GLP1 / GDF15 dual agonist

Abstract

The invention provides a GLP1 / GDF15 dual agonist and a preparation method thereof. In particular, the present invention provides a fusion protein of GLP1 and GDF15. The dual GLP1 / GDF15 agonists of the present invention are long-acting, and comprise a functional Fc monomer.

Description

Technical Field

[0001] The present invention provides a GLP1 / GDF15 dual agonist. Specifically, the present invention provides a fusion protein of GLP1 and GDF15. The GLP1 / GDF15 dual agonist of the present invention is long-acting and comprises a functional Fc monomer. Background Art

[0002] Glucagon-like peptide-1 (GLP-1)

[0003] Glucagon-like peptide-1 (GLP-1) belongs to polypeptide hormones and is produced by enzymatic cleavage of proglucagon (PG). GLP-1 exerts its effects in vivo by targeting the GLP-1 receptor (GLP-1R). The extracellular domain of the receptor binds to the C-terminal α-helix of the ligand to initiate recognition, and then the N-terminal of the ligand binds to the pocket of the transmembrane domain of the receptor to activate the receptor and trigger downstream cascade signals. GLP-1R is widely expressed on various types of cells such as pancreatic β-cells, heart, lung, kidney, gastric pits, small intestinal mucosa, central nervous system, eyes, etc. At the same time, there is evidence that GLP-1R expression can also be detected on human neutrophils and eosinophils. GLP-1 can play roles in blood glucose lowering, fat reduction, cardiovascular regulation, nervous system regulation, anti-inflammatory and immune regulation, and bone metabolism. GLP-1 has the highest content in the pancreas, so GLP-1 mainly plays blood glucose lowering and fat reduction roles in vivo.

[0004] Growth differentiation factor 15 (GDF15)

[0005] Growth differentiation factor 15 (GDF15) is a peptide hormone and a member of the transforming growth factor-β (TGFβ) superfamily. GFRAL, as a transmembrane protein, is currently the only identified endogenous receptor with high affinity for GDF15. After binding to GDF15, the GFRAL-GDF15 complex binds to the tyrosine kinase coreceptor RET. This binding leads to phosphorylation of RET, followed by activation of the intracellular signaling pathways of AKT, ERK1 / 2, and phospholipase C (PLCγ), but does not activate the SMAD pathway. GDF15 may have potential therapeutic effects in weight-related diseases (such as obesity, type 2 diabetes, non-alcoholic steatohepatitis, and non-alcoholic fatty liver disease), cardiovascular diseases, and tumors. GDF15 can also promote the energy consumption of skeletal muscle.

[0006] Combination of GLP-1 and GDF15

[0007] It has been demonstrated in animals that the combination therapy of GLP-1 and GDF15 or the weight loss effect of the fusion protein is superior to that of GLP-1 alone. There is a need for improved drugs with excellent pharmacokinetics and / or pharmacodynamics.

[0008] Functional Fc monomer

[0009] Currently, there are mainly three long-acting technologies for protein drugs: polyethylene glycol (PEG) modification technology, human serum albumin (HSA) fusion technology (US2022 / 0089669A1), and human antibody Fc region fusion technology. Each of these three technologies has its own disadvantages. Generally speaking, the common key disadvantage is that it will greatly increase the molecular weight of the protein drug to be fused or modified, and often significantly reduce the yield and clinical efficacy of the protein drug to be fused. The inventor of the present invention innovatively uses synthetic biology in the field of protein long-acting to develop a novel functional Fc monomer based on antibody IgG Fc, providing a better alternative for the long-acting of protein drugs. Summary of the Invention

[0010] On the one hand, the present invention provides a fusion protein comprising, from the N-terminus to the C-terminus, a GLP-1 peptide, an optional first linker peptide, an Fc monomer polypeptide, an optional second linker peptide, and a GDF15 polypeptide.

[0011] In one embodiment, the GLP-1 peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 34, or an amino acid sequence truncated by 1-5, such as 1, 2, 3, 4 or 5 amino acids from the C-terminus of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 34, consisting essentially of or consisting of the same. In one embodiment, the GLP-1 peptide retains the function of native GLP-1.

[0012] In one embodiment, the GDF15 polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, consisting essentially of or consisting of the same. In one embodiment, the GDF15 polypeptide retains the function of native GDF15.

[0013] In one embodiment, the Fc monomer polypeptide comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In one embodiment, the Fc monomer polypeptide comprises, consists essentially of, or consists of an amino acid sequence that contains arginine (R), histidine (H), lysine (K), and threonine (T) at positions 366, 368, 395, and 409, respectively, according to the EU numbering system, relative to the native Fc sequence (especially IgG Fc, more especially IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc, such as any one of SEQ ID NOs: 28-31). In one embodiment, the Fc monomer polypeptide comprises, consists essentially of, or consists of an amino acid sequence that contains one or more amino acid substitutions at one or more of positions 351, 366, 368, 395, 409, and / or 428 according to the EU numbering system, relative to the native Fc sequence (especially IgG Fc, more especially IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc, such as any one of SEQ ID NOs: 28-31), wherein position 351 is substituted with S, position 366 is substituted with C, G, S, L, N, D, F, I, V, Y, Q, K, E, M, or R, position 368 is substituted with H, Q, N, D, Y, R, C, G, S, F, T, I, V, A, K, or M, position 395 is substituted with N, T, I, S, M, Q, R, L, G, V, A, E, D, Y, F, H, or K, position 409 is substituted with N, S, I, M, E, Q, L, V, A, H, D, Y, F, or T, and position 428 is substituted with Y. In one embodiment, the Fc monomer polypeptide is a functional Fc monomer polypeptide, i.e., retains FcRn binding properties and / or protein A / protein G binding properties. In one embodiment, the Fc monomer polypeptide or a fusion protein comprising the Fc monomer polypeptide can be highly soluble expressed in prokaryotic cells (such as Escherichia coli) and / or eukaryotic cells (such as Expi293). In one embodiment, the Fc monomer polypeptide or a fusion protein comprising the Fc monomer polypeptide has a half-life of up to about two days in an animal body.

[0014] In one embodiment, the GLP-1 peptide is fused to the Fc monomer polypeptide via a first linker peptide. In a further embodiment, the first linker peptide is G n, where n is an integer from 1 to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In a further embodiment, the first linker peptide is G4, i.e., GGGG (SEQ ID NO: 16). In one embodiment, the GDF15 polypeptide is fused to the Fc monomer polypeptide via a second linker peptide. In a further embodiment, the second linker peptide is G p (AP) m G q , where p is an integer from 1 to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, q is an integer from 1 to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, and m is an integer from 1 to 25, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25. In a further embodiment, the second linker peptide is G4(AP) 10 G4, i.e., GGGGGAPAPAPAPAPAPAPAPAPAPGGGG (SEQ ID NO: 17).

[0015] In one embodiment, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32 or SEQ ID NO: 33, consisting essentially of or consisting of the same.

[0016] On the other hand, the present invention provides an oligonucleotide encoding the fusion protein of the present invention.

[0017] On the other hand, the present invention provides an expression vector comprising the oligonucleotide of the present invention.

[0018] On the other hand, the present invention provides a host cell comprising the oligonucleotide of the present invention or the expression vector of the present invention.

[0019] On the other hand, the present invention provides a method for generating the fusion protein of the present invention, which comprises culturing the host cell of the present invention under conditions suitable for the expression of the fusion protein of the present invention.

[0020] On the other hand, the present invention provides a pharmaceutical composition comprising the fusion protein of the present invention, the oligonucleotide of the present invention, the expression vector of the present invention, or the host cell of the present invention, and a pharmaceutically acceptable vehicle.

[0021] On the other hand, the present invention provides the fusion protein of the present invention, the oligonucleotide of the present invention, the expression vector of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention for treating a disease, disorder or condition in a subject in need thereof. On the other hand, the present invention provides the fusion protein of the present invention, the oligonucleotide of the present invention, the expression vector of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention for use in the preparation of a medicament for treating a disease, disorder or condition in a subject in need thereof.

[0022] On the other hand, the present invention provides a method for treating a disease, disorder or condition in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of the fusion protein of the present invention, the oligonucleotide of the present invention, the expression vector of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention. On the other hand, the present invention provides the use of the fusion protein of the present invention, the oligonucleotide of the present invention, the expression vector of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease, disorder or condition in a subject in need thereof.

[0023] In one embodiment, the disease, disorder or condition is selected from obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unregulated cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease, and eczema.

[0024] The fusion protein of the present invention can be produced by a eukaryotic system (such as Expi293 cells), with simple production, few by-products, high yield (milligram per liter level yield can be achieved at the laboratory scale), simple recovery and purification (one-step purification), and the product structure is close to the conformation of the natural protein. The fusion protein of the present invention highly maintains the functions and activities of each component, and realizes the synergistic effects of diet control, weight loss, and blood sugar control. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Showing the SDS-PAGE results of the fusion protein of the present invention.

[0026] Figure 2Show the SEC results of the fusion protein of the present invention.

[0027] Figure 3 Show the denaturation temperature (DSF) of the fusion protein of the present invention.

[0028] Figure 4A and Figure 4B Show the binding (ELISA) of the fusion proteins GLP1-sFc-GDF15 (G4) and G4-M8 of the present invention to GLP-1R, respectively.

[0029] Figure 5A , Figure 5B and Figure 5C Show the binding (ELISA) of the fusion proteins GLP1-sFc-GDF15 (G4), G4-M8 and the reference LY3463251 of the present invention to GFRAL, respectively.

[0030] Figure 6A and Figure 6B Show the binding affinity assay (BLI) of the fusion protein of the present invention and the reference LY3463251 to GFRAL, respectively.

[0031] Figure 7A and Figure 7B Show the binding affinity assay (BLI) of the fusion protein of the present invention and semaglutide to GLP-1R, respectively.

[0032] Figure 8A and Figure 8B as well as Figure 8C and Figure 8D Show the biological functions (reporter gene assay) of the GDF15 end and the GLP-1 end of the fusion protein of the present invention, respectively.

[0033] Figure 9 Show the body weight results of the DIO mouse experiment.

[0034] Figure 10 Show the food intake results of the DIO mouse experiment.

[0035] Figure 11 Show the blood glucose results of the DIO mouse experiment. Detailed implementation mode

[0036] Throughout the specification, a variety of publications, articles and patents are cited; each of these references is incorporated herein by reference in its entirety. The discussions of documents, acts, materials, devices, articles, etc. included in this specification are intended to provide context for the present invention. Such discussions do not admit that any of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms used herein have the meanings as set forth in this specification.

[0038] It should be noted that, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" as used in the specification and claims include plural referents.

[0039] Unless otherwise indicated, any numerical values, such as the concentrations or concentration ranges described herein, should be understood to be modified in all instances by the term "about". Thus, the numerical values generally include the stated value ±10%. As used herein, the numerical ranges expressly include all possible sub-ranges, all individual numerical values within the range, including integers and fractions within such range, unless the context clearly indicates otherwise.

[0040] Unless otherwise indicated, the term "at least" before a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0041] As used herein, the terms "comprising", "including", "having", "containing", or any other variation thereof should be understood to mean including the stated integer or group of integers, but not excluding any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" means inclusive or and not exclusive or. For example, condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0042] As used herein, the conjunctive term "and / or" between a plurality of recited elements is understood to include both a single option and a combined option. For example, in the case where two elements are joined by "and / or", the first option means that the first element applies in the absence of the second element. The second option means that the second element applies in the absence of the first element. The third option means that the first and second elements are suitable for use together. Any of these options is understood to fall within the meaning and thus meet the requirements of the term "and / or" as used herein. The parallel applicability of more than one option is also understood to fall within the meaning and thus meet the requirements of the term "and / or".

[0043] It should also be understood that when referring to the size or characteristics of components of the preferred invention, the terms "about", "approximately", "substantially", "essentially" and similar terms used herein mean that the described size / characteristics are not strict boundaries or parameters and do not exclude minor variations that are functionally the same or similar, as understood by a person of ordinary skill in the art. At a minimum, such references including numerical parameters will include variations using accepted mathematical and industrial principles in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.) that do not change the least significant digit.

[0044] In the present invention, the terms "peptide", "polypeptide" and "protein" are used interchangeably.

[0045] Glucagon-like peptide 1 (GLP1)-Growth differentiation factor 15 (GDF15) fusion protein

[0046] In one general aspect, the present invention relates to a glucagon-like peptide 1 (GLP1)-growth differentiation factor 15 (GDF15) fusion protein. The GLP1-GDF15 fusion protein comprises GLP1 or a GLP1 variant peptide, a first linker peptide, a human functional Fc monomer polypeptide, a second linker peptide, and GDF15 or a GDF15 variant protein.

[0047] GLP1 or GLP1 variant peptide

[0048] Glucagon-like peptide 1 (GLP1) is an insulin secretagogue synthesized in the intestine and released in response to food intake. It is mainly secreted in two forms: GLP1-(7-37) and GLP1-(7-36)NH2, both of which bind to specific GLP1 receptors (GLP1R) present in many tissues and the brainstem, including pancreatic β-cells, where it enhances glucose-stimulated insulin secretion, and in the brainstem where it controls satiety and food intake.

[0049] Many GLP1 analogs and derivatives are known and may be referred to herein as "GLP1 variants".

[0050] It has been demonstrated that GLP1 and GLP1 variant peptides act in multiple ways, which may include but are not limited to reducing food intake, stimulating insulin release, lowering glucagon secretion, inhibiting gastric emptying, and enhancing glucose utilization.

[0051] GLP1R belongs to the class B family of 7-transmembrane heterotrimeric G-protein coupled receptors and is expressed in a wide range of tissues, including but not limited to alpha-cells, beta-cells, and delta-cells of the islets of Langerhans, heart, kidney, stomach, intestine, nodose ganglion neurons of the vagus nerve, and several regions of the central nervous system (CNS), including the hypothalamus and brainstem. GLP1R can couple to Gαs, Gαq, Gαi, and Gαo (Montrose-Rafizadeh et al., Endocrinology, 140:1132-1140, 1999; Hallbrink et al., Biochim Biophys Acta 1546:79-86, 2001), leading to an increase in intracellular calcium, adenylate cyclase, and phospholipase C, as well as the activation of the PKA, PKC, PI-3K, Epac2, and MAPK signal transduction pathways (Drucker et al., PNAS 84:3434-8, 1987; Wheeler et al., Endrocrinology 133:57-62, 1993; Holz et al., JBC 270:17749-57, 1995).

[0052] The present invention provides a GLP1-GDF15 fusion protein, which comprises a first component, and the first component is GLP1 or a GLP1 variant peptide. The GLP1 or GLP1 variant peptide may comprise one of the sequences provided in Table 1. The GLP1 or GLP1 variant peptide may have at least 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% identity with one of the sequences provided in Table 1. The GLP1 or GLP1 variant peptide may be truncated by 1-5, such as 1, 2, 3, 4 or 5 residues from the C-terminus compared with one of the sequences provided in Table 1. The GLP1 or GLP1 variant peptide sequence may be selected based on at least one of the following criteria: (i) expression yield and purity, (ii) in vitro stability, (iii) in vitro potency, (iv) retention of in vitro potency of binding to GDF15 or a GDF15 variant protein, (v) lack of serine xylosylation or serine xylosylation potential, and (vi) characteristics of the GLP1-GDF15 fusion protein (e.g., in vivo stability, in vivo potency (i.e., whether the GLP1 or GLP1 variant peptide and the GDF15 or GDF15 variant protein can respectively have agonist activity against the GLP1R and GDF15R (GFRAL) receptors)).

[0053] The GLP1 or GLP1 variant peptide that constitutes the first component of the fusion protein is intended to cover peptides that have sufficient homology and functionality with native GLP1. The GLP1 or GLP1 variant peptide is designed to be able to bind to GLP1 receptors in tissues (including the pancreas and brainstem), thereby generating the same signal transduction pathway as when native GLP1 binds to GLP1 receptors in these tissues and exhibiting the same or similar physiological activities.

[0054] Table 1: Glucagon-like peptide 1 and its variants

[0055]

[0056] See Cheang and Moyle, ChemMedChem. 2018 Apr 6; 13(7):662-671; Manandhar and Ahn, J Med Chem. 2015 Feb 12; 58(3):1020-37; CN102686607B; and Mahapatra et al., Rev Endocr Metab Disord. 2022 Jun; 23(3):521-539.

[0057] First linker peptide

[0058] The present disclosure provides a GLP1-GDF15 fusion protein, which comprises a second component, and the second component is a first linker peptide (i.e., the linker peptide between the GLP1 peptide and the functional Fc monomer polypeptide).

[0059] The first linker peptide may comprise, for example, from about 1 to about 50 amino acid residues, or any number of amino acid residues therebetween. The first linker peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues.

[0060] In certain embodiments, the first linker peptide may comprise an alanine-proline repeat sequence (i.e., an AP repeat sequence), wherein the AP dipeptide may be referred to as an AP unit. The first linker peptide may comprise, for example, from about 1 to about 25 AP units, or any number of AP units therebetween. The first linker peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 AP units.

[0061] In certain embodiments, the first linker peptide may comprise a glycine-glycine-glycine-glycine-serine repeat sequence (i.e., a G4S repeat sequence), wherein the G4S pentapeptide may be referred to as a G4S unit. The first linker peptide may comprise, for example, from about 1 to about 10 G4S units, or any number of G4S units therebetween. The first linker peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 G4S units.

[0062] In certain embodiments, the first linker peptide may comprise a glycine-glycine-glycine-glycine-alanine repeat sequence (i.e., a G4A repeat sequence), wherein the G4A pentapeptide may be referred to as a G4A unit. The first linker peptide may comprise, for example, from about 1 to about 10 G4A units, or any number of G4A units therebetween. The first linker peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 G4A units.

[0063] In certain embodiments, the first linker peptide can be a polyglycine peptide. The first linker peptide can comprise, for example, from about 1 to about 50 glycine residues, or any number of glycine residues therebetween. The first linker peptide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 glycine residues.

[0064] The above motifs can be combined arbitrarily. For example, one or more AP units are flanked by any two of one or more G4S units, one or more G4A units, and one or more glycine residues (same motif or different motifs, same length or different lengths).

[0065] The first linker peptide sequence can be selected based on at least one of the following criteria: (i) expression yield and purity, (ii) in vitro potency, (iii) in vitro stability, (iv) lack of serine xylosylation or serine xylosylation potential, and (v) properties of the GLP1-GDF15 fusion protein (e.g., in vivo stability, in vivo potency (i.e., whether the GLP1 or GLP1 variant peptide and the GDF15 or GDF15 variant protein can respectively have agonist activity against GLP1R and GDF15R (GFRAL))).

[0066] Functional Fc monomer

[0067] The functional Fc monomer retains the FcRn binding property and protein A / protein G binding property of the antibody Fc region, has the excellent property of being highly soluble and expressible in prokaryotic cells (such as Escherichia coli), and has a half-life of up to about two days in animals. The functional Fc monomer can be of the IgM, IgG, IgA, IgE, or IgD class, especially of the IgG class. Further, the functional Fc monomer can be of the IgG1, IgG2, IgG3, or IgG4 subclass, especially of the IgG1 or IgG4 subclass.

[0068] The present disclosure provides a GLP1-GDF15 fusion protein, which comprises a third component, and the third component is a functional Fc monomer. The functional Fc monomer may comprise one of the sequences provided in Table 2. The functional Fc monomer may have at least 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% identity with one of the sequences provided in Table 2. The functional Fc monomer may comprise, consist essentially of, or consist of an amino acid sequence that contains arginine (R), histidine (H), lysine (K), and threonine (T) at positions 366, 368, 395, and 409, respectively, according to the EU numbering system, relative to a native Fc sequence (especially IgG Fc, more especially IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc, such as any one of SEQ ID NOs: 28-31). The functional Fc monomer may comprise, consist essentially of, or consist of an amino acid sequence that contains one or more amino acid substitutions at one or more of positions 351, 366, 368, 395, 409, and / or 428 according to the EU numbering system, relative to a native Fc sequence (especially IgG Fc, more especially IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc, such as any one of SEQ ID NOs: 28-31), wherein position 351 is substituted with S, position 366 is substituted with C, G, S, L, N, D, F, I, V, Y, Q, K, E, M, or R, position 368 is substituted with H, Q, N, D, Y, R, C, G, S, F, T, I, V, A, K, or M, position 395 is substituted with N, T, I, S, M, Q, R, L, G, V, A, E, D, Y, F, H, or K, position 409 is substituted with N, S, I, M, E, Q, L, V, A, H, D, Y, F, or T, and position 428 is substituted with Y. The functional Fc monomer sequence may be selected based on at least one of the following criteria: (i) expression yield and purity, (ii) in vitro stability, (iii) in vitro potency, (iv) properties of the GLP1-GDF15 fusion protein (e.g., in vivo stability and in vivo potency (i.e., whether the GLP1 or GLP1 variant peptide and the GDF15 or GDF15 variant protein can respectively have agonist activity against the GLP1R and GDF15R receptors)), and (v) half-life.

[0069] The functional Fc monomer that constitutes the third component of the fusion protein is intended to cover peptides that can perform the same or similar functions as the native dimeric / double-stranded Fc region in monomer / single-chain form. The functional Fc monomer is designed to be able to bind to Fc receptors in monomer / single-chain form, thereby extending the half-life of the fusion protein.

[0070] Exemplary native IgG1 Fc sequence (SEQ ID NO: 28)

[0071]

[0072] Exemplary native IgG2 Fc sequence (SEQ ID NO: 29)

[0073]

[0074] Exemplary native IgG3 Fc sequence (SEQ ID NO: 30)

[0075]

[0076] Exemplary native IgG4 Fc sequence (SEQ ID NO: 31)

[0077]

[0078] Table 2: Functional Fc monomers

[0079]

[0080] See EP4238986A1; Wang et al., Front Immunol. 2017 Nov 13; 8:1545; and EP3677595A1.

[0081] Second linker peptide

[0082] Provided herein is a GLP1-GDF15 fusion protein that includes a fourth component, which is a second linker peptide (i.e., a linker peptide between the GDF15 polypeptide and the functional Fc monomer polypeptide).

[0083] The second linker peptide can, for example, include from about 1 to about 50 amino acid residues, or any number of amino acid residues therebetween. The second linker peptide can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues.

[0084] In certain embodiments, the second linker peptide may comprise an alanine-proline repeat sequence (i.e., an AP repeat sequence), wherein the AP dipeptide may be referred to as an AP unit. The second linker peptide may comprise, for example, from about 1 to about 25 AP units, or any number of AP units therebetween. The second linker peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 AP units.

[0085] In certain embodiments, the second linker peptide may comprise a glycine-glycine-glycine-glycine-serine repeat sequence (i.e., a G4S repeat sequence), wherein the G4S pentapeptide may be referred to as a G4S unit. The second linker peptide may comprise, for example, from about 1 to about 10 G4S units, or any number of G4S units therebetween. The second linker peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 G4S units.

[0086] In certain embodiments, the second linker peptide may comprise a glycine-glycine-glycine-glycine-alanine repeat sequence (i.e., a G4A repeat sequence), wherein the G4A pentapeptide may be referred to as a G4A unit. The second linker peptide may comprise, for example, from about 1 to about 10 G4A units, or any number of G4A units therebetween. The second linker peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 G4A units.

[0087] In certain embodiments, the second linker peptide may be a polyglycine peptide. The second linker peptide may comprise, for example, from about 1 to about 50 glycine residues, or any number of glycine residues therebetween. The second linker peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 glycine residues.

[0088] The above motifs can be combined in any manner, such as with one or more AP units flanked by any two of one or more G4S units, one or more G4A units, and one or more glycine residues (same motif or different motifs, same length or different lengths).

[0089] The second linker peptide sequence can be selected based on at least one of the following criteria: (i) expression yield and purity, (ii) in vitro potency, (iii) in vitro stability, (iv) lack of serine xylosylation or serine xylosylation potential, and (v) properties of the GLP1-GDF15 fusion protein (e.g., in vivo stability, in vivo potency (i.e., whether the GLP1 or GLP1 variant peptide and the GDF15 or GDF15 variant protein are able to have agonist activity against GLP1R and GDF15R (GFRAL), respectively)).

[0090] GDF15 or GDF15 variant protein

[0091] Growth differentiation factor 15 (GDF15) is a protein belonging to the transforming growth factor-β (TGF-β) superfamily. GDF15 is a secreted protein that circulates as a 25 kDa dimer. GDF15 is also known as prostate-derived factor (PDF), macrophage inhibitory cytokine-1 (MIC-1), NSAID (non-steroidal anti-inflammatory drug)-activated gene (NAG-1), and placental TGF-β (PTGFβ).

[0092] The function of GDF15 has not been fully elucidated, but it is involved in a variety of biological processes, including but not limited to energy homeostasis, body weight regulation, and cachexia driven by cancer and chronic diseases. It has been demonstrated in mice, rats, and monkeys that pharmacologically administering GDF15 may reduce energy intake and thereby cause weight loss (Johnen et al., Nat Med, 13:1333-1340, 2007; Hsu et al., Nature, 550:255-259, 2017; Mullican et al., Nat Med, 23:1150-1157, 2017; Tsai et al., Int J Obesity, 42:561-571, 2018). GDF15 treatment-mediated weight loss leads to metabolic improvement, including enhanced glucose homeostasis and reduced plasma triglycerides and cholesterol (Xiong et al., Sci Trans Med, 9:412, 2017).

[0093] GDF15 binds to the GDNF family α-like receptor (GFRAL), which is a transmembrane receptor located only in neurons of the brainstem. Upon GDF15 binding, GFRAL complexes with RET, a tyrosine kinase γ that stimulates downstream intracellular phosphorylation cascades, including post-translational modifications of AKT, ERK, and PLC. Although additional molecular and cellular components of this cascade remain to be elucidated, the ultimate effect of GDF15 / GFRAL signaling is reduced food intake and weight loss.

[0094] The present disclosure provides a GLP1-GDF15 fusion protein that includes a fifth component, which is GDF15 or a GDF15 variant protein. The GDF15 protein can include the sequences provided in Table 3, and the GDF15 variant protein can include variants of the sequences provided in Table 3. The GDF15 or GDF15 variant protein can have at least 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% identity to one of the sequences provided in Table 3. The GDF15 and / or GDF15 variant protein sequences can be selected based on at least one of the following criteria: (i) expression yield and purity, (ii) in vitro stability, (iii) in vitro potency, (iv) retention of in vitro potency of binding to GLP1 or a GLP1 variant protein, (v) lack of serine xylosylation or serine xylosylation potential, and (vi) properties of the GLP1-GDF15 fusion protein (e.g., in vivo stability, in vivo potency (i.e., whether the GLP1 or GLP1 variant peptide and the GDF15 or GDF15 variant protein can respectively have agonist activity against the GLP1R and / or GDF15R (GFRAL) receptors)).

[0095] The GDF15 or GDF15 variant protein that constitutes the fifth component of the GLP1-GDF15 fusion protein is intended to encompass peptides that have sufficient homology and functionality to activate the native GDF15R (GFRAL). The GDF15 or GDF15 variant protein is designed to be able to bind to GFRAL in the brainstem, thereby generating the same signaling pathway as when native GDF15 binds to GFRAL in these neurons and exhibiting the same effect on food intake.

[0096] Table 3: GDF15 or GDF15 variant protein

[0097]

[0098] See Hale and Veniant, Mol Metab, 2021 Apr 46; 101117; US2017 / 0327560A1; Zhang et al., Cell Metab. 2023 Feb 7; 35(2):287-298.e4; and US9920118B2.

[0099] Fusion protein

[0100] The present invention provides a GLP1-GDF15 fusion protein, which comprises a first component, a second component, a third component, a fourth component and a fifth component as described above. The first component is GLP1 or a GLP1 variant peptide, the second component is a first linker peptide, the third component is a functional Fc monomer, the fourth component is a second linker peptide, and the fifth component is GDF15 or a GDF15 variant protein. The GLP1-GDF15 fusion protein may comprise one of the sequences provided in Table 4. The GLP1-GDF15 fusion protein may have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with one of the sequences provided in Table 4. The GLP1-GDF15 fusion protein can be selected based on at least one of the following criteria: (i) expression yield and purity, (ii) in vitro potency, (iii) in vitro stability, (iv) lack of serine xylosylation, (v) physical properties of the GLP1-GDF15 fusion protein (e.g., in vivo stability, in vivo potency (i.e., whether the GLP1 or GLP1 variant peptide and GDF15 or GDF15 variant protein can respectively have agonist activity against GLP1R and GDF15R)), (vi) desired balance of in vivo dual agonist pharmacology, and (vi) half-life.

[0101] Table 4: Fusion Proteins

[0102]

[0103]

[0104]

[0105] Polynucleotides and Vectors

[0106] In another general aspect, the present invention relates to an isolated nucleic acid encoding the GLP1-GDF15 fusion protein of the present invention. Those skilled in the art will understand that the coding sequence of a protein can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will understand that the nucleic acid sequence encoding the fusion protein of the present invention can be altered without changing the amino acid sequence of the protein.

[0107] In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid encoding a fusion protein of the present invention. According to the present disclosure, any vector known to those skilled in the art can be used, such as plasmids, cosmid plasmids, phage vectors or viral vectors. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any element that establishes the conventional functions of an expression vector, such as a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible or repressible promoter. A variety of expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to produce the fusion protein in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to the embodiments of the present invention.

[0108] In another general aspect, the present invention relates to a host cell comprising an isolated nucleic acid encoding a fusion protein of the present invention. In view of the present disclosure, any host cell known to those skilled in the art can be used for recombinant expression of the fusion protein of the present invention. In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell, such as Escherichia coli (E. coli) TG1 or BL21 cells, CHO-DG44 or CHO-K1 cells, or HEK293 cells or Expi293 cells. According to specific embodiments, the recombinant expression vector is transformed into the host cell by conventional methods such as chemical transfection, heat shock or electroporation, wherein the recombinant expression vector is stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed.

[0109] In another general aspect, the present invention relates to a method for preparing a fusion protein of the present invention, the method comprising culturing a cell comprising a nucleic acid encoding the fusion protein under conditions for preparing the fusion protein of the present invention, and recovering the fusion protein from the cell or cell culture (such as from the supernatant). The expressed fusion protein can be harvested from the cells and purified according to conventional techniques known in the art and as described herein.

[0110] Pharmaceutical composition

[0111] In another general aspect, the present invention relates to a pharmaceutical composition comprising the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention and a pharmaceutically acceptable vehicle. As used herein, the term "pharmaceutical composition" means a product comprising the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention together with a pharmaceutically acceptable vehicle. The GLP1-GDF15 fusion protein and / or polynucleotide of the present invention and compositions comprising them can also be used to manufacture a medicament for the therapeutic applications described herein.

[0112] As used herein, the term "vehicle" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, or other material known in the art for use in pharmaceutical formulations. It should be understood that the characteristics of the vehicle, excipient or diluent will depend on the route of administration of the particular application. As used herein, the term "pharmaceutically acceptable vehicle" refers to a non-toxic material that does not interfere with the effects of the compositions according to the invention or the biological activity of the compositions according to the invention. According to the present disclosure, any pharmaceutically acceptable vehicle suitable for peptide pharmaceutical compositions can be used in the present invention.

[0113] Pharmaceutically acceptable acid salts / anion salts used in the present invention include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate, ethanedisulfonate, etidronate, esylate, fumarate, glucoheptonate, gluconate, glutamate, p-hydroxymercuribenzoate, hexylresorcinol, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, naphthalenesulfonate, nitrate, pamoate, pantothenate, phosphate / diphosphate, pectinate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theoclate, toluenesulfonate, and triethiodide. Organic acids or inorganic acids also include, but are not limited to, hydroiodic acid, perchloric acid, sulfuric acid, phosphoric acid, propionic acid, glycolic acid, methanesulfonic acid, hydroxyethanesulfonic acid, oxalic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclohexanesulfamic acid, saccharic acid, or trifluoroacetic acid.

[0114] Pharmaceutically acceptable base salts / cation salts include, but are not limited to, aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (also known as tris(hydroxymethyl)aminomethane, tromethamine or "TRIS"), ammonia, benzathine penicillin, tert-butylamine, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium, triethanolamine, or zinc.

[0115] In some embodiments of the present invention, a pharmaceutical preparation is provided, which comprises the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention in an amount of about 0.001 mg / mL to about 100 mg / mL, about 0.01 mg / mL to about 50 mg / mL, or about 0.1 mg / mL to about 25 mg / mL. The pharmaceutical preparation may have a pH of about 3.0 to about 10, such as about 3 to about 7, or about 5 to about 9. The preparation may also comprise at least one component selected from the following: buffer systems, preservatives, tonicity agents, chelating agents, stabilizers, and surfactants.

[0116] Preparations of pharmaceutically active ingredients with pharmaceutically acceptable vehicles are known in the art, such as Remington: The Science and Practice of Pharmacy (e.g., the 21st edition (2005) and any subsequent editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity modifiers, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable vehicles can be used to formulate the pharmaceutical compositions of the present invention.

[0117] In one embodiment of the present invention, the pharmaceutical composition is a liquid preparation. A preferred example of a liquid preparation is an aqueous preparation, i.e., a preparation containing water. Liquid preparations can include solutions, suspensions, emulsions, microemulsions, gels, and the like. Aqueous preparations generally contain at least 50% w / w of water, or at least 60% w / w, 70% w / w, 75% w / w, 80% w / w, 85% w / w, 90% w / w, or at least 95% w / w of water.

[0118] In one embodiment, the pharmaceutical composition can be formulated as an injectable for injection, for example, via an injection device (e.g., a syringe or an infusion pump). The injection can be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, or intravenously.

[0119] In another embodiment, the pharmaceutical composition is a solid preparation, such as a lyophilized or spray-dried composition, which can be used as it is, or a solvent and / or diluent can be added by a physician or a patient before use. Solid dosage forms can include tablets, such as compressed tablets and / or coated tablets, and capsules (e.g., hard gelatin capsules or soft gelatin capsules). The pharmaceutical composition can also be in the form of, for example, sachets for reconstitution, dragees, powders, granules, lozenges, or powders.

[0120] The dosage form can be of the immediate-release type, in which case they can contain a water-soluble or water-dispersible vehicle, or they can be delayed-release, sustained-release, or modified-release, in which case they can contain a water-insoluble polymer that modulates the dissolution rate of the dosage form in the gastrointestinal tract.

[0121] In other embodiments, the pharmaceutical composition can be delivered intranasally, buccally, or sublingually.

[0122] The pH in the aqueous preparation can be between pH 3 and pH 10. In one embodiment of the present invention, the pH of the preparation is from about 7.0 to about 9.5. In another embodiment of the present invention, the pH of the preparation is from about 3.0 to about 7.0.

[0123] In another embodiment of the present invention, the pharmaceutical composition comprises a buffering agent. Non-limiting examples of buffering agents include: arginine, aspartic acid, bis(2-hydroxyethyl)glycine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, triazine and tris(hydroxymethyl)aminomethane, and mixtures thereof. The buffering agent may be present alone or in aggregates at a concentration of from about 0.01 mg / ml to about 50 mg / ml, such as from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific buffering agents constitute alternative embodiments of the present invention.

[0124] In another embodiment of the present invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include: benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butylparaben, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethylparaben, imidurea, methylparaben, phenol, 2-phenoxyethanol, 2-phenylethanol, propylparaben, sodium dehydroacetate, thimerosal and mixtures thereof. The preservative may be present alone or in aggregates at a concentration of from about 0.01 mg / ml to about 50 mg / ml, such as from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific preservatives constitute alternative embodiments of the present invention.

[0125] In another embodiment of the present invention, the pharmaceutical composition comprises an isosmotic agent. Non-limiting examples of such an embodiment include salts (such as sodium chloride), amino acids (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), sugar alcohols (such as glycerol, 1,2-propanediol, propylene glycol), 1,3-propanediol, and 1,3-butanediol), polyethylene glycol (e.g., PEG400), and mixtures thereof. Another example of an isosmotic agent includes sugars. Non-limiting examples of sugars can be monosaccharides, disaccharides, or polysaccharides, or water-soluble dextrans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, amylopectin, dextrin, cyclodextrin, α- and β-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethyl cellulose. Another example of an isosmotic agent is a sugar alcohol, where the term "sugar alcohol" is defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, hexahydric alcohol, xylitol, and arabinitol. Pharmaceutical compositions containing each of the isosmotic agents listed in this paragraph constitute alternative embodiments of the present invention. The isosmotic agent may be present alone or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, such as about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions containing each of these specific isosmotic agents constitute alternative embodiments of the present invention.

[0126] In another embodiment of the present invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, salts of ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agent may be present alone or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, such as about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions containing each of these specific chelating agents constitute alternative embodiments of the present invention.

[0127] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

[0128] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer, wherein the stabilizer is carboxy- / hydroxycellulose and its derivatives (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur-containing substances such as thioglycerol or mercaptoacetic acid. The stabilizer may be present alone or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example about 0.1 mg / ml to about 20 mg / ml. The pharmaceutical compositions comprising each of these specific stabilizers constitute alternative embodiments of the present invention.

[0129] In another embodiment of the present invention, the pharmaceutical composition comprises one or more surfactants, preferably one surfactant, at least one surfactant or two different surfactants. The term "surfactant" refers to any molecule or ion consisting of a water-soluble moiety (hydrophilic) and a lipid-soluble moiety (lipophilic). For example, the surfactant may be selected from: anionic surfactants, cationic surfactants, nonionic surfactants and / or zwitterionic surfactants. The surfactant may be present alone or in aggregates at a concentration of about 0.1 mg / ml to about 20 mg / ml. The pharmaceutical compositions comprising each of these specific surfactants constitute alternative embodiments of the present invention.

[0130] In another embodiment of the present invention, the pharmaceutical composition comprises one or more protease inhibitors, such as for example EDTA and / or benzamidine hydrochloride (HCl). The protease inhibitor may be present alone or in aggregates at a concentration of about 0.1 mg / ml to about 20 mg / ml. The pharmaceutical compositions comprising each of these specific protease inhibitors constitute alternative embodiments of the present invention.

[0131] The pharmaceutical composition of the present invention may comprise an amount of amino acid base sufficient to reduce the formation of polypeptide aggregates during storage of the composition. The term "amino acid base" refers to one or more amino acids (such as methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine) or their analogs. Any amino acid may be present in its free base form or its salt form. Any stereoisomer of the amino acid base may be present (i.e., L, D or a mixture thereof). The amino acid base may be present alone or in combination with other amino acid bases at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example about 0.1 mg / ml to about 20 mg / ml. The pharmaceutical compositions comprising each of these specific amino acid bases constitute alternative embodiments of the present invention.

[0132] The pharmaceutically acceptable salts of the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention include conventional non-toxic salts or quaternary ammonium salts formed from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, benzoate, benzenesulfonate, citrate, camphorate, dodecyl sulfate, hydrochloride, hydrobromide, lactate, maleate, mesylate, nitrate, oxalate, pivalate, propionate, succinate, sulfate, and tartrate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, organic base salts such as dicyclohexylamine salts, and salts of amino acids such as arginine. Additionally, basic nitrogen-containing groups can be quaternized with, for example, alkyl halides.

[0133] The pharmaceutical compositions of the present invention can be administered by any means that can achieve their intended purpose. Examples include administration by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, or ocular routes, etc. Administration by the oral route is possible. Preparations suitable for parenteral administration include aqueous solutions of the active conjugate in water-soluble form (e.g., water-soluble salts), acidic solutions, alkaline solutions, aqueous dextrose solutions, isotonic carbohydrate solutions, and cyclodextrin inclusion complexes.

[0134] The present invention also encompasses methods for preparing pharmaceutical compositions, which include mixing a pharmaceutically acceptable vehicle with any one of the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention. Additionally, the present invention includes pharmaceutical compositions prepared by mixing one or more pharmaceutically acceptable vehicles with any one of the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention.

[0135] Furthermore, the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention may have one or more polymorphic or amorphous crystalline forms and are thus intended to be included within the scope of the present invention. Additionally, the GLP1-GDF15 fusion protein and / or polynucleotide can form solvates (i.e., hydrates) with, for example, water or solvates with common organic solvents. As used herein, the term "solvate" means the physical association of the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate will be able to separate out when, for example, one or more solvent molecules are incorporated into the lattice of the crystalline solid. The term "solvate" is intended to cover both solution-phase solvates and separable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, etc.

[0136] The present invention is intended to encompass within its scope polymorphs and solvates of the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention. Accordingly, in the methods of treatment of the present invention, the term "administering" shall cover methods for using the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention, or a polymorph or solvate thereof, to treat, ameliorate or prevent the syndromes, disorders or diseases described herein, which, although not specifically disclosed, will clearly be included within the scope of the present invention.

[0137] In another embodiment, the present invention relates to the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention for use as a medicament.

[0138] The present invention includes within its scope prodrugs of the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention. Generally speaking, such prodrugs will be functional derivatives of the GLP1-GDF15 fusion protein and / or polynucleotide, which can be readily converted in vivo into the desired GLP1-GDF15 fusion protein and / or polynucleotide. Accordingly, in the methods of treatment of the present invention, the term "administering" shall cover treating the various disorders described with the specifically disclosed GLP1-GDF15 fusion protein and / or polynucleotide, or with a GLP1-GDF15 fusion protein and / or polynucleotide that may not be specifically disclosed but which is converted in vivo into the designated GLP1-GDF15 fusion protein and / or polynucleotide upon administration to a patient. Conventional methods for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", edited by H. Bundgaard, Elsevier, 1985.

[0139] In any method for preparing the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention, it may be necessary and / or desirable to protect any sensitive or reactive groups on any of the relevant molecules. This can be achieved by conventional protecting groups, such as Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; and Protective Groups in Organic Synthesis by T.W. Greene and P.G.M. Wuts, John Wiley & Sons, 1991, each of which is incorporated herein by reference in its entirety for all purposes. The protecting groups can be removed at a convenient subsequent stage using methods known in the art.

[0140] Use

[0141] The present invention relates to methods for preventing, treating or ameliorating GDF15 receptor (GDF15R, GFRAL)-mediated syndromes and / or GLP1 receptor-mediated syndromes, disorders or diseases in a subject in need thereof, the methods comprising administering to the subject in need thereof an effective amount of the GLP1-GDF15 fusion protein, polynucleotide and / or pharmaceutical composition of the present invention.

[0142] The present invention also provides methods for preventing, treating or ameliorating a disorder, disease or condition in a subject in need thereof, or any one or more symptoms of said disorder, disease or condition, or delaying the onset thereof, the methods comprising administering to the subject in need thereof an effective amount of the GLP1-GDF15 fusion protein, polynucleotide and / or pharmaceutical composition of the present invention.

[0143] According to specific embodiments, the disease, disorder or condition is selected from: obesity, type I or type II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unregulated cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease, eczema, sleep apnea, osteoarthritis, polycystic ovary syndrome, chronic kidney syndrome, depression and / or cancer.

[0144] According to specific embodiments, a therapeutically effective amount is a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or ameliorating the severity of or symptoms associated with a disease, disorder, or condition to be treated; (ii) reducing the duration of the treated disease, disorder, or condition or symptoms associated therewith; (iii) preventing the development of the treated disease, disorder, or condition or symptoms associated therewith; (iv) causing regression of the treated disease, disorder, or condition or symptoms associated therewith; (v) preventing the development or onset of the treated disease, disorder, or condition or symptoms associated therewith; (vi) preventing recurrence of the treated disease, disorder, or condition or symptoms associated therewith; (vii) reducing hospitalization of a subject having the treated disease, disorder, or condition or symptoms associated therewith; (viii) reducing the length of hospitalization of a subject having the treated disease, disorder, or condition or symptoms associated therewith; (ix) increasing survival of a subject having the treated disease, disorder, or condition or symptoms associated therewith; (xi) inhibiting or reducing the treated disease, disorder, or condition or symptoms associated therewith in a subject; (xii) enhancing or improving the prophylactic or therapeutic effect of another therapy. and / or (xiii) improving the quality of life of a subject having a disease, disorder, or condition to be treated.

[0145] The therapeutically effective amount or dose can vary depending on various factors such as the disease, disorder, or condition to be treated, the mode of administration, the target site, the physiological state of the subject (including e.g., age, weight, health), whether the subject is human or animal, other drugs administered, and whether it is a prophylactic or therapeutic treatment. The therapeutic dose is optimally titrated to optimize safety and efficacy.

[0146] As used herein, the terms “treatment,” “treating,” and “therapy” are each intended to mean an improvement or reversal of at least one measurable physical parameter associated with a disease, disorder, or condition, which may not necessarily be recognizable in a subject but is capable of being recognized in a subject. The terms “treating” and “treatment” can also refer to causing regression, preventing development, or at least delaying the development of a disease, disorder, or condition. In one specific embodiment, “treatment,” “treating,” and “therapy” mean alleviating, preventing development or onset, or shortening the duration of one or more symptoms associated with a disease, disorder, or condition. In certain embodiments, “treating” and “treatment” mean preventing recurrence of a disease, disorder, or condition. In certain embodiments, “treating” and “treatment” mean increasing survival of a subject having a disease, disorder, or condition. In certain embodiments, “treating” and “treatment” mean elimination of a disease, disorder, or condition in a subject.

[0147] In one embodiment, the present invention provides a method for preventing, treating or improving obesity or any one or more symptoms of obesity, or delaying its onset, in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the GLP1-GDF15 fusion protein, polynucleotide and / or pharmaceutical composition of the present invention. In some embodiments, the weight of the subject is reduced by, for example, between about 0.01% and about 0.1%, between about 0.1% and about 0.5%, between about 0.5% and about 1%, between about 1% and about 5%, between about 2% and about 3%, between about 5% and about 10%, between about 10% and about 15%, between about 15% and about 20%, between about 20% and about 25%, between about 25% and about 30%, between about 30% and about 35%, between about 35% and about 40%, between about 40% and about 45% or between about 45% and about 50% relative to the subject's body weight prior to administration of any one of the GLP1-GDF15 fusion protein, polynucleotide, pharmaceutical composition, form or drug of the present invention described herein, or compared to a control subject not receiving any one of the GLP1-GDF15 fusion protein, polynucleotide, composition, form or drug of the present invention described herein.

[0148] In some embodiments, the reduction in weight persists for, for example, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years or about 20 years.

[0149] The present invention provides a method for preventing, treating or improving a syndrome, disorder or disease, or any one or more symptoms of said syndrome, disorder or disease, or delaying its onset, in a subject in need thereof, wherein the syndrome, disorder or disease is selected from: obesity, type I or type II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unregulated cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and eczema, the method comprising administering to the subject in need thereof an effective amount of the GLP1-GDF15 fusion protein, polynucleotide and / or pharmaceutical composition of the present invention.

[0150] As used herein, metabolic syndrome refers to a subject having one or more of any of the following: hyperglycemia (e.g., high fasting blood glucose), hypertension, abnormal cholesterol levels (e.g., low HDL levels), abnormal triglyceride levels (e.g., high triglycerides), large waist circumference, increased abdominal area fat, insulin resistance, glucose intolerance, elevated C-reactive protein levels (i.e., a pro-inflammatory state), and increased plasma plasminogen activator inhibitor-1 and fibrinogen content (i.e., a pre-thrombotic state).

[0151] The present invention provides a method of reducing food intake in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the GLP1-GDF15 fusion protein, polynucleotide, and / or pharmaceutical composition of the present invention. In some embodiments, the food intake of the subject is reduced, for example, by between about 0.01% and about 0.1%, between about 0.1% and about 0.5%, between about 0.5% and about 1%, between about 1% and about 5%, between about 2% and about 3%, between about 5% and about 10%, between about 10% and about 15%, between about 15% and about 20%, between about 20% and about 25%, between about 25% and about 30%, between about 30% and about 35%, between about 35% and about 40%, between about 40% and about 45%, or between about 45% and about 50% relative to the food intake of the subject prior to administering any one of the GLP1-GDF15 fusion protein, polynucleotide, composition, form, drug, or combination of the present invention described herein, or compared to a control subject not receiving any one of the GLP1-GDF15 fusion protein, polynucleotide, composition, form, drug, or combination of the present invention described herein.

[0152] In some embodiments, the reduction in food intake persists for, for example, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, or about 20 years.

[0153] The present invention provides methods for reducing glycated hemoglobin (A1C) in a subject in need thereof, the methods comprising administering to the subject in need thereof an effective amount of the GLP1-GDF15 fusion protein, polynucleotide, and / or pharmaceutical composition of the present invention. In some embodiments, relative to the A1C of the subject prior to administering any one of the GLP1-GDF15 fusion protein, polynucleotide, composition, form, drug, or combination of the present invention described herein, or compared to a control subject not receiving any one of the GLP1-GDF15 fusion protein, polynucleotide, composition, form, drug, or combination of the present invention described herein, the A1C of the subject is reduced, for example, by about between about 0.001% and about 0.01%, between about 0.01% and about 0.1%, between about 0.1% and about 0.2%, between about 0.2% and about 0.3%, between about 0.3% and about 0.4%, between about 0.4% and about 0.5%, between about 0.5% and about 1%, between about 1% and about 1.5%, between about 1.5% and about 2%, between about 2% and about 2.5%, between about 2.5% and about 3%, between about 3% and about 4%, between about 4% and about 5%, between about 5% and about 6%, between about 6% and about 7%, between about 7% and about 8%, between about 8% and about 9%, or between about 9% and about 10%.

[0154] In other embodiments, methods are provided for reducing the fasting blood glucose level in a subject in need thereof, the methods comprising administering to the subject in need thereof an effective amount of the GLP1-GDF15 fusion protein, polynucleotide, and / or pharmaceutical composition of the present invention. Relative to the fasting blood glucose level of the subject prior to administering any one of the GLP1-GDF15 fusion protein, polynucleotide, composition, form, drug, or combination of the present invention described herein, or compared to a control subject not receiving any one of the GLP1-GDF15 fusion protein, polynucleotide, composition, form, drug, or combination of the present invention described herein, the fasting blood glucose level can be reduced to less than about 140 mg / dL to about 150 mg / dL, less than about 140 mg / dL to about 130 mg / dL, less than about 130 mg / dL to about 120 mg / dL, less than about 120 mg / dL to about 110 mg / dL, less than about 110 mg / dL to about 100 mg / dL, less than about 100 mg / dL to about 90 mg / dL, or less than about 90 mg / dL to about 80 mg / dL.

[0155] The present invention provides methods for modulating GLP1 receptor activity and GDF15 receptor activity in a subject in need thereof, the methods comprising administering to the subject in need thereof an effective amount of a GLP1-GDF15 fusion protein, polynucleotide, and / or pharmaceutical composition of the present invention. As used herein, "modulating" refers to increasing or decreasing receptor activity.

[0156] In some embodiments, an effective amount of a GLP1-GDF15 fusion protein and / or polynucleotide of the present invention or a form, composition, or medicament thereof is administered once daily, twice daily, three times daily, four times daily, five times daily, six times daily, seven times daily, or eight times daily to a subject in need thereof. In other embodiments, an effective amount of a GLP1-GDF15 fusion protein and / or polynucleotide of the present invention or a form, composition, or medicament thereof is administered once every other day, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, twice a month, three times a month, or four times a month to a subject in need thereof.

[0157] Another embodiment of the present invention includes a method for preventing, treating, or ameliorating a disease, disorder, or syndrome in a subject in need thereof or one or more symptoms of any of the diseases, disorders, or syndromes, or delaying its onset, the method comprising administering to the subject in need thereof an effective amount of a GLP1-GDF15 fusion protein, polynucleotide, and / or pharmaceutical composition of the present invention in the form of combination therapy. In certain embodiments, the combination therapy is a second therapeutic agent. In certain embodiments, the combination therapy is a surgical therapy.

[0158] As used herein, in the context of administering two or more therapies to a subject, the term "combination" refers to the use of more than one therapy.

[0159] As used herein, combination therapy refers to administering to a subject in need thereof an effective amount of a GLP1-GDF15 fusion protein and / or polynucleotide of the present invention or a form, composition, or medicament thereof, while administering one or more additional therapeutic agents, or one or more surgical therapies. In some embodiments, one or more additional therapeutic agents or surgical therapies may be administered on the same day as an effective amount of a GLP1-GDF15 fusion protein and / or polynucleotide of the present invention, and in other embodiments, one or more additional therapeutic agents or surgical therapies may be administered in the same week or the same month as an effective amount of a GLP1-GDF15 fusion protein and / or polynucleotide of the present invention.

[0160] The present invention also contemplates using combination therapies to prevent, treat, or ameliorate any one of the diseases, disorders, syndromes, or symptoms described herein in a subject in need thereof, or delay their onset, the combination therapy comprising administering to a subject in need thereof an effective amount of the GLP1-GDF15 fusion protein, polynucleotide, and / or pharmaceutical composition of the present invention in combination with any one or more of the following therapeutic agents: dipeptidyl peptidase-4 (DPP-4) inhibitors (e.g., sitagliptin, saxagliptin, linagliptin, alogliptin, etc.); sodium-glucose cotransporter-2 (SGLT-2) inhibitors (e.g., canagliflozin, dapagliflozin, empagliflozin); bile acid multivalent chelators (e.g., colesevelam, etc.); dopamine receptor agonists (e.g., immediate release bromocriptine); biguanides (e.g., metformin, etc.); insulin; oxyntomodulin; sulfonylureas (e.g., chlorcyclopyramide, glimepiride, glipizide, glibornuride, glibenclamide, gliboruride, gliquidone, glisoxepide, tolazamide, tolbutamide, acetohexamide, carbutamide, etc.); and thiazolidinediones (e.g., pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, nateglinide, liraglitazone, troglitazone, etc.). In some embodiments, when provided in combination with the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention, the dosage of the additional therapeutic agent is reduced. In some embodiments, when used in combination with the GLP1-GDF15 fusion protein and / or polynucleotide of the present invention, the additional therapeutic agent can be used at a lower dosage than when used alone.

[0161] The present invention contemplates using combination therapies to prevent, treat, or ameliorate any one of the diseases, disorders, syndromes, or symptoms described herein in a subject in need thereof, or delay their onset, the combination therapy comprising administering to a subject in need thereof an effective amount of the GLP1-GDF15 fusion protein, polynucleotide, and / or pharmaceutical composition of the present invention in combination with a surgical therapy. In certain embodiments, the surgical therapy can be bariatric surgery (e.g., gastric bypass surgery, such as biliopancreatic diversion with duodenal switch surgery; sleeve gastrectomy; adjustable gastric banding; biliopancreatic diversion; intragastric balloon; gastric plication; and combinations thereof).

[0162] In embodiments in which one or more additional therapeutic agents or surgical procedures are administered on the same day as an effective amount of the GLP1-GDF15 fusion protein and / or polynucleotide of the invention, the GLP1-GDF15 fusion protein and / or polynucleotide of the invention can be administered before, after, or simultaneously with the additional therapeutic agent or surgical procedure. The use of the term "combination" does not limit the order in which the therapies are administered to a subject. For example, the first therapy (e.g., the compositions described herein) can be administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to) the second therapy being administered to the subject, simultaneously therewith, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after).

[0163] Example

[0164] Exemplary fusion proteins of the invention (GLP1-sFc-GDF15 and G4-M8) have amino acid sequences as set forth in SEQ ID NO: 18 and SEQ ID NO: 33, respectively.

[0165] Example 1: Expression and Purification of Fusion Proteins

[0166] The fusion proteins were expressed in HEK Expi293F (ThermoFisher Scientific, #A14527).

[0167] For expression in HEK Expi293F, plasmids encoding the fusion proteins were transfected into the cells by transient transfection according to the manufacturer's recommendations. Briefly, HEK Expi293F cells were passaged by suspension culture in Expi293 expression medium Union-293 (Yonglian Biotechnology Co., Ltd., #UP1000) in an orbital incubator (Zhichu Instruments Co., Ltd., #ZQZY-98B) set at 37 °C, 5% CO2, and 125 RPM. On the day of transfection, the cells were diluted to 2.5×10 per milliliter 6Cells were maintained with a viability of over 95%. The plasmid and the transfection reagent PEI MAX 40K (Polysciences, #24765 - 100) were diluted separately using Opti-MEM medium (Gibco, #31985070). 2.5 μL of the transfection reagent was used for every 1 μg of the plasmid. While gently vortexing the tube containing the plasmid solution, the diluted transfection reagent PEI MAX 40K was added dropwise to the tube, mixed well, and allowed to stand for 20 minutes, then added to HEK Expi293F cells. The cells were cultured for 5 days under the conditions of 37 °C, 5% CO2, and shaking at 125 RPM, and then the culture supernatant was harvested.

[0168] The culture supernatant was collected by centrifugation at 4000×g for 10 minutes and purified in two steps using an affinity purification packing protein G resin (GenScript, #L00209) and a HiTrap Q FF anion exchange chromatography column (Cytiva, #17505301). The specific steps were as follows: 1 mL of the protein G resin was pipetted into an empty chromatography column (BIORAD, #7326008), and 20 mL of a pH 7.4 PBS equilibration system was added; after loading the centrifuged culture supernatant onto the protein G resin, 30 mL of a pH 7.4 PBS equilibration buffer was added to the column to wash away the impurity proteins; 15 mL of an elution buffer (0.1 M glycine, pH 2.0) was added for elution and the eluate was collected; immediately, 1.5 mL of a neutralization buffer (1 M Tris-HCL, pH 8.0) was added to the collected eluate. The HiTrap Q FF anion exchange chromatography column was equilibrated with 20 mL of a buffer (20 mM Tris pH 8.0), the elution sample collected in the previous step was loaded onto the anion exchange chromatography column, then the anion exchange chromatography column was equilibrated again with 15 mL of a buffer (20 mM Tris pH 8.0), a linear gradient elution was performed using 20 mM Tris pH 8.0 (0 - 100% 1 M NaCl), and the elution peak was collected. Finally, the collected protein was exchanged into a storage buffer (PBS, pH 7.4).

[0169] The protein was subjected to SDS-PAGE (SmartPage TMPurity analysis of Precast Protein Gel Plus, Tian Di Ren He Biotechnology Company, #SLE020) and SEC (TSK-GEL G3000SW, Tosoh Bioscience, #0005789). For SDS-PAGE, 5 μg of protein sample was added to 5× reducing loading buffer (Cwbio, #CW0027), mixed well, boiled in boiling water for 5 - 10 minutes and then loaded onto the gel. The electrophoresis was carried out at 120 V for 45 minutes. For SEC, the chromatography conditions were as follows: column temperature 25°C, mobile phase 0.1 mol / L phosphate buffer solution + 0.1 mol / L sodium sulfate (pH 7.2), flow rate 0.5 mL / min, isocratic elution mode, injection volume 5 μg, detection wavelength 280 nm.

[0170] The SDS-PAGE results showed that the bands of the fusion protein of the present invention and the reference LY3463251 were both near 55 kDa. The band position of the fusion protein of the present invention was greater than that of LY3463251, which was close to the theoretical monomer molecular weight of 43 kD (see Figure 1 ). The SEC results showed a single peak, indicating the homogeneity of the protein components (see Figure 2 ). Eukaryotic soluble expression achieved a yield of milligrams per liter.

[0171] Example 2: Detection of the thermal stability of the fusion protein by differential scanning fluorimetry (DSF)

[0172] The fluorescent dye SYPRO Orange (Invitrogen, #S5692) was diluted to 25X with PBS and mixed with 5 μg of protein sample at a ratio of 1:24. The total volume was 25 μL and added to a 96-well plate (BIORAD, #MLL9601). The above-prepared system was placed in a fluorescence quantitative PCR instrument (QuantStudio 6Flex, Applied Biosystems, #4485692), and the settings were as follows: excitation wavelengths, 300 nm and 470 nm; emission wavelength, 570 nm; heating rate, 1°C / 10 s; temperature range, from 25°C to 100°C; fluorescence intensity was measured every 1°C. A curve of fluorescence intensity versus temperature was generated by the system data processing software QuantStudio Software v1.6.1, and the Tm value of the protein sample was calculated.

[0173] The results showed that the Tm value of the fusion protein was 57.1°C (see Figure 3 ).

[0174] Example 3: ELISA detection of the binding of the fusion protein to GLP-1R and GFRAL

[0175] Dilute GLP-1R protein (ACRO, #GLR-H82E3) with PBS to 2 μg / mL, add it to a 96-well plate (Costar, #3690), and incubate overnight at 4°C. The next day, wash the plate three times with 0.05% PBST, block it with 2% BSA-PBS for 1 hour, wash the plate three times with 0.05% PBST, add serially diluted fusion proteins GLP1-sFc-GDF15 (starting from 500 nM, serially diluted 7 times in triplicate to form 8 concentration points) and G4-M8 (starting from 2000 nM, serially diluted 9 times in triplicate to form 10 concentration points), incubate at 37°C for 1.5 hours, wash the plate five times with 0.05% PBST, add HRP-labeled goat anti-human IgG Fc secondary antibody (Sino Biological, #SSA001) diluted 1:10000, incubate at 37°C for 45 minutes, wash the plate five times with 0.05% PBST, develop color for 10 minutes, and after termination, read the OD450 value with SPECTRAMAX i3X (MOLECULAR DEVICES). Plot a graph using the 4-parameter Logistic fitting method based on the OD450 values and concentrations of the 8 or 10 concentration points, and calculate the sample EC 50 The EC50 value of the binding activity between the fusion protein G4-M8 and GLP-1R protein of the present invention is 26.75 nM, and the results are shown in Figure 4A and Figure 4B .

[0176] Dilute GFRAL protein (ACRO, #GFA-H52H3) with PBS to 2 μg / mL, add it to a 96-well plate (Costar, #3690), and incubate overnight at 4°C. The next day, wash the plate three times with 0.05% PBST, block it with 2% BSA-PBS for 1 hour, wash the plate three times with 0.05% PBST, add serially diluted fusion proteins GLP1-sFc-GDF15 (starting from 20 nM, serially diluted 7 times in triplicate to form 8 concentration points) and G4-M8 (starting from 200 nM, serially diluted 9 times in triplicate to form 10 concentration points), incubate at 37°C for 1.5 hours, wash the plate five times with 0.05% PBST, add HRP-labeled goat anti-human IgG Fc secondary antibody (Sino Biological, #SSA001) diluted 1:10000, incubate at 37°C for 45 minutes, wash the plate five times with 0.05% PBST, develop color for 10 minutes, and after termination, read the OD450 value with SPECTRAMAX i3X (MOLECULAR DEVICES). Plot a graph using the 4-parameter Logistic fitting method based on the OD450 values and concentrations of the 8 concentration points, and calculate the sample EC 50 . The results show that the EC 50 values of the binding activity between the fusion protein of the present invention and GFRAL protein are 0.05986 nM and 0.348 nM ([[]] Figure 5A ​and Figure 5B ) is stronger than the control LY3463251EC 50 value of 0.5169 nM ( Figure 5C ).

[0177] Example 4: Determination of the affinity (K D value) of the fusion protein for GFRAL and GLP-1R by Biolayer Interferometry (BLI) technology

[0178] Dilute the GFRAL protein (ACRO, #GFA-H52H3) or GLP-1R protein (ACRO, #GLR-H82E3) to 5 μg / mL with sample dilution buffer (PBS solution containing 0.02% Tween-20 and 0.1% BSA), add it to a black 96-well plate (Greiner bio-one, #655209), place it in an Octet RED96e Protein Interaction Analyzer (Sartorius), and set up the program in Dataacquisition 9 software ( BLI Systems Software) to couple to an NTA sensor (Sartorius, #18-0029). After the signal value reaches 1.0, perform the binding step. Set the starting concentration to 1 μM, add seven protein samples with a three-fold gradient concentration, and set the last point to a 0 concentration point. Bind for 300 s. The dissociation step is set to dissociate with the sample dilution buffer for 300 s. Use Data analysis software for data analysis. Use the sensor corresponding to the 0 concentration point to subtract the background, calculate the binding values of each concentration sample as the ordinate, use the concentration as the abscissa, and fit in a steady-state analysis 1:1 binding mode to calculate K D value.

[0179] The results show that the affinity of the fusion protein of the present invention for GFRAL at the GDF15 end is comparable to that of LY346325 (1.708E-09 M vs. 1.36E-09 M) (see Figure 6A and Figure 6B ), and the affinity for GLP-1R at the GLP1 end is higher than that of semaglutide (7.827E-09 M vs. 3.713E-08 M) (see Figure 7A and Figure 7B ).

[0180] Example 5: Biological activity of the fusion protein

[0181] Seed the hGDF / GFRAL&RET effector reporter cells (Nanjing Kebai, #CBP74186) in a 96-well white flat-bottom plate at 3x10 5Seed the cells at a density of 5×10 cells / mL in a 96-well white flat-bottom plate and culture overnight at 37°C. The next day, add different concentrations of the protein or control protein (starting concentration of 12 nM, serially diluted 3-fold, a total of 11 concentration gradients) to the hGDF / GFRAL&RET effector reporter cells using experimental buffer (Opti-MEM + 0.5% FBS), and co-incubate in a 37°C incubator for 5 hours. Finally, add 100 μL of Ultra Luciferase Detection Kit luciferase detection reagent (Cobioer, #CBPH0001) to the cell-protein mixture system, and read the Luciferase fluorescence signal value using a multimode microplate reader (SpectraMax i3x, Molecular Devices). Plot a graph using the 4-parameter Logistic fitting method based on the read signal values and corresponding concentrations.

[0182] Seed the target cells, 293GLP1R B3 cells (stably expressing GLP1R and CRE-luc2P), in a 96-well white flat-bottom plate at a density of 5×10 5 cells / mL; add different concentrations of the protein GLP1-sFc-GDF15 and control protein Semaglutide (starting concentration of 66.7 nM, serially diluted 3-fold, a total of 10 concentration gradients) or G4-M8 and control protein Semaglutide (starting concentration of 222.33 nM, serially diluted 3-fold, a total of 11 concentration gradients) to the target cells using experimental buffer (DMEM + 10% FBS), and co-incubate in a 37°C incubator for 5 hours. Finally, add ONE-Lite luciferase detection reagent (Vazyme, #1203-03) to the cell-protein mixture system and detect the chemiluminescence signal using a multimode microplate reader (SpectraMax i3x). Plot a graph using the 4-parameter Logistic fitting method based on the read signal values and corresponding concentrations.

[0183] The results showed that the biological activity of the fusion protein of the present invention at the GDF15 end was comparable to that of the control molecule LY346325 (see Figure 8A and 8B ), and the biological activity at the GLP1 end was comparable to that of the control molecule semaglutide (see Figure 8C and 8D ).

[0184] Example 6: Experiment on high-fat diet-induced obesity (DIO) mice

[0185] From the 5th week, 60% high-fat diet (Research Diets, #d12492) was fed. After 25-week-old DIO male C57BL / 6 mice weighing more than 45 g (Jicuiyaokang) entered the animal facility, they were adaptively fed for 3 days or more, and then randomly grouped with body weight as the main grouping index. They were divided into three groups, with 6 animals in each group and 3 animals in each cage. The G1 vehicle group was subcutaneously injected with PBS every other day (Q2D), the G2 control group was subcutaneously injected with 60 nmol / kg semaglutide (Novo Nordisk, Ozempic) daily (QD), the G3 control group was subcutaneously injected with 60 nmol / kg LY3463251 every other day (Q2D), and the G4 drug group was subcutaneously injected with 60 nmol / kg of the fusion protein of the present invention every other day (Q2D). The injection volume was 5 μL / g of mouse body weight for 21 days. The food intake (measured per cage) and body weight (BW) were measured every day. Fasting blood glucose, triglyceride, cholesterol, and insulin were measured on the last day.

[0186] Body weight on the nth day - Body weight on the 1st day

[0187] Body weight change [%] = —————————————— x 100

[0188] Body weight on the 1st day

[0189] The cumulative food intake on the nth day represents the total food intake from the 1st day to the nth day.

[0190] The results showed that the average weight loss effect of the G4 drug group (the fusion protein of the present invention) was significantly better than that of the G2 control group (semaglutide) (see Figure 9 ). In terms of food intake, the G4 drug group was significantly less than the G1 vehicle group, but slightly more than the G2 control group (see Figure 10 ). The fasting (6-hour) blood glucose of the mice was also measured (see Figure 11 ), and the fasting blood glucose at the end point showed that the G4 drug group was lower than the G2 control group (6.8 vs. 7.2, mmol / L).

Claims

1. A fusion protein comprising, from N-terminus to C-terminus, a GLP-1 peptide, an optional first linker peptide, an Fc monomer polypeptide, an optional second linker peptide, and a GDF15 polypeptide.

2. The fusion protein according to claim 1, wherein The GLP-1 peptide comprises, preferably consists of, an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 and 34, or an amino acid sequence truncated by 1 to 5 amino acids at the C-terminus of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 and 34.

3. The fusion protein according to claim 1 or 2, wherein The Fc monomer polypeptide comprises, preferably consists of, an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-15, and is capable of binding to a neonatal Fc receptor (FcRn), or, The Fc monomer polypeptide comprises an amino acid sequence comprising arginine (R), histidine (H), lysine (K) and threonine (T) at positions 366, 368, 395 and 409 according to the EU numbering method, respectively, relative to a native Fc sequence (particularly IgG Fc, more particularly IgG1 Fc, IgG2 Fc, IgG3 Fc or IgG4 Fc, such as any one of SEQ ID NOs: 28-31), and is capable of binding to a neonatal Fc receptor (FcRn), or, The Fc monomer polypeptide comprises a polypeptide corresponding to a native Fc sequence (particularly IgG Fc, more particularly IgG1 Fc, IgG2 Fc, IgG3 Fc or IgG4 Fc, such as SEQ ID NO: 28-31 any one) comprises, consists essentially of, or consists of an amino acid sequence having an amino acid substitution at one or more of positions 351, 366, 368, 395, 409 and / or 428 according to the EU numbering system, respectively, wherein position 351 is substituted with S, position 366 is substituted with C, G, S, L, N, D, F, I, V, Y, Q, K, E, M or R, position 368 is substituted with H, Q, N, D, Y, R, C, G, S, F, T, I, V, A, K or M, position 395 is substituted with N, T, I, S, M, Q, R, L, G, V, A, E, D, Y, F, H or K, position 409 is substituted with N, S, I, M, E, Q, L, V, A, H, D, Y, F or T, and position 428 is substituted with Y, and is capable of binding to neonatal Fc receptor (FcRn).

4. A fusion protein as claimed in any one of the preceding claims, wherein The GDF15 polypeptide comprises, and preferably consists of, an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8.

5. The fusion protein according to any one of the preceding claims, comprising the first linker peptide, preferably, the first linker peptide comprises the amino acid sequence G n Or consisting of it, n is an integer from 1 to 15, for example 4.

6. The fusion protein according to any one of the preceding claims, comprising the second linker peptide, preferably, the second linker peptide comprises the amino acid sequence G p (AP) m G q or consisting thereof, p and q are independently integers from 1 to 15, such as 4, and m is an integer from 1 to 25, such as 10.

7. The fusion protein of claim 1, comprising or consisting of an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-27, 32 and 33. An oligonucleotide encoding the fusion protein according to any one of claims 1 to 7.

9. An expression vector comprising the oligonucleotide according to claim 8.

10. A host cell comprising the oligonucleotide according to claim 8 or the expression vector according to claim 9.

11. A method for producing the fusion protein according to any one of claims 1 to 7, comprising culturing the host cell according to claim 10 under conditions suitable for the expression of the fusion protein according to any one of claims 1 to 7.

12. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 7, the oligonucleotide according to claim 8, the expression vector according to claim 9 or the host cell according to claim 10, and a pharmaceutically acceptable medium.

13. A method of treating a disease, disorder or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the fusion protein of any one of claims 1-7, the oligonucleotide of claim 8, the expression vector of claim 9, the host cell of claim 10 or the pharmaceutical composition of claim 12, optionally the disease, disorder or condition is selected from obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and eczema.

Images