Constructs for modulating the FGF21 / FGFR / β-Klotho signaling pathway and for treating diseases and disorders, and methods thereof.

Abstract

Constructs comprising an FGF21 polypeptide containing residues 186-198 and 200-209 of FGF21 are described herein. The FGF21 polypeptide comprises one or more mutations that confer a stronger affinity to β-Klotho to the FGF21 polypeptide compared to the corresponding wild-type FGF21 polypeptide. Also described herein are compositions comprising the constructs, as well as methods for modulating the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway, and methods for treating, relieving, and / or preventing diseases or disorders associated with misregulation (such as upregulation or downregulation) of said signaling pathway. TIFF2026534020000031.tif64170

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 581,860, filed on 11 September 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence List The XML file named "047162-7483WO1_Seq Listing.xml", created on August 31, 2024, consisting of 81,737 bytes, is thus incorporated entirely by reference. [Background technology]

[0003] background FGF19 and FGF21 (and FGF23) stand out from classical FGF by exhibiting circulating hormone characteristics and are therefore called endocrine FGFs. Both FGF19 and FGF21 function as hormones that specifically bind to receptors located in the liver, adipose tissue, and hypothalamus, regulating metabolic functions (such as bile acid synthesis and lipid production) and also stimulating insulin sensitivity, energy expenditure, and weight loss. Unlike classical FGF, which requires heparan sulfate proteoglycans (HSPGs) to activate FGFR, endocrine FGFs do not have this requirement, but instead specifically depend on Clotot coreceptors for FGFR activation.

[0004] There are two Klotho proteins, encoded by different genes. Alpha-Klotho is required for FGF23-dependent signaling, while beta-Klotho is essential for FGF19-dependent or FGF21-dependent signaling in certain tissues and organs. Although various FGFRs are expressed throughout the body, the expression of Klotho proteins is limited to specific tissues; alpha-Klotho expression is limited to the kidneys and parathyroid glands, while beta-Klotho expression is limited to adipose tissue, the liver, pancreas, and hypothalamus.

[0005] FGF19 or FGF23 binds to FGFR (such as FGFR1c) via the FGF core region, and binds to β-Klotho via the C-terminal tail to form an FGF / FGFR / β-Klotho ternary complex, and results in stimulation of tyrosine kinase activity, which activates downstream signaling events (see, for example, WO 2019 / 010314 A1 (Patent Document 1), which is incorporated herein by reference in its entirety).

[0006] Dysregulation of the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway is associated with various diseases or disorders, including obesity, diabetes, pancreatitis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and acute alcohol toxicity. In addition, certain cancers, such as subtypes of liver cancer, are driven by aberrant activation of the FGF19 / FGFR4 / β-Klotho pathway.

[0007] There is a need for novel compounds and / or constructs that modulate these signaling pathways, as well as compositions, constructs, and / or methods that are capable of treating, ameliorating, and / or preventing diseases or disorders associated with these signaling pathways. The present invention addresses this need.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Summary of the Invention

[0009] Overview Non-Natural Constructs In several aspects, the present invention relates to non-natural constructs, such as non-natural soluble constructs.

[0010] In some embodiments, the construct comprises an FGF21 polypeptide comprising residues 186 to 198 and residues 200 to 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.

[0011] 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.

[0012] In some embodiments, the FGF21 polypeptide comprises residues 186 to 209 of FGF21. In some embodiments, the FGF21 polypeptide lacks all or some of residues 1 to 185 of FGF21. In some embodiments, the FGF21 polypeptide lacks all or some of residues 29 to 185 of FGF21. In some embodiments, the FGF21 polypeptide lacks all or some of residues 169 to 185 of FGF21.

[0013] In some embodiments, the FGF21 polypeptide comprises residues 169 to 209 of FGF21. In some embodiments, the FGF21 polypeptide lacks all or some of residues 1 to 168 of FGF21. In some embodiments, the FGF21 polypeptide lacks all or some of residues 29 to 168 of FGF21.

[0014] In some embodiments, the FGF21 polypeptide comprises residues 29–209 of FGF21. 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 residues 200–209 of FGF21.

[0015] In some embodiments, the FGF21 polypeptide contains mutants L194F, V197I, Q201L, R203W, Y207W, A208E, A208W, and S209G, as well as the sequence RKK conjugated to the C-terminus of residues 200-209 of FGF21.

[0016] In some embodiments, the FGF21 polypeptide includes mutations L194F, V197I, Q201R, R203W, Y207W, A208W, and S209G.

[0017] In some embodiments, the FGF21 polypeptide comprises mutations L194F, V197I, Q201R, R203W, Y207W, and A208W.

[0018] In some embodiments, the FGF21 polypeptide includes mutations L194F, V197L, Q201R, R203W, Y207W, A208W, and S209G.

[0019] In some embodiments, the FGF21 polypeptide includes mutations L194F, V197L, Q201R, R203W, Y207W, and A208W.

[0020] In some embodiments, the FGF21 polypeptide comprises mutations L194F, Q201I, R203W, Y207W, and A208W.

[0021] In some embodiments, the FGF21 polypeptide includes mutants L194F, Q201L, R203W, Y207W, A208E, A208W, and S209G, as well as sequences RKS conjugated to the C-terminus of residues 200-209 of FGF21.

[0022] In some embodiments, the FGF21 polypeptide includes mutants L194F, Q201P, R203W, Y207W, A208W, and S209G, as well as the sequence RRS conjugated to the C-terminus of residues 200-209 of FGF21.

[0023] In some embodiments, the FGF21 polypeptide includes mutants L194F, Q201R, R203W, Y207W, A208W, and S209G, as well as the sequence RRS conjugated to the C-terminus of residues 200-209 of FGF21.

[0024] In some embodiments, the FGF21 polypeptide comprises the mutations L194F, Q201R, R203W, Y207W, and A208W.

[0025] In some embodiments, the FGF21 polypeptide includes mutations L194F, Q201W, R203W, Y207W, A208W, and S209G.

[0026] In some embodiments, the FGF21 polypeptide comprises mutations L194F, R203W, Y207W, and A208W.

[0027] In some embodiments, the FGF21 polypeptide comprises the mutations R203W, Y207W, and A208W.

[0028] In some embodiments, FGF21 is human FGF21 having the protein sequence shown in SEQ ID NO:1.

[0029] In certain embodiments, the FGF21 polypeptide comprises residues 29-209 of SEQ ID NO:1 having one or more mutations as described herein, wherein the FGF21 polypeptide lacks residues 1-28 of SEQ ID NO:1. In another embodiment, the FGF21 polypeptide comprises residues 29-209 having 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 residues 200-209 of FGF21), wherein the FGF21 polypeptide lacks residues 1-28 of SEQ ID NO:1.

[0030] In some embodiments, the construct further includes stability-enhancing domains.

[0031] In some embodiments, the stability-enhancing domain is attached to the N-terminus of the FGF21 polypeptide, or the stability-enhancing domain is attached to the C-terminus of the FGF21 polypeptide.

[0032] In some embodiments, the stability-enhancing domain is at least one selected from the group consisting of albumin, the Fc region of an antibody, glutathione S-transferase, small ubiquitin-like modifiers (SUMOs), the O-glycosylation region of Drosophila Spitz protein, and thioredoxin.

[0033] In some embodiments, the FGF21 polypeptide and the stability-enhancing domain are linked via a linker polypeptide containing approximately 1 to 18 amino acids.

[0034] In some embodiments, the dissociation constant (Kd) of the FGF21 polypeptide relative to β-Klotho is the same as the K of the corresponding wild-type FGF21 polypeptide. d It is less than approximately 20% of that.

[0035] In some embodiments, the K of FGF21 polypeptide against β-Klotho off This is the K of the corresponding wild-type FGF21 polypeptide. off It is less than approximately 20% of that.

[0036] In some embodiments, the construct binds to β-Klotho and segregates β-Klotho from the fibroblast growth factor receptor (FGFR) and downstream pathways of β-Klotho.

[0037] In some embodiments, the construct binds to both FGFR and β-Klotho and activates downstream pathways of both FGFR and β-Klotho.

[0038] In some embodiments, FGFR is FGFR1c.

[0039] composition In some aspects, the present invention relates to compositions.

[0040] In some embodiments, the composition comprises the constructs described herein and a pharmaceutically acceptable carrier.

[0041] Methods for modulating the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway In some respects, the present invention relates to methods for modulating the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway in cells.

[0042] In some embodiments, the method includes the step of bringing the surface of a cell into contact with a construct as described herein.

[0043] In some embodiments, the construct binds to β-Klotho but not to FGFR, thereby sequestering β-Klotho from the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway and downregulating the signaling pathway.

[0044] In some embodiments, the construct binds to both β-Klotho and FGFR, thereby upregulating the FGF21 / FGFR / β-Klotho signaling pathway.

[0045] In some embodiments, the cells are derived from cell lines, primary cells, or cells from within the body of the subject.

[0046] In some embodiments, the cells are cells located in or originating from adipose tissue, the liver, the pancreas, or the hypothalamus.

[0047] Methods for treating, relieving, and / or preventing a disease or disorder. In some aspects, the present invention relates to methods for treating, relieving, and / or preventing diseases or disorders in subjects where such treatment is necessary.

[0048] In some embodiments, the method includes the step of administering an effective amount of a component or composition as specified herein to a subject.

[0049] In some embodiments, the disease or disorder is at least one selected from obesity, diabetes, pancreatitis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), acute alcohol toxicity, and cancer driven by abnormal activation of the FGF19 / FGFR4 / β-Klotho pathway.

[0050] In some embodiments, the construct or composition is administered via at least one route selected from the group consisting of inhalation, oral, rectal, vaginal, parenteral, intracranial, topical, percutaneous, pulmonary, intranasal, buccal, ocular, subarachnoid, and intravenous.

[0051] In some embodiments, the subject is human. [Brief explanation of the drawing]

[0052] The following detailed description of exemplary embodiments will be better understood in conjunction with the accompanying drawings. Non-limiting embodiments are shown in the drawings for illustrative purposes. However, it should be understood that this specification is not limited to the exact arrangement and means of the embodiments shown in the drawings.

[0053] [Figure 1A] Figures 1A-1C are schematic diagrams of FGF21 C-terminal tail variants (CVs) according to several embodiments. The locations of amino acid changes from wild-type FGF21 are indicated by arrows along with the corresponding mutations. [Figure 1B] Please refer to the explanation in Figure 1A. [Figure 1C] Please refer to the explanation in Figure 1A. [Figure 2A] Figures 2A-2B are schematic diagrams of FGF21 protein variants with N-terminal fusion partner proteins according to several embodiments. The fusion partner proteins include histidine tags, SUMO, mouse serum albumin (mSA), Spitz, and Fc regions derived from human IgG1. The size of the regions in all schematic diagrams is proportional to the length of the amino acids, with the exception of the Fc and mSA regions, which have molecular weights of 32 kDa and 65.9 kDa, respectively. [Figure 2B] Please refer to the explanation in Figure 2A. [Figure 3A] Figures 3A–3N show some exemplary sensorgram results from binding kinetic measurements between Fc-FGF21 variants and sKLB using biolayer interferometry (BLI). Fc-FGF21 variants were immobilized on biosensors coated with anti-Fc antibody (AHC) and immersed in wells containing a range of sKLB concentrations. The sensorgrams with reference subtraction (blue line) were fitted to a 1:1 Langmuir binding model (red line) to obtain the reaction rate constants kon and koff, and the dissociation constant KD, which are listed in Figure 3N. [Figure 3B] Please refer to the explanation in Figure 3A. [Figure 3C] Please refer to the explanation in Figure 3A. [Figure 3D]Please refer to the explanation in Figure 3A. [Figure 3E] Please refer to the explanation in Figure 3A. [Figure 3F] Please refer to the explanation in Figure 3A. [Figure 3G] Please refer to the explanation in Figure 3A. [Figure 3H] Please refer to the explanation in Figure 3A. [Figure 3I] Please refer to the explanation in Figure 3A. [Figure 3J] Please refer to the explanation in Figure 3A. [Figure 3K] Please refer to the explanation in Figure 3A. [Figure 3L] Please refer to the explanation in Figure 3A. [Figure 3M] Please refer to the explanation in Figure 3A. [Figure 3N] Please refer to the explanation in Figure 3A. [Figure 4] Figure 4A shows some exemplary BLI sensorgram results from binding kinetic measurements between His-FGF21 wt or His-FGF21 CV16 and sKLB. His-FGF21 variants were immobilized on Amine Reactive 2nd Generation (AR2G) biosensors using a standard amine coupling method utilizing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). The sensorgrams with reference subtraction (blue line) were fitted to a 1:1 Langmuir binding model (red line) to obtain the reaction rate constants kon and koff, and the dissociation constant KD, which are listed in Figure 4B. [Figure 5A]Figure 5A shows the results of several exemplary immunoblotting analyses demonstrating the cellular activity of His-FGF21 wt and His-FGF21 CV16 in L6 cells co-expressing β-Klotho and FGFR1c, according to several embodiments. Cellular activity was revealed by the level of phosphorylated MAPK by immunoblotting with anti-MAPK and anti-pMAPK antibodies. Figure 5B shows the results of glucose uptake assays for His-FGF21 wt (red dots) and His-FGF21 CV16 (blue squares), according to several embodiments. 3T3-L1 fibroblasts were chemically differentiated into adipocytes and treated with either His-FGF21 wt or His-FGF21 CV16. The amount of cellular glucose in each sample was measured as the luminescence level. Data represent the normalized luminescence mean ± SD of four independent experiments. The EC50 values ​​of His-FGF21 wt and His-FGF21 CV16 are 1.84 ± 0.346 nM and 0.29 ± 0.071 nM (value ± SEM), respectively. [Figure 5B] Please refer to the explanation in Figure 5A. [Figure 6A] Figures 6A-6E: Calcium release and MAPK activation in pancreatic acinar (AR42J) cells stimulated with His-FGF21 wt and His-FGF21 CV16, according to several embodiments. Figures 6A-6D: Time course of Ca2+ release in AR42J cells (expressing endogenous FGFR1c and β-Klotho). For Ca2+ release studies, cells were loaded with a fluorescent Ca2+ indicator (Calbryte 520 AM) and then stimulated with His-FGF21 wt or His-FGF21 CV16 at the indicated concentrations. The total amount of Ca2+ release was analyzed as a change in fluorescence. Figure 6E: Analysis of MAPK response. AR42J cells were left unstimulated or stimulated with His-FGF21 wt or His-FGF21 CV16 at the indicated concentrations (10 min, 37°C), and cell activity was monitored by the level of phosphorylated MAPK using an anti-pMAPK antibody. [Figure 6B] Please refer to the explanation in Figure 6A. [Figure 6C]Please refer to the explanation in Figure 6A. [Figure 6D] Please refer to the explanation in Figure 6A. [Figure 6E] Please refer to the explanation in Figure 6A. [Figure 7A] Figure 7A shows several exemplary BLI sensorgram results from binding kinetic measurements between SUMO-FGF21 variants and sKLB, according to several embodiments. SUMO-FGF21 variants were immobilized on Amine Reactive 2nd Generation (AR2G) biosensors using a standard amine coupling method utilizing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Reference-subtracted sensorgrams (blue lines) were fitted to a 1:1 Langmuir binding model (red lines) to obtain the reaction rate constants kon and koff, and the dissociation constant KD, which are listed in Figure 7B. Figure 7C shows several exemplary Western blots demonstrating the cellular activity of SUMO-FGF21 wt and SUMO-FGF21 CV16 in L6 cells co-expressing β-Klotho and FGFR1c, according to several embodiments. Cell activity was monitored by immunoblotting using anti-MAPK antibodies and anti-pMAPK antibodies, specifically by measuring the level of phosphorylated MAPK. [Figure 7B] Please refer to the explanation in Figure 7A. [Figure 7C] Please refer to the explanation in Figure 7A. [Figure 8A]Figure 8A shows some exemplary BLI sensorgram results from binding kinetic measurements between the mSA-FGF21 variant and sKLB. The mSA-FGF21 variant was immobilized on an Amine Reactive 2nd Generation (AR2G) biosensor using a standard amine coupling method utilizing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). The sensorgram with reference subtraction (blue line) was fitted to a 1:1 Langmuir binding model (red line) to obtain the reaction rate constants kon and koff, and the dissociation constant KD, which are listed in Figure 8B. Figure 8C shows some exemplary Western blot results demonstrating the cellular activity of mSA-FGF21 wt and mSA-FGF21 CV16 in L6 cells co-expressing β-Klotho and FGFR1c, according to several embodiments. Cell activity was monitored by immunoblotting using anti-MAPK antibodies and anti-pMAPK antibodies, specifically by measuring the level of phosphorylated MAPK. [Figure 8B] Please refer to the explanation in Figure 8A. [Figure 8C] Please refer to the explanation in Figure 8A. [Figure 9A]Figure 9A shows some exemplary BLI sensorgram results from binding kinetic measurements between Spitz-FGF21 variants and sKLB, according to several embodiments. Spitz-FGF21 variants were immobilized on Amine Reactive 2nd Generation (AR2G) biosensors using a standard amine coupling method utilizing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Reference-subtracted sensorgrams (blue line) were fitted with a 1:1 Langmuir binding model (red line) to obtain the reaction rate constants kon and koff, and the dissociation constant KD, which are listed in Figure 8B. Figure 9C shows some exemplary Western blot results demonstrating the cellular activity of mSA-FGF21 wt and mSA-FGF21 CV16 in L6 cells co-expressing β-Klotho and FGFR1c, according to several embodiments. Cell activity was monitored by immunoblotting using anti-MAPK antibodies and anti-pMAPK antibodies, specifically by measuring the level of phosphorylated MAPK. [Figure 9B] Please refer to the explanation in Figure 9A. [Figure 9C] Please refer to the explanation in Figure 9A. [Figure 10A]Figures 10A–10E show the results of Gaussian-accelerated molecular dynamics simulations of FGF21 wt SIR1 peptides (Figures 10A–10B) and FGF21 wt SIR2 peptides (Figures 10C–10E) according to several embodiments. In the folding free energy landscape (Figure 10A) and conformational population density (Figure 10B) of FGF21 wt SIR1 peptides, the X and Y axes represent the backbone RMSD, which is the distance from the carboxylate side chain to the backbone amide of FGF21 wt D192-V197 and D187-S190, respectively. The dotted line indicates the cutoff between the folded and unfolded states. In the folding free energy landscape (Figure 10C) and conformational population density (Figure 10D) of the FGF21 wt SIR2 peptide, the backbone RMSD of the S204-A208 and S204-Y207 backbone amide distances is used as the metric. The FGF21 wt SIR2 peptide has three populations: folded, intermediate, and unfolded. The activation free energy (ΔG‡) between the folded and unfolded states, estimated from the global minimum of the folding free energy landscape, is approximately 2.5 kcal / mol. The conformational population density plot shows that the folded state of the SIR1 peptide was hardly sampled, while SIR2 has a slight tendency to sample the folded state. Figure 10E demonstrates the conformational population density of the FGF21 CV16 SIR2 variant observed in Gaussian-accelerated molecular dynamics simulations. The conformational state is measured by the backbone RMSD of the backbone amide distances of S204-W208 and S204-W207.FGF21 CV16 SIR2 exhibits a significantly increased population of folded states (39%), suggesting that FGF21 CV16 SIR2 has a greater tendency than FGF21 wt SIR2 to form folded conformations similar to the observed crystallographic conformation (PDB: 5VAQ). [Figure 10B] Please refer to the explanation in Figure 10A. [Figure 10C] Please refer to the explanation in Figure 10A. [Figure 10D] Please refer to the explanation in Figure 10A. [Figure 10E] Please refer to the explanation in Figure 10A. [Figure 11-1] Figure 11A: Crystal structures of sKLB bound to CT16 according to several embodiments. sKLB is shown as a green surface representation, and CT16 is shown as an orange ball-and-stick representation. For clarity, nanobodies used as crystallization chaperones are not shown. Figures 11B-11E: Comparison of the overall conformations of CT16 and CTWT when bound to sKLB according to several embodiments. Site 1 interaction regions (Figures 11B-11C) for the structures of sKLB:CT16 (Figure 11B) and sKLB:CTWT (Figure 11C). Site 2 interaction regions (Figures 11D-11E) for the structures of sKLB:CT16 (Figure 11D) and sKLB:CTWT (Figure 11E). The sKLB molecule is shown in a cartoon representation, with the sKLB:CT16 structure in green and the sKLB:CTWT structure in gray. Amino acid residues in CT16 that differ from those in CTWT are highlighted with red dashed circles. The color scheme used in this diagram will be applied to all structural diagrams unless otherwise specified. [Figure 11-2] Please refer to the explanation in Figure 11-1. [Figure 12]Figure 12A: Structural comparison between sKLB bound to CT16 and sKLB bound to CTWT (PDB ID: 5VAQ) according to several embodiments. sKLB is shown as a Cα trace representation, and the backbone atoms of CT16 and CTWT are shown as rods. The Cα atoms of sKLB are superimposed with RMSDs of 0.29 Å for KL1, 0.46 Å for KL2, and 2.12 Å for both domains. Figures 12B-12C: Side chain atoms of residues at site 1 (Figure 12B) and site 2 (Figure 12C) in contact with CT16 are highlighted as rods. The sKLB molecule is shown as a cartoon representation, while CT16 and CTWT are shown as Cα trace representations. [Figure 13] Figures 13A-13B demonstrate that interactions at site 2 are responsible for the superior binding affinity and higher cellular activity of FGF21 CV16 to β-Klotho compared to FGF21 wt. Figures 13A-13B: Intermolecular contacts contributing to the overall binding affinity between CT16 (orange) and sKLB (green) (Figure 13A), and between CTWT (purple) and sKLB (gray) (Figure 13B) are labeled. [Figure 14] Figure 14 is a schematic diagram illustrating proposed mechanisms of high-affinity binding of FGF21 CV16 to β-Klotho, according to several embodiments. Figure 14A: FGF21 CV16 is in equilibrium in linear and folded conformations. The helical segment at SIR2 may be formed by a π-π interaction between W203 and W208. Figure 14B: The helical conformation at SIR2 facilitates the interaction of β-Klotho with site 2. Figure 14C: The binding process is completed through multiple intramolecular and intermolecular interactions at SIR1 and site 1 of CV16, making the dissociation process less favorable. [Figure 15A]Figures 15A–15D show the results of calcium release activation in Chinese hamster ovary cell lines (CHO) stimulated with FGF21 WT or its FGF21 C-terminal tail variant 16 (FGF21 CV16) according to several embodiments. Figures 15A–15D: For the time course of Ca2+ release in CHO cells (expressing endogenous FGFR1c and β-Klotho), cells were loaded with a fluorescent Ca2+ indicator (Calbryte 520 AM) and then stimulated with increasing concentrations of WT FGF21 or its CV16 variant, as shown, (Figures 15A–15B) with an N-terminal His tag and (Figures 15C–15D) with a human Fc fusion, and Ca2+ release was analyzed as changes in fluorescence. [Figure 15B] Please refer to the explanation in Figure 15A. [Figure 15C] Please refer to the explanation in Figure 15A. [Figure 15D] Please refer to the explanation in Figure 15A. [Figure 16A] Figures 16A–16C demonstrate that several FGF21 constructs according to some embodiments herein are more potent than wild-type FGF21 in activating FGF21 / FGFR / β-Klotho signaling. Specifically, the cellular activity of various FGF21 proteins was measured in L6 cells co-expressing β-Klotho and FGFR1 by detection of phosphorylated MAPK. (Figure 16A) Immunoblotting of His-FGF21 wt (top) and His-FGF21 CV30 (bottom), (Figure 16B) Fc-FGF21 wt (top) and Fc-FGF21 CV16 (bottom), and (Figure 16C) Fc-FGF21 wt (top) and Fc-FGF21 CV30 (bottom) with β-tubulin antibody (loading control) and phosphorylated MAPK antibody. [Figure 16B] Please refer to the explanation in Figure 16A. [Figure 16C] Please refer to the explanation in Figure 16A. [Figure 17A]Figures 17A–17C demonstrate that several FGF21 constructs according to certain embodiments herein are more potent than wild-type FGF21 in activating FGF21 / FGFR / β-Klotho signaling. Specifically, the cellular activity of various FGF21 proteins was measured in L6 cells co-expressing β-Klotho and FGFR1 by detecting phosphorylated MAPKs. (Figure 17A) Graphs from AlphaLISA signals of phosphorylated MAPKs normalized to total MAPK for Fc-FGF21 wt (black circles) and Fc-FGF21 CV25 (white circles), (Figure 17B) FGF21 wt (black circles) and FGF21 CV16 (white circles), and (Figure 18C) FGF21 wt (black circles) and FGF21 CV25 (white circles). [Figure 17B] Please refer to the explanation in Figure 17A. [Figure 17C] Please refer to the explanation in Figure 17A. [Modes for carrying out the invention]

[0054] Detailed explanation The following disclosure provides many different aspects or embodiments to perform various features of the subject matter provided. Specific examples of components and arrangements are described below for the sake of simplicity of this disclosure. These are, of course, merely embodiments and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the following description may include embodiments in which the first and second features are formed in direct contact, as well as embodiments in which an additional feature may be formed between the first and second features such that the first and second features cannot be in direct contact. In addition, the disclosure may repeat reference numbers and / or letters in various embodiments. This repetition is for simplicity and clarity and does not in itself prescribe relationships between the various aspects and / or configurations discussed.

[0055] FGF21 is a crucial regulator of metabolic homeostasis. While most circulating FGF21 is normally produced in the liver, it is also produced in adipose tissue and the pancreas. Furthermore, various nutritional stresses (e.g., alcohol, sugar), as well as fasting and refeeding, induce FGF21 expression in the liver. When pharmacologically administered to diabetic or obese mice, FGF21 induces weight loss, improves insulin sensitivity, and lowers blood glucose and triglyceride levels; similar metabolic effects have also been observed in non-human primates treated with FGF21. Among the indications, there is significant interest in the use of FGF21 analogs as therapeutic agents for treating metabolic diseases such as type 2 diabetes and non-alcoholic fatty liver disease (NASH), pancreatitis, and patients with acute alcohol toxicity.

[0056] FGF21 belongs to the family of endocrine FGFs, which also include FGF19 and FGF23. These act as circulating hormones, specifically binding to Klotho family surface receptors to result in the formation of a ternary complex with the c isoform of FGFR and its selective activation. FGF19 and FGF21 have been shown to interact with and specifically bind to β-Klotho, while FGF23 has been shown to specifically bind to α-Klotho. Endocrine FGFs have been shown to exert their physiological function only in tissues or cells that express Klotho proteins. Importantly, endocrine FGFs contain a unique C-terminal region that specifically and strongly binds to their respective Klotho receptors, providing a "zip code" or targeting signal for FGFR activation by endocrine FGFs. Furthermore, elucidation of the crystal structures of FGF19, FGF21, and FGF23 in complexes with the extracellular domains of α-Kroto and β-Kroto proteins revealed detailed atomic interactions that determine the specificity of endocrine FGFs to Kroto proteins, demonstrating how FGFRs are activated in a Kroto-dependent manner.

[0057] Structural information, which provides detailed molecular interactions between FGF21 and β-klotho, is key to enhancing these interactions and developing FGF21 variants with improved potency. This study presents novel FGF21 variants with extremely high affinity for β-klotho, identified using structural information. These FGF21 variants exhibit greatly improved potency in various cell-based assays. Furthermore, to improve the biochemical and pharmacological properties of the novel variants, we developed various fusion proteins containing potent FGF21 variants.

[0058] In the studies described herein ("the Study"), certain polypeptide constructs were designed by introducing mutations into wild-type fibroblast growth factor 21 (FGF21) fragments. The polypeptide constructs in the Study have a dissociation constant K d When determined by this method, it was confirmed that the polypeptide construct has a stronger (or at least comparable) binding affinity to β-Klotho compared to the corresponding wild-type FGF21 polypeptide. The polypeptide construct in this study also showed increased K off When determined by this method, it was confirmed that the polypeptide possesses stronger or enhanced binding activity to β-Klotho binding compared to the corresponding wild-type FGF21 polypeptide.

[0059] Depending on the composition of the FGF21-derived polypeptide (e.g., the presence or absence of an FGFR-binding core region), the constructs herein may either downregulate FGF / FGFR / β-Klotho signaling by sequestering β-Klotho from endogenous FGF21 and FGF19, or upregulate FGF21 / FGFR / β-Klotho signaling by acting as an FGF21 analog with stronger receptor / co-receptor binding. Accordingly, the constructs herein can be used to treat, alleviate, and / or prevent various diseases or disorders caused by and / or associated with mismodulation of the FGF / FGFR / β-Klotho signaling pathway.

[0060] Therefore, in some respects, the present invention relates to non-natural constructs comprising FGF21 polypeptide.

[0061] The FGF / FGFR / β-Klotho signaling pathway is involved in a variety of diseases and disorders. For example, FGF21 expression in the liver (where β-Klotho is also expressed) is upregulated during various nutritional 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 induces weight loss, improves insulin sensitivity, and lowers blood glucose and triglyceride levels. Therefore, the constructs described herein are expected to be useful as pharmaceutical compositions and for treating a variety of diseases and disorders caused and / or associated with mismodification of the FGF / FGFR / β-Klotho signaling pathway.

[0062] In some aspects, the present invention relates to compositions, such as pharmaceutical compositions, that include the non-natural constructs of the present disclosure.

[0063] In several aspects, the present invention relates to a method for modulating the FGF / FGFR / β-Klotho signaling pathway.

[0064] In some aspects, the present invention relates to methods for treating, relieving, and / or preventing diseases or disorders caused by and / or associated with mismodification of the FGF / FGFR / β-Klotho signaling pathway.

[0065] definition As used herein, each of the following terms has the meaning it has in relation to this section. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art in which this disclosure belongs. Generally, the nomenclature used herein, as well as experimental techniques in animal pharmacology, pharmaceuticals, peptide chemistry, and organic chemistry, are well known and commonly used in the art. It should be understood that the order of steps or the order in which certain actions are performed is not important as long as this teaching remains implementable. Any use of section headings is intended to aid in the reading of the document and should not be construed as limitation; information relating to a section heading may be present in or outside of that particular section. All publications, patents, and patent documents referenced herein are incorporated herein by reference in whole, as they would be incorporated by reference individually.

[0066] In this application, when an element or component is said to be included in and / or selected from the enumerated list of elements or components, it should be understood that the element or component may be any one of the enumerated elements or components, and may be selected from a group of two or more of the enumerated elements or components.

[0067] In the methods described herein, the acts may be performed in any order unless a temporal or operational sequence is explicitly enumerated. Furthermore, the designated acts may be performed simultaneously unless the language of the explicit claims enumerates that they are performed separately. For example, the act of performing claimed X and the act of performing claimed Y may be performed simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0068] In this document, the terms “a,” “an,” or “the” are used to include one or more unless the context clearly indicates otherwise. The term “or” is used to refer to a non-exclusive “or” unless otherwise indicated. The phrases “at least one of A and B” or “at least one of A or B” are synonymous with “A, B, or A and B.”

[0069] When referring to measurable values ​​such as quantity or duration of time, the term "about" as used herein includes variations of ±20% or ±10%, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.1% from the specified value, provided that such variations are appropriate for carrying out the disclosed method.

[0070] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or they can be the immunoreactive portion of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in this invention may exist in various 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).

[0071] The term "antibody fragment" refers to a portion of an intact antibody, and to the antigen-determining variable region 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) consisting of either a VL domain or a VH domain that exhibits sufficient affinity for the target, multispecific antibodies formed from antibody fragments such as camelid antibodies (Riechmann, 1999, J. Immunol. Meth. 231:25-38), camelid VHH domains, and bivalent fragments containing two Fab fragments linked by disulfide bonds in the hinge region, as well as isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger & Hudson, 2005, Nature Biotech. 23:1126-1136). Antigen-binding fragments can also be implanted into polypeptide-based scaffolds such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199 describing fibronectin polypeptide minibodies). Antibody fragments may also include human antibodies or humanized antibodies, or portions of human antibodies or humanized antibodies.

[0072] The term "applicator" means any device, including but not limited to subcutaneous syringes, pipettes, etc., used to administer the compounds and compositions of this disclosure, as the term is used herein.

[0073] As used herein, “disease” means a state of animal health in which the animal is unable to maintain homeostasis and the disease does not go into remission, resulting in a continued deterioration of the animal’s health.

[0074] As used herein, “disorder” in animals is a health condition in which the animal can maintain homeostasis, but the animal’s health is worse than it would be without the disorder. If left untreated, the disorder does not necessarily lead to a further decline in the animal’s health.

[0075] As used herein, the terms “effective dose,” “therapeutic effective dose,” or “pharmaceutical effective dose” of a compound are interchangeable, as they refer to an amount of the compound sufficient to provide a beneficial effect to the subject to which it is administered.

[0076] As used herein, “endogenous” means any material that originates from or is produced within an organism, cell, tissue, or system. As used herein, “exogenous” means any material that is introduced from outside an organism, cell, tissue, or system, or produced outside of it.

[0077] The term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter, as used herein.

[0078] As used herein, the term “FGF21 polypeptide” encompasses naturally occurring FGF21 (e.g., human FGF21), naturally occurring fragments of FGF21 (e.g., human FGF21 fragments), and any variant thereof.

[0079] "Homologous" as used herein refers to subunit sequence identity between two polymer molecules, between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a certain subunit position in both molecules is occupied by the same monomer subunit; for example, if a certain 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 positions that match or are homologous; for example, if half of the positions of two sequences (e.g., five positions in a 10-subunit-length polymer) are homologous, then those two sequences are 50% homologous; if 90% of the positions (e.g., nine out of ten) match or are homologous, then those two sequences are 90% homologous. For example, the DNA sequences 5'-ATTGCC-3' and 5'-TATGGC-3' share 50% homology.

[0080] As used herein, the terms “immunoglobulin” or “Ig” are defined as a class of proteins that function as antibodies. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody found in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus from the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the primary immunoglobulin produced in the primary immune response in most mammals. It is the most efficient immunoglobulin in agglutination, complement binding, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin that does not have known antibody function but can serve as an antigen receptor. IgE is an immunoglobulin that mediates immediate-type hypersensitivity by triggering the release of mediators from mast cells and basophils upon allergen exposure.

[0081] The terms “inhibit” and “antagonize,” as used herein, mean reducing by a measurable amount or completely preventing the expression, stability, function, or activity of a molecule, reaction, interaction, gene, mRNA, and / or protein. An inhibitor is a compound, e.g., an antagonist, that partially or completely blocks, reduces, prevents, delays, inactivates, desensitizes, or downregulates the stimulation of the stability, expression, function, and activity of a protein, gene, and mRNA.

[0082] "Informational materials" include publications, records, figures, or other expressive media that can be used to convey the usefulness of the compositions and / or compounds of the disclosure contained in the kit, as the term is used herein. The informational materials of the kit may, for example, be attached to the container containing the compounds and / or compositions of the disclosure, or shipped together with the container containing the compounds and / or compositions. Alternatively, the informational materials may be shipped separately from the container, with the intention that the recipient will use the informational materials and compounds collaboratively. Delivery of the informational materials may be, for example, by physical delivery of publications or other expressive media conveying the usefulness of the kit, or by computer, such as by electronic transmission, e.g., by email or download from a website.

[0083] "Isolated" means that something has been altered or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting material in its natural state is "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-native environment, such as a host cell.

[0084] "Isolated nucleic acid" refers to a segment or fragment of nucleic acid separated from adjacent sequences in its naturally occurring state, i.e., a DNA fragment removed from sequences normally adjacent to that fragment in the naturally occurring genome. The term also applies to nucleic acids substantially purified from other components naturally associated with nucleic acids, i.e., RNA or DNA or proteins naturally associated within cells. Therefore, the term includes, for example, recombinant DNA incorporated into vectors, autonomously replicating plasmids or viruses, or the genomic DNA of prokaryotes or eukaryotes, or recombinant DNA existing as another molecule unrelated to other sequences (i.e., as cDNA, or as a genome or cDNA fragment produced by PCR or restriction enzyme digestion). This also includes recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0085] As used herein, the term “modulate” refers to a change in a biological state, i.e., an increase, decrease, etc. For example, the term “modulate” is interpreted as the ability to positively or negatively regulate the expression, stability, or activity of a target protein, including but not limited to the transcription of the target protein mRNA, the stability of the target protein mRNA, the translation of the target protein mRNA, the stability of the target protein, the post-translational modification of the target protein, the activity of the target protein, or any combination thereof. Furthermore, the term “modulate” may be used to refer to an increase, decrease, blockade, alteration, inactivation, or restoration of the activity of a target protein, including but not limited to the activity of the target protein.

[0086] "Naturally occurring" means, as applied to substances, the fact that a substance can be found in nature. For example, a polypeptide or polynucleotide sequence found in living organisms (including viruses) that can be isolated from a natural origin and has not been deliberately modified by humans is a naturally occurring sequence.

[0087] The term "functionally linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, if a first nucleic acid sequence is functionally related to a second nucleic acid sequence, then the first nucleic acid sequence is functionally linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, then the promoter is functionally linked to that coding sequence. In general, functionally linked DNA sequences are contiguous and, if two protein-coding regions need to be joined, they are within the same reading frame.

[0088] Parenteral administration of the composition includes, for example, subcutaneous (sc) injection, intravenous (iv) injection, intramuscular (im) injection, or intrasternal injection, or infusion techniques.

[0089] As used herein, the term “pharmaceutical composition” means a mixture of at least one compound useful in this disclosure with other chemical components such as carriers, stabilizers, diluents, dispersants, suspenders, thickeners, and / or excipients. Pharmaceutical compositions facilitate the administration of compounds to living organisms. Multiple techniques for administering compounds exist in the art, including but not limited to intravenous, oral, aerosol, parenteral, intraocular, intrapulmonary, intracranial, and topical administration.

[0090] As used herein, the term “pharmaceutically acceptable” means a material such as a carrier or diluent that does not inhibit the biological activity or properties of a composition and is relatively non-toxic; that is, the material can be administered to an individual without causing undesirable biological effects and without harmfully interacting with any of the components of the composition in which it is contained.

[0091] A “pharmaceutically acceptable carrier” includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid extenders, diluents, excipients, solvents, or capsule materials, that are involved in holding or transporting the compound of the present invention in or to a subject so that it can perform its intended function. Typically, such compounds are held or transported from one organ or part of the body to another organ or part of the body. Each salt or carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the subject. Some examples of materials that can 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; glycerin, sorbitol, mannitol, and polyethylene glycol This includes polyols such as ethyl oleate and ethyl laurate; esters such as agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; diluents; granulators; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavoring agents; fragrances; preservatives; antioxidants; plasticizers; gelling agents; thickeners; curing agents; coagulants; suspending agents; surfactants; wetting agents; carriers; stabilizers; and other non-toxic compatible substances used in pharmaceutical formulations, or any combination thereof. As used herein, “pharmaceutically acceptable carrier” also includes all any coating agents, antimicrobial agents, antifungal agents, and absorption retarders, etc., whose activity and compatibility with the compound make them physiologically acceptable to the subject. Supplementary active compounds may be incorporated into the composition.

[0092] As used herein, the term “pharmaceutically acceptable salt” means a salt of the compound administered, its solvated compound, hydrate, or inclusion compound, prepared from a pharmaceutically acceptable, non-toxic acid, including inorganic and organic acids.

[0093] A "polypeptide" is a polymer composed of amino acid residues, their associated naturally occurring structural variants, and synthetic analogs that do not naturally occur, linked together by peptide bonds. Synthetic polypeptides can be synthesized, for example, using automated polypeptide synthesizers. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides.

[0094] Conventional notation for describing polypeptide sequences is used herein: the left end of a polypeptide sequence is an amino terminus; the right end of a polypeptide sequence is a carboxyl terminus. As used herein, a “peptide mimetic” is a compound containing non-peptide structural elements that can mimic the biological action of a parent peptide. Peptide mimetic compounds may or may not contain peptide bonds.

[0095] As used herein, the terms “prevent” or “prevent” mean, if not yet occurring, that the onset of a disorder or disease will not occur, or, if the onset of a disorder or disease already exists, that further onset of a disorder or disease will not occur. The ability to prevent some or all of the symptoms associated with a disorder or disease will also be considered. Disease and disorder are used interchangeably herein.

[0096] The term "specifically binds," as used herein, means that a first molecule (e.g., an antibody) preferentially binds to a second molecule (e.g., a specific antigenic epitope), but does not necessarily have to bind only to that second molecule.

[0097] As used herein, “Subject” means human or non-human mammal. Non-human mammals include, for example, livestock and pets such as sheep, cattle, pigs, dogs, cats, and mouse mammals. In certain embodiments, the subject is human.

[0098] As used herein, the terms “treatment” or “to treat” are defined as the application or administration of a therapeutic agent, i.e., a composition useful in this disclosure (alone or in combination with another pharmaceutical agent), to a subject who is likely to develop a disease or disorder, symptoms of a disease or disorder, or the disease or disorder, for the purpose of treating, curing, alleviating, reducing, modifying, correcting, relieving, improving, or influencing a disease or disorder, symptoms of a disease or disorder, or the likelihood of developing a disease or disorder, or to a tissue or cell line isolated from the subject (e.g., for diagnostic or ex vivo application). Such treatments may be specifically individualized or modified based on knowledge obtained from the field of pharmacogenomics. The appropriate therapeutic dose in individual cases may be determined by those skilled in the art using routine experiments.

[0099] A “variant” is, as the term is used herein, a nucleic acid or peptide sequence that differs in sequence from a reference nucleic acid or peptide sequence but retains the essential characteristics of the reference molecule. A change in the sequence of a nucleic acid variant may or may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or it may result in amino acid substitutions, additions, deletions, fusions, and shortenings. In some examples, the sequence changes in a peptide variant are limited or conserved, so that the sequences of the reference peptide and the variant are very similar overall and identical in many regions. The variant and the reference peptide may differ in amino acid sequence by any combination of one or more substitutions, additions, or deletions. A variant of a nucleic acid or peptide may be naturally occurring, such as an allele variant, or it may be a variant whose natural occurrence is unknown. Variants of nucleic acids and peptides that do not exist naturally may be created by mutagenesis or by direct synthesis. A change in one or more amino acids in the reference sequence may be a modification selected from the group consisting of substitutions, additions, deletions, and modifications of at least one amino acid in the reference polypeptide. An exemplary variant is a polypeptide containing one or more amino acid changes compared to human FGF21 (SEQ ID NO:1). Another example of a variant is a polypeptide containing one or more amino acid changes compared to a fragment of human FGF21 (SEQ ID NO:1).

[0100] In the context of two or more nucleic acids or two or more polypeptides, the terms “identical” or “percent identity” refer to two or more sequences or subsequences that have the same nucleotides or amino acids, or the same by a specified percentage. For example, approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity across a specified region when compared and aligned for the maximum correspondence across a comparison window or specified region, as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters, or by manual alignment and visual inspection. See, for example, the NCBI website at ncbi.nlm.nih.gov / BLAST. The definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. The algorithm may take gaps, etc., as shown below.

[0101] As used herein, the term “include” means “comprising, consisting of, or essentially consisting of.” For example, the phrase A includes B and C means: in some embodiments A includes B and C (and other elements may also be part of A); in some embodiments A consists of B and C (and A has no further elements other than B and C); in some embodiments A is essentially consisting of B and C (and C may have elements other than B and C, insofar as the additional elements do not substantially affect the basic and novel characteristics of A).

[0102] Throughout this disclosure, various aspects of this disclosure may be presented in scope form. It should be understood that scope form is for convenience and conciseness only and should not be interpreted as an inflexible limitation on the scope of this disclosure. Therefore, scope descriptions should be considered to specifically disclose all possible sub-ranges and individual numbers within that range. For example, a range description such as 1-6 should be considered to specifically disclose sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0103] Non-natural structures In some aspects, the present invention relates to non-naturally soluble structures.

[0104] In some embodiments, the construct includes residues 186-198 and 200-209 of fibroblast growth factor 21 (FGF21).

[0105] In some embodiments, the residue numbers of FGF21 used herein refer to those of the protein sequence shown in SEQ ID NO:1 below. TIFF2026534020000002.tif34146

[0106] The signal sequence for human FGF21 is residues 1-28 of SEQ ID NO:1. Mature human FGF21 lacking the signal sequence (residues 1-28 of SEQ ID NO:1) corresponds to residues 29-209 of SEQ ID NO:1.

[0107] In certain embodiments, the FGF21 polypeptides described herein have an amino acid sequence having 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 with respect to SEQ ID NO:1, or SEQ ID It has an amino acid sequence that has at least approximately 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 with the amino acid sequence of residues 29-209 of NO:1 (mature human FGF21 protein). In a further context, the FGF21 polypeptides described herein have an amino acid sequence having at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:1, or an amino acid sequence having at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of residues 29-209 of SEQ ID NO:1 (mature human FGF21 protein). In an additional aspect, the FGF21 polypeptide has an amino acid sequence having 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 with respect to the specific polypeptides described herein.

[0108] In some embodiments, FGF21 polypeptides containing P199 mutations (such as P199G mutations) and / or A208 mutations (such as A208E mutations) are contemplated herein. Without intending to be limited by any theory, in certain embodiments, P199 mutations, such as P199G mutations, can reduce in vivo cleavage of the FGF21 polypeptide. Without intending to be limited by any theory, in certain embodiments, A208 mutations, such as A208E mutations, can reduce in vivo cleavage of the FGF21 polypeptide.

[0109] In some embodiments, FGF21 polypeptides containing L126 mutations (such as L126R mutations) are intended herein. Without intending to be limited by any theory, in certain embodiments, L126 mutations such as L126R mutations can reduce the aggregation of FGF21 polypeptides.

[0110] The numbering of FGF21 residues used herein is done with reference to SEQ ID NO:1, but it should be noted that the constructs described herein are not limited to human FGF21 having the protein sequence shown in SEQ ID NO:1. For example, orthologues of FGF21 (i.e., FGF21 in other species) may have different sequences and lengths, but those skilled in the art will understand that the residues corresponding to residues 186-198 and 200-209 of SEQ ID NO:1, as well as the residues to be mutated (as described elsewhere herein), can be easily determined by conventional tools such as sequence alignment.

[0111] In some embodiments, the FGF21 polypeptide contains at least one mutation selected from the group consisting of L126, L194, V197, P199, Q201, R203, Y207, A208, and S209.

[0112] In some embodiments, the FGF21 polypeptide contains mutation L126. In some embodiments, the FGF21 polypeptide contains mutation L194. In some embodiments, the FGF21 polypeptide contains mutation V197. In some embodiments, the FGF21 polypeptide contains mutation P199. In some embodiments, the FGF21 polypeptide contains mutation Q201. In some embodiments, the FGF21 polypeptide contains mutation R203. In some embodiments, the FGF21 polypeptide contains mutation Y207. In some embodiments, the FGF21 polypeptide contains mutation A208. In some embodiments, the FGF21 polypeptide contains mutation S209.

[0113] In some embodiments, the FGF21 polypeptide contains 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.

[0114] In some embodiments, the FGF21 polypeptide contains the mutation L126R. In some embodiments, the FGF21 polypeptide contains the mutation L194F. In some embodiments, the FGF21 polypeptide contains the mutation V197I. In some embodiments, the FGF21 polypeptide contains the mutation V197L. In some embodiments, the FGF21 polypeptide contains the mutation P199G. In some embodiments, the FGF21 polypeptide contains the mutation Q201L. In some embodiments, the FGF21 polypeptide contains the mutation Q201I. In some embodiments, the FGF21 polypeptide contains the mutation Q201P. In some embodiments, the FGF21 polypeptide contains the mutation Q201R. In some embodiments, the FGF21 polypeptide contains the mutation Q201W. In some embodiments, the FGF21 polypeptide contains the mutation R203W. In some embodiments, the FGF21 polypeptide contains a mutation selected from the group consisting of Y207W. In some embodiments, the FGF21 polypeptide contains the mutation A208E. In some embodiments, the FGF21 polypeptide contains the mutation A208W. In some embodiments, the FGF21 polypeptide contains the mutation S209G.

[0115] In some embodiments, the segments corresponding to residues 186-198 and 200-209 of the FGF21 polypeptide with SEQ ID NO:1 are part of a continuous polypeptide chain. In some embodiments, the segment corresponding to residues 186-198 of FGF21 with SEQ ID NO:1 is conjugated to the N-terminus of the segment corresponding to residues 200-209.

[0116] In some embodiments, the FGF21 polypeptide comprises residues 186-209 of FGF21, which, according to some embodiments, comprises both a segment corresponding to residues 186-198 and a segment corresponding to residues 200-209 of FGF21.

[0117] In some embodiments, the FGF21 polypeptide comprises residues 169-209 of FGF21, which, according to some embodiments, includes both a segment corresponding to residues 186-198 and a segment corresponding to residues 200-209 of FGF21.

[0118] In some embodiments, the FGF21 polypeptide comprises residues 29-209 of FGF21, which, according to some embodiments, includes both a segment corresponding to residues 186-198 and a segment corresponding to residues 200-209 of FGF21.

[0119] In some embodiments, the FGF21 polypeptide comprises residues 186-209 of FGF21 or residues 169-209 of FGF21, including the C-terminal tail of FGF21 that specifically binds to β-klotho.

[0120] In some embodiments, the polypeptide lacks residues 29-169 of FGF21 (the "core region" responsible for binding to the FGF receptor) or a portion thereof, thereby preventing the FGF21 polypeptide from specifically binding to FGF receptors such as FGFR1c. In some embodiments, the core region-deficient FGF21 polypeptide can bind to β-Klotho and sequester it from the FGF / FGFR / β-Klotho signaling pathway, thereby downregulating this pathway.

[0121] In some embodiments, the FGF21 polypeptide contains residues 29–209 of FGF21. In some embodiments, the FGF21 polypeptide containing residues 29–209 of FGF21 contains both a core region responsible for binding to the FGF receptor and a C-terminal tail region responsible for binding to β-Klotho. In some embodiments, the FGF21 polypeptide containing both the core and tail regions can activate the FGF21 / FGFR / β-Klotho signaling pathway with higher potency than wild-type FGF21.

[0122] In some embodiments, the FGF21 polypeptide further comprises 2 to 5 basic amino acid residues and / or hydrophilic amino acid residues in the C-terminal region of residues 200–209 of FGF21. Those skilled in the art will understand that the basic amino acid residues include, for example, arginine (R) and lysine (K), and the hydrophilic residues include serine (S), threonine (T), and several other amino acid residues. In some embodiments, the amino acid sequence RKK, RRS, or RKS is conjugated to the C-terminus of residues 200–209 of FGF21.

[0123] In some embodiments, the FGF21 polypeptide contains mutations L194F, V197I, Q201L, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutations L126R, P199G, L194F, V197I, Q201L, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutations L194F, V197I, Q201L, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as the corresponding wild-type FGF21. In a further additional embodiment, the FGF21 polypeptide contains mutants L194F, V197I, Q201L, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21 (except for the signal sequence). In a further additional embodiment, the FGF21 polypeptide contains mutants L126R, P199G, L194F, V197I, Q201L, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21. In some embodiments, the FGF21 polypeptide further contains the sequence RKK conjugated to the C-terminus of residues 200-209 of FGF21 (CV3).

[0124] In some embodiments, the FGF21 polypeptide contains mutants L194F, V197I, Q201R, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutants L126R, P199G, L194F, V197I, Q201R, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutants L194F, V197I, Q201R, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV4). In an additional embodiment, the FGF21 polypeptide contains mutations L194F, V197I, Q201R, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21. In a further additional embodiment, the FGF21 polypeptide contains mutations L126R, P199G, L194F, V197I, Q201R, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21.

[0125] In some embodiments, the FGF21 polypeptide contains mutants L194F, V197I, Q201R, R203W, Y207W, and A208W. In some embodiments, the FGF21 polypeptide contains mutants L126R, P199G, L194F, V197I, Q201R, R203W, Y207W, and A208W. In some embodiments, the FGF21 polypeptide contains mutants 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 contains mutants L194F, V197I, Q201R, R203W, Y207W, and A208W, but otherwise has the same sequence as mature human FGF21. In a further embodiment, the FGF21 polypeptide includes mutants L126R, P199G, L194F, V197I, Q201R, R203W, Y207W, and A208W, but otherwise has the same sequence as mature human FGF21.

[0126] In some embodiments, the FGF21 polypeptide contains mutants L194F, V197L, Q201R, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutants L126R, P199G, L194F, V197L, Q201R, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutants L194F, V197L, Q201R, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV11). In an additional embodiment, the FGF21 polypeptide contains mutants L194F, V197L, Q201R, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21. In a further additional embodiment, the FGF21 polypeptide contains mutants L126R, P199G, L194F, V197L, Q201R, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21.

[0127] In some embodiments, the FGF21 polypeptide contains mutants L194F, V197L, Q201R, R203W, Y207W, and A208W. In some embodiments, the FGF21 polypeptide contains mutants L126R, P199G, L194F, V197L, Q201R, R203W, Y207W, and A208W. In some embodiments, the FGF21 polypeptide contains mutants L194F, 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 contains mutants L194F, V197L, Q201R, R203W, Y207W, and A208W, but otherwise has the same sequence as mature human FGF21. In a further embodiment, the FGF21 polypeptide includes mutants L126R, P199G, L194F, V197L, Q201R, R203W, Y207W, and A208W, but otherwise has the same sequence as mature human FGF21.

[0128] In some embodiments, the FGF21 polypeptide contains mutants L194F, Q201I, R203W, Y207W, and A208W. In some embodiments, the FGF21 polypeptide contains mutants L126R, P199G, L194F, Q201I, R203W, Y207W, and A208W. In some embodiments, the FGF21 polypeptide contains mutants 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 contains mutants L194F, Q201I, R203W, Y207W, and A208W, but otherwise has the same sequence as mature human FGF21. In a further embodiment, the FGF21 polypeptide contains mutants L126R, P199G, L194F, Q201I, R203W, Y207W, and A208W, but otherwise has the same sequence as mature human FGF21.

[0129] In some embodiments, the FGF21 polypeptide contains mutants L194F, Q201L, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutants L126R, P199G, L194F, Q201L, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutants 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 contains mutants L194F, Q201L, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21. In further embodiments, the FGF21 polypeptide includes mutants 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 a sequence RKS conjugated to the C-terminus of residues 200-209 of FGF21 (CV18).

[0130] In some embodiments, the FGF21 polypeptide contains mutants L194F, Q201P, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutants L126R, P199G, L194F, Q201P, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutants 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 contains mutants L194F, Q201P, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21. In further embodiments, the FGF21 polypeptide includes mutants L126R, P199G, L194F, Q201P, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21. In some embodiments, the FGF21 polypeptide further includes the sequence RRS (CV21), which is conjugated to the C-terminus of residues 200-209 of FGF21.

[0131] In some embodiments, the FGF21 polypeptide contains mutants L194F, Q201R, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutants L126R, P199G, L194F, Q201R, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutants 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 contains mutants L194F, Q201R, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21. In further embodiments, the FGF21 polypeptide includes mutants L126R, P199G, L194F, Q201R, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21. In some embodiments, the FGF21 polypeptide further includes the sequence RRS conjugated to the C-terminus of residues 200-209 of FGF21 (CV24).

[0132] In some embodiments, the FGF21 polypeptide contains mutants L194F, Q201R, R203W, Y207W, and A208W. In some embodiments, the FGF21 polypeptide contains mutants L126R, P199G, L194F, Q201R, R203W, Y207W, and A208W. In some embodiments, the FGF21 polypeptide contains mutants 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 contains mutants L194F, Q201R, R203W, Y207W, and A208W, but otherwise has the same sequence as mature human FGF21. In a further embodiment, the FGF21 polypeptide includes mutants L126R, P199G, L194F, Q201R, R203W, Y207W, and A208W, but otherwise has the same sequence as mature human FGF21.

[0133] In some embodiments, the FGF21 polypeptide contains mutants L194F, Q201W, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutants L126R, P199G, L194F, Q201W, R203W, Y207W, A208W, and S209G. In some embodiments, the FGF21 polypeptide contains mutants 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 polypeptide contains mutants L194F, Q201W, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21. In a further embodiment, the FGF21 polypeptide includes mutants L126R, P199G, L194F, Q201W, R203W, Y207W, A208W, and S209G, but otherwise has the same sequence as mature human FGF21.

[0134] In some embodiments, the FGF21 polypeptide contains mutants L194F, R203W, Y207W, and A208W. In some embodiments, the FGF21 polypeptide contains mutants L126R, P199G, L194F, R203W, Y207W, and A208W. In some embodiments, the FGF21 polypeptide contains mutants L194F, R203W, Y207W, and A208W, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV30). In additional embodiments, the FGF21 polypeptide contains mutants L194F, R203W, Y207W, and A208W, but otherwise has the same sequence as mature human FGF21. In a further embodiment, the FGF21 polypeptide contains mutants L126R, P199G, L194F, R203W, Y207W, and A208W, but otherwise has the same sequence as mature human FGF21.

[0135] In some embodiments, the FGF21 polypeptide contains mutants R203W, Y207W, and A208W. In some embodiments, the FGF21 polypeptide contains mutants L126R, P199G, R203W, Y207W, and A208W. In some embodiments, the FGF21 polypeptide contains mutants R203W, Y207W, and A208W, but otherwise has the same sequence as the corresponding wild-type FGF21 (CV30). In some embodiments, the FGF21 polypeptide contains mutants R203W, Y207W, and A208W, but otherwise has the same sequence as mature human FGF21. In additional embodiments, the FGF21 polypeptide contains mutants L126R, P199G, R203W, Y207W, and A208W, but otherwise has the same sequence as mature human FGF21.

[0136] In some embodiments, the construct further includes a stability-enhancing domain. In some embodiments, the stability-enhancing domain is attached to the N-terminus of the FGF21 polypeptide. In some embodiments, the stability-enhancing domain is attached to the C-terminus of the FGF21 polypeptide. Non-limiting examples of stability-enhancing domains include albumin, the Fc region of antibodies, glutathione S-transferase, small ubiquitin-like modifiers (SUMOs), the O-glycosylation region of Drosophila Spitz protein, thioredoxin, and the like.

[0137] In some embodiments, the FGF21 polypeptide and the stability-enhancing domain are linked via 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 mobile linker, for example, a peptide rich in glycine (G) and / or serine (S), or a peptide that can be cleaved by an enzyme.

[0138] In some embodiments, the FGF21 polypeptides herein bind β-Klotho more tightly than the corresponding wild-type FGF21 polypeptide. In some embodiments, the dissociation constant (K d ) of the FGF21 polypeptide for β-Klotho is no more than about 20% of the K d of the corresponding wild-type FGF21 polypeptide, for example, no more than 10% or no more than 5%.

[0139] In some embodiments, the FGF21 polypeptides herein bind β-Klotho for a longer duration than the corresponding wild-type FGF21 polypeptide. In some embodiments, the K off of the FGF21 polypeptide for β-Klotho is no more than about 20% of the K off of the corresponding wild-type FGF21 polypeptide, for example, no more than 10% or no more than 5%.

[0140] In some embodiments, the construct binds β-Klotho and sequesters β-Klotho from the downstream pathways of fibroblast growth factor receptor (FGFR) and β-Klotho.

[0141] In some embodiments, the construct binds both FGFR and β-Klotho and activates the downstream pathways of FGFR and β-Klotho. In some embodiments, the FGFR is FGFR1c.

[0142] Compositions In some aspects, the present invention relates to compositions, such as pharmaceutical compositions comprising the non-naturally occurring constructs herein. Details of the formulation of the compositions can be found in the section "Pharmaceutical Compositions and Formulations".

[0143] Methods of Modulating the FGF / FGFR / β-Klotho Signaling Pathway In some aspects, the present research relates to methods of modulating an FGF / FGFR / β-Klotho signaling pathway, such as the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway.

[0144] In some embodiments, the method includes bringing the surface of a cell having the FGF / FGFR / β-Klotho signaling pathway into contact with a construct as described herein. In some embodiments, the construct is the same as or similar to those described in the “Non-Natural Constructs” section.

[0145] As described elsewhere herein, the constructs herein can be configured as positive or negative regulators of the pathway, for example, by adding or removing an FGF core sequence from an FGF21 polypeptide.

[0146] In some embodiments, the construct binds to β-Klotho but not to FGFR, thereby sequestering β-Klotho from the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway and downregulating the signaling pathway.

[0147] In some embodiments, the construct binds to both β-Klotho and FGFR, thereby upregulating the FGF21 / FGFR / β-Klotho signaling pathway.

[0148] In some embodiments, the cells are derived from cell lines, primary cells, or cells within the body of the subject (such as a mammalian subject or a human subject).

[0149] In some embodiments, the cells are cells located in or originating from adipose tissue, the liver, the pancreas, or the hypothalamus.

[0150] Methods for treating, relieving, and / or preventing a disease or disorder. In several aspects, this research concerns methods for treating, relieving, and / or preventing diseases or disorders in subjects where there is a need for them.

[0151] In some embodiments, the disease or disorder is caused and / or associated with misregistration of the 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 and / or associated with upregulated or downregulated FGF / FGFR / β-Klotho signaling pathways.

[0152] In some embodiments, the disease or disorder is at least one selected from obesity, diabetes, pancreatitis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), acute alcohol toxicity, and liver cancer and other cancers driven by abnormal activation of the FGF19 / FGFR4 / β-Klotho pathway in tumor cells.

[0153] In some embodiments, the method includes the step of administering an effective amount of a construct or composition (such as a pharmaceutical composition) as specified herein to a subject.

[0154] In some embodiments, the construct or composition is administered via at least one route selected from the group consisting of inhalation, oral, rectal, vaginal, parenteral, intracranial, topical, percutaneous, pulmonary, intranasal, buccal, ocular, subarachnoid, and intravenous.

[0155] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0156] Pharmaceutical compositions and formulations This disclosure also includes the use of the pharmaceutical compositions of this disclosure to carry out the methods of this disclosure.

[0157] Such pharmaceutical compositions may be provided in a form suitable for administration to a subject and may contain one or more pharmaceutically acceptable carriers, one or more additional components, or any combination thereof. The compositions of the present disclosure may contain physiologically acceptable salts, such as the compounds contemplated in the present disclosure, combined with physiologically acceptable cations or anions, as is well known in the art.

[0158] In certain embodiments, a pharmaceutical composition useful for carrying out the method of this disclosure may be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day. In other embodiments, a pharmaceutical composition useful for carrying out this disclosure may be administered to deliver a dose of 1 ng / kg / day to 500 mg / kg / day.

[0159] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and additional components in the pharmaceutical compositions of this disclosure will vary depending on the identity, size, and condition of the object being treated, and further, on the route through which the composition is administered. For example, a composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0160] Pharmaceutical compositions useful in the methods of this disclosure may be appropriately developed for inhalation, oral, rectal, vaginal, parenteral, topical, intracranial, percutaneous, pulmonary, intranasal, buccal, ocular, subarachnoid, intravenous, or other routes of administration. Other intended formulations include injection nanoparticles, liposome preparations, resealed red blood cells containing active ingredients, and immunological formulations. The route of administration will be readily apparent to those skilled in the art and will depend on numerous factors, including the type and severity of the disease being treated, the type and age of the animal or human patient being treated, and so on.

[0161] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or to be developed in the field of pharmacology. Generally, such preparation methods include the steps of combining the active ingredient with a carrier or one or more other auxiliary components, and then, if necessary or desirable, forming or packaging the product into desired single-dose or multi-dose units.

[0162] As used herein, “unit dose” refers to a specific amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient administered to a subject, or a convenient fraction of such dosage, such as half or one-third of such dosage. A unit dosage form may be for one dose per day or for one dose per day (e.g., about one to four times or more per day). If multiple doses per day are used, the unit dosage form may be the same or different for each dose.

[0163] The descriptions of pharmaceutical compositions provided herein are primarily directed toward pharmaceutical compositions suitable for ethical administration to humans, but it will be understood by those skilled in the art that such compositions are generally suitable for administration to all types of animals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals are well understood, and such modifications can be designed and carried out by ordinary experiments only, if experiments are necessary, by a veterinary pharmacologist of ordinary art. The subjects to which the administration of the pharmaceutical compositions of this disclosure is intended include, but are not limited to, humans and other primates, and mammals, including commercially important mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0164] 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.

[0165] The formulations may be used in combination with conventional excipients known in the art, i.e., pharmaceutically acceptable organic or inorganic carrier substances, suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or other appropriate modes of administration. The pharmaceutical preparations may be sterilized and optionally mixed with adjuvants, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, colorants, flavoring agents, and / or fragrances. They may also be optionally combined with other active agents, such as other analgesics.

[0166] As used herein, “additional components” include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulators and disintegrants; binders; lubricants; sweeteners; flavorings; colorants; preservatives; physiologically degradable compositions such as gelatin; aqueous media and solvents; oily media and solvents; suspending agents; dispersants or wetting agents; emulsifiers, lubricants; buffers; salts; thickeners; bulking agents; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional components” that may be included in the pharmaceutical compositions of this disclosure are known in the art and are described, for example, in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which are incorporated herein by reference.

[0167] Liquid suspensions can be prepared using conventional methods for achieving suspension of active ingredients in aqueous or oily media. Aqueous media include, for example, water and isotonic saline. Oily media include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further contain one or more additional components, including but not limited to suspending agents, dispersing agents or wetting agents, emulsifiers, lubricants, preservatives, buffers, salts, flavoring agents, coloring agents, and sweeteners. Oily suspensions may further contain thickeners. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated fats and oils, sodium alginate, polyvinylpyrrolidone, tragacanth gum, gum arabic, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropyl methylcellulose. Known dispersants or wetting agents include, but are not limited to, naturally occurring phosphatides such as lecithin, condensation products of alkylene oxides with fatty acids, condensation products with long-chain aliphatic alcohols, condensation products of partial esters derived from fatty acids and hexitol, or condensation products of partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifiers include, but are not limited to, lecithin and gum arabic. Known preservatives include, but are not limited to, methyl parahydroxybenzoic acid, ethyl parahydroxybenzoic acid, or n-propyl parahydroxybenzoic acid, ascorbic acid, and sorbic acid. Known sweeteners include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickeners for oily suspensions include, for example, beeswax, solid paraffin, and cetyl alcohol.

[0168] Powdered and granular formulations of the pharmaceutical preparations of this disclosure may be prepared using known methods. Such formulations may be administered directly to a subject or used, for example, to form tablets, to fill capsules, or to prepare aqueous or oily suspensions or solutions by adding an aqueous or oily medium. Each of these formulations may further contain one or more dispersants or wetting agents, suspending agents, and preservatives. Additional excipients such as bulking agents and sweeteners, flavoring agents, or coloring agents may also be included in these formulations.

[0169] The pharmaceutical compositions of this disclosure may be prepared, packaged, or sold in the form of oil-in-water emulsions or water-in-oil emulsions. The oil phase may be a vegetable oil such as olive oil or peanut oil, a mineral oil such as liquid paraffin, or a combination thereof. Such compositions may further contain one or more emulsifiers such as naturally occurring gums such as gum arabic or tragacanth gum, naturally occurring phosphatides such as soy phosphatide 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 oxides such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional components, such as sweeteners or flavorings.

[0170] Methods for impregnating or coating materials with chemical compositions are known in the art and include, but are not limited to, methods for depositing or bonding chemical compositions to a surface, methods for incorporating chemical compositions into the structure of a material during the synthesis of a material (i.e., such as a physiologically biodegradable material), and methods for absorbing aqueous or oily solutions or suspensions into an absorbent material, and then drying or not drying them.

[0171] Administration / Medication The administration plan may affect what constitutes the effective dose. The therapeutic formulation may be administered to the patient either before or after the manifestation of symptoms associated with the disease or condition. Furthermore, several divided and staggered doses may be administered daily or continuously, or the dose may be administered by continuous infusion or bolus injection. In addition, the dosage of the therapeutic formulation may be increased or decreased proportionally, as indicated by the urgency of the therapeutic or preventive situation.

[0172] The administration of the compositions of the present invention to patients, preferably mammals, more preferably humans, may be carried out using known methods in doses and durations effective for treating a disease or condition in the patient. The effective amount of the therapeutic compound required to achieve a therapeutic effect may vary depending on factors well known in the medical field, such as the activity of the specific compound used; the time of administration; the rate of compound elimination; the duration of treatment; other drugs, compounds, or materials used in combination with the compound; and the state of the disease or disorder, the age, sex, weight, condition, overall health, and medical history of the patient being treated. The dosage plan may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be reduced proportionally as indicated by the urgency of the treatment situation. A non-limiting example of the effective dose range of the therapeutic compounds in this disclosure is about 0.01 to 50 mg / kg body weight / day. Those skilled in the art will be able to study the relevant factors and make a determination regarding the effective amount of the therapeutic compound without excessive experimentation.

[0173] The compound may be administered to animals at a frequency such as several times a day, or at a less frequent frequency such as once a day, once a week, once every two weeks, once a month, once every few months, or even once a year or less. The amount of the compound administered daily may be, in non-limiting examples, daily, every other day, every two days, every three days, every four days, or every five days. For example, in the case of every-other-day administration, a dose of 5 mg per day may be started on Monday, the first subsequent dose of 5 mg per day may be administered on Wednesday, the second subsequent dose of 5 mg per day may be administered on Friday, and so on. The frequency of administration will depend on a number of factors, such as the type and severity of the disease being treated, the type and age of the animal, etc., and will not be limited to these, but will be readily apparent to those skilled in the art.

[0174] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may vary to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0175] A physician with ordinary art in this field, such as an internist or veterinarian, can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, an internist or veterinarian can start the dose of the compound of the present disclosure used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0176] In specific embodiments, formulating compounds in dosage units is particularly advantageous for ease of administration and uniformity of dosage. A dosage unit, as used herein, refers to a physically discontinuous unit suitable as a unit dose for a patient being treated; each unit contains a predetermined amount of the therapeutic compound, calculated to produce the desired therapeutic effect, along with the required pharmaceutical medium. The dosage unit forms of this disclosure are indicated by and directly depend on (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the field of compounding / formulating such therapeutic compounds for the treatment of cancer in a patient.

[0177] In certain embodiments, the compositions of the Disclosure are administered to a patient in dosages ranging from one to five times a day or more. In other embodiments, the compositions of the Disclosure are administered to a patient in dosages ranging from once a day, once every two days, once every three days to once a week and once every two weeks, but not limited to these. It will be readily apparent to those skilled in the art that the frequency of administration of the various combination compositions of the Disclosure will vary depending on the subject, due to many factors including, but not limited to, age, the disease or disorder being treated, sex, overall health, and other factors. Therefore, the Disclosure should not be construed as being limited to a specific dosing plan, and the exact dosage and composition to be administered to a patient will be determined by the attending physician, taking into account all other factors relating to the patient.

[0178] The compounds of this disclosure for administration are approximately 1 μg to 7,500 mg, approximately 20 μg to 7,000 mg, approximately 40 μg to 6,500 mg, approximately 80 μg to 6,000 mg, approximately 100 μg to 5,500 mg, approximately 200 μg to 5,000 mg, approximately 400 μg to 4,000 mg, approximately 800 μg to 3,000 mg, approximately 1 mg to 2,500 mg, and approximately 2 mg to 5,000 mg. This may be 2,000 mg, approximately 5 mg to 1,000 mg, approximately 10 mg to 750 mg, approximately 20 mg to 600 mg, approximately 30 mg to 500 mg, approximately 40 mg to 400 mg, approximately 50 mg to 300 mg, approximately 60 mg to 250 mg, approximately 70 mg to 200 mg, approximately 80 mg to 150 mg, and any full or partial increase in between.

[0179] In some embodiments, the dose of the compound disclosed is about 0.5 μg to about 5,000 mg. In some embodiments, the dose of the compound disclosed used in the 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, the dose of the second compound 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 0.5 mg, and any full or partial increase between them.

[0180] In certain embodiments, the present invention relates to a packaged pharmaceutical composition comprising a container for holding a therapeutically effective amount of the compound of the present disclosure, either 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.

[0181] The term “container” includes any container for holding a pharmaceutical composition. For example, in certain embodiments, the container is packaging containing a pharmaceutical composition. In other embodiments, the container is not packaging containing a pharmaceutical composition; that is, the container is a box or vial-like container containing a packaged or unpackaged pharmaceutical composition and instructions for use of the pharmaceutical composition. Furthermore, packaging techniques are well known in the art. Instructions for use of a pharmaceutical composition may be contained within the packaging containing the pharmaceutical composition, and it should be understood that the instructions form an enhanced functional relationship with the packaged product. However, it should be understood that the instructions may also contain information relating to the compound’s intended function, e.g., its ability to treat, prevent, or reduce a disease or disorder in a patient.

[0182] Route of administration Routes of administration of the compositions of this disclosure include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, tongue, (trans) buccal, (trans) urethral, ​​vagina (e.g., transvaginal and perivaginal), nasal (intra) cavity, and (trans) rectal), intravesical, intrapulmonary, intraduodenal, intragastric, subarachnoid, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, intracranial, and topical administration.

[0183] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel capsules, lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magma, lozenges, creams, pastes, ointments, lotions, discs, suppositories, liquid sprays, dry powders or aerosol formulations for inhalation, compositions and formulations for intravesical administration, etc. It should be understood that the formulations and compositions useful in the present invention are not limited to the specific formulations and compositions described herein.

[0184] Oral administration Tablets, sugar-coated tablets, liquids, drops, suppositories, or capsules, caplets, and gel capsules are particularly suitable for oral administration. Other formulations suitable for oral administration include, but are not limited to, powders or granules, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, toothpastes, mouthwashes, coatings, gargles, or emulsions. Compositions for oral use may be prepared by 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 acceptable excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as lactose; granulators and disintegrants such as corn starch; binders such as starch; and lubricants such as magnesium stearate.

[0185] Tablets may be uncoated or coated using known methods to achieve delayed disintegration in the target gastrointestinal tract, thereby providing sustained release and absorption of the active ingredient. For example, materials such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Furthermore, for example, tablets may be coated using the methods described in U.S. Patents 4,256,108; 4,160,452; and 4,265,874 to form osmotic release controlled tablets. Tablets may further contain sweeteners, flavorings, colorings, preservatives, or any combination thereof to provide a pharmaceutically superior and pleasant preparation.

[0186] Hard capsules containing the active ingredient can be prepared using a physiologically biodegradable composition such as gelatin. Such hard capsules contain the active ingredient and may further contain additional components, such as an inert solid diluent, including, for example, calcium carbonate, calcium phosphate, or kaolin.

[0187] Soft gelatin capsules containing an active ingredient can be prepared using a physiologically biodegradable composition such as gelatin. Such soft capsules may contain an active ingredient mixed with water or an oily medium such as peanut oil, liquid paraffin, or olive oil.

[0188] The compounds of this disclosure for oral administration may be in the form of tablets or capsules prepared by conventional means, together with pharmaceutically acceptable excipients such as binders; fillers; lubricants; disintegrants; or wetting agents. Optionally, tablets may be coated by appropriate methods and with coating materials such as the 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 OPADRY® White, 32K18400).

[0189] Liquid preparations for oral administration may be in the form of solutions, syrups, or suspensions. Liquid preparations may be prepared by conventional means using suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated fat); emulsifiers (e.g., lecithin or gum arabic); non-aqueous media (e.g., almond oil, oily esters, or ethyl alcohol); and pharmaceutically acceptable additives (e.g., methyl parahydroxybenzoate, propyl parahydroxybenzoate, or sorbic acid). Liquid formulations of the pharmaceutical compositions of this disclosure suitable for oral administration may be prepared, packaged, and sold in liquid form or in the form of a dried product for regeneration with water or other suitable media before use.

[0190] For example, tablets containing an active ingredient may optionally be prepared by compressing or molding the active ingredient together with one or more additional ingredients. Compressed tablets may optionally be prepared by compressing an active ingredient in a fluid form, such as a powder or granular preparation mixed with one or more of the binders, lubricants, excipients, surfactants, and dispersants, in a suitable device. Molded tablets may be prepared by molding a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least enough liquid to moisten the mixture, in a suitable device. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulators and disintegrants, binders, and lubricants. Known dispersants include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, crystalline 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 binders include, but are not limited to, gelatin, gum arabic, pregelatinized corn starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

[0191] Granulation techniques for modifying active ingredient starting powders or other particulate materials are well known in the pharmaceutical field. Typically, powders are mixed with a binder material to form larger, permanently fluid aggregates or granules, which is called "granulation." For example, "wet" granulation methods using solvents generally involve combining the powder with a binder material, moistening it with water or an organic solvent under conditions that result in the formation of moist granular aggregates, and then evaporating the solvent from it.

[0192] Generally, melt granulation essentially consists of using a material that is solid or semi-solid at room temperature (i.e., having a relatively low softening or melting range) to facilitate the granulation of powders or other materials in the absence of the addition of water or other liquid solvents. When heated to a temperature within its melting point range, the low-melting-point solid liquefies and acts as a binder or granulation medium. The liquefied solid spreads over the surface of the powder material it comes into contact with, and upon cooling, forms a solid granular mass into which the initial materials bind together. The resulting melt granules may then be fed into a tablet press or encapsulated to prepare an oral dosage form. Melt granulation improves the dissolution rate and bioavailability of active substances (i.e., drugs) by forming solid dispersions or solid solutions.

[0193] U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties. Granules are obtained by melting and mixing wax with a certain flow-improving additive, and then cooling and granulating the mixture. In some embodiments, only the wax itself melts in the melted combination of wax and additive, while in other cases, both the wax and additive melt.

[0194] The present invention also includes multilayer tablets comprising a layer providing delayed release of one or more compounds useful in the methods disclosed herein, and a further layer providing immediate release of one or more compounds useful in the methods disclosed herein. A gastric insoluble composition in which the active ingredient is captured and its delayed release is obtained using a mixture of wax / pH-sensitive polymers.

[0195] Parenteral administration As used herein, “parenteral administration” of a pharmaceutical composition includes routes of administration characterized by physical penetration of the target tissue and administration of the pharmaceutical composition through a breakthrough within the tissue. Thus, parenteral administration includes, but is not limited to, the administration of a pharmaceutical composition by injection, application of the composition through surgical incision, and application of the composition through a non-surgical wound that penetrates the tissue. Specifically, parenteral administration includes, but is not limited to, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, and renal dialysis infusion techniques.

[0196] Formulations of pharmaceutical compositions suitable for parenteral administration comprise an active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in forms suitable for bolus or serial administration. Injectable formulations may be prepared, packaged, or sold in unit dosage forms, such as ampoules or multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous media, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further include, but are not limited to, one or more additional components, including suspending agents, stabilizers, or dispersants. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for parenteral administration of the regenerated composition after regeneration in a suitable medium (e.g., sterile pyrogen-free water).

[0197] Pharmaceutical compositions may be prepared, packaged, or sold in the form of sterile, injectable aqueous or oily suspensions or solutions. These suspensions or solutions may be formulated by known techniques and may contain, in addition to the active ingredient, additional components such as dispersants, wetting agents, or suspending agents as described herein. Such sterile, injectable formulations may be prepared, for example, using water or a non-toxic, parenterally acceptable diluent or solvent such as 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride aqueous solutions, and non-volatile oils such as synthetic monoglycerides or diglycerides. Other useful parenterally administered formulations include those containing the active ingredient in microcrystalline form, in liposome preparations, or as a component of biodegradable polymer systems. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0198] Additional dosage forms Additional dosage forms of the present invention include those described in U.S. Patent Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of the present invention also include those described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of the present invention also include those described in PCT applications 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.

[0199] Controlled-release formulations and drug delivery systems Controlled-release or sustained-release formulations of the pharmaceutical compositions of this disclosure can be prepared using conventional technologies. In some cases, the dosage form used may provide a desired release profile of varying rates, for example, by using hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, permeation systems, multilayer coatings, microparticles, liposomes, or microspheres, or combinations thereof, as a sustained-release or controlled-release formulation of one or more active ingredients therein. Controlled-release formulations known to those skilled in the art, suitable for use with the pharmaceutical compositions of this disclosure, including those described herein, can be readily selected. Accordingly, single-unit dosage forms suitable for oral administration, such as tablets, capsules, gel capsules, and caplets adapted for controlled-release, are encompassed in the present invention.

[0200] Most controlled-release pharmaceutical products have the general goal of improving drug therapy beyond what is achieved by their uncontrolled counterparts. Ideally, the use of optimally designed controlled-release preparations in medical procedures is characterized by the use of minimal drug substance to treat or manage a condition in the minimum amount and time. Advantages of controlled-release formulations include extended drug activity, reduced dosing frequency, and increased patient compliance. Furthermore, controlled-release formulations can be used to influence the time of action onset or other characteristics such as drug blood levels, and therefore can influence the incidence of side effects.

[0201] Most controlled-release formulations are designed to release an initial amount of drug to rapidly produce the desired therapeutic effect, and then gradually and continuously release other amounts of the drug to maintain this level of therapeutic effect over a longer period. To maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that replenishes the amount of drug that is metabolized and eliminated from the body.

[0202] The control of the release of the active ingredient can be stimulated by various inducers, such as pH, temperature, enzymes, water, or other physiological conditions, or by compounds. In the context of the present invention, the term "release control component" is defined herein as a compound that facilitates the control of the release of the active ingredient, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres, or combinations thereof.

[0203] In certain embodiments, the formulations of the present invention may be, but are not limited to, short-release formulations, rapid-offset formulations, and controlled-release formulations, such as sustained-release formulations, delayed-release formulations, and pulsed-release formulations.

[0204] The term "sustained-release" is used in its traditional sense to refer to a drug formulation that provides a gradual release of the drug over a long period, resulting in a substantially constant blood drug level over a long period, though not necessarily. The duration may be as long as one month or longer, and should be longer than the release of the same amount of drug administered in bolus form.

[0205] Compounds for sustained release can be formulated using a suitable polymer or hydrophobic material that provides sustained-release properties to the compound. Therefore, compounds for use in the methods of this disclosure may be administered, for example, by injection in the form of microparticles, or by implantation in the form of wafers or disks.

[0206] In a preferred embodiment of the present disclosure, the compounds of the present disclosure are administered to a patient, either alone or in combination with another pharmaceutical agent, using a sustained-release formulation.

[0207] The term "delayed release" is used herein in its conventional sense to refer to a drug formulation that provides an initial release of the drug after some delay following administration, and which, while not always, involves a delay of approximately 10 minutes to approximately 12 hours.

[0208] The term "pulsed release" is used herein in its conventional sense, referring to a drug formulation that provides a drug release that results in a pulsed plasma profile of the drug after administration.

[0209] The term "immediate release" is used in its traditional sense to refer to drug formulations that provide drug release immediately after administration.

[0210] As used herein, short-term means periods up to approximately 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 40 minutes, 20 minutes, or 10 minutes after drug administration, and any full or partial increase of any of these periods.

[0211] As used herein, rapid offset refers to periods up to approximately 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 40 minutes, 20 minutes, or 10 minutes after drug administration, and any full or partial increase within those periods.

[0212] Those skilled in the art can recognize or confirm, using only routine experiments, numerous equivalents of the specific methods, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of the present invention and covered by the appended claims. For example, modifications of reaction and assay conditions using only routine experiments by alternative methods recognized in the art should be understood to be within the scope of this application.

[0213] Wherever values ​​and ranges are provided herein, it should be understood that all values ​​and ranges encompassed within those values ​​and ranges are included within the scope of the present invention. Furthermore, all values ​​within these ranges, as well as any upper or lower limits on the ranges of values, are contemplated in this application.

[0214] The following embodiments further illustrate aspects of the present invention. However, they are not intended to limit the teachings or disclosures of the present invention set forth herein. [Examples]

[0215] This specification will be further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, this specification should not be construed as being limited in any way to the following examples, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0216] Example 1: Identification and Characterization of Novel FGF21 Variants To identify novel FGF21 variants exhibiting enhanced binding affinity to β-Klotho, various mutations were generated in the C-terminal tail (CT) of FGF21, the region responsible for the specific binding of FGF21 to β-Klotho (Figures 1A-1C). Additional variants were obtained by generating constructs that instruct the synthesis of fusion proteins attached to the N-terminal region of FGF21. These fusion proteins included the human Fc domain of Sumo protein, mouse albumin, Spitz, and human IgG1 (Figures 2A-2B). Novel FGF21 variants were generated to enhance binding affinity to β-Klotho, increase expression levels, and improve protein stability, resulting in new FGF21 variants with superior pharmacological properties and activity.

[0217] Schematic diagrams illustrating 14 novel FGF21 variants with mutations in the C-terminal tail region are shown in Figures 1A-1C. The constructs containing the FGF21 variants were generated in the form of genetically engineered fusion proteins containing the human IgG1 Fc region linked to its N-terminus by a mobile linker (Figures 2A-2B). This expression system enabled the efficient production of significant quantities of FGF21 variants with high purity and homogeneity for further molecular characterization. The binding affinity of these Fc-FGF21 variants to the soluble extracellular domain (sKLB) of β-Kroto was measured using biolayer interferometry (BLI). The BLI response, with reference subtraction, was fitted to a 1:1 binding model to obtain dynamic parameters and dissociation constants (Figures 3A-3N).

[0218] This study compared kinetic parameters obtained from BLI measurements to monitor whether the Fc fusion at the N-terminus of FGF21 variants can alter its binding to sKLB. By comparing the binding kinetic parameters between Fc-FGF21 wt (Figure 3N) and His-FGF21 wt (Figure 4B), the overall dissociation constant (K) was determined. D A small and non-significant difference was observed in ), which strongly suggests that the N-terminal Fc fusion on the FGF21 variant does not affect the binding affinity to sKLB.

[0219] The binding kinetic parameters from BLI measurements (Figures 3A-3N) indicate that most Fc-FGF21 variants showed improved binding affinity to sKLB compared to Fc-FGF21 wt. Furthermore, due to their different characteristics (Figures 3A-3M), high-affinity variants can be identified using the features of the BLI sensor gram itself, which is k off This is because the change in velocity reflected an improvement in overall binding affinity. This can be easily seen from the dynamic parameter values ​​listed in Figure 3M; K between Fc-FGF21 variants. D The difference in values ​​is almost exclusively k off The difference in values ​​is the origin.

[0220] To further investigate the properties of FGF21 CV16, this study established and compared the binding affinity of FGF21 wt and FGF21 CV16 to sKLB using an additional approach (Figures 4A-4B). In these experiments, His-FGF21 wt or His-FGF21 CV16 was immobilized on an amine-reactive biosensor AR2G using a standard amine coupling reaction. The sensor was then immersed in wells containing a range of sKLB solutions. Note that the absence of Lys residues in the CT region of all three FGF21 proteins prevented the CT region from covalently binding to the sensor, allowing for proper binding of sKLB after the amine coupling reaction. Binding kinetic parameters from BLI measurements (Figures 4A-4B) showed that FGF21 CV16 exhibited approximately 40-fold higher binding affinity to β-Klotho compared to FGF21 wt, and that the overall K between FGF21 wt and FGF21 CV16 for sKLB was significantly higher. D The difference in values ​​is almost exclusively k off It was confirmed that the difference in values ​​was the origin.

[0221] Example 2: Enhanced cell activity of a novel FGF21 variant To investigate the ability of His-FGF21 wt and His-FGF21 CV16 to stimulate cellular signaling, L6 myoblast cell lines co-expressing FGFR1c and β-Klotho were stimulated with increasing concentrations of either His-FGF21 wt or His-FGF21 CV16. Phosphorylated MAPK (pMAPK) levels were monitored using immunoblotting analysis, with 5 nM FGF1 used as a positive control. The results, shown in Figure 5A, demonstrate that both His-FGF21 wt and His-FGF21 CV16 activated cellular signaling in a dose-dependent manner in L6 myoblast cell lines co-expressing FGFR1c and β-Klotho, as monitored by pMAPK levels, with His-FGF21 CV16 exhibiting higher potency compared to His-FGF21 wt. Rat L6 cells are a desirable host cell line for functional assays of FGFR-mediated signaling activity because they are known to have minimal FGFR expression. FGF21 is selected because it is known to activate signaling via the c-isoform of FGFR1, but not via the b-isoform, and to a lower degree via FGFR2 and FGFR3. It has also been reported that FGFR4-mediated signaling is undetectable upon FGF21 stimulation. All FGF21-mediated signaling requires the presence of β-Klotho.

[0222] To gain further insight into the cellular activity of FGF21 CV16, this study analyzed its ability to stimulate glucose uptake by differentiated 3T3-L1 adipocytes (Figure 5B). Chemical induction of 3T3-L1 fibroblasts into adipocytes significantly increased β-Klotho expression levels, and FGFR signaling could be activated upon addition of human FGF21. FGF21-induced signaling then induces the expression of GLUT4, the major transporter responsible for cellular glucose uptake. Therefore, it was anticipated that the level of cellular glucose uptake in 3T3 adipocytes could be used to assess the efficacy of FGF21 variants in a well-controlled system. In this study, after differentiating 3T3-L1 fibroblasts into adipocytes, cells were treated in quadruple chains with a range of His-FGF21 wt and His-FGF21 CV16 concentrations, and the levels of cellular glucose uptake were compared. As shown in Figure 5B, His-FGF21 wt and His-FGF21 wt CV16 induced glucose uptake in 3T3-L1 adipocytes in a dose-dependent manner, increasing the EC2 levels to 1.84 nM and 0.29 nM, respectively. 50 The value was [value]. This also confirms that the binding affinity of the FGF21 variant to sKLB measured by BLI, as well as the cellular activity monitored at the level of phosphorylated MAPK in L6 cells co-expressing β-Klotho and FGFR1c, can be directly converted to other cellular activities such as the level of glucose uptake in adipocytes.

[0223] This study then investigated the Ca2+ levels in pancreatic acinar (AR42J) cells stimulated with either His-FGF21 wt or His-FGF21 CV16. 2+ The release levels were compared. Ca in AR42J cells (expressing endogenous FGFR1c and β-Klotho) 2+ The temporal changes in release were measured quantitatively. Ca 2+ For the release experiment, fluorescent Ca was added to the cells. 2+ The index was loaded and then stimulated with increasing concentrations of His-FGF21 wt or His-FGF21 CV16. Then Ca2+ The emission level was analyzed as a change in fluorescence emission. The experimental results shown in Figure 6A indicate that Ca 2+ Release was not detected upon stimulation with 0.5 nM His-FGF21 wt, but stimulation of AR42J cells with 0.5 nM His-FGF21 CV16 resulted in strong Ca release in these cells. 2+ This indicates that the release was induced. Also, robust Ca 2+ Release was detected upon stimulation with 2 nM His-FGF21 CV16, but stimulation with 2 nM His-FGF21 wt resulted in a small but detectable level of Ca. 2+ This resulted in the release of Ca by saturating His-FGF21 wt or by His-FGF21 CV16 (50 nM). 2+ Robust Ca similar to release 2+ It induced a release signal.

[0224] The cellular activity of AR42J cells stimulated with either His-FGF21 wt or His-FGF21 CV16 was monitored by the level of phosphorylated MAPK in immunoblotting analysis. The results from the experiment, presented in Figure 6B, clearly demonstrate that the potency of His-FGF21 CV16 in activating the MAPK response in AR42J cells was higher than that of His-FGF21 wt, and the degree of improvement was similar to the enhancement of activity in other stimulated cells, such as L6 cells co-expressing FGFR1c and β-Klotho, as shown in Figure 5A.

[0225] As mentioned, additional variants were obtained by generating constructs that direct the synthesis of fusion proteins linked to the N-terminal region of FGF21, including a small ubiquitin-like modifier (SUMO), mouse serum albumin (mSA), Spitz, and the human Fc region of human IgG1. The sequences of these fusion partners are listed below. TIFF2026534020000003.tif155146

[0226] These fusion constructs of FGF21 variants were generated to improve the expression levels, biochemical properties, and / or superior pharmacological activity of novel FGF21 variants. Other modifications that can be applied to improve pharmacological properties include, but are not limited to, chemical modifications such as PEGylation, lipidation, and glycosylation, as well as other N-terminal fusion constructs such as Fc regions derived from other IgG subtypes, human serum albumin, and albumin-binding domains.

[0227] Binding dynamics parameters from BLI measurements (Figures 3A-3N, 4A-4B, 7A-7C, 8A-8C, and 9A-9C) indicate that all fusion partner proteins on the N-terminus of the FGF21 variant did not alter the binding affinity of the FGF21 variant to sKLB. Importantly, all fusion proteins with FGF21 CV16 retain high affinity binding to sKLB, similar to His-FGF21 CV16. Furthermore, all fusion proteins exhibit cellular activity similar to His-FGF21 CV16 in L6 cells co-expressing FGFR1c and β-Klotho (Figures 7C, 8C, and 9C). This confirms that enhanced binding affinity to β-Klotho directly affects the cellular potency of FGF21, and that FGF21 CV16 exhibits significantly enhanced potency compared to FGF21 wt, regardless of the fusion partner on the N-terminus.

[0228] MAPK activation assayed by immunoblotting: To investigate the ability of other FGF21 variants to stimulate cellular signaling, L6 myoblast cell lines co-expressing FGFR1c and β-Klotho were stimulated with increasing concentrations of the FGF21 variant, and phosphorylated MAPK (pMAPK) levels were monitored by immunoblotting analysis. The results shown in Figure 16A demonstrate that both His-FGF21 wt and His-FGF21 CV30 activated cellular signaling in a dose-dependent manner when monitored by pMAPK levels in L6 myoblast cell lines co-expressing FGFR1c and β-Klotho, with His-FGF21 CV30 exhibiting higher potency compared to His-FGF21 wt. As shown in Figure 16B, both Fc-FGF21 wt and Fc-FGF21 CV16 activated cell signaling in a dose-dependent manner when monitored by pMAPK levels in L6 myoblast cell lines co-expressing FGFR1c and β-Klotho, demonstrating that Fc-FGF21 CV16 exhibited higher potency compared to His-FGF21 wt. As shown in Figure 16C, both Fc-FGF21 wt and Fc-FGF21 CV30 activated cell signaling in a dose-dependent manner when monitored by pMAPK levels in L6 myoblast cell lines co-expressing FGFR1c and β-Klotho, demonstrating that Fc-FGF21 CV30 exhibited higher potency compared to His-FGF21 wt. This confirms that enhanced binding affinity to β-Klotho directly affects the cellular efficacy of FGF21, and that FGF21 CV30 exhibits significantly enhanced efficacy compared to FGF21 wt, regardless of its N-terminal fusion partner. This confirms that Fc-FGF21 CV16 exhibits significantly enhanced efficacy compared to Fc-FGF21 wt.

[0229] MAPK activation assayed by AlphaLISA: To investigate the ability of other FGF21 variants to stimulate cellular signaling, L6 myoblast cell lines co-expressing FGFR1c and β-Klotho were stimulated with increasing concentrations of the FGF21 variant, and phosphorylated MAPK (pMAPK) levels were monitored with AlphaLISA (Revvity). The results shown in Figure 17A demonstrate that both Fc-FGF21 wt and Fc-FGF21 CV25 activated cellular signaling in a dose-dependent manner when monitored by pMAPK levels in L6 myoblast cell lines co-expressing FGFR1c and β-Klotho, with Fc-FGF21 CV25 exhibiting higher potency compared to Fc-FGF21 wt. As shown in Figure 17B, both FGF21 wt (untagged) and FGF21 CV16 activated cell signaling in a dose-dependent manner when monitored by pMAPK levels in L6 myoblast cell lines co-expressing FGFR1c and β-Klotho, demonstrating that FGF21 CV16 exhibited higher potency compared to FGF21 wt. As shown in Figure 17C, both FGF21 wt (untagged) and FGF21 CV25 activated cell signaling in a dose-dependent manner when monitored by pMAPK levels in L6 myoblast cell lines co-expressing FGFR1c and β-Klotho, demonstrating that FGF21 CV25 exhibited higher potency compared to FGF21 wt. This confirms that enhanced binding affinity to β-Klotho directly affects the cellular potency of FGF21, and that FGF21 CV25 exhibits significantly enhanced potency compared to FGF21 wt, with or without a fusion partner at the N-terminus. This confirms that FGF21 CV16 exhibits significantly enhanced potency over FGF21 wt, demonstrating that a fusion partner is not required.

[0230] Example 3: MD simulation results of the C-tail of FGF21 CV16 provide insights into the mechanism of high-affinity binding to β-Klotho. This study applied Gaussian accelerated molecular dynamics (GAMD) simulations, an efficient unconstrained enhanced sampling method that reduces the energy barrier to conformational transitions of simulated systems, to investigate the conformational space of the C-terminal tail (CT) of FGF21 wt (CTWT) and the C-terminal tail (CT16) of FGF21 CV16. Peptide simulations were limited to 10–14 amino acid lengths because peptides longer than 20 amino acids often collapse, forming metastable, partially folded states that reduce sampling efficiency. The first stretch of FGF21, the site-1 interaction region (SIR1), consists of residues 186–198, and the second stretch, the site-2 interaction region (SIR2), consists of residues 200–209.

[0231] Using GAMD, the potential free energy of the simulated system can be derived. The folding free energy can be visualized in the mean force potential (PMF) energy landscape, which shows the energy wells of the states. This landscape was constructed by correlating the energy of the simulated system with the degree of SIR1 peptide folding, i.e., the backbone RMSD of residues D192-V197, and the distance between the carboxylate side chain of D187 and the backbone amide of S190, which form hydrogen bonds that maintain the turn structure in SIR1. The free energy landscape for SIR1 of wild-type FGF21 shows a wide conformational space with multiple shallow metastable energy wells (Figure 10A). Notably, the folded states of both FGF21 CTs were hardly sampled by GAMD even after a total simulation time of 1.2 μs, suggesting that the SIR1 region of FGF21 is intrinsically disordered and does not form the turn conformation observed in atomic structure until it interacts with β-Kroto. This was also observed in the conformational population landscape, which provides information about the sampling frequency of folded, intermediate, and unfolded states during GAMD simulations. SIR1 of wild-type FGF21 sampled mostly unfolded states (67.3%), intermediate states less frequently (30%), and folded states very rarely (1%) (Figure 10B).

[0232] SIR2 in FGF21 wt forms a single α-helical turn when bonded to β-kroto, as shown in the crystal structure. The folding energy theory of SIR2 could be investigated by the difference in folding free energy between the folded and unfolded conformational states. Three distances between backbone amide nitrogen atoms, R203-A208, S204-A208, and S204-Y207, which form backbone amide hydrogen bonds to maintain the α-helical turn structure of the SPS motif, can be used as structural metrics. Using the S204-Y207 backbone amide distance as an example (Figures 10C-10D), the folding free energy landscape of the wild-type FGF21 SIR2 region shows two well-defined energy wells separated by a roughly 2.5 kcal / mol activation free energy barrier (ΔG‡) between the folded and unfolded conformational states. On the other hand, the intermediate state (RMSD of 1.0 Å to 3.0 Å) is energetically similar to the folded state (RMSD < 1.0 Å). In other words, the folded state and the intermediate state are distinct from the unfolded state, even when FGF21-CT is not bound, but they will frequently exchange because there is no significant energy barrier between them. The conformational population landscape of WT FGF21 SIR2 suggests that the unfolded state (58%) is sampled more frequently than the folded state (15%) and intermediate state (27%) combined. This is in contrast to SIR1, where multiple metastable unfolded and intermediate states are found, and the folded state is hardly sampled (Figures 8A-8B). SIR2 has only a few metastable states, and the folded state is frequently sampled, which indicates that the α-helical turn of the SPS motif is intrinsically stable and accessible even in the unbound state.

[0233] Analysis of a computational model of CT16 constructed based on the conformation of the CT region (CTWT) of WT FGF21 bound to sKLB suggests that the two tryptophan substitutions in CT16, R203W and A208W, may interact with each other through edge-to-face π-π stacking between their indole rings and a hydrogen bond between the A208W indole NH and the S204 carbonyl oxygen. It is hypothesized that these two intramolecular interactions may bring R203W and A208W into close proximity and maintain them in such a configuration for a longer period of time, which in turn promotes the tendency of the SPS motif residues between them to adopt an α-helical turn, thus shifting the equilibrium to favor the folded conformational state for β-Klotho binding.

[0234] To test this hypothesis, this study examined the populations of folded and unfolded conformational states sampled during GAMD simulations of SIR2 in FGF21CT16. Analysis of population ratios for GAMD simulations shows that SIR2 in CT16 has a higher population fraction of folded states (39%) and a lower fraction of intermediate and unfolded states combined (61%) than SIR2 in FGF21CTWT shown in Figure 8D. This suggests that amino acid substitutions in CT16 shift the folding equilibrium towards the folded state. Therefore, the R203W / A208W substitution in FGF21 CV16 promotes a tendency for SPS motif folding with specific intramolecular interactions compared to WT, thereby reducing the entropy cost during folding events. This increase in folded FGF21 SPS motifs in FGF21 CV16 may be strongly related to binding to β-Klotho, where folded SPS motifs are more likely to bind to the target than unfolded SPS motifs. Furthermore, the mutation may also reduce the rate of unfolding of folded SPS motifs, and thus retain the folded state population for a longer duration. These retention changes may be related to the dissociation constant of the FGF21-CT-β-Klotho complex, where endogenous unfolding of bound CT may lead to its eventual dissociation. Therefore, reducing the rate of unfolding of bound CT may reduce the overall interaction off-rate between FGF21 and β-Klotho.

[0235] Example 4: Crystal structure of FGF21 CV16 peptide in complex with sKLB To gain insights into how FGF21 CV16 binds to sKLB with high affinity, the present study determined the crystal structure of sKLB in complex with the C-terminal tail peptide (CT16) derived from FGF21 CV16. When sKLB containing two mutations N308Q and N611Q in complex with CT16 and the nanobody Nb30 was screened for crystallization, diffraction-quality crystals were obtained from multiple crystallization conditions. After optimization of each crystallization condition, large plate-like and pyramid-shaped crystals could be obtained by the hanging-drop vapor diffusion method using a solution containing 13% polyethylene glycol 3,350 and 10 mM citric acid / bis-tris (1:1) buffer at pH 5.0. X-ray diffraction data from the crystals were processed and scaled to a resolution cutoff of 3.0 Å. The structure was determined by molecular replacement using the existing model of sKLB in complex with Nb30, followed by iterative refinement steps to generate the model of CT16. The final model (R work / R free =22.8 / 25.7%) exhibits favorable overall geometry and atomic parameters.

[0236] sKLB contains two glycoside hydrolase-like domains, KL1 and KL2 (Figure 11A), each possessing its catalytic Glu residue replaced with either Asn or Ala, thereby becoming an inactive glycoside hydrolase, and the two domains are connected by a linker region. The two domains are intrinsically dynamic, and the interdomain orientation varies depending on crystallization packing conditions and / or the presence of various nanobodies. Just like the C-terminal tail peptide (CTWT) from wild-type FGF21, the C-terminal tail peptide (CT16) from FGF21 CV16 binds to sKLB across the KL1 and KL2 domains (Figure 11A). Comparison of the crystal structures of sKLB in complex with CT16 and sKLB in complex with CTWT (PDB ID: 5VAQ) reveals that the overall conformation of CT16 when bound to sKLB is very similar to that of CTWT. Figures 11B–11C show the site-1 interaction regions (SIR1) for CT16 and CTWT, respectively, while Figures 11D–11E show the site-2 interaction regions (SIR2) for CT16 and CTWT, respectively. Both CT16 and CTWT's SIR1 adopt a unique conformation consisting of multiple turns, which creates a fairly large interface and a KL1-KL2 interface that interacts with the surface of sKLB in the KL1 domain. The amino acid substitution L194F in CT16, within SIR1, did not induce any conformational changes on CT16 compared to CTWT. Both CT16 and CTWT's SIR2 adopt a small helical structure to bind to the sKLB pocket in KL2. Within SIR2, four amino acid substitutions in CT16, Q201I, R203W, Y207W, and E208W, were present, and they did not induce significant conformational changes on CT16 compared to CTWT.The linker regions (residues 197-200) in CT16 and CTWT are structurally the most distinct regions when the overall structures are superimposed. This may be due to the intrinsic flexibility of the linker regions in the peptide and / or the flexibility in the interdomain orientation of β-Klotho.

[0237] The overall structure of sKLB bound to CT16 is similar to that of sKLB bound to WT (Figure 12A); both structures contain two glycoside hydrolase-like domains, KL1 and KL2, which are similarly occupied by the peptide. The superposition of Cα atoms from the overall structure results in a root mean square deviation (RMSD) of 2.12 Å, while the superposition of individual domains results in Cα RMSDs of 0.29 Å and 0.46 Å for KL1 (residues 53-507) and KL2 (residues 515-969), respectively. The results shown in Figure 12B (site 1) and Figure 12C (site 2) demonstrate that each site of sKLB bound to CT16 consists of residues that are nearly identical to those in sites 1 and 2 of sKLB bound to CTWT, and that the structural folds of each site are not altered by the binding of different peptides.

[0238] Due to intrinsic flexibility in interdomain orientation, sKLB can crystallize in various conformations depending on the crystallization packing interface created by molecular interactions facilitated by the utilized nanobodies. The movement of these domains in β-Kroto may be necessary to form sufficient intermolecular interactions for the formation of a signaling complex. A comparison of the structures of sKLB bound to CTWT and sKLB bound to CT16, shown in Figures 12A-12C, suggests that interdomain flexibility may have caused structural differences originating from molecular interactions during the crystallization process. Indeed, analysis of these two structures using DynDom (www.cmp.uea.ac.uk / dyndom) results in a 13.2° change in interdomain angle.

[0239] Results from binding affinity measurements strongly indicate that the binding affinity of FGF21 CV16 and its fusion construct to sKLB is significantly higher than that of wild-type FGF21 and its fusion construct. To understand and identify the possible molecular interactions responsible for such a difference, this study examined molecular interactions at SIR2, where the majority of amino acid changes reside. A detailed comparison of molecular interactions at site 2 for sKLB in complex with CT16 and sKLB in complex with CTWT is shown in Figures 13A–13B. Both CT16 (shown in orange in Figure 13A) and CTWT (shown in purple in Figure 13B) exhibit short α-helical segments surrounding the SPS motif, and both peptides are in contact with the same region in sKLB, as also shown in Figures 12A–12C. Analysis from MD simulations suggests that the entropy contribution of the W203-W208 interaction within CT16 influences the binding affinity to β-Klotho. On the other hand, the intermolecular interactions visualized in the crystal structure explain the enthalpy contribution of the binding affinity changes caused by amino acid substitutions. Each substitution in SIR2 creates a small difference in the intermolecular interaction between CT16 and sKLB versus the interaction between CTWT and sKLB: the R203W substitution creates a hydrophobic interaction with L862 and T860; Y207W strengthens the hydrophobic interaction with P644 and H646; and finally, E208W creates a π-π interaction between the indole ring of W208 (CT16) and the imidazole ring of H646 (sKLB). All of these changes, along with the improvement in hydrophobic interactions due to the L194F substitution, result in a positive contribution to the overall binding affinity of FGF21 CV16 to β-Klotho, leading to a significant improvement in cellular efficacy.

[0240] Example 5: FGF21 expression in the liver is upregulated under various nutritional 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 induces weight loss, improves insulin sensitivity, and lowers blood glucose and triglyceride levels. Therefore, considerable effort has been made to develop FGF21 analogs as a potential novel treatment for metabolic disorders such as type II diabetes and obesity, as well as NASH and NFLD. Evidence also points to the potential use of FGF21 as a therapeutic agent for liver diseases: FGF21 is normally expressed in the liver; recombinant FGF21 has been shown to be non-mitogenic when pharmacologically administered to mice; and transgenic FGF21 mice that produce FGF21 above physiological levels in the liver have an extended lifespan. FGF21 can be used to treat mouse models of pancreatitis and acute alcohol toxicity. Therefore, FGF21 C-tail variants with increased physiological activity and improved pharmacological properties, as well as their fusion constructs, would be extremely useful as therapeutic agents for human diseases.

[0241] This study provides a means to improve the efficacy of wild-type FGF21. Experimental results for FGF21 CV16, one of the FGF21 C-terminal tail variants with superior binding affinity and cellular activity to β-klotho, suggest that a combination of improved interactions between the FGF21 C-tail and β-klotho, as well as improved intramolecular interactions within the C-tail, may significantly influence the in vivo biological activity and therapeutic utility of FGF21. The combined effects of mutations L194F, Q201I, and Y207W, which directly interact with β-klotho and contribute enthalpy, and mutations R203W and A208W, which reduce the entropy cost of binding by increasing the population of folded SPS motifs, may together contribute to an overall improvement in the binding kinetics and cellular activity of FGF21 CV16 to β-klotho, as shown in Figure 14. The observed on-rate is likely the result of cooperative interactions between sites 1 and 2, which occur through a multi-step process. This may explain why changes in on-rate cannot be measured solely through substitutions in SIR2, such as R203W / A208W.

[0242] In summary, this study identified an FGF21 variant that binds to β-klotho with high affinity via an entropy-driven mechanism and enables robust activation of FGFR signaling in the presence of β-klotho.

[0243] Example 6: Materials and Methods Measurement of recombinant protein production and binding affinity All coupling dynamics parameters were derived from biolayer interferometry (BLI) measurements using the Octet RED96 system (ForteBio, Molecular Devices).

[0244] An FGF21 variant fused to the Fc region of human IgG1 at its N-terminal region was expressed in Expi293F cells and purified using protein A sepharose 4B (Thermo-Fisher) followed by size exclusion chromatography. An FGF21 variant with an 8-histidine tag at its N-terminal region (His-FGF21), an FGF21 variant fused to the O-glycosylation region of Drosophila melanogaster Spitz at its N-terminal region (Spitz-FGF21), or an FGF21 variant fused to mouse serum albumin at its N-terminus (mSA-FGF21) was expressed in Expi293F cells and purified using Ni-NTA agarose (Qiagen) or nickel sepharose excel (GE healthcare) followed by size exclusion chromatography.

[0245] sKLB was expressed and purified according to the protocol described in Lee et al. (Nature 553(7689): 501-505, doi: 10.1038 / nature25010. (2018)). Briefly, the DNA encoding amino acids 30-983 (sKLB), followed by the tobacco etch virus (TEV) protease cleavage site, the linker (GGGG), and the region encoding the human IgG1 Fc region were subcloned into the pCEP4 vector (Thermo-Fisher Scientific Inc.) (sKLB-Fc). The resulting plasmid was transfected into HEK293EBNA cells and selected with 250 μg / mL hygromycin B to generate cells that stably express sKLB-Fc. sKLB-Fc was purified from culture medium with recombinant protein A Sepharose 4B (Thermo-Fisher), followed by incubation with TEV enzyme to cleave the C-terminal Fc tag. The Fc fragments and undigested sKLB-Fc were removed by incubation with recombinant protein A Sepharose 4B, and the resulting sKLB was further purified by cation exchange chromatography. The purified protein solution was concentrated to 1 mg / mL and rapidly frozen until ready for further use. For crystallization, sKLB-Fc containing the N308Q / N611Q mutation was expressed in HEK293EBNA cells and purified to resemble wild-type sKLB.

[0246] Fc-FGF21 protein at a concentration of 5 μg / mL was loaded onto an anti-human IgG1 Fc biosensor probe (AHC) in PBS containing 0.01% BSA and 0.002% Tween-20 (PBS-KB). His-FGF21, SUMO-FGF21, Spitz-FGF21, or mSA-FGF21 protein at a concentration of 2 μg / mL was immobilized onto an Amine Reactive 2nd Generation (AR2G) biosensor using a standard amine coupling method utilizing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) in 10 mM sodium acetate buffer at pH 6.0. The FGF21 variant-loaded sensors were briefly washed and then immersed in wells containing a range of purified sKLB concentrations to record the response. The resulting sensorgram is referenced using parallel buffer control subtraction and fitted overall to a 1:1 Langmuir-coupled model using the ForteBio Data Analysis 10.0 software provided by the manufacturer to determine the reaction rate constant (k on and k off ) and the dissociation constant K D We made that decision.

[0247] Untagged FGF21 variants were generated with an N-terminal 6-histidine tag and a tobacco etch virus (TEV) protease moiety (ENLYFQG). The proteins were expressed in Escherichia coli (E. coli), purified by nickel affinity chromatography, and subsequently the 6-histidine tag was cleaved with TEV protease (GenScript). The cleaved tag, histidine-tagged TEV protease, and any uncleaved histidine-tagged FGF21 were removed by repeated nickel affinity chromatography followed by size exclusion chromatography. The protease was cleaved between the Q and G residues of the protease moiety, leaving the N-terminal glycine on the purified FGF21 variant.

[0248] Cell-based activity assays Rattus norvegicus L6 myoblasts 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. The cells were starved overnight in DMEM and stimulated at 37°C for 10 minutes with either 5 nM FGF1 (as control) or an FGF21 variant at concentrations ranging from 0.05 to 10 nM. The cells were washed with cold PBS and lysed in ice-cold lysis buffer containing a protease inhibitor (50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 25 mM NaF, 1 mM MgCl2, 10% glycerol, 1 mM sodium orthovanadate, 1 mM PMSF). Next, the lysates were subjected to SDS-PAGE, followed by immunoblotting with either an anti-phospho-MAPK antibody, an anti-MAPK antibody, or an anti-β-tubulin antibody, according to Lemmon et al. (Cell 141, 1117-1134, doi:10.1016 / j.cell.2010.06.011 (2010)).

[0249] AlphaLISA Assay: To provide additional insights into our cell-based activation assay, this study detected total MAPK and phosphorylated MAPK using an L6 cell line stably co-expressing FGFR1c and β-Klotho and a sandwich immunoassay. The assay was performed by serum-starving L6 cells co-expressing FGFR1c and β-Klotho for 2 hours to reduce background phosphorylation of MAPK, followed by stimulation with FGF21 variants in the protein dilution range of 0.09–24 nM at 37°C for 10 minutes. The 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 ERK1 / 2, a phosphorylated MAPK family member, were measured using the AlphaLISA SureFire Ultra p-ERK1 / 2(Thr202 / Tyr204) assay. The kit specifically measures Thr202 / Tyr204 in phosphorylated ERK1 and Thr185 / Tyr187 in ERK2. Total ERK1 / 2 levels were also measured separately for each sample using a similar sandwich immunoassay (AlphaLISA SureFire Ultra total ERK1 / 2) that detects both phosphorylated and unphosphorylated ERK1 / 2. To adjust for cell number variability between samples, the phosphorylated MAPK signal for each sample was normalized relative to the total MAPK signal. Samples were run in pairs, and the results were averaged. The mean and standard deviation were graphed using GraphPad Prism.

[0250] Glucose uptake assay 3T3-L1 mouse embryonic fibroblasts (ATCCs) were maintained in DMEM containing 10% calf serum (BCS), and differentiation was induced using DMEM containing 10% FBS, 0.25 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 5 μg / mL bovine insulin. 48 hours after induction, the medium was replaced with DMEM containing 10% FBS and 5 μg / mL bovine insulin, and incubated for a further 48 hours. Differentiated cells were maintained in DMEM containing 10% FBS for 10–15 days before treatment with an FGF21 variant.

[0251] 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). Cellular glucose uptake was monitored by detecting the total amount of 2-deoxyglucose-6-phosphate (2DG6P) in the cells using the Glucose Uptake-Glo Assay (Promega).

[0252] Crystallization and structural determination of sKLB bound to CT16 Refined sKLB 2Q This was mixed with nanobodies and purified by size exclusion chromatography. CT16, a peptide corresponding to the C-terminal region (amino acids L174-S209) of FGF21 CV16, was concentrated in sKLB within the complex with the nanobodies. 2Q The mixture was added and incubated at 4°C for 30 minutes, followed by a screening of crystallization. sKLB in complex with nanobody and CT16. 2Qwas mixed with an equal volume of well solution containing 13% PEG 3,350 at pH 5.0, 0.05 M citric acid / 0.05 M BIS-TRIS, and yielded plate-like crystals when equilibrated for 5 to 10 days using the hanging drop vapor diffusion method. The crystals were cryopreserved by gradually transferring the crystals to a mother liquor supplemented with 30% ethylene glycol before being flash-frozen in liquid nitrogen. X-ray diffraction data were collected at the NE-CAT beamline 24-ID-E of the Advanced Photon Source, Argonne, IL. The diffraction data were processed with XDS, the structure was determined by molecular replacement with PHASER, and refinement was performed with PHENIX while manually inspecting the quality in COOT.

[0253] Molecular dynamics (MD) simulation Gaussian accelerated molecular dynamics (GAMD) simulation, which is an unconstrained enhanced sampling method built based on the conventional molecular dynamics (cMD) method implemented in the AMBER simulation package, was used for efficient sampling of large conformational spaces.

[0254] The initial structure of the FGF21 CTWT peptide was either extracted from PDB ID: 5VAQ ("folding") or generated using the Schrodinger Maestro program v2019-1 ("linear"), parameterized with the Amber ff14SB force field, and solvated in a truncated octahedron TIP3P water box using Amber18. The simulation system was brought into charge equilibrium by adding a mono-ion. The SHAKE algorithm was possible for 2-fs simulation time steps. The Particle Mesh Ewald method and a long-range cutoff of 10 Å were used for unbound interactions. For the CT16 and its variant systems, simulations were performed from the linear form, while the double-cysteine ​​variant system started from the folded form. To avoid the peptide collapsing into a "partially folded state" that limits sampling, only short stretches of the peptide, i.e., 10-14 amino acids, were used. The simulation preparation was similar, involving a minimization change over 30,000 steps, followed by heating to 310 K at 400 ps and equilibration at 500 ps. All simulations were run using the PMEMD.CUDA function on an Nvidia Volta V100 GPU.

[0255] A 2 ns cMD simulation was performed to collect initial parameters for the GAMD run, followed by a 20 ns GAMD adjustment run and a 300 ns GAMD production run with a 200 ps window size. The GAMD simulation used dual energy boosts and an upper limit of 6.0 kcal / mol as the standard deviation of the boosts. Two independent trajectories were performed for each system. Post-processing of the GAMD trajectories was performed through CPPTRAJ to calculate structural criteria such as residue distance, torsion angle, and secondary structure defined by DSSP. A total of 600–1,200 ns of independent trajectories were collected, and frames were extracted at 20-ps intervals. The energy data generated by GAMD was reweighted using a cumulant expansion up to the second order using a modified Python script for GAMD to calculate the mean force potential (PMF) of FGF-CT in GAMD. PMF data was plotted with various structural criteria to identify the most relevant metrics and generate a two-dimensional (2D) folding free energy landscape with a maximum energy cutoff of 5.0 kcal / mol. In addition, the 2D conformational population landscape was plotted with structural criteria to visualize the frequency of MD frames falling within the criterion bins. This population information could also be used to calculate the folding free energy difference by comparing the folded and unfolded populations.

[0256] Estimation of free energy for structure formation The difference in folding free energy (ΔG) between a folded conformational state and an unfolded conformational state. folding ) relates to the relative group of conformations through statistical mechanics and can be calculated by equation (1), TIFF2026534020000004.tif8144In formula, ΔG folding P is the free energy of protein folding, allThis represents the total set of all states, and p folded and p unfolded These represent the folded and unfolded states, respectively. T is the simulated temperature of 310 K, and R is 1.987 × 10⁻⁶ -3 It is the gas constant in kcal / K / mol.

[0257] Example 7: Arrangement of structures The protein and nucleic acid sequences of the constructs used in this study are listed below. In certain embodiments, the constructs of the present invention correspond to those listed below minus the signal peptide. TIFF2026534020000005.tif222146TIFF2026534020000006.tif222146TIFF2026534020000007.tif222146TIFF202 6534020000008.tif222146TIFF2026534020000009.tif222146TIFF2026534020000010.tif222146TIFF20265340200 00011.tif222146TIFF2026534020000012.tif222146TIFF2026534020000013.tif222146TIFF2026534020000014.t if222146TIFF2026534020000015.tif222146TIFF2026534020000016.tif222146TIFF2026534020000017.tif222146 TIFF2026534020000018.tif222146TIFF2026534020000019.tif222146TIFF2026534020000020.tif222146TIFF202 6534020000021.tif222146TIFF2026534020000022.tif222146TIFF2026534020000023.tif222146TIFF20265340200 00024.tif222146TIFF2026534020000025.tif222146TIFF2026534020000026.tif221146TIFF2026534020000027.t if222146TIFF2026534020000028.tif221146TIFF2026534020000029.tif222146TIFF2026534020000030.tif209146

[0258] Enumerated aspects In some aspects, the present invention relates to the following non-limiting embodiments. Embodiment 1: A non-naturally soluble construct comprising an FGF21 polypeptide comprising residues 186-198 and 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: A construct of Embodiment 1, wherein the FGF21 polypeptide contains 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: A structure of any one of Embodiments 1 to 2, wherein at least one of the following applies: (a) The FGF21 polypeptide contains residues 186-209 of FGF21; (b) The FGF21 polypeptide comprises residues 169-209 of FGF21; (c) The FGF21 polypeptide contains residues 29 to 209 of FGF21. Embodiment 4: A construct of any one of Embodiments 1 to 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 residues 200 to 209 of FGF21. Embodiment 5: A structure of any one of Embodiments 1 to 4, wherein at least one of the following applies: (a) The FGF21 polypeptide comprises mutants L194F, V197I, Q201L, R203W, Y207W, A208W, and S209G, as well as the sequence RKK conjugated to the C-terminus of residues 200-209 of FGF21; (b) The FGF21 polypeptide comprises mutants L194F, V197I, Q201R, R203W, Y207W, A208W, and S209G; (c) The FGF21 polypeptide comprises mutants L194F, V197I, Q201R, R203W, Y207W, and A208W; (d) The FGF21 polypeptide comprises mutants L194F, V197L, Q201R, R203W, Y207W, A208W, and S209G; (e) The FGF21 polypeptide comprises mutants L194F, V197L, Q201R, R203W, Y207W, and A208W; (f) The FGF21 polypeptide comprises mutants L194F, Q201I, R203W, Y207W, and A208W; (g) The FGF21 polypeptide comprises mutants L194F, Q201L, R203W, Y207W, A208W, and S209G, as well as the sequence RKS conjugated to the C-terminus of residues 200-209 of FGF21; (h) The FGF21 polypeptide comprises mutants L194F, Q201P, R203W, Y207W, A208W, and S209G, as well as the sequence RRS conjugated to the C-terminus of residues 200-209 of FGF21; (i) The FGF21 polypeptide comprises mutants L194F, Q201R, R203W, Y207W, A208W, and S209G, as well as the sequence RRS conjugated to the C-terminus of residues 200-209 of FGF21; (j) The FGF21 polypeptide comprises the mutants L194F, Q201R, R203W, Y207W, and A208W; (k) The FGF21 polypeptide comprises mutants L194F, Q201W, R203W, Y207W, A208W, and S209G; (l) The FGF21 polypeptide comprises mutants L194F, R203W, Y207W, and A208W; (m) The FGF21 polypeptide comprises mutants R203W, Y207W, and A208W. Embodiment 6: A construct according to any one of Embodiments 1 to 5, wherein the FGF21 is human FGF21 having the protein sequence shown in SEQ ID NO:1. Embodiment 7: A construct of any one of Embodiments 1 to 6, further comprising a stability-enhancing domain. Embodiment 8: The construct according to Embodiment 7, wherein the stability-enhancing domain is attached to the N-terminus of the FGF21 polypeptide, or the stability-enhancing domain is attached to the C-terminus of the FGF21 polypeptide. Embodiment 9: A construct of any one of Embodiments 7 to 8, wherein the stability-enhancing domain is at least one selected from the group consisting of albumin, the Fc region of an antibody, glutathione S-transferase, small ubiquitin-like modifier (SUMO), the O-glycosylation region of Drosophila Spitz protein, and thioredoxin. Embodiment 10: A construct according to any one of Embodiments 7 to 9, wherein the FGF21 polypeptide and the stability-enhancing domain are linked via a linker polypeptide containing about 1 to 18 amino acids. Embodiment 11: A structure from any one of Embodiments 1 to 10, wherein at least one of the following applies: (a) The dissociation constant (Kd) of the FGF21 polypeptide relative to β-Klotho is the same as the K of the corresponding wild-type FGF21 polypeptide. d It is less than approximately 20% of that; (b) K of FGF21 polypeptide against β-clotho off However, the K of the corresponding wild-type FGF21 polypeptide off It is less than approximately 20% of that. Embodiment 12: A structure from any one of Embodiments 1 to 11, wherein at least one of the following applies: (a) The construct binds to β-Klotho and segregates β-Klotho from the fibroblast growth factor receptor (FGFR) and the downstream pathway of β-Klotho; (b) The construct binds to both FGFR and β-Klotho and activates the downstream pathways of FGFR and β-Klotho. Embodiment 13: The structure according to Embodiment 12, wherein the FGFR is FGFR1c. Embodiment 14: A composition comprising one construct of any one of Embodiments 1 to 13 and a pharmaceutically acceptable carrier. Embodiment 15: A method for modulating the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway in a cell, comprising the step of bringing the surface of the cell into contact with one of the constructs of Embodiments 1 to 13. Embodiment 16: (a) The construct binds to β-klotho but not to FGFR, thereby sequestering β-klotho from the FGF21 / FGFR / β-klotho signaling pathway or the FGF19 / FGFR / β-klotho signaling pathway and downregulating the signaling pathway; or (b) The construct binds to both β-Klotho and FGFR, thereby upregulating the FGF21 / FGFR / β-Klotho signaling pathway. The method described in aspect 15. Embodiment 17: Any one of embodiments 15 to 16, wherein the cells are derived from a cell line, primary cells, or cells in the body of the subject. Embodiment 18: Any one of embodiments 15 to 17, wherein the cells are cells in or derived from adipose tissue, liver, pancreas, or hypothalamus. Apparatus 19: A method for treating, relieving, and / or preventing a disease or disorder in a subject where such treatment is necessary, A step of administering an effective amount of any one construct from embodiments 1 to 13 or the composition described in embodiment 14 to the subject. Includes, The disease or disorder is at least one selected from obesity, diabetes, pancreatitis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), acute alcohol toxicity, and cancers driven by abnormal activation of the FGF19 / FGFR4 / β-Clotho pathway. The aforementioned method. Embodiment 20: The method according to Embodiment 19, wherein the construct or composition is administered via at least one route selected from the group consisting of inhalation, oral, rectal, vaginal, parenteral, intracranial, topical, transdermal, pulmonary, intranasal, buccal, ocular, subarachnoid, and intravenous. Embodiment 21: Any one of Embodiments 19 to 20, wherein the subject is a human. Embodiment 22: The FGF21 polypeptide has an amino acid sequence of SEQ ID NO:1 having mutations as listed in the following options (a) to (m): (a) The FGF21 polypeptide comprises mutants L194F, V197I, Q201L, R203W, Y207W, A208W, and S209G, as well as the sequence RKK conjugated to the C-terminus of residues 200-209 of FGF21; (b) The FGF21 polypeptide comprises mutants L194F, V197I, Q201R, R203W, Y207W, A208W, and S209G; (c) The FGF21 polypeptide comprises mutants L194F, V197I, Q201R, R203W, Y207W, and A208W; (d) The FGF21 polypeptide comprises mutants L194F, V197L, Q201R, R203W, Y207W, A208W, and S209G; (e) The FGF21 polypeptide comprises mutants L194F, V197L, Q201R, R203W, Y207W, and A208W; (f) The FGF21 polypeptide comprises mutants L194F, Q201I, R203W, Y207W, and A208W; (g) The FGF21 polypeptide comprises mutants L194F, Q201L, R203W, Y207W, A208W, and S209G, as well as the sequence RKS conjugated to the C-terminus of residues 200-209 of FGF21; (h) The FGF21 polypeptide comprises mutants L194F, Q201P, R203W, Y207W, A208W, and S209G, as well as the sequence RRS conjugated to the C-terminus of residues 200-209 of FGF21; (i) The FGF21 polypeptide comprises mutants L194F, Q201R, R203W, Y207W, A208W, and S209G, as well as the sequence RRS conjugated to the C-terminus of residues 200-209 of FGF21; (j) The FGF21 polypeptide comprises the mutants L194F, Q201R, R203W, Y207W, and A208W; (k) The FGF21 polypeptide comprises mutants L194F, Q201W, R203W, Y207W, A208W, and S209G; (l) The FGF21 polypeptide comprises mutants L194F, R203W, Y207W, and A208W; (m) The FGF21 polypeptide comprises mutants R203W, Y207W, and A208W. Furthermore, the FGF21 polypeptide has an amino acid sequence that has at least approximately 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 with respect to SEQ ID NO:1, or SEQ ID The amino acid sequence has at least approximately 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 with the amino acid sequence of residues 29-209 of NO:1 (mature human FGF21 protein). A structure of any one of the embodiments 1 to 4. Embodiment 22: The FGF21 polypeptide has the amino acid sequence of mature human FGF21 (residues 2-209 of SEQ ID NO:1) having mutations as listed in the following options (a) to (m): (a) The FGF21 polypeptide comprises mutants L194F, V197I, Q201L, R203W, Y207W, A208W, and S209G, as well as the sequence RKK conjugated to the C-terminus of residues 200-209 of FGF21; (b) The FGF21 polypeptide comprises mutants L194F, V197I, Q201R, R203W, Y207W, A208W, and S209G; (c) The FGF21 polypeptide comprises mutants L194F, V197I, Q201R, R203W, Y207W, and A208W; (d) The FGF21 polypeptide comprises mutants L194F, V197L, Q201R, R203W, Y207W, A208W, and S209G; (e) The FGF21 polypeptide comprises mutants L194F, V197L, Q201R, R203W, Y207W, and A208W; (f) The FGF21 polypeptide comprises mutants L194F, Q201I, R203W, Y207W, and A208W; (g) The FGF21 polypeptide comprises mutants L194F, Q201L, R203W, Y207W, A208W, and S209G, as well as the sequence RKS conjugated to the C-terminus of residues 200-209 of FGF21; (h) The FGF21 polypeptide comprises mutants L194F, Q201P, R203W, Y207W, A208W, and S209G, as well as the sequence RRS conjugated to the C-terminus of residues 200-209 of FGF21; (i) The FGF21 polypeptide comprises mutants L194F, Q201R, R203W, Y207W, A208W, and S209G, as well as the sequence RRS conjugated to the C-terminus of residues 200-209 of FGF21; (j) The FGF21 polypeptide comprises the mutants L194F, Q201R, R203W, Y207W, and A208W; (k) The FGF21 polypeptide comprises mutants L194F, Q201W, R203W, Y207W, A208W, and S209G; (l) The FGF21 polypeptide comprises mutants L194F, R203W, Y207W, and A208W; (m) The FGF21 polypeptide comprises mutants R203W, Y207W, and A208W. Furthermore, the FGF21 polypeptide has an amino acid sequence that has at least approximately 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 with respect to the amino acid sequence of residues 29-209 of SEQ ID NO:1 (mature human FGF21 protein). A structure of any one of the embodiments 1 to 4.

[0259] The above outlines the features of several embodiments so that those skilled in the art may better understand aspects of this disclosure. Those skilled in the art should understand that this disclosure can be readily used as a basis for designing or modifying other processes and structures to serve the same purposes and / or achieve the same advantages of the embodiments described herein. Those skilled in the art should also recognize that the construction of such equivalents will not deviate from the spirit and scope of this disclosure, and that various changes, substitutions, and modifications described herein can be made without deviating from the spirit and scope of this disclosure.

Claims

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

2. The construct according to 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. A structure according to any one of claims 1 to 2, wherein at least one of the following conditions is met: (a) The FGF21 polypeptide comprises residues 186-209 of FGF21; (b) The FGF21 polypeptide comprises residues 169 to 209 of FGF21; (c) The FGF21 polypeptide comprises residues 29 to 209 of FGF21.

4. The construct according to any one of claims 1 to 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 residues 200 to 209 of FGF21.

5. A structure according to any one of claims 1 to 4, wherein at least one of the following conditions is met: (a) The FGF21 polypeptide comprises mutants L194F, V197I, Q201L, R203W, Y207W, A208W, and S209G, as well as the sequence RKK conjugated to the C-terminus of residues 200–209 of FGF21; (b) The FGF21 polypeptide comprises mutants L194F, V197I, Q201R, R203W, Y207W, A208W, and S209G; (c) The FGF21 polypeptide comprises mutants L194F, V197I, Q201R, R203W, Y207W, and A208W; (d) The FGF21 polypeptide comprises the mutants L194F, V197L, Q201R, R203W, Y207W, A208W, and S209G; (e) The FGF21 polypeptide comprises mutants 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 mutants L194F, Q201L, R203W, Y207W, A208W, and S209G, as well as the sequence RKS conjugated to the C-terminus of residues 200–209 of FGF21; (h) The FGF21 polypeptide comprises mutants L194F, Q201P, R203W, Y207W, A208W, and S209G, as well as the sequence RRS conjugated to the C-terminus of residues 200–209 of FGF21; (i) The FGF21 polypeptide comprises the mutants L194F, Q201R, R203W, Y207W, A208W, and S209G, as well as the sequence RRS conjugated to the C-terminus of residues 200–209 of FGF21; (j) The FGF21 polypeptide comprises the mutations L194F, Q201R, R203W, Y207W, and A208W; (k) The FGF21 polypeptide comprises the mutants L194F, Q201W, R203W, Y207W, A208W, and S209G; (l) The FGF21 polypeptide comprises the mutations L194F, R203W, Y207W, and A208W; (m) The FGF21 polypeptide comprises mutants R203W, Y207W, and A208W.

6. The construct according to any one of claims 1 to 5, wherein the FGF21 is human FGF21 having the protein sequence shown in SEQ ID NO:

1.

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

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

9. The construct according to any one of claims 7 to 8, wherein the stability-enhancing domain is at least one selected from the group consisting of albumin, the Fc region of an antibody, glutathione S-transferase, small ubiquitin-like modifier (SUMO), the O-glycosylation region of Drosophila Spitz protein, and thioredoxin.

10. The construct according to any one of claims 7 to 9, wherein the FGF21 polypeptide and the stability-enhancing domain are linked via a linker polypeptide containing about 1 to 18 amino acids.

11. A structure according to any one of claims 1 to 10, wherein at least one of the following conditions is met: (a) The dissociation constant (Kd) of the FGF21 polypeptide relative to β-Klotho is the same as the K of the corresponding wild-type FGF21 polypeptide. d It is less than approximately 20% of that; (b) K of FGF21 polypeptide against β-Klotho off However, the K of the corresponding wild-type FGF21 polypeptide off It is less than approximately 20% of that.

12. A structure according to any one of claims 1 to 11, wherein at least one of the following conditions is met: (a) The construct binds to β-Klotho and sequesters β-Klotho from the fibroblast growth factor receptor (FGFR) and the downstream pathway of β-Klotho; (b) The construct binds to both FGFR and β-Klotho and activates the downstream pathways of FGFR and β-Klotho.

13. The structure according to claim 12, wherein the FGFR is FGFR1c.

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

15. A method for modulating the FGF21 / FGFR / β-Klotho signaling pathway or the FGF19 / FGFR / β-Klotho signaling pathway in a cell, comprising the step of bringing the surface of the cell into contact with a construct according to any one of claims 1 to 13.

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

17. The method according to any one of claims 15 to 16, wherein the cells are derived from a cell line, primary cells, or cells in the body of the subject.

18. The method according to any one of claims 15 to 17, wherein the cells are cells in or derived from adipose tissue, the liver, the pancreas, or the hypothalamus.

19. A method for treating, relieving, and / or preventing a disease or disorder in a person who needs it, A step of administering an effective amount of the construct according to any one of claims 1 to 13 or the composition according to claim 14 to the subject. Includes, The disease or disorder is at least one selected from obesity, diabetes, pancreatitis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), acute alcohol toxicity, and cancers driven by abnormal activation of the FGF19 / FGFR4 / β-Clotho pathway. The aforementioned method.

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

21. The method according to any one of claims 19 to 20, wherein the subject is a human.

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