º. The results from binding affinity measurements, which strongly indicate that the binding affinities of FGF21 CV16 and its fusion constructs toward sKLB are significantly higher than wild type FGF21 and its fusion constructs. To understand and identify potential molecular interactions that are responsible for such differences, the present study examined the molecular interactions in SIR2, where the majority of the amino acid changes exist. Detailed comparison of molecular interactions in site 2 for sKLB in complex with CT16 and sKLB in complex with CTWT are shown in Figs.13A-13B. Both CT16 colored in orange (Fig.13A) and CTWT colored in purple (Fig.13B) exhibit a short α-helical segment 53 52997671.2 Attorney Docket No.047162-7483WO1 surrounding the SPS-motif, and both peptides make contacts with the same area in sKLB as also illustrated in Figs.12A-12C. Analyses from MD simulation have suggested that the entropic contribution of W203-W208 interactions within CT16 would affect the binding affinities to β-Klotho. On the other hand, inter-molecular interactions visualized in the crystal structure would explain the enthalpic contribution of binding affinity changes caused by amino acid substitutions. Each substitution in SIR2 make small differences in the inter- molecular interactions between CT16 and sKLB vs. interactions between CTWT and sKLB: R203W substitutions would create hydrophobic interactions involving L862 and T860; Y207W would strengthen hydrophobic interactions involving P644 and H646; and finally, E208W would make π-π interactions between indole ring of W208 (CT16) and imidazole rings of H646 (sKLB). All of these changes, together with the improvements in hydrophobic interactions resulting from L194F substitution, makes positive contribution to overall binding affinities of FGF21 CV16 to β-Klotho, resulting in significant improvements in the cellular potency. Example 5: Expression of FGF21 in liver is upregulated upon various nutrient stresses such as starvation, alcohol, and sugar. Studies have shown that FGF21 is an important regulator of metabolic homeostasis. When pharmacologically administered to diabetic or obese animals, FGF21 causes body weight loss, improves insulin sensitivity, and lowers blood glucose and triglyceride levels. Accordingly, there has been enormous efforts in developing FGF21 analogs as a potential novel therapy for treating metabolic disorders such as type II diabetes and obesity as well as NASH and NFLD. Evidences also point out that FGF21 can be used as a therapeutic agent on liver diseases: FGF21 is normally expressed in the livers; recombinant FGF21 is shown to be non-mitogenic when pharmacologically administered in mice; and transgenic FGF21 mice that produce supra-physiological level of FGF21 in liver have extended lifespan. FGF21 can be used in treatment of murine models of pancreatitis and acute alcohol toxicity. Therefore, FGF21 C-tail variants and their fusion constructs with increased bioactivities and improved pharmacological properties would be extremely useful as therapeutic agents for human diseases. The present study provides means to improve the efficacy of wild-type FGF21. The experimental results on one of the FGF21 C-terminus tail variants, FGF21 CV16, that has superior binding affinity to β-Klotho and cellular activities suggest that the combination of improvements in interactions between FGF21 C-tail and β-Klotho as well as intramolecular 54 52997671.2 Attorney Docket No.047162-7483WO1 interactions within C-tail can impact significantly on the in vivo biological activities of FGF21 as well as its therapeutic utility The combined effect of mutations L194F, Q201I, and Y207W that directly interact with β-Klotho to contribute enthalpically and mutations R203W and A208W that reduce the entropic cost of binding by increasing the population of folded SPS motif may contribute together to the overall improvements in the binding kinetics of FGF21 CV16 to β-Klotho and cellular activities as described in Fig.14. The observed on-rate is the consequence of cooperative interactions in site 1 and site 2 that are likely taking place through multi-step processes, and this might be the reason why changes in on-rate cannot be measurable through the substitutions in only SIR2 such as R203W / A208W. In summary, the present study identified FGF21 variants that bind with high affinities to β-Klotho via an entropic-driven mechanism and are capable of robust activation of FGFR signaling in the presence of β-Klotho. Example 6: Materials and Methods Recombinant protein production and binding affinity measurements All binding kinetics parameters were derived from Bio-Layer Interferometry (BLI) measurements using the Octet RED96 system (FortéBio, Molecular Devices). FGF21 variants fused to the Fc region of human IgG1 on their N-terminal regions were expressed in Expi293F cells and purified using recombinant protein A sepharose 4B (Thermo-Fisher) followed by size exclusion chromatography. FGF21 variants with octa- histidine tag on the N-terminal regions (His-FGF21), FGF21 variants fused to the O- glycosylation region of D. melanogaster Spitz on their N-terminal regions (Spitz-FGF21), or FGF21 variants with murine serum albumin fused on the N-termini (mSA-FGF21) were expressed in Expi293F cells and purified using Ni-NTA agarose (Qiagen) or nickel sepharose excel (GE healthcare) followed by size exclusion chromatography. sKLB was expressed and purified following the protocol described in Lee et al. (Nature 553(7689): 501-505, doi: 10.1038 / nature25010. (2018)). Briefly, DNA that encode for amino acids 30-983 (sKLB) followed by regions encoding tobacco etch virus (TEV) protease cleavage site, a linker (GGGG), and human IgG1 Fc region was subcloned into the pCEP4 vector (Thermo-Fisher Scientific Inc.) (sKLB-Fc). The resulting plasmid was transfected to HEK293EBNA cells and selected with 250 μg / mL Hygromycin B to generate cells stably expressing sKLB-Fc. sKLB-Fc was purified from the media with recombinant Protein A Sepharose 4B (Thermo-Fisher), followed by incubation with TEV enzyme to 55 52997671.2 Attorney Docket No.047162-7483WO1 cleave C-terminus Fc tag. The Fc fragment and undigested sKLB-Fc were removed by incubation with recombinant Protein A Sepharose 4B and resulting sKLB was further purified by the cation exchange chromatography. Solution of purified protein was concentrated to 1 mg / mL and flash-frozen until further use. For crystallization, sKLB-Fc containing N308Q / N611Q mutations were expressed in HEK293EBNA cells and purified as the wild-type sKLB. Fc-FGF21 proteins at the concentration of 5 μg / mL in PBS containing 0.01% BSA and 0.002% Tween-20 (PBS-KB) were loaded onto the anti-human IgG1 Fc biosensor probes (AHC). His-FGF21, SUMO-FGF21, Spitz-FGF21 or mSA-FGF21 proteins at the concentration of 2 μg / mL in 10 mM sodium acetate buffers at pH 6.0 were immobilized onto the Amine Reactive 2nd Generation (AR2G) biosensors using standard amine coupling method utilizing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N- hydroxysulfosuccinimide (sulfo-NHS). Sensors loaded with FGF21 variants were briefly washed and subsequently dipped into wells containing a series of concentrations of purified sKLB to record the responses. Resulting sensorgrams were referenced using a parallel buffer control subtraction and fitted globally to a 1:1 Langmuir binding model using FortéBio Data Analysis 10.0 software provided by the manufacturer to determine the kinetic rate constants (kon and koff) and the dissociation constant, KD. Non-tagged FGF21 variants were generated with an N-terminal six-histidine tag and a Tobacco Etch Virus (TEV) protease site (ENLYFQG). The proteins were expressed in E. coli and purified with nickel affinity chromatography followed by cleavage of the six-histidine tag with TEV protease (GenScript). The cleaved tag, histidine tagged TEV protease and any uncleaved histidine tagged FGF21 were removed with repeat nickel affinity followed by size exclusion chromatography. The protease cleaves in between the Q and G residues of the protease site and leaves an N-terminal Glycine on the purified FGF21 variants. Cell-based activity assays Rattus norvegicus L6 myoblast cells stably co-expressing wild-type FGFR1c and wild-type β-Klotho were grown in DMEM supplemented with 10% FBS, 100 U / mL Penicillin-Streptomycin, 0.1 mg / ml hygromycin and 1 μg / ml puromycin. Cells were starved overnight in DMEM and stimulated for 10 minutes at 37˚C with either FGF1 at 5 nM (as a control) or FGF21 variants at concentrations ranging from 0.05 to 10 nM. Cells were washed with cold PBS and lysed with ice-cold lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1mM EGTA, 25mM NaF, 1mM MgCl2, 10% Glycerol, 1 56 52997671.2 Attorney Docket No.047162-7483WO1 mM sodium orthovanadate, 1mM PMSF) with protease inhibitors. The lysates were then subjected to SDS-PAGE followed by immunoblotting with either anti-phospho-MAPK, anti- MAPK, or anti-β-tubulin antibodies according to Lemmon et al. (Cell 141, 1117-1134, doi:10.1016 / j.cell.2010.06.011 (2010)). AlphaLISA assay: To provide an additional readout for our cell-based activation assay, the present study used the L6 cell line stably co-expressing FGFR1c and β-Klotho and a sandwich immunoassay to detect total and phosphorylated MAPK. The assay was executed by serum-starving the L6 cells co-expressing FGFR1c and β-Klotho for 2 hours to reduce the background phosphorylation of MAPK followed by stimulation for 10 minutes at 37°C with a protein dilution range of FGF21 variants between 0.09 and 24 nM. Cells were then placed on ice, washed with cold physiological buffer, and lysed in the presence of phosphatase and protease inhibitors. To measure MAPK activation, levels of phosphorylated MAPK family members ERK1 / 2 were measured with the AlphaLISA SureFire Ultra p-ERK1 / 2 (Thr202 / Tyr204) assay. The kit specifically measures phosphorylated Thr202 / Tyr204 in ERK1 and Thr185 / Tyr187 in ERK2. The total ERK1 / 2 levels are also separately measured for each sample with a similar sandwich immunoassay that detects both phosphorylated and non-phosphorylated ERK1 / 2 (AlphaLISA SureFire Ultra total ERK1 / 2). To control variations in cell number between each sample, the signal for the phosphorylated MAPK for each sample is normalized to the signal for total MAPK. Samples were performed in duplicate, and the results averaged. Average and standard deviation were graphed with GraphPad Prism. Glucose uptake assay 3T3-L1 mouse embryonic fibroblast cells (ATCC) were maintained in DMEM with 10% bovine calf serum (BCS), and the differentiation was induced using DMEM with 10% FBS, 0.25 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 5 μg / mL bovine insulin.48 hours after the induction, the medium was changed to DMEM with 10% FBS and 5 μg / mL bovine insulin and incubated for additional 48 hours. The differentiated cells were maintained in DMEM with 10% FBS for 10-15 days before the treatment with FGF21 variants. The differentiated 3T3-L1 adipocytes were treated with wild-type FGF21 or FGF21 variants at concentrations ranging from 0.01 nM to 100 nM for 24 hours followed by washing with PBS and addition of 2-deoxyglucose (2DG). The cellular glucose uptake was monitored by the detection of the total amount of 2-deoxyglucose-6-phosphate (2DG6P) in the cells using the Glucose Uptake-Glo Assay (Promega). 57 52997671.2 Attorney Docket No.047162-7483WO1 Crystallization and structure determination of sKLB bound to CT16 The purified sKLB2Q was mixed with a nanobody and purified by the size exclusion chromatography. The peptide corresponding to C-terminus region of FGF21 CV16 (amino acids L174-S209) region, CT16, was added to the concentrated sKLB2Q in complex with a nanobody and incubated at 4 degrees for 30 minutes, followed by screening for crystallization. sKLB2Q in complex with a nanobody and CT16 gave plate-like crystals when mixed with equal volume of well solution containing 13% PEG 3,350, 0.05M Citric Acid / 0.05M BIS-TRIS at pH 5.0 and equilibrated for 5-10 days using the hanging drop vapor diffusion method. The crystals were cryopreserved by gradually transferring crystals to the mother liquor supplemented with 30% ethylene glycol before being flash-frozen in liquid nitrogen. X-ray diffraction data was collected at the NE-CAT beamline 24-ID-E at the Advanced Photon Source, Argonne, IL. The diffraction data were processed with XDS, and the structure was determined by molecular replacement with PHASER and the refinement was performed with PHENIX while manually examining the qualities with COOT. Molecular Dynamics (MD) simulation Gaussian Accelerated Molecular Dynamics (GAMD) simulation, an unconstrained enhanced sampling method built upon the conventional Molecular Dynamics (cMD) method implemented in AMBER simulation package, was used for efficient sampling of large conformational space. The initial structure of FGF21 CTWT peptide was either extracted from PDB ID: 5VAQ (“folded”) or generated in Schrödinger Maestro program v2019-1 (“linear”), parameterized with Amber ff14SB force field, and solvated in a box of truncated octahedron TIP3P water using Amber18. The simulation system is charge-balanced by adding mono- ions. SHAKE algorithm was enabled for 2-fs simulation time step. Particle Mesh Ewald method and long-distance cutoff of 10 Å were used for non-bonded interactions. For systems of CT16 and its variants, simulations were done from the linear forms, whereas the systems of double cysteine mutants were started from the folded forms. To avoid the peptides from collapsing into “partially folded state” that limits sampling, only short stretch of peptide, i.e., 10-14 amino acids, were used. Preparation of the simulations were similar, with changes in minimization over 30,000 steps, followed by heating to 310 K in 400 ps and 500 ps of equilibrations. All simulations were run on PMEMD.CUDA function on Nvidia Volta V100 GPUs. 58 52997671.2 Attorney Docket No.047162-7483WO1 2 ns of cMD simulation was run to collect the initial parameters for GAMD run, followed by 20 ns of GAMD adjustment run with a window size of 200 ps and 300 ns of GAMD production run. GAMD simulation used dual energy boost and an upper limit of 6.0 kcal / mol as the standard deviation of boost. For each system, 2 independent trajectories were run. Post-processing of the GAMD trajectories were done through CPPTRAJ to calculate structural metrics such as residue distances, torsion angles, and secondary structures defined by DSSP. A total of 600 to 1,200 ns of independent trajectories were aggregated and frames were extracted at 20-ps interval. Energy data generated by GAMD was reweighted using cumulant expansion to the second order using a modified Python script for GAMD to calculate the potential of mean force (PMF) of FGF-CTs during GAMD. PMF data was plotted with various structural metrics to identify the most relevant measurement metrics and to generate 2-dimensional (2D) folding free energy landscape with a maximum energy cutoff of 5.0 kcal / mol. In addition, 2D conformational population landscape were plotted with the structural metrics to visualize frequency of MD frames that fell within the bins of metrics. This population information could also be used to calculate the folding free energy difference by comparing the populations of the folded and unfolded states. Free energy estimation of structure formation The folding free energy difference (ΔGfolding) between folded and unfolded conformational states is related to the relative population of the conformations through statistical mechanics, which can be calculated with Equation (1), ^ ^ೠ^^^^^^^^ Δ ^^^^^ௗ^^^ൌ െ ^^ ^^ ^^ ^^ ^^ ൌ െ ^^ ^^ ^ ^^ ^^ ^^^^^^^^ೌ^^^ െ ^^ ^^ ^^ೌ^^^^ ൌ െ ^^ ^^ ^^ ^^ ൬^^^^^^^ೠ^^^^^^^^ (1) where ΔGfoldingis the free energy of protein folding, Pallrepresents the total population of all states, pfolded and punfolded represent the population of the folded and unfolded states, respectively. T is the simulation temperature at 310 K and R is the gas constant, 1.987x10-3kcal / K / mol. Example 7: Sequences of constructs The protein sequences and nucleic acid sequences of the constructs used in the present study are listed below. In certain embodiments, the construct of the invention corresponds to one listed below minus the signal peptide. 59 52997671.2 Attorney Docket No.047162-7483WO1 P135. pCEP4 Fc-PrFGF21 wt Protein Sequence: T V S G G C A G G C T C A C G A G T 60 52997671.2 Attorney Docket No.047162-7483WO1 GGCGTTATCCAAATCTTAGGCGTCAAAACTTCACGCTTTTTATGCCAACGTCCT GATGGAGCCCTGTATGGTTCACTGCACTTTGACCCGGAAGCTTGCTCATTCCGT G ) T V I L S C A G G C T C 61 52997671.2 Attorney Docket No.047162-7483WO1 CCCCCCGTACTCGATTCCGACGGATCTTTTTTCCTGTACTCTAAACTTACAGTC GATAAATCACGCTGGCAGCAAGGCAATGTTTTCTCCTGTTCTGTAATGCATGAA C A T A C T V I L S 62 52997671.2 Attorney Docket No.047162-7483WO1 ACCCGAGAACCAGAAGTTACGTGCGTGGTCGTCGACGTTTCACACGAAGATCC AGAAGTTAAATTCAACTGGTACGTGGATGGAGTAGAAGTGCACAATGCCAAAA G G C T C A C A T A T V I L S 63 52997671.2 Attorney Docket No.047162-7483WO1 RGWSPSWWS (SEQ ID NO:12) Italic: Signal peptide from mouse IgG-HV C A G G C T C A C A C A T A C 64 52997671.2 Attorney Docket No.047162-7483WO1 MGWSCIILFLVATATGVHSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITRE PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT T V I L S C A G G C T C A C T T A C 65 52997671.2 Attorney Docket No.047162-7483WO1 CTTTCGCGAACGGCTGCTTGAAGATGGCTACAACGTTTATCAATCGGAAGCAC ATGGTCTCCCACTTCACTTACCAGGCAACAAATCACCACATCGTGACCCTGCTC G C T V I L S C A G G C T C 66 52997671.2 Attorney Docket No.047162-7483WO1 GATAAATCACGCTGGCAGCAAGGCAATGTTTTCTCCTGTTCTGTAATGCATGAA GCTCTCAAATTCCACTATACACAGAAAAGCCTTAGTCTTTCCCCCGGCAAACTC A T A A G T T V I L S C 67 52997671.2 Attorney Docket No.047162-7483WO1 AGAAGTTAAATTCAACTGGTACGTGGATGGAGTAGAAGTGCACAATGCCAAAA CCAAGCCCCGAGAAGAACAGTACAATTCCACATACAGAGTCGTGTCAGTGCTG G C T C A C A A C A A C G T T V I L S 68 52997671.2 Attorney Docket No.047162-7483WO1 Italic: Signal peptide from mouse IgG-HV Bold: Human Fc C A G G C T C A C A C C T A A 69 52997671.2 Attorney Docket No.047162-7483WO1 PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT V I L S C A G G C T C A C A C A C A 70 52997671.2 Attorney Docket No.047162-7483WO1 TGGCCTCCCCTTACACTTACCTGGTAACAAATCTCCACATCGTGACCCTGCCCC TCGCGGTCCAGCACGCTTTCTCCCACTCCCTGGATTACCACCTGCTCTTCCAGA T G T V I L S C A G G C T C A 71 52997671.2 Attorney Docket No.047162-7483WO1 GCTCTCAAATTCCACTATACACAGAAAAGCCTTAGTCTTTCCCCCGGCAAACTC GAGGGCGGCGGAGGATCAGGCGGCGGTGGGAGTCATATGCACCCCATTCCCG C C A C T G T V I L S C A 72 52997671.2 Attorney Docket No.047162-7483WO1 CCAAGCCCCGAGAAGAACAGTACAATTCCACATACAGAGTCGTGTCAGTGCTG ACCGTGCTGCATCAAGACTGGCTTAATGGGAAAGAGTATAAATGTAAAGTGAG C T C A C C A C A C A T T V I L S 73 52997671.2 Attorney Docket No.047162-7483WO1 Bold: Human Fc Underline: FGF21 C A G G C T C A C A G A A T A T 74 52997671.2 Attorney Docket No.047162-7483WO1 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV I L S C A G G C T C A C C T C C 75 52997671.2 Attorney Docket No.047162-7483WO1 CACGTGGTCCCGCCCGTTTCTTGCCATTACCCGGTTTACCTCCTGCTTTACCCGA ACCGCCTGGCATCCTGGCCCCACAGCCACCCGACGTGGGTTCGTCTGATCCATT T V G H C A G G C T C A T 76 52997671.2 Attorney Docket No.047162-7483WO1 AGCGGTGGAGGATCCCCCATACCTGACTCATCCCCCCTTCTCCAATTTGGAGGA CAAGTCCGCCAGCGCTACCTTTATACAGACGACGCCCAACAAACTGAAGCCCA G G T A C C L C A C A C C 77 52997671.2 Attorney Docket No.047162-7483WO1 Protein Sequence: MGWSCIILFLVATATGVHSHHHHHHHHPIPDSSPLLQFGGQVRQRYLYTDDAQQTE L T C T T C T G L A A A 78 52997671.2 Attorney Docket No.047162-7483WO1 AGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTT TAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTC A G C T T T C T G L A A A T A G C 79 52997671.2 Attorney Docket No.047162-7483WO1 AACGCTATTTATACACCGACGATGCACAACAAACAGAAGCACACTTAGAAATT CGTGAAGATGGAACTGTAGGTGGCGCCGCAGATCAAAGCCCAGAATCCTTATT T C L G T D G L Q L A T 80 52997671.2 Attorney Docket No.047162-7483WO1 GATTTGGGAGAACAACATTTCAAAGGCCTAGTCCTGATTGCCTTTTCCCAGTAT CTCCAGAAATGCTCATACGATGAGCATGCCAAATTAGTGCAGGAAGTAACAGA T A T G T T A G C A A G C T G T A C G A A A 81 52997671.2 Attorney Docket No.047162-7483WO1 TCAGGCGGCGGTGGGAGTCATCCAATTCCCGATTCTAGTCCCTTATTGCAATTT GGCGGCCAGGTACGTCAACGCTATTTATACACCGACGATGCACAACAAACAGA A T G G G C C L G T D G L Q F A 82 52997671.2 Attorney Docket No.047162-7483WO1 GGGGTGTGTTTCGCCGAGAAGCACACAAGAGTGAGATCGCCCATCGGTATAAT GATTTGGGAGAACAACATTTCAAAGGCCTAGTCCTGATTGCCTTTTCCCAGTAT A T A T G T T A G C A A G C T G T A C G A A 83 52997671.2 Attorney Docket No.047162-7483WO1 ACGCCTTAGCCCATCACCATCACCATCATCACCACCTCGAGGGCGGCGGAGGA TCAGGCGGCGGTGGGAGTCACCCAATCCCAGATTCTAGCCCACTGTTACAATT G A C A C G C V A C A G C A G A 84 52997671.2 Attorney Docket No.047162-7483WO1 ATCAGAAGCTCATGGTCTTCCTCTTCACCTTCCCGGAAACAAATCACCCCATAG AGATCCCGCACCCAGAGGCCCCGCACGATTCCTTCCTCTCCCAGGTCTCCCCCC G V A C A G G T T C G Enumerated Embodiments 85 52997671.2 Attorney Docket No.047162-7483WO1 In some aspects, the present invention is directed to the following non-limiting embodiments: Embodiment 1: A non-natural soluble construct, which comprises an FGF21 polypeptide comprising residues 186-198 and residues 200-209 of FGF21, wherein the FGF21 polypeptide comprises at least one mutation selected from the group consisting of L126, L194, V197, P199, Q201, R203, Y207, A208, and S209. Embodiment 2: The construct of Embodiment 1, wherein the FGF21 polypeptide comprises at least one mutation selected from the group consisting of L126R, L194F, V197I, V197L, P199G, Q201L, Q201I, Q201P, Q201R, Q201W, R203W, Y207W, A208E, A208W, and S209G. Embodiment 3: The construct of any one of Embodiments 1-2, wherein at least one of the following applies: (a) the FGF21 polypeptide comprises residues 186-209 of FGF21; (b) the FGF21 polypeptide comprises residues 169-209 of FGF21; (c) the FGF21 polypeptide comprises residues 29-209 of FGF21. Embodiment 4: The construct of any one of Embodiments 1-3, wherein the FGF21 polypeptide further comprises at least one sequence selected from the group consisting of RKK, RRS, and RKS conjugated to the C-terminus of the residues 200-209 of FGF21. Embodiment 5: The construct of any one of Embodiments 1-4, wherein at least one of the following applies: (a) the FGF21 polypeptide comprises the mutations L194F, V197I, Q201L, R203W, Y207W, A208W and S209G, and the sequence RKK conjugated to the C-terminus of the residues 200-209 of FGF21; (b) the FGF21 polypeptide comprises the mutations L194F, V197I, Q201R, R203W, Y207W, A208W and S209G; (c) the FGF21 polypeptide comprises the mutations L194F, V197I, Q201R, R203W, Y207W and A208W; (d) the FGF21 polypeptide comprises the mutations L194F, V197L, Q201R, R203W, Y207W, A208W and S209G; (e) the FGF21 polypeptide comprises the mutations L194F, V197L, Q201R, R203W, Y207W and A208W; (f) the FGF21 polypeptide comprises the mutations L194F, Q201I, R203W, Y207W and A208W; (g) the FGF21 polypeptide comprises the mutations L194F, Q201L, R203W, 86 52997671.2 Attorney Docket No.047162-7483WO1 Y207W, A208W and S209G, and the sequence RKS conjugated to the C- terminus of the residues 200-209 of FGF21; (h) the FGF21 polypeptide comprises the mutations L194F, Q201P, R203W, Y207W, A208W and S209G, and the sequence RRS conjugated to the C- terminus of the residues 200-209 of FGF21; (i) the FGF21 polypeptide comprises the mutations L194F, Q201R, R203W, Y207W, A208W and S209G, and the sequence RRS conjugated to the C- terminus of the residues 200-209 of FGF21; (j) the FGF21 polypeptide comprises the mutations L194F, Q201R, R203W, Y207W and A208W; (k) the FGF21 polypeptide comprises the mutations L194F, Q201W, R203W, Y207W, A208W and S209G; (l) the FGF21 polypeptide comprises the mutations L194F, R203W, Y207W and A208W; (m) the FGF21 polypeptide comprises the mutations R203W, Y207W and A208W. Embodiment 6: The construct of any one of Embodiments 1-5, wherein the FGF21 is a human FGF21 having a protein sequence set forth in SEQ ID NO:1. Embodiment 7: The construct of any one of Embodiments 1-6, further comprising a stability enhancing domain. Embodiment 8: The construct of Embodiment 7, wherein the stability enhancing domain is attached to an N-terminus of the FGF21 polypeptide, or wherein the stability enhancing domain is attached to a C-terminus of the FGF21 polypeptide. Embodiment 9: The construct of any one of Embodiments 7-8, wherein the stability enhancing domain is at least one selected from the group consisting of albumin, an Fc region of an antibody, a glutathione S-transferase, a small ubiquitin-like modifier (SUMO), an O- glycosylation region of a Drosophila Spitz protein, and a thioredoxin. Embodiment 10: The construct of any one of Embodiments 7-9, wherein the FGF21 polypeptide and the stability enhancing domain are linked through a linker polypeptide comprising about 1-18 amino acids. Embodiment 11: The construct of any one of Embodiments 1-10, wherein at least one of the following applies: (a) the dissociation constant (Kd) of the FGF21 polypeptide with β-Klotho is about 20% or less of the Kd of a corresponding wild-type FGF21 polypeptide; 87 52997671.2 Attorney Docket No.047162-7483WO1 (b) the Koff of the FGF21 polypeptide with β-Klotho is about 20% or less of the Koffof a corresponding wild-type FGF21 polypeptide. Embodiment 12: The construct of any one of Embodiments 1-11, wherein at least one of the following applies: (a) the construct binds to β-Klotho and sequester β-Klotho from pathways downstream of a fibroblast growth factor receptor (FGFR) and β-Klotho; (b) the construct binds to both an FGFR and β-Klotho and activate pathways downstream of the FGFR and β-Klotho. Embodiment 13: The construct of Embodiment 12, wherein the FGFR is FGFR1c. Embodiment 14: A composition comprising a construct of any one of Embodiments 1-13 and a pharmaceutically acceptable carrier. Embodiment 15: A method of modulating FGF21 / FGFR / β-Klotho signaling pathway or FGF19 / FGFR / β-Klotho signaling pathway in a cell, the method comprising contacting the surface of the cell with the construct of any one of Embodiments 1-13. Embodiment 16: The method of Embodiment 15, wherein (a) the construct binds to β-Klotho but not FGFR, thereby sequestering β-Klotho from the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β- Klotho signaling pathway and down-regulating the signaling pathway; or (b) the construct binds to both β-Klotho and FGFR, thereby upregulating the FGF21 / FGFR / β-Klotho signaling pathway. Embodiment 17: The method of any one of Embodiments 15-16, wherein the cell is from a cell line, a primary cell, or a cell in the body of a subject. Embodiment 18: The method of any one of Embodiments 15-17, wherein the cell is a cell in or derived from adipose tissue, liver, pancreas, or hypothalamus. Embodiment 19: A method of treating, ameliorating and / or preventing a disease or disorder in a subject in need thereof, the method comprising: administering to the subject an effective amount of the construct of any one of Embodiments 1-13 or the composition of Embodiment 14, wherein the disease or disorder is at least one selected from obesity, diabetes, pancreatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), acute alcohol toxicity, and cancers driven by an aberrant activation of FGF19 / FGFR4 / β-Klotho pathway. Embodiment 20: The method of Embodiment 19, wherein the construct or the composition is administered via at least one route selected from the group consisting of inhalational, oral, rectal, vaginal, parenteral, intracranial, topical, transdermal, pulmonary, 88 52997671.2 Attorney Docket No.047162-7483WO1 intranasal, buccal, ophthalmic, intrathecal, and intravenous. Embodiment 21: The method of any one of Embodiments 19-20, wherein the subject is a human. Embodiment 22: The construct of any one of Embodiments 1-4, wherein the FGF21 polypeptide has the amino acid sequence of SEQ ID NO: 1 with mutations as listed in options (a) to (m) below: (a) the FGF21 polypeptide comprises the mutations L194F, V197I, Q201L, R203W, Y207W, A208W and S209G, and the sequence RKK conjugated to the C-terminus of the residues 200-209 of FGF21; (b) the FGF21 polypeptide comprises the mutations L194F, V197I, Q201R, R203W, Y207W, A208W and S209G; (c) the FGF21 polypeptide comprises the mutations L194F, V197I, Q201R, R203W, Y207W and A208W; (d) the FGF21 polypeptide comprises the mutations L194F, V197L, Q201R, R203W, Y207W, A208W and S209G; (e) the FGF21 polypeptide comprises the mutations L194F, V197L, Q201R, R203W, Y207W and A208W; (f) the FGF21 polypeptide comprises the mutations L194F, Q201I, R203W, Y207W and A208W; (g) the FGF21 polypeptide comprises the mutations L194F, Q201L, R203W, Y207W, A208W and S209G, and the sequence RKS conjugated to the C- terminus of the residues 200-209 of FGF21; (h) the FGF21 polypeptide comprises the mutations L194F, Q201P, R203W, Y207W, A208W and S209G, and the sequence RRS conjugated to the C- terminus of the residues 200-209 of FGF21; (i) the FGF21 polypeptide comprises the mutations L194F, Q201R, R203W, Y207W, A208W and S209G, and the sequence RRS conjugated to the C- terminus of the residues 200-209 of FGF21; (j) the FGF21 polypeptide comprises the mutations L194F, Q201R, R203W, Y207W and A208W; (k) the FGF21 polypeptide comprises the mutations L194F, Q201W, R203W, Y207W, A208W and S209G; (l) the FGF21 polypeptide comprises the mutations L194F, R203W, Y207W and A208W; 89 52997671.2 Attorney Docket No.047162-7483WO1 (m) the FGF21 polypeptide comprises the mutations R203W, Y207W and A208W; and wherein the FGF21 polypeptide has an amino acid sequence that has at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, or has an amino acid sequence that has at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of residues 29-209 of SEQ ID NO: 1 (the mature human FGF21 protein). Embodiment 22: The construct of any one of Embodiments 1-4, wherein the FGF21 polypeptide has the amino acid sequence of the mature human FGF21 (residues 2-209 of SEQ ID NO: 1) with mutations as listed in options (a) to (m) below: (a) the FGF21 polypeptide comprises the mutations L194F, V197I, Q201L, R203W, Y207W, A208W and S209G, and the sequence RKK conjugated to the C-terminus of the residues 200-209 of FGF21; (b) the FGF21 polypeptide comprises the mutations L194F, V197I, Q201R, R203W, Y207W, A208W and S209G; (c) the FGF21 polypeptide comprises the mutations L194F, V197I, Q201R, R203W, Y207W and A208W; (d) the FGF21 polypeptide comprises the mutations L194F, V197L, Q201R, R203W, Y207W, A208W and S209G; (e) the FGF21 polypeptide comprises the mutations L194F, V197L, Q201R, R203W, Y207W and A208W; (f) the FGF21 polypeptide comprises the mutations L194F, Q201I, R203W, Y207W and A208W; (g) the FGF21 polypeptide comprises the mutations L194F, Q201L, R203W, Y207W, A208W and S209G, and the sequence RKS conjugated to the C- terminus of the residues 200-209 of FGF21; (h) the FGF21 polypeptide comprises the mutations L194F, Q201P, R203W, Y207W, A208W and S209G, and the sequence RRS conjugated to the C- terminus of the residues 200-209 of FGF21; (i) the FGF21 polypeptide comprises the mutations L194F, Q201R, R203W, Y207W, A208W and S209G, and the sequence RRS conjugated to the C- terminus of the residues 200-209 of FGF21; 90 52997671.2 Attorney Docket No.047162-7483WO1 (j) the FGF21 polypeptide comprises the mutations L194F, Q201R, R203W, Y207W and A208W; (k) the FGF21 polypeptide comprises the mutations L194F, Q201W, R203W, Y207W, A208W and S209G; (l) the FGF21 polypeptide comprises the mutations L194F, R203W, Y207W and A208W; (m) the FGF21 polypeptide comprises the mutations R203W, Y207W and A208W; and wherein the FGF21 polypeptide has an amino acid sequence that has at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of residues 29-209 of SEQ ID NO: 1 (the mature human FGF21 protein). The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure. 91 52997671.2

Claims

Attorney Docket No.047162-7483WO1 CLAIMS What is claimed is:

1. A non-natural soluble construct, which comprises an FGF21 polypeptide comprising residues 186-198 and residues 200-209 of FGF21, wherein the FGF21 polypeptide comprises at least one mutation selected from the group consisting of L126, L194, V197, P199, Q201, R203, Y207, A208, and S209.

2. The construct of claim 1, wherein the FGF21 polypeptide comprises at least one mutation selected from the group consisting of L126R, L194F, V197I, V197L, P199G, Q201L, Q201I, Q201P, Q201R, Q201W, R203W, Y207W, A208E, A208W, and S209G.

3. The construct of any one of claims 1-2, wherein at least one of the following applies: (a) the FGF21 polypeptide comprises residues 186-209 of FGF21; (b) the FGF21 polypeptide comprises residues 169-209 of FGF21; (c) the FGF21 polypeptide comprises residues 29-209 of FGF21.

4. The construct of any one of claims 1-3, wherein the FGF21 polypeptide further comprises at least one sequence selected from the group consisting of RKK, RRS, and RKS conjugated to the C-terminus of the residues 200-209 of FGF21.

5. The construct of any one of claims 1-4, wherein at least one of the following applies: (a) the FGF21 polypeptide comprises the mutations L194F, V197I, Q201L, R203W, Y207W, A208W and S209G, and the sequence RKK conjugated to the C-terminus of the residues 200-209 of FGF21; (b) the FGF21 polypeptide comprises the mutations L194F, V197I, Q201R, R203W, Y207W, A208W and S209G; (c) the FGF21 polypeptide comprises the mutations L194F, V197I, Q201R, R203W, Y207W and A208W; (d) the FGF21 polypeptide comprises the mutations L194F, V197L, Q201R, R203W, Y207W, A208W and S209G; (e) the FGF21 polypeptide comprises the mutations L194F, V197L, Q201R, R203W, Y207W and A208W; (f) the FGF21 polypeptide comprises the mutations L194F, Q201I, R203W, 92 52997671.2Attorney Docket No.047162-7483WO1 Y207W and A208W; (g) the FGF21 polypeptide comprises the mutations L194F, Q201L, R203W, Y207W, A208W and S209G, and the sequence RKS conjugated to the C- terminus of the residues 200-209 of FGF21; (h) the FGF21 polypeptide comprises the mutations L194F, Q201P, R203W, Y207W, A208W and S209G, and the sequence RRS conjugated to the C- terminus of the residues 200-209 of FGF21; (i) the FGF21 polypeptide comprises the mutations L194F, Q201R, R203W, Y207W, A208W and S209G, and the sequence RRS conjugated to the C- terminus of the residues 200-209 of FGF21; (j) the FGF21 polypeptide comprises the mutations L194F, Q201R, R203W, Y207W and A208W; (k) the FGF21 polypeptide comprises the mutations L194F, Q201W, R203W, Y207W, A208W and S209G; (l) the FGF21 polypeptide comprises the mutations L194F, R203W, Y207W and A208W; (m) the FGF21 polypeptide comprises the mutations R203W, Y207W and A208W.

6. The construct of any one of claims 1-5, wherein the FGF21 is a human FGF21 having a protein sequence set forth in SEQ ID NO:

1.

7. The construct of any one of claims 1-6, further comprising a stability enhancing domain.

8. The construct of claim 7, wherein the stability enhancing domain is attached to an N- terminus of the FGF21 polypeptide, or wherein the stability enhancing domain is attached to a C-terminus of the FGF21 polypeptide.

9. The construct of any one of claims 7-8, wherein the stability enhancing domain is at least one selected from the group consisting of albumin, an Fc region of an antibody, a glutathione S-transferase, a small ubiquitin-like modifier (SUMO), an O-glycosylation region of a Drosophila Spitz protein, and a thioredoxin. 93 52997671.2Attorney Docket No.047162-7483WO1 10. The construct of any one of claims 7-9, wherein the FGF21 polypeptide and the stability enhancing domain are linked through a linker polypeptide comprising about 1-18 amino acids.

11. The construct of any one of claims 1-10, wherein at least one of the following applies: (a) the dissociation constant (Kd) of the FGF21 polypeptide with β-Klotho is about 20% or less of the Kd of a corresponding wild-type FGF21 polypeptide; (b) the Koffof the FGF21 polypeptide with β-Klotho is about 20% or less of the Koff of a corresponding wild-type FGF21 polypeptide.

12. The construct of any one of claims 1-11, wherein at least one of the following applies: (a) the construct binds to β-Klotho and sequester β-Klotho from pathways downstream of a fibroblast growth factor receptor (FGFR) and β-Klotho; (b) the construct binds to both an FGFR and β-Klotho and activate pathways downstream of the FGFR and β-Klotho.

13. The construct of claim 12, wherein the FGFR is FGFR1c.

14. A composition comprising a construct of any one of claims 1-13 and a pharmaceutically acceptable carrier.

15. A method of modulating FGF21 / FGFR / β-Klotho signaling pathway or FGF19 / FGFR / β-Klotho signaling pathway in a cell, the method comprising contacting the surface of the cell with the construct of any one of claims 1-13.

16. The method of claim 15, wherein (a) the construct binds to β-Klotho but not FGFR, thereby sequestering β-Klotho from the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β- Klotho signaling pathway and down-regulating the signaling pathway; or (b) the construct binds to both β-Klotho and FGFR, thereby upregulating the FGF21 / FGFR / β-Klotho signaling pathway.

17. The method of any one of claims 15-16, wherein the cell is from a cell line, a primary cell, or a cell in the body of a subject. 94 52997671.2Attorney Docket No.047162-7483WO1 18. The method of any one of claims 15-17, wherein the cell is a cell in or derived from adipose tissue, liver, pancreas, or hypothalamus.

19. A method of treating, ameliorating and / or preventing a disease or disorder in a subject in need thereof, the method comprising: administering to the subject an effective amount of the construct of any one of claims 1-13 or the composition of claim 14, wherein the disease or disorder is at least one selected from obesity, diabetes, pancreatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), acute alcohol toxicity, and cancers driven by an aberrant activation of FGF19 / FGFR4 / β- Klotho pathway.

20. The method of claim 19, wherein the construct or the composition is administered via at least one route selected from the group consisting of inhalational, oral, rectal, vaginal, parenteral, intracranial, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, and intravenous.

21. The method of any one of claims 19-20, wherein the subject is a human. 95 52997671.2

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